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Journal of Nanomaterials
Biocompatibility
and Toxicity of
Nanobiomaterials
Guest Editors: Xiaoming Li, Sang Cheon Lee, Shuming Zhang,
and Tsukasa Akasaka
Biocompatibility and Toxicity of

Nanobiomaterials
Biocompatibility and Toxicity of

Nanobiomaterials
Guest Editors: Xiaoming Li, Sang Cheon Lee, Shuming Zhang,
and Tsukasa Akasaka
Copyright 2012 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in Journal of Nanomaterials. All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.
Editorial Board
Katerina Aifantis, Greece
Nageh K. Allam, USA
Margarida Amaral, Portugal
Xuedong Bai, China
L. Balan, France
Enrico Bergamaschi, Italy
Theodorian Borca-Tasciuc, USA
C. Jerey Brinker, USA
Christian Brosseau, France
Xuebo Cao, China
Shaul Chowdhury, USA
Kwang-Leong Choy, UK
Cui ChunXiang, China
Miguel A. Correa-Duarte, Spain
Shadi A. Dayeh, USA
Claude Estournes, France
Alan Fuchs, USA
Lian Gao, China
Russell E. Gorga, USA
Hongchen Chen Gu, China
Mustafa O. Guler, Turkey
John Zhanhu Guo, USA
Smrati Gupta, Germany
Michael Harris, USA
Zhongkui Hong, USA
Michael Z. Hu, USA
David Hui, USA
Y.-K. Jeong, Republic of Korea
Sheng-Rui Jian, Taiwan
Wanqin Jin, China
Rakesh K. Joshi, India
Zhenhui Kang, China
Fathallah Karimzadeh, Iran
Alireza Khataee, Iran
Do Kyung Kim, Korea

Kin Tak Lau, Australia
Burtrand Lee, USA
Benxia Li, China
Jun Li, Singapore
Shijun Liao, China
Gong Ru Lin, Taiwan
J.-Y. Liu, USA
Jun Liu, USA
Xing-Jie Liang, China
Tianxi Liu, China
Songwei Lu, USA
Daniel Lu, China
Jue Lu, USA
Ed Ma, USA
Gaurav Mago, USA
Santanu K. Maiti, Israel
Sanjay R. Mathur, Germany
A. McCormick, USA
Vikas Mittal, UAE
Weihai Ni, Germany
Sherine Obare, USA
Abdelwahab Omri, Canada
Edward Andrew Payzant, USA
Kui-Qing Peng, China
Anukorn Phuruangrat, Thailand
Ugur Serincan, Turkey
Huaiyu Shao, Japan
Donglu Shi, USA
Suprakas Sinha Ray, South Africa
Vladimir Sivakov, Germany
Marinella Striccoli, Italy
Bohua Sun, South Africa
Saikat Talapatra, USA
Nairong Tao, China

Titipun Thongtem, Thailand
Somchai Thongtem, Thailand
Alexander Tolmachev, Ukraine
Valeri P. Tolstoy, Russia
Tsung-Yen Tsai, Taiwan
Takuya Tsuzuki, Australia
Raquel Verdejo, Spain
Mat U. Wahit, Malaysia
Shiren Wang, USA
Yong Wang, USA
Ruibing Wang, Canada
Cheng Wang, China
Zhenbo Wang, China
Jinquan Wei, China
Ching Ping Wong, USA
Xingcai Wu, China
Guodong Xia, Hong Kong
Zhi Li Xiao, USA
Ping Xiao, UK
Shuangxi Xing, China
Yangchuan Xing, USA
N. Xu, China
Doron Yadlovker, Israel
Ying-Kui Yang, China
Khaled Youssef, USA
William W. Yu, USA
Kui Yu, Canada
Haibo Zeng, China
Tianyou Zhai, Japan
Renyun Zhang, Sweden
Yanbao Zhao, China
Lianxi Zheng, Singapore
Chunyi Zhi, Japan
Contents
Biocompatibility and Toxicity of Nanobiomaterials, Xiaoming Li, Sang Cheon Lee, Shuming Zhang,
and Tsukasa Akasaka
Volume 2012, Article ID 591278, 2 page
Properties of Surfactin C-15 Nanopeptide and Its Cytotoxic Eect on Human Cervix Cancer (HeLa) Cell
Line, Zahra Nozhat, Asadollah Asadi, and Saber Zahri
In Vitro Cytotoxicity Assay on Gold Nanoparticles with Dierent Stabilizing Agents,
S. Vijayakumar and S. Ganesan
Biocompatibility and Toxicity of Magnetic Nanoparticles in Regenerative Medicine, H. Markides,
M. Rotherham, and A. J. El Haj
Silver Nanoparticles in Alveolar Bone Surgery Devices, Stefano Sivolella, Edoardo Stellini, Giulia Brunello,
Chiara Gardin, Letizia Ferroni, Eriberto Bressan, and Barbara Zavan
In Vitro Biocompatibility of Endodontic Sealers Incorporating Antibacterial Nanoparticles,
Itzhak Abramovitz, Nurit Beyth, Guy Weinberg, Alon Borenstein, David Polak, Dana Kesler-Shvero,
and Yael Houri-Haddad
Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929
Cell Line, Hui Peng, Xuehui Zhang, Yan Wei, Wentao Liu, Shenglin Li, Guangyan Yu, Xin Fu, Tong Cao,
and Xuliang Deng
Toxicity of Aqueous Fullerene nC60 to Activated Sludge: Nitrication Inhibition and Microtox Test,
Yongkui Yang, Norihide Nakada, Ryoji Nakajima, Chao Wang, and Hiroaki Tanaka
The Human Stratum Corneum Prevents Small Gold Nanoparticle Penetration and Their Potential Toxic
Metabolic Consequences, David C. Liu, Anthony P. Raphael, Daniel Sundh, Jerey E. Grice, H. Peter Soyer,
Michael S. Roberts, and Tarl W. Prow
Control of In Vivo Transport and Toxicity of Nanoparticles by Tea Melanin, Yu-Shiun Chen,
Yao-Ching Hung, Meng-Yeng Hong, Andrei Aleksandrovich Onischuk, Jin Chern Chiou, Irina V. Sorokina,
Tatiana Tolstikova, and G. Steve Huang
Biocompatibility and Toxicity of Nanoparticles and Nanotubes, Xiaoming Li, Lu Wang, Yubo Fan,
Qingling Feng, and Fu-zhai Cui
Biocompatibility of Nanoporous TiO2 Coating on NiTi Alloy Prepared via Dealloying Method,
Jin Huang, Junqiang Wang, Xiangdong Su, Weichang Hao, Tianmin Wang, Yayi Xia, Guozu Da,
and Yubo Fan
Noninvasive Evaluation of Injectable Chitosan/Nano-Hydroxyapatite/Collagen Scaold via Ultrasound,
Yan Chen, Songjian Li, Xiaoming Li, Yichen Zhang, Zhi Huang, Qingling Feng, Zhilai Zhou, Bomiao Lin,
and Bo Yu
Biocompatible Single-Crystal Selenium Nanobelt Based Nanodevice as a Temperature-Tunable
Photosensor, Yongshan Niu, Aimiao Qin, Wei Song, Menghang Wang, Xuenan Gu, Yangfei Zhang, Min Yu,
Xiaoguang Zhao, Ming Dai, Ling Yan, Zhou Li, and Yubo Fan
In Vitro Biocompatibility and Osteoblast Dierentiation of an Injectable
Chitosan/Nano-Hydroxyapatite/Collagen Scaold, Yan Chen, Zhi Huang, Xiaoming Li, Songjian Li,
Zhilai Zhou, Yichen Zhang, Qing ling Feng, and Bo Yu
Nanotechnology-Based Therapies for Skin Wound Regeneration, Ilaria Tocco, Barbara Zavan,
Franco Bassetto, and Vincenzo Vindigni
Evaluation on Cartilage Morphology after Intra-Articular Injection of Titanium Dioxide Nanoparticles
in Rats, Jiangxue Wang, Yu Gao, Ying Hou, Feng Zhao, Fang Pu, Xiaoyu Liu, Zhihong Wu, and Yubo Fan
Hindawi Publishing Corporation

Volume 2012, Article ID 591278, 2 pages
doi:10.1155/2012/591278
Editorial
Biocompatibility and Toxicity of Nanobiomaterials
Xiaoming Li,1 Sang Cheon Lee,2 Shuming Zhang,3 and Tsukasa Akasaka4
1 Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering,
Beihang University, Beijing 100191, China

of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea
3 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
4 Department of Biomedical Materials and Engineering, Hokkaido University, Sapporo 060-8586, Japan
2 Department
Correspondence should be addressed to Xiaoming Li, x.m.li@hotmail.com

Received 28 November 2012; Accepted 28 November 2012
Copyright 2012 Xiaoming Li et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The nanodimensionality of nature has logically given rise

to the interest in using nanomaterials in biomedical eld
[13]. Over the past decades, nanobiomaterials have played
signicant roles in the elds of biomedical engineering and
pharmaceutics. The wide variety of nanobiomaterials has
encouraged their use in applications including drug delivery
systems, imaging systems for diagnosis, tissue engineering,
and dental/bone implant. For successful clinical applications,
the investigations into biocompatibility and toxicity of
nanobiomaterials are research interests of great signicances.
The same unique physical and chemical properties that
may be benecial in nanobiomaterials may be associated
with potentially deleterious eects on human health. For
example, nanobiomaterials designed for drug delivery deliberately overcome biological barriers or they may cross them
unintentionally as a result of environmental exposure. Once
they have been taken up, they can potentially be deposited
in any region of the body. Due to their small size or specic
functionalization, nanobiomaterials may cross important
biological barriers and cause toxicity to very sensitive systems
such as the brain or the developing fetus. Nanobiomaterials
can be transported through the blood and accumulate in
secondary target tissues and organs such as liver, spleen,
kidney, placenta, the cardiovascular system, and central
nervous system (CNS), where they may cause adverse eects
[46]. But on the other hand, some nanobiomaterials have
been found to be able to concentrate more proteins including
specic proteins and these proteins might not only improve
cell attachment and proliferation but also dierentiate the
inducible cells, which promote the tissue repair. Therefore,
these nanobiomaterials might have the ability to modulate
downstream stem cellular response, without exogenous

growth factors, coatings, or complex ligand incorporation,
and have the potential to greatly facilitate the development
of tissue engineering and cellular therapies [79].
Biocompatibility and toxicity of nanobiomaterials are
tested at three levels. First, tests are performed in vitro in
cell or tissue cultures. If these tests are shown promising,
further testing is performed in vivo in animal models
before clinical studies are performed on humans. Existing
regulatory frameworks in principle cover all important
aspects; however, dierent behavior of nanobiomaterials
from the corresponding bulk material suggests the need
for specic nanopolicies or adaptation of existing frames.
Unfortunately, there is still lack of validated analytical and
biological methods as well as certied reference standards
for exposure and hazard assessment for establishing safe
doses of nanobiomaterials. Well-characterized and dened
nanobiomaterials to be used as certied reference standards
need to be developed as quality controls additional to
standardized test protocols. Furthermore, the underlying
mechanisms, for example, oxidative stress, immunotoxicity,
and genotoxicity, need to be investigated and possible adverse
eects should be considered.
To date, many trials have been performed to estimate in vitro and in vivo biocompatibility and toxicity of
nanobiomaterials. Based on such preclinical evaluation of
biocompatibility and toxicity, current research trends are
focused on whether the nanobiomaterials induce toxic eects
in human body. For example, some nanomaterials may be
uptaken by phagocytic cells of the RES (reticuloendothelial
system), to induce the production of reactive oxygen species
and cause oxidative stress or preinammatory cytotoxic

activity in the lungs, liver, heart, brain, and so forth.
In this special issue, a dozen of articles are devoted to
interaction between nanobiomaterials and cell/tissue, eect
of size, shape, and surface of nanobiomaterials on their
biocompatibility and toxicity, immunogenicity of nanobiomaterials, design of novel biocompatible and biodegradable
nanobiomaterials, instrumentation and technology to analyze toxicity of nanobiomaterials, and challenges to overcome
the toxicity of nanobiomaterials. The readers can nd in this
special issue not only accurate data and updated reviews
on the above-mentioned aspects but also the possibility to
understand accurately how nanobiomaterials interact with
organs, tissues, and cells, and what is their bioavailability
and biopersistence, and self-assembly mechanism of natural
tissues in order to better biomimetically prepare tissue repair
nanobiomaterials, to nd out how the biocompatibility and
toxicity of nanobiomaterials depend on their properties
such as dimensions, size, shape, chemical composition,
surface chemistry, and coatings, and to realize the necessity
to chemically functionalize nanobiomaterials or introduce
various molecules, growth factors, antigens, and specic
DNA sequences, which can subsequently have positive eect
on the desired cellular functions or therapeutic ecacy and
to develop standard toxicological tests for nanobiomaterials
so that one set of experiments can be compared with another.
Xiaoming Li
Sang Cheon Lee
Shuming Zhang
Tsukasa Akasaka
References
[1] X. Li, Q. Feng, X. Liu, W. Dong, and F. Cui, Collagen-based
implants reinforced by chitin bres in a goat shank bone defect
model, Biomaterials, vol. 27, no. 9, pp. 19171923, 2006.
[2] X. Li, H. Gao, M. Uo et al., Eect of carbon nanotubes
on cellular functions in vitro, Journal of Biomedical Materials
Research A, vol. 91, no. 1, pp. 132139, 2009.
[3] X. Li, H. Gao, M. Uo et al., Maturation of osteoblast-like SaoS2
induced by carbon nanotubes, Biomedical Materials, vol. 4, no.
1, Article ID 015005, 2009.
[4] X. Li, X. Liu, J. Huang, Y. Fan, and F.-Z. Cui, Biomedical
investigation of CNT based coatings, Surface and Coatings
Technology, vol. 206, no. 4, pp. 759766, 2011.
[5] X. Li, Y. Fan, and F. Watari, Current investigations into carbon
nanotubes for biomedical application, Biomedical Materials,
vol. 5, no. 2, Article ID 022001, 12 pages, 2010.
[6] M. Dusinska, Z. Magdolenova, and L. Fjellsbo, Toxicological
aspects for nanomaterial in humans, Methods in Molecular
Biology, vol. 948, pp. 112, 2013.
[7] X. Li, C. A. van Blitterswijk, Q. Feng, F. Cui, and F. Watari, The
eect of calcium phosphate microstructure on bone-related
cells in vitro, Biomaterials, vol. 29, no. 23, pp. 33063316, 2008.
[8] X. Li, H. Liu, X. Niu et al., The use of carbon nanotubes
to induce osteogenic dierentiation of human adipose-derived
MSCs in vitro and ectopic bone formation in vivo, Biomaterials, vol. 33, no. 19, pp. 48184827, 2012.
[9] X. Li, H. Liu, X. Niu et al., Osteogenic dierentiation of human
adipose-derived stem cells induced by osteoinductive calcium
phosphate ceramics, Journal of Biomedical Materials Research

B, vol. 97, no. 1, pp. 1019, 2011.

doi:10.1155/2012/526580
Research Article
Properties of Surfactin C-15 Nanopeptide and Its Cytotoxic Effect
on Human Cervix Cancer (HeLa) Cell Line
Zahra Nozhat, Asadollah Asadi, and Saber Zahri
Deptartement of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil 11367-56199, Iran
Correspondence should be addressed to Asadollah Asadi, asad.asady@gmail.com
Received 16 February 2012; Revised 24 October 2012; Accepted 9 November 2012
Academic Editor: Xiaoming Li
Copyright 2012 Zahra Nozhat et al. This is an open access article distributed under the Creative Commons Attribution License,
Surfactin is one of the most powerful biosurfactants that has been known so far. It is an acidic cyclic nonribosomal lipoheptapeptide
that is produced by Bacillus subtilis. In this presentation we investigated dierent properties of surfactin C-15. The nanomicelle
forming ability of surfactin C-15 in dierent aqueous environments with various ionic strengths was studied by scanning electron
microscope. Surfactin second structure was investigated by Far-UV CD spectrum. Its hemolytic activity and cytotoxicity were
measured by hemolysis and MTT assays, respectively. Surfactin formed spherical nanomicelles in distilled water (pH = 7.4) and
amorphous nanomicelles in PBS buer (pH = 7.4). The hemolysis assay results indicated that HC50 of surfactin was 47 M.
Surfactin C-15 arrested growth of human cervix cancer HeLa cell line in a time- and dosage-dependent method, so that its IC50 at
16, 24, and 48h were 86.9, 73.1, and 50.2 M, respectively.
1. Introduction
Surfactin-like lipopeptides produced by the genus Bacillus
are one of the most important classes of these natural
compounds. They have low critical micelle concentration
(CMC), stable emulsication properties, and excellent foamability [1]. Surfactin is an ecient biosurfactant produced by
some Bacillus subtilis strains. It is a cyclic lipoheptapeptide
containing seven amino acids and a -hydroxyl fatty acid.
In aqueous solution the peptide ring of surfactin shows
a horse-saddle topology because of the two negatively
charged amino acid residues l-Glu and l-Asp [2]. Due to its
amphiphilic structure it shows unique surface-, interface-,
and membrane-active properties [3]. It also has hemolytic,
anticoagulant, antimicrobial and antitumoral activities [1, 4
6] inhibition of cAMP phosphodiesterase [7], and antiHIV properties [7, 8]. These activities are related with their
interactions with biomacromolecules such as enzymes and
lipopolysaccharide. The surfactant properties and biological
activities of surfactin analogues appear very interesting in the
perspective of their utilization both in cosmetic and in pharmaceutical elds. Surfactins are a large variety of isoforms
which dier by variation of the length and branching of their
fatty acid components as well as by amino acid replacements
in their peptide ring [1]. How this molecule can be eective
in various biological events is still largely unknown; however,

it is speculated that the structural and lipophilic properties
of surfactin may aect the stability of biological membranes
[9]. With attention to the increasing application of diverse
biocompatible nanoparticles in medicine and considering
that active elds of research are currently also dedicated
to investigate the applications of biosurfactants in pharmaceutical and biomedical sciences and knowing that the
surfactin is one of the most powerful biosurfactants [1015],
in this presentation, after investigation of dierent properties
of surfactin C-15 such as its nanomicellization ability,
secondary structure, and hemolytic activity, we studied its
cytotoxic eect on HeLa cell line.
2. Materials and Methods

2.1. Materials. Surfactin and 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyl Tetrazolium Bromide (MTT) were obtained
from Sigma. RPMI-1640, fetal bovine serum (FBS), and
Trypsin-EDTA 5x were purchased from Gibco, and HeLa cell
line was obtained from Pastor Institute of Iran.
2.2. Surfactin Second Structure Study. The Far-UV CD
spectrum of surfactin was recorded in PBS (pH = 7.4). It
35
[ ] (deg cm 2 dmo 1 )
25
15
5
195
205
215
225
235
245
255
Wavelength (nm)
15
25
srf20
srf40
srf60
srf240
srf500
srf766
Figure 1: CD spectrum of surfactin in PBS.
was measured by AVIV 215 spectropolarimeter in dierent

concentration (20, 40, 60, 240, 500, and 766 M) at 25 C.
The wavelength ranged between 195 and 260 nm.
2.3. Physical Characterization of Surfactin Nanomicelles. For
the preparation of nanomicelles by surfactin, 1 mg surfactin
was sonicated in 1 mL distilled water (pH = 7.4) and 1 mL
PBS (phosphate buer saline, pH = 7.4) for 15 min at 30 C.
The size and shape of nanomicelles were studied by scanning
electron microscope (SEM, LEO1430VP).
2.4. Red Blood Cell Preparation. Human red blood cells
(RBCs) were separated by centrifugation at 2000 g. RBCs
were then washed once in PBS-EDTA and three times in an
isotonic buer (10 mM Tris, 150 mM NaCl adjusted to pH
8.5 with HCl). RBCs were then suspended in this buer at a
cell density of 5 108 cells/mL [16].
2.5. Hemolysis Assay. 850 L of isotonic buer was added to
50 L of a PBS solution containing the copolymer. 100 L of
RBC suspension was added, and the reaction was performed
at 25 C during 30 min. Unaltered RBCs were then removed
by a 10000 g centrifugation, and the absorbance of the
supernatant at 540 nm was compared with two control
samples in order to determine the percentage of hemolysis.
The rst one (100%) was totally hemolysed with distilled
water [16], and the second one (0%) contained 900 L PBS
plus 100 L of RBC suspension. Each test has been carried
out at least twice.
2.6. Cell Culture. Human cervix cancer HeLa cell line was
cultured in RPMI-1640 medium containing 10% fetal bovine
serum, 100 U/mL penicillin, 100 U/mL streptomycin, and
1% glutamine in a humidied cell incubator with an
atmosphere of 5% CO2 at 37 C [17].
2.7. MTT Assay. Cell viability was estimated by MTT assay.
Briey, 3 106 cells/well was plated in 24-well culture
plates. After overnight incubation, the cells were treated with

dierent concentrations of surfactin (0, 20, 40, 60, and
80 M) for 16, 24, and 48 hours. The cells were treated
with 50 L of 5 mg/mL MTT and the resulting formazan
crystals were dissolved in DMSO (500 L). The optical
density (OD) of each well was measured at 570 nm. Each
test was performed in triplicate experiments. The eect
of surfactin and surfactin-loaded nanoparticles on cell
viability was assessed as percentage cell viability compared to
vehicle-treated control cells, which were arbitrarily assigned
100% viability [18, 19].
2.8. Statistical Analysis. All of the experiments were done in
triplicates, and the averages of the data were compared with
independent t-test. A P value of <0.05 was considered as a
statistical signicance.
3. Results
3.1. Surfactin Second Structure Study. Surfactin C-15 in PBS
showed a CD spectrum dominated by a broad negative
band centered at 225 nm and a maximum peak at 195 nm
(Figure 1). Surfactin second structure percents were measured (Table 1).
3.2. Physical Characterization of Surfactin Nanomicelles.
Micellization ability of surfactin C-15 was studied by scanning electron microscope. The results exhibited that the
nanomicelles in distilled water were spherical in shape and
their size was about 100200 nm (Figure 2(a)), while the
nanomicelles shape in PBS was amorphous and their size was
100400 nm (Figure 2(b)).
3.3. Hemolysis Assay. Surfactin C-15 was tested in dierent
concentration for its hemolytic activity. The results were
exhibited in Figure 3. It gives rise to a concentrationdependent hemolysis [10]. HC50 which is dened as the
3
Table 1: Surfactin second structure percents.
-helix
13.2
10.8
8.3
11.1
11.4
12.3
Material
Srf 20 M
Srf 40 M
Srf 60 M
Srf 240 M
Srf 500 M
Srf 766 M
Antiparallel
22.4
22.7
23.2
22.4
21.7
21.9
Parallel
11.9
12.7
13.8
12.6
12.5
12.3
(a)
-turn
16.8
16.1
15.2
16.1
16.2
16.4
Rndm.coil
35.7
37.7
39.5
37.8
38.2
37.1
Total sum
100
100
100
100
100
100
(b)
Figure 2: Scanning electron microscope scan of surfactin nanomicelles in distilled water (a) and PBS (pH = 7.4) (b).
70
Hemolysis (%)
60
50
40
30
20
10
0
0
20
40
60
Concentration (M)
(A)
80
(B)
Figure 3: Surfactin hemolysis assay. (A) HC50 of surfactin C-15 was estimated 47 M; (B) the macroscopic gure of surfactin C-15 hemolysis
assay in (a) negative control, (b) 20 M, (c) 40 M, (d) 60 M, (e) 80 M, and (f) positive control.
concentration of surfactin that bursts 50% of RBC [10] was

47 M.
3.4. MTT Assay. To study the cytotoxic eects of surfactin
C-15, HeLa cell line was treated with dierent concentration
of surfactin C-15 for 16, 24, and 48 h, and then cell viability
was determined by MTT assay. Surfactin C-15 arrested HeLa
cell line growth in a dose- and time-dependent method
(Figure 4), with IC50 at 16, 24, and 48 h of 86.9, 73.1, and
50.2 M, respectively.
4. Discussion and Conclusion

In this study, scanning electron microscope determined
that surfactin C-15 exhibits dierent manners in dierent
ionic aqueous solutions. Surfactin nanomicelles in PBS show

the presence of large and amorphous aggregates. This is
a similar result to that observed by Zou et al. [20]. The
nanomicelles in distilled water are spherical in shape and
smaller in size compared with the nanomicelles in PBA, and
this caused dierent ionic strengths in distilled water and
PBS. The study carried out by Li et al. showed that when
the acyl chain length of the surfactin decreases, the hemolytic
activity under hypotonic conditions decreases [10], and in
this study the HC50 of surfactin C-14 was recorded 300 M.
According to our study, HC50 of surfactin C-15 is 47 M
that is consistent with Li et al. studies. Surfactins have been
considered to be potential antitumoral agents. Recently, it
is reported that the cytotoxic eects of surfactins on tumor
cells are by inducing the apoptosis, which is related with
120
120
100
100
Viability (%)
Viability (%)
80
60
40
80
60
40
20
20
0
0
20
40
60
80
20
Concentration (M)
(a)
40
60
Concentration (M)
80
(b)
120
Viability (%)
100
80
60
40
20
0
0
20
40
60
80
Concentration (M)
(c)
Figure 4: Cell viability assay for surfactin C-15 on HeLa cell line. The cell line was treated by dierent concentration of surfactin for (a) 16 h,
(b) 24 h, and (c) 48 h with IC50 of 86.9, 73.1, and 50.2 M, respectively.
the enzyme activities. In addition, several biological activities

of some lipopeptides are also related with their eects on the
enzyme activities. For instance, the selective inhibitory eect
of surfactin on cytosolic phospholipase A2 contributes to
its anti-inammatory activities. Another study indicates that
the inhibitory eect of surfactin on the alkaline phosphatase
had been attributed to a chelating action of the free carboxyl
groups of the Asp and Glu residues in the peptide moiety
of surfactin [1]. Although the obtained results show the
cytotoxic eect of surfactin C-15 on HeLa cell line in a doseand time-dependent method, more studies are necessary to
conrm its anticancer eects. Also more studies are needed
to determine and clarify the mechanism of surfactin action
and anti-proliferative eects on cancerous cell lines targeted
delivery.
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doi:10.1155/2012/734398
Research Article
In Vitro Cytotoxicity Assay on Gold Nanoparticles with
Different Stabilizing Agents
S. Vijayakumar1 and S. Ganesan2
1 Department
2 Department
of Physics, Sri Ramakrishna Institute of Technology, Pachapalayam, Perurchettipalayam, Coimbatore 641010, India
of Physics, Government College of Technology, Thadagam Road, Coimbatore 641013, India
Correspondence should be addressed to S. Vijayakumar, vijaysk.research@gmail.com

Received 9 April 2012; Revised 16 July 2012; Accepted 20 July 2012
Copyright 2012 S. Vijayakumar and S. Ganesan. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Noble gold nanoparticles (AuNps) are generally nontoxic due to their inert nature. The gold nanoparticles are easily tagged
with various proteins and biomolecules rich in aminoacid leading to important biomedical applications including targeted
drug delivery, cellular imaging, and biosensing. In this study, three cytotoxicity detection assays 3-(4,5-dimethylthiozol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), neutral red cell, and lactate dehydrogenase (LDH) on gold nanoparticles stabilized
with citrate, starch, and gum arabic are used. The assays used are based on dierent mode of detection like LDH release,
MTT metabolism, and neutral red uptake. We found that the AuNps stabilized with citrate are very sensitive to the change of
concentration and time assay compared to starch and gum arabic stabilized gold nanoparticles.
1. Introduction
Gold nanoparticles possess dierent physiochemical characteristics compared to the bulk gold [1, 2]. The six free electrons present in the conduction band of gold nanoparticles
make them a potential candidate to bind with thiols and
amines [3], also gold nanoparticles easily tagged with various
proteins and biomolecules rich in aminoacids leading to
important biomedical applications including targeted drug
delivery [4, 5], cellular imaging [6], and biosensing [7].
The use of nanoparticles for biomedical applications such
as drug and gene delivery, biosensors, cancer treatment, and
diagnostic tools has been extensively studied throughout the
past decade [817].
The toxicity has been expected to be in the following
strategy [18]. The toxic CdSe can release poisonous cadmium
ions inside the living organism and by competing with zinc
for binding sites on metallothionein [19]. The nanoparticles
have been shown to adhere to cell membranes [20] and also
be ingested by cells [21]. The breaching of the cell membrane
and the intracellular storage may have a negative eect on
the cells regardless of the toxicity of the particles and their
subsequent functionality.
Microstructure is essential for inductive bone formation

in calcium phosphate materials after soft-tissue implantation. The increasing surface areas, microstructured calcium
phosphate materials might concentrate more proteins that
dierentiate inducible cells from osteogenic cells that form
inductive bone [22]. The multiwalled carbon nanotubes
(MWNTs) might stimulate inducible cells in soft tissues
to form inductive bone by concentrating more proteins,
including bone-inducing proteins [23]. Distinctly shaped
nanoparticles such as carbon nanotubes can rip cells like
needle [24]. This suggests that nanomaterials of the same
composition would have dierent biologic responses for different morphologies. The advantages of the in vitro approach
includes simplicity, consistency of the experimental setup,
and the reproducibility of the experimental results [25].
Cytotoxicity also depends on the type of cells used. The
33 nm citrate-capped gold nanoparticles were found to be
nontoxic to baby hamster kidney and human hepatocellular
liver carcinoma cells but toxic to a human carcinoma
lung cell lines. AuNps may have advantages over other
metallic nanoparticles in terms of biocompatibility and
noncytotoxicity [26].
2
In this study, gold nanoparticles stabilized with citrate,
starch, and gum arabic are used for cytotoxicity studies. The
assays used are based on dierent modes of detection like
LDH release, MTT metabolism, and Neutral Red uptake.
We found some noticeable dierences in the values for the
cytotoxic eect depending on the assay and nanoparticle
capping agent used. In particular, the citrate stabilized gold
nanoparticles are having little toxicity compared to the starch
and gum arabic stabilized nanoparticles, since we have used
the same size of particles in all the cases.

All chemicals were obtained from Sigma-Aldrich and used as
received. Double-distilled water was used in all experiments.
The PC-3 and MCF-7 cell lines were obtained from the
American Type Culture Collection (ATCC) through Department of Microbiology, PSG Institute of Medical Sciences
and Research, Tamilnadu, India. The cell line (CHO22) was
obtained from R&D-Bio, Coimbatore, India.
2.1. Preparation of Gold Nanoparticles
2.1.1. Preparation of Citrate-Capped Gold Nanoparticles
(Citrate-AuNps). Conventional techniques for aqueous synthesis of gold nanoparticles involve reduction of Au(III)Cl3
with trisodium citrate, a process pioneered by Turkevich and
later rened by Frens [2730]. In this method the citrate salt
initially acts as a reducing agent by forming a layer of citrate
ions over the gold nanoparticles surface, inducing enough
electrostatic repulsion between individual particles to keep
them well dispersed in the medium. This method provides
uniform and fairly spherical nanoparticles.
This reduction process proceeds at a very slow rate and
imparts a wine red color to the solution. 250 mL of 0.25 mM
HAuCl4 was heated to boiling. Then, 4 mL of aqueous
solution of 1% solution of trisodium citrate was added to
the HAuCl4 solution under vigorous stirring and the boiling
was continued for 15 min until it turns to a deep red colour.
The citrate ions serve as both reducing agent for AuNp
formation and a stabilizer, preventing agglomeration of gold
nanoparticles.
2.1.2. Preparation of Starch-Capped Gold Nanoparticles
(Starch-AuNps). Starch (0.0225 g) was dissolved in 6 mL of
doubly ionized water by heating the solution to 90100 C
with continuous stirring. To this hot starch solution, 0.1 mL
of 0.1 M HAuCl4 solution (0.0393 g in 1 mL deionized water)
was added, followed by the addition of 0.02 mL of 0.1 M
trialanine phosphine (THPAL) solution (0.0337 g in 1 mL
DI water) with continuous stirring. When the color of the
solution changed to pinkish purple, stirring was continued
for one minute without heating.
2.1.3. Preparation of Gum Arabic-Capped Gold Nanoparticles
(Gum Arabic-AuNps). Gum Arabic (0.012 g) was dissolved
in 6 mL of doubly ionized water by heating the solution
to 90100 C with continuous stirring. To this hot gum
arabic solution, 0.1 mL of 0.1 M NaAuCl4 solution (0.0338 g
Table 1: Average size, plasmon wavelength, and plasmon width of
gold nanoparticles stabilized with starch, gum Arabic, and citrate.
Sample
name
C-AuNp
S-AuNp
GA-AuNp
Average size Plasmon wavelength

(nm)
max (nm)
21 1.4
523
21 1.5
525
20 2.3
528
Plasmon width
(nm)
90
90
85
in 1 mL DI water) was added, followed by the addition

of 0.02 mL of 0.1 M THPAL solution (0.0338 g in 1 mL DI
water) with continuous stirring (Table 1). When the color
of the solution changed to reddish purple, stirring was
continued for one minute without heating. Table 2 shows
the reaction condition for preparing citrate, starch, and gum
arabic stabilized gold nanoparticles.
2.2. Characterization. Gold nanoparticles stabilized with
citrate, starch, and gum arabic were characterized by UV-Vis
absorption spectroscopy using UV-1700 series spectrometer
(PSG Institute of Advanced Studies, Coimbatore, India) to
study the peak absorption band. About 1 mL solution of
nanoparticles in water was used. The peak absorption band
obtained for citrate, starch, and gum arabic stabilized gold
nanoparticles is 523 nm, 525 nm, and 528 nm, respectively,
(Table 1). TEM images of citrate-, starch- and gum arabiccapped gold nanoparticles were collected on a Philips CM200
(Sophistical Analytical Instrument Facility, Indian Institute
of Technology, Bombay, India). The operating voltage range
Zeta potential
was 20200 kV with a resolution of 2.4 A.
measurements were performed using a Malvern Instruments
Zeta sizer 1000 Hs operating with a variable power (5
50 mW) He-Ne laser at 633 nm (Indian Institute of Science,
Bangalore, India). Measurements were taken at 25 C. Before
and in between the measurements, the cells were washed
with ultrahigh pure water. The AuNp thus, formed were
characterized using UV-Vis spectroscopy, Zeta potential, and
transmission electron microscopy shown in Figures 1, 2, and
3.
2.3. Cytotoxicity Assay
2.3.1. MTT Assay. Cytotoxicity evaluation of citrate-AuNps,
starch-AuNps, and gum arabic-AuNps was performed using
MTT assay as described by Mossman [31]. Approximately
1 105 mL1 cells (MCF-7 and PC-3) in their exponential growth phase were seeded in a at-bottomed 96-well
polystyrene coated plate and were incubated for 24 hrs
at 37 C in a 5% CO2 incubator. Series of dilution (20,
50, 80, 110, and 140 g/mL) of gold nanoparticles in the
medium was added to the plate in hexaplets. After 24
hours of incubation, 10 L of MTT reagent was added to
each well and was further incubated for 4 hours. Formazan
crystals formed after 4 hours in each well were dissolved in
150 L of detergent and the plates were read immediately
in a microplate reader (BIO-RAD microplate reader-550)
at 570 nm. Wells with complete medium, nanoparticles and
MTT reagent, without cells were used as blanks. Untreated
3
90
80
70
Frequency
60
50
40
30
20
10
0
15
17
100 nm
18
19
20
Particle size (nm)
(a)
22
23
(b)
pH
2.4
3.5
4.5
5.5
Zeta potential (mV)
Absorbance
2.2
2
1.8
1.6
10
15
20
25
30
35
1.4
40
1.2
400
450
500
550
600
Wavelength (nm)
650
700
750
First replicate
Second replicate
(c)
(d)
Figure 1: (a) TEM image of citrate stabilized gold nanoparticles. Insight SEAD pattern and an enlarged single nanoparticle. (b) Particle
size histogram and is 21 nm averaged from 350 nanoparticles. (c) Plasmon resonance absorption and is 523 nm with plasmon width () of
90 nm. (d) Zeta potential of gold nanoparticles at dierent pH values.
Table 2: Reaction conditions for producing citrate (C-AuNp), starch (S-AuNp) and gum arabic (GA-AuNp)-capped gold nanoparticles.
Compound
name
Stabilizer (C-AuNp)/(SAuNp)/(GA-AuNp)
Gold precursor
Reducing agent
Time
C-AuNp
4 mL of 38.8 mM in 250 mL of DI
0.25 mM HAuCl4
4 mL of 1% trisodium citrate
1 min
S-AuNp
0.0225 g of starch in 6 mL of DI 0.1 mL of 0.1 M, NaAuCl4

0.0225 g of gum arabic in 6 mL of
0.1 mL of 0.1 M, NaAuCl4
DI
0.02 mL of 0.1 M, THPAL
1 min
Reaction
solution color
Wine-red color
Sodium citrate
Pinkish purple
0.02 mL of 0.1 M, THPAL
1 min
Reddish purple
GA-AuNp
PC-3 and MCF-7 cells as well as the cell treated with (20,
50, 80, 110, and 140 g/mL) concentration of AuNps for
24 hrs were subjected to the MTT assay for cell viability
determination.
2.3.2. Neutral Red Cytotoxicity Assay. CHO22 cells were
seeded at a population of 1.5 104 cells per well in a
96-well plate. The cells were incubated for 24 hours and
reached 8090% conuence. The spent media were removed
and the cells were washed with PBS (0.01 M phosphate
buer, 0.0027 M KCl and 0.137 M NaCl) and 1 L fresh

media was added. The media were then replaced with
test nanoparticles of (20, 50, 80, 110, and 140 g/mL)
concentrations mixed with fresh media. The plates were then
incubated for 24 hrs at 37 in a humidied incubator with
a 5% CO2 environment. Following the incubation periods,
the cells were washed twice with PBS (0.01 M phosphate
buer 0.0027 M KCl and 0.137 M NaCl) and 100 L serumfree media containing neutral red (100 g/mL) was added to
each well and incubated for 2-3 hours.
1.2
Absorbance
1
0.8
0.6
0.4
0.2
0
400
200 nm
450
500
(a)
700
750
(b)
4.5
5.5
28
50
29
40
Frequency (%)
Zeta potential (mV)
3.5
650
60
pH
27
550
600
Wavelength (nm)
30
31
32
30
20
33
10
34
0
12
35
16
19
22
25
Particle size (nm)
29
32
36
First replicate
Second replicate
(c)
(d)
Figure 2: (a) TEM image of Starch stabilized gold nanoparticles. Insight SEAD pattern and an enlarged single nanoparticle. (b) Particle
After the incubation, the cells were washed twice with

PBS (0.01 M phosphate buer 0.0027 M KCl and 0.137 M
NaCl) thereafter 50 L of dye release agent (a solution of 1%
acetic acid: 50% ethanol) was added to each well and the
plates were incubated for further 10 minutes. The plate was
placed on a shaker (Vortex Genie) for 30 minutes after which
the optical density at 540 nm was determined on a multiwall
spectrophotometer.
2.3.3. LDH Assay. Cytotoxicity was assessed using an LDH
cytotoxicity detection kit (Roche applied sciences). This
assay measures the release of cytoplasm enzyme lactate
dehydrogenase (LDH) by damaged cells. Cells cultured in
96 plates were treated with increasing concentrations of
gold nanoparticles (20, 50, 80, 110, and 140 g/mL). After
48 hours of treatment, culture supernatant was collected
and incubated with reaction mixture. The LDH catalyzed
conversion results in the reduction of the tetrazolium salt

to formosan, which can be read at 490 nm absorbance.
These data are measured in LDH activity as a percentage
of the control. Any signicant increase in LDH levels would
indicate cellular disruption or death due to the treatment.
2.3.4. Statistical Analysis. For statistical analysis, all data are
expressed as mean standard deviation (SD). The graphs
and curves are done with SigmaPlot 12.0, Adobe Photoshop
7.0, and Microsoft Oce Excel 2007.
3. Results
In MTT assay only cells that are viable after 24 hours
exposed to the sample were capable of metabolizing a
dye (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium
bromide) eciently and the purple colored precipitate which
5
1.2
Absorbance
1
0.8
0.6
0.4
0.2
0
400
450
500
550
600
650
700
750
Wavelength (nm)
(a)
(b)
30
3.4
25
4.5
5.5
28
29
20
Zeta potential (mV)
Frequency (%)
pH
27
15
10
5
30
31
32
33
34
0
18
21
22
25
27
Particle size (nm)
28
30
35
36
First replicate
Second replicate
(c)
(d)
Figure 3: (a) TEM image of gum arabic stabilized gold nanoparticles. Insight SEAD pattern and an enlarged single nanoparticle. (b) Particle
is dissolved in a detergent was analyzed spectrophotometrically. After 24 hours of posttreatment, PC-3 and MCF-7
cells showed excellent viability even up to the concentration
of 140 g of citrate-, starch-, and gum arabic-capped gold
nanoparticles. These results clearly demonstrate that the
photochemicals within these herbs provide nontoxic coating
on AuNps and corroborate the results of the internalization
studies discussed above. The lack of any noticeable toxicity of
starch and gum arabic stabilized gold nanoparticles provide
new opportunities for the safe application in molecular
imaging and therapy. But the data shows that there is a
marginal cytotoxic eect of citrate stabilized gold nanoparticles with the cell lines used.
The mammalian Chinese hamster ovary (CHO22) cell
line was used in the elucidation of cytotoxicity eects of the
selected gold nanoparticles by neutral red assay. This cell line
has been termed as the mammalian equivalent of the model
bacterium E. coli [32]. For the elucidation of the cytotoxicity
of the gold nanoparticles, the CHO22 cells were treated with
the nanomaterials for 24 hours. The gold nanoparticles were

investigated at ve concentrations. The spectrophotometric
measurements were done for the neutral red dye uptake
and release. The viability assay data for the comparison is
presented in the Figures 4(a), 4(b), and 4(c).
4. Discussion
Comparison of the stabilizing agents revealed that citrate
produced more pronounced response and sensitivity to the
dose changes and time assay. The higher dosage shows
less viability of citrate stabilized nanoparticles than starch
and gum arabic stabilized gold nanoparticles. Cell viability was also determined by an LDH release assay which
was employed to measure the cytotoxicity of the gold
nanoparticles at dierent concentrations. Damaged cells
release cytoplasmic LDH, which catalyzes a conversion of
tetrazolium salt to formazan.
120
120
100
100
Cell viability (%)
Cell viability (%)
80
60
40
20
0
80
60
40
20
0
20
50
80
110
140
Gold nanoparticle concentrations (g/mL)
20
50
80
110
140
Citrate-AuNps
Starch-AuNps
Gum arabic-AuNps
Citrate-AuNps
Starch-AuNps
Gum arabic-AuNps
(a)
(b)
120
Cell viability (%)
100
80
60
40
20
0
20
50
80
110
140
Citrate-AuNps
Starch-AuNps
Gum arabic-AuNps
(c)
Figure 4: Cytotoxic assays by (a) MTT assay, (b) neutral red cell assay, and (c) LDH assay on gold nanoparticles stabilized with citrate, starch
and gum arabic.
The absorbance of the produced formazan at 490 nm

is proportional to the number of damaged or dying cells.
The cytotoxicity of various cells in percent of LDH activity
of the control, after exposure to increasing concentrations
of nanoparticles stabilized with three dierent stabilizing
agents for 24 hours, was analyzed. At each concentration,
there was no signicant cytotoxicity eect produced. The
cell viability results indicate that gold nanoparticles are
nontoxic to the array of cells tested. The incorporation of
surface functionalities via citrate, starch, and gum arabic
renders these nanoparticles highly biocompatible. Noble
metal particles, such as gold are generally nontoxic due to
their inert nature, this has also been seen with their LD50
in toxicity assays, high enough up to 1 mg/mL [33]. The
cell survival at dierent concentrations of gold nanoparticles

stabilized with dierent capping agents showed a small
variation with the increase in the concentration of the
nanoparticles.
The gold nanoparticles of 20 nm are coated with citrate
(citrate-AuNps), starch (starch-AuNps), and gum arabic
(gum arabic-AuNps) are taken with a series of increasing
concentrations (20, 50, 80, 110, and 140 gm/mL) along
with cell cultures and studied under dierent cytotoxicity
assays like MTT, neutral red cell, and LDH. In all the assays,
the nanoparticles show excellent cell viability. The citrate
stabilized gold nanoparticles rapidly show their cytotoxicity
eect compared to starch and gum arabic. The gum arabic
is highly viable than starch and citrate. In our observations,
120
120
100
100
Cell viability (%)
Cell viability (%)
80
60
40
20
0
80
60
40
20
24
36
48
60
Time of exposure (hours)
72
Citrate-AuNps
Starch-AuNps
Gum arabic-AuNps
24
36
48
60
72
Citrate-AuNps
Starch-AuNps
Gum arabic-AuNps
(a)
(b)
120
Cell viability (%)
100
80
60
40
20
0
24
36
48
60
72
Citrate-AuNps
Starch-AuNps
Gum arabic-AuNps
(c)
Figure 5: Cell viability studies at dierent time of exposure of citrate, starch, and gum arabic stabilized gold nanoparticles at a concentration
of 110 g/mL by (a) MTT assay, (b) neutral red cell assay, and (c) LDH assay.
all the gold nanoparticles are viable to cells and because

of the acidic nature of citrate show little less cell viability
compared to other stabilizing agents with gold nanoparticles.
The viability test based on the time of exposure of gold
nanoparticles was also studied with dierent stabilizing
agents at dierent cell assays, Figures 5(a), 5(b), and 5(c).
In this study the starch-AuNps and gum arabic-AuNps show
high viability to cell assays compared to citrate-AuNps,
Figures 6(a), 6(b), and 6(c). IC50 is calculated as 110 g/mL
of gold nanoparticles and this concentration is considered for
the time of exposure assay.
Cell-based cytotoxic assay with dierent concentrations
of gold nanoparticles shows very small variation among
citrate, starch, and gum arabic. The gold nanoparticles used
here were having the same size 20 1 nm, they diers only
in stabilizing agents. The citrate stabilized gold nanoparticles
show less viability than starch and gum arabic, Figures 4(a),
4(b), and 4(c). This is mainly due to the fact that citrate is
acidic in nature. More over the particle sizes are same in
all three cases of citrate, starch and gum arabic. Hence in
this study the size dependent cytotoxicity is ruled out and
it is conrmed that the stabilizing agents are responsible for
cytotoxicity.
These results are consistent with previous investigations
performed with dermal broblasts [34] which demonstrated
that the gold/citrate nanoparticles impaired the proliferation
of dermal broblasts and induced an abnormal formation
of actin laments, causing therefore a reduced cellular
120
120
100
100
80
80
Viability (%)
Viability (%)
60
40
20
60
40
20
0
20
50
80
110
Gold nanoparticle concentration (g/mL)
140
20
MTT assay
Neutral red cell assay
LDH assay
50
80
110
140
MTT assay
LDH assay
(a)
(b)
120
Viability (%)
100
80
60
40
20
0
20
50
80
110
140
MTT assay
LDH assay
(c)
Figure 6: Dierent cell viability assays at dierent concentrations of gold nanoparticles for (a) citrate-capped (b) starch-capped, and (c)
gum arabic capped.
motility and inuencing the cell morphology. On contrary,

[35] reported that citrated and biotinylated 18 nm gold
nanoparticles did not induce toxicity in leukemic cells (cell
line K562), whereas smaller particles were much more toxic.
5. Conclusion
In conclusion, we found that the gold nanoparticles stabilized with citrate, starch, and gum arabic are viable to
dierent cells through dierent assays and with dierent
concentrations of gold nanoparticles. The viability of the
treated cells depending on the stabilizing agent and the
types of cytotoxicity assay used. The cell viability test shows
distinguishable cytotoxic eect for citrate stabilized gold
nanoparticles at higher concentration and this is may be
because the surface coating is acidic in nature compared to
starch and gum arabic.
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doi:10.1155/2012/614094
Review Article
Biocompatibility and Toxicity of Magnetic Nanoparticles in
Regenerative Medicine
H. Markides, M. Rotherham, and A. J. El Haj
Institute of Science and Technology in Medicine, Keele University Medical School, Thornburrow Drive, Hartshill, Stoke-on-Trent,
Staordshire ST4 7QB, UK
Correspondence should be addressed to H. Markides, h.markides@istm.keele.ac.uk
Received 4 May 2012; Revised 28 June 2012; Accepted 16 July 2012
Copyright 2012 H. Markides et al. This is an open access article distributed under the Creative Commons Attribution License,
Regenerative medicine is a pioneering eld aimed at restoring and regenerating the function of damaged cells, organs and tissues
in order to establish normal function. It demands the cross communication of disciplines to develop eective therapeutic stem
cell based therapies. Nanotechnology has been instrumental in the development and translation of basic research to the clinically
relevant therapies. In particular, magnetic nanoparticles (MNPs) have been applied to tag, track and activate stem cells oering
an eective means of monitoring in vitro and in vivo behaviour. MNPs are comprised of an iron oxide core with a biocompatible
biological polymer. Safety is an issue of constant concern and emphasises on the importance of investigating the issue of toxicity.
Any indication of toxicity can ultimately limit the therapeutic eciency of the therapy. Toxicity is highly dependent on the physical,
chemical and structural properties of the MNP itself as well as dose and intended use. Few in vitro studies have reported adverse
eects of MNP on cells at in vitro in therapeutic doses. However, long term in vivo studies have not been studied as extensively.
This review aims to summarise current research in this topic highlighting commonly used toxicity assays to investigate this.
1. Introduction
Nanotechnology is an emerging eld with growing interest
for its numerous applications ranging from information
technologies to medicinal applications [1]. Subsequent social
and economic implications of this eld have resulted in its
increased popularity and demand with a competitive drive
[2]. It is heavily dependent upon the cross-collaboration
of various scientic disciplines to manipulate and alter the
dimensions of materials at an atomic scale resulting in the
formation of nanomaterials [1]. Nanomaterials are dened
as materials with one, two, or three external dimensions
ranging from 1 to 100 nmthe nanoscale [2, 3].
Recently, nanoscale materials have been the centre of
research, particularly in the elds of regenerative medicine
and tissue engineering. Examples include nanoparticles,
nanobers, and nanotubes, all of which can be specically
tailored to their role and function within tissue engineering
[4]. Nanoscale materials in these forms have been used in
conjunction with stem cells to produce stem cell-based therapies with emphasis on replacing and restoring the function of
cells, tissues, or organs in order to establish normal function
[5]. Examples include the transplantation of mesenchymal

stem cells for the treatment of Huntingtons disease [6].
This paper will focus specically on the implications of
applying nanoparticles in regenerative medicine and tissue
engineering.
A nanoparticle can be dened as a material with three
external dimensions of equal nanoscale dimensions [2]. The
major benet of using nanoparticles is that, due to their
size, they can be accurately manoeuvred and targeted to a
specic biological entity or marker [7] and interact on a
cellular (10100 nm), subcellular (20250 nm), protein (3
50 nm), or genetic scale (10100 nm) [8, 9]. Their unique,
electronic, optical, and magnet properties coupled with
their specic dimensions have furthered their attractiveness
in this eld [1012]. Furthermore, nanoparticles can be
customised for a specic biological purpose such as cell
isolation, drug delivery, diagnostics (magnetic resonance
imaging MRI), cellular imaging, and hyperthermia [8, 11,
1315]. Examples of nanoparticles include quantum dots
and magnetic nanoparticles (MNPs) [12, 16]. Magnetic
nanoparticles will be the main focus of this paper.
1.1. Magnetic Nanoparticles (MNPs). Magnetic nanoparticles have a particle size within the nanoscale with magnetic
properties. Various metals can be used to convey the magnetic properties of MNPs; nickel, cobalt, and iron have
demonstrated to be such examples. Organic and inorganic
polymers including RGD peptides, bronectin, and dextran can be used to coat the magnetic core resulting in
improved biocompatibility by protecting biological entities
from adverse toxic reactions [14, 17]. The customisation of
surface coatings to desired specications can be achieved
via surface charge alterations, protein-binding capacity, and
surface topography [18]; this increases functionalization
of the particles encouraging enhanced interaction with
biological entities, with minimal toxic eects [8, 11]. Additional factors that can further inuence the behaviour of
MNPs are the size [19] and magnetisation (paramagnetic,
ferromagnetic, ferromagnetic, and superparamagnetic) of
the particles themselves [20].
Before nanoparticles can be considered for the use in
regenerative medicine, they must full the following specications.
Specications:
(i) biocompatible that is nontoxic to the cells [2123],
(ii) biodegradable [22, 24],
(iii) maintain physical properties after surface modication [11],
(iv) must not aect stem cell characteristics [21],
(v) eective at therapeutic doses [21, 22],
(vi) ideally have regulatory approval [20],
(vii) minimal or no transfer of by-products to surrounding tissue/cells [22, 25],
(viii) chemically stable in physiological conditions [24].
MNPs for the use in biomedical applications are desired
to exhibit superparamagnetic properties (SPIONs) [26].
SPIONs are typically small particles composed of either
a magnetite (Fe3 O4 ) or maghemite (-Fe2 O3 ) core [20,
27] coated with a biocompatible organic/inorganic polymer
[17, 28] or precipitated throughout a porous biocompatible
polymer [10, 20]. Both maghemite and magnetite are traditionally ferromagnetic in nature. However, as they decrease
in size to 30 nm or smaller, they lose their permanent
magnetism and become superparamagnetic [2]. Iron (Fe)
oxide-based MNPs are suitable for biological application for
the following reasons: the superparamagnetic nature implies
that the particles will not be attracted to each other, and so
the risk of agglomeration in a medical setting is minimised
[23]. Fe is a naturally occurring metal in the human
body (ferritin), and hence iron-containing nanoparticles are
biocompatible as the body is adapted to metabolising the
particles into its elements; these can be utilised by the body
in subsequent metabolic processes [19, 2932].
The precedent for using SPIONs in regenerative medicine
comes from the application of SPION-based magnetic
resonance imaging (MRI) contrast agents [33] which are
accepted T2 weighted contrast agents [26]. MR imaging is a
safe and eective noninvasive means of imaging anatomical

tissue [34]. It is thought that these contrast agents can be
used for a variety of purposes in the clinical translation
of stem cell-based therapies for the tagging, tracking, and
activation of stem cells and other cell types [26, 29, 35].
Feridex (USA), also referred to as Endorem (EU) is example
of FDA-approved, iron-based MRI contrast agent which
has recently been taken o the market [20, 36]. These
particles have an iron oxide core and are between 50 and
180 nm in diameter with an overall negative charge along
with biodegradable and biocompatible properties [20, 32].
The dextran coating prevents the coagulation of particles;
however a limitation to using dextran is its inability to enable
the ecient uptake of these particles by cells. In these cases,
transfection agents may be required.
The high resolution and impressive tissue penetration
depth attributed to SPION-based MRI contrast agents
has not only driven diagnostics to higher levels but also
encouraged the multidisciplinary applications of these agents
in elds such as regenerative medicine. These agents are,
however, limited by their poor sensitivity thus preventing
microscopic examination of tissue over time which is a
crucial requirement in the development of stem cell-based
therapies [12]. Other imaging modalities such as positive
emission topography (PET scan), X-ray, ultrasound, and
computed tomography (CT) [34] (available in a clinical
setting) oer greater sensitivity in some cases but are lacking
in other aspects such as the use of radioisotopes which
could result in detrimental side eects in the case of PET
scans [34]. This has therefore encouraged the integration
of imaging modalities [12, 34, 37] and, as a consequence,
the development of multifunctional nanoparticles to be used
in conjunction with various imaging techniques. In the
context of regenerative medicine, this could further our
understanding of biological processes related to stem cellbased therapies [12, 26]. Unlike individual nanoparticles,
multifunctional nanoparticles exploit the benets of the
imaging modalities of choice while minimising the disadvantages. In many instances this is translated in enhanced
cellular tracking with high special resolution and high
anatomical contrast without the issues of ionizing radiation
for extended periods of time [12, 26, 37].
1.2. Multifunctional Nanoparticles. The development of multifunctional nanoparticles requires the reengineering and
modication of particle surfaces to meet a greater variety of
applications. It is highly desirable to incorporate uorescent
properties, near infrared absorption, and photon scattering,
all of which enable in vivo imaging to a greater extent. This
however is no easy task. It is a highly challenging task to
incorporate the properties that make a particle a good MRI
contrast agent and those that allow good NIR response, for
example [12]. It involves novel and intricate approaches to
achieve all the desired characteristics. In a study by Jin et al.,
the surface of nanoparticles was coated with gold (a noble
metal) [12] in an eort to expand on the multifunctional
aspect of MNP and incorporate near infrared absorption and
photon scattering properties. This group successfully created
smooth particles with the core spatially separated from the
Without
transfection
agent
Coated with
transfection agent:
Poly-L-lysine
protamine sulfate
peptides (cholera
toxin, RGD) and
phospholipids
MRI or uorescent tracking:

location, migration, and
number of stem cells
over time
(1) Incubation
(2) Physical
treatment
(electroporation)
Transplantation
+
MRI nanoparticles;
quantum dots
Stem cell
Nanoparticle labelled
stem cell
Figure 1: Schematic diagram highlighting the use of SPIONs in regenerative medicine. Cells requires the internalisation of SPIONS. Labelled
cells are then implanted within the body and visualised by MRI [52].
shell thus resulting in uniform gold-coated MNP while still

maintaining dimensions, electronic, magnetic, and thermal
properties and still being responsive to NIR spectra [12].
Mahmoudi et al. took this one step further and created goldcoated magnetic nanoparticles with jagged edges to allow for
ramen spectroscopy to be employed and in this way imaging
at the molecular level [26]. In another study, Mahmoudi and
Shokrgozar sought to add to the multifunctionality of these
particles by adding and trapping a uorescent polymeric dye
between the core and the gold coating [37]. This simple
addition adds an extra imaging modality for either in vivo
or in vitro imaging.
1.3. Application of SPIONS in Regenerative Medicine. SPIONS are particularly useful and have a multifunctional
aspect within regenerative medicine where their functions
include tagging, tracking, and activation of stem cells both in
vitro and in vivo;this multifunctionality makes them highly
desirable tools in this eld.
1.3.1. Tagging. Incorporating SPIONs into cells allows for
the remote manipulation of cells using an external magnetic
eld gradient. This encourages the precise positioning or
targeting of cells to desired sites for tissue regeneration or
repair [45, 46], allowing them to function as a powerful,
noninvasive tool in stem cell therapy [47, 48].
1.3.2. Tracking. A universal issue central to all cell-based
therapies is the in vivo migration of stem cells within tissues
and on substrates, which is often guided by the presence
of chemical mediators and material topography [49]. The
migration of these cells to and from the target location
requires precise monitoring to determine the eectiveness of
the therapy. The use of nanoparticles can provide answers
to questions such as optimal delivery route; extent of
engraftment; migratory patterns after transplantation; ideal

dosage schemes. Having the answer to these questions will
help in the optimisation of the overall therapy and thus
increase its therapeutic potential [50]. It is thought that
MNP can be employed in conjunction with the use of
magnetic resonance imaging (MRI) to track implanted cells
in vivo (Figure 1). In essence, stem cells are encouraged to
internalise iron-based MNP; in this way magnetic properties
are transferred to the cells. The intracellular iron essentially
disturbs the local magnetic eld thus allowing cells to be
visualised as a lack of signal with MRI [51].
1.3.3. Activation. Mechanical stimulation can be used to
facilitate cell proliferation, dierentiation, and migration of
stem cells [53]; physical forces include uid ow, axial compression, tension, and magnetism [54]. Biological responses
are achieved through the process of mechanotransduction,
whereby cells convert physiological mechanical stimuli into
biochemical signals to activate the biological response [55].
Using MNP functionalised with antibodies or peptides, it is
possible to attach SPIONS to specic mechanosensitive cell
surface receptors and ion channels. This has been shown
to result in membrane polarisation, receptor activation, and
subsequent downstream second messenger signals in hMSC
[56]. Using this technology it has been shown that it is
possible to promote an osteochondral phenotype of hMSC
and human osteoblasts in response to magnetic activation
using an external oscillating magnetic eld [57, 58].
1.3.4. MNP-Mediated Transfection. The transfection of therapeutic genes could play a pivotal role in regenerative
medicine. This technology allows the replacement of defective genes or addition of extra copies of therapeutic genes
that are known to play a crucial part in the regeneration
of tissue. One example is the treatment of ischemia by
4
transfecting human umbilical vein endothelial cells with
vascular endothelial growth factor to promote upregulation
of survival factors and subsequent improved cell viability
[59]. There are many mechanisms of inserting exogenous
genetic material into cells. Transfection with viruses can
be an eective technique but there are safety and ecacy
concerns with this strategy. Nonviral transfection methods,
for example, electroporation, transfection reagents, are considered safer. However these mechanisms often suer from
poor transfection eciency or impact on cell viability to
unacceptable levels depending on cell type. Therefore, a
stable, high transfection system with minimised toxicity that
can successfully deliver genes of interest without compromising gene function is required [60]. MNPs are currently
being considered as alternate gene delivery vehicles; coated
with nucleic acid MNPs have shown the capacity to further
increase transfection eciencies using magnetic elds to
attract the MNP towards the cell membrane; the particles
are then taken up by the cells by various uptake mechanisms
such as endocytosis. Upon uptake the DNA dissociates from
the nanoparticle and can then be expressed. One study has
successfully transfected the MG63 cell line without any major
eects on cell viability [61].
To summarise, all three applications require the labelling
of stem cells with SPIONs. This can be achieved through
either the internalisation of SPIONs or the binding of
SPIONS to cell surface markers, for example, integrins, or
to specic antibodies [50, 62, 63].
2. Safety and Biocompatibility of

Magnetic Nanoparticles
Toxicity issues are a major concern and are important factors
in the context of regenerative medicine and tissue engineering. As mentioned previously, the use of MNPs in regenerative medicine requires the labelling of cells (the therapeutic
agents) with MNPs which may then be implanted within
the body. Employing particles which are toxic in nature
over a long period of time can signicantly diminish the
therapeutic eciency of the cell-based therapy [38]. It is
valid at this point to state that toxicology is dened as the
study of adverse eects of chemical, physical, and biological
agents in people, animals, and the environment [64]. Toxic
cellular eects are translated into impaired mitochondrial
activity, membrane leakage, and morphological changes.This
can have adverse eects on cell viability, proliferation, and
metabolic activity and impair the therapeutic eciency of
the therapy [65]. In cases where the MNPs are incorporated
into the therapy and transplanted within the body, the risk
of MNPs migrating through the organism, entering, and
accumulating within organs is a constant concern. This could
trigger an immunological or an inammatory response
by the body [2, 17, 64]. These are all highly undesirable
consequences. Labelling stem cells for this application
therefore demands the preservation of physiological cellular
properties and the retention of MNPs over prolonged periods
[66]. This formulates the following question: would any of
the properties related to MNPs indicated for regenerative
medicinal use provoke an adverse eect on either the cells
(in vitro applications) or the body (in vivo applications)?
The toxicity of MNPs on biological entities is highly
dependent on a range and combination of factors related
to the properties of the MNP itself; structural properties,
dosage, and the intended use are among dominating factors
[38, 64]. The chemical composition of the particles themselves can be naturally toxic. Interestingly, some materials
which are known to be inert in large quantities are in fact
toxic at nanoscale, such as gold [64]. It has been found
that certain metals such as cobalt, nickel, cadmium, zinc,
and silver are toxic to biological entities and therefore
not suitable for biomedical applications while others such
as titanium and iron oxide-based particles are considered
signicantly less damaging to cells [17, 30, 33]. In the case
of cadmium selenide (CdSe), cadmium ions demonstrated
to cause cell death in primary hepatocytes [38]. Additionally
the location of MNPs in relation to the cells is an extremely
important consideration [38]. For instance, MNPs could
invoke a cytotoxic response when internalised by potentially
interfering with the biological function of the cells but not
when attached to the cell membrane [11, 38]. In other
cases however, SPIONs attached to the surface of cells may
interfere with cell surface interaction [11]. Furthermore,
the physical properties such as the particle size, shape, and
surface coating can also evoke a toxic response by aggregating
and coagulating according to size and shape [38, 64]. When
addressing the possible in vivo application of MNP, it is
equally important to consider the fate of the MNPs after they
have been released by the cells. Further issues arise when
considering the degradation of the MNPs and the outcome
of accumulated MNPs or MNP by-products in various
tissues and organs [2]. Degradation products are thought
to possibly react with various components of the body or
cells. Therefore, the eects of the breakdown products on
the surrounding tissue should be fully investigated [2, 29].
Therefore assumptions on this matter cannot be made, and
appropriate investigations should always be performed.
At the cellular level, oxidative stress is thought to be the
main cause of toxicity by MNPs [40]. Oxidative stress arises
when there is an imbalance between damaging oxidants also
referred to as reactive oxygen species (ROS) such as hydrogen
peroxide, hydroxyl radicals, and the protective antioxidants
of which vitamin C and glutathione are examples. ROS are
primarily formed by the incomplete reduction of oxygen [67]
The accumulation of oxidants eventually leads to destruction
of cellular proteins, enzymes, lipids, and nucleic acids, and
as a consequence the normal cellular processes become
impaired leading to the development of diseases and cell
apoptosis and necrosis [17, 23, 40, 67]. ROS can be generated
from the surface of MNP, the leaching of metal ions from
the core, or release of oxidants by enzymatic degradation
of the MNP [2]. ROS production can be measured using
dichlorouorescein diacetate uorescent probe after SPION
administration [65]. It has been reported that dissociated
iron oxide MNP can promote the formation of ROS and
hydroxyl radicals, and as a result may lead to cellular
toxicity along with impaired cell metabolism and increases in
apoptosis [11]. On the other hand, ROS can be used in cancer

therapies to destroy damaging cancer, for example [67].
3. Studies
Years of research have demonstrated the ability of SPIONs to
be taken up by a wide variety of cells by simple incubation.
Cell types include broblasts, lung cells, liver cells, stem cells,
kidney cells, macrophages, nerve cells, endothelial cells, and
various cancer cells lines [2]. There is also an impressive
portfolio of MNPs that have been used with a variety of cell
types. The degree of toxicity has been known to vary with
cell type, MNPs, and a combination of these two factors. It
is therefore essential to carry out toxicity studies to evaluate
the toxicity of specic MNP on the particular cell type of
interest [29]. For instance, it has been reported that uncoated
SPIONS caused signicant cell death in dermal broblasts
while lung cells appeared not to be aected [2]. This highlights the importance of cell type and MNP relationship. The
general consensus is that labelling cells with SPIONs is safe
validated by the viability, proliferation, and dierentiation
capacity of cells being unaected thus justifying its use in
regenerative medicine [20, 29]. Other studies have shown
that internalised silica-coated magnetic nanoparticles are
biocompatible with stem cells [38]. Silica is a particular benecial coating for nanoparticles since it can easily be functionalised, and it is resistant to degradation within a cellular
environment whilst still being biocompatible [38, 40].
Toxicity is investigated through a series of in vitro and
in vivo experiments, following the general schema below
(Figure 2). In vitro toxicity tests oer a quick and simple
means of gathering preliminary toxicity data which is
also cost eective with minimal ethical issues [17]. When
carrying out in vitro investigations, it is essential to identify
and apply reproducible in vivo environments in terms of
expanding conditions and sample preparation in vitro [2].
Data revealing marginal or no toxicity via in vitro tests can
then be moved onto in vivo studies [68]. In these situations,
small animal experiments are carried out and monitored
over time to investigate the long-term eects of MNPs in a
biological setting. Toxicity validation tests include histology
on injection sites and major metabolic sites (liver, pancreas,
kidney, brain) to look for signs of MNP spreading and
accumulation. Sections are stained for iron by prussian blue
stain and caspase 3 as an indicator for apoptosis within these
areas [69]. It is not unlikely to have contradicting in vitro
and in vivo results. This may be attributed to in vivo bodily
functions/processes such as homeostasis, working to expel
foreign reagents which are not present in vitro, for example,
kidneys acting to lter blood [17]. Should in vivo studies
reveal encouraging results, both the therapeutic eciency
and the safety of the MNP can be fully evaluated, and the
MNP treatment will have to be approved for clinical use by
regulatory bodies such as the Food and Drug Agency (FDA).
This requires human clinical trials. An important question is
whether the particles are regulated in combination with the
therapy or independently. This question highlights the fact
that the regulatory framework relating to these particles is
limited, thus making it dicult to evaluate the safety of the
In vitro tests
Cell interations
In vivo tests
Small animal
work
Clinical trials
Human trials
Regulatory
approval
FDA, EMEA
Figure 2: Schematic highlighting the route of translating stem cellbased therapies (incorporating the use of MNP) from bench to bed
side.
nanomaterials in conjunction with regenerative medicine.

For this reason, extensive safety assessment of these particles
must be carried out to satisfy not only the regulators but
also the patient. Even though SPION-based MRI contrast
agents have been used and approved, the use of these for
stem cell-based therapies still requires FDA approval as their
intended use is dierent from their use as contrast agents
[36]. An essential requirement of contrast agents is to be
excreted relatively quickly and not remain in the body in the
long term. However, for some applications, especially within
the remit of regenerative medicine, long-term treatment of
MNPs and presence in the body may be necessary. Using
FDA-approved contrast agents will however make this task
easier [66].
Table 1 highlights commonly used in vitro tests for MNPmediated toxicity and cell viability. It can be concluded
that MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), PI (Propidium Iodide), for example,
live/dead assay [70] and trypan blue [71] stains are most
commonly used. Other popular tests not mentioned in
this table include BrdU (5-bromo-2 -deoxyuridine) assay
and the LDH (Lactate dehydrogenase) assay for metabolic
activity [10]. Toxicity assays are aimed at investigating vital
cellular activity such as cell death and cell viability. Cell
death or apoptosis is most commonly assessed using the
tetrazolium compound MTT [72, 73], LDH [74], and PI
[75] assay while cell proliferation is assessd using BrdU
[76] and MTT [73]. Figure 3 describes each test in greater
detail. Other notable cytotoxicity assays include cell-life cycle
assay, TUNEL assay (i.e., for apoptosis detection) [77], and
various redox assays [78]. Immunochemistry can also be
performed to look for markers of apoptosis or necrosis [79].
Furthermore, many toxic eects caused by MNP may stop
mitochondrial activity which is measured by assays such as
MTT. Added to this, there are inherent issues associated
with MNP-cell interaction, dosage, and time course, all
of which may impact on results [79]. It should be noted
that in vitro tests that produce specic and quantitative
toxicity read-outs are particularly convenient for the initial
evaluation of toxicity and biocompatibility of new MNP [79].
An important consideration when interpreting the results of
these types of in vitro assays is that they often provide little
information on the mechanism of toxicity or the cause of cell
death. In addition it can be noticed that SPIONs do not aect
cell viability, proliferation, or dierentiation capacity of stem
uncoated
HEDP
Dextran
80150
150 nm
1630 nm
65 nm
hMSC
rMSC
ESCs
rMSCs
mMSCs
Trypan Blue
MTS
50 g/mL
25, 50,
100 g/mL
50250 g/mL Comet assay
Trypan Blue
2.8 g/mL
MTS
[22]
[40]
Cells maintained viability and retained label for

up to 3 months. Dierentiation capacity not
altered
75% cell viability
Limited cell dierentiation in the osteogenic
lineage
Did not aect cell viability

Cell viability not aected at lower
concentrations but viability decreased to 70% at
100 g/mL
Did not aect viability
[44]
[43]
[42]
[41]
[39]
No reduction in viability after 12 hours
Did not aect cell viability
[38]
[19]
[32]
Reference
No reduction in viability after 1 hour. No

reduction in cell proliferation
No loss of cell viability observed after 7 days

with or without either TA
Result
rMSC: rat mesenchymal stem cells; mMSC: mouse mesenchymal stem cell; hMSC: human mesenchymal stem cells; pMSC: porcine mesenchymal stem cells; HUC: human umbilical cells; HEDP: hydroxyethylidene
bisphosphonic acid.
Feridex
Commercially
available
Feridex
Mesoporous
silica
Manganese oxide
nanoparticles
Particle size Cells type
Particle
Toxicity assay
concentration
Feridex (FDA
rMSC &
Live/dead (7 days after labelling) dierentiation
Dextran
80150 nm
25 g/mL
approved)
mMSC
assays
Ferric oxide
MTT (5 days continued culture) cellular
Poly-L-lysine
15 nm
HUC
20 g/mL
(noncommercial)
apoptosisannexin V/PI double stainassay
MTT ( 1 hr and 24 hrs)
Noncommercial
Silica
110 nm
hMSCs
200 g/mL
Trypan Blue
FACS ( uorescence-activated cell sorting) to
Feridex
Dextran
80150 nm hMSC
100 g/mL evaluate cell deathto determine if iron oxide
incorporation aects surface markers expression
Trypan Blue
Iron uorescent
MTT
Polystyrene
900 nm
pMSC
particle
Morphological observations
Dierentiation studies
Biological
polymer
Particle used
Table 1: Summary of in vitro studies performed in recent years highlighting commonly used assays aimed at investigating toxicity of SPIONs on stem cells and the results.
6
PI assay
MTT assay
A uorescent cell viability assay based

on the simultaneous determination of live
and dead cells. Based on the measurement
of intracellular esterase activity and plasma
membrane integrity.
Live cells have intracellular esterase activity
and enzymatically convert the cell-permeant
calcein AM dye to uorescent calcein.
EthD-1 enters cells with damaged membranes,
binds to nucleic acids and increases uorescence.
The tetrazolium compound

MTT is reduced
by metabolically active cells
to formazan which forms
purple dye crystals.
Crystals are solubilised
and the absorbance read
using a spectrophotometer.
BrdU assay
BrdU is incorporated into DNA during
proliferation. This is measured colorimetrically
and is proportional to the quantity of BrdU
incorporated into cells and therefore is an
indication of cell proliferation
Trypan blue
A dye exclusion test used to determine cell

viability. Live cells exclude trypan blue due to
the properties of intact cell membrane. Dead or
dying cells take up the dye.
Evaluation of plasma membrane

damage. Lactate dehydrogenase
(LDH) is an enzyme, present in all
cells, and rapidly released upon
damage of the cell membrane.
LDH oxidizes lactate to generate
NADH which then reacts with WST to
generate a yellow compound in solution.
Absorbance is directly proportional
to the number of dead/dying cells
LDH assay
Figure 3: Overview of in vitro toxicity assays. Cell image adapted from [2]. PI [75], MTT [72, 73], LDH [59], Trypan Blue [71] and BrdU
[76].
cells. In fact it can be said that number of studies reporting

adverse or toxic eects of MNP are few [17].
Despite cost, time, and ethical considerations, in vivo
tests in relevant animal models are crucial for the study
of biological eects that cannot be modelled in vitro. This
includes the pharmacokinetics of MNP in the body, that
is, absorption, distribution, metabolism, and elimination.
These studies allow information about the systemic toxicities
to be elucidated including immunological, neurological,
reproductive, cardiovascular, reproductive, and developmental as well as any carcinogenic eects [79].
As far as in vivo assays are concerned, one of the
important functions to assess is the blood contact properties
of the MNP, that is, blood compatibility. In terms of in
vivo application of MNP, should the MNP be incompatible
with bio uids such as blood, this could lead to haemolysis,
coagulation, and blood clots due to adsorption and/or
activation of biomolecules, for example, plasma protein,
complement factors, and so forth. As such it is critical to
assess these properties before clinical use. One such test is
the haemolysis assay which uses erythrocytes to assess the
MNP toxicity. The coagulation tendencies can be assessed
using common clinical assays including prothrombin time
(PT), activated clotting time (ACT), and activated partial
thromboplastin time (APTT) [79]. Another parameter to
assess is the inammatory response. This can be evaluated
by looking at levels of chemokines and proinammatory

cytokines, for example, IL-8 in the blood as these can indicate
potential oxidative stress toxic eects caused by MNP [34].
Gene expression analysis for transcription factors associated
with oxidative stress can also be examined and may reveal
useful information about the mechanism of toxicity. This
raises the possibility of correlating MNP characteristics with
toxic eects observed in certain cell lines and tissues [79].
Once the MNPs have entered body, MNPs come into
direct contact with biological macromolecules such as proteins, lipids, and enzymes found within the biological uid.
These biomolecules interact with the surface of the MNP
to form a complex layer of molecules over the MNP. This
biological layer is termed corona [80, 81]. The physical
and chemical properties of MNP (size, surface coating, and
functionalization) dictate the binding of molecules to the
particles resulting in either a hard or weak corona which
is signicantly dierent to that of the surface of the MNP
[80, 81]. This aects the initial interaction of the cell with
the MNP as the cells rst point of contact is with the corona
and not the surface of the particle [80]. In turn, this dictates
subsequent cellular and tissue responses [81]. This protein
corona could be responsible for various toxic outcomes [80].
The formation of the corona is a dynamic process, and competitive process involving the attachment and detachment of
various proteins as the particle moves from one biological
8
environment to another [81] Movement of particles from
one biological environment to another causes proteins from
original uid to be replaced by proteins from new uid
leaving evidence of the previously attached proteins [80, 81].
Approximately 1050 proteins with the highest anity for
the surface are bound at one time [81]. The parameters
aecting MNP-protein interaction include physiochemical
properties (such as surface chemistries, particle size, shape,
charge, surface area, surface defects, smoothness/ roughness,
and functional groups) of MNP and the composition of
biological uid (proteins types and ratios) [81].
The interaction of proteins with the surface of MNPs
of dierent types and coatings is also an important consideration when assessing in vitro toxicity. When MNPs are
added to culture media, media proteins and other nutrients
may adsorb onto MNPs and thus are unavailable for cellular
activities. This naturally has implications on cell growth and
viability. It is paramount therefore to test dierent culture
media as MNP-protein interaction is dependent on the in
vitro environment and MNP composition, so dierent media
recipes could inuence the outcome of cytotoxicity assays
[78, 82].
A related concept is the eect of cell vision presented by
Laurent et al. [82]. This refers to the contact point between
MNP and the cell membrane, which is characterised by its
surface molecules, that is, proteins, sugars, and phospholipid
composition. The binding of MNP to these structures
which may be dierent for dierent cell types denes how
individual cell lines see MNPs. As a result the binding of
exogenous objects like MNP to these structures may cause
dierent responses and inuence uptake and metabolism
depending on the cell type. This concept is exemplied by
Laurent et al. who showed that the same concentration of
SPIONS can cause signicant toxicity on neuronal and glial
cells whilst displaying little toxicity on other cell types like
heart and kidney cells [82, 83]. The issues of cell vision and
MNP-media interactions raise questions about the reliability
of toxicity assays when studying the toxic eects of MNP.
This issue is highlighted by the seemingly contradictory
results of some MNP toxicity assays [34]. To address this
issue Mahmoudi et al. have modied the MTT protocol by
exposing SPIONS to culture media thereby allowing MNP
to interact with culture media proteins before adding them
to cell cultures. This led to improvements in the reliability
of in vitro MNP toxicity results. Their results showed that
uncoated and coated SPIONs were less toxic as previously
thought and induced toxicity in various cell lines at greater
doses than permitted for humans [78]. Furthermore another
problem is posed by the sedimentation of MNPs, as this
phenomenon eectively leads to an apparent increase in
MNP concentration at the cell surface and so should be taken
into account when performing toxicity assays [82].
4. Conclusion and Future Perspectives

It has been mentioned that in vitro and in vivo toxicity
results often contradict each other. This can be attributed to
the issue of protein adsorption onto the surface of MNP. It
has also been suggested that various biomaterials intended
for biological/tissue engineering purposes may benet from
exposure to biological uids prior to implantation. This
would allow for a layer of preadsorbed proteins to adhere on
the surface of the material which may oer several advantages
in terms of cellular mediated responses [84]. Following this
theory, in vivo toxicity studies could be mimicked to a closer
extent in vitro with the MNP in question when exposed to
the desired biological uid.
The multifunctional applications of MNP have been
established. However utilizing micro- or nanoporous structures with nanometer superparamagnetic particles embedded within this structure could oer an alternative means
of activating and inducing dierentiation of stem cells
for tissue engineering in vivo. Various biocompatible and
bioactive materials have been used to fabricate these porous
structures and have had impressive results in terms of cellular
compatibility and dierentiation in terms of osteogenesis
and chondrogenesis [84, 85]. Examples of such porous
nanoparticles include nickel titanium nanoparticles coated
with titanium oxide [85] and calcium phosphate ceramics
[84]. Preexposing nanoparticles to solutions of specic
proteins could also warrant increased cell attachment, proliferations, and the dierentiation of stem cells down various
lineages such as osteogenic or chondrogenic pathways [84,
85]. Thus combing the use of biocompatible materials
known to promote or induce dierentiation with magnetic
properties could be of great benet in tissue engineering and
regenerative medicine.
This paper has addressed the relevance of MNP in regenerative medicine, highlighting their diverse and signicant
applications within this eld. MNPs oer a noninvasive,
practical means of monitoring, controlling, and targeting
stem cells to optimise the therapy with clinical adoption
in mind. However, safety concerns could ultimately prevent
the adoption of MNP in regenerative medicine. This would
demand the development of alternative ways of monitoring
and controlling stem cells in vivo. It is essential to conrm
the safety of the procedure prior to in vivo transplantation
despite clinical ecacy of the technique. In reality, there is an
enormous portfolio of particles available both commercially
and in research. This makes it very dicult to give a denitive
answer to the following question: are MNPs toxic? The
toxic eects of MNPs should be evaluated for each specic
purpose. A large number of in vitro toxicity investigations
have shown no adverse side eect of labelling stem cells with
SPIONs. However, long-term in vivo studies have not been
studied as extensively and hence are an area of much needed
research.
Acknowledgments
The authors would like to thank D. Kumar for his contributions to this work. Support was received from the EPSRC
Doctoral Training Centre (EP/F/500491/1) and LOLA BB/G
/010579/1.
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11

doi:10.1155/2012/975842
Review Article
Silver Nanoparticles in Alveolar Bone Surgery Devices
Stefano Sivolella,1, 2 Edoardo Stellini,3 Giulia Brunello,1 Chiara Gardin,4 Letizia Ferroni,4
Eriberto Bressan,2, 5 and Barbara Zavan4
1 Department
of Oral Surgery, University of Padova Institute of Clinical Dentistry, Via Venezia, 90, 35129 Padova, Italy
of Experimental and Diagnostic Medicine, Section of General Pathology,
Interdisciplinary Center for the Study of Inammation (ICSI) and LTTA Center, University of Ferrara, 44121 Ferrara, Italy
3 Department of Prosthodontics, University of Padova Institute of Clinical Dentistry, Via Venezia, 90, 35129 Padova, Italy
4 Department of Biomedical Sciences, University of Padova, Via G. Colombo 3, 35100 Padova, Italy
5 Department of Periodontology, University of Padova Institute of Clinical Dentistry, Via Venezia, 90, 35129 Padova, Italy
2 Department
Correspondence should be addressed to Barbara Zavan, barbara.zavan@unipd.it

Received 15 March 2012; Accepted 11 June 2012
Academic Editor: Sang Cheon Lee
Copyright 2012 Stefano Sivolella et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Silver (Ag) ions have well-known antimicrobial properties and have been applied as nanostrategies in many medical and surgical
elds, including dentistry. The use of silver nanoparticles (Ag NPs) may be an option for reducing bacterial adhesion to dental
implant surfaces and preventing biolm formation, containing the risk of peri-implant infections. Modifying the structure or
surface of bone grafts and membranes with Ag NPs may also prevent the risk of contamination and infection that are common
when alveolar bone augmentation techniques are used. On the other hand, Ag NPs have revealed some toxic eects on cells in
vitro and in vivo in animal studies. In this setting, the aim of the present paper is to summarize the principle behind Ag NP-based
devices and their clinical applications in alveolar bone and dental implant surgery.
1. Introduction
In recent years, silver nanoparticles (Ag NPs) have been
studied and suggested for a variety of medical, surgical, and
biological applications. Due to their antimicrobial activity,
Ag NPs are widely used to reduce burns, chronic ulcers,
and wound infections by means of AgNP-impregnated
wound dressings [1]. Antimicrobial coatings are especially
important in connection with indwelling catheters carrying
a high risk of bacterial line infections, such as vascular and
urinary catheters [2, 3]. Ag NPs are also added to surgical
instruments, prosthetic devices, and bone replacement materials [46].
The oral cavity is populated by a variety of microorganisms. The microbial communities in the oral cavity are
polymicrobial and exist primarily as biolms. These biolms
can be responsible for several local diseases, including
periodontal and peri-implant diseases, which can lead to the
loss of teeth or implants, respectively, [7]. In fact, periimplantitis remains one of the most serious complications
after implant placement [8]. The potential of Ag NPs to
reduce bacterial adhesion to dental implant surfaces and to
prevent biolm formation has been investigated by many
authors [7, 912] with a view to reducing the risk of periimplant infections.
Another interesting application of Ag NPs in dentistry
is for the structural and surface modication of bone grafts
and membranes with a view to preventing the risk of
contamination and associated infection that are common
when bone augmentation techniques such as guided bone
regeneration (GBR) and guided tissue regeneration (GTR)
are used [5, 1317].
Despite the widespread use of Ag NPs, there is still
a shortage of information on their biological eects on
human cells and environments. Some authors have investigated the potential toxicity of Ag NPs in dierent cell
2
systems, including bacteria and mammalian cells [1826].
Such studies have attributed the cytotoxicity of Ag NPs to
several possible mechanisms, including the dissolving or
release of Ag ions from the nanoparticles, the disruption of
cell membrane integrity, oxidative stress, protein or DNA
binding and damage, the generation of reactive oxygen
species, and apoptotic cell death [27].
The toxic mechanism seems likely to depend on the
nanoparticles properties too, for example, surface area, size
and shape, capping agent, surface charge, particle purity,
structural distortion, and the bioavailability of the individual
particles [24].
In the light of the above considerations, the aim of the
present paper was to describe the antibacterial properties,
bone biocompatibility and toxicity of Ag NPs incorporated
in devices used for alveolar bone regeneration and in dental
implants.
2. Alveolar Bone Surgery, Controlling

Bacterial Infection and Ag NPs
2.1. Periodontitis and Peri-Implantitis. The most common
infectious diseases in alveolar bone are periodontitis and
peri-implantitis. Periodontitis is an inammatory disease
caused by infection of the supporting tissue around the teeth.
Bacteria are essential to its onset, but not enough to cause the
disease, which requires a susceptibility of the host to develop
[28].
The prevalence of severe, generalized periodontitis
ranges from 5% to 20% of any population, while mild-tomoderate periodontitis aects the majority of adults [28].
Peri-implant mucositis is an inammatory lesion conned to the mucosa, while peri-implantitis also aects the
supporting bone [29]. Peri-implantitis is seen in up to 43%
of individuals with implants, and peri-implant mucositis in
up to 50% [30].
Bone loss occurring after initial remodeling is assumed
to be due mainly to bacterial infection. The bacterial biolm
forming on implant surfaces is no dierent from the one
that forms on tooth surfaces, but it may be inuenced by
surface roughness [8]. The initial colonizers that adhere
to tooth and implant surfaces include Streptococcus oralis,
Streptococcus sanguis, and Streptococcus mitis. There is also a
predominant component of Gram-negative species, such as
Eikenella corrodens, Veillonella atypica, and Prevotella loeschii.
Coaggregation bridges are common between these early
colonizers and Fusobacterium nucleatum, which then coaggregates with many late colonizers, which are mostly Gramnegative and anaerobic, and include Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema
denticola, Prevotella intermedia, and Tannerella forsythia [31
33].
Treatments for periodontal and peri-implant diseases
usually include local and systemic measures to combat the
infection, and surgical treatment in certain cases to reduce
the inammation and the depth of the periodontal/periimplant pockets, and restore healthy conditions for the soft
and hard tissues surrounding the tooth/implant. In some
cases, regenerative techniques and materials are used in an
eort to regenerate the periodontal or peri-implant soft and
hard tissues. The success of such surgical bone regeneration
procedures may be negatively aected by infections caused
by oral microbia. Chiapasco and Zaniboni [17] conducted
a systematic review on the clinical outcome of guided bone
regeneration (GBR) procedures for correcting peri-implant
dehiscences and fenestrations associated with implant placement. Peri-implant defects were treated with resorbable or
nonresorbable membranes, with or without graft materials.
In the postoperative period, 20% of the nonresorbable
membranes and 5% of the resorbable ones became exposed
and infected. In the studies considered, the membrane had to
be removed in almost all cases of its exposure and infection,
lowering the success rate of the GBR procedure.
2.2. Ag NPs Antimicrobial Strategy. Strategies for reducing
bacterial adhesion to dental implants seem to be one of
the main elds of interest in dental implantology. Ag
NPs are among the products of nanotechnology already
used in dental practice for their antimicrobial properties
and incorporating them in implant coatings may inhibit
biolm formation on the implant surfaces and prevent
dental implant failure [7, 912]. The use of Ag NPs has
also been suggested to reduce the risk of infections after
oral regenerative surgery. The presence of silver ions in
scaolds or membranes may prove a desirable measure for
minimizing the risk of infections [5, 1316].
Nanoparticles are routinely dened as particles in sizes
ranging between around 1 and 100 nm, and with properties
that are not found in bulkier samples of the same material.
Nanoparticles have a greater surface-to-volume ratio (per
unit of mass) than larger-scale particles of the same material,
and are therefore more reactive. Particles smaller than 50 nm
are subject to the laws of quantum physics [34].
Many new methods of synthesis have emerged and are
being assessed for the purpose of Ag NP production for
medical applications. A number of reports are available
in the literature on the synthesis of Ag NPs: from the
chemical reduction of silver ions by sodium citrate or sodium
borohydride [35]; by reduction into reverse micelles [36];
using biological [37], electrochemical [38], or photochemical
methods [39]; radiation [40], laser ablation [41], solvent
reduction in the presence of surfactants [42], and sonochemical [43] methods.
There has recently been a renewed interest in the synthesis of organic-inorganic and inorganic-inorganic nanocomposite materials [44].
Nanotechnology is a rapidly-growing eld, with nanoparticles being produced and used in a broad range of
commercial products all over the world. Several products
containing Ag NPs are already on the market. Over the years,
the antibacterial activity exhibited by silver compounds and
Ag NPs has resulted in their widespread use in bedding,
washing machines, water purication, toothpaste, shampoo,
nursing bottles, fabrics, deodorants, lters, kitchen utensils,
toys, and humidiers [45].
In particular, Ag NPs have lately emerged in a variety
of biomedical applications exploiting their antibacterial
activity. They have proved to be important in improving
wound healing, and, now that several pathogenic bacteria
have become resistant to various antibiotics, they could be
a safer alternative to conventional antimicrobial agents in
topical antimicrobial formulations [1, 46].
Due to their antibacterial properties, Ag NPs are also
useful for reducing bacterial adhesion and preventing biolm
formation on medical devices, such as catheters or dental
implants, where a bacteria-killing activity is highly desirable
[4751].
Ag+ ions and Ag+ salts have been used for decades
as antimicrobial agents in various elds because of their
growth-inhibiting eect on microorganisms, but they are of
only limited use for a number of reasonsincluding the
interfering eects of salts and the antimicrobial mechanism
related to a continuous release of a sucient concentration
of Ag+ ion from the metal formbut such drawbacks can
be overcome by using Ag NPs. It is essential, however, to be
able to prepare the Ag+ with cost-eective methods and to
understand the mechanism behind their antimicrobial eect
[22].
2.3. Ag NPs Mechanism of Action. What gives silver its
inhibitory eects on microorganisms is still not entirely
clear, and further studies are needed to ascertain all of its
properties.
Silver ions inactivate sulfhydryl enzymes when combined
with amino, imidazole, carboxyl, and phosphate groups.
They also aect DNA replication and stop mitosis in
prokaryotes [52].
Some authors believe that silvers antimicrobial activity
depends on its ions, which bind strongly to electron donor
groups in biological molecules containing sulfur, oxygen,
or nitrogen. This may result in defects in the bacterial
cell wall, with a consequent loss of the cells contents. A
complex formed by silver ions and proteins may disturb the
bacterial cells metabolism and their power functions (such
as permeability and respiration), leading to bacterial cell
death. Silver ions can also interact with the DNA of bacteria,
preventing cell reproduction [53, 54].
Silver interacts with sulfhydryl groups of proteins and
DNA, altering hydrogen bonding, respiratory processes,
DNA unwinding, cell wall synthesis, and cell division. It
also induces denaturation and oxidization of the cell wall,
leading to the rupture of the internal cell organelles, and
thus resulting in bacterial cell death. Ag NPs also modulate
the phosphotyrosine prole of putative bacterial peptides,
which could aect bacterial signal transduction and inhibit
the organisms growth [55].
Ag NPs are known to have an antimicrobial activity
against Gram-negative bacteria, creating pits in the wall
of the bacteria. Amro et al. suggested that metal depletion
may cause the formation of irregularly shaped pits in the
outer membrane and change the membranes permeability,
which is due to the progressive release of lipopolysaccharide
molecules and membrane proteins [56].
The specic bactericidal and antiadhesive ecacy of a
hydroxyapatite/titania nanocomposite coating on titanium
3
plates on Gram-negative bacteria known to be periodontal and peri-implantitis pathogens, such as Streptococcus mutans, Porphyromonas gingivalis, and Fusohacterium
nucleatum was tested by Mo et al. in 2007 [57]; they
found that the bactericidal rate on Gram-negative bacteria
reached almost 90% after 3 hours of anaerobic culturing.
According to these authors, adding a suitable amount of Ag+
anatase TiO2 prompted a photocatalytic activity without UV
light. The existence of Ag+ acted benecially both on the
photocatalytic oxidation of TiO2 and as a bactericide. The
experimental group also showed am anti-adhesion ability to
S. mutans and P. gingivalis. After 3 hours of incubation, there
were hardly any bacterial cells on the nAg-HA/TiO2 -coated
plates, unlike the situation in the control group.
Liao et al. [58] found that a Ti-AgNP surface had
remarkable antibacterial and antiadhesive activities in vitro
in relation to Porphyromonas gingivalis and Actinobacillus
actinomycetemcomitan, which are two of the main culprits
responsible for periodontal and peri-implant diseases. Flores
et al. [59] also reported that a modied AgNP-Ti/TiO2
surface exhibited a good resistance to colonization by
Pseudomonas aeruginosa in vitro.
Other studies investigated the eect of Ag NPs, mainly
against Escherichia coli and Staphylococcus aureus. The Ag
NPs antimicrobial activity against E. coli was investigated
by Sondi and Salopek-Sondi as a model of Gram-negative
bacteria [23]. Their study conrmed that the E. coli cells
were damaged, showing the formation of pits in the
cell wall of the bacteria, while the Ag NPs were found
to accumulate in the bacterial membrane. A membrane
with these morphological features becomes signicantly
more permeable, resulting in cell death. The antibacterial
activity and mechanism of action of Ag NPs on Escherichia
coli were also investigated by Li et al. [60], who analyzed
the growth, the permeability, and the morphology of the
bacterial cells after treatment with Ag NPs. The experimental
results indicated an inhibited growth of E. coli cells in
vitro. Meanwhile, the Ag NPs prompted reducing sugar and
protein leakage, and induced respiratory chain dehydrogenase inactivation, suggesting that they were able to disrupt
the bacterial membranes permeability. These results would
indicate that Ag NPs may damage the structure of the
bacterial cell membrane and inhibit the activity of some
membrane enzymes, eventually causing the death of the E.
coli bacteria.
Kim et al. [22] tested Ag NPs in three representative
microorganisms, that is, yeast, E. coli, and Staphylococcus
aureus. They found that yeast and E. coli growth was
eectively inhibited. The Ag NPs antimicrobial activity
against yeast and E. coli was consistent with the ndings
reported by Sondi and Salopek-Sondi [23]. The inhibitory
eect of Ag NPs was milder on S. aureus than on the
other microorganisms, however, giving the impression that
the antimicrobial eects of Ag NPs may be associated with
the particular characteristics of certain bacterial species.
Dierences between bacterial species may inuence their
susceptibility to antibacterial agents.
Gram-positive and Gram-negative bacteria have dierences in their membrane structures, the most distinctive
4
of which is the thickness of the peptidoglycan layer. The
cell walls of Gram-positive species contain 320 times
more peptidoglycan than those of Gram-negative bacteria.
Since peptidoglycans are negatively charged, they probably
bind some portion of the silver ions in the broth; this
would make Gram-positive bacteria generally less susceptible
to antibacterial agents containing silver ions than Gramnegative species [61].
Danilczuk et al. found that Ag+ generated free radicals
by means of an ESR (electron spin resonance) study on
Ag NPs [62]. Kim et al. also observed an Ag+ -specic
ESR spectrum. The Ag NP peak they obtained in an ESR
assay corresponded to the one obtained by Danilczuk et
al. To clarify the relationship between free radical and
antimicrobial activity, Kim et al. used the antioxidant Nacetylcysteine (NAC) to test whether the antioxidant could
inuence AgNP-induced antimicrobial activity. The results
of ESR tests and antioxidant studies suggest that free radicals
may derive from the surface of Ag NPs and be responsible for
their antimicrobial activity in the experimental conditions
considered [22].
Some researchers have reported that the positive charge
on the Ag+ ion is crucial for its antimicrobial activity through
the electrostatic attraction between the microorganisms
negatively charged cell membrane and the positively charged
nanoparticles [6].
The shape of Ag NPs may inuence with their antimicrobial eect. Pal et al. [63] found that triangular Ag NPs
had a greater biocidal action against E. coli than rod-shaped
or spherical nanoparticles. The dierences can be explained
by the proportion of active facets on the nanoparticles of
dierent shapes. An oriented particulate monolayer X-ray
diraction pattern indicated that triangular nanoparticles
have more high-atom-density facets than other shapes,
favoring the silvers reactivity. Pal et al. also speculated that
the action of Ag NPs is broadly similar to that of silver ions.
Sulfur-containing proteins in the membrane or in the cells,
as well as phosphorus-containing elements such as DNA, are
likely to be the preferential binding sites for Ag NPs.
Another factor that may inuence the eectiveness
of these particles antimicrobial activity is their size. In
the report from Baker et al. [64], the Ag NPs exhibited
antibacterial eects at low concentrations these antibacterial
properties correlated with the total surface area of the
nanoparticles, that is, smaller particles with a larger surfaceto-volume ratio provided a more ecient medium for
antibacterial activity. The nanoparticles were found to be
completely cytotoxic for E. coli at surface concentrations as
low as 8 microg of Ag+ /cm2 .
In agreement with Baker et al., Panac ek et al. [53]
reported that the antibacterial activity of Ag NPs depended
on the size of the silver particles: smaller particles with a
larger surface area available for interaction had a greater
bactericidal eect than larger particles.
Many studies investigated the antifungal activity of silver
nanoparticles against the main fungi frequently found in
the oral cavity, but further studies are needed in this
area as regards the formulations and means of delivery
[6567].
Table 1: Synthesis of the various mechanisms of action of Ag+ ions
and Ag NPs.
Author, year
Sondi and Salopek-Sondi, 2004 [23];
Amro et al., 2000 [56];
Li et al., 2010 [60];
Kawahara et al., 2000 [61].
Li et al., 2010 [60];
Pal et al., 2007 [63].
Pal et al., 2007 [63].
Danilczuk et al., 2006 [62].
Action
Cell wall Pit formation
Protein binding
DNA binding
Free radicals formation
Ag NPs have also shown an antiviral potential, proving

active against several types of virus, including human
immunodeciency virus, hepatitis B virus, herpes simplex
virus, respiratory syncytial virus, and monkeypox virus [68].
A summary of the antimicrobial activities of Ag NPs is
given in Table 1.
3. Ag NPs and Devices

The capacity of silver nanoparticles to control the formation
of biolms in the oral cavity, as a result of their antibacterial
activity, has led to their use in prosthetic device coatings, as
topical agents, and in dental materials. Several devices have
recently been proposed for use in the elds of dental implantology, periodontology, and alveolar bone regeneration. The
prevention of dental implant contamination by bacteria and
the need for biocompatible scaolds or membranes for use
in bone grafts with antibacterial properties (achieved by
including silver particles in the scaold, while maintaining
its structure and characteristics) seem to be interesting
applications of Ag NPs. Below is a review of some of the
devices in which Ag NPs have been used, for example,
membranes for guided tissue regeneration (GTR) and guided
bone regeneration (GBR) applications, scaolds for bone
regeneration, and dental implant coatings.
3.1. Membranes. Barrier membranes are devices used in
GTR/GBR procedures to prevent the rapid ingrowth of
broblasts and/or epithelial cells in a bony defect where
slower-growing bone tissue is desired. Barrier membranes
thus keep out the unwanted soft tissues and provide a
secluded space into which osteogenic cells can migrate and
form new bone.
Over the years, numerous resorbable and nonresorbable
membranes have been used for GTR and GBR applications
[69, 70], but they have been unable to reduce the risk of graft
infection, especially when the membranes are exposed to the
oral cavity. Antimicrobial membranes have been developed
to overcome this problem.
Li et al. [14] analyzed a silver ion-substituted nanohydroxyapatite, titania nanoparticles, and polyamide 66 (AgnHA/TiO2 /PA66) membrane, prepared with the intent of
producing an antimicrobial membrane with a gradient
porous structure for GBR with good mechanical properties,
biocompatibility, and antimicrobial activity. Cytocompatibility and bone formation were assessed by both in vitro and
animal experiments. In vitro assays were carried out with
osteoblastlike cells (MG63), and cell viability, alkaline phosphatase activity (ALP), and morphology of cells cultured on
the membrane were ascertained, demonstrating good cell
anity and an increased cell attachment, migration, and
proliferation. In vivo experiments resulted in the complete
closure of 5-mm bone defects created in the skull of SpragueDawley rats 8 weeks after implantation. The same capabilities
were seen for the nHA/PA66 membrane, whereas the cranial
defects were still not fully covered by newly formed bone 8
weeks after implantation of the PA66 or empty membranes.
Ye et al. [16] studied the eects of the Ag-nHAnTiO2 /PA66 membrane on MG63 osteoblastlike cells in vitro;
nHA/PA66 and expanded polytetrauroethylene (e-PTFE)
membranes were used for control purposes in their assays.
The authors found that the Ag-nHA-nTiO2 /PA66 membrane
had no negative eects on the growth of osteoblastlike cells,
while it favored cell adhesion, thus indicating an excellent
tissue compatibility.
Consistently with the previous article, the same group
of researchers found that the osteogenic activity of AgnHA-nTiO2 /PA66 membranes was comparable with that of
conventional e-PTFE membranes in a rat model, proving to
be a safe strategy for reducing inammatory response and
enhancing bone regeneration, and thus suggesting interesting prospects for further research and the development in
antibacterial GBR membranes [71].
The above ndings are summarized in Table 2.
3.2. Scaolds for Bone Regeneration. Bone augmentation procedures have entailed the use of dierent methods, including
GBR and GTR techniques. Especially in nonspace-making
defects, the use of grafts (autografts, allografts, xenografts,
or alloplasts) prevents membrane collapse, and the graft
provides a scaold on which bone cells can adhere, migrate,
grow, and divide, forming new bone. A suitably macroporous
structure is important to ensure rapid vascularization, bone
ingrowth andespecially in the case of implant placement
bone remodeling, with newly formed bone occupying the site
previously taken up by the scaold [73].
To enhance the regeneration of new bone and reduce the
healing time, bone tissue engineering relies on 3-dimensional
scaolds delivering biofactors to assist bone regeneration. In
addition to facilitating new bone deposition, some authors
are interested in producing osteoconductive scaolds with
bactericidal properties with a view to controlling infection
after alveolar bone surgery.
Wu et al. [13] demonstrated that the Ag-nHA/TiO2 /PA66
antimicrobial scaold they developed had a highly porous
structure that would be eective for the sustained Ag+ release
at the bone-implant interface. According to the authors,
these scaolds would provide good mechanical support and
protection for cell adhesion, migration and proliferation, and
hold promise in clinical application in bone augmentation
techniques.
Saravanan et al. [5] analyzed the in vitro antimicrobial
activity of another biocomposite bone tissue engineering
5
scaold containing chitosan/nanohydroxyapatite/nanosilver
(CS/nHAp/nAg). The results suggested that CS/nHAp/nAg
biocomposite scaolds have the potential for controlling
implant-associated bacterial infection during bone reconstruction surgery thanks to the strong antibacterial activity
seen on testing the prepared scaolds with Gram-positive
(S. aureus) and Gram-negative (E. coli) bacterial strains. The
CS/nHAp/nAg scaolds were also found non-toxic for rat
osteoprogenitor cells and human osteosarcoma cell lines.
Finally, Schneider et al. [15] assessed the in vivo performance in sheep of synthetic, cotton woollike nanocomposites consisting of a biodegradable poly(lactide-co-glycolide)
brous matrix containing silver-doped calcium phosphate
nanoparticles (PLGA/Ag-CaP). The area of new bone formation measured histomorphometrically 8 weeks after implantation was very similar for the PLGA/CaP (control) and
the PLGA/Ag-CaP. In fact, the highly porous PLGA/Ag-CaP
scaolds enabled an ecient cell ingrowth, which facilitated
new bone formation everywhere inside the former defect,
and also led to the resorption of the biodegradable polymer
bers. No signs of any inammatory reaction were detected.
These data are summarized in Table 3.
3.3. Dental Implant Surfaces. Peri-implantitis remains one of
the most serious complications in dental implant surgery and
sometimes necessitates implant removal. In fact, the success
and long-term survival of dental implants depend not only
on bone-implant osteointegration, but also on the prevention of bacterial infection after placement. Incorporating Ag
NPs in implant coatings may inhibit biolm formation on
the surfaces and prevent dental implant failure.
Secinti et al. [74] investigated whether Ag NP coatings
could inhibit biolm formation even in slime-forming
bacteria; 20 New Zealand rabbits were randomly divided into
two equal groups and had bacteria applied to surgical sites
on the iliac crests: screws coated with silver using the sol-gel
method were inserted in the rabbits in Group I, uncoated
Ti screws in the rabbits in Group II. The rabbits were
sacriced after 28 days, and the screws and adjacent bone
were examined under transmission (TEM) and scanning
electron microscopy (SEM). The antibacterial eect of Ag
NPs was also conrmed microbiologically. All silver-coated
screws, but only 10% of the uncoated titanium screws, were
sterile. Biolm formation was inhibited on all the silvercoated screws, whereas all the uncoated screws developed a
biolm on their surfaces. These ndings suggest that AgNPcoated screws are as safe as uncoated titanium screws and
that the coating helps to prevent both biolm formation and
infection.
A recent report from Flores et al. [59] describes a method
for modifying Ti/TiO2 surfaces with citrate-capped Ag NPs.
The nanoparticles spontaneously adsorb on the Ti/TiO2 ,
forming nanometer-sized aggregates consisting of individual
Ag NPs that evenly cover the surface. The modied AgNPTi/TiO2 surface exhibits a good resistance to colonization by
Pseudomonas aeruginosa, a model of biolm formation.
Ionita et al. [11] demonstrated the synergetic antibacterial eect of a HA-silver coating with Ag NPs by comparing
the behavior of such a coating with uncoated samples, and
Table 2: Studies about membranes containing Ag NPs proposed in alveolar bone regenerative surgery.
Author, year
Podsiadlo et al., 2005 [72]
Device
In vitro
Layer-by-layer
assembly of nacre-like
nanostructured
composites with
antimicrobial
properties.
In vitro. In vivo (animal)
Antimicrobial
composite membrane
with an asymmetric
porous structure
In vitro
Ag-nHA-nTiO2 /PA66
membrane
Ag-nHA-nTiO2 /PA66
In vivo (animal)
Ag-nHA-nTiO2 /PA66
membrane
Ag-nHA-nTiO2 /PA66
Li et al., 2012 [14]
Ye et al., 2011 [16]
Zhang et al., 2010 [71]
Description of Ag
NPs included
Type of study
Ag NPs
Ag-nHA/TiO2 /PA66
Main results
(i) Excellent structural stability
with no detectable levels of silver
lost over a 1 month period
(ii) Almost complete growth
inhibition of E. coli over an 18 h
period.
(iii) Biocompatibility with the
human osteoblast cell line
(i) In vitro: good cell (osteoblastlike cells) anity and increase of
cell attachment, migration, and
proliferation.
(ii) In vivo: complete closure of 5mm bone defects created in the
skull of Sprague-Dawley rats after
8 weeks of implantation.
(i) No negative eects on growth
of osteoblast-like cells.
(ii) Loose porous structure of the
membrane helped the adhesion
and proliferation of osteoblastlike cells.
(i) Osteogenic activity comparable with e-PTFE.
Table 3: Studies about scaolds containing Ag NPs proposed in alveolar bone regenerative surgery.
Author, year
Type of study
Device
Description of Ag NPs
included
Wu et al., 2010 [13]
In vitro
Antimicrobial
composite scaolds
Ag-nHA/TiO2 /PA66
Saravanan et al., 2011 [5]
In vitro
Bio-composite
scaold for bone
tissue engineering
Chitosan/nanohydroxyapatite/nanosilver
Schneider et al., 2011 [15]
In vivo, animal
Cotton wool-like
exible articial
bone substitutes
PLGA/Ag-CaP
with samples covered with Ag NPs or hydoxyapatite (HA),

or incorporating an antibiotic (Tobrex). The bacteriological
experiments performed in vitro demonstrated the ecacy
of the silver-coated TiAlZr implants against E. coli bacterial
growth. The hydrophilic nature of the coatings investigated
increased in the same direction percentage inhibition. The
Main results
(i) Ag+ release: time and concentration dependent.
(ii) Ag+ release properties inuenced by the immersion medium.
(i) Antibacterial activity against
both Gram-positive and Gramnegative bacteria.
(ii) Non-toxic to rat osteoprogenitor cells and human osteosarcoma cell line.
(i) Bone formation was not negatively inuenced by the 0.4%
silver.
(ii) Area fraction of new bone in
formation after 8 weeks implantation in hole defects of long
bone in sheep was very similar for
PLGA/CaP and PLGA/Ag-CaP.
antibacterial eect of the biomimetic coating with Ag NPs

was high and similar to that of a biomimetic coating with Ag
NPs and antibiotic.
Zhao et al. [9] reviewed the current status of antibacterial
coatings on titanium implants, acknowledging that in vivo
data on these antibacterial coatings were still scarce and that
surfaces with both an excellent tissue-integrating ability and
good antibacterial properties should be explored.
In a recent study by the same authors [10], titania
nanotubes (TiO2 -NTs) incorporated with Ag NPs were fabricated on Ti implants for the purpose of preventing implantassociated infections. The amount of Ag+ introduced in the
NTs was varied by adjusting the processing parameters. The
authors noted in vitro that the NT-Ag killed all the planktonic
bacteria in the suspension over the rst few days, retaining
this ability with no obvious decline for 30 days, which
would normally be long enough to prevent postoperative
infection in the early and intermediate stages and possibly
even late infection around the implant. Although the NT-Ag
samples showed some cytotoxicity, this could be contained
by controlling the Ag+ release rate, and the properties could
be further tailored to achieve both long-term antibacterial
ability and biointegration.
As mentioned earlier, Mo et al. [57] evaluated the
bactericidal and antiadhesive ecacy of rough-surfaced
titanium plates coated with silver-hydroxyapatite/titania
nanocomposites (nAg-HA/TiO2 ) in combating oral bacteria,
and particularly Gram-negative bacteria, which are known
periodontal pathogens and involved in the etiology of
peri-implantitis (Porphyromonas gingivalis, Fusohacterium
nucleatum, and Streptococcus mutans). The viability of each
type of bacteria on the antibacterial lm was suppressed to
about 10% after anaerobic incubation for 3 hours. Adherence
of the bacteria to the nAg-HA/TiO2 -coated surfaces was
considerably lower than for the uncoated surfaces.
Liao et al. [12] investigated AgNP-modied titanium (TiAgNP) surfaces using a silanization method and demonstrated that titanium (Ti) plates with Ag NPs deposited on
them acquired an antibacterial activity. After 24 hours of
incubation, 94% of Staphylococcus aureus and more than
95% of Escherichia coli were killed on the Ti-AgNP surface.
SEM examination of the antiadhesive properties also showed
that there were fewer bacteria attached to the Ti-AgNP
surface than to an untreated Ti control surface.
Since dental implants, being used in the oral cavity,
should also have an antibacterial activity in relation to
oral bacteria, Liao et al. [58] also tested the antibacterial
and antiadhesive action of a silver nanoparticle-modied
titanium (Ti-AgNP) surface in relation to two of the most
common periodontal pathogens, that is, Porphyromonas
gingivalis and Actinobacillus actinomycetemcomitans. Their
ndings indicate that the Ti-AgNP surface had remarkable
anti-bacterial and antiadhesive eects on P. gingivalis and
A. actinomycetemcomitans, suggesting that Ti-AgNP is a
promising implantable biomaterial in terms of its antibacterial properties. The Ti surfaces modied with Ag NPs also
showed no signs of cytotoxicity on cultured human gingival
broblasts (hGFs), suggesting their potential application also
in transgingival abutments, which are an important doorway
for bacteria.
Implants are in contact not only with bone but also
with gingival tissues, and are partially exposed to the oral
cavity. With a view to the rapid achievement of a rm soft
tissue seal around dental implants that can resist bacterial
invasion, Ma et al. [75] created a surface with a lower
7
cytotoxicity while preserving its antibacterial properties by
modifying the Ti surface, immobilizing the AgNP/FGF-2
(broblast growth factor 2) compounds bioactive factors on
a titania nanotube surface. The immobilized AgNP/FGF-2
samples displayed an excellent cytocompatibility, negligible
cytotoxicity, and enhanced hFGF functions such as cell
attachment, proliferation, and ECM-related gene expression.
The absence of any signicant cytotoxicity may be due to
the negligible amounts of Ag+ ions released by the Ag NP
solution, and, as Williams et al. demonstrated [76], small
concentrations of Ag+ released into the extracts are not
cytotoxic. The Ag NPs also exhibited some bioactivity. These
results lead to the same conclusions as the previous paper
and encourage the use of this material in dental implant
abutments.
The data presented above are summarized in Table 4.
4. Ag NP Toxicity
The impact of Ag NPs on cell viability has been investigated
to establish the feasibility of their use in clinical applications.
Several studies have been performed in vitro (on osteoblasts
and other cell lines), and in vivo (in animal models) for this
purpose.
4.1. In Vitro Studies. Mahmood et al. [77] reported on
the in vitro eects of dierent types of nanomaterial
(single-walled carbon nanotubesSWCNTs, hydroxyapatite
nanoparticlesHAP, titanium dioxide nanoparticlesTiO2 ,
and silver nanoparticlesAg NPs) on cell calcication and
mineralization by bone cells. The greatest of cell mineralization enhancement coincided with the use of Ag NPs,
followed by HAP, TiO2 and SWCNTs. Ag NPs were found
to alter microRNA expression; the numerous transcriptional
factors associated with bone formation (Runx2, Dlx3, and
Msx2) were aected by correspondent miRNAs only in the
bone cells exposed to Ag NPs. Exposure to Ag NPs also
resulted in the miRNA regulation of several gene-target
BMPs not found in the controls. The authors also said
that preliminary cytotoxicity studies performed with the
nanoparticle concentrations used in the experiments gave no
indication of any onset of cytotoxic eects.
Studying layer-by-layer (LBL) assemblies of nacrelike
nanostructured composites containing Ag NPs, Podsiadlo et
al. [72] also reported nding no detectable levels in vitro
of silver being lost over a 1-month period, associated with
a bactericidal eect (E. coli growth was almost completely
inhibited over an 18 h period), and the concentration of silver
did not prevent the growth of mammalian tissue cultures.
The LBL composite showed biocompatibility with human
osteoblast cell lines.
On the other hand, Zhao et al. [10] found in vitro that
titania nanotubes (TiO2 -NTs) incorporating Ag NPs showed
some cytotoxicity in primary rat osteoblasts, which could be
reduced by controlling the rate of Ag+ release.
Albers et al. [78] investigated the cytotoxicity of Ag NPs
(50 nm) in vitro on osteoblasts (OBs) and osteoclasts (OCs)
at antibacterial concentrations: they demonstrated that Ag
Table 4: Studies about dental implants surfaces containing Ag NPs.

Author, year
Type of
study
Device
Description of Ag
NPs included
Zhao et al., 2011 [10]
In vitro
Coatings for
titanium
implants
Secinti et al., 2011 [74]
In vivo,
animal
Coatings on
titanium
implants
Ionita et al., 2011 [11]
In vitro
Coatings on
titanium
implants
Liao et al., 2010 [12]
In vitro
Coatings on
titanium
implants
Ag NPs
Liao et al., 2010 [58]
In vitro
Antibacterial
titanium
plate
Ag NPs
Ma et al., 2011 [75]
In vitro
Modied
titanium
implant
surface
Flores et al., 2010 [59]
In vitro
Coatings on
titanium
implants
Mo et al., 2007 [57]
In vitro
Coatings on
titanium
plates
Titania nanotubes
(TiO2 -NTs)
incorporated with
Ag NPs.
Ag NPs
Ag NPs-HA/TiAlZr
TiO2 nanotubular
surface with
immobilized
compound
Ag/FGF-2
(broblast growth
factor-2)
Ag NPs
spontaneously
adsorb on Ti/TiO2
Ag-HA/TiO2
Main results
(i) The NT-Ag structure
shows some cytotoxicity, that
can be reduced by controlling
the Ag+ release rate.
(ii) Long-term antibacterial
ability. Satisfactory
osteoconductivity (osteoblast
from rat calvarial bone).
(i) Biolm formation
inhibition.
(ii) Nontoxic, and no harmful
side eects detected on the
kidney, liver, brain, or cornea.
(i) Inhibition of growth of E.
coli bacteria.
(ii) Antibacterial eect of
biomimetic coating with Ag
NPs is high and close to value
of biomimetic coating with
silver and antibiotic (Tobrex).
(i) Ti-Ag NPs specimens
signicantly inhibited the
growth of both Staphylococcus
aureus and Escherichia coli
than Ti-polished specimen.
(i) Ti-Ag NPs surface:
remarkable antibacterial and
antiadhesive activities to
Porphyromonas gingivalis and
Actinobacillus
actinomycetemcomitans.
(ii) No detectable cytotoxicity
on cultured human gingival
broblasts (hFGFs).
(i) The TiO2 nanotubular
surface with immobilized
compound Ag/FGF-2 has
excellent cytocompatibility
compared to pure Ti.
(i) Good resistance to
colonization by Pseudomonas
aeruginosa.
(i) Antibacterial activity
against: Porphyromonas
gingivalis, Prevotella
intermedia, Fusohacterium
nucleatum, and Streptococcus
mutans.
NPs had cytotoxic eects on both cell lines, as indicated by
dose-dependent decreases in the number of viable cells and
dierentiations. The inhibitory activity of microparticles of
silver (3 m) was signicantly weaker, a nding consistent
with the results of previous studies [7981]. According to
these authors, the size-dependent cytotoxicity is due to the
size- and surface-area-dependent release of silver ions from
the particles. OBs were found more susceptible to the AgNPmediated inhibition of cell viability and dierentiation than
OCs; both OBs and OCs were more susceptible to silver treatment than S. epidermidis. The MICs of Ag+ deriving from
AgNO3 or Ag NPs used to inhibit bacterial growth were 2
4 times higher than the minimal Ag+ concentration needed
to reduce the viability and proliferation of OBs and OCs.
Finally, Albers et al. conrmed the antimicrobial properties
of Ag NPs on S. epidermidis, but they were critical regarding
the biological safety of silver-releasing implantable materials.
Suresh et al. [27] also found that Ag NPs caused celldependent cytotoxicity: their data emphasized the role of
surface coatings or surface charges and particle aggregation
in dictating the cytotoxicity of Ag NPs, but also showed that
the cell type inuenced their cytotoxic eect. Lung epithelial
cells were more resistant than macrophages to the dierently
surface-coated Ag NPs being tested.
The results obtained by Greulich et al. [82] conrmed
a cell-specic uptake of Ag NPs by peripheral blood
mononuclear cells (PBMC) and accordingly dierent cellular
responses after the exposure of monocytes and lymphocytes
(T cells).
Ag NPs revealed no detectable cytotoxicity on cultured
human gingival broblasts (hFGFs) in vitro [58, 75].
5. Conclusions
Ag NPs have been studied as an alternative strategy for
reducing bacterial adhesion and preventing biolm formation thanks to their antimicrobial properties. Ag NPs have
been included in devices used in alveolar bone surgery with
promising results.
Membranes and scaolds for bone regeneration containing Ag NPs have the potential to reduce the incidence
of postoperative bacterial contamination. One of the most
interesting applications of Ag NPs in dentistry is for preventing or delaying peri-implantitis. Ag NP coatings could
be applied to the whole dental implant surface or to selected
areas, such as the most coronal area of the implant or the
inner threaded surface. Another strategy to reduce biolm
formation on dental implants and the related prosthetic
components in the oral cavity might be to apply Ag NPs to
prosthetic devices such as the healing screws, abutments, and
xing screws.
Published data highlight the importance of surface
coatings, surface charge, speed of release, concentration of
Ag+ , and particle aggregation in dictating the cytotoxicity
of Ag NPs, which also seems to depend on the type of cell
aected.
Reproducibility issues arising from the type of Ag NP
application, the tests conducted and the subjects involved
(cell lines or animals) have made any comparison of the
results reported in dierent studies arduous or controversial.
For the time being, most of the studies analyzed here were
conducted in vitro, and there is a clear need for further
clinical trials. In vitro, and animal studies are needed on the
devices described, focusing particularly on the oral microbiota responsible for periodontal and peri-implant diseases.
Conict of Interests
4.2. In Vivo Studies. Other studies have raised concerns
regarding the safety of silver applications in animal models.
Hyun et al. [83] investigated the eects of repeated
exposure to Ag NPs on the histological structure and
mucins of the nasal respiratory mucosa: rats were exposed
to dierent concentrations of silver nanoparticles in an
inhalation chamber for 6 hours a day, 5 times a week for
28 days, after which the animals were sacriced. The study
indicated that the silver nanoparticles inuenced the neutral
mucins in the respiratory mucosa, but not to a toxicologically
signicant degree.
Kim et al. [84] found some signicant dose-dependent
changes in the alkaline phosphatase and cholesterol levels in
male and female rats, which seemed to indicate that exposure
to more than 300 mg of Ag NPs might result in mild liver
damage. Ag NPs were found to induce no genetic toxicity in
male or female rat bone marrow in vivo.
In their in vivo study, Secinti et al. [74] investigated
the antibacterial eect of silver-coated titanium implants in
rabbits, examining their liver, kidney, brain and cornea under
transmission (TEM), and scanning electron microscopy
(SEM). They detected no harmful side eects in these rabbit
organs after the placement of Ag NPs coated screws.
The authors conrm that there is no conicts of interests.
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doi:10.1155/2012/858073
Research Article
In Vitro Biocompatibility of Endodontic Sealers Incorporating
Antibacterial Nanoparticles
Itzhak Abramovitz,1 Nurit Beyth,2 Guy Weinberg,2 Alon Borenstein,2
David Polak,3 Dana Kesler-Shvero,2 and Yael Houri-Haddad2
1 Department
of Endodontics, Hebrew University-Hadassah School of Dental Medicine, 91120 Jerusalem, Israel

of Prosthodontics, Hebrew University-Hadassah School of Dental Medicine, 91120 Jerusalem, Israel
3 Department of Periodontics, Hebrew University-Hadassah School of Dental Medicine, 91120 Jerusalem, Israel
2 Department
Correspondence should be addressed to Nurit Beyth, nuritb@ekmd.huji.ac.il

Received 2 April 2012; Revised 12 June 2012; Accepted 14 June 2012
Copyright 2012 Itzhak Abramovitz et al. This is an open access article distributed under the Creative Commons Attribution
cited.
The main cause of endodontic disease is bacteria. Disinfection is presently achieved by cleaning the root canal system prior
to obturation. Following setting, root canal lling is devoid of any antibacterial eect. Endodontic sealers with antimicrobial
properties yet biocompatible may enhance root canal therapy. For this purpose, quaternized polyethylenimine nanoparticles which
are antibacterial polymers, biocompatible, nonvolatile, and stable may be used. The aim of the present study was to examine the
impact of added QPEI on the cytotoxicity of AH Plus, Epiphany, and GuttaFlow endodontic sealers. The eect of these sealers on
the proliferation of RAW 264.7 macrophage and L-929 broblast cell lines and on the production of TNF from macrophages was
examined. Cell vitality was evaluated using a colorimetric XTT assay. The presence of cytokines was determined by two-site ELISA.
Results show that QPEI at 1% concentration does not impair the basic properties of the examined sealers in both macrophages and
broblast cell lines. Incorporation of 1% QPEI into the sealers did not impair their biocompatibility. QPEI is a potential clinical
candidate to improve antibacterial activity of sealers without increasing cytotoxicity.
1. Introduction
Endodontic disease of human teeth, a common reality, is
caused primarily by bacteria [1, 2]. State-of-the-art disinfection is achieved through biomechanical preparation
that includes mechanical instrumentation and antiseptic
irrigation. Shaping the root canal space allows for a threedimensional obturation and seal of the root canal system.
The endodontic sealer coats the walls of the canals and lls
the space between the root canal lling material and the
root, thus lling the root canal and sealing its ports of entry
and exit. The set root canal lling material is mostly inert.
However, it is expected to physically prevent oral bacterial
contamination as well as regrowth of residual bacterial
contamination thus protecting the tooth supporting tissues
[3, 4]. Regrettably, even when eective instrumentation,
irrigation, and an adequate obturation of the root canal are
performed the irregularity in shape (lateral ducts, and anastomosis) allows for bacterial persistence. In most cases, failure of endodontic treatment is associated to the persistence
of bacteria in the root canal system [5] or their consecutive
coronal leakage. Consequently, bacterial eradication from
the root canal system and prevention of coronal leakage
are of utter importance. Therefore, root canal sealers with
good sealing ability and longer antimicrobial activity are
desirable. Moreover, antibacterial endodontic sealers should
be lethal against contaminating bacteria without harming
tooth-supporting tissues [6].
Research has shown a positive correlation between antibacterial properties of sealers and their cytotoxic eect
[7]. Cytotoxicity becomes even more relevant since extrusion of sealer during root canal obturation is a common
clinical nding [8]. The cytotoxic eect depends on the
leachability of the material [9]. Root canal lling materials
Table 1
Material
AH Plus
Epiphany
Composition
Paste A
(i) Diglycidil-bisphenol-A-ether.
(ii) Calcium.
(iii) Tungsten.
(iv) Zirconium oxide.
(v) Aerosol.
(vi) Iron.
(vii) Oxide.
BisGMA, ethoxylated BisGMA,
UDMA, hydrophilicdifunctional
methacrylates.
Paste B
(i) Amina 1-adamantane.
(ii) N,N dibenzyl-5-oxanonandiamine-1,9.
(iii) TCD-diamine.
(iv) Calcium tungsten.
(v) Zirconium oxide.
(vi) Silicone oxide.
Calcium hydroxide, barium sulphate,

barium glass, bismuth oxychloride, silica.
(i) Polydimethylsiloxane
particles.
GuttaFlow
Manufacturer
AH Plus
Dentsply/Maillefer,
Konstanz, Germany.
Epiphany SE Pentron
Clinical Technologies,
Wallingford, CT, USA.
GuttaFlow
Coltene-whaledent
Langenau, Germany.
(ii) Silicone.
(iii) Paran oil.
(iv) Platinum catalyst.
(v) Zirconium dioxide.
(vi) Nano-silver.
(vii) Gutta-percha powder.
and medicaments may leach through dentin [10] or when

extruded beyond the root apex and cause a cytotoxic damage
[11]. Nonresorbable sealers may cause a cytotoxic eect for
several days due to polymerization derivates release such as
formaldehyde [12]. Regardless, these sealers are widely used
in the clinical daily practice and are accepted owing to their
chemical stability.
Based on the above, the antimicrobial activity of endodontic sealers may be an important tool for infection control.
To reach this goal, antibacterial quaternary ammonium polyethylenimine (QPEI) nanoparticles were incorporated into
conventional endodontic sealers. Previously, we showed that
when these nanoparticles were immobilized into resin-based
materials, they caused a strong, long-lasting antibacterial
eect upon contact without leaching of the nanoparticles
and without compromise of the mechanical properties [13].
Furthermore, dental restorative composites incorporating up
to 2% wt/wt QPEI nanoparticles caused no inammatory in
vivo response [14].
It seems that incorporation of QPEI nanoparticles into
endodontic sealers may be benecial in achieving antibacterial activity [13]. Nonetheless, biocompatibility of modied
endodontic sealers with antibacterial nanoparticles needs to
be established by comparing these new materials to nonmodied sealers. In the present study, the cytotoxic eect was
tested using broblasts and macrophages cells. These cells
have been shown to play a critical role in the biological
response to materials [15]. Due to the fact that macrophages
direct much of the chronic inammatory response, the
ability of a material to alter a cells viability or secretion
function may have signicant consequences on the overall
biological response to a given material [16].
The aim of the present study was to compare the cytotoxic eect of 3 commercially available sealers prior and
following incorporation of 1%, or 2% QPEI on macrophages
and broblast cell lines and on the secretion of TNF from
the macrophages.

2.1. QPEI Nanoparticle Preparation. The synthesis of quaternary ammonium PEI nanoparticle was previously described
by Beyth et al. [13]. Briey, PEI (10 g, 0.23 mol monomer
units) dissolved in 100 mL ethanol was reacted with dibromopentane at a 1 : 0.04 mol ratio (monomer units of
PEI/dibromopentane) under reux for 24 hrs. N-alkylation
was conducted as follows: octyl halide was added at a 1 : 1 mol
ratio (monomer units PEI/octyl halide). Alkylation was carried out under reux for 24 hrs, followed by neutralization
with sodium hydroxide (1.25 equimolar, 0.065 mol) for an
additional 24 hrs under the same conditions. N-methylation
was conducted as follows: 43 mL (0.68 mol) of methyl iodide
were added and methylation was continued at 42 C for
48 hrs followed by neutralization with sodium bicarbonate
(0.23 mol, 19 g) for an additional 24 hrs. The supernatant
obtained was decanted and precipitated in 300 mL of double
distilled water (DDW), washed with hexane and DDW, and
then freeze-dried. The purication step was repeated using
additional amounts of hexane and DDW. The average yield
was 70% (mol/mol).
2.2. Preparation of the Test Samples. The tested materials
AHPlus, Epiphany, and GuttaFlow (Table 1) were prepared
according to the manufacture instructions and applied on
Table 2: Summary of atomic percentage values of elements determined by XPS analysis and DLS measurements.
Tested material
AH Plus
AH Plus + 2% QPEI
I 3d
0.08/0.64
Atomic concentration %/Mass concentration %

O 1s
C 1s
21.79/21.85
59.08/44.48
21.17/21.46
61.09/46.48
Si 2p
19.13/33.67
17.66/31.42
DLS Analysis
AHPlus with and without added QPEI nanoparticles.

a QPEI nanoparticle presence in the modied sealers was estimated using X-ray photoelectron spectroscopy; using Kratos Axis Ultra spectrometer (Kratos
Analytical Ltd., Manchester, UK), using Al K monochromatic radiation X-ray source (1,486.7 eV). AH plus discs incorporating 0% or 2% wt/wt were
prepared and left to dry in the incubator for 1 week.
b Supernatant of AHPlus with and without QPEI nanoparticles was determined using dynamic light scattering analyzer (Zetasizer Nano-S, Malvernd); samples
(n = 8) were incubated for 1 week; supernatant was collected and testedno particles.
specially designed plastic inserts. The nanoparticle powder

was added at 0, 1, or 2% wt/wt to each of the endodontic
sealers and homogeneously mixed according to the manufacturers instructions. These plastic inserts were sterilized
under gas condition for 24 hrs and placed in a 96-well tissue
culture plate directly to the cells. In some of the experiments,
the inserts were immersed in media for another 24 hrs, for
residual materials washing, before challenging the cells.
2.3. Material Characterization. The FT-IR spectra of the
QPEI nanoparticles were recorded on a Perkin-Elmer,
2000 FTIR. FT-IR: 3,440 cm1 (NH), 2,956, 2,926,
and 2,853 cm1 (CH), 1,617 cm1 (NH, small band),
1,465 cm1 (CH), 967 cm1 quaternary nitrogen. 1H-NMR
(DMSO): 0.845 ppm (t, 3H, CH3 , octane hydrogens),
1.24 ppm (m, 10H,CH2 , octyl hydrogens) 1.65 ppm (m,
2H, CH, octyl hydrogens), 3.23.6 ppm (m, CH3 of
quaternary amine, 4H, CH2 , PEI hydrogens and 2H,
CH2 , octyl hydrogens. QPEI nanoparticle presence in the
modied sealers was estimated using X-ray photoelectron
spectroscopy (XPS). X-Ray photoelectron spectra (XPS)
were recorded using Kratos Axis Ultra spectrometer
(Kratos Analytical Ltd., Manchester, UK), using Al K
monochromatic radiation X-ray source (1,486.7 eV). The
emission current was set for 15 mA, and the anode high
voltage to 15 kV. All XPS spectra were collected with 90
take o angle (normal to analyzer), vacuum condition in the
chamber was 1.9109 Torr. The survey XPS spectra were
acquired with pass energy 160 eV and 1 eV step size, and the
high-resolution spectra were collected for C 1s, O 1s, Si 2p,
Zr 3d and I 3d levels, with pass energy 20 eV and 0.1 ev step
size. Additionally, the leaching of QPEI nanoparticles from
the modied sealers was recorded using DLS measurements.
Samples size distributions were evaluated by a dynamic
light scattering analyzer (Zetasizer Nano-S, Malvern.
Surface characterization of the nonmodied versus the
modied sealers is summarized in Table 2. Nanoparticle
electronic microscopy and full characterization of the QPEI
nanoparticles was previously described by Yudovin-Farber
et al. [17].
2.4. Cell Cultures (Macrophages and Fibroblast Cells). The
raw 264.7 macrophage cell line and broblast (CCL-1
L-929 american type culture collection) cell lines were
cultured separately in Petri dishes in Dulbeccos minimum
essential Medium (DMEM) supplemented with 10% fetal

calf serum (FCS), 1% penicillin/streptomycin, and 1%
glutamine. Before the assay, the cells were seeded at a density
of 60,000 cells in 200 L media per well in 96-well tissue
culture plates (NUNC). Twenty-four hours after plating, the
cells were activated by 10 L of heat-killed Porphyromonas
gingivalis 33277 ATCC and exposed to the materials layered
on the special inserts that was placed in the wells. Following
24 hrs of incubation at 37 C in a humidied atmosphere
of 5% CO2 , the plates were analyzed for cell viability and
the secreted levels of TNF of macrophages. Based on our
preliminary data on the kinetics of TNF secretion, the
cytokine level was tested by ELISA following 24 hours of P.
gingivalis challenge.
2.5. Cell Viability. The viability of the cells was evaluated
using a colorimetric XTT assay as described by Scudiero et al.
[18]. At the end of incubation the suspended cells were
exposed to the tested materials, the cells were centrifuged
and all cells were subjected to XTT assay. Results present
the total eect of the materials on the cells in all groups
compared to control. The assay is based on the ability of
metabolically active cells to reduce the tetrazolium salt XTT
to orange-colored compounds of formazan. Following 24 hrs
of incubation, 50 mL of XTT labeling mixture were added
to each well and the microplate was incubated for a further
4 hrs. A Vmax microplate reader (Molecular Devices, Palo
Alto, CA) with a 450 nm optical lter and a 650 nm reference
wavelength was used to measure the absorbance of each well.
2.6. Cytokine Analysis. The presence of cytokines was determined by two-site ELISA [19]. The TNF assay was based
on commercially available antibody pairs (Pharmingen, San
Diego, CA). The 96-well ELISA plates were coated with
1 mg/mL anti-mouse cytokine monoclonal antibodies, and
blocked with 3% bovine serum albumin (BSA). After addition of the samples, a secondary biotinylated antibody was
used as the detecting antibody, followed by a streptavidin
horseradish peroxidase conjugate (Jackson Immunoresearch
Laboratories, West Grove, PA). The substrate used was ophenylenediamine (Zymed, San Francisco, CA). The reaction
was terminated by the addition of 4 N sulfuric acid, and the
optical density was read with the aid of a Vmax microplate
reader (Molecular Devices) at 490650 nm.
140
120
100
(%)
80
60
40
20
0
Control
AH + 0% AH + 1% AH + 2% Epiphany Epiphany Epiphany GuttaFlow GuttaFlow GuttaFlow

0%
1%
2%
0%
1%
2%
Figure 1: The eect of AH+, Epiphany, and GuttaFlow endodontic sealers on the mitochondrial activity of the L-929 broblast cell line.
Mitochondrial activity was normalized to the control without sealers stimulation. Stimulated broblasts (with heat-killed P. gingivalis) with
and without the various endodontic sealers were cultured for 24 hrs. Cell viability was tested using the XTT test. The results are expressed as
the mean + SD. Statistically signicant dierences between the control (media) and the dierent groups are indicated by asterisks (P < 0.05)
and n = 8 wells.
2.7. Heat-killed Bacterium P. gingivalis. P. gingivalis, strain

ATCC 33277, was grown on blood agar plates in an anaerobic
chamber with 85% N2 , 5% H2 , 10% CO2 . After incubation at
37 C for 2-3 days the bacteria were inoculated into peptone
yeast extract and incubated for 3-4 days, under the same
conditions. To obtain heat-killed bacteria, the bacteria were
washed 3 times with PBS and then exposed to 80 C for
10 min [20]. The bacterial concentration was standardized
to an optical density of OD650 = 0.1, corresponding to
1010 CFU/mL [21]. The heat- killed bacteria were stored at
4 C until used when they were resuspended in solution by
brief sonication.
2.8. Statistical Analysis. Data analysis was performed using
SigmaStat statistical software (Jandel Scientic, San Rafael,
CA). One way repeated measure of analysis of variance (RM
ANOVA) was used to test the signicance of the dierences
between the treated groups. If signicance was established,
the intergroup dierences were tested for signicance using
Students t-test with the Bonferroni correction for multiple
testing. The signicance of the dierences between two
treatment groups was evaluated using the t-test. The level of
signicance was set at P < 0.05. All the results are presented
as the mean standard error.
3. Results
3.1. The Cytotoxic Eect on Fibroblasts. The cytotoxicity of
endodontic sealers incorporating 0, 1, or 2% wt/wt was
evaluated using the XTT test. Fibroblasts were exposed
to the tested materials for 24 hrs, and their viability was
normalized according to the control (cells without any

material) mitochondrial activity and evaluated. Results show
that the broblasts viability varied when exposed to the
dierent sealers: in the presence of AHPlus viability of
the cells was not aected when compared to the control,
Epiphany caused signicant decrease 20% in the cell viability and GuttaFlow increased cell viability. The addition
of antibacterial nanoparticles had an insignicant eect on
cell viability when compared to the nonmodied endodontic
sealer material: cell viability was similar in the presence
of Epiphany and GuttaFlow with or without added QPEI
nanoparticles, AHPlus incorporating 2% QPEI nanoparticles
caused some decrease in cell viability only no signicant
decrease was seen in the 1% group (Figure 1).
Immersion of the test materials after their setting in
media for 24 hrs, did not aect the viability of the broblasts.
The same pattern of cytotoxicity of the dierent sealers
independent of nanoparticles incorporation can be seen in
Figure 2. AHPlus sealer incorporating 2% QPEI was the
only group to show a slight decrease in broblast viability
compared to the nonmodied sealer.
3.2. The Cytotoxic Eect on Macrophages. Macrophages were
exposed to the tested materials for 24 hrs, and their viability
was normalized according to the control (cells without
any material) mitochondrial activity. Two of the three
dierent sealers, AH Plus and Epiphany, caused a decrease
in macrophage viability, while GuttaFlow had no eect when
compared to control. The incorporation of the nanoparticles
into the sealers had no eect when added at 1% wt/wt,
whereas when 2% wt/wt of nanoparticles were incorporated
140
120
100
(%)
80
60
40
20
0
Control

0%
1%
2%
0%
1%
2%
Figure 2: The eect of AH+, Epiphany and GuttaFlow endodontic sealers on the mitochondrial activity of the L-929 broblast cell line after
24 hrs immersion in media. Mitochondrial activity was normalized to the control without sealers stimulation. Stimulated broblasts (with
heat-killed P. gingivalis) with and without the various endodontic sealers were immersed in media for 24 hrs, and then transferred to cell
cultured broblasts for another 24 hrs. Cell viability was tested using the XTT. test The results are expressed as the mean + SD. Statistically
signicant dierences between the control (media) and the dierent groups are indicated by asterisks (P < 0.05) and n = 8 wells.
reduced viability of the cells was seen in all three sealers when
modied (Figure 3).
Immersion of the test materials after their setting on the
inserts in media for 24 hrs, reduced the cytotoxicity of all
three materials. The viability of the macrophages was similar
to the control (Figure 4). Incorporation of the nanoparticles
into the sealers had no eect when added at 1% wt/wt,
whereas when 2% wt/wt of nanoparticles were incorporated
reduced viability of the cells was seen with all the tested
materials similarly as in the nonwashed materials (Figure 4).
3.3. TNF Secretion by Macrophages. Macrophages were
exposed to the materials for 24 hrs combined with heat-killed
P. gingivalis challenge to stimulate the cells. The secretion
of TNF by macrophages was measured and normalized
according to the control (cells with bacterial challenge alone).
Challenged macrophages showed reduced secretion of TNF
in the presence of AHPlus and Epiphany. Similar TNF
secretion was seen when nanoparticles were incorporated
into AH Plus with no eect on macrophage activity. However, incorporation of nanoparticles into Epiphany reduced
the levels of TNF to nondetectable (Figure 5). The eect of
GuttaFlow on macrophage activity seemed neglect able. Both
the nonmodied sealer and the sealer incorporating 1% or
2% nanoparticles did not aect TNF, secretion and the levels were similar to the control with no statistical signicance.
4. Discussion
Results show that the incorporation of 1% wt/wt of QPEI
nanoparticles into endodontic sealers has no cytotoxic eect
for all three tested materials. The viability of both cell lines:
broblasts and the macrophages remain stable when compared to the nonmodied sealers. The incorporation of 2%
nanoparticles seems slightly toxic depending on: the tested
material, the culture conditions, and the cell type. This
diverse eect of the materials with dierent cell lines is a very
important outcome for future designing cytotoxic studies.
Endodontic sealers are routinely used to ll gaps within
the solid components of the root canal lling and allow its
adaptation to the dentin walls [4]. Since root canal lling is
the ultimate barrier between the oral cavity and inner body
(e.g., periodontal ligament and bone) an antibacterial eect
is desirable. However, previous attempts to render sealers
with antibacterial properties resulted in sever cytotoxic eect
[22]. This eect was evident when sealer leaching through
dentin various ports of exit from the root canal system to
the tooth surrounding tissues occurred [10, 11, 14]. Such
events were reported to be responsible for cases of pain, nerve
paresthesia, anesthesia and delayed healing due to their cytotoxic eect. More troubling is the possible cytotoxic eect to
remote organs once these materials are dissolved [23].
Several materials constituting endodontic sealers were
recognized as cytotoxic. For example, eugenol that was
proved to leach out of zinc-eugenol bases sealers was found
cytotoxic to nerve cells as well as to human periodontal
ligament (PDL) broblasts [2426]. Another example is
paraformaldehyde that was vastly used in the past and is
a byproduct of contemporary epoxy resin based sealers
[27]. Paraformaldehyde was reported to be cytotoxic and
mutagenic [28]. Another component that is released during
setting is polyketone. It can be found in the polyketonebased cement Diaket which was reported to be cytotoxic to
120
100
(%)
80
60
40
20
0
Control

0%
1%
2%
0%
1%
2%
Figure 3: The eect of AH+, Epiphany and GuttaFlow endodontic sealers on the mitochondrial activity of the RAW macrophage cell line.
Mitochondrial activity was normalized to the control without sealers stimulation. Stimulated macrophages (with heat killed P. gingivalis)
with and without the various endodontic sealers were cultured for 24 hrs. Cell viability was tested using the XTT test. The results are expressed
as the mean + SD. Statistically signicant dierences between the control (media) and the dierent groups are indicated by asterisks (P <
0.05) n = 8 wells.
140
120
100
(%)
80
60
40
20
0
Control

0%
1%
2%
0%
1%
2%
Figure 4: The eect of AH+, Epiphany, and GuttaFlow endodontic sealers on the mitochondrial activity of the RAW macrophage cell line
after 24 hrs immersion in media. Mitochondrial activity was normalized to the control without sealers stimulation. Stimulated macrophages
(with heat-killed P. gingivalis) with and without the various endodontic sealers were immersed in media for 24 hrs and then transferred
to cell cultured macrophages for another 24 hrs. Cell viability was tested using the XTT test. The results are expressed as the mean + SD.
Statistically signicant dierences between the control (media) and the dierent groups are indicated by asterisks (P < 0.05) and n = 8 wells.
HeLa cells and L-645 broblast cells [29]. Another group of

materials that was reported to release cytotoxic components
such as triethylene glycol dimethacrylate monomer, urethane
dimethacrylate (UDMA), HEMA initiators, and silica are
resin-reinforced materials [19].
In the present study, it was observed that the cytotoxic

eect diered between materials. AHPlus caused a moderate
cytotoxic eect that could be attributed to bisphenol A diglycidyl ether release. This resin-based component is known
to be potentially cytotoxic and mutagenic [30]. In addition,
140
120
100
(%)
80
60
40
20
0
Control

0%
1%
2%
0%
1%
2%
Figure 5: The eect of AH+, Epiphany, and GuttaFlow endodontic sealers on TNF secretion in the RAW macrophage cell line.
Mitochondrial activity was normalized to the control without sealers stimulation. Stimulated macrophages (with heat-killed P. gingivalis)
with and without the various endodontic sealers were cultured for 24 hrs in the presence of heat-killed P. gingivalis. The secreted TNF levels
in the media were analyzed by ELISA. The results are expressed as the mean + SD. Statistically signicant dierences between the control
(media) and the dierent groups are indicated by asterisks (P < 0.05) and n = 8 wells.
the cytotoxic eect can be attributed to the release of

small amounts of formaldehyde or amine and epoxy resin
components from the sealer [31]. The resin component urethane dimethacrylate (UDMA) that can be found in EndoRez sealer was reported to cause intracellular glutathione
depletion even at low concentrations within a short period
of time resulting in a cytotoxic eect [32]. Resin-based
endodontic sealers such as Epiphany or Metaseal consist
of polymerized resin components reinforced by inorganic
llers. The resin matrix comprise a mixture of bisphenol
A-glycidyl methacrylate (Bis-GMA), ethoxylated Bis-GMA,
UDMA, and hydrophilic difunctional methacrylates [33],
all of which when leaching out may produce a toxic eect.
Metaseal contains HEMA which is reported to inhibit
intracellular tyrosine phosphorylation in L929 cells [34]. It
also induces cell-growth inhibition and cycle perturbation
as well as glutathione depletion and reactive oxygen species
production [35]. Glutathione depletion was reported to
be responsible for the cytotoxic eect of eugenol [36].
Formaldehyde releasing sealers such as AH26 and N2,
were shown to downregulate alkaline phosphatase causing
inhibition of new bone formation [37].
In vitro cytotoxicity evaluation of endodontic sealers
provides a controlled setup that allows quick evaluation
and comparison between dierent sealers. Previous studies
showed that a silicon-based sealer (Roekoseal) and an epoxy
resin-based sealer (AHPlus) are non-toxic when freshly
prepared or following a setting period of one to seven
days, showing 4060% cells viability as compared to Teon
controls. Under the same conditions Epiphany was found to
be highly cytotoxic showing 0% viability [38]. Similarly, 12week-aged AHPlus retained its ability to suppress cytokine
secretion of monocytic cells without induction of secretion
of TNF, Il, or IL6. One year analysis of elutes of AH
Plus and Roekoseal showed minimal cytotoxic eect on 3t3
broblasts as well as on periodontal ligament broblasts [9].
A study by Al-Hiyasat et al. showed that dierent cytotoxic
eect of AHPlus and Epiphany depends on magnitude of
dilution of the samples [19]. Contradictory to traditional
root canal sealers that polymerize inside the root canal
and may release antibacterial and cytotoxic derivates as
byproducts, QPEI are prepolymerized and do not release
any by products. QPEI was reported to possess an excellent
antibacterial activity [39]. When these polymers are synthesized as insoluble antibacterial nano-sized particles and
are incorporated into resin base materials, potent and longlasting antibacterial surface properties can be attained in
vitro and in vivo [13, 40]. Usage of nanomaterials altering
surface properties has been shown suitable for various
biomedical applications [41, 42]. QPEI is a stable antibacterial compound that does not leach out from the material into
the surrounding environment rendering perpetual antibacterial surface properties. The present study results coincide
with our previous results showing that the incorporation
of QPEI nanoparticles does not aect the base materials
biocompatibility.
Incorporation of QPEI nanoparticles into noncytotoxic
endodontic sealers such as silicon-based sealers may provide
added value of antibacterial properties without compromising the materials biocompatibility. Furthermore, this
antibacterial eect is expected to last longer as QPEI nanoparticles are nonleachable components encapsulated in the
material matrix while the antibacterial eect of traditional
sealer is self-limited to the setting or degradation periods.
[11]
Conict of Interests
[12]
A patent entitled Antimicrobial Nanoparticulate Additives

Forming Non-Leachable Sustained Antimicrobial Polymeric
Compositions is pending approval.
[13]
Authors Contribution
I. Aramovitz and N. Beyth contributed equally to this work.
[14]
Acknowledgments
The authors would like to acknowledge the Dentsply Israeli
branch and Dentorient Fuss Ltd. for kindly supplying the
endodontic sealer materials.
[15]
[16]
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doi:10.1155/2012/160145
Research Article
Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem
Cell-Derived Fibroblasts and an L-929 Cell Line
Hui Peng,1 Xuehui Zhang,1 Yan Wei,1 Wentao Liu,1 Shenglin Li,2, 3 Guangyan Yu,4
Xin Fu,5 Tong Cao,5 and Xuliang Deng1
1 Department
of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, China
Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, China
3 Laboratory of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing 100081, China
4 Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing 100081, China
5 Faculty of Dentistry Research Laboratories, National University of Singapore, Singapore 119083
2 Central
Correspondence should be addressed to Xuliang Deng, kqdengxuliang@bjmu.edu.cn

Received 4 April 2012; Accepted 20 May 2012
Copyright 2012 Hui Peng et al. This is an open access article distributed under the Creative Commons Attribution License,
Consensus about the toxicity of silver nanoparticles (Ag-NPs) has not been reached, even though extensive attention has been
paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety
evaluation. In the present study, human embryonic stem cell-derived broblasts (EBFs), which have been considered a closer
representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (20 nm and 100 nm)
in comparison with L-929 broblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were
examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity
to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared
with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating
that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.
1. Introduction
The unique physicochemical properties of nanomaterials
have allowed their rapid progress and acceptance into
nanobiotechnology and life science. There are reports that
silver nanoparticles (Ag-NPs) have been widely accepted in
catheters [1], wound dressings [2], and the clothing and food
industry [3] due to their ecacy as antimicrobial agents.
Despite this progress, their potential adverse eects on
human health and the environment have not yet been elaborately elucidated, and their biocompatibility remains controversial.
Ag-NPs may translocate to the circulatory system and
distribute throughout the main organs of the body, especially
the kidney, liver, and brain [4], and they can penetrate the
blood-testis and blood-brain barriers [5]. This observation
implies that Ag-NPs could become neurotoxic and genotoxic
[4]. A Previous study has reported that 15 nm Ag-NPs
could lead to drastically reduced mitochondrial function

and cell viability of mouse spermatogonial stem cells at
concentration of 510 g/mL [6]. Hussain et al. [7] reported
that 15 nm Ag-NPs exhibited signicant cytotoxicity at 10
50 g/mL in the BRL 3 A cell line. Other studies have also
reported adverse cellular reactions of Ag-NPs to other cell
types, such as NIH3T3, vascular smooth muscle cells [8], and
mouse embryonic stem cells [9]. But inconsistency, such as
dierent toxic concentration, indeed exists among the results
of numerous cytotoxicity studies, which may be ascribed
to the varied cell models used in the evaluation of Ag-NPs
cytotoxicity to a certain degree.
Recently, embryonic stem cells (ESCs) have gained great
attention and showed multiple potential in tissue regeneration, drug screening, and biomaterial cytotoxicity test [10,
11]. With the development of methodologies for obtaining
cells derived from human ESCs (hESCs), several advantages
of the dierentiated progenies from hESCs have been
2
reported. Unlike immortalized cell lines, which are usually of
cancerous origin, containing chromosomal and genetic aberrations that produce immortality, and highly accustomed to
in vitro culture conditions after countless passages, hESCs
have been demonstrated to be genetically and karyotypically
normal, which would make them more representative of how
a normal cell should behave in vivo. Nowadays, several established hESC lines are readily available, from which an almost
limitless supply of dierentiated somatic progenies can be
obtained. Furthermore, dierentiated somatic progenies are
expected to show little inter-batch variability, provided in
vitro culture and dierentiation protocols are well standardized [12]. Therefore, we proposed the hypothesis that
dierentiated somatic progenies derived from hESCs may be
advantageous in the evaluation of Ag-NPs toxicity and may
be used as a novel option in nanocytotoxicity studies.
This study was planned to study the cytotoxicity of AgNPs (20 nm and 100 nm) to human embryonic stem cellderived broblasts (EBFs) in comparison with L-929 cell line.
Cell proliferation, cell cycle, apoptosis, p53 expression, and
cellular uptake were examined. Whether EBF could be a valid
candidate as a cell model for cytotoxicity screening of Ag-NPs
or not was analyzed.

2.1. Materials and Chemicals. Ag-NPs with two dierent
sizes, that is, Ag-NPs-1 (20 nm) and Ag-NPs-2 (100 nm),
were provided by Hongwu Nanomaterial Co. Ltd. (Xuzhou,
China). L-929 cells were obtained from the American Type
Cell Culture Collection (ATCC, Rockville, MD, USA).
Dulbeccos Modied Eagles Medium (DMEM), RPMI
1640, fetal bovine serum (FBS), antibiotic agent (penicillinstreptomycin, PS), trypsin-EDTA, DMEM/F-12, knockout
serum replacement, nonessential amino acid, and Lglutamine were purchased from Gibco (Grand Island, NY,
USA). Basic broblast growth factor (bFGF) was obtained
from Invitrogen (Carlsbad, CA, USA). -mercaptoethanol
was provided by Sigma-Aldrich (St. Louis, MO, USA).
Annexin-V-FLUOS staining kit was purchased from Roche
Molecular Biochemicals (Mannheim, Germany). Cell
Counting Kit-8 (CCK-8) was purchased from Dojindo
Laboratories (Tokyo, Japan). All other chemical solvents
were of analytical grade with no further purication.
2.2. Characterization of Ag-NPs. Scanning electron microscopy (SEM), transmission electron microscope (TEM), and
energy-dispersive X-ray spectroscopy (EDX) analysis were
used to characterize the size, morphology, distribution, and
chemical purity of the Ag-NPs.
2.3. Cell Culture. The NIH-registered hESC line, H9, was
obtained from the National University of Singapore. H9 cells
were cultured on a layer of mitomycin C-inactivated mouse
embryonic broblasts (MEFs) in ESC medium (Table 1)
at 37 C with 5% CO2 atmosphere and 95% humidity. H9
cells were digested with 1 mg/mL collagenase IV, scraped
down from MEF feeder layers, and cultured as embryonic
bodies (EBs) in suspension with EB medium (Table 1) for
5 days. Then, the EBs were transferred to a new ask coated
with 0.1% gelatin and cultured with dierentiation medium
(Table 1). EBs dierentiated spontaneously to broblast-like
cells, named EBF cells. EBF and L-929 cells were, respectively,
cultured in DMEM and RPMI 1640. Cells were maintained at
37 C in a 5% CO2 atmosphere and 95% humidity. The cells
were subcultivated when they reached 80% conuency.
2.4. In Vitro Assay for Cytotoxic Activity of Ag-NPs
2.4.1. Cell Viability Assay. Cell viability was quantied using
a CCK-8 assay as per manufacturers protocol. In brief, cells
were seeded in 96 well plates at a density of 1 104 cells
(200 L/well) followed by overnight incubation. The supernatant was then aspirated from the well, and fresh aliquots of
growth medium (containing Ag-NPs in concentration of 0.5,
5, 50, and 500 g/mL) after ultrasonic dispersion were added.
After 24, 48, and 72 h, the supernatant was again aspirated,
and cell monolayers were washed with PBS. Subsequently,
water-soluble tetrazolium (WST) reagent (20 L) was added
in each well, incubated for 4 h, centrifuged, the supernatant
obtained, and absorbance at two wavelengths (415 nm and
630 nm) was recorded using a microplate reader (Bio-Rad
680, Microplate Master, Hercules, CA, USA). The eect of
nanoparticles on cells was expressed as the percentage of cell
viability compared with the controls or relative proliferation
rate (RPR), which is calculated as the following formula:
RPR (%) = (A AN ) / AN 100%, where A represents the
absorbance of each dierent concentration group; AN is the
absorbance of negative control group.
2.4.2. Cell Cycle Analysis. Cell cycle analysis was carried out
by propidium iodide (PI) staining followed by ow cytometry measurement of the uorescence. Approximately, 1 105
cells were placed in cell culture ask. Following treatment with Ag-NPs (50 g/mL) for 24, 48, and 72 h, the
medium was removed and stored. Cells were washed in
PBS, trypsinized, harvested in the stored medium, and
centrifuged. The pellet was washed, xed in ice-cold ethanol
(70%), and stored at 20 C. Before ow cytometry analysis,
cells were washed in PBS and stained with PI in RNase
(50 g/mL PI and 0.25 mg/mL RNase A) and incubated at
37 C for 1 h, followed by incubation at 4 C until analysis.
Flow cytometry analysis was performed using BD FACS
Calibur (BD Biosciences) at an excitation wavelength of
488 nm and emission wavelength of 610 nm. Data collected
for 1 106 cells was analyzed using CellQuest software 6.0
(BD Biosciences).
2.4.3. Annexin-V Staining Apoptosis Analysis. Annexin-V
staining was performed to analyze apoptosis induced by AgNPs. Cells were treated with 50 g/mL Ag-NPs for 48 h. Cell
preparation and experimental procedures were as for cell
cycle analysis. The staining was performed as per manufacturers instructions. Data analyses were performed using Cell
Quest software 6.0.
3
Table 1: The component of cells culture medium.
Cells culture medium

ESC medium
EB medium
Dierentiation medium
EBF medium
L-929 medium
Component
DMEM/F-12 supplemented with 20% knockout serum replacement, 4 ng/mL bFGF, 1 mmol/L L-glutamine,
1% non essential amino acid, and 0.1 mmol/L -mercaptoethanol
DMEM/F-12 supplemented with 20% knockout serum replacement, 1 mmol/L L-glutamine, 1% non essential
amino acid, and 0.1 mmol/L -mercaptoethanol
DMEM, 1 mmol/L L-glutamine and 10% FBS
DMEM containing 10 units/mL penicillin, 10 units/mL streptomycin and, 10% FBS
RPMI 1640 containing 10 units/mL penicillin, 10 units/mL streptomycin, and 10% FBS
35
30
(%)
25
20
200 nm
15
10
5
200 nm
100140
50100
4550
4045
3540
3035
2530
2025
1520
1015
Diameter (nm)
(a)
(b)
30
25
(%)
20
500 nm
15
10
1 m
5
180190
170180
160170
150160
140150
130140
120130
110120
100110
90100
8090
7080
6070
Diameter (nm)
(c)
(d)
Figure 1: Characterization of Ag-NPs by SEM, EDX and TEM: (a) Ag-NPs-1 SEM with DEX image, (b) size distribution histogram and
TEM image of Ag-NPs-1, (c) Ag-NPs-2 SEM with DEX images, (d) size distribution histogram and TEM image of Ag-NPs-2.
2.5. Cellular Uptake and Quantitative Determination of the

Uptake of Ag-NPs. Ultrathin sections of cells were analyzed
using TEM to reveal the uptake and distribution of NPs.
Briey, the cells (1.5 106 ) were treated with Ag-NPs
(50 g/mL) for 48 h. At the end of the incubation period,
culture asks were washed many times with PBS to remove
excess unbound NPs. Cells were trypsinized and xed in
2.5% glutaraldehyde for 2 h. Fixed cells were washed with
PBS. Postxation staining was achieved using 1% osmium
tetroxide for 1 h at room temperature. Cells were washed
well, dehydrated in alcohol, and treated with propylene oxide
for 30 min, followed by treatment with propylene oxide,

Spurrs low viscosity resin (1 : 1) for 18 h. Cells were further
treated with pure resin for 24 h and embedded in BEEM capsules containing pure resin. Resin blocks were hardened at
70 C for 2 days. Ultrathin sections (70 nm) were cut using an
ultramicrotome (Lecia EM UC6). The sections were stained
with 1% lead citrate and 0.5% uranyl acetate and analyzed
under transmission electron microscope examination (H7650B, Hitachi, Japan).
Subconuent cells were incubated at 37 C in the presence
or absence of 50 g/mL Ag-NPs for 24 h under cell culture
24 h
48 h
1.2
1
0.8
0.8
0.4
0.2
0.6
0.4
50
500
0.2
Concentration (g/mL)
0.5
5
50
500
0.2
(a)
(b)
(c)
1.2
0.8
0.8
0.8
0.6
0.4
0.6
0.4
0.2
0.2
0.2
50
500
EBF
L-929
RPR (%)
1.2
1
RPR (%)
1.2
0.5
0.5
5
50
500
EBF
L-929
EBF
L-929
EBF
L-929
Ag-NPs-2
RPR (%)
0.4
0
0
0.5
0.6
0.2
0.2
0
0.2
RPR (%)
1
0.6
72 h
1.2
0.8
RPR (%)
Ag-NPs-1
RPR (%)
1.2
0.2
0.6
0.4
0.2
0
0.5
50
500
0.5
50
500
EBF
L-929
EBF
L-929
(d)
0.2
(e)
(f)
Figure 2: Cytotoxicity assay of EBF and L-929 cells after treatment with Ag-NPs (0.5500 g/mL) for 24, 48, and 72 h. (a) Ag-NPs-1 24 h, (b)
Ag-NPs-1 48 h, (c) Ag-NPs-1 72 h, (d) Ag-NPs-2 24 h, (e) Ag-NPs-2 48 h, (f) Ag-NPs-2 72 h. Astatistically signicant dierence between
EBF and L-929 cells (P < 0.05).
conditions. Subsequently, the cells were washed with PBS,

detached with trypsin and suspended in medium. The
uptake of particles into cells was analyzed using ow cytometry. The side scatter data were analyzed using CELL Quest
6.0 software. Calibration reagents and solutions for ow
cytometry were from Becton Dickinson. Ten thousand cells
were acquired for each measurement.
2.6. Real-Time Quantitative-Polymerase Chain Reaction
(RTq-PCR). To assess DNA damage associated with the AgNPs, the level of p53 expression in cells was detected using
RTq-PCR. The cells were seeded with 1 105 , cells/dish and
cultured with the 50 g/mL Ag-NPs solution for 48 h. Total
RNA was isolated with TRIZOL Reagent (Invitrogen) and
was reverse transcribed using an iScript cDNA synthesis
kit (Bio-Rad Laboratories). RTq-PCR was performed with
a two-step RTq-PCR kit (Invitrogen) according to the
manufacturers instructions. The mRNA expression of p53,
an important signaling molecule in checkpoint responses to
DNA damage [13], was detected after treatment by a 7500
real-time PCR System (Applied Biosystems) with the SYBR
Table 2: Primers used for RTq-PCR.

Gene Name
p53-human-R
p53-human-F
p53-mouse-R
p53-mouse-F
GAPDH-human-R
GAPDH-human-F
GAPDH-mouse-R
GAPDH-mouse-F
Sequence
CCGCAGTCAGATCCTAGCG
AATCATCCATTGCTTGGGACG
GTCACAGCACATGACGGAGG
TCTTCCAGATGCTCGGGATAC
AGGGGCCATCCACAGTCTTC
AGAAGGCTGGGGCTCATTTG
GCTCCTGGAAGAGGTGAT
TCGTCCCGTAGACAAAATG
Premix Ex Taq Perfect Real Time kit (Takara Mirus Bio,

Madison, WI). The sequences of the primer pairs are shown
in Table 2.
2.7. Statistical Analysis. All experiments were performed in
duplicate and repeated at least three times. The statistical signicance of the data was expressed as mean SD. Statistical
100
90
100
80
90
80
70
Events (%)
60
50
40
30
50
40
30
20
20
10
24 H
48 H
Ag-NPs-2
Ag-NPs-1
CON
Ag-NPs-2
Ag-NPs-1
CON
Ag-NPs-2
Ag-NPs-1
Ag-NPs-2
Ag-NPs-1
CON
Ag-NPs-2
Ag-NPs-1
CON
Ag-NPs-2
Ag-NPs-1
10
CON
60
CON
Events (%)
70
72 H
24 H
G2/M
S
G0/G1
48 H
G2/M
S
(a)
72 H
G0/G1
(b)
Figure 3: Cell cycle population of EBF (a) and L-929 cells (b) after treatment with Ag-NPs-1 and Ag-NPs-2.
under SEM, with average size of 20 nm 12.6 nm (Ag-NPs-1,

Figure 1(b)) and 100 21.3 nm (Ag-NPs-2, Figure 1(d)), as
measured by software Image J (National Institutes of Health,
USA). EDX results demonstrated that the nanoparticles were
100% pure silver. TEM image showed the even distribution
of Ag-NPs dispersed in water.
18
Relative expression of p53
16
14
12
10
8
3.2. Cytotoxicity of Ag-NPs
6
4
2
0
EBF
L-929
Con
Ag-NPs-1
Ag-NPs-2
Figure 4: Expression of p53 in EBF and L-929 following Ag-NPs

exposure for 48 h. Signicant dierences in comparison with
control group (P < 0.05). Highly signicant dierences in comparison with control group (P < 0.01).
dierences between groups were evaluated by Student t-test

using the software SPSS 13.0 (SPSS Science). P values of less
than 0.05 were considered to be statistically signicant.
3. Results and Discussion

3.1. Characterization of Ag-NPs. Both kinds of Ag-NPs were
observed to be spherical in shape (Figures 1(a) and 1(c))
3.2.1. Cell Viability of EBF and L-929 Cells. Cell viability was
quantied using a CCK-8 assay. Figure 2 displays the RPR
of EBF and L-929 cells after 24, 48, and 72 h exposure to AgNPs-1 and Ag-NPs-2 at concentration of 0.5500 g/mL. The
RPR were decreased in a dose-dependent manner and the
RPR of EBF treated with Ag-NPs of both size were lower than
that of L-929 cells at the same concentration and time point.
50 g/mL was shown the threshold concentration that significantly inhibited EBF proliferation, and this concentration
was used in all subsequent experiments including cell cycle
analysis, apoptosis assay, cellular uptake, and p53 expression.
When the NPs concentration reached 500 g/mL, nearly no
viable cells existed. CCK-8 results also showed apparent
size-dependent cytotoxic eects on cell proliferation. The
RPR of EBF treated with Ag-NPs-1 was lower than that of
Ag-NPs-2 at 550 g/mL, both at 48 and 72 h. However,
the size-dependent cytotoxic eects on L-929 cells occurred
only at 50 g/mL after 72 h exposure in this study. These
results indicated that EBF may be more vulnerable to Ag-NPs
treatment when compared with L-929 cell line.
The dose-dependent toxicity pattern of Ag-NPs was
consistent with previous studies on mammalian germline
stem cells [6] and may be due to the impact of endocytosis
L-929
EBF
100 m
(a)
100 m
(b)
20 m
20 m
100 m
(c)
100 m
(d)
Figure 5: Phase-contrast micrographs (magnication 200x) of EBF and L-929 cells before (a) and (b) and after (c) and (d) treatment with
Ag-NPs-1 at 50 g/mL for 48 h. The insets show the high magnication (magnication 400x).
and metabolism inhibiting normal cellular activity at larger

concentrations of Ag-NPs. The results in our study that
cell proliferation in EBF was more sensitive to Ag-NPs give
evidence for a cell-type-dependent response in biomaterials
cytotoxicity test. Similarly, Cao et al. [14] reported that
mitomycin C decreased EBF viability more obviously than
L-929 cells and considered that EBF were more sensitive than
L-929 cells in cytotoxicity screening tests. Tedja et al. [15]
noticed that the dierent level of biological response should
be primarily attributed to the dierence in the amount of
cellular particle uptake between dierent cell types. Hence,
the cell-type-specic response of cells to Ag-NPs here could
be due to that EBF are genetically and karyotypically normal
and exhibited a stronger capacity to ingest NPs (Figure 7).
Previous studies have also indicated that Ag-NPs have a sizedependent cytotoxicity, with smaller particles being more
toxic [16, 17]. According to Carlson et al. [18], Ag-15 nm
and Ag-30 nm NPs showed more cytotoxicity than that of
Ag-55 nm. Our study revealed similar ndings that the RPR
of EBF and L-929 cells treated with Ag-NPs-1 was lower than
that with Ag-NPs-2. The reason may be that smaller Ag-NPs
have a larger surface area available for interaction and lead to
a stronger eect than larger particles [19].
3.2.2. Cell Cycle Analysis. The inuence of NPs on the cell

cycle was analyzed by subjecting the NP-treated cells to
ow cytometry. In control group, major cell populations
were observed in the G1 phase, whereas in Ag-NPs treated
cells, a decrease in the G1 cell population was accompanied
by an increase in the G2/M cell population (Figure 3).
The G2/M population of Ag-NPs treated EBF cells were
signicantly increased to almost twice the control values at
24, 48, and 72 h (Figure 3(a)). G2/M arrest of L-929 cells was
observed at 72 h (Figure 3(b)). DNA damage was proposed
to be the main cause of cell cycle arrest [20]. AshaRani
et al. [21] reported oxidative stress in Ag-NPs treated cells
indicating the possibility of DNA damage and chromosomal
aberrations which was considered the prime factors resulting
in cell cycle arrest. The results in this study indicated that EBF
cells may be more vulnerable to Ag-NPs than the L-929 cell
line.
3.2.3. Apoptosis and DNA Damage Induced by Ag-NPs.
Annexin-V staining indicated that the rate of EBF cells
apoptosis was increased from 0.77 0.08% (control) to
2.88 0.23% (Ag-NPs-1) and 1.49 0.33% (Ag-NPs-2) at
50 g/mL, whereas that of L-929 cells treated with the
7
Ag-NPs-1
Ag-NPs-1
Ag-NPs-2
Ag-NPs-2
c
100 nm
EBF
c
c
n
c
2 m
500 nm
(a)
500 nm
500 nm
(b)
(c)
(d)
n
100 nm
L-929
c
n
c
c
2 m
500 nm
(e)
(f)
c
500 nm
n
(g)
500 nm
(h)
Figure 6: TEM images of ultrathin sections of EBF (a)-(d) and L-929 (e)-(h) cells treated with Ag-NPs after 48 h. Nanoparticles were showed
inside the cytoplasm (c), but they were not in the nucleus (n) of both cell types. Cells showed large endosomes (a) and (e) and lysosomes (b)
and (f) with nanoparticles inside (red arrow). Invagination (c) and (g) and protrusion (d) and (h) of the plasma membrane were observed
when Ag-NPs attached the cells (yellow arrow).
Table 3: The rate of apoptosis treated with Ag-NPs.
2.5
Scatter intensity
(% of control)
EBF
L-929
1.5
Control
0.77 0.08
0.63 0.13
Ag-NPs-1
2.88 0.23
0.74 0.09
Ag-NPs-2
1.49 0.33
0.67 0.29
P < 0.05 compared with control.
0.5
0
Ag-NPs-1
Ag-NPs-2
Diameter of Ag-NPs (nm)
EBF
L-929
Figure 7: Intracellular occurrence of Ag-NPs agglomerates analyzed using ow cytometric light scatter. Signicant dierences
between EBF and L-929 cells (P < 0.05). # Signicant dierences
between Ag-NPs-1 and Ag-NPs-2 groups.
same concentration of Ag-NPs had no signicant change

compared with the control (Table 3). This result showed that
the impact of early apoptosis, at the concentration of AgNPs selected in our study, was not apparent, but the value
did show that Ag-NPs might cause EBF apoptosis. In the

presence of DNA damage, p53 accumulates and triggers cell
cycle arrest to provide time for the damage to be repaired
[22]. Therefore, p53 expression could be assessed by RTqPCR to monitor the DNA damage indirectly. Data in Figure 4
showed that expression of p53 in EBF cells treated with both
sized Ag-NPs within 48 h increased signicantly. Especially
in Ag-NPs-2 treated EBF cells, their p53 expression even
reached almost 14 times of the control group. While the
changes observed in the L-929 cells were not statistically signicant (Figure 4). Ahamed et al. [9] have also indicated that
Ag-NPs up-regulated p53, Rad51 and phosphorylated-H2AX
expression. This observation supported the hypothesis that
Ag-NPs can cause DNA damage and resulted in G2/M cell
cycle arrest, which may be correlated to long-term eects,
such as mutagenesis or carcinogenesis [23].
3.3. Cellular Uptake and Distribution of Ag-NPs. Under
phase-contrast microscopy, Ag-NPs were observed in the
cytoplasm of both cell types and gathered in the perinucleus.
8
Single or clustered NPs were attached to the cell membrane
and were internalized into cells (Figures 5(c) and 5(d)). TEM
images also showed that the NPs were distributed throughout
the cytoplasm, but they were not observed in the nucleus
of both cell types (Figures 6(a) and 6(e)). According to the
TEM images, there were no signicant dierences in cellular
uptake between the two cell types. Large endosomes and
lysosomes with Ag-NPs were also observed (Figures 6(a),
6(b), 6(e), and 6(f)). Invagination of plasma membranes
(Figures 6(c) and 6(g)) denoted the endocytosis of nanomaterials. The protrusion of the plasma membrane (Figures
6(d) and 6(h)) for uptake of the nanospheres indicated
the characteristics of endocytosis and macropinocytosis.
These results suggested that Ag-NPs were entering the
cells through pinocytosis rather than diusion. Greulich
et al. [24] have reported that the uptake of PVP-coated
Ag-NPs was signicantly inhibited by chlorpromazine and
wortmannin, suggesting endocytosis and macropinocytosis
were the primary uptake mechanisms.
The uptake of nanomaterials depended not only on the
particle size and charge, but also on the cell type [25]. The
quantitative uptake of Ag-NPs was determined by analysis of
the intracellular side scatter signal using ow cytometry. As
Figure 7 demonstrated, EBF had the stronger uptake capacity
of Ag-NPs compared with L-929 cells. The results also
showed that both cell types took up more Ag-NPs-1 particles
than Ag-NPs-2, which might be due to the smaller diameter
and resulted in size-dependent nanotoxicity. The dierences
in uptake capacity between cell lines can be explained by
the dierentiation state of the cells. There was report that
endocytosis was normally downregulated after treatment of
dendritic cells with maturation stimuli [26]. As dierentiated
progenies from hESCs, EBFs were less mature than L-929
immortalized cells. Hence, it could be reasonable to speculate
that the dierence between uptake capacity of EBFs and L929 might be owing to the diering dierentiation state.
The cellular uptake mechanisms, depending on cell type and
particle size, may also be contributed to, or triggered by, the
ability of NPs to penetrate the plasma membrane [27]. The
uptake of the NPs appears to be quite complicated, therefore,
further work is required to elucidate the underling cellular
uptake mechanism elaborately.
4. Conclusion
In this study, the results showed that cytotoxicity of Ag-NPs
was dependent on dose, cell type, and particle size. Ag-NPs
presented higher cytotoxicity to EBF than to L-929. EBF
exhibited an higher G2/M arrest and more upgraded p53
expression after exposed to Ag-NPs for 48 h when compared
with L-929. According to the cellular uptake analysis, the NPs
were found in the cytoplasm and lysosomes, but they were
not observed in the nucleus. EBF demonstrated a stronger
capacity to ingest Ag-NPs. According to the results of this
study, it could be postulated that EBF was more sensitive
to Ag-NPs than L-929. Taking into account of its more
representative of how a normal cell should behave in vivo,
EBF could be considered a promising candidate for cell
model of nanomaterials cytotoxicity screening.
Acknowledgments
The authors would like to thank the National University
of Singapore for the stem cell resources and Center of
Biomedical Analysis at Tsinghua University for the electron
microscopy resources. This work was supported by the
National Basic Research Program of China (2012CB933904),
the National Natural Science Foundation of China (no.
81171000) and International S&T Cooperation Program of
China (ISTCP, 2011DFA32190).
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doi:10.1155/2012/512956
Research Article
Toxicity of Aqueous Fullerene nC60 to Activated Sludge:
Nitrication Inhibition and Microtox Test
Yongkui Yang,1 Norihide Nakada,1 Ryoji Nakajima,2 Chao Wang,1 and Hiroaki Tanaka1
1 Research
2 Testing
Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu 520-0811, Japan
and Analysis Division, Shimadzu Techno Research Inc., 1 Nishinokyo-Shimoaicho, Nakagyo-ku, Kyoto 604-8436, Japan
Correspondence should be addressed to Yongkui Yang, ykyang@biwa.eqc.kyoto-u.ac.jp

Received 3 April 2012; Accepted 11 June 2012
Copyright 2012 Yongkui Yang et al. This is an open access article distributed under the Creative Commons Attribution License,
The increasing production and use of fullerene nanomaterials raised their exposure potential to the activated sludge during
biological wastewater treatment process. In this study, the toxicity of aqueous nanoscaled C60 (nC60 ) to activated sludge was
investigated using nitrication inhibition and Microtox test. The test solutions of nC60 were prepared using two methods:
long stirring (Stir/nC60 ) and toluene exchange (Tol/nC60 ). The nC60 aggregation in test medium was also evaluated for toxicity
assessment. The results showed that the nC60 aggregation behaved dierently in two test mediums during the incubation periods.
The nC60 toxicity was greatly inuenced by the preparation method. Stir/nC60 presented no signicant toxicity to both the
nitrication sludge and bioluminescent bacteria at the maximum concentration studied. In contrast, the EC20 of Tol/nC60 was
obtained to be 4.89 mg/L (3 h) for the nitrication inhibition and 3.44 mg/L (30 min) for Microtox test, respectively.
1. Introduction
Fullerenes, carbon-based nanomaterials, are demonstrating
rapid increase in the commercial and scientic interest by
their unique properties, such as, chemically and thermally
stable, excellent electron acceptor and radical scavenger, and
special optical properties [1, 2]. The production is expected
up to 1500 t in 2007 compared to 400 kg in 2002 by the
largest fullerene production company in the world [3]. In
addition, until 2011 the class of fullerenes and other carbonbased nanomaterials is ranked as the second among all the
nanomaterials used in consumer products available [4].
The C60 , the main type of fullerenes, showed the toxicity
on cell [5], bacterial [6, 7], and sh [8, 9]. Although the
toxicity mechanism is still not clear, the most published
is due to the oxidative stress via reactive oxygen species
dependent [5] or independent [10]. The toxicities suggested
the potential of adverse eect of C60 on the activated sludge
which is important for the removal of organic matters and
nutrient compounds in wastewater. Recent studies pointed
out the toxicity of metal nanoparticles (silver [11, 12],
copper [13], zinc oxide, and titanium dioxide [14]), on the
aerobic/anaerobic activated sludge. However, very limited
studies focused on fullerene C60 toxicity on the activated

sludge. Kang et al. [15] reported about 30% inactivation
of microorganism in wastewater samples after 1 h exposure
to nC60 -coated lter. However, no signicant change was
identied to microbial community structure in the anaerobic
sludge using the denaturing gradient gel electrophoresis
analysis under 50 g/kg biomass [16].
The objective of this study is to assess the toxicity
of aqueous nC60 on the activated sludge. The eect of
nC60 on nitrication activities was investigated using the
cultivated activated nitrifying sludge. The Microtox test was
also conducted as a standardized screening test. To our best
knowledge, this is the rst study on the nC60 toxicity on
activated sludge by checking the nitrication inhibition. This
work is expected to provide useful information to assess the
eect of nC60 on activated sludge and consequent treatment
performance of biological wastewater treatment process.

2.1. Preparation of Aqueous nC60 Suspension. Two types of
aqueous nC60 were prepared using the extended mixing of
3
Stir/nC60
Tol/nC60
260 nm
Zeta potential (mV)
Absorbance
350 nm
2
450550 nm
1
15
30
45
0
200
300
400
500
600
700
800
60
Wavelength (nm)
Figure 3: Zeta potential of prepared nC60. Error bars represent the

standard error (pH 5.6, n = 2).
Tol/nC60
Stir/nC60
Figure 1: UV-visible absorption spectra of prepared nC60 (pH 5.6).
sonication and ltrations. The resulting suspensions were

stored in the dark at 4 C until use. The nC60 concentration
was determined by extracting nC60 into the toluene phase
and quantifying it at 332 nm by the UV/vis spectrometer,
described elsewhere [7, 19, 20]. The calibration curve was
obtained by the stock C60 toluene solution at dierent
concentrations (n = 5).
24
Intensity (%)
18
12
6
0
1
10
100
1000
Size (nm)
Stir/nC60
Tol/nC60
Figure 2: Size distribution of prepared nC60 . Error bars represent

the standard error (pH 5.6, n = 2).
powder C60 in water (Stir/nC60 ) and the toluene-involved

solvent exchange method (Tol/nC60 ), both of which are
widely used for the studies of the C60 toxicity [7, 9, 15]
and its fate [17, 18]. Briey, the Stir/nC60 was produced
by adding 200 mg of powder C60 (purity: 99.9%, SES
research, USA) to 300 mL of ultrapure water (Millipore,
USA) and then mixing with a magnetic stirrer at 500 rpm
for three weeks. And the Tol/nC60 was prepared by following
the previously reported method with minor modications
[7]. Specically, 1000 mg/L C60 solution was obtained by
dissolving the powder C60 into HPLC grade toluene (stock
C60 toluene solution). And then 40 mL of the solution was
added to 100 mL ultrapure water. The toluene was removed
by sonication at 40 C using an ultrasonic cleaner (AS ONE,
Japan) followed by the purge with gentle stream of nitrogen
gas at 0.5 L/min for 1 h. The prepared Stir/nC60 and Tol/nC60
suspensions were, both of yellow colour, sequentially ltered
through a glass lter (pore size: 1 m, Pall Life Sciences, USA)
and cellulose acetate lter (pore size: 0.45 m, Advantec,
Japan). In addition, a blank sample for Tol/nC60 was also
prepared by adding the same amount of toluene (without
C60 ) in the pure water followed by the same procedures of
2.2. Cultivation of Nitrifying Activated Sludge. The seedactivated sludge was collected from the nitrication tank
of a biological wastewater treatment plant. The nitrifying
activated sludge was obtained by cultivating the seed sludge
at 30 C in a 3 L water-jacketed glass reactor. The reactor
was operated at ll and draw mode at a hydraulic retention
time of 12 h and sludge retention time of 20 d in the
dark. Dissolved oxygen was kept at above 1.0 mg/L by
introducing the ltered compressed air via diusers, and
the pH was maintained at 7.5 0.1 with the automatic
addition of 1 M Na2 CO3 . The feed solution only consisted
of the inorganic medium with the 25 mM (NH4 )2 SO4 . The
other nutrient composition, data acquisition, and process
control were described in the previous study [21]. After
reaching the steady state by checking the NH4 + removal
eciency, the mixed liquor in the reactor was collected and
washed three times using 40 mM KH2 PO4 buer (pH 7.8) by
centrifugation (2000 g 5 min).
2.3. Nitrication Inhibition Experiment. The nitrication
inhibition studies were conducted in accordance with the
ISO 9509 test guideline [22]. Batch experiments were carried
out by agitating 100 mL Erlenmeyer asks containing 50 mL
medium solution (2 mM (NH4 )2 SO4 and 6 mM (NaHCO3 )),
determined nitrifying activated sludge and dierent amounts
of nC60 at controlled room temperature (25 C). The experimental conditions, the incubating time of 3 h and MLSS
of 40 mg/L, were determined to ensure about 50% of initial
NH4 + that was left at the end of incubating time to avoid rate
limiting [22]. The dissolved oxygen was kept at above 4 mg/L
by the shaking (150 rpm) on a rotary shaker. The nitrication
inhibition (I) was calculated by the dierence of oxidized N
600
600
400
400
Size (nm)
Size (nm)
200
200
0
5
180
15
30
Contact time (min)
Contact time (min)

Stir/nC60
Tol/nC60
Stir/nC60
Tol/nC60
(a)
(b)
Figure 4: Change in nC60 size in medium for nitrication inhibition (a) and Microtox test (b). Error bars represent the standard error
(n = 2).
50
100
40
Inhibition (%)
Inhibition (%)
75
50
30
20
10
0
25
10
0
0
0.025
0.05
ATU concentration (mg/L)
0.1
Figure 5: Nitrication inhibition as a function of ATU concentration. Error bars represent the standard error (n = 2).
formation (NO3 and NO2 ) between the control and the

nC60 exposure test after 3 h. The equation is given as,
I (%) =
(Nc Nf )
100,
(Nc Ni )
(1)
where Nc (mg-N/L) is the concentration of oxidized N in

the control ask after 3 h, Nf (mg-N /L) is the concentration
of oxidized N in the ask containing nC60 after 3 h, Ni
(mg-N/L) is the concentration of oxidized N in the ask
containing the reference inhibitor of N allylthiourea (ATU).
The EC20 , nC60 concentration with a reduction of oxidized
N formation by 20%, was calculated using the SPSS probit
regression analysis (SPSS, USA).
2.4. Microtox Test. The V. scheri bioassay is also used for
assessing the toxicity of compounds on the activated sludge
[23, 24] which is based on the decrease in bioluminescence
from the bacterium due to the exposure to the toxicants. The
experiment was carried out using a Model 500 luminometer
(Azur Environmental, USA) in accordance with the Microtox
5
nC60 concentration (mg/L)
10
Blank for Tol/nC60

Stir/nC60
Tol/nC60
Figure 6: : Nitrication inhibition of nC60 as a function of preparation method and exposure concentration. Error bars represent the
standard error (n = 2).
acute toxicity procedure [25]. The reagent (freeze-dried

V. scheri) and the solutions (diluent, reconstitution, and
osmotic adjusting solution) were purchased from Strategic
Diagnostics Inc., USA. The EC20 , nC60 concentration with a
reduction of bioluminescence by 20%, was calculated at each
test with dierent exposure periods (5, 15, and 30 min) using
the Microtox Omni Software (Strategic Diagnostics, USA).
The phenol was used for the quality control of this test.
2.5. nC60 Aggregation in the Incubation Medium for the
Toxicity Test. The aggregation of nC60 as a function of
incubation time was determined using the same medium of
the toxicity tests. For the nitrication inhibition test, the nC60
size in medium (2 mM (NH4 )2 SO4 , 6 mM NaHCO3 and
2 mM KH2 PO4 buer) was measured after 5 min (minimum
time for one measurement) and 3 h. And for the Microtox
50
50
40
40
Inhibition (%)
Inhibition (%)
30
20
10
0
30
20
10
0
10
10
2.5
2.5
Blank for Tol/nC60 -30 min

5 min
15 min
30 min
5 min
15 min
30 min
(a)
(b)
Figure 7: Bioluminescence inhibition of Stir/nC60 (a) and Tol/nC60 (b) as a function of exposure time and concentration. The Error bars
represent the standard error (n = 2).
test, the nC60 size in 2% NaCl solution was measured after 5,

15, and 30 min.
2.6. Analysis. The NH4 + , NO3 , and NO2 concentrations
were determined based on the standard methods [26].
The UV absorbance of the stock C60 toluene solution
and nC60 suspension in water was measured using a UV2500 spectrophotometer (Shimadzu Scientic Instruments,
Japan). nC60 size and its distribution were determined by
the dynamic light scattering using a Zetasizer Nano ZS
equipped with a 633 nm laser source and a detection angle
of 173 C (Malvern Instruments, UK). The same instrument
was used to measure the electrophoretic mobility which
was subsequently calculated into Zeta potential using the
Smoluchowski equation. All the tests in this study were
conducted in duplicate.

3.1. Characterization of Prepared nC60 . Figure 1 shows the
UV-visible absorption spectra of Stir/nC60 and Tol/nC60 .
Both nC60 show the absorption peaks at 260 and 350 nm
for the electronic structure of molecular C60 cage [27] and
a broad absorption band at 450550 nm for the aggregated
C60 -C60 interactions [6]. Similar ndings have also been
reported for nC60 prepared via tetrahydrofuran (THF) [28].
However, the ratio of absorbance at 260, 350, and 450 nm
varied with the nC60 types suggesting the dierence in the
structure and composition of nC60 aggregates [29].
Two types of nC60 demonstrated very similar size distribution with an average size of 154 and 144 nm for Stir/nC60
and Tol/nC60 , respectively, as shown in Figure 2. Both nC60
were negatively charged with no signicant dierence at pH
5.6 (Figure 3), which is in agreement with the reported
results of nC60 prepared using similar methodologies [29].
3.2. Aggregation of nC60 in Toxicity Test Medium. The aggregate size is an essential information when assessing the
toxicity of nanoparticles because of proved correlation [7,
30]. Figure 4(a) shows the nC60 size with time in medium
for nitrication inhibition test. After the incubation time
of 3 h the aggregate remained stable with only several nm
of increase in size for both nC60 . It can be explained by
the low ionic strength of this medium (15.8 mM) which
was much lower than the reported threshold destabilization
concentration of 120 mM for the nC60 [31]. In contrast, the
obvious increase in size was found in medium with high ionic
strength (342 mM) for the Microtox test (Figure 4(b)).
Compared to the initial sizes in pure water, the size in
medium increased by 31, 56, and 114% for Stir/nC60 and
55, 97, and 142% for Tol/nC60 after 5, 15, and 30 min. In
addition, the aggregation rate of Stir/nC60 was slower than
that of Tol/nC60 presumably due to the dierence in the
structure and chemistry of nC60 aggregate. Brant et al. found
the hydrophobicity of Stir/nC60 was lower than the nC60
prepared via the organic solvent, such as, toluene and THF
[29].
3.3. Eect of nC60 on Nitrication Activity. The sludge nitrication activity and test performance were conrmed using
the ATU. The EC50 was calculated to be 0.040 mg/L from
the data (Figure 5) which was close to the published
value of 0.025 mg/L [32]. Figure 6 shows the percent
nitrication inhibition by two types of nC60 at varying
concentrations after 3 h, as well as the blank sample for
Tol/nC60 . For the Stir/nC60 , no nitrication inhibition was
observed up to 8.4 mg/L indicating its low toxicity on
nitrifying activated sludge. Previous studies also presented
similar results that no signicant impact was identied
on the microbial community structure in aerobic soil [33]
and anaerobic sludge [16]. In the case of the Tol/nC60 , no
obvious eect was found in the blank sample (less than 2%).
But 40% nitrication was inhibited at 8.40 mg/L, and the
EC20 was calculated to 4.89 mg/L for Tol/nC60 . It clearly
showed that the nC60 toxicity depended on the preparation
method. Zhu et al. [8] compared the toxicity of Stir/nC60
and nC60 produced via THF on Daphnia magna and founded
EC50 (48 h) for the latter was at least one order of magnitude
(0.8 mg/L) less than that for Stir/nC60 (>35 mg/L).
3.4. Eect of nC60 on Bioluminescent Bacteria. Figure 7(a)
shows the percent inhibition due to the exposure to Stir/nC60
at dierent concentrations. No inhibition was observed at
the concentration up to 4.2 mg/L for all the incubation
time. In contrast, the Tol/nC60 showed obvious inhibition at
>1.05 mg/L, and the toxicity increased with the incubation
time (Figure 7(b)). No inhibition was also observed for the
blank sample up to 30 min. The EC20 was calculated to 4.96,
4.98, and 3.44 mg/L for 5, 15, and 30 min, respectively. Both
the facts above conrmed the low toxicity of Stir/nC60 and
the toxicity dependent on the preparation methods. The
result is consistent with that obtained from the nitrication
inhibition test.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
4. Conclusions
The prepared Stir/nC60 and Tol/nC60 showed similar surface
properties, such as, the size distribution, zeta potential, and
UV-vis absorption spectra. However, two types of nC60
presented dierent aggregation rates in test medium during
the incubation periods. Both the nitrication inhibition and
Microtox test showed that the nC60 toxicity was greatly
aected by the preparation method. Stir/nC60 presented no
signicant toxicity to the nitrication sludge and bioluminescent bacteria at maximum concentration studied. In
contrast, the EC20 of Tol/nC60 was obtained to be 4.89 mg/L
(3 h) for the nitrication inhibition and 3.44 mg/L (30 min)
for Microtox test, respectively.
Conict of Interests
[11]
[12]
[13]
[14]
[15]
The authors declare that there is no conict of interests.
Acknowledgments
[16]
This research was partly supported by Grant-in-Aid for

Scientic Research (B), Japan Society for the Promotion
of Science (JSPS) and CREST project, Japan Science and
Technology (JST).
[17]
[18]
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doi:10.1155/2012/721706
Research Article
The Human Stratum Corneum Prevents Small Gold Nanoparticle
Penetration and Their Potential Toxic Metabolic Consequences
David C. Liu,1, 2 Anthony P. Raphael,1 Daniel Sundh,1, 3 Jeffrey E. Grice,2
H. Peter Soyer,1 Michael S. Roberts,2, 4 and Tarl W. Prow1, 2
1 Dermatology
Research Centre, School of Medicine, Princess Alexandra Hospital, The University of Queensland, Brisbane,
QLD 4102, Australia
2 Therapeutics Research Centre, School of Medicine, Princess Alexandra Hospital, The University of Queensland, Brisbane,
QLD 4102, Australia
3 Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, 405 30 Gothenburg, Sweden
4 School of Pharmacy and Medical Science, University of South Australia, Adelaide, SA 5001, Australia
Correspondence should be addressed to Tarl W. Prow, t.prow@uq.edu.au
Received 20 January 2012; Accepted 12 April 2012
Copyright 2012 David C. Liu et al. This is an open access article distributed under the Creative Commons Attribution License,
Nanoparticles are being used in multiple applications, ranging from biomedical and skin care products (e.g., sunscreen) through to
industrial manufacturing processes (e.g., water purication). The increase in exposure has led to multiple reports on nanoparticle
penetration and toxicity. However, the correlation between nanoparticle size and its penetration without physical/chemical
enhancers through the skin is poorly understoodwith studies instead focusing primarily on skin penetration under disrupted
conditions. In this paper, we investigate the penetration and metabolic eects of 10 nm, 30 nm, and 60 nm gold nanoparticles
within viable excised human skin after 24-hour exposure using multiphoton tomograph-uorescence lifetime imaging microscopy.
After 24 hour treatment with the 10, 30, and 60 nm gold nanoparticles, there was no signicant penetration detected below
the stratum corneum. Furthermore, there were no changes in metabolic output (total NAD(P)H) in the viable epidermis
posttreatment correlating with lack of penetration of nanoparticles. These results are signicant for estimating topical nanoparticle
exposure in humans where other model systems may overestimate the exposure of nanoparticles to the viable epidermis. Our data
shows that viable human skin resists permeation of small nanoparticles in a size range that has been reported to penetrate deeply
in other skin models.
1. Introduction
The skin is the largest organ of the body and is the primary
barrier to nanoparticle exposure from naturally occurring
and engineered nanomaterials found in the environment and
workplace [13]. Therefore, it is important to understand the
penetration characteristics of nanoparticles within the skin.
However, accurate assessment of nanoparticle penetration is
challenging, with results dependant on the model used (e.g.,
rat, pig, or human skin), barrier integrity (i.e., skin preparation prior to and during studies), nanoparticle properties
(e.g., size, shape, surface structures, and charge), and degradation kinetics.
A number of reports have shown that nanoparticles penetrate skin into the dermis under various physically disrupted conditions [48]. In addition, nanoparticles have also
been shown to penetrate skin in the presence of chemical

enhancers [3]. We have previously shown ex vivo that skin
treated with gold nanoparticles suspended in toluene results
in nanoparticle penetration into the epidermis due to toluene
acting as a chemical enhancer and disrupting the stratum
corneum [8]. Even though there are multiple reviews on
nanoparticle toxicity and penetration within the skin [913],
the correlation between nanoparticle size and its penetration
without physical/chemical enhancers is poorly understood
with studies instead focusing primarily on skin penetration
under disrupted conditions.
A recent report by Filon et al. (2011) showed 12.9 nm
gold nanoparticle penetration in thawed human skin [14].
The results showed that gold nanoparticle penetration
occurred after 24 hours of treatment in both intact and
damaged skin. Gold nanoparticles were detected in the
2
receiving solution of the Franz cells after 24 hours of skin
exposure. This report supports a previous study conducted
by Sonavane et al. (2008) where 15 nm gold nanoparticles
penetrated through the excised rat skin into the dermis also
after 24-hour treatment [15].
In the last 20 years, multiphoton tomography (MPT) has
developed into a technique that enables the concurrent monitoring of both the organ morphology and solute transported
into that organ, including the skin, with use increasing in
clinical applications [1618]. A benet of MPT is that it can
be used in conjunction with uorescence lifetime imaging
(FLIM). FLIM is a sensitive, time-resolved technique that
enables the detection of the lifetimes and intensity of uorescent molecules within the image plane [19]. MPT-FLIM
is also used to monitor the autouorescence of uorophores
within the skin including NAD/NADP (i.e., NAD[P]H),
avin adenine dinucleotide (FAD), and porphyrins. We have
previously shown that FLIM can also be used to separate
metal nanoparticle signals from skin autouorescence [20].
However, the uorescence lifetime decay curves of gold
nanoparticles and NAD(P)H cannot directly be separated
[8]. Instead, the photon intensity output based on the %
parameters can be used to dierentiate the gold nanoparticle
and NAD(P)H signal.
In this paper, our aim is to investigate the relationship
between gold nanoparticle size and its penetration into
viable human skin following topical exposure without physical/chemical enhancers. Gold nanoparticles (10 nm, 30 nm,
and 60 nm) were applied to freshly excised human skin
for 24 hours. Following application the penetration prole
was analysed using microscopy of whole intact skin and
histology sections. Changes in the metabolic state of viable
skin following gold nanoparticle treatment were evaluated
by analysing the change in NAD(P)H using MPT-FLIM. Our
data showed that there was no signicant penetration of
the dierent sized nanoparticles into the viable tissue even
after 24 hours of treatment. Instead, the nanoparticles were
located on the skin surface, within the stratum corneum
and within the skin furrows. There were also no signicant
metabolic perturbations associated with gold nanoparticle
treatment due to their lack of penetration. Overall, the
data show that excised viable human skin is important
for an accurate and reliable understanding of nanoparticle
penetration.
electron microscopy (TEM), electrospray-dierential mobility analysis (ES-DMA), dynamic light scattering (DLS), and
small-angle X-ray scattering (SAXS). The mean size range
varied slightly between the techniques, resulting in the
average diameter 95% CI:
10 nm particles: 8.5 0.3 nm (AFM), 9.9 0.1 nm
(SEM), 8.90.1 nm (TEM), 11.30.1 nm (ES-DMA),
13.5 0.1 nm (DLS), and 9.1 1.8 nm (SAXS).
(SEM), 27.6 2.1 nm (TEM), 28.4 1.1 nm (ESDMA)), 26.5 28.6 0.9 3.6 nm (DLS), and
24.9 1.2 nm (SAXS).
(SEM), 56.0 0.5 nm (TEM), 56.3 1.5 nm (ESDMA), 55.3 56.6 1.4 8.3 nm (DLS), and
53.2 5.3 nm (SAXS).
The shape of the gold nanoparticles was determined
by SAXs with each measurement calculated from the mean
primary component peak value by tting the I(Q) data
to 2.0 A-1
(10 nm), 0.0001 A
resulting in a Q-range: 0.01 A-1
1 to 1 A-1 (30 nm), and 0.01 A-1 to 2.0 A-1 (60 nm). Particle
aggregation within the sodium citrate buer was determined
by SEM images and analysed with ImageJ v1.37. The data
analysis of the Otsu threshold algorithm was produced
from the binary image, distinguishing particles from the
background. A total of 140 (10 nm), 1185 (30 nm), and 425
(60 nm) particles were analysed within an area of roughly
35 35 pixels (10 nm), 50 50 pixels (30 nm), 100 100 pixels
(60 nm) from 2 (10 nm), 3 (30 nm), and 3 (60 nm) randomly
prepared ampoules.
All other chemicals and solvents were of reagent grade.
2.2. Excised Human Skin Preparation. Human skin was

donated from abdominoplasty patients with the approval of
the Princess Alexandra Hospital Research Ethics Committee
(no. 1997/097). A total of three skin donors were used in
this study. The donor skin was obtained on the day of
removal from the patient, and the experiment was performed
within 24 hours to preserve the viability of the skin. The
subcutaneous fatty tissue was removed from the skin using
a scalpel prior to the start of the experiments. The surface of
skin was cleaned with deionised water, and the excised tissue
was stored covered on saline moistened gauze in a 4 C cold
room until use. Skin handling and experimental design was
conducted to maintain metabolic functionality throughout
the course of these studies [24].
2.1. Materials. Three dierent sized gold nanoparticles

(10 nm [21], 30 nm [22], and 60 nm [23]) suspended in 5 mL
sodium citrate were prepared and purchased from National
Institute of Standards and Technology (NIST) of USA. The
concentration of gold nanoparticles was 5 mg/mL. The gold
nanoparticles were characterized by NIST in relation to their
size and chemical and electrochemical properties (10 nm
[21], 30 nm [22], and 60 nm [23]). The particle size and
morphology were determined by atomic force microscopy
(AFM), scanning electron microscopy (SEM), transmission
2.3. Skin Penetration Studies. The excised skin was cut into
25 mm diameter skin discs and mounted in a Franz diusion
cell with a diusion area of 1.1 cm2 and a receptor chamber
volume of 3.2 mL. High vacuum grease (Ajax, FineChem)
was used to seal the edges of the two chambers. Phosphate
buered saline, pH 7.4, was then pipetted into the receptor
chamber and magnetically stirred at 500 rpm in a water
bath at 35 C. Prior totrans-epidermal water loss (TEWL)
analysis the combined skin/Franz cell was equilibrated for 30
minutes. TEWL was then measured using a Biox AquaFlux
condenser chamber unit (model no. AF200) to evaluate the
surface integrity of the skin surface prior to nanoparticle
treatment [8]. After conrmation of intact viable skin, 500 L
of 5 mg/mL gold nanoparticle suspensions was topically
applied to the skin (n = 3 per group). The two control
groups were topical application of saline and sodium citrate
solution, respectively. The Franz diusion cells were maintained at 35 C throughout the 24-hour exposure period.
After treatment, any excess gold nanoparticle suspension was
drained and the skin removed. The treated skin was then
dried at ambient conditions, and the TEWL was analysed
again to determine any detrimental eect to the stratum
corneum during treatment.
2.4. MPT-FLIM of Viable Treated Skin. Non-invasive microscopy analysis was conducted using MPT-FLIM with a
DermaInspect (JenLab GmbH, Jena, Germany) equipped
with time-correlated single-photon counting, SPC 830 FLIM
detector (Becker & Hickl, Berlin, Germany). The excitation
source consisted of an ultrashort (65-femtosecond pulse
width) pulsed mode-locked tuneable laser at 80 MHz (Mai
Tai, Spectra physics, Mountain View, USA), with a wavelength range of 710920 nm. MPT-FLIM was used to simultaneously image the gold nanoparticles and endogenous
NAD(P)H using an excitation wavelength of 740 nm [10].
The skin was optically sectioned from the stratum corneum
to the stratum basale in 5 m increments using a 40x
objective lens with an acquisition size image size of 210
210 m2 . The image was scanned at 13.4 seconds per frame
at 600 zoom of region of interest (ROI). The emission lter
used was a 350650 nm bandpass lter (BG39) whith a long
wavelength cut-o of <700 nm. A constant incident optical
excitation power of 30 mW at the rear of the objective was
used throughout the experiment. Three replicates of treated
skin were analysed after 24 hours of treatment. FLIM data
were analysed with a SPC 830 2.9 Image software (Becker and
Hickl) to generate uorescence lifetime decay and photon
contribution curves from NAD(P)H autouorescence in
the skin and gold luminescence. The instrument response
function of each FLIM image of treated skin was calibrated
to a sucrose crystal standard (Ajax Finechem Pty Ltd.). Each
FLIM image was calculated using the Decay matrix command, and a selected ROI was cropped and analysed pixel
by pixel and line by line in the selected t parameter shown
in a lifetime distribution histogram. Gold nanoparticle
multiphoton enhanced photoluminescence was quantied
using relative amplitude of pixel intensity/pixel frequency
signal 1 % from 95 to 100%, and metabolic changes were
quantied using 1 /2 ratio and total NAD(P)H lifetime
intensity. The 1 % is dened as the quantity of emission
photons that return to the detector during the short lifetime
decay curve phase, and the 2 % is the long lifetime phase.
The 1 % is not aected by the microenvironment of the
skin. The total NAD(P)H lifetime intensity is the combined
total of free and bound NAD(P)H signals and was quantied
using exported grey-scale image of each FLIM image with
photons per pixel. The grey-scale image was imported to
3
ImageJ 1.43u (National Institutes of Health, USA) at 8-bit
image quality and a selected cell area of 200 200 m2 .
The selected cell area was measured, and the total NAD(P)H
mean intensity was quantied.
2.5. Cryosectioning of Treated Skin. Treated skin was xed in
2% paraformaldehyde in 0.1 M phosphate buer, pH 7.4, for
2 hours at room temperature. After xation, the treated skin
was embedded in OCT (Sakura Tissue-Tek) in a mold block
and frozen at 70 C for 30 minutes. The tissue samples were
sectioned at 10 m at 25 C using a Leica CM1850 cryostat
(Leica microsystems, Heidelberger, Nussloch, Germany).
The sections were mounted in Prolong Gold (Invitrogen,
Mulgrave, Victoria, Australia) prior to MPT-FLIM imaging.
2.6. Statistical Analysis. The dierences in the measured
macroscopic parameters between treated and control groups
were compared by applying the nonparametric MannWhitney U-test. Two-way ANOVA analysis was used to determine the signicance between groups. All calculations were
done using Graphpad Prism version 5.03 (GraphPad software, Inc., USA) analysis. The level of signicance was
accepted at P 0.05.
3. Results
3.1. Surface Barrier Integrity of Excised Viable Human Skin.
Prior to and after treatment, the integrity of the stratum corneum (surface barrier) was analysed using transepidermal
water loss (TEWL). The mean SE of the TEWL was 30.6
4.1, 28.2 1.2, 30.1 0.9, 28.9 1.4, and 27.9 1.1 for
the saline, sodium citrate, 10 nm, 30 nm, and 60 nm gold
nanoparticle groups respectively, before treatment. After 24hour treatment, the TEWL had increased an average of 1.6,
1.8, 1.8, 1.5 and 1.7 times compared to the pre-treatment
values for saline, sodium citrate, 10 nm, 30 nm and 60 nm
gold-nanoparticle-treated groups, respectively. There were
no signicant dierences between any two groups within a
single time point. However, there was a signicant increase
in TEWL for all groups after treatment.
3.2. Gold Nanoparticle Penetration into Skin. To determine
the eect on penetration of the dierent sized nanoparticles,
10, 30, and 60 nm gold nanoparticles were topically applied
to the skin for 24 hours followed by MPT and MPT-FLIM
analysis. Two aspects of nanoparticle penetration within
human skin were studied using MPT. The rst consisted of
en face analysis of whole intact human skin (Figure 1) and
the second analysis of cryosectioned skin (Figure 2). Both
sets of data (Figures 1 and 2) were pseudocoloured based on
1 % 0100% (blue to red). The autouorescent components
of living skin, that is, NAD(P)H and keratin result in 1 %
less than 95% and are subsequently pseudocoloured green,
blue, and yellow (viable skin only) [8]. The gold nanoparticle
luminescence result in high 1 % (95100%) and is pseudocoloured orange to red. To determine any metabolic eects
from the nanoparticles MPT-FLIM analysis was used to
monitor NAD(P)H.
Stratum corneum
Stratum spinosum
Stratum basale
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)
(q)
(r)
(s)
(t)
60 nm AuNP
30 nm AuNP
10 nm AuNP
Sodium citrate
Saline
Stratum granulosum
Figure 1: En face MPT-FLIM images of treated human viable skin. Multiphoton tomography uorescence lifetime microscopy (FLIM)
images showing the layers of excised full-thickness skin (stratum corneum, granulosum, spinosum, and basale) after 24-hour treatment with
gold nanoparticles. Images are pseudocoloured based on 1 % signal (skin autouorescence 095% and AuNPs 95100% signal). No AuNP
signal was detected in the saline control (a)(d); however, deposits of high signal were detected in the stratum corneum of the sodium citrate
control (sodium citrate crystal aggregates).The AuNP-treated groups, 10 nm (i)(l), 30 nm (m)(p), and 60 nm (q)-(t), resulted in signal
throughout the stratum corneum. 10 nm AuNPs were also detected in the furrows. Scale bar indicates 50 m. Color bar, blue to red indicates
1 % 0 100%.
Saline
Sodium citrate
10 nm AuNP
(a)
(b)
(c)
60 nm AuNP
30 nm AuNP
Gold pixel signal ( 1 % 95100%)
(d)
(e)
24-hour exposure
25
20
15
10
0
20
20
40
Depth from surface (m)
60
80
30 nm AuNP
60 nm AuNP
Saline
Sodium citrate
10 nm AuNP
(f)
Figure 2: (a)(e) Gold nanoparticle penetration in cryosectioned skin using MPT-FLIM and (f) gold nanoparticle luminescence skin
depth prole. Cross-sectional images of the skin showing the supercial stratum corneum, viable epidermis and upper dermis. Images
are pseudocoloured based on 1 % signal (skin autouorescence 095% and AuNPs 95100% signal). Both the saline (a) and sodium citrate
buer (b) controls show no AuNP signal. The 10 nm (c), 30 nm (d), and 60 nm (e) AuNP-treated groups resulted in high amounts of AuNP
signal in the stratum corneum with no signal in the lower layers. High signal was detected in the upper dermis corresponding to second
harmonic generation of dermal collagen. Scale bar indicates 50 m. Data from MPT-FLIM depth series (stratum corneum to basale of
excised human skin) were processed to quantify gold nanoparticle luminescence intensity as a function of depth in non-furrow containing
regions (i.e., AuNP penetration) (f). The top of the skin is at 20 m, whereas 0 m is the start of viable epidermis (stratum granulosum).
These data were stacked from a depth series of en face images. Color bar, blue to red indicates 1 % 0100%.
24-hour exposure
NAD(P)H mean intensity
1 %/ 2 %
3
2
1
0
Stratum
granulosum
Stratum
spinosum
Stratum
basale
Viable epidermis layer

Saline
Sodium citrate
10 nm AuNP
30 nm AuNP
60 nm AuNP
(a)
24-hour exposure
200
150
100
50
0
0
20
40
60
Depth from surface (m)
Saline
Sodium citrate
10 nm AuNP
80
30 nm AuNP
60 nm AuNP
(b)
Figure 3: NAD(P)H eects from MPT-FLIM imaging. To assess the metabolic impact of AuNP penetration, NAD(P)H signals were analysed
using 1 %/2 % ratio and total NAD(P)H lifetime intensity using MPT-FLIM. The 1 %/2 % ratio (a) is inversely related to the metabolic
rate and was calculated for each of the treatment groups at 24 hours from stratum granulosum, spinosum, and basale. The total NAD(P)H
lifetime intensities of selected area (b) were examined as a function of depth in non-furrow-containing regions. The beginning of viable
epidermis is at 0 m.
3.3. En Face MPT Analysis of Gold-Nanoparticle-Treated Skin.

Initial analysis of nanoparticle penetration consisted of qualitative MPT analysis of whole intact human skin (Figure 1
(n = 3 per group)). The skin was optically sectioned at 5 m
intervals from the stratum corneum through the stratum
granulosum, stratum spinosum and down to the base of the
stratum basale. To determine if there was any background
noise associated with the nanoparticle buers, both saline
and sodium citrate were applied to the skin in separate
experiments and analysedresulting in low background
signal in all of the layers (Figures 1(a)1(h)). Skin treated
with the 10 nm gold nanoparticles resulted in a signicant increase in high 1 % signals throughout the stratum
corneum compared to the controls (Figure 1(i)). There was
no detection of AuNP signal in the stratum granulosum,
spinosum, or basale. However, AuNP signal was detected on
the surface within the furrows (Figures 1(j)1(l)). Both the
30 and 60 nm gold nanoparticle groups showed similar penetration results to the 10 nm nanoparticles with AuNP signal
detected only in the stratum corneum (Figures 1(i)1(t)).
3.4. Cross-Sectional Analysis of Gold Nanoparticle Penetration
within Skin. A second method using MPT analysis of cryosectioned skin was used to qualitatively assess the gold nanoparticle penetration. Representative images of each group
are shown in Figure 2 resulting in a cross-sectional image
of the stratum corneum (surface) down to the dermis. The
control groups (Figures 2(a) and 2(b)) correlate with the
results observed in Figure 1, with no AuNP signal detected
within the upper layers of the skin. However, 1 % (95
100%) signal was detected within the dermis of not only
the control groups but also the nanoparticle-treated samples
(Figures 2(a), 2(b), 2(d), and 2(e)). This signal is attributed
to second harmonic generation of dermal collagen. The
gold-nanoparticle-treated groups also correlate with the data
observed in Figure 1, with AuNP signal detected only in

the stratum corneum for all samples (Figures 2(c), 2(d),
and 2(e)). However, the nanoparticles were distributed
throughout the entire depth of the stratum corneum but not
into the stratum granulosum. Overall, the two independent
qualitative experiments analysing gold-nanoparticle-treated
skin, resulted in AuNP signal in the stratum corneum layer
with no signicant nanoparticle signal in the lower layers.
3.5. En Face MPT-FLIM Intensity Measurement of Gold
Nanoparticle Luminescence in Human Viable Skin Depth
Study. Having qualitatively assessed the distribution and
penetration prole of gold-nanoparticle-treated skin
(Figures 1 and 2), the related gold uorescence signal was
then quantied using image software, Image J (National
Institutes of Health, Bethesda, Washington, DC, USA) to
analyse the number of pixels corresponding to AuNP signal
and its depth within the skin (Figure 2(f)). This resulted in
detection of a signicantly higher concentration of AuNP
signal within the stratum corneum in the 10 nm nanoparticle-treated group compared to the 30 nm and 60 nm
nanoparticle-treated groups (Figure 2(f)). The AuNP signal
below the stratum corneum did not dier signicantly from
the saline and sodium citrate negative controls. Overall, these
data correlates with the qualitative experiments resulting
in no signicant penetration of the 10, 30, and 60 nm
nanoparticles within the skin.
3.6. MPT-FLIM Analysis of NAD(P)H after Gold Nanoparticle
Treatment. The metabolic prole of the skin was analysed
using MPT-FLIM of total NAD(P)H in the epidermis of
excised viable human skin. A depth study involving a series
of stacked FLIM images of the treated groups was processed
to quantify 1 %/2 % ratio and total NAD(P)H lifetime
intensity. The 1 %/2 % ratio of NAD(P)H has been reported
to be inversely related to the metabolic rate [20]. Overall,
there was no signicant dierence of the total NAD(P)H
lifetime intensities between any of the groups (Figure 3).
4. Discussion and Conclusion

Human skin is regarded as the gold standard for skin
penetration studies compared to animal model investigations
[25]. The present study investigated 10, 30, and 60 nm gold
nanoparticles penetration within excised viable human skin.
An en face penetration prole provided gold nanoparticle
distribution in excised skin from the stratum corneum to
the basale in depth. Analysis of nanoparticle skin penetration
involves a number of factors including the skin model, skin
barrier integrity, and the inherent physicochemical attributes
(size, shape, surface charge, etc.) of the nanoparticles and
vehicle of the formulation. To assess the correlation of
cell viability (metabolism) and nanoparticle penetration,
experiments were conducted with intact human viable skin
in an ex vivo diusion cell system and tested within 24 hours
after excision.
It has been previously reported, using spectrophotometry and TEM, that 15 nm gold nanoparticles suspended
in an aqueous solution were capable of penetrating full
thickness rat skin [15]. These results are consistent with a
recent report by Filon et al. (2011) [14] who showed that
12.9 nm gold nanoparticles were capable of penetrating
into thawed human skin within 24 hours of treatment.
However, our study showed that 24-hour treatment of
excised viable human skin with 10, 30, and 60 nm gold
nanoparticles resulted in nanoparticle penetration into the
stratum corneum only with no signicant penetration into
the lower layers (Figures 1 and 2). Based on the previous
reports showing successful penetration of nanoparticles
smaller than 15 nm, it was hypothesised that the 10 nm
nanoparticle group would result in penetration into the
viable epidermis. We hypothesise that the lack of penetration
observed in our study of the 10, 30, and 60 nm nanoparticles
is associated with the integrity and type of skin sample used
(freshly excised viable human skin). As stated, Filon et al.
used thawed excised human skin. The freezing and thawing
process damaged the viable tissue resulting in increased
penetration. We used MPT to detect gold nanoparticle
penetration. This is a non-invasive method resulting in
minimal disruption of the skin during sample analysis
compared to other assays [26] and further emphasises
the importance of maintaining skin integrity so as to not bias
exposure assessment.
MPT-FLIM was used to assess the eect of gold nanoparticle-penetration on cell metabolism within the viable
epidermis. There was no signicant change in the total
NAD(P)H lifetime intensities for any of the nanoparticle
treated groups compared to the negative controls. These
results (Figure 3) further support the results shown in
Figures 1 and 2 where there was no signicant penetration
of the nanoparticles below the stratum corneum. Our results
show that the stratum corneum protects the viable epidermis
by preventing penetration of 10, 30, and 60 nm gold particles
7
within human skin. When comparing our results to those
in literature, we hypothesise that nanoparticle penetration
occurs more readily in animal models and nonviable human
skin compared to freshly excised viable human skin or in
people. Therefore, there should be careful consideration of
the animal model and viability of tissue prior to conducting
experiments on nanoparticle penetration. This is especially
important for experiments analysing the biocompatibility
and/or toxicity related to nanoparticle exposure.
Acknowledgments
nez and Dr. Xin Liu are acknowledged for
Jenny Ordo
providing valuable laboratory assistance. All members of the
TRC and DRC are thanked for their support. This work was
supported by The United States Air Force, National Health
and Medical Research Council of Australia, EpiDerm, and
The University of Queensland Research Scholarship (DCL).
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doi:10.1155/2012/746960
Research Article
Control of In Vivo Transport and Toxicity of
Nanoparticles by Tea Melanin
Yu-Shiun Chen,1 Yao-Ching Hung,2, 3 Meng-Yeng Hong,1 Andrei Aleksandrovich Onischuk,4
Jin Chern Chiou,5 Irina V. Sorokina,6 Tatiana Tolstikova,6 and G. Steve Huang1
1 Department
of Materials Science and Engineering, National Chiao Tung University, Hsinchu 300, Taiwan
of Obstetrics and Gynecology, China Medical University and Hospital, 91 Hsueh Shih Road, Taichung 404, Taiwan
3 College of Medicine, China Medical University, Taichung 40402, Taiwan
4 Institute of Chemical Kinetics & Combustion, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia
5 Institute of Electrical Control Engineering, National Chiao Tung University, 1001 University Road, EE772, Hsinchu 300, Taiwan
6 Institute of Organic Chemistry, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia
2 Department
Correspondence should be addressed to G. Steve Huang, gstevehuang@mail.nctu.edu.tw

Received 5 April 2012; Accepted 14 May 2012
Copyright 2012 Yu-Shiun Chen et al. This is an open access article distributed under the Creative Commons Attribution
cited.
Nanoparticles are unfamiliar to researchers in toxicology. Toxicity may be generated simply due to the reduction in size.
Compounds that prevent or cure toxic materials may not work on nanoparticles. Furthermore, as there are more and more
applications of nanoparticles in drug delivery and in vivo imaging, controlling the transport and toxicity will be primary concerns
for medical application of nanoparticles. Gold nanoparticles (GNPs) if injected intraperitoneally into mice can enter hippocampus
and induce cognitive impairment. GNPs caused a global imbalance of monoamine levels, specically aecting the dopaminergic
and serotonergic neurons. Pretreatment of tea melanin signicantly prevented the deposition of GNPs in mouse brains, especially
in the hippocampus. Pretreatment of melanin completely alleviated GNP-induced impairment of cognition. Pre-administration
of melanin stably maintained monoamines at normal proles. Melanin completely prevented the invasion of GNPs into the
Cornu Ammonis region of the hippocampus shown by coherent anti-Stoke Raman scattering microscopy. Here we show that
the administration of tea melanin prevented the accumulation of Au in brain, the imbalance of monoamines, and the impairment
of cognition in mice. The current study provides a therapeutic approach to toxicity of nanoparticles and a novel strategy to control
the transport of GNP in mouse brain.
1. Introduction
Nanoparticles provide a novel platform for target-specic
delivery of therapeutic agents [13]. Gold nanoparticles
(GNPs) have recently been developed as an attractive candidate for use as carriers in drug and gene delivery, particularly
because the gold core is essentially inert and nontoxic in
vitro [47]. GNPs have been developed as drug carriers in
pharmaceutical studies. This is largely due to the apparent
benets in targeting and medical image enhancement. Multiple conjugations can be achieved through the tight binding of
sulfhydryl groups. In addition, GNPs are capable of passing
both the blood-brain barrier and the blood-retinal barrier
[8]. However, neurotoxicity of GNPs has been reported

toimpair cognition in mice. In order to enhance the use of
GNPs as drug carriers, the in vivo toxicity of nanoparticles
must be minimized.
In general, GNPs exhibit very low cytotoxicity [916].
The cellular uptake of GNPs likely occurs through endocytosis, and is greatest for 50 nm particles [17]. The rare exception is that GNPs under 2 nm in diameter are toxic to many
cell lines [18]. Furthermore, GNP uptake has been associated
with damage to the cytoskeleton and cell adhesion [19].
GNPs show minor in vivo toxicity [20]. The tissue distribution of GNPs in rats and mice has been examined by
inductively coupled plasma mass spectrometry (ICP-MS)
2
[21, 22]. GNPs 10 nm in diameter were present in the liver,
spleen, kidney, testis, thymus, heart, lung, and brain, whereas
GNPs larger than 50 nm were largely detected in the blood,
liver, and spleen. GNPs ranging from 10 to 50 nm were
found in the brain. In zebrash embryos, silver nanoparticles
produced almost 100% mortality, whereas GNPs induced
minimal mortality at the same time point [23]. Previously,
we have shown that GNPs can pass through the blood-retinal
barrier and impair cognition in mice. To make better use of
GNPs as a drug carrier to target the brain, the neurotoxicity
must be eliminated.
We previously reported a polyphenolic antioxidant, of
melanin nature, found in the black tea Camellia sinensis [24].
Camellia sinensis tea melanin comprises the high molecular
weight portion of tea polyphenols [25]. Tea melanin has
demonstrated a wide range of biochemical and pharmacological activities in animals including antioxidant activity,
free radical scavenging, and immunomodulation [2628].
Melanin also has revealed unexpected protective activity
against various toxic substances, such as, benzidine, hydrazine, snake venoms, cisplatin, and acetaminophen [2932].
In addition to antioxidant properties, one of the most
signicant properties of melanin is its chelating capability
[33]. Melanin is capable of chelating heavy metals. In particular, melanin complexes with gadolinium (Gd) for use in
Magnetic Resonance Imaging (MRI), thereby alleviating the
in vivo toxicity of Gd [27]. In addition, melanin possesses
paramagnetic properties due to a high concentration of free
radicals conjugated to its polymeric matrix. The chelating
and paramagnetic properties render melanin a promising
candidate to scavenge GNPs in vivo.

2.1. Materials. HAuCl4 , sodium citrate, NaBH4 , HCl, HNO3 ,
H2 SO4 , H2 O2 , scopolamine, and other chemicals of analytical grade were purchased from Sigma-Aldrich and Fisher
(United States). H2 O was obtained at >18 M from a MilliQ water purication system. Fully fermented black tea was
purchased from local retail shops (Miaoli, Taiwan).
2.2. Animal Treatment. Animal treatments were performed
following The Guidelines for the Care and Use of Experimental Animals of National Chiao Tung University, Taiwan.
Four-week-old male BALB/c mice were housed at 22 2 C
with a 12 h light/dark cycle and were fed standard rodent
chow and water ad libitum. Mice were randomly assigned
to experimental or control groups consisting of 68 mice.
The control group did not receive any treatment, and the
experimental groups were as follows: a melanin-treated
group, a 17 nm GNP-treated group, a 37 nm GNP-treated
group, a melanin pretreated-plus 17 nm GNP-treated group,
and a melanin pretreated-plus 37 nm GNP-treated group.
GNPs were administered in a single dose intraperitoneally.
For the passive avoidance test, an additional positive control
that received scopolamine (1 mg/kg i.p.) was incorporated.
2.3. Preparation of Gold Nanoparticles. GNPs, with diameters
of 17 and 37 nm, were synthesized as previously reported
[34, 35]. The seed colloids were prepared by adding 1 mL
of 0.25 mM HAuCl4 to 90 mL of H2 O and stirring for
1 min at 25 C. A 2 mL volume of 38.8 mM sodium citrate
was stirred into the solution for 1 min, and then 0.6 mL of
freshly prepared 0.1 M NaBH4 in 38.8 mM sodium citrate
was added. Dierent diameters of GNPs ranging from 3 to
100 nm were generated by altering the volume of added seed
colloid. The solution was stirred for an additional 510 min
at 04 C. Reaction temperatures and times were adjusted to
obtain larger GNPs. All synthesized GNPs were characterized
by UV absorbance. The size of the synthesized GNPs was
veried by electron microscopy and atomic force microscopy.
GNPs were dialyzed against phosphate buered saline (pH
7.4) before injection into the animals.
2.4. Extraction, Fractionation, and Characterization of Tea
Melanin. Isolation of tea melanin was performed according
to a previous report [25]. Monomeric polyphenols were
removed by treating the tea leaves with boiling water at a
ratio of 1 : 10 (m/v) for 10 min followed by ltration. The
obtained solid matter was immersed in 40 C water at a 1 : 10
(solid/liquid) ratio, and the pH was adjusted to 10.5 with
10% NH4 OH. The extraction time was reduced to 12 h to
avoid excessive oxidation of melanin. The extract was ltered
and acidied to pH 2.5 using 2 N HCl and then centrifuged
at 15,000 g for 15 min to form a pellet. Acid hydrolysis
of the pellet was employed to remove carbohydrates and
proteins. Organic solvents (chloroform, ethyl acetate, and
ethanol) were used to remove lipids and related compounds.
In addition, melanin was reduced by treatment with Ti+3 .
The reduced melanin was dialyzed against Milli-Q water to
remove Ti+3 . Finally, the reduced sample was suspended in
distilled water, and 0.1 M NaOH was added drop wise to
dissolve the melanin and to adjust the pH to 7.0. The solution
was ltered through a Nalgene 0.45 m lter. Melanin was
fractioned on a Sephadex G-75 column (1.6 40 cm) at
a 0.5 mL/min ow rate using 50 mM phosphate buer
(pH 7.5). Fractions were monitored by ultraviolet (UV)
absorbance at 280 nm. All operations were conducted under
N2 . The physical and chemical characteristics of melanin
were examined as described. UV absorption spectra were
recorded on a JASCO V-530 UV-Visible Spectrophotometer.
Infrared (IR) spectra were recorded on a Perkin Elmer
spectrometer (Model 1600 FT). The purity of melanin
was examined by thin layer chromatography (TLC) using
silica gel as the stationary phase and chloroform/ethyl
acetate/formic acid (6 : 4 : 1) as the mobile phase. Melanin
was retained at the origin in TLC separation and did
not produce any additional signals on the chromatogram.
Comparisons with TLC separation of caeine, catechin,
epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, and theaavin assured that the melanin
preparation was free from these components.
2.5. Inductively Coupled Plasma Mass Spectrometry (ICP-MS).
For total element determinations, standard solutions were
800
800
600
600
[Au/brain] (ng/g)
[Au/brain] (ng/g)
400
400
200
200
0
0
14
Days
21
28
(a)
21
28
21
28
(b)
200
200
[Au/hippocampus] (ng/g)
14
Days
150
100
50
150
100
50
0
0
14
Days
Control
M
17 nm
21
28
37 nm
17 nm + M
37 nm + M
(c)
14
Days
Control
M
17 nm
37 nm
17 nm + M
37 nm + M
(d)
Figure 1: The eect of tea melanin on the accumulation of GNPs in the brain and hippocampus. Mice were divided into 6 groups: a control
group receiving PBS; a melanin group receiving 0.3 or 0.5 mg/kg tea melanin; a 17 nm group receiving 10 mg/kg of 17 nm GNP; a 37 nm
group receiving 10 mg/kg of 37 nm GNP; a 17 nm + M group receiving 10 mg/kg of 17 nm GNP and pretreated with 0.3 or 0.5 mg/kg melanin;
a 37 nm + M group receiving 10 mg/kg of 37 nm GNP and pretreated with 0.3 or 0.5 mg/kg melanin. Each group included 30 mice. Six mice
from each group were sacriced on day 1, 7, 14, 21, and 28. The brain was carefully dissected and the hippocampus was separated. ICP-MS
was performed to obtain the content of Au. (a) Deposition of Au in the hippocampus for mice receiving 0.3 mg/kg melanin. (b) Deposition
of Au in the brain for mice receiving 0.3 mg/kg melanin. (c) Deposition of Au in the hippocampus for mice receiving 0.5 mg/kg melanin. (d)
Deposition of Au in the brain for mice receiving 0.5 mg/kg melanin.
prepared by dilution of a multielement standard (1,000 mg/L

in 1 M HNO3 ) obtained from Merck (Darmstadt, Germany).
Nitric acid (65%), hydrochloric acid (37%), perchloric acid
(70%), and hydrogen peroxide (30%) of Suprapur grade
(Merck) were used to mineralize the samples. A sizeexclusion column was connected to the ICP-MS apparatus. Brain section samples were homogenized in 25 mM
tris(hydroxymethyl) aminomethane (Tris)12.5 mM HCl
buer solution at pH 8 and centrifuged at 13,000 rpm for 1 h.

The supernatant was applied to the size-exclusion column of
the HPLC system, which had been equilibrated with 25 mM
Tris12.5 mM HCl (containing 20 mM KCl) and eluted with
the same buer at a ow rate of 1 mL/min. The metal
components of metal-binding proteins that were eluted from
the HPLC system were detected by ICP-MS (Perkin Elmer,
SCIEX ELAN 5000). The main instrumental operating
1000
200
600
400
200
100
50
(a)
37 nm + M (0.5 mg/kg)
37 nm + M (0.3 mg/kg)
37 nm + M (0.1 mg/kg)
37 nm GNPs
17 nm + M (0.3 mg/kg)
17 nm + M (0.5 mg/kg)
17 nm + M (0.1 mg/kg)
37 nm + M (0.5 mg/kg)
37 nm + M (0.3 mg/kg)
37 nm GNPs
37 nm + M (0.1 mg/kg)
17 nm + M (0.5 mg/kg)
17 nm + M (0.3 mg/kg)
17 nm GNPs
0
17 nm + M (0.1 mg/kg)
150
17 nm GNPs
[Au/brain] (ng/g)
800
(b)
Figure 2: Dose-dependence for melanin-suppression of Au deposition in the brain and the hippocampus. Mice were pretreated with melanin
at 0.1, 0.3, or 0.5 mg/kg and received 10 mg/kg 17 nm or 37 nm GNPs. The accumulation of Au on day 21 was obtained. (a) Accumulation of
17 nm GNP (left) and 37 nm GNP (right) in the brain. (b) Accumulation of 17 nm GNP (left) and 37 nm GNP (right) in the hippocampus.
conditions were as follows: RF power 1900 W, carrier gas ow

0.8 L/min Ar, and makeup gas ow 0.19 L/min Ar. 197 Au was
used as the internal standard.
2.6. Passive-Avoidance Test. The passive-avoidance apparatus
consisted of two compartments with a steel-rod grid oor
(36 parallel steel rods, 0.3 cm in diameter, set 1.5 cm apart).
One of the compartments (48 20 30 cm) was equipped
with a 20 W lamp located centrally at a height of 30 cm,
and the other compartment was dark and of the same
size, connected through a guillotine door (5 5 cm). The
dark room was used during the experimental sessions that
were conducted between 09:00 and 17:00 h. During the
training trial, the guillotine door between the light and dark
compartment was closed. When the mouse was placed in
the light compartment with its back to the guillotine door,
the door was opened, and the time until the mouse entered
the dark compartment (step-through latency, STL) was
measured with a stopwatch. After the mouse entered the
dark compartment, the door was closed. An inescapable
scrambled foot shock (1 mA for 2 s) was delivered through
the grid oor. The mouse was removed from the dark
compartment 5 s after the shock. Then, the mouse was put
back into the home cage until the retention trial was carried
out 24 hours later. The mouse was again placed in the light
compartment, and as in the training trial, the guillotine door
was opened and the STL was recorded and used as a measure
of retention. If the mouse did not step through the door after
300 s, the experiment was terminated.
2.7. Analysis of Monoamine and Acetylcholine Concentrations

in the Mouse Brain. Monoamine levels were determined
as previously reported [36]. The mice were decapitated,
and their brains were quickly removed. The brain samples
were weighed and homogenized on ice using a polytron homogenizer (Kinematica, Lucern, Switzerland) at a
maximum setting for 20 s in 10 volume equivalents of
0.2 M perchloric acid containing 100 mM Na2 -EDTA and
100 ng/mL isoproterenol. The homogenate was centrifuged
at 15,000 g for 30 min. The pH was adjusted to approximately
3.0 using 1 M sodium acetate. After ltration (0.45 m),
the samples were separated using high-performance liquid
chromatography (HPLC). Monoamines and their metabolites were separated using HPLC at 30 C on a reversephase analytical column (ODS-80, 4.6 mm i.d. 15 cm) and
detected with an electrochemical detector (Model ECD-100,
Eikom Co., Kyoto, Japan). The column was eluted with 0.1 M
sodium acetate-citric acid buer (pH 3.5) containing 15%
methanol, 200 mg/L sodium 1-octanesulfonate, and 5 mg/L
Na2 -EDTA. The following monoamines and their metabolites were measured: norepinephrine (NE), 4-hydroxy3-methoxyphenylglycol (MHPG), dopamine (DA), 3,4dihydroxyphenylacetic acid (DOPAC), 5-hydroxytyramine
(5-HT, serotonin), 5-hydroxyindoleacetic acid (5-HIAA),
epinephrine (EPI), and homovanillic acid (HVA).
2.8. Ex Vivo Coherent Anti-Stoke Raman Scattering (Cars)
Microscopy. Freshly excised hippocampi were dissected into
thin slices, approximately 2 mm in thickness and immersed
180
150
150
120
120
37 nm + M
17 nm + M
37 nm
17 nm
(a)
17 nm
0
37 nm + M
17 nm + M
30
37 nm
30
Scopolamine
60
60
Scopolamine
90
90
Control
Time (s)
180
Control
Time (s)
(b)
Figure 3: The eect of melanin pretreatment on GNP-induced cognition impairment. A passive avoidance test using light/dark rooms was
performed. The latency time proles on day 21 are shown for mice pretreated with 0.3 mg melanin/kg (a) and 0.5 mg melanin/kg (b). Each
group of columns contains, in sequence, the averaged values from the control group (control), the melanin-treated only group (M), the
scopolamine-treated group (scopolamine), the 17 nm GNP-treated group (17 nm), the 37 nm GNP-treated group (37 nm), the melanin
pretreatment-plus 17 nm GNP-treated group (M + 17 nm), and the melanin pretreatment-plus 37 nm GNP-treated group (M + 37 nm).
represents P < 0.05 and represents P < 0.001 from the students t-test.
into a microchamber lled with PBS on a glass slide for

examination.CARS microscopy was performed using a time
constant of 3 ms, a scanning area of 300 300 m, a
step size of 1 m, a scanning size of 300 300 pixels, a
scanning velocity of 1 m/ms, and a sampling rate of 80 kHz.
Laser power was set at 30 mW for 870 nm and 40 mW for
1,064 nm. The wavelengths of the Pump and the Stokes
lasers (Pump = 870 nm and Stokes = 1,064 nm) were tuned
to match a Raman shift (2100 cm1 ) that falls in the
so-called silent region of the vibrational spectra of cells
and tissues.As expected, the CARS images of the untreated
hippocampus did not show appreciable contrast under
the nonresonant condition. The CARS signals from GNPtreated specimens, however, were dramatically enhanced,
that is, they appeared as scattered bright spots. The signal
enhancement presumably resulted from strong scattering
and large third-order polarizability of the GNPs [3740].
2.9. Statistical Analyses. All data are presented as the mean
SD with a minimum of 6 mice per group. Concentrations
of biogenic amines in mouse brains were analyzed using
the unpaired Students t-test. The criterion for statistical
signicance was set at P < 0.05 for all statistical evaluations.

3.1. Pretreatment of Melanin Reduced Au Accumulation in
Brain and Hippocampus. Accumulation of Au in the brain
was investigated (Figure 1). Mice were divided into 6 groups
as follows: a control group receiving PBS; a melanin group

receiving 0.5 mg/kg tea melanin; 17 nm GNP group receiving
10 mg/kg of 17 nm GNPs; 37 nm group receiving 10 mg/kg
of 37 nm GNPs; 17 nm + M group receiving 10 mg/kg of
17 nm GNPs and 0.3 or 0.5 mg/kg melanin 3 hours prior to
GNP treatment; 37 nm + M group receiving 10 mg/kg 37 nm
GNPs and receiving 0.3 or 0.5 mg/kg melanin 3 hours prior
to GNP treatment. Each group included 30 mice. Six mice
from each group were sacriced on day 1, 7, 14, 21, and 28.
The brains were carefully dissected and the hippocampi were
separated. ICP-MS was performed to obtain measurements
for the accumulation of Au. Without melanin pretreatment,
GNPs accumulated rapidly in the brain and reached a plateau
of approximately 770 ng/g brain on day 14 (Figure 1(a)). The
accumulation of 17 nm GNPs was apparently faster than that
of 37 nm GNPs in the rst 14 days. However, both GNPs
reached the same plateau and remained unchanged at later
time points. Pretreatment of 0.3 mg/kg melanin eectively
suppressed the accumulation of Au in brains during the rst
2 weeks. However, an abrupt increase in Au accumulation
was observed in brains at later time points (440 ng/g brain
for 17 nm GNPs and 420 ng/g brain for 37 nm GNPs). The
amount of accumulation for melanin pretreated groups was
approximately 65% compared to the control group on day
14. This ratio was slowly increased to 75% at the end
of the experiment. Administration of 0.5 mg/kg melanin
signicantly reduced the accumulation of GNPs in the brain
from 420 to 100 ng/g for 17 nm and 37 nm GNPs on day 14
(Figure 1(b)). The amount of Au was maintained at this level
until the end of the experiment. Pretreatment of melanin at
180
150
150
120
120
37 nm + M
37 nm + M
120
120
(c)
17 nm
Scopolamine
30
37 nm + M
30
17 nm + M
60
37 nm
60
90
Control
90
17 nm
17 nm + M
150
Scopolamine
17 nm + M
150
Time (sec)
180
37 nm
(b)
180
Control
Time (sec)
(a)
37 nm
37 nm + M
Control
0
17 nm + M
37 nm
30
17 nm
30
Scopolamine
60
60
17 nm
90
Scopolamine
90
Time (sec)
180
Control
Time (sec)
(d)
Figure 4: Time course of the passive-avoidance test performed on mice pretreated with melanin at 0.3 mg/kg. Mice were pretreated with
melanin at 0.3 mg/kg followed by injection with 17 nm GNPs. The passive avoidance test was performed on day 7 (a), day 14 (b), day 21 (c),
and day 28 (d).
0.5 mg/kg eectively suppressed the accumulation of GNPs

in the brain.
In the hippocampus, the Au accumulation prole resembled that of the brain. The Au accumulation increased rapidly
during the rst two weeks and reached a plateau of 160 ng/g
of hippocampus on day 14. Pretreatment of melanin at
0.3 mg/kg decreased accumulated Au to a nondetectable level
in two weeks (Figure 1(c)), but Au increased rapidly to
30 ng/g on day 14, reaching 60 ng/g on day 28. Pretreatment
of melanin at 0.5 mg/kg decreased accumulated Au to a
nondetectable level for the rst two weeks (Figure 1(d)),
but Au increased rapidly to 25 ng/g on day 14, remaining
unchanged until day 28.
The suppression of Au accumulation by pretreatment

of melanin was dosedependent (Figure 2). On day 21, pretreatment of 0.1 mg/kg melanin exhibited no inuence
on the suppression of Au accumulation in the brain
(Figure 2(a)) and hippocampus (Figure 2(b)). Administration of 0.3 mg/kg melanin reduced Au accumulation by 30%
for 17 nm GNPs and by 36% for 37 nm GNPs in the brain.
Melanin at this dosage also reduced 71% Au accumulation
for 17 nm and 37 nm GNPs in the hippocampus. Administration of 0.5 mg/kg melanin reduced 74% of Au accumulation
for 17 nm GNPs and 37 nm GNPs in the brain. Melanin at
this dosage also reduced Au accumulation by 82% for 17 nm
GNPs and by 84% for 37 nm GNPs in the hippocampus.
7
350
100
300
80
ng/g brain
ng/g brain
250
60
40
200
150
100
20
50
0
NE
MHPG
DA
(a)
DOPAC
(b)
120
700
100
600
500
ng/g brain
ng/g brain
80
60
400
300
40
200
20
100
0
0
5-HT
5-HIAA
Control
M
Scopolamine
17 nm
37 nm
17 nm + M
37 nm + M
(c)
EPI
HVA
Control
M
Scopolamine
17 nm
37 nm
17 nm + M
37 nm + M
(d)
Figure 5: Melanin ameliorates the GNP-induced imbalance of neurotransmitter proles. Immediately after the passive avoidance test, brain
tissues were removed and the levels of monoamines were analyzed by HPLC. The levels of neurotransmitters and their metabolites are shown.
(a) Norepinephrine (NE) and 4-hydroxy-3-methoxyphenylglycol (MHPG, metabolite). (b) Dopamine (DA) and 3,4-dihydroxyphenylacetic
acid (DOPAC, metabolite). (c) 5-Hydroxytyramine (5-HT, serotonin) and 5-hydroxyindoleacetic acid (5-HIAA, metabolite). (d)
epinephrine (EPI) and homovanillic acid (HVA, metabolite). Each group of columns contains, in sequence, averaged values from the
control group (control), the melanin-treated only group (M), the scopolamine-treated group (scopolamine), the 17 nm GNP-treated group
(17 nm), the 37 nm GNP-treated group (37 nm), the melanin pretreatment-plus 17 nm GNP-treated group (M + 17 nm), and the melanin
pretreatment-plus 37 nm GNP-treated group (M + 37 nm). represents P < 0.05 and represents P < 0.001 from the students t-test.
3.2. Melanin Ameliorated GNP-Induced Cognition Impairment in Mice. GNP of 17 nm diameter impaired the cognition of mice, which was evident by the reduction in latency
time measured for the passive avoidance test [41]. Pretreatment of melanin eciently suppressed the accumulation
of GNPs in mouse brains and hippocampi. However, it is
not clear if the remaining GNPs would cause any physiological damage to the hippocampus. Mice were pretreated
with 0.3 or 0.5 mg/kg melanin and injected with GNPs at
10 mg/kg. The passive-avoidance test was performed on day

21 (Figure 3). Scopolamine, serving as a positive control,
shortened the latency time from 180 s to 90 s. Injection of
17 nm GNPs shortened the latency time from 180 s to 90 s.
Pretreatment of melanin at a dosage of 0.3 mg/kg recovered
the latency time to 180 s (Figure 3(a)). Melanin at 0.5 mg/kg
also recovered the latency time to 140 s. Melanin is a mild
sedative, and pretreatment with 0.5 mg/kg induced a minor
neuronal disturbance, which was evident in the passive
M
17 nm
37 nm
17 nm + M
37 nm + M
Day 1
Day 7
Day 14
Day 21
Day 28
Figure 6: Time course examination by CARS microscopy of hippocampi isolated from mice in the melanin-treated only group (M), the
17 nm GNP-treated group (17 nm), the 37 nm GNP-treated group (37 nm), the melanin pretreatment-plus 17 nm GNP-treated group (M +
17 nm), and the melanin pretreatment-plus 37 nm GNP-treated group (M + 37 nm). The wavelengths of the Pump and Stokes lasers (Pump
= 870 nm and Stokes = 1,064 nm) were tuned to match a Raman shift (2,100 cm1 ) that fell in the so-called silent region of the vibrational
spectra of cells and tissues. To better visualize the location of GNPs, the enhanced bright spots are colored red in the nal images. The green
uorescence is the autouorescence emitted from the cells of the CA region of the hippocampus.
avoidance test. A 20% reduction in latency was observed

for the melanin-treated group (Figure 3(b); M, M + 17 nm,
and M + 37 nm). Although melanin at 0.5 mg/kg reduced
the accumulation of Au more eciently than melanin at
0.3 mg/kg, the lower dose was selected for the remaining
experiments due to the lower neuronal toxicity.
Mice were treated with melanin at 0.3 mg/kg prior to
the administration of 17 nm or 37 nm GNPs. The passive
avoidance test was performed on day 7, 14, 21, and 28
(Figure 4). On day 14, mice treated with 17 nm GNPs started
to show cognition impairment, which was pronounced by
day 21. Injection of 37 nm GNP, however, never showed
symptoms of neurotoxicity throughout the course of experiment. Pretreatment of melanin was sucient to suppress the
neurotoxicity induced by 17 nm GNP injection.
3.3. Melanin Prevented the GNP-Induced Imbalance of Monoamines in Brain. A global imbalance of neurotransmitters
in the brain has been associated with GNP treatment [41].
In particular, analysis for monoamines from brain tissues
has indicated that treatment with GNPs elevates dopamine
levels and decreases serotonin levels. Administration of GNPs
aected the mouse dopaminergic and serotonergic neurons.
Mice were pretreated with 0.3 mg melanin/kg and then
injected with GNPs at 10 mg/kg. The levels of monoamines
were measured (Figure 5). The levels of norepinephrine (NE)
and epinephrine (EPI) were undisturbed. GNP injection
elevated the level of dopamine (DA), which was alleviated
by the pretreatment of melanin. GNP also decreased the

level of 5-hydroxytyramine (5-HT, serotonin), which was
also brought back to normal with melanin pretreatment
(see Figure S1 in Supplementary Materials available on
line at doi:10.1155/2012/746960). The ameliorating eect of
melanin pretreatment was validated by these monoamine
proles in the brain.
3.4. Melanin Retarded the Invasion of GNPs into the Hippocampus. The hippocampus is located in the medial temporal lobe of the brain, which belongs to the limbic system
and plays major roles in short-term memory and spatial
navigation. GNP injection impaired learning and memory
in mice, and so the GNPs could have been transported
through the blood, across the blood-brain barrier into the
brain and into the hippocampus. Furthermore, 17 nm and
37 nm GNPs showed dierential eects on the cognition
impairment of mice, although the accumulation rate of
both GNPs in the brain and hippocampus were strikingly
similar. To localize GNPs, the freshly dissected hippocampi
were examined using ex vivo CARS microscopy (Figure 6).
GNPs are known to enhance the anti-Stoke Raman signal of
nearby amino acids. Using the proper controls, enhancement
visualized by CARS strongly indicated the presence of GNPs.
Localized enhancement of the anti-Stoke Raman signal at
an excitation wavelength of 817 nm was observed from the
hippocampi removed from 17 nm and 37 nm GNP-treated
mice. The Raman signal was completely absent from control
9
Melanin is known to stimulate the immune system in
mice [24]. The administration of melanin could enhance the
ability of the mouse immune system to protect the invaded
GNPs. However, it remains to be explored if this type of
general immune response could protect the neuronal damage
caused by GNPs invasion into hippocampus.
0.12
0.1
Absorbance
0.08
0.06
4. Conclusion
0.04
0.02
0
300
400
500
600
Wavelength (nm)
GNP
GNP + 0.1 mg/mL melanin
700
800

Figure 7: UV spectra for the titration of 17 nm GNP with melanin.

Melanin is mixed with GNP at nal concentrations of 0, 0.1,
0.2, 0.3, 0.4, and 0.5 mg/mL for melanin and 0.34 mM for GNP.
UV absorbance of the binding reactions is shown. Red-shift of
the absorbance peak indicates the increasing diameter of binding
complex.
Here we demonstrate the protective activity of tea melanin

against GNP-induced neurotoxicity in mice. The administration of tea melanin was capable of retarding the accumulation of Au in the brain, recovering the imbalance of
monoamines and preventing cognition impairment, thereby
preventing GNP-induced neurotoxicity in mice.
Acknowledgments
This study was supported in part by the National Science
Counsel Grant 100-2923-B-009-001-MY3 and by the Aim
for the Top University Plan of the National Chiao Tung
University and Ministry of Education, Taiwan, R.O.C. The
authors also acknowledge funding support from the Air
Force Oce of Scientic Research (AFOSR, FA2386-11-14094).
References
mouse tissues (mice pretreated with melanin). On day 21,
the Raman signal of 17 nm GNPs was localized at the CA
region of the hippocampus inside the cluster of neuronal
cells, whereas 37 nm GNPs were less visible and scattered
throughout the peripheral region. The distribution of 17 nm
and 37 nm GNPs in the hippocampus suggests that the
invasion of GNPs into the cluster of neuronal cells in the
CA might have caused the learning impairment in the
17 nm GNP-treated mice. Accordingly, 37 nm GNPs were
incapable of entering the neuronal cells, therefore causing
only minimal decits in learning and memory. The melaninpretreated groups did not show scattered light throughout
the whole study, which was consistent with our passiveavoidance test.
The in vivo toxicity of GNPs may very likely originate
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complex. Melanin apparently has higher anity than citrate
to GNP binding. The melanin-GNP binding complex was
stable in solution when heated up to 70 C for 10 min.
Melanin is potentially capable of scavenging GNPs from
blood stream.
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11

doi:10.1155/2012/548389
Review Article
Biocompatibility and Toxicity of Nanoparticles and Nanotubes
Xiaoming Li,1 Lu Wang,1 Yubo Fan,1 Qingling Feng,2 and Fu-zhai Cui2

Key Laboratory of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084, China
2 State
Correspondence should be addressed to Xiaoming Li, x.m.li@hotmail.com and Yubo Fan, yubofan@buaa.edu.cn
Received 27 February 2012; Accepted 3 April 2012
Academic Editor: Shuming Zhang
Copyright 2012 Xiaoming Li et al. This is an open access article distributed under the Creative Commons Attribution License,
In recent years, nanoparticles (NPs) have increasingly found practical applications in technology, research, and medicine. The
small particle size coupled with their unique chemical and physical properties is thought to underline their exploitable biomedical
activities. Its form may be latex body, polymer, ceramic particle, metal particles, and the carbon particles. Due to their small size
and physical resemblance to physiological molecules such as proteins, NPs possess the capacity to revolutionise medical imaging,
diagnostics, therapeutics, as well as carry out functional biological processes. But these features may also underline their toxicity.
Indeed, a detailed assessment of the factors that inuence the biocompatibility and toxicity of NPs is crucial for the safe and
sustainable development of the emerging NPs. Due to the unique structure, size, and shape, much eort has been dedicated to
analyzing biomedical applications of nanotubes.This paper focuses on the current understanding of the biocompatibility and
toxicity of NPs with an emphasis on nanotubes.
1. Introduction
First of all, we would better have a clear understanding of the
denition of biocompatibility and toxicity. In 2008, Williams
redened biocompatibility as follows [1]: biocompatibility
refers to the ability of a biomaterial to perform its desired
function with respect to a medical therapy, without eliciting
any undesirable local or systemic eects in the recipient
or beneciary of that therapy, but generating the most
appropriate benecial cellular or tissue response in that
specic situation, and optimising the clinically relevant
performance of that therapy. And his denition has been
recognised. In this context, NP toxicity refers to the ability
of the particles to adversely aect the normal physiology
as well as to directly interrupt the normal structure of
organs and tissues of humans and animals. It is widely
accepted that toxicity depends on physiochemical parameters
such as particle size, shape, surface charge and chemistry,
composition, and subsequent NPs stability.
The size of NPs is not more than 100 nm micro. They can
be obtained by many ways: wet chemical treatment (chemical
reactions in solution), mechanical processing (milling and
grinding technology), vacuum deposition, and gas phase
synthesis. Its form may be latex body, polymer, ceramic

particle, metal particles, and the carbon particles. According
to the dierent preparation methods, those NPs can have
dierent size, chemical composition, and shape and can be
with or without surface coating. Each of these factors can
aect the interactions between the nanomaterials and cells
or tissues. NPs can permeate membrane cells, and spread
along the nerve cells synapses, blood vessels, and lymphatic
vascular. At the same time, NPs selectively accumulate in the
dierent cells and certain cellular structure. NPs of strong
permeability not only provide the eectiveness for the use of
drugs, at the same time, but also give rise to potential threats
on the health of human body.
The development of NPs for biomedical applications
including medical imaging, magnetic hyperthermia, and
gene or drug delivery is currently undergoing a dramatic
expansion. For biomedical applications, emerging nanostructures requires stringent evaluations for their biological
security. There are a number of dierent classes of NPs
promising for biomedical purposes.
Due to the unique structure, size, and shape, much eort
has been dedicated to analyzing biomedical applications of
nanotubes. At present, nanotubes for biomedical application
2
include carbon nanotubes, silicon dioxide nanotubes, boron
nitride nanotubes, titanium dioxide nanotubes, and organic
nanotubes, of which carbon nanotubes are the most widely
used materials.
Here, we will focus on the current understanding of the
biocompatibility and toxicity of NPs and nanotubes. This
paper proceeds as follows. In later sections, the NPs and
nanotubes are reviewed in two separate sections. Section 2
reviews the biocompatibility and toxicity of NPs in the aspect
of hemocompatibility, histocompatibility, cytotoxicity, and
neurotoxicity. Section 3 is a description of main types of
nanotubes. Considering the attention that carbon nanotube
has received, the section of nanotubes is structured a little
dierently from that of the NPs.
2. Biocompatibility and Toxicity

of Nanoparticles
2.1. Biocompatibility of Nanoparticles. For biomedical applications, those NPs enter the body and contact with tissues
and cells directly, thus it is necessary for exploring their
biocompatibility.
2.1.1. Hemocompatibility. NPs are used as vectors for the
applications in drug delivery, gene delivery, or as biosensors,
where a direct contact with blood occurs. Here some NPs are
examined for their blood-compatible behaviors.
Recently, blood cell aggregation and haemolysis studies,
coagulation behaviors experiments have been carried out
evaluating blood compatibility of NPs in vitro conditions,of
which hemolysis is considered to be a simple and reliable
measure for estimating blood compatibility of materials [2].
Chouhan and Bajpai [3] has adopted Hemolysis assay
to judge the in vitro blood compatibility of the prepared
PHEMA NPs. Hemolysis assay experiments were performed
on the surfaces of the prepared particles. The results indicate
that for NPs with 12.37 mM HEMA and 1.06 mM EGDMA
percentage hemolysis is lowest. This clearly suggests a
moderate level of biocompatibility.
Sanoj Rejinold et al. [4] have studied the formulation
of curcumin with biodegradable thermoresponsive chitosang-poly (N-vinyl caprolactam) NPs (TRC-NPs) for cancer
drug delivery. Fresh human blood was used in this study.
Hemolysis assay was carried out to evaluate the blood
compatibility of bare and curcumin-loaded TRC-NPs. The
results showed that the hemolytic ratio of the sample was
within the range of less than 5%, the critical safe hemolytic
ratio for biomaterials according to ISO/TR 7406, which
indicated that the damage of the sample on the erythrocytes
was little.
The blood compatibility of the carrier MSNs-RhB was
evaluated by investigating the hemolysis and coagulation
behaviors in a broad concentration range (50500 mgmL1 )
under in vitro conditions [5]. The results suggested that
MSNs-RhB possessed good blood compatibility and also,
SEM and TEM analyses in Figure 1 indicated that both MSNs
and MSNs-RhB had a subsphaeroidal morphology, a high
dispersivity and uniform particle size of about 400 nm. In
this work, He et al. [5] evaluated the blood compatibility
of SBA-15-type MSNs and MSNs-RhB with negative and
positive surface potentials, respectively, by investigating their
hemolysis and coagulation behaviors. As to their coagulation
behaviors, PT was used to evaluate the extrinsic and common
coagulation pathways, APTT was used to evaluate the
intrinsic and common coagulation pathways, and Fib was
used to evaluate the abnormality of coagulation factor I. The
hemolytic phenomena of SBA-15-type MSNs and MSNsRhB are almost invisible by direct observation.
This is utterly dierent from the dry mesoporous silica
powder previously reported by Dai et al. [6], because all
hydrophilic mesoporous channels of MSNs and MSNsRhB have been fully lled with PBS during experimental
operation in the present study, and no space is left for
further water absorption when mixed with plasma. Thus
both MSNs and MSNs-RhB had not eected the normal
coagulation/anti-coagulation functions of plasma, that is,
the blood compatibility of SBA-15-type MSNs-RhB is satisfactory. The aggregations of the blood cells on interaction
with the NPs are shown for RBCs, WBCs, and platelets.
It revealed no aggregation of blood cells on incubation of
NPs at a higher interaction ratio of 10 mg/mL. Polyethylene
imine (PEI) which was used as positive control showed
aggregation whereas saline used as negative control did
not show any aggregation. Citrate-capped NPs also revealed
no aggregation of blood cells on incubation with blood
as reported earlier [7]. The same was visible with the
haemolytic property of the NPs. The haemolysis induced by
gold NPs is shown which were well within the acceptable
limits of 1% [8]. Stability in physiologically relevant media,
where saline levels are high, is a signicant issue for the use
of gold NPs in biological applications and assays. Therefore,
stability in PBS may be taken as an initial screening test for
compatibility with physiological conditions [9]. Regarding
the application of gold NPs in biomedicine (sensing and drug
delivery applications), they should be easily dispersible at
neutral pH and should be stable.
Most studies are aimed at attempting to understand
the blood compatibility of foreign materials and their
investigation have shown that the blood compatibility was
aected by various properties of the material surface. The
response of blood in contact with the material depends on
physicochemical features such as surface area, surface charge,
hydrophobicity/hydrophilicity, and so forth. As for NPs, the
size eect, structure, and surface are the decisive factors in
these responses [10].
2.1.2. Histocompatibility. Targeted drug delivery is one of the
most intensively explored areas of research and the use of NPs
for diagnostic purposes has already entered the biomedical
eld. The current review is focused on biocompatibility of
several representative types of nanomaterials: super paramagnetic iron oxides (SPION), dendrimers, mesoporous silica
particles, gold NPs, and carbon nanotubes (CNTs).
In general, SPION are classied as biocompatible, showing no severe toxic eects in vitro or in vivo [11]. In primary human macrophages, no immunomodulatory eects
(a)
(b)
(c)
(d)
Figure 1: SEM and TEM images of samples MSNs ((a) and (c), resp.) and MSNs-RhB ((b) and (d), resp.). The scale bars of (a), (b), (c) and
(d) correspond to 1 mm, 1 mm, 100 nm and 100 nm, respectively [5].
were observed when cells were exposed to 30 nm dextrancoated SPION [11]. However, when primary peritoneal
macrophages from rats and mice were exposed to 20 nm
and 60 nm dextran-coated SPION, an increased secretion
of anti-inammatory cytokines, and reduced production
of proinammatory cytokines occurred [12]. In contrast,
an increase in proinammatory cytokines in a murine
macrophage cell line was observed, accompanied by a
decrease in the phagocytic function of these cells upon
exposure to dextran-coated SPION [13]. These studies
underline the importance of using dierent cellular systems
for nanotoxicological studies, including primary human cell
types. Jain et al. [14] have reported that SPION neither
cause any eect in liver function when administered in vivo
in rats nor do the particles induce oxidative stress. Our
own studies demonstrate that dextran-coated SPION are
nontoxic to primary human monocyte-derived macrophages
and dendritic (antigen-presenting) cells (Kunzmann et al.,
unpublished observations).
Dendrimers exhibit a generation-dependent toxicity
with higher generation dendrimers being the most toxic.
The extent of cytotoxicity induced by dendrimers also
depends on surface charge, whereby cationic dendrimers are
more toxic than anionic dendrimers. A marked decrease
in cytotoxicity can also be achieved when the surface is

modied with PEG. Cationic dendrimers induce disruption
including formation of pores in membranes [15]. They
can induce apoptosis caused by mitochondrial dysfunction
[16]. Cationic dendrimers can cause substantial changes in
red blood cell morphology and hemolysis in a generationdependent manner, whereas anionic dendrimers have no
such eect. Cationic dendrimers were shown to induce
caspase-dependent apoptosis and negatively inuence proliferation in a murine macrophage cell line [17]. These
eects could not be observed in two other murine cell lines,
highlighting the importance of cell type specic dierences.
PAMAM dendrimers of generation 3.5 (G3.5) were shown to
aect mitochondrial membrane potential in isolated rat liver
mitochondria [18]. Glucosamine-conjugated dendrimers
inhibit the synthesis of proinammatory cytokines in LPStreated human dendritic cells and macrophages. These
dendrimer conjugates also have an inhibitory eect on tolllike receptor 4 (TLR4), a receptor that triggers LPS-induced
stimulation of immune-competent cells [19]. Shaunak et al.
evaluated the dendrimer conjugates in a rabbit model of
scar tissue formation after glaucoma ltration surgery and
found that the long-term success of the surgery increased
from 30% to 80% [19]. The authors suggested that these
4
dendrimer conjugates could be utilized to prevent scar tissue
formation. The transcriptional prole of monocytes exposed
to phosphorylated dendrimers revealed over expression of
genes involved in anti-inammatory responses [20]. Antiinammatory eects were also reported in vivo when simple
modied PAMAM dendrimers were injected into rats [21].
However, the detailed mechanisms are still unknown.
Silica-NPs-demonstrated a good degree of biocompatibility [22, 23]. Silica-coated NPs, or silica NPs, have been
demonstrated to enter the cell without aecting cell survival.
These insights push research toward the development of
silica NPs based drug delivery systems and biosensors [24
26]. Bardi et al. [27], developed and characterized NH2
functionalized CdSe/ZnS quantum dot (QD)-doped SiO2
NPs with both imaging and gene carrier capabilities. They
show that QD-doped SiO2 NPs are internalized by primary
cortical neural cells without inducing cell death in vitro and
in vivo. Moreover, the ability to bind, transport, and release
DNA into the cell allows GFP-plasmid transfection of NIH3T3 and human neuroblastoma SH-SY5Y cell lines. QDdoped SiO2 NPs properties make them a valuable tool for
future nanomedicine application.
The use of colloidal gold has a long history in coatings
and glassware as a result of their high scattering power,
variability of bright and intense colors, and stability. Furthermore, gold NPs can be readily functionalized with probe
molecules such as antibodies, enzymes, and nucleotides.
These hybrid organic-inorganic nanomaterials are the active
elements of a number of biosensor assays, drug and gene
delivery systems, laser confocal microscopy diagnostic tools,
and other biomaterial-based imaging systems [28]. There
have been many studies on the biocompatibility of gold
NPs. In an attempt to mimic the respiratory tract after
inhalation, Brandenberger et al. [29] devised an epithelialairway model consisting of alveolar epithelial-like cells
(the A549 lung carcinoma cell line), human monocytederived macrophages, and dendritic cells. After exposure
to 15 nm gold NPs using an air-liquid interface exposure
system, no induction of oxidative stress or inammatory
responses was noted. However, the system was responsive to
proinammatory LPS. No synergistic or suppressive eect
was seen in the presence of gold NPs, suggesting that the
gold NPs do not elicit immune reactions. On the other
hand, gold NPs conjugated with peptides were recognized
by primary murine macrophages and induced an immune
response as evidenced by secretion of IL-6, IL-, and TNF [30]. Therefore, the peptide coating on gold NPs is an
important factor to enhance the immune response. Indeed,
recent studies have shown that the conjugation of peptides
on the surface of NPs may enhance the immune response
[30]. Murine bone marrow macrophages were thus found
to be able to recognize gold NP-peptide conjugates, while
peptides or NPs alone were not recognized. The latter studies
shed light on the design of NPs conjugates for modulation of
immune responses in the ght against allergies, cancer, and
autoimmune diseases. The role of plasma proteins attaching
to NPs following entry into circulation also merits attention
as this could potentially interfere with or modulate the
presentation of other ligands attached to the particles.
2.2. Toxicity of Nanoparticles
2.2.1. Toxicology of Nanoparticles [31]. Interaction mechanisms between NPs and living systems are not yet fully
understood. The complexity comes with the particles ability
to bind and interact with biological matter and change
their surface characteristics, depending on the environment
they are in. Scientic knowledge about NPs cell-interaction
mechanisms has been accumulating in recent years, indicating that cells readily take up NPs via either active or
passive mechanisms. Intracellularly, however, mechanisms
and pathways are more dicult to understand. Even particles of the same material can show completely dierent
behaviour due to, for example, slight dierences in surface
coating, charge, or size. This makes the categorisation of
NPs behaviour, when in contact with biological systems,
intricate and thus nanoparticle hazard identication is not
straightforward. This is one of the main distinctions between
nanotoxicology and classical toxicology where, in the latter,
characterisation of toxicants is, in general, protocolised with
a well-established set of methodologies available, employing
a mass-based dose metric. However, with NPs the dose metric is not straightforward, as discussed below. Furthermore
the protocolization of bioassays involving nanomaterials is
still under development and has, in general, not yet been
internationally validated. In addition, there are many more
variables to consider when working with nanomaterials and
these include material, size, shape, surface, charge, coating,
dispersion, agglomeration, aggregation, concentration, and
matrix.
The complexity increases when moving from in vitro to
in vivo models. Hazard identication on the in-vivo level,
with regards to nanomaterials, is still at an early stage. Major
entry routes (lung, gut, and possibly skin) as well as putative
targets (lung, liver, heart, and brain) have been identied.
However, more research is required to understand mechanisms and pathways in the body. What seems clear is that
exposure to insoluble nanoscale particles of b50 nm appears
to be new, when compared to the evolutionary history
of the preindustrial world. Furthermore, such NPs appear
to be able to high jack a preexisting transport mechanism
through the body using endocytotic mechanisms, in the same
manner that viruses employ. Therefore, because widespread
translation of NPs within the body seems to be likely if
the body is exposed, we need to take any toxicological risks
seriously.
2.2.2. Cytotoxicity. Depending on the mode of administration and sites of deposition, toxicity may vary in severity.
Therefore, to maintain clinical relevance, information on
toxicity is presented using a system-based approach focusing
on lung, dermal, liver, and nervous system targets. Figure 2
summarizes the advantages and disadvantages of each of the
routes.
All eukaryotic cells (such as lung cells) contain functionally distinct, membrane-enclosed compartments. The
main types are the nucleus and the organelles which
include mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes, lysosomes, and endosomes. Nucleus and
Oral
Advantage:
Noninvasive
means of NP
delivery
Disadvantage:
First-pass
metabolism in the
liver-potentially
hepatotoxic
Potential for
translocation into
systemic
circulation
Requires intact
intestinal mucosa
for NP uptake
Transdermal
Advantage:
Noninvasive
means of NP
delivery
Large surface area
Local action
Disadvantage:
Local irritation
Potential for
translocation into
systemic circulation
Pulmonary
Advantage:
Noninvasive
means of NP
delivery
Large surface
area
Local action
Avoidance of
rst-pass
metabolism in the
liver
Disadvantage:
Local toxicity
Potential for
translocation into
systemic
circulation
Intravenous
Advantage:
Systemic
delivery of NP
Systemic action
Disadvantage:
First-pass
metabolism in the
liver-potentially
hepatotoxic
Systemic
toxicity
Figure 2: Routes of administration of nanoparticles and their advantages and disadvantages [32].
organelles are enclosed by a lipid bilayer containing distinct

proteins. NPs can cross the membranes of organelles since
they have been localized in lysosomes, mitochondria, and the
nucleus, the mechanisms of internalization are, however, not
known so far.
In this paper we focus on the cytotoxic eects of
frequently used NPs, such as Metal and metallic oxide NPs,
Polymeric NPs, Quantum dots, Silica (SiO2) NPs, to explain
this topic.
Lung Cells. As NPs get in contact with the skin, the gastrointestinal tract, and the respiratory tract, these biological
compartments are designed to act as barriers to the passage
of nanosized materials into the organism. Because the lung is
considered by far the most important portal of entry for NPs
into the human body this overview will mainly focus on the
lung as a potential barrier for inhaled NPs. It should however
be noted that evidence has been published that NPs can also
deposit on the olfactory epithelium and directly be translocated to the brain [33]. Current related researches are mainly
focused on the impact of NPs on alveolar macrophages and
broblasts and bronchial epithelial cells. Studies have already

shown that NPs can remarkably weaken the phagocytic
capacity of macrophages, which causes decline in lungs
clearance ability. Nanocarrier systems (polymeric NPs, silica
(SiO2) NPs, silver NPs, carbon nanotubes) for pulmonary
drug delivery have several advantages which can be exploited
for therapeutic reasons and, thus, intensively studied
Polymeric NPs are biocompatible, surface modiable,
and capable of sustained drug release. They show potential
for applications in the treatment of various pulmonary
conditions such as asthma, chronic obstructive pulmonary
disease (COPD), tuberculosis (TB), and lung cancer as
well as extra pulmonary conditions such as diabetes [34
36]. Already, there is a multitude of organic nanopolymers
including collagen, gelatin, chitosan, alginate, and bovine
serum albumin (BSA). Furthermore, the last three decades
has seen a rise in the development of synthetic polymers
such as the biocompatible and biodegradable poly(lactic-coglycolic acid) (PLGA) for use as drug carrier devices [37,
38]. While such drug-loaded nanocongurations demonstrate promising alternatives to current cancer treatment,
6
cytotoxicity needs to be evaluated. PLGA NPs successfully
improve therapeutic outcome and reduce adverse eects via
sustained and targeted drug delivery. Additionally, the use of
biological capping materials such as chitosan or BSA further
reduce toxicity while their biocompatibility and biodegradative capacity making them an intuitive choice for NPs surface
modication. Romero et al. demonstrated a reduction in
cytotoxicity of PLGA NPs stabilized with BSA compared to
synthetic coating materials in cultured lung cancer cells [37].
Albumin, the most abundant serum protein, was found to be
highly biocompatible making it a useful stabilizer for drug
delivery vehicles. Similarly, chitosan-stabilization resulted in
near-total cellular preservation and improved pulmonary
mucoadhesion in an in vivo lung cancer model [39]. Biological capping materials reduce cytotoxicity by mimicking the
physiological environment, thus hiding from the immune
system. However, the possibility of enzymatic degradation
due to biophysical resemblance needs further investigation.
Silica (SiO2) NPs have found extensive applications in
chemical, mechanical polishing, and as additives to drugs,
cosmetics, printer toners, varnishes, and food. Recently, the
use of silica NPs has been extended to biomedical and
biotechnological elds, such as biosensors for simultaneous
assay of glucose, lactate, l-glutamate, and hypoxanthine levels
in rat striatum, biomarkers for leukemia cell identication
using optical microscopy imaging, cancer therapy, DNA
delivery, drug delivery, and enzyme immobilization. Silica
NPs have been shown to have a low toxicity when administered in moderate doses [40]. Unfortunately, silica NPs also
tend to agglomerate and have been demonstrated to lead to
protein aggregation in vitro at dose of 25 g/mL [41]. Oxidative stress has been implicated as an explanation behind silica
NPs cytotoxicity both in vitro and in vivo [4245]. All these
studies have reported cytotoxicity and oxidative stress, as
determined by increasing lipid peroxidation (LPO), reactive
oxygen species (ROS), and decreasing cellular glutathione
(GSH level), but no similarity exists regarding dose-response.
In the present study, we did not found signicant dierence
in the cytotoxicity and oxidative stress caused by the two sizes
10 nm and 80 nm of amorphous silica NPs. A similar result
was observed for 15 nm and 46 nm silica NPs by Lin et al.
[46]. Present studies suggest that it is theoretically feasible
and within acceptable safety limits to use moderate doses
of silica NPs; however, high-dose toxicity proles warrant
further investigations.
The most common route of pulmonary exposure to silver
NPs (AgNP) is via the occupational inhalation of airborne
particles during manufacturing. The current American Conference of Governmental Industrial Hygienists (ACGIH)
limit for silver dust exposure is 100 g/m3 . In order to
evaluate potentially acute and delayed adverse pulmonary
eects of AgNP, Sung et al. have carried out a series of
inhalation studies focusing on the acute, subacute (28 days)
and subchronic (90 days) toxicity of AgNP in rats [4749].
In the acute setting,rats were exposed to dierent particle
concentrations in a whole-body inhalation chamber for 4
consecutive hours and were subsequently observed for a further 2 weeks. At the highest concentration used (750 g/m3 ;
7.5 times higher than the limit), no signicant body weight
changes or clinical changes were observed. Furthermore,
lung function tests revealed no statistical dierences between
exposed and control groups. Repeated administration of
AgNP for 4 weeks showed similar results. In contrast,
subchronic inhalation for 13 weeks at a maximum concentration of 515 g/m3 (5 times the limit) revealed time- and
dose-dependent alveolar inammatory and granulomatous
changes as well as decreased lung function [50]. Such results
suggest that while high-dose chronic exposure to AgNP
has the potential to cause harm, under current guidelines
and limits, such excessive particle inhalation would seem
unrealistic.
Dermal Cells. The skin is the largest organ of the body
and functions as the rst-line barrier between the external
environment and the internal organs of the human body.
Consequently, it is exposed to a plethora of nonspecic
environmental assaultswithin the air as well as to distinct
and potentially toxic substances within creams, sprays, or
clothing. Topically applied NPs can potentially penetrate the
skin and access the systemic circulation and exert adverse
eects on a systemic scale. In this part, we will explain
cytotoxic eects of TiO2 NPs and gold NPs.
TiO2 NPs have several properties which make them an
advantageous ingredient for commercial sunscreens and cosmetics. They exhibit UV-light blocking properties and confer
better transparency and aesthetics to creams. In vitro studies
demonstrated cell type-dependent TiO2 toxicity aecting
cellular functions such as cell proliferation, dierentiation,
mobility, and apoptosis [51, 52]. Such adverse eects,
however, could not be replicated in vivo. In order to assess
penetrative capacities, dermal inltration studies have been
carried out on human volunteers using dierent investigative
techniques. Lademann et al. investigated the penetrative
eect of repeated administration of TiO2-containing sunscreen on the skin of volunteers [53]. Tape stripping and
histological appraisal of skin biopsies revealed that TiO2
penetrated into the open part of a hair follicle as opposed to
the viable layers of the epidermis or dermis. Furthermore, the
titanium amount in any given follicle was less than 1% of the
applied total amount of sunscreen. Surface penetration via
hair follicles or pores was also suggested by a study conducted
by Bennat and Muller-Goymann where skin permeation
was greater when sunscreen was applied to relatively hairy
skins [54]. Mavon et al. demonstrated near total recovery of
sunscreen after 15 tape strippings with no TiO2 deposition
in hair follicles or skin layers [55]. It could be argued
that dierent degrees of permeation and toxicity correlate
with surface coatings and functionalizations of TiO2 NPs as
well as with the number of follicular pores within the skin
facilitating particle uptake.
Due to facile means of synthesis and the potential for
biofunctionalization, gold NPs (AuNP) are being investigated for clinical applications including dermal drugdelivery [56]. Sonavane et al. demonstrated size-dependent
permeationon excised rat skin after topical application of
dierently sized AuNP (15, 102 and 198 nm) [57]. Smaller
NPs penetrated deeper into the tissue than larger ones which
were mainly accumulated in the more supercial epidermis
and dermis. These ndings may have important implications
with regards to ecient NP-based dermal drug delivery.
Au compounds are generally considered safe and have been
in routine clinical use for many years, for example, in
the treatment of rheumatoid arthritis [58]. However, once
reduced to nanometer scale, particles are known to undergo
profound changes in terms of their biochemical properties
which necessitates renewed investigations into their cytotoxic
prole. Despite the relative wealth of toxicity studies focusing
on AuNP, contradictory results remain the main obstacle
to transition into the clinical setting. Several studies have
demonstrated cellular uptake of AuNP to be a function of
time, particle size, and concentration. In a study by Murphy
et al., human dermal broblasts were exposed to AuNP for a
period of up to 6 days [58]. Three sets of NP concentrations
were obtained for each of two dierent sizes. Larger particles,
45 nm, exhibited marked cytotoxicity at a concentration
of 10 g/mL compared to smaller particles, 13 nm in size,
which only displayed cytotoxic signs at the much higher
concentration of 75 g/mL. These results conict with those
obtained by Mironava et al. who reported maximum toxicity
for a particle size of 1.4 nm [59]. Such dierences may be
explained by the distribution pattern of particles within cells
and require more research.
Liver Cells. Being the site for rst-pass metabolism, the liver
is particularly vulnerable to NP toxicity and has consistently
been shown to accumulate administered substances, even
long after cessation of exposure. Thorough evaluation of NPmediated hepatocellular toxicity thus remains of prevailing
importance. Lipid peroxidation assay, comet assay, and
oxidative DNA damage are commonly used to study the
impact of NPs on liver cells. Gold NPs, silver NPs, silica
NPs, and QDs have been intensively studied for clinically
application reasons. The eect of surface structure and
surface modication of NPs are important factors of their
interaction with cells. Here we use QDs to elaborate it.
Semiconductor nanocrystals, or QDs, may be used in
a variety of biomedical applications. The general structure
of QDs comprises an inorganic core-shell and an organic
coating to which biomolecules may be conjugated to enable
targeting to specic areas within the body. Such close proximity and interaction with the physiological environment
necessitates toxicological evaluation of these particles. Cellbased studies focusing on QD-induced adverse eects that
found that toxicity most likely arises from the liberation of
metal ions released from the heavy metal core [60]. Oxidative
environments further promote degradation and metal-ion
leaching. The liver is of particular importance with regards
to bio-toxicity because of rst-pass metabolism and potential
accumulation and deposition within the organ, as shown
by Derfus et al. [61]. QD size was also postulated to be a
major parameter in organ-specic deposition with smaller
particles (<20 nm) extravagating through capillary fenestrae
that are large enough in the liver (100 nm in size) [62].
The long half-life clearly has implications for organ toxicity,
particularly in view of the livers untoward propensity to
heavy metal ion poisoning which makes exposure to QDs
7
potentially very hazardous. Surface coating to protect the
core from degradation has been shown to reduce toxicity
[63]. Conventionally, QDs are coated with a layer of zinc
sulphide (ZnS) or mercaptoacetic acid. However, evidence of
continued cellular toxicity after prolonged periods of time
suggests either inadequate core coverage or the need for a
dierent type of coating material [64]. Das et al. carried out
a series of experiments assessing additional surface coatings
for their respective cytotoxicities [65]. CdTe/CdSe cored QDs
with a ZnS shell were additionally covered with organic,
carboxylated (COOH), amino (NH2), or poly(ethylene
glycol) (PEG) coatings. Cytotoxicity was tested on exposure
to each type separately by measurement of macrophage cell
viability and LDH release. All QDs were shown to induce
signicant cytotoxicity after 48 h and coating materials as
well as liberated Cd ions were suggested to be the causative
agents. It is likely that a breakdown of physically labile
surface material resulted in ion liberation and subsequent
toxicity. Recently, Seifalian and colleagues have demonstrated
that the novel synthetic nanomaterial polymeric oligohedral silsesquioxane (POSS), when incorporated onto CdTecored QDs, shows signicantly enhanced cytocompatibility
more than conventionally used materials, even without ZnS
shelling (unpublished data). POSS was shown to be nontoxic
by preventing ion leakage from the core. These results
underline the importance of the type of coating material used
and suggest that the most important factor inuencing QD
toxicity remains heavy metal-ion leakage from the core due
to inadequate surface coverage.
2.2.3. Neurotoxicity. The central nervous system is composed
of two parts: the brain and the spinal cord. Both of them
are delicate organs in human body which must be protected
from the injury to xenobiotics. Recent observations suggest
that several NPs, such as polysorbate 80-coated PBCA
NPs and pegylated PLA immunonanoparticles, are able to
cross BBB [66, 67] through intravenous administration and
followed by the accumulation in the brain. However, due
to their special physicochemical properties, such as large
surface area, the NPs may cause neurotoxicity after entering
into the brain. Therefore, the evaluation of the potential
neurotoxic eects of these NPs on CNS function is required,
as specic mechanisms and pathways through which NPs
may exert their toxic eects remain largely unknown. So
far, there are already some reports, but not many, which
observed the neurotoxicity of NPs both in vitro and in
vivo [68, 69]. As a large variety of colloidal dispersions of
super paramagnetic iron oxide nanoparticles (SPIONs) have
been developed and explored for a range of new biological,
biomedical, and diagnostic applications with regard to their
magnetic properties [70, 71]. Here we will give a description
of the toxicity eect of super paramagnetic NPs on the brain.
SPIONs and ultrasmall SPIO nanoparticles (USPIONs)
consist of an iron oxide core and a variable carbohydrate
coating which determines cellular up take and biological
half-life. The degree of surface coverage has been postulated
to be the main parameter in cellular uptake as incomplete
surface coverage was shown to promote opsonization and
rapid endocytosis whereas fully coated SPION escaped
8
opsonization which, as a result, prolonged plasma half-life
[72]. However, more recently, particle size as opposed to
coating degree has been suggested to exert chief inuence
on the rates of uptake by macrophages [73]. Being one of
the few FDA approved NPs for the use in MRI, SPIONs
most commonly nd applications in the imaging of the
vasculature and lymph nodes [7477]. However, recent
reports from both animal models and human subjects have
shown their ecacy in visualizing intracerebral malignancies
and neurological lesions within the CNS [78]. Despite such
routine use of SPION, the long-term eects and potential
neurotoxicity have, as yet, not been evaluated extensively.
The unique physiochemical properties shared by all NPs,
such as nanometer size and a large surface area to volume
ratio, make SPION particularly valuable for novel therapeutic and diagnostic applications. However, such dimensional
reductions may potentially induce cytotoxicity and interfere
with the normal components and functions of the cell
[79]. Previous in vitro studies have shown the capacity for
SPION to induce ROS generation, impair mitochondrial
function and cause leakage of LDH-all of which could
incite neurotoxicity as well as potentially aggravate preexisting neuronal damage [80]. Furthermore, toxicity reports
demonstrated an association between particle size, type of
surface coating and breakdown products, concentration, and
the degree of opsonization and cytotoxicity in cultured cells
[81]. For example, Berry et al. utilized broblast cultures to
demonstrate the ability to tune particle toxicity according
to particle coating. They compared the in vitro toxicity
of plain uncoated magnetic iron oxide NPs (P particles)
with either dextran-derivatized (DD) or albumin-derivatized
(AD) NPs. P particles as well as DD particles exhibited
similar toxicities, whereas AD particles managed to induce
cell proliferation [82]. In a study by Muldoon et al., the distribution, cellular uptake, and toxicity of three FDA-approved
SPION of dierent sizes and surface coatings were compared
to each other and to a laboratory reagent [83]. Firstly,
inoculation of ferumoxtran-10 (USPION: 2050 nm in size,
complete surface coverage with native dextran), ferumoxytol
(USPION: 2050 nm in size, complete surface coverage with
semisynthetic carbohydrate) and ferumoxide (SPION: 60
185 nm in size, incompletely coated with dextran) as well
as the lab reagent MION-46 into tumor-bearing rat brains
demonstrated direct uptake of ferumoxtran-10 into tumor
tissue and long-term retention within the cancerous lesion (5
days). However, uptake seemed NP dependent. Ferumoxide
inoculation did not yield tumor enhancement which suggests
size and surface coverage dependence. The second step
involving osmotic BBB disruption to evaluate transvascular
SPION delivery and neurotoxicity displayed no evidence
of gross pathology implying the feasibility of intracerebral
injection of clinical USPION into humans.
3. Biocompatibility and Toxicity of Nanotubes

At present, nanotubes for biomedical application include
carbon nanotubes, silicon dioxide nanotubes, boron nitride
nanotubes, titanium dioxide nanotubes, and organic
nanotubes, of which carbon nanotubes are the most widely
used materials. So far, there have been many great studies
into it.
3.1. Carbon Nanotubes. Carbon nanotubes are made from
rolled up sheets of graphene and are classied as singlewalled (SWCNTs) or multiwalled carbon nanotubes (MWCNTs) (as shown in Figure 3) depending on the constituent
numbers of graphene layers. Due to their unique size and
shape, much eort has been dedicated to analyzing biomedical applications of CNTs. Such extensive potential requires
the meticulous evaluation of toxicity. This widespread use
of dierent types of NPs in the biomedical eld raises
concerns over their increasing access to tissues and organs
of the human body and, consequently, the potential toxic
eects. Carbon nanotubes (CNTs) are cylindrical graphene
sheets. Due to their unique structure, CNTs can be used
for hyperthermic ablation of cancer cells due of their strong
optical absorption in the NIR wavelength region, as well as
for drug delivery to cancer cells owing to their high surface
areas.
CNTs are hydrophobic in nature and thus insoluble
in water, which limits their application in biomedical and
medicinal chemistry. Therefore, various functionalization
methods like adsorption, electrostatic interaction, and covalent bonding are being utilized with a number of compounds
and polymers to render a hydrophilic character to CNTs so
as to avoid their aggregation and to facilitate their use in
biomedical applications. Recent developments with CNTs
span the areas of gene therapy, drug delivery, thermotherapy,
imaging, and anticancer treatments.
3.1.1. Biocompatibility of Carbon Nanotubes. Carbon nanotubes (CNTs) have attracted broad attention because of
their excellent electrochemical, mechanical, and electrical
characteristics. In recent years, many research eorts have
focused on the exploration of the application of both singlewall (SWCNT) and multiwall (MWCNT) carbon nanotubes
in the biological and biomedical eld as nerve cell stimuli,
diabetes sensors, cancer therapy, drug delivery carrier, bone
scaold materials, and so forth [85, 86].
Interaction with Cells. Among CNTs, single-wall CNTs consist of a single layer of graphite lattice rolled into a perfect
cylinder, whereas sets of concentric cylindrical graphite shells
form multiwall CNTs (MWCNTs). Neurobiology is one of
the elds where the potential applications seem to be very
promising [87]. Neurons and neuronal cell lines can grow
and dierentiate on CNT substrates [88, 89].
It is known that the functionalized CNTs (f-CNTs) can
be used to control the number of growth cones, neurite
outgrowth, length, and branching during neuronal cell
growth on f-CNTs [90]. The neuronal environment with positively charged multiwalled carbon nanotubes (MWCNTs)
has improved neurite outgrowth and branching compared to
neutralor negatively charged MWCNTs [91, 92].
Bardi et al. [93] show that multiwall CNTs (MWCNTs)
coated with Pluronic F127 (PF127) surfactant can be injected
in the mouse cerebral cortex without causing degeneration
(a)
(b)
Figure 3: (a) Single-walled carbon nanotubes (SWCNTs); (b) multiwalled carbon nanotubes (MWCNTs) [84].
of the neurons surrounding the site of injection. These

results suggest that PF127-coated MWCNTs do not induce
apoptosis of cortical neurons. Moreover, the presence of
MWCNTs can reduce PF127 toxicity.
In 2011, to evaluate the eects of the surface roughness and functionalization modications of the SWCNT
papers, Yoon et al. and so forth [94] investigated the neuronal morphology, mitochondrial membrane potential, and
acetylcholine/acetylcholinesterase levels of human neuroblastoma during SH-SY5Y cell growth on the treated SWCNT
papers. Their results demonstrated that the plasma-chemical
functionalization caused changes in the surface charge
states with functional groups with negative and positive
charges and then the increased surface roughness enhanced
neuronal cell adhesion, mitochondrial membrane potential,
and the level of neurotransmitter in vitro. The cell adhesion
and mitochondrial membrane potential on the negatively
charged SWCNT papers were improved more than on the
positively charged SWCNT papers. Also, measurements of
the neurotransmitter level showed an enhanced acetylcholine
level on the negatively charged SWCNT papers compared to
the positively charged SWCNT papers.
It has been demonstrated that CNT support the growth
of osteoblastic cells by stimulating the production of extracellular matrix (ECM), a central step during the formation
of bone tissue [95].
In order to investigate the interaction of cells with
modied multiwalled carbon nanotubes (MWCNTs) for
their potential biomedical applications, the MWCNTs were
chemically modied with carboxylic acid groups (COOH),
polyvinyl alcohol (PVA) polymer and biomimetic apatite
on their surfaces [96]. In this study, human osteoblast
MG-63 cells were cultured in the presence of the surfacemodied MWCNTs. There were no obvious changes in
cell morphology in osteoblastic MG-63 cells cultured in
the presence of these chemically modied MWCNTs. The
surface modication of MWCNTs with apatite achieves an
eective enhancement of their biocompatibility. Recently, a
new study [97] indicated that MWCNTs might stimulate
inducible cells in soft tissues to form inductive bone by
concentrating more proteins, including bone-inducing proteins. In this study, they evaluated human adipose-derived
MSCs (HASCs) cultured on MWCNTs compacts, comparing
on graphite compacts, with and without the adsorption of
FBS and recombinant human bone morphogenetic protein2 (rhBMP-2) in advance in order to nd out how CNTs aect
dierentiation of HASCs. Larger mineral content was found
on the MWNTs compacts than on the GP compacts at day 7.
In vivo experiment showed that the MWNTs could induce
ectopic bone formation in the dorsal musculature of ddy
mice while GP could not.
Surface-coating treatment with multiwalled carbon nanotubes (MWCNTs) was applied to 3D collagen scaold for
bone tissue engineering. In Hirata et al. [98] study, the
eect of the MWCNT coating on dierentiation of rat primary osteoblasts and the tissue response around MWCNTcoated sponges were investigated. Rat primary osteoblasts
(ROBs) were cultured on an MWCNT-coated collagen
sponge (MWCNT-coated sponge) in a 3D dynamic ow cell
culture system and dierentiation markers were measured.
Signicantly more bone formation was observed around the
MWCNT-coated sponges than around the uncoated sponges
and new bone attached to MWCNTs directly at 28 and
56 days after implantation in the femur. Moreover, at 28
days after implantation of the MWCNT-coated sponge with
osteoblasts cultured for 1 day, bone tissues were successfully
formed in the pores according to its honeycomb structure.
Therefore, MWCNT coating appears to be eective for bone
tissue engineering.
As Drug and Vaccine Delivery Vehicles. Carbon nanotubes
(CNTs) could be one of the most advanced nanovectors
for the highly ecient delivery of drugs and biomolecules
owing to their large surface with unique optical and electrical properties. They can be conjugated noncovalently or
covalently with drugs, biomolecules and NPs towards the
development of a new-generation delivery system for drugs
and biomolecules.
CNTs can interact with mammalian cells and enter cells
via cytoplasmic translocation [99102]; they therefore can
deliver a range of therapeutic reagents into the cell. For
example, plasmid DNA has been internalized by the cell,
and the expression of the plasmid-carried marker genes has
been enhanced [102105]. Other macromolecules, including
proteins [106], polymers [107], and single-stranded DNA
[108] have also been internalized by coating onto CNTs and
through the interaction of CNTs with mammalian cells.
10
Based on the dosage dierences in target organelles,
Yang et al. [109] successfully used SWCNTs to deliver
acetylcholine into brain for treatment of experimentally
induced Alzheimer disease with a moderate safety range
by precisely controlling the doses, ensuring that SWCNTs
preferentially enter lysosomes, the target organelles, and not
mitochondria, the target organelles for SWCNT cytotoxicity.
To evaluate the acute response of blood leukocytes to
CNTs in vitro, Medepalli et al. [110] recreated two specic
events: (a) a direct-exposure event that may occur due to
presence of CNTs in circulation and (b) presentation of CNTs
to blood leukocytes via antigen presenting cells. The potential for activation of dierent leukocyte subpopulations was
then evaluated by proling various early activation markers
using ow cytometry. To ensure relevance to gene and
drug delivery, these experiments utilized single-walled CNTs
(SWCNTs) functionalized with single-stranded (ss)-DNA
fragments consisting of guanine-thymine (GT) repeated
sequences, which have potential to serve as a backbone
for transport of biomolecules and also as a surfactant to
prevent aggregation. Results from this study demonstrate
that ss-DNA-functionalized SWCNTs does not elicit an acute
immune response from blood leukocytes through either
direct or indirect interactions as veried by the expression
of early leukocyte activation markers.
As Biosensors. Carbon nanotubes show great potential for

use as highly sensitive electronic (bio)sensors. Single-walled
carbon nanotubes (SWNTs) arguably are the ultimate
nanosensor in this class for a number of reasons: SWNTs
have the smallest diameter (1 nm), directly comparable to
the size of single molecules and to the electrostatic screening
length in physiological solutions [111]. Furthermore, the low
charge-carrier density of SWNTs [112] is directly comparable
to the surface charge density of proteins, which intuitively
makes SWNTs well suited for electronic detection that relies
on electrostatic interactions with analyte (bio)molecules.
Finally, the SWNT consists solely of surface such that every
single carbon atom is in direct contact with the environment, allowing optimal interaction with nearby molecules.
Although an appreciable amount of biosensing studies has
been conducted using carbon nanotube transistors, the physical mechanism that underlies sensing is still under debate
[113]. Several suggested that mechanisms are electrostatic
gating [114], changes in gate coupling, carrier mobility
changes, and Schottky barrier eects [115].
Recently, Zelada-Guillen et al. [116] have reported the
rst biosensor that is able to detect Staphylococcus aureus
in real time. A network of single-walled carbon nanotubes
(SWCNTs) acts as an ion-to-electron potentiometric transducer and anti-S. aureus aptamers are the recognition element. In this study, both biosensor types demonstrated great
versatility in selectivity assays, which suggests the applicability of SWCNT/aptamer-based potentiometric biosensors in
the highly selective identication of S. aureus.
the improvement of the living creature exploitation degree,

and reducing the biological toxicity can be achieved.
CNTs may eliminate quickly in vivo from the blood, is
mainly detained in the liver, the spleen and the lung.
Liver is the dominant site of accumulation after intravenous (iv) or intraperitoneal (ip) administration of CNTs,
but only few studies were conducted in order to establish the
impact of CNTs over the liver [117]. Also, only few studies
have been conducted regarding ip delivery of CNTs [118].
Last year, Clichici et al. and so forth [119] have found that ssDNA-MWCNTs induce oxidative stress in plasma and liver,
with the return of the tested parameters to normal values,
6 h after ip injection of nanotubes, with the exception of
reduced glutathione in plasma. Results demonstrate that ssDNA-MWCNTs produce oxidative stress and inammation,
but with a transient pattern. Given the fact that antioxidants
modify the prole not only for oxidative stress, but also of
inammation, the dynamics of these alterations may be of
practical importance for future protective strategies.
The research [120] indicated that in 2 months watersoluble SWCNTs does not cause the mouse spleen organization rening various biochemistry target change, the histopathology inspects the nonspleen damage; But along with
detection times extension, possibly causes spleens immune
response.
Porter et al. [121] conducted an in vivo dose-response
and time course study of MWCNT in mice in order to
assess their ability to induce pulmonary inammation,
damage, and brosis using doses that approximate estimated
human occupational exposures. The data reported indicate
that MWCNT exposure rapidly produces signicant adverse
health outcomes in the lung. Furthermore, the observation
that MWCNT reach the pleura after aspiration exposure
indicates that more extensive investigations are needed to
fully assess if pleural penetration results in any adverse health
outcomes.
Recently, Pietroiusti et al. [122] have tested the eect of
pristine and oxidized single-wall carbon nanotubes (SWCNTs) on the development of the mouse embryo. No fetal
and placental abnormalities were ever observed in control
animals. In parallel, SWCNT embryo toxicity was evaluated
using the embryonic stem cell test (EST), a validated in vitro
assay developed for predicting embryo toxicity of soluble
chemical compounds, but never applied in full to NPs. The
EST predicted the in vivo data, identifying oxidized SWCNTs
as the more toxic compound.
Toxicity of CNTs and its mechanism have been widely
investigated, which is diering from composition, length,
diameter and sizes. Exposure to pristine CNT has been
shown to cause minimal cytotoxicity at higher concentrations (both in vivo and in vitro), while chemically functionalized CNT enhanced for drug delivery has not demonstrated
any toxicity thus far. However, CNT aggregation has plagued
research in this area and the impact of this key variable is
unclear at this stage.
3.1.2. Toxicity of Carbon Nanotubes. CNTs has certain toxicity, including lung toxicity and embryonic toxicity. Through
covalent modication and adding surfactants to the CNTs,
3.2. Other Nanotubes. Silicon dioxide nanotubes, boron

nitride nanotubes, titanium dioxide nanotubes, organic
nanotubes are emerging used nanotubes. Until now, reports
about their biomedical application are still not sucient.
Simple descriptions of their biomedical studies are given here
to help us get a better understanding about them.
3.2.1. Silicon Dioxide Nanotubes. Silica nanotubes (SNTs)
have become a promising material in biomedical applications, owing to their unique properties. The tube-structured
SNTs are endowed with two physically distinct domains: the
inner void and the outer surface. Dierential functionalization of the inner and outer surfaces of SNT could provide
a facile and eective means to integrate multifunctionality
with SNT technology [123]. For example, various nanosized
biomaterials and therapeutics, such as magnetic particles,
imaging agents, and drugs, can be loaded inside the vacant
inner space of SNT to make them potent multifunctional
materials. Figure 4(a) shows a TEM micrograph of MSNTs
with a length of 610 m and a diameter in the range 400
600 nm. From the TEM image of NH2 -MSNTs (Figure 4(b))
it can be seen that the samples retained the same morphology
as the as prepared MSNTs after modication with APTS.
A TEM micrograph of MSNTs coated with PAH/PSS multilayers is presented in Figure 4(c). Similar results for the
thickness of the ALG/CHI multilayer assembled on NH2 MSNTs can be determined from the TEM micrograph of
Figure 4(d).
Biocompatibility and facile modication through wellknown silane chemistry [125127] make SNTs even more
attractive tools in various biomedical applications, such as
in drug or gene delivery vehicles. Nevertheless, SNTs have
encountered a fundamental impediment in gene delivery
as they acquire a negative charge in aqueous solution due
to the presence of a large number of hydroxyl groups on
their surface. Therefore, to achieve ecient gene delivery, the
surface of the SNT must be rendered positive by conjugating
cationic materials. These cationic materials then condense
and load DNA, having a negatively charged phosphate
backbone and transport it to the target sites, especially
intracellular regions.
The Wu group was the rst to introduce SNT as a
therapeutic cargo for gene delivery [128]. In their report,
the inner surface of SNT was functionalized with 3(aminopropyl)trimethoxysilane to generate a polycationic
surface and, as per confocal microscopy, 60e 70% of the
cells were transfected during incubation. The Ran group
[129] have functionalized SNT with a magnetic-uorescent
nanocomposite and LMW BPEI to construct a device that
can act as an ecient gene delivery carrier and as a MRI
agent. The success of this dual-modality nanoconstruct
should drive further research into multipurpose therapeutic
biomaterials.
3.2.2. Boron Nitride Nanotubes. A boron nitride nanotube
(BNNT) is a structural analog of a carbon nanotube:
alternating B and N atoms entirely substitute for C atoms
in a graphitic-like sheet with almost no change in atomic
spacing [130]. Despite this structural similarity with carbon
nanotubes (CNTs), BNNTs own superior mechanical, chemical, and electrical properties [131, 132]. In the latest years,
several examples of CNT exploitation in biotechnology have
11
been proposed [133], while the biomedical applications of
BNNTs have remained largely unexplored [134], having the
rst study about BNNT-cell interactions been performed by
Ciofani et al. [135].
Figure 5 shows the SEM and TEM images of the asreceived BNNTs SEM image shows nice clean BNNTs,
whereas TEM picture shows the presence of both long
cylindrical tubes and bamboo type structures. BNNTs have
excellent elastic modulus of 1.22 TPa (similar to CNTs)
and are thus a potential candidate as reinforcement. A
recent study through the molecular dynamic approach has
shown the tensile strength of single-walled BNNTs to be P24
GPa [137]. Also, BNNTs are very exible and hence their
reinforcement will not adversely aect the ductility of the
scaolds [138]. BNNTs were rst synthesized in 1995 but
there are very few studies [139144] on BNNT reinforced
composites. Researchers have used BNNTs as reinforcement
in glasses mainly to increase the strength and fracture toughness [139, 140]. Only one report is available on ceramicBNNT composite [141], where enhanced superplasticity in
Al2O3 and Si3N4 with BNNT addition is observed. Few
studies on polymer-BNNT composites have been reported
including nonbiodegradable polymers like polyaniline [142],
polystyrene [143], and copolymer of vinylidene chloride and
acrylonitride [144]. These studies have justied the role of
BNNT in terms of improvement in mechanical and optical
properties. BNNTs have higher chemical stability than CNTs
in oxidative atmosphere, with their oxidation starting at
1223 K compared to CNTs at 773 K [145]. Although human
body temperature is 310 K, the chemical inertness of BNNTs
may still be an added advantage when they are exposed in the
living body.
Given its proposed biomedical application, cytotoxicity
of BNNTs is a very important issue. Recently, Chen et al.
[146] have shown BNNTs to be noncytotoxic to human
embryonic kidney cells (HEK-293) and reported that BNNTs
do not inhibit cell proliferation even after 4 days. Ciofani et
al. [147] demonstrated good cytocompatibility and cellular
uptake of polyethyleneimine- (PEI-) coated BNNTs in a
human neuroblastoma cell line (SH-SY5Y). Both these studies indicate safe use of BNNTs in bioapplication. The application of BNNTs in orthopedic scaold material requires
their cytotoxic behavior to be investigated with bone cells,
for example, osteoblasts. Since PLC matrix is biodegradable,
BNNTs may get exposed to the bloodstream after the
scaold degrades. BNNTs, exposed in the bloodstream,
interact rst with macrophages. Macrophages internalize the
foreign elements entering in the bloodstream to prevent any
harmful reaction. Hence, the cytotoxicity test of BNNTs on
macrophages is also very important. No studies have yet
been performed on cytotoxicity of BNNTs with osteoblasts
or macrophages. It must also be emphasized that no report
exists on any biodegradable polymer-BNNT composites up
to now.
3.2.3. Titanium Dioxide Nanotubes. In 2001, Gong and
coworkers [148] reported the fabrication of vertically
oriented highly ordered TiO2 nanotube arrays up to
12
(a)
(b)
(c)
(d)
Figure 4: TEM images of (a) MSNTs, (b) NH2-MSNTs, (c) PAH/PSS-MSNTs, and (d) ALG/CHI-NH2-MSNTs [124].
(a)
(b)
Figure 5: (a) SEM image of the as-received BNNTs. (b) TEM images of the as-received BNNTs showing the presence of both tubular- and
bamboo-type structures [136].
approximately 500 nm length by anodization of titanium foil

in an aqueous HF electrolyte. Since then, substantial eorts
have been devoted to the self organisation and growth of
TiO2 [149]. Titanium dioxide nanotube layers are used as
photocatalysts in water and environmental purication, as
well as biological and biomedical applications [150152]. In
particular, titanium dioxide nanotubes are used as a biomaterial for implants, drug delivery platforms, tissue engineering,
and bacteria killing [153155]. Another interesting propriety
of TiO2 is its tunable wettability eect [156]. The ability to
modify surface topography and to control wetting behavior
is useful for biomedical applications. Surface roughness,
contact angle, and surface energy are the main factors in

understanding the biology media and material interaction.
We can see the SEM images of the ordered anodized titanium
oxide nanotube arrays in Figures 6(a) and 6(b). Figure 6(c)
shows Silica coated TiO2 nanotubes prepared on the above
anodized TiO2 via a sol-gel method.
In 2010, Feschet-Chassot et al. [158] used the ciliated
protozoan T. pyriformis to predict the toxicity of titanium
dioxide nanotube layers towards biological systems. The
contact angle measurements show clearly the correlation
between surface topography and surface wettability. They
have shown the ability of the titanium dioxide nanotube
13
1 m
500 nm
(a)
(b)
50 nm
(c)
Figure 6: SEM images of TiO2 nanotube arrays anodized at constant potential. (a) Top view, (b) side view, and (c) silica-coated titania
nanotube [157].
layers to degrade AO7. Such surfaces do not show any

characteristic in vitro toxicity eect in a biological system.
3.2.4. Organic Nanotubes. Organic nanotubes (ONTs) selfassembled from amphiphiles are one of the tubular nanomaterials that have attracted many interest in the eld of nanotechnology because of their uniform tubular morphology
with a hollow nanospace and a hydrophilic biocompatible
surface [159]. Application of pristine ONTs has been found
in nanopipettes, photovoltaic devices, and nanocontainer
for and gene. Furthermore, the surface functionalization of
ONTs has become a key methodology to utilize them for
many purposes, such as sensor for guest protein, pathogen
detection chips, and biomimetic catalyst [160].
In the meantime, the use of tubular nanomaterials
as nonviral gene transfer vector has been growing fast.
Inorganic ones, mainly carbon and silica nanotubes, were
functionalized with various positive materials and their gene
transfer ability has been extensively investigated. To the
best of knowledge, two attempts on the usage of ONTs
in gene delivery have been reported. In the rst example,
lipid microtubules were embedded in an agarose gel as a
reservoir for the sustained release of plasmid DNA, but not
as a DNA transfer vector [162]. In the second example,
cationic nanotubes self-assembled from dipeptide were able
to associate single-stranded DNA on their outer surface for
the intracellular delivery [163]; however, they were transformed to spherical vesicles under dilute condition before
cellular internalization. These limited reports indicated that
the utility of ONTs as nonviral tubular gene transfer vectors
has not been fully demonstrated.
The drug loading of ONTs was susceptible to the eect
of ionic strength and H+ concentration in the medium,
and drug release from ONTs was promoted at lower pH,
which is favorable for the release of drugs in the endosome after cellular uptake [161] and Figure 7 shows that
ONT1 was taken up to the cells. Moreover, ONT could
be modied chemically with folate by simply mixing with
a folate-conjugate lipid. Also in 2011, another study [164]
demonstrated the usefulness of functionalized ONT in gene
delivery, and the functionalized ONT represents a novel
type of tubular nonviral gene transfer vector. These novel,
biodegradable organic nanotubes have the potential to be
used as drug carriers for controlled and targeting drug

delivery.
4. Conclusions
Nanoparticles, of large surface area and high specic surface energy, are thermodynamically unstable system. After
the NPs enter the organism by various means, they will
agglomerate, dissolve, and meet with other changes as a
result of the environmental impact of the body (such as
protein concentration and high ionic strength, high acidity).
When NPs agglomerate, the physical and chemical properties
may change, thus aect the biological eects. For metal and
metallic oxide NPs, solubility problems are more important.
The release of metal ions of dissolved NPs, at least part
of them contributes to the toxicity that we observed. The
biological characteristics which NPs demonstrated have
signicant correlations with structures and the nature of
their own. Therefore, people are considering changes in
NPs themselves to reduce their toxicity and improve the
biocompatibility.
Surface modication of NPs and articial control of NPs
size and shape, are eective ways to reduce the toxicity of
NPs. But some scholars believe that articially coated and
modied NPs have lost their original features, which from
a fundamental sense not the original NPs can be compared
with. Surface modication methods can be divided into
the surface coating and chemical modication. Through
the surface modication of NPs, the inherent toxicity of
NPs can be reduced, which also can greatly improve the
biocompatibility of NPs.
To use NPs safely in biomedicine, a detailed understanding of biocompatibility and toxicity of NPs is needed. As
we can see, more and more data are becoming available
regarding NPs toxicity, but highly eort is still required
in order to truly advance our knowledge in this eld.
Currently, researchers may carry out these studies from these
aspects: considering various forms of particles respectively,
considering the dose-response relationship, in vivo and in
vitro experiments, setting about establishing a database of
toxic nanoscale to further clarify the division of nanoscale
toxic nanosize range. Meantime, deep studies about the
14
(a)
(b)
(c)
Figure 7: Transmission electron micrographs showing untreated C26 cells (a) and C26 cells incubated with ONT1 for 24 h (b) and a high
magnication image of the surrounding cell (indicated by an asterisk in (b)) (c). N: nucleus; arrowhead: ONT1, bars = 2 m (a) and (b) and
500 nm (c) [161].
interaction between cells from dierent tissues and NPs are

also necessary.
As a novel kind of nanomaterials with wide potential
applications, adverse eects of carbon nanotubes (CNTs)
have recently received signicant attention after respiratory
exposure. Toxicity of CNTs and its mechanism have been
widely investigated, which is diering from composition,
length, diameter, and sizes. Exposure to pristine CNT
has been shown to cause minimal cytotoxicity at higher
concentrations (both in vivo and in vitro), while chemically
functionalized CNT enhanced for drug delivery have not
demonstrated any toxicity thus far. However, CNT aggregation has plagued research in this area and the impact of this
key variable is unclear at this stage.
Acknowledgments
The authors acknowledge the nancial supports from
the National Natural Science Foundation of China (no.
31000431 and 10925208), the Beijing Nova Program (no.
2010B011), State Key Laboratory of New Ceramic and
Fine Processing (Tsinghua University), and Program for
New Century Excellent Talents (NCET) in University from
Ministry of Education of China.
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doi:10.1155/2012/731592
Research Article
Biocompatibility of Nanoporous TiO2 Coating on
NiTi Alloy Prepared via Dealloying Method
Jin Huang,1 Junqiang Wang,1 Xiangdong Su,2 Weichang Hao,2 Tianmin Wang,2
Yayi Xia,3 Guozu Da,4 and Yubo Fan1

of Material Physics and Chemistry, Beihang University, Beijing 100191, China
3 Orthopedic Institute and The Second Hospital of Lanzhou University, Lanzhou 730030, China
4 Research Division, Gansu Seemine Material High-Tech Co. Ltd., Lanzhou 730030, China
2 Center
Correspondence should be addressed to Weichang Hao, cmpc@buaa.edu.cn and Yubo Fan, yubofan@buaa.edu.cn
Received 2 March 2012; Accepted 30 March 2012
Copyright 2012 Jin Huang et al. This is an open access article distributed under the Creative Commons Attribution License,
This paper investigated the biocompatibility of nanoporous TiO2 coating on NiTi shape-memory alloy (SMA) prepared via
dealloying method. Our previous study shows that the dealloying treatment at low temperature leads to 130 nm Ni-free surface
titania surface layer, which possesses good bioactivity because of the combination of hydroxyl (OH ) group in the process of
dealloying treatment simultaneously. In this paper, the biological compatibility of NiTi alloy before and after dealloying treatment
was evaluated and compared by direct contact method with dermal mesenchymal stem cells (DMSCs) by the isolated culture
way. The interrelation between the biological compatibility and surface change of material after modication was systematically
analyzed. As a consequence, the dealloying treatment method at low temperature could be of interest for biomedical application,
as it can avoid sensitization and allergies and improve biocompatibility of NiTi shape-memory alloys. Thus it laid the foundation
of the clinical trials for surface modication of NiTi memory alloy.
1. Introduction
Nowadays, nickel-titanium (NiTi) shape-memory alloy
(SMA) becomes one of important biomedical metal materials because of its special shape-memory eect, the hyperelasticity, and excellent biocompatibility [1, 2]. However,
NiTi alloy containing high-concentration nickel (atomic
ratio at 50%) can have a large number of nickel ions dissolved
out after corrosion; especially in body uid containing
chlorine ion, point corrosion-resistant performance is not
ideal, which will cause larger chronic host negative response,
such as sensitization, teratogenicity, and even carcinogenic
change [3]. These negative properties make NiTi alloy in
the body of safety questioned, and patients are worried,
which become one of the obstacles for NiTi alloy biomedical
application. In view of the above questions, researchers have
done a lot of work, the use of physical, chemical, and
electrochemical, and so forth diversied method on NiTi
shape-memory alloy surface modication [4, 5]. Recently
our research team have used dealloying technology at low

temperature to realize the surface Ni ions removal and
obtained the titanium oxide of porous structure on the
surface [610]. Li et al. studies show that the formation of the
porous structure is benecial to cell compatibility [11, 12].
The nanoporous surface can obviously improve the cellular
compatibility [13].
Stem cell with the capacity of quick proliferation and
itself renewal can be dierentiated into many dierent target
cells, and its potential of multilineage dierentiation brought
new chance for biological tissue engineering [11, 1416].
Dermal mesenchymal stem cells (DMSCs) as a part of
the stem cells, compared with other mesenchymal stem
cells, are characterized by drawing materials conveniently,
unrestricted position, and the advantages of fast growth and
renewal; yet they are able to keep those characteristics in vitro
culture. Jean et al. have conrmed dermal sources of stem
cells with dierentiation potential to be the fat, cartilage,
2
and bone, which starts the important role of dermal stem
cells in the connective tissue repair for recognizing [1722].
In this paper, rst of all, the dermal stem cells were separated
and subcultured by the traditional program successfully,
then used direct contact method to the evaluation and
comparison of NiTi alloy biological compatibility with that
before and after dealloying treatment and performed a
systematic analysis of the relationship between the nature
change of material surface after modication and the
biological compatibility. Thus it is laid the foundation of the
clinical trials for surface modication of NiTi SMA.
2. Experiments
2.1. Dealloying NiTi Alloy. NiTi shape-memory alloy performance for experiment reaches the country demanding
on using NiTi shape memory alloy to processing material
standard in medical equipment and surgical implants; the
alloy components are Ni: 50.7% and Ti: 49.3%. Sample
was processed into 20 mm 10 mm 2 mm, grinded and
polished gradually with metallographic sand paper from
400 to 1200, and then put, respectively, into acetone
and anhydrous alcohol with ultrasonic cleaning. The 200 mL
self-developed dealloying treatment liquid (a typical formula
as follows: nitro dioctyl phthalate : H2 O2 : HCl : H2 SO4 =
4 : 1 : 2 : 3, volume ratio) was hold in the beaker with sample
and stirred at 50 C, low temperature for 15 h. The sample
was taken out to clean with deionized water and dry. The
dealloyed sample was heated treatment at 400 C for an hour
and prepared for use.
2.2. Structure Characterization. Nano Indenter DCM system
test analyzed mechanical performance to be on submicron
scale on the surface of NiTi memory alloy, taken o the
nickel by process of dealloying. Test adopted the pyramid
pressure head and pressed in the maximum depth of 500 nm.
The maximum load was 500 mN, but the displacement
and loading precision are 0.01 nm and 50 nN, respectively.
The place of 70% maximum load maintained 60 s of the
thermal drift correction after unloading. The Oliver and
Pharr method was used to calculate H and E value.
2.3. Cellular Compatibility Experiments. The New Zealand
rabbit, 25 days old, was twisted o neck to lead to death
and was put on asepsis platform after body disinfection with
75% alcohol. After clipping the whole layer of the backside
skin, we removed the subcutaneous tissue by sterile blade
and operated separation dermis by the mechanical method
(biopsy). DMSCs were separated and subcultured by the
traditional program successfully. DMSCs in passage 3 were
seeded at a nal concentration of 1 105 cells per mL to
the glass slides coated with polylysine in a 6-well plate. Cells
detected positively of Vimentin, cytokeratin, nestin, CD34,
and FVIII factors by the SABC method were considered as
DMSCs.
The NiTi memory alloy after surface treatment by
crystallization at 400 C for 1.5 h was group A, the untreated
alloy was group B, and pure DMSCs was control group.
The sample specications of NiTi memory alloy were width
10 mm, length 15 mm, and height 2 mm, then 40 pieces
sample of group B or 40 pieces sample of group A, and
DMSCs were developed together in vitro. Compared with
the control group, NiTi memory allos from group A and B
were put on the preset against taking o microslides within
12-well plate, respectively. Every hole was covered by cell
suspension with the same volume and density. DMSCs were
counted by the ow cytometry, and cell growth curves were
drawed after cell growth situation was observed for 1 d, 5 d,
and 8 d. The hydroxyproline and alkaline phosphatase of
cells with group A or B were measured for 1 d, 5 d, 8 d (the
kit was provided by the Nanjing Jiancheng Biotechnology
Company). The content of nickel ion in cell culture medium.
The content of nickel ions in cell culture medium from three
groups was determined by AA-800 type graphite furnace
atomic absorption spectrophotometer.
3. The Results and Discussion

3.1. Porous Structure and Properties of TiO2 Coating. The
surface morphology of NiTi alloy after the dierent treatment time via amino oxidation method at 55 C is shown as
Figure 1. It clearly shows that NiTi memory alloy began
gradually to form the porous structure in the dealloying
process for 4 h, at last in the porous alloy surface won a
porous nanogrid structure, which is a typical characteristics
after dealloying. The grid structure is constituted by the rest
undissolved element after one-element selectivity dissolution
from the alloy components. XRD research shows that
because of the thinner membrane formation after dealloying
the diraction peak of alloy substrate phase remained
predominant, but having the titanium dioxide diraction
peak of faint sharp titanium ore phase and rutile phase
appeared after processing, then surface formed the titanium
oxide.
Figure 2 shows mechanical properties of NiTi alloy after
dealloying. We can see that the elastic modulus of 120 nm
depth frontal lm is lower, and the elastic modulus of 50 nm
depth frontal lm is lower than 20 GPa, which is almost
equal to the elastic modulus of human body cortical bone.
Hence, this layer lm with low modulus of elasticity will
benet to weaken stress shelter eect from alloy in the
course of the bone tissue growth. The elastic modulus of
120 nm depth has already reached the highest 71 Gpa, after
that basically remained level, which illustrates that it is
already the elastic modulus value of matrix NiTi austenitic
parent phase. The nanohardness-displacement change on
the surface layer of test sample is seen Figure 2(b). It can
be seen that the nanohardness of alloy increases gradually
from the exterior to the interior and mutations does not
appear, beginning to appear slow at after a depth of about
300 nm and a maximum value 4.7 GPa. From the outside to
the inside, hardness value with the slow continuous increase
the gradient transition is between that membrane layer, and
matrix, and the stress is smaller with good combination. In
the load-displacement curve as shown in Figure 2(c), both
loading curve and unloading curve are nonlinear, then the
0h
(a)
4h
6h
(b)
(c)
9h
15 h
(d)
(e)
Figure 1: SEM images of the surface morphologies of NiTi alloy dealloyed for 0 h, 4 h, 6 h, 9 h, and 15 h.
maximal displacement of the pressure head including both

elasticity and plastic deformation parts, while unloading
produces elastic recovery and elastic recovery reaches 44.4%,
the residual displacement depends on the elastic deformation
mainly, which shows obvious characteristics of ceramic
material. Electrochemical test results in Hanks simulation
body solution (pH = 7.45) are shown in Figure 3. Compared
to the NiTi memory alloys without surface treatment, the
self-corrosion potential of dealloying NiTi sample gets bigger
enhancement from 0.05 V to o.92 V.
3.2. Cells Compatibility
3.2.1. Cell Proliferation. DMSCs grow well in control group,
but DMSCs cultured in groups A and B have dierence forms
as shown in Figure 4. DMSCs in group A have uniform, long
fusiform and their cytoplasm is abundant, then cell culture
ask is almost full of DMSCs after two days, which is close
to control groups. However, most of the DMSCs in group B
are polygonal, with signicant morphological dierences and
less cytoplasm. In Figure 5, the amount of DMSCs increases
sharply in two days and begins to reduce after two days
gradually; moreover all data are signicantly less than those
of control group or group A which have less inuence on
DMSCs proliferation.
3.2.2. Determination of Hydroxyproline and Alkaline Phosphatase in Cell Culture Medium. Hydroxyproline content
analysis results as Table 1 shows. The content of hydroxyproline in the culture medium of group B reduced gradually,
which has close relationship to DMSCs growth. It shows that
cell growth is slow, so absorption of hydroxyproline is also
Table 1: The content of hydroxyproline in cell culture medium

(mg/L).
Group
Initial
content
20.00
20.00
1d
5d
8d
12.07 + 0.13
39.6%
5.34 + 0.21
73.3%
11.40 + 0.08
43%
2.89 + 0.29
85.5%
6.47 + 0.04
67.6%
2.51 + 0.35
87.5%
slow which led to the content decrease slow. However, the

content of hydroxyproline in group A declined dramatically,
which shows that cell growth is quick. The decrease of
hydroxyproline results from its consumption of the collagen
synthesis process.
The results of alkaline phosphatase content analysis as
Table 2 shows. The content of alkaline phosphatase in the
culture medium of group A declined gradually, and in
the culture medium of group B the content of alkaline
phosphatase also gradually declined, but all data of group B
are lower than those of group A, because, after the surface
modication the NiTi memory alloy is the slow release of the
nickel ions which have less inuence on cell metabolism.
3.2.3. Determination of Nickel Ions in Cell Culture Medium.
As indicated in Table 3, the content of nickel ions release
in cell culture medium increases with time; the amount
of nickel ion in group A after 5 days is greatly below the
amount of group B. The change is slight after 8 days, and the
incremental quantity is smaller. In addition, in cell culture
medium of group B, the concentration of nickel ion is about
3.7 times higher than that of the group A signicantly after
80
5
70
50
Hardness (GPa)
Modulus (GPa)
60
40
30
20
2
1
10
0
10
0
0
100
200
300
400
500
100
200
300
400
500
Displacement into surface (nm)

(a)
(b)
16
14
Load on sample (mN)
12
10
8
6
4
2
0
200
400
600

(c)
Figure 2: NiTi memory alloy surface dealloying and surface nanocreasing test.
Table 2: The content of alkaline phosphatase in culture medium
(U/g prot).
Table 3: The content of nickel ions in cell culture medium (ng/mL).
AKP
Group A
Group B
Group
Group A
Group B
Control
1d
8.35 + 0.62
7.06 + 1.13
5d
5.71 + 0.43
4.27 + 1.09
8d
4.25 + 1.30
3.61 + 1.52
5 days, which is a leading cause of aecting cell proliferation.

The much more nickel ions from NiTi shape-memory alloy
in group B may occur in corrosion within the body and have
biological system damage on the body.
3.3. Discussion. After calculated the dealloying conditions
of thermodynamics, a dealloying treatment method was
applied to nearly equiatomic NiTi alloy so as to remove
the harmful element of nickel selectively from NiTi alloys
and form a Ni-free titanium oxide layer on the surface
[23]. The results show that the dealloying treatment at
1d
28.7
31.1
1.97
Time
5d
46.8
173
1.97
8d
50
177.5
1.97
low temperature leads to 130 nm Ni-free surface layer that

possesses a nanometer structure and in situ formation
of titania surface that possesses a degree of bioactivity
because of the combination of hydroxyl (OH ) group in
the process of dealloying treatment simultaneously. This
dealloying treatment can avoid sensitization and allergies
and improve biocompatibility of NiTi shape-memory alloys,
It will be a good news to biomedical application. DMSCs
can timely, accurately reect the early nickel ions adverse
eects which are the inuence of growth and cell metabolism
5
2.2
2
1.8
1.6
E/V (SCE)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0.2
0.4
lgi(nAcm 2 )
NiTi
Uncrystallization
Crystallization
Figure 3: Electrokinetic potential polarization curve of NiTi after dealloying treatment in Hanks simulation body uids (pH = 7.45) and
crystallization in 500 C for 1 h.
Group A
Group B
(a)
(b)
Figure 4: NiTi memory alloy and DMSCs culture in vitro.
nickel ions give to. The NiTi memory alloys after surface
modication or without surface modication and leather
stem cells in culture in vitro observe the inuence on
leather stem cells and determin concentration of nickel ions,
the content of hydroxyproline and alkaline phosphatase in
cell culture medium, thus compare the cell compatibility
dierence between surface modied NiTi memory alloy and
NiTi memory alloy without surface modication.
From cell count detection result in the incubation,
logarithmic growth, and the platform period of the cells,
NiTi memory alloy without the surface modication does
have inuence on the proliferation of the cells, but the
NiTi memory alloy after surface modication reduced greatly
the inuence of cell proliferation. The content of alkaline
phosphatase in cell culture medium of group A declined
gradually and in group B has same trend, but all of values
are below those of group A. The pure TiO2 coating free of
nickel ions within the hundreds of nanometers of surface of
NiTi memory alloy after surface modication blocked the
diusion of nickel ions in the alloy matrix. The low level

of nickel ions in cell culture medium has less eect on cell
metabolism. It has been proved that the NiTi memory alloy
after surface modication at low temperature process can
eectively prevent the spread, dissolution, and release of the
nickel ions.
Hydroxyproline is the main raw material of collagen
synthesis in cells, so the number of hydroxyproline can reect
the collagen synthesis. In the cell culture medium of group
B, the content of hydroxyproline is slow decline, which is
in proportion to cell growth to explain the slow growth of
cells and less collagen synthesis. So the slow absorption of
hydroxyproline in culture liquids results in hydroxyproline
content dropping gradually. The level of hydroxyproline in
group A dropped sharply; it shows the rapid growth of cells
and the more amount of collagen, which also prove the fact
that the modied surface can eectively improve the tissue
compatibility of alloy.
Cell count (105 /mL)
2
0
Cell culture time (day)

Comparing group
Group A
Group B
Figure 5: Flow cytometric cell count analysis.
4. Conclusions
Nanoporous TiO2 coatings on NiTi alloy were prepared
via dealloying method. The hydroxyl (OH ) group was
introduced into the surface of TiO2 during the process
of dealloying treatment simultaneously. The direct contact
method was used for the evaluation and comparison of
biological compatibility between NiTi alloy and samples
after dealloying treatment. Research results indicate that
the dealloying treatment method at low temperature could
be of interest for biomedical application, as it can avoid
sensitization and allergies and improve biocompatibility of
NiTi shape memory alloys. This paper led to the foundation
of the clinical trials for surface modication of NiTi memory
alloy.
Acknowledgments
The word is changed into This work was nancially
supported by the National Basic Research Program of China
(973 program, 2011CB710901), the National Natural Science
Foundation of China (Grants no. 11120101001, 10925208,
51072012), and the Fundamental Research Funds for the
Central Universities (Grant no. YWF-1-03 Q- 079).
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doi:10.1155/2012/939821
Research Article
Noninvasive Evaluation of Injectable
Chitosan/Nano-Hydroxyapatite/Collagen Scaffold via Ultrasound
Yan Chen,1 Songjian Li,2 Xiaoming Li,3 Yichen Zhang,2 Zhi Huang,4 Qingling Feng,5
Zhilai Zhou,2 Bomiao Lin,6 and Bo Yu2
1 Department
of Ultrasonic Diagnosis, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China
of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China
4 School of Geosciences and Info-Physics, Central South University, Changsha 410083, China
5 State Key Laboratory of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084, China
6 Department of Radiology, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China
2 Department
Correspondence should be addressed to Xiaoming Li, x.m.li@hotmail.com and Bo Yu, gzyubo@gmail.com

Received 1 March 2012; Accepted 3 April 2012
Copyright 2012 Yan Chen et al. This is an open access article distributed under the Creative Commons Attribution License,
To meet the challenges of designing an in situ forming scaold and regenerating bone with complex three-dimensional (3D)
structures, an in situ forming hydrogel scaold based on nano-hydroxyapatite (nHA), collagen (Col), and chitosan (CS) was
synthesized. Currently, only a limited number of techniques are available to mediate and visualize the injection process of the
injectable biomaterials directly and noninvasively. In this study, the potential of ultrasound for the quantitative in vivo evaluation
of tissue development in CS/nHAC scaold was evaluated. The CS/nHAC scaold was injected into rat subcutaneous tissue and
evaluated for 28 days. Quantitative measurements of the gray-scale value, volume, and blood ow of the scaold were evaluated
using diagnostic technique. This study demonstrates that ultrasound can be used to noninvasively and nondestructively monitor
and evaluate the in vivo characteristics of injectable bone scaold. In comparison to the CS, the CS/nHAC scaold showed a
greater stiness, less degradation rate, and better blood supply in the in vivo evaluation. In conclusion, the diagnostic ultrasound
method is a good tool to evaluate the in vivo formation of injectable bone scaolds and facilitates the broad use to monitor tissue
development and remodeling in bone tissue engineering.
1. Introduction
Each year, more than a million people worldwide require
bone grafts to create or repair bone. Currently, autografts
are the gold-standard treatment to replace or augment
bone. However, because autografts must be taken from a
separate donor site on the patients body and, therefore,
require two surgical procedures, there is a limited supply
of available bone as well as serious risk of donor-site
pain, infection, and bleeding. To overcome these issues,
there is a signicant clinical need to develop alternatives
to autografts and allografts for bone grafting procedures.
From a clinical perspective, the use of injectable scaolds
is very attractive as it minimizes patient discomfort, risk
of infection, scar formation, and the cost of treatment [1].
These scaolds are liquid before and during the injection,

but they undergo gelation in situ, during the procedure of
surgery, allowing them to ll in a bone defect of any shape.
Chitosan is currently seen as a promising biodegradable
biomaterial, while -glycerophosphate (GP) is a well-known
biocompatible agent [24]. The chitosan solution with
GP salts possesses a physiological pH and can be held
liquid below room temperature for encapsulating living
cells and therapeutic proteins [57]. Recent studies have
demonstrated the application of the C/GP hydrogel as an
injectable scaold for tissue engineering [811].
Bone is mainly composed of nano-hydroxyapatite and
collagen bers, in which the c-axes of the HA are regularly
aligned along the collagen bers [1214]. While considerable
eort has gone into determining the relationship between
2
collagen structure and mineral orientation, synthetic recreation of this most fundamental level of bone structure has
eluded the materials engineer seeking to fabricate bonelike composites. It would be desirable to mimic both the
composition and structure of bone for synthetic bone graft
substitutes [1517]. Biomimetic bone materials can be used
in conjunction with natural bone, to induce new bone tissue
formation and promote bone remodeling. At present this is
the most promising route for the repair of defects in natural
bone [1822]. The ideal bone scaold should promote early
mineralization and support new bone formation while at
the same time allowing for replacement by new bone. A
bone-like nano-hydroxyapatite/collagen (nHAC) composite
by mineralizing type 1 collagen seems to be a very promising
system for bone reconstructive or regenerative surgery.
In our preliminary study, a new type of thermosensitive
material based on CS, HA, and Col was fabricated with
a biomimetic strategy. NHAC uniformly dispersed in the
chitosan matrix and this CS/nHAC composite showed some
features of natural bone. Thus, the CS/nHAC scaolds have
shown to possess promising physicochemical and biological
properties for use in bone tissue engineering approaches [23
26].
In the eld of tissue engineering, there is a growing
need for quantitative methods to analyze in situ and in real
time the tissue development in three-dimensional scaolds.
Ultrasound is a well-known noninvasive and nondestructive
imaging method [2730]. The goal of the current study was
to demonstrate the utility of ultrasound as a noninvasive,
nondestructive tool to evaluate the in vivo development of
the CS/nHAC scaold under subcutaneous implantation on
the back of rats for the period of 4 weeks.

2.1. Materials. Medical grade type 1 collagen powder was
purchased from YierKang Company (China), medical grade
chitosan (Mw 250000 and a degree of deacetylation of
95.6%) was provided by Shandong AKBiotech Ltd (China),
and -glycerophosphate (GP, C3 H7 Na2 O6 P) was from Sigma
(USA). The water used in the experiments was triply distilled.
2.2. Preparation of CS/nHAC and CS. The CS/nHAC composite was prepared by the procedure reported previously.
Firstly, nHAC powder was synthesized by self-assembly of
nanobrils of mineralized collagen and sterilized by ray irradiation (1.5 Mrad). Secondly, chitosan (2 g) was
dissolved in hydrochloric acid solution (98 mL, 0.1 M).
Thirdly, the nHAC powder was added to the chitosan
solution (0.02 g/mL) with the ratio of 1 : 1 in weight. Finally,
the pH of the CS/nHAC solution was adjusted to 7.0 by
adding droplets of -glycerophosphate solution (30% (w/v)).
The CS was prepared similar to the procedure of
CS/nHAC. Chitosan (2 g) was dissolved in hydrochloric acid
solution (98 mL, 0.1 M), and then the pH of the CS solution
was adjusted to 7.0 by adding droplets of -glycerophosphate
solution [30% (w/v)]. The samples for biological tests were
prepared in line with the aseptic technique in an aseptic
manipulation cabinet.
2.3. Implantation Experiments in Rats. All the animals were
operated according to the guidelines for animal experiments.
In this study, ten female Wistar rats (average body weight
300 g) were carried out under general gas anesthesia strictly
following a protocol approved by the Southern medical
University Institutional Animal Care and Use Committee.
Anesthesia consisted of 1% isourane delivered via facemask.
The solutions were prepared by gently mixing 1 mL of CS and
nHAC. A 1 mL syringe with a 26-gauge needle was used to
inject the solution into the subcutaneous dorsum of a rat.
2.4. Ultrasound Image. The implants were imaged using
an ALOKA prosound -10 premier diagnostic ultrasound
system with a 12 MHz transducer using the following
parameters: a dynamic range of 55, a mechanical index of 1.1,
a gain of 80, and a depth of 3 cm. Images were taken at 1 h,
14 days, and 28 days after injection. Anesthetized rats were
laid in the supine position, and an ultrasound transducer
was positioned on the surface of the back. Images were taken
always with the same settings (contrast: 50%, brightness
40%, and zoom: 100%) at dierent time point. At each time
point, ve images from the scaolds in each group were
stored. The images were transferred to an oine computer
(Q-lab, Philips Medical Systems) to measure the gray-scale
value (GV) intensity. A region of interest (ROI) was set to
encompass the entire scaold. Next, the ultrasound system
was set to color Doppler to detect the blood ow surround
the scaold.
2.5. Image Analysis. Ultrasound images are the visualization
of the backscattered signal that arises due to the dierence
in mechanical impedance between dierent materials and
phases. The backscattered signal is displayed as a gray-scale
array with values ranging from 0 to 256, with 0 indicating
a negligible dierence in impedance from the surrounding
media; the development of an ultrasound signal over time
was interpreted as an increase in stiness that may due
to the solidify development of the scaold. The gray-scale
value (GV) was analyzed by measuring the mean GV of
the implants over time by the method of histogram echo
intensity. The measurements from the ve images were
averaged together for each implant. Implant volume was
determined in vivo from the images of the total area of the
scaold. Since the images were binary, the number of implant
pixels could be determined by summation of the pixels. All
image analysis was performed using MATLAB.
2.6. Statistical Analysis. Statistical signicance of dierences
was determined using one-way analysis of variance ANOVA.
If dierences were detected, pairwise comparisons were made
using Turkeys HSD test at a condence interval of 95% (P <
0.05).
(b)
(a)
(a)
(b)
Figure 1: Ultrasound imaging of the injection process of the in situ formation of the CS and CS/nHAC gel: (a) subcutaneous injection of
the CS/nHAC solution under the ultrasound transducer. (b) The injection process was monitored by ultrasound (arrow: the needle of the
syringe).
3. Result and Discussion

Over the past decades, nano-biomaterials have played signicant roles in the eld of biomedical engineering and pharmaceutics. For successful clinical applications, the investigations
into biocompatibility and toxicity of nano-biomaterials are
research interests of great signicances. The use of diagnostic
ultrasound to study the in vivo compatibility of injectable
nano-biomaterials is a novel means in a noninvasive, nondestructive manner. The primary advantage of this technique
is the real-time visualization of the implant formation
process. Long-term information regarding formation and
degeneration behavior for the same implant over time can
be obtained. In the current study, the utility of monitoring
implant formation using ultrasound was demonstrated by
analyzing the formation and degeneration of injectable chitosan and chitosan/nano-hydroxyapatite/collagen scaold.
Figure 1 showed the feasibility of using the ultrasound to
manipulate the CS/nHAC system injecting into the Wistar
back as in situ forming scaold. The CS/nHAC solution
was injected into the subcutaneous dorsum of the Wistar
rat with a 1 mL syringe under the ultrasound transducer
(Figure 1(a)); the needle and the process of the injection
could be easily visualized by the ultrasound (Figure 1(b)).
3.1. Implant Formation. Ultrasound has been used to evaluate tissue properties of articular cartilage with the aim of
developing a noninvasive clinical method to assess articular cartilage degeneration and repair [2729]. Recently, it
was also used to evaluate the in vivo tissue engineering
structures. Kreitz et al. [30] found that the gray-scale value
of the ultrasound demonstrated a good correlation with
the hydroxyapatite content as marker of collagen formation
and with the histological ndings. Oe et al. [31] used the
ultrasound to evaluate the cell numbers in bone marrow
stromal cell/-tricalcium phosphate composites and found
ultrasound eectively responded to the quantity of BMSC/TCP composites and was well correlated to the actual
number of cells contained. Therefore, we chose to use a
Table 1: The grey-scale value of ultrasound image on experimental

group and control group after 0, 14, and 28 days of incubation. The
values are shown as mean standard deviation.
Group
CS
CS/nHAC
CS
CS/nHAC
CS
CS/nHAC
Time (day)
0
0
14
14
28
28
n
5
5
5
5
5
5
GV
52.7 13.0
51.4 11.5
68.6 6.5
81.1 4.6
74.9 7.5
99.8 33.2
P value
1.00
0.80
0.02
medical diagnostic ultrasound device with the advantage of

ensuring the absence of damage to the surrounding tissues.
Ultrasound images of the CS and CS/nHAC scaold taken
at dierent time points (days 0, 14, and 28) are shown in
Figure 2. The average GV was intended to be used as an
index to determine the degree of the hydroxyproline content
as marker of collagen formation and implant stiness, with
lower mean GVs corresponding to a more uid matrix
and larger mean GVs correlating to solid implant. The
ultrasound grey-scale signal increased with time, which
may demonstrate production of extracellular matrix. The
CS/nHAC had a mean GV of 51.7 11 after 1 h of injection;
the mean GV increased to 81.14.6 after 14 days and attained
the highest mean GV of 107.8 22 after 28 days. Signicant
dierences were observed between the GVs on day 1, 14, and
28 in CS/nHAC group. In contrast, the CS scaold had a
mean GV of 52.7 13 after 1 h of injection; the mean GV
increased to 68.5 6.4 after 14 days and had a maximum
GV of 74.8 7.4 after 28 days. But no signicant dierences
were observed between the GVs on day 1, 14, and 28 in CS
group. On day 28, the mean GVs of the CS/nHAC group were
signicantly higher than the CS group (P < 0.05) (Figure 3,
Table 1). Using this measurement, it was observed that the
CS/nHAC scaold attained the highest gray-scale value at day
28, indicating the CS/nHAC scaold had the highest implant
stiness, which maybe related to the early osteogenesis.
(b)
(a)
(a)
(b)
(d)
(c)
(c)
(d)
(e)
(e)
(f)
(f)
Figure 2: The ultrasound images of the scaold taken on day 0 (a) and (d), day 14 (b) and (e), and day 28 (c) and (f). (CS scaolds: (a), (b),
(c); CS/nHAC scaolds: (d), (e), (f); S: scaold; T: surrounding tissues, cross-section area: the blue and yellow dot cross in the scaold).
3.2. Implant Volume. The volumetric changes of the

CS/nHAC and CS scaold were assessed after 1 h, 14
days, and 28 days, respectively. The use of percutaneous
ultrasound would allow volume monitoring over time after
administration of CS and CS/nHAC gel. This procedure
of calculation has been used in radiology ultrasound for
more than 20 years to measure breast cysts, renal cysts,
and more. Volume calculation is reported in the literature
that the detection volume may have 10 percent dierences
in two-dimensional ultrasound, which is at least as good
as that obtained by three-dimensional calculation [3234].
Implant degeneration was measured as a change in crosssectional area over time in both groups. The CS scaold
exhibited decreasing in size more than 80% over the 28 days.

However, the CS/nHAC scaold showed a slight decrease
and remained 62% for the duration of 28 days (Table 2,
Figure 4). Signicant bigger cross-section area was observed
in the CS/nHAC scaold on day 14 (Figures 2(b) and 2(e),
P < 0.05) and day 28 (Figures 2(c) and 2(f) P < 0.01). The
results demonstrated a stable material degeneration rate of
the CS/nHAC scaold, which is suitable to be used as a bone
substitute. However, the CS scaold seemed to degenerate
too fast in the rst 4 weeks to be a good bone substitute.
3.3. Implant Blood Supply. The successful clinical outcome
of the implanted tissue-engineered bone is dependent on
40000
125
Cross sectional area (pixel)
Grey-scale value
100
75
50
25
30000
20000
10000
0
0
14
28
0
Time after scaold injection (day)
CS
CS/nHAC
Figure 3: The grey-scale value of ultrasound image on experimental

group and control group after 0, 14, and 28 days of incubation. The
values are shown as mean standard deviation (n = 5, P < 0.05).
Table 2: The cross-section area of ultrasound image on experimental group and control group after 0, 14, and 28 days of incubation.
The values are shown as mean standard deviation.
Group
Time (day)
CS
0
CS/nHAC
0
CS
14
CS/nHAC
14
CS
28
CS/nHAC
28
14
28
Time after implant formation (day)
n
5
5
5
5
5
5
Pixel
28982.8 6782.9
35662.0 3321.5
17084.8 256.3
35662.0 6341.7
5169.2 613.4
22050.2 3449.4
P value
CS
CS/nHAC
Figure 4: The cross-section area of ultrasound image on experimental group and control group after 0, 14, and 28 days of
incubation. The values are shown as mean standard deviation
(n = 5, P < 0.01, P < 0.05).
way to obtain hemodynamic measurements with little risk to

the experimental subject.
0.16
0.04
0.00
the establishment of a functional vascular network. So it

is very important to evaluate the vascularization of the
implant in vivo. The blood ow surrounding the implant
was shown in Figure 5. In the CS scaold group, no blood
ow was detected by the color Doppler imaging during 28
days after implantation. However sporadic blood ow was
found around the CS/nHAC scaold on day 14 and 28
after implantation (Figure 5) which maybe related to the
material vascularization. Although no blood ow was found
inside the scaold during the 28-day observation, the blood
ow surrounding the materials indicates that there were
new vessels surrounding the scaold and new capillary may
penetrate into the implant and form a network to promote
osteogenesis. The use of ultrasound imaging is of growing
interest as new technologies become available for in vivo
analysis of blood ow. Despite some limitations including
a lower degree of sensitivity to changes in tissue perfusion
and the inability to assess rapid or transient changes in tissue
perfusion, we found that the ultrasound method can be
used successfully to examine regional blood perfusion of the
biomaterials in vivo [35]. As this method does not involve
a surgical procedure, ultrasound oers a rapid, noninvasive
4. Conclusions
This study has demonstrated that it is possible to noninvasively and nondestructively evaluate the volume, stiness,
and blood supply of the injectable bone scaold using
a diagnostic ultrasound, providing a new means for the
biocompatibility study of in situ forming implants in in
vivo applications. The primary advantage of this technique
is the real-time visualization of the implant formation
process. Through the use of quantitative image analysis
techniques, long-term information regarding formation and
degeneration behavior for the same implant over time can
be obtained. In comparison to the CS, the CS/nHAC scaold
showed a greater stiness, less degeneration rate, and better
blood supply in the in vivo biocompatibility evaluation. The
results shown in this paper support the proof of principle of
using ultrasound for the evaluation of tissue development in
hydrogel-based tissue-engineered structures.
Y. Chen and S. Li contributed equally to this paper.
Acknowledgments
The authors are grateful for the nancial support from
National Natural Science Foundation of China (no.
81101348 and no. 31000431), Natural Science Foundation
(b)
(a)
(a)
(b)
Figure 5: The blood supply to the CS/nHAC scaold after 14 (a) and 28 days (b).
of Guangdong Province, China (10451051501004727),

Guangdong Province Science and Technology project,
China (ultrasound evaluation of injectable nanobiomaterials), Doctoral Fund of Ministry of Education,
China (20114433120004), the Beijing Nova Program (no.
2010B011), and State Key Laboratory of New Ceramic and
Fine Processing Tsinghua University, outstanding person
fund of Zhujiang Hospital.
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doi:10.1155/2012/384671
Research Article
Biocompatible Single-Crystal Selenium Nanobelt Based
Nanodevice as a Temperature-Tunable Photosensor
Yongshan Niu,1 Aimiao Qin,2 Wei Song,1 Menghang Wang,1
Xuenan Gu,1 Yangfei Zhang,3 Min Yu,3 Xiaoguang Zhao,4 Ming Dai,1
Ling Yan,1 Zhou Li,1 and Yubo Fan1
1 Key
Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science
and Medical Engineering, Beihang University, Beijing 100191, China
2 Key Lab of New Processing Technology for Nonferrous Metals & Materials Ministry of Education,
School of Materials Science & Engineering, Guilin University of Technology, Guilin 541004, China
3 College of Engineering, Peking Unversity, Beijing 100871, China
4 Mechanical and Manufacturing Engineering, University of New South Wales, Sydney, NSW 2052, Australia
Correspondence should be addressed to Zhou Li, lizhou@buaa.edu.cn and Yubo Fan, yubofan@buaa.edu.cn
Received 1 March 2012; Accepted 19 March 2012
Copyright 2012 Yongshan Niu et al. This is an open access article distributed under the Creative Commons Attribution License,
Selenium materials are widely used in photoelectrical devices, owing to their unique semiconductive properties. Single-crystal
selenium nanobelts with large specic surface area, ne photoconductivity, and biocompatibility provide potential applications
in biomedical nanodevices, such as implantable articial retina and rapid photon detector/stimulator for optogenetics. Here, we
present a selenium nanobelt based nanodevice, which is fabricated with single Se nanobelt. This device shows a rapid photo
response, dierent sensitivities to visible light of variable wave length, and temperature-tunable property. The biocompatibility of
the Se nanobelts was proved by MTT test using two cell lines. Our investigation introduced a photosensor that will be important for
multiple potential applications in human visual system, photocells in energy or MEMS, and temperature-tunable photoelectrical
device for optogenetics research.
1. Introduction
One-dimensional (1D) nanostructures such as nanowires [1,
2], nanobelts [3], and nanotubes [4] showed their fascinating
features and characteristic during the past few decades.
Intensive researches focused on their applications in microcosmic physics and nanodevices [58]. Selenium material is
a crucial semiconductor, widely used in solar cell, photoelectrical element, xerography, and pressure sensors, owing to its
unique photoconductivity, thermoelectric, and piezoelectric
properties [916]. Previously, most research about the selenium mainly focused on bulk or amorphous materials and
hexagonal metallic selenium lm.
Recently, the 1D selenium nanostructures, such as singlecrystalline nanowires [16, 17] and nanobelts [18], become a
new research focus. Numerous works are about synthesis of
1D selenium nanostructures. However, the study about their

properties and applications in photoelectrical nanodevices
is limited. Meanwhile, the biocompatibility and biosafety of
nanostructure is also a serious public concern.
A new single-crystalline selenium nanobelt based photosensor is presented here. This single nanobelt device
exhibits ultrafast photon response and temperature-tunable
properties. We used a cushy and large-scale synthesis method
to prepare the single-crystalline nanobelts and investigated
their cellular level biocompatibility to two dierent cell lines.
This photosensor also presented a variable photoconductivity responding to visible light of dierent wavelengths. Those
novel properties endue the nanodevice multiple potential
applications, such as implantable visual nanodevice as articial retina, rapid photon sensors, photocells, or temperaturetunable photoelectrical element.
Selenium nanobelt
Electric measure system

Light illumination
Quartz glass slide
Silver paste
Silver paste
(a)
(b)
2 m
(c)
Figure 1: Fabrication of a single selenium nanobelt nanodevice. (a) Transfer a single selenium nanobelt to a cleaned quartz glass slide. (b)
Fix both ends of nanobelt by silver paste through suitable masks and connect with electric measure system by enameled copper wire. (c) The
SEM image of fabricated nanodevice. Two ends are silver pasted, and the bright line is a Se nanobelt.
2. Experiment
2.1. Synthesis. Single-crystalline t-selenium nanobelts were
synthesized by solventothermal method. A typical experimental procedure is described as following: 1.3 mmol (0.1 g)
Se powder and 17 mL ethanol were loaded into teonlined autoclave of 25 mL capacity. This autoclave was sealed
and maintained at 200 C for 24 h. Then the autoclave was
taken out and cooled in room temperature. Precipitates were
ltered out, rinsed with absolute alcohol for several times,
and dried naturally. All of chemicals used for the synthesis
were of analytical grade and used as received without further
purication.
2.2. Characterization and Fabrication. The synthesized selenium nanobelts were characterized and analyzed by X-ray
diraction (XRD, Alpha-1) using CuKa radiation, SEM (FESEM, Leon 1530 with EDS), and TEM (TEM, HF-2000).
A single selenium nanobelt was transferred to a cleaned
quartz glass slide to fabricate a nanodevice. Both ends of
the Se nanobelt were xed by silver paste and connected
with copper wire (Figure 1). The electronic transportation
property was conducted on the single nanobelt device by an
electric measure system. Besides, we used uorescent lamp,
ultraviolet lamp, and photoluminescence measure instrument to investigate the photoconductivity of the device.
The electric property was measured in constant temperature
environment. All measurement was carried out and recorded
by same electric measure system.
2.3. Biocompatibility Test. For our study, two cell lines were
utilized [19, 20]. One was HeLa cell (American Type Culture
Collection, ATCC, CCL-2) and the other one was L-929
cell (ATCC, CCL-1). Two kind of cells were cultured in
Dulbeccos modied Eagles medium (DMEM, ATCC) supplemented with 10% fetal bovine serum (FBS, ATCC) and
1% penicillin/streptomycin (ATCC). Cell suspension was

added to 25 cm2 vials and put into an incubator (37 C, 5%
CO2 ).
After being incubated for 24 h, cells were trypsinized with
0.05% trypsin solution and rushed down from the bottom
of 25 cm2 vials when they were in a semiconuent state and
still in log phase of growth. Ten microliter cell suspension
was mixed with 10 L trypan blue, and the total number of
harvest cells was counted under an optical microscope. The
density of the cell was calculated and cellular viability was
greater than 90%. The cell suspension was added to 96-well
plates with 5000 cells per well (90 L). 96-well plates were
incubated in 37 C, 5% CO2 for 12 h.
Se nanobelts were suspended in PBS (10 L) for four
dierent concentrations. These suspensions were added into
96-well plates until the mass concentration of 0.1, 1, 10,
and 100 g/mL, respectively. Some wells were preserved
without adding nanobelts for comparison purpose. After
dispersing nanobelts by micropipettes, the 96-well plates
were incubated in 37 C, 5% CO2 for 12, 24, and 48 h,
respectively.
The biocompatibility of the Se nanobelt was investigated
through an in vitro cell viability test. We measured the activity of mitochondrial enzyme succinate dehydrogenase (SDH)
[21] in two dierent cell lines, after adding Se nanobelts
and incubating for 12, 24, and 48 h.
The Se nanobelts were not removed from each well
and an MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl
tetrazolium bromide)-succinate solution (20 L, 0.5 mg/mL
MTT) was added, and the plates were incubated again at
37 C for 4 h. Then all solution in the 96-well plates was
removed. The MTT-formazan formed by the mitochondrial
enzyme SDH was preserved at the bottom of the well and
dissolved in added dimethyl sulfoxide (DMSO). The 96-well
plates were shaken softly for 5 minutes to dissolve formazan
absolutely. Then the plates were put in a microplate reader
(c)
(b)
101
(a)
10000
Se
8000
Cu
15.33
201
003
112
103
202
210
211
102
111
100
4000
2000
Se
6000
200
O
C
Cu
(d)
110
Cu
Cu
Intensity (a.u.)
Se
Se
0
10
keV
(f)
20
30
40
50
2 (degree)
(g)
60
70
(e)
Figure 2: SEM, HRTEM, EDS, and XRD characterization of selenium nanobelts. ((a), (b)) SEM images of single-crystalline Se nanobelts in
dierent magnications. ((c)(e)) HRTEM images taken from the middle of the Se nanobelt show that the corresponding electron diraction
pattern and its growth direction is [001]. (f) EDS indicates the Se nanobelts with pure composition. (g) XRD pattern conrms that the Se
nanobelts are trigonal structure.
(EL808 IU-PC, BioTek Instruments, Inc.), recording the

photon absorbance of each well at 630 nm wavelength for
investigating the viability of cells.

As shown in the SEM images of dierent magnications, the
size range of the t-Se nanobelts was from 100 to 500 nm in
width and tens to hundreds of micrometers in length. The
ratio of length to diameter was 102 104 (Figures 2(a) and
2(b)). We did chemical element analysis using EDS to test
the purity of Se nanobelt (Figure 2(f)). The XRD showed
the structure of Se nanobelt is trigonal (Figure 2(g)). And
HRTEM study further conrmed that the t-Se nanobelts
had single-crystal structure with growth direction [001]
(Figures 2(c), 2(d), and 2(e)).
In MTT test, we found that single-crystal Se nanobelts
are biocompatible and biosafe to the two cell lines (Figure 3).
Our investigation was designed by time sequence and concentration. The incubation time was 12, 24, and 48 h, and the
concentration was 103 , 102 , 0.1, and 1 mg/mL. The viability of HeLa and L929 cells showed no dierence comparing
with control after incubating with nanobelts. More than 85%

of the HeLa cells and >80% L929 cells were viable after
incubation, and there was no signicant statistic dierence in
viability among the plates of time sequence (tested by SPSS,
paired-sample t-test). Meanwhile, dierent concentrations of
Se nanobelts showed no signicant eect to the viability of
HeLa and L929 cells too (Figure 3).
A single-crystal selenium photosensor device was fabricated (Figure 1). Two ends of the Se nanobelt were xed
by silver paste. The electrical measurement system was connected with the device through enameled copper wires. The
photoconductivity measurement was rst performed in
room temperature and room light (uorescent lamp). The
response time of the Se nanobelt to visible light is calculated.
With the light turned on and o, the current changed immediately. From the I-t curve, it is observed that the current
transported through a single nanobelt drastically increases by
a factor of 10 when the light was turned on. As measured at
half maximum from I-t curve, the increasing and the decay
response time was about 30 and 50 ms, respectively. This
nanobelt based nanodevice presented a high sensitivity and
rapid response to light at room temperature.
1.2
1.2
1
L929 cell viability
HeLa cell viability
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
0
Control
1 mg/mL 0.1 mg/mL 0.01 mg/mL 0.001 mg/mL

Se NWs concentration
12 hours
24 hours
48 hours
Control
1 mg/mL 0.1 mg/mL 0.01 mg/mL 0.001 mg/mL

Se NWs concentration
12 hours
24 hours
48 hours
(a)
(b)
Figure 3: The cell viability tested by MTT method as a function of Se nanobelt concentration and time. (a) Cell viability of HeLa cells in
MTT test, cultured with dierent concentrations of Se nanobelts for 12, 24, and 48 h. (b) Viability of L929 cells in MTT test, cultured with
dierent concentrations of Se nanobelts for 12, 24, and 48 h.
This nanobelt based photosensor also exhibited a temperature-tunable property (Figure 4(a)). In this experiment,
we changed environmental temperature from 80 C to
80 C. The voltage was applied to 1 V. The current change in
volume reduced with the temperature rise. In semiconductive material, the conductivity is mainly determined by the
recombination and trapping of the electron-hole pairs. Generally, when temperature rises, the rate of recombination and
trapping increases, displayed as the increasing of conductivity in semiconductor. This trigonal selenium was accepted as
a p-type extrinsic semiconductor, and conduction occurred
due to valence band hole transportation [22]. The increased
thermal conductivity was likely due to the thermal excitation
of the electron-hole pairs in the valence band as governed
by exp(Eg /KT), where Eg = 1.6 eV was the bandgap of
selenium, K is Boltzmann constant, and T is temperature.
The rate of recombination and trapping in this selenium
nanobelt was shown strongly depending on light and
temperature [22, 23]. The photosensitivity, dened as S =
(I Io )/Io where I and Io were the currents measured when
the light was on and o, respectively, was strongly aected
by temperature. S decreased as temperature increased. In our
experiment, the best photosensitivity was received at temperatures lower than 40 C. The carriers in Se nanobelt were
contributed by photon excitation and thermal excitation.
With the temperature increasing, the thermal excitation was
enhanced and the photosensitivity was relatively reduced.
This property of the nanodevice was a temperature-tunable
ability, which was likely to be important for the applications
in human visual system, implantable light-adjusted medical
devices, and the new scientic eld of optogenetics [2426].
In order to investigate the photon sensitivity of the Se
nanobelt to the light of dierent wavelengths, the photocurrent was measured under a chopped light (75 Watt xenon
lamp) to give monochromatic light. The wave length was

set from UV to infrared light (300 to 800 nm). I-t curve
showed a signicant result as the photosensors response
(Figure 4(b)). The higher response was observed in the
visible wave length, which was in agreement with the
previous report [12]. This was attributed to the particular
photo character of Se nanobelts with some other semiconductors, such as ZnO nanobelts [27, 28]. The maximum
photo response of our single-crystal t-Se nanobelt was at
650 nm in room temperature, which was a little dierent
with previous report: 650 nm at 190 C and 750 nm at
20 C [28]. The sensitive response to visible light and
temperature-tunable ability was superiority for this Se
nanobelt based photosensor applied in implantable biomedical device, such as articial retina and the optogenetics
detector/stimulator.
4. Conclusion
In summary, we synthesized and characterized the singlecrystal selenium nanobelts. We also investigated their biocompatibility to dierent cells. The viability of cells incubated with Se nanobelts showed no dierence with the
control. More than 80% cells were viable indicating the Se
nanobelts behaving a good biocompatibility and biosafety.
A photoelectrical nanodevice was fabricated here, and it
showed a signicant result to visual light of dierent wavelengths. Moreover, this photosensor exhibited a temperaturetunable ability. As a conclusion, this biocompatible singlecrystal Se nanobelt based temperature-tunable photosensor
seems to be important for multiple potential applications,
such as articial retina for human visual system, photocells in
energy or MEMS, and temperature-tunable photoelectrical
device for optogenetics research.
0.9
Light off
0.4
0.3
0.2
0.1
(s)
re
eratu
Temp
0.4
40
0.2
60
(s)
e
Tim
0.6
e
Tim
20
40
60
80
100
120
0.8
Relative in
tensity
0.5
(10 6 A)
0.6
Relative intensity
0.7
Light on
(10 11 A)
1.2
0.8
80
h
lengt
Wave
100
140
80 C
60 C
40 C
20 C
0 C
(a)
300 nm
350 nm
400 nm
600 nm
650 nm
700 nm
450 nm
500 nm
550 nm
750 nm
800 nm
(b)
Figure 4: The I-t curve of Se nanobelt based photosensor. (a) The temperature-tunable property of our photosensor. Relative current
intensity denes as (I I0 ). Temperature changes from 0 C to 80 C. (b) Our photosensor presents sensitive response to the light of dierent
wavelengths, from 300 nm to 800 nm.
Acknowledgments
This work was supported under the Scientic Research
Foundation for the Returned Overseas Chinese Scholars
(20091341) and the Doctoral Fund of the Ministry of
Education of China (20111102120038), the National Natural Science Foundation of China (no. 10925208) and
NSF of Guangxi Province (2010GXNSFC013007), and
National Basic Research Program of China (973 Program,
2011CB710901). Z. Li also thanks the support from the
Excellent Scientist Program of the Beihang University.
Yongshan Niu and Aimiao Qin are equally contributed to this
paper.
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doi:10.1155/2012/401084
Research Article
In Vitro Biocompatibility and Osteoblast Differentiation of
an Injectable Chitosan/Nano-Hydroxyapatite/Collagen Scaffold
Yan Chen,1 Zhi Huang,2 Xiaoming Li,3 Songjian Li,4 Zhilai Zhou,4 Yichen Zhang,4
Qing ling Feng,5 and Bo Yu4
1 Department
of Ultrasonic Diagnosis, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China
of Geosciences and Info-Physics, Central South University, Changsha 410083, China
4 Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China
5 State Key Laboratory of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084, China
2 School
Correspondence should be addressed to Xiaoming Li, x.m.li@hotmail.com and Bo Yu, gzyubo@gmail.com

Received 28 February 2012; Accepted 3 April 2012
Copyright 2012 Yan Chen et al. This is an open access article distributed under the Creative Commons Attribution License,
The purpose of this study was to evaluate the in vitro cell biocompatibility of an in situ forming composite consisting of chitosan
(CS), nano-hydroxyapatite and collagen (nHAC), which has a complex hierarchical structure similar to natural bone. MC3T3E1 mouse calvarial preosteoblasts were cultured on the surface of the injectable CS/nHAC and CS scaold. The proliferations
of seeded MC3T3-E1 were investigated for 10 days. Cytotoxicity, cell proliferation, and cell expression of osteogenic markers
such as alkaline phosphatase (ALP), type 1 collagen (COL-1), RUNX-2, and osteocalcin (OCN) were examined by biochemical
assay and reverse transcription polymerase chain reaction. Cell viability and total cellularity (measured by dsDNA) were similar
between the two scaold groups. However, ALP, COL-1, OCN, and RUNX-2 production were signicantly greater when osteoblasts
were cultured on CS/nHAC scaolds. The increase in osteogenic markers production on CS/nHAC scaolds indicated that these
scaolds were superior to chitosan-only scaolds in facilitating osteoblast mineralization. These results demonstrate the potential
of the CS/nHAC scaolds to be used in bone tissue engineering.
1. Introduction
Annually, more than 2.2 million bone grafting procedures
(autologous bone graft and banked bone) are performed
worldwide to ensure adequate bone healing in many skeletal
problems, such as nonunion fractures, cervical and lumbar
spine fusion, joint arthrodesis, and revision arthroplasty
[1]. Tissue engineering oers a strategy to circumvent
those problems. The concept involves the use of a porous
and biodegradable scaold, allowing cells to adhere and
proliferate, creating conditions for the formation of ECMlike structures [24]. Previous studies have shown that
natural-based polymers such as chitosan [510] have great
potential for bone tissue engineering applications. The main
advantages of these materials include low immunogenic
potential, bioactive behavior, good interaction with host
tissues, chemical versatility, and high availability in nature.

Chitosan is a promising material for bone regeneration
because it is biocompatible and biodegradable with a degradation rate that is dependent on factors such as degree of
deacetylation (DDA) and crystallinity, and it can be easily
formed into beads, bers, or more complex structures [5, 11,
12].
Bone tissues are mainly constructed from nanosized
hydroxyapatite (HA) minerals and type 1 collagen (Col1) matrix with complex hierarchically assembled structures [1316]. The ideal scaolds for bone regeneration
should promote early mineralization and support new bone
formation [1719]. Mineralized collagen brils (nHAC)
composites seem to be very promising biomaterials for bone
regeneration [2023]. In the previouse study, the feasibility
of developing a thermosensitive and injectable chitosan
2
solution in the presence of nHAC was demonstrated [23
25]. Combining nHAC and chitosan has the potential to
maximize the benecial properties of each and creates an
injectable scaold with properties similar to physiological
bone which would undoubtedly aid in the formation of new
bone at the tissue/biomaterial interface [2427].
In the present study, the cytocompatibility of the
injectable CS/nHAC scaolds to act as a bone substitude and
its potential for bone tissue engineering were investigated
in vitro. The ability of the injectable CS/nHAC scaolds to
maintain the viability and functionality of seeded MC3T3-E1
preosteoblasts was investigated in terms of their proliferation
and osteoblastic dierentiation over 10 days of culture.

2.1. Scaold Processing. The CS/nHAC composite was prepared by the procedure reported previously [2427]. nHAC
powder was synthesized by self-assembly of nanobrils of
mineralized collagen and sterilized by -ray irradiation (1.5
Mrad). Chitosan (2 g) was dissolved in hydrochloric acid
solution (98 mL, 0.1 M). The nHAC powder was added to
the chitosan solution (0.02 g/mL). Finally, the pH of the
CS/nHAC solution was adjusted to 7.0 by adding droplets
of -glycerophosphate solution (30% (w/v)). The samples
for biological tests were prepared in line with the aseptic
technique in an aseptic manipulation cabinet. Solutions were
injected into a circular mold (12 mm diameter and 2 mm
thickness) and solidied in an incubator at 37 C for 10 min
to form a hydrogel scaold.
The CS was prepared similar to the procedure of
CS/nHAC. Chitosan (2 g) was dissolved in hydrochloric acid
solution (98 mL, 0.1 M), and then the pH of the CS solution
was adjusted to 7.0 by adding droplets of -glycerophosphate
solution (30% (w/v)). The samples for biological tests were
prepared in line with the aseptic technique in an aseptic
manipulation cabinet.
2.2. Cell Culture Studies. MC3T3-E1 murine calvarial
osteoblasts (subclone 14) cells were purchased from the
cell bank of the Chinese Academy of Sciences (Shanghai,
China). The cells were grown in a culture medium consisting
of -MEM (Sigma, St. Louis, MO), 10% fetal bovine
serum (Biochrom AG, Germany), and 1% of antibioticantimycotic mixture (Sigma, St. Louis, MO) at 37.8 C in
a humidied atmosphere of 5% CO2 . When an adequate
cell number was obtained, cells at passage 2 were detached
with trypsin/EDTA. Cells were seeded at a density of 2
104 cells/scaold under static conditions, by means of a cell
suspension. The culture medium was changed every 2-3 days
until the end of the experiment.
2.3. In Vitro Cytotoxicity Tests. A Cell Count Kit-8 (Cell
Count Kit-8, Beyotime, China) was employed in this
experiment to quantitatively evaluate the cytotoxicity of the
scaold. Cell viability was assessed after MC3T3-E1 were
inoculated on the samples 24-well disks using the CCK-8
kit after 1, 3, 7 and 10 days. CCK-8 reagent (100 L) was
added to MC3T3 in 1 mL medium per well. The plates
were incubated at 37 C for 4 h and shaken for 1 min. After
this, 200 L (n = 5) were transferred to 96-well plates and
the optical density (OD) was measured on a microplate
reader (BioTek, USA) using an absorbance of 490 nm. All the
procedures were replicated 3 times.
2.4. Cell Proliferation by DNA Quantication. MC3T3 proliferation on the CS/nHAC scaolds was determined using
a uorimetric dsDNA quantication kit (PicoGreen, Molecular Probes, Invitrogen, USA). Samples collected at days 1,
3, 7, and 10 were washed twice with a sterile phosphatebuered saline solution and transferred into 1.5 mL microtubes containing 1 mL of ultrapure water. Cell constructs
were cryopreserved at 80 C for further analysis. Prior to
DNA quantication, samples were thawed and sonicated
for 15 min. Standards were prepared with concentrations
ranging between 0 and 2 mg/mL. Per each well of an opaque
96-well plate were added 28.7 L of sample (n = 3) or
standard, 71.3 L of PicoGreen solution, and 100 L of TrisEDTA buer. The plate was incubated for 10 min in the
dark and uorescence was measured using an excitation
wavelength of 260 nm and an emission wavelength of
280 nm. All the procedures were replicated 3 times.
2.5. Alkaline Phosphatase Quantication. After culturing
for 1, 3, 7, and 10 days, the cells were washed and
lysed in 0.1 vol% Triton X-100. The alkaline phosphatase
(ALP) activity in the lysis was determined through a
p-nitrophenyl phosphate (pNPP) liquid substrate system
(Nanjing Jiancheng, China). Five milliliters of each cell
lysate solution was added to 195 L of pNPP substrate and
incubated in the dark at room temperature for 1 min. The
absorbance was read using a plate reader (Molecular Devices,
USA) at 405 nm. The intracellular total protein content was
determined using a Micro BCA Protein Assay Kit (Nanjing
Kaiji, China) and the ALP activity was normalized to it.
2.6. Osteogenic Dierentiation by Reverse Transcriptase PCR.
The expression levels of osteogenesis-related genes were
measured using the quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). The cells were seeded
at a density of 2 104 cells/well, cultured for 1, 3, 7
and 10 days, and then harvested using TRIzol (Gibco) to
extract RNA. An equivalent amount of RNA from each
sample was reverse-transcribed into complementary DNA
(cDNA) using the Superscript II rst-strand cDNA synthesis
kit (Invitrogen). The qRT-PCR analysis of genes including
type 1 collagen (COL-1), osteocalcin (OCN), Runt-related
transcription factor 2 (RUNX-2), and 18S ribosomal RNA
(18 s rRNA) was performed on the Applied Biosystems 7500
using the Quantitect Sybr Green Kit (Qiagen). The primers
for the target genes were listed in Table 1. The Ct values of
target genes were normalized by the Ct values of the TaqMan
Table 1: Primer sequences and product size for real-time PCR

reactions.
OCN
RUNX-2
COLL
18 s rRNA
Sense and anti-sense sequences

5 AGGGCAATAAGGTAGTGAA
5 CGTAGATGCGTCTGTAGGC
5 TGCCCAGTGAGTAACAGAAAGAC
5 CTCCTCCCTTCTCAACCTCTAA
5 CTTCACCTACAGCACCCTTGT
5 AAGGGAGCCACATCGATGAT
5 CCTGGATACCGCAGCTAGGA
5 GCGGCGCAATACGAATGCCCC
bp
0.4
159
123
120
112
O.D. value (490 nm)
Target
0.5
0.3
0.2
0.1
human housekeeping gene 18 s rRNA to obtain the DCt

values. These values were then subtracted by the Ct value
of the cells cultured on the blank disks to obtain the DDCt
values. The fold of change was obtained with n = 3.
2.7. Statistical Analysis. Statistical signicance of dierences
was determined using one-way and two-way analysis of
variance ANOVA. If dierences were detected, pairwise comparisons were made using Tukeys HSD test at a condence
interval of 95% (P < 0.05).
0
1
3.2. Cell Proliferation by DNA Quantication. Over the 10

days of the study, the number of cells, as measured by
dsDNA, was essentially equivalent between both CS and
CS/nHAC scaold groups (Figure 2). Overall, there was not
a signicant amount of cell proliferation detected over the
course of the study. In vitro cell testing of biomaterials is a
well-established method to determine the cytocompatibility
of the materials [2729] by using either cell lines or primary
cells. This study was designed primarily to analyze the ability
of the composite scaold to support osteoblast matrix production, and maintaining similar cell numbers in each group
allowed dierences in these parameters to be studied without
the confounding eects of cell number dierences. The DNA
concentration result of the two groups showed a similar trend
to the CCK-8 result. These results demonstrated that both
the CS and CS/nHAC scaold had good cytocompatibility
and MC3T3-E1 viability, so it highlights the application of
CS/nHAC to be a biomaterial for bone tissue engineering.
10
Time (days)
CS
CS/nHAC
Figure 1: CCK-8 assays of MC3T3-E1 proliferation on experimental group and control group after 1, 3, 7, and 10 days of incubation.
The values are shown as mean standard deviation (n = 5).
3. Result and Discussion
2.5
2
DNA concentration (ug/ul)
3.1. In Vitro Cytotoxicity Tests. The viable cell density

increases over initial days up to a plateau based on the CCK-8
assay (Figure 1). The experimental group and control group
share similar trend of viable cell density over 10 days. the
CS/nHAC group reach a maximum value at day 10, and the
CS group reach a maximum value at day 7; no signicant
growth dierence was observed between the two groups after
10 days of incubation (n = 5, P > 0.05). The results of the
CCK-8 assay reect an overall activity which is aected by
both cell proliferation and cell viability. The cell proliferation
assay showed that cells grew normally with the scaolds and
these scaolds were nontoxic for MC3T3-E1.
1.5
0.5
0
1
10
Time (days)
CS
CS/nHAC
Figure 2: DNA concentration of MC3T3-E1 proliferation on

experimental group and control group after 1, 3, 7, and 10 days
of incubation. The values are shown as mean standard deviation
(n = 5).
3.3. Alkaline Phosphatase Quantication. ALP activity and

calcium mineralization are mostly used as markers for
early and late dierentiation of osteoblast cells, respectively
[21, 22]. Osteoblastic cell dierentiation was assessed by
measuring ALP activity normalized to total protein content
after 1, 3, 7, and 10 days of culture. Figure 3 shows the
400
ALP activity (U/g)
300
200
100
0
1
10
Time (days)
CS
CS/nHAC
Figure 3: ALP assays of MC3T3-E1 proliferation on experimental

group and control group after 1, 3, 7, and 10 days of incubation. The
values are shown as mean standard deviation (n = 3). Bars with
dissimilar letters indicate signicantly dierent values ( P < 0.01).
evolution of ALP activity for MC3T3-E1 cells cultured on

the CS/nHAC surfaces in comparison with CS. The ALP
activity of MC3T3-E1 cells grown on both groups was not
signicantly dierent for day 1 and 3. However, MC3T3-E1
cells cultured on CS/nHAC scaold had signicantly higher
ALP activity than those cultured on CS group on day 7 and
10 (P < 0.001). The ALP activity of CS/nHAC group reaching
a top on day 7 was observed. The following decrease on day
10 is likely due to reaching an advanced cell culture stage.
ALP activity is mostly used as markers for early and late
dierentiation of osteoblast cells, respectively [30, 31]. These
results indicate that the CS/nHAC scaold can stimulate
matrix formation and enhance osteoblast cell dierentiation.
3.4. Osteogenic Dierentiation by Reverse Transcriptase PCR.
A semiquantitative RT-PCR method was used to assess gene
expression of osteoblasts after 1, 3, 7, and 10 days. In
order to know whether primary osteoblast dierentiation
was aected by nano-hydroxyapatite/collegen, we selected
COL-1, OCN, and RUNX-2 as markers for osteoblastic
dierentiation in this study. Among bone matrices, COL-1 is
the most abundant protein synthesized by active osteoblasts
and conductive to mineral deposition. Osteocalcin is secreted
solely by osteoblasts and thought to play a role in the bodys
metabolic regulation and is proosteoblastic or bone-building
[28]. It is also involved in bone mineralization and calcium
ion homeostasis. RUNX-2 gene is a member of the RUNX
family of transcription factors and encodes a nuclear protein
with a Runt DNA-binding domain. The protein can bind
DNA both as a monomer and, with more anity, as a subunit
of a heterodimeric complex. This protein has been implicated

as a key transcription factor associated with osteoblast
dierentiation [29]. The results of real-time PCR for mRNA
expression of osteocalcin and RUNX-2, both of which
are osteoblast markers, are represented as fold increases.
As shown in Figures 4(a) and 4(b), there are signicant
dierences in the osteocalcin expression between CS/nHAC
and CS at day 1, 7, and 10 (P < 0.001) (Figure 4(a)). The
osteocalcin expression was 5.5-fold higher on day 7 and 2.7fold higher on day 10 for the CS/nHAC as opposed to the
CS. Also, the RUNX-2 expression was signicantly higher by
day 7 and 10 for the CS/nHAC as opposed to the CS group.
The COL-1 expression was signicantly lower by day 1 and
3 for the CS/nHAC as opposed to the CS group. However,
signicant higher COL-1 expression of CS/nHAC than CS
was observed at day 7 and 10 (P < 0.01).
Due to the current results in vitro, better osteoblasts
viability, and dierentiation marked by higher OCN, RUNX2 and COL-1 expression were observed on CS/nHAC
surfaces. The addition of nHAC to the chitosan did lead
to signicantly enhanced osteoblasts dierentiation. There
are several factors that could contribute to the enhanced
mineralized matrix production seen when osteoblasts were
cultured on CS/nHAC scaolds. When calcium phosphatecontaining materials are hydrated in cell culture media, a
continuous process of calcium and phosphate ion dissolution
and reprecipitation occurs. This creates a dense carbonated
apatite layer on the surface of the scaold that is similar to
the structure of native bone and may therefore induce more
rapid osteoblast dierentiation and mineralization [30
32]. Taken together, these gene expression data associated
with the osteoblast phenotype provides evidence that the
scaold constitutes a good substrate for MC3T3-E1 cell
dierentiation leading to ECM mineralization.
4. Conclusions
In this study, we conclude that the CS/nHAC scaold has a
suitable biocompatibility for its use as cell culture scaold for
hard tissue regeneration. In vitro experiments revealed more
prominent activation of osteoblast dierentiation in cells
grown on CS/nHAC than in those grown on CS. Collectively,
these data indicates the CS/nHAC may give greater results
concerning cell proliferation and dierentiation compared
to chitosan in this in vitro study. We suggest an excellent
applicability of the CS/nHAC as bone substitutes.
Y. Chen and Z. Huang contributed equally to this paper.
Acknowledgments
The authors are grateful for the nancial support from
National Natural Science Foundation of China (no.
81101348 and no. 31000431), Natural Science Foundation
Relative expression of RUNX-2
Relative expression of OCN
0
1
10
10
Time (days)
Time (days)
CS
CS/nHAC
CS
CS/nHAC
(a)
(b)
Relative expression of COL-1
20
15
10
0
1
10
Time (days)
CS
CS/nHAC
(c)
Figure 4: Real-time PCR analysis of osteoblast-specic gene expression. Values were normalized to 18 s rRNA expression and shown relative
to gene expression on (b) for comparison. (a) OCN, (b) RUNX-2, (c) COL-1. The values are shown as mean standard deviation (n = 3).
Bars with dissimilar letters indicate signicantly dierent values ( P < 0.05, P < 0.01).
of Guangdong Province, China (10451051501004727),

Guangdong Province Science and Technology project,
China (ultrasound evaluation of injectable nanobiomaterials), Doctoral Fund of Ministry of Education,
China (20114433120004), the Beijing Nova Program (no.

2010B011), and State Key Laboratory of New Ceramic and
Fine Processing, Tsinghua University, outstanding person
fund of Zhujiang Hospital.
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doi:10.1155/2012/714134
Review Article
Nanotechnology-Based Therapies for Skin Wound Regeneration
Ilaria Tocco,1 Barbara Zavan,2 Franco Bassetto,1 and Vincenzo Vindigni1
1 Institute
of Plastic Surgery, University Hospital of Padova, 35128 Padova, Italy

of Histology, Microbiology and Biomedical Technologies, University of Padova, 35128 Padova, Italy
2 Department
Correspondence should be addressed to Ilaria Tocco, toccoilaria@gmail.com

Received 8 February 2012; Revised 19 February 2012; Accepted 24 February 2012
Copyright 2012 Ilaria Tocco et al. This is an open access article distributed under the Creative Commons Attribution License,
The cutting-edge combination of nanotechnology with medicine oers the unprecedented opportunity to create materials and devices at a nanoscale level, holding the potential to revolutionize currently available macroscale therapeutics. Nanotechnology
already provides a plethora of advantages to medical care, and the success of nanoparticulate systems suggests that a progressive
increase in the exploration of their potential will take place in the near future. An overview on the current applications of nanotechnology to wound healing and wound care is presented.
1. Introduction
Nobel Prize winner Richard Feynman was the rst in 1959 to
predict the future emergence of a new science which could be
able to deal with structures on a scale 1100 nm. Fifty years
after, the unpredictable result is the ability to manipulate
materials on the same unimaginably small scale which is
used by nature [1]. The repercussions that nanotechnology is
having in our lives nowadays are towering: the current applications to molecular biology are leading to the development
of structures, devices, and systems bearing a capacity to revolutionize medical therapeutics and diagnostics, which has
never been seen before.
Nanodevices are innovative and can provide a wide range
of advantages: from the ability of nanoparticles to enter into
the cytoplasmic space across cellular barriers, like Trojan
horses, and activate specic transport mechanisms [2, 3]; to
the modulation of drugs biocompatibility, bioavailability and
safety proles through nanodelivery systems [4]. Furthermore, therapeutic selection can increasingly be tailored to
each patients prole. Nanotherapies represent a great opportunity to enhance currently available medical treatments, improving standard care and prognosis for challenging healthcare issues, like impaired wound healing.
The public health impact of chronic wounds is staggering. An estimated 1.3 to 3 million US individuals are believed to have pressure ulcers and as many as 10% to 15% of
the 20 million individuals with diabetes are at risk of
developing diabetic ulcers [5]. Nonhealing wounds are the

results of a stop in the progression of the normal sequence
of cellular and biochemical events towards the restoration of
the skins integrity. Factors delaying wound healing include
either preexisting comorbidities (diabetes, chronic peripheral vasculopathy, and immunosuppression), leading to lack
of appropriate metabolism and clearance from toxic substances of the wound, and/or sudden complications, such as
infections, exalting the inammatory state. Nanobiotechnology combined with the knowledge on cellular and subcellular
dysfunctional events occurring in delayed wound healing
oers great opportunities for improving wound care.
This paper will focus on the currently available nanotechnology-based therapies in the eld of wound healing and skin
regeneration. To appreciate the importance of the functions
of the innovative nanosystems, an understanding of the
wound healing process is essential.
1.1. The Wound Healing Process. The wound healing process
is classically dened as a series of continuous, sometimes
overlapping, events. These are haemostasis, inammation,
proliferation, epithelisation, maturation, and remodelling of
the scar tissue [6] (Figure 1).
Haemostatic events occur immediately after injury. The
exposure of subendothelial collagen and the formation of
thrombin lead to the activation of the platelets, located in
Wounding
W-3 days
Haemostasis
Vasoconstriction
Platelet aggregation and
degranulation
Blood clotting
PDGF; FGF;
TGF-
es
Growth
factors
Inammation
Leucocyte migration
Neutrophils, secretion of chemicals killing bacteria
Removal of cellular debris by macrophages
EGF;
VEGF
es
312 days
3 days10 months
Release of growth factors
PDGF; EGF;
Proliferation
FGF; TGF-;
Neovascularisation
TNF
Fibroblasts proliferation and secretion of glycoprotein and collagen
Keratinocytes migration from the wound edge
Granulation tissue
PDGF; FGF;
EGF
Remodelling
Fibroblasts secretion of collagen and matrix proteins to strengthen wound
Wound remodelling
MMPs
Wound contraction
Scar tissue
Figure 1: The main biological phases in wound healing. The events in wound healing and the soluble factors involved in each phase are
well dened. Although presented as separated for absolute clarity, no phase is initiated exactly at the complexion of the previous one, and all
phases overlap to a certain degree (see arrows on the left).
the intravascular space. Activated platelets play a trigger role

in a number of events:
(i) activation of the coagulation cascade. This eventually
leads to the formation of a brin clot that acts as
scaolding for other cells that later enter the wound;
(ii) activation of the complement system;
(iii) platelet degranulation: cells release an array of cytokines, growth factors, and vasoactive substances from
the platelet -granules, such as platelet-derived
growth factor (PDGF), transforming growth factor-
(TGF-), broblast growth factor (FGF), endothelial
growth factor (EGF), platelet-derived angiogenesis factor, serotonin, bradykinin, platelet-activating
factor, thromboxane A2, platelet factor IV, prostaglandins, and histamine. These platelet releasates
initiate the early events of wound healing [6, 7].
The inammatory phase begins immediately after injury
and may continue for up to 6 days [8]. Growth factors released from the platelets diuse into tissues surrounding the
wound and chemotactically draw inammatory cells into the
injured area. Neutrophils are the rst inammatory cells to

enter the wound, followed by monocytes. Once chemotaxis
is completed, local mediators activate the inammatory cells.
Activated neutrophils release a number of lysosomal enzymes
(such as elastase, neutral proteases, and collagenase) which
proteolytically remove damaged components of extracellular matrix (ECM) [8]. Activated monocytes acquire the
macrophage phenotype and aid in host defence [8].
The proliferation phase is characterised by the formation
of the ECM and the beginning of angiogenesis. The primary
cells involved in this phase are broblasts and endothelial
cells. They proliferate in response to growth factors and
cytokines that are released from macrophages, platelets and
mesenchymal cells, or have been stored in the brin clot.
In addition to chemotactically drawing broblasts into the
wound, PDGF, FGF, and EGF induce broblast activation
and proliferation [9]. During the rst 2-3 days after-injury,
broblasts activity predominantly involves migration and
proliferation. After this time, broblasts release collagen and
glycosaminoglycans (mainly hyaluronic acid, chondroitin4-sulphate, dermatan sulphate, and heparin sulphate) in
response to macrophage-released growth factors, hypoxia
and by-products of anaerobic metabolism. The combination
of collagen and bronectin forms the new ECM, which is essential for the development of granulation tissue that eventually lls the wound [6]. Angiogenesis accompanies broblast proliferation and allows nutrients and healing factors to
enter the wound space. It is also essential for the growth of
granulation tissue. The principle growth factors that regulate
angiogenesis are FGF, released by damaged endothelial cells
and macrophages, and vascular endothelial growth factor
(VEGF) which is released by keratinocytes and macrophages
[6].
The maturation phase usually begins 3 weeks after injury
and can take up to 2 years to complete [10]. Unlike uninjured
skin, the arrangement of newly formed collagen bres in the
wound is random and disorganised. The remodelling of collagen bres into a more organised lattice structure gradually
increases the tensile strength of the scar tissue, though this
never exceeds 80 percent of the strength of intact skin. Remodelling of the ECM involves a balance between collagen
synthesis and degradation, which is operated by several enzymes, like matrix metalloproteinases (MMPs), neutrophilreleased elastase and gelatinase, collagenases and stromelysins [11].
Wounds that do not heal within three months are considered chronic. In acute wounds, there is a precise balance
between production and degradation of molecules such as
collagen; in chronic wounds this balance is lost and degradation plays too large a role. Chronic wound bed has been demonstrated to dier from acute wounds for a higher concentrations of proteases (such as MMPs) [12] and lower levels
of growth factors and cytokines [13]. A high and prolonged
proteolytic activity may lead to the degradation of growth
factors, detaining the wound in the inammatory stage for
too long [14].
2. Nanotherapies for Wound Healing

An increasing number of products emerging from the application of nanotechnology to the science of wound healing is
currently under clinical investigation. The current nanoscale
strategies, both carrier, drug related and scaold (Figure 2),
that target the main phases of wound repair will be discussed,
highlighting the cellular signals involved (Table 1).
2.1. Nanoparticle-Bearing Endogenous Molecules. As we
pointed out previously, soluble active proteins (cytokines
and growth factors) govern the progression of the healing
phases through the modulation of the cellular and molecular
components involved [43]. Because of the control they exert
on wound repair, bioactive proteins have gained progressive interest for the treatment of chronic dehiscent wounds.
Clinical trials involving the application of exogenous recombinant growth factors to chronic wounds have been conducted for the past 10 years in anattempt to nd a way to
accelerate healing [44]. Although the results of several pilot
trials have been encouraging, the overall results have been
somewhat discouraging. To date, only a single recombinant
3
growth factorrecombinant human PDGF-BBhas been
approved by the United States Food and Drug Administration, and only for use in diabetic foot ulcers [45]. In fact, the
major limitation to the topical use of growth substances is
that in human plasma the half-life of proteins involved in
signalling cascades is very short (few seconds) due to close
control and inactivation by protease inhibitors [46]. Therefore, the clinical use of endogenous factors as pure compounds is limited by the breakdown by the proteolytic
enzymes that enrich the wound site [47]. Several attempts
have been already made to provide biological molecules
long-term protection from enzymatic degradation (liquids,
gels, collagen sponges, and gene transfer). Recently, the
development of nanoscale systems for drug delivery has
opened new possibilities to enhance the biological ecacy of
molecules through a controlled release for extended periods
of time [48]. Endogenous active molecules that have been engineered nowadays include thrombin, nitric oxide, growth
factors, opioids, and protease inhibitors.
Thrombin (also termed activated factor II or factor IIa) is
a protein involved in the nal stage of the coagulation cascade
activated by wounding. In addition to its well-documented
role in the formation of brin clots and platelet activation,
thrombin has direct eects on inammatory cells, broblasts, and endothelial cells: it stimulates chemotaxis and aggregation of neutrophils, lymphocytes, and monocytes cells,
and the proliferation of broblasts, epithelial and endothelial
cells [49, 50]. Thus, thrombin may play an important role
in initiating early cellular events in tissue repair [49]. Since
1988 researchers suggested that thrombin receptor-activating
peptides could be useful in vivo to mimic the natural eects
of thrombin interaction with receptors on various types of
cells [51], a delivery systems that could provide thrombin a
long-term protection from its natural inhibitors (antithrombin and activated protein C) has been pursued. In recent
years, nanobiotechnology has provided the means to enhance the bioavailability of thrombin by conjugation with
iron oxide nanoparticles (-Fe2 -O3 conjugation) [15]. Animal models were tested for wound response to the treatment
with conjugated thrombin: on a 28-day treated wound, results obtained analysing tensile strength indicated a signicant acceleration of healing process when compared
with free-thrombin-treated wounds and untreated wounds.
Obtaining a greater tensile strength may potentially reduce
surgical complications such as wound dehiscence.
Nitric oxide (NO) is a small radical, formed from the
amino acid L-arginine by three distinct isoforms of nitric
oxide synthase. The inducible isoform (iNOS) is synthesized
in the early phase of wound healing by inammatory cells,
mainly macrophages. However, many cells participate in NO
synthesis during the proliferative phase after wounding. NO
regulates collagen formation, cell proliferation, and wound
contraction in distinct ways in animal models of wound
healing [52]. NO is also a well-known antimicrobial agent,
interfering directly with DNA replication and cell respiration
through the inactivation of zinc metalloproteins [53]. Treatment of acute and chronic wound failure with NO has been
for years a major unresolved goal. Attempts at novel nitric
Remodelling
VAN
EGF
VAN
Embedded
antibiotic
PDGF
Conjugated
nanoparticles
Silver-loaded nanobers
Fe
O
Wound contraction
Lipoxin A
analog
Resolvin D1
Anti-VEGF
TGF-
plasmid DNA
PDGF
Embedded
Embedded growth factors Embedded opioids Embedded DNA protease inhibitors
Embedded
nitric oxide
Nanoscaolds
Embedded
nanoparticles
Inammation
Stem cells-seeded scaolds
2D/3D
nanobrous sheets
Fe
O
O THR
VAN
Conjugated antibiotics
Pure
nanoparticles
Conjugated thrombin
N
H
Silver nanoparticles
Gelatin microparticle
Poly(lactide-co-glycolide)
nanoparticle
Carboxymethyl chitosan
nanoparticle
Polyacrylate nanoparticle
Poly(lactic acid) nanoparticle

Hyaluronan
nanoparticle
Neutrophil-derived
nanoparticle
Solid lipid nanoparticle
Silica nanoparticle
Figure 2: Nanostrategies currently in use for promoting skin wound healing. In the main panel, nanostrategies currently in use to improve
healing are illustrated. The therapeutic potential of nanosphere-based strategies and nanoscaolds is emphasized through correlation with
the healing phase to which the biological action is targeted. In the right panel, materials explored for nanoparticulate-delivery systems are
listed. Two- and three-dimensional nanobrous sheets can be made of both degradable (collagen and chitosan) and nondegradable (PLA,
PLACL, polyurethane, and PVA polymers) nanobers.
oxide therapies, in the form of nitric oxide donors, have

shown limited potential in treating cutaneous infection [54].
A novel approach is oered by recently engineered nanoporous materials, that make possible the storage and delivery of
small gaseous short-lived NO, allowing the free radicals to
exert their antibacterial activity. Using silane hydrogel-based
nanotechnology [23], NO remains trapped and stable within
a dry matrix until the matrix is exposed to moisture. The dry
matrix allows for NO nanoparticles to be easily stored and
applied. Once exposed to moisture, the drug is released from
the nanoparticle over an extended period of time at a relatively xed concentration. This sustained release distinguishes
nanoparticles from other vehicles, such as injections, that
release a large concentration of the drug with a rapid
return to baseline. The ease of storage, application, and

the ability to alter release rate and concentration with
minimal risk of toxicity make this powder formulation
ideal for cutaneous delivery. Authors showed promising
results from NO-releasing nanodelivery systems in preventing and treating skin infections caused by methicillinresistant Staphylococcus aureus (MRSA), which is one
of the major causes of hospital-acquired infections [55,
56]. Moreover, NO-releasing silica nanomolecules have
been demonstrated to exert a bactericidal activity against
Pseudomonas aeruginosa [57] and Acinetobacter baumannii [58]: both microbial agents have become an increasingly prevalent cause of hospital-acquired infections during
the last 15 years, the majority of clinical A. baumannii
5
Table 1: Executive summary table.
Inammation
(i) thrombin is one of the rst products of the coagulation cascade occurring during haemostasis, and is responsible for platelet
activation and aggregation, leading to the formation of the platelet plug and allowing cells and uid to enter the wound bed. In human
plasma, thrombin is rapidly degraded (15 sec). In order to provide long-term protection, it has been conjugated with iron-oxide
nanoparticles for treatment of incisional wounds in rats [15]
(ii) bacterial infection and sepsis exacerbate the inammatory state and cause tissue damage
(a) nanoparticles bearing vancomycin or N-methyltiolated lactams have been developed to act against wound contamination by MRSA
[1619]
(b) silver-based nanoparticles were developed to take advantage on the multilevel antibacterial action of silver and try to reduce the
development of microbial resistance. Pure biostable nanoparticles were produced through photoassisted reduction and ion stabilization.
Silver nanoparticles were also loaded into nanobers [20]. A direct promotion of wound healing by silver nanoparticles through
reduction of the cytokine-modulated inammation and cell migration and proliferation was also demonstrated [21, 22]
(c) donor NO silica nanoparticles showed speed healing by killing both Gram-positive and Gram-negative bacteria and overcoming the
NO deciency [23]. No-releasing nanoparticles may also potentially accelerate healing by a promotion of angiogenesis and tissue
remodelling [24]
Proliferation
(i) the aim of growth factors is to promote cell migration into the wound site, stimulate the growth of epithelial cells and broblasts, start
the formation of new blood vessels, and profoundly inuence the remodelling of the scar. To enhance the in vivo ecacy of growth
factors they have been incorporated into polymer nanocarriers to sustain release. PLA/PLGA/PEG/hyaluronan/gelatin nanoparticles
embedded with dierent growth factors have been successfully applied on skin wounds [2531]
(ii) opioids have been recently indicated as factors promoting keratinocytes migration. Solid lipid nanoparticles were embedded with
opioids, conrming the inuence of these drugs on keratinocytes migration [32, 33]
(iii) nanobrous scaolds: electrospunnanobers networks support cell adhesion, proliferation, and dierentiation mimicking the
brous architecture of the extracellular matrix. Both degradable (collagen and chitosan) and nondegradable (PLA, PVA, PLACL, and
polyurethane) bers are used for 2D and 3D constructs [3436]. Scaolds were also engineered to contain growth factors-releasing
nanoparticles enhancing wound repair [37]
Remodelling
(i) matrix metalloproteinase collagenolytic activity appears to be upregulated in chronic wounds. Protease inhibitors were loaded into
human derived nanoparticles, showing a proresolving action and accelerated healing [38]
(ii) gene therapy: nonviral polymeric gene delivery systems oer increased protection from nuclease degradation, enhanced plasmid
DNA uptake, and controlled dosing to sustain the duration of plasmid DNA administration. Gene delivery systems are formulated from
PLGA polymers, polysaccharides, and chitosan [3941]
(iii) stem cells: cell-based therapies hold the potential to promote vascularization and tissue regeneration. The hVEGF gene was delivered
through biodegradable polymeric nanoparticles: treated stem cells showed the engraftment of the tissue [42]
isolates displaying high-level resistance to common antibiotics [59]. Also, the benecial eects of NO seem to
overcome the antimicrobial activity and involve directly the
healing process. Experiments demonstrated that pharmacological or genetic (NOS knockout) reduction of NO impairs
the speed and eectiveness of wound healing, and that
this process can be reversed by restoring NO production
with increased NOS substrate (arginine), or by transfecting
with the missing NOS gene [24]. Thus, application of NOreleasing nanoparticles may potentially accelerate healing not
only by killing bacteria but also by a promotion of angiogenesis and tissue remodelling.
The clinical use of growth factors in wound healing has
been of great interest recently. They have the potential to accelerate the healing process by attracting cells into the wound
site (TGF-), promoting cell migration, stimulating the proliferation of epithelial cells and broblasts (FGF and PDGF),
as well as initiating the formation of new blood vessels (FGF
and VEGF), and nally participating in the remodelling of
the scar [46]. A mean of enhancing the in vivo ecacy of
growth factors, preventing degradation from proteolytic
enzymes, is to stabilize protein structure and biological activity, prolonging the length of time over which growth factors are released at the delivery site [60, 61]. The period of
drug release from a polymer matrix can be regulated by drug
loading, type of polymer used and the processing conditions.
Delivery systems have been designed in a variety of congurations and have been fabricated from dierent types of
natural and synthetic polymers, both degradable and non degradable. Poly(lactic acid) (PLA) and poly(lactic-co-glycolic
acid) (PLGA) have been demonstrated to be biocompatible
and biodegradable suitable materials. To achieve long-term
in vivo circulation, the surface is generally modied with
polyethylene glycol (PEG), reducing the clearance by the reticuloendothelial system [25]. In biodegradable carriers,
growth factor release is controlled by the polymer matrixs
rate of degradation, which causes changes in the morphological characteristics of the materials, such as porosity and
permeability [26]. Particulate delivery systems explored involve TGF--embedded gelatine microparticles [27], EGF in
PLA microspheres [28], FGF in gelatin microspheres [29],
and PDGF-embedded PLGA nanospheres [30]. In general,
6
porous materials seem to retain a higher specic surface for
the adsorption and release of active components and enhancement of drug release. Nevertheless, the particles show
some limits which are associated with the use of organic solvents in the production process, leading to pollution and toxicity of the product [62]. Therefore, alternative techniques of
fabrications have been proposed, such as spray drying, which
is based on the use of supercritical uids (especially CO2 ) and
oers the advantages of being environmentally safe and inexpensive [63]. A notable recent application of this technology
for the delivery of growth factors in vivo was oered by Zavan
et al. [31]: hyaluronan-based (HYAFF11) porous nanoparticles were embedded with PDGF as a system designed for the
in vivo treatment of skin ulcers. PDGF is known since 1986
to successfully promote chronic wounds healing through a
stimulation of chemotaxis, proliferation and ECM deposition [64]. HYAFF11, the benzyl ester of hyaluronic acid, is
a biopolymer that has found numerous applications for
in vitro reconstruction of skin as well as for the in vivo regeneration of small arteries and veins [65]. HYAFF particles have
the ability to absorb growth factors and to release them in a
temporally and spatially specic event-driven manner. This
timed and localized release of PDGF promoted optimal tissue
repair and regeneration of full-thickness wounds.
Beside the dedicated growth factors, opioids have been
recently indicated as factors promoting keratinocytes migration [66]. This nding is of great interest because of a possible
enhancement of wound healing through topical applications
of opioids at the wound site for pain reduction. Conventional
formulations failed to consistently provide sucient pain
control in patients, possibly due to local drug degradation or
insucient concentrations at the target site [32]. Since long
intervals for painful wound dressing changes are intended,
the formulations should allow for prolonged opioid release.
Bigliardi et al. [32] rst developed nanoparticulate carriers to
increase opioids skin penetration and slow the release of the
loaded drug. Experiments on human keratinocyte-derived
cell line HaCaT showed that opioids stimulated cell migration and closure of experimental wounds. Enhancement of
migration was concentration-dependent and could be blocked by the opioid receptor antagonist naloxone, indicating a
specic opioid-receptor interaction. Kuchler followed [33]
demonstrating that morphine-loaded solid lipid nanoparticles accelerated reepithelialisation on a human-based 3Dwound healing model. On standardised wounds, keratinocytes almost completely covered the dermis equivalent after
4 days, which was not the case when applying the unloaded
particles. In conclusion, acceleration of wound closure, low
cytotoxicity, and irritation as well as possible prolonged morphine release make solid lipid nanoparticles an interesting
approach for innovative wound management.
Matrix metallo proteinases (MMPs) are zinc-dependent
endopeptidases that cleave most macromolecules within the
ECM during the maturation of the wound [67]. The process
of remodelling constitutes a balance between collagen production, breakdown, and remodelling. The biological activity of MMPs is strictly balanced by the presence of specic tissue inhibitors (TIMPs). A tightly coordinated expression of
specic combinations of MMPs and TIMPs appears to be
necessary for proper wound maturation. As previously stated, an uncontrolled proteolytic activity leads to delayed healing through degradation of growth factors. In light of these
considerations, research approaches to improve the remodelling phase are directed to manage enzymatic activity of
MMPs through the topical application of protease inhibitors.
Norling et al. [38] recently took advantage of aspirin-triggered resolving D1 and lipoxin A4 analogs and developed a
carrier providing stable biological activity to these natural
compounds. Human-derived nanoparticles were enriched
with the protease inhibitors, and wound healing reactions
were tested in a murine model. Polymorphonuclear cell inux showed a dramatical reduction in treated wounds, with
shortened resolution intervals, and a proresolving action that
in the end accelerated healing.
2.2. Nanoparticle-Bearing Antibiotics. During the healing
process, infection is an issue potentially compromising the
nal wound closure, exacerbating the tissue damage [68].
Nowadays, wound infection is no longer the ominous event
as at the beginning of the 20th Century, when infection of
wounds, especially burns, was the major cause of morbidity
and mortality (over 50%) [69]. Nevertheless, an appropriate
antimicrobial therapy of the wound controlling colonization
and proliferation of microbial pathogens, including multidrug-resistant organisms, is still required for an appropriate
wound care. Staphylococcus aureusis one of the most common pathogens involved in wound infections. The pharmacological treatment encounters today severe limitations due
to the development of antibiotic resistant strains. For example, penicillinase (an enzyme that breaks down the -lactam
ring of the penicillin molecule) is responsible for the resistance to penicillin of Staphylococcus aureus: the failed treatment of staphylococcal local infections leads to the onset of
serious late complications (bacteraemia, sepsis, toxic shock
syndrome) [69].
The delivery of antibiotic therapy via nanoparticles oers
great potential advantages. Particularly, a controlled release
would decrease the number of doses required to achieve the
desired clinical eect, potentially reducing the risk of development of antibiotic resistance. The physiochemical properties of nanoparticulate drug delivery systems (size, surface
charge, and nature) are determinant in vivo for the factors
delivery. In particular, it is known that 20200 nm particulates are suitable for delivery of therapeutics; larger size particles suer from quick uptake by the reticulo-endothelial
system and rapid clearance from circulation, whereas the
smaller size will tend to cross the fenestration in the hepatic
sinusoidal endothelium, leading to hepatic accumulation
[70].
In the treatment of staphylococcal infections, the latest
generation antibiotic and assumed to be the most useful at
the moment is vancomycin. Several vancomycin-modied
nanoparticles have been developed to enhance the pharmacokinetics and pharmacodynamics of the antibiotic molecule. Hachicha et al. [16] recently proposed the use of vancomycin-conjugated nanoparticles for intraocular continuous release injection for endophthalmitis prophylaxis. The
drug concentration was proved to be maintained above
the minimal inhibitory limit for 24 hours. Chakraborty et al.
[17] developed carboxymethyl chitosan-based nanoparticles,
which were loaded with vancomycin and proved eective
against drug-resistant staphylococcal strains.
The eectiveness of nanobiotechnology in enhancing the
therapeutic properties of antibiotic molecules is emphasized
by the case of N-methylthiolated -lactams: these compounds have been recently identied and proved eective
against Staphylococcus bacteria, including MRSA. These lactams exert growth inhibitory eects on bacteria through a
mode of action that is distinctively dierent from other lactam antibiotics, with peculiar structure-activity patterns.
Nevertheless, their potential application is limited by the
exceeding low water solubility [18]. Thus, a methodology
was developed by Turos et al. [19] to obtain an emulsion of
polyacrylate nanoparticles in which the drug monomer was
incorporated into the polymeric matrix: in vitro screens
conrmed the nanoparticles to be nontoxic to human dermal
broblasts and stable in blood serum for 24 hours.
Within the bacterial species, resistance to all known antibiotic classes has occurred due to mutation and horizontal
gene transfer. This has led to anxiety regarding the futurea
vailability of eective chemotherapeutic options. Due to the
outbreak of infectious diseases caused by dierent pathogenic bacteria and the development of antibiotic resistance,
the pharmaceutical companies and the researchers are now
searching for new antibacterial agents.
2.3. Silver-Based Nanoparticles. The use of silver for the treatment of ulcers is reported since the 5th Century B.C. In the
17th and 18th centuries, silver nitrate was already used for
ulcer treatment. The antimicrobial activity of silver was established in the 19th century. Nevertheless, after the introduction of antibiotics in 1940 the use of silver salts decreased.
Subsequently, silver salts and silver compounds have been
used in dierent biomedical elds, especially in burn treatment [71].
The antimicrobial activity of silver appears high: due to
its multilevel (including multidrug resistance) antibacterial
eects [72] and low systemic toxicity [73], it provides an antibacterial eect that considerably reduce the chances of developing resistance. Evidently, the greatest rate of silver ions
release is wished in order to enhance the clinical eect and
avoid the insurgence of silver-resistant mutant bacteria.
Nanotechnology has provided the means of producing pure
biostable silver nanoparticles, either through photoassisted
reduction and ion stabilization or loading of the metallic particles into nanobers [20]. In all cases, 720 nm silver nanoparticles exhibited antibacterial (especially anti-Gram negative) and antifungal activity, and they are also synergistic to
common antibiotic therapy (streptomycin, kanamycin, and
polymyxyn B) [20]. Studies in vivo demonstrated also a direct
promotion of wound healing by silver nanoparticles through
reduction of the cytokine-modulated inammation: silverinduced neutrophils apoptosis, decreased MMPs activity, and
negative modulation on TGF- resulted in an overall acceleration of wound healing rate and reduction on hypertrophic
scarring [21]. Also, wound healing rate was demonstrated to
be increased by silver ions by a promotion of proliferation
7
and migration of keratinocytes along with a dierentiation
of broblasts into myobroblasts, thereby promoting wound
contraction [22].
Though the progressive expansion of the therapeutic application of silver nanoparticles, the use of a metallic compound carries possible side eects that must be taken into
consideration. Studies have been investigating the biosafety
of silver as a therapeutic agent, reporting an acceptable biocompatibility [74], though the occasional development of
argyria (a cosmetic blue-grey coloration of the skin).
2.4. Nanoparticles and Gene Therapy. Polymeric gene delivery systems oer several advantages for plasmid DNA
delivery, such as protection from degradation by the nuclease
and controlled prolonged release. The potential retained by
the modulation of gene expression in the process of wound
healing lead researchers to apply for an engineered system
for DNA transfection. Actually, transfection capability has
been tested in vitro: biocompatible and biodegradable PLGA
polymers [39] were engineered to obtain high plasmid loading eciency [75] and then loaded withan antiangiogenic
plasmid DNA (pFlt23k). The PLGA nanoparticles were prepared by a supercritical uid extraction of emulsions based
on CO2 : this allowed high loading of pDNA (19.7%, w/w),
high loading eciency (>98%), and low residual solvents
(<50 ppm). The VEGF secretion by epithelial cells was signicantly reduced, showing a potential value in treating wound
disorders in which VEGF is elevated.
In the course of studies on nonviral DNA carriers for
gene delivery and therapy other materials have been applied,
like polysaccharides and other cationic polymers. Chitosan
also was found to be particularly suitable, since it can promote long-term release of incorporated drugs [76]. Masotti
and Ortaggi recently described a nanofabrication method
that may be useful for obtaining small DNA-containing
chitosan nanospheres (38 4 nm) for biomedical applications [40]. Their reported osmosis-based method has general
applicability to various synthetic or natural biopolymers, resulting in nanostructured systems of dierent size and shape
that may be used in several biotechnological applications.
Chellat et al. also recently took advantage of the biochemical
properties of chitosan to test DNA-loaded nanoparticles on
a human macrophage cell line to study the potential modulation of the expression of proinammatory cytokines, metalloproteinases and their specic inhibitors [41]. The secretion
of MMP-9 in cell supernatants increased signicantly after 24
and 48 h in comparison with nontreated cells. MMP-2 secretion was augmented only after 48 h with incubation of the
highest concentrations of nanoparticles (10 and 20 g/mL
DNA content). However, zymography studies showed that
secreted MMPs were in their proactive form, while in the
presence of 10 and 20 g/mL DNA-containing nanoparticles,
the active form of MMP-9, but not MMP-2, was detected in
cell lysates. The results obtained were signicative only for
increased secretion of metalloproteinases, possibly related to
nanoparticles phagocytosis.
2.5. Nanoparticles and Stem Cells. Stem cells are lauded for
their unique ability to develop into every kind of cell. Even if
8
in vivo studies and clinical trials have demonstrated limitations in reconstituting tissue due to the lack of microenvironment-control on proliferation and survival, the successful
use of these cells for investigation towards disease therapy is
still pursued. Last decade has witnessed a growth in the eld
of nanoparticles technology for stem cells isolation, maintenance, and regulation: nanoparticles and nano 3D architectures have been developed to control stem cells proliferation, dierentiation, and maturation [77]. In terms of skin
regeneration, VEGF high-expressing, transiently modied
stem cells have been developed for the purpose of promoting angiogenesis [42]. In order to overcome the insucient
expression of angiogenic factors and low cell viability after
transplantation, nanotechnology has provided nonviral, biodegradable polymeric nanoparticles to deliver hVEGF gene
to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs). Treated stem cells
demonstrated markedly enhanced hVEGF production, cell
viability, and engraftment into target tissues. Implantation
of scaolds seeded with VEGF-expressing stem cells (hMSCs
and hESdCs) led from 2 to 4 fold higher vessel densities 2
weeks after implantation, compared with control cells or cells
transfected with VEGF by using Lipofectamine 2000, a leading commercial reagent. Four weeks after intramuscular injection into mouse ischemic hindlimbs, genetically modied
hMSCs substantially enhanced angiogenesis and limb salvage, while reducing muscle degeneration and tissue brosis
[42]. These results indicate that stem cells engineered with
biodegradable polymer nanoparticles may be therapeutic
tools for vascularizing tissue constructs and treating ischemic
disease.
2.6. Nanobrous Scaolds. The basic strategy of engineered
tissue regeneration is the construction of a biocompatible
scaold that, in combination with living cells and/or bioactive molecules, replaces, regenerates, or repairs damaged
tissues. The scaold should possess suitable properties, like
biocompatibility, controlled porosity and permeability, and,
additionally, support for cell attachment and proliferation.
This articial dermal layer needs to adhere to and integrate
with the wound, which is not always successful for the
current articial dermal analogues available. The high cost of
these articial dermal analogues also makes their application
prohibitive both to surgeons and patients. Engineering dermal substitutes with electrospun nanobres have lately been
of prime importance for skin tissue regeneration. Simple
electro spinning technology served to produce nanobrous
scaolds morphologically and structurally similar to the
extracellular matrix of native tissues. The engineered network has been shown to support cell adhesion, proliferation,
and dierentiation mimicking the brous architecture of
the extracellular matrix [60]. The large surface area and
porosity of electrospun nanobers enables good permeability
for oxygen and water and the adsorption of liquids, and concomitantly protects the wound from bacterial penetration
and dehydration. This feature shows electrospun nanobers
to be a suitable material for wound dressing, especially for
chronic wounds such as diabetic ulcers or burns.
The electro spinning technique can provide both degradable (collagen; chitosan) and nondegradable (PLA, polyvinyl
alcohol (PVA) polymers) nanobers for two-dimensional
nanobrous sheets. Both sorts of biomaterials have been
tested in vivo showing an increased rate of wound epithelialisation and dermis organization [34], as well as good antibacterial activity against the Gram-positive and Gram-negative bacteria [35]. Nanobrous scaolds of poly(L-lactic
acid)-co-poly(-caprolactone) (PLACL) and PLACL/gelatin
complexes were fabricated by Chandrasekaran et al. [36].
These nanobres were characterized by ber morphology,
membrane porosity, wettability, and chemical properties by
FTIR analysis to culture human foreskin broblasts for skin
tissue engineering. The results showed that broblasts proliferation, morphology, and secretion of collagen were significantly increased in plasma-treated PLACL/gelatin scaolds
compared to PLACL nanobrous scaolds. The obtained
results proved that the plasma-treated PLACL/gelatin nanobrous scaold is a potential biocomposite material for skin
tissue regeneration.
Moreover, nanobrous constructs can be obtained with
a 3D prole, even though they scarcely support cells seeding
because of their high porosity. Several strategies have been
developed to improve cell inltration, showing promising
results [79]. Chong et al. [80] proposed a cost-eective composite consisting of a nanobrous scaold directly electrospun onto a polyurethane dressing (Tegaderm, 3 M
Medical)Tegaderm-nanober (TG-NF) constructfor
dermal wound healing. Cell culture was performed on both
sides of the nanobrous scaold and tested for broblast
adhesion and proliferation. Results obtained in this study
suggested that both the TG-NF construct and dual-sided
broblast-populated nanober construct achieved signicant cell adhesion, growth, and proliferation. This was a successful rst step for the nanober construct in establishing
itself as a suitable three-dimensional scaold for autogenous
broblast populations and providing great potential in the
treatment of dermal wounds through layered application.
Steps towards an enhanced regenerative eect will be to
provide scaolds a delivery system for drugs, growth factors
or cytokines that may further promote cell function and tissue regeneration [81]. Jin et al. already worked towards this
direction engineering PLGA microspheres in nanobrous
scaolds to control the release of PDGF in vivo [37]. PDGF
concentration was evaluated in a soft tissue wound repair
model in the dorsa of rats. At 3, 7, 14, and 21 days afterimplantation, the scaold implants were harvested followed
by assessments of cell penetration, vasculogenesis, and tissue
neogenesis. Gene expression proles using cDNA microarrays were performed on the PDGF-releasing NFS. The percentage of tissue invasion into microspheres-containing
nanobrous scaolds at 7 days was higher in the PDGF
groups when compared to controls. Blood vessel number in
the groups containing either 2.5 or 25 g PDGF was increased
above those of other groups at 7d (P < 0.01). Results from
cDNA array showed that PDGF strongly enhanced in vivo
gene expression of the CXC chemokine family members
such as CXCL1, CXCL2, and CXCL5. Thus, sustained release
of rhPDGF-BB, controlled by slow-releasing microspheres
associated with the nanobrous scaold delivery system,
enhanced cell migration and angiogenesis in vivo, and may be
related to an induced expression of chemokine-related genes.
This approach oers a technology to accurately control
growth factor release to promote soft tissue engineering in
vivo.
3. Conclusions
In recent years, nanomedicine has experienced a progressive
expansion: its great potential has attracted considerable
investments from governments and industry, which are predicted to increase in the near future, leading to further enhancement and development. Similarly, the knowledge of the
cellular and molecular processes underlying wound healing
has reached a level which let researchers apply for new therapeutic approaches that act directly on cellular and subcellular events during the healing process. Nanotechnology
today oers the means to overcome the dimensional barrier
of currently used therapies for wounds and ulcers, to
reach a dysfunctional molecular target and exert the therapeutic action straight at the origin of the chronic condition.
Nanocarriers possess an enormous potential, as nanoparticles-based delivery systems can be highly benecial to
augment the therapeutic power of biological and synthetic
molecules. However the promising results brought by new
technologies in therapeutics, the real biological eects of
nanoparticles have to be carefully assessed before introducing
their use in clinical practice. In particular, the release of active
peptides may possibly cause interferences with some biological functions and cellular processes. With regard to this, the
incorporation of a targeting ligand into nanoparticles has
been proposed, in order to give them site-specicity; also, a
wide variety of biomaterials has been prompted to meet the
specic biological requirements [82]. This could be especially
critical for tissue regeneration, where the biomaterial properties could augment the reparative process, or hinder it due
to undesirable attributes of the material.
Despite its recognized importance, there have not been
systemic studies that probe the targeting eciency of nanoparticles nor international standards on their toxicology and
biocompatibility. Our wish is that further research is to be
extended to the applied science of nanotechnology. The revolutionary potential oered by nanoscale therapeutics applied to wound healing involves the need to develop international standards on their biocompatibility. A safe and targeted use is required to prove benecial to such a world-wide
issue, which is chronic wounds and ulcers care.
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doi:10.1155/2012/452767
Research Article
Evaluation on Cartilage Morphology after Intra-Articular
Injection of Titanium Dioxide Nanoparticles in Rats
Jiangxue Wang,1 Yu Gao,1 Ying Hou,1 Feng Zhao,1 Fang Pu,1 Xiaoyu Liu,1
Zhihong Wu,2 and Yubo Fan1
1 Key
Laboratory for Biomechanics and Mechanobiology of the Ministry of Education, School of Biological Science and
Medical Engineering, Beihang University, Beijing 100191, China
2 Department of Orthopedics, Peking Union Medical College Hospital, Beijing 100730, China
Correspondence should be addressed to Yubo Fan, yubofan@buaa.edu.cn
Received 16 November 2011; Accepted 5 January 2012
Copyright 2012 Jiangxue Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
Nanoscale wear particles would generate from orthopedic implants with nanoscale surface topography because of residual stress. In
this study, the eect of TiO2 nanoparticles on articular cartilage was investigated by intra-articular injection in rats. Using contrastenhanced high-resolution microcomputed tomography (micro-CT) technology, the decreased thickness of articular cartilage in
distal femur was determined at 1, 7, 14, and 30 days after nanoparticle exposure. A strong linear correlation (r = 0.928, P <
0.0001) was observed with the results obtained by needle probe testing. After exposure to TiO2 nanoparticles, cartilage thickness
showed time-dependent decrease, and cartilage volume was decreased too. Further, the histopathological examination showed
the edema chondrocyte and shrinked nucleus in the radial and calcied zone of cartilage. The ultrastructure of articular cartilage
implied that the chondrocyte was degenerated, expressing as the condensed chromatin, the dilated endoplasmic reticulum, and
the rich mitochondria. Even, the fragments of ruptured endoplasmic reticulum were observed in the cytoplasm of chondrocytes
at postexposure day 30. Results indicate that potential damage of articular cartilage was induced by particles existed in knee joint
and imply that the biomonitoring should be strengthened in patients with prostheses replacement.
1. Introduction
The nanoscale (less than 100 nm) surface topography endows
nanomaterials as high biological active matrix for protein
adsorption and focal attachment [1, 2], which provides a
forthcoming prospect in tissue regeneration and orthopedic
prostheses [35]. Titanium is widely used in hip and joint
implants and is biocompatible because it spontaneously
forms a protective oxide thin lm (TiO2 coating, typically 4
6 nm thin) at its surface. It is reported that nanoscale coating
creates a conditioned interface for osteoblast and chondrocyte adhesion [610] and promotes the osteointegration
and bone mineralization in vivo [11]. However, because
of corrosion, fretting, friction, and mechanical loss, many
wear particles would generate at the bone-implant interface
or in the joint space [12, 13]. Kuster et al. [14] reported
that wear particles with lamellar, chunky, osseous, elongated,
and rod shapes were observed in healthy and osteoarthritic
human knee joints. Except the dierent shapes, the nanoscale

polyethylene wear particles below 0.05 m and metal wear
particles with sizes from 40 to 120 nm containing Co, Cr, and
Ti are detected in vivo with high-resolution microscopy technology [15, 16]. Many researchers reported that nanoscale
particles have a potential impact on living organism [17,
18]. In vivo, TiO2 nanoparticles would be phagocytosed
by the epithelial and endothelial cells or macrophages, be
translocated into the heart, lung and liver tissues with
the blood circulation and cause the oxidative stress and
inammatory response [19, 20]. For the interaction with
cells, TiO2 nanoparticles are generally studied in vitro that
the DNA damage and cell membrane decomposition are
induced by the photocatalysis of TiO2 [2123]. In our pilot
study, the intra-articular-injected TiO2 nanoparticles have a
potential toxicological eect on the knee joint and could be
disseminated to the major organs of rats from joint cavity
[24]. The aggregated TiO2 nanoparticles deposited in the
2
knee joint induce the synovium hypertrophy, lymphocytes
and plasma cells inltration, broblast proliferation, and
oxidative damage. However, some studies reported that the
inammatory response occurred in synovium involved in
regulating the remodeling of articular cartilage, leading to a
loss of cartilage [25, 26].
Articular cartilage is very important in the joint movement for providing a resilient and low-friction bearing surface. The thickness of articular cartilage is related either
to the age, to the osteoarthritis, or to the mass of donors.
Shepherd and Seedhom [27] reported that thick cartilage
existed in the incongruent knee joint where most body
weight loaded on. Generally, the cartilage thickness is
measured by needle probe, ultrasonic technique, optical
stereomicroscope, and magnetic resonance imaging (MRI)
technique [28]. MRI technique is successfully used for
measuring the articular cartilage thickness of humans [29],
but the resolution of current clinical MRI systems (200 m)
is not enough to analyze the small animal models and limits
its application. With the needle probe method, the intact, in
situ cartilage can be tested.
X-ray microcomputed tomography (-CT) is an X-raybased nondestructive 3D imaging modality with micrometer-level voxel resolutions and quantitative morphological
analysis of electron-dense tissues such as tooth and bone
of rat, mouse, and rabbit. It is widely used for diagnosing
disease in medicine and scientic research in material
science, pharmacy, and biology, and so forth. Golding et al.
[30] proved that -CT is a faster and more accurate spatially
3D technique than histological sections for reconstruction of
molluscan anatomy. For soft tissues, the contrast-enhanced
technique with iodic-contained solution agent is developed
to compensate poor radiopacity and to improve the Xray images. The successful measurement of kidney volume,
length, and thickness in mice was performed in vivo and ex
vivo by Almajdub et al. [31] using the contrast-enhanced
high-resolution -CT technology as well as the liver and
spleen tumor assessment in living mice [32]. Recently, the
equilibrium partitioning of an ionic contrast agent via -CT
(EPIC-CT) is presented as a noninvasive imaging technique
and used to assess the articular cartilage morphology in
rabbit [33] and rat model [34].
In this study, the potential inuence of intra-articular
injected TiO2 nanoparticles on the articular cartilage in distal
femur of rats are investigated at postexposure days 1, 7,
14, and 30. The general approach is to expose rats to the
well-characterized nanoparticles by intra-articular injection,
to estimate the cartilage thickness and volume with time
course using 3D cartilage model which was reconstructed by
contrast-enhanced high-resolution -CT technology and to
assess the potential cartilage injury by morphology analysis.
2. Material and Methods

2.1. Materials. TiO2 nanomaterials (Hangzhou Wan Jing
New Material Co., Ltd.) without any coating were used in this
study. Its purity was higher than 99.8%. The properties such
as size, crystal prole, and structure state of TiO2 were well
characterized previously [24]. Briey, TiO2 nanoparticles
were red blood cells-like wafers with the average diameter of
45.87 7.75 nm, the thickness of 1015 nm, and the average
pore size of 7.50 2.58 nm. The crystal prole was pure
anatase. The surface area was 105.03 m2 /g with the cumulative pore volume of 0.42 cm3 /g, which was determined
under Quadrasorb SI analyzer (Quantachrome Instruments,
USA) by N2 absorption at 77.3 K. In sterile physiological
solution, TiO2 tended to aggregate and clustered from 183.7
to 282.0 nm and from 575.6 to 1018.9 nm.
The contrast agent used in this study was Compound
Meglumine Diatrizoate Injection (CMDI, ionic monomer
iodic contrast agent, iodine concentration = 370 mg/mL;
Shanghai Xudong Haipu Pharmaceutical Co., Ltd, Shanghai)
consisting of 32 mg/mL sodium diatrizoate and 268 mg/mL
meglumine diatrizoate. The ultrapure water was prepared
with a resistivity of 18.2 M cm (PureLab Plus, Pall, USA).
Phenylmethanesulfonyl uoride (PMSF) was provided by
Roche. All other reagents used in this study were at least of
analytical grade.
2.2. Animals. Male Sprague Dawley rats with 180200 g
body weight (about 7-8 weeks old, Experimental animal
center of Peking University) were housed in polycarbonate
cages placed in a ventilated, temperature-controlled room.
The standard conditions were supplied and maintained at
20 2 C room temperature, 60 10% relative humidity,
and 12 h light/dark cycle. The commercial pellet diet and
distilled water for rats were available ad libitum. All procedures used in these animal studies were compliant with
the local approved protocols of the Administration Oce
Committee of Laboratory Animal. Animals were acclimated
to this environment for ve days prior to treatment.
2.3. Experimental Protocol. We prepared TiO2 suspension
using physiological saline solution at 2 mg/mL. Briey, the
powdered TiO2 nanoparticles were dispersed in the fresh
sterilized physiological saline solution, and the suspension
was ultrasonicated for 10 min in 4 C at 200 W to disperse
completely as much as possible.
The animals experiments were set at four time intervals
(postexposure days 1, 7, 14, and 30) to evaluate the change
of articular cartilage thickness and morphology. Based on
our previous study [24], both control and nanoparticlesexposed rats were included (10 rats per group) in each time
interval because the intra-articular nanoparticles would be
disseminated to other tissues. The dosage of 2 mg/kg was
selected, which is lower than the detected Ti particles in
patients [35]. Before treatment, animals were anesthetized
by 30 mg/kg bw i.p. sodium pentobarbital (Germany). The
furs on bilateral hind knees were shaved softly after soaking
with soaps liquid, and the povidone iodine was applied
to prevent infection. The two hind knee joints were intraarticular injected with 100 L of 2 mg/mL TiO2 suspensions
every other day for 4 times, respectively. TiO2 suspension
was vortexed for 3 min before injections. The equal volume
physiological saline solution was given to the control rats.
Following the exposure, all rats were held for drink and
food ad libitum. The daily activity and body weight of all
rats were recorded carefully. At postexposure days 1, 7, 14,
Convert to
longitudinal slice
Segment by
manual
B: transverse slice
Reconstruction
Calculation
Volume
Thickness
A: scout image
E: 3D cartilage model
Figure 1: Scanning, segmenting, and remodeling of articular cartilage in distal femur. (A): scout image of distal femur obtained by -CT;
(B): transverse slice of distal femur; (C): sagittal slice of distal femur; (D): the green part is cartilage; E: 3D cartilage model.
and 30, the hind knee joints were collected both in the
control and exposed group. To cut o the peripheral muscle
and ligament carefully, six distal femurs from three rats per
group were xed in 10% formalin solution for histopathological analysis. Three fresh cartilages from three rats per
group were immediately immersed in 2.5% glutaraldehyde
at 4 C for transmission electron microscopy observation.
The remainder distal femurs were cut transversely at the
midpoint of the femoral neck, wrapped in sterilized gauze
which was soaked with phosphate buered saline (PBS),
and then stored in 20 C. To protect the cartilage from
degeneration, 0.1 mmol/L PMSF was used in PBS.
2.4. Determination of Contrast Agent Concentration. The
concentration of contrast agent is very important for
distinguishing the cartilage and calcied bone tissue, segmenting the cartilage contour accurately, and remodeling
the cartilage. To determining the optimal contrast agent
concentration, the contrast agent CMDI was diluted in
dierent concentration by PBS solution. The four distal
femurs from 10-week-old rats additionally was incubated in
5 mL tube containing 20%, 30%, and 40% CMDI dilution
of PBS for 10 min at 37 C, then immediately transferred to
a -CT system for scanning, respectively. All scanning were
carried out at 70 kV, 142 A, and with 18 m isotropic pixel
size.
2.5. Cartilage Scanning and Remodeling. Based on the above
determined contrast agent concentration, the incubation
in 30% CMDI for 10 min at 37 C was selected as the
best protocol. The freezed distal femurs were thawed at
37 C, incubated in 30% CMDI for 10 min, and then
scanned with 18 m isotropic pixel size using SkyScan 1076
microtomograph (Aartselaar, Belgium) at 70 kV, 142 A. The
whole procedure of scanning, segmenting, and remodeling
of articular cartilage was shown in Figure 1. The specimen
tube was xed on object bed at horizontal level. After
preview, the 35 200 mm area was scanned with the sourcedetector pair rotating with 0.02 /min/step. To enhance tissue
features in image, aluminum 1.0 mm physical lter was
selected to absorb the low-energy X-ray. The transverse

slices of distal femur were reconstructed using cone-beam
reconstruction program and transformed to sagittal slices
using DataViewer software package (Aartselaar, Belgium).
In order to accurately partition the contrast agent, articular
cartilage, and calcied bone, the cartilage contour was
segmented by manual according to the CT value. Finally, the
3D cartilage model was reconstructed (Figure 1(e)). The 3D
cartilage model was imported into the 3D software Geomagic
Studio (Raindrop Geomagic Inc., USA) and to calculate the
volume of articular cartilage. Because the change of articular
cartilage thickness occurred at the femoral weight bearing
sites [33], the cartilage thickness was determined at six points
on the superior load-bearing aspect of the medial condyle
and lateral condyle of femur by virtually sectioning 3D
cartilage model at the desired sagittal plane. All scans and
analyses were performed by a single-experienced operator.
2.6. Needle Probe Testing. The distal femurs used in -CT
scanning were potted in dental resin using a cylindrical pot
and then used to measure cartilage thickness by needle probe
testing. The potted specimen was mounted on a specially
designed apparatus that could adjust the articular cartilage
surface precisely in ve degree-of-freedom directions (x,
y, x, y, and z) to perpendicular to the needle probe
(Figure 2(a)). The apparatus was positioned on the base
groove of an Autograph AG-IS material testing machine
(Shimadzu, Japan). Once positioned, the assembly was
locked strictly to provide enough rigidity through the tests.
Articular cartilage thickness was measured by slow
(0.03 mm/min) insertion of a blunt needle probe (0.5 mm in
diameter) attached to a 50 N load sensor (sensitivity: 0.25 N).
All tests were conducted at room temperature. In whole
test procedure, the cartilage surface was kept hydrated with
PBS-containing PMSF. The load and displacement outputs
were recorded at 0.05 sec interval as the probe penetrated
into the cartilage tissue. A change of the slope of the loaddisplacement curve indicated the probe penetrated from
the cartilage to the calcied bone. Cartilage thickness was
measured using the probe to sense the moments when the
z
Load sensor
Lock
y
z
x
3.0
Load probe
y
2.5
Pot
Femur
Locknut
Locknut
Longitudinal guide
Load (N)
2.0
Hinge
1.5
Bone
surface point
1.0
0.5
Cartilage thickness
Cartilage
surface point
0.0
Lateral guide
0.0
(a)
0.1
0.2
0.3
Displacement (mm)
(c)
0.4
0.5
(b)
Figure 2: (a) the sketch of specially designed apparatus with ve degree-of-freedom direction, (x, y, x, y, and z) adjustment of articular
cartilage surface perpendicular to the load probe (b) the six points on the superior load bearing aspect of the medial condyle and lateral
condyle of distal femur, which is subjected to needle probe tests (c) the representative load-displacement curve.
probe pressed the articular surface and when it contacted

the calcied bone (Figure 2(c)). Needle probe testing was
performed at six points on the medial condyle and lateral
condyle of femur (Figure 2(b)) in corresponding to the sites
on 3D cartilage model.
2.7. Histopathology Examination of Articular Cartilage. The
distal femurs were xed in 10% formalin solution, decalcied
with 10% nitric acid for 24 h, and rinsed by tap water for
4 h. And then, the histopathological tests were performed
using standard laboratory procedures. Briey, the tissues
were dehydrated in graded series of 80%, 90%, 95%, and
100% ethanol, followed by clearing in toluene, inltrated
in hot liquid paran, nally embedded in paran blocks
to allow for 5 m sections, and mounted onto the glass
slides. They were stained with hematoxylin-eosin (H&E) for
microscopic analysis. All sections were observed, and the
photos were taken using optical microscope (Olympus BX51,
USA). The identity and analysis of pathology sections were
blind to the pathologist.
2.8. Ultrastructure of Cartilage by Transmission Electron Microscopy. The fresh cartilage was carefully cut o by scalpel
and immediately immersed in 2.5% glutaraldehyde at 4 C.

After washing with PBS suciently, the cartilage was xed
with 1% osmium tetroxide, dehydrated in a graded series
of ethanol, embedded in araldite, and polymerized for
24 h at 37 C. Ultrathin sections (50 nm) were cut with
ultramicrotome (LKB-V, Sweden), contrasted with uranyl
acetate and lead citrate, and observed with TEM (H-600,
Hitachi).
2.9. Statistical Analysis. For statistical analysis, all data are
expressed as mean standard deviation (SD). The oneway analysis of variance (ANOVA) was performed to analyze
the signicance using the statistical software SPSS 13.0 for
windows. A LSD post hoc multiple comparison test was used
for dierent groups. P < 0.05 was considered as the statistical
signicance.
3. Results
3.1. Concentration of Contrast Agent. According to the dierent X-ray attenuation (CT value) of contrast agent, cartilage
and calcied bone, the optimal contrast agent concentration
was determined. Figure 3 showed the representative saggittal
30% CMDI
150
133
100
78
50
200
X-ray attenuation
X-ray attenuation
221
200
40% CMDI
198
178
150
90
100
50
0
0
Contrast Cartilage Bone
agent
200
X-ray attenuation
20% CMDI
200
148
150
100
106
50
0

agent

agent
Figure 3: The representative sagittal slice of distal femur in 20%, 30%, and 40% CMDI for -CT in the rat model, the average X-ray
attenuation of contrast agent, articular cartilage, and calcied bone in the corresponding CMDI. Yellow, red, and purple arrows refer to the
contrast agent, articular cartilage, and calcied bone, respectively.
slice of distal femur in 20%, 30%, and 40% CMDI for CT in the rat model and the average X-ray attenuation of
contrast agent, articular cartilage, and calcied bone in the
corresponding CMDI. In 20% CMDI, the average X-ray
attenuation of femoral articular cartilage and calcied bone
was 78 and 221, respectively. This contrast dierence was
enough to segment the cartilage from calcied bone, but
not enough to dierentiate the cartilage from contrast agent
accurately. In 40% CMDI, the average X-ray attenuation
of cartilage was 106, which was close to that of calcied
bone (148). Therefore, it was dicult to distinguish between
the cartilage and calcied bone in segmenting by manual.
However, when the distal femur was incubated in 30%
CMDI, the average X-ray attenuation of contrast agent,
cartilage, and calcied bone was 178, 90, and 198, respectively, which provided the appropriate contrast dierence for
accurately segmenting the cartilage from the contrast agent
% reduction =
and calcied bone. In the following scanning, therefore, all

the distal femurs were incubated in 30% CMDI to remodel
the cartilage.
3.2. Thickness and Volume of Articular Cartilage Determined
by -CT. According to the above-determined concentration
of contrast agent, the incubation in 30% CMDI for 10 min
at 37 C was selected as the best protocol. Three distal femurs
per group were scanned to obtain the 3D cartilage model.
The thickness of articular cartilage exposed to TiO2 nanoparticles were calculated and shown in Figure 4. At day 1 after
exposure to TiO2 nanoparticles, the thickness of articular
cartilage was 0.2754 0.0207 mm, which was smaller than
that of the corresponding control (0.2942 0.0150 mm).
The changes in cartilage thickness were calculated comparing
with that in the corresponding control at each particular time
point. The % reduction was calculated as follows.
thickness in the corresponding control thickness in TiO2 exposed rats

100
thickness in the corresponding control
At postexposure days 1, 7, 14, and 30, the thickness

of articular cartilage was reduced with the rate of 6.41%,
4.52%, 8.64%, and 11.03%, respectively. Comparing with
the corresponding control, the signicant dierence was
(1)
detected in rats at days 7, 14, and 30 after exposure to TiO2

nanoparticles (P < 0.05) (Figure 4).
Using the 3D cartilage model, the volume of articular
cartilage covered on the distal femur was measured and
0.35
Thickness of articular cartilage (mm)
Thickness of articular cartilage (mm)
0.35
0.3
0.25
0.2
0.15
0.1
0.3
0.25
0.2
0.15
0.1
0.05
0.05
14
30
(Days)
14
30
(Days)
Control
Exposed
Control
Exposed
Figure 4: Thickness of articular cartilage in the distal femur by

3D cartilage model. P < 0.05 compared with the corresponding
control.
12
Volume of articular cartilage (mm3 )
Figure 6: Thickness of articular cartilage in the distal femur by

needle probe testing. P < 0.05 compared with the corresponding
control.
[34, 35], which could be due to the endochondral bone

development and brillation under function adaptation or
physiological adaptation in rats during normal growth.
10
8
6
4
2
0
1
14
30
(Days)
Control
Exposed
Figure 5: Volume of articular cartilage in the distal femur by

3D cartilage model. P < 0.05 compared with the corresponding
control.
illustrated in Figure 5. At postexposure days 1 and 7, the

volume of cartilage showed a little decrease compared to
the corresponding control (P > 0.05); whereas, at days 14
and 30, the signicant reduced cartilage volume was detected
(P < 0.05). It indicated that the growth of articular cartilage
might be disturbed by TiO2 nanoparticles existed in the joint
cavity.
For the control rats at dierent time points, we determined that the thickness and volume of articular cartilage
decreased with the rat age. This was important in cartilage development and consistent with the reported results
3.3. Thickness of Articular Cartilage Detected by Needle

Probe Testing. In testing, the load-displacement curves were
recorded as the load probe penetrated to the cartilage with
0.03 mm/min. According to the slope change of the loaddisplacement curve, the thickness of articular cartilage in
distal femur of rats in each group was determined and
illustrated in Figure 6. For the control rats, the thickness
of articular cartilage became reduced with the rat age
increase, which was consistent with the results obtained
by the 3D cartilage model. For the rats exposed to TiO2
nanoparticles, the cartilage thickness showed the signicant
decrease compared to the corresponding control (P < 0.05)
at postexposure days 1, 7, 14, and 30, respectively.
These results showed a strong linear correlation (r =
0.928, P < 0.0001, n = 48) with that determined by
the 3D cartilage model (Figure 7), which suggested that the
determination of cartilage thickness obtained both by 3D
cartilage model and by needle probe testing was accurate and
creditable.
3.4. Morphology Change of Articular Cartilage. In whole exposure and postexposure period, animals were given food
and water ad libitum, no abnormal daily activity was observed. After sacricing the rats, the smooth and moist
knee cavity including synovial capsule were observed in the
control rats; whereas, the white particles-xanthoproteic complexes were observed in the synovial joint capsule of exposed
rats, which indicated the deposition of intra-articular TiO2
particles. With the time prolong from the postexposure days
1 to 30, the deposited particles-xanthoproteic complexes
were reduced, as shown in Figure 8.
Thickness by 3D cartilage model (mm)
0.33
0.3
0.27
y = 0.0148 + 0.9436x
r = 0.928
n = 48
P <0.0001
0.24
0.21
0.18
0.15
0.15
0.18
0.21
0.24
0.27
0.3
Thickness by needle probe testing (mm)
0.33
Figure 7: The linear correlation between the values of cartilage

thickness obtained by 3D cartilage model and needle probe testing.
The correlation function y = 0.0148 + 0.9436, and P < 0.0001
showed a strong linear correlation (r = 0.928).
The histopathology of articular cartilage by H&E staining

was shown in Figure 9. Both in the control and exposed
rats, the articular cartilage had the intact perichondrium and
homogenous cartilage matrix. Depending on the arrangement of chondrocytes and collagen bres, articular cartilage
is divided into several zones including the tangential layer,
the transitional zone, the radial zone, and the calcied
cartilage layer. In the control, the matrix of the calcied
cartilage layer stained slightly darker than the matrix of
the other cartilage zones. However, at postexposure day 30,
the calcied cartilage layer was eosin-stained, which stained
lighter than that of the other groups. In the radial and
calcied zone of the cartilage, the chondrocyte was edema,
and the cell nucleus was shrinked. These implied that the
cartilage injury was induced by the intervention of deposited
TiO2 nanoparticles at day 30.
Figure 10 showed the ultrastructure of articular cartilage
in the distal femur observed by TEM. After exposure
to TiO2 nanoparticles, the chromatin was condensed and
distributed over the fringe of nucleus, the nuclear membrane
was invaginated, endoplasmic reticulum was dilated, and
ribosomes were decreased in chondrocyte at day 1. The
rough endoplasmic reticulum had a lamellar arrangement in
the cytoplasm at days 7. The intense axons on the cell surface
were developed, and the mitochondria were rich and became
swollen in chrondrocyte at day 14. At postexposure day 30,
to our surprise, the endoplasmic reticulum ruptured, and the
fragments were distributed in the cytoplasm.
4. Discussion
Herein, the impact of TiO2 nanoparticles on the articular
cartilage in the knee joint was reported. By intra-articular
injecting the nanoscale TiO2 suspension, we observed that
there was some particles deposition in the knee joint of
rats. Using contrast-enhanced high-resolution -CT technology, we determined that the cartilage thickness decreased
signicantly at postexposure days 7, 14, and 30, which
has a strong linear correlation (r = 0.928, P < 0.0001)
with the results obtained by needle probe testing. It is
reported that the cartilage change would occur on the
medial condyle and lateral condyle of femur because of
the compression from weight [36]. Articular cartilage is the
smooth, glistening white tissue that covers the surface of all
the diarthrodial joints. The main structure of cartilage is the
Benningho collagenous bre (mainly type II collagen)
and the hydrated proteoglycan embedded in it to provide
the proper biomechanical function. In our previous study
[24], the intra-articular TiO2 nanoparticles resulted in the
synovium hypotrophy, oxidative damage, and inammation,
such as lymphocytes and plasma cells inltration and broblast proliferation. Some studies reported that the inammatory response occurred in synovium was involved in
regulating the remodeling of articular cartilage and aecting
the chondrocyte function, leading to a loss of cartilage and
erosion and weakness of the bones [25, 26]. It is to say that
the activated synovial broblasts attached to the pannuscartilage interface and released matrix-degrading enzymes,
such as matrix metalloproteinases and the proinammatory
cytokines (TNF- and IL-1) [26, 37]. The matrix-degrading
enzymes would inhibit the synthesis of type II collagen
through regulating the chondrocyte and the aggregation of
proteoglycans [38]. The reduced extracellular matrix would
lead to the thinner articular cartilage. Therefore, in this study,
the signicant decreased articular cartilage thickness was
detected in the distal femur of rats exposed to intra-articular
TiO2 nanoparticles. As determined by the 3D cartilage
model, the cartilage thickness reduced about 4.52%, 8.64%,
and 11.03% at postexposure days 7, 14, and 30, respectively.
It needs to be pointed out that the thickness and volume
of articular cartilage in the control rats also showed a
reduction with age. In the weight-bearing joint, studies
reported that the cartilage thickness reduced with age both
in human [36, 39] and in horse [40]. This is important
in cartilage development and ascribed to the endochondral
bone development and brillation under functional or
physiological adaptation. The fetal cartilage is homogenous,
showing no site-dependent dierences. As the animal gets
older and cartilage maturates, cartilage becomes gradually
heterogeneous under the inuence of joint loading, showing
topographical variations in both thickness and compressive
stiness. It is worth to emphasize that Xie et al. [34] also
showed that the thickness and volume of cartilage in distal
femur decreased in rat during normal growth from 4 to 8
weeks, and even to 16 weeks.
As the only living element of the articular cartilage,
the chondrocyte holds a key position in the development
of cartilage. It produces the components of the matrix,
that is, collagens and proteoglycans. Therefore, besides the
above determination of the articular cartilage thickness,
the histopathology and ultrastructure of articular cartilage
were analyzed in this study to observe the change of
chondrocytes after exposure to TiO2 nanoparticles. Results
showed that the chondrocytes were edema and degenerated
Control
P
P
Exposed
P
F
F
Day 1
Day 7
Day 14
Day 30
Control
Figure 8: Photograph of knee joint cavity in rats after intra-articular injection of TiO2 nanoparticles. Control: the smooth and moist
knee cavity; exposed: the white particles-xanthoproteic complexes (arrows) in the synovial joint capsule of exposed rats, which indicate the
deposition of TiO2 particles. With the time prolong from the postexposure days 1 to 30, the deposited particles-xanthoproteic complexes
were reduced. P: patellar; F: femur.
Exposed
100
100
Day 1
100
Day 7
100
Day 14
100
Day 30
Figure 9: Microphotograph of articular cartilage in the distal femur exposed to TiO2 nanoparticles by H&E staining. Arrow indicates the
edema and degenerated chondrocytes in the radial and calcied zone at postexposure day 30.
in the radial and calcied zone at postexposure day 30. The

ultrastructural study of cartilage suggested the degeneration
of chondrocyte. More importantly, the mitochondria were
rich and became swollen in chondrocyte at postexposure
day 14; the endoplasmic reticulum were ruptured in the
chondrocyte at postexposure day 30. It is well known that
the endoplasmic reticulum plays an important role in the
hydroxylation and glycosylation of procollagen, and the
mitochondrion takes part in oxidative phosphorylation and

functions as the energy factory of cell. The ruptured endoplasmic reticulum would inhibit the synthesis of collagen and
glycosaminoglycans [41]. The reduction in matrix synthesis
may provide a potential explanation for the thinning of
articular cartilage observed in our study. Further, it is
reported that chondrocyte promotes the articular cartilage
loss because the surface receptors for cytokines respond to
Control
ER
10000
M
ER
Exposed
ER
M
ER
ER
ER
8000
10000
Day 1
Day 7
10000
Day 14
10000
Day 30
Figure 10: The ultrastructure of articular cartilage in the distal femur exposed to TiO2 nanoparticles. N: nucleaus; ER: endoplasmic
reticulum; M: mitochondria. After TiO2 nanoparticles exposure, the chromatin was condensed and distributed over the fringe of nucleus,
the nuclear membrane was invaginated, the endoplasmic reticulum was dilated, and the ribosomes were decreased in chondrocyte at
postexposure day 1. At day 7, a lamellar arrangement for rough endoplasmic reticulum was observed in cytoplasm. At day 14, the intense
axons on the cell surface were developed and the mitochondria were rich and became swollen in chondrocyte. At day 30, the endoplasmic
reticulum ruptured, and the fragments were distributed in cytoplasm.
the ligands with the production of prostaglandin E2 and

metalloproteinases in synovitis [42]. Of course, to unveil the
detailed mechanism of cartilage loss, it is necessary to further
research the inuence of nanoparticles on the synthesis of
extracellular matrix in cartilage, including the collagen and
proteoglycan.
5. Conclusion
In conclusion, after intra-articular injection of TiO2 nanoparticles, we determined that the thickness of articular cartilage was decreased using contrast-enhanced high-resolution
-CT technology, which had a strong linear correlation (r =
0.928, P < 0.0001) with the results obtained by needle probe
testing. The cartilage thickness was signicant decreased with
the rat age, and the same trend was observed in cartilage
volume. The analysis of morphology and ultrastructure of
articular cartilage indicated the chondrocyte was degenerated. Results suggested that the articular cartilage is a
potential target for wear particles in knee joint.
Acknowledgments
This paper is nancially supported by the National Natural Science Foundation of China (Grant nos. 30800217,
10925208, and 11032012) and the Postdoctoral Science

Foundation of China (20080430304).
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Journal of Nanomaterials

Biocompatibility and Toxicity of

Biocompatibility and Toxicity of

Copyright 2012 Hindawi Publishing Corporation. All rights reserved.

Do Kyung Kim, Korea

Nairong Tao, China

Hindawi Publishing Corporation

Beihang University, Beijing 100191, China

Correspondence should be addressed to Xiaoming Li, x.m.li@hotmail.com

The nanodimensionality of nature has logically given rise

downstream stem cellular response, without exogenous

and cause oxidative stress or preinammatory cytotoxic

phosphate ceramics, Journal of Biomedical Materials Research

Hindawi Publishing Corporation

in various biological events is still largely unknown; however,

2. Materials and Methods

Figure 1: CD spectrum of surfactin in PBS.

was measured by AVIV 215 spectropolarimeter in dierent

plates. After overnight incubation, the cells were treated with

concentration of surfactin that bursts 50% of RBC [10] was

4. Discussion and Conclusion

ionic aqueous solutions. Surfactin nanomicelles in PBS show

the enzyme activities. In addition, several biological activities

[3] H. Kell, J. F. Holzwarth, C. Boettcher, R. K. Heenan, and J.

Hindawi Publishing Corporation

Correspondence should be addressed to S. Vijayakumar, vijaysk.research@gmail.com

Microstructure is essential for inductive bone formation

2. Materials and Methods

Average size Plasmon wavelength

in 1 mL DI water) was added, followed by the addition

Zeta potential (mV)

0.0225 g of starch in 6 mL of DI 0.1 mL of 0.1 M, NaAuCl4

0.02 mL of 0.1 M, THPAL

0.02 mL of 0.1 M, THPAL

buer, 0.0027 M KCl and 0.137 M NaCl) and 1 L fresh

Zeta potential (mV)

After the incubation, the cells were washed twice with

conversion results in the reduction of the tetrazolium salt

the nanomaterials for 24 hours. The gold nanoparticles were

Cell viability (%)

Cell viability (%)

The absorbance of the produced formazan at 490 nm

cell survival at dierent concentrations of gold nanoparticles

Cell viability (%)

Cell viability (%)

all the gold nanoparticles are viable to cells and because

motility and inuencing the cell morphology. On contrary,

[2] D. L. Feldheim, A. F. Colby, and D. Marcel :, Metal

Hindawi Publishing Corporation

[5]. Examples include the transplantation of mesenchymal

safe and eective noninvasive means of imaging anatomical

MRI or uorescent tracking:

shell thus resulting in uniform gold-coated MNP while still

engraftment; migratory patterns after transplantation; ideal

2. Safety and Biocompatibility of

apoptosis [11]. On the other hand, ROS can be used in cancer

nanomaterials in conjunction with regenerative medicine.

Cells maintained viability and retained label for

Did not aect cell viability