RSC Advances
PAPER
Cite this: RSC Adv., 2015, 5, 84565
Engineered regenerated bacterial cellulose
scaffolds for application in in vitro tissue
regeneration
Shaukat Khan,a Mazhar Ul-Islam,ab Muhammad Wajid Ullah,a Muhammad Ikram,c
Fazli Subhan,c Yeji Kim,a Jae Hyun Jang,a Sik Yoonc and Joong Kon Park*a
In this study, we sought to synthesize regenerated bacterial cellulose (rBC) scaffolds for application in
in vitro tissue regeneration. Bacterial cellulose (BC) was dissolved in N-methyl morpholine-N-oxide
(NMMO), and salt crystals were added as porogens, followed by casting in molds and incubation in
water. The synthesized scaffolds were characterized using Fourier transform infrared (FT-IR)
spectroscopy and field-emission scanning electron microscopy (FE-SEM). The FT-IR spectra exhibited
bands characteristic for BC in rBC scaffolds, indicating no alteration in chemical structure, while FE-SEM
revealed a porous structure of the rBC scaffold. The scaffolds exhibited very high swelling ratio,
indicating enhanced water absorption and nutrient exchange capacity. The in vitro biocompatibility of
the rBC scaffolds was tested based on the adhesion, growth, and proliferation of animal fibroblasts (NIH
Received 22nd August 2015
Accepted 30th September 2015
3T3), and the osteogenesis of animal osteoblasts (MC3T3-E1). Results indicated good cell adhesion,
DOI: 10.1039/c5ra16985b
penetration, and proliferation. Alkaline phosphatase (ALP) activity and Alizarin red staining (ARS) revealed
osteogenic differentiation of animal osteoblasts on the scaffolds. These results demonstrate that the rBC
www.rsc.org/advances
scaffolds are potential candidates for future tissue engineering applications.
Introduction
Tissue engineering offers new perspectives in medical treatment through the reconstruction of damaged and diseased
organs. This approach involves a three dimensional (3-D) matrix
serving as a template to support the inltration, growth,
differentiation, and proliferation of cells to form the targeted
tissue or organ.1 This 3-D matrix, known as a scaffold, must be
nontoxic and biocompatible, with the appropriate surface
chemistry for easy cell adhesion, differentiation, and proliferation; the scaffold morphology should resemble the micro scale
architecture of the native extracellular matrix (ECM).2 Also, the
pores within the 3-D matrix should be uniform and interconnected to facilitate cellular inltration, vascularization,
exchange of nutrients and metabolic wastes, and should also
have adequate mechanical properties to facilitate tissue
formation with structural integrity.3,4
The 3-D scaffolds are commonly fabricated using natural and
synthetic polymers, such as cellulose, gelatin, chitosan, collagen,
a
Department of Chemical Engineering, Kyungpook National University, Daegu,
Republic of Korea. E-mail: parkjk@knu.ac.kr; Fax: +82 539506615; Tel: +82
539505621
b
Department of Chemical Engineering, College of Engineering, Dhofar University,
Salalah, Sultanate of Oman
c
Department of Anatomy, Pusan National University School of Medicine, Yangsan,
Gyeongsangnam-do, Korea
This journal is © The Royal Society of Chemistry 2015
polyglycolic acid (PGA), and polycaprolactone (PCL).5 Cellulose is
the most common and most important biopolymer that is
naturally available. It is considered an unlimited raw material,
meeting the growing demand for green and biocompatible
products as it is safe, stable, biodegradable, nontoxic, inexpensive, and renewable.6 In particular, bacterial cellulose (BC) is
a highly pure biopolymer, produced by a group of acetic acid
bacteria that does not contain biogenic impurities like hemicelluloses, lignin, and pectin.7 The biosynthesis of BC is
a complex process, resulting in a 3-D gelatinous nanobrillar
hydrogel on the surface of the growth medium. The cellulose
chain molecules are connected to each other primarily via strong
hydrogen bonding and van der Waals forces, resulting in
elementary brils that combine to make larger microbrils,
which in turn assemble into bers.8 The microstructures formed
by the ultrane microbrils of BC are 1 to 9 mm in length,
resulting in a dense, reticulated structure that is stabilized by
strong hydrogen bonds. These networks exhibit high crystallinity (around 60%) and a higher degree of polymerization
(16 000 or 20 000) compared to the plant cellulose.9 The regular
crystalline arrangement of the glucan molecules results in the
distinct diffraction pattern, reactivity, and swelling of BC.9
BC has found various applications in food, paper and electronic industries. BC combines unique mechanical and structural properties with high porosity, high water holding capacity,
in situ moldability, permeability for gas and uid exchange,
biocompatibility, and nontoxicity, elevating the demand for BC
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applications, especially in biomedical elds.10 BC has been
patented for use as articial blood vessels and temporary skin
substitutes (Bioll and BASYC). Additional potential biomedical
applications of BC include vascular gras, articial skin, bone
regeneration, cartilage replacement, articial cornea, and as
tissue engineering scaffolds.11 BC has recently had great success
in tissue regeneration studies in vitro and in vivo.
The surface properties of scaffolds, including morphology,
wettability, chemistry, surface charge, and presence of hydrophobic and hydrophilic functional groups, play an important
role in cell–material interactions.12 Surface modication of BC
has led to considerable improvement in its biocompatibility. BC
with its surface modied by treatment with plasma containing
nitrogen exhibited greater cell adhesion properties when
compared to pure BC.13 In another study, the BC surface was
modied by the introduction of cationic and anionic charges.
Improved adhesion of MG63 cells was observed on the cationic
BC, compared to the native and anionic counterparts.14 A BC
surface coated with ECM proteins resulted in increased adhesion of mesenchymal and neuronal cells.15 Improved adhesion
of broblast cells was observed on a BC surface coated with
a cellulose-binding matrix fused with Arg–Gly–Asp.16 Scientists
have also augmented the microporosity of BC for better cell
inltration. For example, in situ production of BC on a honeycombed-patterned agarose lm resulted in the fabrication of
BC with a regular, porous structure.16 Similarly, BC scaffolds
with high porosity and interconnectivity were fabricated by
adding paraffin and starch microparticles to BC harvesting
media.17 However, it is difficult to control the BC to microparticle ratio, as well as the shape of the scaffold produced by this
method.
A porogen/particulate leaching method has also been
applied by several researchers in the fabrication of scaffolds
using biopolymers. Porogens are either placed in a mold followed by polymer casting, or suspended in the polymer solution
and then casted in a mold. This step is followed by porogen
leaching and solvent removal, leaving behind the porous polymer scaffold. Porogen materials normally used include salt,18
paraffin,19 ice,20 gelatin,21 and sugar.22 The primary concern with
this fabrication method is the removal of porogen particles
from the casted scaffolds. Therefore, this method is only
considered suitable for the fabrication of very thin 3-D
membranes. Besides its limitation to very thin scaffolds, its
disadvantages also include the use of toxic solvents, the long
time required for solvent removal (up to weeks), and the
residual porogen particles in fabricated scaffolds.23
In the present study, we report a simple approach for the
preparation of 3-D rBC scaffolds through a combined solvent
casting and particulate leaching method, applying NMMO as
a solvent and salt crystals as porogens. This fabrication route
not only enables us to control the BC to porogen ratio and the
shape of the rBC scaffold, but also ensures the quick and
complete removal of solvent and porogen when incubated in
water. The resulting scaffolds are highly porous and exhibited
an excellent swelling ratio in water. Our in vitro assessment of
their biocompatibility with animal cells had promising
results.
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Materials and methods
Materials
Glucose, sodium hydroxide (NaOH), phosphate buffered saline
(PBS), glutaraldehyde, acetic acid, and succinic acid were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Yeast
extract and peptone were purchased from Becton, Dickinson
and Company (Le Pont de Claix, France). The reagents were
used without any additional processing.
Preparation of BC sheets and synthesis of 3-D rBC scaffolds
Gluconacetobacter hansenii PJK (KCTC 10505BP) was cultured
using a basal medium, while the BC sheets were prepared as
previously reported by our group.10 The concentration of
glucose, yeast extract, peptone, acetic acid and succinic acid in
the basal medium was 10 g L 1, 10 g L 1, 7 g L 1, 1.5 mL L 1
and 0.2 g L 1, respectively. 1 M NaOH solution was used to
adjust the pH of the medium to 5.0. The media was then
sterilized at 121 C for 15 min. G. hansenii PJK colonies were
inoculated in the medium and incubated at 30 C and 150 rpm
for 24 h. Sterilized rectangular container (30 cm 30 cm 10
cm) was used for the preparation of BC sheets. The basal
medium was inoculated with 5% pre-culture and incubated
under static conditions at 30 C for 10 days. The prepared BC
sheets were treated with 0.5 M NaOH at 121 C for 15 min,
followed by washing with distilled water. The 3-D rBC scaffolds
were synthesized through a solution casting method using
sodium chloride (NaCl) crystals as porogens. Briey, BC was
powdered and then dissolved in NMMO (4% by weight). NaCl
was also powdered and sieved (crystal size ¼ 200–400 mm)
before adding to the BC solution (BC to salt ratio was 1 : 3).
The BC–salt mixture was casted into a mold and allowed to
solidify, followed by salt leaching and solvent removal by
incubating in water at room temperature. A BC solution casted
without porogen served as a control (Fig. 1b). The scaffolds
were freeze-dried aer salt leaching and solvent removal was
complete.
Characterization
Fourier transform infrared (FT-IR) spectra of the freeze-dried
rBC and 3-D rBC scaffolds were recorded using a Spectrum GX
& Auto Image. FT-IR spectrophotometer [spectral range: 4000–
400 cm 1; beam splitter: Ge coated on KBr; detector: deuterated
triglycine sulfate; resolution: 0.25 cm 1 (step selectable); PerkinElmer, Melville, NY, USA]. The samples were mixed with KBr
and formed into pellets prior to FT-IR analysis (IR grade; Merck,
Darmstadt, Germany). Field-emission scanning electron
microscopy (FE-SEM) images of the freeze-dried rBC and 3-D
rBC scaffolds were obtained using Hitachi S-4800 (Tokyo, Japan)
and Horiba EDX-350 instruments (Tokyo, Japan). Briey,
samples were xed on a brass holder and then coated with
osmium tetroxide using a VD HPC-ISW osmium coater (Tokyo,
Japan) prior to FE-SEM observations. Scaffold photographs were
obtained using an Olympus digital camera (Model 5050,
Olympus, Tokyo, Japan).
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RSC Advances
having fetal bovine serum (FBS) (10%), penicillin (100 U mL 1)
and streptomycin (100 U mL 1). The in vitro biocompatibility
assessment was carried out by cutting the scaffolds into round
pieces (diameter ¼ 20 mm and thickness ¼ 1 mm) and autoclaved at 121 C for 15 min for sterilization. Aerward, the
samples were incubated in cell culture medium for 24 h. NIH
3T3 (2.7 104 cells per sample) were cultured on the scaffolds,
followed by cell adhesion, proliferation, penetration, and cytotoxicity assessments.
In vitro cell viability and spreading characterization
The attachment and spreading of NIH 3T3 cells on the simple
rBC and 3-D rBC scaffolds were evaluated by FE-SEM. Cell
morphology was observed aer 1 day, 1 week, and 3 weeks of
culture. The cell cultured scaffolds were washed thrice with PBS
and xed with 2.5% glutaraldehyde for 10 min. The scaffolds
were again washed repeatedly with PBS, followed by drying prior
to FE-SEM observations.
Confocal laser scanning microscopy
Fig. 1 Images of freeze dried simple and 3-D rBC scaffolds with cm
scale in background (a). Surface and cross-sectional morphology of
the scaffolds (b).
Swelling behavior and mechanical properties of the scaffolds
The swelling behavior of the scaffolds was examined to determine their water absorption as previously described.24 The
known dry weight of each scaffold (wd) was soaked in water at
room temperature, and weighed (ww) aer soaking for a determined length of time. The swelling ratio (SR) was calculated as:
SR ¼ (ww
wd)/wd 100
where wd is the dry weight and ww is the wet weight of scaffold at
a particular time.24
The mechanical properties of the simple rBC and 3-D rBC
scaffolds were measured using an Instron Universal Testing
machine (Model 4465, USA) according to the procedure
described by the American Society for Testing and Materials
(ASTM D 882). Briey, two metal clamps were placed at either
end of each 10 cm 1 cm rectangular strip of dried sample with
a thickness of 1 mm. The clamps were then mounted on an
Instron 4465 that measured both elongation and maximum
tensile load before fracture. The average values were taken from
at least triplicate experiments.
Cell culture
Animal broblasts (NIH 3T3) were cultured in a 5% CO2, humid
atmosphere at 37 C in minimum essential medium (MEM)
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Confocal microscopy analysis was performed as previously reported.25 Briey, animal broblasts (NIH 3T3) were seeded on
3-D rBC brous scaffolds at 1 104 cells per scaffold, and
cultured for 7 days with occasionally changing media. The
cultured cells were washed with PBS, and xed with 4% paraformaldehyde for 15 min at 4 C. Subsequently, the xative was
removed with cold PBS, followed by permeabilization with 0.1%
Triton X-100 in PBS for 5 min. The cells were again washed with
cold PBS followed by incubation with 1% bovine serum albumin
(BSA, Sigma-Aldrich) for 60 min at ambient temperature. Excess
solution was shaken off, and the cells were incubated for 1 h at
room temperature with uorescein isothiocyanate (FITC)–
phalloidin (diluted 1 : 100; Promega, Madison, WI, USA). The
cells were then rinsed in cold PBS and mounted on glass slides
using Vectashield® containing 4,6-diamidino-2-phenylindole
(DAPI) (Vector Laboratories, Burlingame, CA, USA). Fluorescence of cells was observed using a confocal laser scanning
microscope (Olympus FV1000-IX81).
Confocal image acquisition
Confocal microscope (Olympus FV1000-IX81) equipped with
a 40 oil-immersion objective was used for image recording.
The excitation and emission parameters wavelengths for Alexa
488 were xed as: 488 nm and 500–540 nm, respectively, and for
DAPI as 358 nm and 461 nm, respectively. Gains were adjusted
to avoid saturation in pixel intensity.
3-D reconstruction
3-D reconstruction involves the creation of a 3-D model from
a set of recorded 2-D images. When multiple images are available, then the position of a 3-D reconstruction point is the
intersection of the respective projection rays. The relations
between multiple images show that corresponding sets of
points contain a structure which is related to the calibration
and poses of the camera. The respective 3-D reconstruction
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image of the 3-D rBC scaffold was recorded as; stacks of
confocal images were collected through the scaffold with step
size of 5 mm between adjacent optical planes, starting from one
pole of the scaffold. Aer thresholding, this stack was used to
generate a 3-D animation sequence by using the 3-D projection.
Alkaline phosphatase activity
The differentiation of osteoblast cells was evaluated by the
expression of alkaline phosphatase (ALP) activity as reported
previously.26 Briey, aer 5 and 10 days culture of animal
osteoblast (MC3T3-E1) cells on rBC and 3-D rBC scaffolds, ALP
activity was determined using an ALP assay kit according to the
manufacturer's instructions (Sigma-Aldrich). The cells were
washed twice with PBS, and subsequently lysed through sonication using cell lysis buffer [10 mM Tris–HCl, containing 2 mM
MgCl2 and 0.05% Triton X-100 (pH 8.2)] at 4 C. Cell lysates were
then centrifuged at 10 000 rpm for 10 min at 4 C, and the
supernatant was utilized for the assays. Lysates were incubated
in ALP detection buffer for 30 min at 37 C. Then, 0.1 M NaOH
was added to stop the reaction, and the samples were monitored at 405 nm. Total protein content was measured spectrophotometrically using a Micro-BCA protein assay kit (Pierce,
Rockford, IL, USA) and read at 562 nm. The enzymatic activity of
ALP was normalized to the total protein content of the sample
(405/562 nm).
Alizarin red staining (ARS) of mineralized osteoblast cultures
This assay was performed using a previously reported method.26
Briey, cell cultures were washed two times with PBS, and then
xed with 2.5% glutaraldehyde for 15 min. Aer xation, cells
were washed with water and stained with 0.2% Alizarin red
(Sigma-Aldrich) in 2% ethanol for 15 min. Cells were then
washed four times with water, and dried on 37 C.
Cell toxicity
A 3-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay was performed to nd out the ratio of viable
cells during 1, 3, 5 and 7 days of cell culture on simple rBC and
3-D rBC scaffolds.27 Briey, cultures on the simple rBC and the
3-D rBC scaffolds were rinsed with PBS, followed by addition of
MTT solution (0.5 mg mL 1 in Dulbecco's medium without
phenol red) and incubated at 37 C for 4 h. Later, the supernatant was discarded and 200 mL of DMSO was added for 30 min
at 37 C to dissolve the formazan crystals. The optical density of
the formazan solution was recorded at 540 nm through
a microplate reader. The cells cultured on the simple rBC served
as a control.
Statistical analysis
All quantitative data were expressed as the mean standard
deviation. Statistical comparisons were performed using oneway ANOVA with SPSS 13.0 for Windows soware (SPSS Inc.,
Chicago, IL, USA). P values less than 0.05 were considered
statistically signicant.
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Results and discussion
Synthesis and macro- and microstructural characteristics of
the scaffolds
Highly porous rBC scaffolds were fabricated through a solvent
casting and particulate leaching method using NMMO as
solvent and salt as porogen materials. The inuence of the BC
concentration, the size of the salt crystals, and the BC to salt
ratio on the nal porous structure of the scaffold was preliminarily assessed: the optimal conditions in terms of structural
homogeneity were obtained with 4% BC in NMMO, 200–400 mm
salt crystals, and a BC to salt ratio of 1 : 3. Characterization of
the 3-D rBC scaffolds was carried out as a direct comparison
with simple rBC fabricated with the same BC concentration
without porogens. Fig. 1a depicts the macroscopic images of the
freeze-dried simple and the 3-D rBC scaffolds. The images
highlight the macroscopic morphology of the prepared scaffolds, indicating the greater porosity of 3-D rBC compared to
that of simple BC. The porosity of both samples was further
conrmed through SEM micrographs. The SEM images in
Fig. 1b depict the surface and cross-sectional morphology of the
fabricated scaffolds. The images conrm the compact structure
of simple rBC, due to the absence of porogens in the fabrication
process. In comparison, leaching out of porogens from the 3-D
rBC scaffolds le behind large interconnected pores. These
channels extended throughout the scaffold, as indicated by the
surface and cross-section SEM images (Fig. 1b).
The microstructure design, including pore size and porosity,
are crucial for the determination of cellular activities leading to
tissue regeneration. Uniform and interconnected pores facilitate the diffusion of cells, nutrients, and wastes, and oxygen
exchange, resulting in enhanced cellular ingrowth. Scaffold
porosity and pore size have been reported to have profound
effects on cell proliferation.28 Therefore, controlled porosity and
pore size are important factors in scaffold fabrication for
successful tissue regeneration applications. Among the scaffold
fabrication techniques, electrospinning is of particular interest
as it is an easy and suitable method for continuous and large
scale preparation of micro- and nanobers. Micro- and nanobers of innite length are produced from polymer solutions
under the action of electric eld. This fabrication process is
simple, low cost and offers the possibility to fabricate any
soluble polymer into ultrathin bers.29 However, the required
high voltage and poor cost to yield efficiency limits the applications of this process. Another feasible approach for microbers fabrication is the centrifugal spinning. This approach
applies the centrifugal force to shear the polymer solution into
bers. However, ber uniformity, quality and productivity are
the major issues with this method.30 Another technique
involving production of polymer bers is the solution or melts
blowing method. It involves the extrusion of polymer solution
or melts through an orice under high air pressure. The air
pressure causes the polymer solution or melts to elongate into
microbers. Gyration, the combination of centrifugal spinning
and solution blowing, offers not only the manipulation of the
ber diameter but also ber diameter distribution and ber
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length.30,31 However, both the electrospinning and gyration
methods involve processing of polymer solution at room
temperature. BC/NMMO solution is stable around 50 C and
solidies very quickly room temperature. Therefore, it is very
hard to fabricate rBC scaffolds through electrospinning and
gyration methods.
Our method involves a solvent casting/particulate leaching
method for rBC scaffold fabrication. The most common
problem with solvent-assisted particulate leaching methods is
the difficulty in complete removal of the porogen due to
encapsulation by the hydrophobic polymer. However, in our
case, the hydrophilic nature of BC allows the easy and complete
leaching of NMMO and porogen microparticles in water,
leaving behind a pure and highly porous rBC scaffold. The
macroscopic and SEM images conrm the successful fabrication of highly porous rBC scaffolds through our proposed
solvent casting/particulate leaching (NMMO/salt) method. This
fabrication technique is environmentally friendly, cost effective,
simple, and adaptable to different shapes and geometries.
The FT-IR spectra of native BC, simple rBC, and 3-D rBC are
depicted in Fig. 2. In these spectra, the stretching of –OH
groups, including –CH2–OH and –CH–OH, are observed around
3400 cm 1, while that of aliphatic saturated –C–H groups was
assigned to the peak around 2900 cm 1.32 The stretching band
of H–O–H adsorbed on cellulose is observed around 1650 cm 1,
while the absorption bands for C–H2 symmetric bending and
wagging, –OH wagging, and C–O–C pyranose ring skeletal
vibration are observed around 1417 and 1312, 1165, and
1060 cm 1, respectively.33 The FT-IR spectra of the simple rBC,
and 3-D rBC showed the characteristic absorption bands of
cellulose II. The band for C–H2 symmetric bending and wagging
observed at 1417 and 1312, respectively in BC spectra shied to
lower wave numbers in both the simple and 3-D rBC lms.
These results suggest that the intermolecular hydrogen bonding
involving the exocyclic OH groups is destructed during the
regeneration process. Also, the intensity of the characteristic
band for the amorphous region of BC at 895 cm 1 was increased
in simple and 3-D rBC lms compared to native BC as a result of
regeneration. These results proved the transformation of
cellulose I to cellulose II during the regeneration process.34
However, it is evident from the gure that the FT-IR spectra of
native BC and simple and 3-D rBC lms show absorption bands
for the same functional groups indicating no alterations in the
chemical structure of BC during regeneration. This indicates
that only physical changes occur during the dissolution and
regeneration process.
Swelling studies and tensile properties
The time dependent percent swelling of simple rBC and 3-D rBC
scaffolds is depicted in Fig. 3a. The swelling ratio of the simple
rBC increased to 169%, 251%, 272% and 423% at 10, 20, 30 and
60 min, respectively, and reached a saturation of 461% at 90
min. This gradual increase in the swelling ratio is likely attributed to the compact nature of simple rBC, allowing the water to
penetrate very slowly. In comparison, the swelling ratio of the 3D rBC scaffold increased rapidly to 833% in the initial 10 min,
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RSC Advances
Fig. 2
FT-IR spectroscopy of native BC, simple rBC and 3-D rBC.
and reached a saturation of 1314% in only 30 min. The significant swelling of the 3-D rBC scaffolds indicates the hydrophilic
character of BC and the porous structure of the sample
accommodating free standing water, while the rapid saturation
is attained due to its interconnected porous structure. BC is
known for its high water absorption capacity. It physically
entraps the water molecules on its surface, as well inside the
porous brillar network.35 This property contributes to its
potential numerous applications in the biomedical eld,
including wound healing, the treatment of skin burns, and
tissue regeneration.36 The high water holding capacity of our
scaffolds is highly favorable for biomedical applications,
allowing an easy and continuous transfer of nutrients to cells
throughout the tissue regeneration period. This would also be
helpful in absorbing the wound exudates that help in quick
healing. As the swelling behavior is dependent on the porosity
of rBC lms, it can be easily controlled by maintaining the pore
size for particular application.
Fig. 3b shows the tensile strength results of the simple and 3D rBC scaffolds. The maximum tensile strength at the breaking
point for simple rBC was higher than for the 3-D rBC. The
tensile strength for Simple rBC was found to be 4.39 MPa, while
that of 3-D rBC was 3.9 MPa as shown in Fig. 2. Similarly,
Young's modulus and average % strain for simple rBC was 0.85
GPa and 2.1% while that of 3-D rBC was 0.74 GPa and 1.92%,
respectively.
Mechanical properties such as tensile strength, Young's
modulus and % strain at break are particularly important for
scaffolds used in tissue engineering elds, because they determine the shape-retaining ability of the scaffolds during practical applications. Fig. 3b shows a decrease in the mechanical
properties of 3-D compared to the simple 2-dimensional (2-D)
rBC scaffolds. This decrease can be attributed to the high
porosity of 3-D rBC scaffolds compared to the simple rBC
scaffolds. These results are in complete agreement with
previous studies of Zhang and coworkers who constructed
poly(lactic-co-glycolic acid) (PLGA) 3-D scaffolds and determined their mechanical properties. They also found that the
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Fig. 4 SEM images indicating cell adhesion, growth and proliferation
on the simple and 3-D rBC scaffolds during 1 day, 1 week and 3 weeks
of incubation period.
simple rBC toward animal osteoblast and broblast cells.10,33
Studies have also demonstrated the superiority of 3-D over 2-D
cell cultures. The differentiation of broblast cells and formation of ECM was comparatively evaluated in 2-D and 3-D cell
culture models. It was concluded that the 3-D culture environment enhances cell proliferation and differentiation compared
to their 2-D counterparts.38 In the case of BC, the small pore size
of native BC and simple rBC lms restrict cell penetration and,
in turn, its application as a 3-D scaffold for tissue regeneration
applications. Therefore, we intended to enhance the porosity
and pore size of BC by fabrication of 3-D scaffolds with a solvent
casting/porogen leaching method. Our results revealed the
successful penetration of cells into the 3-D rBC scaffolds, indicating their potential application in the tissue regeneration
eld.
Fig. 3 Swelling ratio profiles of simple rBC and 3-D rBC during
incubation in water at room temperature (a), mechanical properties
including tensile strength, % strain and Young's modulus of Simple rBC
and 3-D rBC scaffolds (b).
Young's modulus decreased as the pore size of scaffold
increased and attributed this trend to the slight reduction in the
scaffolding materials when the pore size was increased.37
Cell adhesion, growth, and proliferation on scaffolds
Porosity and pore size play an important role in cell attachment
and proliferation in 3-D porous scaffolds. Cell adhesion,
growth, and proliferation on the simple and 3-D rBC scaffolds
were assessed by SEM (Fig. 4). The images revealed strong cell
adhesion on the surface of simple rBC aer one day, while cells
were attached inside the pores within the 3-D rBC scaffold aer
one day. Prolonged incubation of one and three weeks resulted
in lopodia formation in both the samples. The cells presented
2-D spreading on the surface of simple rBC, while the cells
occupied the pore walls to form ECM in the porous 3-D rBC
scaffolds. A clear difference was observed between cell growth
patterns on the simple rBC and the 3-D rBC samples presenting
multilayers and a much extended network of lopodia on the
later samples.
The biocompatibility of simple rBC lms is well known.
Previously, we reported the excellent biocompatible behavior of
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Morphological and immunocytological behavior
Morphological and immunocytological observation was carried
out using confocal microscopy to analyze cell morphology and
inltration into the 3-D rBC matrix. Fig. 5a depicts the differential interference contrast (DIC), DAPI, F-actin and DAPI/Factin merged images of stained broblasts cultured for three
weeks, indicating the growth and proliferation pattern in the 3D rBC scaffold. The cells exhibited excellent adhesion, growth,
and proliferation, as well as inltration, inside the 3-D scaffold.
These results are in complete agreement with the SEM images
obtained aer the same incubation time (3 weeks). Optical
slicing of the sample in the Z-direction through Z-stack confocal
microscopy allowed us to observe the penetration of cells into
the 3-D rBC scaffold. Fig. 5b and c contain the Z-stack images of
optical slices from top to bottom with a 5 mm slice thickness and
the reconstructed 3-D projection image of the scaffold. These
images clearly demonstrate the successful growth, proliferation, and inltration (upto 100 mm) of cells inside the 3-D rBC
scaffolds, indicating their suitability for tissue regeneration
applications.
Osteogenesis
ALP activity was used as an indicator of osteoblastic differentiation of MC3T3-E1 cultured on simple and 3-D rBC scaffolds. As
shown in Fig. 6a, the ALP activity of the cells on scaffolds
increased with incubation time. The relative ALP activity of
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simple rBC increased from 100% to 146% 12.6%, while that
of 3-D rBC scaffolds increased from 153% 10.4% to 195%
15.1% when incubated for one week and two weeks, respectively. The degree of ALP activity expressed by the 3-D rBC
scaffold was signicantly higher compared to that expressed by
simple rBC aer two weeks of incubation. We also investigated
the mineralization process using an ARS assay. Fig. 6b, depicts
the Alizarin staining results observed aer one and two weeks of
culture in both simple and 3-D RBC scaffolds. Mineralization
increased with incubation time, and both samples exhibited an
increased level of mineralization at two weeks. ARS levels were
signicantly greater with the 3-D rBC scaffolds compared to the
simple rBC (Fig. 6b). In the 3-D samples, osteoblasts exhibited
signicant calcium production and deposition onto the scaffolds. This data demonstrates the osteogenic nature of the 3-D
rBC scaffolds.
ALP is an enzyme widely recognized as an early marker of
osteogenic activity.39 During bone formation, ALP is responsible
Fig. 5 Confocal images of DAPI and F-actin stained fibroblast cells
cultured on 3-D rBC scaffolds for 3 weeks (a). Z-stack images obtained
from surface to bottom of the scaffold with 5 mm plane thickness are
shown (b). Reconstructed 3-D projection image of the scaffold (c). The
scale bar represents 100 mm.
This journal is © The Royal Society of Chemistry 2015
Determination of osteogenesis and mineralization through ALP
activity and ARS, respectively. Relative ALP activity of simple rBC and 3D RBC scaffolds (a), ARS of the scaffolds (b). Graph shows mean value
SD (P < 0.05).
Fig. 6
RSC Adv., 2015, 5, 84565–84573 | 84571
RSC Advances
for the generation of inorganic phosphate, which is required for
hydroxyapatite crystallization. ALP also facilitates mineral
(calcium) precipitation through the hydrolysis of pyrophosphate,
a mineralization inhibitor.40 BC has been previously investigated
for its osteogenic properties, including ALP activity and mineralization tests. Fang et al.41 investigated the proliferation and
osteoblastic differentiation of human bone marrow stromal cells
(hBMSC) on BC. Signicantly high ALP activity was recorded for
hBMSCs cultured on BC. Saska et al.42 evaluated BC for in vitro
bone regeneration, and reported signicantly high values of ALP
activity for cell cultures grown on pure BC. The same study also
reported a signicant level of mineralization for BC using an ARS
assay.42 It can be inferred from our results that BC scaffolds can
support the proliferation of osteoblasts, as well as the expression
of genes important for osteogenesis such as ALP, osteocalcin,
collagen I, and osteopontin.43 We conclude from our ALP activity
and ARS assay results that 3-D rBC scaffolds can support the
expression of genes important for osteogenesis.
Cell viability
Proliferation of NIH 3T3 cells on simple rBC and 3-D rBC
scaffolds was evaluated using a MTT assay (Fig. 7). The results
indicate an increase in cell numbers for both the samples with
respect to time. There were no signicant differences in cell
viability on simple rBC or 3-D rBC scaffolds on day one and day
three. This signies that during the initial three days, cell
proliferation was unaffected by the morphology differences of
rBC and 3-D rBC. However, on day ve and day seven, a significant increase in cell proliferation on 3-D rBC scaffolds was
recorded compared to simple rBC. This is attributed to the
availability of a larger space for cellular growth and proliferation in the highly porous 3-D rBC scaffolds compared to simple
rBC. Similar results were obtained by Alves et al.44 when they
compared 2-D collagen scaffolds with their 3-D counterparts for
broblast proliferation. Rothan et al.45 evaluated the differentiation of broblasts and the formation of ECM in both 2-D and
Cell proliferation on simple and 3-D rBC scaffolds determined
through MTT assay after 1, 3, 5 and 7 days of cell incubation. Graph
shows mean value SD (P < 0.05).
Fig. 7
84572 | RSC Adv., 2015, 5, 84565–84573
Paper
3-D culture systems consisting of polycaprolactonetriol–citrate
scaffolds. They reported a signicant increase in cell proliferation and differentiation in 3-D culture systems compared to
their 2-D counterparts. These results suggest improved cell
viability in 3-D rBC scaffolds compared to simple rBC, attributed to the 3-D porous structure of the former.
Conclusion
Highly porous 3-D rBC scaffolds were fabricated using an easy
and robust solvent casting/particulate leaching method. Scaffold indicated porous geometry along with transformation from
cellulose I to cellulose II. High swelling ratio (liquid absorbing
capabilities) and reasonable mechanical features encouraged
the applications of synthesized 3-D rBC scaffold in biomedical
elds. The biocompatibility tests conducted with animal cells
indicated strong adhesion, penetration, and proliferation in the
3-D scaffolds. Additionally, the ALP activity and ARS revealed
the osteogenic differentiation and successful mineralization on
the scaffolds. The cell growth in 3-D scaffold, their penetration
and viability was a bright sign for practical applications in
tissue regeneration and wound healing. Furthermore, this study
could be extended for developing composite 3-D scaffolds with
biocompatible materials to further improve the applicability in
biomedical eld.
Acknowledgements
This research was supported by the Basic Science Research
Program through the National Research Foundation (NRF)
funded by the Ministry of Education, Science and Technology,
Korea (NRF-2014-R1A1A2055756) and by the BK21 plus (2014–
2019), Korea (21A.2013-1800001).
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