Ni Dope - 4pdf
Ni Dope - 4pdf
Ni Dope - 4pdf
Master of Technology
In
Biotechnology
By
ANU PRIYA B
213BM2018
May 2014
CERTIFICATE
This is to certify that the research project report entitled “Development of nickel doped
by Anu Priya B, in partial fulfilment of the requirements for the award of the Degree of
To the best of my knowledge, the matter embodied in the thesis has not been submitted to any
other University/ Institute for the award of any Degree or Diploma.
Assistant Professor
I take this opportunity to express my gratitude and heartfelt thanks to every individual who
has taken part in my Report since from the inception of the idea to completion.
N.I.T Rourkela for his esteem guidance and noble supervision during the hours of the project
I am also thankful to Head of the department, Prof. Krishna Pramanik and all the faculty
members of the department of biotechnology and medical engineering, for extending their
Further, I would like to express my thankfulness to Prof. K. Pal for their help and providing
Advanced Studies for providing me with molecular laboratory facility and and HR-TEM
facility, respectively.
I also would like thank Mr. K. Senthil guru, Mr. S.N. Gautham Hari Narayana, Mr.
Soham Mukherjee for their constant encouragement and for their day-to-day support.
Finally, I would like to express my love and respect to my parents Mr. G. Bharathi Rajan
and Mrs. B. Nagalakshmi, family and friends for their encouragement and endless support
that helped me at every step of life. Their sincere blessings and wishes have enabled me to
Anu Priya B
213BM2018
M.Tech. Biotechnology
Department of Biotechnology and Medical Engineering
TABLE OF CONTENTS
Induction of angiogenesis within the artificial bone graft after implantation is of paramount
importance in the field of bone tissue engineering. For this purpose, here, we are developing
nickel ion doped nHAp which improves angiogenesis. nHAp doped with varying
concentrations of nickel were prepared using wet chemical precipitation method using
cationic surfactant (CTAB) as a template and the extent of doping was studied using EDAX.
The prepared particles with ferret diameter of 15-17 nm (TEM analysis), showed almost
similar surface area for all samples (using BET analysis) but there was an increase in surface
charge observed as the doping increases (ZETA potential). Crystalline nature of doped
hydroxyapatite particles were studied using XRD and FTIR. Detailed analysis relevant to the
bone cells (Mg-63) proliferation and differentiation showed that doping of nickel to
hydroxyapatite supports cell growth and proliferation (MTT, live dead assay and cell cycle
analysis) and also differentiation of these cells which were analysed using in vitro Runx2
expression and bone nodule formation by SEM study. VEGF expression by ELISA and HIF-
1α expression studies revealed that doping of nickel greatly supports angiogenesis. This
confirms that nickel ion doped hydroxyapatite improves angiogenesis along with
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List of Tables
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List of figures
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CHAPTER 1
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1.1. Biology of bone:
There are generally two types of bone found in human body: trabecular and cortical bone.
Cortical bones make up to 80 % of the total skeleton and are hard and compact but not much
porous (flat bones and outer part of long bones). These bones have mineralised compartment
and provides mechanical strength. On the other hand, trabecular bones (cancelous or spongy
bones) make up to 20 % of the total skeleton and are highly porous arranged in a honeycomb-
like rods of various sizes called trabeculae. These cancelous bones are fully vascularized and
a) Osteoblasts – These are bone forming cells which initially form non-mineralized
osteoids and later differentiate into osteocytes (mature osteoblasts) that gets
embedded in the bone matrix. Osteoblasts are generally found in the cancelous bones
organic component and dissolving the mineral component of the bone matrix when
needed[2].
c) Osteocytes – These are the terminally differentiated cells which play a role in the
necessary enzymes, controlling bone mineral content and release of calcium ions into
the blood[2].
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1.1.4. Bone formation and remodelling:
The formation of new bone is initiated with the process called cellular condensation in which
the mesenchymal cells migrate from different parts and proliferate. Differentiation of the
mesenchymal stem cells into pre-osteoblasts and osteoblasts occurs via one of the two
ossification, the cartilage plate is first formed which gets replaced by the bone later, and
mesenchymal stem cells directly differentiate into pre-osteoblasts and osteoblasts, and this
mechanism occurs primarily in flat bones of the skull, scapula and mandible[3].
integrity. Remodelling of bone consists of two steps: bone resorption followed by new bone
formation. During bone resorption, osteoclasts attach to the old and damaged bone surface
and initiates the degradation of bone matrix by releasing factors. Osteoblasts then fill up the
resorption area and helps in bone mineralization and formation of the new bone matrix. In a
healthy bone, this process of resorption and formation in remodelling is very well co-
Many bone related diseases like osteoporosis and osteoarthritis, genetic disorders like
osteogenesis imperfecta and bone fractures need treatments which help in regeneration of
bones. As the rate of bone defects and diseases increases, the need for bone substitutes also
increases in the field of the orthopaedic health sector. One of the major concern is the proper
integration of the bone substitutes with its natural counterpart. Conventional methods of
treatment include the use of autografts and allografts that can integrate with the existing
bones perfectly. But they come with limitations such as scarcity of donors, immunorejection,
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donor site morbidity, severe pain and risk of infections and high chances of transmission of
diseases (in case of allografts)[5]. These limitations gave way for the artificial prostheses for
bone fractures (one of the major application of artificial prostheses is the use in hip bone
fracture) but then the wearing of the implant material at times leads to the death of the bone
cells at the site of fracture and also there won’t be any bonding between the chemically inert
prostheses and the biological tissue. These limitations opened the door for the field of bone
tissue engineering which uses a wide range of polymeric and ceramic scaffolds that fits
The focus of bone tissue engineering field is on creating bone constructs that are capable of
improving the osteogenesis in the site of the bone defect. Bone constructs are implanted by
seeding the cells with osteogenic potential into the biodegradable scaffolds in vitro and
directly transplanting in vivo to assess their potential in the formation of bone. Another kind
of bone construct is that after cell seeding into the scaffolds, they were cultured in vitro to
form mature bone-like grafts which are then implanted. The structural properties of the
scaffold have a great influence on the cell growth and proliferation. Bone tissue scaffolds
after implantation should have a macrostructure mimicking the physiological function of the
matrix to maintain the cell’s natural phenotype. Porosity and interconnectivity of the scaffold
(70-80 %) is also necessary for promoting tissue ingrowth and vascularization from
surrounding tissues and also for the removal of the metabolic waste that has penetrated the
scaffold from the surrounding cells. Most importantly scaffold should be bio-compatible and
bio-degradable[7].
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1.4. Biomaterials for bone tissue engineering:
For a successful implantation, the scaffold should be highly biocompatible. The selected
scaffold material should also degrade and resorb at a controllable rate at the same time as the
specific tissue cells seeded into the construct attach, spread and increase in quantity as well as
quality[8]. The design and fabrication of scaffolds in tissue engineering are driven by two
Because of ease of design modification and surface modification, a variety of polymers have
been used to fabricate scaffold. Generally two categories of polymers are found:
for making scaffolds, and they have good mechanical properties and degrade at a controllable
rate. Poly lactic acid (PLA), poly glycolic acid (PGA), collagen and co-poly L-lactide
(CPLA) are some of the examples of biodegradable polymers. One of the important
materials[9]. This gave opportunity for ceramic biomaterial for the application as scaffolds
Ceramic biomaterials based scaffolds are composed of metallic and non-metallic elements
and inorganic ceramic materials. They have good mechanical behaviour such as hardness.
However they are very brittle, requiring less energy to fracture. They can be bioinert,
bioactive or bioresorbable. They do not bond with the bone, instead tissue ingrowth into
ceramic pores happens. Bioactive ceramics is generally used for bone tissue engineering
application and evoke biological response at bone tissue implant surface by exchange of ions
to form a bond between the host tissue and the biomaterial. They can also be used as coating
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on other non-bioactive materials such as metals to induce a strong biological fixation between
1.4.3. Hydroxyapatite:
Hydroxyapatite, with the chemical formulae Ca10(PO4)6(OH)2 is one such bioactive ceramic
which is chemically similar to the inorganic phase of natural bone and used widely in bone
tissue engineering. It is chemically stable at acidity levels less than 4.3 (which is the pH of
blood)[11]. Much like calcium phosphates, HAp also has osteoconductive and osteoinductive
properties. Bioactivity is related to a modification of the surface of the HAp material. Its
bioactivity has been seen to be affected by structural crystallinity. The theortical mole ratio of
Ca and P in HAp is 1.67. However, this is not the value observed in the organism because
small amounts of carbon, nitrogen, iron and other elements are also incorporated. In the
hexagonal systems, the hydroxyl ions (OH-) are located at the centre of Ca+2 triangles along
the c-axes of the hexagonal unit cell. The OH- ions are aligned in columns parallel to the c-
axis along with Ca+2 and (PO4-3) ions. There are various parameters to be considered for
composition and crystallinity. HAp is composed of strongly bonded, large crystal structures
with smaller surface areas than the nano-sixzed, weakly bonded bone mineral crystals
therefore reducing the rate of particle disintegration and crystal dissolution of the
implant[12]. In particular, the high stability and flexibility of the apatite structure accounts
for the great variety of possible cationic and anionic substitutions. Therefore, metal and ion
nowadays.
For the engineered bone tissue to survive and integrate properly, vascularization is a requisite
after implantation as it is necessary for the efficient oxygen and nutrition exchange between
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the tissue. One of the drawbacks in implantation is that the molecular diffusion of oxygen and
nutrients is limited over a short range of 150 – 200 µm and diffusional transport more than
that distance results in acellular regions. Even though cell survival can be enhanced by using
larger scaffolds, vascularization of the implant need to be considered for the successful
Vascularization in bone tissue engineering is of utmost importance for the proper supply of
oxygen, nutrients and growth factors thereby enhancing the new bone formation. To date,
many strategies have been administered for the improvement of vascularization in bone tissue
constructs. They are: i) growth factor delivery ii) co-culturing systems iii) gene delivery iv)
development of microfluidic based network inside the scaffold v) using biomaterials with
appropriate properties.
Cells communicate with each other via a cascade of signalling molecules (growth factors and
proteins) to mediate cellular functions and also migration, proliferation and differentiation of
cells. Some of the growth factors involved in the bone formation are: VEGF (vascular
endothelial growth factor), FGF (fibroblast growth factor), PDGF (platelet derived growth
factor), IGF (insulin-like growth factor), ET-1 (endothelin-1), ET-2 (endothelin-2), BMP-2
(transforming growth factor-β). In the bone micro environment, there is always a cross talk
between the bone cells and the endothelial cells present in the surrounding blood vessels. For
and BMP’s released by the endothelial cells help in osteogenic differentiation. Numerous
researchers around the world working in the field of bone tissue engineering have tried to
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incorporate the idea of delivering exogenous growth factors to control the cellular behaviour
both in vitro and in vivo. But there are many disadvantages. Firstly, delivery of single growth
factor alone cannot elicit a complete regeneration of bone. Secondly, this approach leads to
the nonlocalized delivery of growth factors and transient cellular responses. Finally growth
Another novel approach is to introduce endothelial cells or its progenitors directly into the
scaffold. But this method has its drawback as there is only one cell type used and it is
difficult to develop a complete vascularized tissue as the growth factors and signals from
other cell types (in this case, bone cells) won’t be present. Also, it is an essential from which
source the endothelial cells are taken as its angiogenic nature depends on the source[15]
By now it is understood that both angiogenesis and osteogenesis plays a role in the bone
formation in vivo. Angiogenesis is one of the requisites for the osteogenic differentiation as it
helps in the migration of osteoblast precursor cells to the site of bone formation. Zhou et al.,
and Villers et al., in their research found that co-culturing of human mesenchymal stem cells
(MSCs) and endothelial cells (ECs) within the biomaterial β-TCP resulted in the formation of
vascularized bone tissue. Similarly, Wang et al., demonstrated that the co-culturing of human
osteoblast-like cells and human umblical vein endothelial cells resulted in new bone
formation with blood vessels. However, the co-culturing system becomes more complex to
study and understand. Also research on co-culture system to date are very conflicting as in
few studies though they showed the vessel formation there was no evidence for the integrity
and functionality of the vessels within the grafts (Fuchs et al., and Rouwkema et al.,)[15].
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1.6.4. Gene delivery:
Limitations of gene delivery include: i) safety concerns for viral gene deliveries ii) non-viral
delivery faces the problem of insufficient transport of pDNA into the nucleus and iii) lastly,
Recent advancement in this field is the development of microfluidic based network for
vascularization within the scaffold. These microfluidic channels mimic the micro
vasculatures present surrounding the bone. Growing the endothelial cells within these
microfluidic networks helped in developing the 3D vascular network within the scaffold.
Even though this system is gaining much attention, it is very difficult to create such a
complex array of system as it requires much sophisticated system and costly infrastructure
and also it is very important to study the nature of the materials used for creating such
channels[17].
triggers the cells to produce vast quantities of angiogenic factors in vivo which is normally
achieved by creating tissue hypoxic condition in vivo. It is already a known fact that hypoxia
(decreased oxygen level) is one of the potent inducers of angiogenesis. Here, hypoxia
inducible factor – 1α (HIF-1α) is the key regulator of angiogenesis. HIF-1 consists of two
subunits: HIF-1β and HIF-1α. HIF-1β (also called aryl hydrocarbon receptor nuclear
translocator (ARNT)) is constantly expressed and present within the nucleus. On the other
hand, HIF-1α is present in the cytosolic region, and it is oxygen sensitive. Under the
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hydroxylase (PHD) (which becomes active when its co-factors iron, 2-oxoglutarate and
oxygen gets bound to it). Von-Hippel-Lindau protein (E3 ubiquitin ligase) then initiates its
proteosomal degradation. But under hypoxic condition, since prolyl hydroxylase cannot be
activated, accumulation of HIF-1α happens which moves into the nucleus and forms dimer
with HIF-1β subunit which forms a complex with co-activator p-300 and trans activates the
HIF-1 responsive genes by binding to the hypoxia responsive elements (HRE) region in those
genes. One such gene is VEGF, which in turn activates the VEGF protein (a potent mitogen)
that helps in the migration and proliferation of endothelial cells forming tubular structures.
Also, it is known that in the bone remodelling compartment, expression of VEGF helps in
osteoblast and osteoclast differentiation thereby enhancing the bone resorption and bone
formation[18]. It is quite evident from the previous researches that even under normoxia,
metal ions like nickel, cobalt, and copper inactivates the PHD enzyme by replacing iron (a
co-factor required for the activation of PHD) which thereby stabilizes HIF-1α. Wu et al.,
exploited these strategies and showed that copper and cobalt doped mesoporous bioactive
glass scaffolds can effectively induce cellular VEGF production and angiogenesis[19, 20].
But the stabilization of HIF-1α and subsequent activation of VEGF was very strong in the
exploit this area. However, high concentration of nickel may induce nickel toxicity. So a
controlled ion release system is very much essential for vascularized bone tissue engineering
strategy.
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CHAPTER 2
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2.1. Objectives:
To prepare and physicochemically characterize nickel doped nano hydroxyapatite. Further
biologically characterize the prepared nickel doped nano hydroxyapatite to evaluate the
2.3. Rationale:
As the scope of bone tissue engineering is rising in most parts of the world, the need
for vascularization of the bone grafts is of paramount importance for successful implantation.
Although there are many pre-vascularization techniques available, they come with their
limitations. Hence this study is aimed at developing a pre-angiogenic nickel doped nano
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CHAPTER 3
MATERIALS AND METHODS
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3.1. Materials:
bromide (CTAB) and ammonia solution (25%) were purchased from Merck, India.
solution, and MTT assay kit were purchased from Himedia, India. Human VEGF ELISA kit
was purchased from Abcam, UK. mRNA isolation kit (RNeasy Mini) and cDNA synthesis kit
(MuLV Reverse Transcriptase Plus Kit) were bought from Qiagen and BioBharti
LifeSciences Pvt. Ltd., India, respectively. The MG-63 cell line was procured from NCCS,
India.
The well-known wet chemical precipitation method was employed here to synthesis
the hydroxyapatite[21]. In this method, 200 ml of 0.05M Calcium nitrate tetrahydrate was
taken in a beaker and placed in a sonicator bath at 80°C and the pH of the system was
(CTAB) in the ratio 1:1 was added drop-wise at a constant flow rate of approximately
1.5ml/min from a burette. The slurry was allowed to age for 24 hours and then repeated
washing was done by centrifuging at 6000 RPM for 10 minutes to remove the ammonia
byproducts and unwanted CTAB. The precipitate was then dried at 55°C for 24 hours to get
the HAP powders. Nickel doped nano-hydroxyapatite samples were prepared using the above
method in which different doping percentage of Nickel(II) nitrate hexahydrate(1%, 5%, and
10%) was first added to the calcium nitrate solution and then reaction was started. The molar
The phase content of the samples was studied using XRD method. Philips XRD-
PW1700, Rockville USA instrument was used for this analysis. The scanning was done over
FTIR analysis was done to determine the functional groups present in the HAP
Samples. It was performed using an AKTR FTIR Spectrophotometer instrument with the
scanning range of 400 cm-1-4000cm-1, and the pellets were obtained using the KBr pelleting
technique[23].
was used to determine the nature of the synthesized HAP particles. Sample specimen were
EDAX analysis was performed to determine the amount of nickel doped onto each
sample[25].
BET surface area analyser (Quantachrome Instruments) was used to determine the
surface area, pore size and pore volume of the synthesized HAP particles the analysis was
The ZETA potential of the HAP samples was determined using Marvelin DLS and
ZETA analyser using water as the dispersion medium. The samples were dispersed in10 ml
of water taken in a test tube and sonicated for 10 -15 min so that there wont be any
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3.6. Protein Adsorption Study:
The adsorption of proteins by the samples can be studied by comparing it with the
adsorption of BSA (Bovine Serum Albumin) by the samples. The residual protein
concentration in the supernatant was determined using Bradford assay. The adsorption of the
proteins by the samples were determined by subtracting the residual protein concentration
Cell proliferation of the samples were studied using MTT Assay on MG-63 cell line
(from NCCS, Pune, India). 1 x 104 cells/well were added in 96 well plate and incubated for
24 hrs for the cells to adhere properly. After 24 hours of incubation, the cells were treated
with HAP samples at a concentration of 100µg/ml and then again incubated. After 1st, 3rd and
Cell cycle analysis was performed used FACS. 1 X 106 cells were seeded into 6 well
plate and then treated with the samples at a concentration of 100µg/ml and then incubated.
After 24 hours of sample treatment, the cells were trypsinized, washed with ice cold PBS and
then fixed with 70% ice cold ethanol. After 24 hours, fixed cells were centrifuged at
3000RPM and the pellets were suspended in 100µl of PBS and 500µl of PI staining dye and
then allowed to stain for 30 min at room temperature[]. The samples were then analyzed
Live/Dead assay was perfoemed using FACS for cell viability study. 5 X 105 MG-63
cells were seeded onto the 6 well plate and left for adhereing at 37°C, 5% CO 2 and 95%
humidity. After 12 hours of incubation, the cells were treated with 100µg/ml of sample and
further incubated. After one day of sample addition, the cells were trypsinized (also the
supernatant was taken) and washed with ice cold PBS and then suspended in 500µl of PI
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buffer (50µg/ml PI and 100 U/ml RNase) and incubated for 30 min at room temperature[31].
The samples were analysed using FACS Accuri C6 (BD Biosciences). The same protocol was
Field Emission Scanning Electron Microscopy (TOEL India TSM 6480 LV) was used to
determine the effect of the samples on MG-63 cells. 5 X 104 cells were seeded onto the glass
slides kept inside each well of the 12 well plate and left to adhere for 12 hours in the
incubator at 37°C, 5% CO2. Thereafter, the cells were treated with the samples at a
concentration of 100µg/ml and incubated at 37°C, 5% CO2. After 3 days of sample treatment,
the cells were taken out and washed with SEM buffer at pH 7.2 (3 X 5min) and fixed with
SEM grade gluteraldehyde for 2 hours. After 2 hours, the cells were again washed and fixed
with osmium tetroxide for 2 hours. The cells were then washed and serially dehydrated with
ethanol from 30% -100 % each (10 min) and then allowed to dry[32].
Expression of the differentiation marker Runx2 and angiogenic promoter HIF-1α was studied
using immunocytochemistry analysis.5 X 104 MG-63 cells were seeded and incubated at
37°C, 5% CO2 and 95% humidity for the cells to adhere properly. After the incubation
period, the standard ICC protocol was followed. The cells were fixed with 4%
(PBS with 0.25% TritonX) for 10 min at room temperature and then incubated with blocking
buffer (1% BSA in PBST + 0.3M Glycine) for 30 min at room temperature to block any
unspecific binding of the antibodies. After this, the cells were incubated for 1 hour at room
temperature with the primary antibody which was followed by washing with PBS (3 X 5min)
then incubated with secondary antibody tagged with Alexa Flour 488 (1:1000 dilution) for 1
17 | P a g e
hour at room temperature. Further, the cells were washed again with PBS (3 X 5min), stained
Expression of VEGF was determined using human VEGF ELISA kit (Abcam 100662). For
this purpose, 2 X 105 MG-63 cells were seeded onto a 12 well plate and incubated at 37°C,
5% CO2 and 95% humidity for the cells to adhere properly. After 12 hours of incubation, the
cells were treated with 100µg/ml of samples. After 48 hours of sample treatment, the
supernatant was transferred to a sterile vial and then ELISA was performed as mentioned in
the protocol from the kit. To summarize the process, 100µl of the samples were added to the
8-well ELISA plate which were pre-coated with VEGF capture antibody and incubated for
2.5 hours which was followed by washing, incubation with 100µl of 1X biotinylated VEGF
detection antibody for 1 hour, subsequent washing and then incubation with 100µl of 1X
HRP Streptavidin solution for 45min at room temperature. Further, it was incubated with
100µl of TMB onestep substrate reagent for 30 min at room temperature for the colour
development. Subsequently stop solution was added, and the absorbance was measured at
450nm[33].
Expression of VEGF, Runx2, HIF-1α was checked using RT-PCR. For this purpose 1 x 106
cells were seeded on to each well of a 6-well plate and cultured following the protocol
mentioned above. After 24 h of incubation, samples (100µg/ml) were added to the cells, and
it was further incubated for 48 h. After incubations, cells were lysed using lysis buffer.
Extraction of mRNA from the cell lysate was done using RNA isolation kit (QIAGEN
RNeasy Mini Kit) following the manual provided by the supplier. At the end of the process,
18 | P a g e
the concentration of mRNA was estimated spectrophotometrically (Nanodrop) and
appropriate dilution was made to keep the mRNA concentration same for all the samples.
Then mRNA were converted to cDNA and subjected to PCR using BioBharti MuLV
ReverseTranscriptase Plus Kit. For PCR, cDNA were mixed with primers (listed below) and
'PCR mix’ following appropriate stoichiometry as per the instruction given in the manual.
After completion of the PCR, sample were analyzed using 1.5 % agarose gel[34].
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CHAPTER 4
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4.1. Preparation of Ni+2 doped nano hydroxyapatite
Synthesis of nano hydroxy apatite and Ni+2 doped nano hydroxy apatite was done by
ammoniacal precipitation method (Table.1 and Fig.1). To control the size of the nanoparticle,
a cationic surfactant CTAB was used as a capping agent [35]. Three different Ni+2 doped
nano hydroxy apatite samples were prepared by varying the doping concentration. The yield
of nano HAp powder was between ~ 0.9 to 1.0 g in all the cases. To break the agglomerate
of the synthesized nano hydroxy apatite, the dried samples were ball- milled. Physical
inspection of the powder revealed that all powders were free flowing. Pure nano hydroxy
apatite was white coloured, but the colour changed from white to whitish- green with Ni
doping because of hexaaquanickel ion[36]. ) and Yan Li et al., (2010) when they doped Co+2
Table 1. Composition, yield and physical appearance of nHAp and Ni +2 doped nHAp
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Fig. 1 nHAp and Ni+2 doped nHAp prepared using ammoniacal precipitation method.
4.2. EDAX
The extent of doping is a major concern for the metal doped bio ceramics, especially for
metal ion doped hydroxyapatite. So far, many research groups have reported the synthesis of
different metal ions (example cobalt, magnesium, zinc, iron) doped hydroxyapatite. A critical
analysis of their results revealed that in most of the cases actual doping was less than the
expected value (theoretical percentage doping). In the present study, the presence of dopant
and extent of doping was confirmed by EDAX. The result showed that ammoniacal
precipitation method can be successfully used for the doping of nickel in hydroxyapatite. It
was observed that though there was a gradual increase in percentage doping with an increase
of Ni+2 concentration in the precursor, however, the actual doping was less in comparison to
the theoretical value (Fig.2). Analysis showed that in all cases the actual loading was less
than 30% of the theoretical doping. In recent reports Guerra Lopez et.al, has shown that
percentage doping for transition metals including nickel generally less than 1/3rd of the
theoretical doping. This happens probably because of the formation of stable amine co-
ordination complex of Ni+2 at basic pH. That complex ions are generally much bigger in size,
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4.3. TEM
The size of the hydroxyapatite nano crystal is very important for bone tissue application. In
vivo, the nano hydroxyapatite is synthesized in situ during bone remodelling inside the grove
of the collagen helices. The native size of hydroxyapatite nanocrystals lies between (2nm-
10nm)[39].Here, TEM micrographs showed that the size of the synthesized doped HAp
approximately(Fig.2).All the nanoparticles were found elliptical. The average ferret diameter
for HAP was 17.2 ± 3.9 nm while the same for N1-HAP, N5-HAP and N10-HAP was 15.6 ±
3.3, 16.8± 3.0 and 15.1 ± 3.0 nm, respectively. Such a close value of the average ferret
diameter of all the doped samples clearly implies that the doping of nickel has no significant
DataData
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Please
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banerjeei@nitrkl.ac.in
banerjeei@nitrkl.ac.in
for details
for details
Fig. 2. [A] FESEM micrographs of nHAp and Ni+2 doped nHAp. [B] TEM Images of nHAp and Ni+2
doped nHAp. [C] Nano particle size distribution. TEM Image based analysis was done using NIH-ImageJ
software. For each analysis, 25 individual particles were considered (p< 0.05). [D] Percentage of Ni +2
doping with respect to calcium content (w/w). Results were expressed as Mean ± S.D of three independent
experiments (p <0.05).
23 | P a g e
4.4. XRD
Analysis of the XRD profiles (Fig.3) of the doped HAp showed that there were no additional
peaks corresponding to nickel. The characteristic peaks of HAp corresponding to 32°, 33°
and 34° 2θ (for planes (211), (300) and (302) respectively) were found in all samples.
Analysis of the crystal structure of the samples (Table.2) showed that the magnitude of the
lattice parameter 'c' for HAP, N1-HAP and N5-HAP and N10-HAP were almost same.
Further analysis showed that there was a decrease in crystallinity with an increase in doping
percentage. In this present study, no such peak broadening was observed which implies that
Ni+2 doping did not distort the apatite crystal much. However, the variation in crystal
parameter observed was probably because of the smaller ionic radii of Ni+2 (0.72Å) in
Table 2. Crystal parameters of nHAp and Ni+2 doped nHAp. Percentage crystallinity and d spacing were
calculated using the XRD and TEM diffraction data corresponding to 002 planes. For the analysis of
crystal lattice parameters, ‘an’ and ‘c’, 300 and 002 planes are considered.
4.5. FT-IR
The FTIR analysis of the samples revealed the characteristic peaks of hydroxyapatite (Fig.3)
No major peak shift was observed in the nickel doped samples. The peaks at 630cm-1, 600cm-
1
and 550cm-1 corresponding to free OH and υ4 vibrational bands of a phosphate group
24 | P a g e
(PO4)3- respectively appears less prominent as the doping increases and becomes merged with
N10-HAP. Peaks for other bands like υ3 stretching of (PO4)3- (1100 - 1000 cm-1) and
υ2bending of (PO4)3- (470cm-1) also became less prominent as the doping increases. These
results were found to be in accordance with Guerra-López et al. and G. Gergely et al.[38].
This maybe because of the mild variation in that ionic environment in the crystal lattice
which leads to alteration in the stretching field. All the samples show a common peak of H-
bonded OH stretching at 3570cm-1. However in case of N5-HAP and N10-HAP, a new peak
appeared at 3640cm-1 that could be attributed to the stretching of free OH, which becomes
more intense with the increasing doping. Bands at 1470cm-1 and 870cm-1 corresponding to
(CO3)2- was seen due to the CO2 adsorption from the atmosphere. Adsorbed water gave two
kinds of the band, O-H bending (1640cm-1) and O-H stretching (3450cm-1) which was also
due to the hydroxyl group present in the apatite. The peak at 1640cm-1 became wider in N10-
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for details
Fig. 3. [A] XRD analysis of nHAp and Ni+2 doped nHAp. Important characteristic peaks are marked in
the figure, and its corresponding crystal planes were mention in the embedded table. [B] FT-IR analysis
of nHAp and Ni+2 doped nHAp. Important characteristic peaks are marked in the figure, and its
Large surface area is one of the key parameters why nanomaterials are highly reactive when
compared micro or macro scale materials. For this reason, we analysed the surface area of the
synthesized HAp samples. The surface area of pure HAP was found to be 109.120m2/g and
was in accordance with the previously reported value[40]. The same for N1-HAP, N5-HAP,
and N10-HAP were 107.591m2/g,111.732m2/g and 83.165m2/g respectively (Table 3). Our
TEM study showed that the size of the nano particles for all samples were close. So it is
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expected that the surface area of the nanomaterial should also be same unless there is a
variation in the porosity of the synthesized materials. The BET study showed that there was
no significance variation in surface area with doping which implies the doping has no
The zeta potential of the nano materials influences the biological performance of the
materials in a number of ways. It is already reported that in physiological condition, the zeta
potential modulate protein adsorption, cell material interaction, and cellular uptake of the
nano material. Doping of metal ions can change the normal zeta potential of hydroxyapatite
by two ways: i) By altering the surface charge density ii) By imparting strain in the crystal
that could lead to differential ion dissolution from a crystal. Here, the zeta potential of HAP
was found to be -3.54mV and was found in accordance with former reports. It is also reported
that the metal ion doping causes a variation in the zeta potentialin hydroxyapatite. However,
the extent of such variation depends on the nature of the metal ion and extent of doping.
Here, we have observed that with Ni+2 doping, the zeta becomes more positive in comparison
to the HAP, and however there is no proportional increase in zeta with the increase in doping
concentration (Table.3).
The protein adsorption onto the implant plays a vital role in determining the host response
towards the implant. There are number of plasma proteins (albumin, immunoglobins and
fibrinogen, fibronectin) that interact with the biomaterial surface. Hence, BSA (Bovine
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Serum Albumin) was used as a model protein to study the protein adsorption on the Ni doped
hydroxyapatite. As reported earlier, protein adsorption by HAp depends on surface area and
zeta potential more specifically on the charge distribution of the Ca/Ni and P ions over the
crystal surface since Ca and P sites are the sites for the electrostatic adsorption of proteins
[41]. Both HAp and doped Hap samples showed similar protein adsorption onto their surface
(Table.3). The average protein adsorption was found in the range 900µg/10mg of the
samples. The result clearly implied that the doping of nickel did not influence the protein
Table 3. Analysis of surface area, pore volume, zeta potential and protein absorption.
The data were mean of three independent experiments and presented as Mean ± S.D.
S.No Samples Surface area Pore volume Zeta potential Protein adsorption
During development, osteoblast cells are the one which first proliferates, then differentiates
into osteocytes and produce an extracellular bone matrix. It is now well established that
osteoblast proliferation is essential for the bone regeneration and remodelling. In this present
study, the MTT assay was performed for the cells treated with Ni doped HAp samples
(Fig.4). Pure HAp showed the highest cell proliferation in comparison to all other samples.
The extent of cell growth was very much similar for all the samples on day 1. The
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proliferation rate becomes slow from day 3 with N10-HAp giving the least cell growth. N1-
HAP and N5-HAP showed similar proliferation rate as pure HAP on day 3 but on day 5
proliferation reduced when compared to pure HAP. This behaviour may be attributed to
either nickel doping being cytotoxic to cells, or the cells are getting differentiated after day 3.
Cell cycle analysis (Fig.4) showed that nickel doping did not perturb normal cell cycle
progression. For all the treatment groups, distribution of cell populations in different phases
of cell cycle was similar to that of control (TCP) which indicates that nickel doping has no
detrimental effect on viability and cell proliferation. For all the cases, highest cell population
was observed in G0/G1 phase (between 55-65%) except the cells treated with N5- HAP where
percentage cell population in G0/G1 was even little higher. More interestingly, N5- HAP has
least cell population in S phase (12%). On the other hand, least population in G2/M phase was
found in N10-HAP treated group. In bone physiology, a high population of cells in G0/G1
often indicates their commitment towards differentiation. Therefore, a higher value G0/G1 in
case of N5-HAP treated group could be associated with differentiation potential of the
material. FACS analysis also showed that the percentage cell population in pro G0/G1 (a
phase associated with apoptosis) was negligible for all groups which imply that nickel doping
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Data protected for publication
Please contact
banerjeei@nitrkl.ac.in for details
Fig. 4. [A] Study of cell proliferation by MTT assay. Data were expressed as Mean ± S.D (n=3). Statistical
significance was checked for p< 0.005. [B] Study of the cell cycle after 24 h of treatment. Experiments
were done in triplicate. Representative histograms for each sample was presented.
It was evident from the literature that hydroxyapatite and doped hydroxyl apatite support
osteoblast cell viability and proliferation in vitro for longer period. Our MTT analysis
apparently showed Ni doped hydroxy apatite samples help in MG-63 cell proliferation.
metabolic state of the cells and often fails to indicate cell proliferation effectively. Therefore,
it was important to quantify the variation in a live population of cells. For this purpose, flow
cytometry based Live / Dead assay was performed to determine the percentage of live cells in
the total cell population after the treatment (Table.4). The result showed that the percentage
of live cells in the total cell population for all the samples were more than 90%. This clearly
suggests that Ni+2 doped hydroxyl apatite were as cytocompatible as control (synthetic
hydroxyl apatite).Our MTT study showed that there was a decrease in cell proliferation rate
with time. As mentioned earlier, the two possibilities could be either cell death or cell
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differentiation. It was evident from the live dead assay that there was no significant cell death
at the later phase of culture which clearly implied that the decrease in cell proliferation could
Table 4. Analysis of the percentage of live cells after sample treatment. The assay was performed by flow
Osteoblast differentiation into osteocytes and its maturation were the main processes
involved in the effective bone remodelling after bone matrix deposition and osteoblast
proliferation. The inorganic components of bone (hydroxyapatite), extra cellular matrix and
its components along with many different kinds of growth factors like bone morphogenic
proteins (BMPs, epidermal growth factors (EGFs) , fibroblast growth factors(FGFs), and
many more widely affect the osteoblast differentiation[41]. Transcription factor runx2 is one
among the signature protein which is a pre-requisite for the mesenchymal stem cells to
Therefore, Runx2 expression always remains in basal level in MG-63 in comparison to its
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progenitor. A qualitative analysis of immuno-cytochemistry micrograph showed that as per
expectation Runx2 is not localized on cell nucleus (as the cells has passed their early
differentiation phase) and distributed all over the cells. Quantitative Image analysis using
NIH-Image J showed that variation in expression level of Runx2 in all samples were
statistically insignificant. When checked at mRNA level by RT-PCR, it was observed that
there was no sample specific variation in the expression of Runx2 (Fig.5). These two
experiments altogether showed that the presence of nickel doesn’t affect the expression of
runx2.
SEM micrograph taken after 3 days of sample treatment showed clear evidence of bone
matrix deposition and nodule formation in all samples (Fig.5). Previously, Gough et.al
outlined that the formation of the nodule is a signature of osteoblast differentiation. N5-HAP
showed bigger nodule formation when compared to all samples which implied its greater
osteogenic property.
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Data protected for publication
Please contact
banerjeei@nitrkl.ac.in for details
Fig. 5. [A] SEM images of cells cultured in presence of nHAp and Ni +2doped nHAp samples at day 3. [B]
Confocal micrographs of cells cultured in presence of nHAp and Ni+2doped nHAp samples at day 3. Red
[F actin], Blue [DAPI] and Green [Runx2]. [C] Quantitative image analysis of Runx 2 expression.
Intensity was calculated for 10 cells. Statistical significance was checked for p< 0.005. [D] Study of Runx2
expression by RT-PCR.
The expression of VEGF measured by ELISA (Fig.6) showed that there were a 2.3 fold, 3.5
fold and 3.8 fold increase in VEGF expression for N1-HAP, N5-Hap, N10-HAP treated
group respectively, with respect to pure HAP after 48 h of treatment. Statistical analysis
revealed that such increase in cellular VEGF expression with the increase in doping
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concentration is statistically significant (p<0.005) for any two samples including control.
RT-PCR based analysis of VEGF expression at mRNA level (Fig.6) further confirmed the
increase in VEGF gene expression with the increase in Ni+2 doping concentration. An image
based analysis of the intensity of the PCR bands (amplicons) showed that, in comparison to
pure HAp, maximum expression was obtained for N10-HAP ( fold ) followed by N5-HAP (
In this study, the immunocytochemistry profile of HIF-1α showed that the nickel doped HAp
samples had a significantly higher expression of HIF-1α. In case of bone formation during
embryogenesis, vascularisation of cartilage analague is a key step for further bone formation.
Bivalent metal ions such as nickel, cobalt, copper, etc. mimics hypoxia condition by
replacing Fe2+ ions, the key cofactor of the prolyl hydroxylase enzyme and thereby
inactivating the enzyme and stabilizing HIF-1α. Interestingly, in this study, we observed that
such expression level of HIF-1α did not show a linear trend with respect to nickel doping
concentration (Fig.6). Generally, the release of ions and the degradation of HAp depends on
its crystal structure (Ref.). Hence, the variation among the crystal structure of doped HAp
might lead to variation in the nickel release and further HIF-1α expression. In case of HIF-1α
expression, N5 showed the highest expression followed by N10, N1 and HAP, respectively.
But in case of VEGF expression, the increase was linear with respect to nickel doping
concentration. This might be explained on the basis that VEGF expression does not merely
depend on the HIF-1α expression level. Apart from this, Lee et al. (2012) stated that Runx2
also stabilizes HIF-1α by competing with the Von Hippel-Lindau Protein (pVHL) (protein
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that aids in proteosomal degradation of HIF-1α) and stabilizes HIF-1α protein. From the
followed by N10. It was also reported for cu and co doped proangiogenic biomaterials
Fig. 6. [A] Study of VEGF expression by MG-63 cells. Cells were treated with Ni+2 doped nHAp for 48 h.
Data are expressed as Mean ± S.D. [B] Study of VEGF mRNA expression by RT-PCR. [C] Study of
micrographs of the cells [Red (F actin), Blue (DAPI) and Green (HIF-1α)]. [D] Quantitative image
analysis of HIF-1α expression. Intensity was calculated for 10 cells. Statistical significance was checked
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CHAPTER 5
CONCLUSION
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5. Conclusion:
Here we have reported the synthesis and characterization of Ni+2 doped nHAp and evaluation
of its osteoconductive and proangiogenic properties. Bivalent nickel has been known as an
nHAp might convert it into a proangiogenic-osteogenic biomaterial. Our study showed that
Ni+2 doped nHAp particles of the narrow size distribution could effectively be prepared by
wet chemical precipitation method using CTAB. Doping of Ni+2 upto 3.3 % w/w of Ca+2 did
not cause crystal deformation. Similarly, physical properties like surface area, zeta potential
and protein absorption remained almost same with Ni+2 doping. All the doped samples were
bone cell compatible and osteoconductive. Analysis showed that Ni+2 doped nHAp samples
were a potent inducer of cellular VEGF, and the expression of VEGF was directly
proportional to the doping concentration of Ni+2. A mechanistic analysis further implied the
Comparison of these results with the relevant literature revealed number of important points.
Firstly, although biological performance of the Ni+2 doped nHAp was similar to the other
proangiogenic bio-ceramics (metal ion doped), i.e. all are osteoconductive as well as potent
VEGF inducer, the relative VEGF expression per unit quantity of doping was higher in the
present case. Secondly, the extent of nickel doping was less than all the reported values and
below the toxic level. Finally, particle size was found within the range of 14-19 nm, which is
good for bone tissue engineering. In conclusion, Ni+2 doped nHAp is a proangiogenic–
osteogenic material and could be used in bone tissue engineering. The response of endothelial
cells (viability and tube formation) against these materials in vitro has to be studied.
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CHAPTER 6
BIBLIOGRAPHY
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