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DEVELOPMENT OF NICKEL DOPED NANO HYDROXYAPATITE AS

PROANGIOGENIC MATERIAL FOR BONE TISSUE ENGINEERING

A PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

Master of Technology
In

Biotechnology

By

ANU PRIYA B

213BM2018

Under the Supervision of

Dr. INDRANIL BANERJEE

Department of Biotechnology and Medical Engineering

National Institute of Technology

Rourkela-769008, Orissa, India

May 2014
CERTIFICATE

This is to certify that the research project report entitled “Development of nickel doped

nano hydroxyapatite as proangiogenic material for bone tissue engineering” submitted

by Anu Priya B, in partial fulfilment of the requirements for the award of the Degree of

Master of Technology in Biotechnology and Medical Engineering with specialization in

Biotechnology at National Institute of Technology Rourkela is an authentic work carried out

by her under my supervision and guidance.

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.

Dr. Indranil Banerjee

Assistant Professor

Department of Biotechnology and Medical Engineering

National Institute of Technology, Rourkela


ACKNOWLEDGEMENTS

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.

I am privileged to express my deep sense of gratitude and profound regards to my supervisor

Dr. I. Banerjee, Asst. Professor, Department of Biotechnology and Medical Engineering,

N.I.T Rourkela for his esteem guidance and noble supervision during the hours of the project

since from the needs of the project to results of it.

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

cooperation and helping hand in completing my project work.

Further, I would like to express my thankfulness to Prof. K. Pal for their help and providing

access to their lab.

I consider it a privilege to express my gratefulness to Prof.T.K.Maiti, Department of

Biotechnology, Indian Institute of Technology Kharagpur and PSG Institute of

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

complete my work successfully.

Anu Priya B
213BM2018
M.Tech. Biotechnology
Department of Biotechnology and Medical Engineering
TABLE OF CONTENTS

Chapters Description Page


Abstract i
List of Tables ii
List of Figures iii
Chapter 1. INTRODUCTION AND LITERATURE REVIEW 1
1.1 Biology of bone 2
1.1.1. Bone structure 2
1.1.2. Bone cells 2
1.1.3. Bone formation and remodelling 3
1.2. Need for bone tissue engineering 3
1.3. Bone tissue engineering – current trends 4
1.4. Biomaterials used for bone tissue engineering 5
1.4.1. Polymeric Biomaterials 5
1.4.2. Ceramic Biomaterials 5
1.4.3. Hydroxyapatite 6
1.5. Vascularization of bone grafts 6
1.6. Present strategies for improving vascularization in bone 7
tissue constructs
1.6.1.Growth factor delivery 7
1.6.2. Culturing Endothelial cells inside the scaffold 8
1.6.3. Co-culturing system 8
1.6.4. Gene delivery 9
1.6.5. Development of microfluidic based network 9
1.7. Development of pro-angiogenic biomaterial 9
Chapter 2. OBJECTIVES AND WORK PLAN 11
2.1 Objective of the work 12
2.2 Plan of work 12
2.3. Rationale 12

Chapter 3. MATERIALS AND METHODS 13


3.1 Materials 14
3.2. Synthesis of nano-hydroxyapatite and nickel doped 14
nano-hydroxyapatite
3.3. XRD and FT-IR 15
3.4. TEM and EDAX 15
3.5. BET and Zeta potential analysis 15
3.6. Protein adsorption study 16
3.7. Cytocompatibility study 16
3.8. FESEM study 17
3.9. Expression of Runx2 and HIF-1α 17
3.10. VEGF expression study 18
3.11. RT-PCR analysis 18
Chapter 4. RESULTS AND DISCUSSION 20
4.1 Preparation of Ni+2 doped nHAp 21
4.2 EDAX 22
4.3. TEM 23
4.4. XRD 24
4.5. FT-IR 24
4.6. BET surface area analysis 26
4.7. Zeta potential analysis 27
4.8. Protein adsorption analysis 27
4.9. MTT assay 28
4.10. Cell cycle analysis 29
4.11. Live-Dead Assay 30
4.12. Differentiation of osteoblasts 31
4.13. Expression of VEGF 33
4.14. Expression of HIF-1α 34
Chapter 6. CONCLUSION 36-37
Chapter 7. REFERENCES 38-42
ABSTRACT

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

osteogenesis pinpointing its potential in bone tissue engineering application.

i|Page
List of Tables

S.No. Description Page No.

1 Composition, yield and physical appearance of nHAp and Ni+2 doped 21


nHAp

Crystal parameters of nHAp and Ni+2 doped nHAp. Percentage


2 crystallinity and d spacing were calculated using the XRD and TEM 24
diffraction data corresponding to 002 plane. For the analysis of crystal
lattice parameters, ‘a’ and ‘c’, 300 and 002 planes are considered.

3 Analysis of surface area, pore volume, zeta potential and protein


absorption. The data were mean of three independent experiments and 28
presented as Mean ± S.D.
Analysis of the percentage of live cells after sample treatment. The assay
4 31
was performed by flow cytometry using PI as a probe. Data were
expressed as Mean± S.D (n=3).

ii | P a g e
List of figures

S.No. Description Page No.


1 nHAp and Ni+2 doped nHAp prepared using ammoniacal precipitation 22
method
[A] FESEM micrographs of nHAp and Ni+2 doped nHAp. [B] TEM
Images of nHAp and Ni+2 doped nHAp. [C] Nano particle size
2 distribution. TEM Image based analysis was done using NIH-ImageJ
23
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).
[A] XRD analysis of nHAp and Ni+2 doped nHAp. Important
characteristic peaks are marked in the figure, and its corresponding
3 crystal planes were mention in the embedded table. [B] FT-IR analysis of 26
nHAp and Ni+2 doped nHAp. Important characteristic peaks are marked
in the figure, and its corresponding bond perturbation were mention in
the embedded table.
[A] Study of cell proliferation by MTT assay. Data were expressed as
4 Mean ± S.D (n=3). Statistical significance was checked for p< 0.005. [B] 30
Study of the cell cycle after 24 h of treatment. Experiments were done in
triplicate. Representative histograms for each sample was presented.
[A] SEM images of cells cultured in the presence of nHAp and
Ni+2doped nHAp samples at day 3. [B] Confocal micrographs of cells
cultured in the presence of nHAp and Ni+2doped nHAp samples at day 3.
5 33
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.
[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 HIF-1α
6 expression by MG-63 cells after 12 h post treatment by immuno-
35
cytochemistry. Confocal 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 for p< 0.005. [E] Study of HIF-1α mRNA expression by
RT-PCR.

iii | P a g e
CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1|Page
1.1. Biology of bone:

1.1.2. Bone structure:

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

help in the metabolic functions[1].

1.1.3. Bone cells:

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

where the metabolic rate is higher[2].

b) Osteoclasts – These multinucleated cells helps in bone resorption by degrading the

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

normal metabolism of bone by transducing signals from mechanical stress, secreting

necessary enzymes, controlling bone mineral content and release of calcium ions into

the blood[2].

2|Page
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

mechanisms: endochondral ossification and intramembranous ossification. In endochondral

ossification, the cartilage plate is first formed which gets replaced by the bone later, and

mostly bone formation is through this process. In intramembranous ossification,

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].

Bone is a connective tissue which undergoes continuous remodelling to maintain structural

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-

ordinated and balanced[4].

1.2. Need for bone tissue engineering:

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,

3|Page
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

perfectly into the bone defects and helps in osteointegration[6].

1.3. Bone tissue engineering – current trends:

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].

4|Page
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

main categories: polymers and ceramics.

1.4.1. Polymeric biomaterials:

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:

biodegradable and non-biodegradable polymers. Biodegradable polymers are generally used

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

limitations of biodegradable polymers is that endothelial cells cannot adhere to these

materials[9]. This gave opportunity for ceramic biomaterial for the application as scaffolds

for bone tissue engineering.

1.4.2. Ceramic biomaterials:

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

5|Page
on other non-bioactive materials such as metals to induce a strong biological fixation between

the host tissue and implant material[10].

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

usage of hydroxyapatite such as size, morphology, appropriate stoichiometry, phase

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

substituted hydroxyapatite is very much used in bone tissue engineering applications

nowadays.

1.5. Vascularization of bone grafts:

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
6|Page
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

implantation of the bone construct[13].

1.6. Present strategies for improving vascularization in bone tissue constructs:

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.

1.6.1. Growth factor delivery:

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

(bone morphogenetic protein-2), BMP-7 (bone morphogenetic protein-7), TGF-β

(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

instance, VEGF produced by osteoblasts help in promoting angiogenesis in endothelial cells

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

7|Page
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

factor delivery is time and dose dependent[14].

1.6.2. Culturing Endothelial cells inside the scaffold:

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]

1.6.3. Co-culturing system:

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].

8|Page
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,

many clinical trials of gene deliveries failed[16].

1.6.5. Development of microfluidic based network:

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].

1.7. Development of pro-angiogenic biomaterial:

Nowadays, development of pro-angiogneic biomaterial is gaining much attention as it

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

normoxic condition, HIF-1α undergoes prolyl hydroxylation by the enzyme prolyl

9|Page
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

case of nickel ions. Therefore, we hypothesize to incorporate nickel in a biomaterial to

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.

10 | P a g e
CHAPTER 2

OBJECTIVES AND WORK PLAN

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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

osteogenic and angiogenic property of the material invitro.

2.2. Work Plan:

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

hydroxyapatite for bone tissue engineering application.

12 | P a g e
CHAPTER 3
MATERIALS AND METHODS

13 | P a g e
3.1. Materials:

Calcium nitrate tetrahydrate (Ca(NO3)2.4H2O), diammonium hydrogen phosphate

((NH4)2HPO4), nickel nitrate hexahydrate (Ni(NO3)2.6H2O), cetyltrimethylammonium

bromide (CTAB) and ammonia solution (25%) were purchased from Merck, India.

Dulbecco’s Modified Essential Media (DMEM), Dulbecco’s Phosphate Buffer Saline

(DPBS) solution, Trypsin-EDTA solution, Fetal Bovine Serum, antibiotic-antimycotic

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.

3.2. Synthesis of nano-hydroxyapatite and nickel doped nano-hydroxyapatite:

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

maintained at 10-12 by addition of ammonium hydroxide. To this solution, 200ml of solution

containing 0.03M diammonium hydrogen phosphate and cetyltrimethylammonium bromide

(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

ratio of Ca/P in all the cases were maintained at 1.67.


14 | P a g e
3.3. XRD and FT-IR:

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

a range of 2θ = 20°-60° at a scanning rate of 2° per minute[22].

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].

3.4. TEM and EDAX:

JOEL JEM 2100 High –Resolution Transmission Electron Microscope (HR-TEM)

was used to determine the nature of the synthesized HAP particles. Sample specimen were

prepare using Copper grid with Formware –C Coating[24].

EDAX analysis was performed to determine the amount of nickel doped onto each

sample[25].

3.5. BET and Zeta Potential Analysis:

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

done at 5-point fast scan[26].

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

agglomeration of HAP particles[27].

15 | P a g e
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

from total protein concentration[28].

3.7. Cytocompatibility Study:

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

5thday, MTT assay was performed[29].

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

using FACS Accuri C6 (BD Biosciences)[30].

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
16 | P a g e
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

followed to determine the amount of live cells on day 3 and day 5.

3.8. FESEM study

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].

3.9. Expression of Runx2 and HIF-1α

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%

paraformaldehyde (10 min, room temperature), permeabilized with permeabilization buffer

(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

with TRITC (tetramethylrhodamineisothiocyanate) phalloidin and Hoechst dyes to view

under confocal microscope (TCS-SP8, Leica, Germany)[33].

3.10. VEGF Expression Study:

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].

3.16. RT-PCR analysis:

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].

19 | P a g e
CHAPTER 4

RESULTS AND DISCUSSION

20 | P a g e
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

and Cu+2, respectively in hydroxyapatite [25, 37].

Table 1. Composition, yield and physical appearance of nHAp and Ni +2 doped nHAp

S.No Samples Composition [Ca(NO3)2 .4H2O (NH4)2HPO4 Yield Colour


+ g/200ml (g)
Ni(NO3)2.6H2O]
g/200ml
1. HAP Hydroxyapatite 2.36 + 0.0 0.79 1.00±0.03 Milk
[Ca/P: 1.67] White
Data protected for publication
2. N1-HAP Hydroxyapatite + 2.36 + 0.0236 0.796 0.97±0.01 Creamy
Please contact
1%(w/w)Nickel White
banerjeei@nitrkl.ac.in for details
[(Ca+Ni)/P: 1.67]
3. N5-HAP Hydroxyapatite + 2.36 + 0.118 0.822 0.99±0.09 Greenish
5%(w/w)Nickel White
[(Ca+Ni)/P: 1.67]
4. N10-HAP Hydroxyapatite + 2.36 + 0.236 0.854 1.00±0.01 Greenish
10%(w/w)Nickel White
[(Ca+Ni)/P: 1.67]

21 | P a g e
Data protected for publication
Please contact
banerjeei@nitrkl.ac.in for details

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,

therefore, failed to get accommodated in the crystal lattice of hydroxyapatite[38]

22 | P a g e
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

particles were all in nano range with an average diameter of15-17nm

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

impact on the size of the nano crystals.

DataData
protected
protected
for publication
for publication
Please
Please
contact
contact
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

comparison to Ca+2 (0.99Å).

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.

S.No Samples % Crystallinity Crystal lattice ‘d’-spacing


parameters (nm)
Data protected for ‘a.'
publication ‘c’
1. HAP 42 contact
Please 9.37 6.88 0.3644
banerjeei@nitrkl.ac.in for details
2. N1-HAP 42 9.43 6.90 0.3642
3. N5-HAP 64 9.37 6.88 0.3624
4. N10-HAP 22 9.43 6.90 0.3578

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-

HAP and N1-HAP, and this may be due to crystal deformation.

25 | P a g e
Data protected for publication
Please contact banerjeei@nitrkl.ac.in
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

corresponding bond perturbation were mention in the embedded table.

4.6. BET Surface Area Analysis:

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

26 | P a g e
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

significant effect on the porosity of the material.

4.7. Zeta potential of HAP particles:

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).

4.8. Protein adsorption:

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

27 | P a g e
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

adsorption capacity of hydroxyapatite nanoparticles.

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

(m2/g) (cc/g) (mV) (mg)


Data protected for publication
1. HAP 109.1 0.041 -3.5 881±19.5
Please contact
2. N1-HAP banerjeei@nitrkl.ac.in
107.6 0.040 for details
-1.0 885±7.4

3. N5-HAP 111.7 0.046 3.2 873±18.4

4. N10-HAP 83.2 0.034 1.1 878.5±1.2

4.9. MTT assay

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

28 | P a g e
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.

4.10. Cell cycle analysis

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

has no adverse effect on the cell viability.

29 | P a g e
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.

4.11. Live/Dead Assay:

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.

However, it is important to mention that, MTT assay is actually a measurement of the

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

30 | P a g e
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

be because of differentiation of cells in presence of doped nano hydroxy apatite.

Table 4. Analysis of the percentage of live cells after sample treatment. The assay was performed by flow

cytometry using PI as a probe. Data were expressed as Mean± S.D (n=3).

Live Dead Assay: Percentage of live cells

Day Control HAP N1-HAP N5-HAP N10-HAP

Day1 94.95±1.23 94.95±1.01 93.45±1.49 92.76±1.48 89.99±2.53


Data protected for publication
Please contact banerjeei@nitrkl.ac.in
Day3 94.20±0.91 for
91.73±0.48
details 93.37±1.09 92.95±0.07 94.54±1.10

Day5 95.08±1.83 92.07±1.48 92.14±1.14 91.13±0.87 92.28±0.14

4.12. Differentiation of osteoblasts

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

differentiate to osteoblast cells. It is important to mention that MG -63 is a mature osteoblast.

Therefore, Runx2 expression always remains in basal level in MG-63 in comparison to its

31 | P a g e
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.

32 | P a g e
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.

4.13. Expression of VEGF

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
33 | P a g e
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 (

fold ) and N1-HAP ( fold ) respectively.

4.14. Expression of HIF-1α

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.

Mesenchymal condensation Generally, under normoxic conditions, prolyl hydroxylase

enzyme activates the proteosomal degradation pathway of HIF-1α through ubiquitination.

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

34 | P a g e
that aids in proteosomal degradation of HIF-1α) and stabilizes HIF-1α protein. From the

Runx2 immunocytochemistry expression profile, N5 showed better expression of Runx2

followed by N10. It was also reported for cu and co doped proangiogenic biomaterials

VEGF expression profile not always follow the HIF-1α profile.

Data protected for publication


Please contact
banerjeei@nitrkl.ac.in for details

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

HIF-1α expression by MG-63 cells after 12 h post treatment by immuno-cytochemistry. Confocal

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

for p< 0.005. [E] Study of HIF-1α mRNA expression by RT-PCR.

35 | P a g e
CHAPTER 5

CONCLUSION

36 | P a g e
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

inducer of cellular VEGF. We hypothesized that incorporation of Ni+2 into osteoconductive

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

involvement of Ni+2 in HIF-1α stabilization followed by increased VEGF expression.

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.

Moreover, it is important to assess the biological performance of the materials in vivo.

37 | P a g e
CHAPTER 6

BIBLIOGRAPHY

38 | P a g e
6.BIBLIOGRAPHY

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11. Kay, M., R. Young, and A. Posner, Crystal structure of hydroxyapatite. 1964.

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