Materials Science and Engineering C 58 (2016) 648–658

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Cobalt doped proangiogenic hydroxyapatite for bone tissue engineering application Senthilguru Kulanthaivel a, Bibhas Roy b, Tarun Agarwal a, Supratim Giri c, Krishna Pramanik a, Kunal Pal a, Sirsendu S. Ray a, Tapas K. Maiti b, Indranil Banerjee a,⁎ a b c

Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha 769008, India Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India Department of Chemistry, National Institute of Technology, Rourkela, Odisha 769008, India

a r t i c l e

i n f o

Article history: Received 24 August 2014 Received in revised form 5 August 2015 Accepted 25 August 2015 Available online 15 September 2015 Keywords: Angiogenesis Cobalt Hydroxyapatite HIF-1α VEGF Bone tissue engineering

a b s t r a c t The present study delineates the synthesis and characterization of cobalt doped proangiogenic–osteogenic hydroxyapatite. Hydroxyapatite samples, doped with varying concentrations of bivalent cobalt (Co2+) were prepared by the ammoniacal precipitation method and the extent of doping was measured by ICP–OES. The crystalline structure of the doped hydroxyapatite samples was confirmed by XRD and FTIR studies. Analysis pertaining to the effect of doped hydroxyapatite on cell cycle progression and proliferation of MG-63 cells revealed that the doping of cobalt supported the cell viability and proliferation up to a threshold limit. Furthermore, such level of doping also induced differentiation of the bone cells, which was evident from the higher expression of differentiation markers (Runx2 and Osterix) and better nodule formation (SEM study). Western blot analysis in conjugation with ELISA study confirmed that the doped HAp samples significantly increased the expression of HIF-1α and VEGF in MG-63 cells. The analysis described here confirms the proangiogenic–osteogenic properties of the cobalt doped hydroxyapatite and indicates its potential application in bone tissue engineering. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, bone tissue engineering has emerged as a promising approach to treat fracture or bone defects [1]. This approach involves the application of biologically active osteogenic implants (porous scaffold with/without bone cells) to the injury or defect site and its subsequent integration with the native osteo-chondral system [2]. Success of bone tissue engineering typically relies on the survival of the bone cells within the scaffold post-implantation and their commitment towards the regeneration process. In practice, at the early stage of implantation, due to the absence of the proper vasculature and limited diffusional transport of nutrients and oxygen (diffusion limit of solute and gases inside the tissue is 150–200 μm from the surface), bone cells present in the interior of the scaffold often dies via necrosis [3]. The importance of angiogenesis in tissue engineering; especially, in osteogenesis and bone tissue engineering was reviewed by Moon et al. [5] and Auger et al. [4]. The literature clearly suggests that early restoration of the vasculature at the site of implantation is essential for the success of bone tissue engineering based therapies. In the last couple of years, numerous attempts have been made to create the vasculature inside the implanted construct to facilitate the supply of nutrient and oxygen to the cells within a clinically relevant ⁎ Corresponding author. E-mail address: [email protected] (I. Banerjee).

http://dx.doi.org/10.1016/j.msec.2015.08.052 0928-4931/© 2015 Elsevier B.V. All rights reserved.

time scale [6]. Initial approaches were based on improving the cell adhesion properties of the implants to support the growth of endothelial cells and further vasculature formation. These approaches involved incorporation of extracellular matrix (ECM) proteins (collagen, fibronectin, fibrin, laminin, etc.) [7–8] and ECM derived peptides (RGDS and REDV peptides from fibronectin [9–11], YIGSR peptide from laminin [12]) to mimic the in vivo microenvironment and support the adhesion and growth of endothelial cells over the implants. Later, to improve the quality and rate of angiogenesis, different signaling molecules especially angiogenesis inducers like the vascular endothelial growth factor (VEGF) [13], platelet-derived growth factor (PDGF) [14], and basic epidermal growth factor (bEGF) [15] were incorporated into the implants. Apart from these approaches, various micropatterning and micromolding techniques such as photolithography and laser printing were also endeavored to produce pre-vasculature networks in vitro prior to implantation [16–18]. Alternatively, ventures have been taken up to trigger the cellular angiogenic growth factor secretory mechanism through genetic manipulation [19]. Although the aforesaid approaches have shown certain promises at a laboratory scale, there exists a number of issues which limit their routine clinical applications. Most of the angiogenic growth factors are very expensive. Moreover, when applied in vivo, it is difficult to control their biofunctionality in the complex in vivo environment [20]. It is now evident from the literature that any biomaterial capable of stimulating the cellular processes pertaining to the secretion of

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angiogenic growth factors is advantageous for bone tissue engineering [21]. Metal ions which are commonly found as cofactors for cellular enzymes inside the body play a vital role in various cell signaling processes [22]. It has already been reported that some metal ions can create oxidative stress in cells (mimicking hypoxia conditions) and induce the expression of angiogenic growth factors. In particular, transition metals such as chromium, nickel [23], vanadium [24], copper [25] and cobalt [26] generally stabilize hypoxia inducible factor 1 alpha (HIF-1α) and induce the expression of VEGF in the bone cells. The released signaling molecules (VEGF) further activate the cell signaling cascade pertaining to angiogenesis. Apart from these transition metals, a recent study showed that other metals such as magnesium ions also can induce angiogenesis through the HIF-1α pathway in human bone marrow derived stromal cells [27]. In this regard, metal ion doped ceramic/ composite materials have started gaining importance in the field of bone tissue engineering. Among these metal ions, cobalt +II (especially cobalt chloride) has long been used to upregulate and stabilize HIF-1α which, in turn, promotes angiogenesis by creating a hypoxia mimicking conditions [28]. Hypoxia (low oxygen pressure) plays an important role in coupling angiogenesis to osteogenesis [29]. Under normoxic conditions, the prolyl hydroxylase (PH) enzyme hydroxylates the proline residues of HIF-1α thereby making it a target for ubiquitination followed by proteosomal degradation. Cobalt ions stabilize HIF-1α by decreasing the hydroxylation and ubiquitination activities of PH. It also reduces the activity of FIH (factor inhibiting HIF-1α) by depleting Fe2 + ions and ascorbate which are the key cofactors of PH and FIH. This process leads to the formation of a complex transcription factor with HIF-1β which, in turn, increases the expression of VEGF gene. As a result, cobalt stimulates the secretion of VEGF through the HIF-1α pathway and induces angiogenesis [30]. The aforesaid facts are indicative of the potential of bivalent cobalt (Co2+) ion for use in biomaterials to improve their angiogenesis inducing potential. In a recent report, Wu et al. have also shown that proangiogenic mesoporous bioglass could be made by selective cobalt doping [26]. Calcium hydroxyapatite [Ca10(PO4)6(OH)2], often referred to as synthetic hydroxyapatite (HAp), has widely been accepted as a biomaterial of choice for bone tissue engineering because of its close structural resemblance with the normal bone apatite, non-toxicity, osteoconductive property and non-immunogenicity [31]. Interestingly, the high flexibility and stability of the apatite crystal structure allow the selective replacement of cations (especially Ca2 +) and anions (OH − and (PO 4) 3 −). Exploiting this property of the apatite structure, different metal ion (e.g. Mg2 +, Zn 2 +, Mn2 +, Cd 2 + , and Y3 +) [32–36] doped HAp samples have been prepared for various applications including bone tissue engineering. Keeping that perspective in mind, we hypothesize that doping of the bivalent cobalt (Co2 +) ions into calcium HAp will impart an angiogenesis inducing potential to HAp. To date only a few reports pertaining to the synthesis and application of cobalt doped HAp have been published that include antibacterial bone implants and carbon monoxide (CO) gas sensor applications [37]. However, no comprehensive work related to angiogenesis and bone tissue engineering applications of cobalt doped HAp has been published yet. The present study delineates the synthesis of cobalt doped HAp and its physico-chemical and biological characterizations. A number of methodologies such as solid state synthesis, hydrothermal synthesis, wet chemical method, and sol–gel and biosynthesis have been employed for the preparation of HAp and doped HAp [38]. Among them, the wet chemical method has been used by many groups for its ease of synthesis and process controllability [39]. Therefore, here we synthesized cobalt doped HAp by the wet chemical precipitation method. The materials were first subjected to XRD, FTIR, and ICP–OES studies to evaluate their physico-chemical properties. Osteoconductivity and angiogenic properties of the materials were tested by checking the expression of bone differentiation markers (Runx2 (Runt-related transcription factor 2) and Osterix) and angiogenic factors (HIF-1α and

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VEGF), respectively, in human bone cell line MG-63 cultured in the presence of the doped HAp. 2. Materials and methods 2.1. Materials Calcium nitrate tetra-hydrate (Ca(NO3)2·4H2O), di-ammonium hydrogen phosphate ((NH4)2HPO4), cobalt chloride hexahydrate (CoCl2·6H2O), cobalt nitrate hexahydrate (Co(NO3)2·6H2O), Dulbecco's Modified Eagle Media (DMEM), Dulbecco's Phosphate Buffer Saline (DPBS), Trypsin–EDTA solution, Fetal Bovine Serum (FBS), Antibiotic– Antimycotic solution, and MTT assay kits were purchased from Himedia, Mumbai, India and ammonia solution was procured from Merck chemicals, India. For cell lysis, Pierce IP lysis buffer was procured from Thermo Scientific, US. Antibodies against HIF-1α, Runx2 and Osterix, secondary antibodies (anti-Rabbit, Alexa Flour 488 tagged) and human VEGF ELISA (Enzyme-linked immunosorbent assay) kit were procured from Abcam, UK. 2.2. Method HAp was prepared using the ammoniacal precipitation method [40]. 200 ml of 0.05 M of Ca(NO3)2·4H2O solution was taken in a 500 ml beaker and to it, 200 ml of 0.03 M of (NH4)2HPO4 solution was added dropwise at a rate of ~2 ml/min from a burette. The process conditions were maintained at a calcium to phosphate ratio (Ca/P) of 1.67, pH of 10–12 (using NH4OH) and a temperature of 80 °C under sonication. At room temperature (30 ± 5 °C), the slurry was aged for 24 h and then centrifuged. The obtained precipitate was washed several times with distilled water for the removal of residual ammonia and the unreacted reactants (Ca(NO3)2·4H2O and (NH4)2HPO4) and then dried at 50 ± 5 °C for 24 h to get HAp powder. Cobalt doped HAp samples were prepared by the addition of cobalt sources (cobalt chloride and cobalt nitrate) along with calcium nitrate solution using the abovementioned method. Doping was done at different weight percentages (10%, 5%, 1% and 0.5% w/w) of cobalt to calcium. The molar ratios of (Ca + Co)/P were set to 1.67 for all the cases. The different doping percentages and their notations are given in Table 1. HA1 is the commercial HAp procured from Acros-organics, Thermo Fisher scientific (Mumbai, India). HA2 is the pure HAp prepared by the ammoniacal precipitation method. 2.3. Characterization 2.3.1. Physical characterization The characterization of the phase content of the samples was done by the X-ray diffraction (XRD) method using a Philips XRD-PW1700 diffractometer which produces a monochromatic CuKα radiation of wavelength (λ = 1.541 Å). The samples were scanned over a range of 2θ from 20° to 60° at a scanning rate of 2° (2θ)/min [41]. Functional groups present in the doped HAp samples were analyzed by FTIR (Fourier

Table 1 Stoichiometry, color and yield of cobalt doped HAp samples. S. no.

Sample

Notation

Color

Yield (g)

1 2 3 4 5 6 7 8 9 10

STD HAp Pure HAp 0.5% CoCl2-HAp 0.5% Co(NO3)2-HA 1% CoCl2-HAp 1% Co(NO3)2-HAp 5% CoCl2-HAp 5% Co(NO3)2-HAp 10% CoCl2-HAp 10% Co(NO3)2-HAp

HA1 HA2 HAC1 HAN1 HAC2 HAN2 HAC3 HAN3 HAC4 HAN4

White White White White Light gray Light gray Dark green Dark gray Dark green Dark gray

– 0.79 0.94 0.8 0.79 0.83 0.85 0.8 0.9 0.88

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Transformed Infrared Spectroscopy). The FTIR study was done using Shimadzu/IR prestige 21 over the scanning range of 400–4000 cm−1 using the KBr pellet method [42]. 2.3.2. Chemical characterization The extent of cobalt doping was measured using inductively coupled plasma optical emission spectrometry (ICP–OES) (Perkin Elmer optima 5300 DV). The samples were dissolved in acid solutions of conc. nitric acid (HNO3) (69%) and conc. perchloric acid (70%) in the ratio of 2:1. The dissolved sample solutions were placed over a hot plate and continuously heated at 50 °C to evaporate the concentrated acids. The samples were then diluted using 1% HNO3. Heating and dilution steps were repeated for 4 to 5 times until major portions of the concentrated acids were evaporated. The final volume was reduced to 1 ml, which was subsequently diluted to 25 ml using distilled water. The pH of the final digestion solution was set to ~7 for ICP–OES analysis [43]. The release of cobalt ions from the sample was quantified using the same technique mentioned above (ICP–OES). For the release study, 10 mg of HAp samples was suspended in 25 ml of phosphate buffer saline (PBS) (pH 7.4) and kept at a constant shaking of 100 rpm in an orbital shaker at 37 °C for 60 days. After the incubation period, the samples were centrifuged and the supernatant was taken for ICP–OES analysis, to determine the amount of cobalt released [44]. 2.3.3. Biological characterization The protein adsorption behavior of the samples was studied taking bovine serum albumin (BSA) as model protein. HAp and doped HAp samples (10 mg) were added separately to the test tubes containing 1 ml aqueous solution of BSA (1200 μg/ml). Samples were incubated at 37 °C for a period of 24 h to allow protein adsorption. Thereafter, the reaction slurry was centrifuged at 8000 rpm (REMI-Cooling Centrifuge) for 10 min and the supernatant was collected [45]. The residual protein concentration in the supernatant was determined using the Bradford assay [46]. The extent of protein absorption was determined by subtracting the residual protein from the total protein. The hemolysis study of the samples was performed as per the American Society for Testing Materials (ASTM F 756-00, 2000) protocol [47]. To prevent coagulation, tri-sodium citrate (3.8 g w/v %) was added to the blood. The blood was diluted with normal saline (0.9% NaCl) in the ratio of 8:10 (v/v) as the working standard. 10 mg of the samples was taken in a clean test tube and 0.5 ml of blood was added to it. The sample volume was made up to 10 ml with normal saline (0.9% NaCl) and kept at 37 °C for 1 h. 0.5 ml of saline and 0.5 ml of 0.1 M HCl were taken as negative and positive controls, respectively. Thereafter, the solutions were centrifuged at 4000 rpm for 10 min and the absorbance of the supernatant was measured at 545 nm. The samples having percentage hemolysis less than 5% are considered as highly hemocompatible and the samples having percentage hemolysis less than 10% is hemocompatible. The samples which have percentage hemolysis more than 20% are not hemocompatible [48]. The percentage hemolysis is given by Eq. (1). % Hemolysis ¼ ½ðODTest –OD−ive Þ=ðODþive –OD−ive Þ  100

ð1Þ

The biocompatibility studies were performed using the MG-63 cell line, procured from NCCS, Pune, India. The cells were maintained in DMEM with 10% FBS at 37 °C, 5% CO2 and 95% humidity. During passaging, the cells were washed and harvested using DPBS and Trypsin–EDTA solution, respectively. The viable cell density was assessed using the Trypan Blue dye exclusion method and the cells were then seeded onto a 96 well plate at a cell concentration of 5 × 104 cells/well. The seeded plate was placed in the incubator for the next 12 h for cell adhesion. After that, cells were incubated with the samples at 100 μg/ml concentration for a period of 2 to 7 days and the cell viability was assessed using the MTT assay [49].

For cell cycle analysis, MG-63 cells at 70% confluence in a T-25 flask were treated with the 7 day releasate of HAp samples prepared in DMEM media, at a concentration of 100 μg/ml for a period of 36 h. HAp treated cells were trypsinized and washed with ice-cold DPBS. Harvested cells were fixed with 70% ice-cold ethanol. Fixed cells were centrifuged at 3000 rpm and finally suspended in 500 μl of propidium iodide (PI) staining buffer (50 μg/ml PI, and 100 U/ml RNase) and stained for 30 min at room temperature [50]. The samples were analyzed using FACS (Fluorescent Activated Cell Sorting) Accuri C6 (BD Biosciences) and FACS Accuri C6 software. The osteogenic property of the doped HAp samples was analyzed by checking the expression of the differentiation markers, Runx-2 and Osterix using immunocytochemistry. MG-63 cells (4 × 105 cells/well) were seeded in a glass bottom petri dish. The seeded cells were kept in the incubator for a period of 12 h for adherence. Thereafter, the adhered cells were treated with different doped HAp samples at a concentration of 100 μg/ml. For Runx-2 and Osterix expression studies, cells were given HAp treatment for a period of 3 and 9 days, respectively. After HAp treatment, cells were first fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X and then blocked with 2% BSA prepared in PBST (0.1% Triton X in PBS). Thereafter, the cells were treated with primary antibodies of Runx-2 (rabbit, anti-human, dilution: 1:1000) and Osterix (rabbit, anti-human, dilution: 1:200) and incubated overnight at 4 °C with gentle shaking. All the antibody solutions were prepared in 1% BSA PBST. After the incubation, the antibody solution was removed and washed thrice with PBS to remove the unbound antibodies. Cells were then treated with secondary antibodies tagged with Alexa Fluor 488 (dilution: 1:1000) with gentle shaking for a period of 1 h at room-temperature. Finally, samples were washed thrice with PBS and counter-stained with DAPI (4′,6-diamidino-2-phenylindole) and TRITC (tetramethylrhodamine isothiocyanate) Phalloidin. The expressions of Runx-2 and Osterix were visualized using a confocal microscope (Olympus Fluoview 1000) [51]. The angiogenic property of the doped HAp samples was analyzed by estimating HIF-1α and VEGF expression levels. The expression of HIF1α was analyzed by western blotting. For this, 10 mg of HAp samples was sterilized and suspended in 1 ml of DMEM media for a period of 7 days at 37 °C. Therafter, the releasate of HAp samples at 100 μg/ml concentration was added to MG-63 cells (seeded at the concentration of 1.3 × 105 cells/well in a six well plate, 12 h prior to sample loading). For deliberate HIF1α expression, 200 μM CoCl2 was used as the positive control. After 24 h of incubation, cells were trypsinized and the cell lysate was prepared using cell lysis buffer (Thermo Scientific, US). The protein concentration was measured using a Bradford assay kit. 20 μg of the total protein from each sample was fractionated with 7% SDSPAGE and transferred onto a PVDF membrane using an electrotransfer apparatus. The membrane was blocked with 5% BSA solution. The blocked membrane was immunoblotted with a primary antibody against HIF-1α (dilution: 1:200, mouse anti-human) and incubated overnight at 4 °C with gentle shaking. The antibody solution was removed and the membrane was washed thrice with PBST buffer. Thereafter, the membrane was incubated with the secondary antibody (antimouse HRP conjugated, dilutions: 1: 1000) for 1 h at room-temperature with gentle shaking. The chemiluminescence ECL kit was used to detect the immunostained samples [52]. The membrane was exposed to X-ray film to obtain bands. β-Actin was used as control for this analysis [26]. VEGF expression of MG-63 cells was analyzed using a Human VEGF ELISA kit (Abcam 100662). In a twelve well plate, 1.5 × 105 cells/well were seeded and kept for a period of 12 h at 37 °C and 5% CO2. The cells were then treated with the 7 day releasate of the HAp samples at a concentration of 100 μg/ml for 24 h. 200 μM cobalt chloride was used as a positive control. The reaction volume was limited to 600 μl to avoid dilution of VEGF. After 24 h of incubation, the supernatant was collected and ELISA was performed following manufacturer's instructions. In brief, 100 μl samples were added to each well of a precoated ELISA plate and incubated for 2.5 h. The supernatant was

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removed after 2.5 h. The sample incubation step was followed by primary antibody incubation, washing, secondary antibody (100 μl of biotinylated VEGF) incubation and subsequent washing. Then 100 μl of the 1× HRP streptavidin solution was added to the wells. After 45 min of incubation, 100 μl of TMB substrate was added to each well and samples were kept for 30 min for color development. The stop solution was added subsequently and the absorbance was measured immediately at 450 nm [53]. 2.4. Statistical analysis All the experiments were run in triplicate. Statistical analysis was carried out using Student's independent “t test”. A “p” value of 0.05 or less was taken as significant. 3. Results 3.1. Preparation of pure and cobalt doped HAp samples In the present study, cobalt doped HAp samples were synthesized by the ammoniacal precipitation method. Doping was performed with an intention to replace only the cation (using cobalt nitrate) and both the cation and anion (using cobalt chloride) from the calcium HAp crystal. The yield of HAp powder was between ~ 0.8 and 1.0 g in all the cases. The highest yield was obtained for HAC1 (0.94 g) followed by HAC3 (0.85 g), whereas the yield was lowest for HA2 and HAC2 (0.79 g) (Table 1). A color difference was observed between the pure HAp and with cobalt nitrate and cobalt chloride doped HAp samples (Annexure I, Fig. A.1). The pure HAp sample was milky white in color, whereas cobalt nitrate doped HAp samples were gray colored and cobalt chloride doped HAp appeared greenish. Doping of cobalt in the synthesized material was confirmed by ICP–OES (data is given in Section 3.3). 3.2. Structural study XRD is often used to identify and analyze apatite structures [54]. In this case, the XRD pattern of the doped samples (Fig. 1) was found similar to that of crystalline HAp (Ca10(PO4)6(OH)2) as per the standard data sheet (JCPDS No. 09-0432) [55]. The XRD profiles of the doped HAp were devoid of any additional peaks corresponding to cobalt and did not differ much from pure HAp (HA1) except a slight broadening of the peak at 32°, 33° and 34° 2θ. In the case of cobalt chloride doped

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HAp, the characteristic peaks of HAp (32°, 33° and 34° 2θ) were significantly broadened and merged. The third intense peak of HAp at 26° 2θ, observed in all the cases without much variation in the intensity corresponded to the (0 0 2) plane of apatite crystal. The other characteristic peaks of HAp correspond to the crystal planes (3 1 0), (2 2 2), (2 1 3), (4 0 2) and (0 0 4) at 40°, 47°, 50°, 52° and 53° 2θ that were also observed in all the samples. For the general hexagonal crystal system, the relationship between the crystal plane index and interplanar distance (dhkl) is given by the following equation: h

i1=2  2 2 2 1=dhkl ¼ 4 h þ k þ hk =3a2 þ l =c2

ð2Þ

where ‘h’, ‘k’, and ‘l’ are the miller indices of the crystal plane. The lattice parameters of the HAp samples were calculated using the following formula derived from the above equation (Eq. (2)) [56]. For calculating parameters ‘a’ and ‘c’, plane indices ‘300’ and ‘002’ were selected, respectively.   a ¼ 2  √3  d300 c ¼ 2  d002 The percentage crystallinity of the samples were calculated by KA ¼ β1=2  ðXcÞ1=3 : where β1/2 is the full width at half maximum corresponding to the plane '002', KA is the constant (KA=0.24) and Xc is the degree of crystallinity. The values of the lattice parameters and the percentage crystallinity were given in Table 2. Analysis of the lattice parameter showed that there was no significant variation between HAN and HAC groups. Lattice parameter ‘a’ was same for all the samples (around ~9.4 Å). The value of c was found close to 6.9 Å for all the samples. The percentage crystallinity decreased with increasing doping percentage. HAC3 was less crystalline (8.26%) in comparison to other HAp samples followed by HAC1 (12.5%). HAC2 and HA2 were more crystalline (20.3%) (Table 2). The FTIR spectrum of the pure and the cobalt doped HAp (Fig. 2) showed the presence of the characteristic bands of HAp. The bands at 1035 cm− 1 and 1095 cm− 1 were due to the ν3 (P\\O) stretching of

Fig. 1. XRD analysis of cobalt doped HAp samples.

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Table 2 Crystal parameters of the cobalt doped HAp samples. S. no.

1 2 3 4 5 6 7 8

Samples

HA1 HA2 HAC1 HAN1 HAC2 HAN2 HAC3 HAN3

Crystal lattice parameters (Å) a

c

9.4109 9.4358 9.4428 9.4299 9.4023 9.4019 9.415 9.3974

6.9162 6.9124 6.879 6.9116 6.9004 6.8984 6.8794 6.8982

Percentage crystallinity [Xc (%)]

17.14 20.35 12.5 14.57 20.35 17.14 8.26 14.57

The parameters ‘a’, ‘c’ and ‘Xc’ were calculated from XRD patterns data. For calculation of ‘a’ the ‘300’ plane and for ‘Xc’ and ‘c’ ‘002’ plane were considered.

the phosphate groups (PO4)3− [55]. The band at 473 cm−1 corresponds to ν2 bending of the phosphate group. The ν4 vibrational band of the phosphate groups (PO4)3− appeared at three different wave numbers (566, 633 and 603 cm− 1) of the FTIR spectrum [57]. The bands at 1470 cm−1 and 870 cm−1 were assigned to (CO3)2− ions due to the adsorption of carbon dioxide from the atmosphere [54]. The bands at 3450 cm−1 and 3572 cm− 1 correspond to O\\H stretching of the absorbed water [58] and hydroxyl group (OH−) of HAp. The band at 1640 cm−1 corresponds to O\\H bending of the absorbed water. Although, the characteristic bands of HAp were observed in all the samples, the intensities of the bands varied among the doped samples. 3.3. Chemical study ICP–OES analysis revealed that there was an increase in the percentage doping of cobalt in HAp with the increase in initial dopant concentration up to a threshold value (5% w/w) above which no significant increase in doping was observed (Table 3). Here, HAN3 showed the

highest doping percentage of about 1.2%, followed by HAC3 (~1.19%), where HAN1 showed the least doping percentage of about 0.15%. Since 10% doping (theoretical) did not show any further increase in the actual doping percentage in comparison to 5% doping, 10% cobalt doped samples were not considered for further study. The extent of leaching of the cobalt ions in physiological buffer was carried out for 60 days. The release study was carried out in phosphate buffered saline (pH 7.4) and the analysis was done by ICP–OES study. It was observed that (Table 4) the release of cobalt ions from HAC1 and HAN1 was the least (0.004 mg/l) in comparison to the other doped HAp samples. Further, the release of cobalt ions increased with an increase in the doping concentration. HAC2 and HAN2 samples showed a cumulative cobalt release of 0.008 mg/l, whereas the same for HAC3 and HAN3 was about 0.136 mg/l and 0.059 mg/l, respectively. 3.4. Biological characterization 3.4.1. Protein adsorption and hemolysis study BSA was used as the model protein for the adsorption study. Among the pure and doped HAp samples, HAC1 showed the maximum protein adsorption of 75.34 μg/mg (Fig. 3). The protein adsorption of HAN samples was higher than that of the pure HAp sample (HA1), but in the case of the HAC samples, the protein adsorption decreased with an increase in the doping percentage and HAC3 showed the least protein adsorption (42 μg/mg). HAC1 had significantly higher protein adsorption in comparison with all other samples (p b 0.05). However, there was not much variation in the protein adsorption profiles among the other doped samples. The hemolysis study (Annexure I Fig. A.2) showed that all the samples, except HAC1 did not cause hemolysis of more than 5%, which means that they are hemocompatible. It was observed that HAC3 and HA2 followed the basal level comparable to the negative control (Table 5). HAC1 caused a significantly higher hemolysis of about 5.4% as compared to HA1 and HA2 (p b 0.05), followed by HAC2 (4.8%). All

Fig. 2. FTIR analysis of cobalt doped HAp samples.

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Table 3 ICP–OES analysis: Extent of cobalt ion doping in HAp samples. S. no.

Samples

Theoretical concentration (ppm)

Experimental concentration (ppm)

Theoretical percentage of doping (%)

Experimental percentage of doping (%)

1 2 3 4 5 6 7 8 9 10

HA1 HA2 HAC1 HAN1 HAC2 HAN2 HAC3 HAN3 HAC4 HAN4

0 0 10 10 20 20 50 50 50 50

0.141 0.023 3.490 2.984 6.586 7.338 11.88 11.99 6.648 7.743

0 0 0.5 0.5 1 1 5 5 10 10

0 0 0.175 0.15 0.33 0.37 1.19 1.2 1.32 1.54

the three HAN samples induced hemolysis, but within the acceptable limits (b5%). Among the HAN samples, HAN3 showed a maximum hemolysis of about 3.3%, followed by HAN1 and HAN2 of about 0.86% and 0.32% respectively. 3.4.2. Osteoblast proliferation The cell proliferation study showed that on day 2, all the doped HAp samples favored the proliferation of MG-63 cells (Fig. 4(a)). Among them, HAN1 induced the highest proliferation followed by HAC1. HAN1 and HAC1 caused 1.5 and 1.27 fold increases in the cell proliferation in comparison with control (tissue culture plate (TCP)) (p b 0.05). In the case of HAN3, the viability index was close to that of control. On day 7, all the doped samples showed less proliferation as compared to the pure HAp (HA1 & HA2). HAC2 and HAN1 showed a higher proliferation among the doped samples and both showed a 1.2 fold increase in comparison with control. Both HA1 and HA2 enhanced the proliferation significantly (p b 0.05) compared to TCP and all other doped HAp samples. In the case of HAN2 the viability index was close to TCP, whereas HAN3 failed to support cell proliferation. Cell cycle analysis by FACS (Fig. 4(b)) showed that the percentage of the cells in the S phase varied with different HAp treatments. It was observed that the percentage of the cells in the S phase was highest in the case of HA2 treated cells (~ 28.9% cells) followed by HAN1 (27.2%), whereas HAN2 (24.9%) and HAN3 (21.5%) showed the least for the same. FACS analysis also showed that percentage apoptosis was negligible for all the samples (Annexure 1, Fig. A.3). The SEM (scanning electron microscope) micrograph (Fig. 5(a)) of the osteoblast cells cultured on doped HAp (at day 9) showed that the cells spreaded well and maintained their characteristic shape. It was also observed that there was a significant bone matrix deposition and formation of nodules in the HAN2, HAC3 and HAN3 samples. 3.4.3. Differentiation of osteoblast In this study, we have shown that there was a differential Runx2 expression with the cobalt doping percentage. However, such variation did not show any general trend (Fig. 5(b)). Immunocytochemistry

Table 4 ICP–OES analysis: Extent of cobalt ion leaching from HAp samples (cumulative). S. no.

1 2 3 4 5 6 7 8

Sample

HA1 HA2 HAC1 HAN1 HAC2 HAN2 HAC3 HAN3

Experimental doping percentage (%)

Cobalt ion released (60 days) (ppm)

Cobalt ion released (60 days) (%)

0 0 0.175 0.15 0.33 0.37 1.19 1.2

0 0.002 0.004 0.004 0.008 0.008 0.136 0.059

– – 0.571 0.667 0.606 0.54 2.857 1.229

Fig. 3. Protein adsorption profile of cobalt doped HAp samples. Data were expressed as mean ± S.D. Statistical significance was checked for p b 0.05 by Student's ‘t test’.

data showed that the expression was highest in HAN2 followed by HAC2. Both HAN1 and HAC1 did not induce any significant Runx2 expression and almost follow the basal level (control HAp). Interestingly, in the case of 5% cobalt doping (HAN3 and HAC3) the expression of Runx2 was less than that observed in the 1% doped sample (HAN2 and HAC2), but in the case of HAC3, localization to the nucleus was more. Immunocytochemistry data showed that the Osterix (late differentiation marker) expressions also did not follow any general trend in accordance with the cobalt doping percentage. HAN2 showed the highest expression of Osterix followed by HAC2 as similar to the Runx2 expressions (Fig. 5(c)). The least expression of Osterix was observed for HAC3. 3.4.4. HIF-1α expression The HIF-1α expression profile of HAp treated MG-63 cells was studied by western blot analysis (Fig. 6(a)). In this study, the positive control (200 μM cobalt chloride solution) showed the highest expression of HIF-1α. Among different doped samples, HAN2 showed the highest expression of HIF-1α whereas the lowest expression was observed in HA2. The HAC1, HAC2 and HAN3 samples also showed a significant expression of HIF-1α next to the HAN2 sample. However, the other doped samples failed to stimulate HIF-1α expression. 3.4.5. VEGF expression The relative expression level of VEGF measured by ELISA showed a differential expression pattern with respect to cobalt doping (Fig. 6(b)). In this study, 200 μM cobalt chloride solution (positive control) showed a 1.5 fold increase in VEGF compared to control. HAN3 and

Table 5 Hemolysis study of the cobalt doped HAp samples (hemolysis caused by 0.1 N HCl was considered as 100% hemolysis and values for samples were normalized on that basis). S. no

Sample

% hemolysis

1 2 3 4 5 6 7 8 9

HA1 HA2 HAC1 HAC2 HAC3 HAN1 HAN2 HAN3 0.1 N HCl

1.16 ± 0.05 (~0) 5.43 ± 0.75 4.87 ± 0.61 (~0) 0.87 ± 0.18 0.32 ± 0.03 3.35 ± 0.50 100

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Fig. 4. (a) Viability of MG-63 cells treated with cobalt doped HAp samples at a concentration of 100 μg/ml for 2 and 7 days. Cell viability was assessed by MTT assay. Data were expressed as mean ± S.D. Statistical significance was checked for p b 0.05 by Student's ‘t test’. (b) Cell cycle analysis of MG-63 cells treated with the seven day releasate of cobalt doped HAp samples (at a concentration of 100 μg/ml) through flow cytometry. (Control — cells cultured on tissue culture plate without HAp samples).

HAN2 showed the highest expression of VEGF among the HAp samples and showed expression of about 1.35 and 1.22 fold increase in comparison to the control (HA2). HAN3 had an expression significantly higher than HAN2 and other doped HAp samples (p b 0.05). The general trend was HAN3 N HAN2 N HAC3 N HAC2 N HAC1 N HAN1. 4. Discussion Here, we have used the wet chemical method to synthesize cobalt doped HAp. In the case of cobalt chloride doping, there was a possibility of both anion replacement and cation replacement. Calcium ions of HAp could be replaced by cobalt ions and also a portion of the hydroxyl ions of HAp might be replaced by the chloride ions of cobalt chloride. But in the case of cobalt nitrate doped HAp only cation replacement was possible. As mentioned in the Results section, we observed a color variation with varying cobalt ion doping concentrations. In the case of doped HAp such color differences were found to be directly proportional to the doping concentration which indirectly gave a clue about cobalt doping. The difference in color may also be due to the variation in the ion displacement pattern and the extent of doping. Wakamura et al. earlier reported

that cobalt ions tend to form a color ammine complex (hexaamminecobalt) with NH4OH in basic pH during HAp preparation [59]. In accordance with this, during the synthesis process, appearance of dark bluish-green color was observed in the reaction solution (calcium nitrate/cobalt ion solution), when ammonia solution was added to set the pH to basic (~10–12). Hence, such color appearance of the samples might be due to the formation of the hexaamminecobalt color complex and its subsequent adsorption/doping into the crystal lattice of HAp. Tank et al. [60] and Yan Li et al. [61] also reported the same phenomenon while doping cobalt and copper into HAp, respectively. The XRD profile (Fig. 1) of the doped HAp samples did not reveal any additional peaks corresponding to cobalt, which indicates that cobalt might have replaced calcium ions from the apatite crystal frame perfectly and did not cause much distortion of the crystal structure [62]. HAC samples showed the maximum peak broadening (2θ = 32°, 33° and 34°) in comparison with HAN (cobalt nitrate doped HAp) samples and pure HAp. The parameters which affect the peak broadening are percentage crystallinity, crystallite size and crystal lattice distortion [63]. The peak broadening could be due to the poor crystallinity of samples. The reason for such decrease in crystallinity might be due to the possible anionic replacement of OH− ions present in the HAp by chloride (Cl−) ions of cobalt chloride in addition to the replacement of calcium by cobalt. Broadening of the aforementioned peaks with increasing percentage of doping of cobalt chloride strongly supports such possibility. Tank et al. also observed the similar phenomenon of peak broadening with an increase in the doping percentage of cobalt while preparing cobalt doped HAp [60]. Therefore, it can be said that doping of cobalt chloride resulted in smaller crystal size and less crystallinity compared to pure HAp and cobalt nitrate doped HAp samples. This was clearly evident from the percentage crystallinity data (Table 2) where HAC1 and HAC3 were less crystalline (12.5% and 8.26%, respectively) in comparison to other HAp samples. The variations in the values of lattice parameters ‘a’ and ‘c’ might be due to little distortion caused by incorporation of cobalt ions into the HAp lattice. In the FTIR spectra (Fig. 2), the ν4 vibrational band of the (PO4)3− group at the three sites (566, 633 and 603 cm−1) is generally considered as the FTIR signature mark of HAp. In all the cases, irrespective of doping percentage and doping type, the aforesaid bands were observed. Earlier, Kannan et al. reported that anionic replacement of OH− ions by fluorine and chlorine ions in the HAp can be noticed by the disappearance of the (O\\H) stretching band at 3570 cm−1 [64]. In the case of HA1 and HA2, the band at 3572 cm− 1 was prominent. The same band was present in HAN samples (more prominent in HAN2) but not observed in the case of HAC samples. Uysal et al. also reported the same phenomenon while incorporating fluorine ions into HAp crystal [65]. This suggests that there might be a possible replacement of OH− of HAp by Cl− ions of CoCl2. The hump ranging 3700–2700 cm−1 showed a significant increase in intensity with an increase in cobalt doping concentration. This increase in intensity may be attributed to the increase in the water adsorption capability of the HAp samples with an increase in the doping concentration [60]. The band at 1640 cm−1 became more prominent with an increase in the cobalt doping percentage due to the increased water adsorption of cobalt doped HAp as compared to pure HAp [60]. In every case, it was observed that the actual doping of cobalt always remains less in comparison to its theoretical value (Table 3). A similar trend was reported by Venkatasubbu et al. for zinc doped apatite [66]. It was observed that for a specific doping percentage, the anion replacement did not have any influence over the extent of cobalt (cation) doping. In the ammoniacal precipitation method, the maximum cobalt doping was observed for 5% (w/w) Co:Ca. The release of cobalt ion from the doped samples was estimated by ICP–OES analysis (Table 4). In general, the leaching of the foreign ions depends upon the amount of ions doped and the crystallinity and crystal lattice parameters of the apatite formed. Analysis showed that crystal structure distortion (as per the XRD data analysis) had direct influence

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Fig. 5. (a) FESEM micrographs of MG-63 cells cultured with cobalt doped HAp samples at a concentration of 100 μg/ml for 9 days. (b) Runx2 and (c) Osterix expression profiles of cobalt doped HAp sample (at a concentration of 100 μg/ml) treated MG-63 cells. In the confocal micrographs (b & c), green corresponds to Runx2 and Osterix and blue (DAPI) and red (TRITC Phalloidin) correspond to the nucleus and F-actin.

over cobalt leaching. Distortion of the crystal structure was maximum in HAC3 among all the doped HAp samples and so was the release of the cobalt ion [67]. On the other hand, both HAC2 and HAN2 having almost regular apatite structure showed a very less amount of cobalt release. The release of cobalt ions from the materials is very important in the context of the so called ‘cobalt toxicity’. It is already reported that excess consumption of cobalt causes nausea, hypothyroidism and goiter. Interestingly, we all need cobalt at a trace amount for normal biological function. Apart from being an essential component of vitamin B12 cyanocobalamin, cobalt is also a part of metalloprotein enzymes such

Fig. 6. (a) Western blot profile of HIF-1α of MG-63 cells treated with the seven day releasate of cobalt doped HAp samples. (b) VEGF expression of MG-63 cells treated with the seven day releasate of cobalt doped HAp samples measured by ELISA (CC—CoCl2). Data were expressed as mean ± S.D. Statistical significance was checked for p b 0.05 by Student's ‘t test’. For these studies, the cells were treated with seven day releasate of cobalt doped HAp samples at a concentration of 100 μg/ml for 24 h.

as methionine aminopeptitase-2 which aid in post-translational modification of proteins [68]. The dietary requirement of inorganic cobalt is about ~ 0.1 μg per day. Taylor and Marks reported that the normal level of cobalt present in the human body is about 1.1 mg, of which 43% is being stored in muscles, 14% in bone and the rest in soft tissues [69]. Bansal also reported that the normal cobalt level stored in the human body is about 1.5 mg [70]. Finely et al. conducted studies over cobalt doses and reported that the chronic oral reference dose of 0.03 mg per kg of body weight per day is considered to be protective without any negative health effects [71]. Traditionally, cobalt was used for the treatment of anemia in pregnant women [72–73]. Our release study showed that the extent of release of cobalt from all the doped HAp was much less than the cytotoxic level reported. Therefore, it can be concluded that the cobalt doped HAp reported here is safe for in vivo application. Cellular response to any implant device depends on the protein absorption on its surface. This is the event that influences the integration of the implant with the host system [74]. The adsorbed protein's composition, concentration and conformation determine the cellular response such as cell attachment, proliferation and differentiation [75]. Therefore, the estimation of the protein adsorption is essential to evaluate the potential of an implant. Among various body proteins, albumin (a serum protein) is a common one and is found in high concentration. Structural characterization of HAp reported earlier, confirmed the presence of two crystal faces: one comprised of ac and bc planes (Ca2+ ions called C site) and the other one holds the ab plane (oxygen atoms from phosphate ions called P site) [76]. The calcium sites (C sites) and the phosphate sites (P sites) are the preferential sites for protein adsorption [77]. Earlier, it was reported that protein adsorption increased with an increase in the surface area of the samples when the crystal size was small [78]. Protein adsorption is also influenced by the charge distribution of Ca2+/Co2+, OH− and (PO4)3− ions over the crystal surfaces [74], so it might be concluded that Ca/P crystal surface defects and vacancies may affect protein adsorption. From XRD, the pattern of HAC samples exhibited an increase in the peak broadening with an increase in the doping percentage. Peak broadening indicates the occurrence of crystal deformation or unevenness in the crystal structures. Protein adsorption of HAC samples (Fig. 3) also decreased with an increase in the doping percentage. Therefore, it can be said that the deformed crystals of HAC samples having irregular surface charge distribution are unfavorable for protein adsorption. It is also important to mention that in the case of CoCl2 doping, peak broadening was due to the partial replacement

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of OH− ions by Cl− ions as in the case of natural bones. But with an increase in doping concentration, Cl− ions increase the local acidic environment of the surface which might have led to the repulsion of the negatively charged BSA protein. Therefore, protein adsorption of HAC samples decreased with an increase in the doping percentage. The hemolysis study gives an indication about the hemocompatibility of the sample. It was already reported that Co2 + ions increase the cell number of red blood cells through HIF-1α activation, which is followed by activation of the EPO (Erythropoietin) gene leading to erythropoiesis [79]. Hence, the concentration of cobalt would not play a significant role in hemolysis. However, doping of cobalt changed the crystalline nature and the degree of crystallinity of HAp, which could affect hemolysis [80]. It was observed that doped HAp samples with lower crystallinity like HAC3 showed a very less amount of hemolysis (Table 5). On the other hand, samples which were less distorted and more crystalline (as per XRD data) like HAC1, HAC2 and HAN3 showed higher amounts of hemolysis. Bone physiology involves proliferation and differentiation of osteoblast cells [81]. Osteoblast proliferation is important to maintain the cellular homeostasis during bone remodeling. It has already been known that different metal ions increase the osteoblast cell proliferation. The same trend was observed by Fielding et al. while studying the proliferation of the human preosteoblast cell line in the presence of divalent metal ion (SiO2, SrO, MgO and ZnO) doped tricalcium phosphate [82]. In this study, the MTT assay of the samples (Fig. 4(a)) performed on day 2 showed that all HAp samples supported the proliferation of MG-63 cells as compared to the control (TCP). Among the doped samples HAN1 favored better cell proliferation. On day 7, the cell proliferation study showed that the extent of the cell proliferation was decreased with respect to day 2. HAN3, HAC1 and HAN2 showed a significant decrease in the viability index on day 7 as compared to day 2. HAC2 was the only doped sample which favored cell proliferation on both days 2 and 7. From the SEM micrograph (Fig. 5(a)) on the ninth day, it was evident that the HAN2 sample showed a better nodule formation and bone matrix deposition among all the samples which is generally considered as a sign of cell differentiation. It was also observed from the MTT data that there was a decrease in the cell proliferation with respect to time. This could be justified considering the differentiation of the cells on cobalt doped HAp (generally proliferation decreases when differentiation starts). In the case of bone repair or bone tissue engineering applications, in vitro formation of nodules is considered important since it shows the ability of a material to induce osteoconductive cellular response. Gough et al. also reported the nodule formation and their importance while culturing human primary osteoblast cells over porous bioactive glass scaffolds [83]. Cell cycle analysis (Fig. 4(b)) also followed the same trend as the cell proliferation study. Generally, in the case of somatic cells, arrest at the G0/G1 phase or prolongation of the G0/G1 phase might result in the differentiation of the cells, apart from apoptosis. Hence, HAN2 and HAN3 samples might have induced cell differentiation so the only showed the least proliferation rate and least percentage of the cells in the S phase, whereas other doped samples might have enhanced the cell proliferation [84]. Cheng et al. also reported that osteoblast-like cells treated with fluoridated HAp showed an increase in the percentage of cells in the S phase [50]. These results clearly suggest that the incorporation of cobalt has no detrimental effect on the osteoconductivity of HAp. Osteoblasts are responsible for the secretion of the bone matrix which includes collagen and non-collagen proteins and inorganic HAp mineral matrix [81]. After the bone matrix deposition, osteoblasts differentiate into osteocytes [85]. The transitional stages of preosteoblasts into osteocytes involve a spatio-temporal expression of different proteins which include majorly Runx2, Osterix and β catenin [86]. Osteoblast differentiation is influenced by the extracellular matrix and its composition, which may include growth factors such as epidermal growth factors, the insulin like growth factor and inorganic components of the matrix [87].

Runx2 is a key transcription factor that stimulates the differentiation of mesenchymal stem cells (MSC) into a lineage of osteoblast cells and it also blocks the differentiation of MSC into other cell lineages like adipocytic or chondrocytic lineages [86]. Runx2 activates a number of bone matrix protein genes such as Col1a1, osteocalcin and osteopontin which in turn govern the cell differentiation. For this reason, the expression of Runx2 is often considered as an early indication of bone cell differentiation [88]. In an earlier report, Stein et al.have shown that the incorporation of different bivalent metal ions in calcium phosphate materials has a definite role in osteoblast differentiation which can be correlated with the Runx2 expression profile [89]. Here, the cobalt doped HAp samples induced the expression of Runx2 as compared to HA2 (Fig. 5(b)). A possible explanation for such an effect of cobalt over Runx2 expression could be the activation of the calcium dependent Wnt5 signaling pathway. Possibly, cobalt ions could also act as an agonist to Ca2+ ions and activate the Wnt5 signaling cascade leading to the expression of Runx2 [82]. The result also highlighted a possibility of an antagonistic effect of cobalt ions to Ca2+ ions at a high cobalt concentration and reduction in the expression of Runx2. Hence, the highest doping percentage (HAC3 and HAN3) showed the least expression of Runx2. As we observed in the Runx2 expression profile, cobalt ion doping also had an influence over the Osterix expression (Fig. 5(c)). The reason might be that Osterix is also governed by the Wnt5 signaling pathway and further it is a down-regulator of Runx2 [90]. Hence, the particular doped HAp sample (HAN2) that had shown the highest expression of Runx2 also showed a better expression of Osterix and osteoblast differentiation. HAN2 showed better nodule formation among the other doped samples as evident from the SEM micrograph. Nodule formation is a sign of bone differentiation. The faster nodule formation in the case of HAN2 further confirms its differentiation potential. This supports the conclusion conferred from immunocytochemistry analysis. These facts together imply that cobalt doping improves the osteoconductive properties of the bone cells and the best results were obtained in the case of HAN2. Western blot analysis showed that the extent of cobalt doping had an influence on the expression of HIF-1α (Fig. 6(a)). Earlier reports proved that cobalt ions can mimic the hypoxia conditions by inactivating the PH enzyme and stabilizing HIF-1α [26]. Interestingly, HIF-1α expression did not show dose dependency as expected. This might be due to the crystal structure and the variation in the release of the cobalt ions from the samples. Apart from inhibiting PH and stabilizing HIF-1α, cobalt ions can also trigger the transcriptional activation of HIF-1α through the Wnt5 signaling pathway by regulating nuclear factor kappa B (NFkB) expression. Wu et al. reported that there was an enhanced expression of HIF-1α while culturing human bone marrow stromal cells over cobalt ion incorporated mesoporous bioactive glass [26]. Considering all the postulates, it could be said that HAN2 had a cobalt ion release rate, which might be optimal to mimic hypoxia conditions and lead to better expression of HIF-1α. The earlier reports proved that the VEGF gene is one of the immediate downregulators of HIF-1α [26]. In the case of HIF-1α expression, HAN2 showed the highest expression followed by HAC1 and HAN3. On the contrary HAN3 showed the highest VEGF expression followed by HAN2 (Fig. 6(b)). Here, HAC1 failed to stimulate VEGF expression. Earlier reports stated that VEGF expression does not depend on the mere expression level of HIF-1α; since it also depends upon the kinase activity of an individual cell and the phosphorylated activation of HIF1α [91]. The HAN3 sample might have favored the kinase activity in comparison to the other samples and lead to better expression of VEGF. Wu et al. also observed an enhanced VEGF expression level in human bone marrow stromal cells cultured over cobalt and copper incorporated mesoporous bioactive glass [26,92]. All these biological characterizations together implied that doping of Co2+ in HAp improves its osteo-conductive property [as evident from cell proliferation data (Fig. 4(a))] and capability of inducing the

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differentiation of bone cells (showed in Fig. 5) and secretion of the angiogenic factor, VEGF, especially in the case of HAN2 and HAN3. It is already established that the aforesaid properties contribute towards the success of scaffold mediated bone tissue engineering. Therefore, Co2+ doped HAp can be used as proangiogenic–osteogenic biomaterial for bone tissue engineering. 5. Conclusion Cobalt doped HAp was successfully prepared by the ammoniacal precipitation method. However, the studies proved that the existing methodology is inadequate to increase the doping percentage over a threshold level. It was evident from the study that the replacement of the anion from the apatite crystal during HAp preparation has no influence over the extent of cobalt ion doping. The study showed that the amount of cobalt released from the doped samples over a period of 60 days was below the systemic toxic limit. Cellular studies conferred that there is no direct proportionality between the extent of cobalt doping and osteogenic and angiogenic properties of doped HAp samples. Only a certain doping percentage (0.37%) corresponding to 1% theoretical loading (HAN2) induced favorable cellular response. Interestingly, the study also revealed that the cobalt induced HIF-1α expression profile and VEGF expression profile were not exactly corresponding to each other, though HIF-1α is the transcriptional factor for VEGF. These studies confirmed that cobalt doping into HAp structure imparted an excellent proangiogenic property without compromising the osteogenic property of HAp. The present study clearly implies that cobalt doped HAp may be a potential alternative to the existing methodologies for inducing angiogenesis in bone tissue engineering. However, further in vivo studies are required to confirm the pro-angiogenic properties of the materials. Profiling of HAp desorption in the presence of osteoclast is also needed for better understanding of its osteogenic properties of cobalt doped HAp. Acknowledgment 1. SAIF IIT Madras for ICP–OES analysis 2. DBT, Govt of India (RGYI scheme project no. BT/PR6230/GBD/27/ 391/2012) 3. DBT, Govt of India (RGYI scheme project no. BT/PR6227/GBD/27/ 390/2012)

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