Bone cell–material interactions on metal-ion doped polarized hydroxyapatite

Materials Science and Engineering C 31 (2011) 755–761

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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c

Bone cell–material interactions on metal-ion doped polarized hydroxyapatite Subhadip Bodhak 1, Susmita Bose, Amit Bandyopadhyay ⁎ W. M. Keck Biomedical Materials Research Laboratory, School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164-2920, USA

a r t i c l e

i n f o

Article history: Received 19 March 2010 Received in revised form 30 December 2010 Accepted 11 January 2011 Available online 15 January 2011 Keywords: Polarized hydroxyapatite Metal ion doping In vitro bone cell–material interactions Human osteoblast cells Stored surface charge density

a b s t r a c t The objective of this work is to study the influence of Mg2+ and Sr2+ dopants on in vitro bone cell–material interactions of electrically polarized hydroxyapatite [HAp, Ca10(PO4)6(OH)2] ceramics with an aim to achieve additional advantage of matching bone chemistry along with the original benefits of electrical polarization treatment relevant to biomedical applications. To achieve our research objective, commercial phase pure HAp has been doped with MgO, and SrO in single, and binary compositions. All samples have been sintered at 1200 °C for 2 h and subsequently polarized using an external d.c. field (2.0 kV/cm) at 400 °C for 1 h. Combined addition of 1 wt.% MgO/1 wt.% SrO in HAp has been most beneficial in enhancing the polarizability in which stored charge was 4.19 μC/cm2 compared to pure HAp of 2.23 μC/cm2. Bone cell–material interaction has been studied by culturing with human fetal osteoblast cells (hFOB) for a maximum of 7 days. Scanning electron microscope (SEM) images of cell morphology reveal that favorable surface properties and dopant chemistry lead to good cellular adherence and spreading on negatively charged surfaces of both Sr2+ and Mg2+ doped HAp samples over undoped HAp. MTT assay results at 7 days show the highest viable cell densities on the negatively charged surfaces of binary doped HAp samples, while positive charged doped HAp surfaces exhibit limited cellular growth in comparison to neutral surfaces. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Bone injuries are frequently caused by trauma, disease, and developmental deformity, and are continuously growing with an increasingly active and aging population [1]. Currently, repairing such bone sites involves surgical techniques, including the use of autogenous grafts, allogenous grafts, internal and external fixation devices, electrical stimulation, and replacement implants [2,3]. To this end, synthetic hydroxyapatite [HAp, Ca10(PO4)6(OH)2] ceramics have attracted special attention as an implant material, typically as artificial teeth and bone grafts [4]. Synthetic HAp exhibits good affinity for living bone tissues, and has the ability to promote cellular functions due to its close resemblance in composition to the inorganic ingredients of teeth and bones of vertebrate animals [5]. However, existing clinical uses of synthetic HAp based bone implants are still plagued with different challenges such as slow tissue in-growth processes compared to natural bone tissue, prolong healing time as well as implant's primary stability period due to inferior osteoconductivity than that of natural bone tissue [6]. Overall, enhancing osseointegration of HAp ceramics is still a significant challenge due to complex interactions of chemistry and surface properties, mechanical stress environment and tissue–material

⁎ Corresponding author. Tel.: + 1 509 335 4862; fax: +1 509 335 4662. E-mail address: [email protected] (A. Bandyopadhyay). 1 Currently working as a JSPS Postdoctoral Fellow at Biomaterials Center, National Institute for Materials Science (NIMS), Tsukuba, Ibaraki 305-0044, Japan. 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2011.01.003

interactions. Therefore, there is a growing interest to develop a new generation of HAp based biomaterials, which enhances the growth of new bone leading to faster healing [7]. Recently, significant research has been invested on improving the osteoconductivity of HAp using electro-thermal polarization treatment as a surface modification technique [8–20]. The original concept of polarized HAp to be used as a bone graft material was first introduced by Yamashita et al. [8] during the late 1990s. It was shown that the HAp can be an excellent electret which stored a surface charge between 0.08 μC/cm2 and 1.2 mC/cm2 depending on polarization conditions and successfully demonstrated that this induced surface charge of polarized HAp can significantly accelerate or decelerate bone like apatite deposition in simulated body fluid (SBF) depending on surface charge amount and its polarity [8–10]. Following this, a number of studies have investigated the effect of electrical charges and polarization conditions on the biological response to polarized HAp towards orthopedic and reconstructive implant applications [11–20]. In our earlier works, we have shown that bulk sintered HAp wettability and surface energy can be tailored by inducing surface charge through electro-thermal polarization treatment without introducing any volumetric effects in the material [11,12]. We have successfully demonstrated that tailoring the combined effect of stored surface charge, surface wettability and charge polarity on the polarized HAp surface, accelerated apatite mineralization as well as improved bone cell adhesion, proliferation, and differentiation on a negatively charged HAp surface [11,12]. In recent years several studies have also been reported on the synthesis of metal ion substituted HAp and shown to improve its

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structural stability and cellular biocompatibility properties [21–23]. Among various metal ions dopants, Sr2+ and Mg2+ were found very effective in enhancing the structural stability and biological properties of synthetic HAp. Earlier in vitro and in vivo studies have also indicated that magnesium and strontium influence the mineral metabolism during the bone remodeling process and enhance the osteoclast apoptosis as well as preosteoblastic cell proliferation [21–25]. However, there is no comprehensive study on bone cell–material interactions of electrically polarized HAp ceramics doped with different metal ion dopants which can show additional advantage of matching bone chemistry along with the original benefits of electrical polarization treatment. Based on literature review and our own research [20–25], we have selected MgO and SrO as dopants in single and binary dopant compositions. In this work, we have made an attempt to study the influence of metal ion dopants on osteoblast cell adhesion and proliferation on polarized HAp surfaces. Experiments in this research will fill an important knowledge gap by determining the extent of influence of electrical polarization on bulk sintered HAp with trace elements, i.e., Mg2+ and Sr2+ towards in vitro biological properties. 2. Materials and methods

where, Qp denotes the stored charge density, and β and J(T) are the heating rate and the current density, respectively. The detail of the stored charge measurement technique was already discussed in our previous reports [11,12,27]. 2.3. In vitro bone cell–material interactions The in vitro cytotoxicity behavior of polarized as well as nonpolarized doped and undoped HAp samples was evaluated for 3, and 7 days of incubation using human fetal osteoblast cells (hFOB). The cells used were derived from an immortalized, osteoblastic cell line, established from human fetal bone tissue. All samples were sterilized by autoclaving at 121 °C for 20 min prior to the cell culture experiment. Following this, cells were seeded onto HAp samples polarized at 400 °C, placed into the wells of a 24-well plate with either the negatively charged (‘N’ poled) or positively charged (‘P’ poled) surfaces facing upwards. Nonpolarized undoped as well as doped HAp samples were used as controls in our investigation. Initial cell density was 1.0× 104 cells/wall. 1 ml of DMEM media enriched with 10% fetal bovine serum was added to each well. Cultures were maintained at 34 °C under an atmosphere of 5% CO2 and 95% air in an incubator. The culture media were changed every alternate day during the duration of the experiment.

2.1. Sample preparation and characterization Synthetic HAp powder was obtained from Berkeley Advanced Biomaterials Inc., CA, USA. High purity strontium oxide (SrO, 99.9% purity, Aldrich, MO, USA) and magnesium oxide (MgO, Puratronic, 99.998%, Alfa Aesar, MA, USA) were procured from different venders. All other chemicals were of analytical grade and used without further purification. Four different HAp compositions were prepared: (i) pure undoped HAp, (ii) 1.0 wt.% SrO doped HAp, (iii) 1.0 wt.% MgO doped HAp, and (iv) HAp doped with binary dopants consisting of 1.0 wt.% SrO and 1.0 wt.% MgO. The amount of single and binary dopant compositions was optimized based on our previous research [22–25]. A definite amount of dopants was added to HAp powders and the mixtures were ball milled in ethanol media for 24 h at 70 rpm. After ball milling, powders were then placed in an oven at 80 °C for 72 h for drying. Dried powders were then pressed using a uniaxial press to form discs (12 mm in diameter × 1 mm in height) and sintered at 1200 °C for 2 h in a conventional muffle furnace. The relative density for each composition was measured by Archimedian's method. Surface morphologies of disc compacts of all compositions were observed under a field emission SEM (FEI Inc., OR, USA) following gold sputter-coating (Technics Hummer V, CA, USA). 2.2. Electro-thermal polarization and stored surface charge calculations Electro-thermal polarization was carried out on sintered and polished undoped and doped HAp samples, in air, using platinum electrodes, with Keithley 6487 picoammeter (Keithley Instruments Inc., OH, USA) and ceramic strip heater (OMEGA Engineering Inc., CT, USA). During polarization, the samples were slowly heated from room temperature to polarization temperature (Tp) with a controlled heating rate of 5 °C/min and soaked at Tp for 1 h before applying a d.c. voltage. A d.c. electrical field (Ep) of 2.0 kV/cm was applied for 1 h at Tp and maintained until the sample cooled to room temperature. In order to estimate the stored static charge due to polarization operation, the thermally stimulated depolarization current (TSDC) technique was used [26]. During TSDC analysis, polarized samples were heated at a rate of 5 °C/min up to 550 °C and the thermally stimulated release depolarization current was measured using Keithley 6487 picoammeter. Finally the stored surface charge density was calculated using Eq. (1) [9]. Qp =

1 ∫J ðT ÞdT β

ð1Þ

2.3.1. Cell morphology Cell morphology was assessed under SEM observation after 3 and 7 days of incubation period. Cultured samples for SEM observation were rinsed with 0.1 M phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde/2% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4 °C. Following this, post fixation for each sample was made with 2% osmium tetroxide (OsO4) for 2 h at room temperature. Fixed samples were then dehydrated in an ethanol series 30%, 50%, 70%, 95% and 100% three times, followed by a hexamethyldisilane (HMDS) drying procedure. Dried samples were then mounted on aluminum stubs, gold coated and observed under a field emission SEM (FEI Inc., OR, USA). 2.3.2. Cell proliferation using MTT assay The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma Inc., St. Louis, MO) was performed for 3 and 7 days of incubation to determine hFOB cell proliferation on polarized as well as nonpolarized doped and undoped HAp surfaces. Triplicate samples per group were evaluated and three data points were measured from each sample. An MTT (Sigma, St. Louis, MO) solution of 5 mg/ml was prepared by dissolving MTT in PBS, and was filtersterilized using a filter paper of a 0.2 μm pore. The MTT was diluted (100 μL into 900 μL) in a DMEM/F12 medium. A 1 ml diluted MTT solution was then added to each sample in 24-well plates. After 2 h of incubation, 1 ml of solubilization solution made up of 10% Triton X-100, 0.1 N HCl, and isopropanol was added to dissolve the formazan crystals. 100 μL of the resulting supernatant was transferred into a 96-well plate, and read by a plate reader at 570 nm. 2.4. Statistical analysis Triplicate samples were used in all the experiments to ensure reproducibility. Data for the MTT assay are presented as mean± standard deviation. Statistical analysis was performed on MTT assay results using Student's t-test and a P value b 0.05 was considered significant. 3. Results and discussion 3.1. Densification and stored surface charge calculation Our experimental results showed that dopants have a significant influence on relative density, HAp phase thermal stability as well as

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Table 1 Calculated relative densities as well as estimated maximum current densities and corresponding temperatures along with the calculated stored charge values obtained from different doped and undoped sintered HAp samples. [Polarization condition: All samples in our present research were sintered in air and electro-thermally polarized at 400 °C under an electric field of 2.0 kV/cm for 1 h.] Samples

Undoped HAp 1 wt.% SrO doped HAp 1 wt.% MgO doped HAp 1 wt.% MgO–1 wt.% SrO doped HAp

Relative density (% theoretical)

Electrical polarization results Maximum current density (nA/cm2)

Temperature at maximum current density (°C)

Stored charge density (Qp) (μC/cm2)

93.11 ± 1.95 93.34 ± 1.29 98.21 ± 1.16 97.66 ± 0.85

1.66 1.61 3.58 3.95

465.4 448.7 484.3 490.9

2.23 2.37 3.86 4.19

polarizability and/or charge storage ability of sintered HAp samples. Table 1 presents the densification results (% theoretical density) for sintered doped and undoped HAp samples. It can be seen that undoped HAp has the lowest relative density (93.11 ± 1.96%). It is suggested that during sintering HAp has been partially decomposed into the β-TCP phase (JCPDS No. 09-0169) due to dehydroxylation (removal of OH−) which is a common phenomenon often encountered in sintering of commercial HAp powder. The high temperature HAp to β-TCP phase transition caused the lattice volume expansion and lowered the density of undoped HAp samples [27]. However, addition of Mg2+ and Sr2+ to HAp improved the thermal stability of the HAp crystal structure. A maximum density of 98.2 ± 1.16% has been achieved for 1 wt.% MgO doped HAp samples. Binary doped HAp (1 wt.% Mgo–1 wt.% SrO) samples also exhibited considerable improvement in density (97.6 ± 0.84%) over undoped HAp. It can be suggested that during sintering the bivalent smaller ionic radius Mg2+ ion (0.66 Å) substituted a higher ionic radius Ca2+ ion (0.99 Å) in the HAp crystal lattice structure. This led to shrinking of the unit cell of HAp lattice and increases the density of Mg2+ ion doped HAp samples. However, the relative density of 1.0 wt.% SrO doped HAp (93.34 ±

1.29%) was found to be comparable with undoped HAp, in spite of stabilizing the HAp phase during sintering. This observation can be explained from the higher ionic radius (1.13 Å) of Sr2+ than Ca2+ ion (0.99 Å) which led to volume expansion when substituted into the HAp crystal lattice structure. Undoped and doped HAp samples were analyzed by SEM to understand the influence of dopants on their microstructure. Fig. 1 shows the SEM micrographs of the top surface of the sintered HAp samples. Overall, all samples exhibited a dense microstructure with no significant difference in grain morphology or grain size (~3–4 μm). However, occasional residual pores were observed in undoped HAp and SrO doped HAp (Fig. 1a and b). To understand the influence of different dopants on the polarizability and charge storage ability of sintered HAp ceramics, the thermally stimulated depolarization current (TSDC) technique was used [11,12,27]. Table 1 summarizes the electrical polarization results as measured for different polarized HAp samples. The value of stored surface charge density for the undoped HAp sample was estimated as 2.23 μC/cm2. A small increase of around 6% in stored charge density (~2.37 μC/cm2) was measured for Sr2+ doped HAp. However, considerable change in the polarizability behavior of doped HAp can be

Fig. 1. SEM micrographs showing sintered surface microstructures for (a) undoped HAp, (b) 1.0 wt.% SrO doped HAp, (c) 1.0 wt.% MgO doped HAp, and (d) 1.0 wt.% SrO + 1.0 wt.% MgO doped HAp samples.

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Fig. 2. SEM micrographs showing the cell adhesion after 3 days of culture on poled and unpoled (a–c) undoped HAp, (d–f) SrO doped HAp, (g–i) MgO doped HAp, and (j–l) binary doped HAp surfaces. [‘N’ — negative, and ‘P’ — positive].

noticed for small addition of Mg2+ and combined incorporation of Mg2+–Sr2+ dopants. Interestingly, the stored charge density of doped HAp was observed to increase from 2.23 μC/cm2 to 3.86 μC/cm2 with the doping of Mg2+, which was nearly 63% increase in comparison to undoped HAp. Evidently, binary doped HAp samples exhibited the highest stored charge density of 4.19 μC/cm2, where combined incorporation of Mg2+–Sr2+ dopants resulted in about 88% increment of charge storage as compared to undoped HAp. Consequently, the maximum current density also increased from 1.66 nA/cm2 to 3.95 nA/cm2 for the combined addition of Mg2+ and Sr2+ dopants in HAp. It can be recalled that the polarization of HAp has been attributed to the ionic conduction mechanism, where protons (H+) and oxide ions (O2−) present in the hexagonal unit cell of HAp are considered as potential mobile charge carriers [9–12,18]. However, α-TCP cannot be polarized due to the absence of any hydroxyl group

in the crystal lattice [9,10]. Therefore a decrease in charge carrier density results in lowering the polarizability of undoped HAp samples. Moreover, the presence of a higher volume fraction of residual pores also acts as a barrier against charge carrier migration and consequently leads to lower charge accumulation in the surface of the poorly sintered undoped HAp surfaces. In contrast, dense and phase pure doped HAp samples exhibited significantly better polarizability and resulted in higher surface charge storage [11,12,27]. 3.2. In vitro bone cell–material interactions 3.2.1. Cell morphology Fig. 2 shows bone cell attachment, and spreading after 3 days of culture. It can be seen that depending on the surface charge, charge polarity, and dopant chemistry, HAp samples exhibited different levels

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of bioactivity. In all cases negatively poled surfaces demonstrated pronounced cell viability and superior cell attachment where osteoblasts occupied with flatten morphology and cells were seen to adhere to each other with cellular micro extensions as compared to unpoled HAp surfaces irrespective of dopant chemistry. In contrast, cells on a positive surface exhibited less adhesion and spreading as they assumed a round shape with diffused or limited establishment. This observation is in good agreement with our previous in vitro cell culture results on pure polarized bulk and HAp coated surfaces [11,12]. A significant increase in cell attachment and growth on negatively charged HAp surfaces when compared to the electrically neutral surfaces has also been reported [13,14]. However, some apparent contradiction was present on the biological responses of positively charged HAp surfaces, where both positive and negatively charged HAp surfaces exhibited equally enhanced in vitro osteoblastic activities over neutral HAp

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surfaces for longer incubation time [19,20]. Furthermore, the benefit of dopant incorporation on improving the biological properties of HAp can be best realized by comparing the negatively polarized doped and undoped HAp samples as shown in Fig. 2. Because of favorable surface properties as well as matching bone chemistry, negatively charged surfaces exhibited superior cell viability and better concentration of cell banding suggesting good cell–material interactions and cell-to-cell communication for Sr2+ (Fig. 2e), Mg2+ (Fig. 2h), and Sr2+–Mg2+ ion (Fig. 2k) doped HAp samples as compared to undoped HAp samples (Fig. 2b). Fig. 3 shows the hFOB cells spreading and growth on different HAp surfaces after 7 days of culture. Although a similar trend was noticed even after 7 day culture time, a significant improvement in cell growth behavior can be observed depending on surface charge and dopant chemistry. After 7 days, cells appeared to be more elongated

Fig. 3. SEM micrographs illustrating the cell spreading and growth after 7 days of culture on poled and unpoled (a–c) undoped HAp, (d–f) SrO doped HAp, (g–i) MgO doped HAp, and (j–l) binary doped HAp surfaces. [‘N’ — negative, and ‘P’ — positive].

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and in a higher number on the negatively charged undoped HAp surface as shown in Fig. 3b than the unpoled HAp surface (Fig. 3a), while cell numbers were evidently less on the positively charged undoped surface (Fig. 3c). In the case of single Sr2+ and Mg2+ ion doped HAp samples, both negatively charged and neutral doped HAp surfaces showed extensive flaky layers of adhered cells with rough cellular surfaces. However, because of the combined influence of surface charge and dopant chemistry, negatively charged doped HAp surfaces exhibited better surface coverage of viable cells in comparison to unpoled doped HAp surfaces, while the positively poled surface exhibited limited bone cell growth. In particular, the negatively charged surfaces of Sr2+–Mg2+ binary doped HAp samples were found to considerably improve the bioactivity of HAp with thick confluent cellular coverage suggesting enhanced viability of osteoblast cells on these sample surfaces (Fig. 3k). It is believed that the superior biological properties can be achieved by the higher surface charge storage of Mg2+ (~3.86 μC/cm2) and Sr2+–Mg2+ (~4.19 μC/cm2) binary doped samples which accelerated the mineralization process and favored adsorption of cell adhesive proteins (e.g., fibronectin, vitronectin, etc.) for osteoblast cell adhesion on negatively charged surfaces [11,12]. Our results are also in good agreement with previous investigations which revealed that the presence of Mg2+ and Sr2+ can enhance osteoblast growth and favor the bone regeneration process by accelerating mineral metabolism and promoting catalytic reactions during the bone remodeling process [21–25]. 3.2.2. Cell proliferation The MTT assay study was used to quantitatively determine the cell viability and proliferation of hFOB cells on both doped and undoped HAp surfaces in poled as well as unpoled conditions. Fig. 4 shows a comparison of optical densities which are linearly proportional to the viable cell densities, illustrating hFOB cell proliferation on different samples after 3 and 7 day culture period. Overall, bone cells proliferated more rapidly on negatively charged HAp surfaces compared to unpoled HAp for both undoped and doped HAp samples. Opposite growth kinetics was observed for positively charged surfaces where cell growth was suppressed irrespective of dopant chemistry. On the other hand, it can be clearly seen from the MTT assay results that the total number of viable cells is always higher on single as well binary doped unpoled HAp samples as compared to undoped and unpoled HAp samples for all culture periods. Interestingly, the combined influence of surface charge and dopant chemistry was found to be more pronounced during early stage of culture period. Evidently, after a 3 day incubation period metal-ion doped HAp showed better cell adhesion and growth over undoped HAp, while binary doped negatively charged HAp samples exhibited maximum viable cell densities among all samples (Fig. 4a). However, with increasing culture time all samples have started responding in quite a similar manner as can be observed from MTT assay results (Fig. 4b). In particular, the cell proliferation rate on negatively charged binary doped HAp was found to drop to some extent between 3 and 7 day culture periods as compared to negatively charged undoped HAp samples. We speculate that with the increasing time period the formation of confluent cellular layers on highly bioactive negatively charged binary doped HAp surfaces could have possibly lowered the growth rate of osteoblast cells on these surfaces. However, it is important to note here that due to the early stage stimulation, the number of cells on the negatively charged surfaces of both single and binary doped HAp samples was always higher in comparison to negatively charged undoped HAp samples and the difference was statistically significant as confirmed by Student's ‘t’ test (p b 0.05) for both 3 day and 7 day culture periods. Evidently, the highest viable cell densities were measured on negatively charged surfaces of binary doped HAp samples after 7 days (Fig. 4b). Therefore, our research findings clearly suggest that incorporation of the trace elements into polarized HAp ceramics as dopants can help us in developing a highly

Fig. 4. Optical density measurement illustrating hFOB cell proliferation on undoped and various doped HAp surfaces under poled as well as unpoled condition after (a) 3, and (b) 7 days of culture time. Statistical analysis indicates that the differences in cell densities among various samples are significant (* = P b 0.05, n = 5).

bioactive polarized HAp surface with tailored adhesion and adsorption properties which leads to an early stage that can potentially improve bone cell–material interaction towards faster bone tissue repairing and regeneration. 4. Conclusions In this study, commercial HAp powder was doped with Sr2+ and Mg to study the influence of dopants on the polarization behavior and biological properties of pure HAp. It has been observed that small addition of Mg2+ and combined addition of Mg2+–Sr2+ are most beneficial in enhancing density, polarizability and charge storage ability. In vitro cell–material interaction study showed that presence of Mg2+ and Sr2+ can significantly enhance the initial day osteoblast response and activities on a negatively charged surface. MTT results showed that due to the early stage stimulation the total number of viable cells was always higher on doped HAp samples for all culture periods, while the highest viable cell densities were measured on the 2+

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negatively charged surfaces of binary doped HAp samples after 7 day incubation. Our research findings established that the combined influence of metal ion dopants and polarization can potentially assist in designing bone graft materials that can be used for tissue in-growth for the HAp based orthopedic implants focusing on faster healing.

[9] [10] [11] [12] [13] [14] [15]

Acknowledgements The authors would like to acknowledge financial support from the Office of Naval Research under grant no. N00014-01-05-0583 and the National Institutes of Health under grant no. NIH-R01-EB-007351.

[16] [17] [18] [19] [20]

References [1] M. Doblare, J.M. Garcia, M.J. Gomez, Eng. Frac. Mech. 71 (2004) 1809. [2] A.S. Greenwald, S.D. Boden, V.M. Goldberg, Y. Khan, C.T. Laurencin, R.N. Rosier, J. Bone Joint Surg. Am. 83 (2001) S98. [3] J.A. Spadaro, Clin. Orthop. 122 (1977) 325. [4] M. Jarcho, Clin. Orthop. Relat. Res. 157 (1981) 259. [5] J.C. Elliott, Structure and Chemistry of the Apatites and Other Calcium Orthophosphates, Elsevier Science, Amsterdam, 1994, p. 74. [6] A.J. Salgado, O.P. Coutinho, R.L. Reis, Macromol. Biosci. 4 (2004) 743. [7] J.W. Calvert, L.E. Weiss, M.J. Sundine, Clin. Plast. Surg. 30 (2003) 641. [8] K. Yamashita, N. Oikawa, T. Umegaki, Chem. Mater. 8 (1996) 2697.

[21] [22] [23] [24] [25] [26] [27]

761

S. Nakamura, H. Takeda, K. Yamashita, J. Appl. Phys. 10 (2001) 5386. M. Ueshina, S. Nakamura, K. Yamashita, Adv. Mater. 14 (2002) 591. S. Bodhak, S. Bose, A. Bandyopadhyay, Acta Biomater. 5 (2009) 2178. S. Bodhak, S. Bose, A. Bandyopadhyay, Acta Biomater. 6 (2010) 641. M. Ohgaki, T. Kizuki, M. Katsura, K. Yamashita, J. Biomed. Mater. Res. A 3 (2001) 366. N. Dekhtyar, N. Polyaka, R. Sammons, 14th Baltic Conference on Biomedical Engineering and Medical Physics, vol. 20, Springer, Berlin, Heidelberg, 2008. R. Okabayashi, M. Nakamura, T. Okabayashi, Y. Tanaka, A. Nagai, K. Yamashita, Biomed. Mater. Res. B Appl. Biomater. 90B (2009) 641. T. Kobayashi, S. Nakamura, K. Yamashita, J. Biomed. Mater. Res. 57 (2001) 477. P. Calvert, S. Mann, Nature 386 (1997) 127. J.P. Gittings, C.R. Bowen, I.G. Turner, F.R. Baxter, J.B. Chaudhuri, Mater. Sc. Forum 587 (2008) 587. T. Kizuki, M. Ohgaki, M. Katsura, S. Nakamura, K. Hashamito, Y. Toda, S. Udagawa, K. Yamashita, Biomaterials 24 (2003) 941. D. Kumar, J.P. Gittings, I.G. Turner, C.R. Bowen, A. Bastida-Hidalgo, S.H. Cartmell, Acta Biomater. 6 (2010) 1549. C.C. Liu, J.K. Yeh, J.F. Aloia, J. Bone Mineral. Res. 3 (1988) S104. W. Xue, H.L. Hosick, A. Bandyopadhyay, S. Bose, C. Ding, K.D. Luk, K.M. Cheung, W.W. Lu, Surf. Coat. Tech. 201 (2007) 4685. W. Xue, J. Moore, H.L. Hosick, S. Bose, A. Bandyopadhyay, W.W. Lu, K. Cheung, K. Luk, J. Biomed. Mater. Res. A 79A (2006) 804. S.S. Banerjee, S. Tarafder, N.M. Davies, A. Bandyopadhyay, S. Bose, Acta Biomater. 6 (2010) 4167. A. Bandyopadhyay, S. Bernard, W. Xue, S. Bose, J. Am. Ceram. Soc. 89 (2006) 2675. C. Bucci, R. Fieschi, G. Guidi, Phys. Rev. 148 (1966) 816. S. Bodhak, S. Bose and A. Bandyopadhyay, J. Am. Ceram. Soc. (in press). doi: 10.1111/j.1551-2916.2010.04228.x.