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Surface & Coatings Technology
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Colloidal-sol gel derived biphasic FHA/SrHA coatings
Ping Yin a, FangFang Feng b, Ting Lei b, XinChun Jian a,⁎a Xiangya Hospital, Central South University, Changsha 410008, Chinab State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, China
Fluoridated hydroxyapatite/strontium-substituted hydroxyapatite (FHA/SrHA) biphasic coatings are preparedon titanium substrate via colloidal-sol gel method, where FHA acts as the dissolution-resistant matrix andSrHA serves as the soluble phase. Surface characterization, adhesion strength and solubility aswell as in vitro cel-lular responses of FHA/SrHAbiphasic coatingswere investigated. FHA/SrHA biphasic coating displays unique sur-face characteristics in comparison with single phase coating. The dissolution rate of FHA/SrHA biphasic coatingsis much slower than that of pure SrHA coating, which increases progressively with increasing amount of SrHAand is between that of pure FHA and SrHA coating. The adhesion strength between coating and Ti substratedecreases with increasing amount of SrHA. The cultured osteoblastic MG63 cells attached, spread, and grewfavorably on the FHA/SrHA2 (molar ratio of FHA and SrHA is 1:2) biphasic coating film.
Hydroxyapatite (HA, Ca10(PO4)6OH2) is a bioceramic that is chemi-cally and biologically similar to the inorganic minerals of human hardtissues [1]. Hence, HA coated titanium alloy has been widely used asimplant materials in orthopedic and dental applications [2]. However,pure HA coating has a relatively high dissolution rate in the biologicalenvironment, which affects its long-term stability and makes the inter-face between bone and implant unstable [3,4]. On the other hand,natural bone is a kind of calcium-deficient HA, which contains consider-able amounts of additional elements including F, Mg, Sr and Zn, etc. [5].Accordingly, modification of HA by incorporation of these elements isrequired to make apatite more biocompatible and more resemblinghuman bone in terms of composition for clinical applications.
It is known that fluorine is an essential trace element in bonetissues and teeth, which can promote the mineralization and crystal-lization of calcium phosphate in the process of bone formation [6,7].Fluorine-doped hydroxyapatite Ca10(PO4)6(OH)2−xFx (FHA, x is thedegree of fluoridation) is reported to possess lower solubility andcomparable bioactivity and biocompatibility over HA [8]. Also, FHAcoatings are found to strongly adhere to the Ti6Al4V substrate [9],which is important to reduce debris detachment and can further en-sure the long-term stability of coatings.
Sr is also known to be one of the most important cations in bonetissues, acting to stimulate bone cell growth and restrain bone resorp-tion as well as suppressing osteoporosis [10,11]. Sr-substituted HA(SrHA) shows osteoinductive activity as well as higher solubility thanHA [12,13]. Therefore, it is reasonable to deduce that incorporation of
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F and Sr ions into one coating layer simultaneously could be favorablefor the improvement of both bioactivity and long term stability of thecoating.
Biphasic coatings of FHA/β-TCP and FA/HA were synthesized by thesol–gel method [14–16] and their solubility and biological propertiesinvestigated. In this contribution, FHA/SrHA biphasic coatings wereprepared on titanium substrate by sol–gel and dip-coating processes,whereby FHA is considered the dissolution-resistant matrix and SrHAthe soluble phase. In such a constructed biphasic structure, the SrHAphase is intended to induce rapid cellular responses at initial periodsafter implantation due to its high dissolution rate and high bioactivity,and the FHA phase, because of its low solubility, is expected to last forlong periods, giving the coating longevity and stability. The characteris-tics, bonding strength, dissolution rate and in vitro cellular responses ofthe coatings were also evaluated.
2. Experimental
2.1. Materials
Commercially pure titaniumplates (99.9% purity, Kermel Co., China)of 20×40×10 mm size were used as the substrate materials. Calciumnitrate tetrahydrate (Ca(NO3)2·4H2O, Sigma-Aldrich, AR), phosphorouspentoxide (P2O5, Merck, GR) and hexafluorophosphoric acid (HPF6,Sigma-Aldrich, GR) were selected as Ca-precursor, P-precursor andF-precursor, respectively.
2.2. Preparation of FHA and SrHA sol–gel
In this study, the degree of substitution of OH− by F− was designedto be 50% to obtain FHA with general formula of Ca10(PO4)6F(OH), and
strontium-substituted hydroxyapatite (SrHA) with 10 mol% Ca2+
replaced by Sr2+ was also designed. The detailed preparation processof FHA and SrHA by the sol–gel method has been documented in theliterature [15,17,18]. To prepare the FHA solution, Ca(NO3)2·4H2Owas dissolved in absolute ethanol to form a 2 mol/L Ca-precursor;P2O5 was dissolved in absolute ethanol to form a 2 mol/L solutionfollowed by a refluxing process for 24 h to obtain the P-precursor solu-tion. A designed amount of HPF6 based on Ca/F molar ratio of 10:1, wasadded drop-wise into the P-precursor solution. After that, the Caprecursor was added into the P–F mixture in Ca/P molar ratio of 1.67.This mixed solution was refluxed for 24 h and then aged overnight toobtain the FHA sol.
Similarly, in brief, analytical grade Ca(NO3)2·4H2O, Sr(NO3)2(Sigma-Aldrich, AR) and P2O5 were dissolved in absolute alcohol toform the Ca+Sr precursor (molar ratio of Ca:Sr=9:1) and P precur-sor solutions. After that, the P precursor was added into the Ca+Srprecursor in (Ca+Sr)/P molar ratio of 1.67 and then refluxed for24 h followed by aging overnight to obtain the SrHA sol.
2.3. Deposition of biphasic FHA/SrHA coatings
Before the dip-coating experiment, the Ti substrate surface waspolished incrementally with 400-grit to 1000-grit emery paper, andthen washed with acetone and distilled water in an ultrasonic cleaner,followed by etching treatment in 1 mol/L HCl solution for 30 min to re-move the air-formed oxide layer and subsequently ultrasonically wash-ing in deionized water. Afterwards, the samples were treated byalkali-heat-treatment process: soaking in boiling 5 mol/L NaOH solu-tion for 15 min, ultrasonically washing with acetone and distilledwater, and finally drying at 150 °C for 4 h.
SrHA powders were obtained after drying of the aged sols at 80 °Cfor 24 h, and further heat treatment at 600 °C for 1 h in air. After-wards, SrHA powders were directly added to the refluxed FHA sol inSrHA powder/FHAsol molar ratio of 1/1, 2/1 and 3/1 and ultrasonicallydispersed for 20 min to obtain the colloidal sols.
The substrates were dipped vertically into the colloidal sols andwithdrawn at a rate of 8 cm/min. The as-dipped coatings were driedin an oven at 150 °C for 15 min followed by firing in a furnace at600 °C for 15 min, and then cooling down to room temperature to ob-tain a single biphasic coating layer. Such dipping–drying–firing pro-cess was repeated 4 times to increase coating thickness to 4 μm. Theas-prepared biphasic coatings were designated FHA/SrHA1, FHA/SrHA2 and FHA/SrHA3, respectively. As a control, single FHA andSrHA coating were also prepared following the same procedure.
2.4. Coating characterization and adhesion test
For the characterization of the coating morphology and composi-tion a field-emission scanning electron microscope (Nova NanoSEM230) equipped with energy dispersive X-ray (EDX) analyzer wasused. The phase composition of the as-prepared biphasic coatingswere analyzed by X-ray diffractometry (XRD: D/MAX-255) with Cu–Kα1 radiation (wavelength λ=1.5406 Å). The tube voltage and thetube electric current of XRD were 40 kW and 250 mA, respectively.
The adhesion strength between the coating and substrate wasevaluated using a scanning scratch tester (Shimadzu, SST-101). Aspherical Rockwell C diamond tip of 10 μm radius with a progressiveload from 0 to 30 N was used. The tip scanned the coating surface at aspeed of 2 μm/s with a scanning amplitude of 50 μm perpendicular tothe scratching direction. The load at which the coating is completelypeeled off from the substrate is called the “upper critical load”. This“upper critical load” was used as a measurement of the adhesionstrength between the coating and substrate [16,17]. Five readings ofsuch critical load values were averaged for each sample, and the stan-dard deviation determined.
2.5. Evaluation of the solubility of the coatings
Aqueous tris(hydroxymethyl)aminomethane ((HOCH2)3CNH2, Tris)solution was employed in the immersion test to evaluate the solubilityof the biphasic coatings. Tris solution can provide the same pH value asbody fluid and contains only one cationic H+ and one anionic Cl−with-out any other ionic components, and thus can avoid interference bychemical reaction of other ions [19]. The other ions found in Tris solu-tion after an immersion test is coming from the dissolved coating.Accordingly, the cumulative concentration of dissolved ions can bemea-sured and used to evaluate the dissolution rate of coatings. A 0.05 mol/LTris solution was prepared in deionized water and 1.00 mol/L HCl wasadded to adjust pH value to 7.25. The coated samples were immersedin Tris solution at 37±0.5 °C in a ratio of 20 mL/cm2 (solution volumeto sample surface area). After soaking for 14 days without agitatingand replenishing the solution, the samples were cleaned and dried inair, and then the surface of the samples was examined by SEM. Mean-while, an amount of 5 mL of immersion solution was taken to measureCa, Sr and P concentrations by an inductively coupled plasma emissionspectrometer (ICP-OES, JOBYVON 70 plus).
2.6. Cell and culture conditions
Human osteosarcoma MG63 cells purchased from American TypeCulture Collection (ATCC, Rockville, MD) were used to evaluate theosteoblastic cell response on specimens [20,21]. The cells were cul-tured at 37 °C in a humidified 5% CO2 atmosphere in a standardculture medium containing Dulbecco's Modified Eagle's Medium(DMEM, HyClone) supplemented with 10% Fetal Bovine Serum (FBS,HyClone) and 1% penicillin/streptomycin (GIBCO). For the cell assay,the coated samples were sterilized in an autoclave at 121 °C for 20 minprior to the cell culture tests.
Cells at a density of 4.0×l05 cells mL−1 were seeded on the coatingsurface to observe their morphology. After incubation for 1 day, cellswere fixed with 2.5% glutaraldehyde for 1 h at room temperaturefollowed by dehydration with a series of graded ethanol/water solu-tions (50%, 70%, 80%, 95% and 100%, respectively). Specimens werecritical point dried using liquid CO2 and then sputter-coated withgold film for cell morphology observation by SEM.
3. Results and discussion
3.1. Microstructures of the biphasic coatings
Fig. 1 shows the XRD analysis of pure FHA, SrHA and biphasic FHA/SrHA coatings derived from colloidal sols after heat treatment of eachcoating at 600 °C for 15 min in air. All coatings have almost the iden-tical XRD patterns of apatite besides strong diffraction peaks of Ti. Noimpurity phase is detected in these coatings. A sodium titanate(Na2Ti5O11) peak appears between 28° and 30° in 2θ, which is inagreement with the results reported by Kim et al. [22]. It is worth-while to note that the FHA and SrHA peaks cannot be distinguisheddue to their similar crystallographic structures. The Ti peaks originat-ed from the substrate. The X-ray penetrated through the coating layerand gave rise to diffraction of titanium surface. On the other hand, incomparison with FHA coating, the diffraction peaks of SrHA coating atcrystal plane (112) and (300) shift to lower angles, which is in goodagreement with previous report [12] and can be explained by thecrystal lattice distortion caused by Sr2+ or F− incorporation.
Since all the colloidal-sols are prepared based on exact the sameas-refluxed sol, the colloidal-sol derived biphasic coatings are sup-posed to have an identical matrix of FHA. It is also noted that thepeak intensity for biphasic coating becomes increasingly higherwith increasing amount of SrHA, indicating that the SrHA amount inthe coatings can be tailored through variation of the amount of powdersin the sols.
20 25 30 35 40 45 50
0
2000
4000
6000
8000
10000
Rel
ativ
e in
tens
ity/a
.u.
2θ/ο
FHA
FHA/SrHA1
FHA/SrHA2
FHA/SrHA3
SrHA
TiApatiteNa2Ti5O11
Fig. 1. X-ray diffraction patterns of pure FHA, SrHA and FHA/SrHA biphasic coatings.
610 P. Yin et al. / Surface & Coatings Technology 207 (2012) 608–613
The SEMmorphologies of coatings on the Ti substrate are presentedin Fig. 2. For comparison, the surface appearance of Ti substrate afteralkali-heat-treatment is shown in Fig. 2a. As observed in the SEMmicro-graphs, the treated Ti surface exhibits a porous network structure, whilepure FHA, SrHA coatings and the biphasic FHA/SrHA coatings show dif-ferent surface morphology. The surface appearances of pure FHA andSrHA coatings are relatively smoothwith visiblemicro-pores and cracksas shown in Fig. 2b and c. In contrast, all the biphasic coatings showmuchhigher roughnesswith larger cracks and voids and pits. Increasingamount of SrHA leads to the roughness increase and more particles or
Fig. 2. SEM micrograph of the surface morphology of (a) Ti substrate after alkali-heat-trea
agglomerates as indicated by arrows are observed due to the thickeningeffect of SrHA addition. Similar observations were reported on FHA/β-TCP and FA/HA biphasic coatings [14,15], respectively.
3.2. Adhesion strength of the biphasic coatings
The adhesion strength between coating and Ti substrate for all coat-ings is depicted in Fig. 3. All coatings show comparable adhesionstrength, with the highest adhesion strength of 4.77 N observed forpure FHA coating. However, with increasing SrHA amounts in FHA,the adhesion strengths decrease monotonically. Similar trends werereported for FHA/β-TCP biphasic coating, the adhesion strength ofwhich varied with the amount of β-TCP powder dispersed in the sol[14]. Pure SrHA coating exhibits the poorest adhesion strength likelydue to the coefficient of thermal expansionmismatch between the coat-ing and Ti substrate.
The maximum adhesion strength of FHA can be attributed to twoeffects. On the one hand, FHA has much closer coefficient of thermalexpansion (9.1×10−6) to that of the Ti substrate (8.9×10−6) [23,24].As a result, incorporation of fluorine in HA reduces the thermalmismatch and thus residual stress, which in turn contributes to betteradhesion strength. On the other hand, oxides form on titanium surfacein the process of alkali-heat-treatment, leading to formation of Ti\OHbonds on the outmost surface [25]. In dipping and firing process, fluo-rine ions released by FHA easily form hydrogen bond with H in OHgroup [26]. The higher the fluorine concentration, the more hydrogenbonds form. Accordingly, the formation of interfacial chemical bondingcould relieve the thermal mismatch between coating and substrate andthus reduce internal stresses and consequently improve the adhesionstrength. With the increase of SrHA amount in the biphasic coatings,the interfacial chemical bonding is weakened,which in turn contributesnegatively to adhesion strength.
tment (b) pure FHA; (c) pure SrHA; (d) FHA/SrHA1; (e) FHA/SrHA2; (f) FHA/SrHA3.
FHA FHA/SrHA1 FHA/SrHA2 FHA/SrHA3 SrHA0
1
2
3
4
5C
ritic
al lo
ad/ N
Sample
Fig. 3. Adhesion strength of pure FHA, SrHA and FHA/SrHA biphasic coatings.
The solubilities of all coatings in Tris solution after heat treatment at600 °C for 15 min are shown in Fig. 4. The cumulative concentration ofCa2+, PO4
3− and Sr2+ ions released from the coating layer was moni-tored after soaking for up to 14 days.
As expected, the dissolution rate of pure SrHA coating was consider-ably higher than that of the pure FHA coating. Interestingly, all biphasicFHA/SrHA coatings showed lower solubility than FHA coating. Theconcentration of Ca2+ released from the biphasic coating into the solu-tion is increasing progressively with the SrHA amount in FHA. The Sr2+
and PO43− concentrations exhibit the same trend as that of Ca2+ con-
centration. The observed dissolution rates of biphasic coatings suggestthe possibility of tailoring the solubility of the biphasic coating layersthrough SrHA amount. It is also suggested that the coexistence of F−
and Sr2+ ions in biphasic coatings may restrain the dissolution rate ofapatite phase,which is beneficial for the improvement of coating stability.Accordingly, it is reasonable to deduce that the solubility of the biphasiccoatings is retarded and controllable by changing the relative phaseproportions,which is of importance to obtain implantswith good bioac-tivity and longevity.
The XRD patterns of all coatings after soaking in Tris solution for14 days are shown in Fig. 5. The diffraction peaks correspond toapatite phases, calcium phosphates rich in Ca and P elements and
SrHA FHA/SrHA1 FHA/SrHA2 FHA/SrHA3 FHA0
20
40
60
80
100
Dis
solu
tion
amou
nt/ 1
0-3m
g/cm
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Ca
P
Sr
Fig. 4. Dissolution rate of pure FHA, SrHA and FHA/SrHA biphasic coatings after immer-sion in Tris solution for 14 days.
TiO2 phase from Ti substrate. Since the Tris solution is free of Ca2+,Sr2+ and PO4
3+ ions, the newly formed calcium phosphates must beproducts of re-deposition of ions dissolved during the soaking period.A similar phenomenon was observed on HA coated porous NiTi alloy[19]. It suggests that in immersion test, the apatite phases in the coat-ing tend to dissolve in the solution leading to an increase of Ca2+ andPO4
3− concentrations at the initial stage of immersion. Then, with im-mersion time increasing, Ca2+ and PO4
3− concentrations increase andreach their saturation. As a result, the Ca2+ and PO4
3− ions in the so-lution may re-deposit on the substrate as calcium phosphates untilfinally reaching a precipitation–dissolution equilibrium.
The dissolved surface morphologies of all coatings are shown inFig. 6a–e. After immersion in Tris solution for 14 days, the surfacesof pure FHA and SrHA coatings become more dense and rougher asshown in Fig. 6a and b, the reason for which is likely filling andhence repair of cracks by re-deposition of calcium phosphate precip-itates. Similarly, after immersion for 14 days, all biphasic coating sur-faces became much rougher and less porous. A close observation athigher magnification (inset) indicated a lamellar-like structure of allcoatings, likely due to the dissolution–precipitation process. The asso-ciated EDS analysis results show the formation of compounds rich inCa and P (data not shown here). Further calculation of the Ca/P or(Ca+Sr)/P atomic ratios gives values of 1.57, 1.58, 1.50, 1.48, 1.47for FHA, SrHA, FHA/SrHA1, FHA/SrHA2 and FHA/SrHA3 coating, respec-tively, all of which are smaller than the stoichiometric ratio of 1.66 forpure HA, indicative of the retention of Ca2+, Sr2+ and PO4
3− ions in Trissolution. These results are in accord with the solubility test and XRDanalysis.
3.4. Cell responses
The in vitro cell responses to pure FHA and biphasic coatings on Tiwere assessed using osteoblastic MG63 cells. Fig. 7 shows typical SEMmicrographs of MG63 cells after 1 day of culture on FHA, FHA/SrHA1,FHA/SrHA2 and FHA/SrHA3 coatings. No apparent difference wasobserved in cell morphology among the coatings. As observed, theosteoblast cells have attached and flattened well across the coatingsurface. Under higher magnification as shown in the inset of Fig. 7,the cells were seen to flatten and attach tightly to the coating surfaceswith their filopodia and lamellipodia, suggesting good cell viabilityand a highly biological affinity to the coatings. Similar cell growth mor-phologies have been observed on pure FHA coating previously [27].Among the FHA and biphasic coatings, comparable improvements in
Fig. 5. X-ray diffraction patterns of pure FHA, SrHA and FHA/SrHA biphasic coatingsafter soaking in Tris solution for 14 days.
Fig. 6. SEMmicrograph and insetwith highermagnification of the surfacemorphology of coating after soaking in Tris solution for 14 days for (a) pure FHA; (b) pure SrHA; (c) FHA/SrHA1;(d) FHA/SrHA2; (e) FHA/SrHA3.
612 P. Yin et al. / Surface & Coatings Technology 207 (2012) 608–613
cell proliferation were observed. The cell density on all biphasic coat-ings, regardless of SrHA content, was much higher than that on pureFHA coating, which is likely associated with increased surface rough-ness owing to enhanced dissolution of the coating layer. Thus, the bi-phasic coatings with dispersion of SrHA in FHA matrix are considered
Fig. 7. SEM micrograph of the MG63 cells growing on coatings after culturing
to be effective for promoting the osteoblast–coating interactionsresulting in the enhanced bioactive properties. Themaximumcell num-bers are observed on FHA/SrHA2 coating surface as shown in Fig. 7c, im-plying that the concentration of Ca2+, Sr2+ and PO4
3− ions releasedfrom biphasic FHA/SrHA2 coating layer is favorable for cell growth.
for 1 day: (a) pure FHA. (b) FHA/SrHA1; (c) FHA/SrHA2; (d) FHA/SrHA3.
The difference observed in cell responses for different FHA/SrHA bi-phasic coatings suggests that the bioactivity of biphasic coatings canbe controlled by varying the phase constituent in coatings. Overall, thebiphasic coating structure provides a promising approach to improvethe biological performance of coatings in terms of its dissolution behaviorand in vitro cell responses.
4. Conclusion
FHA/SrHA biphasic coatings were prepared on Ti substrates usinga sol–gel dip-coating method. The colloidal sols consisted of FHA asthe matrix and SrHA as the soluble phase. The relative phase propor-tion of biphasic coatings can be tailored by the amount of SrHA in thecolloidal sol. The surface morphology of biphasic coatings becomesincreasingly rougher with increasing SrHA contents. The dissolutionrate and adhesion strength of FHA/SrHA biphasic coatings were be-tween those of pure FHA and SrHA coating. Moreover, the study hasfurther clarified that the dispersion of SrHA in FHA has promotedgrowth and attachment of osteoblast cells, which confirmed theimproved bioactivity of the FHA/SrHA biphasic coating film on Ti sub-strate with the sol–gel coating treatment. As a consequence, FHA/SrHAbiphasic coatings on Ti substrate provide a promising approach, whichcould be advantageous in achieving more stable, more bioactive andmore biocompatible implants for clinical applications by varying therelative phase proportion.
Acknowledgment
This work was supported by National Natural Science Foundation ofChina (grant no. 51021063) and Open Project of State Key Laboratoryfor Powder Metallurgy of CSU.
References
[1] L.L. Hench, J. Wilson, An Introduction to Bioceramics, World Scientific Press,Singapore, 1993.
[2] J.L. Ong, D.L. Carnes, K. Bessho, Biomaterials 25 (2004) 4601.[3] L. Gineste, M. Gineste, X. Ranz, A. Ellefterion, A. Guilhem, N. Rouquet, P. Frayssinet,
J. Biomed. Mater. Res. 48 (1999) 224.[4] S. Overgaard, M. Lind, K. Josephsen, A.B. Maunsbach, C. Bnger, K. Soballe, J. Biomed.
877.[9] K. Cheng, S. Zhang, W. Weng, X. Zeng, Surf. Coat. Technol. 198 (2005) 242.
[10] G.X. Ni, W.W. Lu, B. Xu, K.Y. Chiu, C. Yang, Z.Y. Li, et al., Biomaterials 27 (2006)5127.
[11] S.G. Dahl, P. Allain, P.J. Marie, Y. Mauras, G. Boivin, P. Ammann, et al., Bone 28(2001) 446.
[12] E. Landi, A. Tampieri, G. Celotti, S. Sprio, M. Sandri, G. Logroscino, Acta Biomater. 3(2007) 961.
[13] K.K. Johal, G. Mendoza-Suarez, J.I. Escalante-Garcia, J. Mater. Sci. Mater. Med. 13(2002) 375.
[14] K. Cheng, S. Zhang, W.J. Weng, Thin Solid Films 515 (2006) 135.[15] K. Cheng, S. Zhang, W.J. Weng, J. Mater. Sci. Mater. Med. 18 (2007) 2011.[16] K. Cheng, S. Zhang, W.J. Weng, K.A. Khor, Thin Solid Films 516 (2008) 3251.[17] S. Zhang, X.T. Zeng, Y.S. Wang, K. Cheng, W.J. Weng, Surf. Coat. Technol. 200
(2006) 6350.[18] Y.F. Li, Q. Li, S.S. Zhu, E. Luo, J.H. Li, G. Feng, Y.M. Liao, J. Hu, Biomaterials 31 (2010)
9006.[19] J.X. Zhang, R.F. Guan, X.P. Zhang, J. Alloys Compd. 509 (2011) 4643.[20] K. Cheng, W.J. Weng, H.M. Wang, et al., Biomaterials 26 (32) (2005) 6288.[21] Y.L. Cai, J.J. Zhang, S. Zhang, S.S. Venkatraman, X.T. Zeng, H.J. Du, D. Mondal,
Biomed. Mater. 5 (2010) 054114.[22] H.M. Kim, F. Miyaji, T. Kokubo, Nakamura, J. Biomed. Mater. Res. 32 (1996) 409.[23] E.J. Lee, S.H. Lee, H.W. Kim, Y.M. Kong, H.E. Kim, Biomaterials 26 (2005) 3843.[24] J.M. Gomez-Vega, E. Saiz, A.P. Tomsia, G.W. Marshall, S.J. Marshall, Biomaterials
21 (2000) 105.[25] D.M. Brunette, P. Tengvall, M. Textor, P. Thomsen, in: Springer, Berlin, 2001,
p. 171.[26] A. Montenero, G. Gnappi, F. Ferrari, M. Cesari, E. Salvioli, L. Mattogno, S. Kaciulis,
M. Fini, J. Mater. Sci. 35 (2000) 2791.[27] H.W. Kim, Y.M. Kong, C.J. Bae, Y.J. Noh, H.E. Kim, Biomaterials 25 (2004) 2919.
