Preparation And Biocompatibility Analysis Of Biphasic FHA/SrHA Coatings

Hydroxyapatite (HA, Ca10(P04)6OH2) coated titanium metal implants have been applied extensively due to their outstanding biological responses in the physiological environment. Hence, HA coated titanium alloy has been widely used as implant materials in orthopedic and dental applic-ations. However, pure HA coating has a relatively high dissolution rate in the biological environment, which affects its long-term stability and makes the interface between bone and implant unstable. On the other hand, natural bone is a kind of calcium-deficient HA, which contains considerable amounts of additional elements including F, Mg, Sr and Zn, etc. Accordingly, modification of HA by incorporation of these elements is required to improve both bioactivity and long term stability of the apatite in terms of composition for clinical applications. Fluorine-doped hydroxyapatite Ca10(PO4)6(OH)2-xFx (FHA, x is the degree of fluoridation) is reported to possess lower solubility and comparable bioactivity and biocompatibility over HA. Sr-substituted HA(SrHA) shows osteoinductive activity as well as higher solubility than HA.The aim of this study is to investigate the biological properties of biphasic coatings FHA/SrHA prepared by sol-gel method, which would be advantageous in achieving more stable, more bioactive and more biocompa-tible implants for clinical applications.Chapter Ⅰ:The fabrication and characterization of biphasic coatings FHA/SrHA on titanium by sol-gel methodObjective:FHA/SrHA biphasic coatings with different doses of strontium were prepared on titanium substrate by sol-gel processes. The characteristics, bonding strength, dissolution rate and in vitro bioactivity in simulated body fluid of the coatings were also evaluated.Methods:FHA/SrHA biphasic coatings (FHA/SrHA1, FHA/SrHA2and FHA/SrHA3) with different doses of strontium were prepared on titanium substrate by sol-gel processes. The surface morphology of the coatings was analyzed by scanning electron microscope (SEM). The element composition and phase component were analyzed with energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). The adhesion strength between the coating and substrate was evaluated using a scanning scratch tester. Dissolution behaviors were analyzed in TRIS solution and CPBS solution. The ability to induce apatite precipitation on coating surfaces was evaluated in vitro by immersion in simulated body fluid (SBF).Results:1. FHA/SrHA biphasic coatings with different SrHA amount were successfully prepared on Ti substrate by sol-gel method. All FHA/SrHA biphasic coatings exhibited stronger adherence to Ti substrate than pure SrHA coating, but inferior adherence to pure FHA. The solubility of biphasic coatings and bonding strength between biphasic coatings and Ti substrate were adjustable and controllable by changing the addition amount of SrHA.2. All FHA/SrHA biphasic coatings exhibited a process of dissolving and re-precipitating calcium phosphate in TRIS solution and CPBS solution. The dissolution rate changed with the variation of phase constitution. However, biphasic coatings displayed good stability in comparison with pure phase coatings.3. All FHA/SrHA biphasic coatings exhibited the inducing ability for bone-like apatite formation upon soaking in SBF for14d. The XRD pattern indicated the diffraction peak intensity of apatite increased with increasing the SrHA amount. These findings implied that FHA/SrHA biphasic coatings were bioactive with biomineralization ability. Conclusion:It is easy to prepare FHA/SrHA biphasic coatings by sol-gel method. Their dissolution rates are controllable by changing the addition amount of SrHA. FHA/SrHA biphasic coatings exhibit good stability and bioactivity, which is meaningful for the improvement in early bioactivity and long-term stability of Ti plants in clinic application.Chapter Ⅱ. Osteoblastic cell response on biphasic coatings FHA/SrHAObjective:To investigate the osteoblast biocompatibility of the single FHA coating and biphasic coatings FHA/SrHA.Methods:In vitro cell responses were evaluated using osteoblast-like MG63cells in terms of cell initial cell attachment (cell counting), morphology (inverted microscope and SEM), proliferation(MTT)and differentiation (alkaline phosphatase activity and osteocalcin level).Results:1. Initial cell attachment:After1h culture, the mean numbers of attached cells between single FHA coating and biphasic coatings FHA/SrHA had no significant deviation (P>0.05).2. Evaluated by inverted microscope and SEM:The cell growth morphology on biphasic coatings was quite similar to that on single FHA and the cells grew favorably on all the coatings surface.3. Osteoblast proliferationAfter1,3, and5days of culture, the MTT assay observation demonstrated that single FHA and FHA/SrHA biphasic coatings promoted cell proliferation. Moreover, all the biphasic coatings, regardless of SrHA amount, showed much higher cell proliferation activity after5days than single FHA coating. Meanwhile, the relatively higher OD values of FHA/SrHA2biphasic coating after5days of incubation indicated a superior proliferation (P<0.05).4. The intracellular alkaline phosphatase (ALP) expressionA relatively higher ALP level for FHA/SrHA2was observed and there were no significant differences in total ALP activity among other coatings in the first7days in culture(P>0.05). After14days, cells on biphasic coatings had a significant higher ALP activity than those on pure FHA(P<0.05). Meanwhile, cells on FHA/SrHA2coating had significantly higher total ALP activity than those on single FHA and other biphasic coatings(P<0.05).After21days, from day14to day21, a down-regulation was seen for all the coatings:significant decrease for FHA/SrHA1, FHA/SrHA2and FHA/SrHA3(P<0.05)and insignificant change for FHA(P>0.05).5. Oteocalcin (OCN) expressionAfter7days, all coatings had similar level of OCN synthesized by the osteoblasts (P>0.05).After14and21days, a significant increase in OCN levels was observed for all the coatings(P<0.05). Cells on biphasic coatings had a significant higher OCN levels than those on single FHA(P<0.05) ompared with single FHA and other biphasic coatings, FHA/SrHA2coating stimulated significantly higher OCN levels (P<0.05).Finally, all coatings reached maximum OCN expression after21ays in culture.Conclusion:Compared with single FHA, biphasic coatings FHA/SrHA have better surface properties which favor the cell proliferation and different-ation of osteoblast, especially at FHA/SrHA2.