| Objective:This study investigated the characterization of porous titanium coated with different concentrations of TGF-β1 loaded gelatin microspheres, and estimate the effects on function of osteoblast cells after surface modificated with TGF-β1 loaded gelatin microspheres in vitro.Materials and methods:Porous titanium with porosity of 60% and cross-connection pore structure were prepared by metal injection molding.Gelatin microspheres were prepared by improved emulsified cold condensation method. The porous titanium were coated with TGF-β1 loaded gelatin microspheres, MTT assay method was used to evaluate the cytotoxicity of coating to the rat fibroblast cells L929. The encapsulation rate, drug content were test with TGF-β1 ELISA kit.Assessed the influence of different concentrations of TGF-β1 loaded gelatin microspheres on proliferation and differentiation of osteoblast cells MG63 in vitro and select the best concentrations of TGF-β1 comparative studied influence of porous titanium coated with TGF-β1 loaded gelatin microspheres(group A), porous titanium with TGF-β1 (group B), porous titanium (group C)on adhesion, proliferation and differentiation of osteoblast cells MG63.Result:Porous titanium implants were produced by MIM, which microstructure characters were 60% porosity with cross-connection pore structure and pore size range of 50-300μm. The size of gelatin microspheres were (20.33±3.67)μm in average.The MTT test showed the leaching liquor with 100%,50%,10% concentrations of porous titanium coated with gelatin microspheres was nontoxic to L929.The porous titanium implants were pretreated by 5wt% gelatin and coated with 20mg/ml TGF-β1 loaded gelatin microspheres were appropriate,the structure of pores were kept completely. In vitro,20-30% of TGF-β1 released from gelatin microspheres coating in 24h and releasing rat gradually decreased and became stable as time increased. Finally,93% of TGF-β1 were released during 12days. The concentrations of TGF-β1 effected proliferation and differentiation of osteoblast cells, they appeared a positive dose-effect relationship while the value between 0.025-2.5ng/mg, especially significant on the value of 2.5ng/mg. Values lower than 0.025ng/mg has no significant effect, higher than 2.5ng/mg inhibit cells'proliferation, but in favor of differentiation.After cultured 3,7 days it showed the amount of cells in A group>B group>C group, and the difference was remarkable. on 14th day, the amount of cells in A group> B group and C group,there was no significant difference between group B and group C (P>0.05). (P<0.05); Observing cell culture in vitro on 7th day by SEM, we comed to a conclusion that:The quantity, morphology and growing status in interspaces of MG63 A were different among 3 groups. Samples on group A own more cells than group B and C. MG63 in group A had irregular shapes and more pseudopod, they laid on the surface of materials, most of cells adhered to the border of pores while a few of them have migrated inside. MG63 in group B spreaded well, they adhered to the border and even grow into the pores vertically, trended to migrate inside. MG63 in group C is spindle shaped, laid on the surface of porous titanium and had few pseudopod, they got no trend of migrating inside. Observed by SEM in 14th day,3 groups of cells develop well, they had no differences in appearance,most of cells are in irregular polygon shaped.MG63 in group A formed an net work. MG63 in group B laminated lay on and inside pores, can barely show surface structure of porous titanium. Most of cells in group C grew vertically to the bottom of pores in reticular structure.After cultured 3,7days, no difference in ALP and BGP production was detected on group A and group B (P>0.05). But there was significant (P<0.05) between other groups. No difference in BGP production was detected on 5% and 30% group (P>0.05). But the difference was remarkable (P<0.05) on the other three groups,in 14th day,it showed the amount in A group>B group>C group, and the difference was remarkable.Conclusion: 1) In vitro, porous titanium coated with TGF-β1 loaded gelatin microspheres could controlledly release, TGF-β1 was released for 12 days.2) Proliferation and differentiation of osteoblasts were effected by the concentration of TGF-b1, they appeared a positive dose-effect relationship while the value between 0.025-2.5ng/mg, especially significant on the value of 2.5ng/mg. Values lower than 0.025ng/mg has no significant effect, higher than 2.5ng/mg inhibit cells'proliferation, but in favor of differentiation.3) Porous titanium with TGF-β1 is propitious to osteoblast cell's proliferation on 3th and 7th has no significant effect on 14th,but it is propitious to proliferation. Porous titanium coated with TGF-β1 loaded gelatin microspheres is propitious to osteoblast cell's adhesion, proliferation and differentiation on the three time slot.
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