| Objective:This study investigated the optimal concentration of IGF-1and TGF-β1loaded gelatin microspheres, investigated the influence of porous titanium coated with alone or in combination of IGF-1and TGF-β1loaded gelatin microspheres on adhesion, proliferation and differentiation of MG63cells and explored the possible mechanisms of action on the MG63cells by sustained-release of IGF-1and TGF-β1loaded gelatin microspheres.Methods:Porous titanium with porosity of60%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 gelatin microspheres, MTT assay method was used to evaluate the cytotoxicity of coating to the rat fibroblast cells L929. Optimized the optimal concentration of IGF-1、 TGF-β1in gelatin release microspheres in vitro experiments. Comparatively studied the influence of the separate and combined application of IGF-1and TGF-β1released coating on the function of MG63cells.Result:1)The MTT test showed the leaching liquor with100%,50%、10%concentrations of porous titanium coated with gelatin microspheres was nontoxic to L929.2)The results of optimizing the concentration of IGF-1loaded gelatin sustained-release microspheres showed that:The concentrations of IGF-1effected proliferation and differentiation of MG63cells, they appeared a positive dose-effect relationship while the value between0.1-10ng/mg, especially significant in the value of10ng/mg. The results of optimizing the concentration of TGF-β1loaded gelatin sustained-release microspheres showed that:The concentrations of TGF-β1effected proliferation and differentiation of MG63cells, they appeared a positive dose-effect relationship while the value between0.025-2.5ng/mg, especially significant in the value of2.5ng/mg. 3)The experiment used the optimal concentration of IGF-1with different concentrations of TGF-β1in combination and found that:10ng/mg of IGF-1in combination with2.5ng/mg of TGF-β1had the best role in promoting proliferation and differentiation of MG63cells. The experiment used the optimal concentration of TGF-β1with different concentrations of IGF-1in combination and found that:2.5ng/mg of TGF-β1in combination with lOng/mg of IGF-1had the best role in promoting proliferation and differentiation of MG63cells.4) IGF-1loaded gelatin sustained-release microspheres to promote proliferation of MG63cells was better than TGF-β1, but weaker than TGF-β1in promoting differentiation of MG63cells. The combined use of IGF-1and TGF-β1loaded gelatin release microspheres had a synergistic effect on proliferation and differentiation of MG63cells, the combined group to promote the proliferation and differentiation effects of MG63cells was more obvious than a single application of IGF-1or TGF-β1group.5)Porous titanium sample groups of different sustained-release coatings cultured with MG63cells for7and14days, the test results of proliferation showed that:Contained IGF-1and TGF-β1in microsphere complex coating group> Contained IGF-1in microsphere coating group> Contained TGF-β1in microsphere coating group> Without growth factor microspheres coating group, and the difference was remarkable (P>0.05). Results suggested that:IGF-1microspheres coating group to promote the proliferation of MG63cells was more effective than TGF-β1microspheres coating group, the group of the composite coating of IGF-1and TGF-β1to promote proliferation of MG63cells was most effective. After cultured3、7、14days, results of detection the content of ALP and BGP suggested that:The effect of promoting cells’differentiation by porous titanium coated with TGF-β1microspheres group was higher than the porous titanium coated with IGF-1group, the complex coating group to promote the differentiation effects of MG63cells was most significant, and the difference was remarkable (P>0.05).6)Samples of different sustained-release coating cultured with MG63cells. After7、14days the samples were fixed、 dried and sprayed gold treatment. Observing cell culture in vitro on7th day by SEM, we comed to a conclusion that:MG63cells in group C had irregular shapes and more pseudopodium, they laid on the surface of materials, most of cells adhered to the border of pores while a few of them have migrated inside. MG63cells in group A and B spreaded well, they adhered to the border and even grow into the pores vertically, trended to migrate inside. MG63cells in group D were spindle shaped, laid on the surface of porous titanium and had few pseudopodium, they got no trend of migrating inside. Observed by SEM in14th day, cells of four groups developed well, they had no differences in appearance, most of cells were in irregular polygon shaped, laminated lay on and inside pores. The cells of group C connected into anetwork structure in the pores; cells of group A and B imbricated paved on porous titanium, covering almost all porous titanium’s surface structure; cells of group D grew close to the hole wall, partially covering the surface of porous titanium.Conclusion:1)The concentrations of IGF-1effected proliferation and differentiation of MG63cells, they appeared a positive dose-effect relationship while the value between0.1-10ng/mg, especially significant in the value of lOng/mg. The concentrations of TGF-β1effected proliferation and differentiation of MG63cells, they appeared a positive dose-effect relationship while the value between0.025-2.5ng/mg, especially significant in the value of2.5ng/mg, higher than2.5ng/mg inhibited cells’proliferation, but in favor of differentiation. The joint application of two growth factors in the optimal concentration to promote proliferation and differentiation of MG63cells was superior than a single application of IGF-1and TGF-β1group.2) The effect of promoting cells’differentiation by porous titanium coated with TGF-β1microspheres group was higher than the porous titanium coated with IGF-1group, but the proliferative effect was weaker than IGF-1group. IGF-1and TGF-β1loaded gelatin sustained-release microspheres complex coating had a synergistic effect on proliferation and differentiation of MG63cells, the complex coating group to promote the proliferation and differentiation effects of MG63cells were more obvious than a single application of IGF-1or TGF-β1loaded gelatin sustained-release microspheres coating group.3) The effect of porous titanium coated with IGF-1and TGF-β1loaded gelatin sustained-release microspheres to promote adhesion of MG63cells on the surface was better than porous titanium with a single application of IGF-1or TGF-β1loaded gelatin release microspheres coating. |
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