3D打印海藻酸钠-明胶皮肤支架及其与成纤维细胞的体外生物相容性研究

3D printing of SA-gelatin scaffolds and its biocompatibility on fibroblasts in vitro

目的利用3D打印技术将海藻酸钠(SA)与明胶制备成不同规格的SA-明胶皮肤支架,评价其生物相容性及对大鼠皮肤成纤维细胞生物学行为的影响。方法采用挤压式3D打印技术,将3%、4%、5%的SA与明胶制成生物墨水,打印获得不同规格的SA-明胶皮肤支架。通过扫描电镜观察其形态结构;将大鼠皮肤成纤维细胞与其共培养,于第2天进行Live/Dead染色观察皮肤支架表面细胞生长状态,第5天扫描电镜观察细胞生长黏附情况及形态,第1、3、5天使用CCK-8法检测细胞增殖情况,第1、2、3天检测胶原蛋白及IL-6的表达水平。结果扫描电镜观察显示,3组皮肤支架均为多孔网状立体结构,且孔隙均匀,3%SA组皮肤支架的表面较另2组明显粗糙。Live/Dead染色结果表明,细胞在3组皮肤支架表面均能较好地粘附与生长,但5%SA组细胞数量少于另外两组;扫描电镜下,可见细胞能在皮肤支架表面粘附与生长,且4%SA组细胞数量多于另2组。CCK-8检测结果表明,4%SA组细胞增殖率显著高于另2组(P<0.05)。4%SA组的胶原蛋白分泌量显著高于另2组(P<0.05);其IL-6表达水平则明显低于另2组(P<0.05)。结论4%SA-明胶的皮肤支架具有较好的生物相容性和胶原蛋白分泌效应,并可降低IL-6的表达水平,从而有效抑制炎症反应的发生,有望为创面修复提供一种可降解的3D打印皮肤支架。

Objective To prepare SA-Gelatin skin scaffolds of different kinds with sodium alginate(SA)and gelatin by 3D bio-printing,and to evaluate the biocompatibility of the scaffolds and their effects on the biological behavior of rat skin fibroblasts.Methods Using extruding 3D bio-printing technology,3%,4%,5%SA-Gelatin were made into biological ink,and different kinds of SA-Gelatin skin scaffolds were obtained by printing.The structure was observed by scanning electron microscope.Rat skin fibroblasts were co-cultured with them,and cell growth on the skin scaffold surface was observed by Live/Dead staining on the 2nd day.Cell growth adhesion and morphology were observed by scanning electron microscopy on the 5th day.Cell proliferation was detected by CCK-8 method on the 1st,3rd and 5th days,and expression levels of collagen and IL-6 were detected on the 1st,2nd and 3rd days.Results Scanning electron microscopy:The skin scaffolds in the three groups were all of porous reticular stereoscopic structure with uniform pores.The surface of skin scaffolds in the 3%SA group was obviously rougher than that in the other two groups.Live/Dead staining:Cells adhered and grew well on the surface of skin scaffolds in all three groups,but the number of cells in 5%SA group was less than that in the other two groups.Scanning electron microscopy:The cells could adhere to and grow on the surface of skin scaffold,and the number of cells in 4%SA group was higher than that in the other two groups.CCK-8:The cell proliferation rate of 4%SA group was significantly higher than that of the other two groups(P<0.05).Collagen secretion:4%SA group was significantly higher than that in the other two groups(P<0.05).IL-6 expression level:4%SA group was significantly lower than the other two groups(P<0.05).Conclusion The skin scaffold with 4%SA-Gelatin has good biocompatibility and collagen secretion effect,and can reduce the expression level of IL-6 and effectively inhibit the occurrence of inflammatory reaction,which is expected to provide a biodegradable 3D bio-printing skin scaffold for wound repair.