不同孔径梯度纳米羟基磷灰石三维支架制备及对MC3T3-E1生物学性能的影响

Effect of hydroxyapatite three-dimensional scaffolds with different apertures on the biological properties of MC3T3-E1 cells

背景:骨组织工程支架孔径是评估其物理性能的重要因素,寻求孔径及力学性能二者的最佳结合点一直是骨组织工程亟待解决的问题。目的:制备不同孔径梯度的三维支架,并研究其对MC3T3-E1细胞生物学行为的影响。方法:将直径300,500,800μm的蜡球造孔剂分别与羟基磷灰石混合,采用改良型粒子浸出法制备孔径均一的纳米羟基磷灰石三维支架,分别记为D300、D500、D800组支架;将3种直径的蜡球从小到大依次堆积,采用改良型粒子浸出法制备孔径梯度纳米羟基磷灰石三维支架,记为D-Gradient组支架,检测4组支架的孔隙率。将MC3T3-E1细胞分别接种于4组支架上,以细胞单独培养的为空白对照组,检测细胞增殖活性、碱性磷酸酶活性、细胞黏附及细胞骨架重排与成骨分化标志性因子转录水平。结果与结论:(1)4组支架的孔隙率为60%-80%;(2)接种24 h后,各组支架均可促进MC3T3-E1细胞的增殖,增殖指数大小为:D-Gradient组>D500组>D800组>D300组>空白对照组;D300组、D500组、D-Gradient组支架均可促进MC3T3-E1细胞碱性磷酸酶的分泌,碱性磷酸酶分泌量多少为:D300组>D500组>D-Gradient组>D800组≈空白对照组;D300组、D500组、D-Gradient组支架均可促进MC3T3-E1细胞骨桥蛋白与骨钙素m RNA的表达,两种基因表达量由高到低为:D300组>D500组D-Gradient组>D800组≈空白对照组;(3)接种48 h后的免疫荧光染色显示,与空白对照组相比,4支架上的MC3T3-E1细胞均呈现出较好的黏附及运动活性,黏着斑肌动蛋白荧光强度由高到低为:D-Gradient组>D500组>D800C组>D300组>空白对照组;(4)结果表明,孔径梯度纳米羟基磷灰石三维支架可促进MC3T3-E1细胞的生长、增殖、黏附及成骨分化。

BACKGROUND:The aperture of bone tissue engineering scaffold is one of the important indexes to evaluate its biological performance,and seeking the best combination of aperture and mechanical properties has always been an urgent problem to be solved in bone tissue engineering.OBJECTIVE:To prepare three-dimensional scaffolds with different aperture gradients and to study their effects on the biological behavior of MC3 T3-E1 cells.METHODS:The three-dimensional nano-hydroxyapatite scaffolds with uniform pore size were prepared by mixing wax spheres of specific diameter(300,500,800μm)with hydroxyapatite as pore-forming agent using the modified particle leaching method,which were recorded as the D300,D500,and D800 scaffolds,respectively.The wax spheres of three diameters were piled up in order from small to large.The pore gradient nano-hydroxyapatite three-dimensional scaffold was prepared by the modified particle leaching method,which was recorded as the D-Gradient scaffold.The porosity of scaffolds of the four groups was measured.MC3 T3-E1 cells were inoculated on scaffolds of the four groups.The cells cultured separately were used as the blank control group.Cell proliferation activity,alkaline phosphatase activity,cell adhesion,cytoskeleton rearrangement and the transcription level of osteogenic differentiation marker factors were detected.RESULTS AND CONCLUSION:(1)The porosity of scaffolds of the four groups was 60%-80%.(2)After 24 hours of inoculation,the scaffolds in each group could promote the proliferation of MC3 T3-E1 cells.The proliferation index was:D-Gradient group>D500 group>D800 group>D300 group>blank control group.The scaffolds in the D300 group,D500 group,and D-Gradient group promoted the secretion of alkaline phosphatase in MC3 T3-E1 cells.The amount of alkaline phosphatase secretion was:D300 group>D500 group>D-Gradient group>D800 group≈blank control group.The scaffolds in the D300 group,D500 group,and D-Gradient group promoted the expression of osteopontin and osteocalcin m RNA in MC3 T3-E1 cells.The expression levels of the two genes from high to low were:D300 group>D500 group>D-Gradient group>D800 group≈blank control group.(3)Immunofluorescence staining after 48 hours of inoculation showed that compared with the blank control group,MC3 T3-E1 cells on the four scaffolds showed better adhesion and locomotor activity.The fluorescence intensity of focal adhesion actin from high to low was:D-Gradient group>D500 group>D800 C group>D300 group>blank control group.(4)It is concluded that the aperture gradient scaffolds promote the growth,proliferation,adhesion,and osteogenic differentiation of MC3 T3-E1 cells.