“壳-芯”结构电纺纤维膜对MC3T3-E1细胞体外早期成骨分化的影响

Effects of PLGA/PCL-BMP-2 core-shell electrospun nanofibrous composite membranes on the early osteogenic differentiation of MC3T3-E1 cells in vitro

目的研究载有骨形态发生蛋白-2(bone morphogenetic protein 2,BMP-2)的聚乳酸-羟基乙酸共聚物(poly(lactic-coglycolic acid),PLGA)、聚己内酯(polycaprolactone,PCL)"壳芯结构"静电纺丝纤维膜(PP-B)对MC3T3-E1细胞体外早期成骨分化的影响。方法通过同轴静电纺丝的方法制备PLGA/PCL-BMP-2纤维膜(PP-B),以不含BMP-2的PLGA/PCL电纺纤维膜(PP)为对照组。通过透射电子显微镜观察PP-B膜的"壳-芯"结构;用ELISA法检测BMP-2的体外释放行为;收集PP-B膜的浸出液培养细胞,用碱性磷酸酶(ALP)活性检测法检测其释放的BMP-2对MC3T3-E1细胞的影响;将MC3T3-E1细胞接种到纤维膜上,通过CCK-8法检测1 d、3 d、5 d、7 d的增殖情况;以碱性磷酸酶(ALP)活性为早期骨向分化指标,检测其7 d的活性;用实时荧光定量逆转录PCR检测细胞接种7 d时成骨指标Bmp2、Alp、Col1、Msx2、Runx2的表达。结果透射电镜结果表明,同轴静电纺丝法制备的PP-B静电纺丝可形成连续均一的"壳-芯"结构;ELISA结果显示,PP-B膜可以实现BMP-2的持续释放;ALP活性检测结果证实本实验条件下制备的负载BMP-2的同轴静电纺丝支架能保持生长因子的部分活性。CCK-8结果显示PP-B膜和PP膜上的MC3T3-E1细胞在1、3、5、7 d都持续增殖;ALP活性检测及染色结果显示PP-B膜上的MC3T3-E1细胞的ALP活性高于PP膜(P<0.05);实时荧光定量逆转录PCR结果显示接种在PP-B膜上的MC3T3-E1细胞的相关成骨基因Bmp2、Alp、Col1、Msx2、Runx2的表达高于PP膜(P<0.05)。结论本实验制备的PP-B膜能促进MC3T3-E1细胞在体外的早期成骨分化。

Objective To study the effects of bone morphogenetic protein 2（ BMP-2）/poly（ lactic-co-glycolic acid）,（ PLGA）/polycaprolactone（ PCL） core-shell nanofibrous composite membranes（ PP-B） on the early osteogenic differentiation of MC3 T3-E1 cells in vitro. Methods PLGA/PCL-BMP-2 core-shell nanofibrous composite membranes（ PP-B） were fabricated by coaxialelectrospinning. Electrospun PLGA/PCL nanofibrous membranes（ PP） were set as control group. The core-shell structure of PP-B electrospun fibers was assessed by Transmission electron microscope（ TEM）. In vitro releasing behavior of BMP-2 in PP-B membranes was detected by ELISA. The extracts of PP-B nanofibrous composite membranes were collected to culture MC3 T3-E1 cells,and the alkaline phosphate（ ALP） activity of the cells was evaluated. MC3 T3-E1 cells were seeded onto the nanofibrous composite membranes of the two groups.CCK-8 kit was used to observe cell proliferation. The ALP activity was measured to present the early osteogenic differentiation. Gene expression of Bmp2,Alp,Col1,Msx2,Runx2 was detected by reverse transcriptase-quantitive polymerase chain reaction（ RT-q PCR）. Results The TEM image confirmed that PP-B electrospun fibers fabricated by coaxial-electrospinning formed uniform core-shell structure. The results of ELISA showed BMP-2 contained in PP-B composite membranes released sustainedly in vitro. BMP-2 remained active in PP-B nanofibrous composite membranes. The data of CCK-8 continually increased in both groups within 7 days.BMP-2 remained active in PP-B nanofibrous composite membranes.The ALP activity of MC3 T3-E1 cells on PP-B nanofibrous composite membranes increased（ P〈0. 05）. RT-qP CR results revealed a significant increase in the expression of Bmp2,Alp,Col1,Msx2,Runx2 in MC3 T3-E1 cells cultured on PP-B nanofibrous composite membranes（ P〈0. 05）. Conclusions PP-B nanofibrous composite membranes can improv the early osteogenic differentiation of MC3 T3-E1 cells in vitro.