可长时间缓释骨形态发生蛋白2的聚己内酯复合支架的制备及应用

Preparation and application of polycaprolactone composite scaffold with long-term slow-release of bone morphogenetic protein 2

目的制备一种可长时间缓释骨形态发生蛋2(BMP-2)的聚己内酯(PCL)复合支架,并通过检测其对人源骨髓间充质干细胞(BMSC)成骨分化的影响探讨其在骨组织工程中的应用。方法将磷脂(PL)和BMP-2混合形成的BMP-2/PL混合物(B/P)分散在二氯甲烷中,与PCL混合后,采用相分离法制备负载BMP-2的三维PCL-B/P复合支架和PCL-B传统支架,通过酶联免疫吸附试验(ELISA)检测两种支架的BMP-2缓释效果。将BMSC种植在PCL-B传统支架和PCL-B/P复合支架中,分别采用CCK-8法和实时定量PCR(qPCR)检测两种支架上BMSC的增殖和成骨分化能力。结果与PCL-B传统支架对比,PCL-B/P复合支架对BMP-2缓释效果更佳,缓释时间更长,可达22d。在BMSC培养的第7、14和21天,PCL-B/P复合支架上BMSC的增殖能力均优于PCL-B传统支架(P<0.05),且PCLB/P复合支架上BMSC中碱性磷酸酶和Ⅰ型胶原蛋白、骨钙、骨桥蛋白3种成骨基因mRNA的表达均高于PCL-B传统支架(P<0.05,P<0.01)。结论成功地制备出一种可长时间缓释BMP-2的高分子三维PCL-B/P复合支架,其较PCL-B传统支架能更好地诱导BMSC的增殖和成骨分化。

Objective To prepare a polycaprolactone（PCL）composite scaffold which can slowly release bone morphogenetic protein 2（BMP-2）for a long time,and to explore the application value of PCL composite scaffold in bone tissue engineering through studying osteogenic differentiation level of human bone mesenchymal stem cells（BMSCs）.Methods The BMP-2/PL compounds（B/P）,formed by the BMP-2and soybean phospholipid（PL）,were dispersed in methylene dichloride,and then we mixed it with PCL and prepared the PCL-B/P composite scaffold and PCL-B traditional scaffold loaded with BMP-2by phase separation method.The slow-release of BMP-2from two kinds of scaffolds was observed with ELISA assay.The human BMSCs were cultured in the media containing PCL-B/P composite scaffold or PCL-B traditional scaffold,and the proliferation abilities and osteogenic differentiation levels of BMSCs on two kinds of scaffolds were detected and analyzed by CCK-8 assay and qPCR,respectively.Results Compared with the PCL-B traditional scaffold,PCL-B/P composite scaffold in this study showed a better slow-release effect of BMP-2 and a longer slow-release time of 22 days.The proliferation abilities of BMSCs on PCL-B/P composite scaffold were better than that on PCL-B traditional scaffold on the 7^th day,14 th day and 21st day of cell culture（P〈0.05）.The alkaline phosphatase content and mRNA expressions of collagen typeⅠ,osteocalcin and osteopontin of BMSCs in the PCL-B/P composite scaffolds were significantly higher than those in the PCL-B traditional scaffold（P〈0.05,P〈0.01）.Conclusion We have successfully prepared a PCL-B/P composite scaffold loaded with BMP-2.The PCL-B/P composite scaffold can slowly release BMP-2 for a long time and induce the proliferation and osteogenic differentiation of human BMSCs.