载血小板衍生生长因子3D生物打印半月板支架的制备流程

Preparation of platelet-derived growth factor loaded three-dimensional bio-printed Meniscus scaffold

背景:基于原位组织工程再生理念应用于半月板再生领域中的需要,可利用3D生物打印技术构建一种有效结合招募性生物活性因子和优良材料的支架。目的:优化负载有血小板衍生生长因子BB的聚己内酯/甲基丙烯酸酯化明胶/半月板细胞外基质3D生物打印支架制备流程,检测其生长因子缓释性能及对滑膜间充质干细胞迁移和增殖的作用。方法:利用甲基丙烯酸酯化明胶/半月板细胞外基质制备光敏性生物墨水,采用3D生物打印技术制备单纯聚己内酯支架及聚己内酯/甲基丙烯酸酯化明胶/半月板细胞外基质仿生支架,最终负载血小板衍生生长因子BB于生物墨水内构建聚己内酯/甲基丙烯酸酯化明胶/半月板细胞外基质/血小板衍生生长因子BB功能化支架,检测支架的力学性能与缓释性能。分离培养新西兰大白兔滑膜间充质干细胞,通过Transwell和CCK-8实验探究3种支架对滑膜间充质干细胞的迁移和增殖行为的影响,以单纯培养基培养的细胞为阴性对照。将第3代滑膜间充质干细胞分别接种于3种支架上,利用共聚焦显微镜和扫描电镜观察3种支架上细胞的活性、黏附和生长状态。结果与结论:①仿生支架的拉伸模量及压缩模量与单纯聚己内酯支架比较差异无显著性意义(P>0.05);②功能化支架具有良好的缓释性能,可持续释放血小板衍生生长因子BB长达28 d;③相对于阴性对照组,仿生支架及功能化支架可促进滑膜间充质干细胞的迁移,其中以功能化支架促迁移效果更显著;④相对于阴性对照组,仿生支架及功能化支架培养4,7 d的细胞增殖较快(P<0.001),其中功能化支架的促增殖作用更明显;⑤死活染色显示,各组细胞活性良好,且功能化支架上的细胞数量最高;⑥扫描电镜显示,细胞均附着于3组支架表面生长,其中仿生支架及功能化支架上的细胞黏附数量较多,并分泌大量细胞外基质;⑦结果表明,负载血小板衍生生长因子BB的3D生物打印半月板支架具有良好的力学性能和生物相容性,可促进滑膜间充质干细胞的迁移和增殖。

BACKGROUND:Based on the need of in situ tissue engineering regeneration in the field of meniscus regeneration,three-dimensional(3D)bioprinting technology can be used to construct a scaffold which can effectively combine recruitment bioactive factors and excellent materials.OBJECTIVE:To optimize the preparation process of polycaprolactone/methacrylate anhydride gelatin/meniscus extracellular matrix(PCL/GelMA/MECM)3D bioprinting scaffold loaded with platelets derived growth factor-BB(PDGF-BB),and to test the sustained release properties of the growth factor and its effect on the migration and proliferation of the synovial mesenchymal stem cells.METHODS:Photosensitive bio-ink was prepared by GelMA/MECM.PCL scaffold and PCL/GelMA/MECM bio-scaffold were prepared by 3D bioprinting technology.Finally,PDGF-BB was loaded to fabricate functional bioprinting PCL/GelMA/MECM/PDGF scaffold,and then its mechanical properties and sustained release properties were tested.New Zealand white rabbit synovial mesenchymal stem cells were isolated and cultured.Transwell and CCK-8 assays were used to explore the effects of PCL scaffold,PCL/GelMA/MECM scaffold and PCL/GelMA/MECM/PDGF scaffold on the migration and proliferation of synovial mesenchymal stem cells,and single medium cultured cells were used as negative control.The third passage of synovial mesenchymal stem cells was inoculated on PCL scaffold,PCL/GelMA/MECM scaffold and PCL/GelMA/MECM/PDGF scaffold respectively.The viability,adhesion and growth of the cells on the three scaffolds were observed by confocal microscope and scanning electron microscope.RESULTS AND CONCLUSION:(1)There was no significant difference in tensile modulus and compression modulus between PCL/GelMA/MECM scaffolds and PCL scaffolds(P>0.05).(2)PCL/GelMA/MECM/PDGF scaffolds had good sustained release performance,and PDGF-BB could be released continuously for up to 28 days.(3)Compared with the negative control group,PCL/GelMA/MECM scaffolds and PCL/GelMA/MECM/PDGF scaffolds could promote the migration of synovial mesenchymal stem cells,among which PCL/GelMA/MECM/PDGF scaffolds were more effective.(4)Compared with the negative control group,the cells cultured with PCL/GelMA/MECM scaffolds and PCL/GelMA/MECM/PDGF scaffolds proliferated faster after 4 and 7 days(P<0.001),and the proliferative effect of PCL/GelMA/MECM/PDGF scaffolds was more obvious.(5)Dead/Live staining showed that the cells in each group had good activity,and the number of cells on the PCL/GelMA/MECM/PDGF scaffold was the highest.(6)Scanning electron microscope showed that the cells were attached to the surface of the three groups of scaffolds,and the cells on PCL/GelMA/MECM scaffolds and PCL/GelMA/MECM/PDGF scaffolds adhered to a large number of cells and secreted a large amount of extracellular matrix.(7)These results showed that the 3D bioprinted meniscus scaffold loaded with PDGF-BB had good mechanical properties and biocompatibility and could promote the migration and proliferation of synovial mesenchymal stem cells.