骨髓间充质干细胞来源细胞外基质/胶原蛋白支架促进成纤维细胞运动和迁入

Bone marrow stem cells-derived extracellular matrix/collagen scaffold promotes motility and infiltration of fibroblasts

目的探讨小鼠骨髓间充质干细胞来源细胞外基质增强成纤维细胞运动和迁入胶原蛋白支架的功能。方法采用细胞裂解和DNA酶解的方法制备出骨髓间充质干细胞来源细胞外基质,通过酶解获得骨髓间充质干细胞来源细胞外基质溶液。将I型胶原蛋白的醋酸溶液和骨髓间充质干细胞来源细胞外基质溶液机械混匀、冻干、饱和戊二醛蒸汽交联后,得到骨髓间充质干细胞来源细胞外基质/胶原蛋白支架,同时,制备纯胶原蛋白支架。扫描电镜观察支架微观结构,检测支架的傅里叶变换红外吸收光谱。将支架埋植于小鼠皮下5 d、10 d后,观察内源细胞迁入支架情况,通过Transwell实验和划痕愈合实验测试支架对人真皮成纤维细胞的运动和迁移的影响。结果骨髓间充质干细胞来源细胞外基质/胶原蛋白支架为连通多孔支架,除含有蛋白质的主要成分外,还含有糖胺聚糖等物质。苏木精-伊红染色显示,相比纯胶原蛋白支架,骨髓间充质干细胞来源细胞外基质/胶原蛋白支架显著促进内源细胞迁入支架内部。ER-TR7免疫染色显示,迁入支架的细胞为成纤维细胞。与纯胶原蛋白支架相比,在骨髓间充质干细胞来源细胞外基质/胶原蛋白支架作用下,穿过Transwell嵌套膜的细胞数均显著增多,划痕伤口愈合率均显著提高。结论通过引入骨髓间充质干细胞来源细胞外基质使胶原蛋白支架具有诱导组织修复细胞运动和迁入支架的活性,是一种潜在的促软组织原位修复新材料。

【Objective】To explore the function of mouse bone-marrow stem cells derived extracellular matrix in promoting fibroblast motility and infiltration into collagen scaffolds.【Methods】By the methods of cell lysing and enzyme hydrolysis of DNA,the solution of bone-morrow mesenchymal stem cells-derived extracellular matrix(BM-ECM)was harvested.Acetic acid solution of type I collagen and BM-ECM solution were homogenized mechanically,freeze-lyophilized,and crosslinked with saturated glutaraldehyde vapor,and BM-ECM/collagen scaffolds were obtained.The microscopic structure of the scaffolds was observed by scanning electron microscope,and Fourier-transform infrared spectroscopy spectra of the scaffolds were analyzed.Infiltration of endogenous cells into the scaffolds were observed on 5 and 10 days after subcutaneous implantation of the scaffolds in mice.The influence of the scaffolds on the motility and migration of human dermal fibroblasts was tested through Transwell experiment and scratch wound healing experiment.【Results】The extracellular matrix derived from bone marrow mesenchymal stem cells is a connected porous scaffold,which contains glycosaminoglycans and other substances in addition to the main components of protein.Hematoxylin-eosin staining shows that compared with pure collagen protein scaffold,bone marrow mesenchymal stem cell-derived extracellular matrix/collagen scaffold significantly promotes the migration of endogenous cells into the scaffold.ER-TR 7 immunostaining shows that the cells that migrate into the scaffold are fibroblasts.Compared with pure collagen scaffold or blank scaffold,extracellular matrix/collagen scaffold derived from bone marrow mesenchymal stem cells has significantly increased the number of cells passing through the Transwell nested membrane,and the scratch healing rate has been significantly improved.【Conclusion】The introduction of the extracellular matrix derived from bone marrow mesenchymal stem cells enables the collagen scaffold to induce tissue repair cell movement and migration into the scaffold.It is a potential new material for in situ repair of soft tissues.