可注射性甲基丙烯酸酐改性明胶/软骨源性基质微粒复合水凝胶支架的制备及生物相容性

Fabrication and biocompatibility of injectable gelatin-methacryloyl/cartilage-derived matrix particles composite hydrogel scaffold

背景:关节软骨的再生修复目前仍是充满挑战的医学难题,构建可仿生细胞生长微环境的可注射水凝胶支架对关节软骨的再生具有重要意义。目的:制备可注射甲基丙烯酸酐改性明胶/软骨源性基质微粒(gelatin-methacryloyl/cartilage-derived matrix particles,Gel MA/CDMPs)复合水凝胶支架,并评价其生物相容性。方法:(1)通过物理粉碎和化学酶法制备软骨源性基质微粒,通过甲基丙烯酸酐接枝明胶制备甲基丙烯酸酐改性明胶,并以两者为原料,制备5种不同质量浓度的Gel MA/CDMPs复合水凝胶支架(其中软骨源性基质微粒的质量浓度分别为0,5,10,20,30 g/L),通过力学测试确定力学性能最佳的软骨源性基质微粒质量浓度用于细胞实验;(2)将兔骨髓间充质干细胞接种至Gel MA/CDMPs复合水凝胶支架中,置于成软骨培养基中共培养(实验组);将兔骨髓间充质干细胞接种至甲基丙烯酸酐改性明胶水凝胶支架中,置于成软骨培养基共培养(对照组),通过细胞死活染色和CCK-8细胞增殖实验评估两组支架内的细胞活性及细胞增殖能力;通过细胞骨架荧光染色观察支架内细胞的形态和伸展扩散能力;通过组织学染色和糖胺多糖定量评估支架促进细胞分泌软骨基质的能力。结果与结论:(1)力学测试表明,Gel MA/CDMPs 10 g/L复合水凝胶支架的压缩模量高于其他组(P<0.05),后续实验选择该质量浓度;(2)活死染色显示,两组支架内培养1,5 d的细胞活性均在90%以上;CCK-8细胞增殖实验显示,实验组培养3,5 d的细胞增殖快于对照组(P<0.05);(3)培养7 d的细胞骨架荧光染色显示,对照组的细胞为球形形态,只有少部分细胞形成丝状伪足,实验组的细胞呈伸展的不规则多边形;(4)培养21 d的组织学与免疫组化染色显示,两组支架内的细胞形态与软骨细胞相似,实验组可见更多的软骨基质分泌;(5)实验组培养7,21 d的糖胺多糖含量高于对照组(P<0.05);(6)结果表明,Gel MA/CDMPs复合水凝胶支架可更好地模拟软骨细胞生长微环境,进而促进骨髓间充质干细胞的生长、增殖和成软骨分化。

BACKGROUND: Articular cartilage regeneration and repair is still a challenging medical problem. The injectable hydrogel scaffold that constructs a biomimetic microenvironment for cell growth is of great significance for the regeneration of articular cartilage. OBJECTIVE: To prepare injectable gelatin-methacryloyl/cartilage-derived matrix particles(GelMA/CDMPs) composite hydrogel scaffold, and evaluate its biocompatibility.METHODS:(1) CDMPs were prepared by physical pulverization and chemical enzymatic methods, and GelMA was prepared by the reaction of methacrylic anhydride grafted gelatin. The two were used as raw materials to prepare five different GelMA/CDMP composite hydrogel scaffolds(where the CDMP mass concentrations were 0, 5, 10, 20, and 30 g/L), and the mass concentration of CDMPs with the best mechanical properties was determined through mechanical tests.(2) Rabbit bone marrow mesenchymal stem cells were encapsulated in the GelMA/CDMPs composite hydrogel scaffolds(as the experimental group);bone marrow mesenchymal stem cells were encapsulated in GelMA hydrogel scaffolds(as the control group);both of which were cultured in the chondrogenic medium. The cell viability and cell proliferation of the two groups of stents were evaluated through cell live/dead staining and CCK-8 cell proliferation experiments. The morphology and spreading ability of cells in the scaffolds were observed by cytoskeleton fluorescence staining. The secretion of cartilage matrix of bone marrow mesenchymal stem cells in the scaffolds was evaluated by histological staining and quantitative glycosaminoglycan test. RESULTS AND CONCLUSION:(1) Mechanical tests showed that compressive modulus of GelMA/CDMPs 10 g/L composite hydrogel scaffolds was significantly higher than that of other groups(P < 0.05). And this concentration was selected for the subsequent experiments.(2) The cell live/dead staining demonstrated that cell viability of bone marrow mesenchymal stem cells in the two groups of scaffolds was above 90% after 1 and 5 days of culture. After 3-and 5-day culture, the cell proliferation of the experimental group was faster than that of the control group(P < 0.05).(3) Fluorescence staining of the cytoskeleton after culture for 7 days showed that the cell morphology in the control group was spherical;and only a small part of the cells formed filopodia. The cell morphology in the experimental group was stretched and showed irregular polygons.(4) After 21 days of culture, histological and immunohistochemical staining showed that the cell morphology of bone marrow mesenchymal stem cells in the two groups of scaffolds was similar to chondrocytes morphology;and more cartilage matrix was secreted by bone marrow mesenchymal stem cells in the experimental group.(5) After 7-and 21-day culture, the content of glycosaminoglycan was significantly higher in the experimental group than that in the control group(P < 0.05).(6) These results suggest that GelMA/CDMPs composite hydrogel scaffold can better mimic the microenvironment of chondrocyte growth, and then promote the growth, proliferation and chondrogenesis of bone marrow mesenchymal stem cells.