目的探究大鼠尾腱细胞外基质制备方法及其复合脂肪间充质干细胞构建微组织,为组织工程法修复肌腱损伤所需生物材料提供实验基础。方法取20只150~200 g SD大鼠鼠尾作为实验材料,分别抽取出大鼠尾腱,经PBS彻底清洗后反复冻融3次,然后经过1...目的探究大鼠尾腱细胞外基质制备方法及其复合脂肪间充质干细胞构建微组织,为组织工程法修复肌腱损伤所需生物材料提供实验基础。方法取20只150~200 g SD大鼠鼠尾作为实验材料,分别抽取出大鼠尾腱,经PBS彻底清洗后反复冻融3次,然后经过1%SDS于室温下处理24 h,并用大量PBS清洗1周除去细胞残渣,最后^(60)Co消毒备用。对制备的大鼠尾腱细胞外基质进行病理学染色,观察细胞残留情况。同时,检测该来源的生物源性材料残留α-Gal含量。此外,观察制备的大鼠尾腱细胞外基质复合脂肪间充质干细胞对细胞增殖的影响。同时,用CCK-8试剂盒检测该生物材料的细胞毒性。结果大体观察可见,经脱细胞处理过的尾腱体积增大,呈乳白色匀浆状。HE以及DAPI染色结果显示,经脱细胞处理后的尾腱细胞核大量减少,细胞数量显著降低,每个高倍镜视野内细胞残余量不大于5个。脱细胞尾腱残留α-Gal含量与正常尾腱相比有统计学差异,脱细胞尾腱残留DNA极少。复合脂肪间充质干细胞培养7 d相比培养1 d活细胞数量明显增多。CCK-8毒性测试结果显示这种脱细胞大鼠尾腱的生物相容性较好,没有细胞毒性。结论经过脱细胞方法制备的大鼠尾腱细胞外基质可有效除去细胞成分,并且与脂肪间充质干细胞有较好的生物相容性,可作为一种生物材料用于肌腱损伤修复。展开更多
Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derive...Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and translucent cartilage-like tissue after 3 weeks of culture.We observed evenly distributed cartilage ECM proteoglycans and collagen type Ⅱ around seeded BMSCs on the surface and inside the pores throughout the scaffold.Conclusion This study stuggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.展开更多
基金This study was funded by the National Natural Science Foundation of China （Nos. 31000432, 30930092 and 81272046） and National Technology Research and Development Program of China （No. 2012AA020502, 2012CB518106）.Acknowledgments： We thank HUANG Jing-xiang, T1AN Yue, and SUI Xiang for kind assistance in cell culture and histology.
文摘Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and translucent cartilage-like tissue after 3 weeks of culture.We observed evenly distributed cartilage ECM proteoglycans and collagen type Ⅱ around seeded BMSCs on the surface and inside the pores throughout the scaffold.Conclusion This study stuggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.
