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组织工程软骨的体外构建被认为是一种有希望治疗关节软骨缺损的有效途径.为评估载脂肪干细胞(Adipose-derived stem cells,ADSCs)壳聚糖/明胶水凝胶支架,在体外动态构建组织工程软骨相对传统静态培养的优势,本研究用壳聚糖/明胶制备了软骨仿生支架,并检测其物理性质.在制备的水凝胶支架上以1×107 cells/mL 密度接种 ADSCs 后,分别置于转瓶及 T-瓶的软骨诱导基中培养两周,通过试剂染色、代谢检测和电镜观察,考察了细胞的软骨分化能力、活性、生长分布、渗透深度、增殖及胞外基质分泌情况.结果表明,壳聚糖/明胶支架的平均孔径为118．25±19．51μm,孔隙率为82．60±2．34％,吸水率为361．28±0．47％,弹性模量为61．2±0．16 kPa,具有良好生物相容性.ADSCs 生长状态良好,可向软骨细胞分化,适于作为组织工程软骨构建的种子细胞.表征结果显示,转瓶内水凝胶支架中细胞蛋白多糖的表达更显著,细胞生长分布更加均匀,细胞外基质分泌基本填满整个支架.因此,转瓶载壳聚糖/明胶支架所提供的三维动态环境,是体外构建组织工程软骨的良好方法.

It was considered to be promising in treating articular cartilage defect by fabricating tissue engineered cartilage in vitro.To assess the advantages of chitosan/gelatin hydrogel scaffold seeded with ADSCs to construct tissue engineered cartilage dynamically in vitro in contrast to traditional static culture,chitosan/gelatin hybrid was chosen as cartilage biomimetic scaffold and its physical properties were subsequently tested.After that,adi-pose-derived stem cells (ADSCs)were inoculated into the chitosan/gelatin hydrogel scaffold at density of 1 × 10 7 cells/mL and cultured in a spinner flask and T-flask with chondroinductive media for two weeks,respective-ly.Chondrogenic differentiation ability of ADSCs within hydrogel scaffold was investigated with Toluidine Blue and Safranine O staining,the cell metabolism of glucose and lactic acid was analyzed through detecting kits, while the cell distribution,adhesion and extracellular matrix secretion within scaffold were observed by Dead/Live staining and scanning electron microscopy (SEM),respectively.The results showed that the average pore size,porosity,swelling rate and elasticity modulus of chitosan/gelatin hydrogel scaffold with good biocompati-bility were 1 18.25±1 9.5 1 μm,82.60±2.34%,361.28±0.47%,and 61.2±0.1 6 kPa,respectively.The ADSCs, in good conditions,could differentiate into chondrocytes,thus were suitable as seeding cells in tissue engineered cartilage construction.In addition,it was confirmed that the induced cells could express more proteoglycans in the dynamic spinner flask,where the cell distribution within the scaffold was more uniform and the scaffold could be filled mostly by extracellular matrix.The spinner flask with immobilized scaffold enhanced prolifera-tion and chondrogenic differentiation of ADSCs within chitosan/gelatin hydrogel scaffolds,thus accelerated the dynamic fabrication of cell-hydrogel constructs,which could be an excellent method to construct tissue engi-neered cartilage in vitro.