犬半月板脱细胞基质支架的制备及其特性的研究

Preparation and characterization of decellularized scaffold derived from canine meniscus

半月板是膝关节中的基础纤维软骨组织。损伤半月板会损害正常膝关节功能和易患骨关节炎。人和其他动物半月板的脱细胞处理均有文献报道,但犬半月板的脱细胞支架制备鲜有文献报道。本研究采用高低渗反复冻融和1%十二烷基硫酸钠溶液（SDS）脱细胞处理,制备犬半月板脱细胞基质支架。通过HE染色、天狼猩红染色、阿尔新蓝染色和DNA含量测定检测细胞脱除成度。组织学观察和扫描电镜结果显示犬半月板的胶原纤维主要呈线性沿圆周方向排列,鲜有径向排列,细胞陷窝沿纤维束排列。力学性能检测显示,脱细胞基质与天然半月板相比压缩模量有所降低,这与阿尔新蓝染色显示的脱细胞后蛋白多糖丧失具有一定的关系。体外细胞毒性测定结果表明半月板脱细胞支架对犬骨髓间充质干细胞（BMSCs）没有毒性。细胞增殖、扫描电镜观察和活/死细胞染色试验表明犬BMSCs可粘附于支架并且不断增殖。这说明犬半月板脱细胞基质支架可作为构建组织工程半月板的支架。

Meniscus is the basic fibrous cartilage tissue in the knee joint.The injury meniscus can damage normal knee joint function and make the patient sufferring from osteoarthritis.The decellularization of animal and human meniscies has been reported in the literature,but the preparation of decellularized scaffolds for the canine meniscus is rarely reported. METHODS：In this study,the high and low permeability repeating freezing and thawing process,1%（w/w） sodium dodecyl sulfate solution（SDS）being used as a decellularizer and enough washing steps were used to prepare a canine meniscus decellularized scaffold.HE staining,Sirius red staining,Alcian blue staining and DNA content determination were used to detect the degree of cells removal.In result,histology and scanning electron microscopy observation showed that the collagen fibers of the canine meniscus were mainly linearly arranged in the circumferential direction,with little radial arrangement,and cell lacunaes were arranged along the fiber bundles.The mechanical properties test showed that the compressive modulus of the decellularized meniscus was reduced compared to the native meniscus,which was associated with the loss of proteogly-cans after decellularization.In vitro cytotoxicity assay results indicate that the meniscal decellularized scaffold is not toxic to canine bone marrow mesenchymal stem cells（BMSCs）.Further proliferation assays,scanning electron microscopy assays,and live/dead cell staining assays also showed that canine BMSCs can adhere to and proliferate on the surface of the scaffold. In conclusion,the canine meniscus decellularized scaffold can be used as a scaffold to construct tissue engineering meniscus.