目的·利用软骨脱细胞基质（acellular cartilage matrix，ACM）与复合天然丝素蛋白（silk fibroin，SF）生物材料，构建具有双网络交联的生物活性组织工程支架，用于软骨组织再生。方法·用核酸酶消化法消化掉软骨组织中细胞相关的免疫原性成分，并将细胞外基质相关糖蛋白、胶原结构保留，用DNA、组织糖胺多糖、胶原定量试剂盒通过分光光度仪检测软骨组织脱细胞效率。将ACM和SF配置成混合溶液，通过加入乙二醇二缩水甘油醚与两者所含羟基、氨基发生亲核交联反应，冷冻干燥制成多孔仿生支架（n=5），同时以相同方法制备仅含有ACM或SF的多孔支架（n=5）。扫描电子显微镜（scanning electron microscope，SEM）观察支架微观孔隙结构，并通过力学测试对不同组支架的力学强度、弹性模量以及回弹性进行评估，以吸水率反映支架的内外物质交换能力。分离并培养兔耳软骨细胞，接种于ACM-SF支架上，SEM观察培养1、4、7 d后细胞在支架上的黏附、分布与基质分泌情况，活/死细胞双染色观察细胞活力状况，通过CCK-8检测支架的细胞毒性，并于裸鼠皮下植入细胞支架复合物，体内培养4周、8周后，行组织学检测。各组间比较采用单因素方差分析。P<0.05为差异具有统计学意义。结果·经酶消化后的软骨脱细胞基质几乎无细胞残留，并保留了软骨细胞外基质活性成分。ACM-SF制备的复合支架具有相互连通的微孔结构和良好的弹性，湿态下多次压缩后能恢复原状，ACM-SF的吸水率达到了近20倍，为细胞黏附环境提供了有效的物质交换条件。此外，该支架无生物毒性，具有促进软骨细胞增殖的能力；组织学检测显示，ACM-SF支架可在体内再生均质、典型的软骨组织。结论·ACM-SF复合多孔支架具有良好的仿生微环境，可应用于组织工程软骨再生。

Objective ·To construct a bioactivity tissue engineering scaffold with double network cross-linking for cartilage tissue regeneration using an acellular cartilage matrix (ACM) with a natural silk fibroin (SF) biomaterial. Methods ·The cell-associated immunogenic components were removed by nuclease digestion, and the extracellular matrix-associated glycoproteins and collagen structures were retained, The efficiency of cartilage tissue decellularization was measured by spectrophotometry by using DNA, histoglycosaminoglycan and collagen quantification kits. ACM and SF were configured into a mixed solution, and the nucleophilic cross-linking reaction with the hydroxyl and carboxyl groups contained in both was carried out by adding ethylene glycol diglycidyl ether. Then it was freeze-dried to make porous bionic scaffolds (n=5). At the same time, porous scaffolds containing only ACM or SF were prepared by the same method (n=5). The microstructure of the scaffolds was observed by scanning electron microscopy (SEM), and the mechanical strength, elastic modulus and resilience of different groups of scaffolds were evaluated by mechanical tests. The internal and external nutrient exchange capacity of the scaffolds was reacted by water absorption rate. Chondrocytes from rabbit ears were isolated, cultured, and seeded on ACM-SF scaffolds. After 1, 4, and 7 days of culture, the adhesion, distribution, and matrix secretion of the cells on the scaffolds were observed by SEM, and the viability status of the cells was determined by double-staining of live and dead cells. CCK-8 method was used to determine the cytotoxicity of the scaffolds. The cells were implanted subcutaneously in nude mice, cultured in vivo for 4 and 8 weeks, and finally removed for histological testing. Differences between groups were tested by One-Way ANOVA. Statistical significance was accepted at a value of P<0.05. Results ·After enzymatic digestion, almost no cells remained in the acellular matrix, and the active components of the extracellular matrix were retained. The composite scaffold prepared by ACM-SF has interconnected microporous structure and good elasticity, and could recover its original shape after repeated compression in the wet state. The water absorption rate of ACM-SF reached nearly 20 times, which provided an effective material exchange condition for the cell adhesion environment. Histological tests showed that the ACM-SF scaffold regenerated homogeneous, typical cartilage tissue in vivo. Conclusion ·ACM-SF composite porous scaffold has a good bionic microenvironment and can be applied to tissue engineering cartilage regeneration.