软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究

Fabrication and characterization of a novel cartilage acellular matrix/chitosan hybrid scaffold for cartilage tissue engineering

目的探讨采用壳聚糖与脱细胞软骨基质复合制备组织工程软骨支架的可行性，检测其理化性能和细胞相容性。方法取天然人软骨粉碎，取100nm～5μm软骨微丝，脱细胞处理后制备为质量浓度1％悬液，与质量浓度2％壳聚糖醋酸溶液按1：1（重量比）充分搅拌混合，冷冻干燥制备复合支架。对支架交联，并进行组织学、扫描电镜、孔隙率及吸水性测定、生物力学评估，MTF法分析支架浸提液毒性。分离培养犬软骨细胞，种植到支架上，倒置显微镜、电镜观察细胞在支架的生长、分化情况。结果组织学显示支架中无细胞碎片残留，Ⅱ型胶原免疫组化染色阳性。扫描电镜显示支架内孔洞相互连通似海绵状，孔径为（136．2±34．9）μm，孔隙率为81．4％±3．5％，吸水性约为1525．7±129．3％。支架纵向弹性模量为（1．9400±0.335）MPa。MTT法显示不同浓度支架浸提液与对照培养液吸光度值比较，差异无统计学意义（P〉0．05）。倒置显微镜观察，细胞在支架上粘附良好，扫描电镜下细胞在支架上均匀分布，呈圆形或椭圆形，有基质分泌。结论软骨细胞外基质和壳聚糖复合制备的仿生三维多孔双相支架，具有较高的孔隙率和吸水性，良好的生物力学特性，无毒，生物相容性良好，是组织工程软骨的良好支架载体。

Objective To explore the feasibility of fabricating a novel cartilage acellular matrix/chi- tosan hybrid scaffold for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and made into 1% suspension after decellularization. The sus- pension was mixed with 2% chitosan acetic acid solution, and then hybrid scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-linked and were investigated by histological staining, SEM observation, porosity measurement, water absorption rate, biomechanical properties, and biocompatibili- ty analysis. MTT test was also done to assess the cytotoxicity of scaffold leaching liquor. Canine chondrocytes were isolated and seeded into the scaffold. Cell proliferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed no chondrocyte fragments remained in the scaffolds, and anti-col II immunohistochemistry staining were positive. SEM observation show the scaffold has good pore intereonnectivity with pore diameter （136.2±34.9） μm, 81.4%±3.5% porosity and 1525.7%±129.3% water absorption rate. The longitudinal elastic modulus of the scaffold was （1.940：e0.335） MPa. MTT test showed that the scaffold leaching liquor did not exert any eytotoxie effect on BMSCs. Inverted micro- scope and SEM micrographs indicated that cells covered the scaffolds uniformly, and majority of the cells showed the round or elliptic morphology with much matrix secretion. Conclusion Novel cartilage acellular matrix/chitosan hybrid scaffold had similar extracellular matrix as cartilage, good pore diameter and porosity, appropriate biomechanical character, non-toxicity and good biocompatibility, which make it a suitable candi- date as an alternative cell-carrier for cartilage tissue engineering.