牛关节软骨细胞外基质材料的制备

Preparation of Extracellular Matrix Material of Bovine Articular Cartilage

目的探讨将关节软骨粉碎颗粒后再溶解脱细胞的复合方式制备关节软骨细胞外基质(articular cartilage extracellular matrix,ACECM)的可行性,以提供理想的软骨组织工程支架材料。方法无菌条件下,切取分离出牛关节软骨,液氮冷冻后立即粉碎,400目筛网过滤后得到直径<150μm的软骨颗粒;应用胰酶-EDTA溶液对其进行消化,去除细胞成分后冲洗,梯度冻存后对脱细胞软骨颗粒冷冻干燥,制备为ACECM。对制备的ACECM行大体观察和组织学检测,并利用扫描电镜(SEM)观察基质材料微观结构;将分离培养的兔软骨细胞接种于ACECM,SEM观察细胞生长粘附状态,MTT法检测细胞的增殖活性。结果制备的ACECM材料主要含有胶原及氨基葡聚糖等细胞外基质成分,无细胞成分残留,基质材料微观孔隙结构有利于接种的软骨细胞粘附和生长。结论通过颗粒脱细胞方式获取的软骨基质含有天然ACECM成分,是一种体外培养软骨细胞的良好支架。

Objective The feasibility of preparing articular cartilage extracellular matrix（ACECM） by complexing cartilage granules and re-dissolving and decellularizing the cartilage is discussed to provide an ideal scaffold for cartilage tissue engineering. Methods Under aseptic conditions, the cartilage of bovine articular cartilage was harvested and separated. Immediately after freezing in liquid nitrogen,cartilage particles with a diameter of less than 150 μm were obtained by filtration through a 400-mesh sieve. The cartilage particles were digested with trypsin-EDTA solution, and the cellular components were removed and washed. The acellular cartilage particles were freeze-dried after being frozen and prepared as ACECM. Gross observation and histological examination were performed on the prepared ACECM, and the microstructure of the matrix material was observed by scanning electron microscopy（SEM）; rabbit chondro-cytes isolated and cultured were inoculated into ACECM; SEM was used to observe the cell growth and adhesion status; MTT assay was used to detect cell proliferation activity. Results The prepared ACECM material mainly contains extracellular matrix components such as collagen and aminodextran. No cell components remain, and the microscopic pore structure of the matrix material is favorable for the adhesion and growth of inoculated chondrocytes. Conclusion The cartilage matrix obtained by particle decellularization contains natural ACECM components and is a good scaffold for the culture of chondrocytes in vitro.