脱细胞半月板细胞外基质/脱钙骨基质双相半月板支架的制备及其生物相容性的研究

Preparation and biocompatibility research on the decellularized meniscal extracellular matrix/demineralized bone matrix diphasic meniscal scaffold

目的成功制备脱细胞半月板细胞外基质/脱钙骨基质双相半月板支架,并与半月板纤维软骨细胞结合研究其生物相容性。方法联合利用湿法粉碎、差速离心等物理方法和胃蛋白酶等化学方法制备脱细胞半月板细胞外基质,利用改良Urist法制备脱钙骨基质,并利用灌注、冷冻干燥等方法分别制备脱细胞半月板细胞外基质支架、脱钙骨基质支架以及脱细胞半月板细胞外基质/脱钙骨基质双相支架等三种不同的支架,并从组织学、分子生物学、生物力学等方面研究三种支架的异同;原代培养兔半月板纤维软骨细胞,并将P3代的纤维软骨细胞分别种植在以上三种支架上,利用扫描电镜、死/活细胞染色等观察细胞生长情况,并分别在3、7、14 d时检测纤维软骨细胞的增殖情况以及分泌胶原和糖胺多糖的含量。结果物理化学联合法脱细胞可以去除半月板中绝大部分的细胞成分,并且很好地保留正常半月板细胞外基质的胶原以及糖胺多糖成分;脱细胞半月板基质支架、脱钙骨基质支架以及脱细胞半月板细胞外基质/脱钙骨基质双相支架三种支架均具有良好的孔隙率,合适的孔径大小,扫描电镜结果显示纤维软骨细胞可以很好地在支架上生长,死/活细胞染色结果显示三种支架均可以维持良好的细胞活性,但是脱细胞半月板细胞外基质/脱钙骨基质双相支架具有更好的生物力学特性,脱细胞半月板基质支架和脱钙骨基质/脱细胞半月板基质双相支架在促进纤维软骨细胞增殖和维持细胞表型方面要比单纯脱钙骨基质支架更优。结论脱细胞半月板细胞外基质/脱钙骨基质双相支架具有较好的生物力学特性,良好的生物相容性,可以促进纤维软骨细胞增殖,同时也可以维持纤维软骨细胞的表型,是一种可以应用于组织工程半月板再生的支架。

Objective To manufacture a decellularized meniscal extracellular malrix/demineralized bone matrix diphasic meniscal scaffold and then integrate it with fibrochondrocytes to investigate the biocompatibility of the materials. Methods The decellularized meniscal extracellular matrix was prepared by physical methods such as the waterproof pulverization and differential centrifugation, as well as some chemical approaches like the pepsin digestion. The demineralized bone matrix was prepareded by the improved Urist method. We utilized infusing and lyophilization methods to construct the decellularized meniscal extracellular matrix scaffold （DMECMS）, demineralized bone matrix scaffold （DBMS）, and decellularized meniscal extracellular matrix/demineralized bone matrix diphasic scaffold （DMECMS/DBMS）, respectively, and then examined the histology, molecular biology, and biomechanical characters of the three different scaffolds. The rabbit meniscus fibrochondrocytes of passage 3 were seeded in the three different scaffolds, and then evaluated the biocompatibility by scanning electron microscopy （SEM） and Live/Dead staining. The collagen and GAGs secreted by fibrochondrocytes on the three scaffolds were detected on the 3rd, 7th, and 14th day. Results The physico-chemical decellularization method eliminated most of the cellular elements, and preserved the collagen and GAGs components of the meniscal extracellular matrix. The DMECMS, DBMS and DMECMS/DBMS had appropriate porosity and pore size. The SEM and Live/Dead staining results showed that the fibrochondrocytes could grow in the three different scaffolds and maintain good viability. However, the DMECMS/DBMS had the best biomeehanics property, and the DMECMS and the DMECMS/DBMS promoted the fibrochondrocytes proliferation and collagen and GAGs secretion. Conclusion The DMECMS/DBMS has good biomechanics property and biocompatibility, and can promote fibrochondrocytes proliferation and collagen and GAGs secretion. In conclusion, it is a good scaffold for meniseal tissue engineering.