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目的:探讨应用壳聚糖支架三维立体培养人骨髓基质干细胞(mesenchymalstemcells,MSCs),体外诱导其分化成为软骨细胞的可行性。方法:实验于2004-02/11在中山大学附属第三医院中心实验室完成。相分离法制备三维多孔壳聚糖支架,检测支架的孔隙率、孔径。抽取人骨髓,密度梯度离心法分离纯化,体外培养扩增。将第3代MSCs复合于支架中培养,应用无血清诱导液诱导细胞向软骨细胞分化,通过组织学染色及扫描电镜观察细胞的形态、增殖及功能的改变。结果:通过相分离法可制备出高孔隙率的三维壳聚糖支架,孔隙率达86.5%,孔隙分布均匀且相互连通,平均孔径182μm。诱导分化的MSCs在支架中贴附良好,呈现典型的软骨细胞形态,并有细胞外基质分泌。结论:人MSCs在三维立体培养环境下可诱导分化为软骨细胞,作为种子细胞在组织工程软骨的构建及软骨损伤的修复中有良好的应用价值。
OBJECTIVE: To investigate the feasibility of using chitosan scaffolds for three-dimensional culture of mesenchymal stem cells (MSCs) to induce their differentiation into chondrocytes in vitro. Methods: The experiment was performed at the Central Laboratory of the Third Affiliated Hospital of Sun Yat-sen University from February to February 2004. Three-dimensional porous chitosan scaffolds were prepared by phase separation method. The porosity and pore size of the scaffolds were measured. Human bone marrow was harvested, purified by density gradient centrifugation and cultured in vitro. The 3rd generation MSCs were cultured in the scaffold, and the cells were induced to differentiate into chondrocytes by using serum-free induction liquid. The morphology, proliferation and function of the cells were observed by histological staining and scanning electron microscopy. Results: The three-dimensional chitosan scaffold with high porosity could be prepared by phase separation method. The porosity was 86.5%. The pores were evenly distributed and communicated with the average pore size of 182 μm. Differentiated MSCs adhere well to the scaffolds, showing typical chondrocyte morphology with extracellular matrix secretion. CONCLUSION: Human MSCs can differentiate into chondrocytes under the condition of three-dimensional culture. As the seed cells, they have good application value in the construction of tissue engineering cartilage and the repair of cartilage damage.