胶原复合梯度磷酸三钙负载软骨细胞的体外培养

目的 观察新型三维支架材料胶原复合梯度磷酸三钙在体外与软骨细胞的相容性和黏附性,评价其作为软骨组织工程支架的可行性.方法 取8周龄新两兰大白兔膝关节软骨,以酶消化法获得高纯度软骨细胞,培养3代后与三维支架材料胶原复合梯度磷酸三钙在体外复合培养.用倒置相差显微镜、HE染色、免疫组织化学及扫描电镜观察软骨细胞形态、Ⅱ型胶原表达及成软骨能力,同时观察支架材料与软骨细胞的相容性.结果 扫描电镜观察显示支架材料具有疏松多孔结构,孔隙结构规则,孔径100～150 μm,材料内部孔与孔之间贯通良好.支架亲水性好.软骨细胞吸附于支架表面,增殖并逐渐顺孔隙迁徙至支架内部,在孔壁贴附良好,表型维持稳定,可分泌细胞外基质.结论 胶原复合梯度磷酸三钙三维支架具有良好的细胞相容性.

Collagen complex gradient tricalcium phosphate composite scaffold with chondrocytes cultured in vitro

Objective To evaluate the feasibility of collagen complex gradient tricalcium phosphate composite scaffolds for cartilage tissue engineering by observing the compatibility and adhesion of chondrocytes. Methods High purified chondrocytes from knee joints of 8-week-old New Zealand rabbits were obtained by enzyme digestion, then the chondrocytes of the third generation were cultured combined with collagen complex gradient tricalcium phosphate composite scaffolds in vitro.Chondrocyte morphology, collagen Ⅱ expression, chondrogenesis, the compatibility of chondroeyte and scaffolds were evaluated with inverted microscope, HE staining, scanning electron microscope (SEM) and immunohistochemistry.Results By SEM, the scaffold had regular, porous architecture of communicated microholes.The average pore diameter was 100-150 μm.Observation revealed good hydrophilicity of the composite scaffold.The chondrocytes adhered to the surface, proliferated and migrated into the inside of the scaffold.Chondrocytes attached to wall of microholes of the scaffold had largely maintained morphology and could secrete the extracellular matrix on the porous scaffold.Conclusions Collagen complex gradient tricalcium phosphate composite scaffold possesses good cellular compatibility.