柱状分层的胶原-羟基磷灰石复合支架负载软骨细胞体外培养

目的 观察具有柱状分层结构的胶原-羟基磷灰石（hydroxyapatite，HA）复合支架对软骨细胞的吸附作用及对软骨细胞生物学性状的影响，评价其作为软骨组织工程支架的可行性及价值。方法 以胶原复合HA逐层制备胶原-HA复合支架，体外培养新西兰大白兔关节软骨细胞并扩增，吸附于多孔胶原-HA复合支架材料上进行三维立体培养3周，通过倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测支架对软骨细胞的表型、增殖及功能的影响。结果 胶原-HA复合支架为柱状，支架表层为胶原。平均孔径为147μm，孔隙率89％；中层为多孔胶原-HA复合；底层为HA，平均孔径为85μm，孔隙率85％。胶原-HA复合支架亲水性好，软骨细胞吸附于支架表面24h后，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，分泌细胞外基质，Ⅱ型胶原免疫组织化学染色阳性。结论 具有柱状分层结构的胶原-HA复合支架其细胞相容性良好，较胶原纤维具有更强的力学性能，有望成为一种比较理想的软骨组织工程支架材料。

CULTURE OF CHONDROCYTES USING COLLAGEN-HYDROXYAPATITE COMPOSITE SCAFFOLDS IN VITRO

OBJECTIVE: To evaluate the feasibility and the value of the layered cylindric collagen-hydroxyapatite composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affects the cell behaviors. METHODS: The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen/hydroxyapatite composite scaffold and were cultured in a three-dimensional environment for 3 weeks. The effects of the composite scaffold on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by the phase-contrast microscopy, histology, scanning electron microscopy, and immunohistochemistry. RESULTS: The pore diameter of the upper layer of the collagen-hydroxyapatite composite scaffold was about 147 microm, and the porosity was 89%; the pore diameter of the bottom layer was about 85 microm and the porosity was 85%. The layered cylindric collagen-hydroxyapatite composite scaffold had good hydrophilia. The chondrocytes that adhered to the surface of the scaffold, proliferated and migrated into the scaffold after 24 hours. The chondrocytes attached to the wall of the microholes of the scaffold maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold. CONCLUSION: The layered cylindric collagen-hydroxyapatite composite scaffold has a good cellular compatibility, and it is stronger in the mechanical property than the pure collagen. It will be an ideal scaffold for the cartilage tissue engineering.