组织工程椎间盘的构建及体外观察

目的探讨构建组织工程椎间盘的可行性,修复退变椎间盘。方法应用热致分相法制备聚乳酸-羟基乙酸三维多孔支架,酶消化法体外分离培养20周龄意外流产胎儿椎间盘细胞,PKH-26荧光标记后接种于支架,体外培养48h,通过MTT摄取、激光共聚焦荧光显微镜及扫描电镜方法进行体外观察。结果原代细胞形态多为卵圆形或三角形,中央有圆形核,胞质向外伸出多个长短不同的突起。多孔支架超微结构显示孔隙由中心向外呈放射状分布,形态、取向规则有序,孔隙直径50～300μm。细胞-支架复合体加入MTT溶液2h后呈深蓝着色,激光共聚焦荧光显微观察可见大量红色荧光着色的细胞贴附于支架结构内。结论应用组织工程技术构建的细胞-支架复合体具备理想的三维微观结构及具有生命力的功能细胞,有可能在椎间盘再生研究中发挥重要作用。

IN VITRO STUDIES ON TISSUE ENGINEERED INTERVERTEBRAL DISC

OBJECTIVE: To investigate the feasibility of tissue engineered intervertebral disc for regeneration of discs. METHODS: A three-dimensional porous poly (L-lactic-co-glycolic acid) (PLGA) scaffold was fabricated by temperature induced phase separation method. Human fetal disc cells were isolated and cultured in vitro. The disc cells labeled with a PKH-26 fluorescent dye were seeded into a three-dimensional porous scaffold. The proliferation of disc cells with PKH-26 fluorescent labels was assessed by using MTT uptake, laser fluorescence microscopy and SEM. RESULTS: Human fetal disc cells displayed a polygonal shape in primary monolayer culture. A regular arrangement and microtubules orientation-structure scaffold with 50-300 microm in diameter was fabricated by thermal-induced phase separation technique. MTT uptake and fluorescent microscopy examination indicated that the seeded disc cells were viable and showed proliferation activity within a porous scaffold. CONCLUSION: The above findings support potential applications of tissue engineered disc in treatment of disc degenerative diseases.