软骨细胞三维培养扩增与液态凝胶复合构建软骨的实验研究

目的通过在微载体上进行三维培养扩增软骨细胞，并结合液态胶原构建组织工程软骨。方法比较兔软骨细胞在单层培养与微载体上进行三维培养扩增软骨细胞的保持表型能力。幼兔软骨细胞分别进行单层和微载体三维培养扩增，并进行体外球型培养评价软骨细胞保持表型能力和糖胺多糖的定量生化分析。三维培养扩增软骨细胞与液态鼠尾胶原复合构建组织工程软骨，分别以低细胞密度（2×10^6个／mL）和高细胞密度（1．2×10^7个／mL）两种细胞密度接种，培养14d后通过组织学特种染色鉴定构建组织特性。结果微载体培养的软骨细胞可以保持良好活力和保持表型能力，与单层培养体系相比较，细胞糖胺多糖的定量生化分析的差异具有统计学意义（P〈0．05）。三维培养扩增软骨细胞复合液态鼠尾胶原构建组织工程软骨，体外14d后发现高细胞密度接种时可形成形态稳定的软骨组织。组织学染色显示为透明软骨样组织。结论在微载体上进行三维培养扩增软骨细胞可以加强细胞保持表型能力。软骨细胞与液态胶原合成后，以高细胞密度（1．2×10^7个／mL）可以在体外形成形态稳定的组织工程软骨。

Chondrocytes Expansion in Three-dimensional Environment and Engineering Cartilage with Liquid Collagen

Objective This study focuses on rabbit articular chondrocytes expansion on three-dimensional environment microcarriers and engineering cartilage with liquid collagen. Methods Compare the post-expansion phenotype of cartilage chondrocytes expanded in monolayer and on eytodex-3 microearrier cultures. The effect of expansion were assessed via cell viability analysis,yielded doubling times ,SEM observation,immumolocalization of collagen type it . Furthermore ,redifferentiation was evaluated in vitro via pellet cultures,which were observed by biochemical analysis in quantification of glycosaminoglcans （GAG）. Engineering a cartilage in vitro via chondrocytes mixed with liquid collagen. Chondrocytes were expansed on microcarriers in three-dimensional environment The constructions were detected after 14d culture period,their paraffin sections were stained by toluidine blue. Results Chondrocytes remained viable and ability of redifferentiation during microearrier culture ,yielded doubing times （2.77±0. 267） d ,the quantification of glycosaminoglcans （30. 417±1. 116 ug proteoglycan/mg sample） comparable to monolayer culture （3.10±1.92） d and （45. 122±1. 239 ug proteoglyean/mg sample）. Conclusion Expansion of ehondrocytes on microcarriers culture systems can enhanced redifferentiation,and the mixture of ehondroctes and collagen can formed stable shaped tissue engineering cartilage in vitro.