软骨细胞微载体（Cytodex-3）平板三维培养扩增的实验研究

目的通过在微载体上进行平板培养扩增软骨细胞，并结合液态壳聚糖构建组织工程软骨。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再分化能力。通过酶解法消化幼兔膝关节软骨后，得到种子细胞，分别进行单层和微载体三维培养扩增。通过评价细胞活性，倍增时间分析培养效果。并进行体外球型培养评价软骨细胞再分化能力，进行了糖胺多糖的定量生化分析。三维培养扩增软骨细胞与壳聚糖复合构建组织工程软骨，培养21天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再分化能力，与单层培养体系相比较，糖胺多糖的定量生化分析（30．417±1．116ugGAG／mg样本）和（45．122±1．239ugGAG／mg样本）的差异具有统计学意义（P〈0．05）。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再分化能力。软骨细胞与壳聚糖合成后，可以在体外形成形态稳定的组织工程软骨。

Three-dimensional chondrocytes culture on flat plate

Objectis This study expanded the rabbit articular chondrocytes on three-dimensional environment microcarriers and established tissue engineering cartilage with chitosan hydrogel. Methods Compare the post - expansion phenotype of cartilage chondrocytes expanded in monolayer and on cytodex-3 microcarrier cultures. The effect of expansion were assessed via cell viability analysis, yielded doubling times.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 chitosan hydrogel. Chondrocytes were expansed on microcarriers in three-dimensional environment The constructions were detected after 21d culture period and characterized by special histological staining. Results Chondrocytes remained viable and ability of redifferentiation during microcarrier culture, yielded doubing times （2.77 ± 0.267d）, the quantification of glycosaminoglcans （30.417 ± 1.116 ug proteoglycan/mg sample） comparable to monolayer culture （3.10 ± 1.92d） and （45.122 ± 1.239ug proteoglycan/mg sample）. Conelusions Expansion of chondrocytes on microcarriers culture systems can enhanced redifferentiation, and the mixture of chondroctes and chitosan hydrogel can formed stable shaped tissue engineering cartilage in vitro.