不同种植密度对大鼠脂肪间充质干细胞三维培养下成软骨能力的影响

背景:软骨组织工程要求植入的软骨细胞在三维支架材料中能够合成与软骨相同的软骨基质,而植入密度是成功的关键点之一.目的:探讨壳聚糖一胶原一硫酸软骨素支架上不同种植密度对大鼠脂肪间充质干细胞成软骨能力的影响.设计、时间及地点:细胞一支架学体外观察,于2007-11/2008-07在中国医科大学细胞生物实验室完成.材料:清洁级雄性SD大鼠6只,由中国医科大学实验动物中心提供.方法:室温下以乙酸为溶剂的5 g/L Ⅰ型胶原溶液与20 g/L壳聚糖按7:3体积比置于预冷的模具中混合,冷冻干燥后将支架切成5 mm×5 mm×2 mm,浸入含20 g/L硫酸软骨素的乙醇中室温交联,双蒸水冲洗至中性,再次冷冻干燥即为壳聚糖-胶原-硫酸软骨素支架.切取大鼠腹股沟脂肪组织,通过胰蛋白酶和Ⅰ型胶原酶消化后得到脂肪间充质干细胞.将制备的壳聚糖-胶原-硫酸软骨素支架分为3组,分别调整第3代细胞密度为2×109L-1,2×1010L-1,2×1011L-1,吸取细胞悬液50 μL均匀的种植于每个支架上,加入成软骨诱导培养基培养3周.主要观察指标:取样制成切片进行苏木精-伊红染色、Ⅱ型胶原免疫组化染色,RT-PCR检测软骨特异性基因的表达.结果:诱导培养3周后,各组细胞在支架中生长黏附良好,且高种植密度2×1011L-1组细胞排列紧密.有较多的基质形成,并有软骨陷窝样结构:各组Ⅱ型胶原均呈阳性表达,并随细胞种植密度的升高呈递增趋势.RT-PCR结果显示.随着细胞种植密度的升高,蛋白聚糖、Ⅱ型胶原mRNA的表达逐渐增强,而Ⅹ型胶原mRNA的表达逐渐下降.结论:壳聚糖-胶原-硫酸软骨素复合支架材料可为脂肪间充质干细胞生长分化及组织形成提供一个良好的环境,2×1011L-1高密度种植有利于脂肪间充质干细胞的成软骨分化.

Effect of different cell seeding concentrations on chondrogenic differentiation of adipose derived sromal cells in three-dimensional culture

BACKGROUND: The implanted cartilage calls can synthesize cartilage matrix as cartilage in cartilage tissue enginsedng, and the density of implanted cells is the key point.OBJECTIVE: To evaluate the effect of cell seeding concentration on the chondrogenic differentiation of the adipose dadved sromal cells (ADSCs).DESIGN, TIME AND SETTING: The in vitro cellular-scaffold observation was performed at the cytobiological laboratory of China Medical University from November 2007 to July 2008.MATERIALS: Six male SD rata with clean grade were supplied by the Experimental Animal Center of China Medical University.METHODS: Totally 5 g/L type ; collagen solution and 20 g/L chitosan was mixed in a mould with volume ratio of 7:3, after lyophillization, it was cut into pieces with 5 mm ～ 5 mm x 2 mm, followed by crosslinking with ethanol contained of 2% chondroitic acid at room temperature. After washing with double distilled water and freeze drying, the chitosan-collagen-chondroitin sulfate copolymar matrices scaffolds were harvested. ADSCs isolated from rat inguinal fat pads were digested with collagenase and trypsase. The prepared scaffolds were randomly divided into 3 groups, and the third passage cells with density of 2×10 9/L,2×10 109/L, and 2×10 11/L were seeded into chitosan-coflagen-chondroitin sulfate scaffolds, and cultured in chondrogenic medium for 3 weeks.MAIN OUTCOME MEASURES: The expression of cartilage specificity gene was detected by hematoxylin-eosin staining, type Ⅱ collagen immunohistochemical staining and RT-PCR.RESULTS: Hematoxylin-eosin staining showed that after 3 weeks of culture, the cell proliferated and differentiated well, especially in 2x101～/L group, more extrocelluer matrices were produced and cartilage lacuna-structure could be seen. The type Ⅱ collagen was positive expressed in each group, which showed a gradually increasing tendency with the cell seeding concentration increasing. RT-PCR showed that the expression of proteoglycen and type Ⅱ collagen mRNA were slowly increased. However the expression of Ⅹ collagen mRNA was decreased with increasing cell seeding concentration.CONCLUSION: The chitosan-collagen-chondroitin sulfate copolymer matrices can provide an appropdate environment for the generation of cartilage-like tissues and high call seeding concentration of 2×1010/L facilitate ADSCs to differentiate into cartilage.