组织工程化软骨的低温保藏

背景：如何保存大量的具有生物活性的组织工程化软骨，并且长时间保持组织工程化软骨的生物活性，建立组织工程软骨库，是组织工程尚待解决的课题。目的: 观察低温冻存对组织工程化软骨生物活性的影响。设计、时间及地点：随机对照动物实验，于2006-02/2008-12在解放军第四军医大学唐都医院中心实验室完成。材料：聚羟基乙酸无纺网为美国Albany公司产品。2周龄新西兰白兔5只，成年新西兰白兔20只由解放军第四军医大学实验动物中心提供。方法:体外培养2周龄新西兰兔关节软骨，取第2代对数生长期培养软骨细胞，制成细胞悬液，调整软骨细胞悬液浓度约为5×1010 L-1，接种于聚羟基乙酸三维支架材料上，复合物体外培养1周后冻存，冻存4，8，12周后解冻复苏。1周后接种于成年新西兰兔皮下，术后12周取材。主要观察指标：采用光镜及扫描电镜观察冻存复苏后的组织工程化软骨的生长情况。采用四唑盐比色法测定细胞存活率。体内实验观察软骨细胞形态、软骨分泌胶原情况、基质分泌酸性黏多糖情况。结果: 经低温冻存的组织工程化软骨生长良好，冻存4，8，12周细胞存活率差异无显著性意义。组织学观察冻存组织工程骨植入区有软骨生成、胶原和黏多糖生成丰富。结论: 深低温冻存对组织工程化软骨的生物活性无明显的影响，可用于保存组织工程化软骨。

Cryopreservation of tissue-engineered cartilage

BACKGROUND: It is still problems that how to preserve tissue-engineered cartilage with bioactivity, maintain the bioactivity for a long period, and built a bank of tissue-engineered cartilage.OBJECTIVE: To study the effect of cryopreservation on the bioactivity of tissue-engineered cartilage. DESIGN, TIME AND SETTING: A randomized controlled animal study was performed at the Experimental Center of Tangdu Hospital, the Fourth Military Medical University of Chinese PLA from February 2006 to December 2008. MATERIALS: Polyglycolic acid (PGA) was provided by Albany Company, USA; 5 New Zealand white rabbit aging 2 weeks and 20 adult rabbits were provided by the Experimental Animal Center of the Fourth Military Medical University of Chinese PLA.METHODS: Cartilage was collected from 2-week-old rabbits. The cartilage cells in the second logarithmic phase were obtained to make cell suspension which was adjusted to the density of 5×1010/L and then inoculated on the three-dimensional PGA scaffold. The composite was cultured in vitro for 1 week, cooled for 4, 8, and 12 weeks, and then thawed. One week later, the composite was subcutaneously inoculated in the adult rabbits for 12 weeks. MAIN OUTCOME MEASURES: The growth of thawed tissue-engineered cartilage was observed under optic microscope and scanning electron microscope; the survival rate was measured using MTT colorimetry; cartilage morphology, collagen secreted from cartilage, and acid mucopolysaccharide secreted from matrix were detected in vivo. RESULTS: The tissue-engineered cartilage grew well following cryopreservation, and there was no significant difference in survival rate at 4, 8, and 12 weeks after cryopreservation. Histological examination demonstrated that cartilage formed, and collagen and acid mucopolysaccharide were rich in implanted region of thawed tissue-engineered cartilage. CONCLUSION: Cryopreservation does not have any effect on bioactivity of tissue-engineered cartilage, so it can be used to preserve tissue-engineered cartilage.