课题背景：实验为国家自然科学基金课题“大网膜包盖促进血管再生，不同发展程度的组织工程气管原位重建的动物实验研究”的一部分。课题侧重探讨组织工程人工气管植入受体后的早期血管再生机制和不同发展程度的组织工程气管的利弊。

应用要点：实验采用大鼠剑突软骨细胞种植于新型生物材料DegraPol管状支架，在体外静态培养条件下，获得了管状的工程化组织，组织学和力学检测显示DegraPol材料与大鼠剑突软骨细胞有良好的生物相容性，同时工程化组织具备一定力学强度。

同行评价：人工生物气管研究已取得很大进展，远远超出了细胞培养的水平，已有动物模型移植成功的报道，临床也有其他种类人工气管移植成功的个案报道。本文提出了新的获取软骨细胞的方法，并获得成功，为人工生物气管的研究提供了新的方法。

摘要
背景：前期实验已证实人气管软骨细胞在DegraPol片状支架上保持了正常的形态和活力，同时分泌软骨特有基质成分。
目的：以大鼠剑突软骨细胞为种子细胞种植于DegraPol管状泡沫支架，观察新形成的工程化组织在体外静态培养条件下的组织和力学特点。
设计、时间及地点：单一样本观察，于2006-12/2008-02在深圳市人民医院医学研究中心完成。
材料：DegraPol管状材料为瑞士联邦理工大学聚合材料研究所提供；实验动物为2周龄体质量50~60 g的SPF级SD幼鼠。
方法：收集第3代幼鼠剑突软骨细胞接种于DegraPol管状支架，形成软骨细胞-支架复合物，体外静态培养6周。
主要观察指标：① AO/PI荧光染色观察软骨细胞在DegraPol管状泡沫支架中的存活情况，于培养3，6周取出软骨细胞-DegraPol支架复合体，MTT法测定细胞增殖情况。②培养3，6周，应力-应变机械力学方法测定最大应变和应力的变化。③培养3，6周，扫描电镜观察软骨细胞在DegraPol支架中培养后的超微结构。
结果：①AO/PI荧光染色显示软骨细胞在DegraPol支架内保持良好的活性，MTT法结果显示培养6周组A值高于培养3周组(P < 0.05)。②应力-应变机械力学测定结果显示，培养3周组应力值和应变值高于培养6周组 (P < 0.05)。③扫描电镜结果显示DegraPol支架与大鼠剑突软骨细胞有良好的生物相容性，培养6周后获得更多的软骨细胞增殖和更多的细胞外基质。
结论：DegraPol管状泡沫支架有良好的生物相容性并且软骨细胞在支架上保持正常活性，但由于工程化复合物的生物力学强度较差，培养条件和培养方式有待进一步改善。
关键词：组织工程；剑突软骨细胞；管状支架；生物相容性；生物材料

杨林，武延格，纪玲，王正.管状支架体外静态培养组织工程气管软骨[J].中国组织工程研究与临床康复，2008，12(19):3601-3604 [www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3601(ps).pdf]

Static culture of tissue-engineered tracheal cartilage with scaffolds in vitro

Abstract

BACKGROUND:The human cartilage tracheal chondrocytes can retain their normal morphous and activity on the DegraPol scaffold, and simultaneously excrete cartilage specific matrix.
OBJECTIVE: Rat xiphoid chondrocytes were seeded and cultivated on DegraPol scaffolds, to aim directly at the histology and mechanical characteristic of tissue-engineered tracheal cartilage during the static culture in vitro.
DESIGN, TIME AND SETTING: A single sample observation was carried out in the Medical Science Research Center of Shenzhen People's Hospital (Shenzhen, Guangdong, China) from December 2006 to February 2008.
MATERIALS: DegraPol scaffolds were provided by Swiss Federal Institute of Technology (Zurich). SD rats of SPF grade and aged 2 weeks, were provided by the Experimental Animal Center of Guangdong Province (China).
METHODS: The third passage of rat xiphoid chondrocytes were seeded onto DegraPol scaffolds to develop the chondrocyte-scaffold composites, which were cultured for 6 weeks in vitro.
MAIN OUTCOME MEASURES: The survival status of chondrocytes was examined by AO/PI fluorescent stain, and MTT test was used to determine viable cells at weeks 3 and 6 when the chondrocyte-scaffold composites were took out. The stress-strain mechanical force analysis of maximal stress and maximal strain, together with the ultrastructure of the chondrocytes on DegraPol scaffold was observed under scanning electron microscope.
RESULTS: AO/PI stain showed chondrocytes possess favorable activity on DegraPol scaffolds. MTT assay showed the A values at week 3 of the culture was lower than that at week 6 (P < 0.05). The maximal strain load strength test was (1.43±0.58) mm/mm at week 3，(0.48±0.07) mm/mm at week 6. The stress-strain mechanical force analysis showed that, the maximal stress and the maximal strain were higher at week 3 of the culture compared with week 6 (P < 0.05). Scanning electron microscopy showed more cell proliferation and more extracellular matrix were found at week 6, which indicated the good histocompatibility between DegraPol scaffolds and rat xiphoid chondrocytes.
CONCLUSION: DegraPol scaffolds have favorable biocompatibility, and chondrocytes seeded on scaffolds possess normal viability. However, the biomechanical strength of engineered composites is so weak that the culture condition and style need to be improved.

Yang L, Wu YG, Ji L, Wang Z. Static culture of tissue-engineered tracheal cartilage with scaffolds in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(19):3601-3604
[www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3601(ps).pdf]