课题背景：疾病导致气管节段切除端端吻合的安全长度为6 cm。超过此长度时，如何修复缺损气管一直困扰着胸外科医生。曾有自体或同种异体组织及人工替代材料应用于临床，但在解剖结构、免疫排斥及与机体融合、感染等方面存在不足。组织工程技术有望解决这一难题。本实验为构建初级化工程化气管软骨及初级化工程气管打下基础。

应用要点：①目前工程化气管研究热点为支架材料、种子细胞两大方面。支架材料分为天然材料和人工合成两类。应用较广泛的为人工合成的材料,有聚乳酸、聚羟基乙酸、二者的聚合物聚乳乙醇酸。②种子细胞研究热点集中在干细胞方面，因为其具有获取方便、易扩增、可定向分化、不具有免疫原性等特点。③目前需要解决的问题有：工程化气管的血管化、上皮化。

同行评价：文章选题是目前胸外科基础研究的热门课题，组织工程化气管是组织工程研究的一个重点方向。应用骨髓间充质干细胞接种于聚羟基乙酸支架上，植入自体猪皮下，进行体内培养8周后，可生成组织工程化软骨。课题有一定的创新性和临床应用前景。

摘要
目的：如何修复气管缺损是一项医学难题，组织工程技术有望解决这一难题。要构建工程化气管软骨，种子细胞是关键因素之一。探讨利用组织工程方法,将猪骨髓间充质干细胞在体外诱导、培养后，构建软骨组织的可能性。
方法：实验于2006-10/2007-05在北京协和医院中心实验室完成。①实验材料：小型猪6只由中国农业大学提供，雌雄不限，体质量15~20 kg；聚羟基乙酸纤维（Equl.com.美国）。②实验过程及评估：抽取猪的胸骨骨髓, 经密度梯度离心法在体外进行分离、纯化，得到骨髓间充质干细胞, 并在含有转化生长因子β１的特定培养基内进行培养、诱导，免疫组织化学检测诱导细胞Ⅱ型胶原分泌。将诱导分化的骨髓间充质干细胞接种于聚羟基乙酸支架上，将其作为实验组；对照组为未接种细胞的单纯聚羟基乙酸支架；将两组标本环行包裹于直径为0.4 cm的离心管外表面，植入自体猪皮下，进行体内培养，分别于6，8，10周后取材，进行大体观察和组织学检测，评估工程化软骨的形成。
结果：①通过密度梯度离心法可获取骨髓间充质干细胞，对其进行培养扩增后可获得较多的细胞。②猪骨髓间充质干细胞通过特定的诱导后可向软骨细胞分化，诱导细胞Ⅱ型胶原免疫组织化学检测结果为阳性。③猪骨髓间充质干细胞-聚羟基乙酸复合物经过体内第6，8，10周培养后,标本出现软骨组织外观,进行组织学切片可见软骨陷窝，Ⅱ型胶原蛋白的免疫组织化学检测为阳性。
结论：猪骨髓间充质干细胞在成软骨诱导剂作用下，经体外和体内培养后，可生成组织工程化软骨。
关键词：间质干细胞；软骨；组织工程；组织构建

韩志军，刘晓峥，任华.猪骨髓间充质干细胞体外诱导构建组织工程化软骨[J].中国组织工程研究与临床康复，2008，12(2):209-212 [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-209(ps).pdf]

Constructing tissue-engineered cartilage with porcine bone marrow mesenchymal stem cells in vitro

Abstract

AIM：Repair of trachea is disturbing the surgeon. Tissue engineering technology will probably resolve this problem. Seed cell is one of the key factors in engineered tracheal cartilage construction. This study investigated the feasibility of constructing tissue-engineered cartilage from porcine bone marrow mesenchymal stem cells (MSCs) cultured and induced in vitro using tissue engineering technique.
METHODS: The experiment was performed in the Central Laboratory of Peking Union Medical College Hospital between October 2006 and May 2007. ①By density gradient centrifugation, the MSCs were isolated and purified from porcine bone marrow. The MSCs had been cultured and induced in the defined medium mainly including transforming growth factor-β1, and then the type Ⅱ collagens were detected by immunohistochemical assay. The induced MSCs were seeded onto polyglycolic acids (PGA) scaffold as experimental group, and PGA scaffold were implanted into subcutaneous tissue as control group. The cell-scaffold construct was wrapped around a silicon tube (0.4 cm in diameter) and implanted into subcutaneous tissue of porcine. All specimens were harvested after in vivo culture for 6, 8 and 10 weeks and evaluated by gross view, histology, and immunohistochemistry.
RESULTS: ①The MSCs were obtained by density gradient centrifugation method, and abundant seed cells were obtained after culture and amplification. ②The MSCs differentiated towards chondrocyte when cultured in the specific medium in vitro and were verified by the positive result of collagen type Ⅱ through immunohistochemistry. ③After implanted into subcutaneous tissue for 6, 8 and 10 weeks , the cell-scaffold formed a tubular cartilage, which was very similar to normal porcine tracheal cartilage in both gross view and histology. And the result of collagen type Ⅱ through immunohistochemistry was positive.
CONCLUSION: The in vivo and vitro cultured MSCs from porcine bone marrow can generate tissue-engineered cartilage under chondrogenic induction.

Han ZJ, Liu XZ, Ren H.Constructing tissue-engineered cartilage with porcine bone marrow mesenchymal stem cells in vitro .Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2):209-212(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-209(ps).pdf]