兔骨髓间充质干细胞/壳聚糖-胶原支架复合体构建软骨组织工程支架

背景:软骨组织工程的核心是利用少量的细胞经体外培养、扩增后,在一定环境下附着在三维多孔支架上,再将细胞/支架复合体移植到体内形成新的组织.目的:拟构建兔骨髓间充质干细胞/壳聚糖-胶原支架复合体,探讨该支架作为软骨组织工程支架的可行性.设计、时间及地点:细胞-支架学体外观察,于2008-03/2009-02在武警医学院生物化学教研室完成.材料:日本大耳白兔6只用于分离培养骨髓间充质干细胞.医用壳聚糖粉末为山东奥康生物科技有限公司产品.Ⅰ型胶原为Sigma公司产品.方法:取3%的壳聚糖和2%的胶原混合的醋酸溶液,倒入72孔板内,-20℃预冷冻10 h,冻干机冷冻抽干24 h制作成壳聚糖-胶原支架.取第3代兔骨髓间充质干细胞,以1×109L-1密度接种于支架内,构建细胞/支架复合体.主要观察指标:傅里叶红外光谱、扫描电镜、液体置换法测定支架的理化性质,观察三维培养后细胞在支架上的生长情况.结果:壳聚糖-胶原支架孔径为160～380 μm,平均为270 μm,孔相通性好,支架孔隙率为(86.00±5.12)%.傅里叶红外光谱仪测定数据表明复合支架组成物有典型的壳聚糖、胶原峰,未发现有聚乙二醇峰.细胞/支架共培养24,48,72 h后,骨髓间充质干细胞可渗入支架多孔结构内,并黏附在支架上成簇生长,部分细胞已与支架融合.结论:壳聚糖-胶原支架基本符合软骨组织工程支架要求,能够作为种子细胞的承载体.

Construction of cartilage tissue engineered scaffold by rabbit bone marrow mesenchymal stem cells/chitosan-collagen composite

BACKGROUND: The core of cartilage tissue engineering is that a few of cells following in vitro culture and amplification are attached to three-dimensional porous scaffold, and cell/scaffold composite is then implanted to form new tissues. OBJECTIVE: To study the feasibility of rabbit bone marrow mesenchymal stem cells (BMSCs)/chitosan-collagen scaffolds to considered as cartilage tissue engineered scaffold. DESIGN, TIME AND SETTING: An in vitro cell-scaffold observation was performed at Research Room of Biochemistry, Medical College of Chinese People's Armed Police Force from March 2008 to February 2009. MATERIALS: Six Japanese rabbits were used for separation and culture BMSCs. Chitosan was provided by Aokang Biotechnology Co., Ltd., Shangdong; type I collagen was provided by Sigma, USA. METHODS: 3% chitosan and 2%collagen were mixed in acetic acid solution, and the mixture was then put in 72-well plate, pre-frozen at -20 ℃ for 10 hours, and frozen and dried for 24 hours to obtain chitosan-collagen scaffold. The third-passage rabbit BMSCs were inoculated in the scaffold at the density of 1×10~9/L to construct cell/scaffold composite. MAIN OUTCOME MEASURES: Physico-chemical properties of scaffold were detected using Fourier transform infrared spectroscopy, scanning electron microscopy, and liquid substitution method, while cell growth was observed after three-dimensional culture. RESULTS: Pore of chitosan-collagen scaffold was 160-380 urn, with the mean value of 270 urn. the permeability was well, and the porosity was (86.00?.12)%. Fourier transform infrared spectroscopy indicated that the products in the cell/scaffold composite group contained chitosan and collagen peaks, but polyethylene glycol peak was not found. At 24, 48, and 72 hours after cell/scaffold co-culture, BMSCs permeated into porous structure of the scaffold and adhered to the scaffold. Partial BMSCs were in fusion with the scaffold. CONCLUSION: The novel chitosan-collagen scaffold is satisfactory for cartilage tissue engineered scaffold, thus it can be used as a vector of seed cells.