聚羟基丁酸酯-聚羟基己酸酯共聚物/氧化异丙烯F-127复合支架构建人组织工程软骨

Construction of human tissue-engineered cartilage with polyhydroxybutyrate-polyhydroxyhexanoate and Pluronic F-127 complex scaffolds

目的:探讨聚羟基丁酸酯-聚羟基己酸酯共聚物/氧化异丙烯F-127复合支架作为软骨组织工程支架的可行性。方法:实验于2004-09/2005-04在中国医科大学整形外科医院外耳整形与再造中心完成。①选取4周龄无胸腺雄性裸鼠8只,左侧背部皮下为软骨细胞-支架复合物组,右侧背部皮下为单纯支架对照组。人软骨细胞来源于中国医科大学整形外科医院外耳整形与再造中心临床小耳畸形患者行肋软骨移植时剩余的边脚料分离软骨细胞。聚羟基丁酸酯-聚羟基己酸酯共聚物泡沫材料由清华大学化工系提供,1.0cm×1.0cm×0.3cm大小,环氧乙烷消毒后备用。②消化、分离、培养人肋软骨细胞,将第3代细胞制成悬液,细胞浓度为5×1010L-1。取1mL软骨细胞悬液与少量30%氧化异丙烯F-127溶液(4℃)均匀混合后接种至聚羟基丁酸酯-聚羟基己酸酯共聚物支架上,形成细胞-生物材料复合物,体外培养1周后植入软骨细胞-支架复合物组,单纯支架对照组植入单纯聚羟基丁酸酯-聚羟基己酸酯共聚物/氧化异丙烯F-127复合支架。③术后12周取材,行大体观察。利用苏木精-伊红染色、甲苯胺蓝染色、阿尔辛蓝-丽春红染色对再生的软骨组织进行组织学检测。结果:实验选取4周龄无胸腺雄性裸鼠8只,全部进入结果分析。①术后12周大体标本观察结果:软骨细胞-支架复合物组形成了与支架形态相同的乳白色类似软骨样组织,表面光滑有弹性。单纯支架对照组呈褐色,质地较软。②再生的软骨组织术后12周组织学检查结果:组织学检查证实软骨细胞-支架复合物组有软骨细胞存在,并且有基质分泌。单纯支架对照组无新生软骨组织形成。结论:利用聚羟基丁酸酯-聚羟基己酸酯共聚物/氧化异丙烯F-127复合支架可在裸鼠体内形成人组织工程化软骨。

AIM： To investigate the feasibility of using polyhydroxybutyrate- polyhydrnxyhexanoate （PHB-PHH） and Pluronic F-127 serving as the scaffolds for cartilage tissue engineering. METHODS： The experiment was conducted in the External Ear Plastic Center, Plastic Surgery Hospital, Chinese Academy of Medical Sciences between September 2004 and April 2005. （1）Eight male nude mice of 4 weeks old without thymus were selected, whose subcutaneous part of left back served as the chondrocytes-scaffold complex group, and the right part as the simple control group. The chondrocytes were dissociated from the rib cartilage of patients given ear reconstruction in the External Ear Plastic Center of Plastic Surgery Hospital, Chinese Academy of Medical Sciences. The PHB-PHH foam material of 1.0 cm×1.0 cm×0.3 cm was provided by the Department of Chemical Engineering, Tsinghua University and disinfected with epoxy ethane. （2）The 3^rd generation chondrocytes were collected after digestion, isolation and culture to make suspension with cell concentration of 5×10^10L^-1. Chondrocytes suspension 1 mL was mixed with 30% Pluronic F-127, then the compounds were seeded evenly into PHBPHH scaffold at 4℃ to form cell-biological material complex. After incubating for 1 week in vitro, the complex was implanted into the chondrocytes-scaffold complex group, while the PHB-PHH/Pluronic F-127 complex scaffolds without cells were implanted into the control .group. （3） After implantation for 12 weeks, the specimens were harvested for gross observation. The HE stain, toluidine blue stain and ponceau stain were used for histological detection of regenerated cartilages. RESULTS： All the 8 nude rats were involved in the result analysis. （1） Result of gross observation after 12 weeks of operation： New cream white cartilage-like tissues with similar shapes to the scaffolds and smooth surface were formed in the chondrecytes-scaffold complex group, which was brown and soft in the control group. （2）Histological results of regenerated cartilage tissue after 12 weeks of operation： The results suggested there was chondrecytes and matrix secreted in the complex group, but no cartilages were found in the control group. CONCLUSION： The findings suggest that PHB-PHH/Pluronic F-127 complex scaffold can bc used as a favorable scaffold for human cartilage tissue engineering.