兔韧带-骨界面移行结构的组织工程学构建

The construction of a tissue-engineered tendon mimicking the transitional architecture at the ligament-bone interface in rabbit

目的 探讨在脱细胞兔跟腱内构建一种具有连续异质性结构的组织工程学肌腱的方法.方法 体外培养家兔上皮组织成纤维细胞CRL-2087,并制备胶原水凝胶.实验分为单纯支架组、成纤维细胞＋软骨细胞组（Fb＋ CC组）、成纤维细胞＋成骨细胞组（Fb＋OB组）、成纤维细胞＋软骨细胞＋成骨细胞组（Fb＋ CC＋ OB组）.将异质性细胞群：成纤维细胞、软骨细胞、成骨细胞依次连续种植于支架内预先设定的3个功能区域（成纤维区、成软骨区及成骨区）,构建一种具有纤维形成、软骨形成及骨形成3个功能区域的“移行结构化”组织工程学肌腱,并对脱细胞支架行形态学、显微结构及细胞兼容性进行检测.采用组织学及免疫荧光分析3个功能区域内特定组织的梯度形成情况.结果 脱细胞跟腱的形态学、显微结构及细胞兼容性检测结果显示,经脱细胞处理后,腱细胞消失,胶原纤维排列疏松,腱束间可见较多孔隙存在,兔成纤维细胞在单纯支架表面或胶原水凝胶复合支架内培养时呈平稳增加.细胞支架复合共培养后,组织形态学及免疫荧光检测结果显示,软骨标志物糖胺聚糖、小鼠抗兔Ⅱ型胶原2A1只在软骨段内生成,而骨标志物钙结节、山羊抗兔骨钙素只在成骨段内生成,纤维生成段新生的胶原纤维相互连接,有效地修复了支架的结构.在连续种植成纤维细胞、软骨细胞、成骨细胞的脱细胞跟腱内,表现为纤维-纤维软骨-骨的结构渐变现象,而对照组无此现象.结论 脱细胞兔跟腱内层次性种植异质性细胞后,体外成功复制了一种拟生理的纤维-纤维软骨-骨的过渡界面结构,其具有细胞间相互作用可控性及基质不均匀性的多组织过渡特征.

Objective To investigate a method that constructing a tissue-engineered tendon with a continuous and heterogeneous transition region.Methods Fibroblasts derived from rabbit epithelial tissue were cultured in vitro and collagen gel was prepared.The experimental groups were scaffold only group,fibroblasts ＋ chondrocytes group （Fb ＋ CC group）,fibroblasts ＋ osteoblasts group （Fb ＋ OB group）,fibroblasts ＋ chondrocytes ＋ osteoblasts group （Fb ＋ CC ＋ OB group）.Heterogeneous cell populations （fibroblasts,chondrocytes and osteoblasts） with collagen gel were seeded within three predesigned specific regions （fibrogenesis,chondrogenesis,and osteogenesis）of decellularized rabbit achilles tendons to fabricate a stratified scaffold containing three biofunctional regions supporting fibrogenesis,chondrogenesis,and osteogenesis.The tests of morphology,architecture and cytocompatibility of the scaffolds were performed.Gradient tissue-specific matrix formation was analysed within the predesignated regions via histological staining and immunofluorescence assays.Results The HE staining and scanning electron microscopy analysis demonstrated that no major cell fragments or nuclear material was evident,and increased intra-fascicular and inter-fascicular spaces were found,the cytocompatibility of the scaffolds showed that the numbers of viable cells on the scaffold surfaces increase steadily,no significant differences were found between the scaffold only containing ordinary culture medium and scaffold containing gel groups.Histological staining and immunofluorescence assays demonstrated that the cartilage-related markers （GAG,COL2A1） were found only in the chondrogenesis region,but bone-related proteins only in the osteogenesis region of bone tunnel,and fibrosis was remarkable for the fibrogenesis region in the joint cavity.The transitional architecture with ligament-fibrocartilage-bone was constructed in the ligament-bone tunnel interface.Conclusions A transitional interface （fiber-fiberocartilage-bone） could be replicated in a decellularized tendon through stratified tissue integration in vitro.The cell-tendon complex offers the advantages of a multitissue transition involving controlled cellular interactions and matrix heterogeneity.