移行结构化细胞-肌腱复合物重建韧带-骨连接体的实验研究

Reconstruction of ligament-bone interface by a tissue-engineered cell-tendon complex with characteristics of transitional architecture and its efficacy in rabbits

目的观察体外构建的具有连续异质性移行结构的细胞-肌腱复合物重建兔前交叉韧带(anterior cruciate ligament,ACL)在动物体内的愈合效果。方法通过胶原水凝胶将异质性细胞群种植于脱细胞兔跟腱支架内特定的区域,构建一种具有纤维形成、软骨形成及骨形成3个功能区域的细胞-肌腱复合物重建兔ACL,8周后进行组织学、显微CT、免疫荧光分析检测移植物与骨隧道间预先设定区域内特定组织的梯度形成情况。结果组织学及免疫荧光结果显示,软骨标志物GAG、COL2A1只在软骨段内生成,而骨标志物钙结节、OCN只在成骨段内生成,关节腔内纤维生成段内新生的胶原纤维相互连接,有效地修复了支架的胶原结构。显微CT结果显示,与对照组相比,实验组骨隧道腔内大量新生矿化组织形成。该细胞-肌腱复合物重建兔ACL 8周后,在骨隧道-关节腔界面表现出纤维-纤维软骨-骨的结构渐变现象。结论该细胞-肌腱复合物重建兔ACL,骨隧道-肌腱界面愈合良好,微观结构与生理性ACL-骨界面极其相似,有望应用于ACL的损伤后重建。

Objective To reconstruct anterior cruciate ligament by a composite cell-tendon scaffold with a continuous and heterogeneous transitional region to mimic native ligament insertion site in rabbits and observe the outcome. Methods Heterogeneous cell populations with collagen gel were seeded within specific regions of decellularized rabbit Achilles tendons to fabricate a stratified scaffold containing 3 biofunctional regions to support fibrogenesis,chondrogenesis,and osteogenesis. A total of 24 male rabbits were inflicted with ACL resection in one hind limb and then followed grafted with our obtained ACL,and the other hind limb receiving sham operation served as control. In 8 weeks later,histological staining,micro-CT scanning and immunofluorescence assay were carried out to observe the growth of the graft with bone tunnel. Results Histological staining and immunofluorescence assay demonstrated that the cartilage-related markers,glucosamine glycolic acid and collagen 2A1,were found only in the chondrogenesis region,but bone-related protein,osteocalcin,was only in the osteogenesis region of bone tunnel,and fibrosis was remarkable for the fibrogenesis region in the joint cavity. Micro-CT scanning showed that the newly formed mineralized tissue was detected in the bone tunnels in the experimental group compared with controls. These results suggest that the ACL was reconstructed with the cell-tendon complex in 8 weeks after transportation,and the transitional architecture with ligament-fibrocartilage-bone was constructed in the ligament-bone tunnel interface.Conclusion Our ACL is reconstructed successfully with the cell-tendon complex after 8 weeks. The transitional architecture with ligament-fibrocartilage-bone is constructed in the ligament-bone tunnel interface,which can be applied for the regeneration of the ligament-bone interface.