生物型人工韧带的制备及体外检测

目的探讨应用新的组织处理技术研制的生物型人工韧带应用在膝关节交叉韧带重建的可行性。方法以猪肌腱韧带为原料应用环氧化物作为交联剂加上专门的除抗原技术及蛋白修饰技术制作生物型人工韧带,在体外应用组织学检查、扫描电镜、细胞培养和力学测试分析生物型人工韧带的组织结构、生物力学和细胞的相容性。结果生物型人工韧带的外观与正常肌腱外观相似。组织学检查显示韧带为无细胞的胶原纤维。电镜检查显示韧带为走向一致的发状纤维样物紧密平行排列,表面有棉絮样物附着,未见细胞。人工韧带支架经生理盐水多次洗涤,洗去抗菌保存液后将异种骨髓基质细胞于支架中培养2周可见细胞成活。培养3周,韧带材料表面可见细胞粘附,内部未见细胞。扫描电镜观察,韧带材料中细胞培养3周,细胞呈球形粘附在韧带材料表面及孔隙侧壁,并可见细胞分泌的基质沉积于细胞周围,但基质分泌量较少。力学测试人工韧带直径约5mm,最大拉力为927.19N,抗拉强度47.22N/mm,速度100mm/min。正常山羊的前交叉韧带(ACL)直径约5mm,最大拉力为807.50N,抗拉强度41.13N/mm。结论应用新的组织处理技术研制的生物型人工韧带有效地去除了猪肌腱的异种蛋白的免疫原性,具有良好的生物力学特性,韧带材料的细胞相容性较好。有望应用在膝关节叉韧带重建,成为生物性人工韧带的理想产品。

Preparation of xenogeneic ligamentous matrix and its properties in vitro

Objective To investigate the feasibility of reconstructing anterior cruciate ligament(ACL) and posterior cruciate ligament(PCL) of knee joint with xenogeneic tendon from ligament tissue engineering.Methods Fresh ligaments were harvested from porcine tendon.For the preparation of xenogeneic ligamentous matrix,antigenic elements were extracted using chemical methods with epoxy compound cross-linking.Scanning electron microscopy,histological examination and mechanical testing was used to study the properties of the xenogeneic ligaments.Bone marrow derived stroma cells(BMSCs) were seeded on the graft and incubated for 3 weeks.Cell proliferation and morphological changes were observed by light and scanning electron microscopy.Results The appearance of the xenogeneic ligament grafts was similar to intrinsical ACL of goat.Histological examination of the xenogeneic ligaments showed that these grafts were consisted of collagenous fibers,between which no cells could be seen.Scanning electron micrographs demonstrated that collagenous fibers were arranged uniformly.At the 3rd weeks after seeding cells added into the xenogeneic ligament grafts,BMSCs attaching to the surface of the grafts could be seen,which largely maintained a rounded morphology,could proliferate slightly,and secreted matrix on the scaffolds.The failure force for the xenogeneic ligament graft,with a diameter of 5mm was 927.19 N,while the failure force for the intrinsical goat ACL with the same diameter was 807.5 N.Conclusion It was obviously demonstrated from in vitro study that heterologous protein immunogenicity of porcine tendon is removed successfully by antigen-extracting treatment.The grafts possess a favourable,excellent mechanical function and biocompatible characteristic.It may be served as a useful naturally occurring extracellular matrix scaffolds for ACL reconstruction.