组织工程人工肌腱的实验研究

Experimental study of tissue engineering endon

目的 为构建组织工程肌腱积累经验。方法 应用显微外科技术剥除人胚肌腱外膜组织后 ,经过胰蛋白酶及胶原酶分步消化分离出人胚腱细胞 ,使用F12培养液将其传代培养。该细胞贴壁生长 ,具有接触抑制的性质。细胞群体倍增时间大致为 4d。通过冻存后复苏的细胞继续培养后发现 ,冻存对其生长、形态、遗传学等特征无明显影响。经测定培养细胞分泌的羟脯氨酸含量 ,证实培养的人胚腱细胞与体内腱细胞同样具有合成胶原的能力 ,并且这种能力不为传代所改变。将肌腱细胞与碳纤维、PGA、胶原及其共混编织材料进行体外联合培养 ,观察细胞与材料的相容性以及细胞在材料上的生长情况 ,筛选出最佳支架材料 ;并用该材料与肌腱细胞复合后植入兔体内 ,分期观察肌腱细胞的形态学改变及合成胶原的类型。结果 在体外培养条件下 ,碳纤维增强PGA加胶原表面涂层材料与肌腱细胞的相容性最好。肌腱细胞与碳纤维增强PGA加胶原表面涂层材料复合植入体内后肌腱细胞能继续增殖 ,并能合成Ⅰ型胶原。肌腱细胞在植入 2周起在整个支架材料立体空间内增殖、合成胶原 ,其胶原纤维相互衔接 ,形成了致密的组织结构。在肌腱缝合处 ,胶原纤维互相衔接 ,表明植入体与受体肌腱已愈合。扫描电镜下观察 ,见肌腱细胞在碳纤维间排列整齐、均匀 。

Objective To obtain artificial tendon by tissue engineering. Methods Tenocytes were isolated from the human embryonic tendons by digesting it with trypsin and collagenase. These cells were cryopreserved until they were cultured by F12 culture medium. Then the cells were subcultured to 13th generation. These cells were able to grow adhering to the wall and stop growing with contact inhibition. The time of cell group duplication was 4 days, which was similar to the peak time of its mitosis. After the cells were subcultured, the frozen storage didn't influence their growth, morphology, and genetic characteristics. Hydroxyproline content was detected in different generation cells and the results showed that the cultured human embryonic tenocytes had same collagen synthesis capacity. There was no change in biologic characteristics with cell subculture. The tenocytes were co-cultured with carbon fiber, polylatic acid (PGA) and collagen scaffolds. The optimal material was chosen based on the observation of histocompatibility and cell growth. Then, the tenocyte-scaffold complexes were implanted into rabbits to repair Achilles tendon defects. Morphological changes of tenocytes and synthesis of type Ⅰ collagen were observed by stages. Results In in vitro culture, the histocompatibility between tenocytes and carbon fiber strengthened, collagen coated PGA was best. After in vivo implantation of the complex, the tenocytes could proliferate and synthesize type I collagen. Two weeks after implantation, the tenocytes proliferated inside the steric frame and synthesized collagen. The collagen fibers fused to form dense tendon structure. At the suture site, the collagen fibers of the implant fused with the recipient tendon collagen fibers, indicating healing between the implant and recipient tendon. Scanning electron microscopic observation revealed that the tenocytes aligned regularly and evenly among the carbon fibers. The collagen fibers formed a network, its main direction being aligned with the carbon fibers. Normal nucleus, nucleolus, and organelles were found under transmission electron microscope. Conclusions Combined culture of tenocyte with mix woven of carbon fiber and PGA scaffold is able to make tissue engineering tendon.