软骨细胞膜片修复山羊气管的实验研究

Reconstruction of Goat Trachea via Chondrocytes Cell-Sheet Approach

目的探讨软骨细胞膜片技术构建的组织工程软骨,修复大型哺乳动物气管缺损的方法。方法取6头山羊耳廓软骨细胞为种子细胞,利用细胞膜片技术构建软骨组织。膜片体外培养6周,回植到动物腹部皮下;3例在皮下埋置8周后包裹于硅胶管上,移植到颈旁进行再血管化和塑形,8周后带肌肉蒂修复气道缺损(带蒂移植组);另3例在皮下埋置16周后,直接用于气道缺损修复(游离移植组)。动物气管缺损范围均为3个气管环长的全段缺损。结果所有膜片在体外培养6周后,均能形成较为成熟的软骨样组织。带蒂移植组在皮下埋置8周后能形成成熟软骨组织,移植到颈旁肌肉能成功再血管化和塑形。气管缺损修复术后,带蒂移植组均能带T型管长期存活(14周),但拔出T型管后均在2周内死亡。取材时发现,带蒂移植组修复段组织工程软骨仍然存活,并在相应部位维持了很好的管壁外形,但是没有软骨外壁的部位发生明显塌陷;内壁结缔组织增生严重,阻塞管腔。游离移植组在皮下埋置16周后,也形成了成熟软骨组织。移植修复后,在T型管存在的情况下,均在术后2周内死亡。取材发现,修复段气道均发生了严重的组织坏死感染,并波及周边组织;组织学观察仅能发现少数散在的组织工程软骨组织。结论软骨细胞膜片技术是稳定可靠的组织工程软骨构建方法;在气道复杂环境中,游离移植的软骨补片会在短时间内发生坏死;带蒂移植能保证修复段组织的存活,但是完整的软骨外壁和内壁提前(或尽快)上皮化是修复成功的关键。

Objective To investigate proper trachea reconstruction approaches of goat by using chondrocytes cell-sheet technology. Methods Tissue engineering (TE) cartilage was constructed via cell-sheet technology. Six goats were involved in this study. After 6-week-culture in vitro, cell-sheets were implanted into superficial fascia layer of the goat ’s abdomen. Eight weeks later, cartilage-like tissue wrapped around a silica tube was transplanted into peritrachea muscles in 3 goats. Finally, the TE cartilage with muscle pedicle was used to repair goat trachea defect (pedicled transplantation group, PT group) after cultured in vivo for 8 weeks. The remains were auto-transplanted to reconstruct trachea defect after 16-week-culture in vivo (dissociated transplantation group, DT group). Three intact cartilage rings were resected in all animals. Results After 6 weeks cultured in vitro, all sheets were charactered as cartilage features. In PT group, 8 weeks later after implantation, matured TE cartilage were observed. After 8-week-culture in peritrachea muscle, TE cartilage was well vascularized and reformed. Animals in PT group had lived for a long time (14 weeks) until the T tube was removed. The gross view and histology showed the survived TE cartilage with well remained tube shape. But collapse was observed on local site where not surrounded by TE cartilage. In the meanwhile, serious hyperplasia of fibrillar connective tissue that obstacled the lumina was observed. In DT group, typical cartilage features were presented in TE cartilage after 16-week-cultur in vivo. But animals all died in two weeks after transplantation even with T tube. The gross view and histology indicated serious necrosis of repaired segment with tiny sporadic cartilage like tissue. Conclusion Cell-sheet technology is a stable approach for TE cartilage reconstruction; Dissociated TE cartilage transplant cannot survive in perplexing trachea environment; In the oppo-site, vascularized TE cartilage could be an ideal approach for trachea reconstruction, and the integrated cartilage exterior lay-er and epithelized lumina are both crucial to successful operation.