自体BMSCs复合胶原膜修复猪全层皮肤缺损

REPAIR OF SWINE FULL-THICKNESS CUTANEOUS DEFICIENCY BY AUTOGENIC BMSCs COMPOUNDED WITH COLLAGEN MEMBRANE

目的探讨自体BMSCs与胶原膜复合构建组织工程皮肤修复小型香猪全层皮肤缺损,为组织工程皮肤的临床应用提供实验依据。方法取4只贵州小型香猪骨髓各20mL培养BMSCs,传至第3代。取新鲜牛肌腱通过化学交联的方法制备成直径为5cm的圆形Ⅰ型胶原膜。将密度为2×107个/mL第3代BMSCs用DAPI标记后种植到Ⅰ型胶原膜上孵育24h,制备组织工程皮肤。在猪背部正中线两侧分别由前向后切取5个直径5cm、深达筋膜的圆形全层皮肤缺损创面,随机分为两组(n=10)。对照组行单纯Ⅰ型胶原膜修复,实验组行组织工程皮肤修复。于术后3、8周,观察创面愈合情况,并取材行HE染色及透射电镜观察,并于术后3周对实验组行荧光显微镜及糖原染色观察。结果动物均存活至实验完成。术后3周对照组创面约40%皮肤发黑、结痂、坏死,8周时仍见坏死组织且瘢痕挛缩明显;术后3周实验组创面皮肤成活,无明显瘢痕形成,8周创面愈合良好;两组创面愈合率差异有统计学意义(P<0.01)。组织学观察:术后3周对照组可见肉芽组织增生,无表皮层覆盖;实验组具有良好的表皮和真皮结构,糖原染色可见基底膜呈完整连续深红染色。术后8周两组均形成正常皮肤样结构。透射电镜观察:术后3周对照组真皮层有大量中性粒细胞和成纤维细胞,未见表皮超微结构;实验组可见大量表皮细胞并以桥粒相连,表皮基底细胞与基膜以半桥粒连接;真皮层可见大量成纤维细胞,以及细胞外大量胶原微纤维沉积,可见内皮细胞形成的毛细血管。术后3周实验组荧光显微镜观察,带有DAPI标记的BMSCs定位于重建的表皮和真皮组织中,表皮主要定位于基底膜附近,真皮层可见大量荧光标记的BMSCs。结论以自体BMSCs为种子细胞与胶原膜复合构建组织工程皮肤,可有效修复猪全层皮肤缺损,有望成为一种较理想的组织工程皮肤。

Objective To supply references to tissue-engineered skin clinical applications with autogenic BMSCs composited collagen membrane to repair swine full-thickness cutaneous de ciency. Methods Twenty mL bone marrow were obtained respectively from 4 swine,autogenic BMSCs were cultured and passed to the 3rd passage. The fresh bovine tendon treated by means of chemically cross-linked was made 5 cm diameter collagen I （Col I） membrane. The 2×10^7/mL P3 swine autogenic BMSCs labeled DAPI were planted to sterile Col I membrane for 24 hours incubation,then the tissue-engineered skin was constructed. The ve full-thickness skin defect of 5 cm diameter was excised to the muscle from forward to backward on the back midline two sides of swine. The tissue-engineered skin were implanted in theffexperimental group,while Col I membrane was implanted in control group. After 3 and 8 weeks of implantation,the two swine wound surface healing circumstance was observed and further evaluated with histology analysis and TEM. After 3 weeks of implantation,theffexperimental group were observed with uorescence microscopy and staining for glycogen. Results After 3 weeks of implantation,the wound surface of control group were observed nigrescence,scab and putrescence,and after 8 weeks of implantation,also evident putrescence and scar. The wound surface of experiment group was alive after 3 weeks implantation,appearance was leveled o and exible without evident scar. The wound surface recovered well after 8 weeks of implantation,wound surface healing rate was signi cantly di erence between the two groups （P 〈 0.01）. After 3 weeks of implantation,control group were observed acestoma hyperplasia and no epidermal coverage by histology analysis. Theffexperimental group was showed integrity epidermis and dermis structure. The basal layer was crimson and continuously positive with glycogen staining. After 8 weeks of implantation,theffexperimental group and control group wereffemerged normal skin structure. After 3 weeks of implantation in control group,a lot of neutrophilic granulocytes and broblasts were noticed,but no epidermal structure was observed under TEM. In theffexperimental group,a lot of epidermal cells were observed,dermatome connection among epidermal cells and hemidermosome connection between basilar membrane cells and basal membrane were observed in epidermis. In the dermis experimental group,blood capillary endothelial cells were noticed. Furthermore,considerable collagen ber deposit was found in the surrounding tissue of broblasts. After 3 weeks of implantation,BMSCs labeled with DAPI were located reconstructed epidermal basement membrane and dermis by uorescence microscopy. Conclusion Tissue-engineered skin which is composited with autogenic BMSCs as seed cells and collagen membrane were potential prospects in application of repairing swine full-thickness cutaneous de ciency.