骨髓间充质干细胞—聚乳酸聚乙醇酸共聚复合物支架修复组织损伤的实验研究

Repair of skin damage with mesenchymal stem cells-poly (lactic-co-glycolic acid) scaffolds: experimental study with rabbits

目的探讨骨髓间充质干细胞-聚乳酸聚乙醇酸共聚复合物(PLGA)的构建及对损伤组织的修复效果。方法(1)分离培养新西兰幼兔骨髓间充质干细胞(MSC),4,6联脒2苯基吲哚(DAPI)标记后接种于PLGA支架上。扫描电镜和免疫荧光显微镜观察不同时间段MSC在支架上的生长情况。(2)分别将MSC-PLGA复合物及PLGA支架移植于新西兰大白兔背部皮肤创面。术后观察创面愈合情况,5周后取移植物行HE染色、VG染色及细胞角蛋白AE1/AE3免疫荧光组化检测。结果MSC在支架上生长良好,细胞数量随时间增长而逐渐增加,第10天细胞达到90%融合。MSCPLGA复合物所修复创面,随着聚合材料的降解,逐渐形成新生皮肤,HE染色及VG染色示新生皮肤与正常皮肤结构相似,且荧光显微镜下可见胞核呈蓝色荧光的角化层细胞及毛囊细胞,其胞浆同时表达角蛋白。PLGA支架所修复创面则新生皮肤仅见少量毛囊,并见增厚的纤维瘢痕。结论骨髓间充质干细胞复合PLGA聚合物是良好的移植替代物。

Objective To evaluate the feasibility and effect of mesenchymal stem cells （MSGs）poly （lactic-co-glycolic acid） （PLGA） scaffold as transplant in repair of skin damage. Methods MSCs were isolated from the bone marrow of femur of a one-month-old New Zealand rabbit, cultured, and labeled with diamidino-phenyl-indole （DAPI）. Porous foam scaffolds were made with PLGA. MSCs of 2 - 3 passages were seeded on the scaffolds. Fluorescence microscopy and scanning electron microscopy were used to observe the growth of the MSCs. Six pieces of skin 2 cm × 2 cm in size were cut from the hacks of five 5- month-old new Zealand rabbits and then 4 pieces of MSCs- PLGA scaffolds and 2 pieces of porous foam PLGA scaffolds of the size similar to these of the cut skin were transplanted to the skin wounds. The wound healing was observed. Five days after the operation, samples of newly-grown skin were taken to undergo HE staining, VG staining, and microscopy. Immunofluorescence histochemistry was used to detect the cytokeratin AE1/AE3. Results Scanning electron microscopy showed that holes were distributed evenly on the surface of and inside the porous foam PLGA scaffolds Fluorescence microscopy and scanning electron microscopy showed that the MSCs grew well on the porous foam PLGA scaffolds and the number of MSCs increased gradually. Animal experiment showed that with the degradation of the polymer scaffolds the wounds were gradually covered by newly grown skin similar to the normal skin. Immunofluorescence histochemistry showed fluorescence positive cells in the stratum corneum and follicles. The wounds transplanted only with porous foam PLGA scaffolds formed new skin too, however, in the dermis of the new skin only thickened fibrous scars and a few follicles were seen. Conclusion The compound of MSCs- PLGA polymer is effective in wound healing.