组织工程皮肤修复全层皮肤缺损的实验研究

EXPERIMENTAL STUDY ON REPAIRING FULL-THICKNESS CUTANEOUS DEFICIENCY WITH TISSUE ENGINEERED SKIN

目的探索由人表皮干细胞、成纤维细胞与纤维蛋白支架构建的组织工程皮肤修复全层皮肤缺损的可行性。方法将人全层皮肤采用胰蛋白酶消化法使表皮干细胞和真皮成纤维细胞分离后,分别在无血清培养基中行原代及传代培养。将体外扩增培养至第3、4代的表皮干细胞(5×104/mL)和真皮成纤维细胞(1×104/mL)共0.5mL与纤维蛋白支架(0.5mL)混合凝固构建组织工程皮肤。取4～5周龄雄性裸鼠45只,体重19.5～20.3g,平均20.0g,制备背部全层皮肤缺损模型。随机分为5组,每组9只,分别为空白对照组(C组),创面仅覆盖凡士林油纱,自然愈合;单纯支架组(F组),创面移植无细胞的纤维蛋白支架;表皮支架组(S组),创面移植含有表皮干细胞的纤维蛋白支架复合物;成纤维细胞支架组(Fb组),创面移植含有成纤维细胞的纤维蛋白支架复合物;组织工程皮肤组(T组),创面移植组织工程皮肤。术前及术后1、3、6、8周行全身及移植部位大体观察;移植后3、6、8周,取材行组织学、免疫组织化学及扫描电镜观察。结果?细胞培养4周后,表皮干细胞培养皿内可见圆形细胞,在加有BrdU的培养液中培养6d后可见BrdU染色阳性细胞;成纤维细胞培养皿内可见梭形细胞。构建的组织工程皮肤可见CK19免疫荧光染色阳性细胞、Nestin染色阳性细胞。移植后T组新生皮肤较其余各组生长快、瘢痕轻。术后6周,C、F、S、Fb及T组皮肤厚度分别为:(0.460±0.049)、(0.480±0.055)、(0.540±0.043)、(0.510±0.032)及(0.660±0.047)mm,T组明显厚于其余各组,且差异有统计学意义(P<0.05)。术后3、6、8周,HE染色及扫描电镜示,T组新生表皮层数及真皮成纤维细胞、血管数量均多于其余各组,且T组真皮血管及胶原纤维排列均较其余各组整齐;术后3周,Ⅳ型胶原免疫组织化学染色显示,T组已形成连续的染色带,而其余各组均不连续;术后6周,CK14免疫组织化学染色显示,T组可见阳性细胞,其余各组均未见。结论用表皮干细胞、成纤维细胞及纤维蛋白支架构建的组织工程皮肤移植后能使创面迅速愈合,可望成为一种较理想的组织工程皮肤。

Objective To search for a feasibility of repairing full-thickness cutaneous deficiency with tissue engineered skin substitute composited by human epidermal stem cells and fibroblasts in fibrin frame. Methods Epidermal stem cells and fibroblasts were harvested from human epidermis and dermis by trypsin digestion. Cells were cultured and subcuhured in non-serum medium. Epidermal stem cells （5×10^4/mL） and dermal fibroblasts （1×10^4/mL） in 0.5 mL medium were coagulated in 0.5 mL fibrin frame to construct tissue engineered skin substitute. The tissue engineered skin substitute was grafted onto full-thickness cutaneous deficiency of nude mice. Forty-five male mice, 4-5 week old, weighted 20 g on average, were randomly divided into 5 groups. Oil yarn （group C）, fibrin frame membrane without cell inoculation （group F）, composite skin substitute with epidermal stem cells （group S） and composite skin substitute with fibroblasts （group Fb） were used as controls, while tissue engineered skin substitute （group T） was experimental group. The wounds were observed 1, 3, 6, 8 weeks after surgery. Samples were harvested 3, 6, 8 weeks after surgery, and were examined by means of histology, immunohistochemistry and scanning electron microscopy （SEM）. Results Four weeks after cell culture, there were some round cells in the culture capsule of epidemic cells, and some fusiform cells in the culture capsule of fibroblast. Six days after cells were cultured in the BrdU culture medium, there were some BrdU positive cells appeared. There were some CK19 positive cells and Nestin positive cells appeared in the chaff of group T before transplanting. The new formed skin of group T grew faster and had less scar than other groups. Six weeks after surgery, the average thickness of new formed skin was （0.460 ± 0.049） mm in group C, （0.480 ± 0.055） mm in group F, （0.540 ± 0.043） mm in group S, （0.510 ± 0.032） mm in group Fb, （0.660 ± 0.047） mm in group T. The thickness of new formed skin in group T was thicker than other groups （P 〈 0.05）. By histology and SEM observation, 3, 6, 8 weeks after surgery, the new formed cuticular layer, fibroblast and blood vessels in the group T were more than those in the other groups. The alignment of blood vessels and collagen fibers in group T were much regular than those in the other groups. Three weeks after surgery, the new formed skin of group T had a continuous color zone of positive collagen IV staining, while no continuous color zone was found in the other groups. Six weeks after surgery, CK14 positive cells appeared in the new formed skin of group T, while no positive cell was found in the other groups. Conclusion Tissue engineered skin substitute which is composited with epidermal stem cells and fibroblasts in fibrin frame has potential prospects in application of repairing fullthickness cutaneous deficiency with advantage of faster wound healing.