体外构建组织工程皮肤修复Ⅲ度烫伤切痂创面的实验研究

In vitro construction of tissue engineered skin for wound repair after escharectomy of third degree scald: An experimental study

目的观察体外构建的组织工程皮肤修复大鼠Ⅲ度烫伤切痂创面的可行性和效果。方法酶消化法获取SD乳鼠表皮细胞和成纤维细胞(Fb)后进行体外培养,同时用高渗盐水/氢氧化钠法制备无细胞毒性猪去细胞真皮基质(PADM),然后将Fb与Ⅰ型牛胶原混合种植于PADM的表面,以SD乳鼠的第3代表皮细胞种植在真皮基质胶原面获取组织工程皮肤,以SD乳鼠的第3代表皮细胞制备表皮细胞膜片。对48只SD大鼠Ⅲ度烫伤切痂创面分别行组织工程皮肤移植(实验组)和单纯表皮细胞膜片移植(对照组),术后行大体观察和组织学观察,比较各组创面愈合率、收缩率。抗CD34单克隆抗体免疫组化染色标记血管内皮细胞后行创面微血管计数,抗Laminin免疫组化染色观察基底膜中层黏连蛋白的表达情况。结果两组创面术后均未见对移植物的急性期免疫排斥反应,第2、4、6周实验组移植物成活率分别为75.05%±3.69%、83.12%±3.13%、92.03%±3.87%,与对照组(分别为77.63%±3.23%、83.17%±3.92%、9 1.0 9%±3.3 5%)比较差异无统计学意义(P>0.0 5)。术后第2、4、6周实验组移植物收缩率分别为9.1 3%±2.2 7%、18.52%±3.40%、23.92%±3.01%,明显低于对照组(分别为14.21%±3.05%、29.12%±3.02%、39.78%±3.42%,P<0.05)。术后6周实验组基底膜结构清晰、连续,对照组基底膜区模糊、不连续。第4、6周后实验组微血管数目分别为37.5±3.9、46.9±3.5个/HP,明显高于对照组(分别为23.0±2.0、27.5±2.7个/HP,P<0.05)。结论以Fb-胶原-PADM活性复合真皮基质为载体,与表皮细胞构建的组织工程皮肤,适用于修复Ⅲ度烫伤切痂创面,能改善创面愈合质量。

Objective To observe the practicability and effect of tissue engineered skin for repairing the wound after escharectomy of third degree scald （TDSE） in rat model. Methods Epithelial cells and fibroblasts from newborn SD rats were isolated by enzyme digestion method and cultured in vitro, and porcine acellular dermal matrix （PADM） without cytotoxicity was prepared by hyperosmotic saline/sodium hydroxide method. The fibroblasts were mixed with bovine type I collagen and inoculated on the surface of PADM. Third passage of cultured epidermal cells from newborn SD rats were inoculated on the collagen surface of the dermal matrix to obtain tissue engineered skin, and it was used to prepare epidermal cell sheet. Forty-eight SD rats with TDSE wound were randomly divided into two groups, then tissue engineered skin （experiment group）, and epidermal cell sheet （control group） graftings were performed to cover the wounds respectively. Finally, gross observation and histological changes were observed in grafted area. The wound healing rate and wound contraction rate were compared between the two groups. Microvessel count （MVC） was performed with anti- CD34 monoclonal antibody immunohistochemical staining technique, and vascular endothelial cells were labeled. Basal membrane of the skin was identified by immunohistochemical anti-Laminin staining technique. Results There was no obvious sign of acute rejection of the graft in both groups. The graft survival rate was 75.05% ~ 3.69%, 83.12% ±3.13% and 92.03% ± 3.87% at the 2th, 4th and 6th week respectively in the experimental group. The graft survival rate was 77.63%± 3.23%, 83.17% ± 3.92% and 91.09% ±3.35% at the 2th, 4th and 6th week in the control group. There was no significant difference between the two groups （P〉0.05）, but the contraction rate of the grafts was 9.13% ± 2.27%, 18.52% ±3.40%, 23.92% ±3.01% at the 2th, 4th, 6th week, respectively in the experimental group,and 14.21% ± 3.05%, 29.12% ＋ 3.02% and 39.78% ＋ 3.42% at the 2th, 4th and 6th week in the control group. It was significantly lower than that of the control group （P〈0.05）. At the 6th post-grafting, the basement membrane was clear and continuous in the experimental group. In contrast, the basement membrane was blurred and discontinuous in the control group. The microvessel count was 37.5 ± 3.9 and 46.9：1：3.5 per high-power visual field at the 4th and 6th week in the experimental group, and 23.0 ± 2.0 and 27.5± 2.7 per high-power visual field at the 4th and 6th week in the control group, and the count was significantly higher in the experimental group than the control group （P〈0.05）. Conclusion Tissue engineered skin prepared by the dermal matrix containing fibroblasts-collagen-PADM combined with epidermal cells is suitable for repairing TDSE wound, and it improves wound healing qualit）n