含有生肌液的组织工程皮肤修复兔皮肤缺损

Repair of rabbit skin defect by tissue-engineered skin loaded with Shengji liquid

背景:在皮肤开放创面上进行缺损修复与重建是组织修复领域的研究焦点。构建含有中药的组织工程皮肤修复创面缺损的研究并不多见。目的:将含有生肌液的皮肤支架与自体皮肤细胞复合,构建含药组织工程皮肤,修复兔皮肤缺损。设计:自体对照动物实验。单位:实验于2005-02/08在天津市天津医院骨科研究所细胞工程室完成。材料:同窝大耳白兔8只,体质量1.6～1.8kg。自行提取生肌液(1500g/L),成分为当归、生地、龟板、象皮、血余、生石膏、炉甘石;明胶-壳聚糖支架材为料天津大学提供,分别修剪成1.3cm直径的圆片,60Co照射灭菌;DR无细胞真皮基质由江苏省启动市医疗用品研究所提供。方法:分离培养兔皮肤细胞,获取第3代角质形成细胞与成纤维细胞。延兔脊柱两侧制作直径1.5cm的圆形全层皮肤缺损创面4个。分为4组,阴性对照组(明胶-壳聚糖支架贴附于创面后滴加生理盐水),人工真皮组(DR无细胞真皮基质贴附于创面后接种细胞悬液),非含药组织工程皮肤组(明胶-壳聚糖支架贴附于创面后接种细胞悬液),含药组织工程皮肤组(生肌液-明胶-壳聚糖支架贴附于创面后接种细胞悬液)。主要观察指标:①术后13d测量剩余创面面积。②术后7d、10d取材行组织学与免疫组织化学染色检测。结果:①术后13d各组创面面积均有不同程度的缩小,剩余面积阴性对照组>人工真皮组>非含药组织工程皮肤组>含药组织工程皮肤组,非含药组织工程皮肤组、含药组织工程皮肤组与阴性对照组比较差异有显著性意义(P<0.05)。②术后10d,阴性对照组、人工真皮组、非含药组织工程皮肤组均有炎性肉芽组织增生,无表皮组织形成;含药组织工程皮肤组形成接近正常的上皮组织及幼稚的真皮组织。非含药组织工程皮肤组与人工真皮组BrdU标记弱阳性,含药组织工程皮肤组BrdU标记强阳性。结论:实验采用的方法使种子细胞在含有生肌液的明胶-壳聚糖支架上增殖,在体修复皮肤损伤,能够有效扩展皮肤缺损处的覆盖面积,缩短皮肤缺损的愈合时间。

BACKGROUND： Defect reparation and reconstruction on skin defect are focuses in tissue reparation field. There are few studies on skin defect reparation by the construction of tissue-engineered skin loaded with Chinese medicine. OBJECTIVE： To construct Chinese medicine-loaded tissue-engineered skin with skin scaffold loaded with Shengji liquid and autogenous skin cells to repair rabbit skin defect. DESIGN： A self-controlled animal experiment. SETTING： The experiment was performed at the Cell Engineering Laboratory of Orthopedic Institute, Tianjin Orthopedic Hospital from February to August 2005. MATERIALS： Eight brood rabbits, weighing 1.6k- 1.8 kg, were selected. Shengji liquid （1 500 g/L） was prepared, which was composed of Danggui, Shengdi, Guiban, Xiangpi, Xueyu, gypsum, and Luganshi. Gelatin-chitosan scaffold was provided by Tianjin University, and cut into 1.3-cm round patches, and exposed to 60Co irradiation. DR acellular dermal matrix was provided by Institute of Medical Reagent of Jiangsu ProvinceMETHODS： Rabbit skin cells were isolated and cultured, and the 3rd passage of keratinocyte and fibroblast were obtained. Four 1.5-cm round full-thickness skin wounds were made along bilateral spinal column. Four groups were set up： negative control group （gelatin-chitosan scaffold and normal saline）; artificial derma group （DR acellular dermal matrix and cell suspension）; tissue-engineered skin without drug group （gelatin-chitosan scaffold and cell suspension） and drug-loaded tissue-engineered skin group （Shengji liquid, gelatin-chitosan scaffold and cell suspension）. MAIN OUTCOMIE MEASURES： General state of wounds was observed daily. Wound diameter and wounds area 13 days after operation was determined. Histological and immunohistochemistrical staining was performed at 7 and 10 days after operation. RESULTS： Wound area was decreased at 13 days after surgery, and the remnant area of negative control group〉artificial derma group〉tissue-engineered skin group〉drug-loaded tissue-engineered skin group. Significant differences were found in remnant wound area between negative control group and two tissue-engineered skin groups （P 〈 0.05）. Inflammatory granulation tissues were proliferated in negative control, artificial derma and tissue-engineered skin groups 10 days after operation with no epidermal tissue. Normal epithelial tissue and immature dermal tissue were observed in drug-loaded tissue-engineered skin group. Weak positive of BrdU immunohistochemistrical staining was observed in tissue-engineered skin and artificial derma group, and strong positive was observed in drug-loaded tissue-engineered skin group. CONCLUSION： Seed cells proliferate on gelatin-chitosan scaffold loaded with Shengji liquid to repair skin injury, and shorten skin recovery time.