生肌液-明胶-壳聚糖载药皮肤支架的细胞相容性(英文)

Cellular biocompatibility of shengji solution-gelatin-chitosan drug-loading skin scaffold

背景:组织工程皮肤对于大面积皮肤缺损的修复与重建具有重要作用,含有中药的组织工程皮肤支架对于细胞黏附及创面感染控制的作用研究并不多见。目的:利用角质形成细胞与成纤维细胞筛选生肌液应用浓度,观察生肌液-明胶-壳聚糖载药皮肤支架对细胞黏附及生物相容性影响。设计、时间及地点:以角质形成细胞与成纤维细胞为观察对象,随机对照实验,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:健康大耳白兔1只,用于获取皮肤种子细胞;生肌液、当归提取液及生地提取液(自行提取,1.5g/mL),明胶-壳聚糖支架及生肌液-明胶-壳聚糖支架由天津大学提供。方法:①以dispaseⅡ-胰蛋白酶-EDTA消化法获取第3代角质形成细胞与成纤维细胞。②实验分为5组,对照组为普通培养液(含体积分数为0.1胎牛血清的基础培养基);生肌液组为含有生肌液质量浓度分别为5,6,8,12g/L的培养液;当归组为含有当归提取液质量浓度为8g/L的培养液;生地组为含有生地提取液质量浓度为8g/L的培养液;表皮生长因子组为含有10μg/L表皮生长因子的培养液;分别用于培养第3代角质形成细胞和成纤维细胞,于1,3,5,7d用MTT法检测细胞的增殖情况。③制备基质中生肌液质量浓度分别为50,60,80,120g/L的载药支架组,将上述细胞在支架上培养,分别于7,14d用半定量方法观察细胞与支架的黏附情况。④60,80g/L载药支架组种植角质形成细胞,分别于4,7,14d取材,进行苏木精-伊红染色和扫描电镜观察。主要观察指标:①生肌液对皮肤种子细胞增殖的影响。②载药支架对细胞黏附的影响。③细胞与载体的相容性。结果:①MTT检测结果表明,5、6、8g/L的生肌液组细胞增殖较快,与对照组相比,差异有显著性意义(P<0.05)。②第3代角质形成细胞与成纤维细胞在60、80g/L的载药支架上可获得较多增殖。③支架上的角质形成细胞形态良好,与支架紧密贴附,药物对细胞无明显毒性作用。结论:生肌液质量浓度为60,80g/L的明胶-壳聚糖载药支架可有效促进角质形成细胞和成纤维细胞的黏附和增殖。

BACKGROUND： Tissue-engineered skin plays an important role in the repair and reconstruction of large-area skin lesion. Little is known about the effects of Chinese medicine-loading tissue-engineered skin scaffold on cell adhesion and wound surface infection. OBJECTIVE： To screen the application concentration of shengji solution, which can promote tissue regeneration, using keratinocytes and fibroblasts, and to observe the effects of shengji solution-gelatin-chitosan drug-loading skin scaffold on cellular adhesion and biocompatibility. DESIGN, TIME AND SETTING： Taking keratinocytes and fibrobtasts as subjects, the present randomized and controlled experiment was performed at the Laboratory of Cell Engineering, Orthopedic Institute, Tianjin Hospital between February and August 2005. MATERIALS： One healthy big-ear rabbit was included for harvesting skin seed cells. Shengji solution, Danggui （Radix Angelicae Sinensis） and Shengdi （rehmannia dride rhizome） extract （self-extracted, 1.5 g/mL）, gelatin-chitosan scaffold and shengji solution-gelatin-chitosan scaffold were provided by Tianjin University, China. METHODS： Passage 3 keratinocytes and fibroblasts were harvested by dispase Ⅱ- trypsase- ethylenediamine tetraacetic acid（EDTA） digestion. The prepared cells were divided into 5 groups： control, shengji solution, Danggui, Shengdi, and epidermal growth factor （EGF）. Common culture solution containing 0.1 volume fraction of fetal bovine serum, shengjisolution （5, 6, 8, and 12 g/L）, Dangguiextract （8 g/L）, Shengdiextract （8 g/L）, and EGF culture medium （10 μg/L） were used in corresponding groups for cell culture. At 1,3, 5, and 7 days, cellular proliferation was detected by methyl thiazolyl tetrazolium （M-IT） assay. Keratinocytes and fibroblasts were cultured in different concentrations of shengji solution （50, 60, 80, and 120 g/L） and grouped according to different concentrations. At 7 and 14 days, cellular adhesion was observed by semi-quantitative method. At 4, 7, and 14 days, keratinocytes cultured by 60 and 80 g/L shengji solution were harvested for hematoxylin-eosin staining and subsequent scanning electron microscope observation. MAIN OUTCOME MEASURES： Effects of shengji solution on skin seed cell proliferation; effects of drug-loading scaffold on cellular adhesion; and cell-carrier biocompatibility. RESULTS： MTT detection results demonstrated that cells significantly proliferated after treatment of 5, 6, and 8 g/L shengji solution compared to the control group （P 〈 0.05）. Obvious proliferation of passage 3 keratinocytes and fibroblasts was found on 60 and 80 g/L drug-loading scaffold. Keratinocytes on the drug-loading scaffold exhibited good cellular morphology and closely adhered to the scaffold. Shengji solution had no apparent toxic effects on cells. CONCLUSION： Shengji solution （60 and 80 g/L）-gelatin-chitosan scaffold can effectively promote the adhesion and proliferation of keratinocytes and fibroblasts.