Picture Overviews - Skingraft on base of hAM

Allogenic graft material and tissue engineering have recently shown promising results for the improvement of both aesthetic and functional outcomes in the treatment of large skin defects. We chose human amniotic membranes (hAM) as a cellular scaffold in order to develop a skin substitute for later in-vivo uses. Various methods of de-epithelialization of the hAM were evaluated by histological analysis including hematoxylin-eosin and laminin staining, optic coherence tomography (OCT), and scanning electron microscopy with 0.25/0.02% trypsin/EDTA treatment and mechanical cell removal showing a complete loss of the epithelium and a mainly intact basement membrane. Novel examination of hAM by OCT was feasible, but difficulties were experienced in handling and interpretation of the tissue as no comparable data exist. Subsequently, we developed an air-liquid-interface cell culture to cultivate keratinocytes and fibroblasts on the de-epithelialized hAM. When comparing various air-liquid cell-culture media and resulting cell viability, we found that a mainly keratinocyte-based medium containing a low amount of serum provided good keratinocyte and fibroblast viability. We achieved a mostly keratinized surface on the epidermal side with a confluent fibroblast network on the chorion side.

异体移植材料与组织工程领域近期在治疗大型皮肤缺损方面展现出良好前景，可同时改善治疗后的美学与功能预后效果。本研究选用人羊膜（human amniotic membranes, hAM）作为细胞支架，以开发可用于后续体内实验的皮肤替代物。本研究通过组织学分析（包括苏木精-伊红染色、层粘连蛋白染色）、光学相干断层扫描（optical coherence tomography, OCT）以及扫描电镜，对多种人羊膜脱上皮方法进行了评估；结果显示，采用0.25/0.02%胰蛋白酶/乙二胺四乙酸（trypsin/EDTA）联合机械去除细胞的方法，可完全去除上皮层且基底膜基本保持完整。采用OCT对hAM进行新型检测具备可行性，但由于缺乏可参照的同类数据，该组织样本的处理与结果解读存在一定难度。随后，本研究构建了气液界面细胞培养体系，以在脱上皮后的hAM上培养角质形成细胞与成纤维细胞。通过对比多种气液界面细胞培养基及对应的细胞存活情况，本研究发现：采用含低浓度血清的角质形成细胞基础培养基，可获得良好的角质形成细胞与成纤维细胞存活率。最终，在表皮侧构建了基本角化的细胞表面，而绒毛膜侧则形成了融合的成纤维细胞网络。