Golinski P A, Zöller N, Kippenberger S, Menke H, Bereiter-Hahn J, Bernd A
Zentrum der Dermatologie und Venerologie, Goethe-Universität, Frankfurt/M.
Handchir Mikrochir Plast Chir. 2009 Dec;41(6):327-32. doi: 10.1055/s-0029-1234132. Epub 2009 Aug 26.
A cell-based wound coverage with keratinocytes and fibroblasts on the basis of a commercially available dermal substitute (Matriderm ((R)), Kollagen/Elastin matrix) was generated, in order to treat wide burn wounds. First the expansion of keratinocytes was optimised and the culturing time was minimised. Raw material was 1-2 cm (2) split skin. Dermis and epidermis were separated by enzymatic treatment with thermolysin. After treatment of both compartments with trypsin and collagenase I, keratinocytes and fibroblasts were isolated and expanded in collagen I coated dishes. After 10 days fibroblasts were seeded on Matriderm ((R)). After cultivation of the fibroblasts-containing matrix for one week keratinocytes were seeded on top. After an additional week of submersed cultivation the matrix was lifted up to the air-liquid interface to initiate epidermal cell differentiation. After 16 days in the air-liquid interphase the matrix was fixed and underwent immunohistochemical and electron microscopic analysis. Histological analysis showed a regularly stratification of the epidermal part. We observed collagen IV, a marker for the basement membrane, between epidermis and dermis. Desmoglein and the differentiation markers involucrine and cytokeratin 10 were found in the suprabasal layers of the epidermis. Electron microscopic analysis showed the basement membrane in the epidermal junction zone as well as cell-cell connections in the form of desmosomes. Late differentiation characteristics, like granular structures and the cornified layer, were found in the stratum granulosum and stratum corneum. Our results demonstrate that a skin equivalent can be generated by using a collagen/elastin matrix, with an expansion rate of 50-100-fold. This skin equivalent may be useful for covering deep wounds.
为治疗大面积烧伤创面，在市售真皮替代物（Matriderm ((R))，胶原蛋白/弹性蛋白基质）的基础上构建了含角质形成细胞和成纤维细胞的细胞基创面覆盖物。首先优化角质形成细胞的扩增并尽量缩短培养时间。原材料为1 - 2平方厘米的断层皮片。通过嗜热菌蛋白酶的酶处理分离真皮和表皮。用胰蛋白酶和胶原酶I处理两个部分后，分离角质形成细胞和成纤维细胞，并在涂有I型胶原的培养皿中进行扩增。10天后，将成纤维细胞接种到Matriderm ((R)) 上。在含成纤维细胞的基质培养一周后，将角质形成细胞接种在其上方。再经过一周的浸没培养后，将基质提升至气液界面以启动表皮细胞分化。在气液界面培养16天后，将基质固定并进行免疫组织化学和电子显微镜分析。组织学分析显示表皮部分有规则的分层。我们在表皮和真皮之间观察到了作为基底膜标志物的IV型胶原。在表皮的基底上层发现了桥粒芯糖蛋白以及分化标志物内披蛋白和细胞角蛋白10。电子显微镜分析显示表皮连接区有基底膜以及桥粒形式的细胞间连接。在颗粒层和角质层发现了晚期分化特征，如颗粒结构和角质层。我们的结果表明，使用胶原蛋白/弹性蛋白基质可以构建皮肤替代物，扩增率为50 - 100倍。这种皮肤替代物可能有助于覆盖深度创面。
