Braziulis Erik, Biedermann Thomas, Hartmann-Fritsch Fabienne, Schiestl Clemens, Pontiggia Luca, Böttcher-Haberzeth Sophie, Reichmann Ernst, Meuli Martin
Department of Surgery, Tissue Biology Research Unit, University Children's Hospital Zurich, Steinwiesstrasse 75, Zurich, Switzerland.
Pediatr Surg Int. 2011 Mar;27(3):241-7. doi: 10.1007/s00383-010-2777-0.
Extended full thickness skin defects still represent a considerable therapeutic challenge as ideal strategies for definitive autologous coverage are still not available. Tissue engineering of whole skin represents an equally attractive and ambitious novel approach. We have recently shown that laboratory-grown human skin analogues with near normal skin anatomy can be successfully transplanted on immuno-incompetent rats. The goal of the present study was to engineer autologous porcine skin grafts for transplantation in a large animal model (pig study = intended preclinical study).
Skin biopsies were taken from the pig's abdomen. Epidermal keratinocytes and dermal fibroblasts were isolated and then expanded on culture dishes. Subsequently, highly concentrated collagen hydrogels and collagen/fibrin hydrogels respectively, both containing dermal fibroblasts, were prepared. Fibroblast survival, proliferation, and morphology were monitored using fluorescent labelling and laser scanning confocal microscopy. Finally, keratinocytes were seeded onto this dermal construct and allowed to proliferate. The resulting in vitro generated porcine skin substitutes were analysed by H&E staining and immunofluorescence.
Dermal fibroblast proliferation and survival in pure collagen hydrogels was poor. Also, the cells were mainly round-shaped and they did not develop 3D-networks. In collagen/fibrin hydrogels, dermal fibroblast survival was significantly higher. The cells proliferated well, were spindle-shaped, and formed 3D-networks. When these latter dermal constructs were seeded with keratinocytes, a multilayered and partly stratified epidermis readily developed.
This study provides compelling evidence that pig cell-derived skin analogues with near normal skin anatomy can be engineered in vitro. These tissue-engineered skin substitutes are needed to develop a large animal model to establish standardized autologous transplantation procedures for those studies that must be conducted before "skingineering" can eventually be clinically applied.
大面积全层皮肤缺损仍然是一个巨大的治疗挑战，因为目前仍没有理想的确定性自体覆盖策略。全层皮肤组织工程是一种同样具有吸引力且雄心勃勃的新方法。我们最近发现，具有近乎正常皮肤解剖结构的实验室培养的人类皮肤类似物能够成功移植到免疫缺陷大鼠身上。本研究的目的是构建自体猪皮移植物，用于大型动物模型（猪研究 = 预期的临床前研究）。
从猪腹部获取皮肤活检样本。分离表皮角质形成细胞和真皮成纤维细胞，然后在培养皿中进行扩增。随后，分别制备了均含有真皮成纤维细胞的高浓度胶原蛋白水凝胶和胶原蛋白/纤维蛋白水凝胶。使用荧光标记和激光扫描共聚焦显微镜监测成纤维细胞的存活、增殖和形态。最后，将角质形成细胞接种到这种真皮构建物上并使其增殖。通过苏木精和伊红染色以及免疫荧光分析所产生的体外培养的猪皮肤替代物。
真皮成纤维细胞在纯胶原蛋白水凝胶中的增殖和存活情况较差。此外，细胞主要呈圆形，未形成三维网络。在胶原蛋白/纤维蛋白水凝胶中，真皮成纤维细胞的存活率显著更高。细胞增殖良好，呈纺锤形，并形成三维网络。当将角质形成细胞接种到这些真皮构建物上时，很容易形成多层且部分分层的表皮。
本研究提供了令人信服的证据，表明具有近乎正常皮肤解剖结构的猪细胞来源的皮肤类似物能够在体外构建。需要这些组织工程皮肤替代物来建立大型动物模型，以便为那些在“皮肤工程”最终能够临床应用之前必须进行的研究建立标准化的自体移植程序。
