Pajoum Shariati Seyed Ramin, Shokrgozar Mohammad Ali, Vossoughi Manouchehr, Eslamifar Ali
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.
Biomedical Group, Dept. of Chemical Engineering and Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran.
Iran Biomed J. 2009 Jul;13(3):169-77.
Extensive full-thickness burns require replacement of both epidermis and dermis. In designing skin replacements, the goal has been to re-create this model and make a product which has both essential components.
In the present study, we developed procedures for establishing confluent, stratified layers of cultured human keratinocytes on the surface of modified collagen-chitosan scaffold that contains fibroblasts. The culture methods for propagation of keratinocytes and fibroblasts isolated from human neonatal foreskin were developed. The growth and proliferation of normal human keratinocytes were evaluated in serum-free (keratinocyte growth medium) and our modified medium. Characterization of human keratinocytes was determined by using pan-keratin and anti-involucrin monoclonal antibodies. For fabrication of relevant biodegradable and biocompatible collagen-chitosan porous scaffold with improved biostability, modified method of freeze-gelation was used. In generating organotypic co-cultures, epidermal keratinocytes were plated onto the upper surface of scaffold containing embedded fibroblasts.
The results showed that the growth of isolated human skin fibroblasts and keratinocytes in our modified medium was more than that in the serum-free medium. The different evaluations of collagen-chitosan scaffold showed that it is relevant to growth of cells (fibroblast and keratinocyte) and has a good flexibility in manipulation of the living skin equivalents.
These findings indicate that the integration of collagen-chitosan scaffold with co-cultured keratinocyte and fibroblast in vitro provides a potential source of living skin for grafting in vivo.
大面积全层烧伤需要替换表皮和真皮。在设计皮肤替代物时，目标是重新创建这种模型并制造出包含两种基本成分的产品。
在本研究中，我们开发了在含有成纤维细胞的改良胶原 - 壳聚糖支架表面建立汇合、分层的培养人角质形成细胞层的程序。开发了从人新生儿包皮分离的角质形成细胞和成纤维细胞的培养方法。在无血清（角质形成细胞生长培养基）和我们的改良培养基中评估正常人角质形成细胞的生长和增殖。使用泛角蛋白和抗内披蛋白单克隆抗体对人角质形成细胞进行表征。为了制造具有改善的生物稳定性的相关可生物降解和生物相容性的胶原 - 壳聚糖多孔支架，采用了改良的冷冻凝胶化方法。在生成器官型共培养物时，将表皮角质形成细胞接种到含有嵌入成纤维细胞的支架上表面。
结果表明，分离的人皮肤成纤维细胞和角质形成细胞在我们的改良培养基中的生长比在无血清培养基中更多。对胶原 - 壳聚糖支架的不同评估表明，它与细胞（成纤维细胞和角质形成细胞）的生长相关，并且在操纵活皮肤替代物方面具有良好的灵活性。
这些发现表明，胶原 - 壳聚糖支架与体外共培养的角质形成细胞和成纤维细胞的整合为体内移植提供了潜在的活皮肤来源。
