Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents
Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents
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DOI：
10.1016/j.ejcb.2006.12.005
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发表时间：
2007-12-01
影响因子：
6.6
通讯作者：
Stark, Hans-Juergen
中科院分区：
文献类型：
--
作者：
Boehnke, Karsten;Mirancea, Nicolae;Stark, Hans-Juergen
体外生成的皮肤模型作为再生医学基础研究和临床应用的有前景的工具越来越受到人们的关注。在这里，我们提出了改进的长期皮肤当量(SE)的进一步细节，使皮肤重建和表皮功能的机制研究成为可能。通过分析成纤维细胞在三维支架中的生长条件，为常规组织重建和角质形成细胞的功能提供真实的真皮基质，从而优化真皮微环境。这些SE表现出持续的表皮活性-超过12周-规则的分化，所有分化产物的体内样图案证实了这一点，这里的例子是角化的包膜成分loricrin和repen。ZO-1在颗粒层中所有主要紧密连接成分的连续表达，与表皮屏障脂类的存在相一致，以及板层小体的超微结构积累，共同表明了适当的表皮屏障结构。值得注意的是，共培养的角质形成细胞对定植于支架上的成纤维细胞起到持续的增殖刺激作用，可与成纤维细胞生长因子的混合物相媲美。因此，在基础或富含生长因子的培养液中预培养真皮替代物(DES)对共培养的表皮再生质量的影响很小。至于成纤维细胞数量的作用，完全缺乏真皮细胞会导致上皮萎缩，但细胞数量低至5×10(4)个/厘米(2)的细胞对表皮组织质量的影响与标准密度(2×10(5)个/厘米(2))相当。令人惊讶的是，成纤维细胞在DES中预培养7天(标准)并没有显示出比2天更好的表皮组织形成效果，而14天的预培养会导致萎缩的表皮和真皮组织发育。这些数据表明：(1)表皮组织的再生严格依赖于成纤维细胞的存在；(2)角化细胞与成纤维细胞相互作用对细胞增殖和器官发生有重要作用；(3)适宜的微环境对表皮组织的功能和体外干细胞生态位的建立具有重要意义。(C)2007年爱思唯尔股份有限公司。版权所有。
In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured keratinocytes. These SEs demonstrate sustained epidermal viability - over 12 weeks - with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured keratinocytes exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast growth factors. Consequently, precultivation of dermal equivalents (DEs) in basal or growth factor-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5 x 10(4) cells/cm(2) on epidermal tissue quality equalled that of the standard density (2 x 10(5) cells/cm(2)). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to 2 days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual keratino cyte-fi bro blast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro. (c) 2007 Elsevier GmbH. All rights reserved.