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真皮替代物中的真实成纤维细胞基质可使器官型共培养中的表皮组织发生和真皮表皮交界处正常化。

Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture.

作者信息

Stark Hans-Jürgen, Willhauck Michael J, Mirancea Nicolae, Boehnke Karsten, Nord Iris, Breitkreutz Dirk, Pavesio Alessandra, Boukamp Petra, Fusenig Norbert E

机构信息

German Cancer Research Center (DKFZ), Heidelberg, Germany.

出版信息

Eur J Cell Biol. 2004 Dec;83(11-12):631-45. doi: 10.1078/0171-9335-00435.

Abstract

Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin, tenascin), with time collagens of mature dermis (type I and III) were accumulating giving rise to an in vivo-like matrix with regularly organised bundles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche.

摘要

除了作为复合皮肤移植的医学应用外,体外构建的皮肤替代物(SEs)或器官型共培养物是皮肤生物学的宝贵工具。传统模型采用胶原蛋白水凝胶作为真皮替代物(DEs),其主要缺点是稳定性较差且寿命有限,将研究限制在皮肤再生的早期阶段。在此,我们展示了一种改进的体外稳定模型,该模型实际上为类似皮肤的内环境稳定提供了基础。角质形成细胞生长在由改性透明质酸纤维增强的真皮替代物(Hyalograft-3D)上,并接种皮肤成纤维细胞,产生真正的真皮型基质。这些SEs形成了具有正常分化和超微结构的优质表皮结构,其发生速度也比基于胶原蛋白-DEs的SEs更快。早期仍不平衡的分化关键方面被完美地重新归一化,最显著的是角蛋白K1/K10的共表达以及再生相关角蛋白如K16的下调。整合素和K15分布以及角质形成细胞增殖仅限于基底层,这突出了恢复的组织极性,而生长速率降至生理水平则意味着最终实现了内环境稳定。这与更快的基底膜(BM)形成以及超微结构定义的真皮-表皮连接相关,包括丰富的锚定纤维以实现牢固的组织连接。支架中的成纤维细胞最初分泌典型的临时再生基质(纤连蛋白、腱生蛋白),随着时间的推移,成熟真皮的胶原蛋白(I型和III型)逐渐积累,形成具有规则排列的条纹状胶原纤维束的类似体内的基质。与传统DEs中更多的分解代谢状态相反,真正真皮组织的从头重建似乎是维持角质形成细胞长期正常增殖(随访长达8周)、满足组织内环境稳定标准并可能提供干细胞微环境的关键因素。

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