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首页> 外文期刊>Tissue engineering, Part A >Making More Matrix: Enhancing the Deposition of Dermal-Epidermal Junction Components In Vitro and Accelerating Organotypic Skin Culture Development, Using Macromolecular Crowding
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Making More Matrix: Enhancing the Deposition of Dermal-Epidermal Junction Components In Vitro and Accelerating Organotypic Skin Culture Development, Using Macromolecular Crowding

机译：制作更多矩阵：使用大分子拥挤增强体外表皮结合点成分的沉积并促进器官型皮肤培养的发展

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摘要

Skin is one of the most accessible tissues for experimental biomedical sciences, and cultured skin cells represent one of the longest-running clinical applications of stem cell therapy. However, culture-generated skin mimetic multicellular structures are still limited in their application by the time taken to develop these constructs in vitro and by their incomplete differentiation. The development of a functional dermal-epidermal junction (DEJ) is one of the most sought after aspects of cultured skin, and one of the hardest to recreate in vitro. At the DEJ, dermal fibroblasts and epidermal keratinocytes interact to form an interlinked basement membrane of extracellular matrix (ECM), which forms as a concerted action of both keratinocytes and fibroblasts. Successful formation of this basement membrane is essential for take and stability of cultured skin autografts. We studied interactive matrix production by monocultures and cocultures of primary human keratinocytes and fibroblasts in an attempt to improve the efficiency of basement membrane production in culture using mixed macromolecular crowding (mMMC); resulting ECM were enriched with the deposition of collagens I, IV, fibronectin, and laminin 332 (laminin 5) and also in collagen VII, the anchoring fibril component. Our in vitro data point to fibroblasts, rather than keratinocytes, as the major cellular contributors of the DEJ. Not only did we find more collagen VII production and deposition by fibroblasts in comparison to keratinocytes, but also observed that decellularized fibroblast ECM stimulated the production and deposition of collagen VII by keratinocytes, over and above that of keratinocyte monocultures. In confrontation cultures, keratinocytes and fibroblasts showed spontaneous segregation and demarcation of cell boundaries by DEJ protein deposition. Finally, mMMC was used in a classical organotypic coculture protocol with keratinocytes seeded over fibroblast-containing collagen gels. Applied during the submerged phase, mMMC was sufficient to accelerate the emergence of collagen VII along the de novo DEJ, together with stronger transglutaminase activity in the neoepidermis. Our findings corroborate the role of fibroblasts as important players in producing collagen VII and inducing collagen VII deposition in the DEJ, and that macromolecular crowding leads to organotypic epidermal differentiation in tissue culture in a significantly condensed time frame.
机译：皮肤是实验生物医学科学中最容易接近的组织之一，培养的皮肤细胞代表了干细胞疗法中运行时间最长的临床应用之一。然而，培养物产生的模拟皮肤的多细胞结构的应用仍然受到体外开发这些构建体所花费的时间及其不完全分化的限制。功能性皮肤-表皮连接（DEJ）的开发是培养皮肤最需要的方面之一，也是最难在体外重建的方面之一。在DEJ，真皮成纤维细胞和表皮角质形成细胞相互作用形成细胞外基质（ECM）的相互连接的基底膜，形成角质形成细胞和成纤维细胞的协同作用。该基底膜的成功形成对于培养的皮肤自体移植物的摄取和稳定性至关重要。我们研究了原代人角质形成细胞和成纤维细胞的单培养和共培养的交互式基质生产，以试图提高使用混合大分子拥挤（mMMC）培养的基底膜的生产效率;所得的ECM富含胶原蛋白I，IV，纤连蛋白和层粘连蛋白332（层粘连蛋白5）的沉积，还富含胶原蛋白VII（锚定的原纤维成分）。我们的体外数据表明成纤维细胞而不是角质形成细胞是DEJ的主要细胞来源。与角质形成细胞相比，我们不仅发现成纤维细胞产生更多的胶原蛋白VII和沉积，而且还观察到脱细胞的成纤维细胞ECM刺激角质形成细胞产生胶原蛋白VII的产生和沉积，超过了角质形成细胞单培养。在对抗性培养中，角质形成细胞和成纤维细胞通过DEJ蛋白沉积显示出自发的分离和细胞边界的分界。最后，将mMMC用于经典的器官型共培养方案，将角质形成细胞接种在含成纤维细胞的胶原蛋白凝胶上。在浸没阶段应用mMMC足以促进从头开始的DEJ胶原VII的出现，同时在新表皮中具有更强的转谷氨酰胺酶活性。我们的发现证实了成纤维细胞在DEJ中产生胶原VII和诱导胶原VII沉积方面起着重要作用，并且大分子拥挤在明显浓缩的时间框架内导致组织培养中的器官型表皮分化。

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