Background The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes (hbTESSs) designed for the treatment of severely burned patients. These are the scaffold-free self-assembled skin substitute (SASS) and the human plasma-based skin substitute (HPSS). Methods Fibroblasts and keratinocytes from three humans were extracted from skin biopsies (N = 3) and cells from the same donor were used to produce both hbTESS models. For SASS manufacture, keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced (n = 12), while for HPSS production, keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial (Fibrin), plasma combined with hyaluronic acid (Fibrin-HA) or plasma combined with collagen (Fibrin-Col) (n/biomaterial = 9). The production time was 46-55 days for SASSs and 32-39 days for HPSSs. Substitutes were characterized by histology, mechanical testing, PrestoBlue™-assay, immunofluorescence (Ki67, Keratin (K) 10, K15, K19, Loricrin, type IV collagen) and Western blot (type I and IV collagens). Results The SASSs were more resistant to tensile forces (p-value < 0.01) but less elastic (p-value < 0.001) compared to HPSSs. A higher number of proliferative Ki67+ cells were found in SASSs although their metabolic activity was lower. After epidermal differentiation, no significant difference was observed in the expression of K10, K15, K19 and Loricrin. Overall, the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs. Conclusions This study demonstrates, for the first time, that both hbTESS models present similar in vitro biological characteristics. However, mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.

中文翻译：

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人体血浆基皮肤替代品和自组装组织工程皮肤替代品的体外比较：两种不同的制造工艺用于治疗深层且难以愈合的损伤。

背景 这项体外研究的目的是并排比较两种设计用于治疗严重烧伤患者的人体双层组织工程皮肤替代品 (hbTESS) 模型。它们是无支架自组装皮肤替代品（SASS）和人血浆皮肤替代品（HPSS）。方法 从皮肤活检中提取三名人类的成纤维细胞和角质形成细胞 (N = 3)，并使用来自同一供体的细胞来生成两个 hbTESS 模型。对于 SASS 制造，角质形成细胞被接种在三个自组装真皮片上，该真皮片包含成纤维细胞及其产生的细胞外基质 (n = 12)，而对于 HPSS 生产，角质形成细胞在水凝胶上培养，该水凝胶由嵌入任一血浆中的成纤维细胞作为独特的生物材料（纤维蛋白）组成。 ）、血浆结合透明质酸（Fibrin-HA）或血浆结合胶原蛋白（Fibrin-Col）（n/生物材料 = 9）。SASS 的生产时间为 46-55 天，HPSS 的生产时间为 32-39 天。通过组织学、机械测试、PrestoBlue™ 测定、免疫荧光（Ki67、角蛋白 (K) 10、K15、K19、Loricrin、IV 型胶原）和蛋白质印迹（I 型和 IV 型胶原）对替代品进行表征。结果 与 HPSS 相比，SASS 更能抵抗拉力（p 值 < 0.01），但弹性较差（p 值 < 0.001）。在 SASS 中发现了更多数量的增殖性 Ki​​67+ 细胞，尽管它们的代谢活性较低。表皮分化后，K10、K15、K19和Loricrin的表达无显着差异。总体而言，SASS 中 I 型和 IV 型胶原蛋白的产生以及真皮-表皮连接处的粘合强度更高。结论 本研究首次证明两种 hbTESS 模型表现出相似的体外生物学特性。然而，机械性能有所不同，未来的体内实验将旨在比较它们的伤口愈合潜力。