Department of Tissue Engineering & Regenerative Medicine (TERM), University Hospital Würzburg , Würzburg 97070, Germany.
Translational Center Würzburg 'Regenerative Therapies in Oncology and Musculoskeletal Diseases', Würzburg Branch of the Fraunhofer Institute for Interfacial Engineering and Biotechnology , Würzburg 97070, Germany.
ACS Appl Mater Interfaces. 2017 Jun 21;9(24):20417-20425. doi: 10.1021/acsami.7b04017. Epub 2017 Jun 9.
Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as nonanimal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically cross-link the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a noncytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), cross-linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air-liquid interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.
全厚皮肤等效物作为治疗大面积皮肤缺损或慢性创面的皮肤移植物或非动物测试平台，越来越受到关注。然而，其成纤维细胞介导的收缩和较差的机械稳定性导致其在体外和体内的重现性和适用性受到限制。为了克服这些缺陷，我们旨在通过化学交联由胶原 I 水凝胶组成的全厚皮肤模型的真皮层。使用非细胞毒性的四臂琥珀酰亚胺戊二酸聚乙二醇（PEG-SG），可以在细胞接种的胶原水凝胶中实现交联。PEG-SG/胶原 0.5mg/mg 的浓度导致与未交联的胶原水凝胶相当的活力，且细胞内乳酸脱氢酶的释放没有增加。与未交联的胶原水凝胶相比，交联的胶原水凝胶具有更高的机械稳定性，并且通过胶原酶降解的倾向更小。值得注意的是，在 21 天内，交联的胶原水凝胶保持其初始表面积，而标准的真皮模型收缩了 50%。最后，通过在等效物表面接种人表皮角质形成细胞并在气液界面培养这些等效物来生成全厚皮肤等效物。交联模型的免疫组织化学染色显示了定义明确的表皮层，包括完整的角质层和真皮部分，其中均匀分布有人真皮成纤维细胞。这些结果表明，PEG-SG 交联胶原可以减少胶原水凝胶的收缩，从而增加这些模型作为功效和安全性评估的附加工具或新一代皮肤移植物的适用性。
