Layered protein fiber scaffolds for skin tissue engineering

由于人口的快速老龄化以及糖尿病和肥胖症发病率的增加，世界范围内皮肤伤口的数量急剧增加。目前治疗皮肤缺陷的方法有多种缺点，包括可获得的供体细胞有限或体外扩增细胞耗时。因此，皮肤再生仍然是再生医学的主要挑战，需要新的支架材料。当开发新的皮肤模型时，特别重要的是模仿具有不同孔隙度和厚度的天然皮肤的分层结构。同时，皮肤替代品需要通过选择性靶向不同的整合素来支持不同细胞类型的同时生长。然而，尽管在皮肤再生方面进行了广泛的研究，但在皮肤组织工程所需的体外多层蛋白支架研究方面仍然存在知识空白。因此，我们的项目目标是建立具有纳米纤维结构的分层纤维蛋白原-胶原支架，作为皮肤组织工程的新型无细胞支架。我们的项目将基于一个关键问题：“具有分层3d结构的纳米纤维蛋白原-胶原复合材料是否适合用于皮肤组织工程的支架材料？”，我们将在三个连续的工作包（wp）中回答这个问题。WP 1：我们将纤维蛋白原和胶原纳米纤维的不同自组装程序结合成一个新的工艺，建立新的层状蛋白支架。一个重要的焦点将是裁剪单个蛋白质层的厚度和孔隙度，以密切模仿天然皮肤的生物物理特征。最后，我们将开发一个分离程序，以可重复地制造具有分层结构的独立蛋白质支架。WP 2：为了使层状蛋白支架作为湿润环境下的皮肤模型得以应用，需要对其力学特性和肿胀特性进行定制。此外，为了研究纤维蛋白原-胶原支架在空气-液界面（ALI）共培养装置中的相互作用，支架在再水化时需要具有机械稳定性。因此，我们将通过拉伸试验结合膨胀行为研究来分析复水合蛋白支架的力学性能。WP 3：成功的皮肤修复需要不同类型细胞的共同培养。因此，我们将利用HaCaT角化细胞和人真皮成纤维细胞，研究它们在体外ALI条件下在层状蛋白支架上的生长和浸润。评估不同共培养设置的重要参数将是细胞增殖和迁移以及细胞特异性形态和不同蛋白质的表达。基于这一工作计划，我们的项目将引入一种新的皮肤模型进行体外研究，这将为皮肤细胞与分层支架的相互作用提供基本的见解。

Due to the rapidly aging population and increasing incidence of diabetes and obesity, the number of skin wounds has increased tremendously worldwide. Current approaches to treat skin defects bear manifold disadvantages including limited availability of viable donor cells or time-consuming in vitro expansion of harvested cells. Therefore, skin regeneration remains a major challenge in regenerative medicine that requires novel scaffold materials. When developing new skin models, it is particularly important to mimic the layered architecture of native skin with varying porosity and thickness. At the same time, skin substitutes need to support the simultaneous growth of different cell types by selectively targeting different integrins. Yet, despite extensive research in skin regeneration there is still a knowledge gap when it comes to multi-layered protein scaffolds for in vitro studies as required for skin tissue engineering. Therefore, the aim of our project is to establish layered fibrinogen-collagen scaffolds with nanofibrous architecture as new cell-free scaffolds for skin tissue engineering. Our project will be based on the key question “Are nanofibrous fibrinogen-collagen composites with layered 3D-architecture suitable scaffold materials for skin tissue engineering?”, which we will answer in three consecutive work packages (WPs). WP 1: We will establish new layered protein scaffolds by combining different self-assembly routines for fibrinogen and collagen nanofibers into a new process. An important focus will be on tailoring the thickness and porosity of individual protein layers to closely mimic the biophysical features of native skin. Finally, we will develop a detachment routine to reproducibly fabricate free-standing protein scaffolds with layered architecture. WP 2: To facilitate the application of layered protein scaffolds as skin models in a moist environment their mechanical characteristics and swelling properties need to be tailored. Moreover, to study the interaction of fibrinogen-collagen scaffolds in a co-culture setup at the air-liquid-interface (ALI) the scaffolds need to be mechanically stable when being rehydrated. Therefore, we will analyze the mechanical properties of rehydrated protein scaffolds by tensile testing in combination with studies of the swelling behavior. WP 3: Successful skin repair requires the co-cultivation of different cell types. Therefore, we will use HaCaT keratinocytes and human dermal fibroblasts to study their growth and infiltration on layered protein scaffolds at the ALI under in vitro conditions. Important parameters to evaluate different co-culture setups will be cell proliferation and migration as well as cell-specific morphology and expression of different proteins. Based on this work program, our project will introduce a new skin model for in vitro studies that will provide fundamental insights into the interaction of skin cells with layered scaffolds.