Mete Gunaydin Hilal, Fogg Saskia M, Moakes Richard J A, Grover Liam M
School of Chemical Engineering, University of Birmingham, Edgbaston, UK.
Methods Mol Biol. 2025;2922:173-186. doi: 10.1007/978-1-0716-4510-9_13.
Although it is now possible to use tissue engineering approaches to regenerate the dermis and epidermis of the skin, replicating the cellular framework and functions of the skin's three primary layers-epidermis, dermis, and hypodermis remains challenging. Here, we describe the use of a bioprinting method known as suspended layer additive manufacturing (SLAM) to produce a structure with a gradient of mechanical properties, supporting the growth of various cell types, to create a continuous tri-layered skin substitute that closely mimics human skin. SLAM utilizes fluid gels as a temporary support matrix to facilitate the extrusion bioprinting of soft hydrogel-based bioinks into complex, functional, three-dimensional (3D) tissue structures. The printed structures are initially designed using computer-aided design (CAD) and may be printed using a variety of bioinks that can replicate the three primary skin layers: epidermal, dermal, and hypodermal. To accurately reproduce the cellular framework and functions of human skin, it is crucial to use materials that can duplicate the natural extracellular matrix (ECM) structure. This structure provides structural support and appropriate biochemical cues to the embedded cells. Here we describe a method that uses pectin to mimic ECM polysaccharides, along with collagen, which is a major component of human skin ECM. The resulting structure is supported within an agarose support-bed and ionically cross-linked prior to removal. To accurately replicate the cellular framework of human skin, human epidermal keratinocytes (hEKs), human dermal fibroblasts (HDFs), and adipose-derived stem cells (ADSCs) were integrated into the scaffold. A 21-day culture of the skin construct showed that the cellular components were crucial in remodelling the printed structure into architectures resembling those of healthy skin. Integration of the implant, demonstrated by the mobilization of adipose tissue from the surrounding area into the construct, occurred within 7 days post-ex vivo implantation into a simulated porcine wound.
尽管现在可以使用组织工程方法来再生皮肤的真皮和表皮，但复制皮肤的三个主要层——表皮、真皮和皮下组织的细胞框架和功能仍然具有挑战性。在这里，我们描述了一种称为悬浮层增材制造（SLAM）的生物打印方法，用于生产具有机械性能梯度的结构，支持各种细胞类型的生长，以创建一种紧密模仿人类皮肤的连续三层皮肤替代物。SLAM利用流体凝胶作为临时支撑基质，以促进基于软质水凝胶的生物墨水挤出生物打印成复杂的、功能性的三维（3D）组织结构。最初使用计算机辅助设计（CAD）设计打印结构，并可以使用多种能够复制皮肤三个主要层（表皮、真皮和皮下组织）的生物墨水进行打印。为了准确再现人类皮肤的细胞框架和功能，使用能够复制天然细胞外基质（ECM）结构的材料至关重要。这种结构为嵌入的细胞提供结构支持和适当的生化信号。在这里，我们描述了一种方法，该方法使用果胶来模拟ECM多糖，以及胶原蛋白，胶原蛋白是人类皮肤ECM的主要成分。所得结构在琼脂糖支撑床内得到支撑，并在去除之前进行离子交联。为了准确复制人类皮肤的细胞框架，将人表皮角质形成细胞（hEKs）、人真皮成纤维细胞（HDFs）和脂肪来源干细胞（ADSCs）整合到支架中。皮肤构建体的21天培养表明，细胞成分对于将打印结构重塑为类似于健康皮肤的结构至关重要。植入物的整合表现为周围区域的脂肪组织动员到构建体中，在体外植入模拟猪伤口后7天内发生。
