The skin is the largest human organ, serving a fundamental role in protecting the human body from harmful external stimuli by acting as a physical and biological barrier. The human skin can be divided into three layers, the epidermal layer being of interest in this study. The human epidermis, mainly populated by keratinocytes, can in turn be divided into 5 cell layers, each distinguished by the keratinocyte differentiation state. In addition, laminins are of interest in this study. Laminins are large glycoproteins that are secreted, as heterotrimers, by e.g., keratinocytes. They are a major component of the basal lamina of in vivo skin and are involved in various signalling pathways and regulate the adhesion, migration, differentiation, and apoptosis of keratinocytes.
Human skin equivalents (HSEs) aim to represent the human skin, allowing us to study its physiology and relation to disease. Although various biomaterials have been used to construct HSEs, many of them suffer from poor mechanical integrity, elasticity, fragile ECM structure and/or require functionalization. FN-4RepCT, a recombinantly produced spider silk protein, forms a biomaterial that overcomes these shortcomings. It contains four repetitive polyalanine blocks and four glycine-rich units, a C-terminal domain, and a fibronectin derived RGD motif. Protein solutions of FN-4RepCT can be used to produce 3D-scaffolds of various formats, that are protein-permeable, biodegradable, elastic, mechanically robust, and support cell proliferation. Within this project FN-silk membranes, with nanofibrillar structures, are formed under ambient conditions and are used to construct an epidermal equivalent integrated with laminins and keratinocytes. The method presented herein for forming such membranes, and coating them with key laminins, is easy and may be repeated without the use of complex laboratory equipment.
The results presented herein provides further support for FN-4RepCT as a biomaterial within the field of tissue engineering. It is also demonstrated that laminins, combined with the silk, promote the proliferation and differentiation of keratinocytes, and that coating FN-silk membranes with laminins is beneficial for construction of a physiologically relevant epidermal equivalent. The epidermal constructs were evaluated with immunofluorescence staining of the keratinocyte differentiation markers keratin 5, keratin 10, and involucrin. The results reveal that the construct is built up by multiple cell layers with distinct morphology and expression of the aforementioned differentiation markers. Overall, it can be concluded that FN-4RepCT, in combination with laminins, is a promising option for tissue engineering of epidermal constructs.