Biologically well-tolerated materials are at the core of intense research. In this study, we report both the intrinsic features of formic acid (FA)-crosslinked three-dimensional (3D) nonwoven scaffolds consisting of sericin-deprived, Bombyx mori silk fibroin (SF) in β-sheet form and the long-term co-culture of adult human epidermal keratinocytes (HEKs) and dermal fibroblasts (HDFs) on them. The results of scanning electron microscopy, differential scanning calorimetry, and thermogravimetric and tensile strength studies showed that such scaffolds are made by a composite material, in which anisotropic SF fibres are enclosed within an isotropic matrix of SF in film form. Both fibres and films are firmly crosslinked by FA treatment and water-insoluble owing to their β-sheet crystalline structure. Moreover, while a certain strength of the material in the dry state may favour its manufacturing, handling, and shaping, the dramatic loss of hardiness under wet conditions renders the scaffolds softer, mechanically more compliant and, hence, more apt for implantation. Normal HEKs and HDFs could be successfully co-cultured on such nonwovens for up to 75-95 days in vitro, thus forming a novel kind of dermo-epidermal equivalent, in which the cells were metabolically active and performed specific functions (e.g. the de novo production and assembly of collagen fibres), but never released urea nitrogen (an index of protein catabolism) or proinflammatory interleukin-1β (IL-1β). In keeping with the results of previous studies in animals (Dal Pra I, et al, Biomaterials 26: 1987-1989, 2005), these findings support the view that 3D SF-based nonwovens may be excellent candidates for beneficial applications in the field of human tissue engineering/regeneration/repair.

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