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A powerful replica molding methodology to transfer on-demand functional topographies to the surface of bacterial cellulose nanofiber textures is presented. With this method, termed guided assembly-based biolithography (GAB), a surface-structured polydimethylsiloxane (PDMS) mold is introduced at the gas-liquid interface of an Acetobacter xylinum culture. Upon bacterial fermentation, the generated bacterial cellulose nanofibers are assembled in a three-dimensional network reproducing the geometric shape imposed by the mold. Additionally, GAB yields directional alignment of individual nanofibers and memory of the transferred geometrical features upon dehydration and rehydration of the substrates. Scanning electron and atomic force microscopy are used to establish the good fidelity of this facile and affordable method. Interaction of surface-structured bacterial cellulose substrates with human fibroblasts and keratinocytes illustrates the efficient control of cellular activities which are fundamental in skin wound healing and tissue regeneration. The deployment of surface-structured bacterial cellulose substrates in model animals as skin wound dressing or body implant further proves the high durability and low inflammatory response to the material over a period of 21 days, demonstrating beneficial effects of surface structure on skin regeneration.

Full-thickness skin defects can be covered with dermal skin substitutes in combination with split-thickness skin grafts. However, slow vascularization of the matrices bears the risk of wound infection and extends the length of hospitalization. To overcome these problems, we describe a promising vascularization strategy. Green fluorescent protein adipose tissue-derived microvascular fragments (ad-MVF) were isolated from epididymal fat pads of C57BL/6-Tg(CAG-EGFP)1Osb/J mice. ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects in the dorsal skinfold chamber of wild-type C57BL/6 mice. Nonseeded matrices served as controls. Vascularization, lymphangiogenesis, and integration of the implants were studied by using intravital fluorescence microscopy, histology, and immunohistochemistry over 14 days. ad-MVF rapidly reassembled into microvascular networks within the implants, which developed interconnections to the host microvasculature. Accordingly, vascularization of the implants was markedly accelerated, as indicated by a significantly higher microvessel density when compared with controls. Moreover, dense lymphatic networks originating from the green fluorescent protein ad-MVF developed within the implants. This was associated with an improved implant integration. Hence, seeding ad-MVF on collagen-glycosaminoglycan matrices represents a potential strategy to reduce morbidity and hospitalization of patients undergoing the treatment of full-thickness skin defects.

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Vascularization is a key process in skin tissue engineering, determining the biological function of artificial skin implants. Hence, efficient vascularization strategies are a major prerequisite for the safe application of these implants in clinical practice. Current approaches include (i) modification of structural and physicochemical properties of dermal scaffolds, (ii) biological scaffold activation with growth factor-releasing systems or gene vectors, and (iii) generation of prevascularized skin substitutes by seeding scaffolds with vessel-forming cells. These conventional approaches may be further supplemented by emerging strategies, such as transplantation of adipose tissue-derived microvascular fragments, 3D bioprinting and microfluidics, miRNA modulation, cell sheet engineering, and fabrication of photosynthetic scaffolds. The successful translation of these vascularization strategies from bench to bedside may pave the way for a broad clinical implementation of skin tissue engineering.

The micron-scale surface topography of implanted materials represents a complementary pathway, independent of the material biochemical properties, regulating the process of biological recognition by cells which mediate the inflammatory response to foreign bodies. Here we explore a rational design of surface modifications in micron range to optimize a topography comprised of a symmetrical array of hexagonal pits interfering with focal adhesion establishment and maturation. When implemented on silicones and hydrogels in vitro, the anti-adhesive topography significantly reduces the adhesion of macrophages and fibroblasts and their activation toward effectors of fibrosis. In addition, long-term interaction of the cells with anti-adhesive topographies markedly hampers cell proliferation, correlating the physical inhibition of adhesion and complete spreading with the natural progress of the cell cycle. This solution for reduction in cell adhesion can be directly integrated on the outer surface of silicone implants, as well as an additive protective conformal microstructured biocellulose layer for materials that cannot be directly microstructured. Moreover, the original geometry imposed during manufacturing of the microstructured biocellulose membranes are fully retained upon in vivo exposure, suggesting a long lasting performance of these topographical features after implantation.

This new model allows for repetitive analysis of the microcirculation during skin graft healing. It provides ideal in vivo conditions to further delineate the exact mechanisms of blood vessel interconnection during the complex process of angiogenesis, and may also allow study of the vascularization of tissue-engineered skin substitutes.

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