CELL SHEET-BASED SKIN SUBSTITUTE TO MODULATE VASCULATURE AND INVESTIGATE WOUND-HEALING ASSOCIATED ANGIOGENESIS

Jun 30, 2022, 2:50 PM
10m
Room: S1

Room: S1

Speaker

Muller, Laurent (CIRB-Collège de France)

Description

"Vasculature plays an essential role in skin physiology and its architecture and function are altered in aged and diseased skin. There is thus a need to develop innovative 3D in vitro models with adjustable and amenable vasculature. Several in vitro skin models co-seeding endothelial cells with fibroblasts and keratinocytes have been proposed using scaffolds or bioprinting. However, they all fail in faithfully mimicking native skin microenvironment, including its complex ECM, papillary and reticular specificity or wound-healing associated microenvironment. Indeed, despite cellular remodeling of the scaffold material at the cellular level, exogenous proteins remain as the unique major component thus limiting assessment of the actual dermis microenvironment dynamics.
The aim of this work was to develop a vascularized skin substitute in a dermal microenvironment generated by fibroblasts and displaying plasticity in response to angiogenic factors or physiologic processes. We thus used the scaffold-free approach of cell sheet co-culture to produce the skin microenvironment. Skin primary fibroblast cell sheets co-seeded with endothelial cells or keratinocytes were cultured and superimposed to generate vascularized full-thickness skin substitutes. Using immunofluorescence and transmission electron microscopy, we confirmed the presence of a fully differentiated epidermis and well-structured dermal-epidermal junction. Whole-mount immunofluorescence demonstrated that endothelial cells organized into a dense vascular network throughout the dermis. Capillaries displayed a lumen and were stabilized by a basement membrane and the recruitment of perivascular cells. Modulating Vascular Endothelial Growth Factor (VEGF) concentration in the ng/ml range and time of application differentially affected angiogenesis in our model, resulting in distinct vascular network length and branching. Interestingly, these variations also impacted epidermis differentiation and proliferation. Furthermore, applying a full thickness wound to the skin substitute resulted in wound closure mimicking the time frame and ordered physiological process. In this context, we could follow centripetal revascularization by sprouting angiogenesis from the wound boundaries.
We have thus implemented a novel skin substitute displaying vascular plasticity in response to subtle angiogenic stimuli and wound healing. This model is of interest to mimic physiological and compromised skin conditions involving the vascular component (aging, neuro-inflammatory diseases, etc) and to evaluate the capacity of natural active molecules to restore skin vascular homeostasis."
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