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BACKGROUND: To date, the reconstructive approach addressing chronic non-healing wounds, deep tissue damage, and severe skin defects often relies upon avascular dermal grafts, either autologous or engineered. All such grafts are limited by their reliance upon host cellular invasion for neovascularization and incorporation, which makes them impractical for use in all but the most optimal wound beds. Herein, we fabricate a pre-vascularized full-thickness cellularized skin equivalent containing a three-dimensional hierarchical vascularized network of interconnected macro and microchannels lined with vascular and mural cells and, within a collagen neodermis containing encapsulated fibroblasts, and an epidermis comprised of human keratinocytes that may be anastomosed to provide immediate perfusion of the construct.
METHODS: Pluronic F127 was used for network preparation: 1.5 mm diameter “U” shaped macrofibers and 100-500 µm-interwoven microfibers were heat extruded and then embedded within Type I collagen into which CFP-tagged human placental pericytes (HPLP-CFP) and human foreskin fibroblasts (HFF1) at a density of 1x10^6 cells/mL, respectively had been encapsulated. After pluronic sacrifice, channels were intraluminally seeded with 5x10^6 cells/mL RFP-tagged human aortic smooth muscle cells (HASMC-RFP), 5x10^6 cells/mL GFP-tagged human umbilical vein endothelial cells (HUVEC-GFP). The construct was then topically seeded with 1x10^6 cells/mL human epidermal keratinocytes (HEK). Constructs were incubated for 14 and 28 days and analyzed using multiphoton microscopy (MPM) or fixed and processed for histology. Additionally, after 14 and 28 days of culture, endothelial cells were extracted from the construct using collagenase digestion and RT-qPCR was performed to analyze expression of markers of angiogenesis and maturation of the vascular network.
RESULTS: Following 14 and 28 days of culture, MPM imaging demonstrated a hierarchical vascular network of interconnected microchannels successfully lined by endothelial and smooth muscle cells, supported by perivascular pericytes, all in appropriate microanatomic arrangement. Neodermal HFF1 proliferated throughout the observation period and the HEK neoepidermis developed into a stratified epithelium along the superficial aspect of the construct. MPM imaging revealed sprouting angiogenesis through 14 days of culture, and RT-qPCR demonstrated increased endothelial expression of Jagged 1, a marker of angiogenic activity. Following 28 days of culture, MPM images revealed stabilized neovessels within our skin flap environment, which was further delineated through increase endothelial cell expression of Dll4, a marker of microvascular stabilization and maturation. Self-assembly of a stable microvascular network was further demonstrated by migration of pericytes from an initially homogenous distribution within the bulk to a predominantly perivascular location, with 74% of pericytes found within 200 µm of a microchannel.
CONCLUSIONS: We have successfully fabricated the first ever tissue-engineered pre-vascularized full thickness skin flap, which recapitulates the inherent hierarchical vasculature found within the human skin and is suitable to provide whole tissue perfusion. We provide the platform for an on-demand, geometrically tunable tissue engineered equivalent with an anastomosable vascular network which we believe will serve as a next generation skin equivalent.