Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Tissue-Engineered, Vascularized Skin Flaps Containing Dermal Appendages
Rachel C. Hooper, MD, Adam M. Jacoby, BA, Ope A. Asanbe, MD, Wilmina N. Landford, BA, Peipei Zhang, PhD, Hector L. Osoria, BS, Alice Harper, BA, Jason A. Spector, MD, FACS.
Weill Cornell Medical College, New York, NY, USA.

Purpose: Reconstruction of large areas of skin loss is challenging, especially as sites for autologous tissue harvest are limited. Despite widespread use, contemporary xenografts, allografts and autografts are avascular and rely upon non-guided host cellular invasion for neovascularization and incorporation. This occurs over several weeks, especially in patients with significant co-morbidities. Furthermore, none of these materials have dermal appendages. Here, we fabricate full-thickness skin equivalents with a hierarchical vascular supply consisting of an interweaving network of microvessels in circuit with macrovessels, also with and without dermal appendages.
Methods: Vascular networks were fabricated using one of two methods: 1) creating “U-shaped” 1.5 mm diameter Pluronic F127 macrofibers or 2) bridging these U-shaped fibers by fusing manually extruded 100-500 µm Pluronic F127 “mesh” or “checkerboard.” Pluronic F127 fibers were sacrificed in type I collagen with encapsulated human foreskin fibroblasts (HFF1) at a density of 1 x106 cells/mL. Twenty-four hours following fiber sacrifice, 5 x106 cells/mL of human aortic smooth muscle cells (HASMC) were seeded into the patent luminal space. The next day, 5 x106 cells/mL of human umbilical vein endothelial cells (HUVEC) were seeded. Lastly, 1 x106 cells/mL of human epidermal keratinocytes (HEK) were topically seeded onto scaffolds. Hair follicles from adult rats were implanted into the surface of a subset of scaffolds. Scaffolds underwent daily media changes and were fixed and processed for histology after 7 or 14 days of culture.
Results: Macrochannels were successfully lined with HUVEC and HASMC, generating anatomically appropriate neointimal and neomedial layers by day 7 and maintained by day 14, while smaller bridging microchannels predominantly contained HUVEC. Proliferation of HFF1 was evident after 7 days and increased after 14 days. HEK proliferated and increased in thickness in a time-dependent manner, leading to the formation of a stratified “epidermal-like” layer along the construct surface. Immunohistochemical analysis revealed CD31+ HUVEC along the luminal surface of the macrochannel and the one cell layer thick microvessel linings, α-SMA expressing HASMC in the subendothelial plane, fibroblast specific-1-expressing fibroblasts within the collagen bulk, and involucrin+ keratinocytes along the scaffold surface after both 7 and 14 days. Scaffolds with implanted rat hair follicles demonstrated appendages with intact inner root sheaths and hair shafts, with surrounding viable cells by day 7.
Conclusions: We successfully fabricated tissue-engineered, vascularized skin flaps with and without hair follicles. We successfully incorporated an inherent vascular network that recapitulates the hierarchical organization of an arteriole, venule, and capillary bed that is suitable for microanastomosis and immediate perfusion. With a built-in vascular network, vital epidermal (HEK) and dermal (HFF, collagen) components, and dermal appendages, these constructs hold tremendous promise for the future of full-thickness, tissue-engineered skin equivalents.


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