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Fabrication of a Tissue-Engineered Pre-Vascularized Perfusable Skin Flap
Ross H. Weinreb, BS1, Kerry A. Morrison, BA1, Julia Jin, BS1, Xue Dong, BA1, Yoshiko Toyoda, BA1, Adam Jacoby, MD1, Sushmita Mukherjee, Ph.D.2, Jason A. Spector, MD1.
1Laboratory of Bioregenerative Medicine & Surgery, Weill Cornell Medical Center, New York, NY, USA, 2Department of Biochemistry, Weill Cornell Medical Center, New York, NY, USA.

Introduction
Fabrication of tissues with an inherent hierarchical vascular network remains the holy grail of tissue engineering. Herein, we fabricate a pre-vascularized full-thickness cellularized skin equivalent containing a three-dimensional vascularized network of interconnected macro and microchannels lined with vascular cells, within a collagen neodermis containing encapsulated fibroblasts, and an epidermis comprised of human keratinocytes.
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), and the construct was then topically seeded with 1x10^6 cells/mL human epidermal keratinocytes (HEK). Multiphoton microscopy (MPM) and histology were conducted after 7 and 14 days of culture.
Results
MPM imaging demonstrates a hierarchical vascular network containing macro and microvessels lined by endothelial and smooth muscle cells and supported by perivascular pericytes, all in appropriate microanatomic arrangement. Neodermal HFF1 proliferated throughout the observation period and the HEK neoepidermis remained stable along the superficial aspect of the construct.
Conclusion
We have successfully fabricated a tissue-engineered pre-vascularized full thickness skin flap construct with stable and anatomically appropriate vascularity. Our platform provides tremendous promise in furthering the development of tissue-engineered skin and other types of pre-vascularized flaps.


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