Plug 'N Play: Induction of Biomimetic and Hierarchical Angiogenesis Under Stimulation with S1P
Justin Buro, BS, Andrew Abadeer, MD, Yoshiko Toyoda, BA, Karel-Bart Celie, Alexandra Lin, MD, Jason A. Spector, MD.
Weill Cornell Medical College, Laboratory of Bioregenerative Medicine and Surgery, New York, NY, USA.
Introduction:The field of tissue engineering has long held the promise of an "off-the-shelf" vascularized free flap as a means of true dermal replacement. Our current state of dermal replacement utilizes an acellular dermal matrix that, while promising and effective, is severely limited by the wound bed. A Tissue engineered dermal replacement that includes an inherent vascular network would obviate this limitation. Our previous experiments have demonstrated reliable endothelial cell sprouting from a monolayer under stimulation by a gradient of S1P, VEGF, and FGF, and we have even demonstrated the self-assembly of endothelial cells within a collagen matrix. The task remains to anastomose this microsurgically relevant monolayer with the capillary-like self-assembled vessels. It is our hypothesis that the induction of such sprouts into a self-assembled network will anastomose to the network, thereby achieving hierarchical vascular organization that can be utilized in the production of tissue engineered pre-vascularized free flaps. Methods: Invasivity assays were created by injecting Type 1 Collagen impregnated with 1 μM S1P into well plates. A monolayer of GFP-tagged Human Umbilical Vein Endothelial Cells (HUVECs) were topically seeded, covered with Endothelial Cell media enhanced with VEGF and FGF, and cultured for 1, 3, and 5 Days. Self-assembled network assays were created by seeding a monolayer of GFP-tagged HUVECs as above, followed by injecting a second layer of collagen above the monolayer and coverage with growth factor enhanced media. After optimization, combination cultures were created by layering a collagen base layer, a YFP-Tagged HUVEC network second layer, an S1P impregnated collagen third layer, and a topically seeded GFP-Tagged HUVEC monolayer. All constructs were confocal imaged for analysis. Results: Invasivity assays demonstrated robust sprouting with an average of 46.89 ± 2.54 by Day 1, 131.4 ± 10.34 by Day 3, and 321.3 ± 39.31 by Day 5 per 0.6 mm˛. [AA1] Self-assembled network assays demonstrated biologically appropriate inter-capillary distance less than 50μm by Day 5. Combination cultures demonstrated robust sprouting from both the network and monolayer and anastomosis as evidenced by YFP and GFP multi-fluorescent channels, however quantification of anastomoses are still in progress. Conclusions:In this project we have successfully demonstrated the ability of endothelial cells to form a hierarchical vascular network that mimics that of normal human tissue in a way that is readily scalable and easily reproducible. Future iterations of this project will focus on a straight channel design and maintenance of the channel and network with flow. <!--EndFragment-->
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