Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Optimization of Functional, Perfusable Vascular Networks Within Tissue Engineered Hydrogels
Rachel C. Hooper, MD, FACS, Karina A. Hernandez, DO, Tatiana Boyko, MD, Jeremiah Joyce, BA, Adam Jacoby, BA, Ope Asanbe, MD, Kadria N. Derrick, MD, Jason A. Spector, MD, FACS.
Weill Cornell Medical College, New York, NY, USA.

PURPOSE:
New blood vessels are formed de novo via vasculogenesis or through sprouting from existing blood vessels via angiogenesis. A layer of endothelial cells comprising the tunica intima provides a non-thrombogenic surface and allows continuous blood flow within these new blood vessels. The ability to precisely replicate the intricate design of the vascular system de novo is beyond any current tissue-engineering techniques and remains a major challenge towards creating surgically relevant constructs for clinical application. Here we describe the synthesis of tissue-engineered hydrogels, containing an internal microvessel with “neointima” and “neomedia”.
METHODS:
Pluronic F127 fibers were sacrificed in type I collagen, creating a central “loop” microchannel, 1.5 mm in diameter. A cell suspension of 5 x106 cells/mL human umbilical vein endothelial cells (HUVEC) was injected into the inlet of a loop microchannel. For co-culture scaffolds, a cell suspension of 5 x106 cells/mL human aortic smooth muscle cells (HASMC) was injected initially and after 24 hours, a HUVEC cell suspension was seeded as above. Following 7 and 14 days of static culture, constructs were injected with 10 µg/mL low-density lipoprotein acetylated-dil complex (Dil-Ac-LDL) to demonstrate HUVEC-receptor mediated endocytosis. Whole constructs were subsequently imaged via multiphoton microscopy and immunohistochemical staining was performed for 4’,6-diamidino-2-phenylindole (DAPI), CD31, von Willebrand Factor (vWF), collagen IV, and alpha smooth muscle actin (α-SMA) in order to determine the density and spatial relationship between cell types. Additionally, Lycopersicion esculentum staining was performed to identify the presence of synthesized heparan sulfate, an extracellular matrix protein.
RESULTS:
Microchannels were successfully seeded with HUVEC and HUVEC/HASMC. Multiphoton microscopy of microchannels seeded with HUVEC/HASMC after 7 days demonstrated a confluent concentric endothelial lining, which was maintained after 14 days. However, HUVEC-only seeded microchannels demonstrated delamination, which continued through the 14-day time point. HUVEC demonstrated functionality via receptor-mediated uptake of Dil-Ac-LDL along the microchannels seeded with HUVEC alone and in co-culture with HASMC after 7 and 14 days. Histological analysis confirmed co-culture microchannels with HUVEC and HASMC organized in concentric layers with elaboration of additional extracellular matrix (ECM) proteins. HASMC/HUVEC-seeded constructs exhibited CD31+ and vWF expressing HUVEC along the luminal surface of the microchannel forming a “neointima” with α-SMA expressing HASMC in the subendothelial plane, forming the neomedia. Additionally, after 14 days HUVEC/HASMC-seeded constructs demonstrated elaboration of heparan sulfate, a component of the glycocalyx and ECM as well the deposition of basal lamina protein collagen IV along the abluminal surface of CD31+ HUVEC.
CONCLUSION:
<brWe have successfully created tissue-engineered scaffolds with microchannels that support engraftment of smooth muscle and endothelial cells, forming neointimal and neomedia with receptor-mediated endocytosis demonstrating functionality of the seeded endothelial cells. With a preformed vascular network lined by endothelial cells these constructs recreate the architecture found in vivo and provide a surface for thrombosis-free blood flow, thus allowing for surgical implantation via microanastomosis.


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