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In Vivo Microanastomosis of Microvessel Containing Tissue-Engineered Constructs: The Final Frontier
Rachel C. Hooper, MD, FACS, Karina A. Hernandez, DO, Tatiana Boyko, MD, Jeremiah Joyce, BA, Adam Jacoby, BA, Jason A. Spector, MD, FACS.
Weill Cornell Medical College, New York, NY, USA.
Although autologous tissue transfer has been established as a reliable approach to the reconstruction of complex defects, there are associated consequences including donor site pain, functional loss, paresthesias, dysthesthia, and scarring. The ability to synthesize vascularized constructs for the management of these complex wounds would represent a quantum leap in the field of tissue engineering. In previous work we synthesized and performed an in vivo microvascular anastomosis of a collagen construct containing an unseeded internal longitudinal microchannel with inlet and outlet. Here we fabricate and microsurgically anastomose collagen constructs containing an internal endothelialized microchannel.
Pluronic F127 microfibers were embedded in neutralized type I collagen, then sacrificed leaving a central “loop” microchannel, 1.5 mm in diameter. Constructs contained an inlet and outlet and were reinforced with polyglactone mesh for tensile strength at the anastomotic site. Microchannels were seeded with 5 x106 cells/mL human umbilical vein endothelial cells (HUVEC) or a co-culture of HUVEC and human aortic smooth muscle cells (HASMC) and cultured for 7 days with daily media changes. Seeded and unseeded constructs were microsurgically anastomosed to the femoral artery and vein of RNU 316 nude rats. Following completion of anastomoses, patency was evaluated via venous strip tests and in vivo microdoppler assessment. Unseeded constructs were perfused for 2.5 and 5 hours. Seeded constructs were perfused for up to 24 hours. Following perfusion, all constructs were fixed in 10% formalin, embedded, stained and analyzed.
Polyglactone mesh provided the necessary tensile strength, allowing microchannel-containing constructs to be successfully anastomosed to the femoral artery and vein of nude rats. In vivo gross inspection and H&E staining of seeded and unseeded constructs following harvest revealed intact microchannels capable of withstanding physiologic perfusion pressures. Patency was confirmed via venous strip tests and auscultation of continuous pulsatile blood flow via microdoppler. Post-perfusion analysis of unseeded constructs demonstrated microchannels with adherent host inflammatory cells along the luminal surface. Post-harvest histology following perfusion of HUVEC-seeded microchannels demonstrated areas of delamination whereas HUVEC/HASMC-seeded microchannels maintained concentric confluent “neointimal” and “neomedial” layers comprised of endothelial and smooth muscle cells respectively. Immunohistochemical analysis of HUEVC-only seeded microchannels revealed CD31 expressing cells whereas the co-culture-seeded microchannels demonstrated both CD31 and α-SMA expressing HUVEC and HASMC.
We have successfully created custom vascularized biodegradable, biocompatible constructs that support microchannel endothelialization and microsurgical anastomosis in vivo. Constructs with their own inherent vascular network can be directly anastomosed to host vasculature providing immediate perfusion, thus increasing the survival of cellular constituents within the scaffold as well as the rate of incorporation into the host. This represents a major advance in tissue engineering and opens the door to the creation and application of larger, more complex surgically relevant constructs.
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