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BACKGROUND: Oxygen is a potent modulator of cell function and wound repair in vivo. Hypoxia can enhance the production of specific extracellular matrix components and induce angiogenesis through the hypoxia-inducible factor-1 pathway. However, in vivo, very few cells within the body experience ambient (21%) oxygen tension. As such, exploring the effect of decreased oxygen tension is of particular interest in tissue engineering, where vascularization poses a significant obstacle to the development of large scale tissue-engineered constructs. Herein, we sought to utilize and analyze the effects of oxygen tension on our novel, pre-vascularized tissue engineered constructs containing a 1 mm diameter microchannel lined with human vascular and mural cells.
METHODS: Vascular networks were fabricated by sacrificing Pluronic F127 macrofibers in type I collagen with encapsulated human foreskin fibroblasts (HFF1) and human placental pericytes (HPPL) at a density of 1 x10^6 cells/mL, respectively. Twenty-four hours following fiber sacrifice, 5 x10^6 cells/mL of human aortic smooth muscle cells (HASMC) and 5x10^6 cells/mL of human umbilical vein endothelial cells (HUVEC) were seeded sequentially into the patent luminal space. Subsequently, 48 hours after fiber sacrifice, 1 x10^6 cells/mL of human epidermal keratinocytes (HEK) were topically seeded onto scaffolds. Concurrently, endothelial cells were also cultured in vitro at varying cell densities on 24-well plates. Both scaffolds and well plates were incubated at 1.5%, 5.0%, or 20.0% oxygen and underwent daily media changes. Scaffolds were analyzed after 7 and 14 days in culture. Well plates were analyzed after 3 days of culture, and the cell proliferation index was calculated with a ratio of Ki-67 (cellular marker of proliferation) to DAPI (cell nuclei stain). RESULTS: Macrochannels were successfully lined with HUVEC and HASMC, generating anatomically appropriate neointimal and neomedial layers by as early as day 7. The most robust cellular linings were seen in constructs incubated in 5.0% ambient oxygen. Immunohistochemical analysis revealed CD31+ HUVEC along the luminal surface of the macrochannel, and α-SMA expressing HASMC in the subendothelial plane. Furthermore, proliferation of HFF1 within the collagen bulk was evident as early as 7 days after seeding. HEK proliferated leading to the formation of a stratified epidermal layer along the construct surface and fibroblast specific-1-expressing fibroblasts within the “neo dermis.” Finally, in vitro 3-day cultures of endothelial cells demonstrated that continuous 5.0% oxygen had a higher proliferation index than that of both 1.5% and 20.0% oxygen, respectively.
CONCLUSIONS: Moderately hypoxic (5.0%) conditions promote increased angiogenesis and vascular stability in our tissue engineered, pre-vascularized skin flaps as compared to normoxic conditions, without detrimental effects on other flap cellular constituents. Although seemingly counter-intuitive, these data corroborate the observation that most cells in vivo “live” and even thrive under conditions of relative hypoxia. Further investigations are warranted to determine the optimal ambient oxygen tension for the fabrication of vascularized tissue engineered constructs.