Assessing the Minimum Time-Period of Normoxic Preincubation for Stable Adipose Stromal Cell-Derived Vascular Networks.

Abstract

Pre-vascularization of tissue engineered grafts is a promising strategy to facilitate their improved viability following in vivo implantation. In this process, endothelial cells (ECs) form capillary-like networks that can anastomose with host vasculature. Adipose-derived stromal cells (ASCs) are a commonly used cell population for tissue engineering and contain a subpopulation of ECs capable of assembling into robust vascular networks and anastomosing with the host. However, their initial vascular assembly is significantly impaired in hypoxic conditions (2% O2). In this study, we explored the minimum period of normoxic (20% O2) pre-treatment required to enable the formation of stable vascular networks. ASC-derived vascular structures were allowed to preassemble in fibrin hydrogels in normoxia for 0, 2, 4, or 6days and then transplanted into hypoxic environments for 6days. Total vascular length, pericyte coverage, cell proliferation, apoptosis rates, and ECM production was assessed. Vascular assembly increased with time over the 6days of culture. We found that 4days was the minimum period of time required for stable vascular assembly. We compared the major differences in cell behavior and network structure at Days 2 and 4. Neither proliferation nor apoptosis differed, however, the Day 4 time-point was associated with a significant increase in pericyte coverage (46.1±2.6%) compared to Day 2 (24.3±5.3%). These data suggest oxygen tension may be a mediator of EC-pericyte interactions during vascular assembly. Pre-vascularization strategies should incorporate a normoxic period of to enable successful vascular formation and development.