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Oxygen is the single most important molecule for sustaining life and, therefore, an important variable in tissue engineering and regenerative medicine. It has been shown that the change in oxygen concentration in an artificial or tissue-engineered graft affects cell survival, differentiation, and tissue growth in profound ways. However, at present, there are no reliable methods to map partial oxygen pressure (pO 2 ) in growing artificial tissues. Here, we adapt and test the suitability of electron paramagnetic resonance oxygen imaging (EPROI) in assessing tissue graft oxygenation in vitro. EPROI is an established method to assess absolute pO 2 and has been widely applied to study tumor hypoxia in small animals. In this study, we demonstrate the feasibility of EPROI in evaluating oxygen dynamics in tissue grafts. We measured oxygen concentration in mesenchymal stem cell (MSC)-seeded polylactic-co-glycolic acid (PLGA) scaffolds with variable porosity. The pO 2 maps of these scaffolds showed that the mean pO 2 inside the scaffolds was smaller than the ambient air pO 2 (21% oxygen, 160 torr) and was gradually increased with increasing pore size. We assessed the local oxygen dynamics of the MSC-seeded osteogenic scaffold made from collagen-chitosan hydrogels in a partially sealed Eppendorf tube. The change in pO 2 values as a function of time inside the graft showed that the cells had used available oxygen within first 2 h of the experiment and then went to a dormant low oxygen consumption state until the oxygen supply was reestablished. Collectively, these data suggest that EPROI could be successfully used for mapping pO 2 in tissue-engineered grafts. The knowledge of tissue graft oxygenation may be used to improve scaffold design and to assess the tissue viability and growth.

Noninvasive Absolute Electron Paramagnetic Resonance Oxygen Imaging for the Assessment of Tissue Graft Oxygenation