Hypoxic Preconditioned Nanofat at 1% O₂ for 24 h Loses Its Regenerative In Vivo Vascularization Capacity

Hypoxic preconditioning is increasingly explored to enhance the survival and vascularization of fat grafts. In this study, nanofat from donor mice was exposed to hypoxia (1% O₂) for 24 h to investigate the effects of this preconditioning protocol on the viability, gene expression and vascularization capacity of this mechanically processed fat derivative. Ex vivo analyses revealed that hypoxic preconditioning does neither affect apoptotic nor necrotic cell death within nanofat but significantly upregulates the expression of hypoxia-inducible factor (HIF)-1α and stromal cell-derived factor (SDF)-1 compared to non-preconditioned nanofat. Moreover, preconditioned nanofat exhibited a pro-angiogenic protein expression profile. For in vivo analyses, dermal substitutes were either seeded with preconditioned or non-preconditioned nanofat and transferred into dorsal skinfold chambers of mice to assess their vascularization by intravital fluorescence microscopy. Unexpectedly, implants seeded with preconditioned nanofat exhibited a significantly reduced functional microvessel density when compared to non-preconditioned controls. Immunohistochemical analyses also confirmed a lower microvessel density within the implants of the preconditioned group. These findings suggest that hypoxic preconditioning at 1% O₂ for 24 h cannot be recommended for enhancing the regenerative in vivo vascularization capacity of nanofat. Therefore, milder preconditioning protocols with shorter periods of hypoxia or higher oxygen levels should be alternatively tested in future studies.