Vitamin D Acts As a Metabolic Lipoprotectant Enhancing Fat Graft Viability Under Hypoxic Conditions

Goals/Purpose: Post-operative reabsorption of engrafted adipose results in up to 50% tissue loss in the first post-operative year and is limits reliable volumetric results. We have previously demonstrated the efficacy of vitamin D as a lipoprotectant to enhance adipose retention in in vitro, ex vivo, and xenograft human-to-murine in vivo models. To demonstrate translatability of this work and identify a viable mechanism we have concurrently performed further in vitro and in vivo studies. In vitro we have evaluated how VD3 affected the metabolism and proliferative indices of ASCs and adipocytes. In vivo we have adapted our studies to a clinically relevant porcine subcutaneous fat graft model to examine the efficacy of vitamin D as an orally bioavailable lipoprotectant.

Methods/Technique: In vitro, lipoaspirate was either a) enzymatically dissociation to free the stromal vascular fraction (SVF) or maintained ex vivo for 7-days. A fraction of these SVF was assessed for general metabolic and proliferative indices utilizing MTT assay under hypoxic or normoxic conditions. Calcein/PI cytometry was utilized to assess cell size and survival. ASCs and differentiated adipocytes were treated and analyzed without and without CoCl2 using a seahorse bioanalyzer (Agilent) for real time kinetics of metabolic flux in live cells. For in vivo porcine studies female 3–4-month-old Yorkshire swine were sacrificed to provide fresh adipose which was milled under sterile conditions to for injection through an 18-gauge cannula. Recipients were herd matched to allogeneic donors. Each donor was used to provide adipose for a matched pair of recipients. Treatment groups receive oral vitamin D post-engraftment compared to matched-chow controls. Grafts were followed serially with ultrasound. After 8- or 12-weeks post-procedure subjects were sacrificed for terminal adipose weights, volumes, and histology.

Results/Complications: In vivo we have found that vitamin D treated demonstrates improved viability and proliferation (p<0.05). This effect was most evident under persistent hypoxic stress and remained for up to a week post-harvest (p<0.05). When we examined O2 under conditions of hypoxic stress we note reduced oxygen consumption rates in vitamin D treated SVF with increased cellular energetic reserve capacity. In vitro, as with prior xenografted murine work we were able to successfully transplant herd-matched allogeneic adipose tissue with maintained survival up to 8-12 weeks post engraftment with oral vitamin D tolerance noted in all cases.

Conclusion: We have previously demonstrated that Vitamin D offers a safe and effective option for the prevention of graft loss in vitro and in vivo lower animals. Here we perform validation in the clinically translatable porcine model. Further, we demonstrate cell and hypoxia dependent changes in metabolism and proliferation with vitamin D supporting a more proliferative and energetic profile in those adipose stem cells necessary for fat graft renewal.