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Vitamin D3 Enhances Adipose Stromal Cell Bioenergetics Capacity Andproliferation Under Hypoxic Conditions And Improves Fat Grafting Outcomes
Anya Singh-Varma, BS1, Shawn Jeffrey Loder, MD1, Wayne Vincent Nerone, BS1, Phoebe Lee, BS1, David Guerrero, BS1, J Peter Rubin, MD2, Lauren Kokai, PhD1.
1University of Pittsburgh, Pittsburgh, PA, USA, 2University of Pittsburgh, PITTSBURGH, PA, USA.

PURPOSE: Reabsorption of autologous fat grafts is often unpredictable and thus volumetric results are not reliable. We have previously demonstrated the efficacy of vitamin D as a lipoprotectant to enhance adipose tissue survival in vitro, ex vivo, and in xenograft human-to-murine in vivo models. Our preliminary results showed that VD3 primarily affected adipose stromal cells and thus in this study, we evaluated the impact of VD3 on human adipose stromal cell (ASC) proliferation and metabolism. In addition, we tested oral VD3 efficacy in a clinically relevant porcine model of subcutaneous fat grafting.
METHODS: In vitro, lipoaspirate was either a) enzymatically dissociation to free the stromal vascular fraction (SVF) or maintained in culture as tissue particles for 7-days. For SVF cultures, cells were assessed under normoxic or hypoxic conditions in the presence of VD3 and general metabolic and proliferative indices were measured with Seahorse Bioanalyzer and MTT, respectively. For tissue particles, stromal cells and adipocytes were isolated and viability and size were measured with Calcein/PI cytometry. In vivo, female 3-4-month-old Yorkshire swines were sacrificed to provide fresh adipose which was milled under sterile conditions and injected subcutaneously on the dorsum with 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 received oral vitamin D post-engraftment and 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: VD3 improves viability and proliferation of adipose stromal cells (p<0.05, N = 6 unique donors). This effect was most evident under persistent hypoxic stress and remained for up to a week post-harvest (p<0.05). Metabolically, VD3 significantly increased cellular energetic reserve capacity under hypoxia which increases the cellular ATP supply (p = 0.0001 for VD3 concentrations versus control). In vivo, 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. Volume retention studies are on-going.
CONCLUSION: We have demonstrated that Vitamin D is a safe and effective option for prevention of fat graft loss in vitro and in mice. Here we perform validation in a clinically translatable porcine model. Further, we have demonstrated hypoxia dependent changes in human adipose stromal metabolism and proliferation with vitamin D supporting a more proliferative and energetic profile in those adipose stem cells necessary for fat graft renewal.


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