12. Optimizing VD3 Timing and Dosing for Significantly Increasing Fat Graft Retention in a Swine Model (2023)

PURPOSE: A major limitation of fat grafting is unpredictable retention. We previously demonstrated that bioactive Vitamin D (VD3), calcitriol, improves human fat graft retention in a xenograft mouse model, however, high dose calcitriol incurs safety risks. Additionally, we have verified that inactive VD3, cholecalciferol, similarly protects adipose stromal cell viability during vascular insufficiency post-grafting and improves long term retention in a porcine model. Within our overall goal of defining a clinical protocol for direct translation of VD3 into practice, we currently aim to elucidate optimal timing and dosing of VD3 administration in our porcine model. METHODS: Human adipose lipoaspirate was collected from discarded surgical samples under IRB exemption and cultured ex vivo for 7-days with or without VD3. Residual tissue was enzymatically digested to release mature adipocytes and the stromal vascular fraction (SVF), which was evaluated for viability, identity, proliferation, and mitochondrial stress with Seahorse Bioanalyzer. An immunocompromised xenograft mouse model was used to determine survival, retention, and histologic characteristics of VD3 treated vs. untreated fat. Effects of oral VD3 treatment vs no treatment were assessed in a porcine model of engraftment using female Yorkshire swine over a 3-month study period. Graft volume and retention were serially assessed by ultrasound and confirmed by gas pycnometry after sacrifice. Terminal VD3-treated adipose tissue was harvested, processed, and re-injected into a second cohort of swine that compared timing of VD3 administration. Graft volume and retention were assessed in a similar manner. Terminal VD3-treated adipose was processed for re-injection into a third cohort of swine investigating optimal dosing of oral VD3, which is currently underway. RESULTS:Ex vivo, adipose tissue cultures demonstrated greater viability when treated with VD3; cytometric analysis demonstrated significant enrichment of endothelial and pericyte populations. SVF treated with VD3 demonstrated enhanced spare mitochondrial respiratory capacity and survival under acute hypoxic conditions. Xenografted lipoaspirate demonstrated enhanced survival of human cells, reduced fibrosis, increased host-derived vascularity and increased host-derived adipocyte replacement at 12 weeks with intraperitoneal VD3. Treatment with oral VD3 demonstrated significantly enhanced retention of allografted fresh adipose on ultrasound throughout the 3-month study period and by graft volume assessment at sacrifice, compared to no VD3 therapy. Significant improvement in graft volume and retention was shown in pigs that received oral VD3 therapy prior to engraftment, compared to after engraftment. Serologic analysis of VD3 metabolism and histologic comparison of tissue specimens collected throughout the study period are being completed. Dosing comparison studies are underway. CONCLUSIONS: Inactive VD3 significantly increases viability of the vascular and perivascular components of hypoxic adipose tissue. In vivo, VD3 significantly improved graft retention in both xenograft-murine and porcine models, likely through increased recipient bed revascularization and adipocyte replacement. Additionally, data shows improved graft volume and retention for oral VD3 administration prior to fat grafting. These findings support use of VD3 to minimize graft resorption and improve clinical outcomes.