46. “Off-The-Shelf” Fat Graft: Supercritical CO2for the Fabrication of Human-Derived Injectable Adipose Scaffold

PURPOSE: Autologous fat grafting is a versatile soft tissue augmentation technique employed in a myriad of reconstructive scenarios. Because of unpredictable volume retention, plastic surgeons seek alternative methods. Tissue-engineered decellularized adipose extracellular matrix (ECM) sourced from allograft donors holds promise for inducing tissue regeneration as cells are recruited to the scaffold by the bioactive matrix within. However, conventional decellularization methods employ harsh chemicals that can denature bioactive factors and leave toxic residues in the ECM - potentially damaging native tissue architecture/bioactivity and triggering a destructive immune response. We have engineered an adipose derived injectable ECM utilizing supercritical carbon dioxide (scCO2), an emerging decellularization method that forgoes use of abrasive chemical agents, thereby promising a gentler, non-toxic cleansing of matrix components with greater potential for post-implantation tissue regeneration. METHODS: Adipose tissue removed from abdominoplasty procedures was homogenized via mechanical processing. Lipid was removed with isopropanol at 37°C, after which the tissue was subjected to NovaSterilis’ scCO2 cleaning process. All samples were then decellularized with two cycles of scCO2 decellularization process. Processed tissue was further homogenized through cryomilling. Once achieving the desired form, the tissue was sterilized with scCO2 terminal sterilization process for tissue products. Samples underwent histochemical analysis and DNA quantification to verify sufficient removal of residual nucleic acid levels (~50 ng/mg tissue). Adipose ECM was reconstituted at 0.5g sample/mL PBS, and 0.4mL was injected into subcutaneous dorsal flank and cranial pockets of immunocompetent C57BL/6 mice through a 20-gauge catheter. Commercial-grade hyaluronic acid was injected (0.4mL, dorsal flank) as a control. Samples were explanted after one month and underwent microCT volume analysis. RESULTS: Processing methods resulted in a significant (>95%) volume loss from initial tissue to resultant ECM. Delipidated, decellularized, adipose ECM demonstrated a mean DNA content of 44 ng of double-stranded DNA per mg of sample. For comparison, the DNA content of delipidated, non-decellularized adipose ECM was 125 ng/mg, and AlloMax™ (CR Bard/Davol Inc, Cranston, RI) decellularized human dermis was 195 ng/mg. Hematoxylin and eosin (H&E) staining of adipose ECM revealed compact milled particles, and DAPI staining confirmed the absence of cell nuclei. After one month, mean adipose graft volume was 478mm3 (120% of initial volume) at dorsal flank, 468mm3 (117%) at dorsal cranial, and hyaluronic acid volume was 1,381mm3 (345%), due to its hygroscopic nature. Cranial-site adipose grafts appeared more highly vascularized than flank-site, consistent with greater regional blood flow. Minimal inflammation was observed. Histologic analysis is ongoing. CONCLUSION: We report an innovative scCO2-based decellularization process to efficiently fabricate adipose ECM scaffold. We demonstrate adequate decellularization and rapid reconstitution into a homogenous, injectable form that demonstrates excellent volume retention and unremarkable immunogenicity after one month in vivo. By optimizing adipose scCO2-decellularization, we anticipate the development of an “off-the-shelf” fat graft alternative without potentially toxic residues that retains bioactive moieties critical to host cell repopulation. Further study is ongoing to explore longer term in vivo outcomes.