Fabrication of a Bioactive Human Adipose Extracellular Matrix Allograft Using Supercritical Carbon Dioxide

Autologous fat grafting is widely used for soft tissue augmentation but hindered by unpredictable volume retention and donor site morbidity. Decellularized adipose extracellular matrix (DAM) represents a promising alternative. Herein we evaluate an injectable, human-derived DAM decellularized via supercritical carbon dioxide (scCO₂, sDAM), promising a gentler, more eco-friendly isolation of matrix components than conventional chemical decellularization.

Human adipose tissue was homogenized, delipidated, then underwent scCO₂ cleansing, two cycles of scCO₂ decellularization, cryomilling, and scCO₂ terminal sterilization. The sDAM was injected into dorsal flank and cranial subcutaneous pockets of immunocompetent C57BL/6 mice (400 mm³ of 0.5 g sample/mL PBS). Commercially available hyaluronic acid (HA) was injected (400 mm³) for comparison. Samples were explanted after 1, 3 and 6 months.

sDAM demonstrated a mean of 47 ng DNA/mg of dry weight, below the 50 ng/mg "complete" decellularization threshold. After 6 months, adipose flank volume was 307 mm³ (77% of initial volume), adipose cranial was 273 mm³ (68%), and HA flank was 1393 mm³ (348%), due to its hydrophilic nature. sDAM explants demonstrated significantly higher cellular density over HA explants (p < 0.0001). Perilipin expression revealed adipocytes developing within sDAM after 3 months, increasing in density and size by 6 months; few cells were noted in HA explants. Similarly, CD31 expression demonstrated neo-vascularization in sDAM only. Picrosirius red staining of sDAM demonstrated deposition of new fibrillar collagen after 1 month, which became more aligned over time.

We present the first description of scCO₂ DAM preparation foregoing the use of detergents and enzymatic chemicals with successful volume preservation and adipose tissue regeneration in vivo.