Selection of Mechanical Fragmentation Methods Based on Enzyme-Free Preparation of Decellularized Adipose-Derived Matrix

Background: The decellularized adipose-derived matrix (DAM) has emerged as a promising biomaterial for inducing adipose tissue regeneration. Various methods have been employed to produce DAM, among which the enzyme-free method is a relatively recent preparation technique. The mechanical fragmentation step plays a crucial role in determining the efficacy of the enzyme-free preparation. Methods: The adipose tissue underwent fragmentation through the application of ultrasonication, homogenization, and freeze ball milling. This study compared the central temperature of the mixture immediately following crushing, the quantity of oil obtained after centrifugation, and the thickness of the middle layer. Fluorescence staining was utilized to compare the residual cell activity of the broken fat in the middle layer, while electron microscopy was employed to assess the integrity and properties of the adipocytes among the three methods. The primary products obtained through the three methods were subsequently subjected to processing using the enzyme-free method DAM. The assessment of degreasing and denucleation of DAM was conducted through HE staining, oil red staining, and determination of DNA residues. Subsequently, the ultrasonication-DAM (U-DAM) and homogenation-DAM (H-DAM) were implanted bilaterally on the back of immunocompromised mice, and a comparative analysis of their adipogenic and angiogenic effects in vivo was performed. Results: Oil discharge following ultrasonication and homogenization was significantly higher compared to that observed after freeze ball milling (p < 0.001), despite the latter exhibiting the lowest center temperature (p < 0.001). The middle layer was found to be thinnest after ultrasonication (p < 0.001), and most of the remaining cells were observed to be dead following fragmentation. Except for DAM obtained through freeze ball milling, DAM obtained through ultrasonication and homogenization could be completely denucleated and degreased. In the in vivo experiment, the first adipocytes were observed in U-DAM as early as 1 week after implantation, but not in H-DAM. After 8 weeks, a significant number of adipocytes were regenerated in both groups, but the U-DAM group demonstrated a more efficient adipose regeneration than in H-DAM (p = 0.0057). Conclusions: Ultrasonication and homogenization are effective mechanical fragmentation methods for breaking down adipocytes at the initial stage, enabling the production of DAM through an enzyme-free method that facilitates successful regeneration of adipose tissues in vivo. Furthermore, the enzyme-free method, which is based on the ultrasonication pre-fragmentation approach, exhibits superior performance in terms of denucleation, degreasing, and the removal of non-adipocyte matrix components, thereby resulting in the highest in vivo adipogenic induction efficiency.