Decellularization of rat and human omentum to develop novel scaffolds for regenerative medicine

Background: Homologous tissues may be an interesting source of decellularized scaffold maintaining a complex physiological 3D structure, completed with vessels, to be recellularized by autologous cells. In particular, tissue-engineered adipose substitutes such as the omentum would have numerous applications in the field of reconstructive surgery. Methods: Adult rat and human omenta were treated with an adapted decellularization protocol involving freeze-thawing cycles that break down adipose and endothelial cells, enzymatic digestion involving trypsin, deoxyribonuclease, lipase and ribonuclease, and lipids polar solvent extraction to yield a collagenous natural matrix, i.e., decellularized adipose tissue (DAT). The scaffolds obtained were studied with histological (haematoxylineosin, azan-Mallory, Van Gieson, Oil Red and Sudan) and immunohistochemical (anti-CD31, -laminin, -collagen IV) stainings to highlight the persistence of cells, the presence of lipids, the architecture and composition of the material, the persistance of frames of the vascular network. The study was performed in accordance with the Italian Public Health Office regulations. Results: Histological stainings confirmed the effectiveness of the decellularization protocol, resulting in a cell-free scaffold with no residual cells present in the matrix. Oil red and Sudan stainings denoted that no lipids were found in the decellularized adipose tissue. Anti-CD31 showed the absence of endothelial cells. Conversely, azan Mallory and Van Gieson stainings identified a large amount of collagen and elastic fibers, organized in a quite complex three-dimensional network. The volume of the original omentum sample was quite preserved during the decellularizing passages. Immunostaining for laminin and collagen type IV showed that both basement membrane components were still present in the network-type regions of the DAT scaffolds, as well as along the lumens of the decellularized vascular structures, that persisted after the decellularization method. Conclusion: The fat-rich and well vascularized omental adipose tissue may be decellularized to realize complex tridimensional scaffolds suitable for recellularization. Further analysis will have to verify the possibility of recolonization of the scaffold by autologous cells after in vivo reimplantation, as already known for homologus skin implants in regenerative processes.