Supercritical Carbon Dioxide Decellularization of Porcine Nerve Matrix for Regenerative Medicine

Tissue engineering scaffolds are often made from the decellularization of tissues. The decellularization of tissues caused by prolonged contact with aqueous detergents might harm the microstructure and leave cytotoxic residues. In this research, we developed a new technique to use supercritical carbon dioxide (Sc-CO2)-based decellularization for porcine nerve tissue. The effect of decellularization was analyzed by histological examination, including Hematoxylin and Eosin, Masson's Trichrome staining, and 4 ',6-diamidino-2-phenylindole staining. Moreover, biochemical analysis of the decellularized tissues was also performed by measuring DNA content, amount of collagen, and glycosaminoglycans (GAGs) after decellularization. The results showed that the tissue structure was preserved, cells were removed, and the essential components of extracellular matrix, such as collagen fibers, elastin fibers, and GAG fibers, remained after decellularization. In addition, the DNA content was decreased compared with native tissue, and the concentration of collagen and GAGs in the decellularized nerve tissue was the same as in native tissue. The in vivo experiment in the rat model showed that after 6 months of decellularized nerve implantation, the sciatic function index was confirmed to recover in decellularized nerve. Morphological analysis displayed a range of infiltrated cells in the decellularized nerve, similar to that in native tissue, and the number of Schwann cells that play essential for motor function and sensory in the decellularized nerve was confirmed. These findings indicate that tissue decellularization using Sc-CO2 has been successfully used in tissue engineering. Impact statement The nervous system is pivotal in regulating bodily functions, encompassing a complex network of central and peripheral regions. Injuries to these systems can lead to substantial mental and physical disabilities, profoundly impacting health care systems. Utilizing a novel approach involving supercritical carbon dioxide for decellularization, this research focuses on creating acellular scaffolds with intact neuronal structures. The promising outcomes of this technique, demonstrated through mechanical testing, histological analyses, and transplantation on the rat model, offer new prospects for human nerve tissue transplantation.