Supercritical CO₂-Mediated Decellularization of Bovine Spinal Cord Meninges: A Comparative Study for Decellularization Performance

The extracellular matrix (ECM) of spinal meninge tissue closely resembles the wealthy ECM content of the brain and spinal cord. The ECM is typically acquired through the process of decellularizing tissues. Nevertheless, the decellularization process of the brain and spinal cord is challenging due to their high-fat content, in contrast to the spinal meninges. Hence, bovine spinal cord meninges offer a promising source to produce ECM-based scaffolds, thanks to their abundance, accessibility, and ease of decellularization for neural tissue engineering. However, most decellularization techniques involve disruptive chemicals and repetitive rinsing processes, which could lead to drastic modifications in the tissue ultrastructure and a loss of mechanical stability. Over the past decade, supercritical fluid technology has experienced considerable advancements in fabricating biomaterials with its applications spreading out to tissue engineering to tackle the complications mentioned above. Supercritical carbon-dioxide (scCO₂)-based decellularization procedures especially offer a significant advantage over classical decellularization techniques, enabling the preservation of extracellular matrix components and structures. In this study, we decellularized the bovine spinal cord meninges by seven different methods. To identify the most effective approach, the decellularized matrices were characterized by dsDNA, collagen, and glycosaminoglycan contents and histological analyses. Moreover, the mechanical properties of the hydrogels produced from the decellularized matrices were evaluated. The novel scCO₂-based treatment was completed in a shorter time than the conventional method (3 versus 7 days) while maintaining the structural and mechanical integrity of the tissue. Additionally, all hydrogels derived from scCO₂-decellularized matrices demonstrated high cell viability and biocompatibility in a cell culture. The current study suggests a rapid, effective, and detergent-free scCO₂-assisting decellularization protocol for clinical tissue engineering applications.