Development of an in vitro fibrotic scar model of spinal cord injury using macromolecular crowding

Spinal cord injury (SCI) results in a cascade of cellular and molecular events that lead to permanent tissue damage and functional impairment. A key consequence of this injury is the formation of both glial and fibrotic scars, which pose significant barriers to regeneration. The fibrotic scar that forms following SCI remains a significant therapeutic challenge. One major obstacle in developing anti-fibrotic compounds is the absence of a comprehensive in vitro screening system. In this study, we employed a macromolecular crowding (MMC) technique to accelerate ECM deposition. Leptomeningeal (LPG) cells were cultured in media supplemented with the MMC Ficoll (FC). To mimic the injury environment in vivo, the cells were exposed to either physical or chemical injury. The growth and metabolic activity of the LPG cells remained unchanged under these different injuries and treatments. Groups supplemented with the MMC FC exhibited higher deposition of ECM proteins involved in fibrotic scar formation, including fibronectin, collagen IV, collagen I, and laminin, compared to those without FC. A key limitation of conventional cell culture in aqueous media is its clear difference from the naturally ‘crowded’ tissue environment, resulting in a slow rate of ECM protein deposition. Using the MMC approach, we successfully accelerated ECM protein deposition within an in vitro model of the fibrotic scar. Supplementing LPG culture media with MMCs can effectively mimic the fibrotic scar environment, providing a valuable refinement in developing SCI in vitro models for drug screening and therapeutic applications.