Fabrication and characterization of Schwann cell-derived acellular matrix and chemical-ionic dual crosslinking strategy to produce bioinks for peripheral nerve regeneration

Peripheral nerve injury (PNI) is a common neurological problem that can hamper the quality of life causing sensory and motor dysfunction. While small peripheral nerve defects get repaired by themselves, larger defects require tissue engineering substitutes to bridge the nerve gap. One of the recent technologies and strategies to address the peripheral nerve regeneration is through 3D bioprinting. In this study, we show an effective method to fabricate acellular matrix (ACM) from rat Schwann RSC96 cells and incorporate it into alginate hydrogel. We observed that ACM showed presence of ~400 µg/ml of proteins and specific Schwann cell marker (S100) and myelination markers (PMP22, MPZ) with significant DNA reduction, indicating effective decellularization and minimal immunogenicity. Cytochemical and immunofluorescence staining confirmed presence of extracellular matrix (ECM) proteins, collagen I and fibronectin. We also developed a novel dual crosslinking strategy using glutaraldehyde (pre-print crosslinking) and calcium chloride (post-print crosslinking) to facilitate efficient bioprinting. Further, rat Schwann RSC96 cells were blended with alginate/ACM hydrogel to prepare a functional bioink. Our results demonstrated good cell viability and functionality in the printed structures, highlighting the potential role of rat Schwann RSC96-derived ACM as well as a dual crosslinking strategy for peripheral nerve tissue regeneration.