Neural differentiation of mesenchymal cells after 3D bioprinting using a decellularized spinal cord-based biomaterial

Three-dimensional (3D) bioprinting combines biomimetic materials and cells to promote the functional recovery of tissues and organs. This technique can be applied to overcome challenges in the regeneration of tissues with a poor intrinsic regeneration capacity, such as the nervous tissue. In this context, the aim of this study was to produce a bioink using rat Decellularized Spinal Cord Tissue (DSCT) and mesenchymal cells (MSCs) for nervous tissue 3D bioprinting. The bioink was produced with 1.5% DSCT, 3% gelatin, 4% alginate, 0.1 mg/mL PEDOT:PSS, a conductive polymer, and 1X106 MSC/mL. MSCs were isolated from human deciduous teeth and were characterized using flow cytometry. The material physical properties were evaluated using electrical conductivity measurements, rheological characterization, and scanning electron microscopy (SEM). The swelling ratio and degradation were analyzed for a duration of 4 weeks. Cell viability was analyzed using MTT and live/dead assays. The bioprinted cells were submitted to a neural differentiation protocol and neural markers were analyzed using flow cytometry. The isolated cells were positively identified as MSCs by characteristic stem cell markers. The addition of PEDOT:PSS to the hydrogel increased its electrical conductivity. The hydrogel presented shear thinning behavior and a low G’’/G’ ratio, allowing for good printability without significantly compromising cell viability. SEM images showed a highly porous three-dimensional structure. The hydrogel reached its peak swelling ration at week 1 after printing and lost 24% of its weight at week 2, maintaining a constant weight until week 4. The MTT assay on day 1 indicated no viability reduction compared to the control. The live/dead assay showed more than 75% cell viability at the week 1 timepoint. Flow cytometry indicated an increased ꞴIII-tubulin and glial fibrillary acidic protein expression. The data mentioned above indicate that the bioink holds great promise as an easily available biomaterial for neural tissue engineering via 3D bioprinting.