Development of a 3D in vitro Model of Spinal Cord Injury Using Additive Manufacturing Technologies

Spinal cord injury (SCI) is a disabling pathology that occurs in the spinal cord (SC) following traumatic events such as accidents or falls, or non-traumatic causes such as tumors or infections. The primary phase, corresponding to the initial injury, is followed by a secondary acute-to-chronic inflammatory phase that can last for months and is characterized by neuronal and glial cell death, resulting in glial scar formation that hinders recovery. Once the inflammatory phases are complete, the result is a sensory and/or motor paralysis which can affect only the trunk and legs (paraplegia) or arms, body and legs (tetraplegia). The World Health Organization reports that SCI incurs significant lifetime costs, due to medical care and loss of productivity. Today, there is no efficient therapy for SCI, mainly because many of the inflammatory mechanisms that occur after injury are not fully understood. Therefore, it is of paramount importance to better analyze what leads to glial activation and to the subsequent glial scar formation, which, while partially protecting the intact SC from further damage, is one of the major obstacles to regeneration. Therefore, in vitro models that mimic the SCI environment are needed to better understand the inflammatory pathways and to develop high-throughput drug testing systems to reduce the number of animal models in preclinical testing, in line with the principles of the 3Rs. Based on this, the tissue engineering approach seems to be the perfect candidate to achieve this goal. Specifically, 3D bioprinting technology combined with appropriate biochemical cues can be used to recreate the neural microenvironment and culture cells with appropriate mechanical and biochemical stimuli. In this work, the melt electrowriting technique (MEW) was used to obtain a polycaprolactone (PCL) scaffold with aligned fibers. Meanwhile, neural stem cells (NE-4C cell line) were encapsulated in a gelatin methacrylate hydrogel used as a bioink and 3D bioprinted over the PCL scaffold. This complex architecture allows cells to proliferate and organize along aligned fibers in a physiologically relevant structure. The constructs were maintained in culture, allowing the cells to differentiate into neurons and astrocytes. On the day 17 of culture, a lesion was induced mechanically and/or with pro-inflammatory cytokines. Such a lesion model was then maintained in culture for a further 4 days. In vitro assays were performed to monitor the viability and morphology of the cells and the expression of specific proteins. All results were compared to the not-injured counterpart of the model. The test results showed an accumulation of dead cells and the presence of GFAP-expressing cells in the perilesional areas. These results indicated that the astrocytes were transitioning to their reactive phenotype, which is a prerequisite for glial scar formation. A preliminary study was also conducted to evaluate the possibility of including another cell type (NSC-34 cell line), which differentiates into motor neurons, in this SC model. Preliminary in vitro assays were performed to evaluate cell viability and protein expression. There are still many steps to be taken to obtain a faithful model of SCI, such as increasing the complexity of the model by including all cell types involved in the injury. However, this model is a good starting point because it was demonstrated to recapitulate some key features of the SCI microenvironment.