Resolving Differential Gene Expression After Therapeutic Hypoxia Treatment for Spinal Cord Injury for the Development of a Biomimetic Hydrogel Testing Platform

Introduction:: Spinal cord injury (SCI) affects over 200,000 people in the United States[1]. This results in irreversible damage to the spinal cord and dysfunction downstream of the injury site. While there is no cure, therapeutic acute intermittent hypoxia shows promise in increasing neuroplasticity [2],[3]. The spinal cord extracellular matrix (ECM) undergoes extensive remodeling following injury, changing the cellular binding domains, mechanics, and overall function of the tissue as scar tissue develops [4]. Here, we use QuantSeq mRNA sequencing to determine changes in cell function in the spinal cord following injury and use that information to further test the impact of hypoxia on human cell function and ECM interaction in a spatial hydrogel mimic of spinal cord extracellular matrix. New 3D printing technologies that jam spherical microgel (uGel) particles together at specific concentrations to form liquid-like solids, which exhibit liquid like properties under shear, have been utilized for 3D cell culture [5]. Constructing a biomimetic model of the injured human spinal cord is crucial for the future of testing regenerative therapeutics for spinal cord injury.

Materials and Methods:: Sprague-Dawley rats were anesthetized and a C2 complete transection of the left spinal cord was performed, resulting in a spinal cord injury. Tissue samples were collected and sequenced using QuantSeq mRNA. Reads were trimmed and aligned to the rat reference genome. Transcript read counts were analyzed with DESeq2 for differential expression. Genes were considered significantly upregulated or downregulated with a fold change +/- 2 log 2 and p-value less than 0.05. Genes up and down regulated in each condition were further analyzed using Cytoscape and STRING DB for network construction. GO biological processes and cellular components were used to narrow the scope of networks to astrocyte associated. Custom radial transwells were constructed to facilitate 3D printing and imaging for the embedded 3D printing SCI testbed platform using polyethylene glycol (PEG) as a support bath. Spinal cord testbeds and gradients were printed utilizing mechanically tunable glycidyl methacrylate-hyaluronic acid (GMHA) at 10 mg/mL. Normal Human Astrocytes were used as a model cell to determine the potential for uGel as a spinal cord tissue testbed platform.

Results, Conclusions, and Discussions::

Gene networks were constructed for each condition (normoxia, DAIH, mild CIH, and mod CIH) (Figure 1). Astrocyte associated characteristics, indicated by the pink rings, are shown. Conditions omitted (upregulated DAIH, upregulated mild CIH, downregulated mild CIH, upregulated mod CIH and downregulated mod CIH) did not show any clear patterns or genes associated with astrocyte functions. Of note, downregulated in both normoxia and mild CIH is GFAP. Glial fibrillary acidic protein is found in CNS astrocytes and is a marker for reactive astrocytes. Overall, the down regulated pathways in normoxia are consistent with literature and serve as a point to explore the implications of downregulation of other pathways in DAIH.

3D printing technology allows for the ability to selectively deposited ECM to match the spatiotemporal changes following SCI. Each print is designed to be adjusted to incorporate a lesion site and the print path in the grey matter region is altered to allow for the addition of a glial scar mimic. Addition of astrocytes into the embedded 3D printed system allows for the exploration of the impact of hypoxia on the cell-ECM interaction in the glial scar environment. Glial scar formation is noted as a key player in the prevention of axonal regeneration after SCI, and this remodeling forms a gradient of CSPGs from the lesion [6]. The spatiotemporal alterations to the ECM landscape are mimicked within the 3D printed testbed by altering the bioink formulation and feature size along the length of the print to form a gradient of material deposition. In vitro testbeds allow for the exploration of specific cell-ECM interactions in a controlled environment. The SCI testbed system exhibits the modularity required to study spatiotemporal changes and SCI therapeutics. Bioinformatics-informed design of a biomimetic hydrogel for spinal cord injury is essential for a complete and more accurate biomaterial. Here, we determined transcripts that are highly altered and important in incorporating or exploring in a complete biomimetic hydrogel testbed system.

Acknowledgements (Optional): :

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