Development of an iPSC Loaded Biomimetic Biomaterial Scaffold for Spinal Cord Repair Applications

<b>Introduction</b>: Following spinal cord injury, a lesion cavity develops preventing axonal regrowth. Biomaterial implants that bridge the cavity and encourage axonal growth while delivering trophic cells to restore lost tissue may have potential for repair. Therefore, by identifying neurotrophic extracellular-matrix proteins to incorporate into biomimetic hyaluronic acid biomaterial scaffolds and by tuning the biomaterial stiffness, we aimed to develop a novel platform for spinal cord repair. Furthermore, we wished to determine if the developed biomimetic biomaterial scaffolds were capable of delivering and modulating the trophic capacity of induced pluripotent stem cell (iPSC)-derived progenitors for spinal cord repair applications.<br/><br/><b>Methods</b>: Spinal cord astrocytes and neurons were cultured on a range of extracellular matrix-proteins to identify the optimal neurotrophic substrate combination. Following the incorporation of the neurotrophic substrate into freeze-dried 3D hyaluronic acid scaffolds of varying stiffnesses, scaffold properties were characterized. Next, spinal cord astrocytes, neurons, dorsal root ganglia (DRG) and iPSC-derived astrocyte progenitors were cultured in the scaffolds for up to 21 days and the effect of scaffold physicochemical properties was assessed. Thereafter, the impact of scaffold properties on the trophic capacity of iPSC-derived progenitors was assessed <i>in vitro</i> and <i>ex vivo</i> using neurons, DRG and spinal cord explants.<br/><br/><b>Results</b>: A combination of collagen-IV (CIV) and fibronectin (FN) synergistically enhanced neurite outgrowth by 203% while also promoting astrocyte process extension. Subsequently, hyaluronic acid biomaterial scaffolds functionalized with CIV/FN were successfully manufactured with different stiffnesses ranging from soft/biomimetic to stiff/supraphysiological (0.8-3.5 kPa). Spinal cord astrocytes cultured in soft-CIV/FN functionalized biomaterial scaffolds matching cord stiffness, displayed positive stellate morphologies and increased pro-regenerative IL-10 release. Furthermore, soft scaffolds significantly enhanced neurite outgrowth from neurons and adult mouse DRG explant cultures compared to stiffer scaffolds. Soft-CIV/FN scaffolds also enhanced iPSC-progenitor neurotrophic and functional capacity while encouraging the growth of iPSC-derived spheroids that subsequently formed extensive neuronal/astrocytic cellular tracts, colonizing the biomaterial scaffold. Conditioned media from soft-CIV/FN but not other, iPSC-loaded scaffolds, and applied to growing neurons enhanced neurite outgrowth by over 280%. Super-resolution microscopy also confirmed similar neurite enhancement (170%) in mouse primary spinal cord neurons treated with media from soft-CIV/FN scaffolds only without eliciting negative inflammatory and/or reactive responses from healthy and injured astrocytes. Furthermore, analysis of media from stiffer or non-CIV/FN-containing scaffolds showed increased pro-inflammatory IL-1β release compared to soft CIV/FN scaffolds. Finally, when <i>ex vivo</i> DRGs or spinal cord explants were cultured directly on CIV/FN iPSC-loaded biomaterial scaffolds up to 21 days, only soft biomaterials enhanced cellular infiltration, astrocytic process extension and directed axonal outgrowth between DRG and iPSC-spheroids.<br/><br/><b>Conclusion</b>: Overall, this research describes the development and utilization of a novel, biomimetic biomaterial scaffold-based platform for spinal cord repair and demonstrates that biomimetically inspired biomaterials can enhance the growth of cord resident neurons and astrocytes and promote the neurotrophic potential of implanted progenitor cells to act as a novel, therapeutic platform for spinal cord injury repair applications.<br/><br/><b>Acknowledgements</b>: This work is funded by the Anatomical Society, Irish Rugby Football Union Charitable Trust, and the Science Foundation Ireland Advanced Materials for Bioengineering Research (AMBER) centre.