Following spinal cord injury, a complex scar forms around a lesion cavity, preventing axonal regrowth. Despite ongoing development of stem cell treatments for spinal cord injury, effective repair of the cord remains a challenge in part due to the lack of a supportive environment and cell death. Therapeutics that physically bridge the cavity with a neurotrophic environment while simultaneously delivering stem cells to restore lost tissue may have potential. Building off success in our lab in peripheral nerve repair, we aimed to identify neurotrophic extracellular matrix (ECM) proteins to develop novel, biomimetic functionalized hyaluronic acid scaffold implants and to determine their trophic capacity for spinal cord applications using multiple cell models. By optimizing scaffold stiffness and matrix composition for stem cell delivery, it was hypothesized that the pro-regenerative signaling properties of trophic induced pluripotent stem cell (iPSC) derived astrocyte progenitors could be enhanced by scaffold physiochemical properties to promote cord repair.
To identify neurotrophic ECM proteins, astrocytes and various neuronal cells were cultured on a range of ECM combinations to identify a novel neurotrophic substrate. Following incorporation of the neurotrophic substrate mix into freeze-dried 3D hyaluronic acid scaffolds of varying stiffness, scaffold physicochemical properties were characterized. Astrocytes, neurons, dorsal root ganglia (DRG) and iPSC-derived astrocyte progenitors were cultured in scaffolds up to 21 days and the effect of scaffold stiffness and matrix composition was assessed. Additionally, the impact of scaffold properties on the therapeutic effectiveness of iPSC-derived progenitors was assessed using various models.
Screening of central nervous system ECM components revealed that a combination of collagen-IV (Coll-IV) and fibronectin (FN) synergistically enhanced neuronal and astrocyte outgrowth compared to control substrate poly L-lysine. Subsequently, hyaluronic acid scaffolds functionalized with Coll-IV/FN were manufactured using different concentrations of hyaluronic acid to produce scaffolds of varied stiffnesses ranging from soft to stiff (0.8-3kPa). Astrocytes cultured in soft, Coll-IV/FN functionalized scaffolds, increased secretion of IL-10 and exhibited morphologies typical of resting phenotypes. Furthermore, soft CIV/FN scaffolds significantly enhanced neurite outgrowth from DRG explants (a model of axonal growth) compared to stiffer scaffolds. Soft, but not stiff Coll-IV/FN scaffolds also promoted iPSC progenitor infiltration, differentiation and glutamate uptake (a measure of functional capacity) while encouraging iPSC-derived spheroid growth. Furthermore, conditioned media taken from soft, CIV/FN iPSC-loaded but not stiffer scaffolds significantly enhanced neurite outgrowth 2.8 fold. Finally, mouse spinal cord and DRG explants cultured on soft, Coll-IV/FN iPSC scaffolds promoted astrocyte migration and long axonal extensions between DRG and iPSC neurospheres within scaffolds.
These data indicate that by appropriately tuning the physicochemical properties of scaffolds to mimic that of the uninjured spinal cord, significantly enhances astrocyte responses while promoting neurite extension. Furthermore, biomimetic scaffolds promote the paracrine activity of patient-derived progenitor cells, enhancing their therapeutic capacity. Overall, the impact of biomaterial properties on the therapeutic effectiveness of stem cells has significant implications for spinal cord repair applications.
This work is funded by the IRFU-Charitable Trust, Anatomical Society and SFI-AMBER centre.
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