Speaker
Description
The injured spinal cord (SC) generates a unique and complex pathophysiology which presents a multifaceted challenge for repair. Broadly, normally supportive astroglia become reactive following injury and form a glial scar. This scar in turn inhibits recovery by releasing an impenetrable inhibitive extracellular matrix (ECM), which prevents axonal regrowth. Tissue engineering approaches aim to overcome these barriers by bridging the cyst with a physical neurotrophic substrate optimised for axonal growth. However, reactive astrocytes rapidly encapsulate these scaffolds, forming a physical barrier to regeneration. Our previous experience in peripheral nerve repair demonstrates that composition and stiffness of scaffolds play an essential role in neural tissue regeneration, directing the behaviour of axons and supporting cells1. Considering the physicochemical properties of SC tissue, we hypothesized that comprehensive optimization of the structure, stiffness and composition of matrix-based biomaterial scaffolds for SC applications could provide potent neurotrophic signalling and intrinsic immunomodulatory properties. Scaffolds with such innate signalling present a simplified alternative to traditional biochemical approaches to addressing the key clinical challenges of astrogliosis, the CNS foreign body response and cross-injury axonal regrowth.
To assess the trophic and immunomodulatory properties of native spinal cord materials, neurons and primary human astrocytes were cultured for 7 days on ECM-coated (5 µg/ml) coverslips, or with increasing gradients of biochemical factors and analyzed using immunohistochemistry. To produce biomimetic scaffolds of varying mechanical stiffnesses, soft (3mg/ml), medium (5 mg/ml) stiff (10 mg/ml) hyaluronic acid (Hya) hydrogels were prepared, after which the optimal ECM composition was mixed throughout the hydrogel. The resulting slurry was freeze-dried at -40˚C to form macroporous scaffolds, whose physicochemical properties were subsequently analyzed. Astrocytes and adult dorsal root ganglia (DRGs) were then seeded individually on the HyA scaffolds of varying stiffness and cultured for 1 - 3 weeks. ELISA analysis was performed to examine the release of inflammatory markers IL-6 and IL-10 from the scaffold-resident astrocytes. Immunohistochemistry was performed to examine cell morphology, nuclear:cytoplasmic ratio and axonal extension.
A novel mix of collagen-IV and fibronectin (Coll-IV/FN) was found to optimally enhance neuronal, astrocyte and DRG growth and induce morphological features typical of resting astrocytes. When incorporated into porous hyaluronic acid scaffolds of varying stiffness (0.8-3KPa), Coll-IV/Fn matrix-enhanced scaffolds promoted a range of growth-promoting behaviors in seeded cells in a stiffness-dependent manner. Softer scaffolds with properties similar to the native tissue were shown to mediate astrocyte polarization and significantly increased IL-10 production. Furthermore seeded DRGs exhibited alignment of neurites through the aligned pore structure and significantly increased axonal extension. These results indicate that the interaction of stiffness and the biomaterial surface plays an essential role in mediating repair-critical cellular responses and demonstrates the benefits of a biomimetic approach to the design of scaffolds for SCI repair2.
References:
1. Hibbitts, A. J. et al. Matrix Biol. (2022)
2. Woods, I. et al. Adv. Healthc. Mater.2101663.
Funding Acknowledgements:
Funded through a joint initiative by The Irish Rugby Football Union Charitable Trust/SFI AMBER Centre (SFI/12/RC/2278) as well as by an Irish Research Council Postgraduate Fellowship (Government of Ireland), Grant Number: GOIPD/2021/262.
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