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The extracellular matrix (ECM) of the central nervous system is a specialized, ultra-soft structure that provides crucial biochemical and mechanical support for neuronal survival, differentiation, and synaptic connectivity. It consists of a network of biomolecules, including glycosaminoglycans like hyaluronic acid, as well as structural and adhesive proteins such as laminins. Laminins play a pivotal role for spinal cord neurons by promoting cell adhesion, neurite outgrowth, and synapse formation, making it essential for proper neuronal network formation. Laminin-111 is expressed during embryonic development and is involved in axonal guidance and synapse formation. Laminin-211 and 221 are expressed in the neuromuscular system, and laminin-211 enhances neuronal differentiation and survival, while laminin-221 promotes motor axon outgrowth and cell adhesion.[1] Astrocytes, are regulators of the ECM, they modulate its composition by secreting ECM components, including laminins, and influence neuronal adhesion and connectivity through biochemical signaling and direct interactions.
We previously developed a three-dimensional (3D) spinal cord model using MEW-printed scaffolds as reinforcing structures, combined with Matrigel as commercially available ECM.[2] This study demonstrated the importance of the third dimension for neuronal models as not only protein expression differed compared to 2D, but also functional activity in the developed 3D samples started much earlier. To overcome the Matrigel limitations of batch-to-batch variations due to mouse sarcoma identity, and to improve our model we shifted to a thiolate hyaluronic acid (HA-SH)-polyethylene glycol (PEG) hydrogel, which provides a biocompatible ECM-like environment. Previous studies could already show that HA-SH hydrogels, included astrocytes and reinforced with PCL scaffolds successfully supported the network formation of cortical neuron.[3, 4] In addition to a hyaluronic acid based ECM including astrocytes, we added the three mentioned laminin isoforms into our system.
The study investigates how astrocytes and ECM molecules like laminins influence the network formation and function of spinal cord neurons. We compared three conditions: spinal cord neuron-only cultures, spinal cord neuron-astrocyte co-cultures, and laminin-supplemented spinal cord neuron-astrocyte co-cultures. Cell viability was assessed using live/dead assays, while immunocytochemistry was employed to analyze neuronal morphology and synaptic organization. Neuronal network activity was examined via calcium imaging, and the mechanical properties of the constructs were characterized through cyclic compression and tension tests. This model provides a platform to investigate how astrocytes and ECM composition influence spinal cord network formation in a 3D environment. 3D models of the spinal cord will be of interest in future studies to model e.g. neurodegenerative disease mechanisms or in testing therapeutic strategies.
- Colognato, H. and P.D. Yurchenco, Form and function: the laminin family of heterotrimers. Dev Dyn, 2000. 218(2): p. 213-34.
- Fischhaber, N., et al., Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix-A Model System to Study Disease Mechanisms. Adv Healthc Mater, 2021. 10(19): p. e2100830.
- Janzen, D., et al., Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation. Adv Healthc Mater, 2022. 11(21): p. e2201826.
- Andrade Mier, M.S., et al., Primary Glial Cell and Glioblastoma Morphology in Cocultures Depends on Scaffold Design and Hydrogel Composition. Adv Biol (Weinh), 2023. 7(10): p. e2300029.
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