Applications for 2D and 3D configurations of hybrid OPF+ scaffolds to investigate neuroregeneration in vitro and in vivo following spinal cord injury
1Siddiqui A, 1Madigan N, 2Brunner R, 1Liu X, 3Schwarzbauer J, 3Harris G, 3Schwartz J, 1Lu L, 1Yaszemski M, 1Windebank A
1Mayo Clinic, Rochester, Minnesota, United states; 2Paracelsus Medical University, Salzburg, , Austria; 3Princeton University, Princeton, New jersey, United states
Objective: Spinal cord injury (SCI) occurs due to traumatic force applied to the cord leading to paralysis. The spinal cord has a limited ability to regenerate and current therapies are not effective in promoting regeneration. This may be due to events that occur in the secondary injury phase, such as axonal loss, demyelination, cyst formation, and extracellular matrix (ECM) remodeling. We have developed a novel scaffold biomaterial that is fabricated from biodegradable hydrogel oligo(poly(ethylene glycol) fumarate) (OPF). We have shown that positively charged OPF+ fabricated in an open spaced, multichannel design loaded with glial cell derived neurotrophic factor (GDNF) secreting Schwann cells enhanced functional and anatomical recovery after SCI. However, the axons were not only found passing through the channels of the scaffold but also on the outside surface. Therefore, we hypothesize that surface area available for the axon plays an important role in regeneration.
Methods: We have developed a hybrid OPF+ biomaterial that increases the surface area available for attachment and that contains an aligned microarchitecture with an ECM to better support axonal regeneration. OPF+ was fabricated as a 0.08mm thick sheet containing 100μm high polymer ridges that can self-assemble into a spiral morphology when hydrated. The ridges on the sheets were space 0.2mm, 0.4mm, or 1mm apart. The sheets were coated with different ECM molecules (laminin, fibronectin, and collagen). Then whole dorsal root ganglions (DRGs), dissociated DRGs, or Schwann cells were seeded on top. Cell attachment, neurite extension, and alignment were measured. Schwann cells and dissociated DRGs were co-cultured on top of differently spaced scaffolds to look at alignment of myelinated axons per area.
Results: We found that laminin or fibronectin ECM coatings enhanced cell attachment and outgrowth on the scaffold surface. In addition, the ridges aligned axons in a longitudinal bipolar orientation. Decreasing the space between the ridges increased the number of cells and aligned neurites in the direction of the ridge. These scaffolds can be loaded with cells and configured into a 3D structure for transplantation into the spinal cord which may enhance axonal outgrowth and reconnection following SCI. Schwann cells seeded on laminin coated OPF+ sheets align along the ridges over a 6 day period. When co-cultured with dissociated DRGs on the scaffolds, the Schwann cell myelinate the neurites over a 3 week period. Reducing the ridge spacing is able to increase the amount of myelinated neurites in a given area. These scaffolds are being further modified to incorporate a striped, cell attractive chemical interface interfaced with decellularized ECM to provide further ultrastructural alignment.
Conclusion: This study provides a synthetic material with a matrix architecture containing adhesion molecules and growth factors to provide an environment favorable for cell survival, migration, and regrowth. Our in vitro results suggest that this novel scaffold design with closer spaced ridges and Schwann cells would be ideal for in vivo transplantation to promote regeneration after SCI.
Support: This work is generously funded by the New Jersey Commission on Spinal Cord Research.
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