Defining spatial relationships between spinal cord axons and blood vessels in hydrogel scaffolds

Abstract

1Spinal cord injury (SCI) results in lifelong paralysis due to the poor regenerative capability of the 2 central nervous system and the hostile microenvironment that is created from processes such as 3 inflammation, scarring, axonal dieback, and demyelination. Hydrogel scaffolds facilitate a 4 permissive regenerative environment and overcome these barriers by reducing scarring. One 5 important other consideration for axonal regeneration is the availability of nutrients and oxygen, 6 making it crucial to further investigate vascularization characteristics in the regenerating spinal 7 cord. 8 We previously described the close relationship between blood vessel formation and axonal 9 regeneration. In this study, we focused on identifying the vascular -axonal relationship, as well 10 describing novel techniques to analyze their interactions after a complete T9 spinal cord 11 transection in rats. Following implantation of positively charged oligo-polyethylene glycol 12 fumarate (OPF+) scaffolds containing Matrigel-only (MG), Schwann cells (SCs), or SCs with 13 rapamycin-eluting poly-lactic-co-glycolic acid (PLGA) microspheres (RAPA), stereological 14 methods were applied to measure core area, blood vessel number, volume, diameter, inter-vessel 15 distances, total vessel surface and cross-sectional areas, and radial diffusion distances in each 16 group 6 weeks after implantation.17Immuno-histochemical and stereological analysis demonstrated a significantly larger core area in 18 the RAPA group and found a total of 2,494 myelinated and 4,173 unmyelinated axons at 10 19 micron circumferential intervals around 708 individual blood vessel profiles within scaffold 20 channels. We found that axon number and surface density in the SC group exceeded that seen in 21 the MG and RAPA groups and that higher axonal densities correlated with smaller vessel cross-22 sectional areas. Generally, axons were concentrated within a concentric distance of 200 microns 23 from the blood vessel wall, but were excluded from a 25 micron zone immediately adjacent to 1 the vessel. Using a statistical spatial algorithm to generate cumulative distribution functions of 2 axons within each scaffold channel type, we demonstrated that axons located around blood 3 vessels were not randomly distributed. 4By providing methods to quantify the axonal-vessel relationship, our results refine spinal cord 5 tissue engineering strategies to optimize the regeneration of complete neurovascular bundles in 6 their relevant spatial relationships, and further provide better understanding of axon regeneration 7 in relation to revascularization in SCI.

Newly regenerated axons through a cell-containing biomaterial scaffold promote reorganization of spinal circuitry and restoration of motor functions with epidural electrical stimulation

“…Reconnection across complete SCI has been achieved with a nerve autograft and scaffolds 21,22 , particularly when delivering cell types and growth factors [22][23][24][25][26][27][28] . Although the functional outcome in this study was better in rats who received combined therapy, the number of axons through scaffolds was not different between the groups and was in between previously reported with scaffold loaded GDNF secreting Schwann cells 25 or rapamycin with Schwann cells 29 . Rapamycin may reduce the number of regenerating axons, although previous study demonstrated improvement when compared with OPF+ scaffolds loaded with Schwann cells alone 14 .…”

Defining Spatial Relationships Between Spinal Cord Axons and Blood Vessels in Hydrogel Scaffolds

“…In addition, finding a proper combination of biomaterials, cell types, and small molecules remain to be elucidated. Our recent findings demonstrated the close relationship between blood vessel formation and the number of regenerating axons (21). We demonstrated that the formation of a capillary structure in turn supported higher numbers of regenerating axons, implicating the importance of vascular normalization in the regenerating spinal cord.…”

“…25 mg of PLGA microspheres were added to 250 ul OPF+ liquid polymer thereafter. Scaffold fabrication was as described previously (7) via injection of the liquid polymer with suspended microspheres into a tubular glass mold containing seven parallel aligned wires as placeholders for the channel spaces.…”

“…SCs were cultured from the sciatic nerve of 2-5 day old rat pups, as we have previously described (7). Cells were grown in SC media (DMEM/F12 medium containing 10% fetal bovine serum, 100 units/mL penicillin/streptomycin (Gibco, Grand Island, New York, USA), 2 μM forskolin (Sigma-Aldrich), and 10 ng/mL recombinant human neuregulin-1-β1 extracellular domain (R&D Systems, Minneapolis, Minnesota, USA)).…”

“…This model allows for precise management of the local injury conditions, clear discrimination between true regeneration and remodeling or distal axonal sprouting (2)(3)(4)(5) and reliable quantification (6). Positively-charged multichannel oligo(poly(ethylene glycol) fumarate) (OPF+) hydrogel scaffolds provide an ideal platform to control the micro-environment of the regenerating spinal cord and is amongst the most effective polymers in terms of supporting axonal regeneration (7) as it creates a microenvironment permissive for axonal regeneration (8). OPF+ was found to have enhanced neuron adherence, sustained neuron and Schwann cell viability for three weeks and promoted axon certified by peer review) is the author/funder.…”