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

• Published in: Tissue engineering. Part A
• Main Authors: Siddiqui, Ahad Mahmood, Oswald, David, Papamichalopoulos, Sophia, Kelly, Domhnall, Summer, Priska, Polzin, Michael, Hakim, Jeffery, Schmeichel, Ann M, Chen, Bingkun, Yaszemski, Michael J, Windebank, Anthony J, Madigan, Nicolas Niall
• Format: Journal Article
• Language: English
• Published: United States 01.06.2021
• ISSN: 1937-335X
• DOI: 10.1089/ten.tea.2020.0316

Positively charged oligo-polyethylene glycol fumarate (OPF+) hydrogel scaffolds, implanted into a complete transection spinal cord injury (SCI), facilitate a permissive regenerative environment and provide a platform for controlled observation of repair mechanisms. Axonal regeneration after SCI is critically dependent upon nutrients and oxygen from a newly formed blood supply. Our objective was to investigate fundamental characteristics of revascularization in association with the ingrowth of axons into hydrogel scaffolds, thereby defining spatial relationships between axons and the neovasculature. A novel combination of stereologic estimates and precision image analysis techniques quantitate neurovascular regeneration in rats. Multichannel hydrogel scaffolds containing Matrigel-only (MG), Schwann cells (SCs), or SCs with rapamycin-eluting poly(lactic co-glycolic acid) (PLGA) microspheres (RAPA) were implanted for 6 weeks following complete spinal cord transection. Image analysis of 72 scaffold channels identified a total of 2,494 myelinated and 4,173 unmyelinated axons at 10 micron circumferential intervals centered around 708 individual blood vessel profiles. Blood vessel number, density, volume, diameter, inter-vessel distances, total vessel surface and cross-sectional areas, and radial diffusion distances were compared. Axon number and density, blood vessel surface area, and vessel cross-sectional areas in the SC group exceeded that in the MG and RAPA groups. Individual axons were concentrated within a concentric radius of 200-250 microns from blood vessel walls, in Gaussian distributions which identified a peak axonal number (Mean Peak Amplitude) corresponding to defined distances (Mean Peak Distance) from each vessel. The highest concentrations of axons were largely excluded from a 25-30 micron zone immediately adjacent to the vessel, and from vessel distances of >150 microns. Higher axonal densities correlated with smaller vessel cross-sectional areas. A statistical spatial algorithm was used to generate cumulative distribution F- and G-functions of axonal distribution in the reference channel space. Axons located around blood vessels were definitively organized as clusters and were not randomly distributed. A scoring system stratifies 5 direct measurements and 12 derivative parameters influencing regeneration outcomes. By providing methods to quantify the axonal-vessel relationships, these results may refine spinal cord tissue engineering strategies to optimize the regeneration of complete neurovascular bundles in their relevant spatial relationships after SCI.