Defining spatial relationships between spinal cord axons and blood vessels in hydrogel scaffolds
Authors: Siddiqui Ahad M. , Oswald David , Papamichalopoulos Sophia , Kelly Domnhall , Summer Priska , Polzin Michael , Hakim Jeffrey , Chen Bingkun , Yaszemski Michael J. , Windebank Anthony J. , Madigan Nicolas N. 
Published: October 2019

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 the availability of nutrients and oxygen from a newly formed blood supply. In this study, the objective was to investigate fundamental characteristics of revascularization in association with the ingrowth of axons into hydrogel scaffolds, and to define the spatial relationships between axons and the neovasculature. A novel combination of stereologic estimates and precision image analysis techniques are described to 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 in each group were measured. 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. Axons were concentrated within a concentric radius of 200-250 microns from the blood vessel wall in Gaussian distributions which identified a peak axonal number (mean peak amplitude) corresponding to defined distances (mean peak distance) from each vessel. Axons were largely excluded from a 25 micron zone immediately adjacent to the vessel. 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. 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.

Impact Statement

Vascular disruption and impaired neovascularization contribute critically to the poor regenerative capacity of the spinal cord after injury. In this study, hydrogel scaffolds provide a detailed model system to investigate the regeneration of spinal cord axons as they directly associate with individual blood vessels, using novel methods to define their spatial relationships and the physiologic implications of that organization. These results refine future tissue-engineering strategies for spinal cord repair to optimize the re-development of complete neurovascular bundles in their relevant spatial architectures.