Regenerating axons and blood vessels in tissue engineered scaffolds have defined spatial relationships after complete spinal cord injury in rats.
1Madigan N, 2Oswald D, 3Kelly D, 1Hakim J, 1Chen B, 1Yaszemski M, 1Windebank A
1Mayo Clinic, Rochester, Minnesota, USA; 2Paracelsus Medical University, Salzburg, , Austria; 3National University of Ireland, Galway (NUIG), Galway, , Ireland
We have previously demonstrated that positively-charged oligo-polyethylene glycol fumarate (OPF+) hydrogel scaffolds implanted after complete thoracic spinal cord transection in rats facilitate a permissive regenerative environment by reducing scarring, and that there is a close relationship between new blood vessel formation and the number of regenerating axons. In this study we used multichannel OPF+ hydrogel scaffolds to provide a platform to control the micro-environment of the regenerating spinal cord. Our objective was to assess the spatial relationships between blood vessel formation and axonal regeneration in the core area of scaffold channels eight weeks after implantation. Poly-lactic-co-glycolic acid (PLGA) microspheres were embedded within Schwann cell (SC)-loaded OPF+ scaffolds for sustained release of the anti-fibrotic and immunosuppressant drug rapamycin. Animals were implanted with scaffolds containing Matrigel alone (n=5), SCs plus empty PLGA microspheres (n=6), or SCs plus rapamycin-eluting microspheres (n=6). Stereologic methods were applied to measure core channel area, blood vessel number, volume, diameter, intervessel distances, total vessel surface and cross-sectional areas, and radial diffusion distances in each group. Neurolucida software, immunohistochemistry and confocal imaging were used to perform a Sholl analysis to count a total of 2,494 myelinated and 4,173 unmyelinated axons at 10 micron circumferential intervals around 708 individual blood vessel profiles within scaffold channels. Patterns in the Gaussian distribution of axons with respect to their distance from and density around blood vessels were measured. Despite an equivalent number of new blood vessels in each group, axon number and surface density in the SC group exceeded that seen in the Matrigel and SC plus rapamycin animal groups. Higher axonal densities correlated with smaller vessel cross-sectional areas (Spearman r = -0.1431, p=0.0257). Axons in each group were concentrated within a concentric distance of 200 microns from the blood vessel wall, but were excluded from a 25 micron zone immediately adjacent to the vessel. Finally we used a statistical spatial algorithm to generate cumulative distribution functions of axons within each scaffold channel type to demonstrate that axons located around blood vessels were definitively clustered and not randomly distributed. Our results further refine spinal cord tissue engineering strategies to optimize the regeneration of complete neurovascular bundles in their relevant spatial relationships.
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