This review highlights current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury. The concept of developing 3-dimensional polymer scaffolds for placement into a spinal cord transection model has recently been more extensively explored as a solution for restoring neurologic function after injury. Given the patient morbidity associated with respiratory compromise, the discrete tracts in the spinal cord conveying innervation for breathing represent an important and achievable therapeutic target. The aim is to derive new neuronal tissue from the surrounding, healthy cord that will be guided by the polymer implant through the injured area to make functional reconnections. A variety of naturally derived and synthetic biomaterial polymers have been developed for placement in the injured spinal cord. Axonal growth is supported by inherent properties of the selected polymer, the architecture of the scaffold, permissive microstructures such as pores, grooves or polymer fibres, and surface modifications to provide improved adherence and growth directionality. Structural support of axonal regeneration is combined with integrated polymeric and cellular delivery systems for therapeutic drugs and for neurotrophic molecules to regionalize growth of specific nerve populations.

Objective: To determine the safety of intrathecal autologous adipose-derived mesenchymal stromal cell treatment for amyotrophic lateral sclerosis (ALS).Methods: Participants with ALS were enrolled and treated in this phase I dose-escalation safety trial, ranging from 1 3 10 7 (single dose) to 1 3 10 8 cells (2 monthly doses). After intrathecal treatments, participants underwent standardized follow-up, which included clinical examinations, revised ALS Functional Rating Scale (ALSFRS-R) questionnaire, blood and CSF sampling, and MRI of the neuroaxis.Results: Twenty-seven patients with ALS were enrolled and treated in this study. The safety profile was positive, with the most common side effects reported being temporary low back and radicular leg pain at the highest dose level. These clinical findings were associated with elevated CSF protein and nucleated cells with MRI of thickened lumbosacral nerve roots. Autopsies from 4 treated patients did not show evidence of tumor formation. Longitudinal ALSFRS-R questionnaires confirmed continued progression of disease in all treated patients.Conclusions: Intrathecal treatment of autologous adipose-derived mesenchymal stromal cells appears safe at the tested doses in ALS. These results warrant further exploration of efficacy in phase II trials. Classification of evidence:This phase I study provides Class IV evidence that in patient with ALS, intrathecal autologous adipose-derived mesenchymal stromal cell therapy is safe.

The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.

Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

Positively-charged oligo[poly(ethylene glycol)fumarate] (OPF ) is a biodegradable hydrogel used for spinal cord injury repair. We compared scaffolds containing primary Schwann cells (SCs) to scaffolds delivering SCs genetically modified to secrete high concentrations of glial cell-derived neurotrophic factor (GDNF). Multichannel OPF scaffolds loaded with SCs or GDNF-SCs were implanted into transected rat spinal cords for 4 weeks. GDNF-SCs promoted regeneration of more axons into OPF scaffolds (2773.0 ± 396.0) than primary SC OPF scaffolds (1666.0 ± 352.2) (p = 0.0491). This increase was most significant in central and ventral-midline channels of the scaffold. Axonal remyelination was quantitated by stereologic analysis. Increased myelination of regenerating axons was observed in the GDNF-SC group. Myelinating cell and axon complexes were formed by host SCs and not by implanted cells or host oligodendrocytes. Fast Blue retrograde tracing studies determined the rostral-caudal directionality of axonal growth. The number of neurons that projected axons rostrally through the GDNF-SC scaffolds was higher (7929 ± 1670) than in animals with SC OPF scaffolds (1069 ± 241.5) (p < 0.0001). The majority of ascending axons were derived from neurons located more than 15 mm from the scaffold-cord interface, and were identified to be lumbosacral intraspinal motor neurons. Transected animals with GDNF-SC OPF scaffolds partially recovered locomotor function at weeks 3 and 4 following surgery. Copyright © 2017 John Wiley & Sons, Ltd.

The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF(+)) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF(+) scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF(+) scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.

Functionalized biomaterial scaffolds targeted at improving axonal regeneration by enhancing guided axonal growth provide a promising approach for the repair of spinal cord injury. Collagen neural conduits provide structural guidance for neural tissue regeneration, and in this study it is shown that these conduits can also act as a reservoir for sustained gene delivery. Either a G-luciferase marker gene or a neurotrophin-3-encoding gene, complexed to a non-viral, cyclized, PEGylated transfection vector, was loaded within a multichannel collagen conduit. The complexed genes were then released in a controlled fashion using a dual release system both in vitro and in vivo. For evaluation of their biological performance, the loaded conduits were implanted into the completely transected rat thoracic spinal cord (T8-T10). Aligned axon regeneration through the channels of conduits was observed one month post-surgery. The conduits delivering neurotrophin-3 polyplexes resulted in significantly increased neurotrophin-3 levels in the surrounding tissue and a statistically higher number of regenerated axons versus the control conduits (P<0.05). This study suggests that collagen neural conduits delivering a highly effective non-viral therapeutic gene may hold promise for repair of the injured spinal cord.

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