Spinal Cord Injury Regeneration Research Articles

Hydrogels and Cell Based Therapies in Spinal Cord Injury Regeneration.

Spinal cord injury (SCI) is a central nervous system- (CNS-) related disorder for which there is yet no successful treatment. Within the past several years, cell-based therapies have been explored for SCI repair, including the use of pluripotent human stem cells, and a number of adult-derived stem and mature cells such as mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although promising, cell transplantation is often overturned by the poor cell survival in the treatment of spinal cord injuries. Alternatively, the therapeutic role of different cells has been used in tissue engineering approaches by engrafting cells with biomaterials. The latter have the advantages of physically mimicking the CNS tissue, while promoting a more permissive environment for cell survival, growth, and differentiation. The roles of both cell- and biomaterial-based therapies as single therapeutic approaches for SCI repair will be discussed in this review. Moreover, as the multifactorial inhibitory environment of a SCI suggests that combinatorial approaches would be more effective, the importance of using biomaterials as cell carriers will be herein highlighted, as well as the recent advances and achievements of these promising tools for neural tissue regeneration.

Biomaterial scaffolds used for the regeneration of spinal cord injury (SCI).

This review presents a summary of various types of scaffold biomaterials used alone or together with therapeutic drugs and cells to regenerate spinal cord injury (SCI). The inhibitory environment and loss of axonal connections after SCI give rise to critical obstacles to regeneration of lost tissues and neuronal functions. Biomaterial scaffolds can provide a bridge to connect lost tissues, an adhesion site for implanted or host cells, and sustained release of therapeutic drugs in the injured spinal cord. In addition, they not only provide a structural platform, but can play active roles by inhibiting apoptosis of cells, inflammation and scar formation, and inducing neurogenesis, axonal growth and angiogenesis. Many synthetic and natural biomaterial scaffolds have been extensively investigated and tested in vitro and in animal SCI models for these purposes. We summarized the literature on the biomaterials commonly used for spinal cord regeneration in terms of historical backgrounds and current approaches.

Beneficial Effects of Melatonin Combined with Exercise on Endogenous Neural Stem/Progenitor Cells Proliferation after Spinal Cord Injury

Endogenous neural stem/progenitor cells (eNSPCs) proliferate and differentiate into neurons and glial cells after spinal cord injury (SCI). We have previously shown that melatonin (MT) plus exercise (Ex) had a synergistic effect on functional recovery after SCI. Thus, we hypothesized that combined therapy including melatonin and exercise might exert a beneficial effect on eNSPCs after SCI. Melatonin was administered twice a day and exercise was performed on a treadmill for 15 min, six days per week for 3 weeks after SCI. Immunohistochemistry and RT-PCR analysis were used to determine cell population for late response, in conjunction with histological examination and motor function test. There was marked improvement in hindlimb function in SCI+MT+Ex group at day 14 and 21 after injury, as documented by the reduced size of the spinal lesion and a higher density of dendritic spines and axons; such functional improvements were associated with increased numbers of BrdU-positive cells. Furthermore, MAP2 was increased in the injured thoracic segment, while GFAP was increased in the cervical segment, along with elevated numbers of BrdU-positive nestin-expressing eNSPCs in the SCI+MT+Ex group. The dendritic spine density was augmented markedly in SCI+MT and SCI+MT+Ex groups. These results suggest a synergistic effect of SCI+MT+Ex might create a microenvironment to facilitate proliferation of eNSPCs to effectively replace injured cells and to improve regeneration in SCI.

Telemedicine and digital management in repair and regeneration after nerve injury and in nervous system diseases.

Telemedicine and digital management in repair and regeneration after nerve injury and in nervous system diseases.

Identification and distribution of rRNH1, a gene upregulated after spinal cord primary neuron injury

Our previous study identified and characterized one differentially expressed gene, Rattus norvegicus ribonuclease/angiogenin inhibitor 1 (rRNH1). Transcriptional activity of lots of genes involves in spinal cord injury (SCI) and regeneration, but the mechanisms remain unknown. In the present study, we analyzed the sequences of rRNH1 and examined the expression pattern of rRNH1 in different adult rat tissues. We found the sequences of rRNH1 show high homology to Homo sapiens ribonuclease/angiogenin inhibitor 1; it encoded a protein of 461 amino acid residues and contained 13 leucine-rich repeat motifs. Using real-time quantitative PCR (q-PCR), rRNH1 mRNA was widely expressed in adult rat tissues, especially in the central nervous system. Moreover, rRNH1 protein was found to be upregulated after SCI. Although the precise function of rRNH1 is unknown, its unique expression pattern and upregulation after SCI suggest that rRNH1 might be involved in the succeeding injury and/or regeneration processes of injured spinal cord. All these data make rRNH1 a new interesting start to study the mechanisms of SCI and neuron regeneration.

Therapeutic effects of NogoA vaccine and olfactory ensheathing glial cell implantation on acute spinal cord injury

Therapeutic effects of NogoA vaccine and olfactory ensheathing glial cell implantation on acute spinal cord injury Zhicheng Zhang, Fang Li, Tiansheng Sun, Dajiang Ren, Xiumei Liu PLA Institute of Orthopedics, Beijing Army General Hospital, Beijing, People's Republic of China Background: Many previous studies have focused on the effects of IN-1, a monoclonal antibody that neutralizes Nogo (a neurite growth inhibitory protein), on neurologic regeneration in spinal cord injury (SCI). However, safety problems and the short half-life of the exogenous antibody are still problematic. In the present study, the NogoA polypeptide was used as an antigen to make a therapeutic NogoA vaccine. Rats were immunized with this vaccine and were able to secrete the polyclonal antibody before SCI. The antibody can block NogoA within the injured spinal cord when the antibody gains access to the spinal cord due to a compromised blood–spinal cord barrier. Olfactory ensheathing glial cell transplantation has been used in a spinal cord contusion model to promote the recovery of SCI. The present study was designed to verify the efficacy and safety of NogoA polypeptide vaccine, the effects of immunotherapy with this vaccine, and the synergistic effects of the vaccine and olfactory ensheathing glial cells in repair of SCI. Methods: A 13-polypeptide fragment of NogoA was synthesized. This fragment was then coupled with keyhole limpet hemocyanin to improve the immunogenicity of the polypeptide vaccine. Immunization via injection into the abdominal cavity was performed in rats before SCI. The serum antibody level and ability of the vaccine to bind with Nogo were detected by enzyme-linked immunosorbent assay. The safety of the vaccine was evaluated according to the incidence and severity of experimental autoimmune encephalomyelitis. Olfactory ensheathing glia cells were obtained, purified, and subsequently implanted into a Wistar rat model of thoracic spinal cord contusion injury. The rats were divided into four groups, ie, an SCI model group, an olfactory ensheathing glia group, a vaccine group, and a combined treatment group. Animal behavior, histopathology, and axonal regeneration were compared between the four treatment groups. Results: The antibody against the polypeptide was detected in rat serum by enzyme-linked immunosorbent assay. Experimental autoimmune encephalomyelitis was not found in the immunized rats. Three months after injury, Basso, Beattie, Bresnahan scores and nerve fiber counts in biotinylated dextran amine nerve tracing studies were significantly better in the combined treatment group than in the other groups. Conclusion: The polypeptide NogoA vaccine can stimulate the humoral immune system to produce antibodies against the NogoA polypeptide. The binding reaction between the antibody and antigen was shown in ex vivo experiments. No evidence was found to suggest a relationship between NogoA vaccination and increased risk of experimental autoimmune encephalomyelitis. The combined strategy of olfactory ensheathing glial cell implantation and NogoA vaccination may promote regeneration of axons and functional recovery in the spinal cord contusion injury model. This study may provide a new strategy for combining modalities to enhance axonal regeneration and a better balance of the CNS microenvironment after SCI. Keywords: spinal cord injury, immunotherapy, cell transplantation, olfactory ensheathing glial cells, NogoA, vaccine

Development and Characterization of a PHB‐HV‐based 3D Scaffold for a Tissue Engineering and Cell‐therapy Combinatorial Approach for Spinal Cord Injury Regeneration

Spinal cord injury (SCI) leads to devastating neurological deficits. Several tissue engineering (TE)-based approaches have been investigated for repairing this condition. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) is found to be particularly attractive for TE applications due to its properties, such as biodegradability, biocompatibility, thermoplasticity and piezoelectricity. Hence, this report addresses the development and characterization of PHB-HV-based 3D scaffolds, produced by freeze-drying, aimed to SCI treatment. The obtained scaffolds reveal an anisotropic morphology with a fully interconnected network of pores. In vitro studies demonstrate a lack of cytotoxic effect of PHB-HV scaffolds. Direct contact assays also reveal their ability to support the culture of CNS-derived cells and mesenchymal-like stem cells from different sources. Finally, histocompatibility studies show that PHB-HV scaffolds are well tolerated by the host tissue, and do not negatively impact the left hindlimb locomotor function recovery. Therefore results herein presented suggest that PHB-HV scaffolds may be suitable for SCI treatment.

Introduction to the Guidelines for the Management of Acute Cervical Spine and Spinal Cord Injuries

Introduction to the Guidelines for the Management of Acute Cervical Spine and Spinal Cord Injuries

Combining Adult Stem Cells and Olfactory Ensheathing Cells: The Secretome Effect

Adipose-derived adult stem cells (ASCs), bone marrow mesenchymal stem cells (bmMSCs), and human umbilical cord perivascular cells (HUCPVCs) tissue have been widely tested for regenerative applications, such as bone regeneration. Moreover, olfactory ensheathing cells (OECs) show promise in promoting spinal cord injury (SCI) regeneration. Our group recently proposed the use of a hybrid scaffold targeting both vertebral bone repair and SCI regeneration. According to this concept, both MSCs and OECs should be in close contact to be influenced by the factors that are involved in secretion. For this reason, here we studied the effects of the OEC secretome on the metabolic activity and proliferation of ASCs, bmMSCs, and HUCPVCs. The stem cells' secretome effects on metabolic activity and proliferation of the OECs were also considered. In co-cultures of OECs with ASCs, bmMSCs, or HUCPVCs, the metabolic activity/viability, proliferation, and total cell numbers were measured after 2 and 7 days of culture. The results demonstrated that the secretome of OECs has a positive effect on the metabolic activity and proliferation of MSCs from different origins, especially on ASCs. Furthermore, in general, the stem cells' secretome also had a positive effect on the OECs behavior, particularly when ASCs were in co-culture with OECs. These results suggest that the most suitable combination of cells to be used in our hybrid scaffold is the OECs with the ASCs. Finally, this work adds new knowledge to the cell therapy field, bringing new information about paracrine interactions between OECs and distinct mesenchymal stems.

Genetic modification of mesenchymal stem cells in spinal cord injury repair strategies

Spinal cord injury (SCI) is a serious injury of the central nervous system and up until now there is no evident effective treatment for SCI. Axonal regeneration is the only way to restore functions after serious SCI that interrupt the long tracts mediating motor and sensory function. The hurdles for axonal regeneration in SCI include: glial scar tissue and molecular barriers, the inhibiting microenvironment, and the lack of sufficient neurotrophic support. Therefore, the key point of applying stem cells to treat SCI is to build a microenvironment conducive to the survival and differentiation of stem cells and regulate neurotrophic factor expression. Adult mesenchymal stem cells (MSCs) have been applied in experimental animal models and clinical trials of SCI. Genetic modification of MSCs can increase secretion of peptides or total length proteins with potential to repair SCI and promote survival of themselves and survival or regeneration of neurons. There are many proteins that have been applied to modified MSCs, such as neurotrophic factors (neurotrophin 3, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, nerve growth factor, and MNTS1), receptor tyrosine kinases (tropomyosin-related kinase C), and hepatocyte growth factor. In the future, there will be more molecules acting as transgenes in MSCs for treatment of SCI.

Transection method for shortening the rat spine and spinal cord

Previous studies have presented evidence which indicates that the regeneration of axons in the spinal cord occurs following spinal cord transection in young rats. However, in a transection-regeneration model, the completeness of the transection is often a matter of dispute. We established a method for shortening the rat spine and spinal cord to provide a spinal cord injury (SCI) model in which there was no doubt about whether the axonal transection was complete. In the future, this model may be applied to the chronic period of complete paralysis following SCI. Adult, female Wistar rats (220–250g) were used in the study. The spinal cord was exposed and a 4-mm-long segment of the spinal cord was removed at Th8. Subsequently, the Th7/8 and Th8/9 discs were cut between the stumps of the spinal cord to remove the Th8 vertebra. The stitches which had been passed through the 7th and 9th ribs bilaterally were tied gradually to bring together the stumps of the spinal cord. Almost all the rats survived until the end of the experiment. Uncoordinated movements of the hind limbs in locomotion were observed at 4 weeks after surgery. However coordinated movements of the hind limbs in locomotion were not observed until the end of the experiment. After 12 weeks, an intracardiac perfusion was performed to remove the thoracic spine and the spinal cord. There were no signs of infection. The bone fusion of the Th7 and Th9 vertebrae was observed to be complete in all specimens and the alignment of the thoracic spine was maintained. The spinal canal was also correctly reconstituted. The stumps of the spinal cord were connected. Light microscopy of the cord showed that scar tissue intervened at the connection site. Cavitation inhibiting the axonal regeneration was also observed. This model was also made on the assumption that glial scar tissue inhibits axonal regeneration in chronic SCI. Axonal regeneration was not observed across the transected spinal cord in this model. Attempts should be made to minimize the damage to the spinal cord and the surgery time for successful axonal regeneration to occur. The model developed in this study may be useful in the study of axonal regeneration in SCI.

FM19G11 Favors Spinal Cord Injury Regeneration and Stem Cell Self-Renewal by Mitochondrial Uncoupling and Glucose Metabolism Induction

Spinal cord injury is a major cause of paralysis with no currently effective therapies. Induction of self-renewal and proliferation of endogenous regenerative machinery with noninvasive and nontoxic therapies could constitute a real hope and an alternative to cell transplantation for spinal cord injury patients. We previously showed that FM19G11 promotes differentiation of adult spinal cord-derived ependymal stem cells under hypoxia. Interestingly, FM19G11 induces self-renewal of these ependymal stem cells grown under normoxia. The analysis of the mechanism of action revealed an early increment of mitochondrial uncoupling protein 1 and 2 with an early drop of ATP, followed by a subsequent compensatory recovery with activated mitochondrial metabolism and the induction of glucose uptake by upregulation of the glucose transporter GLUT-4. Here we show that phosphorylation of AKT and AMP-activated kinase (AMPK) is involved in FM19G11-dependent activation of GLUT-4, glucose influx, and consequently in stem cell self-renewal. Small interfering RNA of uncoupling protein 1/2, GLUT-4 and pharmacological inhibitors of AKT, mTOR and AMPK signaling blocked the FM19G11-dependent induction of the self-renewal-related markers Sox2, Oct4, and Notch1. Importantly, FM19G11-treated animals showed accelerated locomotor recovery. In vivo intrathecal sustained administration of FM19G11 in rats after spinal cord injury showed more neurofilament TUJ1-positive fibers crossing the injured area surrounded by an increase of neural precursor Vimentin-positive cells. Overall, FM19G11 exerts an important influence on the self-renewal of ependymal stem progenitor cells with a plausible neuroprotective role, providing functional benefits for spinal cord injury treatment.

Peripheral mineralization of a 3D biodegradable tubular construct as a way to enhance guidance stabilization in spinal cord injury regeneration

Spinal cord injuries (SCI) present a major challenge to therapeutic development due to its complexity. Combinatorial approaches using biodegradable polymers that can simultaneously provide a tissue scaffold, a cell vehicle, and a reservoir for sustained drug delivery have shown very promising results. In our previous studies we have developed a novel hybrid system consisting of starch/poly-e-caprolactone (SPCL) semi-rigid tubular porous structure, based on a rapid prototyping technology, filled by a gellan gum hydrogel concentric core for the regeneration within spinal-cord injury sites. In the present work we intend to promote enhanced osteointegration on these systems by pre-mineralizing specifically the external surfaces of the SPCL tubular structures, though a biomimetic strategy, using a sodium silicate gel as nucleating agent. The idea is to create two different cell environments to promote axonal regeneration in the interior of the constructs while inducing osteogenic activity on its external surface. By using a Teflon cylinder to isolate the interior of the scaffold, it was possible to observe the formation of a bone-like poorly crystalline carbonated apatite layer continuously formed only in the external side of the tubular structure. This biomimetic layer was able to support the adhesion of Bone Marrow Mesenchymal Stem Cells, which have gone under cytoskeleton reorganization in the first hours of culture when compared to cells cultured on uncoated scaffolds. This strategy can be a useful route for locally stimulate bone tissue regeneration and facilitating early bone ingrowth.

Beneficial effects of melatonin combined with exercise on endogenous neural stem/progenitor cells regeneration after spinal cord injury

After spinal cord injury (SCI), as a protective response, endogenous adult neural stem/progenitor cells (NSPCs) proliferate and differentiate into neurons and glial cells to facilitate healing process. We have shown that melatonin treatment combined with exercise had a synergistic effect in functional recovery after SCI. In this study, we have examined the combined effects of melatonin and exercise (SCI+MT+Ex) on NSPCs, at early and late phases of spinal injury. Compared to the SCI group, there was marked improvement in hind limb function in SCI+MT+Ex group after 14 and 21, as documented by the reduced size of the spinal lesion and a higher density of dendritic spines and axons; such functional improvements were associated with increased numbers of BrdU‐positive progenitor cells. Furthermore, neuronal marker MAP‐2 was increased in the injured thoracic segment, while astrocyte marker GFAP was increased in the cervical segment, along with elevated numbers of BrdU‐positive nestin expressing neural precursor cells in the SCI+MT+Ex group. These results suggest that the synergistic effect of melatonin combined with exercise might create a microenvironment to facilitate proliferation of nestin‐positive endogenous NSPCs to effectively replace injured cells and to improve regeneration of SCI.

Interactions between Schwann and olfactory ensheathing cells with a starch/polycaprolactone scaffold aimed at spinal cord injury repair

Spinal cord injury (SCI) represents a major world health problem. Therefore it is urgent to develop novel strategies that can specifically target it. We have previously shown that the implantation of starch-based scaffolds (SPCL) aimed for spine stabilization on SCI animals leads to motor skills improvements. Therefore, we hypothesize that the combination of these scaffolds with relevant cell populations for SCI repair will, most likely, lead to further improvements. Therefore, in this work, the ability of SPCL scaffolds to support the 3D culture of olfactory ensheathing cells (OECs) and Schwann cells (SCs) was studied and characterized. The results demonstrate for the first time that SPCL scaffolds were able to support the growth and migration of OECs and SCs. Moreover, the results indicate that two weeks of in vitro culture is the ideal time to reach a high number of transplantable cells. Future work will focus on the spine stabilization of SCI animals using SPCL scaffolds loaded with OECs or SCs for SCI regeneration.

Transplantation of neurospheres derived from bone marrow stromal cells promotes neurological recovery in rats with spinal cord injury

Previous studies have revealed that cell therapy using bone marrow stromal cells (BMSCs) could promote motor functional recovery in animals with spinal cord injury (SCI). We describe here the development of cell biology technique and the experimental study of regeneration in SCI. The aim of this study was to investigate the potential for neurological recovery by transplantation neurospheres (NS) derived from BMSCs into thoracic SCI. Adult Fisher rats were used: 45 were subjected to complete thoracic SCI performed by the balloon compression method. BMSCs were cultured in vitro to obtain NS. Seven days after thoracic SCI, groups of 15 rats each received transplants of BMSCs-NS (group A), BMSCs (group B), or injection of medium only (group C) into the SCI lesion. Rats from each group were evaluated and compared longitudinally for motor function recovery. The spinal cords (SC) of injured rats were harvested at day 21 or day 42 and prepared for histological analysis. Five weeks after transplantation, many neuronal or axonal sproutings were observed and replaced by host cells in the SCI lesion of group A. Also, transplanted BMSCs-NS expressed neuronal lineage markers. Transplanted rats could walk with weight bearing and showed recovered motor evoked potentials (MEPs).

Development of Complete Thoracic Spinal Cord Transection Model in Rats for Delayed Transplantation of Stem Cells

In vivo study of a rat spinal cord injury model. To develop complete transection model of thoracic spinal cord using a polymer sheet and a microtube relevant for delayed transplantation of stem cells. Stem cell transplantation for the regeneration of spinal cord injuries has used animal models. However, current models suffer from inflammation and leakage, which lessens their usefulness in studying delayed stem cell transplantation. Thoracic spinal cord at T9 level of adult Sprague-Dawley rats was exposed and a 50:50 sheet of poly(D,L-lactic-coglycolic acid) was inserted, exposed spinal cord was completely transected, and collagen was filled between the gap between the proximal and distal stumps of transected spinal cord. A microtube was placed and fixed between the polymer surfaces facing each other. Behavior testing, magnetic resonance imaging, and myelography were performed to characterize the new complete transection with a gap formation and polymer insertion (GAP) model and to compare the GAP model with the control models. Human mesenchymal stem cells (hMSCs) were transplanted into 3 models and immunohistochemistry and western blot were performed. The inserted poly(D,L-lactic-coglycolic acid) sheet was completely disappeared 10 weeks after operation, but the inserted microtube remained firmly fixed in its original position. Myelography of the GAP model showed no leakage of contrast medium around the injured spinal cord, whereas magnetic resonance imaging of the severe contusion and simple transection models showed some leakage of contrast medium. Immunohistochemistry and western blot after hMSCs transplantation indicated that transplanted hMSCs survived and migrated well in the GAP model, and the deposition of inflammatory cells in GAP model was less than a simple transection model or severe contusion model. The developed GAP model is more relevant for delayed transplantation of stem cells for the study of regeneration of spinal cord injury of rats.

Review of "Scar-Busting Chondroitinase With Peripheral Nerve Grafting Promotes Axonal Regeneration in Chronic Spinal Cord Injury"

Review of "Scar-Busting Chondroitinase With Peripheral Nerve Grafting Promotes Axonal Regeneration in Chronic Spinal Cord Injury"

Transplantation of Nanostructured Composite Scaffolds Results in the Regeneration of Chronically Injured Spinal Cords

The destruction and hollowing of entire tissue segments represent an insurmountable barrier to axonal regeneration and therapeutics in chronic spinal cord injury. To circumvent this problem, we engineered neural prosthetics, by assembling electrospun nanofibers and self-assembling peptides into composite guidance channels and transplanted them into the cysts of a postcontusive, chronic spinal cord injury rat model, also providing delivery of proregenerative cytokines. Six months later conspicuous cord reconstruction was observed. The cyst was replaced by newly formed tissue comprising neural and stromal cells. Nerve fibers were interspersed between and inside the guidance channels, spanning the lesion, amidst a well-developed vascular network, basal lamina, and myelin. This was accompanied by a significant improvement in the activity of ascending and descending motor pathways and the global locomotion score. Thus by engineering nanostructured matrices into neuroprosthetics, it is possible to recreate an anatomical, structural, and histological framework, which leads to the replacement of large, hollow tissue gaps in the chronically injured spinal cord, fostering axonal regeneration and neurological recovery.

Polylaminin, a polymeric form of laminin, promotes regeneration after spinal cord injury

Regeneration of spinal cord injury (SCI) is a major topic of biomedical research. Laminin is an extracellular matrix protein implicated in neural development and regeneration, but despite that, there are no reports of exogenous laminin contributing to improve the outcome of experimental SCI. Here we investigated whether a biomimetic polymer of laminin assembled on pH acidification, henceforth called polylaminin, could be used to treat SCI in rats. Acute local injection of polylaminin, but not of nonpolymerized laminin, improved motor function after thoracic compression, partial or complete transection. In the latter case, the BBB score for open field locomotion 8 wk after lesion increased from 4.2 ± 0.48 to 8.8 ± 1.14 in animals treated with polylaminin of human origin. Accordingly, neurons retrogradely labeled from the sublesion stump were detected in the spinal cord and brain stem, indicating regrowth of short and long fibers across a complete transection. Polylaminin also played an unsuspected anti-inflammatory role, which underlies the early onset of its positive effects on locomotion from the first week after treatment. The beneficial effects of polylaminin were not observed in animals treated with the nonpolymerized protein or vehicle only. We propose that polylaminin is a promising therapeutic agent to treat human SCI.