Bioprinted Schwann and Mesenchymal Stem Cell Co-Cultures for Enhanced Spatial Control of Neurite Outgrowth.

Bioprinting nerve conduits supplemented with glial or stem cells is a promising approach to promote axonal regeneration in the injured nervous system. In this study, we examined the effects of different compositions of bioprinted fibrin hydrogels supplemented with Schwann cells and mesenchymal stem cells (MSCs) on cell viability, production of neurotrophic factors, and neurite outgrowth from adult sensory neurons. To reduce cell damage during bioprinting, we analyzed and optimized the shear stress magnitude and exposure time. The results demonstrated that fibrin hydrogel made from 9 mg/mL of fibrinogen and 50IE/mL of thrombin maintained the gel's highest stability and cell viability. Gene transcription levels for neurotrophic factors were significantly higher in cultures containing Schwann cells. However, the amount of the secreted neurotrophic factors was similar in all co-cultures with the different ratios of Schwann cells and MSCs. By testing various co-culture combinations, we found that the number of Schwann cells can feasibly be reduced by half and still stimulate guided neurite outgrowth in a 3D-printed fibrin matrix. This study demonstrates that bioprinting can be used to develop nerve conduits with optimized cell compositions to guide axonal regeneration.

Efficacy of Nerve-Derived Hydrogels to Promote Axon Regeneration Is Influenced by the Method of Tissue Decellularization.

Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.

Human Embryonic Stem Cell-derived Neural Crest Cells Promote Sprouting and Motor Recovery Following Spinal Cord Injury in Adult Rats.

Spinal cord injury results in irreversible tissue damage and permanent sensorimotor impairment. The development of novel therapeutic strategies that improve the life quality of affected individuals is therefore of paramount importance. Cell transplantation is a promising approach for spinal cord injury treatment and the present study assesses the efficacy of human embryonic stem cell-derived neural crest cells as preclinical cell-based therapy candidates. The differentiated neural crest cells exhibited characteristic molecular signatures and produced a range of biologically active trophic factors that stimulated in vitro neurite outgrowth of rat primary dorsal root ganglia neurons. Transplantation of the neural crest cells into both acute and chronic rat cervical spinal cord injury models promoted remodeling of descending raphespinal projections and contributed to the partial recovery of forelimb motor function. The results achieved in this proof-of-concept study demonstrates that human embryonic stem cell-derived neural crest cells warrant further investigation as cell-based therapy candidates for the treatment of spinal cord injury.

In vivo Diffusion Tensor Imaging, Diffusion Kurtosis Imaging, and Tractography of a Sciatic Nerve Injury Model in Rat at 9.4T.

Peripheral nerve injuries result in severe loss of sensory and motor functions in the afflicted limb. There is a lack of standardised models to non-invasively study degeneration, regeneration, and normalisation of neuronal microstructure in peripheral nerves. This study aimed to develop a non-invasive evaluation of peripheral nerve injuries, using diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), and tractography on a rat model of sciatic nerve injury. 10 female Sprague Dawley rats were exposed to sciatic nerve neurotmesis and studied using a 9.4 T magnet, by performing DTI and DKI of the sciatic nerve before and 4 weeks after injury. The distal nerve stump showed a decrease in fractional anisotropy (FA), mean kurtosis (MK), axonal water fraction (AWF), and radial and axonal kurtosis (RK, AK) after injury. The proximal stump showed a significant decrease in axial diffusivity (AD) and increase of MK and AK as compared with the uninjured nerve. Both mean diffusivity (MD) and radial diffusivity (RD) increased in the distal stump after injury. Tractography visualised the sciatic nerve and the site of injury, as well as local variations of the diffusion parameters following injury. In summary, the described method detects changes both proximal and distal to the nerve injury.

Regenerative effects of human embryonic stem cell-derived neural crest cells for treatment of peripheral nerve injury.

Surgical intervention is the current gold standard treatment following peripheral nerve injury. However, this approach has limitations, and full recovery of both motor and sensory modalities often remains incomplete. The development of artificial nerve grafts that either complement or replace current surgical procedures is therefore of paramount importance. An essential component of artificial grafts is biodegradable conduits and transplanted cells that provide trophic support during the regenerative process. Neural crest cells are promising support cell candidates because they are the parent population to many peripheral nervous system lineages. In this study, neural crest cells were differentiated from human embryonic stem cells. The differentiated cells exhibited typical stellate morphology and protein expression signatures that were comparable with native neural crest. Conditioned media harvested from the differentiated cells contained a range of biologically active trophic factors and was able to stimulate in vitro neurite outgrowth. Differentiated neural crest cells were seeded into a biodegradable nerve conduit, and their regeneration potential was assessed in a rat sciatic nerve injury model. A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells. Moreover, the up-regulation of several regeneration-related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals. Our results demonstrate that the differentiated neural crest cells are biologically active and provide trophic support to stimulate peripheral nerve regeneration. Differentiated neural crest cells are therefore promising supporting cell candidates to aid in peripheral nerve repair.

Effects of early nerve repair on experimental brachial plexus injury in neonatal rats.

Obstetrical brachial plexus injury refers to injury observed at the time of delivery, which may lead to major functional impairment in the upper limb. In this study, the neuroprotective effect of early nerve repair following complete brachial plexus injury in neonatal rats was examined. Brachial plexus injury induced 90% loss of spinal motoneurons and 70% decrease in biceps muscle weight at 28 days after injury. Retrograde degeneration in spinal cord was associated with decreased density of dendritic branches and presynaptic boutons and increased density of astrocytes and macrophages/microglial cells. Early repair of the injured brachial plexus significantly delayed retrograde degeneration of spinal motoneurons and reduced the degree of macrophage/microglial reaction but had no effect on muscle atrophy. The results demonstrate that early nerve repair of neonatal brachial plexus injury could promote survival of injured motoneurons and attenuate neuroinflammation in spinal cord.

Trimethylene carbonate-caprolactone conduit with poly-p-dioxanone microfilaments to promote regeneration after spinal cord injury.

Spinal cord injury (SCI) is often associated with scarring and cavity formation and therefore bridging strategies are essential to provide a physical substrate for axonal regeneration. In this study we investigated the effects of a biodegradable conduit made from trimethylene carbonate and ε-caprolactone (TC) containing poly-p-dioxanone microfilaments (PDO) with longitudinal grooves on regeneration after SCI in adult rats. In vitro studies demonstrated that different cell types including astrocytes, meningeal fibroblasts, Schwann cells and adult sensory dorsal root ganglia neurons can grow on the TC and PDO material. For in vivo experiments, the TC/PDO conduit was implanted into a small 2-3 mm long cavity in the C3-C4 cervical segments immediately after injury (acute SCI) or at 2-5 months after initial surgery (chronic SCI). At 8 weeks after implantation into acute SCI, numerous 5HT-positive descending raphaespinal axons and sensory CGRP-positive axons regenerated across the conduit and were often associated with PDO microfilaments and migrated host cells. Implantation into chronically injured SCI induced regeneration mainly of the sensory CGRP-positive axons. Although the conduit had no effect on the density of OX42-positive microglial cells when compared with SCI control, the activity of GFAP-positive astrocytes was reduced. The results suggest that a TC/PDO conduit can support axonal regeneration after acute and chronic SCI even without addition of exogenous glial or stem cells. STATEMENT OF SIGNIFICANCE: Biosynthetic conduits can support regeneration after spinal cord injury but often require addition of cell therapy and neurotrophic factors. This study demonstrates that biodegradable conduits made from trimethylene carbonate and ε-caprolactone with poly-p-dioxanone microfilaments alone can promote migration of different host cells and stimulate axonal regeneration after implantation into acute and chronic spinal cord injury. These results can be used to develop biosynthetic conduits for future clinical applications.

Long-Term Effects of Fibrin Conduit with Human Mesenchymal Stem Cells and Immunosuppression after Peripheral Nerve Repair in a Xenogenic Model.

INTRODUCTION: Previously we showed that a fibrin glue conduit with human mesenchymal stem cells (hMSCs) and cyclosporine A (CsA) enhanced early nerve regeneration. In this study long term effects of this conduit are investigated. METHODS: In a rat model, the sciatic nerve was repaired with fibrin conduit containing fibrin matrix, fibrin conduit containing fibrin matrix with CsA treatment and fibrin conduit containing fibrin matrix with hMSCs and CsA treatment, and also with nerve graft as control. RESULTS: At 12 weeks 34% of motoneurons of the control group regenerated axons through the fibrin conduit. CsA treatment alone or with hMSCs resulted in axon regeneration of 67% and 64% motoneurons respectively. The gastrocnemius muscle weight was reduced in the conduit with fibrin matrix. The treatment with CsA or CsA with hMSCs induced recovery of the muscle weight and size of fast type fibers towards the levels of the nerve graft group. DISCUSSION: The transplantation of hMSCs for peripheral nerve injury should be optimized to demonstrate their beneficial effects. The CsA may have its own effect on nerve regeneration.

The neurotrophic effects of different human dental mesenchymal stem cells.

The current gold standard treatment for peripheral nerve injury is nerve grafting but this has disadvantages such as donor site morbidity. New techniques focus on replacing these grafts with nerve conduits enhanced with growth factors and/or various cell types such as mesenchymal stem cells (MSCs). Dental-MSCs (D-MSCs) including stem cells obtained from apical papilla (SCAP), dental pulp stem cells (DPSC), and periodontal ligament stem cells (PDLSC) are potential sources of MSCs for nerve repair. Here we present the characterization of various D-MSCs from the same human donors for peripheral nerve regeneration. SCAP, DPSC and PDLSC expressed BDNF, GDNF, NGF, NTF3, ANGPT1 and VEGFA growth factor transcripts. Conditioned media from D-MSCs enhanced neurite outgrowth in an in vitro assay. Application of neutralizing antibodies showed that brain derived neurotrophic factor plays an important mechanistic role by which the D-MSCs stimulate neurite outgrowth. SCAP, DPSC and PDLSC were used to treat a 10 mm nerve gap defect in a rat sciatic nerve injury model. All the stem cell types significantly enhanced axon regeneration after two weeks and showed neuroprotective effects on the dorsal root ganglia neurons. Overall the results suggested SCAP to be the optimal dental stem cell type for peripheral nerve repair.

Differentiation of Pre- and Postganglionic Nerve Injury Using MRI of the Spinal Cord.

Brachial plexus injury (BPI) is a devastating type of nerve injury, potentially causing loss of motor and sensory function. Principally, BPI is either categorized as preganglionic or postganglionic, with the early establishment of injury level being crucial for choosing the correct treatment strategy. Despite diagnostic advances, the need for a reliable, non-invasive method for establishing the injury level remains. We studied the usefulness of in vivo magnetic resonance imaging (MRI) of the spinal cord for determination of injury level. The findings were related to neuronal and glial changes. Rats underwent unilateral L4 & L5 ventral roots avulsion or sciatic nerve axotomy. The injuries served as models for pre- and postganglionic BPI, respectively. MRI of the L4/L5 spinal cord segments 4 weeks after avulsion showed ventral horn (VH) shrinkage on the injured side compared to the uninjured side. Axotomy induced no change in the VH size on MRI. Following avulsion, histological sections of L4/L5 revealed shrinkage in the VH grey matter area occupied by NeuN-positive neurons, loss of microtubular-associated protein-2 positive dendritic branches (MAP2), pan-neurofilament positive axons (PanNF), synaptophysin-positive synapses (SYN) and increase in immunoreactivity for the microglial OX42 and astroglial GFAP markers. Axotomy induced no changes in NeuN-reactivity, modest decrease of MAP2 immunoreactivity, no changes in SYN and PanNF labelling, and a modest increase in OX42 and SYN labeling. Histological and radiological findings were congruent when assessing changes after axotomy, while MRI somewhat underestimated the shrinkage. This study indicates a potential diagnostic value of structural spinal cord MRI following BPI.

Chitosan polyplex mediated delivery of miRNA-124 reduces activation of microglial cells in vitro and in rat models of spinal cord injury.

Traumatic injury to the central nervous system (CNS) is further complicated by an increase in secondary neuronal damage imposed by activated microglia/macrophages. MicroRNA-124 (miR-124) is responsible for mouse monocyte quiescence and reduction of their inflammatory cytokine production. We describe the formulation and ex vivo transfection of chitosan/miR-124 polyplex particles into rat microglia and the resulting reduction of reactive oxygen species (ROS) and TNF-α and lower expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia cells. Alternatively particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury 72 h post injection. Microinjections of chitosan/miR-124 particles significantly reduced the number of ED-1 positive macrophages in the injured spinal cord. Taken together, these data present a potential treatment technique to reduce inflammation for a multitude of CNS neurodegenerative conditions. FROM THE CLINICAL EDITOR: The treatment of spinal cord injury remains an unresolved problem. Secondary damage is often the result of inflammation caused by activated microglia and/or macrophages. In this article, the authors developed their formulation of chitosan/miR-124 polyplex particles and investigated their use in the suppression of neuronal inflammation. This exciting data may provide a new horizon for patients who suffer from spinal cord injury.

Intrinsic mechanisms underlying the neurotrophic activity of adipose derived stem cells.

Adipose derived stem cells (ADSC) can be differentiated into Schwann cell-like cells which enhance nerve function and regeneration. However, the signalling mechanisms underlying the neurotrophic potential of ADSC remain largely unknown. In this study, we hypothesised that ADSC, upon stimulation with a combination of growth factors, could rapidly produce brain derived neurotrophic factor (BDNF) with a similar molecular mechanism to that functioning in the nervous system. Within 48 h of stimulation, ADSC demonstrated potent neurotrophic effects on dorsal root ganglion neurons, at a magnitude equivalent to that of the longer term differentiated Schwann cell-like cells. Stimulated ADSC showed rapid up-regulation of the neuronal activity dependent promoter BDNF exon IV along with an augmented expression of full length protein encoding BDNF exon IX. BDNF protein was secreted at a concentration similar to that produced by differentiated Schwann cell-like cells. Stimulation also activated the BDNF expression gating transcription factor, cAMP responsive element binding (CREB) protein. However, blocking phosphorylation of CREB with the protein kinase A small molecule inhibitor H89 did not suppress secretion of BDNF protein. These results suggest rapid BDNF production in ADSC is mediated through multiple compensatory pathways independent of, or in addition to, the CREB neuronal activation cascade.

The therapeutic effects of human adipose-derived stem cells in a rat cervical spinal cord injury model.

Spinal cord injury triggers a cascade of degenerative changes leading to cell death and cavitation. Severed axons fail to regenerate across the scar tissue and are only capable of limited sprouting. In this study, we investigated the effects of adult human adipose-derived stem cells (ASC) on axonal regeneration following transplantation into the injured rat cervical spinal cord. ASC did not induce activation of astrocytes in culture and supported neurite outgrowth from adult rat sensory dorsal root ganglia neurons. After transplantation into the lateral funiculus 1 mm rostral and caudal to the cervical C3-C4 hemisection, ASC continued to express brain-derived neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor-2 for 3 weeks but only in animals treated with cyclosporine A. Transplanted ASC stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone with some terminal arborizations reaching the caudal spinal cord. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure of the lesion scar with numerous astrocytic processes extended into the middle of the trauma zone in a chain-like pattern and in close association with regenerating axons. The density of the astrocytic network was also significantly decreased. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the activity of OX42-positive microglial cells was markedly reduced. However, ASC did not support recovery of forelimb function. The results suggest that transplanted ASC can modify the structure of the glial scar and stimulate axonal sprouting.

Stimulating the neurotrophic and angiogenic properties of human adipose-derived stem cells enhances nerve repair.

In future, adipose-derived stem cells (ASC) might be used to treat neurological disorders. In this study, the neurotrophic and angiogenic properties of human ASC were evaluated, and their effects in a peripheral nerve injury model were determined. In vitro growth factor stimulation of the cells resulted in increased secretion of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor-A (VEGF-A), and angiopoietin-1 proteins. Conditioned medium from stimulated cells increased neurite outgrowth of dorsal root ganglia (DRG) neurons. Similarly, stimulated cells showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. ASC were seeded into a fibrin conduit that was used to bridge a 10 mm rat nerve gap. After 2 weeks, the animals treated with control or stimulated ASC showed an enhanced axon regeneration distance. Stimulated cells evoked more total axon growth. Analysis of regeneration and apoptosis-related gene expression showed that both ASC and stimulated ASC enhanced GAP-43 and activating transcription factor 3 (ATF-3) expression in the spinal cord and reduced c-jun expression in the DRG. Caspase-3 expression in the DRG was reduced by stimulated ASC. Both ASC and stimulated ASC also increased the vascularity of the fibrin nerve conduits. Thus, ASC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for nerve regeneration.

Neuronal myosin-X is upregulated after peripheral nerve injury and mediates laminin-induced growth of neurites.

The successful outcome of peripheral neuronal regeneration is attributed both to the growth permissive milieu and the intrinsic ability of the neuron to initiate appropriate cellular responses such as changes in gene expression and cytoskeletal rearrangements. Even though numerous studies have shown the importance of interactions between the neuron and the extracellular matrix (ECM) in axonal outgrowth, the molecular mechanisms underlying the contact between ECM receptors and the cellular cytoskeleton remain largely unknown. Unconventional myosins constitute an important group of cytoskeletal-associated motor proteins. One member of this family is the recently described myosin-X. This protein interacts with several members of the axon growth-associated ECM receptor family of integrins and could therefore be important in neuronal outgrowth. In this study, using radioactive in situ hybridization, we found that expression of myosin-X mRNA is upregulated in adult rat sensory neurons and spinal motoneurons after peripheral nerve injury, but not after central injury. Thus, myosin-X was upregulated after injuries that can be followed by axonal regeneration. We also found that the protein is localized to neuronal growth cones and that silencing of myosin-X using RNA interference impairs the integrin-mediated growth of neurites on laminin, but has no effect on non-integrin mediated growth on N-cadherin.

Effect of delayed peripheral nerve repair on nerve regeneration, Schwann cell function and target muscle recovery.

Despite advances in surgical techniques for peripheral nerve repair, functional restitution remains incomplete. The timing of surgery is one factor influencing the extent of recovery but it is not yet clearly defined how long a delay may be tolerated before repair becomes futile. In this study, rats underwent sciatic nerve transection before immediate (0) or 1, 3, or 6 months delayed repair with a nerve graft. Regeneration of spinal motoneurons, 13 weeks after nerve repair, was assessed using retrograde labeling. Nerve tissue was also collected from the proximal and distal stumps and from the nerve graft, together with the medial gastrocnemius (MG) muscles. A dramatic decline in the number of regenerating motoneurons and myelinated axons in the distal nerve stump was observed in the 3- and 6-months delayed groups. After 3 months delay, the axonal number in the proximal stump increased 2-3 folds, accompanied by a smaller axonal area. RT-PCR of distal nerve segments revealed a decline in Schwann cells (SC) markers, most notably in the 3 and 6 month delayed repair samples. There was also a progressive increase in fibrosis and proteoglycan scar markers in the distal nerve with increased delayed repair time. The yield of SC isolated from the distal nerve segments progressively fell with increased delay in repair time but cultured SC from all groups proliferated at similar rates. MG muscle at 3- and 6-months delay repair showed a significant decline in weight (61% and 27% compared with contra-lateral side). Muscle fiber atrophy and changes to neuromuscular junctions were observed with increased delayed repair time suggestive of progressively impaired reinnervation. This study demonstrates that one of the main limiting factors for nerve regeneration after delayed repair is the distal stump. The critical time point after which the outcome of regeneration becomes too poor appears to be 3-months.

Aging effect on neurotrophic activity of human mesenchymal stem cells.

Clinical efficacy of stem cells for nerve repair is likely to be influenced by issues including donor age and in vitro expansion time. We isolated human mesenchymal stem cells (MSC) from bone marrow of young (16-18 years) and old (67-75 years) donors and analyzed their capacity to differentiate and promote neurite outgrowth from dorsal root ganglia (DRG) neurons. Treatment of MSC with growth factors (forskolin, basic fibroblast growth factor, platelet derived growth factor-AA and glial growth factor-2) induced protein expression of the glial cell marker S100 in cultures from young but not old donors. MSC expressed various neurotrophic factor mRNA transcripts. Growth factor treatment enhanced the levels of BDNF and VEGF transcripts with corresponding increases in protein release in both donor cell groups. MSC in co-culture with DRG neurons significantly enhanced total neurite length which, in the case of young but not old donors, was further potentiated by treatment of the MSC with the growth factors. Stem cells from young donors maintained their proliferation rate over a time course of 9 weeks whereas those from the old donors showed increased population doubling times. MSC from young donors, differentiated with growth factors after long-term culture, maintained their ability to enhance neurite outgrowth of DRG. Therefore, MSC isolated from young donors are likely to be a favourable cell source for nerve repair.

Neuroprotective effects of N-acetyl-cysteine and acetyl-L-carnitine after spinal cord injury in adult rats.

Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4-L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury.

Fibrin conduit supplemented with human mesenchymal stem cells and immunosuppressive treatment enhances regeneration after peripheral nerve injury.

To address the need for the development of bioengineered replacement of a nerve graft, a novel two component fibrin glue conduit was combined with human mesenchymal stem cells (MSC) and immunosupressive treatment with cyclosporine A. The effects of MSC on axonal regeneration in the conduit and reaction of activated macrophages were investigated using sciatic nerve injury model. A 10mm gap in the sciatic nerve of a rat was created and repaired either with fibrin glue conduit containing diluted fibrin matrix or fibrin glue conduit containing fibrin matrix with MSC at concentration of 80×10(6) cells/ml. Cells were labeled with PKH26 prior to transplantation. The animals received daily injections of cyclosporine A. After 3 weeks the distance of regeneration and area occupied by regenerating axons and ED1 positives macrophages was measured. MSC survived in the conduit and enhanced axonal regeneration only when transplantation was combined with cyclosporine A treatment. Moreover, addition of cyclosporine A to the conduits with transplanted MSC significantly reduced the ED1 macrophage reaction.

Adipose tissue and bone marrow-derived stem cells react similarly in an ischaemia-like microenvironment.

Mesenchymal stem cells (MSCs) from adipose tissue and bone marrow are promising cell sources for autologous cell therapy of nerve injuries, as demonstrated by their intrinsic neurotrophic potential. However, extensive death of transplanted cells limits their full benefits. This study investigated the effects of ischaemia (metabolically induced by sodium azide and 2-deoxyglucose) and serum-derived mitogens on the viability and functional profile of MSCs in vitro. MSCs were more susceptible to combined, rather than individual, blockade of glycolysis and oxidative phosphorylation. Apoptosis and autophagy were involved in ischaemia-induced cell death. Chemical ischaemia alone and serum withdrawal alone induced a similar amount of cell death, with significantly different intracellular ATP maintenance. Combined ischaemia and serum deprivation had additive effects on cell death. Expression of the extracellular matrix (ECM) molecules laminin and fibronectin was attenuated under ischaemia and independent of serum level; however, BDNF and NGF levels remained relatively constant. Strong upregulation of VEGF and to a lesser extent angiopoietin-1 was observed under ischaemia but not in serum withdrawal conditions. Importantly, this study demonstrated similar reactions of MSCs derived from adipose and bone marrow tissue, in ischaemia-like and mitogen-deprived microenvironments in terms of viability, cellular energetics, cell death mechanisms and expression levels of various growth-promoting molecules. Also, the results suggest that ischaemia has a larger impact on the ability of MSCs to survive transplantation than withdrawal of mitogens.