Combining chitin biological conduits with small autogenous nerves and platelet-rich plasma for the repair of sciatic nerve defects in rats.

AIMS: Peripheral nerve defects are often difficult to recover from, and there is no optimal repair method. Therefore, it is important to explore new methods of repairing peripheral nerve defects. This study explored the efficacy of nerve grafts constructed from chitin biological conduits combined with small autogenous nerves (SANs) and platelet-rich plasma (PRP) for repairing 10-mm sciatic nerve defects in rats. METHODS: To prepare 10-mm sciatic nerve defects, SANs were first harvested and PRP was extracted. The nerve grafts consisted of chitin biological conduits combined with SAN and PRP, and were used to repair rat sciatic nerve defects. These examinations, including measurements of axon growth efficiency, a gait analysis, electrophysiological tests, counts of regenerated myelinated fibers and observations of their morphology, histological evaluation of the gastrocnemius muscle, retrograde tracing with Fluor-Gold (FG), and motor endplates (MEPs) distribution analysis, were conducted to evaluate the repair status. RESULTS: Two weeks after nerve transplantation, the rate and number of regenerated axons in the PRP-SAN group improved compared with those in the PRP, SAN, and Hollow groups. The PRP-SAN group exhibited better recovery in terms of the sciatic functional index value, composite action potential intensity, myelinated nerve fiber density, myelin sheath thickness, and gastrectomy tissue at 12 weeks after transplantation, compared with the PRP and SAN groups. The results of FG retrograde tracing and MEPs analyses showed that numbers of FG-positive sensory neurons and motor neurons as well as MEPs distribution density were higher in the PRP-SAN group than in the PRP or SAN group. CONCLUSIONS: Nerve grafts comprising chitin biological conduits combined with SANs and PRP significantly improved the repair of 10-mm sciatic nerve defects in rats and may have therapeutic potential for repairing peripheral nerve defects in future applications.

Cell-enrichment with olfactory ensheathing cells has limited local extra beneficial effects on nerve regeneration supported by the nerve guide Perimaix.

The reconstruction of peripheral nerve injuries is clinically challenging, and today, the autologous nerve transplantation is still considered as the only gold standard remedy for nerve lesions where a direct nerve coaptation is not possible. Nevertheless, the functional merits of many biomaterials have been tested as potential substitutes for the autologous nerve transplant. One of the strategies that have been pursued is the combination of bioengineered nerve guides with cellular enrichment. In this present study, we combined the previously evaluated collagen-based and microstructured nerve guide Perimaix with olfactory ensheathing cell enrichment. Rat sciatic nerve defects of 20 mm were either bridged by a cell-seeded or nonseeded nerve guide or an autologous nerve transplant. Animals were monitored for 12 weeks for structural and functional parameters. Seeded cells survived on Perimaix, and following implantation aligned along the microstructured Perimaix framework. Axonal densities within the cell-seeded nerve guides were higher than in the nonseeded nerve guides and were comparable to the autograft. Additionally, cell-seeding had local beneficial effects on myelination within the nerve guide, as myelin sheath thickness was enhanced when compared with the empty scaffold. Nevertheless, for bridging the nerve gap of 20 mm, both the cell-seeded as well as nonseeded scaffolds were equally efficient regarding the functional outcome, which did not differ between the autograft, seeded or nonseeded groups. Our data demonstrate that olfactory ensheathing cell enrichment has local effects on nerve regeneration in combination with the Perimaix nerve guide. Surprisingly, for traversing the lesion gap, additional cell-seeding is not crucial.

Olfactory ensheathing glia cell therapy and tubular conduit enhance nerve regeneration after mouse sciatic nerve transection.

The regenerative potential of the peripheral nervous system (PNS) is widely known, but functional recovery, particularly in humans, is seldom complete. Therefore, it is necessary to resort to strategies that induce or potentiate the PNS regeneration. Our main objective was to test the effectiveness of Olfactory Ensheathing Cells (OEC) transplantation into a biodegradable conduit as a therapeutic strategy to improve the repair outcome after nerve injury. Sciatic nerve transection was performed in C57BL/6 mice; proximal and distal stumps of the nerve were sutured into the collagen conduit. Two groups were analyzed: DMEM (acellular grafts) and OEC (1×105/2µL). Locomotor function was assessed weekly by Sciatic Function Index (SFI) and Global Mobility Test (GMT). After eight weeks the sciatic nerve was dissected for morphological analysis. Our results showed that the OEC group exhibited many clusters of regenerated nerve fibers, a higher number of myelinated fibers and myelin area compared to DMEM group. The G-ratio analysis of the OEC group showed significantly more fibers on the most suitable sciatic nerve G-ratio index. Motor recovery was accelerated in the OEC group. These data provide evidence that the OEC therapy can improve sciatic nerve functional and morphological recovery and can be potentially translated to the clinical setting.

Does the preclinical evidence for functional remyelination following myelinating cell engraftment into the injured spinal cord support progression to clinical trials?

This article reviews all historical literature in which rodent-derived myelinating cells have been engrafted into the contused adult rodent spinal cord. From 2500 initial PubMed citations identified, human cells grafts, bone mesenchymal stem cells, olfactory ensheathing cells, non-myelinating cell grafts, and rodent grafts into hemisection or transection models were excluded, resulting in the 67 studies encompassed in this review. Forty five of those involved central nervous system (CNS)-derived cells, including neural stem progenitor cells (NSPCs), neural restricted precursor cells (NRPs) or oligodendrocyte precursor cells (OPCs), and 22 studies involved Schwann cells (SC). Of the NSPC/NPC/OPC grafts, there was no consistency with respect to the types of cells grafted and/or the additional growth factors or cells co-grafted. Enhanced functional recovery was reported in 31/45 studies, but only 20 of those had appropriate controls making conclusive interpretation of the remaining studies impossible. Of those 20, 19 were properly powered and utilized appropriate statistical analyses. Ten of those 19 studies reported the presence of graft-derived myelin, 3 reported evidence of endogenous remyelination or myelin sparing, and 2 reported both. For the SC grafts, 16/21 reported functional improvement, with 11 having appropriate cellular controls and 9/11 using proper statistical analyses. Of those 9, increased myelin was reported in 6 studies. The lack of consistency and replication among these preclinical studies are discussed with respect to the progression of myelinating cell transplantation therapies into the clinic.

Olfactory ensheathing cells (OECs) degrade neurocan in injured spinal cord by secreting matrix metalloproteinase-2 in a rat contusion model.

The mechanism by which olfactory ensheathing cells (OECs) exert their potential to promote functional recovery after transplantation into spinal cord injury (SCI) tissue is not fully understood, but the relevance of matrix metalloproteinases (MMPs) has been suggested. We evaluated the expression of MMPs in OECs in vitro and the MMP secretion by OECs transplanted in injured spinal cord in vivo using a rat SCI model. We also evaluated the degradation of neurocan, which is one of the axon-inhibitory chondroitin sulfate proteoglycans, using SCI model rats. The in vitro results showed that MMP-2 was the dominant MMP expressed by OECs. The in vivo results revealed that transplanted OECs secreted MMP-2 in injured spinal cord and that the expression of neurocan was significantly decreased by the transplantation of OECs. These results suggest that OECs transplanted into injured spinal cord degraded neurocan by secreting MMP-2.

CD44 is required for the migration of transplanted oligodendrocyte progenitor cells to focal inflammatory demyelinating lesions in the spinal cord.

Remyelination of chronically demyelinated axons in multiple sclerosis (MS) requires the recruitment of endogenous cells or their replacement by transplanted, exogenous oligodendrocyte progenitor cells (OPCs). We have previously shown that an OPC line, CG4, preferentially migrates after transplantation toward focal areas of inflammatory demyelination and axon loss created by injection of zymosan in the rat spinal cord. Here we show that many transplanted CG4 cells had already migrated into the inflammatory lesion after 1 day. We demonstrate that a large number of CG4 cells that had migrated, expressed the adhesion protein, CD44, and that CD44's main ligand, hyaluronic acid (HA) was robustly expressed in the inflammatory lesion. In an in vitro migration assay, migration declined significantly following blocking of CD44 expression on CG4 cells. Likewise, migration of CG4 cells toward a zymosan lesion in vivo was inhibited when transplanted cells were exposed to a CD44 blocking antibody prior to transplantation. These findings suggest that CD44 is a key molecule in the migration of OPCs toward the focal inflammatory demyelinated lesion induced by zymosan, and may be an important in OPC repair in MS.

Motor outcome and allodynia are largely unaffected by novel olfactory ensheathing cell grafts to repair low-thoracic lesion gaps in the adult rat spinal cord.

Olfactory ensheathing cells (OEC) are a promising graftable cell population for improving functional outcomes after experimental spinal cord injury. However only few studies have focused on experimental models with large cavitations, which require bridging substrates to transfer and maintain the donor cells within the lesion site. Here, a state-of-the-art collagen-based multi-channeled three dimensional scaffold was used to deliver olfactory ensheathing cells to 2 mm long unilateral low-thoracic hemisection cavities. For a period of 10 weeks, allodynia of the hindpaws was monitored using the von Frey hair filament test, while an extensive analysis of motor ability was performed with use of the CatWalk gait analysis system and the BBB locomotor scale. No substantial improvement or deterioration of motor functions was induced and there was no effect on lesion-induced allodynia. On the basis of these data, we conclude that relatively large spinal cord lesions with cavitation may present additional hurdles to the therapeutic effect of OEC. Future studies are needed to address the nature that such lesion cavities place on cell grafts.

Combined transplantation of neural stem cells and olfactory ensheathing cells improves the motor function of rats with intracerebral hemorrhage.

OBJECTIVE: To investigate the effects of combined transplantation of neural stem cells (NSC) and olfactory ensheathing cells (OEC) on the motor function of rats with intracerebral hemorrhage. METHODS: In three days after a rat model of caudate nucleus hemorrhage was established, NSCs and OEC, NSC, OEC (from embryos of Wistar rats) or normal saline were injected into hematomas of rats in combined transplantation group, NSC group, OEC group, and control group, respectively. Damage of neural function was scored before and in 3, 7, 14, 30 days after operation. Tissue after transplantation was observed by immunocytochemistry staining. RESULTS: The scores for the NSC, OEC and co-transplantation groups were significantly lower in 14 and 30 days after operation than in 3 days after operation (P < 0.05). The scores for the NSC and OEC groups were significantly lower than those for the control group only in 30 days after operation (P < 0.05), while the difference for the NSC-OEC group was significant in 14 days after operation (P < 0.05). Immunocytochemistry staining revealed that the transplanted OEC and NSC could survive, migrate and differentiate into neurons, astrocytes, and oligodendrocytes. The number of neural precursor cells was greater in the NSC and combined transplantation groups than in the control group. The number of neurons differentiated from NSC was significantly greater in the co-transplantation group than in the NSC group. CONCLUSION: Co-transplantation of NSC and OEC can promote the repair of injured tissue and improve the motor function of rats with intracerebral hemorrhage.

Effects of 660-nm gallium-aluminum-arsenide low-energy laser on nerve regeneration after acellular nerve allograft in rats.

PURPOSE: The purpose of this study was to explore and discuss the effects of 660-nm gallium-aluminum-arsenide low-energy laser (GaAlAs LEL) irradiation on neural regeneration after acellular nerve allograft repair of the sciatic nerve gap in rats. METHODS: Eight male and female Sprague-Dawley rats were used as nerve donors, and 32 healthy Wistar rats were randomly divided into four groups: normal control group, acellular rat sciatic nerve (ARSN) group, laser group, and autograft group. Twelve weeks after surgery, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, myelinated nerve number, and calcitonin gene-related peptide (CGRP) protein and mRNA expression of the spinal cord and muscle at the injury site were quantified and statistically analyzed. RESULTS: Compared with the ARSN group, laser therapy significantly increased nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, myelinated nerve number, and CGRP protein and mRNA expression of the L(4) spinal cord at the injury site. CONCLUSIONS: These findings demonstrate that 660-nm GaAlAs LEL therapy upregulates CGRP protein and mRNA expression of the L(4) spinal cord at the injury site and increases the rate of regeneration and target reinnervation after acellular nerve allograft repair of the sciatic nerve gap in rats. Low-energy laser irradiation may be a useful, noninvasive adjunct for promoting nerve regeneration in surgically induced defects repaired with ARSN.

Transplanted embryonic spinal tissue promotes severed sciatic nerve regeneration in rats.

The effects of transplanted embryonic spinal tissue on host motor nerve regeneration and target muscle reinervation were investigated in severed sciatic nerves of rats. The electromyogram (EMG) responses and number of motor end plates (MEP) in target muscles, number of nerve axons, and retrogradely labeled motor neurons were examined in transplantation-, anastomosis without transplantation-, and naïve groups of the animals. The EMG patterns of the transplantation group returned to nearly normal at the 8th week, but those of the anastomosis group did not. MEP counts in the transplantation group were significantly higher than in the anastomosis group. The myelinated axon counts and myelin sheath thickness in the transplantation group were significantly higher than those in the anastomosis group. The number of retrogradely labeled motor neurons was significantly higher in the transplantation group. We conclude that transplanted embryonic spinal tissue can promote both host motor nerve regeneration and target muscle reinnervation.

Repair of spinal cord injury by co-transplantation of embryonic stem cell-derived motor neuron and olfactory ensheathing cell.

BACKGROUND: The failure of regeneration after spinal cord injury (SCI) has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells (OEC) and embryonic stem (ES) cell-derived motor neurons (ESMN) on contused SCI. METHODS: OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups. RESULTS: The purity of OEC culture was 95%. Motor neuron progenitor markers (Olig2, Nkx6.1 and Pax6) and motor neuron markers (Isl1, Isl2 and Hb9) were expressed. Histological analysis showed that significantly more (P<0.001) spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups (P< 0.05). The numbers of ESMN in co-transplanted group were significantly higher than ESMN group (P<0.05). A significant (P<0.05) recovery of hindlimb function was observed in rats in the transplanted groups. CONCLUSION: We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery.

Cellular remyelinating therapy in multiple sclerosis.

Demyelination is a pathological hallmark of multiple sclerosis (MS), a chronic autoimmune disorder of the central nervous system (CNS) that affects mainly young people in western countries. Recent studies have shown that remyelination can be accomplished by supplying demyelinated regions with myelinogenic cells or neural stem cells via transplantation. The remyelinating effect of these cells may be via one or more mechanisms, including: 1) as an immunomodulator by producing soluble factors; 2) direct cell replacement by differentiating into neural and glial cells in the lesion; and 3) indirect action by promoting neural and glial differentiation of endogenous cells. Identifying these mechanisms will help in choosing an optimal and more effective approach for cell-based therapy. Here we present a brief view focusing on myelin-forming cell types that can be used for cell transplantation and draw on a variety of recent experimental findings to speculate on the likely future evolution of remyelinating therapies.

Rescue of congenital hypomyelination by progenitor cell transplantation.

Replacement of myelin-forming cells is an attractive but unproven therapy for inherited and acquired myelin diseases. In this issue of Cell Stem Cell, Windrem et al. (2008) describe the first progenitor cell transplantation paradigm that rescues the neurological phenotypes and increases life spans of mice with inherited myelin disease.

Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse.

Congenitally hypomyelinated shiverer mice fail to generate compact myelin and die by 18-21 weeks of age. Using multifocal anterior and posterior fossa delivery of sorted fetal human glial progenitor cells into neonatal shiverer x rag2(-/-) mice, we achieved whole neuraxis myelination of the engrafted hosts, which in a significant fraction of cases rescued this otherwise lethal phenotype. The transplanted mice exhibited greatly prolonged survival with progressive resolution of their neurological deficits. Substantial myelination in multiple regions was accompanied by the acquisition of normal nodes of Ranvier and transcallosal conduction velocities, ultrastructurally normal and complete myelination of most axons, and a restoration of a substantially normal neurological phenotype. Notably, the resultant mice were cerebral chimeras, with murine gray matter but a predominantly human white matter glial composition. These data demonstrate that the neonatal transplantation of human glial progenitor cells can effectively treat disorders of congenital and perinatal hypomyelination.

Olfactory ensheathing glia graft in combination with FK506 administration promote repair after spinal cord injury.

The aim of this study was to determine whether a combination of olfactory ensheathing cell (OEC) graft with the administration of FK506, two experimental approaches that have been previously reported to exert protective/regenerative effects after spinal cord injury, promotes synergic restorative effects after complete or partial spinal cord injuries. In partial spinal cord injury, combination of an OEC graft and FK506 reduced functional deficits evaluated by the BBB score, motor-evoked potentials (MEPs) and H reflex tests, diminished cavitation, astrogliosis and increased sparing/regeneration of raphespinal fibers compared to untreated and single-treatment groups of rats. After complete spinal cord transection, the combined treatment significantly improved functional outcomes, promoted axonal regeneration caudal to the lesion, and diminished astrogliosis compared only to non-transplanted animals. Slightly, but non-significant, better functional and histological results were found in OEC-grafted animals treated with FK506 than in those given saline after spinal cord transection. Nevertheless, the combined treatment increased the percentage of rats that recovered MEPs and promoted a significant reduction in astrogliosis. In conclusion, this study demonstrates that OEC grafts combined with FK506 promote additive repair of spinal cord injuries to those exerted by single treatments, the effect being more remarkable when the spinal cord is partially lesioned.

Effects of COX-2 and iNOS inhibitors alone or in combination with olfactory ensheathing cell grafts after spinal cord injury.

STUDY DESIGN: We studied the effects of inhibitors of COX-2 (NS398) and iNOS (aminoguanidine) alone or in combination with olfactory ensheathing cell (OEC) grafts after spinal cord injury in the rat. OBJECTIVE: To assess the role exerted by COX-2 and iNOS after spinal cord injury and an OEC transplant. SUMMARY OF BACKGROUND DATA: COX-2 and iNOS exert a detrimental effect after spinal cord injury. In contrast, OECs grafted into the injured spinal cord mediate neuroprotection and also promote the up-regulation of COX-2 and iNOS. METHODS: Photochemical injury was induced at T8 spinal cord segment. Rats received local injection of OECs (n = 15) or vehicle (DMEM; n = 15). Six subgroups of rats (n = 5 rats each) were given NS398 (DM-NS; OEC-NS), aminoguanidine (DM-AG; OEC-AG), or saline (DM-SS; OEC-SS). Locomotor ability, pain sensibility, tissue sparing, and density of blood vessels were evaluated. RESULTS: Two weeks following injury, motor skills and nociceptive response were significantly higher in DM-NS and DM-AG than in DM-SS rats. The area of preserved spinal cord parenchyma was higher in treated animals than in those given saline. In contrast, functional outcome, tissue sparing, and density of blood vessels were lower in OEC-NS and OEC-AG than in OEC-SS animals. CONCLUSIONS: These results suggest that, although COX-2 and iNOS exert a detrimental role after spinal cord injury, they may play an important role in the neuroprotective mechanisms induced by OEC grafts after spinal cord injury.

Olfactory ensheathing glial co-grafts improve functional recovery in rats with 6-OHDA lesions.

Olfactory ensheathing cells (OEC) transplanted to the site of a spinal cord injury can promote axonal sparing/regeneration and functional recovery. The purpose of this study was to investigate if OEC enhance the effects of grafted dopamine-neuron-rich ventral mesencephalic tissue (VM) in a rodent model of Parkinson's disease. We co-grafted VM with either OEC or astrocytes derived from the same olfactory bulbs as the OEC to rats with a unilateral 6-hydroxydopamine lesion of the nigrostriatal system. Co-grafting fetal VM with OEC, but not with astrocytes enhanced dopamine cell survival, striatal reinnervation and functional recovery of amphetamine- and apomorphine-induced rotational behaviour compared with grafting embryonic VM alone. Grafting OEC or astrocytes alone had no effects. Intriguingly, only in the presence of OEC co-grafts, did dopamine neurons extend strikingly long neurites that reached peripheral striatal compartments. Comparable results were observed in a co-culture system where OEC promoted dopamine cell survival and neurite elongation through a mechanism involving both releasable factors and direct contact. Cell type analysis of fetal VM grafts suggested that dopamine neurons of the substantia nigra rather than of the ventral tegmental area were increased in the presence of OEC co-grafts. We conclude that the addition of OEC enhances efficacy of grafted immature dopamine neurons in a rat Parkinson's disease model.

Autologous olfactory ensheathing cell transplantation in human spinal cord injury.

Olfactory ensheathing cells transplanted into the injured spinal cord in animals promote regeneration and remyelination of descending motor pathways through the site of injury and the return of motor functions. In a single-blind, Phase I clinical trial, we aimed to test the feasibility and safety of transplantation of autologous olfactory ensheathing cells into the injured spinal cord in human paraplegia. Participants were three male paraplegics, 18-55 years of age, with stable, complete thoracic injuries 6-32 months previously, with stable spinal column, no implanted prostheses, and no syrinx. Olfactory ensheathing cells were grown and purified in vitro from nasal biopsies and injected into the region of damaged spinal cord. The trial design includes a matched injury group as a control for the assessors, who are blind to treatment status. Assessments, made before transplantation and at regular intervals subsequently, include MRI, medical, neurological and psychosocial assessments, and standard American Spinal Injury Association and Functional Independence Measure assessments. One year after cell implantation, there were no medical, surgical or other complications to indicate that the procedure is unsafe. There is no evidence of spinal cord damage nor of cyst, syrinx or tumour formation. There was no neuropathic pain reported by the participants, no change in psychosocial status and no evidence of deterioration in neurological status. Participants will be followed for 3 years to confirm long-term safety and to compare neurological, functional and psychosocial outcomes with the control group. We conclude transplantation of autologous olfactory ensheathing cells into the injured spinal cord is feasible and is safe up to one year post-implantation.

Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation.

Human embryonic stem cells (hESCs) demonstrate remarkable proliferative and developmental capacity. Clinical interest arises from their ability to provide an apparently unlimited cell supply for transplantation, and from the hope that they can be directed to desirable phenotypes in high purity. Here we present for the first time a method for obtaining oligodendrocytes and their progenitors in high yield from hESCs. We expanded hESCs, promoted their differentiation into oligodendroglial progenitors, amplified those progenitors, and then promoted oligodendroglial differentiation using positive selection and mechanical enrichment. Transplantation into the shiverer model of dysmyelination resulted in integration, differentiation into oligodendrocytes, and compact myelin formation, demonstrating that these cells display a functional phenotype. This differentiation protocol provides a means of generating human oligodendroglial lineage cells in high purity, for use in studies of lineage development, screening assays of oligodendroglial-specific compounds, and treating neurodegenerative diseases and traumatic injuries to the adult CNS.

CNS axons retain their competence for myelination throughout life.

An important question relevant to developing remyelination therapies is whether axons that remain without myelin sheaths after an episode of demyelination retain myelination competence. To resolve this, we have developed a model of transplantation into the nerve fibre layer of the adult rat retina, where the axons are unmyelinated. In the adult, these axons can be myelinated by transplantation of both the oligodendrocyte progenitor cells (OPCs) and an OPC line (CG4). The extent of myelination achieved following transplantation of OPCs is the same in young adult recipients (2 months old) as that which occurs in old adult recipients (12-18 months old), indicating that there are no changes in axons remaining unmyelinated for many months that would prevent effective remyelination. This finding suggests that chronically demyelinated regions of axons such as those in seen in multiple sclerosis are likely to remain competent to be remyelinated.