Olfactory ensheathing cell (OEC) transplantation has been widely shown to support axonal regeneration, remyelination, and functional recovery after central nervous system injury; however, autologous approaches are limited by low cell yields from patient biopsies, which may be insufficient for large spinal cord lesions. This study evaluated whether cryopreservation could provide a scalable alternative by preserving the therapeutic potential of mucosa-derived OECs. Using a rat dorsal root injury model, cryopreserved mucosa-derived OECs (CmOECs) were thawed and assessed for viability, phenotype, and efficacy following transplantation. Although total viable cell yield was reduced compared with primary cultures, the relative proportion of OECs remained stable, and cells retained characteristic morphology and marker expression in vitro. In vivo, transplantation of CmOECs resulted in significant functional recovery in climbing and forepaw fault tasks compared with injured controls, with outcomes comparable to primary mucosal OEC transplantation. Immunohistochemical analysis confirmed the survival and integration of transplanted cells at the dorsal root entry zone, alongside evidence of axonal regeneration and astrocytic remodeling. These findings demonstrate that mucosa-derived OECs retain therapeutic efficacy following cryopreservation and support the development of standardized OEC biobanks as a scalable strategy for spinal cord repair.

Schwann cell (SC) transplantation is considered as a promising strategy for spinal cord injury. However, SCs show less capability in assisting the regenerative axons to penetrate through astrocyte (AS)-formed scar barrier. Anosmin-1, an extracellular matrix glycosylated adhesion protein expressed in the olfactory bulb, is involved in olfactory ensheathing cells and reborn olfactory nerve axons continually penetrating the glial barrier and targeting the olfactory bulb. In this study, we employ a dorsal root crush injury model treated with anosmin-1. A vertical climbing test was used for behavioral analysis and immunohistochemical study for SC/AS interaction in regenerative axon targeting. Anosmin-1 improved rat forepaw grasping as revealed by forelimb proprioception assessment. After treated with anosmin-1, p75+ immature SCs and P0+ mature SCs mingled well with ASs at the peripheral/central glial interface, reforming the glial barrier from a tight to loose structure. Furthermore, regenerated axons traced by BDA staining revealed proper axonal targeting to the dorsal horn of the spinal cord. These results suggest that anosmin-1 can regulate SC/AS interactions at the peripheral/central boundary site to open the glial barrier for regenerating axons crossing, targeting, and establishing functional neuronal circuits. Anosmin-1 might have a potential application in repair of spinal cord injuries, particularly in combination with SCs for autologous cell transplantation.

Chitosan coatings could effectively increase the biostability and biocompatibility of biomaterials while maintaining their structural integrity. In this study, electrospun fibrous polyhydroxybutyrate (PHB) membranes were pre-treated with potassium hydroxide (KOH) or hydrogen peroxide (H2O2) and then modified with dopamine (DA) and glutaraldehyde (GA) to improve their adhesion with chitosan (CS). Scanning electron microscopy (SEM), water contact angles (WCA), and Fourier transform infrared spectroscopy (FTIR) were used to demonstrate the successful generation of DA and GA-modified PHB fibers. KOH pre-treated PHB membranes exhibited superior binding efficiency with CS at low concentrations compared to their H2O2 pre-treated counterparts. The thermal analysis demonstrated a considerable decrease in the degradation temperature and crystallinity of KOH pre-treated membranes, with temperatures dropping from 309 °C to 265.5 °C and crystallinity reducing from 100% to 25.59% as CS concentration increased from 0 to 2 w/v%. In comparison, H2O2 pre-treated membranes experienced a mild reduction in degradation temperature, from 309 °C to 284.4 °C, and a large decrease in crystallinity from 100% to 43%. UV-vis analysis using Cibacron Brilliant Red 3B-A dye (CBR) indicated similar binding efficiencies at low CS concentrations for both pre-treatments, but decreased stability at higher concentrations for KOH pre-treated membranes. Mechanical testing revealed a considerable increase in Young’s modulus (2 to 14%), toughness (31 to 60%), and ultimate tensile stress (UTS) (14 to 63%) for KOH-treated membranes compared with H2O2 pre-treated membranes as CS concentration increased from 0 to 2 w/v%.

In the past decades, the properties of olfactory ensheathing cells (OECs) have been widely investigated. Studies have shown that transplantation of OECs cultured from the olfactory bulb mediates axonal regeneration, remyelination and restores lost functions in experimental central nervous system (CNS) injury models. Autologously sourcing the cells from the nasal mucosa or the olfactory bulb to treat patients with spinal cord injuries would be ideal, but the cell yield achieved may be inadequate to cover the surface area of the lesions typically encountered in human spinal cord contusion injuries. Therefore, banking allogenic cryopreserved olfactory bulb cells from donors or generating cell lines could provide a marked increase in cell stock available for transplantation. This study is undertaken in two control and two intervention groups. The control groups have lesions alone and lesions with collagen gel but without cells. The intervention groups have either transplantation of primary cultured olfactory bulb OECs (bOECs) encapsulated in collagen gel or cryopreserved bulb OECs (CbOECs) encapsulated in collagen gel. Here, we report that transplantation of cryopreserved rat bOECs encapsulated in collagen restored the loss of function in a vertical climbing test in a unilateral C6-T1 dorsal root injury model. The loss of function returns in 80% of rats with injuries in about 3 weeks comparable to that we observed after transplantation of primary cultured bOECs. The regeneration axons induced by the transplant are identified by neurofilament antibodies and ensheathed by OECs. Our results indicate that cryopreserved OECs retain their properties of inducing axon regeneration and restoring loss of function in the experimental model. This is a step forward to translate the research into future clinical applications.

Peripheral nerve injury (PNI) is a neurological disorder that causes more than 9 million patients to suffer from dysfunction of moving and sensing. Using biodegradable polymers to fabricate an artificial nerve conduit that replicates the environment of the extracellular matrix and guides neuron regeneration through the damaged sites has been researched for decades and has led to promising but primarily pre-clinical outcomes. However, few peripheral nerve conduits (PNCs) have been constructed from controllable biodegradable polymeric materials that can maintain their structural integrity or completely degrade during and after nerve regeneration respectively. In this work, a novel PNC candidate material was developed via the electrospinning of polyhydroxy butyrate/chitosan (PHB/CS) composite polymers. An SEM characterisation revealed the resultant PHB/CS nanofibres with 0, 1 and 2 wt/v% CS had less and smaller beads than the nanofibres at 3 wt/v% CS. The water contact angle (WCA) measurement demonstrated that the wettability of PHB/CS electrospun fibres was significantly improved by additional CS. Furthermore, both the thermogravimetric analysis (TGA) and differentiation scanning calorimetry (DSC) results showed that PHB/CS polymers can be blended in a single phase with a trifluoracetic solvent in all compositions. Besides, the reduction in the degradation temperature (from 286.9 to 229.9 °C) and crystallinity (from 81.0% to 52.1%) with increasing contents of CS were further proven. Moreover, we found that the degradability of the PHB/CS nanofibres subjected to different pH values rated in the order of acidic > alkaline > phosphate buffer solution (PBS). Based on these findings, it can be concluded that PHB/CS electrospun fibres with variable blending ratios may be used for designing PNCs with controlled biodegradability.

Background/Aim: The regeneration of a completely damaged spinal cord is still a challenge in modern medicine. A promising treatment method is autologous transplantation of olfactory ensheathing cells (OECs). This study aimed primarily to test methods of culturing OECs with the use of materials and reagents that are certified for pharmaceutical use in the production of an advanced cell therapy product intended for humans. Materials and Methods: The culture of OECs was performed using various modifications of the surface of the culture vessels (with fibronectin and poly-D-lysine). The number of cells was assessed after immunofluorescence staining using anti-fibronectin and anti-p75 NGF receptor antibodies. The study compared, in terms of surgical manipulations, scaffolds with OECs prepared based on 3 types of collagen: Acid Solubilized Telo Collagen and Pepsin Solubilized Atelocollagen, and the popular Corning collagen. Results: We have shown that when suspending OECs in collagen gel, it is much better to use acid-solubilized collagen (ASC) than pepsin-solubilized collagen (PSC) because the 3D collagen scaffold from ASC provides much easier handling of the product during a surgical procedure. We also found that the OEC cultures should be grown on the surface modified with fibronectin. Furthermore, we have also shown that the optimal concentration of fetal bovine serum (FBS) for culturing these cells should be around 10%. Conclusion: The culture of OECs based on reagents intended for human use can be successfully carried out, obtaining sufficient OECs content in the heterogeneous cell culture to produce a functional advanced therapy medicinal product.

In a previous study, we reported that no axons were crossing from the severed dorsal roots to the spinal cord using the rat dorsal rhizotomy paradigm. The injury caused ipsilateral deficits of forepaw function. An attempt to restore the function by transplanting cells containing 5% olfactory ensheathing cells (OECs) cultured from the olfactory mucosa did not succeed. However, obtaining OECs from the olfactory mucosa has an advantage for clinical application. In the present study, we used the same rhizotomy paradigm, but rats with an injury received cells from a modified mucosal culture containing around 20% OECs mixed in collagen. The forelimb proprioception assessment showed that 80% of the rats receiving the transplants had functional improvement over six weeks of the study. The adhesive removal test showed that the time taken for the rats to notice the adhesive label and remove it almost returned to the normal level after receiving the transplants. Transplanted cells were identified with the expression of green fluorescent protein (ZsGreen). Some regeneration fibres immunostained for neurofilament (NF) or traced by biotinylated dextran amine (BDA) in the injury area were associated with the transplanted cells. The evidence in this study improves the prospect of clinical application using OECs from the olfactory mucosa to treat CNS injuries.

Purpose: To determine whether transplantation of olfactory mucosal cells (OMCs) is able to rescue the loss of optic nerve axons after the intraocular pressure (IOP) is elevated in rats. Methods: The IOP was raised by injection of magnetic microspheres into the anterior chamber of the eye. OMCs cultured from the adult olfactory mucosa were transplanted into the region of the optic disc. Results: We demonstrated that although the raised IOP returned to its normal level at six weeks, there was an irreversible 58% loss of optic nerve axons in the control group. However, the loss of the axons was reduced to 23% in the group with the transplanted OMCs. The Pattern Electroretinograms (pERG) showed that the decrement of the voltage amplitudes in association with the raised IOP was significantly alleviated in the group with transplantation of OMC. Conclusions: Transplantation of OMCs is able to rescue loss of optic nerve axons induced by raised IOP in the rats. The pERG recording suggested that the functional activities of the axons are also protected. Translational relevance: The results demonstrated the ability of the transplanted OMCs to protect against the loss of the optic nerve axons and the loss of function caused by raised lOPs. The findings provide a basis for future human clinical trials by autografting OMCs from autologous nasal epithelial biopsies to treat or delay glaucoma diseases.

Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive "off-the-shelf" alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycERTAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycERTAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75NTR, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycERTAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.

The dorsal root entry zone is often used in research to examine the disconnection between the central and peripheral parts of the nervous system which occurs following injury. Our laboratory and others have used transplantation of olfactory ensheathing cells (OECs) to repair experimental spinal cord injuries. We have previously used a four dorsal root (C6–T1) transection model to show that transplantation of OECs can reinstate rat forelimb proprioception in a climbing task. Until now, however, we have not looked in detail at the anatomical interaction between OECs and the peripheral/central nervous system regions which form the transitional zone. In this study, we compared short- and long-term OEC survival and their interaction with the surrounding dorsal root tissue. We reveal how transplanted OECs orient toward the spinal cord and allow newly formed axons to travel across into the dorsal horn of the spinal cord. Reconstruction of the dorsal root entry zone was supported by OEC ensheathment of axons at the injured site and also at around 3 mm further away at the dorsal root ganglion. Quantitative analysis revealed no observable difference in dorsal column axonal loss between transplanted and control groups of rats.

Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version “Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)”. The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility.

Olfactory ensheathing cells (OECs) are a promising potential cell therapy to aid regeneration. However, there are significant challenges in isolating and characterizing them. In the current study, we have explored methods to enhance the recovery of cells expressing OEC marker p75NTR from rat mucosa. With the addition of a 24-hour differential adhesion step, the expression of p75NTR was significantly increased to 73 ± 5% and 46 ± 18% on PDL and laminin matrices respectively. Additionally, the introduction of neurotrophic factor NT-3 and the decrease in serum concentration to 2% FBS resulted in enrichment of OECs, with p75NTR at nearly 100% (100 ± 0% and 98 ± 2% on PDL and laminin respectively), and candidate fibroblast marker Thy1.1 decreased to zero. Culturing OECs at physiologically relevant oxygen tension (2–8%) had a negative impact on p75NTR expression and overall cell survival. Regarding cell potency, co-culture of OECs with NG108-15 neurons resulted in more neuronal growth and potential migration at atmospheric oxygen. Moreover, OECs behaved similarly to a Schwann cell line positive control. In conclusion, this work identified key bioprocessing fundamentals that will underpin future development of OEC-based cell therapies for potential use in spinal cord injury repair. However, there is still much work to do to create optimized isolation methods.

Trauma causes spinal cord injury, and the devastating consequences of the injury are due to the failure of the damaged central nervous system (CNS) axons to regenerate. Previous studies have shown that olfactory ensheathing cells (OECs) are a unique type of glial cell and they can promote regeneration of CNS axons to aid recovery after spinal cord injury. Transplantation of OECs, in particular from the olfactory bulb (OB), is considered one of the most promising therapeutic strategies for the repair of CNS injuries, including spinal cord injury. Transplantation of OECs can be autologous or allogenic. Here we focused on the less invasive and more error-proof allograft approach which needs a collection of donor OB tissue for OEC production. In this study, we investigated the effects on the yield and proportions of OECs and olfactory nerve fibroblasts (ONFs) from storing OB tissue in various media for periods of 24 and 48 hours. The OEC yield contributes to the viability of a successful cell transplant. We concluded that storing OB tissue for a period longer than 24 hours negatively impacted the total cell number and subsequently the OEC population. This study provides useful information for future clinical applications.

Ying Li,1 Pawel Tabakow,2 Daqing Li,1 Hongyun Huang3 On behalf of the International Association of Neurorestoratology 1Spinal Repair Unit, Department of Brain Repair and Rehabilitation, UCL Institute of Neurology, London, UK; 2Department of Neurosurgery, Wroclaw Medical University, Wroclaw, Poland; 3Institute of Neurorestoratology, General Hospital of Armed Police Forces, Beijing, People’s Republic of China One year ago (January 27, 2017), the sad news that Life Honorary President of the International Association of Neurorestoratology (IANR), Geoffrey Raisman (Figure 1) had passed away shocked all our association members. We could hardly hide our grief. Besides expressing our grief and condolences to his wife, Vivian; his daughter, Ruth; and all his family, the Core Administration members communicated among each other via email and made a proposal to rename the Youth Forum of the Annual Conference, to the Raisman Youth Forum of the Annual Conference. The fourth IANR Council Board meeting in Argentina on September 29, 2017 approved this motion, and decided to establish the Raisman Neurorestoratology Foundation and to write an article commemorating him to be published in the Journal of Neurorestoratology. This commemorative article is divided into three parts: 1) his students and colleagues Daqing Li and Ying Li introduce his life and work; 2) his collaborating partner Pawel Tabakow introduces the process of their clinical trial; and 3) Hongyun Huang on behalf of the IANR introduces Geoffrey Raisman’s contribution to neurorestoratology and IANR. All individuals in the included images have provided written informed consent for the images to be published.

Transplanted human olfactory ensheathing cells (hOECs) were mixed with collagen into a unilateral transection of four dorsal roots (C6-T1) in a rat model. By mixing with collagen, the limited numbers of hOEC were maximized from an olfactory bulb biopsy and optimize cavity filling. Cyclosporine was administered daily to prevent immune rejection. Forelimb proprioception was assessed weekly in a vertical climb task. Half of the rats receiving hOEC transplants showed some functional improvement ("responders") over six weeks of the study while the other half did not ("nonresponders") and performed similarly to "injured only" rats. Transplanted cells were seen at both one week and six weeks after the surgical procedure; many were concentrated within the lesion cavity, but others were found with elongated processes in the overlying connective tissue. There were some fibers in the injury area associated with transplanted cells that were immunostained for neurofilament and TUJ1. Responder and nonresponder rats were compared with regard to microglial activation within the deep dorsal horn of cervical levels C7, C8 and also axon loss within the cuneate fasciculus at cervical level C3. Little difference was seen in microglial activation or axonal loss that could account for the improved proprioception in the responders group. This preliminary study is the first to transplant human olfactory bulb cells into a rat model of dorsal root injury; by refining each component part of the procedure, the repair potential of OECs can be maximized in a clinical setting.

The central branches of the C7 and C8 dorsal roots were avulsed close to their entry point into the spinal cord in adult rats. The forepaw responses to heat and cold stimuli were tested at 1, 2, and 3 weeks after injury. Over this period, the paws were sensitive to both stimuli at 1-2 weeks and returned toward normal at 3 weeks. Immunohistology showed no evidence of axonal regeneration into the spinal cord in a control group of rats with avulsion only, implying that adjacent dorsal roots and their corresponding dermatomes were involved in the recovery. In a further group of rats, a mixture of bulbar olfactory ensheathing cells and olfactory nerve fibroblasts were transplanted into the gap between the avulsed roots and the spinal cord at the time of avulsion. These rats showed no evidence of either loss of sensation or exaggerated responses to stimuli at any of the time points from 1 to 3 weeks. Immunohistology showed that the transplanted cells formed a complete bridge, and the central branches of the dorsal root fibers had regenerated into the dorsal horn of the spinal cord. These regenerating axons, including Tuj1 and CGRP immunoreactive fibers, were ensheathed by the olfactory ensheathing cells. This confirms our previous demonstration of central regeneration by these transplants and suggests that such transplants may provide a useful means to prevent the development of abnormal sensations such as allodynia after spinal root lesions.

Cell transplantation is one of the most promising strategies for repair of human spinal cord injuries. Animal studies from a number of laboratories have shown that transplantation of olfactory ensheathing cells cultured from biopsies of the olfactory bulb mediate axonal regeneration and remyelination and restore lost functions in spinal cord injuries. For translation from small laboratory experimental injuries to the large spinal cord injuries encountered in human patients the numbers of cells that can be obtained from a patient's own olfactory bulb becomes a serious limiting factor. Furthermore, removal of an olfactory bulb requires invasive surgery and risks unilateral anosmia. We here report that xenografted mouse bulbar olfactory ensheathing cells immunoprotected by daily cyclosporine restore directed forepaw reaching function in rats with chronic C1/2 unilateral corticospinal tract lesions. Once function had been established for 10 days, cyclosporine was withdrawn. Thirteen out of 13 rats continued to increase directed forepaw reaching. Immunohistochemistry shows that in all cases neurofilament-positive axons were present in the lesion, but that the grafted cells had been totally rejected. This implies that once grafted cells have acted as bridges for axon regeneration across the lesion site their continued presence is no longer necessary for maintaining the restored function. This raises the possibility that in the future a protocol of temporary immunoprotection might allow for the use of the larger available numbers of immunoincompatible allografted cells or cell lines, which would avoid the need for removing a patient's olfactory bulb.

In 1969 a paper entitled Neuronal plasticity in the septal nuclei of the adult rat proposed that new synapses are formed in the adult brain after injury (Raisman, 1969). The quantitative electron microscopic study of the timed responses to selective partial denervation of the neuropil of the adult rat septal nuclei after distant transection of the hippocampal efferent axons in the fimbria showed that the new synapses arise by sprouting of surviving adjacent synapses which selectively take over the previously denervated sites and thus restore the number of synapses to normal. This article presents the evidence for the role of perisynaptic astrocytic processes in the removal and formation of synapses and considers its significance as one of the three major divisions of the astrocytic surface in terms of the axonal responses to injury and regeneration. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

A radial array of fortified astrocytes (FASTs) is the load bearing structure of the rat optic nerve head (ONH). At the retinal end the ONH is suspended on a fluid filled extracellular space occupied by modified pigment cells which generate a glomerular‐like formation of villi. We propose that regulation of fluid in and out of this space may contribute to buffering the normal fluctuations of intraocular pressure. The energy requirement for the fluid transfer process is provided by the dense vascularity of the ONH and is reflected in the giant mitochondria of the FASTs. We propose that glaucoma occurs when a maintained rise in pressure overwhelms the capacity of this regulatory system. Under these circumstances the FAST array becomes detached from its anchorage in the surrounding ONH sheath. Progressively driven backwards by the pressure, the FASTs degenerate. We propose that the degeneration of the FASTs is associated with ischemic damage caused by the backward stretching of their blood supply. Retraction of the FAST processes deprives the retinal ganglion cell axons of their energy support, resulting in axotomy. We consider that our previously observed rescue of axons and FASTs by transplantation of olfactory ensheathing cells is due to replacement of this lost energy source. GLIA 2015;63:1537–1552

In an ongoing clinical trial, a spinal injured patient who received a transplant of autologous cells cultured from the olfactory bulb is showing greater functional benefit than three previous patients with transplants of mucosal origin. Previous laboratory studies of transplantation into rat spinal cord injuries show that the superior reparative benefits of bulbar over mucosal cultures are associated with regeneration of severed corticospinal tract fibers over a bridge of olfactory ensheathing cells (OECs) formed across the injury site. In a rat rhizotomy paradigm, we reported that transplantation of bulbar cell cultures also enables severed axons of the C6–T1 dorsal roots to regenerate across a bridge of OECs into the spinal cord and restore electrophysiological transmission and forepaw grasping during a climbing test. We now report a repeat of the same rhizotomy procedure in 25 rats receiving cells cultured from olfactory mucosal biopsies. In no case did the transplanted cells form a bridging pathway. No axons crossed from the severed roots to the spinal cord, and there was no restoration of forepaw grasping. This suggests that the superior clinical benefit in the patient receiving bulbar cell transplants is due to regeneration of severed fibers across the injury site, and this correlates with imaging and the pattern of functional recovery. Using present culture protocols, the yield of OECs from bulbar biopsies is around 50%, but that from mucosal biopsies is less than 5%. Improving the yield of OECs from mucosal biopsies might avoid the necessity for the intracranial approach to obtain bulbar cells.