Differentiated adipose-derived stem cells promote peripheral nerve regeneration.

INTRODUCTION: Many reports have indicated that adipose-derived stem cells (ADSCs) are effective for nerve regeneration. We investigated nerve regeneration by combining a polyglycolic acid collagen (PGA-c) tube, which is approved for clinical use, and Schwann cell-like differentiated ADSCs (dADSCs). METHODS: Fifteen-millimeter-long gaps in the sciatic nerve of rats were bridged in each group using tubes (group I), with tubes injected with dADSCs (group II), or by resected nerve (group III). RESULTS: Axonal outgrowth was greater in group II than in group I. Tibialis anterior muscle weight revealed recovery only in group III. Latency in nerve conduction studies was equivalent in group II and III, but action potential was lower in group II. Transplanted dADSCs maintained Schwann cell marker expression. ATF3 expression level in the dorsal root ganglia was equivalent in groups II and III. DISCUSSION: dADSCs maintained their differentiated state in the tubes and are believed to have contributed to nerve regeneration.

Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells into Schwann-Like Cells Improves Neuromuscular Function in a Mouse Model of Charcot-Marie-Tooth Disease Type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited motor and sensory neuropathy, and is caused by duplication of PMP22, alterations of which are a characteristic feature of demyelination. The clinical phenotype of CMT1A is determined by the degree of axonal loss, and patients suffer from progressive muscle weakness and impaired sensation. Therefore, we investigated the potential of Schwann-like cells differentiated from human tonsil-derived stem cells (T-MSCs) for use in neuromuscular regeneration in trembler-J (Tr-J) mice, a model of CMT1A. After differentiation, we confirmed the increased expression of Schwann cell (SC) markers, including glial fibrillary acidic protein (GFAP), nerve growth factor receptor (NGFR), S100 calcium-binding protein B (S100B), glial cell-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), which suggests the differentiation of T-MSCs into SCs (T-MSC-SCs). To test their functional efficiency, the T-MSC-SCs were transplanted into the caudal thigh muscle of Tr-J mice. Recipients' improved locomotive activity on a rotarod test, and their sciatic function index, which suggests that transplanted T-MSC-SCs ameliorated demyelination and atrophy of nerve and muscle in Tr-J mice. Histological and molecular analyses showed the possibility of in situ remyelination by T-MSC-SCs transplantation. These findings demonstrate that the transplantation of heterologous T-MSC-SCs induced neuromuscular regeneration in mice and suggest they could be useful for the therapeutic treatment of patients with CMT1A disease.

Multichannel polymer scaffold seeded with activated Schwann cells and bone mesenchymal stem cells improves axonal regeneration and functional recovery after rat spinal cord injury.

The adult mammalian CNS has a limited capacity to regenerate after traumatic injury. In this study, a combinatorial strategy to promote axonal regeneration and functional recovery after spinal cord injury (SCI) was evaluated in adult rats. The rats were subjected to a complete transection in the thoracic spinal cord, and multichannel scaffolds seeded with activated Schwann cells (ASCs) and/or rat bone marrow-derived mesenchymal stem cells (MSCs) were acutely grafted into the 3-mm-wide transection gap. At 4 weeks post-transplantation and thereafter, the rats receiving scaffolds seeded with ASCs and MSCs exhibited significant recovery of nerve function as shown by the Basso, Beattie and Bresnahan (BBB) score and electrophysiological test results. Immunohistochemical analyses at 4 and 8 weeks after transplantation revealed that the implanted MSCs at the lesion/graft site survived and differentiated into neuron-like cells and co-localized with host neurons. Robust bundles of regenerated fibers were identified in the lesion/graft site in the ASC and MSC co-transplantation rats, and neurofilament 200 (NF) staining confirmed that these fibers were axons. Furthermore, myelin basic protein (MBP)-positive myelin sheaths were also identified at the lesion/graft site and confirmed via electron microscopy. In addition to expressing mature neuronal markers, sparse MSC-derived neuron-like cells expressed choline acetyltransferase (ChAT) at the injury site of the ASC and MSC co-transplantation rats. These findings suggest that co-transplantation of ASCs and MSCs in a multichannel polymer scaffold may represent a novel combinatorial strategy for the treatment of spinal cord injury.

A novel growth-promoting pathway formed by GDNF-overexpressing Schwann cells promotes propriospinal axonal regeneration, synapse formation, and partial recovery of function after spinal cord injury.

Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function.

Poly (dextrogyr-levogyr) lactide acid-triiodothyronine scaffold for peripheral nerve regeneration.

OBJECTIVES: To evaluate the poly(dextrogyr-levogyr) lactide acid-triiodothyronine (PDLLA-T3) seeded with Schwann cells conduit for repairing sciatic nerve defect. MATERIALS & METHODS: The rats were divided into three groups: autologous nerve transplantation (Group A), PDLLA-T3 + Schwann cells (Group B) and PDLLA + Schwann cells (Group C). RESULTS: Myelin sheath thickness was significantly greater in Group A compared with Group B and Group C. The regenerated nerves had nearly normal structure in Group A, and in Groups B and C nerve tissues filled in the anastomotic site and angiogenesis was noted. The mean number of myelinated nerve fibers and neurons in Group B was greater than in Group C. CONCLUSIONS: PDLLA-T3 is superior to PDLLA alone for repairing nerve defects.

Regenerative medicine for the treatment of spinal cord injury: more than just promises?

Spinal cord injury triggers a complex set of events that lead to tissue healing without the restoration of normal function due to the poor regenerative capacity of the spinal cord. Nevertheless, current knowledge about the intrinsic regenerative ability of central nervous system axons, when in a supportive environment, has made the prospect of treating spinal cord injury a reality. Among the range of strategies under investigation, cell-based therapies offer the most promising results, due to the multifactorial roles that these cells can fulfil. However, the best cell source is still a matter of debate, as are clinical issues that include the optimal cell dose as well as the timing and route of administration. In this context, the role of biomaterials is gaining importance. These can not only act as vehicles for the administered cells but also, in the case of chronic lesions, can be used to fill the permanent cyst, thus creating a more favourable and conducive environment for axonal regeneration in addition to serving as local delivery systems of therapeutic agents to improve the regenerative milieu. Some of the candidate molecules for the future are discussed in view of the knowledge derived from studying the mechanisms that facilitate the intrinsic regenerative capacity of central nervous system neurons. The future challenge for the multidisciplinary teams working in the field is to translate the knowledge acquired in basic research into effective combinatorial therapies to be applied in the clinic.

Transplantation of microencapsulated Schwann cells and mesenchymal stem cells augment angiogenesis and improve heart function.

Because of their plasticity and availability, bone-marrow-derived mesenchymal stem cells (MSC) are a potential cell source for treating ischemic heart disease. Schwann cells (SC) play a critical role in neural remodeling and angiogenesis because of their secretion of cytokines such as vascular endothelial growth factor (VEGF). Cell microencapsulation, surrounding cells with a semipermeable polymeric membrane, is a promising tool to shelter cells from the recipient's immune system. We investigated whether transplantation of microencapsulated SC (MC-SC) and MSC together could improve heart function by augmenting angiogenesis in acute myocardial infarction (AMI). Sprague-Dawley rats with ligation of the left anterior descending artery to induce AMI were randomly divided for cell transplantation into four groups-MC-SC+MSC, MC+MSC, MSC, MC-SC, and controls. Echocardiography was performed at 3 days and 2 and 4 weeks after AMI. Rat hearts were harvested on day 28 after transplantation and examined by immunohistochemistry and western blot analysis. Echocardiography revealed differences among the groups in fractional shortening and end-systolic and end-diastolic dimensions (P < 0.05). The number of BrdU-positive cells was greater with MC-SC+MSC transplantation than the other groups (P < 0.01). The vessel density and VEGF level in the infarcted zone was significantly increased with MC-SC+MSC transplantation (P < 0.05). These results show that transplanting a combination of MC-SC and MSC could augment angiogenesis and improve heart function in AMI.

Sequential oxygen supply system promotes peripheral nerve regeneration by enhancing Schwann cells survival and angiogenesis.

Local hypoxia in cellular grafts remains a challenge during the repair of peripheral nerve injury. Oxygen carriers (perfluorotributylamine, PFTBA) have been shown to provide oxygen to Schwann cells (SCs) for a short period. However, the limited oxygen supply from oxygen-carrying materials hinders the ability of such systems to counteract hypoxia over an extended period and limits their therapeutic potential. In this study, PFTBA/VEGF core-shell fibers were fabricated through coaxial electrospinning to construct an oxygen supply system that can sequentially provide oxygen, first via the oxygen carrier and subsequently by promoting angiogenesis via VEGF. Then, the oxygen release and proangiogenic effects of the PFTBA/VEGF core-shell fibers were examined in vitro. Furthermore, sequential oxygen supply conduits prepared using the fibers and filled with SCs were used to bridge 15-mm-long sciatic nerve defects in rats. The PFTBA-VEGF system was confirmed to protect SCs from hypoxia and promote angiogenesis in vitro. Subsequent in vivo studies showed that after the oxygen carried by PFTBA was exhausted, the VEGF could induce neovascularization, and the nascent blood vessels acted as sequential oxygen suppliers for SCs during nerve regeneration. In addition, rats transplanted with the sequential oxygen supply system showed significant morphological and functional improvements in axonal regeneration, the sciatic function index, and the muscle wet weight ratio. The final functional outcomes were similar after treatment with the sequential oxygen supply conduits and autografts. Western blots revealed that the VEGF in the system could upregulate p-AMPK, contributing to axon regeneration after sciatic nerve injury. The sequential oxygen supply system offers essential insights into the oxygen regulation of biomaterials and highlights the potential of oxygen supply strategies as therapeutic approaches for repairing defects in peripheral nerves and other aerobic tissues.

Methylcobalamin-Loaded PLCL Conduits Facilitate the Peripheral Nerve Regeneration.

The feasible fabrication of nerve guidance conduits (NGCs) with good biological performance is important for translation in clinics. In this study, poly(d,l-lactide-co-caprolactone) (PLCL) films loaded with various amounts (wt; 5%, 15%, 25%) of methylcobalamin (MeCbl) are prepared, and are further rolled and sutured to obtain MeCbl-loaded NGCs. The MeCbl can be released in a sustainable manner up to 21 days. The proliferation and elongation of Schwann cells, and the proliferation of Neuro2a cells are enhanced on these MeCbl-loaded films. The MeCbl-loaded NGCs are implanted into rats to induce the regeneration of 10 mm amputated sciatic nerve defects, showing the ability to facilitate the recovery of motor and sensory function, and to promote myelination in peripheral nerve regeneration. In particular, the 15% MeCbl-loaded PLCL conduit exhibits the most satisfactory recovery of sciatic nerves in rats with the largest diameter and thickest myelinated fibers.

Enhanced sciatic nerve regeneration by poly-L-lactic acid/multi-wall carbon nanotube neural guidance conduit containing Schwann cells and curcumin encapsulated chitosan nanoparticles in rat.

The main aim of this study was to improve the efficacy of peripheral nerve regeneration by an artificial neural guidance conduit (NGC) as a carrier to transplant allogeneic Schwann cells (SCs) and curcumin encapsulated chitosan nanoparticles (nanocurcumin). The conduit was prepared by poly-L-lactic acid (PLLA) and surface-modified multi-wall carbon nanotubes (mMWCNT) and filled with SCs and nanocurcumin. SCs play an important role in the regeneration of injured peripheral nerve and controlled curcumin release can decrease SCs apoptosis, and enhance the regeneration and functional recovery of injured peripheral nerves. The mechanical properties, contact angle, and cell biocompatibility experiments showed that the optimized concentration of mMWCNT inside PLLA wall of conduits was 0.15 wt%. The drug release experiments showed slower release of curcumin from nanocurcumin samples compared to nanocurcumin encapsulated inside NGC wrapped fibrin gel sample. It was found that simultaneous using of both SCs and curcumin inside NGC had a significant role in sciatic nerve regeneration in vivo. Histological examination revealed a significant increase in the number of axons in injured sciatic nerve following treatment by SCs and nanocurcumin compared to negative control group. Histological evaluation also revealed a significant decrease in the number of vessels in fibrin groups compared to positive control group. The results showed that there was no significant difference between the reaction time and sciatic functional index (SFI) values of rats with injured sciatic nerve treated by NGC/SCs/nanocurcumin sample and autograft sample. In conclusion, our results strongly showed that PLLA/mMWCNT nanofibrous conduit filled with fibrin gel containing SCs and nanocurcumin is a proper strategy for improving nerve regeneration after a nerve transaction in the rat.

Designing ideal conduits for peripheral nerve repair.

Nerve tubes, guides, or conduits are a promising alternative for autologous nerve graft repair. The first biodegradable empty single lumen or hollow nerve tubes are currently available for clinical use and are being used mostly in the repair of small-diameter nerves with nerve defects of < 3 cm. These nerve tubes are made of different biomaterials using various fabrication techniques. As a result these tubes also differ in physical properties. In addition, several modifications to the common hollow nerve tube (for example, the addition of Schwann cells, growth factors, and internal frameworks) are being investigated that may increase the gap that can be bridged. This combination of chemical, physical, and biological factors has made the design of a nerve conduit into a complex process that demands close collaboration of bioengineers, neuroscientists, and peripheral nerve surgeons. In this article the authors discuss the different steps that are involved in the process of the design of an ideal nerve conduit for peripheral nerve repair.

Combinatorial tissue engineering partially restores function after spinal cord injury.

Hydrogel scaffolds provide a beneficial microenvironment in transected rat spinal cord. A combinatorial biomaterials-based strategy provided a microenvironment that facilitated regeneration while reducing foreign body reaction to the three-dimensional spinal cord construct. We used poly lactic-co-glycolic acid microspheres to provide sustained release of rapamycin from Schwann cell (SC)-loaded, positively charged oligo-polyethylene glycol fumarate scaffolds. The biological activity and dose-release characteristics of rapamycin from microspheres alone and from microspheres embedded in the scaffold were determined in vitro. Three dose formulations of rapamycin were compared with controls in 53 rats. We observed a dose-dependent reduction in the fibrotic reaction to the scaffold and improved functional recovery over 6 weeks. Recovery was replicated in a second cohort of 28 animals that included retransection injury. Immunohistochemical and stereological analysis demonstrated that blood vessel number, surface area, vessel diameter, basement membrane collagen, and microvessel phenotype within the regenerated tissue was dependent on the presence of SCs and rapamycin. TRITC-dextran injection demonstrated enhanced perfusion into scaffold channels. Rapamycin also increased the number of descending regenerated axons, as assessed by Fast Blue retrograde axonal tracing. These results demonstrate that normalization of the neovasculature was associated with enhanced axonal regeneration and improved function after spinal cord transection.

Biodegradable poly-beta-hydroxybutyrate scaffold seeded with Schwann cells to promote spinal cord repair.

Cavity formation is an important obstacle impeding regeneration after spinal cord injury and bridging strategies are essential to provide physical substrate allowing axons to grow across the lesion site. In this study we evaluated effects of biodegradable tubular conduit made from poly-beta-hydroxybutyrate (PHB) scaffold with predominantly unidirectional fiber orientation and supplemented with cultured adult Schwann cells on axonal regeneration after cervical spinal cord injury in adult rats. After transplantation into the injured spinal cord, plain PHB conduit was well-integrated into posttraumatic cavity and induced modest astroglial reaction. Regenerating axons were found mainly outside the PHB with only single fibers crossing the host-graft interface. No host Schwann cells migrated into the graft. In contrast, when suspension of adult Schwann cells was added to the PHB during transplantation, neurofilament-positive axons filled the conduit and became associated with the implanted cells. Although rubrospinal fibers did not enter the PHB, numerous raphaespinal and CGRP-positive axons were found within the conduit. Modification of PHB surface with fibronectin, laminin or collagen significantly increased Schwann cell attachment and proliferation in vitro. However, transplantation of PHB conduit pre-coated with fibronectin and seeded with Schwann cells did not alter axonal growth response. The results demonstrate that a PHB scaffold promotes attachment, proliferation and survival of adult Schwann cells and supports marked axonal regeneration within the graft.

Polyglycolic acid filaments guide Schwann cell migration in vitro and in vivo.

Nerve conduits filled with longitudinal aligned filaments have demonstrated a better regenerative outcome for bridging large peripheral nerve gaps than hollow nerve conduits. In the present study, we investigated the in vitro and in vitro cellular behavior of Schwann cells on polyglycolic acid (PGA) filaments by immunocyto/histochemistry and light/electron microscopy. After 1-3-week culture of rat dorsal root ganglia (DRGs) onto PGA filaments, Schwann cells from rat DRGs adhered to and migrated along PGA filaments. Twenty-four rats received implantation of chitosan conduits inserted with PGA filaments to bridge 10-mm-long sciatic nerve gaps. At 1, 2, 3 and 4 weeks post-implantation (n = 6, each time point), Schwann cells were found to migrate along PGA filaments and form cell columns resembling bands of Büngner. These results suggest that PGA filaments may play a contact guidance role in Schwann cell migration and thus serve as a promising conduit-filling material to facilitate peripheral nerve repair.

[Fibrin matrix enhances adherence of peripheral nerve regenerative cells]. / Fibrinmatrix erhöht adhärenz von regenerativen peripheren nervenzellen.

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.

Dental Pulp Stem Cells - Exploration in a Novel Animal Model: the Tasmanian Devil (Sarcophilus harrisii).

Dental pulp stem cells (DPSC) are a heterogeneous population of highly proliferative stem cells located in the soft inner pulp tissue of the tooth. Demonstrated to have an affinity for neural differentiation, DPSC have been reported to generate functional Schwann cells (SC) through in vitro differentiation. Both DPSC and SC have neural crest origins, recently a significant population of DPSC have been reported to derive from peripheral nerve-associated glia. The predisposition DPSC have towards the SC lineage is not only a very useful tool for neural regenerative therapies in the medical field, it also holds great promise in the veterinary field. Devil Facial Tumour (DFT) is a clonally transmissible cancer of SC origin responsible for devastating wild populations of the Tasmanian devil. Very few studies have investigated the healthy Tasmanian devil SC (tdSC) for comparative studies between tdSC and DFT cells, and the development and isolation of a tdSC population is yet to be undertaken. A Tasmanian devil DPSC model offers a promising new outlook for DFT research, and the link between SC and DPSC may provide a potential explanation as to how a cancerous SC initially arose in a single Tasmanian devil to then go on to infect others as a parasitic clonal cell line. In this review we explore the current role of DPSC in human regenerative medicine, provide an overview of the Tasmanian devil and the devastating effect of DFT, and highlight the promising potential DPSC techniques pose for DFT research and our current understanding of DFT.

Schwann Cell Transplantation Methods Using Biomaterials.

Biomaterials can be utilized to assist in the transplantation of Schwann cells to the central and peripheral nervous system. The biomaterials can be natural or man-made, and can have preformed shapes or injectable formats. Biomaterials can play multiple roles in cellular transplantation; for example, they can assist with cellular integration and protect Schwann cells from cell death initiated by the lack of a substrate, an occurrence known as "anoikis." In addition, biomaterials can be engineered to increase cell proliferation and differentiation by the addition of ligands bound to the substrate. Here, we describe the incorporation of Schwann cells to both man-made and natural matrices for in vitro and in vivo measures relevant to Schwann cell transplantation strategies.

Skin derived precursor Schwann cell-generated acellular matrix modified chitosan/silk scaffolds for bridging rat sciatic nerve gap.

Extracellular/acellular matrix has been attracted much research interests for its unique biological characteristics, and ACM modified neural scaffolds shows the remarkable role of promoting peripheral nerve regeneration. In this study, skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) were used as parent cells to generate acellular(ACM) for constructing a ACM-modified neural scaffold. SKP-SCs were co-cultured with chitosan nerve guidance conduits (NGC) and silk fibroin filamentous fillers, followed by decellularization to stimulate ACM deposition. This NGC-based, SKP-SC-derived ACM-modified neural scaffold was used for bridging a 10 mm long rat sciatic nerve gap. Histological and functional evaluation after grafting demonstrated that regenerative outcomes achieved by this engineered neural scaffold were better than those achieved by a plain chitosan-silk fibroin scaffold, and suggested the benefits of SKP-SC-derived ACM for peripheral nerve repair.

Schwann cell graft: a method to promote sensory responses of osseointegrated implants.

Osseointegrated dental implants have been widely used in clinics to restore the missing teeth of patients. Since there are no periodontal ligament and associated Ruffini endings in the peri-implant tissues, sensory thresholds of the implant are much higher than those of natural teeth, and its self-protective reflex is quit poor. Implant fracture or aggressive bone loss sometimes occurs because the patient cannot feel the overloads exerted on the implant. Until now, no available method has been issued to solve such a problem. Schwann cell is the glial cell of peripheral nerve system. It has been widely accepted to play indispensable roles during neural development and regeneration. Its mechanism includes forming Büngner's band, producing neurotrophic factors, synthesizing surface cell adhesion molecules, and elaborating basement membrane. Furthermore, Schwann cell is quite important for the periodontal Ruffini endings. Applying these functions of Schwann cells, we put forward a hypothesis that transplanting Schwann cells into the implant site can be a method to promote sensory responses of the dental implants.

Schwann cells, acutely dissociated from a predegenerated nerve trunk, can be applied into a matrix used to bridge nerve defects in rats.

BACKGROUND: The gold standard to reconstruct a nerve defect is a conventional autologous nerve graft. There may be a lack of such grafts in severe nerve injuries. Alternatives to autologous nerve grafts are needed. METHODS: We have developed a technique where mainly Schwann cells are acutely dissociated from the ends of the severed nerve trunk after nerve injury. The technique does not require long-term cell culture procedures. The obtained cells, which can be dissociated within a few hours, are applied to a silicone tube or a tendon autograft used to bridge a nerve defect. FINDINGS: Dissociated cells from the ends of the severed nerve ends consist of more than 85% of Schwann cells. The remaining cells are ED1 stained macrophages. The cells survive transfer to a silicone tube or a tendon autograft which bridge the nerve defect. Axons do grow through such a graft filled with dissociated cells. CONCLUSION: Our novel model to obtain mainly Schwann cells by dissociation of the cells from the severed nerve ends after injury and add them to a matrix, thereby creating an artificial nerve graft, may be a new technique with potential clinical application in nerve reconstruction.