Rejection and regeneration through peripheral nerve allografts: immunoperoxidase studies with laminin, S100 protein and neurofilament antisera

The pattern and temporal sequence of histopathological events in a rat nerve allograft model were evaluated. Following grafting and varying survival periods (from 1 to 30 weeks), the host and donor nerve were removed and assessed by light and electron microscopy. Nerve allografts underwent Wallerian degeneration and rejection. Wallerian degeneration was the dominant pathologic process at weeks 1 and 2 after engraftment. Histologic rejection started as an epineurial process at weeks I and 2, became progressively endoneurial and was most prominent at 4 and 6 weeks after engraftment. Rejection was accompanied by evidence of graft Schwann cell and endoneurial tube loss. The rejection process delayed, but did not prevent, nerve regeneration by the host. Regeneration of fine neurofilament-positive axonal sprouts into the proximal portions of the graft was observed as early as week 2. Subsequently, regeneration occurred through the periphery and around the exterior of the rejected nerve allograft fascicle. Regenerating axons were accompanied by S100 protein reactive Schwann cells and newly synthesized laminin-positive endoneurial tubes. Regenerating axons reinnervated the distal host segment at week 8 and increased in number and myelination thereafter. The observations of rejection and regeneration through nerve allograft segments are discussed in reference to previous studies.     

Agmatine treatment and vein graft reconstruction enhance recovery after experimental facial nerve injury

The rate of nerve regeneration is a critical determinant of the degree of functional recovery after injury. Here, we sought to determine whether treatment with the neuroprotective compound, agmatine, with or without nerve reconstruction utilizing a regional autogenous vein graft would accelerate the rate of facial nerve regeneration. Experiments compared the following seven groups of adult male rats: (A) Intact untreated controls. (B) Sham operation with interruption of the nerve blood supply (controls). (C) Transection of the mandibular branch of the facial nerve (generating a gap of 3 mm) followed by saline treatment. (D) Nerve transection with unsutured autogenous vein (external jugular) graft reconstruction plus saline treatment. (E) Nerve transection with sutured vein graft approximation (coaptation of the proximal and distal nerve stumps) plus saline. (F) Nerve transection with sutured vein graft followed by agmatine treatment (four daily intraperitoneal injections of 100 mg/kg agmatine sulfate). (G) Nerve transection with unsutured vein graft followed by agmatine treatment. Functional recovery, as assessed by grading vibrissae movements and by recording nerve conduction velocity and numbers of regenerated axons, indicated that either vein reconstruction or agmatine treatment resulted in accelerated and more complete recovery as compared with controls. But best results were observed in animals that underwent combined treatment, i.e., vein reconstruction plus agmatine injection. We conclude that agmatine treatment can accelerate facial nerve regeneration and that agmatine treatment together with autogenous vein graft offers an advantageous alternative to other facial nerve reconstruction procedures.     

Processed allografts and type I collagen conduits for repair of peripheral nerve gaps

Autografting is the gold standard in the repair of peripheral nerve injuries that are not amenable to end‐to‐end coaptation. However, because autografts result in donor‐site defects and are a limited resource, an effective substitute would be valuable. In a rat model, we compared isografts with Integra NeuraGen® (NG) nerve guides, which are a commercially available type I collagen conduit, with processed rat allografts comparable to AxoGen's Avance® human decellularized allograft product. In a 14‐mm sciatic nerve gap model, isograft was superior to processed allograft, which was in turn superior to NG conduit at 6 weeks postoperatively (P < 0.05 for number of myelinated fibers both at midgraft and distal to the graft). At 12 weeks, these differences were no longer apparent. In a 28‐mm graft model, isografts again performed better than processed allografts at both 6 and 22 weeks; regeneration through the NG conduit was often insufficient for analysis in this long graft model. Functional tests confirmed the superiority of isografts, although processed allografts permitted successful reinnervation of distal targets not seen in the NG conduit groups. Processed allografts were inherently non‐immunogenic and maintained some internal laminin structure. We conclude that, particularly in a long gap model, nerve graft alternatives fail to confer the regenerative advantages of an isograft. However, AxoGen processed allografts are superior to a currently available conduit‐style nerve guide, the Integra NeuraGen®. They provide an alternative for reconstruction of short nerve gaps where a conduit might otherwise be used. Muscle Nerve, 2008     

Costimulation blockade inhibits the indirect pathway of allorecognition in nerve allograft rejection

Nerve allografts provide a temporary scaffold for host nerve regeneration. The need for systemic immunosuppression limits clinical application. Characterization of the immunological mechanisms that induce immune hyporesponsiveness may provide a basis for optimizing immunomodulating regimens. We utilized wild-type and MHC class II-deficient mice, as both recipients and donors. Host treatment consisted of triple costimulatory blockade. Quantitative assessment was made at 3 weeks using nerve histomorphometry, and muscle testing was performed on a subset of animals at 7 weeks. Nerve allograft rejection occurred as long as either the direct or indirect pathways were functional. Indirect antigen presentation appeared to be more important. Nerve allograft rejection occurs in the absence of a normal direct or indirect immune response but may be more dependent on indirect allorecognition. The indirect pathway is required to induce costimulatory blockade immune hyporesponsiveness.     

Prolonged cold-preservation of nerve allografts

The goal of this study was to determine the effect of varying durations of cold-preservation on the immunogenicity of nerve allografts and their subsequent ability to facilitate neuroregeneration across a short nerve gap. Allografts preserved for 1, 4, and 7 weeks were compared to untreated allografts and isografts. There was a shift from an interferon-gamma-producing cellular response (untreated allografts) to an absence of response (7-week cold-preserved allografts and isografts). There were no detectable alloantibodies by flow cytometry. Histomorphometry distal to the graft showed robust regeneration in the isograft and 7-week cold-preserved groups when compared to the untreated allograft group. Increasing duration of cold-preservation diminished the cellular immune response. This cold-preservation does not preclude subsequent nerve regeneration across a short nerve graft. Prolonged cold-preservation of nerve allograft tissue could serve as a means to produce unlimited graft material for use in peripheral nerve reconstruction.     

A double-transgenic mouse used to track migrating Schwann cells and regenerating axons following engraftment of injured nerves

We propose that double-transgenic thy1-CFP(23)/S100-GFP mice whose Schwann cells constitutively express green fluorescent protein (GFP) and axons express cyan fluorescent protein (CFP) can be used to serially evaluate the temporal relationship between nerve regeneration and Schwann cell migration through acellular nerve grafts. Thy1-CFP(23)/S100-GFP and S100-GFP mice received non-fluorescing cold preserved nerve allografts from immunologically disparate donors. In vivo fluorescent imaging of these grafts was then performed at multiple points. The transected sciatic nerve was reconstructed with a 1-cm nerve allograft harvested from a Balb-C mouse and acellularized via 7 weeks of cold preservation prior to transplantation. The presence of regenerated axons and migrating Schwann cells was confirmed with confocal and electron microscopy on fixed tissue. Schwann cells migrated into the acellular graft (163+/-15 intensity units) from both proximal and distal stumps, and bridged the whole graft within 10 days (388+/-107 intensity units in the central 4-6 mm segment). Nerve regeneration lagged behind Schwann cell migration with 5 or 6 axons imaged traversing the proximal 4 mm of the graft under confocal microcopy within 10 days, and up to 21 labeled axons crossing the distal coaptation site by 15 days. Corroborative electron and light microscopy 5 mm into the graft demonstrated relatively narrow diameter myelinated (431+/-31) and unmyelinated (64+/-9) axons by 28 but not 10 days. Live imaging of the double-transgenic thy1-CFP(23)/S100-GFP murine line enabled serial assessment of Schwann cell-axonal relationships in traumatic nerve injuries reconstructed with acellular nerve allografts.     

Promoting axonal regeneration following nerve surgery:a perspective on ultrasound treatment for nerve injuries

Nerve injury is often associated with limited axonal regeneration and thus leads to delayed or incomplete axonal reinnervation.As a consequence of slow nerve regeneration,target muscle function is often insufficient and leads to a lifelong burden.Recently,the diagnosis of nerve injuries has been improved and likewise surgical reconstruction has undergone significant developments.However,the problem of slow nerve regeneration has not been solved.In a recent meta-analysis,we have shown that the application of low-intensity ultrasound promotes nerve regeneration experimentally and thereby can improve functional outcomes.Here we want to demonstrate the experimental effect of low intensity ultrasound on nerve regeneration,the current state of investigations and its possible future clinical applications.     

Chondroitinase treatment increases the effective length of acellular nerve grafts

Acellular nerve allografts have been explored as an alternative to nerve autografting. It has long been recognized that there is a distinct limit to the effective length of conventional acellular nerve grafts, which must be overcome for many grafting applications. In rodent models nerve regeneration fails in acellular nerve grafts greater than 2 cm in length. In previous studies we found that nerve regeneration is markedly enhanced with acellular nerve grafts in which growth-inhibiting chondroitin sulfate proteoglycan was degraded by pretreatment with chondroitinase ABC (ChABC). Here, we tested if nerve regeneration can be achieved through 4-cm acellular nerve grafts pretreated with ChABC. Adult rats received bilateral sciatic nerve segmental resection and repair with a 4 cm, thermally acellularized, nerve graft treated with ChABC (ChABC graft) or vehicle-treated acellularized graft (Control graft). Nerve regeneration was examined 12 weeks after implantation. Our findings confirm that functional axonal regeneration fails in conventional long acellular grafts. In this condition we found very few axons in the distal host nerve, and there were marginal signs of sciatic nerve reinnervation in few (2/9) rats. This was accompanied by extensive structural disintegration of the distal graft and abundant retrograde axonal regeneration in the proximal nerve. In contrast, most (8/9) animals receiving nerve repair with ChABC grafts showed sciatic nerve reinnervation by direct nerve pinch testing. Histological examination revealed much better structural preservation and axonal growth throughout the ChABC grafts. Numerous axons were found in all but one (8/9) of the host distal nerves and many of these regenerated axons were myelinated. In addition, the amount of aberrant retrograde axonal growth (originating near the proximal suture line) was markedly reduced by repair with ChABC grafts. Based on these results we conclude that ChABC treatment substantially increases the effective length of acellular nerve grafts.     

Nerve allograft regeneration and immunological tolerance of rats with sciatic nerve transplantation after intragastric pretreatment with ultraviolet B-irradiated donor splenocytes★

BACKGROUND: Nerve allograft rejection is an unavoidable problem for nerve allografts. Traumatic peripheral nerve injuries are commonly reconstructed using autogenous nerve grafts. However, this form of reconstruction is limited by insufficient autologous nerves for large gap repairs and by morbidity at the nerve donor site. OBJECTIVE: To examine sciatic nerve regeneration and immune tolerance reaction after intragastric administration of ultraviolet B-irradiated (UVB) donor splenocytes. DESIGN, TIME AND SETTING: A complete randomized grouping design and controlled experiment. The experiments were conducted in the Department of Orthopedics, the First Affiliated Hospital to Shanxi Medical University, China, between March and October 2007. MATERIALS: Fourteen adult male SD rats and fourteen male Wistar rats, weighing 250–300 g, were randomly matched as donors and acceptors. A further seven male SD rats (weight 250–300 g, age 12–16 weeks) were used for nerve isografts. Immune preparations and the Epics XL flow cytometer were purchased from B-D Company, USA. A computer-assisted electromyograph machine was provided by Keypoint P, Dantel Company, Denmark. METHODS: Splenocytes from Wistar rats were isolated, purified, and cultured, and then irradiated with ultraviolet B. In the first control group (Group 1), the SD rats received a syngeneic SD nerve isograft. In the second control group (Group 2), the SD rats received a nerve allograft from Wistar rats without pretreatment. In the oral-tolerance treated group (Group 3), the SD recipient rats were inoculated with 2.5×107 Lewis UVB-irradiated donor splenocyte cells by intestinal tract administration at seven days before transplantation. MAIN OUTCOME MEASURES: (1) The recent end and the middle and distal end of the transplanted nerve were cut at 8 and 12 weeks after operation. Recovery of nerve regeneration was measured with HE staining using the total number, density, and diameter of the nerve fibers. (2) The level of CD25+T lymphocytes in peripheral blood was detected with the Epics XL flow cytometer at one week after operation. (3) The bilateral sciatic nerves were exposed from the sciatic notch up to 0.5 mm beyond the distal graft site at eight weeks post-engraftment. Bipolar platinum stimulating electrodes were placed under the sciatic nerve proximally and the mean conduction velocity was recorded with recording electrodes on the posterior tibial nerve at 0.3 cm distal to the nerve graft. RESULTS: Eight weeks after operation, total axon number and fiber density in Group 3 were higher than that in Group 1 (P < 0.05), neural regeneration in Group 3 was lower than that in Group 1 (P < 0.05) , The level of CD25+T lymphocytes in peripheral blood of Group 3 was significantly lower than that of Group 2 (P < 0.05). There was no significant difference between Group 3 and Group 1 (P > 0.05). At eight weeks post-engraftment the mean conduction velocity of Group 3 approximated that of Group 1. The untreated allografts in Group 2 demonstrated no measurable recovery of conduction velocity. CONCLUSION: Pretreatment with a single intragastric dose of UVB-modified donor antigen specifically induced tolerance to peripheral nerve allografts in rats. Key Words: sciatic nerve; transplantation, homologous; immune tolerance     

Effects of motor versus sensory nerve grafts on peripheral nerve regeneration

Autologous nerve grafting is the current standard of care for nerve injuries resulting in a nerve gap. This treatment requires the use of sensory grafts to reconstruct motor defects, but the consequences of mismatches between graft and native nerve are unknown. Motor pathways have been shown to preferentially support motoneuron regeneration. Functional outcome of motor nerve reconstruction depends on the magnitude, rate, and precision of end organ reinnervation. This study examined the role of pathway type on regeneration across a mixed nerve defect. Thirty-six Lewis rats underwent tibial nerve transection and received isogeneic motor, sensory or mixed nerve grafts. Histomorphometry of the regenerating nerves at 3 weeks demonstrated robust nerve regeneration through both motor and mixed nerve grafts. In contrast, poor nerve regeneration was seen through sensory nerve grafts, with significantly decreased nerve fiber count, percent nerve, and nerve density when compared with mixed and motor groups (P < 0.05). These data suggest that use of motor or mixed nerve grafts, rather than sensory nerve grafts, will optimize regeneration across mixed nerve defects.     

Prevention of macrophage invasion impairs regeneration in nerve grafts

The importance of cell invasion for regeneration in nerve segments was investigated in rats. The regeneration distance of axons in predegenerated nerve segments was compared to the outgrowth in nerve segments where cell invasion had been prevented. A 10 mm long nerve segment, which was predegenerated (preserved or impaired blood circulation) or kept in a Millipore chamber (pore size 0.22 μm), was sutured as a nerve graft at the contralateral side three days or two weeks after the initial procedure. At two weeks immunocytochemical staining and routine histologic analysis revealed pronounced myelin breakdown and presence of ED1 and ED2 positive macrophages in the predegenerated nerve segment. Nerve segments, which were kept in the Millipore chamber, showed no invasion of macrophages and the myelin sheaths were preserved. The regeneration distances of axons in the nerve segments, evaluated with the pinch reflex test, were significantly longer in the predegenerated nerve segments compared to the nerve segments kept in Millipore chambers. Nerve grafts, which were taken from predegenerated nerves with intact blood circulation, showed the longest regeneration distances. It is suggested that the regeneration process can be impaired in nerve segments where cell and macrophage invasion as well as myelin breakdown are prevented and that preservation of the blood circulation during the degeneration process is important.     

The impact of motor and sensory nerve architecture on nerve regeneration

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerve's Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p < 0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p < 0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.     

Regeneration across preserved peripheral nerve grafts

The potential to store nerve grafts for a prolonged period of time was assessed in a rat sciatic nerve model. Three-centimeter syngeneic nerve grafts were stored in Belzer/University of Wisconsin cold storage solution at different temperatures (5°C, 22°C, or 37°C) for varying time periods (6 h, 24 h, or 3 weeks) prior to transplantation. Functional assessment using serial walking track analyses revealed no difference between storage times and temperatures. At 14 months postengraftment, the conduction velocities and the number of myelinated fibers that had regenerated across all grafts stored at 5°C for all time periods tested were superior to grafts stored at either 22°C or 37°C. Nerve grafts stored for up to 3 weeks at 5°C acted as effective conduits for proximal regenerating fibers and resulted in histologic, electrophysiologic, and functional results equivalent to fresh nerve grafts. Nerve graft storage may be applicable to nerve allografts and potentially provide allograft material that requires reduced or no associated host immunosuppression.© 1995 John Wiley & Sons, Inc.     

Nerve grafts with various sensory and motor fiber compositions are equally effective for the repair of a mixed nerve defect

Autologous, cellular nerve grafts are commonly used to bridge nerve gaps in the clinical setting. Sensory nerves are most often selected for autografting because of their relative ease of procurement and low donor site morbidity. A series of recent reports conclude that sensory isografts are inferior to motor and mixed nerve isografts for the repair of a mixed nerve defect in rat. The aim of the present study was to determine if the disparity reported with cellular graft subtypes exists for detergent decellularized, chondroitinase ABC processed nerve grafts. We hypothesized that processing removes or neutralizes the inferior properties attributed to sensory nerve grafts. Saphenous (cutaneous branch), femoral quadriceps (muscle branch) and tibial (mixed trunk) nerve grafts 5 mm in length were used in tensionless reconstruction of syngenic rat tibial nerves. Nerve regeneration through the grafts and into the recipient distal nerve was evaluated 21 days after grafting by two methods, toluidine blue staining of semi-thin sections (myelinated axons) and neurofilament-immunolabeling (total axons). Contrary to previous reports using this grafting scheme, we found no significant difference in the myelinated axon counts for the three cellular graft subtypes. Moreover, total axon counts indicated cellular saphenous nerve grafts were more effective than the quadriceps and tibial nerve grafts. A similar though less pronounced trend was found for the decellularized processed grafts. These findings indicate that nerve graft composition (sensory and motor) has no substantial impact on the short-term outcome of nerve regeneration in a mixed nerve repair model.     

The role of vascularization in nerve regeneration of nerve graft

Vascularization is an important factor in nerve graft survival and function. The specific molecular regulations and patterns of angiogenesis following peripheral nerve injury are in a broad complex of pathways. This review aims to summarize current knowledge on the role of vascularization in nerve regeneration, including the key regulation molecules, and mechanisms and patterns of revascularization after nerve injury. Angiogenesis, the maturation of pre-existing vessels into new areas, is stimulated through angiogenic factors such as vascular endothelial growth factor and precedes the repair of damaged nerves. Vascular endothelial growth factor administration to nerves has demonstrated to increase revascularization after injury in basic science research. In the clinical setting, vascularized nerve grafts could be used in the reconstruction of large segmental peripheral nerve injuries. Vascularized nerve grafts are postulated to accelerate revascularization and enhance nerve regeneration by providing an optimal nutritional environment, especially in scarred beds, and decrease fibroblast infiltration. This could improve functional recovery after nerve grafting, however, conclusive evidence of the superiority of vascularized nerve grafts is lacking in human studies. A well-designed randomized controlled trial comparing vascularized nerve grafts to non-vascularized nerve grafts involving patients with similar injuries, nerve graft repair and follow-up times is necessary to demonstrate the efficacy of vascularized nerve grafts. Due to technical challenges, composite transfer of a nerve graft along with its adipose tissue has been proposed to provide a healthy tissue bed. Basic science research has shown that a vascularized fascial flap containing adipose tissue and a vascular bundle improves revascularization through excreted angiogenic factors, provided by the stem cells in the adipose tissue as well as by the blood supply and environmental support. While it was previously believed that revascularization occurred from both nerve ends, recent studies propose that revascularization occurs primarily from the proximal nerve coaptation. Fascial flaps or vascularized nerve grafts have limited applicability and future directions could lead towards off-the-shelf alternatives to autografting, such as biodegradable nerve scaffolds which include capillary-like networks to enable vascularization and avoid graft necrosis and ischemia.     

Histomorphological and functional impacts of postoperative motor training in rats after allograft sciatic nerve transplantation under low‐dose FK 506

This study aimed to determine the effect of motor training on recovery after nerve transplantation under low‐dose FK 506. Rats (n = 30) of two strains were randomly assigned to three groups. Group I served as untreated controls; groups II and III received allograft transplants for reconstruction of the sciatic nerve and FK 506 (0.1 mg/kg/d). Nonoperated limbs served as intra‐animal controls. Group III received postoperative motor training. Functional and histomorphological outcomes were assessed by walking track analysis and by blob analysis for myelinization of nerve sections. Regeneration occurred in both groups II and III. The control sections of the nonoperated limbs in group III showed significantly higher myelinization compared with group I and II; regeneration of the operated side was superior in group II. With regard to postoperative motor training, no benefit could be seen; however, the impact of postoperative motor training on the nonoperated limb were identified. Muscle Nerve, 2009     

Failure of host axons to regenerate through a once successful but later rejected long nerve allograft

Host axons will regenerate through a long nerve allograft in an immunologically tolerant rat. However, if tolerance is abolished, rejection occurs and allogeneic cells (e.g., Schwann, vascular, perineurial, etc.) as well as regenerated host axons disappear from the allograft. Because following tolerance abolition host axons begin to regenerate into the connective tissue remnants of the rejected nerve allografts, the extent of this renewed axonal growth was investigated. It was found that in a tolerance-abolished rat, host axons only regrew into the proximal 1 cm of a 4-cm allograft which in a fully tolerant recipient would have had numerous allogeneic Schwann cell-myelinated axons throughout its length. It is concluded that viable allogeneic cells (i.e., Schwann, fibroblast, and vascular) together with their connective tissue matrix provide the best way to aid host nerve fiber regeneration through a long nerve allograft.     

Acellular nerve allografts in peripheral nerve regeneration: a comparative study

Introduction: Processed nerve allografts offer a promising alternative to nerve autografts in the surgical management of peripheral nerve injuries where short deficits exist. Methods: Three established models of acellular nerve allograft (cold‐preserved, detergent‐processed, and AxoGen‐processed nerve allografts) were compared with nerve isografts and silicone nerve guidance conduits in a 14‐mm rat sciatic nerve defect. Results: All acellular nerve grafts were superior to silicone nerve conduits in support of nerve regeneration. Detergent‐processed allografts were similar to isografts at 6 weeks postoperatively, whereas AxoGen‐processed and cold‐preserved allografts supported significantly fewer regenerating nerve fibers. Measurement of muscle force confirmed that detergent‐processed allografts promoted isograft‐equivalent levels of motor recovery 16 weeks postoperatively. All acellular allografts promoted greater amounts of motor recovery compared with silicone conduits. Conclusion: These findings provide evidence that differential processing for removal of cellular constituents in preparing acellular nerve allografts affects recovery in vivo. Muscle Nerve, 2011     

Remnant neuromuscular junctions in denervated muscles contribute to functional recovery in delayed peripheral nerve repair

Schwann cell proliferation in peripheral nerve injury(PNI)enhances axonal regeneration compared to central nerve injury.However,even in PNI,long-term nerve damage without repair induces degeneration of neuromuscular junctions(NMJs),and muscle atrophy results in irreversible dysfunction.The peripheral regeneration of motor axons depends on the duration of skeletal muscle denervation.To overcome this difficulty in nerve regeneration,detailed mechanisms should be determined for not only Schwann cells but also NMJ degeneration after PNI and regeneration after nerve repair.Here,we examined motor axon denervation in the tibialis anterior muscle after peroneal nerve transection in thyl-YFP mice and regeneration with nerve reconstruction using allografts.The number of NMJs in the tibialis anterior muscle was maintained up to 4 weeks and then decreased at 6 weeks after injury.In contrast,the number of Schwann cells showed a stepwise decline and then reached a plateau at 6 weeks after injury.For regeneration,we reconstructed the degenerated nerve with an allograft at 4 and 6 weeks after injury,and evaluated functional and histological outcomes for 10 to 12 weeks after grafting.A higher number of pretzel-shaped NMJs in the tibialis anterior muscle and better functional recovery were observed in mice with a 4-week delay in surgery than in those with a 6-week delay.Nerve repair within 4 weeks after PNI is necessary for successful recovery in mice.Prevention of synaptic acetylcholine receptor degeneration may play a key role in peripheral nerve regeneration.All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University on 5 July 2017,30 March 2018,and 15 May 2019(A2017-311C,A2018-297A,and A2019-248A),respectively.     

Neural crest derived stem cells from dental pulp and tooth-associated stem cells for peripheral nerve regeneration

The peripheral nerve injuries, representing some of the most common types of traumatic lesions affecting the nervous system, are highly invalidating for the patients besides being a huge social burden. Although peripheral nervous system owns a higher regenerative capacity than does central nervous system, mostly depending on Schwann cells intervention in injury repair, several factors determine the extent of functional outcome after healing. Based on the injury type, different therapeutic approaches have been investigated so far. Nerve grafting and Schwann cell transplantation have represented the gold standard treatment for peripheral nerve injuries, however these approaches own limitations, such as scarce donor nerve availability and donor site morbidity. Cell based therapies might provide a suitable tool for peripheral nerve regeneration, in fact, the ability of different stem cell types to differentiate towards Schwann cells in combination with the use of different scaffolds have been widely investigated in animal models of peripheral nerve injuries in the last decade. Dental pulp is a promising cell source for regenerative medicine, because of the ease of isolation procedures, stem cell proliferation and multipotency abilities, which are due to the embryological origin from neural crest. In this article we review the literature concerning the application of tooth derived stem cell populations combined with different conduits to peripheral nerve injuries animal models, highlighting their regenerative contribution exerted through either glial differentiation and neuroprotective/neurotrophic effects on the host tissue.