Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

The influence of cultured Schwann cells on regeneration through acellular basal lamina grafts

Acellular basal lamina grafts have been shown to be less immunogenic in comparison to cellular grafts, but possess a limited potential for supporting axonal regeneration through them. The present study describes the effect of cultured Schwann cells on enhancing regeneration through acellular grafts. 2 cm long acellular grafts, and in vitro Schwann cell populated acellular grafts were used to repair a surgically created gap in the host peroneal nerve. The transplants were analyzed at 1, 2, 4 and 8 weeks to determine their ability to support axonal regeneration. Host axonal regeneration through Schwann cell cocultured acellular grafts occurred rapidly and was significantly better as compared to non-cultured acellular grafts. The results demonstrate a beneficial effect of Schwann cell culture pretreatment on regeneration through acellular grafts and an improved recovery of the target muscle. The procedure of first preparing acellular grafts with subsequent coculture with Schwann cells offers a novel approach for the repair of injured nervous tissue.     

Optic axons regenerate into sciatic nerve isografts only in the presence of Schwann cells

Optic axons regenerate into normal but not acellular peripheral nerve (PN) grafts. The first axons penetrate the PN graft before 5 days and grow inside the basal lamina tubes amongst the Schwann cells. By 30 days, 4% of the surviving retinal ganglion cells (RGC) regenerate axons for at least 10 mm into the PN graft. Laminin rich basal lamina tubes persist in the acellular PN transplants but only a few axons penetrate the most proximal parts of the tubes by 5 days and none grow farther into the graft by 30 days. RGC counts demonstrate that 34% of the normal RGC population survive 30 days after anastomosing a normal PN to the transected optic nerve. After anastomosing acellular PN grafts, 25% of RGCs survive compared with 10% after optic nerve section. These findings demonstrate that laminin does not promote regeneration of axons and that Schwann cells play the primary role of offering trophic support and even a substrate for growth. RGC survival is also enhanced by PN grafts even when Schwann cells are absent. This latter result suggests that RGC survival is promoted by a trophic substance released from axons and/or Schwann cells in the PN grafts which survives the thawing/freezing procedure (used to kill the Schwann cells) and is active in the grafts in the immediate post operative period.     

The role of Schwann cells and basal lamina tubes in the regeneration of axons through long lengths of freeze-killed nerve grafts

The ability of long acellular nerve grafts to support axonal regeneration was examined using inbred rats. Grafts (40 mm long) of tibial/plantar nerves were used either as live grafts or after freeze-drying to render the grafts acellular. The grafts were sutured to the proximal stump of severed tibial nerves in host animals which were then killed 1-12 weeks later. Axons rapidly regenerated through the living grafts but only extended 10-20 mm into the acellular grafts. This distance was achieved by 6 weeks and thereafter no significant further axonal extension occurred in the acellular grafts. A few naked axons lacking Schwann cell contact were identified in all acellular grafts, but became more numerous near the distal extent of axonal penetration into 6-12 week grafts. These axons contained large numbers of neurofilaments. When the distal 20 mm of 6 week acellular grafts (segments into which axons had not penetrated) were sutured to freshly severed tibial nerves, axons grew readily into the grafted tissue to a maximum distance of 9 mm. It is therefore likely that the limits to axonal regeneration through initially acellular grafts were set by factors intrinsic to the severed nerve. It is suggested that the limited migratory powers of Schwann cells may be one such factor. The concept that basal lamina tubes are not essential for axonal regeneration but may act as low resistance pathways for both axonal elongation and Schwann cell migration is discussed.     

Are Schwann cells essential for axonal regeneration into muscle autografts?

When axons regenerate through frozen–thawed (FT) muscle grafts, they are accompanied by co–migrating Schwann cells derived from the nerve stumps. Although acellular, FT muscle grafts contain an internal scaffold of basal laminae rich in components capable of supporting neurite outgrowth in vitro such as laminin and fibronectin: it is not known whether Schwann cells are essential for axonal regrowth within these grafts. In this paper we test the hypothesis that sarcolemmal basal laminae will support axonal regeneration in the absence of Schwann cells. Two groups of 12 adult Wistar rats were used. All rats received a 0.5 cm FT muscle graft, and 12 rats also received a subperineurial injection of the anti–mi to tic agent mitomycin C (400 μg/ml in physiological saline) prior to grafting. Previous studies have shown that this dose effectively depresses cell proliferation within the endoneurium for 3–4 weeks [17, 18, 28]. Rats were killed ( n = 3) 1, 2, 3 or 4 weeks later. The spatio–temporal sequence of axonal regeneration into the grafts was assessed histologically, by immunofluorescence using antibodies against GAP–43; S–100; RT97; laminin and macrophages (EDI), and by transmission electron microscopy. Outgrowth of almost all axons from the mitomycin C–treated proximal stumps was delayed for up to 3 weeks, after which time vigorous regeneration occurred into the persisting tubes of sarcolemmal basal lamina. All axons regenerating within the grafts (irrespective of mitomycin C–treatment) were accompanied by co–migrating Schwann cells. The results suggest that Schwann cells play an important role in axonal regeneration across FT muscle autografts and that sarcolemmal basal laminae alone are insufficient to support axonal regeneration.     

REGENERATION IN CELLULAR AND ACELLULAR AUTOGRAFTS IN THE PERIPHERAL NERVOUS SYSTEM

The regeneration that occurs in cellular autografts of sciatic nerve has been compared with that seen in acellular models prepared either by cycles of alternating freezing and thawing, or by detergent-extraction. The responses to either fresh or pre-degenerate grafts (cellular and acellular) have been examined electron microscopically. It was found that whereas neurites grew into a fresh autograft and rapidly re-established functional relationships with vital Schwann cells lying in bands of Büngner within the graft, penetration of acellular grafts was less efficient. Many basal lamina tubes in the acellular grafts remained either empty or filled with debris-laden macrophages for the first 2 weeks after suture, although subsequently reinnervation did occur. The roles of Schwann cells, macrophages and basal laminae during reinnervation are discussed.     

Schwann cell migration through freeze-killed peripheral nerve grafts without accompanying axons

Summary Freeze-dried tibial nerve grafts were anastomosed to either the proximal stump or the distal stump of severed tibial nerves in adult inbred Fischer rats. In the case of grafts attached to the proximal stump the tibial nerve was ligated three times, the most distal ligature from the spinal cord being 1 cm from the site of anastomosis. In both types of experiment Schwann cells were, therefore, free to enter the initially acellular grafts without accompanying axons. The grafts were examined 17 days to 12 weeks after operation. Immunofluorescence for S-100 protein was used to evaluate the distance migrated by the Schwann cells and electron microscopy was used to examine the morphology of the cells which invaded the grafts. Schwann cell migration was similar from the proximal and distal stumps. The migrating Schwann cells formed columns which resembled bands of Bungner. They were found mainly, but not exclusively, inside the pre-existing basal lamina tubes left behind by the killed nerve fibres. Some Schwann cells secreted a thin, patchy basal lamina even though they lacked axonal contact. Schwann cell columns became partially compartmentalized by fibroblast processes. Myelin and other debris were removed most rapidly in those parts of the grafts penetrated by large numbers of Schwann cells. The maximum distance the Schwann cells penetrated into the grafts was 8.5 mm and this was achieved by 6 to 8 weeks after operation. This is about half the maximum distance migrated by Schwann cells accompanying regenerating axons through similar grafts. The reasons why Schwann cells migrate shorter distances without axons and the significance of these results for the interpretation of axonal regeneration experiments using acellular grafts are discussed.Supported by a grant from the Medical Research Council     

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.     

Influence of insulin-like growth factor-I (IGF-I) on nerve autografts and tissue-engineered nerve grafts

To overcome the problems of limited donor nerves for nerve reconstruction, we established nerve grafts made from cultured Schwann cells and basal lamina from acellular muscle and used them to bridge a 2-cm defect of the rat sciatic nerve. Due to their basal lamina and to viable Schwann cells, these grafts allow regeneration that is comparable to autologous nerve grafts. In order to enhance regeneration, insulin-like growth factor (IGF-I) was locally applied via osmotic pumps. Autologous nerve grafts with and without IGF-I served as controls. Muscle weight ratio was significantly increased in the autograft group treated with IGF-I compared to the group with no treatment; no effect was evident in the tissue-engineered grafts. Autografts with IGF-I application revealed a significantly increased axon count and an improved g-ratio as indicator for "maturity" of axons compared to autografts without IGF-I. IGF-I application to the engineered grafts resulted in a decreased axon count compared to grafts without IGF-I. The g-ratio, however, revealed no significant difference between the groups. Local administration of IGF-I improves axonal regeneration in regular nerve grafts, but not in tissue-engineered grafts. Seemingly, in these grafts the interactive feedback mechanisms of neuron, glial cell, and extracellular matrix are not established, and IGF-I cannot exert its action as a pleiotrophic signal.     

DIABETIC NEUROPATHY: ABNORMALITIES OF SCHWANN CELL AND PERINEURIAL BASAL LAMINAE. IMPLICATIONS FOR DIABETIC VASCULOPATHY

During Wallerian degeneration, the Schwann cell basal laminal ensheathment around myelinated nerve fibres remains after the removal of myelin and axonal debris, forming a corrugated tube within which Schwann cell proliferation takes place. In nerve biopsies from patients with diabetic neuropathy, such residual basal laminal tubes tend to be circular rather than corrugated and appear to be more persistent during regeneration; this suggests increased rigidity and durability. These changes could be the result of increased cross-linkage of type IV collagen or alterations to other components of the basal lamina. A similar mechanism may be responsible for the thickening of perineurial basal laminae and the reduplication of basal laminae around endoneurial capillaries in diabetic patients; such reduplication may lead to reduced compliance of the vessel walls and impaired vascular perfusion.     

GDNF‐treated acellular nerve graft promotes motoneuron axon regeneration after implantation into cervical root avulsed spinal cord

T‐H. Chu, L. Wang, A. Guo, V. W‐K. Chan, C. W‐M. Wong and W. Wu (2012) Neuropathology and Applied Neurobiology 38, 681–695 GDNF‐treated acellular nerve graft promotes motoneuron axon regeneration after implantation into cervical root avulsed spinal cord It is well known that glial cell line‐derived neurotrophic factor (GDNF) is a potent neurotrophic factor for motoneurons. We have previously shown that it greatly enhanced motoneuron survival and axon regeneration after implantation of peripheral nerve graft following spinal root avulsion. Aims: In the current study, we explore whether injection of GDNF promotes axon regeneration in decellularized nerve induced by repeated freeze‐thaw cycles. Methods: We injected saline or GDNF into the decellularized nerve after root avulsion in adult Sprague–Dawley rats and assessed motoneuron axon regeneration and Schwann cell migration by retrograde labelling and immunohistochemistry. Results: We found that no axons were present in saline‐treated acellular nerve whereas Schwann cells migrated into GDNF‐treated acellular nerve grafts. We also found that Schwann cells migrated into the nerve grafts as early as 4 days after implantation, coinciding with the first appearance of regenerating axons in the grafts. Application of GDNF outside the graft did not induce Schwann cell infiltration nor axon regeneration into the graft. Application of pleiotrophin, a trophic factor which promotes axon regeneration but not Schwann cell migration, did not promote axon infiltration into acellular nerve graft. Conclusions: We conclude that GDNF induced Schwann cell migration and axon regeneration into the acellular nerve graft. Our findings can be of potential clinical value to develop acellular nerve grafting for use in spinal root avulsion injuries.     

Hyperbaric oxygen treatment has different effects on nerve regeneration in acellular nerve and muscle grafts

Effects of hyperbaric oxygen treatment (HBO) on nerve regeneration in acellular nerve and muscle grafts were investigated in rats. Nerve and muscle grafts were made acellular by freeze-thawing and the obtained grafts were used to bridge a 10-mm gap in the sciatic nerve on the left and right sides, respectively. Rats were treated with HBO (100% oxygen for 90 minutes at 2.5 atmospheres absolute pressure ATA) twice a day for 7 days. Axonal outgrowth, Schwann cell migration and invasion of macrophages were examined 10 days after the graft procedure by staining neurofilaments, S-100 proteins and the macrophage antibodies ED1 and ED2, respectively. Axonal outgrowth and Schwann cell migration in acellular nerve grafts were superior to that found in the acellular muscle grafts. However, there was no difference between HBO-treated and nontreated rats in acellular nerve grafts. Such a difference was found in acellular muscle grafts concerning both axonal outgrowth and Schwann cell migration from the proximal nerve end. No differences in the content of macrophages or neovascularization (alkaline phosphatase staining) in either of the grafts and treatments were seen. It is concluded that there is a differential effect of HBO-treatment in acellular nerve and muscle grafts and that HBO-treatment has no effect on the regeneration process in acellular nerve grafts, in contrast to fresh cellular nerve grafts where a beneficial effect has previously been reported.     

Axonal outgrowth in muscle grafts made acellular by chemical extraction

Purpose: To compare nerve regeneration in autologous detergent extracted and freeze-thawed muscle grafts and to electrophoretically characterize the grafts. Methods: Autologous acellular muscie grafts were created either by freeze/thawing or by detergent extraction and then used to bridge a 10 mm gap in rat sciatic nerve. The autologous grafts were compared with respect to protein content, using electrophoresis preimplantation, and axonal outgrowth, Schwann cell and macrophage content, using immunocytochemistry (neurofilaments, S-100 protein, ED 1 macrophages) at 5-20 days postimplantation. Results: The extracted muscle grafts were elastic, but the amount of several proteins was reduced and laminin was still present at a position of basal laminae of the muscle fibers. The freeze/thawed grafts were brittle and lacked elasticity, but resulted in minor changes in major proteins. The axons regenerated through both types of grafts (initial delay 6 days and rate 0.7-0.8 mm/day), which shrunk in length by 25%. There were no apparent differences with respect to Schwann cells and macrophages. Conclusions: The results suggest that detergent extracted muscle tissue, in which some basal lamina proteins remain but cells are removed, could present a new favourable option for nerve grafting.     

THE EFFECT OF INHIBITING SCHWANN CELL MITOSIS ON THE RE-INNERVATION OF ACELLULAR AUTOGRAFTS IN THE PERIPHERAL NERVOUS SYSTEM OF THE MOUSE

Reactive gliosis in the zone immediately proximal to transection of the sciatic nerve has been inhibited by intraneural injection of mitomycin C, an anti-mitotic agent known to arrest Schwann cell division after transection, crush or demyelination. Mitomycin C-pretreated proximal stumps were subsequently sutured to cellular or acellular autografts (0.5 cm long) and neurite growth into and within the grafts was examined during a 5-week post-operative period. Neurites grew into cellular autografts and became associated with the resident population of Schwann cells within the grafts, to the extent that remyelination was well established in the majority of Schwann cell basal lamina tubes by week 5 post-suture. In marked contrast, very few neurites grew into acellular grafts during this time, and where axons and Schwann cells were seen they tended to be grouped in 'minifascicles'. The results suggest that neurite outgrowth from proximal stumps is dependent upon active Schwann cell participation.     

Peripheral nerve allograft prepared by an acellular,hypotonic tissue engineering method

BACKGROUND:Tissue engineered acellular nerves are good autologous nerve substitutes. Acellular peripheral nerves prepared using a conventional chemical extraction method cause a great deal of damage to nerve structures, and the allograff affects the nerve regeneration following transplantation.OBJECTIVE:To prepare peripheral nerve grafts through an acellular tissue engineering method, and observe their histology, ultrastructure, protein components and histocompatibility.DESIGN, TIME AND SETTING:A randomized, controlled, in vivo nerve tissue engineering experiment was performed at the Department of Biochemistry and Molecular Biology, Shenyang Medical College, China, from September 2006 to June 2007.MATERIALS:Triton X-100, Pepstatin A, Aprotinin and Leupeptin were purchased from Sigma, USA; Tris (hydroxymethyl) aminomethane was purchased from Gibco, USA.METHODS:The bilateral sciatic nerves of Wistar rats were harvested, treated with 0.05 mol/L Tris-HCI buffer, followed by proteinase inhibitor and Triton X-100 to prepare acellular peripheral nerves. The nerves were implanted in the quadriceps femoris muscle of healthy Wistar rats.MAIN OUTCOME MEASURES:Tissue structure and ultrastructure of acellular peripheral nerves were observed by optical microscopy and scanning electron microscopy. Growth associated proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Nerve allograft and the surrounding muscles were observed by hematoxylin-eosin staining.RESULTS:Acellular treatment eliminated Schwann cells, epineurium or perineurium cells, myelin sheaths and axons of nerve fibers in normal peripheral nerves, while the spatial structure, comprising basement membrane tubes of Schwann cells and the extracellular matrix of perineurium and nerve fascicles was maintained. Protein bands at the region of 30 kD were no longer visible, had slightly decreased at 43 kD and remained unchanged at 65 kD. Following implantation for 7 days, epineurium cells were absorbed. However, increased fibroblasts, decreased newly-generated capillaries and maturation of granulation tissue were observed.CONCLUSION:The acellular nerve allograft prepared through the use of a hypotonic, acellular method displays good histocompatibility, eliminates immune substances and retains growth associated proteins that induce the growth of the neural axis. In addition, this method provides an ideal scaffold to construct artificial nerves.     

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.     

Coincidence of Schwann cell-derived basal lamina development and loss of regenerative specificity of spinal motoneurons

The Schwann cell-derived basal lamina forms a tube around single peripheral axons or small groups of axons that is continuous from the spinal cord to the target. In bullfrog tadpoles (Rana catesbeiana), motor axons transected at early developmental stages regenerate to the appropriate hindlimb region. In the present paper, we found that at these stages Schwann tubes are absent by morphological criteria, and individual axons are separated only by occasinal extensions of support cells. At stages when axons no longer regenerate to the correct hindlimb region, every axon is encased in a basal lamina tube. Schwann tubes persist in the distal stump after nerve transection, and regenerating axons grow within these tubes. These findings are consistent with previous results showing that the errors regenerating axons make in older animals are not random, but depend upon the course of the denervated Schwann tubes to which they ahve acces. In order to determine whether formation of the Schwann tube itself or interaction of its molecular constituents with growing axons was associated with loss of regenerative specificity, the expression during development of two major constituents of the basal lamina, laminin and heparan sulfate proteoglycan, was investigated. Immunoreactivity to both constituents was present both before and after the transition from specific to nonspecific regeneration, indicating that their expression per se was not sufficient to limit regnerative specificity. These data support the hypothesis that the physical constraint imposed by the Schwann cell-derived basal lamina prevents regenerative specificity. © 1993 Wiley-Liss, Inc.     

Cryopreservation of peripheral nerve grafts

The utilization of viable biological nerve graft substitutes and nerve allografts raises the problem of nerve storage. To clarify this, rat sciatic nerve segments were harvested and stored in Dulbecco's modified eagle medium. The segments were divided into three groups. In the first group, no cryoprotectant was added, whereas the second had 10% dimethyl sulfoxide (DMSO) added as cryoprotectant. These two groups of nerve segments were subjected to controlled freezing. In a third group, segments were frozen uncontrolled in liquid nitrogen (-196 degrees C). All nerves were replanted orthotopically. Fresh conventional autografts (fourth group) served as control group. Histologically, freezing did not affect the structural elements such as basal lamina tubes and perineurial tissue. Morphometrically, all cryopreserved grafts had significantly reduced axon counts and less myelinization than did controls. Cryoprotected nerves (group 2) showed no different morphometric parameters compared with the group without DMSO (group 1). Controlled freezing was superior to uncontrolled freezing (group 3). Impaired regeneration was attributed mainly to delayed Wallerian degeneration and slower revascularization. Moreover, decreased survival of resident Schwann cells in the graft may impair regeneration due to the lack of neurotrophic, neurotropic, and attachment factors in early regeneration. Grafts subjected to controlled freezing support axonal regeneration to a certain extent, but further studies are required to assess various cooling patterns, cryoprotectants, and graft revascularization.     

A systematic evaluation of Schwann cell injection into acellular cold-preserved nerve grafts

Peripheral nerve regeneration after injury depends on environmental cues and trophic support. Schwann cells (SCs) secrete trophic factors that promote neuronal survival and help guide axons during regeneration. The addition of SCs to acellular nerve grafts is a promising strategy for enhancing peripheral nerve regeneration; however, inconsistencies in seeding parameters have led to varying results. The current work sought to establish a systematic approach to seeding SCs in cold-preserved acellular nerve grafts. Studies were undertaken to (1) determine the needle gauge for optimal cell survival and minimal epineurial disruption during injection, (2) track the seeded SCs using a commercially available dye, and (3) evaluate the seeding efficiency of SCs in nerve grafts. It was determined that seeding with a 27-gauge needle resulted in the highest viability of SCs with the least damage to the epineurium. In addition, Qtracker(?) dye, a commercially available quantum dot nanocrystal, was used to label SCs prior to transplantation, which allowed visualization of the seeded SCs in nerve grafts. Finally, stereological methods were used to evaluate the seeding efficiency of SCs in nerve grafts immediately after injection and following a 1- or 3-day in vitro incubation in SC growth media. Using a systematic approach, the best needle gauge and a suitable dye for SC visualization in acellular nerve grafts were identified. Seeding efficiency in these grafts was also determined. The findings will lead to improvements ability to assess injection of cells (including SCs) for use with acellular nerve grafts to promote nerve regeneration.     

Mechanisms of Disease: what factors limit the success of peripheral nerve regeneration in humans?

Functional recovery after repair of peripheral nerve injury in humans is often suboptimal. Over the past quarter of a century, there have been significant advances in human nerve repair, but most of the developments have been in the optimization of surgical techniques. Despite extensive research, there are no current therapies directed at the molecular mechanisms of nerve regeneration. Multiple interventions have been shown to improve nerve regeneration in small animal models, but have not yet translated into clinical therapies for human nerve injuries. In many rodent models, regeneration occurs over relatively short distances, so the duration of denervation is short. By contrast, in humans, nerves often have to regrow over long distances, and the distal portion of the nerve progressively loses its ability to support regeneration during this process. This can be largely attributed to atrophy of Schwann cells and loss of a Schwann cell basal lamina tube, which results in an extracellular environment that is inhibitory to nerve regeneration. To develop successful molecular therapies for nerve regeneration, we need to generate animal models that can be used to address the following issues: improving the intrinsic ability of neurons to regenerate to increase the speed of axonal outgrowth; preventing loss of basal lamina and chronic denervation changes in the denervated Schwann cells; and overcoming inhibitory cues in the extracellular matrix.