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Spatial-Temporal Progress of Peripheral Nerve Regeneration Within a Silicone Chamber: Parameters for a Bioassay
Abstract
The spatial-temporal progress of peripheral nerve regeneration across a 10-mm gap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7-14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1-5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.
... The process of nerve regeneration in tubulation is well described by Williams et al. [4]. The first stage of nerve regeneration in a nerve conduit is the formation of a fibrin matrix within the chamber space between the transected nerve stumps joined to either end of the conduit. ...
... The first stage of nerve regeneration is fibrin matrix formation in the conduit space between the nerve stumps joined to the ends of the conduit [4]. The distance across which axons can regenerate is determined by the length of the fibrin matrix through which capillaries can extend. ...
... Axon extension is thus accompanied by capillary extension, and it is highly likely that improved vascularization within the chamber space is followed by a promotion of axon extension. In a tubulation model using a silicone tube, capillaries extended into the fibrin matrix only from the nerve stumps joined to either end of the tube [4]. Previous researchers have attempted to promote capillary extension in tubulation in several ways: by transplantation of a thin vascular pedicle into the conduit lumen [6,7,[15][16][17][18][19]; by the use of capillary permeable tubes [8,20,21]; by the creation of prefabricated vascularized tubes [22,23]; and by intrachamber administration of chemical factors promoting capillary proliferation [24,25]. ...
There are many commercially available artificial nerve conduits, used mostly to repair short gaps in sensory nerves. The stages of nerve regeneration in a nerve conduit are fibrin matrix formation between the nerve stumps joined to the conduit, capillary extension and Schwann cell migration from both nerve stumps, and, finally, axon extension from the proximal nerve stump. Artificial nerves connecting transected nerve stumps with a long interstump gap should be biodegradable, soft and pliable; have the ability to maintain an intrachamber fibrin matrix structure that allows capillary invasion of the tubular lumen, inhibition of scar tissue invasion and leakage of intratubular neurochemical factors from the chamber; and be able to accommodate cells that produce neurochemical factors that promote nerve regeneration. Here, we describe current progress in the development of artificial nerve conduits and the future studies needed to create nerve conduits, the nerve regeneration of which is compatible with that of an autologous nerve graft transplanted over a long nerve gap.
... The spatiotemporal sequence of cellular events that characterize silicone conduit colonization was described several years ago through conventional light and electron microscopy analyses. Conduits are first bridged by a mesh of fibrin deposits containing many erythrocytes, neutrophils, platelets, and macrophages [20,21]. Perineurial cells are the first cell type from both nerve stumps colonizing the conduit. ...
... Perineurial cells are the first cell type from both nerve stumps colonizing the conduit. After initial perineurial tube formation, nerve fibroblasts, endothelial cells, and Schwann cells [21,22] migrate in the conduits, followed by axon growth and myelination [20][21][22][23]. While these studies reported only qualitative observations, Li and colleagues [24] further quantified the percentage of different cell types that colonize a silicone conduit at different time points using electron microscopy, showing a large presence of capillaries; however, only general information on the interactions between the different cell types was reported. ...
... Moreover, the gap nerve repair with a conduit needs microsurgery, while nerve bridge formation is a spontaneous event. The colonization of silicon conduits by different cell populations was described both qualitatively and quantitatively by several authors through light and electron microscopy analysis, but the relationship between Schwann cells and endothelial cells has not been described [20][21][22][23][24]. ...
The repair of severe nerve injuries requires an autograft or conduit to bridge the gap and avoid axon dispersion. Several conduits are used routinely, but their effectiveness is comparable to that of an autograft only for short gaps. Understanding nerve regeneration within short conduits could help improve their efficacy for longer gaps. Since Schwann cells are known to migrate on endothelial cells to colonize the “nerve bridge”, the new tissue spontaneously forming to connect the injured nerve stumps, here we aimed to investigate whether this migratory mechanism drives Schwann cells to also proceed within the nerve conduits used to repair large nerve gaps. Injured median nerves of adult female rats were repaired with 10 mm chitosan conduits and the regenerated nerves within conduits were analyzed at different time points using confocal imaging of sequential thick sections. Our data showed that the endothelial cells formed a dense capillary network used by Schwann cells to migrate from the two nerve stumps into the conduit. We concluded that angiogenesis played a key role in the nerve conduits, not only by supporting cell survival but also by providing a pathway for the migration of newly formed Schwann cells.
... Once axons have reached the distal stump, typically after two to four weeks [9], the process of re-innervation begins. The Schwann cells then switch phenotype and begin to myelinate the axons, typically after 6-16 weeks [22]. Hollow conduits provide an enclosed environment for this process to occur and a small amount of guidance to growing axons by keeping the nerve stumps aligned and in place resulting in sprouting axons that can better navigate towards their native endoneurial tube [23]. ...
... Air bubbles were removed from the suspension by centrifuging at 2500 rpm for 30 s. After cooling the suspension at 4 • C overnight, films of micron-scale thickness were cast by reverse pipetting 200 µL of slurry per well into a CytoOne TCtreated 48-well plate (STARLAB, Milton Keynes, UK) and drying for 48 h in a laminar flow cabinet [21,22]. ...
... Like for non-crosslinked films, these stronger chemical interactions also explain why dissociation was proportionally increased when higher dendrimer quantities were initially integrated into the collagen films. These studies show that while EDC/NHS crosslinked collagen has been shown to have superior mechanical stability relative to non-crosslinked collagen, albeit at the expense of reduced bioactivity [21,22,33,34], when bioactivity is restored (i.e., by attaching IKVAVcapped dendrimers), these biochemical motifs only remain unequivocally integrated within the collagen films when they are permanently attached. The different dendrimer-collagen interactions are illustrated in Figure 11. Figure 11. ...
Collagen-based scaffolds offer the potential to bridge gaps in damaged nerves via infiltration of cells along oriented pore structures which can guide sprouting axons and direct Schwann cell migration. Neural cells have been shown to be highly mechanosensitive and hence substrate stiffness is an important factor in the repair process. However, the competition between neural cell attachment and fibrotic scar tissue formation also needs to be addressed. The goal of this thesis was to investigate the effects of mechanically and biochemically tailored collagen scaffolds on the attachment, migration and proliferation of neural- and fibroblast cells. By assessing cell behaviour in contact with scaffolds and thin films (as a surrogate for scaffold struts) it was possible to deconvolute the effects of two- and three dimensional architectures. Chemical cross linking and incorporation of elastin in collagen films were investigated by atomic force- and optical microscopy, and tensile mechanical testing. 10% elastin incorporation was shown to increase the surface roughness of the films three-fold and decrease the stiffness from 10s of MPa to 100s of kPa. Carbodiimide cross linking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of an N-hydroxy-succinimide (NHS) catalyst resulted in dose-dependent stiffness. Cross linking was varied from 0% to 100% (defined as molar ratio EDC:NHS:COOH[from collagen] of 5:2:1), resulting in an increase in modulus from 7 MPa to 65 MPa for collagen films and from 340 kPa to 740 kPa for collagen-10% elastin films. It was concluded that control of composition and degree of cross linking can be used to tailor film stiffness and surface roughness. Three dimensional collagen scaffolds with aligned pore structures were produced by a unidirectional freezing process. The pore morphology, including pore size and percolation diameter was characterised by micro computed tomography and scanning electron microscopy, with median pore sizes of approximately 50 μm. Carbodiimide cross linking and elastin incorporation were investigated to create structures with physiologically relevant stiffness. The biological response to 2D films was assessed using rat Schwann cells (RSC96), model neuronal (PC-12) cells and human dermal fibroblasts (HDF). Fluorescence imaging and lactate dehydrogenase assays revealed that elastin incorporation and lower levels of cross linking led to an increase in the attachment of RSC96 and PC-12 cells. Elastin incorporation alone led to a doubling of PC-12 attachment. It was concluded that this was due to changes in film mechanical properties as the cells were found to bind non-specifically to the films. HDFs showed minimal sensitivity to elastin incorporation, but binding was reduced by up to 45% at higher cross linking densities due to specific integrin binding motifs being consumed by the cross linking process. Schwann cell infiltration and proliferation in 3D scaffolds was investigated with fluorescence microscopy and a PrestoBlue assay. The cells appeared to be insensitive to the degree of cross linking and elastin incorporation and full penetration of the scaffolds occurred by Day 5 with a relatively high degree of proliferation. The results suggested that the scaffolds provided sufficient guidance to Schwann cells to allow their migration and ultimately help to support tissue regeneration.
... Like the majority of the peripheral nerves in mammals, the rodent sciatic nerve is under tension and therefore a nerve gap will be generated upon transection injury due to the retraction of the nerve ends (Chen et al., 2019;Dun & Parkinson, 2018a). In our experience, we normally create a nerve gap varying between 1 and 2.5 mm (Figure 1a-c) (Chen et al., 2019;Dun et al., 2019;Dun & Parkinson, 2015, 2018a, 2018b and these gaps may first be bridged by a fibrin deposit (Schroder, May, & Weis, 1993;Williams, Longo, Powell, Lundborg, & Varon, 1983). ...
... We, and others, typically use mouse and rat sciatic nerve transection as a research model to create nerve gaps and study the molecular and cellular events regulating axon regeneration across such peripheral nerve gaps (Chen et al., 2019;Dun et al., 2019;Dun & Parkinson, 2015, 2018aParrinello et al., 2010;Savastano et al., 2014). Following a sciatic nerve transection, numerous erythrocytes, granulocytes, thrombocytes, and macrophages are localized into the nerve gap (Schroder et al., 1993;Weis et al., 1994;Williams et al., 1983). These cells are the earliest cell types arriving into the nerve bridge, and they are released from the transected endoneurial and epineurial blood vessels of the nerve rather than migration into the nerve gap from either nerve stump. ...
... Thus, macrophages, neutrophils, perineurial cells, nerve fibroblasts, endothelial cells, and Schwann cells are the major cell types forming the nerve bridge and they regulate each other's recruitment, migration, and organization in the nerve bridge during regeneration (Cattin et al., 2015;Dun et al., 2019;Kucenas, 2015;Parrinello et al., 2010;Weis et al., 1994;Williams et al., 1983). The formation of the nerve bridge requires an initial fibrin deposit and then involves the coordinated migration of multiple cell types. ...
Schwann cells within the peripheral nervous system possess a remarkable regenerative potential. Current research shows that peripheral nerve‐associated Schwann cells possess the capacity to promote repair of multiple tissues including peripheral nerve gap bridging, skin wound healing, digit tip repair as well as tooth regeneration. One of the key features of the specialized repair Schwann cells is that they become highly motile. They not only migrate into the area of damaged tissue and become a key component of regenerating tissue but also secrete signaling molecules to attract macrophages, support neuronal survival, promote axonal regrowth, activate local mesenchymal stem cells, and interact with other cell types. Currently, the importance of migratory Schwann cells in tissue regeneration is most evident in the case of a peripheral nerve transection injury. Following nerve transection, Schwann cells from both proximal and distal nerve stumps migrate into the nerve bridge and form Schwann cell cords to guide axon regeneration. The formation of Schwann cell cords in the nerve bridge is key to successful peripheral nerve repair following transection injury. In this review, we first examine nerve bridge formation and the behavior of Schwann cell migration in the nerve bridge, and then discuss how migrating Schwann cells direct regenerating axons into the distal nerve. We also review the current understanding of signals that could activate Schwann cell migration and signals that Schwann cells utilize to direct axon regeneration. Understanding the molecular mechanism of Schwann cell migration could potentially offer new therapeutic strategies for peripheral nerve repair.
... Unmodified and modified NiTi were thus coated on poly--L-lysine-coated coverslips to test their efficacy in supporting neurite extension and survival of cultured rat B35 neuroblastoma cells, an established model of neuronal morphology and differentiation [50,51]. Upon differentiation in culture, B35 cells extend long neurites, a process that is essential to neurogenesis [50] and neuroregeneration [52]. Neurite extension depends on specific interactions between cellular ligands and the extracellular matrix, which must provide the cell with an appropriate terrain for adhesion, elongation, and wayfinding [52] to support growth and differentiation [53,54]. ...
... Upon differentiation in culture, B35 cells extend long neurites, a process that is essential to neurogenesis [50] and neuroregeneration [52]. Neurite extension depends on specific interactions between cellular ligands and the extracellular matrix, which must provide the cell with an appropriate terrain for adhesion, elongation, and wayfinding [52] to support growth and differentiation [53,54]. Normal survival and neurite extension by B35 cells grown on NiTi coatings suggest their high biocompatibility, and their potential efficacy in supporting neural regeneration on implants. ...
The challenges facing metallic implants for reconstructive surgery include the leaching of toxic metal ions, a mismatch in elastic modulus between the implant and the treated tissue, and the risk of infection. These problems can be addressed by passivating the metal surface with an organic substrate and incorporating antibiotic molecules. Nitinol (NiTi), a nickel-titanium alloy, is used in devices for biomedical applications due to its shape memory and superelasticity. However, unmodified NiTi carries a risk of localized nickel toxicity and inadequately supports angiogenesis or neuroregeneration due to limited cell adhesion, poor biomineralization, and little antibacterial activity. To address these challenges, NiTi nanoparticles were modified using self-assembled phosphonic acid monolayers and functionalized with the antibiotics ceftriaxone and vancomycin via the formation of an amide. Surface modifications were monitored to confirm that phosphonic acid modifications were present on NiTi nanoparticles and 100% of the samples formed ordered films. Modifications were stable for more than a year. Elemental composition showed the presence of nickel, titanium, and phosphorus (1.9% for each sample) after surface modifications. Dynamic light scattering analysis suggested some agglomeration in solution. However, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy confirmed a particle size distribution of <100 nm, the even distribution of nanoparticles on coverslips, and elemental composition before and after cell culture. B35 neuroblastoma cells exhibited no inhibition of survival and extended neurites of approximately 100 μm in total length when cultured on coverslips coated with only poly-l-lysine or with phosphonic acid-modified NiTi, indicating high biocompatibility. The ability to support neural cell growth and differentiation makes modified NiTi nanoparticles a promising coating for surfaces in metallic bone and nerve implants. NiTi nanoparticles functionalized with ceftriaxone inhibited Escherichia coli and Serratia marcescens (SM6) at doses of 375 and 750 μg whereas the growth of Bacillus subtilis was inhibited by a dose of only 37.5 μg. NiTi-vancomycin was effective against B. subtilis at all doses even after mammalian cell culture. These are common bacteria associated with infected implants, further supporting the potential use of functionalized NiTi in coating reconstructive implants.
... Studies on nerve conduits began in 1880 with attempts to create nerve conduits using arteries, veins, muscles, cartilage, organic and non-organic materials (gelatin, metal, plastic) [6,[16][17][18]. Williams et al. [18] presented a study on a silicon nerve conduit for a 1-cm sciatic nerve defect that was important in clarifying the mechanism of nerve regeneration in the nerve conduits. ...
... Studies on nerve conduits began in 1880 with attempts to create nerve conduits using arteries, veins, muscles, cartilage, organic and non-organic materials (gelatin, metal, plastic) [6,[16][17][18]. Williams et al. [18] presented a study on a silicon nerve conduit for a 1-cm sciatic nerve defect that was important in clarifying the mechanism of nerve regeneration in the nerve conduits. This study shed light on other nerve conduit studies that had been carried out. ...
Nerve conduits could be used to provide a bridge between both nerve endings. In this study, the tuba uterina of female rats were prepared in a vascularized pedicled flap model and it used as a nerve conduit. The aim was to investigate the effectiveness of a vascularized pedicle nerve conduit and its ciliated epithelium in a sciatic nerve defect. The study was conducted between May and August 2018, and used a total of 60, 14–16-week-old female Wistar albino rats. Six groups were created; Cut and Unrepaired Group, Nerve Graft Group, Flap-Forward Group (Tuba uterina tubular flap, forward direction), Flap-Reversed Group (Tuba uterina tubular flap, reverse direction), Graft-Forward Group (Tuba uterina tubular graft, forward direction) and Graft-Reverse Group (Tuba uterina tubuler graft, reverse direction). Nerve regeneration was evaluated 3 months (90 days) after the surgery by the following methods: (1) Sciatic Functional Index (SFI) measurement, (2) Electromyographic (EMG) assessment, (3) Microscopic assessment with the light microscope and (4) Microscopic assessment with the electron microscope. According to the SFI, EMG and microscopic assessments with the light and electron microscope, it was observed that the transfer of tuba uterina tubular conduit as a graft was statistically better in its effect on nerve regeneration than flap transfer, but also indicated that the direction of the ciliated structures had no significant effect. We believe that as this model is improved with future studies, it will shed light on new models, ideas and innovations about nerve conduits.
... Our data show that, while the expression of ErbB2 and ErbB3 is up-regulated in the autograft 7, 14, 28 days after injury, as occurs in the distal portion of the nerve after a crush injury or after an end-to-end repair [17], in the chitosan conduit ErbB2 and ErbB3 expression starts to be detectable 14 days after nerve repair and becomes high only 2 weeks later. The absence of ErbB expression one week after injury is consistent with the fact that the conduit needs time to be colonized by repair Schwann cells [28], which in the autograft play a key role either in the Wallerian degeneration, either in nerve regeneration, while in the conduit are mainly involved in nerve regeneration. Conversely, NRG1 expression is strongly detectable in both autograft and chitosan 7 days after nerve injury and repair. ...
... We further confirmed this by analysis, at mRNA level, the expression of Schwann cell markers, together with a marker of nerve fibroblasts, which are supposed to colonize tubular nerve conduits earlier, as in the first stages conduits are filled with extracellular matrix and fibrin cables [28]. The high expression of NRG1 and of a fibroblast marker together with the low expression of Schwann cell markers led us to speculate that nerve fibroblasts might be the main source of soluble NRG1 detected within the conduit. ...
Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration (Schwann cells, fibroblasts, endothelial cells, macrophages) while in the autograft many cells are resident and just need to be activated. Since it is known that soluble Neuregulin1 (sNRG1) is released after injury and plays an important role activating Schwann cell dedifferentiation, its expression level was investigated in early regeneration steps (7, 14, 28 days) inside a 10 mm chitosan conduit used to repair median nerve gaps in Wistar rats. In vivo data show that sNRG1, mainly the isoform α, is highly expressed in the conduit, together with a fibroblast marker, while Schwann cell markers, including NRG1 receptors, were not. Primary culture analysis shows that nerve fibroblasts, unlike Schwann cells, express high NRG1α levels, while both express NRG1β. These data suggest that sNRG1 might be mainly expressed by fibroblasts colonizing nerve conduit before Schwann cells. Immunohistochemistry analysis confirmed NRG1 and fibroblast marker co-localization. These results suggest that fibroblasts, releasing sNRG1, might promote Schwann cell dedifferentiation to a “repair” phenotype, contributing to peripheral nerve regeneration.
... More recent studies considering this same topic have revealed the role of the immune system [10,13,14,[18][19][20][21]. Immune cells, such as macrophages, are among the first cells to be recruited to an injury site and repopulate acellular scaffolds, where they play a critical role in facilitating regeneration [7,[22][23][24]. Infiltrating macrophages will react to the hypoxic environment within acellular scaffolds by secreting pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), to promote angiogenesis. ...
The use of acellular nerve allografts (ANAs) to reconstruct long nerve gaps (>3 cm) is associated with limited axon regeneration. To understand why ANA length might limit regeneration, we focused on identifying differences in the regenerative and vascular microenvironment that develop within ANAs based on their length. A rat sciatic nerve gap model was repaired with either short (2 cm) or long (4 cm) ANAs, and histomorphometry was used to measure myelinated axon regeneration and blood vessel morphology at various timepoints (2-, 4- and 8-weeks). Both groups demonstrated robust axonal regeneration within the proximal graft region, which continued across the mid-distal graft of short ANAs as time progressed. By 8 weeks, long ANAs had limited regeneration across the ANA and into the distal nerve (98 vs. 7583 axons in short ANAs). Interestingly, blood vessels within the mid-distal graft of long ANAs underwent morphological changes characteristic of an inflammatory pathology by 8 weeks post surgery. Gene expression analysis revealed an increased expression of pro-inflammatory cytokines within the mid-distal graft region of long vs. short ANAs, which coincided with pathological changes in blood vessels. Our data show evidence of limited axonal regeneration and the development of a pro-inflammatory environment within long ANAs.
... As SCs enter the defect, they form cords between the stumps pulling axons in with them (Cattin et al., 2015). By weeks 3-4 in rat models, a 10 mm defect can be bridged completely with myelination actively proceeding (Williams et al., 1983). Signalling, cellular infiltration and neovascularization depends on both the proximal and distal stumps. ...
From the first surgical repair of a nerve in the 6th century, progress in the field of peripheral nerve surgery has marched on; at first slowly but today at great pace. Whether performing primary neurorrhaphy or managing multiple large nerve defects, the modern nerve surgeon has an extensive range of tools, techniques and choices available to them. Continuous innovation in surgical equipment and technique has enabled the maturation of autografting as a gold standard for reconstruction and welcomed the era of nerve transfer techniques all while bioengineers have continued to add to our armamentarium with implantable devices, such as conduits and acellular allografts. We provide the reader a concise and up-to-date summary of the techniques available to them, and the evidence base for their use when managing nerve transection including current use and applicability of nerve transfer procedures.
... Fibrin is particularly suitable for peripheral nerve tissue engineering, since the formation of a fibrin cable between the lesioned nerve stumps during the initial stages of nerve repair provides an important provisional matrix for SC proliferation and migration, leading to the formation of bands of Büngner that bridge the defect. 45,46 In fact, early in vivo studies suggested that SC proliferation and migration was inhibited due to the lack of fibrin cable formation in larger defects, thereby hindering nerve regeneration. 47,48 Although several approaches attempted to create aligned SC constructs to mimic bands of Büngner structures, mainly based on seeding of cells on structured surfaces, [21][22][23][24][25] we, to the best of our knowledge, for the first time report the successful establishment of 3D tissue-engineered bands of Büngner-like tissue structures by incorporating active mechanical stimulation in a 3D SC culture model. ...
Treatment of peripheral nerve lesions remains a major challenge due to poor functional recovery; hence, ongoing research efforts strive to enhance peripheral nerve repair. In this study, we aimed to establish three-dimensional tissue-engineered bands of Büngner constructs by subjecting Schwann cells (SCs) embedded in fibrin hydrogels to mechanical stimulation. We show for the first time that the application of strain induces (i) longitudinal alignment of SCs resembling bands of Büngner, and (ii) the expression of a pronounced repair SC phenotype as evidenced by upregulation of BDNF, NGF, and p75NTR. Furthermore, we show that mechanically aligned SCs provide physical guidance for migrating axons over several millimeters in vitro in a co-culture model with rat dorsal root ganglion explants. Consequently, these constructs hold great therapeutic potential for transplantation into patients and might also provide a physiologically relevant in vitro peripheral nerve model for drug screening or investigation of pathologic or regenerative processes.
... This conduit was previously tested in vivo to bridge a limiting gap lesion (15 mm) in the rat sciatic nerve with very promising results including good morphology of regenerated nerve and muscle reinnervation. However, the study reported a better functional recovery outcome for the animal treated with autograft, mainly because our conduit design is based on a hollow tube surrounded by a highly porous chitosan matrix, unlike the autograft, whose best regenerative performances can be attributed to the presence of native Schwann cells and a connective tissue network that promptly supports nerve regeneration [55]. Moreover, the reported pore morphology of Chi@PCL NCs displayed pores with high dimensions, which may not have sufficiently hindered the infiltration of fibrotic tissue and cells from the external to the internal lumen of the conduit, as shown by the quantity of connective tissue and cellular infiltrates found within chitosan matrix [21]. ...
Nerve conduits may represent a valuable alternative to autograft for the regeneration of long-gap damages. However, no NCs have currently reached market approval for the regeneration of limiting gap lesions, which still represents the very bottleneck of this technology. In recent years, a strong effort has been made to envision an engineered graft to tackle this issue. In our recent work, we presented a novel design of porous/3D-printed chitosan/poly-ε-caprolactone conduits, coupling freeze drying and additive manufacturing technologies to yield conduits with good structural properties. In this work, we studied genipin crosslinking as strategy to improve the physiochemical properties of our conduit. Genipin is a natural molecule with very low toxicity that has been used to crosslink chitosan porous matrix by binding the primary amino group of chitosan chains. Our characterization evidenced a stabilizing effect of genipin crosslinking towards the chitosan matrix, with reported modified porosity and ameliorated mechanical properties. Given the reported results, this method has the potential to improve the performance of our conduits for the regeneration of long-gap nerve injuries.
... Pure Zn filaments (of a size similar to our already tested Mg filaments) remained intact after implantation in animal arterial tissues for at least 3-4 months and retained 60% of their structure after a year [27,28]. The time that a scaffold must remain intact for nerve repair is weeks rather than months, as non-neurons form a tissue strand across a 10 mm gap inside a silicone conduit by three weeks and axons cross by four weeks [29]. The retention of the metal inside the naturally restricted space of a peripheral nerve for a year would limit the number of axons regenerating and cause tissue friction and damage. ...
Peripheral nerve damage that results in lost segments requires surgery, but currently available hollow scaffolds have limitations that could be overcome by adding internal guidance support. A novel solution is to use filaments of absorbable metals to supply physical support and guidance for nerve regeneration that then safely disappear from the body. Previously, we showed that thin filaments of magnesium metal (Mg) would support nerve regeneration. Here, we tested another absorbable metal, zinc (Zn), using a proprietary zinc alloy with 2% iron (Zn-2%Fe) that was designed to overcome the limitations of both Mg and pure Zn metal. Non-critical-sized gaps in adult rat sciatic nerves were repaired with silicone conduits plus single filaments of Zn-2%Fe, Mg, or no metal, with autografts as controls. After seventeen weeks, all groups showed equal recovery of function and axonal density at the distal end of the conduit. The Zn alloy group showed some improvements in early rat health and recovery of function. The alloy had a greater local accumulation of degradation products and inflammatory cells than Mg; however, both metals had an equally thin capsule (no difference in tissue irritation) and no toxicity or inflammation in neighboring nerve tissues. Therefore, Zn-2%Fe, like Mg, is biocompatible and has great potential for use in nervous tissue regeneration and repair.
... A porous structure of suitable pore size and connectivity was shown to be a determining factor for completion of the biological functions of NGCs, promoting the early adhesion, spreading, proliferation, and differentiation of Schwann cells to form cord-like structures (Bungner bands), promote vascularization, and reduce the formation of fibrous scars [11]. The concept of a nonpermeable silicone "nerve regeneration chamber" presented by Lundborg et al. [12] in the 1990s must be innovated, although its closed space can enrich bioactive factors (such as PDGF, FGF, and TGF-β secreted by Schwann cells or macrophages) and effectively prevent the invasion of fibrous scars. In vivo studies [13,14] have shown that the peripheral nerve repair effects of hollow impermeable conduits are outmatched by those of porous, permeable conduits. ...
Porous structure is an important three-dimensional morphological feature of the peripheral nerve guidance conduit (NGC), which permits the infiltration of cells, nutrients, and molecular signals and the discharge of metabolic waste. Porous structures with precisely customized pore sizes, porosities, and connectivities are being used to construct fully permeable, semi-permeable, and asymmetric peripheral NGCs for the replacement of traditional nerve autografts in the treatment of long-segment peripheral nerve injury. In this review, the features of porous structures and the classification of NGCs based on these characteristics are discussed. Common methods for constructing 3D porous NGCs in current research are described, as well as the pore characteristics and the parameters used to tune the pores. The effects of the porous structure on the physical properties of NGCs, including biodegradation, mechanical performance, and permeability, were analyzed. Pore structure affects the biological behavior of Schwann cells, macrophages, fibroblasts, and vascular endothelial cells during peripheral nerve regeneration. The construction of ideal porous structures is a significant advancement in the regeneration of peripheral nerve tissue engineering materials. The purpose of this review is to generalize, summarize, and analyze methods for the preparation of porous NGCs and their biological functions in promoting peripheral nerve regeneration to guide the development of medical nerve repair materials.
... The typical regeneration steps reported in silicone NGC-based studies were perineurial cell formation followed by fibroblast nerve generation, endothelial and SCs cell migration, axonal growth, and myelination. 292 Newly formed blood vessels can contribute towards cell migrations. In a recent study, the progression of SCs was monitored using immunofluorescence analysis on a weekly basis to understand how the formation of new blood vessels was relevant in directing nerve growth. ...
At present, peripheral nerve injuries (PNIs) are one of the leading causes of substantial impairment around the globe. Complete recovery of nerve function after an injury is challenging. Currently, autologous nerve grafts are being used as a treatment; however, this has several downsides, for example, donor site morbidity, shortage of donor sites, loss of sensation, inflammation, and neuroma development. The most promising alternative is the development of a nerve guide conduit (NGC) to direct the restoration and renewal of neuronal axons from the proximal to the distal end to facilitate nerve regeneration and maximize sensory and functional recovery. Alternatively, the response of nerve cells to electrical stimulation (ES) has a substantial regenerative effect. The incorporation of electrically conductive biomaterials in the fabrication of smart NGCs facilitates the function of ES throughout the active proliferation state. This article overviews the potency of the various categories of electroactive smart biomaterials, including conductive and piezoelectric nanomaterials, piezoelectric polymers, and organic conductive polymers that researchers have employed latterly to fabricate smart NGCs and their potentiality in future clinical application. It also summarizes a comprehensive analysis of the recent research and advancements in the application of ES in the field of NGC.
... Nerve regeneration using tube-like materials is known as tubulation [6]. According to a study on the process of nerve regeneration via tubulation, a fibrin matrix containing various neurochemical factors is formed within the tube during the first step [7]. Next, the capillary extension of both the nerve stumps into the fibrin matrix was performed. ...
A conduit for peripheral nerve regeneration (NERBRIDGE®; Toyobo Co., Japan) in cases of disconnection or deficiency of the peripheral nerve was approved in Japan in 2013. NERBRIDGE® is a polyglycolic acid-collagen (PGA) product derived from porcine skin. The use of NERBRIDGE® has been reported mainly in orthopaedic surgery. Reports on its use for sensory nerve injuries in the oral region are scarce. This case report describes a case of significant sensory recovery obtained by nerve repair using NERBRIDGE® in a patient with schwannoma-induced inferior alveolar nerve resection. At the 1-year postoperative evaluation, sensory nerve recovery using the two-point discrimination test was confirmed.
... We chose 6 weeks to compare with our previous studies that showed substantial gaps in similar Mg filaments at 6 weeks [10,11]. By this time, we expected axons to have regenerated across the gap, based on both previous work and our own findings [10,11,51]. We showed that neither polishing nor PEO anodization slowed degradation sufficiently to preserve the full filament length; all three types of filaments had substantially degraded. ...
In vivo use of biodegradable magnesium (Mg) metal can be plagued by too rapid a degradation rate that removes metal support before physiological function is repaired. To advance the use of Mg biomedical implants, the degradation rate may need to be adjusted. We previously demonstrated that pure Mg filaments used in a nerve repair scaffold were compatible with regenerating peripheral nerve tissues, reduced inflammation, and improved axonal numbers across a short—but not long—gap in sciatic nerves in rats. To determine if the repair of longer gaps would be improved by a slower Mg degradation rate, we tested, in vitro and in vivo, the effects of Mg filament polishing followed by anodization using plasma electrolytic oxidation (PEO) with non-toxic electrolytes. Polishing removed oxidation products from the surface of as-received (unpolished) filaments, exposed more Mg on the surface, produced a smoother surface, slowed in vitro Mg degradation over four weeks after immersion in a physiological solution, and improved attachment of cultured epithelial cells. In vivo, treated Mg filaments were used to repair longer (15 mm) injury gaps in adult rat sciatic nerves after placement inside hollow poly (caprolactone) nerve conduits. The addition of single Mg or control titanium filaments was compared to empty conduits (negative control) and isografts (nerves from donor rats, positive control). After six weeks in vivo, live animal imaging with micro computed tomography (micro-CT) showed that Mg metal degradation rates were slowed by polishing vs. as-received Mg, but not by anodization, which introduced greater variability. After 14 weeks in vivo, functional return was seen only with isograft controls. However, within Mg filament groups, the amount of axonal growth across the injury site was improved with slower Mg degradation rates. Thus, anodization slowed degradation in vitro but not in vivo, and degradation rates do affect nerve regeneration.
... Nerve regeneration through a tube-like material is called tubulation [1,2]. Williams et al. studied the process of nerve regeneration via tubulation [3]. According to their study, a fibrin matrix containing various neurochemical factors is formed within the tube in the first step of nerve regeneration via tubulation. ...
To promote nerve regeneration within a conduit (tubulation), we have performed studies using a tube model based on four important concepts for tissue engineering: vascularity, growth factors, cells, and scaffolds. A nerve conduit containing a blood vascular pedicle (vessel-containing tube) accelerated axon regeneration and increased the axon regeneration distance; however, it did not increase the number or diameter of the axons that regenerated within the tube. A vessel-containing tube with bone-marrow-derived mesenchymal stem cell (BMSC) transplantation led to the increase in the number and diameter of regenerated axons. Intratubularly transplanted decellularized allogenic nerve basal lamellae (DABLs) worked as a frame to maintain the fibrin matrix structure containing neurochemical factors and to anchor the transplanted stem cells within the tube. For the clinical application of nerve conduits, they should exhibit capillary permeability, biodegradability, and flexibility. Nerbridge® (Toyobo Co. Ltd., Osaka, Japan) is a commercially available artificial nerve conduit. The outer cylinder is a polyglycolic acid (PGA) fiber mesh and possesses capillary permeability. We used the outer cylinder of Nerbridge as a nerve conduit. A 20-mm sciatic nerve deficit was bridged by the PGA mesh tube containing DABLs and BMSCs, and the resulting nerve regeneration was compared with that obtained through a 20-mm autologous nerve graft. A neve-regeneration rate of about 70%–80% was obtained in 20-mm-long autologous nerve autografts using the new conduits.
Graphical Abstract
... Due to those shortcomings, synthetic nerve conduits emerged as the first clinically available alternatives to autografts. Synthesized from biocompatible polymers such as polyglycolic acid, collagen, or polycaprolactone (PCL) (Meek and Coert, 2008;Reid et al., 2013), these hollow tube conduits offered a passive mechanical bridge between the distal and proximal stump whose regenerative function depended on the locally secreted growth factors from the severed nerve ends (Williams et al., 1983;Danielsen and Varon, 1995). As such, they are only effective over a short nerve gap (maximum of 3 cm) and their use has been largely limited to the reconstruction of non-critical sensory nerves (i.e., nerves that do not supply the thumb or index finger, namely the radial sensory nerve). ...
Peripheral nerve injuries remain a challenging problem in need of better treatment strategies. Despite best efforts at surgical reconstruction and postoperative rehabilitation, patients are often left with persistent, debilitating motor and sensory deficits. There are currently no therapeutic strategies proven to enhance the regenerative process in humans. A clinical need exists for the development of technologies to promote nerve regeneration and improve functional outcomes. Recent advances in the fields of tissue engineering and nanotechnology have enabled biomaterial scaffolds to modulate the host response to tissue repair through tailored mechanical, chemical, and conductive cues. New bioengineered approaches have enabled targeted, sustained delivery of protein therapeutics with the capacity to unlock the clinical potential of a myriad of neurotrophic growth factors that have demonstrated promise in enhancing regenerative outcomes. As such, further exploration of combinatory strategies leveraging these technological advances may offer a pathway towards clinically translatable solutions to advance the care of patients with peripheral nerve injuries. This review first presents the various emerging bioengineering strategies that can be applied for the management of nerve gap injuries. We cover the rationale and limitations for their use as an alternative to autografts, focusing on the approaches to increase the number of regenerating axons crossing the repair site, and facilitating their growth towards the distal stump. We also discuss the emerging growth factor-based therapeutic strategies designed to improve functional outcomes in a multimodal fashion, by accelerating axonal growth, improving the distal regenerative environment, and preventing end-organs atrophy.
... There were limitations of the in vivo experiment reported in this chapter. Firstly, the transplantation duration was only 3 weeks, selected because it was a minimum time point sufficient for regenerating neurites to reach the distal stump in a 10-mm nerve gap (Williams et al., 1983). The approach to investigate host and transplanted neurites at the interface was a snapshot in time, so any temporal changes could not be examined. ...
Following peripheral nerve injury, the axons in the distal nerve between the injury site and the muscle degenerate. When the injured site is very proximal, functional recovery from nerve repair is a clinical challenge since neuronal regeneration rate is limited, resulting in muscle atrophy due to the delay in reinnervation, even where the ‘gold standard’ autograft is used. Much research focuses on developing biomaterial scaffolds that mimic the autograft and promote host neurite regeneration from proximal to distal stump, whereas here, we aim to improve long distance repair by populating constructs with functional neurons and glial cells. With an engineered living scaffold populated with neurons exhibiting long neurite extensions supported by glial cells, the gap between proximal stump and muscle could potentially be reconnected promptly once the challenge of integration is overcome. To test the concept, a method was developed using tethered aligned engineered neural tissue (TaeNT) formed from simultaneous self-alignment of Schwann cells and collagen fibrils in a fully-hydrated tethered gel resulting in an anisotropic tissue-like structure. The in vitro results showed neurite elongation and alignment in the co-culture of neurons and Schwann cells in TaeNT, indicating that TaeNT could be an appropriate substrate for growing long neurites with a view to generating therapeutic constructs containing long functional neurons. The implantation of TaeNT containing neurons and Schwann cells in a 10mm-gap rat sciatic nerve for 3 weeks provided information about host-transplant cell interaction including Schwann cell migration and alignment inside the conduit, and neurite elongation across the conduit interface. Furthermore, in an attempt to induce longer neurite growth, TaeNT was proposed as a substrate that could be combined with mechanical tension application using a 3D-printed mould developed to stretch the cellular gels in a controlled manner. A series of newly designed protocols for mechanical tension application to induce growth response for enhanced neural regeneration was developed and discussed correspondingly. In summary, the findings represent the development and investigation of the regenerative potential for engineered living scaffolds containing neurons and Schwann cells suitable for stretch-growth to provide an elongated functional nerve graft. With a view to translation for clinical use, investigating the source of therapeutic cells in the conduit and the functional integration of host and transplanted cells is an important step towards optimising the regenerative potential of the engineered living scaffold.
... Thus, the fluorescent fibrin appears to have condensed and/or been incorporated into a fibrin-rich tissue cable, 17 the timing of which is consistent with classic findings that peripheral nerves undergoing gap regeneration in a hollow conduit pass through a fibrinogen-rich "fluid" phase and a fibrin-rich "matrix" phase by the second week after injury. 18,19 These results suggest that exogenous fibrin made from fluorophore-conjugated fibrinogen responds to tissue remodeling, thus providing a visual tool to track endogenously formed tissue structures. ...
Significance:
Exogenous extracellular matrix (ECM) proteins, such as fibrinogen and the thrombin-polymerized scaffold fibrin, are used in surgical repair of severe nerve injuries to supplement ECM produced via the injury response. Monitoring the dynamic changes of fibrin during nerve regeneration may shed light on the frequent failure of grafts in the repair of long nerve gaps.
Aim:
We explored whether monitoring of fibrin dynamics can be carried out using nerve guidance conduits (NGCs) containing fibrin tagged with covalently bound fluorophores.
Approach:
Fibrinogen was conjugated to a near-infrared (NIR) fluorescent dye. NGCs consisting of silicone tubes filled with the fluorescent fibrin were used to repair a 5-mm gap injury in rat sciatic nerve ( n = 6 ).
Results:
Axonal regeneration in fluorescent fibrin-filled NGCs was confirmed at 14 days after implantation. Intraoperative fluorescence imaging after implantation showed that the exogenous fibrin was embedded in the early stage regenerative tissue. The fluorescent signal temporarily highlighted a cable-like structure within the conduit and gradually degraded over two weeks.
Conclusions:
This study, for the first time, visualized in vivo intraneural fibrin degradation, potentially a useful prospective indicator of regeneration success, and showed that fluorescent ECM, in this case fibrin, can facilitate imaging of regeneration in peripheral nerve conduits without significantly affecting the regeneration process.
... Williams et al. classified nerve regeneration in the 10 mm rat sciatic nerve gap into five stages: the fluid phase, matrix phase, cellular phase, axonal phase, and myelination phase. 24 There is a tendency that when the nerve defect size gets longer, the fibrin cable formation is compromised during the matrix phase, and the regrowing axon alignment is poorly constructed. Therefore, it is necessary to organize and guide the structure inside the lumen well. ...
... Con la propuesta de reconstrucción con injerto compuesto y el uso de vendaje semioclusivo, impresiona que, o bien nunca hubo una muerte celular, o bien la hubo, pero, por algún mecanismo, estas células fueron remplazadas, y pensamos que estas repoblaron una zona con estructura tisular conservada. Algo parecido a lo que ocurre con otros procesos biológicos, como la repoblación de tejido celular neural, en la que, frente a la perdida de tejido, los injertos de nervio proveen un exoesquelto tisular que conduce el repoblamiento celular, 14 o como ocurre con un aloinjerto óseo, en el que el tejido estructural óseo provee el soporte para que se produzca la colonización de células óseas y la consolidación. 15,16 Sin embargo, proponemos que, al momento de colocar el injerto compuesto, se retire parte del tejido del pulpejo, para disminuir el grosor de este y asegurar una menor área de isquemia. ...
Resumen
La reconstrucción de una amputación distal de dedo en un niño es un desafío. Los procedimientos propuestos son muchos, y los resultados no han sido buenos. La reconstrucción con reposición del segmento a modo de injerto compuesto, o con técnicas microquirúrgicas, parece ofrecer la mejor de las posibilidades, pues se conservan estructuras irremplazables, como el lecho ungueal y el hiponiquio, lo que permite que los niños mantengan un pulpejo anatómico y con función normal. Presentamos una serie de tres pacientes pediátricos tratados con una nueva técnica, que combina la reposición del segmento, como un injerto compuesto, y el uso de curación semioclusiva (composite autograft and semi-oclussive dressing, CASOD, en inglés). Hemos observado buenos resultados.
... In the initial phase of nerve regeneration through a bioinert hollow conduit like a silicone tube, fibrin matrix is formed between the nerve stumps that are joined to either end of the conduit. Next, capillaries extend in the fibrin matrix from both nerve stumps, followed by Schwann cell migration and axon extension [23]. ...
Previously, we showed silicone nerve conduits containing a vascular bundle and decellularized allogenic basal laminae (DABLs) seeded with bone marrow-derived mesenchymal stem cells (BMSCs) demonstrated successful nerve regeneration. Nerve conduits should be flexible and biodegradable for clinical use. In the current study, we used nerve conduits made of polyglycoric acid (PGA) fiber mesh, which is flexible, biodegradable and capillary-permeable. DABLs were created using chemical surfactants to remove almost all cell debris. In part 1, capillary infiltration capability of the PGA tube was examined. Capillary infiltration into regenerated neural tissue was compared between the PGA tube with blood vessels attached extratubularly (extratubularly vascularized tube) and that containing blood vessels intratubularly (intratubularly vascularized tube). No significant difference was found in capillary formation or nerve regeneration between these two tubes. In part 2, a 20 mm gap created in a rat sciatic nerve model was bridged using the extratubularly vascularized PGA tube containing the DABLs with implantation of isogenic cultured BMSCs (TubeC+ group), that containing the DABLs without implantation of the BMSCs (TubeC- group), and 20 mm-long fresh autologous nerve graft (Auto group). Nerve regeneration in these three groups was assessed electrophysiologically and histomorphometrically. At 24 weeks, there was no significant difference in any electrophysiological parameters between TubeC+ and Auto groups, although all histological parameters in Auto group were significantly greater than those in TubeC+ and TubeC- groups, and TubeC+ group demonstrated significant better nerve regeneration than TubeC- group. The transplanted DABLs showed no signs of immunological rejection and some transplanted BMSCs were differentiated into cells with Schwann cell-like phenotype, which might have promoted nerve regeneration within the conduit. This study indicated that the TubeC+ nerve conduit may become an alternative to nerve autograft.
... In the 1980s, Lundborg and his group emphasized the importance of the distal nerve stump for nerve regeneration after segmental injury, although fibrin's pivotal role was not the focus of their extensive work (Lundborg et al. 1982a, b, c, d;Lundborg 1982a, b). Williams and colleagues extensively studied the spatiotemporal process of peripheral nerve regeneration within a silicon chamber (Williams et al. 1983). After a nerve is cut, a clot mainly consisting of fibrin and fibronectin is formed to connect the two severed nerve ends (Williams and Varon 1985;Williams 1987;Le Beau et al. 1988;Liu 1992;Hoffman-Kim et al. 2010). ...
... Regeneration in tubular guides is dependent on the formation of a connective cable that bridges the gap between the nerve stumps. Inside the tube with the attached nerve stumps at the ends, an initial fibrin clot is formed, and provides a guiding surface for the ingrowth of fibroblasts and Schwann cells migrating from both proximal and distal stumps (Liu, 1992;Williams et al., 1983). The intratubular cable is then enriched with ECM components, mainly collagen fibrils longitudinally oriented, fibronectin and laminin. ...
Peripheral nerve injuries result in the loss of the motor, sensory and autonomic functions of the denervated segments of the body. Neurons can regenerate after peripheral axotomy, but inaccuracy in reinnervation causes a permanent loss of function that impairs complete recovery. Thus, understanding how regenerating axons respond to their environment and direct their growth is essential to improve the functional outcome of patients with nerve lesions. Schwann cells (SCs) play a crucial role in the regeneration process, but little is known about their contribution to specific reinnervation. Here, we review the mechanisms by which SCs can differentially influence the regeneration of motor and sensory axons. Mature SCs express modality-specific phenotypes that have been associated with the promotion of selective regeneration. These include molecular markers, such as L2/HNK-1 carbohydrate, which is differentially expressed in motor and sensory SCs, or the neurotrophic profile after denervation, which differs remarkably between SC modalities. Other important factors include several molecules implicated in axon-SC interaction. This cell-cell communication through adhesion (e.g., polysialic acid) and inhibitory molecules (e.g., MAG) contributes to guiding growing axons to their targets. As many of these factors can be modulated, further research will allow the design of new strategies to improve functional recovery after peripheral nerve injuries.
... In addition, axons are known to grow only a short distance beyond their own reparative matrix while an intact endoneurium is associated with better functional outcomes. Therefore, the formation of a properly aligned extracellular matrix scaffold is essential in enhancing the proliferation and migration of seeded cells, through which blood vessels and other cell types can immigrate and form a new nerve supportive structure [40,58]. In this present study, the CEANA was used to bridge a 1cm sciatic nerve defect. ...
Background:
To date, it has repeatedly been demonstrated that infusing bone marrow-derived stem cells (BMSCs) into acellular nerve scaffolds can promote and support axon regeneration through a peripheral nerve defect. However, harvesting BMSCs is an invasive and painful process fraught with a low cellular yield.
Methods:
In pursuit of alternative stem cell sources, we isolated stem cells from the inguinal subcutaneous adipose tissue of adult Sprague-Dawley rats (adipose-derived stem cells, ADSCs). We used a co-culture system that allows isolated adult mesenchymal stem cells (MSCs) and Schwann cells (SCs) to grow in the same culture medium but without direct cellular contact. We verified SC phenotype in vitro by cell marker analysis and used red fluorescent protein-tagged ADSCs to detect their fate after being injected into a chemically extracted acellular nerve allograft (CEANA). To compare the regenerative effects of CEANA containing either BMSCs or ADSCs with an autograft and CEANA only on the sciatic nerve defect in vivo, we performed histological and functional assessments up to 16 weeks after grafting.
Results:
In vitro, we observed reciprocal beneficial effects of ADSCs and SCs in the ADSC-SC co-culture system. Moreover, ADSCs were able to survive in CEANA for 5 days after in vitro implantation. Sixteen weeks after grafting, all results consistently showed that CEANA infused with BMSCs or ADSCs enhanced injured sciatic nerve repair compared to the acellular CEANA-only treatment. Furthermore, their beneficial effects on sciatic injury regeneration were comparable as histological and functional parameters evaluated showed no statistically significant differences. However, the autograft group was roundly superior to both the BMSC- or ADSC-loaded CEANA groups.
Conclusion:
The results of the present study show that ADSCs are a viable alternative stem cell source for treating sciatic nerve injury in lieu of BMSCs.
... Cattin et al [37] showed Schwann cells appear unable to migrate within the 3D matrix but situation improved efficiently when physical surface of blood vessels were present. Without sufficient biomolecule and cellular support, it can lead to reduce functional regeneration [46,47]. Therefore, SAT limited the degree of nerve regeneration resulted in random clusters of extracellular matrix tissue in most cases (supplement figure 1 is available online at stacks.iop.org/BMM/15/035003/mmedia). ...
Artificial nerve guidance conduits (NGCs) are being investigated as an alternative to autografts, since autografts are limited in supply. A polycaprolactone (PCL)-based spiral NGC with crosslinked laminin on aligned nanofibers was evaluated in vivo post a successful in vitro assessment. PC-12 cell assays confirmed that the aligned nanofibers functionalized with laminin were able to guide and enhance neurite outgrowth. In the rodent model, the data demonstrated that axons were able to regenerate across the critical nerve gap, when laminin was present. Without laminin, the spiral NGC with aligned nanofibers group resulted in a random cluster of extracellular matrix tissue following injuries. The reversed autograft group performed best, showing the most substantial improvement based on nerve histological assessment and gastrocnemius muscle measurement. Nevertheless, the recovery time was too short to obtain meaningful data for the motor functional assessments. A full motor recovery may take up to years. An interesting observation was noted in the crosslinked laminin group. Numerous new blood capillary-like structures were found around the regenerated nerve. Owing to recent studies, we hypothesized that new blood vessel formation could be one of the key factors to increase the successful rate of nerve regeneration in the current study. Overall, these findings indicated that the incorporation of laminin into polymeric nerve conduits is a promising strategy for enhancing peripheral nerve regeneration. However, the best combination of contact-guidance cues, haptotactic cues, and chemotactic cues have yet to be realized. The natural sequence of nerve regeneration should be studied more in-depth before modulating any strategies.
... The nerve bridge, a newly formed tissue connecting the proximal and distal nerve stumps, consists of macrophages, endothelial cells, fibroblasts, Schwann cells, and perineurial cells (Williams et al., 1983;Schröder et al., 1993;Weis et al., 1994;Parrinello et al., 2010;Cattin et al., 2015). As Slit3 and Robo1 are highly expressed in the nerve bridge, we next used different cell markers to identify Slit3-and Robo1-positive cell types in the nerve bridge. ...
The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors. Although we previously reported that Slit1-3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system, how this expression changes after injury has not been well studied. Herein, we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve. At 14 days after injury, quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1-3 and Robo1-2 in L4-5 spinal cord and dorsal root ganglia, as well as the sciatic nerve. Immunohistochemical analysis was performed to examine Slit1-3, Robo1-2, neurofilament heavy chain, F4/80, and vimentin in L4-5 spinal cord, L4 dorsal root ganglia, and the sciatic nerve. Co-expression of Slit1-3 and Robo1-2 in L4 dorsal root ganglia was detected by in situ hybridization. In addition, Slit1-3 and Robo1-2 protein expression in L4-5 spinal cord, L4 dorsal root ganglia, and sciatic nerve were detected by western blot assay. The results showed no significant changes of Slit1-3 or Robo1-2 mRNA expression in the spinal cord within 14 days after injury. In the dorsal root ganglion, Slit1-3 and Robo1-2 mRNA expression were initially downregulated within 4 days after injury; however, Robo1-2 mRNA expression returned to the control level, while Slit1-3 mRNA expression remained upregulated during regeneration from 4-14 days after injury. In the sciatic nerve, Slit1-3 and their receptors Robo1-2 were all expressed in the proximal nerve stump; however, Slit1, Slit2, and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury. Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury. Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge. Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury. Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration, and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury. This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No. 30/3203) on April 12, 2014.
The effective treatment of long-gap peripheral nerve injury (PNI) remains a challenge in clinical settings. The autograft, the gold standard for the long-gap PNI therapy, has several limitations, including a limited supply of donor nerve, size mismatch between the donor and recipient sites, functional loss at the donor site, neuroma formation, and the requirement for two operations. With the increasing abundance of biocompatible materials with adjustable structures and properties, tissue engineering provides a promising avenue for bridging peripheral nerve gaps and addressing the above issues of autograft. The physical cues provided by tissue engineering scaffolds, essential for regulating the neural cell fate and microenvironments, have received considerable research attention. This review elaborates on three major physical cues of tissue engineering scaffolds for peripheral nerve regeneration: topological structure, mechanical support, and electrical stimulation. These three aspects are analogs to Lego bricks, wherein different combinations result in diverse functions. Innovative and more effective bricks, along with multi-level and all-around integration, are expected to provide new advances in tissue engineering for peripheral nerve generation.
Peripheral nerve damages cause loss of sensorimotor and autonomic functions, resulting in a significant burden for the patients. Nerve injuries above a limiting gap length require surgical repair. Although autograft...
Nerve injuries may result in neurological dysfunction or even permanent disability, which poses various challenges to physicians. In the peripheral nervous system (PNS), only small nerve injuries can be regenerated spontaneously in vivo, while larger nerve injuries must be treated surgically with biomaterials or nerve grafts harvested. Attributed to the influence of inhibiting factors such as inflammation and microenvironment changes, a solution to completely repair central nervous system (CNS) injuries has not been discovered. Hence, most bioengineering strategies for PNS have been focused on the guidance of regenerative nerves, whereas the efforts for CNS have been focused on creating a suitable regenerating microenvironment in vivo. Recent advances in neurology, tissue engineering, biomaterials, gene transfection, and multifactor combinations offer optimistic prospects for the development of nerve regeneration. In this chapter, we firstly examine the current understanding of the neurophysiology and factors that are critical for nerve regeneration, and discuss their implications for promoting axon regeneration. Then, the current approaches, challenges, and future perspectives of biomaterials being explored to aid PNS and CNS regeneration are highlighted.
Appropriate animal models, mimicking conditions of both health and disease, are needed to understand not only the biology and the physiology of neurons and other cells under normal conditions but also under stress conditions, like nerve injuries and neuropathy. In such conditions, understanding how genes and different factors are activated through the well-orchestrated programs in neurons and other related cells is crucial. Knowledge about key players associated with nerve regeneration intended for axonal outgrowth, migration of Schwann cells with respect to suitable substrates, invasion of macrophages, appropriate conditioning of extracellular matrix, activation of fibroblasts, formation of endothelial cells and blood vessels, and activation of other players in healthy and diabetic conditions is relevant. Appropriate physical and chemical attractions and repulsions are needed for an optimal and directed regeneration and are investigated in various nerve injury and repair/reconstruction models using healthy and diabetic rat models with relevant blood glucose levels. Understanding dynamic processes constantly occurring in neuropathies, like diabetic neuropathy, with concomitant degeneration and regeneration, requires advanced technology and bioinformatics for an integrated view of the behavior of different cell types based on genomics, transcriptomics, proteomics, and imaging at different visualization levels. Single-cell-transcriptional profile analysis of different cells may reveal any heterogeneity among key players in peripheral nerves in health and disease.
Myelin-associated glycoprotein (MAG), released from pre-degenerated distal nerves following axotomy, blocks the regrowth of sprouts and naked axons. Ensheathed axons, however, continue to elongate and reach MAG-releasing distal nerves. To determine the regenerative mechanism of ensheathed axons without navigators of axonal growth cones by the film model method, we inserted a MAG-releasing distal nerve segment treated with liquid nitrogen (N2DS) between the two films, facing the proximal end of the common peroneal nerves in mice transected 4 days earlier for axons to become ensheathed. On the third postoperative day (Day 3), axon fascicles, subjected to silver staining, extended toward N2DS but with few branches, forming terminal swellings called Cajal’s gigantic clubs (CGCs) that are filled with axonal growth cones. Filter paper wetted with either 250 pg/ml MAG or N2DS showed the same configurations when inserted between the two films. This effect was lost following anti-MAG treatment; fascicles strayed near the parent nerve with numerous branches, formed a net of axons and tapered toward thin tips at their ends, just like controls without N2DS. Schwann cell bundles on Day 3 detected with anti-S100, formed sheaths of CGCs at their ends and connected to pioneer Schwann cells (pSCs). To analyze the physiology of Schwann cells, independent of axons, the parent nerve transected 4 days prior was crushed. On Day 2, with pSCs ahead, Schwann cell bundles extended toward N2DS. On Day 4, main bundles regressed, leaving pSCs motionless. Thus, MAG is a candidate chemoattractant for both pSCs and CGCs.
Graphical Abstract
Purpose:
While there are advantages and disadvantages to both processed nerve allografts (PNA) and conduits, a large, well-controlled prospective study is needed to compare the efficacy and to delineate how each of these repair tools can be best applied to digital nerve injuries. We hypothesized that PNA digital nerve repairs would achieve superior functional recovery for longer length gaps compared with conduit-based repairs.
Methods:
Patients (aged 18-69 years) presenting with suspected acute or subacute (less than 24 weeks old) digital nerve injuries were recruited to prticipate at 20 medical centers across the United States. After stratification to short (5-14 mm) and long (15-25 mm) gap subgroups, the patients were randomized (1:1) to repair with either a commercially available PNA or collagen conduit. Baseline and outcomes assessments were obtained either before or immediately after surgery and planned at 3-, 6-, 9-, and 12-months after surgery. All assessors and patients were blinded to the treatment arm.
Results:
In total, 220 patients were enrolled, and 183 patients completed an acceptable lasta evaluable visit (at least 6 months and not more than 15 months postrepair). At last follow-up, for the short gap repair groups, average static two-point discrimination was 7.3 ± 2.8 mm for PNA and 7.5 ± 3.1 mm for conduit repairs. For the long gap group, average static two-point discrimination was significantly lower at 6.1 ± 3.3 mm for PNA compared with 7.5 ± 2.4 mm for conduit repairs. Normal sensation (American Society for Surgery of the Hand scale) was achieved in 40% of PNA long gap repairs, which was significantly more than the 18% observed in long conduit patients. Long gap conduits had more clinical failures (lack of protective sensation) than short gap conduits.
Conclusions:
Although supporting similar levels of nerve regeneration for short gap length digital nerve repairs, PNA was clinically superior to conduits for long gap reconstructions.
Type of study/level of evidence:
Therapeutic I.
Every year, there are approximately 500 000 peripheral nerve injury (PNI) procedures due to trauma in the US alone. Autologous and acellular nerve grafts are among current clinical repair options; however, they are limited largely by the high costs associated with donor nerve tissue harvesting and implant processing, respectively. Therefore, there is a clinical need for an off-the-shelf nerve graft that can recapitulate the native microenvironment of the nerve. In our previous work, we created a hydrogel scaffold that incorporates mechanical and biological cues that mimic the peripheral nerve microenvironment using chemically modified hyaluronic acid (HA). However, with our previous work, the degradation profile and cell adhesivity was not ideal for tissue regeneration, in particular, peripheral nerve regeneration. To improve our previous hydrogel, HA was conjugated with fibrinogen using Michael-addition to assist in cell adhesion and hydrogel degradability. The addition of the fibrinogen linker was found to contribute to faster scaffold degradation via active enzymatic breakdown, compared to HA alone. Additionally, cell count and metabolic activity was significantly higher on HA conjugated fibrinogen compared previous hydrogel formulations. This manuscript discusses the various techniques deployed to characterize our new modified HA fibrinogen chemistry physically, mechanically, and biologically. This work addresses the aforementioned concerns by incorporating controllable degradability and increased cell adhesivity while maintaining incorporation of hyaluronic acid, paving the pathway for use in a variety of applications as a multi-purpose tissue engineering platform.
Engineered grafts constitute an alternative to autologous transplant for repairing severe peripheral nerve injuries. However, current clinically available solutions have substantial limitations and are not suited for the repair of long nerve defects. A novel design of nerve conduit is presented here, which consists of a chitosan porous matrix embedding a 3D‐printed poly‐ε‐caprolactone mesh. These materials are selected due to their high biocompatibility, safe degradability, and ability to support the nerve regeneration process. The proposed design allows high control over geometrical features, pores morphology, compression resistance, and bending stiffness, yielding tunable and easy‐to‐manipulate grafts. The conduits are tested in chronic animal experiments, aiming to repair a 15‐mm long gap in the sciatic nerve of rats, and the results are compared with an autograft. Electrophysiological and nociception tests performed monthly during a 4‐month follow‐up show that these conduits allow a good degree of muscle functional recovery. Histological analyses show abundant cellularization in the wall and in the lumen of the conduits and regenerated axons within all rats treated with these grafts. It is suggested that the proposed conduits have the potential to repair nerves over the limiting gap length and can be proposed as strategy to overcome the limitations of autograft.
Background:
Peripheral nerve injuries affect over 2% of trauma patients and can lead to severe functional impairment and permanent disability. Autologous nerve transplantation is still the gold standard in the reconstruction of nerve defects. For small defects, conduits can be considered for bridging. Lately, the combined use of conduits and electrical stimulation has gained attention in the treatment of peripheral nerve injury. This review aimed to present the currently available data on this topic.
Methods:
PubMed, Embase, Medline and the Cochrane Library were searched for studies on electrical stimulation through nerve conduits for nerve defects in in vivo studies.
Results:
Fifteen studies fit the inclusion criteria. All of them reported on the application of nerve conduits combined with stimulation for sciatic nerve gaps in rats. Functional, electrophysiological and histological evaluations showed improved nerve regeneration after electrical stimulation. High variation was observed in the treatment protocols.
Conclusion:
Electrically stimulated conduits could improve peripheral nerve regeneration in rat models. The combined application of nerve guidance conduits and electrical stimulation shows promising results and should be further evaluated under standardized conditions.
This chapter focuses on surgical strategies in nerve repair. Surgical treatment should be adapted to the type and proximity of the peripheral nerve lesion and the time interval since trauma. The gold standard for bridging a segmental nerve defect gap is still autologous interfascicular nerve grafting according to Millesi. However, sources of autologous nerve grafts are limited and therefore the use of nerve allografts and nerve conduits is discussed. If there is a loss of the proximal nerve stump and/or the calculated time interval for successful reinnervation seems to be too long, nerve transfers and end-to-side coaptation are possible treatment approaches. The main goal of our treatment is to provide a maximum of possible nerve fibers for reinnervation. This concept implies that peripheral nerve fiber transfer as the single source for reinnervation is not adequate. If possible, the nerve lesion must be explored and the reconstruction of the lesion should be part of the treatment. Muscle-tendon transfer after nerve reconstruction is an essential part of surgical strategy. Neurolysis is a procedure to decompress nerve fascicles after external compression and neural fibrotic changes. If this procedure is performed in a methodical way and the decompression of fascicles is the main goal, it is a useful therapeutic tool for all nerve compression syndromes. The importance of the gliding tissues around the nerve and their reconstruction in this type of pathology is comprehensively discussed. Finally, some basic strategies are provided for different types of brachial plexus injury, including functional free muscle transfer.
The immune system has garnered attention for its role in peripheral nerve regeneration, particularly as it pertains to regeneration across segmental injuries. Previous work demonstrated that eosinophils are recruited to regenerating nerve and express interleukin-4, amongst potential cytokines. These results suggest a direct role for eosinophils in promoting nerve regeneration. Therefore, we further considered eosinophils roles in nerve regeneration using a segmental nerve injury and Gata1 knockout (KO) mice, which are severely eosinophil deficient, compared to wild-type BALB/c mice (WT). Mice receiving a sciatic nerve gap injury demonstrated distinct cytokine expression and leukocytes within regenerating nerve. Compared to controls, Gata1 KO regenerated nerves contained decreased expression of type 2 cytokines, including Il-5 and Il-13, and decreased recruitment of eosinophils and macrophages. At this early time point during ongoing regeneration, the macrophages within Gata1 KO nerves also demonstrated significantly less M2 polarization compared to controls. Subsequently, motor and sensory axon regeneration across the gap injury was decreased in Gata1 KO compared to WT during ongoing nerve regeneration. Over longer observation to allow for more complete nerve regeneration, behavioral recovery measured by grid-walk assessment was not different comparing groups but modestly delayed in Gata1 KO compared to WT. The extent of final axon regeneration was not different amongst groups. Our data provide additional evidence suggesting eosinophils contribute to nerve regeneration across a nerve gap injury, but are not essential to regeneration in this context. Our evidence also suggests eosinophils may regulate cytokines that promote distinct macrophage phenotypes and axon regeneration.
The treatment of peripheral nerve defects has always been one of the most challenging clinical practices in neurosurgery. Currently, nerve autograft is the preferred treatment modality for peripheral nerve defects, while the therapy is constantly plagued by the limited donor, loss of donor function, formation of neuroma, nerve distortion or dislocation, and nerve diameter mismatch. To address these clinical issues, the emerged nerve guide conduits (NGCs) are expected to offer effective platforms to repair peripheral nerve defects, especially those with large or complex topological structures. Up to now, numerous technologies are developed for preparing diverse NGCs, such as solvent casting, gas foaming, phase separation, freeze‐drying, melt molding, electrospinning, and three‐dimensional (3D) printing. 3D printing shows great potential and advantages because it can quickly and accurately manufacture the required NGCs from various natural and synthetic materials. This review introduces the application of personalized 3D printed NGCs for the precision repair of peripheral nerve defects and predicts their future directions. The personalized nerve guide conduits are quickly and accurately manufactured from various natural and synthetic materials by a three‐dimensional printing technique, which meets personalized imaging data to repair nerve defects of complex anatomical structures with the assistance of different growth factors and cells.
Commercial nerve guidance conduits (NGCs) for repair of peripheral nerve discontinuities are of little use in gaps larger than 30 mm, and for smaller gaps they often fail to compete with the autografts that they are designed to replace. While recent research to develop new technologies for use in NGCs has produced many advanced designs with seemingly positive functional outcomes in animal models, these advances have not been translated into viable clinical products. While there have been many detailed reviews of the technologies available for creating NGCs, none of these have focussed on the requirements of the commercialisation process which are vital to ensure the translation of a technology from bench to clinic. Consideration of the factors essential for commercial viability, including regulatory clearance, reimbursement processes, manufacturability and scale up, and quality management early in the design process is vital in giving new technologies the best chance at achieving real-world impact. Here we have attempted to summarise the major components to consider during the development of emerging NGC technologies as a guide for those looking to develop new technology in this domain. We also examine a selection of the latest academic developments from the viewpoint of clinical translation, and discuss areas where we believe further work would be most likely to bring new NGC technologies to the clinic.
Statement of Significance
: NGCs for peripheral nerve repairs represent an adaptable foundation with potential to incorporate modifications to improve nerve regeneration outcomes. In this review we outline the regulatory processes that functionally distinct NGCs may need to address and explore new modifications and the complications that may need to be addressed during the translation process from bench to clinic.
Synthetic nerve guidance conduits (NGCs) offer an alternative to harvested nerve grafts for treating peripheral nerve injury (PNI). NGCs have been made from both naturally derived and synthesized materials. While naturally derived materials typically have an increased capacity for bioactivity, synthesized materials have better material control, including tunability and reproducibility. Protein engineering is an alternative strategy that can bridge the benefits of these two classes of materials by designing cell-responsive materials that are also systematically tunable and consistent. Here, we tested a recombinantly derived elastin-like protein (ELP) hydrogel as an intraluminal filler in a rat sciatic nerve injury model. We demonstrated that ELPs enhance the probability of forming a tissue bridge between the proximal and distal nerve stumps compared to an empty silicone conduit across the length of a 10 mm nerve gap. These tissue bridges have evidence of myelinated axons, and electrophysiology demonstrated that regenerated axons innervated distal muscle groups. Animals implanted with an ELP-filled conduit had statistically higher functional control at 6 weeks than those that had received an empty silicone conduit, as evaluated by the sciatic functional index. Taken together, our data support the conclusion that ELPs support peripheral nerve regeneration in acute complete transection injuries when used as an intraluminal filler. These results support the further study of protein engineered recombinant ELP hydrogels as a reproducible, off-the-shelf alternative for regeneration of peripheral nerves.
Peripheral nerve injuries commonly result in a surgically irreducible gap and represent a serious problem in surgery. Clinically, autologous nerve grafts are the most effective means of promoting axonal regeneration across the space, but normally produce donor site morbidity. Other variations of experimental models have been used with the aim of directing rapid nerve regeneration, including entubation, artery and vein grafts and Millipore. However, each of these have significant limitations which affect complete regrowth. Fibronectin (Fn), a large extracellular matrix cell adhesion glycoprotein has been made into three-dimensional mats with a predominant fibre direction and shown to successfully enhance peripheral nerve regeneration in a rat model. The aim of this study was to identify the main features of Fn-implants which are important for rapid nerve repair. This involved testing implanted versions of Fn-mat from rat and monkey models, assessment of cell-matrix interaction in vitro and modification of Fn-materials by chemical and growth factor addition. The results from this study show that new materials can be made from fibronectin which have a potential use in repair of long peripheral nerve lesions. These fibronectin-based materials may be stabilised with micromolar concentrations of copper and zinc which also support strong growth of Schwann cells within these materials in culture. Fibronectin mats may also be soaked with neurotrophins to enhance nerve cell survival and enhance nerve regeneration. The speed of Schwann cell migration and alignment (upto 50 μm away from the original fibre) was increased. Migration speed was further enhanced when the fibres were treated with micromolar concentrations of copper or made, with a substantial content of fibrinogen (optimum 50:50). Taken together, all these results suggest a first design for an ideal conduit material for peripheral nerve repair. This would be (a) dimensions (fibrous), (b) fibre orientation (c) 50:50 fibronectin: fibrinogen, (d) treated with micromolar concentrations of copper, (e) seeded with Schwann cells in culture, and (f) soaked with a 'cocktail' of neurotrophic growth factors including nerve growth factor and neurotrophin-3.
This thesis examines both the survival of grafts of myenteric plexus and intestinal smooth muscle implanted in the adult rat striatum and the interaction between the graft and the surrounding striatum. Pieces of adult rat ileal muscularis externa (myenteric plexus sandwiched between smooth muscle layers) were implanted into the corpus striatum and examined by electron microscopy. Enteric neurons and glial cells survived for at least 6 weeks and remained morphologically similar to those seen in situ. Bundles of small unmyelinated axons, interpreted as CNS axonal sprouts, were seen in the striatum surrounding the grafts. Some passed between the brain and the grafts. Grafts of freeze-killed muscularis externa produced little axonal sprouting in the striatum and were not invaded by axons. Similar live grafts were examined immunohistochemically using an antibody against tyrosine-hydroxylase (TH). Grafts were invaded by TH-containing fibres of CNS origin. Pieces of freshly dissected myenteric plexus from young donors were implanted in quinolinic acid-lesioned and unlesioned adult rat striatum. Some grafts were histochemically stained for NADPH-diaphorase, three and six weeks after implantation. NADPH-diaphorase-containing enteric neurons were identified within the grafts and extended long processes into the surrounding lesioned or unlesioned striata. The remaining grafts were examined electron microscopically three and six weeks after implantation and were found to contain healthy-looking enteric neurons and glia. Dissected enteric ganglia also appeared to stimulate a sprouting response in both the lesioned and unlesioned striatum adjacent to the grafts. Once again, grafts were invaded by oligodendrocyte-myelinated and non-myelinated CNS axons. As part of the investigation of the mechanisms involved in the sprouting response of the striatum, freshly dissected colonic smooth muscle was implanted in the unlesioned striatum and examined electron microscopically, three and six weeks after implantation. A sprouting response was once again observed in the striatum around the grafts. Putative CNS sprouts, associated with CNS glia and Schwann cells, invaded the grafts.
Peripheral nerve injury can result in debilitating outcomes including loss of function and neuropathic pain. Although nerve repair research and therapeutic development are widely studied, translation of these ideas into clinical interventions has not occurred at the same rate. At the turn of this century, approaches to peripheral nerve repair have included microsurgical techniques, hollow conduits, and autologous nerve grafts. These methods provide satisfactory results; however, they possess numerous limitations that can prevent effective surgical treatment. Commercialization of Avance, a processed nerve allograft, sought to address limitations of earlier approaches by providing an off‐the‐shelf alternative to hollow conduits while maintaining many proregenerative properties of autologous grafts. Since its launch in 2007, Avance has changed the landscape of the nerve repair market and is used to treat tens of thousands of patients. Although Avance has become an important addition to surgeon and patient clinical options, the product's journey from bench to bedside took over 20 years with many research and commercialization challenges. This article reviews the events that have brought a processed nerve allograft from the laboratory bench to the patient bedside. Additionally, this review provides a perspective on lessons and considerations that can assist in translation of future medical products.
The origin, termination, and length of axonal growth after focal central nervous system injury was examined in adult rats
by means of a new experimental model. When peripheral nerve segments were used as "bridges" between the medulla and spinal
cord, axons from neurons at both these levels grew approximately 30 millimeters. The regenerative potential of these central
neurons seems to be expressed when the central nervous system glial environment is changed to that of the peripheral nervous
system.
Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.
The leading tips of elongating nerve fibers are enlarged into "growth cones" which are seen in tissue culture to continually undergo changes in conformation and to foster numerous transitory slender extensions (filopodia) and/or a veillike ruffling sheet. After explantation of 1-day-old rat superior cervical ganglia (as pieces or as individual neurons), nerve fibers and tips were photographed during growth and through the initial stages of aldehyde fixation and then relocated after embedding in plastic. Electron microscopy of serially sectioned tips revealed the following. The moving parts of the cone, the peripheral flange and filopodia, contained a distinctive apparently filamentous feltwork from which all organelles except membranous structures were excluded; microtubules were notably absent from these areas. The cone interior contained varied forms of agranular endoplasmic reticulum, vacuoles, vesicles, coated vesicles, mitochondria, microtubules, and occasional neurofilaments and polysomes. Dense-cored vesicles and lysosomal structures were also present and appeared to be formed locally, at least in part from reticulum. The possible roles of the various forms of agranular membranous components are discussed and it is suggested that structures involved in both the assembly and degradation of membrane are present in the cone. The content of these growing tips resembles that in sensory neuron growth cones studied by others.
The centrally directed neurite of the dorsal root neuroblast has been described from the period of its initial entrance into the neural tube until a well-defined dorsal root is formed. Large numbers of microtubules, channels of agranular reticulum, and clusters of ribosomes are found throughout the length of the early axons. The filopodia of the growth cone appear as long thin processes or as broad flanges of cytoplasm having a finely filamentous matrix material and occasionally small ovoid or elongate vesicles. At first the varicosity is a small expansion of cytoplasm, usually containing channels of agranular reticulum and a few other organelles. The widely dilated cisternae of agranular reticulum frequently found within the growth cone probably correspond to the pinocytotic vacuoles seen in neurites in tissue culture. The varicosities enlarge to form bulbous masses of cytoplasm, which may measure up to 5 micro in width and 13 micro in length. They contain channels of agranular reticulum, microtubules, neurofilaments, mitochondria, heterogeneous dense bodies, and a few clusters of ribosomes. Large ovoid mitochondria having ribonucleoprotein particles in their matrix are common. Dense membrane specializations are found at the basal surface of the neuro-epithelial cell close to the area where the early neurites first enter the neural tube.
The failure of axons to elongate in the injured central nervous system (CNS) of adult mammals restricts drastically the establishment of connections with target tissues situated more than a few millimetres away. Mechanisms that include a primary inability of some nerve cells to support renewed axonal growth, a premature formation of synapses on nearby neurones1, an obstruction caused by the formation of a glial scar2,3 and other influences of the microenvironment4-7 are presumed to contribute to the failure of nerve fibres to regenerate as effectively in the CNS as in the peripheral nervous system (PNS). Support for the hypothesis that conditions in the glial environment of injured fibres have a decisive role in successful axonal elongation has recently come from studies using transplants containing either central glia or peripheral nerve segments as conduits of axon growth7,8. While CNS glial grafts have been shown to prevent growth of PNS fibres7-9, experiments which used labelling techniques to trace the source of axons growing into PNS grafts provided evidence that processes from nerve cells in the spinal cord and medulla oblongata of adult rats may increase in length by 1 or more centimetres when the CNS glial environment is replaced by that of peripheral nerves10,11. Here we report for the first time the extensive elongation of axons from neurones in the brain of adult rats through PNS grafts introduced into the cerebral hemispheres.
The purpose of studying repair of nerves in experimental animals, such as the rat, is to perform model experiments in which the phenomena inaccessible to large scale investigation in human subjects may be analyzed under controlled conditions. It is neither intended nor to be expected that the results obtained in such model experiments will become immediately applicable for clinical use. What is to be expected, however, is that the lessons learned from those experiments, when properly interpreted in terms of the conditions prevailing in the human body, may furnish directives for clinical research and possibly clinical practice. In a preceding article 1 a method of reuniting severed nerves by means of arterial cuffs was outlined, and the superior results of nerve regeneration following "sleeve splicing" were described. Since that publication, the method has proved its value on several hundred experimental animals, including rats, chickens, rabbits, cats and monkeys. We have
Following injury to unmyelinated nerve fibers, differentiation between degenerative and regenerative changes is difficult. Therefore, in order to study unmyelinated nerve fiber regeneration more precisely, siliconized rubber tubes with central dividing walls were inserted between the proximal and distal stumps of transeeted rabbit anterior mesenterie nerves. Nerve fibers which grew into the tube from the proximal nerve stumps were examined by phase contrast and electron microscopy at intervals from 2 days to 13 weeks after anterior mesenteric nerve transection. Axons and Schwann cells were evaluated quantitatively.
The origin, termination, and length of axonal growth after focal central nervous system injury was examined in adult rats by means of a new experimental model. When peripheral nerve segments were used as "bridges" between the medulla and spinal cord, axons from neurons at both these levels grew approximately 30 millimeters. The regenerative potential of these central neurons seems to be expressed when the central nervous system glial environment is changed to that of the peripheral nervous system.
Using negatively staining techniques fibrin clots have been examined by electron microscopy, and finer detail of the clot structure has been observed than previously reported. From these observations a model of fibrinogen is proposed having different dimensions from those which are generally accepted and it is suggested that the fibrin clot is formed by end-to-end polymerization of the monomer with a one third molecular length overlap between laterally adjacent fibrils.
The behavior of the connective tissue during the healing of transsected nerves was studied. Experiments were carried out with rahbits. The sciatic nerve was trans-sected and repaired using different techniques. Postoperatively the animals were all treated in exactly the same way. The clinical course was registrated carefully, with special attention to the return of the motor function and the occurence and extension of decubital ulcers. Finally the animals were sacrified, and the site of nerve repair was excised and histologically examined using Hmatoxilin, Eosin. Van Gieson, Kresylviolett, Klver-Barriera, Sudan-Schwarz B, Bodian staining.Two different techniques were compared by operating on the two sciatique nerves of the same animal. Individual factors could be excluded as the result of each two operations was compared in the same individual.
A.
In 32 rabbits, 64 sciatic nerves were repaired by epineural end-to-end suture (Mersilen 60) in order to study the timing of the healing process.
1.
After 3 days the circumference of the suture site was bridged by a fibrin membrane. Proliferation of connective tissue was seen mainly in the epineurium and the external layers of the perineurium for a short distance both proximal and distal to the suture line. The connective tissue advanced in the direction of the suture line. Obstacles were avoided by going round about on the external side.
After 7 days a layer of connective tissue was formed at the circumference of the suture site. This layer originated from the epineurium proximal to the suture line, passed the sutures at their external side and reached the epineurium of the distal stump distal to the suture line.
At the 3rd day the first regenerating axons were seen in the proximal stump. Some of them had reached the distal stump at day 7.
2.
After 14 days there was a firm connective tissue layer developed. There was fibrous tissue around the suture material. According to the tendency of the connective tissue to advance around the external site of obstacles, all the suture material was inside the connective tissue layer and shifted towards the center of the cross section.
3.
After 21 days there was progressive maturation of the connective tissue. Many regenerated axons had reached the distal stump. There was no tendency to aberration if the straight course of advancement was not blocked e.g. by suture material etc.
4.
After 5 weeks the perineurium was re-established. Between the two stumps there was a more-or-less broad endoneural scar. Many axons are to be seen within the distal stump having already crossed the suture line; some of them show thickening, loss of contour and fragmentation. As the remnants of the original axons following Wallerian degeneration had already disappeared within the 2nd week, these damaged axons are regenerated axons.
5.
After 2 months the scar formation is terminated.
B.
The influence of tension on the connective tissue proliferation. In 26 rabbits, a section of 5mm length (8%) was excised from the sciatic nerve and the two stumps united by end-to-end suture under moderate tension.
The gap between the two stumps became filled by scar tissue, which was in these cases much wider. The connective tissue proliferation was increased. There was much more endoneural scar tissue formed, and the perineurium was thickened. Within the proximal and distal stump stretching of the nerve fibres and endoneural fibrosis was present. Many more of the regenerated axons within the distal stumps presented signs of degeneration.
The forces which are needed to unite the stumps of a trans-sected nerve were determined.
To unite the stumps of a nerve without any defect a force of 5–6 g is necessary. The force increases slowly as the defect increases up to 2 or 3 mm. If a larger defect is present the amount of tension necessary to unite the stumps increases rapidly (see Fig. 3).
The functional result after suture under tension was much inferior to the controls (see Figs. 4 and 5).
These experiments demonstrate the deterioration of the functional results if a nerve suture is carried out under tension. This corresponds to reports in the literature referring to interior clinical results if the size of nerve defects which are united by end-to-end suture exceed a certain limit.
The reason for the inferior results is not only blocking of the suture line by scar tissue but to a certain degree secondary damage to regenerated axons, apparently by shrinkage of the scar tissue.
In 10 human cases trans-sected peripheral nerves had been united by end-to-end suture under moderate tension 6–12 months previously. No function returned. Therefore the suture sites were resected and the nerve defects bridged by nerve grafts. The specimens were studied carefully. In all of them much scar tissue was found between the nerve stumps. Suture material with granulomatas could be seen within the cross sections. In 5 cases only a few axons were present in the distal stumps; in 3 cases a larger number were present. Many of them showed signs of recent damage. Since the primary nerve lesion 6–12 months had passed, and this proves that the damaged axons or regenerated axons having suffered secondary damage. The results of the experiments in rabbits are confirmed by this study in human specimens.
C.
By reducing the tension at the suture site connective tissue proliferation and scar formation could be reduced.
D.
In 32 rabbits a section 5 mm in length of one sciatic nerve was resected and regrafted into the defect as a free graft. In this experiment the regenerating axons had to cross two suture lines in the nerve and were under the same tension as in the case of end-to-end suture without a defect.
In 25 rabbits the sciatic nerve was trans-sected. A nerve graft of 5 mm, provided from the contralateral sciatic nerve was introduced as a free graft. In this group the axons had to cross two suture lines, but the nerve was under reduced tension.
In 3 rabbits the total gap between the nerve stumps due to retraction after trans-section (6–8 mm) was bridged by grafts. These nerves were under no tension at all.
The connective tissue proliferation decreases with decreasing tension. The endoneural scars were very thin, and in cases with long grafts hardly to be detected.
The functional results demonstrated that under favorable conditions the regeneration after grafting is equal to end-to-end suture, and much better than after end-to-end suture under tension (Figs. 4 and 5).
E.
Wrapping of the suture site by collagen, millipore or silastic membranes increased connective tissue proliferation between nerve and sheath. The functional results were inferior to simple suture.
F.
Suture material causes granulomatas and fibrosis. Due to the advancement of the connective tissue along the outer site of the sutures they are shifted towards the centre. Therefore in a part of the cross section the axon growth is impeded. The connective tissue proliferation is reduced if only a very small amount of very fine suture material is used.
The use of cyanoacrylate glues lead to a high degree of fibrosis and inferior results.
Homogenous and autogenous plasma was used by different authors, but severe reactions against fibrin and deviation of the growing axons was observed.
G.
Natural union. To reduce the operative trauma and to avoid any foreign body reaction nerve grafts were introduced into the gap resulting from retraction of the nerve stumps after trans-section without using any suture, glue or anything else to secure the union. The nerve stumps and the ends of the graft were united very carefully. As the graft was exactly as long as, or even a bit longer than, the gap, there was no tendency to separate. After 20 minutes the ends adhered strongly enough to permit the total nerve, including the graft, to be excised and removed without occurence of separation.
H.
From the experiments the conclusion was drawn that, when uniting peripheral nerves, tension should be avoided. If there is a defect, nerve grafts yield better results than suture under tension.
The following points can be established.
1.
The origin of the main part of the connective tissue after trans-section of a peripheral nerve is the epineurium. Connective tissue proliferation of the surrounding connective tissue does not play any role if approximation of the stumps in secured.
2.
The circumference of the suture line is bridged by a fibrin membrane after 3 days and by a layer of connective tissue after 7 days.
3.
The amount of connective tissue proliferation depends of the tension at the suture line. It is secondarily affected by the operative trauma and by the amount and quality of the suture material. Wrapping does not decrease the connective tissue proliferation.
4.
Without obstacles such as scars and suture granulomatas the regenerating axons proceed straight ahead without any tendency to aberrate. Wrapping seems therefore not be of any advantage.
5.
The connective tissue proliferation can be decreased by: Avoiding tension. Reducing the operative trauma. Reducing amount of suture material. Resection of the epineurium as main source of the connective tissue proliferation.
6.
If approximation without tension is not possible, nerve grafting should be carried out.
On the basis of the experimental experience the following operative technique of nerve grafting was developed:The epineurium of the two nerve stumps is resected. A dissection of the fasciculi of the nerve stumps is carried out. Major fasciculi are isolated individually. Minor fasciculi are isolated in groups of 3–5. The ends are resected at different levels according to the amount of trauma or fibrosis present. A sketch is made of the fascicular structure of the two nerve stumps. Using this sketch we try to define the corresponding fasciculi or groups of fasciculi. Between the corresponding fasciculi or groups of fasciculi nerve grafts (sural nerve) are introduced. The grafts are larger than the defect in position of function. Under microscopic control a careful adaptation is achieved. One suture (Nylon 100) at each end of each graft is used to secure the union. Under certain conditions we have relied only on the natural union and did not use any suture at all. As the fasciculi respectively groups of fasciculi had been resected at different levels, stumps and grafts are interdigitated with each other. For a median nerve 4–6, and for an ulnar nerve 3–5 individual nerve grafts were used.This technique was used in more than 200 cases with satisfactory results. The results even after bridging long defects (more than 5 cm) are good, and much better than the results published by Asworth, Boyes and Stark (1971) who used end-to-end suture to overcome large defects of peripheral nerves.
Between seven days and six weeks after division the internal architecture of rat sciatic nerves is altered, their original mono- or di-fascicular configuration being replaced by a collection of small fascicles each surrounded by perineurium. This change, called by us compartmentation, has a minimum retrograde extent of 3.5 mm and is brought about by changes in Schwann cells and endoneurial fibroblasts, which undergo circumferential elongation to surround groups of axons and so come to resemble perineurial cells. Ultrastructural changes occur in these cells during compartmentation. There is a marked rise in the number of endoneurial fibroblasts in the distal segments of the proximal stump. The stimulus to the development of compartmentation is considered to be disturbance of the endoneurial environment following rupture of the perineurium. Changes in the structure and appearance of endoneurial cells suggest that metaplasia occurs between Schwann cells, endoneurial fibroblasts and perineurial cells, and it is concluded that these cell types in the endoneurium have a common origin from embryonic ectoderm. This suggests that the surgical treatment of peripheral nerve injuries should be primarily directed to the reconstitution of the endoneurial environment.
Severed peripheral nerves and spinal cords have a strong regenerative potential when gaps in these structures are shielded within a porous cellulose acetate plastic tube, Millipore. Experiments, utilizing 1- to 2.5-cm gaps in the cat's sciatic nerve demonstrated more successful results when a single sling stitch was used to unite the severed nerve ends within the tube. Addition of a preformed clot of cat plasma to the region of the gap did not significantly change the pattern of neural regeneration. The substitution of nonporous for standard porous Millipore tubes seemed to retard the progress of cell migration across the gap. The plastic filter material served as a satisfactory substrate for migration of cells of leptomeningeal origin across the gaps in spinal cords, while apparently meeting their nutritional requirements by passing adequate amounts of fluid from the tissue bed outside the tube. The following factors attributable to the physical properties of Millipore have been identified as favoring neural regeneration: (a) Millipore is inert in tissues; (b) a shield is provided which limits the regenerating tissue to a directed pathway and prevents invasion by extraneural tissue; (c) nutrition by exchange of extracellular fluids through the pores supplies the metabolic requirements of the early phases of neural regeneration; (d) a substrate for initial orientation of regenerating structures is offered by the inner walls of the tube.
The presence of neuronotrophic factors (NTFs) in noninjured sciatic nerve extract and the course of their accumulation from 3 h to 30 days after nerve transection was examined. Rat sciatic nerves were transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers leaving a 10-mm interstump gap. Previous studies had shown that regeneration occurs in chambers containing both stumps but is absent in chambers lacking the distal stump. Chambers became completely filled with fluid 10 to 12 h after implantation. Fluid from chambers without nerve stumps (open-ended) implanted adjacent to nerve-containing chambers had markedly lower trophic activities than those containing one or both stumps. In fluid collected from chambers containing both proximal and distal nerve stumps, the highest titers of NTFs directed to sensory neurons were measured at 3 h posttransection whereas the highest titers of NTFs directed to sympathetic and spinal cord neurons were detected at 1 and 3 days, respectively. Chambers containing only the proximal or only the distal stumps showed similar temporal dynamics for sensory and sympathetic NTFs. Sensory and sympathetic neuronotrophic activity in extracts of proximal and distal stumps followed a similar temporal course to those in chamber fluid. Extracts of nonlesion nerve segments 5 mm from the transection site contained higher sensory and lower sympathetic trophic activity than extracts including the transection site. Spinal cord activity was undetectable in all extracts. Antiserum to nerve growth factor had no effect on fluid or extracts containing high sensory or sympathetic activities. These observations suggested that (i) some NTFs may be present in normal nerves and others may be synthesized or accumulated in response to nerve injury, (ii) sensory, sympathetic, and spinal cord NTFs are separate agents and immunochemically distinct from nerve growth factor, (iii) NTFs predominantly originate from nerve stumps rather than from surrounding fluid, and (iv) proximal and distal nerve stumps accumulate and release NTFs at similar rates.
Rat sciatic nerves can be transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers. Anatomical regeneration has been demonstrated across 10 mm long chambers containing both stumps, although little or no axonal outgrowth occurs in chambers omitting the distal stump or exceeding the 10 mm length. We have previously shown that chambers containing both proximal and distal stumps accumulate within days of implantation a clear fluid containing neuronotrophic factors (NTFs) directed to neurons from neonatal mouse dorsal root ganglia. We report here that these chamber fluids also have considerable neuronotrophic activity for chick embryo neurons from embryologic day 4 (E4) lumbar spinal cord, E12 sympathetic ganglia, E12 (but not E8) dorsal root ganglia and E8 ciliary ganglia. Thus, the neuronal types supported by trophic factors of these fluids include all those which contribute axons to the sciatic nerve, namely sensory, spinal motor, and sympathetic. In fluid collected 1 week after implantation, NTF levels directed to different neurons varied independently from one another in chambers with different nerve insertions, suggesting that these activities reside in separate factors. Fluid collected from chamber arrangements allowing little proximal fiber regrowth did not always contain correspondingly lower titers of NTFs. However, generally higher titers of all NTFs were found in chambers containing either or both nerve stumps that in nerve-free chambers. Fluids collected from nerve-containing chambers were subjected to heat, dialysis or trypsin treatments. The behavior of their neuronotrophic activities suggests their association with proteins.
Interactions between PNS axons and CNS glia were studied morphologically by transplanting optic nerves into peripheral nerves in groups of rats and mice. Four to 11 months after grafting, small numbers of axons from the peripheral nerves had penetrated the CNS grafts where they became ensheathed and myelinated by CNS glia. Glial protuberances observed at the CNS-PNS interfaces suggested that there had been an active glial response to innervation by PNS axons. These findings provide experimental evidence that denervated CNS glia can be reinnervated and form myelin.
Labeling regenerating axons with axonally transported radioactive proteins provides information about the location of the entire range of axons from the fastest growing ones to those which are trapped in the scar. We have used this technique to study the regeneration of motor axons in the rat sciatic nerve after a crush lesion. From 2 to 14 days after the crush the lumbar spinal cord was exposed by laminectomy and multiple injections of [3H]proline were made stereotactically in the ventral horn. Twenty-four hours later the nerves were removed and the distribution of radioactivity along the nerve was measured by liquid scintillation counting. There was a peak of radioactivity in the regenerating axons distal to the crush due to an accumulation of label in the tips of these axons. After a delay of 3.2 +/- 0.2 (S.E.) days, this peak advanced down the nerve at a rate of 3.0 +/- 0.1 (S.E.) mm/day. The leading edge of this peak, which marks the location of the endings of the most rapidly growing labeled fibers, moved down the nerve at a rate of 4.4 +/- 0.2 mm/day after a delay of 2.1 +/- 0.2 days; this is the same time course as that of the most rapidly regenerating sensory axons in the rat sciatic nerve, measured by the pinch test. Another peak of radioactivity at the crush site, presumed to represent the ends of unregenerated axons or misdirected sprouts, declined rapidly during the first week, and more slowly thereafter.
Continuing from earlier work which demonstrated the peripheral axonal regulation of Schwann cell myelination, this study has investigated the possibility that a peripheral axon can stimulate oligodendrocyte myelination. To test this hypothesis, regenerating PNS axons were allowed to interact with uncommitted oligodendrocytes by transecting a rat peroneal nerve and inserting a segment of the autologous optic nerve between the cut ends. Grafts were maintained for 4-28 weeks and then examined by light and electron microscopy. A few regenerating peripheral myelinated nerve fibers penetrated the optic nerve graft. Some axons penetrated the outer margin of the graft, were myelinated by Schwann cells, and surrounded by astrocyte processes bordered by basal lamina. More centrally in the optic nerve graft, regenerating peripheral axons displayed myelin of CNS type. The outer myelin lamella abutted directly on the plasmalemma surface of surrounding astrocytic processes and was expanded focally to form a glial tongue. These observations demonstrate the experimental induction of central myelination by regenerating peripheral axons and suggest the existence of a common neuronal mechanism to stimulate myelin formation by both the Schwann cell and the oligodendrocyte.
Developmental changes in relative amounts of peripheral nerve proteins and glycoproteins have been correlated with the degree of morphological myelination at various ages during the first 25 postnatal days in rat sciatic nerve. At birth there is virtually no major myelin glycoprotein (P0), but there is a protein which migrates to the same point on sodium dodecyl sulphate (SDS) polyacrylamide gels as the small myelin basic protein (P2). During the time myelin is being formed in the nerve, the P0 protein increases and the P2 protein appears to decrease in relative amount in the nerve. The accumulation of P0 protein in the nerve correlates extremely well with the degree of myelination in sciatic nerve.
At 4–6 days postnatal, smooth membrane profiles are observed which are located within axons and in the inner Schwann cell cytoplasm. Such profiles are also observed to fuse with the axolemma-Schwann cell interface. The profiles may represent membrane material being added to or deleted from the axolemma or myelin during myelination.
The regeneration of transected peripheral nerves of mice was studied using autoradiographical and electron microscopical techniques. In general, maximal proliferation occurred between the 5th and 7th day after dissection and stopped when the cells emigrating from the proximal and distal stumps of the nerve started to contact one another. Special attention was paid to the reaction of the connective tissue cells of the endo-, epi- and perineurium. The perineurial cells seemed to dedifferentiate between the 3rd and 5th day after the transection and then started to proliferate into the defect. Labelled perineurial cells were completely absent, when the minifascicles were fully developed in the neuroma. The epineurial fibroblasts started to proliferate during the 1st day. Even 6 weeks after transection the multiplication rate was about ten fold that of the controls. The results are discussed with special reference to clinical nerve repair.
Following injury to unmyelinated nerve fibers, differentiation between degenerative and regenerative changes is difficult. Therefore, in order to study unmyelinated nerve fiber regeneration more precisely, siliconized rubber tubes with central dividing walls were inserted between the proximal and distal stumps of transected rabbit anterior mesenteric nerves. Nerve fibers which grew into the tube from the proximal nerve stumps were examined by phase contrast and electron microscopy at intervals from 2 days to 13 wk after anterior mesenteric nerve transection. Axons and Schwann cells were evaluated quantitatively. By 2 days after injury, small axons approximately 0.4 microns in diameter were present. Initially as many as 200 of these axons were observed in relation to individual Schwann cells. Such axonal bundles were progressively partitioned into smaller groups by Schwann cells and their processes. However, axon Schwann cell ratios remained higher than normal throughout the period of study. Thus, regeneration of transected unmyelinated nerve fibers simulates fetal patterns of neurogenesis. Regenerating axons remained smaller than normal, however, suggesting that axonal maturation cannot occur when longitudinal growth is obstructed.
Our preliminary electron microscopic observations of embryonic chick spinal cord indicate that the majority of synapses in the cervical cord at the fourth and sixth days of incubation are found in the marginal layer rather than the mantle layer. These findings raise two basic questions about the type of connections in the marginal zone during the first week of development: are all these early synapses axondendritic, and are synaptic junctions present on axonal and dendritic growth cones? Since criteria for distinguishing between axonal and dendritic growth cones are scarce or nonexistent, serial section electron microscopy was employed to definitively identify the two types of terminals.
Two types of dendritic growth cones are present in the marginal layer during the first week of development. The first type is distinguished by a dendritic stalk which expands into a club‐shaped or bulb‐like terminal. A characteristic feature of this type of dendritic ending is the presence of numerous vesicles which vary in diameter from about 1000 Å to 2500 Å. Its cytoplasm has a floccular appearance and is frequently electron lucent. In contrast, the second type of dendritic terminal is electron dense, has a consistently homogeneous appearance, and usually lacks the large vesicles of the first type. The shape of the second type is a finger‐like process (filopodium) roughly of the same diameter as the main dendritic trunk. Evidence is presented that some dendritic growth cones observed in this study represent growing, motile processes rather than quiescent enlargements. Synapses on dendritic growth cones are quite numerous and, in addition, the first synapses on dendrites appear to form on growth cones. The hypothesis is put forward that synapses on dendritic growth cones. The hypothesis is put forward that synapses on dendritic growth cones are incorporated onto the dendritic trunk as the terminal continues to grow.
Axonal growth cones may be distinguished from dendritic growth cones on the basis of their overall morphology and their cytoplasmic components. An axonal growth cone is, in general, a larger and more irregular structure than the dendritic terminal. This irregularity is due to the large number of processes radiating away from the terminal enlargement or varicosity. While dendritic terminals almost always have finger‐like projections, processes of axonal growth cones are more often sheet‐like extensions of cytoplasm (folipodia). Another important difference between axonal and dendritic growth cones is that ribosomes in axonal varicosities are infrequently encountered and rarely aggregated into polysomes as in dendritic terminals. Axonal growth cones are occasionally observed to be presynaptic to a dendrite during the first week of development.
In the first six days after division myelinated axons in the proximal stump of rat sciatic nerves produce collateral and terminal sprouts. These are present as circumscribed "groups" which are positively distinguishable from clusters of non-myelinated axons. Two types of "groups" are identifiable, and their distribution in some of the nerve segments is analysed. Their evolution was followed in sequential nerve segments, the initial 'tight' structure becoming looser between 7 and 10 days, and myelinated axons appeared in them during this time. At this stage a complete basal lamina was present surrounding the entire "group". Some of the cells in the "groups" did not have the characteristics of Schwann cells. Between 7 and 10 days after division alveolate vesicles and densely staining material in the cisternae of the rough surfaced endoplasmic reticulum were prominent in Schwann cells in the distal part of the proximal stump. It is thought that both types of "group" are developed from single myelinated axons and the name "regenerating unit" is proposed for both types. Their relationship to "clusters", seen in the distal stump of regenerating peripheral nerves, and "onion bulbs", present in some peripheral neuropathies, is discussed.
Scctioncd lumbosacral ventral roots in 18 cats were anatomically repaired by tubulation with Millipore filter. The anastomosis included both straight reunion of roots (i.e. L7 to L7) and coupling of roots one or two spinal segments apart (notably L6 to L7, L7, to S1, L6 to S1 and L5 to L7). After regeneration periods of four to thirty weeks, the tubulated root segments were studied by histological techniques. It was found that:90% of the fibres had grown down into the distal stump after straight reunion.After heterogenous anastomosis 65 % of the fibres grew into the distal stump.Ip lo 23 weeks after surgery the fibre diameter spectrum was unimodal but in one of the longest surviving animals (30 weeks) a tendency lo restitution of a normal bimodal fibre pattern was encountered.
Extraocular muscles of the rat possess numerous nerves suitable for the study of fine structure. In these muscles, small nerves made up of one to ten myelinated and unmyelinated nerve fibers are surrounded by two or three layers of perineurium. The perineurium is arranged in concentric sleeves, each one cell thick. Continuous boundary membranes separate the perineural sleeves from the epineural and endoneural tissue space, but the boundary membranes may be spotty or absent between individual sleeves. The presence of boundary membranes around perineural cells distinguishes them from nearby fibroblasts which lack similar membranous investment. Tight intercellular junctions join the cells comprising each sleeve so that the nerves are completely ensheathed in perineurium. The number of sleeves decreases as the nerve becomes smaller, either by the termination of the innermost sleeve or by the loss of a sleeve as the nerve branches. The last sleeve ends shortly before the termination of the nerve. The perineurium is thus open-ended peripherally and, at these places, the epineurium and endoneurium are continuous. Continuities between the epineurium and endoneurium also exist at the entrance and exit of blood vessels supplying the nerve and at points where reticular fibers pierce the perineurium. These structural features correlate well with the action of the perineurium as a diffusion barrier and as a pathway in the transmission of infections.
The authors believe that the silicone chamber model discussed promises to be an outstanding new tool for neurobiological investigation of neural regeneration: One can contrast properties and behaviors in situations favoring or hindering the repair of motor and sensory (and sympathetic) nerves and, thus begin to recognize physical, biochemical, and cellular influences that bear on the outcome of a regenerative process. One can analyze the composition of the fluid environment; the matrix in which nerve fibers will or will not grow; the temporal changes occurring in the proximal and distal components; the reactions of the source neurons in the spinal cord and dorsal root ganglia, and of their partner glial cells in their various locations; and, the roles and responses of the end organs addressed by the nerve. One can introduce and investigate the effects of (among several other possible manipulations) different factors, antibody against them, hormones, nutrients, drugs; glial cells of PNS or CNS origin, as well as connective or other cells; and semisolid matrices or experimental terrains for neurite promotion and/or directional guidance. Obviously, a vast amount of work needs to be carried out before the model described here will fulfill even part of its promises. Eventually, however, one should be able to explore the applications of this model to selected problems of interest to both neurobiologists and neuropathologists, for example: Myelin degeneration and regeneration, peripheral neuropathies (including genetic defects expressed in mouse neurological mutants), compression damage on regenerated nerves, or the role of electrical stimulation in nerve repair. Lastly, one may be able to adapt this PNS model to CNS tissues such as optic nerve and spinal cord: if so, what one will have learned about repair processes and the humoral or cellular influences which control them in the PNA might become applicable at least in part to the much greater complexity of CNS regeneration.
Newly transected or denervated segments of isogeneic rat tibial nerve were implanted into the rat midbrain and sampled at weekly intervals up to 6 weeks post-operation. By 3 weeks, the peripheral nervous system (PNS) grafts were well-vascularized and contained Schwann cells, axons associated with Schwann cell processes, and macrophages. From 3 to 6 weeks, many axons within both the fresh and predegenerated grafts were myelinated by Schwann cells. The nerve fiber arrangement within the implant was similar to that of regenerating peripheral nerve in situ. The central nervous system (CNS) border of the implant was clearly demarcated by a rim of astrocytes behind which was a layer of regenerating oligodendrocytes and axons. Extending from the CNS margin were radial bridges of astroglial tissue which apprarently guided regenerating axons into the implant. Between the CNS and the PNS implant, abundant collagen deposition was present. The findings suggest that regenerating CNS axons grow via astroglial bridges into transplanted PNS tissue and are capable of stimulating the implanted Schwann cells to form myelin. Even Schwann cells deprived of axonal contact for prolonged periods were still capable of PNS myelin formation.
The proximal stump of a transected rat sciatic nerve has been observed to regenerate through a cylindrical silicone chamber across a 10 mm gap to the distal stump. The fluid filling such in vivo chambers contains trophic factors that ensure in vitro survival and growth of at least sensory neurons from rodent dorsal root ganglia--as already demonstrated for fluid generated in vitro from Schwann and other cell cultures.
Polyglactin 910, a resorbable synthetic material, was used as a mesh-tube to bridge defects (7 to 9 mm in length) in a sectioned rabbit tibial nerve. After absorption of the mesh a new nerve sheath was formed which enclosed numerous minute fascicles of regenerating axons. The polyglactin tube influenced the direction taken by the regenerating axons and guided them into the distal segment. The tube also reduced the formation of neuromas and the growth of scar tissue from surrounding structures.
We describe an experimental in vivo system for studying peripheral nerve regeneration, in which the proximal stump of a transected nerve regrows through a transparent silicone chamber toward the distal stump. Physical separation permits examination of the effects of the humoral and/or cellular influences from the distal stump on regenerating fibers before they invade the distal segment itself. A small segment of the rat sciatic nerve was resected, leaving a 6 mm gap which was then encased by a cylindrical silicone chamber. Within the first weeks, a nerve trunk regenerated along the central axis of the chamber bridged the gap between the proximal and distal stumps. When the distal nerve stump was omitted from the distal opening of the chamber, only a thin structure with a few small-caliber fibers extended across the gap. In each instance regenerating nerve appeared as a cord-like structure completely surrounded by clear fluid, a feature which permits easy collection of the extracellular fluid for analysis of its chemical properties and biological activity. This feature also allows in vivo manipulation of the humoral environment in which nerve regeneration occurs.
The range of growth-promoting influences from a distal nerve stump on a regenerating proximal stump was determined using an experimental system in which a gap between cross-anastomosed rat sciatic nerves was encased by a cylindrical silicone chamber. Two arrangements were examined after 1 month in situ: A proximal-distal (PD) system in which both proximal and distal stumps were introduced into the ends of the chamber, and a proximal-open (PO) system in which the distal stump was omitted. When the gap was 6 mm long, a regenerated nerve extended all the way through the chamber in both the PD and PO systems. When the gap was increased to 10 mm, a similar regrowth occurred in the PD chamber, whereas in the PO chamber proximal regrowth was partial or nonexistent. When the gap was increased to 15 mm, no regeneration occurred, even in the presence of the distal stump. These observations confirm that the distal stump influences proximal regeneration and indicate that this influence can act only over a limited distance or volume. Such an influence could consist of humoral agents which support nerve growth and/or outgrowth from the distal stump.
Regeneration of severed peripheral nerves is unfortunately often incomplete, due to loss of nerve fibers and neuroma formation. A new approach is presented with the intention of improving the conditions for nerve repair. In the first of the two stages, a pseudosynovial tube is formed around a silicone rubber rod, surrounded by a stainless steel spiral, which was placed in the backs of rats. This tube, in the second stage, is used as a free "tube graft" to bridge gaps of about 10-12 mm lengths in the severed sciatic nerve. The tube was kept open by the metal spiral. Regenerating nerve fibers with their sprouts grew into the initially open space in the tube. A new nerve trunk was formed, comprised of closely packed myelinated and unmyelinated axons, organized into fascicles. Demonstration by electron microscopy and by EMG recording of reinnervation of foot muscles supported successful long-term results. The fascicles were delimited by perineurial and epineurial sheaths and, furthermore, showed signs of maturation. It was also demonstrated that the nerve-fiber regeneration ceased after a few weeks if there was no distal nerve inserted into the tube. The importance of optimizing the interaction between local factors and regenerating nerve fibers for reestablishment of functionally valuable motor units is discussed.
Axons in the peripheral nervous system (PNS) and central nervous system (CNS) form sprouts after injury. Elongation of regenerating axonal sprouts has been observed as the exception within the adult mammalian CNS but is the rule in the PNS of mammals as well as in the CNS of some fish and amphibians. The relative importance of intrinsic neuronal properties and axonal environment in determining the extent of axonal regrowth is unknown. Neuroglial cells, nerve growth factor and target tissues such as smooth muscle are known to influence neuronal responses to injury. Here we have examined the capacity of transected axons originating in the CNS to regrow into nerve grafts containing Schwann cells.