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A collagen-based nerve guide conduit for peripheral nerve repair: An electrophysiological study of nerve regeneration in rodents and nonhuman primates
Abstract
When a peripheral nerve is severed and left untreated, the most likely result is the formation of an endbulb neuroma; this tangled mass of disorganized nerve fibers blocks functional recovery following nerve injury. Although there are several different approaches for promoting nerve repair, which have been greatly refined over recent years, the clinical results of peripheral nerve repair remain very disappointing. In this paper we compare the results of a collagen nerve guide conduit to the more standard clinical procedure of nerve autografting to promote repair of transected peripheral nerves in rats and nonhuman primates.
In rats, we tested recovery from sciatic nerve transection and repair by (1) direct microsurgical suture, (2) 4 mm autograft, or (3) entubulation repair with collagen‐based nerve guide conduits. Evoked muscle action potentials (MAP), were recorded from the gastrocnemius muscle at 4 and 12 weeks following sciatic nerve transection. At 4 weeks the repair group of direct suture demonstrated a significantly greater MAP, compared to the other surgical repair groups. However, at 12 weeks all four surgical repair groups displayed similar levels of recovery of the motor response.
In six adult male Macaca fascicularis monkeys the median nerve was transected 2 cm above the wrist and repaired by either a 4 mm nerve autograft or a collagen‐based nerve guide conduit leaving a 4 mm gap between nerve ends. Serial studies of motor and sensory fibers were performed by recording the evoked MAP from the abductor pollicis brevis muscle (APB) and the sensory action potential (SAP) evoked by stimulation of digital nerves (digit II), respectively, up to 760 days following surgery. Evoked muscle responses returned to normal baseline levels in all cases. Statistical analysis of the motor responses, as judged by the slope of the recovery curves, indicated a significantly more rapid rate of recovery for the nerve guide repair group. The final level of recovery of the MAP amplitudes was not significantly different between the groups. In contrast, the SAP amplitude only recovered to the low normal range and there were no statistically significant differences between the two groups in terms of sensory recovery rates.
The rodent and primate studies suggest that in terms of recovery of physiological responses from target muscle and sensory nerves, entubulation repair of peripheral nerves with a collagen‐based nerve guide conduit over a short nerve gap (4 mm) is as effective as a standard nerve autograft. Furthermore, preliminary results show that entubulation repair with this material can support axon regeneration and maturation over a nerve gap distance of at least 15 mm.
... The fibrillar structure of the collagen is maintained throughout the manufacturing process, permitting the construction of a biocompatible tubular matrix that has sufficient mechanical strength, defined permeability and a controlled rate of resorption. [63][64][65] However, this material is not expected to fully resorb for a period of up to 4 years post implantation. 26 The manufacturers advise prehydration of the tube for pliability and resilience in order to permit ease of handling provided in the manufacturers instructions for use (IFU). ...
... The first trials using NeuraGen 1 were conducted by Mackinnon et al. 66 and showed no occurrence of compression neuropathy in comparison with other more rigid or non-biodegradable (silicon) materials. Archibald et al. 63 later reported on the entubulation repair of peripheral nerves with NeuraGen 1 to be as effective as a standard nerve autograft. This study compared NeuraGen 1 to autograft and direct suturing of transected peripheral nerves in both rats (sciatic) and nonhuman primates (median nerve of six adult male Macaca fascicularis monkeys). ...
... In these studies, there was no reported scar tissue or inflammatory response with complete absorption minimizing the possibility of nerve entrapment. 63,65 Tyner et al. 67 used NeuraGen 1 in a rat sciatic nerve model in an effort to stimulate linear neuronal outgrowth and reduce random axon sprouting. 13% (1 of 8) of animals receiving the conduit developed autotomy in comparison to 88% (7 of 8) to the control (neurectomy) group, which was significantly less (P < 0.01). ...
... Clinical trials in the field of peripheral nerve regeneration must be preceded by long-lasting preclinical research, which usually starts with the manufacturing of a biomaterial with precise characteristics (Archibald et al., 1991;Hashimoto et al., 2002;Ahmed et al., 2003;Jansen et al., 2004;Sundback et al., 2004;Yang et al., 2007a;Xie et al., 2008;He et al., 2009). When finalized, this biomaterial is primarily tested on in vitro models to assess its biocompatibility, cytotoxicity, genotoxicity, the absence of toxic degradation products, the interaction between cells and the biomaterial, its degradation rate, cell proliferation upon the biomaterial and so on. ...
... The different binding domains on it give the possibility to create topographical cues that guide the axon regrowth (Stang et al., 2005a,b) facilitating cell adhesion, survival and migration (Alberti et al., 2014;Sulong et al., 2014;Drobnik et al., 2017a,b). Many researchers have shown that collagen has biological properties superior to other materials available in the market for nerve scaffold fabrication and it is also effective over a nerve gap distance of at least 15 mm (Archibald et al., 1991). 10 mm long hollow conduits reported better results in rat nerve regeneration and muscle re-innervation if compared to collagen polyglycolic acid (PGA)-filled conduits (Saltzman et al., 2019). ...
Peripheral nerve injury treatment is a relevant problem because of nerve lesion high incidence and because of unsatisfactory regeneration after severe injuries, thus resulting in a reduced patient’s life quality. To repair severe nerve injuries characterized by substance loss and to improve the regeneration outcome at both motor and sensory level, different strategies have been investigated. Although autograft remains the gold standard technique, a growing number of research articles concerning nerve conduit use has been reported in the last years. Nerve conduits aim to overcome autograft disadvantages, but they must satisfy some requirements to be suitable for nerve repair. A universal ideal conduit does not exist, since conduit properties have to be evaluated case by case; nevertheless, because of their high biocompatibility and biodegradability, natural-based biomaterials have great potentiality to be used to produce nerve guides. Although they share many characteristics with synthetic biomaterials, natural-based biomaterials should also be preferable because of their extraction sources; indeed, these biomaterials are obtained from different renewable sources or food waste, thus reducing environmental impact and enhancing sustainability in comparison to synthetic ones. This review reports the strengths and weaknesses of natural-based biomaterials used for manufacturing peripheral nerve conduits, analyzing the interactions between natural-based biomaterials and biological environment. Particular attention was paid to the description of the preclinical outcome of nerve regeneration in injury repaired with the different natural-based conduits.
... Collagen fibers within gels can be longitudinally aligned using magnetic fields and result in an improved neurite outgrowth when compared to that observed with randomly oriented collagen fibers [108]. Moreover, collagen NGCs containing a porous collagen glycosaminoglycan matrix promoted regeneration levels similar to those resulting from nerve autograft [109,110]. Finally, commercial collagen type III membranes (commercially available as GentaFleece ® , Baxter, Nuremberg, Germany) were demonstrated to promote the regeneration of rat sciatic nerves undergoing neurotmesis [104] and axonotmesis [111]. ...
... • NeuraGen ® (approved in 2001) was demonstrated not to cause neuropathy by compression [112], a common observation when using rigid materials, and sustained nerve repair in the period of 4 weeks [110]. ...
... The predominantly preferred treatment is still an autograft [1]. A tissue engineered NGC is a viable clinical alternative for clinicians to treat peripheral nerve injury [2][3][4][5]. This construct has the potential to replace the use of autografts, thus eliminating some of the limitations, such as limited supply, diminished Schwann cell viability after harvest, size mismatch, and donor site morbidity [6]. ...
... It was suggested that a long-term study should take place in order to obtain meaningful data. Yet, a full reinnervation may take up to years [5]. Following nerve healing, the gastrocnemius muscle undergoes reinnervation. ...
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.
... Collagen is a major extracellular matrix structural protein and is abundantly available. 37,38 This is the material of choice and has yielded numerous commercial NGCs: Neura-Gen, Neuroflex, NeuroMatrix, NeuraWrap, and NeuroMend. 39 There are 44 different known Collagen genes which develop 28 different kinds of Collagen fibrils of which Type I, II, and III are most abundant. ...
... 46 Gastrocnemius muscle measurement The mass of the gastrocnemius muscle is proportional to the degree of sciatic nerve innervation and is an indicator of the functional activity of sciatic nerves. 37 The relative gastrocnemius muscle weight (RGMW) is the ratio of the gastrocnemius muscle weight from the experimental (right) side to that of the normal (left) side. RGMW is used as a parameter to represent the "functional" consequences of sciatic nerve regeneration. ...
Nerve guidance conduits (NGCs) are artificial substitutes for autografts, which serve as the gold standard in treating peripheral nerve injury. A recurring challenge in tissue engineered NGCs is optimizing the cross‐sectional surface area to achieve a balance between allowing nerve infiltration while supporting maximum axonal extension from the proximal to distal stump. In this study, we address this issue by investigating the efficacy of an NGC with a higher cross‐sectional surface composed of spiral structures and multi‐channels, coupled with inner longitudinally aligned nanofibers and protein on aiding nerve repair in critical sized nerve defect. The NGCs were implanted into 15‐mm‐long rat sciatic nerve injury gaps for 4 weeks. Nerve regeneration was assessed using an established set of assays, including the walking track analysis, electrophysiological testing, pinch reflex assessment, gastrocnemius muscle measurement, and histological assessment. The results indicated that the novel NGC design yielded promising data in encouraging nerve regeneration within a relatively short recovery time. The performance of the novel NGC for nerve regeneration was superior to that of the control nerve conduits with tubular structures. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B, 2018.
... In such context, the restoration of dentin and the recovery of nutrition supplied to nerves and blood vessels are the essential factors for pulp regeneration [108]. Collagen is a common substance for nerve regeneration [109,110]. Lee et al. [111] developed a straightforward cell printing method to form neural cell patterns in multilayered collagen hydrogel. In this design, collagen layers were first printed to provide a scaffold for the cells. ...
Oral diseases have emerged as one of the leading public health challenges globally. Although the existing clinical modalities for restoration of dental tissue loss and craniomaxillofacial injuries can achieve satisfactory therapeutic results, they cannot fully restore the original complex anatomical structure and physiological function of the tissue. 3D printing of biological tissues has gained growing interest in the field of oral medicine with the ability to control the bioink component and printing structure for spatially heterogeneous repairing constructs, holding enormous promise for the precise treatment of oral disease. Particularly, collagen-based materials have been recognized as promising biogenic bioinks for the regeneration of several tissues with high cell-activating and biocompatible properties. In this review, we summarize 3D printing methods for collagen-based biomaterials and their mechanisms. Additionally, we highlight the animal sources of collagen and their characteristics, as well as the methods of col-lagen extraction. Furthermore, this review provides an overview of the 3D bioprinting technology for the regenera-tion of the pulpal nerve and blood vessels, cartilage, and periodontal tissue. We envision that this technique opens up immense opportunities over the conventional ones, with high replicability and customized function, which can ultimately promote effective oral tissue regeneration.
... Compared to many synthetic polymers, biomaterials made from collagen offer several advantages, including biocompatibility, non-toxic degradation products, and minimal foreign body response (69). Collagen conduits have been reported to be as effective as autografts when repairing small peripheral nerve defects up to 4 mm in length (70). ...
... Collagen-based biomaterials and scaffolds have been used to study the cells proliferation, differentiation as well as phenotype expression. Collagen-based biomaterials are reported (12,13) for bone and cartilage tissue regeneration and promising nerve guide (14,15). Recent findings show that collagen-based biomaterials are leading materials for cardiovascular regenerative medicine (16,17). ...
Collagen is the most abundant protein in humans and animals, comprising of one third of the total proteins that accounts for three quarters of the dry weight skin in humans. Collagen containing a range of proteins has been reported for tissue engineering applications, but, only a small number of studies related to chemical structure evaluation of collagen are found in the literature. Collagen can be obtained from both the natural and synthetic sources and offers a wide range of biomedical applications due to its excellent biocompatibility and low immunogenicity. Hence, it is important to identify chemical structural properties of collagen and Fourier transform infrared (FTIR) appears to be a technique of choice to study their chemical structure. This review aims to highlight the use of FTIR to study collagen-based biomaterials, using it for characterization of collagen extracted from various sources. Characterization of collagen-based materials used in wound healing, skin substitutes, derma fillers, and aging of skin, collagen containing drug delivery agents, collagen-based materials used in tissue engineering, bone regeneration, and osteogenic differentiation is discussed in detail. FTIR analysis of collagen-containing materials used for dental applications, cleft-palate, and in alveolar-ridge preservation has also been highlighted.
... APPROACHES FOR NEUROVASCULARIZATION Tissue engineering approaches that address both innervation and vascularization hold great potential to promote craniofacial tissue regeneration 158 as opposed to targeting only vascularization 159−161 or innervation. 162−165 These approaches can be adopted from traditional tissue engineering approaches and include cells, 166,167 synthetic 168−170 and natural 166,171,172 scaffolds, growth factors, 173,174 and combinations therein, 127,175 which are detailed in the following sections. ...
... A nerve guidance conduit aims to suppress the invasion of inflammatory tissue by covering the nerve defect portion with a tubular structure and also provides an environment suitable for nerve reconstruction via the biological characteristics of the materials. Previously, nerve guidance conduits using various materials were fabricated, and their efficacy to facilitate peripheral nerve regeneration was shown by transplantation into laboratory animals such as rats and dogs (Archibald et al. 1991;Whitworth et al. 1995;Nicoli Aldini et al. 1996;Hadlock et al. 1998;Matsumoto et al. 2000;Nakamura et al. 2004). However, their recovery score did not replace that of autografting. ...
The nervous system is an ensemble of organs that transmit and process external information and are responsible for the adaption to the external environment and homeostasis control of the internal environment. The nervous system of vertebrates is divided into the central nervous system (CNS) and peripheral nervous system (PNS) due to its structural features. The CNS, which includes the brain and the spinal cord, processes information from external stimuli and assembles orders suitable for these stimuli. The CNS then sends signals to control other organs/tissues. On the other hand, the PNS connects the CNS to other organs/tissues and functions as a signal pathway. Therefore, the decline and loss of various functions due to injuries of the nervous system cause an impaired quality of life (QOL) and eventually the termination of life activities. Here, we report mainly on decellularized neural tissue and its application as a substrate for the regeneration of the nervous system.
... Hollow collagen-based NGC have proven efficacious in promotion of axonal regeneration and functional recovery across short and long gap repairs (Archibald et al., 1991, Saltzman et al., 2019, whilst devices such as NeuraGen® have been approved for clinical use in humans (Kehoe et al., 2012). Moreover, due to its presence in the endoneurium (Luque et al., 1983, Nath et al., 1997 and role in the native peripheral nerve regeneration response (Siironen et al., 1992a, Siironen et al., 1996, collagen I has been frequently employed as a luminal filler as a hydrogel, either acellular (Yoo et al., 2020) or as a support cell delivery system, or as aligned fibres to provide adhesive guidance cues for axonal regeneration (Saeki et al., 2018, Kazuya et al., 2000, Toba et al., 2002. ...
Peripheral nerve injury poses a serious clinical problem, with sensory and motor deficits resulting in significant reductions in patient quality of life. Nerve guidance conduits aim to overcome issues with the current gold standard for repair, the autograft. Recent advances in the field of tissue engineering have allowed for the devlopment of biochemically and physically complex constructs that aim to bridge the injury site to provide an environment that favours regeneration. This thesis explores the possibility of extracellualr matrix (ECM) hydrogels derived from decellularised tissues (dECM-h) and their potential for the maintenance of Schwann cells, ability to form anisotropic cellular tissue, and their subsequent ability to promote in vitro and in vivo neurite extension. A number of tissues were decellularised, biochemical properties assessed, and formed into hydrogels that were mechanically characterised. In vitro screening was then performed to assess Schwann cell metabolic activity, contraction, and alignment within three selected dECM-h. Additionally, stabilised dECM-h seeded with Schwann cells were formed and seeded with dorsal root ganglia to assess their in vitro capabilities to promote neurite extension. Hydrogels derived from decellularised cancellous bone (B-ECM) were found to be appropriate to be taken forward into a rat sciatic nerve transection model to be compared to the currently used purified collagen I derived from rat tails. In vivo axonal regeneration was found to be comparable between the two groups, however did not match that observed in nerve autografts. This study brought a portfolio of decellularised materials from generation, through characterisation and in vitro screening, to selction of one candidate that was taken forward into an in vivo model. This has shown, for the first time, that alternatives to the currently used collagen I hydrogels may be employed in the production and utilisation of engineered neural tissue (EngNT).
... 17 Artificial nerve conduit to bridge the gap between severed nerve stumps is widely accepted as a useful alternative that provides structural support and a favorable microenvironment for axonal regeneration. 18 The great advantage of collagen is very resorbable, and Archibald et al. 19,20 described excellent regeneration following median nerve repair in monkeys as by. Its structure also offers biochemical support for nerve regeneration, as it has been showed that collagen is one of extracellular matrix (ESM) factors that has been shown to play a significant role in the process of nerve regeneration. ...
... As well known, the length of the gap is the main discriminating factor for the application of the most suitable strategy. In particular, for not excessively long gaps, tissue engineering approaches, based on the use of nerve guides or decellularized heterologous nerves, proved to be very effective in inducing the tissue regeneration and in restoring the functionality, albeit partial, of the injured tract [5,38,39] . Col appears as the best biomaterial for the manufacture of hollow or internally filled neural guides [2,6,40,41] . ...
Peripheral nerve injuries may lead to a significant function loss, which deeply affect patient’s quality of life. In this context, tissue engineered collagen-based nerve guides are a promising alternative to autografts. To enhance the regeneration of the injured nerve tract, several bioactive molecules can be adopted as further components of the collagen-based conduits. Herein, sericin (Ser), a waste product of textile industry, was combined with type I collagen (Col), for the development of a bioactive substrate potentially able to support and enhance the regeneration of the peripheral nervous system. In particular, in order to identify an optimal substrate composition in terms of physicochemical and biological properties, thin Ser-Col films with different Ser:Col ratios were produced by air-drying and subsequently crosslinked through two different crosslinking methods. Then, Ser:Col films were characterized from a physicochemical point of view by examining secondary protein structure modifications via FTIR spectroscopy, swelling ratios, degradation rates and Ser release kinetics. Moreover, Ser:Col films ability to promote adhesion and proliferation of Schwann cells was evaluated in vitro. The results obtained in this study, although preliminary, suggest that Ser:Col blends could represent a promising strategy to enhance the repair of damaged peripheral nerves, by providing a sustained release of Ser from collagen-based nerve substrates.
... NeuraGen® (Integra, FDA 510 (k) approval 2001; CE mark 2003), NeuroMatrix®, and NeuroFlex® (Stryker, FDA 510(k) approval 2014) 10 . In vivo studies with NeuraGen® demonstrated equivalent functional performance in entubulation repair compared to use of standard nerve autograft and direct suture repair 14 . NeuraGen® clinical data from several prospective and retrospective studies have been published demonstrating clinical safety and efficacy [15][16][17] . ...
Background: Peripheral nerve injuries are common, with approximately 9,000 cases in the UK annually. Young working individuals are predominantly affected, leading to significant health and social implications. Functional recovery is often poor with impaired hand sensation, reduced motor function and pain and cold intolerance. Where a nerve gap exists, nerve grafting remains the gold-standard treatment but creates a second surgical site, sensory deficit at the donor site, possible neuroma formation and has limited availability. Current commercially available synthetic and resorbable nerve conduit alternatives are reported to be rigid and inflexible. This study will set out to examine the first-in-man use of a new nerve conduit device ‘Polynerve’ to repair small nerve gaps in digital sensory nerves of the hand. Polynerve is a degradable co-polymer of poly-ε-caprolactone and poly-l-lactic acid, which is shaped as a cylinder that has greater tensile strength, flexibility and less acidic degradation compared with current commercially available synthetic nerve conduits. In addition, it has a novel micro-grooved internal lumen that aids Schwann cell ingress and alignment to improve nerve regeneration.
Methods: In total, 17 eligible participants will be recruited to undergo repair of a transected sensory nerve of the hand using the Polynerve device. All participants that receive the nerve conduit device will be followed for a period of 12 months post-surgery. The primary endpoint is safety of the device and the secondary endpoint is degree of sensory nerve regeneration through the conduit assessed using standard sensory testing (2-PD, WEST monofilament testing and locognosia).
Discussion: The ‘UMANC’ trial is a single-centre UK-based, prospective, unblinded, phase I clinical trial of a novel nerve conduit device. We aim to demonstrate the safety of Polynerve as a synthetic, biodegradable nerve conduit and improve the treatment options available to patients with significant nerve injuries.
Registration: Clinicaltrials.gov: NCT02970864 ; EudraCT: 2016-001667-37.
... Type I collagen along with type III are the important types which constitutes the nerves and thus play an important role in peripheral nerve regeneration (Keilhoff et al., 2003). Collagen has been used for fabrication of nerve conduits and widely experimented over the past years due to its high biocompatibility and minimal foreign body reaction ( Colin and Donoff, 1984;Archibald et al., 1991;Yao et al., 2010). Though it degrades very fast in the body, its degradability can be controlled by crosslinking with certain ...
Treating peripheral nerve injury is a major clinical challenge owing to the limited ability of the peripheral nerves to regenerate itself. Conventional treatment methods such as coaptation, autografting and xenografting possess inherent drawbacks such as reduced nerve stretching capacity, neuroma formation etc. To overcome these limitations, artificial nerve grafts employing natural or synthetic biomaterials are being developed. Axonal guidance structures and luminal fillers such as Schwann cells, stem cells, or nerve growth factors are also incorporated in the grafts to enhance their functionality. This paper reviews some of the biomaterials used in peripheral nerve regeneration, various modifications incorporated in them and their efficiency in aiding nerve repair.
... Although the median CMAP amplitude area recorded in the CNG[F]s ranged slightly above that one recorded from the corrCNG[F] reconstructed forelimbs, motor skills returned to a higher rate in the second group. This indicates again that CMAP amplitude recovery is not necessarily accompanied by precise regeneration of all motor axons (Archibald et al., 1991;Fugleholm et al., 2000;Valero-Cabre et al., 2001;Navarro and Udina, 2009;Pfister et al., 2011). Additionally, it demonstrates that also stimulation of finally misdirected axons contributes to the value of CMAP amplitude areas and these axons do not compulsorily lead to regeneration of especially fine motor skills (Galtrey and Fawcett, 2007). ...
Tension-free surgical reconstruction of transected digital nerves in humans is regularly performed using autologous nerve grafts (ANGs) or bioartificial nerve grafts. Nerve grafts with increased bendability are needed to protect regenerating nerves in highly mobile extremity parts. We have recently demonstrated increased bendability and regeneration supporting properties of chitosan nerve guides with a corrugated outer wall (corrCNGs) in the common rat sciatic nerve model (model of low mobility). Here, we further modified the hollow corrCNGs into two-chambered nerve guides by inserting a perforated longitudinal chitosan-film (corrCNG[F]s) and comprehensively monitored functional recovery in the advanced rat median nerve model. In 16 adult female Lewis rats, we bilaterally reconstructed 10 mm median nerve gaps with either ANGs, standard chitosan nerve guides (CNGs), CNGs (CNG[F]s), or corrCNG[F]s (n = 8, per group). Over 16 weeks, functional recovery of each forelimb was separately surveyed using the grasping test (reflex-based motor task), the staircase test (skilled forelimb reaching task), and non-invasive electrophysiological recordings from the thenar muscles. Finally, regenerated tissue harvested from the distal part of the nerve grafts was paraffin-embedded and cross-sections were analyzed regarding the number of Neurofilament 200-immunopositive axons and the area of newly formed blood vessels. Nerve tissue harvested distal to the grafts was epon-embedded and semi-thin cross-sections underwent morphometrical analyses (e.g., number of myelinated axons, axon and fiber diameters, and myelin thicknesses). Functional recovery was fastest and most complete in the ANG group (100% recovery rate regarding all parameters), but corrCNG[F]s accelerated the recovery of all functions evaluated in comparison to the other nerve guides investigated. Furthermore, corrCNG[F]s supported recovery of reflex-based grasping (87.5%) and skilled forelimb reaching (100%) to eventually significantly higher rates than the other nerve guides (grasping test: CNGs: 75%, CNG[F]s: 62.5%; staircase test: CNGs: 66.7%, CNG[F]s: 83.3%). Histological and nerve morphometrical evaluations, in accordance to the functional results, demonstrated best outcome in the ANG group and highest myelin thicknesses in the corrCNG[F] group compared to the CNG and CNG[F] groups. We thus clearly demonstrate that corrCNG[F]s represent promising innovative nerve grafts for nerve repair in mobile body parts such as digits.
... Initially, both appeared to have only a limited benefit on nerve regeneration (Eppley and Delfino, 1988;Ruskin, 1991), probably due to the lack of advanced micromanufacturing technologies, but now their use appears indispensable. Collagen prostheses are known to promote nerve regeneration by increasing re-myelination and neurite extension in rats, and improving lost sensation in humans, compared to autografting (Archibald et al., 1991). On bridging a gap of 20 mm in a rat sciatic nerve, collagen prostheses had significantly higher numbers and diameters of regenerated myelinated axons compared to autografts (Yoshii et al., 2001). ...
Peripheral nerve injuries are relatively common and can be caused by a variety of traumatic events such as motor vehicle accidents. They can lead to long-term disability, pain, and financial burden, and contribute to poor quality of life. In this review, we systematically analyze the contemporary literature on peripheral nerve gap management using nerve prostheses in conjunction with physical therapeutic agents. The use of nerve prostheses to assist nerve regeneration across large gaps (> 30 mm) has revolutionized neural surgery. The materials used for nerve prostheses have been greatly refined, making them suitable for repairing large nerve gaps. However, research on peripheral nerve gap management using nerve prostheses reports inconsistent functional outcomes, especially when prostheses are integrated with physical therapeutic agents, and thus warrants careful investigation. This review explores the effectiveness of nerve prostheses for bridging large nerve gaps and then addresses their use in combination with physical therapeutic agents.
... Tubulization alone was able of promoting nerve regeneration and functional recovery, although these results are found in 16 weeks (Beigi et al., 2014;di Summa et al., 2011). Literature points evidences of tubulization using stem cells derived from other tissues, such as axonal regeneration at 2 and 6 weeks (di Summa et al., 2011;Hadlock et al., 2000), electrophysiological improvement of tissue in 4 weeks (Archibald et al., 1991) and improved motor function at 4 and 6 weeks after peripheral nerve injury (Pereira Lopes et al., 2006 ...
Peripheral nerve injury is an important cause of incapability and has limited available treatment. Autologous donor nerve implant is the golden standard treatment, however, may cause secondary deficits. Stem cells show positive results in preclinical settings, preserving tissue and function. We tested the efficacy of stem cells derived from human exfoliated deciduous teeth seeded in poly (lactide-co-glycolide) scaffolds in sciatic nerve transection model. Seventy-two adult male Wistar rats had 7-mm nerve gap bridge using scaffolds with (or without) stem cells. Animals were randomly divided into: sham-operated; sham-operated without scaffold; sham-operated + scaffold + stem cells; sciatic transection + no treatment; sciatic transection + acellular scaffolds; sciatic transection + scaffold + stem cells. Sciatic Functional Index and Ladder Rung Walking tests were performed before (−1), 14 and 28 days after surgery. Morphometric nerve measurement and muscle weights were assessed. Scaffolds with stem cells improved function in Sciatic Functional Index. Acellular scaffold was effective, promoting functional recovery and nerve regeneration following nerve injury. Scaffolds provide better nerve regeneration and functional recovery after sciatic transection. Despite cell therapy promoting faster recovery after sciatic transection in the Sciatic Index Score, stem cells did not improve functional and morphological recovery after nerve injury. This is the first study testing the potential use of scaffolds combined with stem cells in the early stages after injury. Scaffolds with stem cells could accelerate nerve recovery and favor adjuvant therapies, evidencing the need for further studies to increase the knowledge about stem cells’ mechanisms.
... It can be used as both conduit material as well as luminal filler. One of the earlier successful studies using collagenbased nerve guide conduits showed that it was as effective as nerve autograft for repairing short sub-critical nerve gap (4 mm) in rat (Archibald et al., 1991;Li et al., 1992). More recent endeavors have been focused on treating critical nerve defect by using collagen-based scaffold as cell delivery platform to improve nerve regeneration. ...
Nerve injuries can be life-long debilitating traumas that severely impact patients' quality of life. While many acellular neural scaffolds have been developed to aid the process of nerve regeneration, complete functional recovery is still very difficult to achieve, especially for long-gap peripheral nerve injury and most cases of spinal cord injury. Cell-based therapies have shown many promising results for improving nerve regeneration. With recent advances in neural tissue engineering, the integration of biomaterial scaffolds and cell transplantation are emerging as a more promising approach to enhance nerve regeneration. This review provides an overview of important considerations for designing cell-carrier biomaterial scaffolds. It also discusses current biomaterials used for scaffolds that provide permissive and instructive microenvironments for improved cell transplantation.
... Neurogen ® (figure 4(a)) is a semi-permeable, collagen I nerve guide with a degradation period of 9-12 months. Pre-clinical studies have shown success in defects up to 40 mm [86], while clinical studies have shown results comparable to an autograft in defects up to 20 mm [87]. Neurolac ® (figure 4(b)) is a biodegradable, transparent, nerve guide made from PLCL and degrades within approximately 16-months. ...
After many decades of biomaterials research for peripheral nerve regeneration, a clinical product (the nerve guide), is emerging as a proven alternative for relatively short injury gaps. This review identifies aspects where 3D printing can assist in improving long-distance nerve guide regeneration strategies. These include (1) 3D printing of the customizable nerve guides, (2) fabrication of scaffolds that fill nerve guides, (3) 3D bioprinting of cells within a matrix/bioink into the nerve guide lumen and the (4) establishment of growth factor gradients along the length a nerve guide. The improving resolution of 3D printing technologies will be an important factor for peripheral nerve regeneration, as fascicular-like guiding structures provide one path to improved nerve guidance. The capability of 3D printing to manufacture complex structures from patient data based on existing medical imaging technologies is an exciting aspect that could eventually be applied to treating peripheral nerve injury. Ultimately, the goal of 3D printing in peripheral nerve regeneration is the automated fabrication, potentially customized for the patient, of structures within the nerve guide that significantly outperform the nerve autograft over large gap injuries.
... Early guides were made of Silicone and were not biodegradable; 9 they supported regeneration but were considered responsible for stenosis of the regenerated nerve and their removal was required in some instances. 10,11 Degradable guides were developed using poly(glycolic acid), 12 treated bovinecollagen, 13 or other biodegradable polymers. 14 An overview of the clinical outcome in peripheral nerve gap injuries showed that artificial nerve guides perform at least as good as autografts in gaps not longer than 20 mm, bearing the significant advantage of no donor site morbidity. ...
Background We describe the development of a new surgical procedure to be used in the treatment of disruptive brachial plexus (BP) lesions. It is centered on an artificial device designed to assist nerve regeneration by providing a confined and protected environment. Nerve fibers can repair inside the device, while the adverse massive scar-tissue formation is limited to the outside of the device.
Methods Steps in the development of the procedure were (1) definition of the rationale, (2) design of the device, (3) choice of an in vivo translational model, (4)refinement of the surgical procedure, and (5) performance of an in vivo pilot study as a proof of concept. An interdisciplinary team from several laboratories was involved in this work over a period of 6 years.
Results Results showed the absence of significant scar tissue in the regenerate and the presence of myelinated fibers aligned proximodistally between the stumps. This surgical approach can be seen not only as a definitive treatment but also as an early examination and stabilization before some different surgery will be later performed. It may also be used as additional protection for traditional surgery like end-to-end coaptation.
Conclusions We conclude that the availability of a suitable device-assisted early treatment, even if not to be considered definitive, could help in addressing the BP lesions at an earlier stage and this may improve the final outcome. Our evidence justifies further experimentation on this approach.
... Scaffolds can be incorporated with nerve-compatible biomaterials, neurotrophic factors, and/or support cells. Common biomaterials incorporated into conduits include collagen, fibronectin, laminin, alginate, silk, Matrigel, and fibrin (Archibald et al., 1991;Akassoglou et al., 2002;Verdu et al., 2002;Yang et al., 2007;Alluin et al., 2009). These materials have all demonstrated an allowance for increased axonal regeneration and can provide an adherent substance on which regenerating nerve may migrate. ...
Damage to peripheral nerve tissue may cause loss of function in both the nerve and the targeted muscles it innervates. This study compared the repair capability of engineered nerve conduit (ENC), engineered fibroblast conduit (EFC), and autograft in a 10-mm tibial nerve gap. ENCs were fabricated utilizing primary fibroblasts and the nerve cells of rats on embryonic day 15 (E15). EFCs were fabricated utilizing primary fibroblasts only. Following a 12-week recovery, nerve repair was assessed by measuring contractile properties in the medial gastrocnemius muscle, distal motor nerve conduction velocity in the lateral gastrocnemius, and histology of muscle and nerve. The autografts, ENCs and EFCs reestablished 96%, 87% and 84% of native distal motor nerve conduction velocity in the lateral gastrocnemius, 100%, 44% and 44% of native specific force of medical gastrocnemius, and 63%, 61% and 67% of native medial gastrocnemius mass, respectively. Histology of the repaired nerve revealed large axons in the autograft, larger but fewer axons in the ENC repair, and many smaller axons in the EFC repair. Muscle histology revealed similar muscle fiber cross-sectional areas among autograft, ENC and EFC repairs. In conclusion, both ENCs and EFCs promoted nerve regeneration in a 10-mm tibial nerve gap repair, suggesting that the E15 rat nerve cells may not be necessary for nerve regeneration, and EFC alone can suffice for peripheral nerve injury repair.
Oral diseases have emerged as one of the leading public health challenges globally. Although the existing clinical modalities for restoration of dental tissue loss and craniomaxillofacial injuries can achieve satisfactory therapeutic results, they cannot fully restore the original complex anatomical structure and physiological function of the tissue. 3D printing of biological tissues has gained growing interest in the field of oral medicine with the ability to control the bioink component and printing structure for spatially heterogeneous repairing constructs, holding enormous promise for the precise treatment of oral disease. Particularly, collagen-based materials have been recognized as promising biogenic bioinks for the regeneration of several tissues with high cell-activating and biocompatible properties. In this review, we summarize 3D printing methods for collagen-based biomaterials and their mechanisms. Additionally, we highlight the animal sources of collagen and their characteristics, as well as the methods of collagen extraction. Furthermore, this review provides an overview of the 3D bioprinting technology for the regeneration of the pulpal nerve and blood vessels, cartilage, and periodontal tissue. We envision that this technique opens up immense opportunities over the conventional ones, with high replicability and customized function, which can ultimately promote effective oral tissue regeneration.
Graphical Abstract
Peripheral nerve injury (PNI) is frequent and many patients suffer lifelong disabilities in severe cases. Although the peripheral nervous system is able to regenerate, its potential is limited. In this study, we tested in a nerve regeneration model in rat the potential beneficial effect of a short mimetic peptide, named PSELT, which derives from SELENOT, an essential thioredoxin‐like selenoprotein endowed with neuroprotective and antioxidant activities. For this purpose, the right facial nerve of female Long–Evans rats was axotomized then bridged with a free femoral vein interposition graft. PSELT (1 μM) was injected into the vein immediately and 48 h after the injury, and the effects observed were compared to those found after an end‐to‐end suture used as a gold standard treatment. Whisking behavior, electrophysiological potential, and histological analyses were performed 3 months after injury to determine the effects of these treatments. These analyses revealed that PSELT‐treated animals exhibit a better motor recovery in terms of protraction amplitude and velocity of vibrissae compared to control and end‐sutured nerve animal groups. Moreover, administration of PSELT following injury enhanced muscle innervation, axonal elongation, and myelination of newly formed nerve fibers. Altogether, these results indicate that a PSELT‐based treatment is sufficient to enhance facial nerve myelination and regeneration and could represent a new therapeutic tool to treat PNI. We demonstrate that administration of a novel selenopeptide named PSELT to a lesioned facial nerve improved its regeneration and ameliorated motor performances in rat, strongly suggesting that PSELT is a valuable therapeutic candidate to treat peripheral nerve injuries.
The nervous system consists of neurons and glial cells, blood vessels enfolded by
pericytes, and extracellular matrix (ECM) that have structurally and functionally
interactions with each other. In nervous system, blood brain barrier (BBB) is made by
endothelial tight junctions and controls the transportation of substances to the brain and
spinal cord. Astrocytes and pericytes around blood vessels contribute to regulation of
BBB functions. In mammalian embryos, the formation of the nervous and vascular
systems occurs coordinated with each other. As a result, angiogenesis and neurogenesis
are regulated by common cues and are closely linked, and it is possible that factors
derived from blood vessels and their microenvironment control adult neurogenesis.
The use of autografted nerve in surgical repair of peripheral nerve injuries (PNI) is severely limited due to donor site morbidity and restricted tissue availability. As an alternative, synthetic nerve guidance channels (NGCs) are available on the market for surgical nerve repair, but they fail to promote nerve regeneration across larger critical gap nerve injuries. Therefore, such injuries remain unaddressed, result in poor healing outcomes and are a limiting factor in limb reconstruction and transplantation. On the other hand, a myriad of advanced biomaterial strategies to address critical nerve injuries are proposed in preclinical literature but only few of those have found their way into clinical practice. The design of synthetic nerve grafts should follow rational criteria and make use of a combination of bioinstructive cues to actively promote nerve regeneration. To identify the most promising NGC designs for translation into applicable products, thorough mode of action studies, standardized readouts and validation in large animals are needed. We identify design criteria for NGC fabrication according to the current state of research, give a broad overview of bioactive and functionalized biomaterials and highlight emerging composite implant strategies using therapeutic cells, soluble factors, structural features and intrinsically conductive substrates. Finally, we discuss translational progress in bioartificial conduits for nerve repair from the surgeon’s perspective and give an outlook toward future challenges in the field.
Peripheral nerve injury is a frequent clinical problem and causes loss of motor and sensory functions for patients. Though some inherent regeneration capacity exists, this is often very limited in the cases of nerve tissue gaps. The design of a nerve conduit that results in successful nerve axon regeneration across these gaps and recovery of normal functions is a significant challenge for researchers in the fields of tissue engineering and regenerative medicine. This chapter includes a historic background to this research area and summarizes the current knowledge of various biomaterials, fabrication techniques and structural modifications, and the inclusion of cell and molecular therapies used in the production of nerve conduits.
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.
Peripheral nerve injury and regeneration continue to be extensively studied through basic science research using animal models. A translational gap remains between basic science research and clinical application. The importance of peripheral nerve regeneration in basic science research depends on the design of the study, the outcome measures, and the time of regeneration selected. The purpose of this article is to provide an overview of the importance of the design and outcome measures of peripheral nerve basic science research, for hand surgeons to understand for potential clinical translation.
Biopolymers are naturally found material and most of the materials are made in nature during the life cycles of plants, animals, fungi and bacteria. For any pharmaceutical formulation the two main ingredients are active pharmaceutical ingredient and excipients. As excipients in any kind of dosage form, the biopolymers play a vital function. Biopolymers are pharmacologically inert ingredients formulated along with the active ingredient to increase the volume; they help in the formulating dosage form and also simultaneously can improve the physicochemical parameters of the dosage form and so are widely used in the development of new drug delivery system. The biopolymers which are obtained from animal sources are usually non-toxic, biocompatible, stable and economic; and can control the release pattern of the drug. Natural polymers have more preponderant effects on fast dissolving tablets than synthetic polymers. Now-a-days, because of many problems regarding drug release and adverse effects of synthetic polymers, manufacturers are going towards using natural polymers. In this review article we mainly discussed about types of polymer, different alignment of natural polymer, advantages of natural polymers over synthetic polymers, drawbacks of natural polymers, mechanism of drug release of polymer, different methods of preparation of biopolymers, role of polymer in pharmaceutical industry and drug delivery systems. Keywords: Natural polymers, Chitin, Collagen, Sources, Preparation.
Le système nerveux périphérique a des capacités intrinsèques de régénération, mais la récupération motrice est insuffisante dans les cas de lésions les plus graves. Si la chirurgie reste l'approche thérapeutique "de référence", son efficacité est souvent limitée et diverses stratégies alternatives ont été développées, comme la thérapie à base de cellules souches. Cependant, on observe également une régénération incomplète des nerfs, et plusieurs approches visent à améliorer le potentiel de ces cellules. Parmi celles-ci, la modulation de l'environnement redox à l'aide de molécules antioxydantes est l'une des plus prometteuses. La sélénoprotéine T (SELENOT) est une oxydoréductase qui fait partie de la famille des sélénoprotéines, l'une des plus importantes familles d'enzymes antioxydantes dans l'organisme. Récemment, un peptide mimétique (PSELT) dérivé de SELENOT a montré un effet cardioprotecteur et neuroprotecteur dans un environnement nocif. La première partie de cette étude visait à évaluer le potentiel thérapeutique du PSELT dans un modèle de lésion du nerf facial chez le rat. Puis, nous avons recherché si le PSELT pouvait potentialiser l’effet thérapeutique d’une thérapie basée sur les cellules souches dérivées des capsules frontières (CF), une population de cellules souches située dans le derme qui a montré un fort potentiel neurogènique. Immédiatement et 48 heures après l'opération, le PSELT a été injecté dans une chambre de régénération d’origine veineuse dont le rôle était de réunir les deux rameaux d’un nerf facial axotomisé au niveau de la sortie du foramen stylo-mastoïdien. L’évaluation motrice des vibrisses, réalisée trois mois après la lésion, a montré que PSELT améliorait la récupération motrice de manière plus efficace comparée aux animaux témoins et à ceux ayant reçu un traitement chirurgical standard. Ces bénéfices cliniques ont été confirmés par des analyses électromyographiques et histologiques qui ont révélé une meilleure repousse nerveuse, une meilleure myélinisation et une réinnervation plus fine des muscles cibles. La combinaison de PSELT avec des cellules souches dérivées des CF n'a pas potentialisé la régénération nerveuse par rapport à la thérapie basée sur le PSELT seul, tant au niveau clinique qu'histologique. Dans l'ensemble, nos résultats indiquent que le PSELT offre un avantage thérapeutique pour traiter les lésions des nerfs périphériques par rapport aux approches de chirurgie et de thérapie cellulaire, et constitue donc un candidat précieux pour la médecine régénérative.
Nerve trunks are composed of bundles of axons organized by connective tissues into fascicles. Nonneural glial (Schwann) cells, fibroblasts, and endothelial cells are also present. Nerves can contain motor, sensory, or mixed fibers, depending on their target and related function. Motor fibers conduct electrical impulses at a more rapid rate than sensory fibers. The presence of myelin profoundly enhances the velocity of impulse transmission by allowing saltatory conduction. A myelinated fiber has a compact myelin sheath, a lipid and protein bilayer formed by a Schwann cell wrapped spirally around the axon. Individual Schwann cells meet along fibers at the nodes of Ranvier; here, small gaps in the myelin exist and the axon is surrounded only by the Schwann cell basal lamina. Small bundles of nonmyelinated axons can be encircled by a single Schwann cell, but no myelin is present.
Poly-3-hydroxybutyrate (PHB) conduits are an alternative to nerve autografting and support regeneration across long nerve gaps, although to suboptimal levels. The aim of this study was to improve these results by combining PHB with glial growth factor (GGF), enhancing nerve regeneration by contact guidance and an improved trophic microenvironment. Two and 4cm gaps in the rabbit common peroneal nerve were bridged using PHB-GGF conduits. The rate and quantity of axonal and Schwann cell (SC) regeneration were assessed by quantitative immunohistochemistry at 21, 42 and 63 days, and compared to empty and alginate filled conduits. Addition of GGF improved axonal and SC regeneration, which was sustained up to 63 days independent of gap length. The distance and quantity of axonal regeneration were increased by up to 53% and 4317% respectively. At 120 days axonal and SC regeneration within the PHB-GGF grafts remained superior to the controls resulting in enhanced motor organ reinnervation, as was demonstrated by an improved recovery of muscle mass compared to the controls. In both the short and long term studies the alginate filled conduits resulted in regeneration inferior to both the GGF and empty tubes. As a result alginate fibres were assessed in vitro and in vivo as an alternative to alginate hydrogel with a potential to deliver GGF. However, regeneration in vivo in alginate fibre filled conduits was inferior to conduits filled with alginate hydrogel. Polyhydroxyalkatone (PHA) was also evaluated as a conduit material, as GGF linkage and release from its walls is a feasible option. Four different PHA configurations were used to bridge a 1cm rat sciatic nerve gap. All 4 PHA configurations, accelerated axonal regeneration rate to 1mm/day versus 0.7mm/day with PHB conduits and resulted in a quantity of axonal regeneration superior to that seen in the autograft repairs. In conclusion, GGF improves axonal and SC regeneration across short and long gaps through PHB conduits, but alginate hydrogel appears to limit the trophic effect of GGF. Alginate fibres provide no improvement, however alginate's limitations may be overcome and regeneration further improved by using PHA as a bioconstruct releasing GGF into the conduit microenvironment.
Bridging critical‐sized defects in peripheral nerves to achieve functional recovery is a challenge for orthopedic and hand surgeons. Inadequate regeneration of peripheral nerve axons often results in long‐term partial or total sensory and/or motor impairment. Currently, the best treatment available for long‐gap peripheral nerve regeneration is autologous nerve transplantation, while the successful implementation of this approach requires for secondary surgery and donor nerves. The nerve guide conduit (NGC) serves as an alternative to autograft of nerve, as it connects the proximal and distal ends of nerve defects and provides physical and biochemical guidances for axon regeneration. Functionalized NGCs enhance nerve regeneration by providing neuroprotection, antioxidation, vascular regeneration enhancement, and immune regulatory effects. In this review, the authors summarize the latest advances in functional polymer‐based NGCs for peripheral nerve regeneration and present the perspectives on the development of peripheral NGCs for potential clinical applications. Functional polymer‐based nerve guide conduits connect the proximal and distal ends of nerve defects and provide physical and biochemical guidance for axon regeneration, which enhances nerve regeneration by providing neuroprotection, antioxidation, angiogenesis, and immune regulation effects, indicating their great potential in clinical application.
A number of natural polymer biomaterial‐based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross‐linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross‐linking agents. In this study, collagen‐based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross‐linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross‐linking is developed, by incorporating genipin into solution prior to freeze‐drying the NGCs. This leads to successful cross‐linking as demonstrated by higher cross‐linking degree and similar tensile strength of genipin cross‐linked conduits compared to formaldehyde cross‐linked conduits. Genipin cross‐linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross‐linked conduits, supports the viability of Schwann cells. Moreover, genipin cross‐linked conduits direct macrophages away from a pro‐inflammatory and toward a pro‐repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti‐inflammatory alternative to formaldehyde for cross‐linking clinical grade NGCs.
Painful neuroma formation is a common and debilitating sequela of traumatic or oncologic nerve amputations. Studies suggest that isolating transected nerve stumps within protective caps during amputation surgery or revision procedures may assist in preventing symptomatic nerve-end neuroma formation. This study evaluated the local effects of two porcine small intestine submucosa (pSIS) nerve caps of differing configurations on a terminal nerve end in an animal model. The tibial nerves of 57 Sprague Dawley rats were transected and transposed to the lateral hind leg. The nerves were treated with one of three SIS materials, including (i) a nerve cap with spiraling chambering, termed spiral nerve cap (SNC), (ii) a nerve cap with bifurcated chambers termed chambered nerve cap (CNC), or (iii) an open tube. The surgical control consisted of nerve stumps that were not treated. Overall tissue response, axonal swirling, optical density of axons, and behavioral pain response were quantified at 8 and 12 weeks postoperatively. There were no notable differences between the performance of the SNC and CNC groups. The pSIS nerve caps mitigated aberrant axonal regeneration and decreased neuroma formation and associated pain response. These findings suggest that nerve caps with internal chambers for axonal outgrowth may improve axonal alignment, therefore reducing the likelihood of symptomatic neuroma formation.
Impact statement
This study provides evidence for using nerve caps with internal structure on nerve stumps after amputation surgeries to reduce or prevent symptomatic neuromas. This study showed that porcine small intestine submucosa had a favorable remodeling profile and tissue response, illustrating that this device can be used to (i) minimize soft tissue attachments around the nerves that are capped, (ii) align axonal outgrowth to guide nerve regeneration away from aberrant neuroma formation, and (iii) act as a barrier between the nerve and external stimuli ultimately remodeling into a new soft tissue layer around the nerve stump thus decreasing symptomatic neuroma formation.
The successful introduction of innovative treatment strategies into clinical practise strongly depends on the availability of effective experimental models and their reliable pre-clinical assessment. Considering pre-clinical research for peripheral nerve repair and reconstruction, the far most used nerve regeneration model in the last decades is the sciatic nerve injury and repair model. More recently, the use of the median nerve injury and repair model has gained increasing attention due to some significant advantages it provides compared to sciatic nerve injury. Outstanding advantages are the availability of reliable behavioural tests for assessing posttraumatic voluntary motor recovery and a much lower impact on the animal wellbeing. In this article, the potential application of the median nerve injury and repair model in pre-clinical research is reviewed. In addition, we provide a synthetic overview of a variety of methods that can be applied in this model for nerve regeneration assessment. This article is aimed at helping researchers in adequately adopting this in vivo model for pre-clinical evaluation of peripheral nerve reconstruction as well as for interpreting the results in a translational perspective.
Peripheral nerve injury can considerably affect the daily life of affected people through reduced function and permanent deformation of the nerve. One of the conventional treatments used for the management of the disease is the application of autograft, which is recognized as a golden standard method; however, the process of gaining access to autograft has posed a significant challenge to its use. Nerve guidance channels (conduits), which are made in different methods, can act as an alternative therapy for patients that have undergone nerve injury; but, achieving these conduits has always been a major dilemma to be applied for patients with nerve injury. In this study, a novel conduit based on polymer blend nanocomposites of polyglycolic acid (PGA), collagen, and nanobioglass (NBG) were prepared by electrospinning technique and then compared with PGA/collagen and PGA conduits that were made in previous studies. Additionally, their various properties were characterized by scanning electron microscopy (SEM), X‐ray diffraction (XRD), contact angle, dynamic mechanical thermal analysis (DMTA), tensile strength, Fourier‐transform infrared (FTIR), and the porosity and degradation. The results showed that the mechanical, chemical, biocompatibility, and biodegradability properties of PGA/collagen/NBG conduits were more favorable in comparison with other materials. According to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and 4′,6‐diamidino‐2‐phenylindole (DAPI) staining technique, nanofibrous electrospun PGA/collagen/NBG conduits are more suitable for cell adhesion and proliferation in comparison with either PGA or PGA/collagen conduits and can have potential for nerve regeneration.
Contemporary reconstructive modalities focus on breast anatomy and attempt to reconstruct breasts that are soft, of adequate shape, size, and symmetry. However, a functional component, i.e. sensation, has largely been ignored. Flap neurotization addresses this shortcoming. While we are still in search of the ideal surgical technique to achieve this goal, a novel approach that limits nerve harvest to the sensory branch only, thus, minimizing abdominal donor‐site morbidity, is presented.
Trauma-associated peripheral nerve defect is a widespread clinical problem. Autologous nerve grafting, the current gold standard technique for the treatment of peripheral nerve injury, has many internal disadvantages. Emerging studies showed that tissue engineered nerve graft is an effective substitute to autologous nerves. Tissue engineered nerve graft is generally composed of neural scaffolds and incorporating cells and molecules. A variety of biomaterials have been used to construct neural scaffolds, the main component of tissue engineered nerve graft. Synthetic polymers (e.g. silicone, polyglycolic acid, and poly(lactic-co-glycolic acid)) and natural materials (e.g. chitosan, silk fibroin, and extracellular matrix components) are commonly used along or together to build neural scaffolds. Many other materials, including the extracellular matrix, glass fabrics, ceramics, and metallic materials, have also been used to construct neural scaffolds. These biomaterials are fabricated to create specific structures and surface features. Seeding supporting cells and/or incorporating neurotrophic factors to neural scaffolds further improve restoration effects. Preliminary studies demonstrate that clinical applications of these neural scaffolds achieve satisfactory functional recovery. Therefore, tissue engineered nerve graft provides a good alternative to autologous nerve graft and represents a promising frontier in neural tissue engineering.
La Ingeniería de Tejidos (IT) constituye un área multidisciplinaria que involucra varias ramas del conocimiento, tales como la biología, la medicina, la bioingeniería, la química, entre otras; su fin último es lograr la recuperación de un tejido u órgano con funcionalidad reducida a causa de enfermedades, accidentes u otros factores. La IT llega donde la medicina por sí sola no es suficiente para el mejoramiento de la calidad de vida de pacientes afectados.
El interés del libro Ingeniería de tejidos y aplicaciones presenta al lector, familiarizado o no con la aplicación de la ingeniería en problemas biomédicos, una perspectiva analítica recurriendo a fuentes originales, los conceptos básicos de las diferentes estructuras, técnicas y aplicaciones basadas en la ingeniería de tejidos, que se han utilizado en el tratamiento de diversas patologías o afecciones. Asimismo, se profundiza en conceptos como andamios tridimensionales, matrices descelularizadas, biomateriales y tipos de injertos, así como el trasfondo biológico de sus mecanismos de actuación en tejidos cardiovascular, nervioso, de piel y óseo.
Tissue Engineering (TE) is a multidisciplinary area that involves many branches of knowledge such as biology, medicine, bioengineering, chemistry, etc. The TE aims to restore the organ or tissue with reduced functionality due to illness, accident or other external factors. The TE overcomes the limitations of the medicine related with improving quality of life in patients.
The goal of the book Tissue Engineering and Applications is to show to the expert and inexperienced reader, an analytical perspective of the treatments used for a wide range of diseases, based on well supported evidence in therapeutic techniques and applications of TE reported in scientific literature. This book delves into concepts such as scaffolding, decellularized matrix, biomaterials and implants, as well as the biological support of the actuation mechanisms in cardiovascular, neural, skin and bone tissues.
Peripheral nerve injury is a common disease that affects more than 20 million people in the United States alone and remains a major burden to society. The current gold standard treatment for critical-sized nerve defect is autologous nerve graft transplantation; however, this method is limited in many ways and does not lead to satisfactory outcomes. The limitations of autografts have prompted investigations into artificial neural scaffolds as replacements, and some neural scaffold devices have progressed to widespread clinical use, scaffold technology overall has yet to be shown to be consistently on par with or superior to autografts. Recent advances in biomimetic scaffold technologies have opened many new and exciting opportunities, and novel improvements in material, fabrication techniques, scaffold architecture, and lumen surface modifications that better reflect biological anatomy and physiology have all independently been shown to benefit overall nerve regeneration. Furthermore, biomimetic features of neural scaffolds have also been shown to work synergistically with other nerve regeneration therapy strategies such as growth factor supplementation, stem cell transplantation, and cell surface glycoengineering. This review summarizes the current state of neural scaffolds, highlights major advances in biomimetic technologies, and discuss future opportunities in the field of peripheral nerve regeneration.
Introduction:
The safety and efficacy of using artificial collagen nerve conduits filled with collagen filaments to treat nerve defects has not been fully studied in humans. We conducted a multicenter, controlled, open-label study to compare the safety and efficacy of artificial nerve conduit grafts with those of autologous nerve grafts.
Methods:
We included patients with a sensory nerve defect of ≤30 mm, at the level of the wrist or a more distal location, with the first-line surgical methods selected according to a patient's preference. We compared sensory recovery using static two-point discrimination and adverse events between the artificial collagen nerve conduit and autologous nerve grafting.
Results:
The artificial nerve conduit group included 49 patients, with a mean age of 42 years and nerve defect of 12.6 mm. The autologous nerve graft group included 7 patients, with historical data of an additional 31 patients, with a mean age of 36 years and nerve defect of 18.7 mm. The rate of recovery of sensory function at 12 months was 75% (36/49) for the artificial nerve conduit group and 73.7% (28/38) in the autologous nerve group. No serious adverse events directly associated with use of the artificial nerve conduit were identified.
Conclusions:
The treatment of nerve defects ≤30 mm using artificial collagen nerve conduits was not inferior to treatment using autologous nerve grafts. Based on our data, the new artificial collagen nerve conduit can provide an alternative to autologous nerve for the treatment of peripheral nerve defects.
Synthetic nerve guidance channels are used to better understand the cellular and molecular events controlling peripheral nerve regeneration. In the present study, the contribution of wound-healing molecules to peripheral nerve regeneration was assessed by varying the molecular weight cutoff of the tubular membrane. Nerve regeneration through polysulfone tubular membranes with molecular weight (Mw) cutoffs of 10(5) and 10(6) Da was analyzed in a transected hamster sciatic nerve model. Cohorts of 6 animals received tubes of either type for 4 or 8 weeks with the distal end of the polymer tube capped. Other cohorts of 6 animals received tubes of either type for 4 weeks with the distal nerve stump secured within the guidance channel so as to create a 4 or 8 mm gap between both nerve stumps. Both types of channels contained regenerated tissue cables extending to the distal end of the guidance channel at both 4 and 8 weeks in the absence of a distal nerve stump. The cables regenerated in the 10(5) Da channels were composed of nerve fascicles surrounded by a loose epineurial sheath, whereas those regenerated in the 10(6) Da channels were composed mainly of granulation tissue. The numbers of myelinated and unmyelinated axons were significantly greater in the 10(5) Da than in the 10(6) Da channels at both 4 and 8 weeks. Both types of channel contained regenerated tissue cables with numerous nerve fascicles when the distal nerve stump was present with either gap length. However, when the gap distance was 8 mm, the 10(6) Da channels contained significantly fewer myelinated axons than the 10(5) Da channels. The present study reveals that the Mw cutoff of a semipermeable guidance channel strongly influences the outcome of peripheral nerve regeneration, possibly by controlling the exchange of molecules between the channel's lumen and the external wound-healing environment. These results suggest that the wound-healing environment secretes humoral factors that can either promote or inhibit the nerve-regeneration process.
We have investigated the repair of peripheral nerves in animal models using tubular guiding conduits. The materials used to fabricate the nerve conduits and their physicochemical and mechanical characteristics can influence the extent, rate and morphology of regeneration. Permeability of the conduit membranes is one parameter which seems to play an important role in nerve regeneration. In the present study, two types of nerve conduits were developed from bovine tendon collagen with distinctly different permeabilities. The permeability of the conduit membranes was determined by diffusion of various sized molecules across these membranes. One type of conduit had pores which only allowed small molecules such as glucose to pass (small pore collagen conduits). The other type had pores which were readily permeable to macromolecules such as bovine serum albumin (large pore collagen conduits). The large pore collagen conduits supported nerve regeneration to a greater degree than the small pore collagen conduits when tested in mice to bridge 4 mm gaps of the sciatic nerve. Studies in rats and primates suggested that large pore collagen conduits worked as effectively as nerve autografts in terms of physiological recovery of motor and sensory responses. The results of in vitro and in vivo studies of these conduits represent a significant step towards our specific aim of developing suitable off-the-shelf prostheses for clinical repair of damaged peripheral nerves.
This volume presents a solid roster of Japanese investigators and their work in the area of connective tissue, a field that, after a slow start, has been increasingly cultivated in Japan since 1960. Regrettably, much of this work has not entered the general literature and it is to be hoped that the present offering will help to correct this imbalance. However, the last quoted papers are from 1971, which is an indication of considerable publication lag.This is a collection of research papers, eclectic in nature, dealing with more or less familiar topics, although work on the mucopolysaccharides of the otolaryngological division seems to have been preempted by Japanese workers. One finds some of the familiar shibboleths. Study of ultrastructure by the approved methods of fixation and staining appears to have reached a point of diminishing conceptual returns—in the West as well as in the Far East.This is a
We have developed a quantitative model which allows assessment of the number of primary motor and sensory neurons which regenerate an axon through a tubular prosthesis bridging a 4–5 mm gap of peripheral nerve (1). Labeled somas of motor and sensory neurons are quantified following retrograde transport of horseradish peroxidase (HRP) applied to the distal stump beyond the bridged transection site. The number of myelinated axons at mid-guide level is also quantified. We have studied biodegradable and nin-biodegradable materials as well as the effects on nerve regeneration of basement membrane materials, and specific proteins added to the nerve guide., Adding a laminin-oontaining gel (Matrigel, Collaborative Research, MA) to the lumen of polyethylene tubes increases the rate of nerve regeneration as well as the maximum distance that can be successfully bridged. Similar effects are seen with collagen-based nerve guides when the laminin-cxitaining gel is incorporated into the tube wall. Addition of highly purified acidic fibroblast growth factor (aFGF) to the lumen of polyethylene tubes filled with collagen significantly increases the number of myelinated axons at mid-tube level and the number of primary sensory neurons labeled with HRP.
We coxpared collagen-based nerve guides of different wall porosities. Tubes freely permeable to bovine serum albumin (BSA) contain significantly more myelinated axons (4 week survival) than identical collagen tubes permeable to glucose-size molecules. These and additional experiments demonstrate that manipulation of the extracellular microenvironment of regenerating PNS axons can significantly enhance the early regenerative response.
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.
Single nerve fiber recordings were obtained from traumatically induced neuromas of the superficial radial nerve in baboons 1–7 months after injury. Eight to 18% of the fibers had spontaneous activity, and 67% of these were unmyelinated. Myelinated as well as unmyelinated fibers responded to mechanical stimulation of the neuroma whereas no fibers responded to similar stimulation of the normal nerve. Apparent crosstalk of action potential activity between fibers at the neuroma was observed which could be due to electrical coupling, though retrograde sprouting is another possible explanation. These abnormalities in neural activity originating from a neuroma in the primate are qualitatively similar to those noted in other species and may provide an explanation for certain abnormal sensory phenomena associated with peripheral nerve injury.
Collagenases produced by human polymorphonuclear leukocytes, human lung fibroblasts, and rabbit pulmonary alveolar macrophages were compared in their ability to digest soluble native type I and type III collagens. While the fibroblast and macrophage collagenases attacked the two substrates at approximately equal rates, the leukocyte collagenase attacked type I collagen preferentially (15:1) in comparison to type III collagen. This was true with human or rabbit collagen substrates. Thus, proteolysis of collagen, particularly in acute inflammation, may have a significant role in controlling the types of collagen present in connective tissue.
In three patients sequential studies were performed of sensory and motor conduction after complete section and suture of the median nerve at the wrist and in one patient after partial section of the nerve. The sensory potential evoked by stimuli to digits III and I and recorded proximal to the suture line at the wrist appeared after a delay of three to four months, corresponding to a growth rate of 1.5-2.0 mm per day. From early in the course of regeneration the sensory potential was dispersed in 40 components. In the adult patient the cumulative amplitude increased for two years slowly and thereafter at a two times faster rate. Amplitude and tactile sensibility were normal after 40 months, but the sensory potential was still five times more dispersed than normal. The overall increase in the amplitude of the sensory potentials in children aged 10 and 12 years was three times faster than in adults. In the adults and in the children the maximum sensory conduction velocity was 10-25% of normal. It then increased at 3% per month during the first two years, and thereafter 10 times slower. Forty months after suture in the adults and 13-19 months after suture in the children the conduction velocity had reached 65-75% of normal. The pattern of discrete electrical activity during voluntary effort and the prolonged duration of motor unit potentials indicate persistent enlargement of the reinnervated motor units by peripheral sprouting. The sensory potential recovered five times faster after a compressive nerve lesion than after section and suture as seen in another patient with an affection of the ulnar nerve at the elbow. Normal tactile sensibility was attained 10 times faster than after section and suture. Maximum sensory and motor condution velocity recovered within one year from 60-70% to 80-90% of normal.
This is a report of a study in which cuffs of biodegradable copolymers were placed about ulnar and peroneal nerves in legs of ten adult mongrel dogs. The results were evaluated by clinical response, electromyographic observations, nerve conduction studies, and light microscopic examination.
A 22-year-old male was studied 3 1/2-4 1/2 years after a traumatic section-avulsion amputation of the left upper extremity at the level of the distal humerus. The arm was reattached after a cold ischemia time of 4-5 hours and good vascularization was obtained. The ulnar nerve was repaired early with an end-to-end juncture while the median and radial nerves were repaired after seven months delay using a combination of vascularized radial nerve and nonvascularized sural nerve grafts. Some intrinsic hand muscle function had recovered. Pin-prick and touch sensation was present in all digits, although localization of touch stimulation was poor. Evoked motor responses had recovered by 25-50% of control amplitude in ulnar-innervated and by 10-25% in median-innervated muscles. Amplitudes of sensory responses from digit V had recovered by 25% and from digits I and III by 1-5%. Fast-adapting touch receptors had become reinnervated. There was electrophysiological evidence of aberrant sensory regeneration and of abnormal connections between sensory and motor fibers. Digital blood flow measurements suggested the presence of vascular obstruction in vessels of the replanted upper extremity. However, the digital vasoconstriction during cold exposure indicated regeneration of sympathetic nerve fibers.
Arrays of chronically implanted electrodes were used to examine the time course of elongation and maturation of peripheral nerve fibers in the cat after crush of the tibial nerve in the proximal calf. Regeneration after crush alone was compared with crush 5 mm proximal to a tight constriction of the nerve. Regeneration was monitored by the progression of excitability along the electrode arrays on the tibial and plantar nerves. The sensitivity was sufficient to record the averaged activity in single nerve fibers allowing detection of the earliest regeneration. The diameters of the fastest regenerating fibers were estimated from the conduction velocity proximal to the site of crush. Both after crush alone, and after crush constriction, small myelinated fibers regenerated in front of large fibers. The rate of elongation after crush alone was 3.2 mm/day, whereas it was slower (P less than 0.02) distal to crush + constriction (2.2 mm/day). In both lesions, the extrapolated delay to onset of regeneration was 8 days. In observations up to 300 days after crush, maturation was delayed or impaired by the constriction, and the compound nerve action potential had a smaller amplitude and a dispersed shape. Transverse sections of nerves after crush + constriction showed a diminished number of large and an increased number of small fibers compared with crush alone, possibly due to persistent branching of regenerated fibers. After both crush alone and crush + constriction, regenerated fibers had similar g ratios, suggesting that myelination developed fully in fibers of diminished diameters.
The presence of a distal nerve segment is considered to be essential for peripheral nerve regeneration through impermeable synthetic guidance channels. The use of a perm-selective material may provide a more appropriate regenerating environment by allowing solute exchange across the wall of the channel. We compared perm-selective acrylic copolymer (AC) channels with impermeable silicone elastomer (SE) channels in terms of regeneration in the absence of a distal nerve stump. Cohorts of 6 animals received AC and SE channels for either 4 or 8 weeks, with the distal end of the polymer tube left open in half of the animals, and plugged with the same polymer ('capped') in the other half. Capped and uncapped AC channels contained regenerated nerve cables which extended fully to the distal end of the channel, whereas capped SE channels contained only 1 mm long granulomatous tissue cables, and uncapped SE channels showed small cables with only a few myelinated axons. The nerve cables regenerated in uncapped AC channels were smaller and contained fewer myelinated axons than those observed in capped AC channels. Capped AC channels sleeved with a tight-fitting silicone tube to render them impermeable, showed no regenerated tissue within their lumen. The use of a perm-selective channel may have allowed the influx of nutrients and growth factors from the external environment while concentrating factors released by the proximal nerve stump.
The degree of regeneration in surgically repaired sciatic nerves in rats was measured using a simple new electrophysiologic method: comparison of the size of nerve responses evoked by stimulation distal and proximal to the anastomosis. Five different repair procedures were evaluated. After simple end-to-end suture anastomosis, about 40% of the severed parent fibers regenerated past the suture line. The result was substantially improved when the anastomosis was covered with a newly designed thinwall silicone sheath which incorporated a narrow longitudinal slit. The presence of suture material at the point of anastomosis had no effect. Finally, regeneration across a 5-mm gap ensheathed in silicone (67%) was better than regeneration through a 5-mm autograft (45%).
The regeneration of the sciatic nerve in Wistar rats across a 10-mm gap, within a semipermeable chamber was examined. The sequence of regeneration can be summarized as follows: (i) formation of a fibrin-containing matrix; (ii) invasion of this matrix by fibroblasts, forming two populations defined by their relative position within the matrix; (iii) the appearance of blood vessels; (iv) bridge formation; and (v) colonization of the bridge by nerve fibers. After 4 weeks, the number of myelinated fibers and the regenerate surface were studied at midtube level. Two and four weeks after the operation, both myelinated and unmyelinated leading fibers were detected and their distances from proximal stump measured. The results approach those reported in the literature for impermeable tubes, but not those obtained for fully permeable ones.
An experimental reason for placing stumps of a transected nerve in an impermeable tube is that factors and soluble substances from the nerve stumps are pooled and separated from cells and soluble substances in the body in general. Previous work showed that certain parameters of regeneration were improved, however, when the impermeable tube was made completely permeable by cutting macroscopic holes in its side. To begin exploring the reasons for these improvements, we covered the holes in the permeable tubes with filters of two different pore sizes, and found that the improvements resulted when the pore size was large enough to allow both fluid and cells to exchange but not when the pore size allowed only fluid to exchange. These findings suggest that cells from the general connective tissue should be given consideration when designing experimental procedures to maximize the regeneration potential of regenerating axons.
Piezoelectric nerve guidance channels made of polyvinylidene fluoride (PVDF) were evaluated in a transected mouse sciatic nerve model. Poled PVDF channels were compared to unpoled PVDF channels after 4 and 12 weeks of implantation. In all animals, the proximal and distal nerve stumps were bridged by a continuous nerve cable. Nerves regenerated in poled channels contained a higher number of myelinated axons than those regenerated in unpoled channels at both time periods. We conclude that piezoelectric nerve guidance channels enhance peripheral nerve regeneration and provide a tool to investigate the influence of electrical activity on nerve regeneration.
The recovery of axons regenerated through silicone tube splices was studied with electron microscopic and morphometric methods. Regenerated nerves contained both myelinated and unmyelinated axons of near normal morphology. The number and diameter of axons increased with postoperative time, and size-frequency histograms demonstrated that regeneration occurred in all major fiber groups. Remyelination occurred between about 4 and 6 weeks. Some of the smallest regenerated axons had unusually thick myelin sheaths, but overall regenerated axons had a slightly thinner sheath compared with similar-size normal fibers, although the ratio of sheath thickness to axon size was within the normal limits of g = 0.65 to 0.8 by 6 weeks. Axons did not, however, regain their normal size within 10 months of surgery. This deficit was apparently the primary factor limiting conduction velocity in these regenerated axons.
Changes in conduction properties of axons regenerating across a 10-mm gap bridged by a silicone cuff were investigated from compound action potential responses. Compound action potentials were detected as early as 6 weeks after surgery, and were small and slowly conducted at maximum velocities of about 3 m/s. With longer regeneration time, the potentials increased in size, velocity, and complexity. Conduction velocities increased rapidly at first than slowly and asymptotically approached rates that were approximately 40% below normal after 10 months. One component of the compound action potential, the refractory period, decreased from 5 ms to near normal value after only 3 months. The time constant of excitation changed more rapidly, and after 2 months approximated values near those for controls. The properties of axons regenerated across an epineural suture with no gaps. The database for the time course of events established here will be useful in guiding studies using the silicone cuff technique as an in situ experimental chamber for studies of regeneration and remyelination.
The sciatic nerve of adult mice was transected and proximal and distal nerve stumps were sutured into a nontoxic bioresorbable nerve guide. Nerve guide lumens were either empty or filled with a gel containing 80% laminin and additional extracellular matrix components. Two weeks later cells in the L3 through L5 dorsal root ganglia and the ventral horn of the spinal cord were retrogradely filled with horseradish peroxidase. All animals with the laminin-containing gel but none with empty nerve guides displayed labeled cells. This suggests that the laminin-containing gel significantly hastened axonal regeneration in vivo.
In a primate model a histologic assessment of neuroma formation is reported. Three experimental groups were defined. Transected sensory nerves left adjacent to the incisional wound in an area of movement (wrist) were considered the control group. In the "proximally cut" group the same sensory nerves were positioned well proximal to the incisional wound. In the "muscle-implantation" group these nerves were placed in adjacent muscles. At 6 months a histologic assessment of the neuroma formation in the three experimental groups was carried out. Implantation of the sensory nerve into muscle significantly altered the regenerative potential of that nerve. The muscle completely surrounded the sensory nerve. The minimal neuroma that formed had significantly less scar tissue and contained nerve fibers that were of a smaller diameter and decreased density than either the control or the proximally cut group. There were no histologic differences between these latter two groups. However, regeneration into the overlying skin that was noted in the control neuromas was not seen in those nerves which had been proximally cut.
An in vivo preparation is presented to study the rate and time course of motor and sensory axonal regeneration. The cut ends of a transected sciatic nerve were inserted into each end of a 5-6 mm non-toxic and bioresorbable nerve guide tube to create a 4 mm nerve gap in adult mice. Subsequently, cell bodies in the ventral spinal cord and L3-L5 dorsal root ganglia that had regenerated axons across the gap were retrogradely labeled with horseradish peroxidase (HRP). The HRP was applied 3 mm distal to the nerve guide and was accessible only to axons that had regenerated through the nerve guide. Labeled cells were counted in 40 micron serial sections at 2, 4 and 6 weeks after initial nerve transection. The results indicate a significant increase in the number of labeled motor and sensory cell bodies over time. By 6 weeks after transection, approximately two thirds as many ventral horn motor cells and one third as many dorsal root ganglion sensory cells were labeled as in control non-transected animals. These data serve as a baseline to compare differential effects of additives to the nerve guide lumen in terms of sensory and motor neuron response.
One approach to repair of transected nerves is to attempt extrinsic guidance of axons across the gaps. We inserted the proximal and distal stumps of severed mouse sciatic nerves into opposite ends of biodegradable polyester tubes. The nerves and ensheathing tubes were examined after postoperative survival times of as long as 2 years. Myelinated fiber number in each successfully regenerated nerve was measured and correlated with the tube's residual lumen size. In selected regenerated nerves axonal sizes and myelin sheath widths were sampled and compared with control values. Swelling and deformation of tube walls occurred in nearly all tubes. Successful regeneration was obtained through more than half of the implants, and was more probable in tubes with larger initial lumens. Myelinated fiber number in regenerated nerves ranged from 231 to 3561 (normally 3900 to 4200); larger values again were found in tubes with larger initial lumens. Mean axonal areas in regenerated nerves were roughly half of normal, though myelin sheaths became appropriately thick. We concluded that the more biodegradable a tube, the more likely it was to incur distortion and luminal narrowing. Tube composition per se seemed of importance mainly as it related to maintenance of adequate luminal size over the length of the degrading tubes; luminal adequacy, not tube composition, seemed paramount in determining the extent of nerve regeneration.
Nontoxic, bioresorbable "nerve guide" tubes were used to bridge the transected optic nerves of adult rats. Nerve guides were fabricated as polymers of synthetic poly D,L-lactates with 2% triethyl citrate added as a plasticizer. The local environment was manipulated further by the addition of the proteins collagen, fibrinogen, and anti-Thy-1 antibody to the nerve guide lumens at the time of operation. Neovascular growth through the nerve guide lumens was quantified with the aid of a computer-controlled microscope. Neovascular growth was greater in the nerve guides to which proteins had been added, compared with initially empty nerve guides. These experiments demonstrated the effectiveness of these nerve guide tubes in supporting and directing neovascular growth in the mammalian central nervous system, and suggested that specific alterations of the local environment within the nerve guide lumen can affect the extent of neovascular growth.
Present techniques of nerve repair by suture are based on an anatomical approach. The severed layers of connective tissue are reapproximated. Another approach to nerve repair is to separate the specific cellular components of the peripheral nerve that contribute to fibrous healing and nerve regeneration. The perineurium normally separates the extrafascicular epineurium of mesodermal origin from the intrafascicular endoneurium of ectodermal origin. A cellular approach to nerve repair would use a tube around the fascicle as an artificial perineurium to separate fibrous healing from axonal regeneration until the perineurium reestablishes its continuity. Fascicular tubulization with polyglycolic acid tubes was studied in 25 rats. The polyglycolic acid tube is resorbed after the perineurium has reestablished its continuity. The repairs by fascicular tubulization demonstrated improved organization of the repair site compared to suture repairs. The diameter histograms of the regenerated myelinated axons were similar in repairs by tube and suture techniques. The total regenerated cross-sectional area of the myelinated axons was similar in the repairs by fascicular tubulization to repairs by fascicular suture.
An experimental model of a free, empty perineurial tube for use as a nerve graft is presented as an alternative to existing methods. The technique is described and the model compared with conventional nerve graft in the sciatic nerve of a rabbit. Results are evaluated with EMG studies, angiography, and histology. The experimental model compared favorably with the standard graft. Further avenues of investigation and clinical use are suggested.
This report describes the use of a porous polymeric sleeve (Gore-tex) to direct nerve fiber growth after axotomy. Select nerves of the triceps surae muscles in 5 adult cats were surgically isolated, sectioned, and crossed or self- reunited . A piece of Gore-tex, 15 mm in length, was compressed to 5 mm and sleeved over each distal nerve end. The appropriate proximal and distal ends were stitched together, and the Gore-tex stretched back to its original length over the suture junction. The effectiveness of the Gore-tex sleeve was assessed 4-15 months post-operatively. Electrophysiological measurements of muscle force and dorsal root volleys revealed a complete absence of unintended reinnervation and a regeneration that was more substantial for motor than sensory axons. Finally, serial histological cross-sections were prepared for each nerve above, below and at the cross union. There was no evidence of nerve tissue invading the Gore-tex wall.
Nerve grafting was performed in a series of patients, 81% of whom had associated severe soft tissue injuries in the area in which nerve grafting was done. Other factors that have been shown to have an adverse effect on nerve grafting results were analyzed and were not thought to be major factors influencing results. Results were worse than those of previous reports in which the initial injury was less severe. The initial soft tissue injury is very important in predicting how well a nerve graft will function. Nerve grafting is a valuable procedure even in the face of severe soft tissue injuries, since it alone can restore protective sensation.
Defects in a sectioned tibial nerve were bridged by a new method using a polyglactin mesh-tube and compared with conventional nerve grafting in the rabbit. The capability of healing was evaluated by morphometrical observations and repeated EMG-recordings. Only minor differences between the two different techniques were observed and the possible advantage of the polyglactin method is discussed.
Using the transected sciatic nerve model in adult mice, regeneration of a large bundle of axons organized into the form of a nerve with myelinated and unmyelinated axons, Schwann cells, fibroblasts, collagen, blood vessels, and connective tissue sheaths has been achieved with bioresorbable microtubular guidance channels over gaps of 5 mm in nonimmobilized animals. After 4-6 wks postoperatively, the regenerated nerve cable contains on the order of 40% as many myelinated axons as were measured in the proximal nerve stumps. With the channels used so far in this model, regenerating axons pass into the distal stump in about 3-6 wks postoperatively. The guidance channels used consist of synthetic polyesters and/or polyester composites including glycolic and lactic acid polymers, and polyesters derived from Krebs Cycle dicarboxylic acids. Inflammatory response to these materials has been minimal. Biodegradation/resorption rates can be controlled so as to be compatible with axon growth rates.
The superficial sensory branch of the radial nerve appears prone to develop painful neuromas out of proportion to its likelihood for injury. Based on cadaver dissections and intraoperative observations, an anatomical mechanism for this "predisposition" is suggested. Exit of this nerve beneath dense fascia and the tendons of brachioradialis and extensor carpi radialis longus provide a proximal tethering against which tension develops as the distal fixation point (neuroma) is pulled through the long excursion of wrist arc of motion. This long excursion and proximal tethering are not present anatomically for the dorsal cutaneous branch of the ulnar nerve nor the palmar cutaneous branch of the median nerve.
The regeneration of transected mouse sciatic nerves using semipermeable acrylic copolymer tubes to enclose both stumps has been qualitatively assessed from 1 to 30 weeks post-operative. Quantitative morphometric analysis of electron micrograph montages of complete transverse sections of the segment regenerated between stumps has permitted determinations of the percents of total area occupied by the various tissue constituents--blood vessels, epineurium, perineurium, endoneurium, myelinated axon/Schwann cell units, and unmyelinated axon/Schwann cell units. Significant differences were found in the total cross-sectional area of segments regenerated through tubes of 1.0 mm versus 0.5 mm internal diameters. Segments regenerated with the distal stump inserted in the tube contained significantly greater percentages of neural units and were significantly larger at 8 weeks post-operative compared to segments regenerated for 9-10 weeks with the distal stump avulsed. The morphometric method permits rapid quantitation of sizeable electron micrograph montages which at 1300 X permit all types of tissue components, including the unmyelinated axons, to be visualized.
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.
To evaluate the usefulness of nerve grafting we studied 38 patients having 11 median, 7 ulnar, and 33 digital nerve grafts. Group funicular (interfascicular) grafting using magnification was performed in all patients. We followed 12 patients with 8 median and 5 ulnar nerve grafts for at least one year and 18 patients with 27 digital nerve grafts for at least six months. Medical Research Council criteria were used for evaluation of nerve function. Results in our patients and in previously reported patients having nerve grafting or repair were compared. Sensory function following ulnar nerve grafting was significantly better than that following nerve repair. Sensory function following median and digital nerve grafting was as good as that following nerve repair. Motor function following ulnar nerve grafting was as good as that following nerve repair. Previously reported patients having median nerve repairs or grafts had significantly better motor function than our patients.
Consequences of disruption of the endoneurium and perineurium: Neuroma formation Nerves and Nerve Injuries
- S Sunderland
Sunderland, S. (1978) Consequences of disruption of the endoneurium and perineurium: Neuroma formation. In Sir S. Sunderland (ed): Nerves and Nerve Injuries. New York: Churchill Livingstone, pp. 188-200.
Evaluation of Sensibility of Re-education of Sensation in the Hand
- A. L. Dellon