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Exogenous matrix percursors promote functional nerve regneration across a 15-mm gap within a silicone chamber in the rat
Abstract
When silicone regeneration chambers are implanted empty, axonal regeneration fails if the interstump gap length is greater than 10 mm. Previous experiments using the 10-mm gap model demonstrated that regeneration success correlated with the dimension and/or consistency of the naturally formed acellular fibrin matrix. Both spatial and temporal parameters of regeneration could be stimulated through modifications of the fibrin matrix by prefilling the chambers at the time of implantation either with phosphate-buffered saline or plasma dialyzed against phosphate-buffered saline. In the present experiments, similar modification of matrix formation was found to promote successful regeneration across 15-mm and 20-mm interstump gap lengths. In addition, prefilling 15-mm-gap chambers with dialyzed plasma resulted in a 3.5-fold increase in the incidence of functional restitution detected at 8 weeks after implantation over the outcome with chambers prefilled with phosphate-buffered saline.
... [5][6][7][8][9] Although numerous studies have been yet performed on repairing nervous defect and developing an appropriate conduit for nerve growth, but just few investigations have been done on effectiveness of different materials on fibroblasts function, collagen production and scar formation. [1][2][3][4][5][6][7][10][11][12][13] In some of these studies, various materials such as muscle, fat tissue, amnion, vein have been used as biologic barriers or absolvable materials such as collagen and polyglycolic have been applied as conduit. [5][6][7]9,10,12,[14][15][16][17][18] Although most of them were effective in nerve repair, some adverse effects have been reported. ...
... Therefore, multiple investigations have been done for adjusting fibroblast activities and prevention scar formation. 5,6,8,9,[11][12][13]18,22 Numerous investigation have been designed on animal model for finding more effective materials as conduits for reconstructing nerves, so far. 7,9,12,[23][24][25] In this study, we evaluated silicon gel efficacy on rat sciatic nerve repair. ...
... More studies and molecular investigations on probable role of silicone on myelin production by Schwann cells In several studies, silicone has been compared with other materials for nerve reconstruction. 13,[15][16][17][18][20][21][22][23][24][25] For instance, Feng and colleagues evaluated nerve graft, silicone and chistosan-PLA for rat sciatic nerve reconstruction. They reported that nerve graft and chistosan-PLA were more effective than silicone. ...
Peripheral nerve repair is often complicated by fibroblastic scar formation, nerve dysfunction, and traumatic neuroma formation. Use of silicone may improve outcomes of these repairs. In this study, we tried to evaluate effectiveness of silicone gel on rats' sciatic nerve repair, axon regeneration and scar formation around and in the nervous tissues.
This experimental study was performed on 18 rats. They underwent bilateral sciatic nerve dissection. Then, right and left damaged sciatic nerves were sutured. In left side, silicone gel was applied. Two months later, both sides were evaluated regarding to myelin fiber diameter (µm), total fascicular area (mm(2)), axon diameter (µm), myelin thickness (µm), G- ratio (axon diameter/myelin thickness), connective tissue area, ratio of connective tissue area/fascicular area, neuroma and foreign body formation in liver and lungs and spleen reaction. Results of right and left sides were compared.
Silicone was significantly more effective in increasing myelin thickness in the side that silicone was applied) than the control side. It was not associated with inflammation, scar formation, granuloma, and neuroma formation. No foreign body reaction occurred in liver, spleen and lungs after silicone application; but axonal regeneration did not improve with after its use.
According to our findings, it seems that silicone application in the cases with significant complications or in the cases that nerve graft is not possible would be an ideal option.
... In the rat sciatic nerve, this critical distance measures > 10 mm, and in the primate ulnar nerve it is > 3 cm. 23,31,[47][48][49]52,[81][82][83] Critical distances can be overcome by adding exogenous growth factors and/or tissues to the tube environment. In the rat, a 13-mm gap can be successfully bridged using silicone chambers only when exogenous fibrin matrix precursors or a small segment of degenerated peripheral nerve is added to the conduit. ...
... In the rat, a 13-mm gap can be successfully bridged using silicone chambers only when exogenous fibrin matrix precursors or a small segment of degenerated peripheral nerve is added to the conduit. 23,40,47,48,[81][82][83] More specifically, some authors have demonstrated no evidence of regeneration within an empty or saline-filled 15-mm silicone tube (13-mm nerve gap) at 16 weeks, but the addition of a short, interposed, 2-mm nerve segment resulted in significant regeneration similar to that of an autograft. In the monkey, a 3-cm gap can be repaired with a bioabsorbable polyglycolic acid tube. ...
Object:
Segmental nerve defects pose a daunting clinical challenge, as peripheral nerve injury studies have established that there is a critical nerve gap length for which the distance cannot be successfully bridged with current techniques. Construction of a neural prosthesis filled with Schwann cells (SCs) could provide an alternative treatment to successfully repair these long segmental gaps in the peripheral nervous system. The object of this study was to evaluate the ability of autologous SCs to increase the length at which segmental nerve defects can be bridged using a collagen tube.
Methods:
The authors studied the use of absorbable collagen conduits in combination with autologous SCs (200,000 cells/μl) to promote axonal growth across a critical size defect (13 mm) in the sciatic nerve of male Fischer rats. Control groups were treated with serum only-filled conduits of reversed sciatic nerve autografts. Animals were assessed for survival of the transplanted SCs as well as the quantity of myelinated axons in the proximal, middle, and distal portions of the channel.
Results:
Schwann cell survival was confirmed at 4 and 16 weeks postsurgery by the presence of prelabeled green fluorescent protein-positive SCs within the regenerated cable. The addition of SCs to the nerve guide significantly enhanced the regeneration of myelinated axons from the nerve stump into the proximal (p < 0.001) and middle points (p < 0.01) of the tube at 4 weeks. The regeneration of myelinated axons at 16 weeks was significantly enhanced throughout the entire length of the nerve guide (p < 0.001) as compared with their number in a serum-only filled tube and was similar in number compared with the reversed autograft. Autotomy scores were significantly lower in the animals whose sciatic nerve was repaired with a collagen conduit either without (p < 0.01) or with SCs (p < 0.001) when compared with a reversed autograft.
Conclusions:
The technique of adding SCs to a guidance channel significantly enhanced the gap distance that can be repaired after peripheral nerve injury with long segmental defects and holds promise in humans. Most importantly, this study represents some of the first essential steps in bringing autologous SC-based therapies to the domain of peripheral nerve injuries with long segmental defects.
... 45,46 In fact, early in vivo studies suggested that SC proliferation and migration was inhibited due to the lack of fibrin cable formation in larger defects, thereby hindering nerve regeneration. 47,48 Although several approaches attempted to create aligned SC constructs to mimic bands of Büngner structures, mainly based on seeding of cells on structured surfaces, [21][22][23][24][25] we, to the best of our knowledge, for the first time report the successful establishment of 3D tissue-engineered bands of Büngner-like tissue structures by incorporating active mechanical stimulation in a 3D SC culture model. We showed that the type of tensile stress applied strongly influenced SC alignment, as static strain induced more pronounced alignment than cyclic strain. ...
Treatment of peripheral nerve lesions remains a major challenge due to poor functional recovery; hence, ongoing research efforts strive to enhance peripheral nerve repair. In this study, we aimed to establish three-dimensional tissue-engineered bands of Büngner constructs by subjecting Schwann cells (SCs) embedded in fibrin hydrogels to mechanical stimulation. We show for the first time that the application of strain induces (i) longitudinal alignment of SCs resembling bands of Büngner, and (ii) the expression of a pronounced repair SC phenotype as evidenced by upregulation of BDNF, NGF, and p75NTR. Furthermore, we show that mechanically aligned SCs provide physical guidance for migrating axons over several millimeters in vitro in a co-culture model with rat dorsal root ganglion explants. Consequently, these constructs hold great therapeutic potential for transplantation into patients and might also provide a physiologically relevant in vitro peripheral nerve model for drug screening or investigation of pathologic or regenerative processes.
... Incorporation of the fibrous, blood-clotting ECM protein fibrin 3 into NGCs has been shown to promote regeneration in long nerve gaps. 4,5 During regeneration in an empty NGC, a transient network of fibrin is formed in the inter-stump gap space from infiltrated fibrinogen. 6,7 The fibrin is subsequently used as a biophysical guide for nerve-related cells to migrate, evidently serving as an indispensable promoter of axonal regrowth. ...
Significance:
Exogenous extracellular matrix (ECM) proteins, such as fibrinogen and the thrombin-polymerized scaffold fibrin, are used in surgical repair of severe nerve injuries to supplement ECM produced via the injury response. Monitoring the dynamic changes of fibrin during nerve regeneration may shed light on the frequent failure of grafts in the repair of long nerve gaps.
Aim:
We explored whether monitoring of fibrin dynamics can be carried out using nerve guidance conduits (NGCs) containing fibrin tagged with covalently bound fluorophores.
Approach:
Fibrinogen was conjugated to a near-infrared (NIR) fluorescent dye. NGCs consisting of silicone tubes filled with the fluorescent fibrin were used to repair a 5-mm gap injury in rat sciatic nerve ( n = 6 ).
Results:
Axonal regeneration in fluorescent fibrin-filled NGCs was confirmed at 14 days after implantation. Intraoperative fluorescence imaging after implantation showed that the exogenous fibrin was embedded in the early stage regenerative tissue. The fluorescent signal temporarily highlighted a cable-like structure within the conduit and gradually degraded over two weeks.
Conclusions:
This study, for the first time, visualized in vivo intraneural fibrin degradation, potentially a useful prospective indicator of regeneration success, and showed that fluorescent ECM, in this case fibrin, can facilitate imaging of regeneration in peripheral nerve conduits without significantly affecting the regeneration process.
... It has been shown that each animal species has its own upper limit for the maximum nerve gap distance that can be effectively repaired by this type of empty acellular conduit (Lundborg et al., 1982a,b). In the rat sciatic nerve, this critical distance measures approximately 13 mm, and in the primate ulnar nerve, it is 3 cm (Williams et al., 1987;Hess et al., 2007). Nerve injuries with long segmental defects in humans pose a similar challenge in which gap lengths of more than 6 cm have extremely poor clinical outcomes (Pan et al., 2020). ...
Peripheral nerve injury (PNI) is found in a relatively large portion of trauma patients. If the injury is severe, such as with the presence of a long segmental gap, PNI can present a challenge for treatment. The current clinical standard of nerve harvest for the repair of long segmental gap PNI can lead to many potential complications. While other methods have been utilized, recent evidence indicates the relevance of cell therapies, particularly through the use of Schwann cells, for the treatment of PNI. Schwann cells (SCs) are integral in the regeneration and restoration of function following PNI. SCs are able to dedifferentiate and proliferate, remove myelin and axonal debris, and are supportive in axonal regeneration. Our laboratory has demonstrated that SCs are effective in the treatment of severe PNI when axon guidance channels are utilized. However, in order for this treatment to be effective, optimal techniques for cellular placement must be used. Thus, here we provide relevant background information, preclinical, and clinical evidence for our method in the treatment of severe PNI through the use of SCs and axon guidance channels.
... Parallel fibers are involved in the differentiation of stem cells and Schwann cells [42,66,67]. For nerve defects > 10 mm, it is difficult for fibrin cables to support nerve regeneration [68]. Therefore, in this study, the chitin conduit was filled with ACG to repair 15-mm sciatic nerve defects in rats. ...
Autologous nerve transplantation, which is the gold standard for clinical treatment of peripheral nerve injury, still has many limitations. In this study, aligned chitosan fiber hydrogel (ACG) grafted with a bioactive peptide mixture consisting of RGI (Ac-RGIDKRHWNSQGG) and KLT (Ac-KLTWQELYQLKYKGIGG), designated as ACG-RGI/KLT, was used as nerve conduit filler to repair sciatic nerve defects in rats.
Methods: Chitosan nanofiber hydrogel was prepared by a combination of electrospinning and mechanical stretching methods, and was then grafted with RGI and KLT, which are peptides mimicking brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), respectively. The physicochemical properties of ACG-RGI/KLT were fully characterized. In vitro, the distribution, proliferation, and secretory activity of Schwann cells were analyzed. Next, the in vivo repair potential for 15-mm rat sciatic nerve defects was examined. The recovery of regenerated nerve, muscle, and motor function was evaluated by neuromuscular histology, electrophysiology, and catwalk gait analysis.
Results: We first constructed directionally aligned chitosan nanofiber hydrogel grafted with RGI/KLT peptide mixture (ACG-RGI/KLT). ACG-RGI/KLT oriented the Schwann cells, and promoted the proliferation and secretion of neurotrophic factors by Schwann cells. At an early injury stage, ACG-RGI/KLT not only enhanced nerve regeneration, but also promoted vascular penetration. At 12 weeks, ACG-RGI/KLT facilitated nerve regeneration and functional recovery in rats.
Conclusions: Aligned chitosan nanofiber hydrogel grafted with RGI/KLT peptide provides an effective means of repairing sciatic nerve defects and shows great potential for clinical application.
... Peripheral nerves have a unique vascular network due to their anatomical compartmentalisation (Ubogu, 2013). This vascular network, the vasa nervorum, consists of two separate, functionally independent, but anastomosing, systems: an extrinsic epineurial system and an intrinsic endoneurial system (Wigley, 2008) The circulation of peripheral nerves derives from regional extrinsic vessels (EV) which give rise to branch radicular vessels (RV) that supply the intrinsic circulation of the vasa nervorum. The intrinsic circulation consists of longitudinally oriented vessels that run through the epineurium (Epi), down to the perineurium (Peri) and join with vessels in the endoneurium (Endo) via transperineurial connections (arrow). ...
There has been an increased interest in the development of nerve repair devices to improve peripheral nerve regeneration following injury. The aim of this study was to investigate whether nerve repair devices containing genetically modified cells overexpressing VEGF-A165 would augment regeneration. An in vitro proof-of-concept study was carried out to deliver marker genes (luciferase and eGFP) to a rat Schwann cell line (SCL4.1/F7) using a lentiviral vector. The transduced cells were used to produce engineered neural tissue and bioluminescence imaging was used to assess cell viability in the constructs. It was initially thought the presence of the luciferase gene in the expression cassette would allow real-time and sustained imaging of the cells in the engineered neural tissue. However, while bioluminescence imaging provided an indication of cell viability in vitro, it proved to be ineffective for in vivo and ex vivo imaging. Having established that SCL4.1/F7 cells were amenable to lentiviral transduction, a lentiviral vector delivering the VEGF-A165 gene was designed and produced. The VEGF-A165 produced by the transduced SCL4.1/F7 cells increased endothelial cell viability, migration and tube formation in vitro. It also increased SCL4.1/F7 cell proliferation and migration. SCL4.1/F7 cells overexpressing VEGF-A165 were found to increase endothelial cell network formation and neurite length in 3D co- culture models. Foetal human neural stem cells and rat adipose derived stem cells were also successfully transduced with the lentiviral vector delivering VEGF-A165. Based on the in vitro results, it was postulated that EngNT made from SCL4.1/F7 cells overexpressing VEGF-A165 implanted into a rat model of sciatic nerve injury would enhance regeneration. Unexpectedly, a pilot study revealed that this did not result in improved functional recovery or increased axon and blood vessel counts compared to controls. The results from this study highlight that attention needs to be paid to the dose and duration of expression of VEGF-A165 to optimise both its angiogenic and neurotrophic effects.
... In hollow tubes of various materials, it is crucial that a fibrin matrix is formed, facilitated by the tube to a certain extent, between the proximal and distal nerve ends inside the tube [32][33][34] . If the distance between the nerve ends is too long, no matrix is formed, hindering the regeneration process across a nerve defect [35][36][37][38][39] . This is observed also clinically and is considered in clinical practice concerning various conduits and other materials, such as nerve allografts 40 . ...
Electrospinning can be used to mimic the architecture of an acellular nerve graft, combining microfibers for guidance, and pores for cellular infiltration. We made electrospun nerve guides, from polycaprolactone (PCL) or poly-L-lactic acid (PLLA), with aligned fibers along the insides of the channels and random fibers around them. We bridged a 10 mm rat sciatic nerve defect with the guides, and, in selected groups, added a cell transplant derived from autologous stromal vascular fraction (SVF). For control, we compared to hollow silicone tubes; or autologous nerve grafts. PCL nerve guides had a high degree of autotomy (8/43 rats), a negative indicator with respect to future usefulness, while PLLA supported axonal regeneration, but did not outperform autologous nerve grafts. Transplanted cells survived in the PLLA nerve guides, but axonal regeneration was not enhanced as compared to nerve guides alone. The inflammatory response was partially enhanced by the transplanted cells in PLLA nerve grafts; Schwann cells were poorly distributed compared to nerve guide without cells. Tailor-made electrospun nerve guides support axonal regeneration in vivo, and can act as vehicles for co-transplanted cells. Our results motivate further studies exploring novel nerve guides and the effect of stromal cell-derived factors on nerve generation.
... Fibrin is natural wound healing ECM protein that has inherent cell-binding sites. Longitudinal fibrin cables form spontaneously during peripheral nerve regeneration for short gap injuries (Williams et al., 1987;Williams and Varon, 1985). In addition, these cables also form in empty nerve conduit, which can direct the migration of SCs and promote axonal growth. ...
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.
... nerve from which the conduit is made. The NGF and BDNF promote peripheral nerve regeneration, and laminin aides in axonal lengthening and peripheral nerve adhesion (Madison et al., 1987;Williams et al., 1987;Labrador et al., 1998). ...
The use of autologous nerve grafts remains the gold standard for treating nerve defects, but current nerve repair techniques are limited by donor tissue availability and morbidity associated with tissue loss. Recently, the use of conduits in nerve injury repair, made possible by tissue engineering, has shown therapeutic potential. We manufactured a biodegradable, collagen-based nerve conduit containing decellularized sciatic nerve matrix and compared this with a silicone conduit for peripheral nerve regeneration using a rat model. The collagen-based conduit contains nerve growth factor, brain-derived neurotrophic factor, and laminin, as demonstrated by enzyme-linked immunosorbent assay. Scanning electron microscopy images showed that the collagen-based conduit had an outer wall to prevent scar tissue infiltration and a porous inner structure to allow axonal growth. Rats that were implanted with the collagen-based conduit to bridge a sciatic nerve defect experienced significantly improved motor and sensory nerve functions and greatly enhanced nerve regeneration compared with rats in the sham control group and the silicone conduit group. Our results suggest that the biodegradable collagen-based nerve conduit is more effective for peripheral nerve regeneration than the silicone conduit.
... Предложена конструкция, в которой выровненные полимерные волокна представляют рельефные стимуляторы, облегчающие регенерацию периферических нервов при большом диастазе нерва [10]. Мнение о том, что регенерация улучшается, если периферическую матрицу, заполняющую трубчатый трансплантат, выровнять по оси трубки, высказано в более ранних работах [18,19]. Для этих целей можно использовать магнитные поля [20]. ...
Вступ. Метою експериментального дослідження була гістоморфометрична оцінка ефективності комбінованої пластики в цілому та порівняння ефективності варіантів комбінованої пластики сідничного нерва (СН) при його великому дефекті у щурів в експерименті.Матеріали і методи. З метою оцінки стану нейрофібрил використовували імпрегнацію сріблом за Більшовським–Грос, для ідентифікації мієлінових волокон – фарбування за Шпільмейєром.Висновки. 1. Результати морфометричного дослідження СН при заміщенні його дефекту з використанням методів комбінованої пластики наближаються до результатів, отриманих при заміщенні дефекту СН з використанням «золотого стандарту» відновної хірургії периферійних нервів (ПН) при їх дефектах — методу аутонейропластики. 2. Використання тубажу СН щура за допомогою трубчастого протеза, заповненого гелевим композитом Neurogel™, насиченого фактором росту нервів NGF-B щура, забезпечує найвищий ступінь мієлінізації аксонів, на 60-ту добу експерименту кількість мієлінізованих аксонів на 47% більша, ніж у тварин, в яких з метою заміщення дефекту СН використовували аутонейропластику – «золотий стандарт».
... An additional beneficial characteristic of hAM includes its composition of extracellular matrix proteins, such as fibronectin and laminin [68]. These are axon-promoting proteins abundantly present in the basal lamina of Schwann cells [69,70]. Thus, the ECM of hAM could represent a guiding structure for the elongating axons stumps. ...
Statement of significance:
Abnormal fibrotic bonding of tissues, frequently involving peripheral nerves, affects millions of people worldwide. These so-called adhesions usually cause severe pain and drastically reduce quality of life. To date, no adequate treatment exists and none is routinely used in the clinical practice. In this study, vital human amnion, a part of the placenta, was transplanted in a rat model of peripheral nerve scarring and recurring adhesions as novel therapeutic approach. Amniotic cells have already demonstrated to feature stem-cell like properties and produce pro-regenerative factors, which makes the amnion an increasingly promising biomaterial for regenerative medicine. We identified that its transplantation was very effective against peripheral nerve scarring and distinctly reduced recurring adhesions. Moreover, we identified a pro-regenerative effect. This study showed that the amnion is a highly promising novel therapeutic approach for adhesion-related disorders.
... Prefilling tubes with various materials and then grafting then into peripheral nerve gaps improve the number of axons that grow across nerve gaps. [300][301][302][303][304] However, in spite of these approaches axons generally do not regenerate through tubes longer than 2 cm. ...
Spinal cord trauma immediately kills neurons and their processes and sequelae triggered by trauma typically cause the death of large additional numbers of neurons. The smaller the number of neurons killed and connections lost, the smaller the neurological losses and the number of neural circuits that must be re-established to restore normal neurological function. The inability to induce neurological recovery is not only due to spinal cord complexity in terms of number and types of neurons, other cells, and their connections, but also the cellular environment of the spinal cord contains factors that inhibit axon regeneration, it lacks factors necessary to promote axon regeneration, to induce target recognition and to induce the formation of appropriate functional synaptic connections. The literature is full of promising approaches, some providing neuroprotection in the immediate aftermath of trauma, others for neutralizing axon regeneration-inhibiting factors, whereas others induce and direct axon regeneration. Although each technique separately is generally inadequate for the required task, when viewed in a unifying light, combining various techniques should lead to clinically applicable tools for promoting axon regeneration leading to neurological recovery. This article examines spinal cord trauma and its sequelae, neurotoxicity following trauma, factors involved in the inhibition of axon regeneration, techniques providing neuroprotection and for promoting axon regeneration with the aim of inducing neurological recovery and their applicability for clinical use.
... Fibrin was chosen as the base material for the delivery system because it is the natural biomaterial of nerve regeneration and is readily penetrated by the proteolytic activity of the neurite growth cone as it extends through the three-dimensional fibrin matrix (16). Others have previously demonstrated that the addition of fibrin matrices to nerve guide tubes enhanced peripheral nerve regeneration (17,18). Our goal was to enhance the ability of fibrin matrices to promote nerve regeneration by incorporating a drug delivery system into the matrix, thus allowing the fibrin matrix to serve not only as a wound-healing scaffold but also as vehicle for controlled drug release. ...
The goal of this research was to develop an approach to growth factor delivery that would allow the stable incorporation of growth factors within a cell in‐growth matrix in a manner such that local enzymatic activity associated with tissue regeneration could trigger growth factor release. We investigated this approach in the context of peripheral nerve regeneration by designing modified beta‐nerve growth factor (β‐NGF) fusion proteins and testing their ability to promote neurite extension. Fibrin was selected as the cell in‐growth matrix, and the transglutaminase activity of factor XIIIa was utilized to covalently incorporate β‐NGF fusion proteins within fibrin matrices. Novel β‐NGF fusion proteins, which contained an exogenous factor XIIIa substrate to allow incorporation into fibrin matrices, were expressed recombinantly. An intervening plasmin substrate domain was placed between the factor XIIIa substrate and the NGF domain to allow cell‐mediated growth factor release in response to plasmin, which is generated by invading cells. Immobilized NGF fusion protein with an intervening functional plasmin cleavage sequence enhanced neurite extension from embryonic chick dorsal root ganglia by 50% relative to soluble native β‐NGF and by 350% relative to the absence of NGF. These results suggest that this novel approach to growth factor delivery, in which the factor is delivered upon cellular demand, could enhance nerve regeneration and may be useful in tissue engineering.
... In peripheral nerve tissue engineering, hydrogels are applied as NGC fillers, serving to physically bolster the nerve conduit lumen, and to act as delivery vehicles for bioactive molecules and cells (Lin and Marra, 2012). Tubes filled with fibrin matrices (Williams et al., 1987), laminin containing gels (Madison et al., 1985), collagen (Ciardelli and Chiono, 2006;Cordeiro et al., 1989), hyaluronic acid (Seckel et al., 1995) have shown faster axonal regeneration compared to empty tubes or tubes containing physiological saline solution. Despite many studies demonstrated that NGC fillers can enhance the regeneration process, hydrogels with unsuitable physicochemical properties could represent a physical barrier to axonal growth impeding regeneration. ...
... Mesenchymal stem cells also promote enhanced axon regeneration by promoting Schwann cells within the central and distal nerve stumps to migrate into the nerve gap where they proliferate and release additional axon regenerationpromoting factors [254,[262][263][264][265][266][267][268][269][270][271]. These and other mesenchymal stem cell-released factors simultaneously act directly on axons to induce and promote their regeneration [28,221]. ...
Platelet-rich plasma (PRP) has been tested in vitro, in animal models, and clinically for its efficacy in enhancing the rate of wound healing, reducing pain associated with injuries, and promoting axon regeneration. Although extensive data indicate that PRP-released factors induce these effects, the claims are often weakened because many studies were not rigorous or controlled, the data were limited, and other studies yielded contrary results. Critical to assessing whether PRP is effective are the large number of variables in these studies, including the method of PRP preparation, which influences the composition of PRP; type of application; type of wounds; target tissues; and diverse animal models and clinical studies. All these variables raise the question of whether one can anticipate consistent influences and raise the possibility that most of the results are correct under the circumstances where PRP was tested. This review examines evidence on the potential influences of PRP and whether PRP-released factors could induce the reported influences and concludes that the preponderance of evidence suggests that PRP has the capacity to induce all the claimed influences, although this position cannot be definitively argued. Well-defined and rigorously controlled studies of the potential influences of PRP are required in which PRP is isolated and applied using consistent techniques, protocols, and models. Finally, it is concluded that, because of the purported benefits of PRP administration and the lack of adverse events, further animal and clinical studies should be performed to explore the potential influences of PRP.
... Im Vergleich zu matrixfreien und mit NaCl angereicherten Konduiten wurde in mit Fibrin gefüllten Nervenleitschienen eine gesteigerte Regeneration nachgewiesen [28]. Fibrin stellt ein optimales Medium für die Kultivierung und Transplantation von Zellen dar, insbesondere von Schwannzellen; Untersuchungen von unterschiedlichen Arbeitsgruppen konnten einen proportionalen Zusammenhang zwischen der Fibrin-Konzentration und der Axonwachstumslänge von Spinalzellen nachweisen [1,10,23]. ...
... 39 Insoluble extracellular matrix molecules including laminin, fibronectin and some forms of collagen are known to promote axon extension 15 , and researchers have shown that their incorporation in the lumen of guidance channels may result in improved functional restitution. 40 Researchers have also investigated the use of biological cells as growth-promoting agents. Lopes et al. (2006) investigated the use of bone marrow stromal cells (BMCs) in resorbable guidance tubes implanted to repair sciatic nerve injuries in mice. ...
... The extracellular matrix molecules are important for axonal extension and act as guiding the nerve regeneration. It has been found that ECM molecules including fibronectin, collagen and laminin incorporated into the conduit, act as a guidance channel and have had variable results [45,46]. ...
Peripheral Nerve Injuries are one of the most common causes of hand dysfunction caused by upper limb trauma but still current management has remained suboptimal. This review aims to explain the traditional view of pathophysiology of nerve repair and also describe why surgical management is still inadequate in using the new biological research that has documented the changes that occur after the nerve injury, which, could cause suboptimal clinical outcomes. Subsequently presentation and diagnosis will be described for peripheral nerve injuries. When traditional surgical repair using end-to-end anastomosis is not adequate nerve conduits are required with the gold standard being the autologous nerve. Due to associated donor site morbidity and poor functional outcome documented with autologous nerve repair several new advancements for alternatives to bridge the gap are being investigated. We will summarise the new and future advancements of non-biological and biological replacements as well as gene therapy, which are being considered as the alternatives for peripheral nerve repair.
... Previous studies demonstrate the effectiveness of the fibrin as intraluminal filler. In these studies, silicone-based NC were filled with exogenous fibrin hydrogel promoting an increase of the nerve regeneration across 1 cm [121] and 2 cm [122] of nerve gap. Similarly, fibrin was used to fill synthetic permeable and biodegradable NC, showing a significant increase of the nerve regeneration and the mechanical properties of these NC [123,124]. ...
The structure and function of peripheral nerves can be affected by a range of conditions with severe consequences in these patients. Currently, there are several surgical techniques available to treat peripheral nerve defects. Direct repair is the preferred treatment for short nerve gaps, and nerve autografting is the gold standard in critical nerve defects. The autografting is not always available, and the use of allograft, decellularized allograft and nerve conduits are often used with variable success. During the recent years, several outcomes were achieved in peripheral nerve tissue engineering. Promising experimental results have been demonstrated with this novel generation of nerve conduits, mainly composed by biodegradables materials in combination with intraluminal fillers, growth factors and different cell sources.
... Various tissue engineering strategies have been used to influence different aspects of the regenerative process with hope for functional recovery using natural and synthetic tubular scaffolds. The design criteria for fabrication of scaffolds should address various factors including composition [75] and dimensions of the tubular scaffold [76], the addition of exogenous factors such as fibrin precursors [77] and growth factors, the incorporation of glial cells, most often Schwann cells and fibroblasts [78,79], the elastic modulus, permeability, topography, swelling ratio, degradation rate, size, and clearance of the degradation products [80]. In particular the elastic modulus of the scaffold should be at least 1,200 kPa in order to resist compressive and tensile forces that are generated both during the surgery as well as from surrounding tissue after implantation [81]. ...
Biodegradable and biocompatible poly(amidoamine)-(PAA-) based hydrogels have been considered for different tissue engineering applications. First-generation AGMA1 hydrogels, amphoteric but prevailing cationic hydrogels containing carboxylic and guanidine groups as side substituents, show satisfactory results in terms of adhesion and proliferation properties towards different cell lines. Unfortunately, these hydrogels are very swellable materials, breakable on handling, and have been found inadequate for other applications. To overcome this problem, second-generation AGMA1 hydrogels have been prepared adopting a new synthetic method. These new hydrogels exhibit good biological properties in vitro with satisfactory mechanical characteristics. They are obtained in different forms and shapes and successfully tested in vivo for the regeneration of peripheral nerves. This paper reports on our recent efforts in the use of first-and second-generation PAA hydrogels as substrates for cell culturing and tubular scaffold for peripheral nerve regeneration.
... Moreover, addition of these substrates to a hollow tube can increase the capability of the tube to sustain regeneration and, thus, it is a first step toward engineering an artificial guide that mimics the autograft (Fig. 10.3). In fact, the sole addition of plasma, an important source of fibrin, into a silicone tube increased the gap length permissive for regeneration (Williams, Danielsen, Müller, & Varon, 1987). On the other hand, collagen-based (Chamberlain, Yannas, Arrizabalaga, et al., 1998;Chamberlain, Yannas, Hsu, Stritchartz, & Spector, 1998;Labrador, Butí, & Navarro, 1998;Rosen et al., 1990) and laminin-based matrices (Bailey, Eichler, Villadiego, & Rich, 1993;Labrador et al., 1998) have been successfully used to enhance axonal regeneration in nerve guides. ...
Injured axons of the peripheral nerve are able to regenerate and, eventually, reinnervate target organs. However, functional recovery is usually poor after severe nerve injuries. The switch of Schwann cells to a proliferative state, secretion of trophic factors, and the presence of extracellular matrix (ECM) molecules (such as collagen, laminin, or fibronectin) in the distal stump are key elements to create a permissive environment for axons to grow. In this review, we focus attention on the ECM components and their tropic role in axonal regeneration. These components can also be used as molecular cues to guide the axons through artificial nerve guides in attempts to better mimic the natural environment found in a degenerating nerve. Most used scaffolds tested are based on natural molecules that form the ECM, but use of synthetic polymers and functionalization of hydrogels are bringing new options. Progress in tissue engineering will eventually lead to the design of composite artificial nerve grafts that may replace the use of autologous nerve grafts to sustain regeneration over long gaps.
... Ainsi, les polymères synthétiques ont été principalement utilisés pour étudier les facteurs pouvant influencer la régénération axonale de fibres nerveuses périphériques lésées (Lundborg, 1982a(Lundborg, , 1982b(Lundborg, , 1982cLundborg et al., 1982aLundborg et al., , 1982bLundborg et al., , 1982cHall, 1986 ;Berry et al., 1992 ;Guenard et al., 1992 ;Hall et al., 1992 ;Liu, 1992aLiu, , 1992bLabrador et al., 1995 ;Liu et al., 1995 ;Buti et al., 1996). Ces polymères synthétiques peuvent être remplis de composants non neuronaux tels que ceux de la matrice intratubulaire (fibrine, laminine et collagène) ou des cellules dissociées (fibroblastes, astrocytes et cellules de Schwann) (Jenq et Coggeshall, 1985a, 1985b, 1985c, 1985dWilliams, 1987 ;Williams et al., 1987 ;Montgomery et Robson, 1993). L'objectif des chercheurs et des cliniciens est d'obtenir des conduits biocompatibles imitant l'anatomie du nerf (Heath et Rutkowski, 1998). ...
Introduction
Nerve injury compromises sensory and motor functions. Techniques of peripheral nerve repair are based on our knowledge regarding regeneration. Microsurgical techniques introduced in the late 1950s and widely developed for the past 20 years have improved repairs. However, functional recovery following a peripheral mixed nerve injury is still incomplete.
State of art
Good motor and sensory function after nerve injury depends on the reinnervation of the motor end plates and sensory receptors. Nerve regeneration does not begin if the cell body has not survived the initial injury or if it is unable to initiate regeneration. The regenerated axons must reach and reinnervate the appropriate target end-organs in a timely fashion. Recovery of motor function requires a critical number of motor axons reinnervating the muscle fibers. Sensory recovery is possible if the delay in reinnervation is short. Many additional factors influence the success of nerve repair or reconstruction. The timing of the repair, the level of injury, the extent of the zone of injury, the technical skill of the surgeon, and the method of repair and reconstruction contribute to the functional outcome after nerve injury.
Conclusion
This review presents the recent advances in understanding of neural regeneration and their application to the management of primary repairs and nerve gaps.
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.
Various artificial materials have been fabricated as alternatives to autologous nerve grafts in peripheral nerve regeneration, and these afford positive recovery effects without the disadvantages of the gold standard. In this study, we prepared a three-dimensional functionalized self-assembling peptide nanofiber hydrogel containing two neurotropic peptides (CTDIKGKCTGACDGKQC and RGIDKRHWNSQ derived from NGF and BDNF, respectively), which reflected the structure and properties of the neural extracellular matrix. The material was used to promote axonal regrowth and functional recovery. Scanning electron microscopy revealed a three-dimensional porous matrix within the hydrogel. Circular dichroism spectroscopy and atomic force microscopy confirmed that the peptides displayed β-sheet structure and self-assembled into long nanofibers. Rheology measurements and atomic force microscopy indicated that the elasticity of the peptide hydrogels were close to that of the nerve tissue matrix. In vitro work with Schwann cells and dorsal root ganglia showed the hydrogels exhibited good cell compatibility. Furthermore, the hydrogel containing CTDIKGKCTGACDGKQC and RGIDKRHWNSQ promoted the neurite outgrowth of PC12 cells significantly compared with non-functionalized peptide. In vivo, the hydrogels were placed into chitosan tubes and used to bridge 10-mm-long sciatic nerve defects in rats. We found that the combination of CTDIKGKCTGACDGKQC and RGIDKRHWNSQ accelerated axonal regeneration and afforded good functional recovery, suggesting that they synergistically facilitate peripheral nerve regeneration.
Biofabrication techniques have endeavored to improve the regeneration of the peripheral nervous system (PNS), but nothing has surpassed the performance of current clinical practices. However, these current approaches have intrinsic limitations that compromise patient care. The “gold standard” autograft provides the best outcomes but requires suitable donor material, while implantable hollow nerve guide conduits (NGCs) can only repair small nerve defects. This review places emphasis on approaches that create structural cues within a hollow NGC lumen in order to match or exceed the regenerative performance of the autograft. An overview of the PNS and nerve regeneration is provided. This is followed by an assessment of reported devices, divided into three major categories: isotropic hydrogel fillers, acting as unstructured interluminal support for regenerating nerves; fibrous interluminal fillers, presenting neurites with topographical guidance within the lumen; and patterned interluminal scaffolds, providing 3D support for nerve growth via structures that mimic native PNS tissue. Also presented is a critical framework to evaluate the impact of reported outcomes. While a universal and versatile nerve repair strategy remains elusive, outlined here is a roadmap of past, present, and emerging fabrication techniques to inform and motivate new developments in the field of peripheral nerve regeneration.
Statement of significance:
In peripheral nervous system defect repair, a wide variety of strategies have been proposed for preparing functionalized nerve guidance conduits (NGC) with more complex configurations to obtain optimal repair effects. Longitudinally oriented fibrin cables were reported to form spontaneously during the initial stages of peripheral nerve regeneration in an empty NGC, which can direct the migration and proliferation of Schwann cells and promote axonal regrowth. Therefore, based on the biomimetic idea, we prepared a three-dimensional hierarchically aligned fibrin nanofiber hydrogel (AFG) through electrospinning and molecular self-assembly, resembling the architecture and biological function of the native fibrin cable and serving as an intraluminal filling to accelerate axon regeneration. We found that the AFG was a beneficial microenvironment to support SCs cable formation and accelerate axonal regrowth with improved motor functional recovery.
Objective:
To investigate the protective effect of autologous venous ensheathment on sutured rat facial nerve and to test whether the ensheathment could improve the functional recovery of repaired nerve and accuracy of axonal growth.
Study design:
In vivo study.
Methods:
Forty-six rats were examined, with six rats serving as normal controls and 40 receiving facial nerve transection and suture repair (SR) or transection and suture repair with an additional venous ensheathment (VE). The rats were then subjected to functional testing, histological assessment of nerve specimens, or retrograde tracing, respectively.
Results:
At the postoperative day (POD) 60, the venous ensheathment showed no adhesion at the surrounding tissues. No significant difference in neuroma formation was found between the two surgical manipulations (SR and VE groups) (P < 0.05). Retrogradely labeled motoneurons in facial nuclei were extremely disorganized after the facial nerve undertook surgical manipulation. In all manipulated groups, double retrogradely labeled neurons, indicative of aberrant axonal branching during regeneration, could be observed after peripheral manipulation across all time points. With the two facial surgical manipulations, the average count of double-labeled neurons at POD 60 was significantly less than at POD 21 (P < 0.05).
Conclusion:
Autologous venous ensheathment could not help with the functional recovery of facial nerve or improve the accuracy of axonal regeneration. Further studies are warranted to elucidate the effects of venous ensheathment in other motor and sensory nerve models.
Level of evidence:
NA. Laryngoscope, 2017.
Die Überbrückung kurzer Defekte von Nerven durch Röhrchen (Entubulation) ist eine einfache und elegante Lösung für ein komplexes Problem. Diese Technik erbringt eine praktische Lösung für viele mechanische-, zell- und biochemischen Forderungen, die überwunden werden müssen, um einen durchtrennten Nerven effektiv wieder zu vereinen, wobei dies nicht nur durch die klassische mikrochirurgische Neurorrhaphie gelöst wird.
It can be presumed that any materials interposed into traumatic nerve gaps serve only as guiding vehicles for regenerating axons growing toward the periphery, and many ideas about using guides or conduits (tubes) other than a piece of donor nerve have therefore been discussed. Reviews are provided by Weiss as early as 1944 [17] and recently by Fields Ellisman in 1989 [3] and by ourselves in 1992 [16]. Still, the procedure of microsurgical autologous nerve grafting remains the clinically routine method in dealing with peripheral nerve lesions caused by transection trauma and subsequent gap.
Abstract Background and Objectives: Collagen gel can specifically play role in the nerve regeneration. The aim of this research was to study the effect of collagen gel-coated within collagen tube on sciatic nerve regeneration. Material & Methods: This research is an experimental study that 48 male rats (200-250 gr) were used. After axotomy, 1cm segment of the sciatic nerve was removed and the animals were divided into four groups (collagen tube, collagen tube + collagen gel-coated, autograft, and sham surgery). All animals were evaluated by sciatic functional index (SFI), electrophysiology and histology. Results: At 49th and 60th days post operation, the mean of SFI in Collagen + Collagen gel-coated were superior to other experimental groups (collagen and autograft) (P< 0.05). At 12 weeks post operation, the mean nerve conduction velocity and the mean number of myelinated axons in collagen + collagen gel-coated group were superior to other experimental groups (collagen and autograft) (P< 0.05). Conclusion: In this study positive effect of collagen gel on nerve regeneration through a collagen tube shows that it may be useful in treating peripheral nerve injuries.
Despite improved microsurgical techniques, the results of peripheral nerve repair very often remain unsatisfactory [1–4]. Therefore, studies aimed at a better identification and potential manipulation of the cellular and molecular events in PNS regeneration are still meaningful to both scientists and clinicians [5].
Thousands of American people are admitted to the hospitals everyday due to organ failure and tissue loss. It is estimated that over 8 million surgical procedures are performed annually in the United States to treat the millions of Americans who experience organ failure or tissue loss. Physicians treat these patients by transplanting organs from one individual to another, performing reconstructive surgery, or using lifesaving mechanical devices such as kidney dialyzers and mechanical heart valves. However, many of the patients who need organ transplantation (like heart, liver, and kidney transplantations) will die because of the lack of suitable transplant organs. Fortunately, through the developments in the new field of tissue engineering, patients who need new vital tissue and organs have new hope.
Despite recent efforts, there is still no clinically attractive alternative to nerve or vein autografts for repair of peripheral nerve defects. Much research is being devoted to the creation of the optimum nerve guidance channel, which will most likely incorporate multiple forms of stimuli for the regenerating nerve. The synthesis of additional novel and interactive biomaterials will open many doors for tissue engineering efforts in the nervous system. Biotechnology will also play a larger role, either by incorporation of cells genetically engineered to secrete neurotrophic or survival factors, or by direct incorporation of specifically tailored recombinant peptides or proteins (for example, adhesive ligand sequences, growth factors, enzyme pockets, and catalytic or inhibitory antibody fragments). In addition, drug delivery technology will become more critical for the design of the ideal nerve guidance channel (NGC). Techniques by which a series of bioactive molecules could be released over time in sequence with stages of regeneration may ultimately enhance repair in the PNS (peripheral nervous system), and potentially even in the CNS (central nervous system). In addition, more quantitative and uniform methods of analyzing regeneration need to be realized. Quantitative molecular analyses must be conducted in parallel with more phenomenological studies, so that ultimately, mechanistic models can be developed to make a priori predictions based on the biological responses of the regenerating nerve to stimulatory and inhibitory cues.
The goal of this work was to improve the potential of fibrin to promote nerve regeneration by enzymatically incorporating exogenous neurite-promoting heparin-binding peptides. The effects on neurite extension of four different heparin-binding peptides, derived from the heparin-binding domains of antithrombin III, neural cell adhesion molecule and platelet factor 4, were determined. These exogenous peptides were synthesized as bi-domain peptide chimeras, with the second domain being a substrate for factor XIIIa. This coagulation transglutaminase covalently bound the peptides within the fibrin gel during coagulation. The heparin-binding peptides enhanced the degree of neurite extension from embryonic chick dorsal root ganglia through 3-dimensional fibrin gels, and the extent of enhancement was found to correlate positively with the heparin-binding affinity of the individual domains. The enhancement could be inhibited by competition with soluble heparin, by degradation of cell-surface proteoglycans, and by inhibition of the covalent immobilization of the peptide. These results demonstrate an important potential role for proteoglycan-binding components of the extracellular matrix in neurite extension and suggest that fibrin gels modified with covalently bound heparin-binding peptides could serve as a therapeutic agent to enhance peripheral nerve regeneration through nerve guide tubes. More generally, the results demonstrate that the biological responses to fibrin, the body's natural wound healing matrix, can be dramatically improved by the addition of exogenous bioactive peptides in a manner such that they become immobilized during coagulation.-Sakiyama, S. E., Schense, J. C., Hubbell, J. A. Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. [on SciFinder (R)]
The role of nerve growth factor (NGF) was examined in the neural repair of adult rabbit facial nerves using an in vivo preparation. A 35-μl nerve growth chamber was created by suturing the proximal and distal ends of a transected facial nerve (superior buccal branch) into a silicone tube. A gap of 8 mm in the chamber remained after removal of a 5-mm piece of nerve and insertion of the proximal and distal stumps into the tube. Animals were operated bilaterally; one side of the chamber was filled with NGF and the contralateral side was filled with Ringer's solution. Regeneration of the nerves was examined 1 to 5 weeks following the surgery. The caliber of the nerve bundle, the distribution pattern of regenerating motoneurons, axon number per fascicle, size distribution, and the total number of cells were compared to the preoperative morphology pattern found for that animal. Each buccal branch served as its own control. The NGF-filled chambers demonstrated an overall larger caliber of nerve regeneration at 5 weeks and a higher density distribution of axon growth at 3 and 5 weeks. In the early regeneration case (3 weeks), the axon growth profile exhibited more fascicles and less axons than the preoperative controls. In the more advanced stage (5 weeks), the fascicle number was reduced and the axon number was increased. After 5 weeks of regeneration the number of fascicles was still more than that found in the preoperative state. Axon size at 5 weeks was 80% that of the preoperative controls and the thickness of the myelin sheath was less than the preoperative level. The histogram of the size distribution revealed the same distribution as in the preoperative control section.
Aim of this study is to evaluate if regeneration in repair of nerve defects can be improved by combination of a poly-dl-lactide-ɛ-caprolactone conduit (PLC) with long-term release of anti-inflammatory Interleukin 10 (IL10), which is known to reduce intraneural scarring in nerve regeneration through its anti-inflammatoric properties.
Experiments were performed at 30 female Lewis rats. Conduits filled with fibrin (PLC-group n = 10) and fibrin loaded with IL10 (IL10-group n = 10) were compared to autologs nerve grafts (NG-group n = 10) in a 15 mm sciatic nerve gap lesion. Sciatic function index (SFI) and electrophysiological analyses were performed 16 weeks after surgery prior to histological evaluation. In histological analyses total nerve count, total nerve area, myelination index, and N-ratio were measured. Additionally, gastrocnemius muscle was weighed.
SFI (NG-group:-50.68 ± 7.03%; PLC-group:-56.48 ± 2.30%; IL10-group:-56.54 ± 8.22%) and nerve conduction velocity (NG-group: 92.52 ± 4.64 m/s; PLC-group: 92.77 ± 5.07 m/s; IL10-group: 93.78 ±3.63 m/s) showed no significant differences after 16 weeks (P > 0.05). Significant higher axon count (17.592 ± 483) were observed in the NG-group compared to PLC- (6.722 ± 553) and IL10-group (6.842 ± 681) (P < 0.001). NG-group had significant highest nerve cross sections (604.214 ± ±15.217 µm(2) ) as compared to PLC- (245.669 ± ±28.034 µm(2) ) and IL10-group (244.698 ± 26.772 µm(2) ) (P < 0.001). Comparison of myelination index showed significant higher values for NG-group (0.46 ± 0.02) than PLC- (0.64 ± 0.01) and IL10-group (0.62 ± 0.01) (P < 0.001). N-ratios in PLC-group (0.21 ± 0.01) and IL10-group (0.24 ± 0.01) were lower than in NG-group (0.51 ± 0.03) (P < 0.001). Between PLC- and IL10-group no differences were observed (P > 0.05). Gastrocnemius muscle was heavier in NG-group (0.86 ± 0.21g) as compared to PLC- (0.26 ± 0.05g) and IL-10 group (0.29 ± 0.06 g) (P < 0.05).
Bridging critical nerve defects through fibrin-filled PLC conduits is possible. Although, autologs nerve graft showed superior histological results. Long-term release of IL10 in the conduit did not improve regeneration of critical nerve defects. © 2015 Wiley Periodicals, Inc. Microsurgery 00:000-000, 2015.
© 2015 Wiley Periodicals, Inc.
Peripheral nerve regeneration over longer distances through conduits is limited. In the presented study, critical size nerve gap bridging with a poly-DL-lactide-ε-caprolactone (PLC) conduit was combined with application of C3 toxin to facilitate axonal sprouting.
The PLC filled with fibrin (n = 10) and fibrin gel loaded with 1-μg C3-C2I and 2-μg C2II (n = 10) were compared to autologous nerve grafts (n = 10) in a 15-mm sciatic nerve gap lesion model of the rat. Functional and electrophysiological analyses were performed before histological evaluation.
Evaluation of motor function and nerve conduction velocity at 16 weeks revealed no differences between the groups. All histological parameters and muscle weight were significantly elevated in nerve graft group. No differences were observed in both PLC groups.
The PLCs are permissive for nerve regeneration over a 15-mm defect in rats. Intraluminal application of C3 toxin did not lead to significant enhancement of nerve sprouting.
Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear-thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin-based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar–gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear-thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two- and three-dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.
The inability to identify the optimal construction of a nerve guidance conduit (NGC) for peripheral nerve regeneration is a challenge in the field of tissue engineering. This is attributed to the vast number of parameters that can be combined in varying quantities. A pre-existing normalization standard is applied in this paper which uses a calculated ratio of gap length divided by the graft's critical axon elongation denoted as L/Lc. This allows for a direct comparison of the nerve regenerative activity, a measure of performance, of any NGC across an array of gap lengths relative to a standard nerve conduit. Data was extracted from a total of 28 scientific publications that compared the nerve regenerative activity of experimental NGCs relative to standard NGCs. Of the extracted data, 40 parameters were identified that impacted the performance of the experimental conduits. We demonstrate how bootstrap aggregated neural networks provides substantial increases in accuracy in predicting the performance of a NGC over a single neural network and previous prediction attempts by the SWarm Intelligence based Reinforcement Learning (SWIRL) system. The improved accuracy will provide for a better understanding and insight for theorizing successful strategies for NGC development.
Wallerian degeneration, axonal reaction and chromatolysis, axonal growth, target reinnervation and maturation of regenerated axons are sequential processes that occur after a peripheral nerve injury. In all these steps there is a close relationship between neurons and Schwann cells. In response to nerve injury, Schwann cells divide rapidly, migrate to the lesion zone, phagocyte myelin and axonal debris and create endoneurial bands of Büngner for regenerating axons to follow. Schwann cells secrete a number of neurotrophic factors, cell adhesion molecules and basement membrane components that can promote axonal growth. Early genes are expressed in both neurons and Schwann cells after nerve lesions, regulating the expression of later genes, that code for proteins responsible for phenotypic changes in each phase of nerve regeneration. The first steps that prepare surviving neurons for growth are mediated by cell-to-cell interactions and by soluble factors secreted by Schwann cells. Axonal regrowth is dependent on guiding substrates provided by extracellular matrix components and reactive Schwann cells in the distal degenerated nerve. Peripheral reinnervation is mediated by interactions between neuron and target cells, but Schwann cells also participate in the reformation and maintenance of synapses. Whilst during axonal growth neuronal gene expression is controlled by factors liberated by Schwann cells, in the final reinnervation and maturation steps Schwann cell and target cell gene expression is controlled by the regenerated neurons. The potential of Schwann cells to allow and promote regeneration may be applied in the development of cellular prostheses, being composed of a nerve chamber filled with an exogenous extracellular matrix and seeded with Schwann cells, which may provide an artificial substitute to the traditional autologous graft used to repair severe nerve lesions.
Nerve bridging is to suture a biomaterial-made conduit and to overpass the damaged nerve end to end with microsurgery. Poly L-lactide (PLLA) is an excellent biomaterial that has biocompatible, biodegradable and good mechanical properties; it is thus potential to be engineered as nerve conduits and manufactured as scaffolds for nerve tissue replacement. On the other hand, chitosan provides cell affinity and considerably promotes nerves regeneration. This study is to apply plasma processing for PLLA film modification, graft the plasma-modified film with vaporized acrylic acid (AAc) monomers and then immobilize chitosan by amide bonding on the pAAc-grafted surface. This work using plasma-activation and subsequent evaporation of AAc greatly avoids PLLA thermal cracking and remaining the PLLA film in good mechanical properties. Surface morphologies are evaluated by Nano Focus. Electron Spectroscopy for Chemical Analysis (ESCA) and Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR) are respectively employed for determining elements’ functionalities and chemical structures. Moreover, biological functionalities of the chitosan-immobilized PLLA films are thereafter assessed by antibacterial test and in vitro fibroblastic cell growth assay. The result exhibits that chitosan is immobilized on the modified PLLA films, which is plasma-activated subsequent to the evaporation of AAc. The process does not induce thermal cracking. In vitro fibroblastic cell growth assay on the chitosan-immobilized PLLA films has demonstrated that fibroblast cells on the surface become circular in shape. It decreases cell growth rate and the development of scar tissues, which may thereafter promote the effect of nerve repairing.
In recent times, tissue engineering researchers have been attempting to provide the scientific and medical communities with improvements in the repair of peripheral nerve injuries using synthetic grafts. Although the nerve autograft still remains the clinical gold standard in bridging nerve injury gaps, many advances on several fronts have been made in developing a more effective nerve tubular construct to guide regenerating axons across the lesion. This review discusses several strategies that have been employed to enhance the regenerative effectiveness of artificial nerve guidance channels. These strategies include the use of scaffolds, the integration of contact-mediated cues within the tubular construct, and incorporation or delivery of exogenous growth factors into the conduit lumen uniformly or in a gradient form. Animal and clinical studies are reviewed to explain some of the ideas involved in developing a guidance channel of the future.
The authors present the case of a 20-year-old man who, 3 months after his initial injury, underwent repair of a 1.7-cm defect of the ulnar nerve at the wrist; repair was performed with an acellular nerve allograft. Given the absence of clinical or electrophysiological recovery at 8 months postrepair, the patient underwent reexploration, excision of the "regenerated cable," and rerepair of the ulnar nerve with sural nerve autografts. Histology of the cable demonstrated minimal axonal regeneration at the midpoint of the repair. At the 6- and 12-month follow-ups of the sural nerve graft repair, clinical and electrophysiological evidence of both sensory and motor reinnervation of the ulnar nerve and associated hand muscles was demonstrated. In this report, the authors describe a single case of failed acellular nerve allograft and correlate the results with basic science and human studies reporting length and diameter limitations in human nerve repair utilizing grafts or conduits devoid of viable Schwann cells.
In the United States more than 200 000 people per year are treated for severe peripheral nerve injuries that require surgical intervention. Functional recovery of motor and sensory capability is limited after autografting, the most common surgical intervention for severe peripheral nerve injuries. The process of peripheral nerve regeneration has been studied extensively in a variety of animal models using a tubular conduit. This model has been used to generate a large base of data from a wide variety of experimental devices; however, this data has not been analyzed comparatively due to a lack of standardization of experimental conditions, assays, and reported measures of the quality of regeneration. As a result, progress in understanding conditions for optimal nerve regeneration has been stunted and the optimal characteristics for such an implant have not been identified. So while tubulation repair of a transected peripheral nerve presents an attractive alternative to autograft, it has not yet shown the ability to satisfactorily restore lost function. In this article, we provide an overview of mammalian wound healing following severe injury, the physiology of the peripheral nervous system, the standardized wound models used to study peripheral nerve regeneration, and the critical axon elongation criteria and how it can be used to directly compare results from dissimilar studies. We complete this review article with a description of the critical features of tubular implants used to induce peripheral nerve regeneration that can be optimized in order to improve the quality of regeneration.
With increasing fetal development, the mechanism of mammalian wound healing transitions from regeneration to a repair process characterized by organized wound contraction and scar synthesis. Recently, a variety of tissue-engineering constructs have been developed to block the contraction and scar formation mechanisms of repair, and to induce regeneration following injury with similar mechanisms of action observed for both the skin and peripheral nervous system (PNS). Such constructs, mostly scaffolds that are analogs of extracellular matrix and possess specific biological activity, have become the basis of studies of in vivo synthesis of tissues and organs. In this article, we provide an overview of mammalian wound healing processes following severe injury as well as a description of the tissue triad and the regenerative capacity of the three distinct tissue types that comprise the triad. We also discuss the critical structural elements of an active extracellular matrix analog that induces regeneration, and describe the use of standardized wound models for study of in vivo regeneration processes. We conclude this review describing recent data from studies utilizing active extracellular matrix analogs (scaffolds) that have shown regenerative activity.
Cellular engineering, principally the control and regulation of cell proliferation, differentiation, and function, is vital to the success of cell-based therapeutic applications and technologies. Despite significant advances in cell and tissue engineering, current approaches to the engineering of cell-surface interactions and extracellular matrix design fall short of mimicking the complexity of those relationships in the physiological microenvironment. When considering the bioengineering of complex systems, such as tissue or organs, we must reduce the complexity of the system while remaining in control of cell behavior and function. Therefore, a thorough understanding of the cellular bioprocesses and assembly, and how factors or stimuli affect them, will significantly aid future biomedical engineering research. Optimizing the combinations of cells, matrices, and locally and systemically active stimuli is a complex process characterized by an inter-reliant set of variables with a potentially infinite scope of combinations.
To optimize the internal environment of a collagen nerve tube, we designed a Schwann cell-seeded intrinsic framework and its biocompatibility was investigated. We fixed 6-0 polyglactin woven filaments (Vicryl) or polydioxanone monofilaments (PDS) on a silicone ring in a net fashion. It was coated with matrigel and then incubated with cultured newborn or adult Schwann cells. Furthermore, we implanted 1.5-cm-long filament-filled collagen tubes in a rat model. Using a live/dead fluorescent assay and electron microscopy, we found that adherent Schwann cells onto filaments remained viable and oriented longitudinally along filaments. The preliminary in vivo study indicated that a mild inflammatory reaction was present around the tube wall. However, nerve regeneration occurred around and between filaments. We concluded that the arrangement of Schwann cell columns onto filaments was achieved, mimicking Bünger bands. It was shown that the biomaterials did not impede nerve regeneration. © 2001 Wiley-Liss, Inc. MICROSURGERY 21:6–11 2001
Proliferation of Schwann cells in neonatal mouse sciatic nerve was studied radioautographically in 1-micro glycol methacrylate sections. 28 mice were injected with thymidine-(3)H, 4 microc/g, 48 hr after birth, and were killed serially over the next 4 days. For the cell cycle following injection, the generation time was approximately 24 hr as determined by grain-count halving data; the duration of synthesis phase was 8 hr as determined from a curve constructed from the per cent of mitotic figures containing label; and the labeling index was 9% at 2 hr after injection. With these estimates, the per cent of Schwann cells proliferating was calculated to be 27%. In addition, roughly 25% of dividing cells appeared to cease division during the cell cycle under study. The relationship of these findings to other events during maturation of nerve is discussed.
Highly porous collagen-glycosaminoglycan (CG) membranes have been useful in the treatment of skin injuries including full-thickness burns in humans. It has been recently shown that by seeding these CG membranes with autologous epidermal cells, large full-thickness wounds of guinea pigs can be closed in less than two weeks. Studies with similar CG polymeric materials were used to bridge gaps of 15 mm in the rat sciatic nerve. Silicone tubes 25 mm length were either filled with a porous CG polymer (test grafts) or were left empty (control grafts). The CG polymer used in the sciatic nerve study differed from that used for full-thickness skin wounds in that the pore structure was oriented parallel to the axis of the tube (rather than random) and the degree of crosslinking was decreased. These grafts were vascularized along their length and were encased in dense multiple wrappings of perineurial tissue.
The spatial-temporal progress of nerve regeneration was examined in silicone chambers of three different volume capacities: 11, 25, and 75 μl. In all chambers, the stumps of a transected rat sciatic nerve were sutured into the ends of the chamber leaving a 10 mm gap between the stumps. Chambers were implanted empty (E chambers) or prefilled with saline (PF chambers). A coaxial and continuous fibrin matrix had formed in all chambers by 1 week. In E chambers, the matrices had a proximal-distal taper that was more pronounced in E25 and E75 chambers due to significantly larger matrix diameters in the proximal region. At 3 weeks, vascular and Schwann cell migration and axonal regeneration were less advanced in the E25 and E75 than in the control E11 chambers. The retardation correlated with the presence of an avascular organization of circumferential cells. Saline prefill-ing affected the caliber and density of fibrin fibers in the 1 week matrices of PF25 and PF75 chambers. The matrices did not have a prominent taper and diameters were progressively larger with increasing chamber volume. Saline prefilling did not affect regeneration progress in 3 week PF11 chambers but did enhance regeneration in the PF25 chambers; a 1.5-fold larger diameter nerve formed at 3 weeks that contained 2,6-fold more axons. Progress in the PF75 chamber was retarded. We conclude that the volume, timing, and nature of the fluid filling a silicone chamber have significant influence on the formation of fibrin matrices. Alterations in matrix formation correlate with substantial changes in the subsequent progress of intrachamber regeneration events.
An experimental model is presented for studying axonal growth after experimental hyperthyroidism and hypothyroidism. The left sciatic nerve of the rat was transected and transposed to the back. The proximal nerve stump was inserted into a 50-mm-long mesothelial chamber leaving the distal end of the chamber open. Different groups of young adult rats were given daily injections of thyroxine ( body weight) or the goitrogen, thiamazol, in the drinking water (0.125 g/liter) for 12 weeks. Thyroxine treatment increased significantly the extent of axonal outgrowth from the proximal nerve stump compared with untreated rats. Experimental hypothyroidism (thiamazol treatment), evidenced by a retarded body growth, did not affect the extent of axonal outgrowth. In other experiments the left proximal nerve stump was crossanastomosed with the right distal nerve stump. The two nerve stumps were bridged with a mesothelial chamber leaving a 15-mm gap. This gap distance is known from our previous studies to inhibit axonal overgrowth to the distal nerve stump. As evidenced by histological evaluation, in three of six thyroxine-treated rats, axons had bridged the 15-mm gap. We conclude that experimentally induced hyperthyroidism enhances axonal growth in mesothelial chambers.
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.
The regeneration of transected peripheral nerves of mice was studied using autoradiographical and electron microscopical techniques. In general, maximal proliferation occurred between the 5th and 7th day after dissection and stopped when the cells emigrating from the proximal and distal stumps of the nerve started to contact one another. Special attention was paid to the reaction of the connective tissue cells of the endo-, epi- and perineurium. The perineurial cells seemed to dedifferentiate between the 3rd and 5th day after the transection and then started to proliferate into the defect. Labelled perineurial cells were completely absent, when the minifascicles were fully developed in the neuroma. The epineurial fibroblasts started to proliferate during the 1st day. Even 6 weeks after transection the multiplication rate was about ten fold that of the controls. The results are discussed with special reference to clinical nerve repair.
In this laboratory, a silicone chamber model for peripheral nerve regeneration in adult rats has been developed and used to define basic principles of the regenerative events, such as the sequential stages being followed during 'spontaneous' regeneration in vivo and the role of neuronotrophic- and neurite-promoting factors as well as extracellular matrix molecules. Each of the defined stages seems amenable to experimental modulation. Previous attempts to enhance regeneration included increasing the volume of the nerve chambers along with the modification of fibrin matrix formation by prefilling with saline (PBS) or matrix precursors. We present here the results of a series of experiments on the effects of exogenous biochemical agents applied by multiple injections into these in vivo chambers. Out of a variety of agents screened, a mixture of laminin (L), testosterone (T), ganglioside GM 1 (G), and catalase (C) was shown to advance substantially the progress of regeneration in 16 day chambers, as compared to PBS-prefilled and PBS-injected controls. LTGC-treatment at day 0, 6, and 10 postimplantation caused an increasingly frequent occurrence of cellular elements in cross-sections obtained from the middle (S5) of the chambers (i.e. 5 mm from the proximal stump), which was 2-fold for vessels, 3-fold for Schwann cells, and 10-fold for axons. When only sections containing axons 3 mm from the proximal stump (S3) were compared in experimental and control groups, computerized area measurements also revealed an average 2-fold difference for the cross-sectional size of the whole regenerate, the endoneurium and the space occupied by blood vessels.
The silicone chamber model permits the investigation of the cellular and molecular events underlying successful regeneration of the rat sciatic nerve across a 10 mm gap. When 25 microliter chambers are implanted prefilled with phosphate-buffered saline (PBS), it takes 5-7 days before sufficient fibrin matrix (derived from plasma precursors) accumulates naturally to form a complete bridge across the chamber gap; at 1 week postimplantation, cellular migration into the matrix from the nerve stumps is just beginning. The temporal progress of regeneration might be stimulated if a fibrin matrix, conducive to cell migration, was provided to the nerve stumps at or shortly after the time of chamber implantation. To test this hypothesis, chambers were prefilled, at the time of implantation, with different preparations of homologous plasma. A solution of 90% platelet-free plasma dialyzed against PBS (DP) formed a fibrin matrix by 24 hours postimplantation that, like the naturally formed matrix, had a predominantly longitudinal orientation. The temporal progress of regeneration was stimulated in the DP-prefilled chambers; at 17 days postimplantation, the extents of Schwann cell migration and axonal elongation were significantly greater than in the control system. In contrast, prefilling chambers with either non-citrated plasma or DP + calcium resulted in the generation of a matrix within 8 minutes that was composed of randomly oriented fibrin polymers. These matrices significantly retarded the progress of regeneration.
To compare peripheral nerve regeneration across bridging synthetic tubular implants with ordinary autologous transplantation, we recorded evoked muscle action potentials 3 months after transection, following direct stimulation of rat tibial and peroneal nerves. Significantly, with autologous transplantation we were able to evoke compound muscle action potentials in all but one case (this having a 15-mm gap). Pooling the groups together, compound action potentials could be recorded in 15 of 45 cases that had regenerated through 13- to 16-mm gaps. Potentials in the synthetic implant-treated group tended to show more temporal "scattering" in the late phases of the EMG response. No differences were found in compound muscle action potential amplitudes and duration, or terminal motor conduction velocity, between both experimental groups, between different artificial tubuli, between different gap lengths, or between the peroneal and tibial nerves. The overall values were remarkably lower than in nontransected controls, about 25% of the compound muscle action potential normal amplitude voltage and 60% of the terminal motor conduction velocity.
The present study determines the numbers of axons that regenerate after sciatic nerve transection in the rat. The transections are done by removing either 4 mm or 8 mm of the nerve. The axons are counted in the gap and distal stump of the sciatic nerve and in 5 of its tributaries. Survival time is 9 months which we define as long-term to allow comparison with short-term data obtained after a much shorter survival. The first findings is that the numbers of axons in the gap and distal stump are different in the 2 transection paradigms. For the 4 mm paradigm, more axons than normal appear in the gap and only a fraction of these pass into the distal stump. For the 8 mm paradigm, the numbers of axons in the gap are normal and the numbers in the distal stump do not deviate far from these. Thus by changing only the length of the segment of removed nerve, one causes major differences in the numbers of axons that regenerate. Second the numbers of axons that regenerate in tributary nerves that innervate muscle have a different pattern than the numbers that regenerate into cutaneous nerves. Thus the factors control axonal numbers must be different in the 2 types of nerves. Finally, axons that regenerate into tributary nerves do not, by and large, regenerate in concert with those in the distal stump of the parent nerve. Thus the factors that control axonal numbers in the tributary nerves must be different from those that control the numbers in the distal stump of the parent nerve.
In the first six days after division myelinated axons in the proximal stump of rat sciatic nerves produce collateral and terminal sprouts. These are present as circumscribed "groups" which are positively distinguishable from clusters of non-myelinated axons. Two types of "groups" are identifiable, and their distribution in some of the nerve segments is analysed. Their evolution was followed in sequential nerve segments, the initial 'tight' structure becoming looser between 7 and 10 days, and myelinated axons appeared in them during this time. At this stage a complete basal lamina was present surrounding the entire "group". Some of the cells in the "groups" did not have the characteristics of Schwann cells. Between 7 and 10 days after division alveolate vesicles and densely staining material in the cisternae of the rough surfaced endoplasmic reticulum were prominent in Schwann cells in the distal part of the proximal stump. It is thought that both types of "group" are developed from single myelinated axons and the name "regenerating unit" is proposed for both types. Their relationship to "clusters", seen in the distal stump of regenerating peripheral nerves, and "onion bulbs", present in some peripheral neuropathies, is discussed.
We re-examined the hypothesis of Cajal³, later refuted by Weiss and Taylor²⁰, that cells in distal stumps of transected peripheral nerves exert an attractive (tropic) effect on regenerating axons. This question was re-assessed in vivo using surgical materials and assay procedures not available to those workers. Proximal stumps of transected rat sciatic or cat peroneal nerves were inserted into the single inlet end of a hollow, Y-shaped Silastic implant. Regenerating axons were provided with alternative targets consisting of a vacant arm vs one occupied by a sciatic nerve graft (rat), or a tibial (Tout) vs peroneal (Pout) distal nerve stump (cat). In some cases Pout was rendered metabolically compromised relative to Tout by exposing the former to dry ice and inhibitors of DNA and RNA synthesis. At 4.5 or 6 weeks postoperatively, the number of regenerating axons in each fork of the implant was assessed by morphometric analysis (total number of non-myelinated and myelinated axons greater than 1 μm in diameter at 4.5 weeks, and total number of myelinated axons at 6 weeks postoperatively), or by quantification of an axonally transported label.
The successful regeneration of a multifascicular, complete peripheral nerve through a tubular synthetic biodegradable nerve guide across a gap of 10 mm in the rat sciatic nerve is reported. The importance of the distal nerve as a source of target-derived neuronotrophic factors necessary for the successful regeneration of the proximal regenerating nerve is emphasized. A simplified research model for further investigation into and manipulation of the biological processes of nerve regeneration is described. The potential clinical utilization of this model in the management of peripheral nerve injuries and, ultimately, central nervous system lesions is mentioned.
The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)
The spatial-temporal progress of peripheral nerve regeneration across a 10-mm gap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7-14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1-5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.
An experimental model is presented for studying nerve regeneration over gaps of various lengths between the both ends of a severed nerve. After transferring left and right sciatic nerves of rat to the back, the gap between the two nerve ends could be bridged by a preformed, tube-shaped mesothelial chamber of a desired length. When the gap length was 10 mm or less, a well developed nerve structure was generated in the chamber between the nerve ends, and axons from the left sciatic nerve reinnervated muscles in the right limb via the right sciatic nerve. When the gap length was extended to 15 mm or more no such regeneration occurred. When no distal nerve end was introduced into the chamber, there was a limited growth into this chamber over only 5-6 mm. This "limited growth phenomenon" is discussed with respect to a lack of "trophic" or cellular support from a distal nerve segment. It is also proposed that the termination of growth, seen under these circumstances, may suggest a new principle for avoiding the development of neuromas after nerve transections.
A new peripheral nerve forms across a 10 mm gap within a silicone chamber regeneration model when the distal segment of a transected sciatic nerve, connected to its end organs, is sutured into the distal end of the chamber. We have tested the ability of other tissue inserts to support axonal regeneration in the chamber. When an isolated 2 mm piece of sciatic nerve was sutured into the distal end, fibrin matrix formation, cell immigration and axonal regeneration were identical to those occurring in the control. When the distal nerve insert was replaced with a 2 mm piece of skin or a ligation, a matrix did not form and subsequent cell immigration and axonal regeneration did not occur. When a 2 mm piece of tendon was inserted, a matrix did form at 1 week, but a structure across the gap was observed at later time periods in only 2 out of 7 chambers. The matrix either dissolved before cells could enter the chamber or did not promote cellular immigration and subsequent axonal regeneration. When the distal end was left open, a matrix formed and cells from the reactive tissue outside the chamber entered the matrix and formed a granulation tissue bridge across the gap. This tissue failed to support axonal regeneration; at 3 weeks, axons stopped 1 mm beyond the proximal stump at the interface with the granulation tissue. Thus, matrix formation and a cellular bridge are necessary but not sufficient to ensure regeneration. Successful regeneration across the silicone chamber gap requires humoral and/or cellular contributions available from peripheral nervous tissue and not from the other tested tissues.
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.
Modification of the microenvironment allows axonal regeneration across a 20 mm nerve gap using entubulation repair
- Madison R.
Madison, R., C.F. Da Silva, and P. Dikkes (1985) Modification of the mi-croenvironment allows axonal regeneration across a 20 mm nerve gap using entubulation repair. SOC. Neurosci. Abstr. 12:1253.
Axonal growth in mesothelial chambers
- N Danielsen
- L B Dahlin
- Y F Lee
- G Lundborg
Danielsen, N., L.B. Dahlin, Y.F. Lee, and G. Lundborg (1983) Axonal growth in mesothelial chambers. Scand. J. Plast. Reconstr. Surg. 17:119-125.
Evoked muscle action potentials from regenerated rat tibia1 and peroneal nerves: Syn-thetic versus autologous interfascicular grafts
- H Miiller
- K Shibib
- H Friedrich
- M Modrack
Miiller, H., K. Shibib, H. Friedrich, and M. Modrack (1987a) Evoked muscle action potentials from regenerated rat tibia1 and peroneal nerves: Syn-thetic versus autologous interfascicular grafts. Exp. Neurol. 95.2-33.
Regeneration of a transected peripheral nerve: An autoradiographic and electron microscopic study
- Jurecka
The fine structure of fibrin of the rat
- Kisch B.
Axonal growth in mesothelial chambers
- Danielsen
A study of degeneration and regeneration in the divided rat sciatic nerve based on electronmicroscopy. II. The development of the “Regeneration Unit”
- Morris J. H.