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Sciatic nerve regeneration navigated by laminin-fibronectin double coated biodegradable collagen grafts in rats
Abstract
Biodegradable type I collagen tube grafts filled longitudinally with laminin and fibronectin double coated collagen fiber bundles (L-F grafts) were implanted to promote sciatic nerve regeneration in rats. Grafts filled with uncoated collagen fibers were used as control. A 1 cm defect on the right sciatic nerve was filled with a graft in the manner of bridging. Thirty days after implantation, several newly developed nerve fasciculi were found at the middle portion of the L-F grafts in contrast to no developed nerves in the controls. After 60 days, the middle and distal portions of both grafts included well-developed nerve tissues with prominent myelinated and unmyelinated nerve fibers surrounded by perineural cells, but the control distal portion showed fewer nerve fibers. All artificial collagen elements were completely degraded and absorbed at 30 days, and new nerve tissues surrounded by an epineurium successfully connected the proximal stump to the distal stump of the initially separated nerve. Descending and ascending action potentials were evoked in all grafts at 60 days. These results indicated that laminin and fibronectin may promote the growth of axons in biodegradable collagen grafts, which guided nerve regeneration well and allowed the formation of epineurium.
... Veins, which are abundantly available and have less donor site morbidity, have been found effective as nerve conduits for bridging short nerve gaps (Chiu, Janecka et al. 1982;Chiu, Lovelace et al. 1988;Suematsu, Atsuta et al. 1988;Risitano, Cavallaro et al. 1989;Wang, Costas et al. 1993a;Wang, Costas et al. 1993b;Benito-Ruiz, Navarro- Monzonis et al. 1994;Wang, Costas et al. 1995;Colonna, Anastasi et al. 1996;Ferrari, De Castro Rodrigues et al. 1999;Pu, Syed et al. 1999;Kelleher, Al-Abri et al. 2001;Rodrigues Ade and Silva 2001;Strauch, Rodriguez et al. 2001;Zhang, Blain et al. 2002). Nerve growth factors NGF have been used and found beneficial in enhancing the axonal regeneration within venous nerve conduits (Cordeiro, Seckel et al. 1989;Derby, Engleman et al. 1993) Other investigators tried to bridge the nerve gap with a three-dimensional materials that would work as a scaffold for the regenerating axons as muscle fibers (Norris, Glasby et al. 1988;Gattuso, Glasby et al. 1989;de Blaquiere, Curtis et al. 1994;Di Benedetto, Lassner et al. 1994;Whitworth, Dore et al. 1995;Roganovic, Savic et al. 1996;Fullarton, Glasby et al. 1998;Liu, Arai et al. 2001), collagen fiber bundles (Tong, Hirai et al. 1994), fibronectin-mats (Whitworth, Brown et al. 1995), polyamide sutures (Lundborg, Dahlin et al. 1997), and collagen matrix (Tranquillo, Girton et al. 1996 We used the rat sciatic nerve, which is the most widely used model for the evaluation of nerve function (Dijkstra, Meek et al. 2000). A 10-mm gap was chosen, as larger gap would have made the microsurgical anastomosis difficult. ...
... Different strategies have been investigatedto provide the tube conduit with some internal orientation. In one set of experiments, biodegradable collagen tube grafts filled longitudinally with laminin and fibronectin double coated collagen fiber bundles were found to enhance sciatic nerve regeneration in rat model(Tong, Hirai et al. 1994). Fibronectin-mats were also found to promote the growth and produce better orientation of regenerating axons and Schwann cells with formation of a completely new epineurial sheath (Whitworth,Brown et al. 1995). ...
... Common grafting in surgery implies autograft or nerve removal from elsewhere in the body. Unfortunately, autografts have limitations such as body injury, repeated surgery and disproportion of grafted nerve tissue in terms of size and structure [8,9] while transplantation of allograft or xenograft are associated with complications such as stimulation of the immune system [10][11][12][13][14]. The studies were conducted on the application of artificial neural tube to form neural cords [15][16][17]. ...
A nanofibrous silk nerve conduit has been evaluated for its efficiency based on the promotion of peripheral nerve regeneration in rats. The designed tubes with or without Schwann cells were implanted into a 10 mm gap in the sciatic nerves of the rats. Four months after the surgery, the regenerated nerves were monitored and evaluated by macroscopic assessments and histology. The results demonstrated that the nanofibrous grafts, especially in the presence of Schwann cells, enabled reconstruction of the rat sciatic nerve trunk with a restoration of nerve continuity and formation of nerve fibres with myelination. Histological data demonstrated the presence of Schwann and glial cells in regenerated nerves. This study strongly supports the feasibility of using artificial nerve grafts for peripheral nerve regeneration by bridging large defects in a rat model.
... Common grafting in surgery was autograft or nerve removal from elsewhere of the body. Unfortunately, autografts had limitations such body injury, repeated surgery and disproportion of grafted nerve tissue in terms of size and structure of nerve tissue [8,9] also; transplantation of allograft or xenograft had similar problems in addition to the stimulation of the immune system [10][11][12][13][14]. ...
The aim of this study was to produce a nanofibrous biodegradable silk nerve substrate. The woven and oriented artificial scaffolds were designed by electrospinning method, and were evaluated by microscopic, mechanical analyses, and cell culture assays with Schwann cells. Results of analyses were showed a good resilience and compliance with movement as a neural graft. Cellular experiments showed a better cell adhesion and growth on the oriented nanofibrous scaffolds compared to woven nanofiber ones. This neural conduit appears to have the right organization for testing in vivo nerve tissue engineering studies.
... Among them, poly-glycolic acid (PGA), poly-lactic acid (PLA), poly-lacticco-glycolic-acid (PLGA) and polycaprolactone (PCL) have shown positive effects in PNS regeneration attempts (Evans et al., 2002;Schmidt and Leach, 2003;Schlosshauer et al., 2003;Yannas and Hill, 2004). The tubulization technique also enables the use of extracellular matrix molecules, cells and neurotrophic factors directly involved in the regenerative microenvironment (Fields et al., 1989;Tong et al., 1994;Labrador et al., 1998;Chen et al., 2000;Rodrígues et al., 2000;Bloch et al., 2001;Schmidt and Leach, 2003;da Silva et al., 2003;Oliveira et al., 2004;Pierucci et al., 2004). ...
The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL) - based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs.
Copyright © 2015. Published by Elsevier Inc.
... A different approach may be the addition of micro-fibres or -filaments in the conduit lumen, which also enhance the scaffold permeability, reducing the risk of cell necrosis in the centre of the nerve guidance (Gu et al., 2011). These micro-filaments can be then further coated with ECM components in order to enhance cell-surface interactions, hence promoting higher regeneration of the injured tissue (Tong et al., 1994). ...
Peripheral nerve injury is a serious problem affecting significantly patients' life. Autografts are the "gold standard" used to repair the injury gap, however, only 50% of patients fully recover from the trauma. Artificial conduits are a valid alternative to repairing peripheral nerve. They aim at confining the nerve environment throughout the regeneration process, and providing guidance to axon outgrowth. Biocompatible materials have been carefully designed to reduce inflammation and scar tissue formation, but modifications of the inner lumen are still required in order to optimise the scaffolds. Biomicking the native neural tissue with extracellular matrix fillers or coatings showed great promises in repairing longer gaps and extending cell survival. In addition, extracellular matrix molecules provide a platform to further bind growth factors that can be released in the system over time. Alternatively, conduit fillers can be used for cell transplantation at the injury site, reducing the lag time required for endogenous Schwann cells to proliferate and take part in the regeneration process. This review provides an overview on the importance of extracellular matrix molecules in peripheral nerve repair.
... Another important ECM protein, fibronectin, has also been studied and demonstrated to be capable of promoting peripheral nerve regeneration and preventing invasion by fibroblasts [192,193]. ...
Central and peripheral neural injuries are traumatic and can lead to loss of motor and sensory function, chronic pain, and permanent disability. Strategies that bridge the site of injury and allow axonal regeneration promise to have a large impact on restoring quality of life for these patients. Engineered materials can be used to guide axonal growth. Specifically, nanofiber structures can mimic the natural extracellular matrix, and aligned nanofibers have been shown to direct neurite outgrowth and support axon regeneration. In addition, cell-seeded scaffolds can assist in the remyelination of the regenerating axons. The electrospinning process allows control over fiber diameter, alignment, porosity, and morphology. Biodegradable polymers have been electrospun and their use in tissue engineering has been demonstrated. This paper discusses aspects of electrospun biodegradable nanofibers for neural regeneration, how fiber alignment affects cell alignment, and how cell-seeded scaffolds can increase the effectiveness of such implants.
... Hence, prefilling the tube with extracellular matrix components may improve the regenerative process [51]. Several molecules, including collagen, laminin, hyaluronic acid, fibronectin and, more recently, glycosaminoglycans alone or associated with collagen, are useful for this purpose [6,10,11,25,35,38,47,59,69,70]. The use of these substances, resulted in a greater number of regenerating fibers and allowed the gap between the stumps to be increased. ...
Transection of a peripheral nerve results in a loss of function at the target organ that can rarely be recovered without surgical repair. Such an intervention usually involves nerve autografting but is complicated by problems such as the need for secondary surgery, a limited donor nerve supply and loss of sensitivity in the donor nerve area. An alternative approach involving repair by nerve tubulization has been extensively used to study substances that may improve the regenerative process. An interesting feature of the tubulization technique is the possibility of filling the tube with substances that can enhance regeneration. Such substances include collagen, laminin, hyaluronic acid, fibronectin and, more recently, glycosaminoglycans alone or with collagen. Biopolymers, purified glial cells, and neurotrophic factors have also been tested. By using the tubulization technique, it has been possible to increase the number of regenerating fibers and the gap between the stumps. In this review, we discuss some of the basic concepts of this technique, as well as recent advances in this field.
... Unfortunately, autografts had limitations such body injury, repeated surgery and disproportion of grafted nerve tissue in terms of size and structure of nerve tissue 8,9 also transplantation of allograft or xenograft had similar problems in addition to the stimulation of the immune system. [10][11][12][13][14] Therefore, the studies were conducted on the application of artificial neural tubes to form neural cords. [15][16][17] Clinical investigations were showed functional improvement and regeneration of peripheral nerve tissue with a gap of 3-5 mm by silicone tube. ...
... However, autografts have limitations for example body injury, repeated surgeries and disproportion of grafted nerve tissue in size and structure [8][9] . Similar problems have been encountered in allografting or xenografting, for example, stimulation of the immune system is required [10][11][12][13][14] . Many studies in which artificial neural tubes are used to repair nerve defects have been performed [15][16][17] . ...
It has been confirmed that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit can promote peripheral nerve regeneration in rats. However, its efficiency in repair of over 30-mm-long sciatic nerve defects needs to be assessed. In this study, we used a nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit to bridge a 30-mm-long gap in the rat sciatic nerve. At 4 months after nerve conduit implantation, regenerated nerves were cally observed and histologically assessed. In the nanofibrous graft, the rat sciatic nerve trunk had been reconstructed by restoration of nerve continuity and formation of myelinated nerve fiber. There were Schwann cells and glial cells in the regenerated nerves. Masson's trichrome staining showed that there were no pathological changes in the size and structure of gastrocnemius muscle cells on the operated side of rats. These findings suggest that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit is suitable for repair of long-segment sciatic nerve defects.
... Common grafting in surgery was autograft or nerve removal from elsewhere of the body. Unfortunately, autografts had limitations such body injury, repeated surgery, and disproportion of grafted nerve tissue in terms of size and structure of nerve tissue [8,9] also; transplantation of allograft or xenograft had similar problems such as stimulation of the immune system [10][11][12][13][14]. The studies were conducted on the application of artificial neural tube to form neural cords [15][16][17]. ...
The aim of this study was to produce an oriented biodegradable poly (3-hydroxybutyrateco-3-hydroxyvalerate) (PHBV) nerve guide. Oriented porous micropatterned artificial nerve guide designed onto the micro-patterned silicon wafers, then their surfaces modified with to oxygen plasma for cell attachment increasing. Afterwards, the neural guides were evaluated by microscopic, structural, physical, and mechanical analyses, and cell culture assays with Schwann cells. Results of structural, physical, and mechanical analyses showed a good resilience and compliance with movement as a neural graft. Cellular experiments showed a better cell adhesion, growth, and proliferation inside the plasma-modified neural guides compared to un-modified ones, also Schwann cells were well attached on plasma-modified surface. This neural guide appears to have the right organization for testing in vivo nerve tissue engineering studies.
... Biodegradable nerve guides must be preferred because no foreign body material will be left in the host after the device has fulfilled its task. [5][6][7][8][9][10][11][12][13][14] Nerve guides have been fabricated from a variety of materials including gelatin, 15 hyaluron, 16 lactosorb, 17 fibronectin, 18 and polycaprolactone. 19 Among these synthetic polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microbial polyester can be noted as a biocompatible and biodegradable copolymer. ...
An oriented poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nerve conduit has been used to evaluate its efficiency based on the promotion of peripheral nerve regeneration in rats. The oriented porous micropatterned artificial nerve conduit designed onto the micro-patterned silicon wafers, then their surfaces modified with to oxygen plasma for cell adhesion increasing. The designed conduits were investigated by cell culture analyses with Schwann cells. The conduits were implanted into a 30 mm gap in sciatic nerves of rats. Four months after surgery, the regenerated nerves were monitored and evaluated by macroscopic assessments and histology and behavioral analyses. Results of cellular analyses were showed suitable properties of designed conduit for nerve regeneration. The results demonstrated that in the polymeric graft with Schwann cells, the rat sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. Histological results demonstrated the presence of Schwann and glial cells in regenerated nerves. Functional recovery such walking, swimming and recovery of nociceptive function were illustrated for the all grafts especially conduits with Schwann cells. This study proves the feasibility of the artificial nerve graft filled Schwann cells for peripheral nerve regeneration by bridging a longer defect in an animal model.
... Unfortunately, autografts had limitations such body injury, repeated surgery and disproportion of grafted nerve tissue in terms of size and structure of nerve tissue 8,9 also transplantation of allograft or xenograft had similar problems in addition to the stimulation of the immune system. [10][11][12][13][14] Therefore, the studies were conducted on the application of artificial neural tubes to form neural cords. [15][16][17] Clinical investigations were showed functional improvement and regeneration of peripheral nerve tissue with a gap of 3-5 mm by silicone tube. ...
The aim of this study was to produce a chitosan-cross-linked nanofibrous biodegradable poly (3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit. The artificial nerve scaffold designed by electrospinning method and cross-linked with chitosan by chemical method. Afterwards, the scaffolds were evaluated by microscopic, physical, and mechanical analyses and cell culture assays with Schwann cells. The conduits were implanted into a 10 mm gap in the sciatic nerves of the rats. Four months after surgery, the regenerated nerves were evaluated by macroscopic assessments and histology. This polymeric conduit had sufficiently good mechanical properties to serve as a nerve guide. Cellular experiments showed a better cell adhesion, growth, and proliferation inside the cross-linked nanofibrous scaffolds compared with un-cross-linked ones, also Schwann cells well attached on chitosan-cross-linked nanofibrous surface. The in vivo results demonstrated that in the nanofibrous graft, the sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. This neural conduit appears to have the right organization for testing in vivo nerve tissue engineering studies.
... Fibronectin was also tested with laminin in double-coated collagen fiber bundles inserted into collagen tubes. The results were better than when using uncoated collagen fibers, revealing that laminin and fibronectin together may act positively on axonal growth (Tong et al., 1994). ...
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.
... Les conduits biodégradables élaborés à partir de certaines protéines matricielles doivent permettre d'offrir un support physique et trophique favorisant la régénération des fibres nerveuses (Mackinnon et Dellon, 1990a, 1990bMadorsky et al., 1998 ;Lundborg, 2000aLundborg, , 2000bLundborg, , 2000c. Plusieurs études animales montrent une régénération axonale conséquente lorsque les conduits sont de faible longueur (Kim et al., 1994 ;Tong et al., 1994 ;Archibald et al., 1995). Chez le primate, il a même été montré que cette repousse nerveuse pouvait atteindre 2 cm (Mackinnon et Dellon, 1990b). ...
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.
... Over the years, several extracellular matrix (ECM) compounds have been used to increase the number of regenerated axons after peripheral nerve lesion and tubulization repair. The most studied ECM molecules include collagen, laminin, hyaluronic acid, fibronectin and more recently, glycosaminoglycans alone or associated with collagen [3,4,6,13,14,18]. The use of these substances has made it possible to increase the number of regenerating fibers and to fill gap between nerve stumps [9]. ...
After axotomy, regeneration can be enhanced by bridging the transected nerve with a biocompatible tube, and the effect of trophic substances or molecules from the extracellular matrix can be investigated by filling the prosthesis. In this study, we assessed the importance of the molecular organization and aggregational state of collagen type I in axonal regeneration and guidance. Two types of collagen were used, namely, a collagen gel derived from bovine tendon that displays supraorganization after extrusion, and collagen from rat tail which does not self-organize under such conditions. Adult male Wistar rats were divided into four groups. In the first group (n=3), the polyethylene tube was filled with bovine collagen, while in the second (n=3), the prosthesis was filled with rat-derived collagen. In the third group (n=3), the tube was left empty, and the fourth group (n=3), consisted of unoperated rats. Six weeks after tubulization, the number of axons was significantly higher with bovine collagen than with rat collagen (7,661 ± 1,018 versus 4,110 ± 1,027, p
... They could also be seen under nerve cuff bridging conditions. [60][61][62] Scaffolds reinforced by fibers or tubes for tendon repair Tendon disorders are frequent and are responsible for much morbidity both in sport and the workplace. 63,64 Incomplete healing of tendon injuries can lead to marked dysfunction and disability, with compromised joint biomechanics and debilitating pain. ...
As a dynamic and hierarchically organized composite, native extracellular matrix (ECM) not only supplies mechanical support which the embedded cells needs but also regulates the functions of various cellular through interaction with them. Based on the ECM-mimetic principle, good biocompatibility and appropriate mechanical properties are the two basic requirements that the ideal scaffolds for the tissue engineering or regenerative medicine need. Some fibers and tubes have been shown effective to reinforce scaffolds for tissue engineering or regenerative medicine. In this review, three parts, namely properties affected by the addition of fibers or tubes, scaffolds reinforced by fibers or tubes for soft tissue repair and scaffolds reinforced by fibers or tubes for hard tissue repair, are stated, which shows that tissue repair or regeneration efficacy was enhanced significantly by fiber or tube reinforcement. Also, it indicates that these reinforcing agents can improve the biocompatibility and biodegradation of the scaffolds in most cases. However, there are still some concerns, such as the homogeneousness in structure or composition throughout the reinforced scaffolds, the adhesive strength between the matrix and the fibers or tubes, cytotoxicity of nanoscaled reinforcing agents, etc., which were also discussed in the conclusion and perspectives part.
... This success percentage is signi"cantly higher than that of the empty tubes (60%). This result is consistent with that reported by Tong et al. that the presence of laminin and "bronectin in collagen grafts can dramatically increase the ability of neural components to regenerate e!ectively over long nerve gaps [19]. Wang et al. also found that the number of regenerated axons was signi"cantly decreased as compared to normal controls if the laminin or the "bronectin was depleted in nerve grafts [34]. ...
A 10 mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with an extracellular gel containing collagen, laminin and fibronectin. Empty silicone rubber tubes were used as controls. Six weeks after implantation, all extracellular elements were completely degraded and absorbed, and 90% of the animals from the extracellular gel group exhibited regeneration across the nerve gaps, whereas only 60% in the control group. Both qualitative and quantitative histology of the regenerated nerves revealed a more mature ultrastructural organization with 28% larger cross-sectional area and 28% higher number of myelinated axons in the extracellular gel group than the controls. These results showed that the gel mixture of collagen, laminin and fibronectin could offer a suitable growth medium for the regeneration of axons.
Peripheral nerve damages cause loss of sensorimotor and autonomic functions, resulting in a significant burden for the patients. Nerve injuries above a limiting gap length require surgical repair. Although autograft...
Synthetic nerve guidance conduits (NGCs) offer an alternative to harvested nerve grafts for treating peripheral nerve injury (PNI). NGCs have been made from both naturally derived and synthesized materials. While naturally derived materials typically have an increased capacity for bioactivity, synthesized materials have better material control, including tunability and reproducibility. Protein engineering is an alternative strategy that can bridge the benefits of these two classes of materials by designing cell-responsive materials that are also systematically tunable and consistent. Here, we tested a recombinantly derived elastin-like protein (ELP) hydrogel as an intraluminal filler in a rat sciatic nerve injury model. We demonstrated that ELPs enhance the probability of forming a tissue bridge between the proximal and distal nerve stumps compared to an empty silicone conduit across the length of a 10 mm nerve gap. These tissue bridges have evidence of myelinated axons, and electrophysiology demonstrated that regenerated axons innervated distal muscle groups. Animals implanted with an ELP-filled conduit had statistically higher functional control at 6 weeks than those that had received an empty silicone conduit, as evaluated by the sciatic functional index. Taken together, our data support the conclusion that ELPs support peripheral nerve regeneration in acute complete transection injuries when used as an intraluminal filler. These results support the further study of protein engineered recombinant ELP hydrogels as a reproducible, off-the-shelf alternative for regeneration of peripheral nerves.
The failure of severed CNS axons to regenerate and to reconnect after injury is thought to be due to both insufficient expression of pro-regenerative genes by central neurons, and to an inhibitory microenvironment at the lesion site. Regarding the latter, astroglial scarring and presence of inhibitory myelin proteins are believed to represent major obstacles to axon regeneration. In addition, the inflammatory response within the CNS white matter is slow to develop, especially with regard to macrophage recruitment, and it is likely too weak to trigger sufficient local production of required growth-promoting molecules (Perry et al. (1987); Schwartz et al. (1999)). Transplantation of fetal neural tissue has been studied as one method for filling cystic spinal cord lesions, and has shown some promise for promoting functional recovery (Diener and Bregman (1998); Bregman et al. (1997); Anderson et al. (1995), Reier et al. (1994); Bregman et al. (1989)). However, while fetal tissue transplantation may allow filling of a lesion cavity, host fibers in adult animals tend to terminate within fetal transplants, rather than grow across them (Reier et al. (1983)). The same problem may also arise with neural stem cell transplants, which are increasingly being investigated for their benefit in spinal cord repair (Cao et al. (2001); Liu et al. (2000)).
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.
This book details polysaccharides and other important biomacromolecules covering their source, production, structures, properties, and current and potential application in the fields of biotechnology and medicine. It includes a systematic discussion on the general strategies of isolation, separation and characterization of polysaccharides and proteins. Subsequent chapters are devoted to polysaccharides obtained from various sources, including botanical, algal, animal and microbial.
In the area of botanical polysaccharides, separate chapters are devoted to the sources, structure, properties and medical applications of cellulose and its derivatives, starch and its derivatives, pectins, and exudate gums, notably gum arabic. Another chapter discusses the potential of hemicelluloses (xylans and xylan derivatives) as a new source of functional biopolymers for biomedical and industrial applications. The algal polysaccharide, alginate, has significant application in food, pharmaceuticals and the medical field, all of which are reviewed in a separate chapter. Polysaccharides of animal origin are included with separate chapters on the sources, production, biocompatibility, biodegradability and biomedical applications of chitin (chitosan) and hyaluronan. With the increasing knowledge and applications of genetic engineering there is also an introduction in the book to nucleic acid polymers, the genome research and genetic engineering.
Proteins and protein conjugates are covered, with one chapter providing a general review of structural glycoproteins, fibronectin and laminin, together with their role in the promotion of cell adhesion in vascular grafts, implants and tissue engineering. Another chapter discusses general aspects of a number of industrial proteins, including casein, caseinates, whey protein, gluten and soy proteins, with emphasis on their medical applications, and with reference to the potential of bacterial proteins. Another natural polymer resource, microbial polyesters, although small compared with polysaccharides and proteins, is also gaining increasing interest in biomedical technology and other industrial sectors. One chapter, therefore, is devoted to microbial polyesters, with comprehensive coverage of their biosynthesis, properties, enzymic degradation and applications.
By dealing with biopolymers at the molecular level, the book is aimed at the biomedical and wider materials science communities and provides an advanced overview of biopolymers at the graduate and postgraduate level. In addition it will appeal to both academic and industrial life scientists who are involved in research and development activities in the medical and biotechnology field.
Tissue is frequently damaged or lost in injury and disease. There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Tissue engineering is an exciting strategy being explored to deal with damaged or lost tissue. It is the science of generating tissue using molecular and cellular techniques, combined with material engineering principles, to replace tissue. This could be in the form of cells with or without matrices. There have been developments in almost all surgical disciplines, including plastic surgery. Unfortunately significant hurdles remain to be overcome in many areas before tissue engineering becomes more routinely used in clinical practice. In this paper, the tissue engineering approaches relevant to plastic surgery for tendons and skin are discussed.
Tissue is frequently damaged or lost in injury and disease. There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Tissue engineering is an exciting strategy being explored to deal with damaged or lost tissue. It is the science of generating tissue using molecular and cellular techniques, combined with material engineering principles, to replace tissue. This could be in the form of cells with or without matrices. There have been developments in almost all surgical disciplines, including plastic surgery. Unfortunately significant hurdles remain to be overcome in many areas before tissue engineering becomes more routinely used in clinical practice. In this paper the tissue engineering approaches relevant to plastic surgery for tendons and skin are discussed.
Spontaneous regeneration occurs for nerve gaps that are less than 10 mm; however, injuries that are greater than this length require a graft that will bridge the two nerve ends. Currently, nerve autografts are the ‘gold standard’; however, their limited availability and efficacy remain a limitation in bridging larger gaps. Therefore, other biological grafts have been investigated, including nerve allografts, veins grafts, artery grafts, and muscle grafts. However, their immunogenicity and inability to match autograft mediated axonal regeneration lead to the development of synthetic nerve conduits.
Tissue engineering in the peripheral nervous system unites efforts by physicians, engineers, and biologists toward a common goal to create either natural or synthetic tubular nerve guidance channels as alternatives to nerve autografts for the repair of peripheral nerve defects. Guidance channels help direct axons sprouting from the regenerating nerve end, provide a conduit for diffusion of neurotropic and neurotrophic factors secreted by the damaged nerve stumps, and minimize infiltration of fibrous tissue. In addition to efforts to control these physical characteristics of nerve guidance channels, researchers are optimizing the incorporation of biologic factors and engineering interactive biomaterial that can specifically stimulate the regeneration process. It is believed that current and future research will ultimately result in biologically active and interactive nerve guidance channels that can support and enhance peripheral nerve regeneration over longer, more clinically relevant defect lengths.
We evaluated facial nerve regeneration using a collagen tube as a nerve conduit in five cats. In three 5 mm of the facial nerve were resected, a collagen tube was implanted, and a 5 mm segment of the opposite facial nerve was resected, reversed 180°, and sutured back as an autologous nerve graft. In one a collagen tube was implanted on one side, and in the remaining one a 5 mm nerve segment was reversed. Histological, electrophysiological, and horseradish peroxidase labelling examinations were carried out 4-24 weeks postoperatively. Histological study showed that the nerve was well vascularised and regenerated. Electrophysiological examination confirmed the recovery of evoked electromyograms through to the regenerated axons. Horseradish peroxidase examination also confirmed restoration of the whole facial nerve. The collagen tube is an efficient nerve conduit.
The use of autologous sural nerve grafts is still the current gold standard for the repair of peripheral nerve injuries with wide substance losses, but with a poor rate of functional recovery after repair of mixed and motor nerves, a limited donor nerve supply, and morbidity of donor site. At present, tubulization through the muscle vein combined graft, is a viable alternative to the nerve autografts and certainly is a matter of tissue engineering still open to continuous development, although this technique is currently limited to a critical gap of 3 cm with less favorable results for motor function recovery. In this report, we present a completely new tubulization method, the amnion muscle combined graft (AMCG) technique, that consists in the combination of the human amniotic membrane hollow conduit with autologous skeletal muscle fragments for repairing the substance loss of peripheral nerves and recover both sensory and motor functions. In a series of five patients with loss of substance of the median nerve ranging 3–5 cm at the wrist, excellent results graded as S4 in two cases, S3+ in two cases, and S3 in one case; M4 in four cases and M3 in one case were achieved. No iatrogenic damage due to withdrawal of a healthy nerve from donor site was observed. This technique allows to repair extensive loss of substance up to 5 cm with a good sensory and motor recovery. The AMCG thus may be considered a reasonable alternative to traditional nerve autograft in selected clinical conditions. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014.
Restoration of function following complete nerve injuries with subsequent nerve repair is still not satisfactory and in many cases poor, especially when a gap has to be bridged by a graft. In such situations, there may be insufficient access to autologous graft material. Alternatives have to be developed and a close collaboration between basic scientists and clinicians is required. In the present article, current studies on experimental nerve grafts are discussed and some new alternatives to autologous nerve grafts are reviewed.
A modified nanofibrous poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nerve conduit has been used to evaluate its efficiency based on the promotion of peripheral nerve regeneration in rats. The authors used a gelatin-modified nanofibrous poly (3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit to bridge a 30 mm long gap in the rat sciatic nerve. At four months after nerve conduit implantation, regenerated nerves were macroscopically observed and histologically assessed. In the nanofibrous graft, the rat sciatic nerve trunk had been reconstructed by restoration of nerve continuity and formation of myelinated nerve fiber. There were Schwann cells and glial cells in the regenerated nerves. These findings suggest that modified nanofibrous poly(3-hydroxybutyrate-co-3- hydroxyvalerate) nerve conduit is suitable for repair of long-segment sciatic nerve defects.
Autografts or allografts are commonly used in the repair of damaged peripheral nerves. However, similar problems have been encountered in allografting or xenografting. Previous studies concerning artificial neural tubes to repair nerve defects mainly focus on peripheral nerve defects less than 30 mm. Dr. Esmaeil Biazar and colleagues from Islamic Azad University, Iran investigate the feasibility of poly(3-hydroxy- butyrate-co-3-hydroxyvalerate) conduits in the repair of 30-mm sciatic nerve gap in a rat model. The researchers found that at 4 months after nerve conduit implantation, regenerated nerves were macroscopically observed and histologically assessed.
In the nanofibrous graft, the rat sciatic nerve trunk had been reconstructed by restoration of nerve continuity and formation of myelinated nerve fiber. There were Schwann cells and glial cells in the regenerated nerves. Masson's trichrome staining showed that there were no pathological changes in the size and structure of gastrocnemius muscle cells on the operated side of rats. These findings, published in the Neural Regeneration Research (Vol. 8, No. 24, 2013), suggest that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit is suitable for repair of long-segment sciatic nerve defects. Results from Dr. Esmaeil Biazar and colleagues offer a novel solution for repair of long-segment peripheral nerve defects in the clinic.
The aim of this study was to produce a chitosan-crosslinked nanofibrous biodegradable poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nerve conduit. The artificial scaffold was designed by electrospinning method, and cross-linked with chitosan by chemical method. The scaffolds were evaluated by microscopic, physical, and mechanical analyses, and cell culture assays with Schwann cells. Results of analyses showed a good resilience and compliance with movement as a neural graft. Cellular experiments showed a better cell adhesion and growth inside the crosslinked nanofibrous scaffolds compared with un-crosslinked ones. This neural conduit appears to have the right organization for testing in vivo nerve tissue engineering studies.
Polymers are being extensively investigated to help facilitate nerve regeneration. Entubulization methods involving polymers where a conduit is used to connect the nerve endings has great potential as a repair method for peripheral nerve regeneration. The conduit allows for neurotropic and neurotrophic communication between the nerve stumps and provides physical guidance for the regenerating axons similar to the grafts. The closely fitting tubes facilitate axonal regeneration by inducing rapid development of a highly organized capsule that isolates the repair site and guides and aligns endoneurial components. Entubulization minimizes unregulated axonal growth at the site of injury by providing a distinct environment, and allows for trophic factors emitted from the distal stump to reach the proximal segment, which enhances physiological conditions for nerve regeneration. The spatial cues also induce a change in tissue architecture, with the cabling of cells within the microconduit. The conduits can also be environmentally enhanced with chemical stimulants like laminin and nerve growth factor (NGF), biological cues such as from Schwann cells and astrocytes, the satellite cells of the peripheral and central nervous systems, and lastly, physical guidance cues.
Cell transplantation using porous polymer scaffolds is now being explored as therapeutic approach to replace malfunctioning tissues and organs. This strategy which relies upon the creation of completely natural tissue from the host is the most promising alternative to whole organ transplantation. Biocompatible and biodegradable synthetic polymers play an important role in the design of temporary substrates to which cells can adhere, grow and retain their differentiated function. This review gives a survey of novel manufacturing methods to develop reproducibly polymer scaffolds with high pore volume for cell seeding and high surface area for cell attachment. Contribution of these scaffolds to cell transplantation for the engineering of new tissues is largely illustrated.
Although autogenous nerve grafting remains the gold standard for repair of peripheral nerve defects, the use of various conduits can be a substitute provided these conduits meet the above-mentioned prerequisites. For the moment, autogenous vein grafts or denatured muscle grafts can be used to bridge short defects, especially in distal sensory nerves. Incorporation of muscle into a vein graft expands its application to longer defects in bigger nerves. PGA conduits have also been clinically proven to be reliable in reconstruction of digital nerve defects. Although nonabsorbable conduits cause irritation and nerve compression that necessitates secondary surgery removal, silicone tubes or Goretex tubes can be used in selected cases until absorbable conduits large enough for major peripheral nerves are available. To date, 3 cm seems to be the barrier for conduits. Incorporation of trophic factors and Schwann cells into the conduits will make their way into the clinic if problems like controlled release of trophic factors, obtaining and sustenance of an appropriate number of viable Schwann cells, are solved.
A nanofibrous PHBV nerve conduit has been used to evaluate its efficiency based on the promotion of nerve regeneration in rats. The designed conduits were investigated by physical, mechanical and microscopic analyses. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the regenerated nerves were evaluated by macroscopic assessments and histology. This polymeric conduit had sufficiently high mechanical properties to serve as a nerve guide. The results demonstrated that in the nanofibrous graft with cells, the sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. For the grafts especially the nanofibrous conduits with cells, muscle cells of gastrocnemius on the operated side were uniform in their size and structures. This study proves the feasibility of artificial conduit with Schwann cells for nerve regeneration by bridging a longer defect in a rat model.
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
AIM: To explore the effect of nerve regeneration through repairing the defect of sciatic nerve in rats with the combination of optimized acellular allogeneic nerve and autologous neuroma. MATeRIAL and MeTHodS: 30 SD rats were randomly divided into two groups A and B, with 15 in each, which were used in preparing the models of the autologous neuroma and the defect of sciatic nerve. In the group A, the combination of allogeneic nerve and autologous neuroma was transplanted; in the group B, the autogenous nerve was transplanted. 15 Wistar rats were used to provide acellular allogeneic nerve, which came from the sciatic nerve in one side of the leg. The electrophysiology examination, the evaluation of sciatic nerve function index and the histological examination were done at the 8th weeks and the 16th weeks after the operation. ReSULTS: At the 8th week, the limb escape response appeared in all rats; at the16th week, there were many nerve fibers passing through the transplant in group A and group B. There was no significant difference in the number of the regenerated nerve fiber, diameter and the thickness of medullary sheath. At the 8th week, the conduction velocity of the regenerative nerve in group A were lower than that in group B (p<0.05), and there was no statistical difference in two groups at the 16th week; there was no significant deviation in the function index of sciatic nerve in group A and group B(P >0.05). CoNCLUSIoN: The combination of allogeneic nerve and autologous neuroma, which repairs the defect of peripheral nerve, can promote the regeneration of nerve, and the function of nerve conduction can be recovered, which is a good substitute of nerve grafts.
Facial nerve paralysis due to resection of tumors or as a consequence of trauma is a frequently observed complication. Thus, in the present study, we evaluated a collagen nerve guide in facial nerve regeneration across a 5-mm nerve gap. This biological tube was manufactured from 3% collagen, coated over a Teflon tube used only as a template and submitted to thermal dehydration at 105C for 24h. The collagen tube was implanted at the dorsal ramous of the facial nerve of five adult cats over a gap of 5mm. The facial nerve of the contralateral side was kept intact and used as control. Electrophysiological study was performed from 3 weeks after surgery, and histological and horseradish peroxidase labeling examination was carried out 8 weeks after implantation. Electrophysiological study confirmed the recovery of electrical activity of the collagenimplanted regenerated nerve. Light-microscopic examination of collagen tube-implanted specimens revealed a well vascularized regenerated nerve, which under an electron microscope showed many myelinated axons surrounded by Schwann cells and unmyelinated axons. Horseradish peroxidase staining demonstrated labeling of facial motoneurons in the brainstem and facial nerve terminals in the neuromuscular junction, also confirming restoration of the whole facial nerve tract from the reinnervated muscles, passing through the regenerated site to the brainstem. The collagen tube was very efficient as a nerve guide over a 5mm facial nerve gap and shows great promise as a nerve conduit.
In order to assess the usefulness of collagen and laminin gels prefilling nerve chambers to enhance nerve regeneration, we compared reinnervation of target organs after sciatic nerve resection leaving gaps of 4 or 6 mm followed by repair with silicone tubes in different groups of mice. Tubes were prefilled with saline solution, collagen gels, or laminin-containing gels at different concentrations. Functional reinnervation was assessed by noninvasive methods to quantitate recovery of sweating, nociceptive, sensory, and motor functions in the hindpaw repeatedly during 4–5 months postoperation. The increase in gap length between nerve stumps delayed the beginning and reduced the degree of functional recovery achieved. Reinnervation started earlier and achieved slightly higher levels with collagen gel diluted at 1.28 mg/ml than with more concentrated (1.92 and 2.56 mg/ml) collagen gels and with saline-prefilled tubes bridging a 4-mm gap. Recovery was also better with diluted (4 mg/ml) than with concentrated (12 mg/ml) laminin-containing gel, although lower than with collagen gels and saline. By prefilling silicone tubes bridging a 6-mm gap, a length considered limiting for regeneration in the mouse sciatic nerve, with diluted collagen or laminin gels, both matrices allowed for higher levels of recovery and for successful regeneration in a higher proportion of mice than saline solution. The laminin gel performed slightly better than the collagen gel.
Nerve guides are increasingly being used surgically to repair acute peripheral nerve injuries. This is not only due to an increase in the number of commercially available devices, but also clinical acceptance. However, regeneration distance is typically limited to 20-25 mm, in part due to the basic tubular design. A number of experimental studies have shown improvements in nerve regeneration distance when conduits incorporate coatings, internal scaffolds, topographical cues, or the delivery of support cells. Current studies on designing nerve guides for maximizing nerve regeneration focus both on cell-containing and cell-free devices, the latter being clinically attractive as "off the shelf" products. Arguably better results are obtained when conduits are used in conjunction with support cells (e.g., Schwann cells or stem cells) that can improve regeneration distance and speed of repair, and provide informative experimental data on how Schwann and neuronal cells respond in regenerating injured nerves. In this review we discuss the range of current nerve guides commercially available and appraise experimental studies in the context of the future design of nerve guides for clinical use.
Neural tissue engineering may be a promising option for neural repair treatment, for which a well-designed scaffold is essential. Smart materials that can stimulate neurite extension and outgrowth have been investigated as potential scaffolding materials. A piezoelectric polymer polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) was used to fabricate electrospun aligned and random scaffolds having nano- or micron-sized fiber dimensions. The advantage of using a piezoelectric polymer is its intrinsic electrical properties. The piezoelectric characteristics of PVDF-TrFE scaffolds were shown to be enhanced by annealing. Dorsal root ganglion (DRG) neurons attached to all fibrous scaffolds. Neurites extended radially on random scaffolds, whereas aligned scaffolds directed neurite outgrowth for all fiber dimensions. Neurite extension was greatest on aligned, annealed PVDF-TrFE having micron-sized fiber dimensions in comparison with annealed and as-spun random PVDF-TrFE scaffolds. DRG on micron-sized aligned, as-spun and annealed PVDF-TrFE also had the lowest aspect ratio amongst all scaffolds, including non-piezoelectric PVDF and collagen-coated substrates. Findings from this study demonstrate the potential use of a piezoelectric fibrous scaffold for neural repair applications.
Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.
Electron microscopic immunostaining of rat duodenum and incisor tooth was used to examine the location of four known components of the basement-membrane region: type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. Antibodies or antisera against these substances were localized by direct or indirect peroxidase methods on 60-microns thick slices of formaldehyde-fixed tissues. In the basement-membrane region of the duodenal epithelium, enamel-organ epithelium, and blood-vessel endothelium, immunostaining for all four components was observed in the basal lamina (also called lamina densa). The bulk of the lamina lucida (rara) was unstained, but it was traversed by narrow projections of the basal lamina that were immunostained for all four components. In the subbasement-membrane fibrous elements or reticular lamina, immunostaining was confined to occasional "bridges" extending from the epithelial basal-lamina to that of adjacent capillaries. The joint presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basal lamina indicates that these substances do not occur in separate layers but are integrated into a common structure.
Skeletal muscle grafts, when thawed after freezing, can be used to repair peripheral nerves. This method was used after transection of the median nerve in the upper arm in marmosets. Examination at 28 days showed total denervation of flexor carpi radialis; at 150 days electrophysiological evidence of recovery of nerve conduction across the graft and of muscle activation was seen. Sections at this time showed nerve fibres and new functional neuromuscular junctions in the muscle. It is concluded that effective reinnervation of target muscles is possible after peripheral nerve repair using skeletal muscle autografts.
Neuronal responses to extracellular matrix (ECM) constituents are likely to play an important role in nervous system development and regeneration. We have studied the interactions of a neuron-like rat pheochromocytoma cell line, PC12, with ECM protein-coated substrates. Using a quantitative cell attachment assay, PC12 cells were shown to adhere readily to laminin (LN) or collagen IV (Col IV) but poorly to fibronectin (FN). The specificity of attachment to these ECM proteins was demonstrated using ligand-specific antibodies and synthetic peptides. To identify PC12 cell surface proteins that mediate interactions with LN, Col IV, and FN, two different antisera to putative ECM receptors purified from mammalian cells were tested for their effects on PC12 cell adhesion and neuritic process outgrowth. Antibodies to a 140-kD FN receptor heterodimer purified from Chinese hamster ovarian cells (anti-FNR; Brown, P. J., and R. L. Juliano, 1986, J. Cell Biol., 103:1595-1603) inhibited attachment to LN and FN but not to Col IV. Antibodies to an ECM receptor preparation purified from baby hamster kidney fibroblastic cells (anti-ECMR; Knudsen, K. A., P. E. Rao, C. H. Damsky, and C. A. Buck, 1981, Proc. Natl. Acad. Sci. USA., 78:6071-6075) inhibited attachment to LN, FN, and Col IV, but did not prevent attachment to other adhesive substrates. In addition to its effects on adhesion, the anti-ECMR serum inhibited both PC12 cell and sympathetic neuronal process outgrowth on LN substrates. Immunoprecipitation of surface-iodinated or [3H]glucosamine-labeled PC12 cells with either the anti-FNR or anti-ECMR serum identified three prominent cell surface glycoproteins of 120, 140, and 180 kD under nonreducing conditions. The 120-kD glycoprotein, which could be labeled with 32P-orthophosphate and appeared to be noncovalently associated with the 140- and 180-kD proteins, cross reacted with antibodies to the beta-subunit (band 3) of the avian integrin complex, itself a receptor or receptors for the ECM constituents LN, FN, and some collagens.
Electron microscopic immunostaining of rat duodenum and incisor tooth was used to examine the location of four known components of the basement-membrane region: type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. Antibodies or antisera against these substances were localized by direct or indirect peroxidase methods on 60-microns thick slices of formaldehyde-fixed tissues. In the basement-membrane region of the duodenal epithelium, enamel-organ epithelium, and blood-vessel endothelium, immunostaining for all four components was observed in the basal lamina (also called lamina densa). The bulk of the lamina lucida (rara) was unstained, but it was traversed by narrow projections of the basal lamina that were immunostained for all four components. In the subbasement-membrane fibrous elements or reticular lamina, immunostaining was confined to occasional "bridges" extending from the epithelial basal-lamina to that of adjacent capillaries. The joint presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basal lamina indicates that these substances do not occur in separate layers but are integrated into a common structure.
A protein with properties of laminin has been isolated from human placental extracts by using monoclonal antibodies. Placental tissue was extracted with 0.5 M NaCl and high molecular weight proteins were isolated from the extract by salt precipitation and gel filtration on Sepharose 6B. The resulting protein fraction which contained material cross-reactive with anti-sera to rat laminin was used as immunogen to prepare hybridomas. Thirteen hybrids produced antibodies which reacted with basement membrane-associated antigens in indirect immunofluorescence of tissues. One of these, 4E10, was characterized in detail. This monoclonal antibody reacted with human laminin as shown by several lines of evidence. Immunoprecipitation from metabolically labeled culture media of a human amniotic epithelial cell line with the 4E10 antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed polypeptides with Mr similar to those of rat laminin. Immunochromatography of placental extracts obtained by limited pepsin digestion yielded material with main polypeptides at 160 and 130 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. These peptic fragments cross-reacted with rat laminin in immunodiffusion and enzyme immunoassay, and a polyclonal antiserum against the fragments reacted with basement membranes in tissues in a manner identical with the 4E10 antibody. Electron microscopic images of the human peptic fragments showed structures similar to the cross-shaped images of murine laminins, although the short arms were truncated to various degrees or even absent. The isolated peptic fragments also displayed biological activity similar to that of murine laminins in that the outgrowth of neurites by neuronal cells was promoted on plates coated with the fragments.
In an initial report (Carbonetto, S. T., M. M. Gruver, and D. C. Turner (1982) Science 216: 897-899) we described the use of 2-hydroxyethylmethacrylate (HEMA) in preparing defined culture substrates for studying nerve fiber growth. In those studies fibronectin and collagen were conspicuous, among a variety of HEMA-embedded proteins, in supporting fiber growth of embryonic neurons from chick dorsal root ganglia. Here we further document and extend our preliminary studies, especially with regard to the interaction of growing nerve fibers with fibronectin. HEMA substrates were prepared with proteolytic fragments of fibronectin, each of which had one or more of the functional sites of the intact molecule. Only those fragments of fibronectin that retained the region of the molecule known to mediate myoblast attachment were active in supporting nerve fiber growth. When added in excess to the culture medium, the smallest of the active fragments inhibited fiber growth on substrates that contained intact fibronectin. In culture medium depleted of serum fibronectin, HEMA gels containing collagens purified from connective tissues (types I and III) or from basement membranes (type IV) were about as effective as HEMA substrates containing fibronectin in supporting fiber growth. Nerve fiber growth on collagen substrates proceeded in the absence of fibronectin in the culture medium. Several glycosaminoglycans (heparin, chondroitin sulfate, hyaluronic acid) were ineffective as substrates for fiber growth. Treatment of HEMA/ fibronectin gels with heparin or incorporation of heparin along with fibronectin in the gel profoundly diminished the efficacy of fibronectin in supporting nerve fiber growth. Our studies suggest that the growth of nerve fibers on fibronectin substrates results from direct interaction with a specific portion of the fibronectin molecule and that this interaction can be inhibited by heparin and possibly other glycosaminoglycans.
Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.
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.
Nerve segments approximately 6-7mm long were excised from the predegenerated sciatic nerves of mice, and treated 5 times by repetitive freezing and thawing to kill the Schwann cells. Such treated nerve segments were grafted into the original place, being in contact with the proximal stump of the sciatic nerve. The animals were sacrificed 2, 3, 5, 7 and 10 days, 2, 3, 5 and 8 weeks after the grafting. The grafts were examined at the middle level, i. e., about 3-4mm distal to the proximal end of the graft, by light and electron microscopy.
Within 2-3 days after the grafting, the dead Schwann cells were disintegrated into fragments and gradually phagocytized by macrophages. Howere, the basal laminae of the Schwann cells remained as empty tubes (basal lamina scaffolds). The notable finding was that the regenerating axons always grew through these basal lamina scaffolds. New Schwann cells seemed to migrate along these axons from the proximal stumps. The number of axons growing through the basal lamina scaffolds gradually increased with time. These axons were surrounded in a bundle by Schwann cells. About 1 week after the grafting, axons began to be segregated into smaller bundles by Schwann cells. Axons with a relatively large diameter (about 2μm) tended to be sorted out and surrounded by their own Schwann cells. The myelination began about 2 weeks after the grafting on such large diameter axons. The basal lamina scaffolds, through which the regenerating axons had grown, were gradually disintegrated into fragments by the expansive forces due to the increase in number and volume of the regenerating axons and Schwann cells. Groups of axons, which had been derived from the same basal lamina scaffolds, were enclosed with the cells resembling perineurial epithelial cells. These perineurial epithelial cells proliferated and further separated groups of axons into smaller ones or even into single axons. The number of myelinated axons increased with the advancement of regeneration.
These results show that the basal lamina scaffolds of Schwann cells serve as efficient conduits for the elongation, maintenance and maturation of regenerating axons.
The effect of mouse nerve growth factor (NGF) on cultured human fetal sensory neurons was assayed by measuring neurite length, density and rate of growth. Addition of NGF increased adhesion of dissociated sensory neurons cultured on collagen coated surfaces. Almost all neurons of 9 to 10 week old fetuses are postmitotic, contain neuron-specific enolase, (an enzyme linked to differentiation), and require NGF for optimal neurite growth. Sensory ganglia re-explanted on collagen showed maximal neurite length and density when treated with 1 ng/ml of NGF. Neurite density was reduced considerably in the absence of mouse NGF and was almost abolished by addition of antimouse NGF antibodies. Surfaces coated with the matrix glycoproteins laminin or fibronectin further stimulated neurite growth of ganglia in the presence of NGF. Increasing amounts of matrix proteins could partly compensate for the absence of mouse NGF or the inhibition of NGF activity by antibodies. Stimulation of neurite growth by matrix proteins was time-dependent, and neurites showed maximum length at 10 days (2 to 3 mm). Neurite growth was more pronounced with laminin than with fibronectin and collagen, and antibodies to laminin suppressed all neurite growth. In the presence of a constant amount of NGF, mean neurite growth reached 26 μm/hr (at 1 day), and was 2.1 and 1.7 times faster on laminin and fibronectin (respectively) than on collagen. Thus, laminin, and to a lesser degree fibronectin, may enhance neurite growth of human sensory neurons in synergy with NGF.
The response of growth cones from embryonic chick dorsal root ganglia to a patterned substrate of adsorbed nerve growth factor (NGF) was studied. The patterned substrate presented growth cones with an adsorbed NGF pattern and NGF-free substrate. NGF-responsive growth cones from 7 and 9 day ganglia could not proceed onto NGF-free substrate, reproducing the adsorbed NGF pattern. NGF-unresponsive growth cones from 17 day ganglia did not display any preference for adsorbed NGF or NGF-free substrata, which resulted in neurites not reproducing the adsorbed NGF pattern. Neurite outgrowth from NGF responsive 7-day ganglia onto a patterned NGF substrate, in NGF-containing medium, was radially symmetrical, exhibiting no growth cone response to the patterned NGF substrate. The lack of NGF-responsive growth cone extension onto NGF-free substrate indicates that NGF is a requirement for neurite elongation. If NGF is withdrawn from growth cones by microperfusion, neurite elongation ceases. Thus, an adsorbed pattern of NGF may be duplicated because growth cones are not able to extend onto NGF-free substrate, since NGF is a requirement for neurite elongation. These results indicate that substrate adsorbed NGF can support neurite formation and elongation as well as guide the direction of neurite elongation.
Wistar rats (close cloned strain) were used to investigate the effect of endogenous laminin and fibronectin on axons, Schwann cells and perineurial cells in the regenerating peripheral nervous system (PNS). Sciatic nerve grafts obtained from donor rats were frozen, thawed and treated with rabbit anti-rat laminin or anti-fibronectin antiserum. Control grafts were treated with normal rabbit serum alone. One cm long portions of the sciatic nerve of the recipient rats were replaced with grafts. At 15 days after transplantation the number of regenerated axons in the laminin- and fibronectin-depleted grafts was half of that in the control. The growing axons in the laminin-depleted grafts did not recognize the basal lamina scaffolds (BLS) remaining in the basal lamina tubes, while in the control and fibronectin-depleted grafts 90% or more of axons grew inside the BLS. Elongation of axons always preceded migration of Schwann cells with the latter subsequently adhering to and wrapping around the former. Perineurium-forming fibroblastic cells recognized the combination of axons and Schwann cells and formed perineurial fasciculi around them. These fibroblastic cells did not recognize empty BLS but responded to them only when fibronectin was depleted. Macrophages sometimes closely faced the naked axons which elongated outside the BLS. These results suggest that in the early stages of nerve regeneration endogenous laminin and fibronectin not only regulate the growth of regenerating nerve fibers, but also exert a positive influence on perineurial cells and macrophages, both of which play important roles in nerve tissue injury and repair.
Regeneration of the sciatic nerve in transplanted nerve grafts in which laminin was inactivated was examined electron microscopically. Nerve grafts for transplantation were obtained from close cloned donor Wistar rats; 1-cm nerve segments of the sciatic nerve were frozen and thawed to kill the Schwann cells. Control recipient rats received grafts treated with normal rabbit serum to repair the artificially-made complete defect of the right sciatic nerve, and the experimental group of rats received grafts doubly treated with normal serum and rabbit anti-laminin antiserum. In the control grafts regenerating axons grew almost completely through the inside of the basal lamina scaffolds (92%) and adhered to the structure, while in the anti-laminin antiserum treated grafts the axons were present outside (52%) and inside (48%) the scaffolds simultaneously. In this case, the adhesion of axons to the scaffolds was obscure. Axons were associated with and without Schwann cells both inside and outside the basal lamina scaffolds. No unassociated Schwann cells were observed. The maximal number of axons in a 2 mm portion of the antiserum-treated grafts was approximately 250 axons per 100 x 100 microns square and 520 in the control at 15 days. At 30 days, almost the same number of axons was found at the distal (8 mm) portion of both groups. The growth in the former was delayed for 3 days. These results indicate that regenerating peripheral nerve axons may enter the basal lamina scaffolds and grow well because of the neurotrophic function of laminin present at the inner side of Schwann cell basal lamina.
Autologous muscle grafts were used to repair 12 mixed peripheral nerves (9 posterior tibial, 3 median) in 10 patients with leprosy who had total anaesthesia and analgesia of the area supplied by the nerve. Postoperatively, 7 patients reported improved sensation in the foot or hand, with a return of vibration sense and joint position sense in 11 and of perception of a 10 g pin in 5; the ability to sweat in the affected area was also restored in 7.
While distal influences acting both tropically and trophically upon regenerating nerve fibres are not in doubt, the specificity of such influences is unresolved. The present experiments were designed to test whether pioneering axons regenerating through a large muscle graft showed specificity for their homonymous distal stumps if faced with a choice of pathways. Regeneration was assessed with respect to nerve conduction, the electromyograph, isometric tension and morphological indices of nerve regeneration in the median and ulnar nerves of non-human primates. Reinnervation of both distal stumps with functional neuromuscular transmission was found to take place by growth of nerve fibres from either proximal nerve into either distal stump. There was thus no evidence of specific homonymous reinnervation. The implications of this finding are discussed from a mechanistic and a clinical point of view.
Repair of digital nerves and the larger nerves supplying the hand is a common clinical problem. A number of options exist for either direct repair or reconstitution using an interposed graft. Each of these options is associated with a set of problems peculiar to itself and to nerve repair in general. The heterogeneity of nerve injuries and of the various methods of repair and assessment has not led to the establishment of clear comparisons and guidelines for surgical intervention. Recently, freeze-thawed coaxially aligned skeletal muscle autografts have been added to the list of available grafting techniques. The present article discusses the genesis of the muscle graft technique and examines steps which have been taken to elucidate its performance in controlled laboratory conditions. The extent to which these experimental observations provide a foundation for the clinical use of muscle grafts is discussed.
Microneurosurgical techniques to reconstruct nerve gaps with nerve grafts frequently fail to achieve excellent functional results and create donor-site morbidity. In the present study, 15 patients had gaps of 0.5 to 3.0 cm (mean 1.7 cm) in digital nerves reconstructed by one surgeon with a bioabsorbable polyglycolic acid (PGA) tube. A final evaluation of sensibility was done by a second surgeon at a mean postoperative interval of 22.4 months (range 11 to 32 months). These were all secondary reconstructions. The evaluation included a digital nerve block with local anesthetic for the intact (not reconstructed) digital nerve. Excellent functional sensation (moving two-point discrimination less than or equal to 3 mm and/or static two-point discrimination less than or equal to 6 mm) was present in 33 percent and good functional sensation (moving two-point discrimination of 4 to 7 mm and/or static two-point discrimination of 7 to 15 mm) in 53 percent of the digital nerve reconstructions. One patient with poor sensory recovery and one with no recovery were judged as functional failures (14 percent). Absence of pain at the site of reconstruction was judged by the patient to be excellent in 40 percent, good in 33 percent, and poor in 27 percent. We conclude that reconstruction of nerve gaps of up to 3.0 cm with a bioabsorbable PGA tube gives clinical results at least comparable to the classic nerve graft technique while avoiding donor-site morbidity.
An exogenous fibronectin-laminin (FN-LAM) solution was added into silicone chambers to determine the effects on peripheral nerve regeneration across 18-mm long gaps. The sciatic nerves of adult rats were sutured into silicone chambers 20 mm in length, creating an 18-mm gap between the proximal and distal nerve stumps. The chambers were filled with either a mixture of fibronectin and laminin (500 micrograms/ml each) or a solution of cytochrome C (1 mg/ml) as the control. After six weeks, the animals were killed and the chambers were examined for regeneration. Seventy percent of the animals from the FN-LAM group demonstrated regeneration across the 18-mm gaps, compared to only 30% in the control group. The combination of FN-LAM significantly increased the number of axons that grew into the distal end of the chamber (FN-LAM, 1325 +/- 522; cytochrome C, 153 +/- 104; p = 0.03). Examination of the distal tributaries of the sciatic nerve revealed axons only in the FN-LAM group; none were found in the control group. Quantitative analysis of neurons, retrogradely labeled with horseradish peroxidase via injection of the sciatic nerve distal to the regenerated segment, revealed a greater number of sensory and motor neurons in the FN-LAM group compared to the control group. Morphometric studies revealed that the mean area of the regenerated segment in the FN-LAM group was 37% larger than the controls, and ultrastructural analysis demonstrated a more mature regenerated nerve. This is the first in vivo demonstration that this combination of fibronectin and laminin significantly enhances the regeneration of myelinated axons across a long nerve gap in the rat sciatic nerve.
A study was conducted to compare the regeneration of rat peroneal nerves across 0.5-cm gaps repaired with artificial nerve grafts versus sutured autografts. The artificial nerve graft model is composed of a synthetic biodegradable passive conduit made of polyglycolic acid filled with a collagen extracellular matrix (predominantly Type I collagen, derived from calf skin, and with the telopeptide ends left intact). Axonal regeneration was studied in 22 long-term animals (11 or 12 months). The nerves were studied by qualitative and quantitative histological and electrophysiological methods, and by functional analysis in 9 of the animals. The axonal regeneration of the artificial nerve graft is equal to sutured autografts as measured by axonal counts, and by physiological and functional methods, although the sutured autografts demonstrated statistically superior axonal diameters.
In our previous paper, we reported a marked advantage in using collagen gel matrix available for cell culture in combination with a silicone tube as an effective environment for axonal sprouting during peripheral nerve regeneration. In the present experiment, collagen film was substituted for the silicone tube because of its non-toxicity, biocompatibility and better availability. Also, the surgical procedure was simplified, using a fibrin adhesive system instead of suturing. In the second postoperative week, severed proximal and distal stumps became joined together concomitantly with absorption of the collagen matrix and film. On size-frequency histograms, the diameters of both myelinated and unmyelinated axons at 8 weeks after surgery had recovered to their normal ranges. These findings demonstrate that this procedure of enveloping a collagen matrix and severed nerve stumps in bioresorbable collagen film would be an ideal way of forming a perfect perineurium. The regeneration of peripheral nerve axons resulted in normal thickness of the original nerve bundle without exception, unlike the axons on the control side.
We have tested the ability of a gentle gradient of neurite-promoting activity to orient the extension of embryonic growth cones. Gradients of neurite-promoting activity were made with biologically active, tritium-labeled laminin. The distributions of laminin bound to glass substrata were visualized by autoradiography and quantified with an image processing system. Embryonic chick sympathetic ganglia were explanted onto laminin gradients and cultured. No tendency for neurites to be oriented up-gradient was detected by examining the morphology of explants. Time-lapse studies of individual growth cones detected no up- or down-gradient bias in growth cone motility. These results suggest that growth cone orientation is relatively insensitive to a graded distribution of a naturally occurring neurite-promoting molecule.
Nerve regeneration across a vascularized pseudosheath and a sural nerve graft was compared in a primate model using histologic and electrophysiologic parameters for regeneration. At 12 months, regeneration was noted across both the vascularized pseudosheath and the sural nerve graft. Although histologic and electrophysiologic properties of the regenerated nerve were significantly different from the normal ulnar nerve, the quality of regeneration noted between experimental groups was similar.
Laminin is reportedly an extremely potent neurite-promoting agent in vitro. We investigated the effect of laminin on regeneration of axons in vivo, using a cord of polyester filaments coated with laminin as a guide, replacing a 10-mm segment of the rat sciatic nerve. After 4 weeks the cross section of the nerve guide at its midportion was examined under an electron microscope. Many regenerated axons were seen in the laminin-coated group, whereas no axon was observed in the controls. We conclude that laminin guides the regeneration of axons in vivo.
Semipermeable guidance channels were filled with saline, collagen-, or laminin-containing gels and used to repair a 4-mm sciatic nerve gap in mice. After 12 weeks, nerve cables regenerated in gel-filled channels displayed fewer myelinated axons than saline-filled channels. Remnants of the exogenous substrates were still in evidence, in amounts related to the initial collagen or laminin gel concentration. The impairment of nerve regeneration by collagen or laminin-containing gels suggests that the regenerative environment created within semipermeable channels is not improved by the addition of growth substrates in a gel form.
The suitability of muscle basal lamina as a graft material for the repair of peripheral nerves was investigated. Grafts were prepared by evacuating the myoplasm from muscles excised from rats and rabbits. This produced a material consisting mainly of basal lamina and connective tissue, with the basal lamina arranged as parallel tubes. Rat- and rabbit-derived graft material in 0.5-cm lengths was sutured into rat sciatic nerves, and 4-cm lengths of rabbit-derived graft material were interposed into rabbit sciatic nerves. For controls, 0.5-cm nerve autografts were grafted into rats and 4-cm autografts into rabbits. After 2 to 3 months, the success of the grafts was assessed functionally, electrophysiologically, and anatomically. By all these criteria the basal lamina grafts were as successful as nerve autografts; essentially the same number of axons of the same size grew through both graft types, animals recovered their limb function equally well, and the nerve conduction velocities and relative refractory periods were the same in both groups of animals. In rats, following both basal lamina and nerve autografts, the number of axons distal to the grafts was approximately the same as that proximal to them, but axon diameter and speed of conduction were significantly less than normal. The authors conclude that muscle basal lamina grafts are as effective as nerve autografts for repairing severed rat or rabbit peripheral nerves, and suggest that grafts prepared in this way may prove to be useful for nerve repair in humans.
These experiments present quantitative data concerning peripheral nerve regeneration in vivo. We used entubulation repair as a model to compare two different types of tubular prostheses, one nonbiodegradable and the other biodegradable. We modified the microenvironment of the regenerating axons within the tubular prostheses by adding a laminin-containing gel to the interior of the tube at the time of initial implantation. The data demonstrate that specific manipulations to the microenvironment of regenerating peripheral axons have quantitative effects on the rate and extent of nerve regeneration. Such effects were dependent on the composition of the tubular prosthesis and varied according to the survival time of the animals. For instance, the laminin gel within the biodegradable tubes enhanced nerve regeneration at 2 weeks but was inhibitory at 6 weeks. Furthermore, such manipulations may have different effects on the number of myelinated axons found within the regenerating nerve cable versus the number of primary motor and sensory neurons giving rise to such axons. We concluded that: the presence of a laminin-containing gel significantly increased the initial rate at which axons from primary sensory and motor neurons cross a transection site; an initial delay in axonal outgrowth at early time points did not necessarily predict diminished outgrowth at later times; and because of the potential for axonal branching the number of myelinated axons found in the midportion of a tubular prosthesis did not always correlate with the number of primary motor and sensory neurons which gave rise to those axons.
The sciatic nerve of adult mice was transected and proximal and distal nerve stumps were sutured into a nontoxic bioresorbable nerve guide. Nerve guide lumens were either empty or filled with a gel containing 80% laminin and additional extracellular matrix components. Two weeks later cells in the L3 through L5 dorsal root ganglia and the ventral horn of the spinal cord were retrogradely filled with horseradish peroxidase. All animals with the laminin-containing gel but none with empty nerve guides displayed labeled cells. This suggests that the laminin-containing gel significantly hastened axonal regeneration in vivo.
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 localization of laminin and fibronectin was examined on the basal laminae of Schwann cells. Basal laminae from sciatic nerves were isolated by sonication, and the localization of laminin and fibronectin on such isolated basal laminae was studied by immunoferritin histochemistry. Laminin was localized mainly on the cellular side (i.e. the side originally facing the Schwann cell plasma membrane) of the basal laminae. On the other hand, fibronectin was found to be present as aggregates only on the interstitial side (i.e., the side originally facing the endoneurial connective tissue) of the basal laminae. Thus, the locations of laminin and fibronectin were distinctly different. It is presumed that laminin might be involved in the attachment of axons and Schwann cells to the basal laminae, while fibronectin mediates the adhesion of the basal laminae to connective tissue elements, including the collagen fibrils. These findings are discussed from a standpoint of nerve regeneration through the basal laminae scaffolds of Schwann cells.
Severe nerve injuries may require microsurgical grafting to span a defect. Introduction of graft material into a highly vascular recipient bed is documented to aid in early regeneration of neuronal blood supply.
A silicone rod (SR)-induced fibrovascular sheath was employed to evaluate the regeneration of rat tibial nerve through 2-mmdiameter collagen tubes (CT) or contralateral nerve autografts (AUTO). At first operation, 5 mm of right tibial nerve was resected from 30 retired male breeder Sprague-Dawley rats. Resected nerve was replaced with either a 5 x 2 mm SR or the nerve ends were sutured to the intermuscular fascia.
Four weeks later, animals were repaired by replacing the SR with either a CT or a contralateral AUTO from the left tibial nerve. Three months later, EMG testing was performed, and histologic sections were prepared.
The EMG latency and the size of the compound action potential for sheathed or non-sheathed CT or AUTO were statistically superior to controls at the 95% confidence level. All other intergroup comparisons of latency and action potential size were statistically insignificant. The proportion of nerve fibers traversing the surgical sites was not influenced by the method of repair or by the presence or absence of sheathing. Tubulized repairs most closely resembled unoperated nerves, and autografted repairs had a large diameter, but much fibrosis, whereas controls displayed immaturity and disorganization.
Our observations suggest that there was no difference between repairs performed with or without a vascular pseudosheath. However, CT supported regeneration better than did AUTO repair.
Nerve segments approximately 7 mm long were excised from the predegenerated sciatic nerves of mice, and treated 5 times by repetitive freezing and thawing to kill the Schwann cells. Such treated nerve segments were grafted into the original places so as to be in contact with the proximal stumps. The animals were sacrificed 1, 2, 3, 5, 7 and 10 days after the grafting. The grafts were examined by electron microscopy in the middle part of the graft, i.e. 3-4 mm distal to the proximal end and/or near the proximal and distal ends of the graft. In other instances, the predegenerated nerve segments were minced with a razor blade after repetitive freezing and thawing. Such minced nerves were placed in contact with the proximal stumps of the same nerves. The animals were sacrificed 10 days after the grafting. Within 1-2 days after grafting, the dead Schwann cells had disintegrated into fragments. They were then gradually phagocytosed by macrophages. The basal laminae of Schwann cells, which were not attacked by macrophages, remained as empty tubes (basal lamina scaffolds). In the grafts we examined, no Schwann cells survived the freezing and thawing process. The regenerating axons always grew out through such basal lamina scaffolds, being in contact with the inner surface of the basal lamina (i.e. the side originally facing the Schwann cell plasma membrane). No axons were found outside of the scaffolds. One to two days after grafting, the regenerating axons were not associated with Schwann cells, but after 5-7 days they were accompanied by Schwann cells which were presumed to be migrating along axons from the proximal stumps. Ten days after grafting, proliferating Schwann cells observed in the middle part of the grafts had begun to sort out axons. In the grafts of minced nerves, the fragmented basal laminae of the Schwann cells re-arranged themselves into thicker strands or small aggregations of basal laminae. The regenerating axons, without exception, attached to one side of such modified basal laminae. Collagen fibrils were in contact with the other side, indicating that these modified basal laminae had the same polarity in terms of cell attachment as seen in the ordinary basal laminae of the scaffolds.(ABSTRACT TRUNCATED AT 400 WORDS)
Using the transected sciatic nerve model in adult mice, regeneration of a large bundle of axons organized into the form of a nerve with myelinated and unmyelinated axons, Schwann cells, fibroblasts, collagen, blood vessels, and connective tissue sheaths has been achieved with bioresorbable microtubular guidance channels over gaps of 5 mm in nonimmobilized animals. After 4-6 wks postoperatively, the regenerated nerve cable contains on the order of 40% as many myelinated axons as were measured in the proximal nerve stumps. With the channels used so far in this model, regenerating axons pass into the distal stump in about 3-6 wks postoperatively. The guidance channels used consist of synthetic polyesters and/or polyester composites including glycolic and lactic acid polymers, and polyesters derived from Krebs Cycle dicarboxylic acids. Inflammatory response to these materials has been minimal. Biodegradation/resorption rates can be controlled so as to be compatible with axon growth rates.
Nerve segments approximately 6-7 mm long were excised from the predegenerated sciatic nerves of mice, and treated 5 times by repetitive freezing and thawing to kill the Schwann cells. Such treated nerve segments were grafted into the original place, being in contact with the proximal stump of the sciatic nerve. The animals were sacrificed 2, 3, 5, 7 and 10 days, 2, 3, 5 and 8 weeks after the grafting. The grafts were examined at the middle level, i.e., about 3-4 mm distal to the proximal end of the graft, by light and electron microscopy. Within 2-3 days after the grafting, the dead Schwann cells were disintegrated into fragments and gradually phagocytized by macrophages. However, the basal laminae of the Schwann cells remained as empty tubes (basal lamina scaffolds). The notable finding was that the regenerating axons always grew through these basal lamina scaffolds. New Schwann cells seemed to migrate along these axons from the proximal stumps. The number of axons growing through the basal lamina scaffolds gradually increased with time. These axons were surrounded in a bundle by Schwann cells. About 1 week after the grafting, axons began to be segregated into smaller bundles by Schwann cells. Axons with a relatively large diameter (about 2 microns) tended to be sorted out and surrounded by their own Schwann cells. The myelination began about 2 weeks after the grafting on such large diameter axons. The basal lamina scaffolds, through which the regenerating axons had grown, were gradually disintegrated into fragments by the expansive forces due to the increase in number and volume of the regenerating axons and Schwann cells. Groups of axons, which had been derived from the same basal lamina scaffolds, were enclosed with the cells resembling perineurial epithelial cells. These perineurial epithelial cells proliferated and further separated groups of axons into smaller ones or even into single axons. The number of myelinated axons increased with the advancement of regeneration. These results show that the basal lamina scaffolds of Schwann cells serve as efficient conduits for the elongation, maintenance and maturation of regenerating axons.
This study was performed to determine whether vein grafts might serve as a conduit for nerve regeneration. A 1 cm segment of sciatic nerve was removed bilaterally in 12 Sprague-Dawley rats. On one side the gap was not repaired, and on the other side a segment of femoral vein was used to bridge the nerve gap. Nerve conduction studies and necropsies were performed at intervals. Reconstitution of nerve trunk continuity and healing of plantar ulcers occurred only in the vein-grafted side. Histologic examination revealed orderly growth of nerve fibers within the lumen of the vein grafts as early as 1 month after repair. Most regenerating nerve fibers passed through the proximal junction in an orderly pattern and reached the distal stumps within 2 months after repair. Results of nerve conduction study at 4 months after operation demonstrated restoration of conduction through the vein-grafted sciatic nerves with muscle reinnervation. Nearly normal muscle fibers in the gastrocnemius on the repaired side were confirmed at necropsy. This study demonstrated that autogenous vein grafts can serve as a conduit for nerve regeneration in rodents.
The effect of mouse nerve growth factor (NGF) on cultured human fetal sensory neurons was assayed by measuring neurite length, density and rate of growth. Addition of NGF increased adhesion of dissociated sensory neurons cultured on collagen coated surfaces. Almost all neurons of 9 to 10 week old fetuses are postmitotic, contain neuron-specific enolase, (an enzyme linked to differentiation), and require NGF for optimal neurite growth. Sensory ganglia re-explanted on collagen showed maximal neurite length and density when treated with 1 ng/ml of NGF. Neurite density was reduced considerably in the absence of mouse NGF and was almost abolished by addition of antimouse NGF antibodies. Surfaces coated with the matrix glycoproteins laminin or fibronectin further stimulated neurite growth of ganglia in the presence of NGF. Increasing amounts of matrix proteins could partly compensate for the absence of mouse NGF or the inhibition of NGF activity by antibodies. Stimulation of neurite growth by matrix proteins was time-dependent, and neurites showed maximum length at 10 days (2 to 3 mm). Neurite growth was more pronounced with laminin than with fibronectin and collagen, and antibodies to laminin suppressed all neurite growth. In the presence of a constant amount of NGF, mean neurite growth reached 26 microns/hr (at 1 day), and was 2.1 and 1.7 times faster on laminin and fibronectin (respectively) than on collagen. Thus, laminin, and to a lesser degree fibronectin, may enhance neurite growth of human sensory neurons in synergy with NGF.
Vein grafts have been used as conduits for nerve regeneration in both research protocols and clinical situations. Results to date are encouraging. This study investigated several variations in the vein graft model in an attempt to enhance peripheral nerve regeneration. Results showed that the jugular vein, with an improved suture technique made possible by its larger diameter, is superior to the femoral vein as a conduit for regeneration of the sciatic nerve in the rat. The larger diameter of the jugular vein and its lack of valves provided less hinderance to regenerating axons. Results also showed that coating the luminal epithelium of the jugular vein with type I collagen gel greatly improved regeneration.
We report a vascularized sciatic nerve graft in the rat, based upon the femoral popliteal superior muscular artery pedicle. The pedicle may be raised from the common femoral artery via the popliteal artery, with accompanying venous drainage to the femoral vein. We have characterized two vessels: the middle tibial artery to the posterior tibial nerve and the arteria comitas nervus peroneus to the peroneal nerve. Plastic monomer and Evans blue labeled albumin injections established the anatomic model. The graft was reliably perfused at the time of isolation and 24 hours post-transplantation. The model was used to evaluate regeneration across allogeneic (ACI to Lewis, n = 12) versus syngeneic (Lewis to Lewis, n = 6) nerve grafts. Electrophysiologic and histomorphometric assessments demonstrated that the vascularized immunosuppressed allograft was similar to the vascularized syngeneic graft. Both were superior to the vascularized allograft without immunosuppression.
A new experimental model was designed in which the regeneration of rat femoral nerve across a 8 mm excised gap was investigated after insertion of the distal and proximal stumps into the anatomically and functionally intact femoral artery (AIAB). This model was compared with groups of free artery and autologous nerve grafting. After a period of 12 weeks, a histological and electrophysiological analysis was carried out, which demonstrated that the AIAB and autologous nerve grafting group had a significantly higher percentage of regeneration compared with the free artery-graft group. The nerve regeneration and intraneural vascular reconstruction that occurred within AIAB group were more successful than those that occurred in the artery and nerve-grafting groups.
We investigated the role of laminin in functional recovery of a peripheral nerve injury using electrophysiological and behavioral approaches on the rat sciatic nerve in vivo. These studies were complemented by neurofilament protein immunocytochemistry on the sciatic nerve 20 days after an operation, in which an 8-mm piece of the nerve was removed and replaced by a graft of laminin, its neurite outgrowth-promoting peptide, a control peptide, collagen, or by resuturing of the removed piece of the nerve. Electrophysiological measurements of muscle strength 4 months after the sciatic nerve transection showed that a laminin graft was as effective as neurorrhaphy in supporting functional recovery of an injured peripheral nerve. A laminin graft also significantly reduced autotomy in the operated animals. Immunocytochemistry confirmed that both a laminin graft and resuturing supported growth of the 200-kDa neurofilament-positive axons into the distal stump of the nerve within 20 days of operation. A graft with a neurite outgrowth-promoting peptide of the B2 chain of laminin supported similar axon growth, whereas another peptide graft also derived from laminin or a collagen graft did not support axon growth. All grafts allowed Schwann cell growth into the distal stumps of the nerves, but neurites accompanied them only in the regeneration-supporting grafts and in the resutured nerves. The Schwann cells of the regenerating nerves expressed high levels of the neurite outgrowth-promoting domain of the B2 chain of laminin, whereas the Schwann cells of the degenerating nerves failed to express this domain in the distal stumps of the degenerating nerves. These results provide the first in vivo evidence for the functional role of laminin in peripheral nerve regeneration. As the neurite outgrowth-promoting domain of the B2 chain of laminin is as efficient as laminin or resuturing in supporting a short-term recovery of an injured sciatic nerve, this area may be a regeneration-promoting domain of this glycoprotein. More importantly, as grafting significantly reduces post-traumatic pain behavior in the operated animals, the laminin graft surgery may provide a useful method for clinical restoration of the injured peripheral nerves.
Autogenous vein graft was used to fill 18 digital nerve defects between 0.5 to 5.8 cm in length during flexor tendon surgery in zone 2. The vein was taken from the forearm and reversed to bridge the digital nerve. For nerves with defects over 2.0 cm, normal nerve slices were inserted inside vein conduits. Recovery of sensibility was evaluated by von Frey test, pin-prick detection, localization of stimulus, moving two-point discrimination and sweating on the finger pulp. Follow-up revealed excellent recovery in two digital nerves, good in nine, fair in five and poor in two. The results suggest that vein graft provides a simple and practical method to reconstruct a digital nerve defect during tendon repair in zone 2.
The use of vein or muscle grafts to bridge nerve defects longer than 1-1.5 cm gives poor results. Veins collapse and in muscle grafts axons may regrow outside the graft. We used veins (to guide regeneration) filled with muscle (to avoid vein collapse). Nerve regeneration through 1 and 2 cm grafts made of vein plus muscle was compared with similarly long traditional nerve grafts, free fresh muscle grafts, and empty vein grafts. Regeneration was assessed clinically and histologically (qualitative and quantitative evaluation) in the graft and distal nerve stumps. Vein plus muscle grafts were superior to vein and fresh muscle grafts both functionally and histologically. Functional results were similar to those found in traditional nerve grafts, but axon number was superior in the veins filled with muscle. This suggests that vein filled with muscle might serve as a grafting conduit for the repair of peripheral nerve injuries and could give better results than traditional nerve grafting.
Laminin gel stimulates axonal regeneration in vivo
- Da Silva
- C Dikkes
- P Madison
- R Greatorex
- D Sid-Man
Da Silva, C., Dikkes, P., Madison, R., Greatorex, D. and Sid-man, R., Laminin gel stimulates axonal regeneration in vivo, Soc. Neurosci. Abstr., 10 (1984) 283.
Quantitative effects of a laminin-containing gel, collagen, or empty polyethylene tube on peripheral nerve regeneration in vivo
- Da Silva
- C Madison
- R Greatrex
- D Dikkes
- P Sidman
Da Silva, C., Madison, R., Greatrex, D., Dikkes, P. and Sidman, R., Quantitative effects of a laminin-containing gel, collagen, or empty polyethylene tube on peripheral nerve regeneration in vivo, Soc. Neurosci. Abstr., 11 (1985) 1253.
What Makes Nerves Regenerate? Many experimental strategies revolve around a central issue: what conditions promote regeneration and functional recovery in mammalian nerves?
- Barnes