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Increased Rate of Peripheral Nerve Regeneration Using Bioresorbable Nerve Guides and a Laminin Containing Gel
Abstract
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.
... Haptotactic cues are the incorporation of contact-mediated cues such as insoluble ECM proteins to guide axonal elongation to the target organ [8]. ECM proteins such as collagen, fibronectin, and laminin have considerable positive effects on nerve regeneration [9][10][11][12][13][14]. For example, laminin is one of the series of structural proteins that activates the β1-integrin receptor and is a critical player of the peripheral nerve basal lamina, which is required to promote Schwann cell migration and axonal elongation [15][16][17][18][19]. ...
... Laminin can be found in the peripheral nerve basal lamina that is effective in promoting Schwann cell migrations and neurite outgrowth [32][33][34]. It interacts with Schwann cell integrins, which is essential for successful nerve regeneration [9][10][11][12][13][14]. Armstrong et al [9] demonstrated that Schwann cells proliferation was influenced more by laminin than collagen and fibronectin. ...
Artificial nerve guidance conduits (NGCs) are being investigated as an alternative to autografts, since autografts are limited in supply. A polycaprolactone (PCL)-based spiral NGC with crosslinked laminin on aligned nanofibers was evaluated in vivo post a successful in vitro assessment. PC-12 cell assays confirmed that the aligned nanofibers functionalized with laminin were able to guide and enhance neurite outgrowth. In the rodent model, the data demonstrated that axons were able to regenerate across the critical nerve gap, when laminin was present. Without laminin, the spiral NGC with aligned nanofibers group resulted in a random cluster of extracellular matrix tissue following injuries. The reversed autograft group performed best, showing the most substantial improvement based on nerve histological assessment and gastrocnemius muscle measurement. Nevertheless, the recovery time was too short to obtain meaningful data for the motor functional assessments. A full motor recovery may take up to years. An interesting observation was noted in the crosslinked laminin group. Numerous new blood capillary-like structures were found around the regenerated nerve. Owing to recent studies, we hypothesized that new blood vessel formation could be one of the key factors to increase the successful rate of nerve regeneration in the current study. Overall, these findings indicated that the incorporation of laminin into polymeric nerve conduits is a promising strategy for enhancing peripheral nerve regeneration. However, the best combination of contact-guidance cues, haptotactic cues, and chemotactic cues have yet to be realized. The natural sequence of nerve regeneration should be studied more in-depth before modulating any strategies.
... In the animal model, a critical distance of 10 millimeters between nerve endings was identified (Lundborg et al., 1982c). This distance can be enlarged by predenervation of the distal stump to 15 millimeters , and by changing the biochemical milieu of the chamber, to 20 millimeters (Madison et al., 1985;Müller et al., 1987). ...
... However, it was only the use of modern procedures of biochemical and electrophysiological analysis which led to knowledge of the cellular and molecular interplay which takes place during the regeneration process. Advances in manufacturing techniques have made it possible to integrate specific molecules, for example laminin, in the wall of synthetic chambers (Madison et al., 1985) and via their pore size, to determine the permeability for cells and substances (Jenq and Coggeshall, 1987;Aebischer et al., 1988;Aebischer et al., 1989). Using the fiber count, Jenq and Coggeshall (Jenq and Coggeshall, 1987) were able to demonstrate a stimulating effect of the external matrix on the growth behavior of the axon sprouts. ...
Autologous nerve grafts are steadily regarded as the method of choice for bridging the nerve gaps resulting after peripheral nerve lesions with substance defects. Microsurgical techniques and the perineurial suture of corresponding fascicles have improved the functional results following peripheral nerve graft.
However, regeneration success is often disappointing, despite the most thorough technique and expertise. The loss of spinal motoneurons associated with a nerve lesion and the growth of axon sprouts in inadequate endoneurial sheaths were held responsible as the reason for the lowered muscular strength, limited movement coordination and fine motor skills, poor differentiation and localization of sensory stimuli and for the lack of tactile gnosis.
In this experimental study, it is assumed that the central effects at the level of the spinal motoneuron nuclei show an image of the peripheral misinnervation in topographical-morphological terms, and can supply an explanatory model for the functional motor deficits after peripheral nerve graft.
On the other hand, the plastic changes of a motor cell column in the reinnervation process influence the structural-functional relationships of the motor units in a variety of clinically relevant ways.
... In an attempt to expedite regeneration and synthetically mimic the natural fibrin matrix, many nerve regeneration studies use prefilled nerve conduits with a three-dimensional inner lumen hydrogel matrix derived from biopolymers such as laminin, alginate, or collagen. [5][6][7][8][9][10][11][12][13][14] The chemical attachment of a cell-signaling or neurite-promoting moiety to the filler matrix can enhance regeneration further. Among many others, brain-derived neurotrophic factor, 15 platelet-derived growth factor, 16 and glial growth factor 17,18 have been explored. ...
... Although many nerve repair studies have used a conduit filled with a cell-friendly matrix, [5][6][7][8][9][10][11][12][13][14] our study suggests that the effects of using such a filler may only be beneficial if conduits are impermeable to infiltrating fibrous tissue. When porous conduits are filled with a hydrogel, a balance must be achieved between the conduit porosity and the bioactivity of the filler matrix to encourage faster axonal regeneration than fibrotic tissue infiltration. ...
Nerve conduits pre-filled with hydrogels are frequently explored in an attempt to promote nerve regeneration. This study examines the interplay between the porosity of the conduit wall and the level of bioactivity of the hydrogel used to fill the conduit. Nerve regeneration in porous (P) or non-porous (NP) conduits that were filled with either collagen-only or collagen enhanced with a covalently attached neurite-promoting peptide mimic of the glycan Human Natural Killer Cell Antigen-1 (m-HNK) were compared in a 5 mm critical size defect in the mouse femoral nerve repair model. While collagen is a cell-friendly matrix that does not differentiate between neural and non-neural cells, the m-HNK-enhanced collagen specifically promotes axon growth and appropriate motor neuron targeting. In this study, animals treated with non-porous (NP) conduits filled with collagen grafted with m-HNK (CollagenHNK) had the best functional recovery, commensurate with a substantial improvement in all tested histomorphometric parameters (number of axons, degree of raw tissue area, percentage of myelinated nerve fibers, cross-sectional area of myelinated nerve fibers) relative to all other conduit conditions. Our data indicate that under some conditions, the use of generally cell friendly fillers such as collagen, may limit nerve regeneration. This finding is significant, considering the frequent use of collagen based hydrogels as fillers of nerve conduits.
... Another more complex hydrogel addition to nerve guidance conduits is Matrigel, a basement membrane hydrogel containing laminin, collagen IV, heparan sulphate proteoglycans and growth factors, which is commonly used as a cell carrier and coating for neural stem cells (Rodriguez et al., 2000;Kim et al., 2007;Koutsopoulos and Zhang, 2013;Li et al., 2018;Liu et al., 2020). Including Matrigel in collagen conduits has been shown to improve regeneration compared with collagen alone, which was not improved with the addition of Schwann cells (Udina et al., 2004), as well as encouraging Schwann cell infiltration into the proximal and distal device of a hollow conduit, resulting in improved axonal regeneration in short gap repair models (Madison et al., 1985;Labrador et al., 1998). Although Matrigel supports neural cells and Schwann cell migration in vitro it is unclear if this translates well to in vivo peripheral nerve regeneration as in other studies the inclusion of Matrigel decreased axon number compared to the empty tube control or autograft (Guenard et al., 1992). ...
Nerve tissue function and regeneration depend on precise and well-synchronised spatial and temporal control of biological, physical, and chemotactic cues, which are provided by cellular components and the surrounding extracellular matrix. Therefore, natural biomaterials currently used in peripheral nerve tissue engineering are selected on the basis that they can act as instructive extracellular microenvironments. Despite emerging knowledge regarding cell-matrix interactions, the exact mechanisms through which these biomaterials alter the behaviour of the host and implanted cells, including neurons, Schwann cells and immune cells, remain largely unclear. Here, we review some of the physical processes by which natural biomaterials mimic the function of the extracellular matrix and regulate cellular behaviour. We also highlight some representative cases of controllable cell microenvironments developed by combining cell biology and tissue engineering principles.
... Earlier reports on the effects of peroral administration of drugs known to speed up axonal regrowth have shown that this treatment leads to reduced postoperative hyperinnervation of muscles (Angelov et al., 1996(Angelov et al., , 1997. Alternatively, extracellular matrix (ECM) proteins, such as collagen Type I (Dohm et al., 2000;Rosen et al., 1990), fibronectin, laminin (Madison et al., 1985;Madison et al., 1987;Borkenhagen et al., 1998;Dohm et al., 2000), tenascin-R (Dohm et al., 2000;Pesheva et al., 1994;Taylor et al., 1993), neuropilin-1/Fc chimera, and semaphorin 3A/Fc chimera (Hristov et al., 2005) have been applied locally to evaluate whether axonal regrowth stimulation might prevent the appearance of supernumerary axonal branches and hence improve target reinnervation. ...
Insufficient recovery after injury of a peripheral motor nerve is due to (1) inappropriate pathfinding as a result of axonal regrowth to inappropriate targets, (2) excessive collateral axonal branching at the lesion site, and (3) polyinnervation of the neuromuscular junctions (NMJs). The rat facial nerve model is often used because of its simple and reliable readout to measure recovery of function (vibrissal whisking). Over the last decades scientists have concentrated their efforts to combat mostly NMJ polyinnervation, because it turned out to be very difficult to reduce collateral axonal branching and impossible to navigate thousands of axons toward the original fascicles. In the past, several groups of scientists concentrated their efforts to reduce the activity‐dependent polyinnervation of NMJs by electrical stimulation of the muscles (square 0.1 msec pulses at 5 Hz). The results showed no recovery of functions and a severe reduction in the number of innervated NMJs to approximately one fifth of those observed in intact animals. More recent experiments, however, have shown that motor recovery improved significantly following mechanical stimulation of the denervated facial muscles (vibrissal and orbicularis oculi) and that restored functions could invariably be linked to reduced polyinnervation at the NMJ while the number of innervated NMJ remained the same. These results suggest that clinically feasible and effective therapies could be developed and tested in the near future. Anat Rec, 302:1287–1303, 2019. © 2019 Wiley Periodicals, Inc.
... At body temperature, 37°C, Matrigel® gels and forms a structure identical to three-dimensional ECM, supporting cell adhesion and differentiation and providing the ideal environment to promote in vivo regeneration of different tissues. Effectively, the use of Matrigel® promotes attachment and differentiation of neurons [53,54] and supports in vivo peripheral nerve regeneration [55][56][57][58]. ...
Peripheral nerve injury remains a clinical challenge with severe physiological and functional consequences. Despite the existence of multiple possible therapeutic approaches, until now, there is no consensus regarding the advantages of each option or the best methodology in promoting nerve regeneration. Regenerative medicine is a promise to overcome this medical limitation, and in this work, chitosan nerve guide conduits and olfactory mucosa mesenchymal stem/stromal cells were applied in different therapeutic combinations to promote regeneration in sciatic nerves after neurotmesis injury. Over 20 weeks, the intervened animals were subjected to a regular functional assessment (determination of motor performance, nociception, and sciatic indexes), and after this period, they were evaluated kinematically and the sciatic nerves and cranial tibial muscles were evaluated stereologically and histomorphometrically, respectively. The results obtained allowed confirming the beneficial effects of using these therapeutic approaches. The use of chitosan NGCs and cells resulted in better motor performance, better sciatic indexes, and lower gait dysfunction after 20 weeks. The use of only NGGs demonstrated better nociceptive recoveries. The stereological evaluation of the sciatic nerve revealed identical values in the different parameters for all therapeutic groups. In the muscle histomorphometric evaluation, the groups treated with NGCs and cells showed results close to those of the group that received traditional sutures, the one with the best final values. The therapeutic combinations studied show promising outcomes and should be the target of new future works to overcome some irregularities found in the results and establish the combination of nerve guidance conduits and olfactory mucosa mesenchymal stem/stromal cells as viable options in the treatment of peripheral nerves after injury.
... Regeneration is optimal if a conduit is in situ for an appropriate length of time so as to support axonal guidance but is reabsorbed before effects of chronic constriction take place. Several materials have been used to construct such tubes: copolymers of glycolic and lactic acids (Molander et al., 1982;Nyilas et al., 1983;Seckel et al., 1984;Madison et al., 1985;da Silva et al., 1985); collagen alone (Archibald and Madison, 1984;Colin and Donoff, 1984;Toyota et al., 1988) and in conjunction with other materials (Yannas et al., 1985;Kiyotani et a l, 1995;Ellis and Yannas, 1996); copolymers of lactic acid and caprolactone (den Dunnen et al., 1993(den Dunnen et al., , 1995(den Dunnen et al., and 1996; maxon (glycolide trimethylene carbonate) (Mackinnon and Dellon, 1990); poly (organo)phosphazene compounds (PEIP) (Langone et al., 1995) and polytetrafluoroethylene (PTFE) (Zetti et a l, 1991). ...
Following trauma and loss of peripheral nerve tissue, an autologous nerve graft is the current gold standard for surgical repair. The alternative use of synthetic conduits has not yet offered suitable materials for wide clinical application. Two novel materials, poly-3-hydroxybutyrate (PHB) and polydioxanone (PDS), are bioresorbable and potentially useful as nerve conduits. The aim of this study was to examine peripheral nerve regeneration in vivo following grafting of PHB or PDS conduits, either alone or in combination with glial growth factor (GGF). Schwann cells are essential for nerve regeneration. GGF delivery to the injured nerve may increase their number within the graft and, indirectly enhance nerve regeneration. Nerve regeneration at the repair site was assessed by quantitative immunohistochemistry up to 1 year post-operatively. Centrally, primary sensory neuron survival and peripherally, target organ reinnervation were also studied morphologically following injury and repair. The use of PHB wrap-around for primary nerve repair was compared with direct epineural suture in the cat superficial radial nerve model. The two methods showed comparable values of axonal counts, diameter, myelin thickness and g-ratio. The results demonstrated that PHB wrap-around is a suitable alternative to epineural suture in primary repair. PHB tubes tested for nerve gap repair in the rat sciatic nerve model exhibited axonal regeneration comparable to that in nerve autografts. GGF administration in PHB tubes produced progressive increase of axonal growth with time, secondary to GGF-induced Schwann cell proliferation. PDS tubes also supported axonal regeneration, although these presented problems of material fragmentation and intense inflammatory reaction. Microgeometry of the internal surface of PDS tubes did not significantly improve nerve regeneration, peripheral target organ reinnervation or sensory neuron survival. The results of this study demonstrated PHB and PDS conduits sustain successful peripheral nerve regeneration, which can be further improved by targeted administration of growth factor.
... Silicone tubes packed with a degradable coUagen-glycosaminoglycan polysaccharide matrix was shown to support regeneration across a 15 mm gap in the rat sciatic nerve (Yannas, 1985;Yannas et al., 1985). DaSilva et al., 1985 andMadison et al., 1985, induced sciatic nerve regeneration across a 20 mm gap through tubes lined with either a laminin-containing gel or a collagen matrix. ...
Peripheral nerve injuries commonly result in a surgically irreducible gap and represent a serious problem in surgery. Clinically, autologous nerve grafts are the most effective means of promoting axonal regeneration across the space, but normally produce donor site morbidity. Other variations of experimental models have been used with the aim of directing rapid nerve regeneration, including entubation, artery and vein grafts and Millipore. However, each of these have significant limitations which affect complete regrowth. Fibronectin (Fn), a large extracellular matrix cell adhesion glycoprotein has been made into three-dimensional mats with a predominant fibre direction and shown to successfully enhance peripheral nerve regeneration in a rat model. The aim of this study was to identify the main features of Fn-implants which are important for rapid nerve repair. This involved testing implanted versions of Fn-mat from rat and monkey models, assessment of cell-matrix interaction in vitro and modification of Fn-materials by chemical and growth factor addition. The results from this study show that new materials can be made from fibronectin which have a potential use in repair of long peripheral nerve lesions. These fibronectin-based materials may be stabilised with micromolar concentrations of copper and zinc which also support strong growth of Schwann cells within these materials in culture. Fibronectin mats may also be soaked with neurotrophins to enhance nerve cell survival and enhance nerve regeneration. The speed of Schwann cell migration and alignment (upto 50 μm away from the original fibre) was increased. Migration speed was further enhanced when the fibres were treated with micromolar concentrations of copper or made, with a substantial content of fibrinogen (optimum 50:50). Taken together, all these results suggest a first design for an ideal conduit material for peripheral nerve repair. This would be (a) dimensions (fibrous), (b) fibre orientation (c) 50:50 fibronectin: fibrinogen, (d) treated with micromolar concentrations of copper, (e) seeded with Schwann cells in culture, and (f) soaked with a 'cocktail' of neurotrophic growth factors including nerve growth factor and neurotrophin-3.
... The concept of providing a three-dimensional filling substance to the nerve guidance is Sciatic nerve regeneration in vein grafts-Review of Literatures 52 not a new one. Filling the tube with a substance, which form a loose matrix of a substrate compatible with axonal regeneration, can promote the regeneration of nerves across gaps within a guidance nerve conduit and it has been shown to enhance structural stability as well as act as a scaffold to support cell migration (Madison, daSilva et al. 1985;Seckel 1990). Insoluble extracellular matrix molecules including laminin, fibronectin, and some forms of collagen, promote axonal extension and, therefore, are excellent candidates for incorporation into the lumen of guidance channels(Evans 2001). ...
... Laminin-111, a 800-kDa protein, is the prototype of the family and the best characterized laminin isoform [1,3] It is adhesive for most cell types, promotes cell survival in vitro and has various biological key activities [3][4][5], including cell adhesion, proliferation, differentiation and migration [1,6]. Laminins are frequently used for in vitro and in vivo neuronal cell cultivation [7][8][9][10][11], angiogenesis [5,12], wound healing [6,[13][14][15], or stem cell studies [16,17]. ...
Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield. Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells. In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.
... 13 In other words, laminin has been shown to increase axonal regeneration. 14 Schwann cell basal membrane includes NPFs, like laminin. There are specifi c receptors for laminin in growth cone. ...
... Laminin-111, a 800-kDa protein, is the prototype of the family and the best characterized laminin isoform [1,3] It is adhesive for most cell types, promotes cell survival in vitro and has various biological key activities [3][4][5], including cell adhesion, proliferation, differentiation and migration [1,6]. Laminins are frequently used for in vitro and in vivo neuronal cell cultivation [7][8][9][10][11], angiogenesis [5,12], wound healing [6,[13][14][15], or stem cell studies [16,17] , . ...
Isolation of human protein from placenta
... [138,139] This was first applied to optic nerve regeneration in rabbits followed by a 5 mm PNI defect in mice. [139] Initial results showed improved regeneration over a 2 week period compared to a hollow tube, [140] though a follow-up study revealed that long-term growth (6 weeks) was impeded. [141] Valentini et al. later confirmed this using different concentrations of either Matrigel and collagen-only gels for a 5 mm defect in the same animal model over a 12 week period. ...
Biofabrication techniques have endeavored to improve the regeneration of the peripheral nervous system (PNS), but nothing has surpassed the performance of current clinical practices. However, these current approaches have intrinsic limitations that compromise patient care. The “gold standard” autograft provides the best outcomes but requires suitable donor material, while implantable hollow nerve guide conduits (NGCs) can only repair small nerve defects. This review places emphasis on approaches that create structural cues within a hollow NGC lumen in order to match or exceed the regenerative performance of the autograft. An overview of the PNS and nerve regeneration is provided. This is followed by an assessment of reported devices, divided into three major categories: isotropic hydrogel fillers, acting as unstructured interluminal support for regenerating nerves; fibrous interluminal fillers, presenting neurites with topographical guidance within the lumen; and patterned interluminal scaffolds, providing 3D support for nerve growth via structures that mimic native PNS tissue. Also presented is a critical framework to evaluate the impact of reported outcomes. While a universal and versatile nerve repair strategy remains elusive, outlined here is a roadmap of past, present, and emerging fabrication techniques to inform and motivate new developments in the field of peripheral nerve regeneration.
... Experimenters have preloaded the tube with biological materials such as fibronectin, laminin, collagen, and Nerve Repair: Biomaterials glycosaminoglycans, which are naturally occurring extracellular matrix molecules. Prefilling the tube speeds up regeneration (Madison et al., 1985) and supports regeneration across longer gaps because the initial matrix and its components that would first need to be formed or synthesized within the tube are already present. Besides its biological characteristics, the physical characteristics of the matrix play an important role in promoting regeneration. ...
Nerve guidance channels fabricated from different biomaterials with different physical and chemical properties have been designed to enhance regeneration of severed peripheral nerves. The goal is to produce a readily available implant to replace missing nerve cable when the two ends cannot be sutured together, thus eliminating the need to harvest a nerve segment from the patient. Currently, there are several clinically approved implants made mostly from singular material components that in general show recovery equivalent to nerve grafts for short gaps. The challenge remains in bridging longer gaps. This may someday be overcome by combining different tube properties along with engineering the constituents placed within the tube.
... In particular, a 3D culture technology using a collagen gel as a cell culture substrate is effective to reconstruct a model of angiogenesis (Velazquez et al. 2002;Tsujii et al. 1998), cancer invasion model (Tsujii et al. 1998;Kusunoki et al. 2002), epithelial mesenchymal models (Bell et al. 1981) such as seen in a skin. Furthermore, a Matrigel matrix basement with extracellular matrix proteins that extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma is useful for culturing hepatocytes (Vukicevic et al. 1992) and epithelial cells (Martin-Belmonte et al. 2007), and regenerating in vivo peripheral nerves (Madison et al. 1985). ...
In order to hourly monitor the variation of specific cell growth, a grid mesh for the recording of adhesion positions on the surface of glass slides and dishes is printed. However, since the focal length of a lens, used for making observation, changes with the film thickness when using a protein matrix basement membrane, the surface of the basement membrane and the bottom glass plate cannot simultaneously be observed. In one way to address this problem, the author considered patterning fine structures directly on the surface of the protein matrix basement membrane by employing a thermal imprinting technology. As a mold, a 100-μm-wide grid and images of area-numbers were processed using laser lithography and reactive-ion-etching on a 1-inch Si wafer. For a gelatinous protein, we chose a mixture basement membrane BioCoat Matrigel, which was coated on a 35-mm-diameter dish. A protein thin layer was gelled on a dish according to the manufacturer’s specs; the dish was then placed on the bottom loading stage of our thermal nanoimprint system. After that, a Si mold was pressed against a Matrigel matrix basement by a servo motor of the thermal nanoimprint system for 30–90 s at a press force of 1 kN. And then the mold was then released from the Matrigel without cooling when a press time reached to a set value. When an imprint temperature was 75 °C, an imprinted pattern could barely be observed. On the other hand, if the mold was heated up to 100 °C or more, the imprinted grid mesh and the printed number on the Matrigel could be clearly observed. However, a heating at 125 °C seemed to have discolored white a part of the protein. Therefore, under our experimental conditions, it can be said that the optimal heating temperature exists around 100 °C. Although in the past, in conventional thermal nanoimprint lithography, a thermoplastic bearing a glass transition temperature has been targeted, the work presented here sets an example of direct patterning on biological materials such as proteins.
... The use of a matrix inside an NC without a growth factor may either enhance ( Madison et al., 1985;Chen et al., 2000;Toba et al., 2002), conditionally enhance (depending on the time point of assessment and conduit type) ( Madison et al., 1987), or impede nerve regeneration ( Valentini et al., 1987;Terris et al., 1999;Mohanna et al., 2005) as compared with an empty NC. It has been shown that relatively weak collagen or laminin hydrogel matrices mediate superior nerve regeneration than denser hydrogels ( Labrador et al., 1998). ...
... For example, gene therapy studies have been used to successfully treat epidermolysis bullosa, 293,294 while biomaterials coated with laminin have been used for nerve regeneration. [295][296][297][298] ...
Musculoskeletal injuries and degenerative conditions constitute a bottleneck in the healthcare system. Tissue grafts and scaffolds, based on extracellular matrix (ECM) molecules, have received much attention as they closely imitate the structural, biochemical, biophysical, and biological properties of the tissue to be replaced. This chapter summarizes the most abundant ECM components that are used in tissue-engineering applications for bone, intervertebral discs, and tendon.
... In peripheral nerve tissue engineering, hydrogels are applied as NGC fillers, serving to physically bolster the nerve conduit lumen, and to act as delivery vehicles for bioactive molecules and cells (Lin and Marra, 2012). Tubes filled with fibrin matrices (Williams et al., 1987), laminin containing gels (Madison et al., 1985), collagen (Ciardelli and Chiono, 2006;Cordeiro et al., 1989), hyaluronic acid (Seckel et al., 1995) have shown faster axonal regeneration compared to empty tubes or tubes containing physiological saline solution. Despite many studies demonstrated that NGC fillers can enhance the regeneration process, hydrogels with unsuitable physicochemical properties could represent a physical barrier to axonal growth impeding regeneration. ...
Background
Autologous nerve grafting, the criterion standard for bridging peripheral nerves, can cause complications at the donor site. We investigated a novel approach to reconstruct the nerve gap with a split cross-sectional unmatched semifascicle autograft, which was harvested from the distal part of the injured nerve.
Methods
A patient diagnosed with left-sided frontal branch facial nerve dissection underwent nerve bridging emergency surgery using a semifascicle nerve graft. A sciatic nerve model was used to validate the feasibility and mechanism of this method. Male Sprague-Dawley rats (n = 36) were randomized into (A) intact fascicle, (B) semifascicle, and (C) semifascicle + conduit groups and further subdivided into 4- and 8-week groups for histological analysis of the neurotissue area, fibers, and Schwann cells. The 8-week groups underwent weekly pain and temperature tests; the wet weight of the gastrocnemius muscle was measured after euthanasia.
Results
The frontalis of the patient's injured side exhibited movement at 2 months postsurgery and recovered a symmetrical appearance at 13 months. Group A exhibited more neurotissue areas and fibers than groups B and C at week 4; group B had more neurotissue than group C. Group A had greater neurotissue areas than groups B and C at week 8; groups B and C exhibited no differences. The groups displayed no differences regarding nerve fiber, pain, and temperature analysis at week 8. Muscle wet weight of groups A and B exhibited no differences and was higher than that of group C.
Conclusion
We demonstrated the clinical translational value of semifascicle nerve grafts; the injured site was both the donor and recipient, thereby avoiding donor site damage and associated complications.
Three-dimensional (3-D) neural cultures represent a promising platform for studying disease and drug screening. Tools and methodologies for measuring the electrophysiological function in these cultures are needed. Therefore, the purpose of this work was primarily to develop a methodology to interface engineered 3-D dissociated neural cultures with commercially available 3-D multi-electrode arrays (MEAs) reliably over 3 weeks to enable the recording of their electrophysiological activity. We further compared the functional output of these cultures to their structural and synaptic network development over time. We reliably interfaced a primary rodent neuron-astrocyte (2:1) 3-D co-culture (2500 cells/mm3 plating cell density) in Matrigel™ (7.5 mg/mL) that was up to 750 µm thick (30–40 cell-layers) with spiked 3-D MEAs while maintaining high viability. Using these MEAs we successfully recorded the spontaneous development of neural network-level electrophysiological activity and measured the development of putative synapses and neuronal maturation in these co-cultures using immunocytochemistry over 3 weeks in vitro. Planar (2-D) MEAs interfaced with these cultures served as recording controls. Neurons within this interfaced 3-D culture-MEA system exhibited considerable neurite outgrowth, networking, neuronal maturation, synaptogenesis, and culture-wide spontaneous firing of synchronized spikes and bursts of action potentials. Network-wide spikes and synchronized bursts increased rapidly (first detected at 2 days) during the first week in culture, plateaued during the second week, and reduced slightly in the third week, while maintaining high viability throughout the 3-week culturing period. Early electrophysiology activity occurred prior to neuronal process maturation and significant synaptic density increases in the second week. We successfully interfaced 3-D neural co-cultures with 3-D MEAs and recorded the electrophysiological activity of these cultures over 3 weeks. The initial period of rapid increase in electrophysiological activity, followed by a period of neuronal maturation and high-level of synapse formation in these cultures suggests a developmental homeostatic process. This methodology can enable future applications both in fundamental investigations of neural network behavior and in translational studies involving drug testing and neural interfacing.
Peripheral nerve injury can result in debilitating outcomes including loss of function and neuropathic pain. Although nerve repair research and therapeutic development are widely studied, translation of these ideas into clinical interventions has not occurred at the same rate. At the turn of this century, approaches to peripheral nerve repair have included microsurgical techniques, hollow conduits, and autologous nerve grafts. These methods provide satisfactory results; however, they possess numerous limitations that can prevent effective surgical treatment. Commercialization of Avance, a processed nerve allograft, sought to address limitations of earlier approaches by providing an off‐the‐shelf alternative to hollow conduits while maintaining many proregenerative properties of autologous grafts. Since its launch in 2007, Avance has changed the landscape of the nerve repair market and is used to treat tens of thousands of patients. Although Avance has become an important addition to surgeon and patient clinical options, the product's journey from bench to bedside took over 20 years with many research and commercialization challenges. This article reviews the events that have brought a processed nerve allograft from the laboratory bench to the patient bedside. Additionally, this review provides a perspective on lessons and considerations that can assist in translation of future medical products.
Functionalized neurotube are a third generation of conduits with chemical or architectural bioactivity developed for axonal proliferation. The goal of this review is to provide a synopsis of the functionalized nerve conduits described in the literature according to their chemical and architectural properties and answer two questions: What are their mechanisms of action? Has their efficacy been proven compared to the autologous nerve graft? Our literature review relates all kind of conduits corresponding to functionalized neurotubes in peripheral nerve regeneration found in Medline and PubMed Central. Studies developing nerve gaps, chemotactic or structural features promoting each conduit, results, efficiency were selected. Fifty-five studies were selected and classified in: (a) intraluminal neurotrophic factors; (b) cell-based therapy (combined-in-vein muscles, amniotic membrane, Schwann cells, stem cells); (c) extracellular matrix proteins; (d) tissue engineering; (e) bioimplants. Functionalized neurotubes showed significantly better functional results than after end-to-end nerve suture. No studies can be able to show that neurotube results were better than autologous nerve graft results. We included all studies regardless of effectives to evaluate quality of reinnervation with modern tubulization. Functionalized neurotubes promote basic conduits for peripheral nerve regeneration. Thanks to bioengineering and microsurgery improvement, further neurotubes could promote best level of regeneration and functional recovery to successfully bridge a critical nerve gap.
Peripheral nerve injury is a complex condition with a variety of signs and symptoms such as numbness, tingling, jabbing, throbbing, burning or sharp pain. Peripheral nerves are fragile in nature and can easily get damaged due to acute compression or trauma which may lead to the sensory and motor functions deficits and even lifelong disability. After lesion, the neuronal cell body becomes disconnected from the axon's distal portion to the injury site leading to the axonal degeneration and dismantlement of neuromuscular junctions of targeted muscles. In spite of extensive research on this aspect, complete functional recovery still remains a challenge to be resolved. This review highlights detailed pathophysiological events after an injury to a peripheral nerve and the associated factors that can either hinder or promote the regenerative machinery. In addition, it throws light on the available therapeutic strategies including supporting therapies, surgical and non-surgical interventions to ameliorate the axonal regeneration, neuronal survival, and reinnervation of peripheral targets. Despite the availability of various treatment options, we are still lacking the optimal treatments for a perfect and complete functional regain. The need for the present age is to discover or design such potent compounds that would be able to execute the complete functional retrieval. In this regard, plant-derived compounds are getting more attention and several recent reports validate their remedial effects. A plethora of plants and plant-derived phytochemicals have been suggested with curative effects against a number of diseases in general and neuronal injury in particular. They can be a ray of hope for the suffering individuals.
Regardless of the intervention for peripheral nerve repair, slow rates of axonal regeneration often result in poor clinical outcomes. Thus, using new materials such as biologically inspired, biocompatible, organic rosette nanotubes (RNTs) could provide a tailorable scaffold to modulate neurite extension and attachment for improved nerve repair. RNTs are obtained through the spontaneous self-assembly of a synthetic DNA base analogue featuring the hydrogen bond triads of both guanine and cytosine, the GC base. Here we investigated the potential of RNTs functionalized with Lysine and Arg-Gly-Asp-Ser-Lys (RGDSK) peptide to support neural growth. We hypothesized that (a) due to their dimensions, the RNTs would support neuron attachment, and (b) their conjugation to the integrin-binding peptide RGDSK would further enhance neurite outgrowth compared to unfunctionalized RNT. Neurite extension was examined on a variety of RNT structures including RNT with a Lysine side chain (K1), a mixture of the K1 and a free RGDS peptide, RNT alone, an RGDSK-functionalized RNT, in addition to poly-D-Lysine and laminin controls. Both whole dorsal root ganglion (DRG) and single dissociated DRG neurons were seeded onto RNT-coated substrates containing various ratios of peptides. Analysis of neuron morphometrics showed that RNT blends support DRG neuron attachment and neurite extension, with RGDS presentation increasing neurite outgrowth from whole DRG by up to 47% over a 7-day period compared to K1 alone (p<0.013). In addition, while RNTs increased the sprouting of primary neurites extending from dissociated DRG neurons, the total neurite outgrowth per neuron remained the same. These results show that functionalized biomimetic RNTs provide a support for neurite growth and extension and have the ability to modulate neuronal morphology. These results also pave the way for the design of injectable RNT-based nanomaterials that support guided neural regeneration following traumatic injury.
Supported by the Muscular Dystrophy Association, NIH Grants 10161 and 11255 and a grant from the Florence and Marie Hall Endowment for Programs of Excellence in Education in the Medical Sciences.
Controlling interactions between cells and engineered materials is a critical aim in biotechnology. Frequently, biomaterials are designed to provide cell-instructive signals through engagement of cell surface receptors. Thus, several strategies are employed to intentionally display ligands at interfaces with retained activity. This chapter discusses extracellular matrix (ECM) proteins and ECM-derived peptides that might be immobilized on biomaterials, and surveys immobilization methodologies available for accomplishing this goal. We describe several functional parameters, including spatial arrangement and dynamic, time-dependent display of ligands, and provide examples of peptide-modified surfaces in several biomedical applications.
Musculoskeletal injuries and degenerative conditions constitute a bottleneck to the healthcare system. Tissue grafts and scaffolds based on extracellular matrix molecules have received much attention since they closely imitate the structural, biochemical, biophysical and biological properties of the tissue to be replaced. This review summarises the most abundant extracellular matrix components that are used in tissue engineering applications for bone, intervertebral disc, cartilage and tendon.
It is clear that angiogenesis is a critical component in almost all forms of tissue repair aside from cartilage and some cartilage-like tissues. The most widely studied example is in dermal repair (1,2) but others include repair of bone, tendon, and peripheral nerve. New microvessels form as part of the early granulation tissue, through invasion of the initial fibrin-based clot. In dermal repair, the fine, apparently random network of vessels at this stage (2) is remodeled in the maturing scar tissue to give microvascular loops reaching from the deeper layers to the dermal-epidermal junction (3).
Extracellular matrix components, such as laminin and fibronectin, promote cellular adhesion, growth, migration, and differentiation of glioma cells [1,2]. Matrigel is a biopolymer of natural constituents containing 60% laminin, 30% type IV collagen, 5% nidogen, 3% heparin sulfate proteoglycan, and 1% entactin.
Despite improved microsurgical techniques, the results of peripheral nerve repair very often remain unsatisfactory [1–4]. Therefore, studies aimed at a better identification and potential manipulation of the cellular and molecular events in PNS regeneration are still meaningful to both scientists and clinicians [5].
We have studied the effects of variations in the structure of a collagen-glycosaminoglycan (CG) copolymer matrix on the regeneration of transected rat sciatic nerves. Silicone tubes ensheathing 10-mm lengths of CG copolymer were grafted between the transected sciatic nerve stumps. Empty and saline-filled silicone tubes, as well as autografts, were studied as controls. The mean pore diameter and the degradation rate of the copolymer in collagenase were independently varied to investigate how each affects regeneration. Electrophysiological properties of the regenerating motor nerve fibers innervating the plantar flexor muscles, were serially monitored over about 40 weeks following surgery. Rapidly degrading CG copolymers with pore channels oriented predominantly along the axes of the tubes induced regeneration with a success rate of 100% (n = 35). Although CG copolymers with axially-oriented pore channels that degraded slowly had a success rate as high as 96% (n = 23), the long-term electrophysiological results were markedly inferior to those obtained with the rapidly degrading grafts. In another study of axially-oriented pore structures, the level of recovery of long-term electrophysiological results was observed to increase monotonously as preliminary results showed that CG copolymers with pore channels predominantly oriented along the radial direction of the tubes had a success rate of only 50% (n = 6). Control groups of empty and saline-filled tubes had an aggregated success rate of 29% (n = 21). The ongoing study has shown that systematic physiochemical manipulation of simple chemical analogs of the extracellular matrix can be used to define substrate features which encourage regeneration.
Peripheral nerve injury is a common condition in both civil and military circumstances, which results on morbidities that are usually both permanent and significant. Typical symptoms are sensory and motor function defects that could result in complete paralysis of an affected limb or development of intractable neuropathic pain. Approximately 100,000 patients undergo peripheral nerve surgery in the United States and Europe annually. While direct end-to-end nerve repair yields the best outcomes, other means of nerve repair have to be employed if tension-free or minimal tension coaptation of nerve stumps cannot be achieved.
Control of cell–material interactions is a critical aim of much work in the field of biomaterials and biotechnology. These interactions are often governed by cell receptor engagement with many plasma proteins adhering to biomaterials surfaces. While cell receptors prefer cell adhesive proteins typically found in extracellular matrix (ECM), other biomacromolecules bound to both the cell outer membrane and the biomaterial surface can facilitate nonspecific cell adhesion as well. Subsequent recognition of surface-immobilized ligands by cell-surface receptors results in specific and important cell behaviors including adhesion, migration, proliferation, and differentiation. In applications where biomaterials are designed to provide cell-instructive signals, several strategies are used to display ligands at interfaces with retained activity. This chapter discusses ECM proteins and ECM-derived peptides, like the arginine-glycine-aspartic acid (RGD) cell adhesive motif, that might be immobilized on biomaterials, and also surveys immobilization methodologies available for accomplishing this goal. We also describe several functional parameters for immobilized peptides and proteins (e.g., ligand surface density, spatial arrangement) that directly influence application outcomes. Examples of peptide-modified surfaces in several biomedical applications are also discussed.
Facial paralysis can have a profound effect on the patient from both an aesthetic and functional point of view. Just as there are numerous etiologies of facial paresis, there are as many therapeutic options and variations of these options. This article reviews the most current surgical options for neural reanimation of a damaged facial nerve, including recent advances in nerve repair, conduit technology, and nerve transfers, as well as emerging technology in translational research with biomedical engineering and tissue engineering.
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.
Nanotechnology is the creation and utilization of materials, devices and systems through the control of substance in a nanometer scale. Nanobiotechnology creates a better understanding of cell biology because molecules in the cells are organized in nanoscale dimensions and they function as nanomachines. Nanomedicine is the process of diagnosing, treating and preventing diseases and traumatic injuries, relieving pain and improving human health. The high cost, together with a limited space for significant economies in the mass scale production of tissue engineered products has hindered widespread clinical application. In addition, presently available tissue engineered products still share some of the concepts of substitution medicine, where a laboratory grown 'spare part' is implanted in the body to compensate for lost tissue. Several of these recent developments in electrospun nanofibers are already at advanced phases of commercialization or clinical trials. By building pioneering achievements in tissue engineering, advanced therapies in the regeneration of pathological tissues to treat, modify and prevent disabling chronic disorders such as diabetes, osteoarthritis, diseases of cardiovascular and central nervous system are achievable. The vision for nano-assisted regenerative medicine ideally involves the development of cost-effective disease modifying therapies that will allow for in situ tissue regeneration. This article summarises the recent developments in electrospun nanofibers for healthcare applications.
The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co-purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.
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.
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We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.
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.
Tetramethyl benzidine (TMB) is a presumptively non-carcinogenic chromogen which yields a blue reaction-product at sites of horseradish peroxidase activity. Sixty-six distinct procedures were performed in rats and monkeys in order to determine the optimal incubation parameters for TMB. As a result, a procedure is recommended whose sensitivity greatly surpasses that of a previously described benzidine dihydrochloride method. Indeed, the sensitivity of this new method in demonstrating retrograde transport is markedly superior to that of the previously described benzidine dihydrochloride method. Furthermore, as a consequence of this enhanced sensitivity, many efferent connections of the injection site are also visualized. The injection site demonstrated by this TMB procedure is significantly larger than the one demonstrated when benzidine dihydrochloride or diaminobenzidine is used as a chromogen. Finally, this TMB procedure has been compared to two other TMB procedures and found to provide superior morphology and sensitivity.
An in vivo preparation is presented to study the rate and time course of motor and sensory axonal regeneration. The cut ends of a transected sciatic nerve were inserted into each end of a 5-6 mm non-toxic and bioresorbable nerve guide tube to create a 4 mm nerve gap in adult mice. Subsequently, cell bodies in the ventral spinal cord and L3-L5 dorsal root ganglia that had regenerated axons across the gap were retrogradely labeled with horseradish peroxidase (HRP). The HRP was applied 3 mm distal to the nerve guide and was accessible only to axons that had regenerated through the nerve guide. Labeled cells were counted in 40 micron serial sections at 2, 4 and 6 weeks after initial nerve transection. The results indicate a significant increase in the number of labeled motor and sensory cell bodies over time. By 6 weeks after transection, approximately two thirds as many ventral horn motor cells and one third as many dorsal root ganglion sensory cells were labeled as in control non-transected animals. These data serve as a baseline to compare differential effects of additives to the nerve guide lumen in terms of sensory and motor neuron response.
Nontoxic, bioresorbable "nerve guide" tubes were used to bridge the transected optic nerves of adult rats. Nerve guides were fabricated as polymers of synthetic poly D,L-lactates with 2% triethyl citrate added as a plasticizer. The local environment was manipulated further by the addition of the proteins collagen, fibrinogen, and anti-Thy-1 antibody to the nerve guide lumens at the time of operation. Neovascular growth through the nerve guide lumens was quantified with the aid of a computer-controlled microscope. Neovascular growth was greater in the nerve guides to which proteins had been added, compared with initially empty nerve guides. These experiments demonstrated the effectiveness of these nerve guide tubes in supporting and directing neovascular growth in the mammalian central nervous system, and suggested that specific alterations of the local environment within the nerve guide lumen can affect the extent of neovascular growth.
Laminin is a high mol. wt. non-collagenous matrix glycoprotein, confined in adult tissues to basement membranes. In normal rat brain we found laminin mainly in vessel walls but, after injury, induced by stereotaxic injection of a neurotoxin, laminin immunoreactivity appeared also in reactive astrocytes, which are characteristically positive for the glial fibrillary acidic protein (GFAP). Laminin was first detected in GFAP-immunoreactive glial cells 24 h after injury. Four days later the majority of reactive astrocytes in the gray matter were positive for laminin and the laminin immunoreactivity, but not that of GFAP, gradually subsided within a month. Fibronectin, the other major matrix glycoprotein, was found only in capillary structures both in normal and lesioned brain tissue. The results indicate that mature astrocytes have the potential to produce laminin and suggest a role for this glycoprotein in brain regeneration.
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 spatial-temporal progress of peripheral nerve regeneration across a 10-mm gap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7-14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1-5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.
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.
Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.
Fetal mouse retinal ganglion cell axons have been shown to ramify within co-cultured basement membrane secreting tumor explants and upon isolated basement membranes. Here we report that laminin, a glycoprotein found in basement membranes and adhesion sites of a variety of cell types, acted as a substrate for retinal explant attachment and axonal outgrowth. When axons were given a direct choice, laminin was preferred over collagen. Under suitable conditions (air-dried upon underlying collagen gels), laminin was more effective than fibronectin for promoting axon emergence from retinal explants. These findings have implications for the study of molecular mechanisms underlying CNS axonal outgrowth.
The part played by basement membrane in the guidance of peripheral nerve growth in vivo has been assessed by examining the capacity of degenerating mouse muscle to support the regeneration of the cut sciatic and saphenous nerves. Ethanol and formaldehyde-fixed gluteus maximus muscles were implanted around the contralateral cut nerves. The subsequent nerve growth into the degenerating muscle was assessed by silver staining after 3, 4 and 10 days. By 4 days, linear axonal growth was seen, parallel to the length of the muscle fibres, and coinciding with the onset of degeneration of the sarcoplasm. Transverse sections of the 10 day preparations showed that over 90% of linearly growing axons were located inside the remaining sheaths of muscle fibre basement membrane. This relationship was confirmed by electron microscopy of ruthenium red-stained preparations. Both motor and sensory axons were able to grow in this manner, for electrophysiological testing revealed the presence of motor axons from the sciatic nerve, while the saphenous nerve contains only sensory axons. Identical growth was seen at 10 days in muscles caused to degenerate by incubation in distilled water. However, linear growth did not occur in live-innervated and glutaraldehyde-fixed muscles, in which muscle fibre architecture was preserved. It is concluded that basement membrane derived from muscle can promote peripheral nerve regeneration. Furthermore, both motor and sensory axons show a strong preference for growth along its inner surface, the basal lamina.
A moderately compressive tubulation procedure has been devised to improve regeneration that follows fascicular neurorrhaphy. The early phases of the regenerative process were analyzed to identify the underlying cellular mechanisms. Adult Sprague-Dawley rats were subjected to bilateral fascicular neurorrhaphy involving tubulation on one side with a dura mater sheet closely apposed to the suture site. One to 16 days postoperatively the nerves were processed for histological analysis. Compared with nontubulated nerves, the cuffed side displayed a longer extent of retrograde myelin and axonal degeneration, a faster rate of orthograde remyelination, axonal invasion of the suture plane at about the same period (fifth postoperative day), larger contingent of regenerating fibers invading the distal stump, more longitudinally oriented fibers at the repair level, no escape of regenerating fibers into the extraneural tissue through the repair borders, and less intraneural edema. These findings have implications for the potentially beneficial effects of mechanically restricting post-traumatic intraneural edema buildup during nerve regeneration.
The regeneration of transected mouse sciatic nerves using semipermeable acrylic copolymer tubes to enclose both stumps has been qualitatively assessed from 1 to 30 weeks post-operative. Quantitative morphometric analysis of electron micrograph montages of complete transverse sections of the segment regenerated between stumps has permitted determinations of the percents of total area occupied by the various tissue constituents--blood vessels, epineurium, perineurium, endoneurium, myelinated axon/Schwann cell units, and unmyelinated axon/Schwann cell units. Significant differences were found in the total cross-sectional area of segments regenerated through tubes of 1.0 mm versus 0.5 mm internal diameters. Segments regenerated with the distal stump inserted in the tube contained significantly greater percentages of neural units and were significantly larger at 8 weeks post-operative compared to segments regenerated for 9-10 weeks with the distal stump avulsed. The morphometric method permits rapid quantitation of sizeable electron micrograph montages which at 1300 X permit all types of tissue components, including the unmyelinated axons, to be visualized.
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.
The proximal stump of a transected rat sciatic nerve has been observed to regenerate through a cylindrical silicone chamber across a 10 mm gap to the distal stump. The fluid filling such in vivo chambers contains trophic factors that ensure in vitro survival and growth of at least sensory neurons from rodent dorsal root ganglia--as already demonstrated for fluid generated in vitro from Schwann and other cell cultures.
The range of growth-promoting influences from a distal nerve stump on a regenerating proximal stump was determined using an experimental system in which a gap between cross-anastomosed rat sciatic nerves was encased by a cylindrical silicone chamber. Two arrangements were examined after 1 month in situ: A proximal-distal (PD) system in which both proximal and distal stumps were introduced into the ends of the chamber, and a proximal-open (PO) system in which the distal stump was omitted. When the gap was 6 mm long, a regenerated nerve extended all the way through the chamber in both the PD and PO systems. When the gap was increased to 10 mm, a similar regrowth occurred in the PD chamber, whereas in the PO chamber proximal regrowth was partial or nonexistent. When the gap was increased to 15 mm, no regeneration occurred, even in the presence of the distal stump. These observations confirm that the distal stump influences proximal regeneration and indicate that this influence can act only over a limited distance or volume. Such an influence could consist of humoral agents which support nerve growth and/or outgrowth from the distal stump.
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.
Regeneration of severed peripheral nerves is unfortunately often incomplete, due to loss of nerve fibers and neuroma formation. A new approach is presented with the intention of improving the conditions for nerve repair. In the first of the two stages, a pseudosynovial tube is formed around a silicone rubber rod, surrounded by a stainless steel spiral, which was placed in the backs of rats. This tube, in the second stage, is used as a free "tube graft" to bridge gaps of about 10-12 mm lengths in the severed sciatic nerve. The tube was kept open by the metal spiral. Regenerating nerve fibers with their sprouts grew into the initially open space in the tube. A new nerve trunk was formed, comprised of closely packed myelinated and unmyelinated axons, organized into fascicles. Demonstration by electron microscopy and by EMG recording of reinnervation of foot muscles supported successful long-term results. The fascicles were delimited by perineurial and epineurial sheaths and, furthermore, showed signs of maturation. It was also demonstrated that the nerve-fiber regeneration ceased after a few weeks if there was no distal nerve inserted into the tube. The importance of optimizing the interaction between local factors and regenerating nerve fibers for reestablishment of functionally valuable motor units is discussed.
Laminin gel stimulates axonal regeneration in vivo
- da Silva
DA SILVA, C. F., P. DIKKES, R. MADISON, D. GREATOREX, AND R. L. SIDMAN. 1984. Laminin gel stimulates axonal regeneration in vivo. Sot. Neurosci. Abstr. 10: 283.
Bioresorbable nerve guides bridge transected optic nerve
- Madison
MADISON, R., R. L. SIDMAN, T.-H. CHIU, AND E. NYILAS. 1983. Bioresorbable nerve guides bridge transected optic nerve. Sot. Neurosci. Abstr. 9: 770.
Neovascular growth through nontoxic bioresorbable nerve guides that bridge transected adult rat optic nerve
- Greatorex
GREATOREX, D., R. MADISON, R. L. SIDMAN, E. NYILAS, AND T.-H. CHIU. 1983. ET AL. Neovascular growth through nontoxic bioresorbable nerve guides that bridge transected adult rat optic nerve. Sot. Neurosci. Abstr. 9: 1223.
Isolation and characterization of Type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma
- Kleinman
Laminin in rat sciatic nerve undergoing Wallerian degeneration
- Bignami