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Demonstration of typical nodes of Ranvier on regenerated axons. (A) The nodes showed high density of sodium channel NaV1.6 (B) Determination of muscle weight after injury and regeneration of tibial nerve defect by either autologous nerve transplant or spider silk construct implantation in comparison to normal muscle weight of uninjured gastrocnemius muscle in adult sheep (C). Scale bar in B = 6 mm. doi:10.1371/journal.pone.0016990.g004
Source publication
Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein...
Contexts in source publication
Context 1
... of Ranvier were observed on the regenerated axons within the nerve grafts as ''breaks'' in myelin staining between SC segments. A putative node of Ranvier is indicated in a single regenerated axon within the regenerated and myelinated axons within the construct (Fig. 4A). Immunostaining for the voltage- gated sodium channel NaV 1.6 demonstrated high expression of this sodium channel on the axon at these gap regions indicating the nodal nature of these regions of axon membrane and that appropriate sodium channel subtype is present on the regenerated axons ( ...
Context 2
... between normal and the operated groups including the autologous and construct implanted animals: gastrocnemius weight reduction could be observed in the transplant group contrast to uninjured control group, but no statistically significant differences in weight of the muscle were measured between autologous and construct implanted animals ( Fig. 4C) ten months after surgery and nerve defect regeneration. This is in accordance to the axon counts where the spider silk construct resulted in comparable myelinated axon counts as the autologous nerve transplant as described in Figure ...
Citations
... Natural spider silk can be obtained with special devices (dragline silk) or via harvesting as an egg sac silk, which can be used as naturally or as regenerated silk [89]. This dragline silk was used in nerve and skin tissue engineering [90,91]. Although the mechanical behavior of spider silk is comparable to that of ligaments and tendons, the production of a complete scaffold for ACL tissue engineering seems rather unsuitable due to the small quantities obtained. ...
Silk has a long history as an exclusive textile, but also as a suture thread in medicine; nowadays, diverse cell carriers are manufactured from silk. Its advantages are manifold, including high biocompatibility, biomechanical strength and processability (approved for nearly all manufacturing techniques). Silk’s limitations, such as scarcity and batch to batch variations, are overcome by gene technology, which allows for the upscaled production of recombinant “designed” silk proteins. For processing thin fibroin filaments, the sericin component is generally removed (degumming). In contrast to many synthetic biomaterials, fibroin allows for superior cell adherence and growth. In addition, silk grafts demonstrate superior mechanical performance and long-term stability, making them attractive for anterior cruciate ligament (ACL) tissue engineering. Looking at these promising properties, this review focusses on the responses of cell types to silk variants, as well as their biomechanical properties, which are relevant for ACL tissue engineering. Meanwhile, sericin has also attracted increasing interest and has been proposed as a bioactive biomaterial with antimicrobial properties. But so far, fibroin was exclusively used for experimental ACL tissue engineering approaches, and fibroin from spider silk also seems not to have been applied. To improve the bone integration of ACL grafts, silk scaffolds with osteogenic functionalization, silk-based tunnel fillers and interference screws have been developed. Nevertheless, signaling pathways stimulated by silk components remain barely elucidated, but need to be considered during the development of optimized silk cell carriers for ACL tissue engineering.
... Spider silk fibers extending from the electrode contacts to the modiolus could provide such support. Natural spider silk is known for its biocompatibility [5] and support of neurite outgrowth and has successfully been used as longitudinal guiding fibers in nerve guidance conduits to bridge longdistance peripheral nerve defects by regenerating neurites [6,7,8,9]. In case of the CI, the fibers would need to be fixed to the electrode, should protrude modiolar from the contacts, and must not impede electrical stimulation by covering the contacts or impairing the insertion behavior. ...
... This indicates an excellent compatibility of the spider silk with the spiral ganglion tissue and suitability as matrix to support regenerating neurites even in a liquid environment. These findings are in accordance with previous studies showing high suitability of natural spider silk as cell and neurite growth and guiding matrix for human model neurons (NT2) [6] and primary dorsal root ganglia neurons [7] in vitro or in nerve conduits for peripheral nerve regeneration in vivo [8,9] and patients [13]. In contrast to peripheral nerve regeneration where several cm long defects have to be bridged by the neurons of a relatively compact nerve, in case of the CI a relatively short distance of about 2 mm [2] has to be overcome by regenerating neurites of the several thousand neurons of the spiral ganglion over the whole length of the electrode array (about 2.6 cm [14]). ...
The cochlear implant (CI) restores hearing to patients with severe to profound sensorineural hearing loss by stimulating the spiral ganglion neurons (SGN). Due to the inner ear anatomy, a fluid-filled gap remains between the SGN and the electrode array. This gap impedes focused stimulation and thus optimized performance with the CI. Regenerating neurites may bridge this fluid-filled gap for a direct nerveelectrode- link but require a supportive matrix. Spider silk might be a suitable candidate for this support. Therefore, silk fibers were tested for biocompatibility with inner ear neuronal tissue, suitability as neurite outgrowth matrix and applicability to the CI-electrode array. Dragline silk from female Trichonephila spiders was woven around a frame and autoclaved. Spiral ganglia of young rats were prepared, cut and placed in a drop of medium on the parallel silk fibers for pre-cultivation to support adherence before remaining medium was added. After 5d of cultivation, cells were fixed and immunocytologically stained. For CIapplication, silk was wrapped around a silicone dummy and a one-sided spacer. Opposite the spacer, alginate-hydrogel was applied for silk-fixation and -shielding. After gelation, silk loops were cut in the middle to form protruding threads. Cells showed a high degree of migration and neurite regeneration along the silk fibers and in some cases the outgrown neurites directly contacted the silk. It was possible to fix the silk to the dummy as protruding fibers. The tested spider silk is compatible with the inner ear tissue in culture, supports cell growth and seems to be an attractive material for neurite contacting. Application to the CI as protruding fibers is possible, making it a promising candidate for structural support to bridge the nerve-electrode-gap.
... There are two types of spider silk proteins: natural and recombinant ones. Natural spider silk proteins can be received by harvesting spider silk webs and egg sacs or milking spider silk from the appropriate spiders [72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91]. In general, collecting spider silk webs/egg sacs has several disadvantages. ...
Biomaterials are an indispensable part of biomedical research. However, although many materials display suitable application-specific properties, they provide only poor biocompatibility when implanted into a human/animal body leading to inflammation and rejection reactions. Coatings made of spider silk proteins are promising alternatives for various applications since they are biocompatible, non-toxic and anti-inflammatory. Nevertheless, the biological response toward a spider silk coating cannot be generalized. The properties of spider silk coatings are influenced by many factors, including silk source, solvent, the substrate to be coated, pre- and post-treatments and the processing technique. All these factors consequently affect the biological response of the environment and the putative application of the appropriate silk coating. Here, we summarize recently identified factors to be considered before spider silk processing as well as physicochemical characterization methods. Furthermore, we highlight important results of biological evaluations to emphasize the importance of adjustability and adaption to a specific application. Finally, we provide an experimental matrix of parameters to be considered for a specific application and a guided biological response as exemplarily tested with two different fibroblast cell lines.
... Spider silk also works well as an alignment material for nerve regeneration 7 . Schwann cells migrate and proliferate on spider silk bers 8 and axons grow, align and myelinate on natural spider silks 9 . Surgical sutures made from natural spider silks had been shown to exhibit better tensile properties than conventional ones, and therefore could be applied in exor tendon repair 10 . ...
Spider silk is a promising material with great potential in biomedical applications due to its incredible mechanical properties and resistance to bacterial degradation, particularly commercially available strains. However, little is known about the bacterial communities that may inhabit spider webs and how these microorganisms interact with spider silk. In this study, we exposed two exopolysaccharide-secreting bacteria, isolated from webs of an orb spider, to major ampullate (MA) silk from host spiders. The naturally occurring lipid and glycoprotein surface layers of MA silk were experimentally removed to further probe the interaction between bacteria and silk. Extensibility of major ampullate silk produced by Triconephila clavata that was exposed to either Microbacterium sp. or Novosphigobium sp. was significantly higher than that of silk that was not exposed to bacteria. This strain-enhancing effect was not observed when the lipid and glycoprotein surface layers of MA silks were removed. The presence of exopolysaccharides was detected through NMR from MA silks exposed to these two bacteria but not from those without exposure. Here we report for the first time that exopolysaccharide-secreting bacteria inhabiting spider webs can enhance extensibility of host MA silks and silk surface layers play a vital role in mediating such effects.
... The augmentation of empty conduits with dragline spider silk as a fibrous luminal filling has led to promising results [21][22][23]. Even for larger nerve defects of 60 mm, NGCs filled with dragline silk could measure up to nerve autografts [24]. It has been shown that the success of dragline fibers can be related to their remarkable support of Schwann cell (SC) adhesion, movement, and proliferation, as well as formation of structures similar to bands of Büngner [25]. ...
... In search for the optimum luminal filling for NGCs, the success of the dragline silk of spider genus Trichonephila was confirmed [24,71]. In fact, the dragline fibers were capable of bridging a critical length gap of 60 mm, which has not been achieved with other materials [72]. ...
Dragline silk of Trichonephila spiders has attracted attention in various applications. One of the most fascinating uses of dragline silk is in nerve regeneration as a luminal filling for nerve guidance conduits. In fact, conduits filled with spider silk can measure up to autologous nerve transplantation, but the reasons behind the success of silk fibers are not yet understood. In this study dragline fibers of Trichonephila edulis were sterilized with ethanol, UV radiation, and autoclaving and the resulting material properties were characterized with regard to the silk's suitability for nerve regeneration. Rat Schwann cells (rSCs) were seeded on these silks in vitro and their migration and proliferation were investigated as an indication for the fiber's ability to support the growth of nerves. It was found that rSCs migrate faster on ethanol treated fibers. To elucidate the reasons behind this behavior, the fiber's morphology, surface chemistry, secondary protein structure, crystallinity, and mechanical properties were studied. The results demonstrate that the synergy of dragline silk's stiffness and its composition has a crucial effect on the migration of rSCs. These findings pave the way towards understanding the response of SCs to silk fibers as well as the targeted production of synthetic alternatives for regenerative medicine applications.
... Spider silk is characterized by a high toughness and elasticity and-in contrast to other bioengineering materials for nerve regeneration like surgical suturing materials (e.g., polydiaxanone monofilaments, PDS) or silkworm silk-spider silk degrades in a physiological pH diminishing a potentially impaired neuroregeneration in an acid environment [9]. Spider silk interposition has already provided promising results in peripheral nerve reconstruction in vitro and in vivo with axonal regeneration comparable to autologous nerve transplantation-both Radtke et al. and Kornfeld et al. demonstrated equal electrophysical results in sheep models after spider silk implantation compared to the gold standard of a full-thickness nerve graft [10,11]. However, the impact on autonomic neuroregeneration in humans is unknown. ...
... While different types of silk are produced by the female spider, the major-ampullate-dragline functions as the safety line that attaches the spider with the connected surrounding and is therefore characterized by a high level of stability and elasticity [12]. Up to 500 m of dragline silk can be harvested in one process without harm to the animal using a computerized reeling machine with subsequent sterilization on the collector (Fig. 1b) [10,11]. ...
Purpose
To investigate the safety and feasibility of spider silk interposition for erectile nerve reconstruction in patients undergoing robotic radical prostatectomy (RARP).
Methods
The major-ampullate-dragline from Nephila edulis was used for spider silk nerve reconstruction (SSNR). After removal of the prostate with either uni- or bilateral nerve-sparing, the spider silk was laid out on the site of the neurovascular bundles. Data analysis included inflammatory markers and patient reported outcomes.
Results
Six patients underwent RARP with SSNR. In 50% of the cases, only a unilateral nerve-sparing was performed, bilateral nerve-sparing could be performed in three patients. Placement of the spider silk conduit was uneventful, contact of the spider silk with the surrounding tissue was mostly sufficient for a stable connection with the proximal and distal ends of the dissected bundles. Inflammatory markers peaked until postoperative day 1 but stabilized until discharge without any need for antibiotic treatment throughout the hospital stay. One patient was readmitted due to a urinary tract infection. Three patients reported about erections sufficient for penetration after three months with a continuous improvement of erectile function both after bi- and unilateral nerve-sparing with SSNR up to the last follow-up after 18 months.
Conclusion
In this analysis of the first RARP with SSNR, a simple intraoperative handling without major complications was demonstrated. While the series provides evidence that SSNR is safe and feasible, a prospective randomized trial with long-term follow-up is needed to identify further improvement in postoperative erectile function due to the spider silk-directed nerve regeneration.
... However, injuries of the forelimb nerves lead to more functional deficits, since compensation for animal weight support and movement is more effective in the hindlimbs [37]. The sciatic and the tibial nerves have also been subjected to gap lesions and repair with different conduits bridging a 1 cm gap [44][45][46], and with recellularized allografts in a 2 cm gap [47] in comparison with autologous nerve grafts. The sheep peroneal nerve has been used in a few studies prior to the present study. ...
... Considering a rate of regeneration of 1-2 mm/day, similar to humans, 6 to 12 months would be needed for regaining muscle innervation, and even longer for reinnervation of the dorsum of the hindfoot. Our evidence of TA muscle reinnervation by nerve conduction tests at 6.5 months suggests a regeneration rate~2 mm/day, in line with previous results reported by Strasberg et al. [32], Radtke et al. [45], and Roballo et al. [37], who performed similar electrophysiological tests. Indeed, an average axonal regeneration velocity of 1.57 mm/day was estimated in sheep that received an autologous nerve graft in the tibial nerve [46]. ...
... At 9 months, all sheep but one showed positive CMAPs with increasing amplitude and decreasing latency, indicating regeneration and myelination of motor axons. Based on our electrophysiological findings, 9 months appeared to be the minimum timepoint to reliably evaluate different therapies after long nerve gap neurotmesis in the sheep, in agreement with other studies [32,37,45], and the amplitude of the CMAP is the most valuable parameter [50]. Complementing electrophysiology, we used high-resolution echography to evaluate the degree of atrophy of the TA muscle as an indirect measure of reinnervation. ...
Despite advances in microsurgery, full functional recovery of severe peripheral nerve injuries is not commonly attained. The sheep appears as a good preclinical model since it presents nerves with similar characteristics to humans. In this study, we induced 5 or 7 cm resection in the peroneal nerve and repaired with an autograft. Functional evaluation was performed monthly. Electromyographic and ultrasound tests were performed at 6.5 and 9 months postoperation (mpo). No significant differences were found between groups with respect to functional tests, although slow improvements were seen from 5 mpo. Electrophysiological tests showed compound muscle action potentials (CMAP) of small amplitude at 6.5 mpo that increased at 9 mpo, although they were significantly lower than the contralateral side. Ultrasound tests showed significantly reduced size of tibialis anterior (TA) muscle at 6.5 mpo and partially recovered size at 9 mpo. Histological evaluation of the grafts showed good axonal regeneration in all except one sheep from autograft 7 cm (AG7) group, while distal to the graft there was a higher number of axons than in control nerves. The results indicate that sheep nerve repair is a useful model for investigating long-gap peripheral nerve injuries.
... Suggested applications include different kinds of wearables 12 such as space suits 13 , as durable components in robotics 14 , other high-end textiles 15,16 , and as biomaterials for medical devices [17][18][19][20][21] . This wide range of applications is attributed to the distinctive property portfolio of spider silk, which combines strength and extensibility, with biocompatibility and biodegradability [22][23][24] . ...
Artificial spider silk has emerged as a biobased fiber that could replace some petroleum-based materials that are on the market today. Recent progress made it possible to produce the recombinant spider silk protein NT2RepCT at levels that would make the commercialization of fibers spun from this protein economically feasible. However, for most applications, the mechanical properties of the artificial silk fibers need to be improved. This could potentially be achieved by redesigning the spidroin, and/or by changing spinning conditions. Here, we show that several spinning parameters have a significant impact on the fibers’ mechanical properties by tensile testing more than 1000 fibers produced under 92 different conditions. The most important factors that contribute to increasing the tensile strength are fast reeling speeds and/or employing post-spin stretching. Stretching in combination with optimized spinning conditions results in fibers with a strength of >250 MPa, which is the highest reported value for fibers spun using natively folded recombinant spidroins that polymerize in response to shear forces and lowered pH.
... Spider silk fibers support the growth of dorsal root ganglia and human model neurons, as well as the adhesion and proliferation of Schwann cells, the primary structural and functional cells responsible for peripheral nerve regeneration [104,[107][108][109][110][111][112]. The use of decellularized vein grafts filled with Trichonephila clavipes dragline silk as a guiding material has successfully bridged sciatic nerve gaps of 20 mm in rats [113] and tibial nerve gaps of 60 mm in adult sheep [114,115]. The effects of spider silk in nerve regeneration are likely due to a synergy of properties of the fiber. ...
... Depending on factors such as implantation site, mechanical environment, diameter, and variables related to the spider's health and silk's physiological status, the fibers are slowly absorbed in vivo by proteolytic degradation triggered by a foreign body reaction [114,115]. In vivo, native dragline silk showed a constant rate of degradation with complete disappearance approximately four months after subcutaneous implantation in animal experiments [105,106,120] and six to eight months in cases where silk was implanted for nerve grafts enclosed in decellularized veins [113][114][115]. ...
... Depending on factors such as implantation site, mechanical environment, diameter, and variables related to the spider's health and silk's physiological status, the fibers are slowly absorbed in vivo by proteolytic degradation triggered by a foreign body reaction [114,115]. In vivo, native dragline silk showed a constant rate of degradation with complete disappearance approximately four months after subcutaneous implantation in animal experiments [105,106,120] and six to eight months in cases where silk was implanted for nerve grafts enclosed in decellularized veins [113][114][115]. This slow absorption in the body is crucial, as the rate of degradation of a scaffold should not exceed the rate of tissue repair [127]. ...
Spider silk has fascinated mankind for millennia, but it is only in recent decades that scientific research has begun to unravel all its characteristics and applications. The uniqueness of spider silk resides in its versatility, in which a combination of high strength and extensibility results in extraordinary toughness, superior to almost all natural and man-made fibers. Dragline silk consists of proteins with highly repetitive amino acid sequences, which have been correlated with specific secondary structures responsible for its physical properties. The native fiber also shows high cytocompatibility coupled with low immunogenicity, making it a promising natural biomaterial for numerous biomedical applications. Recently, novel technologies have enabled new insights into the material and biomedical properties of silk. Due to the increasing interest in spider silk, as well as the desire to produce synthetic alternatives, we present an update on the current knowledge of silk fibers produced by the spider genus Trichonephila.
... However, the bridging of large nerve defects requires the utilization of different materials for the preparation of artificial nerve grafts that are seeded with OECs. Examples of these include, PLGA (Li et al., 2010), acellularized vein filled with spider silk (Radtke et al., 2011), muscle-stuffed vein (Lokanathan et al., 2014), collagen (Goulart et al., 2016), as well as silicone and nanofibrous CNT/PLLA (Kabiri et al., 2015). ...
This review describes the heterogeneity of peripheral glial cell populations, from the emergence of Schwann cells (SCs) in early development, to their involvement, and that of their derivatives in adult glial populations. We focus on the origin of the first glial precursors from neural crest cells (NCCs), and their ability to differentiate into several cell types during development. We also discuss the heterogeneity of embryonic glia in light of the latest data from genetic tracing and transcriptome analysis. Special attention has been paid to the biology of glial populations in adult animals, by highlighting common features of different glial cell types and molecular differences that modulate their functions. Finally, we consider the communication of glial cells with axons of neurons in normal and pathological conditions. In conclusion, the present review details how information available on glial cell types and their functions in normal and pathological conditions may be utilized in the development of novel therapeutic strategies for the treatment of patients with neurodiseases.
