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Highly permeable polylactide-caprolacton nerve guides enhance peripheral nerve regeneration through long gaps
Abstract
We compared regeneration and functional reinnervation after sciatic nerve resection and tubulization repair with bioresorbable guides of poly(L-lactide-co-epsilon-caprolactone) (PLC) and permanent guides of polysulfone (POS) with different degrees of permeability, leaving a 6 mm gap in different groups of mice. Functional reinnervation was assessed to determine recovery of motor, sensory and sweating functions in the hindpaw during four months postoperation. Highly permeable PLC guides allowed for faster and higher levels of reinnervation for the four functions tested than impermeable or low-permeable PLC guides, while semipermeable 30 and 100 kDa POS tubes yielded very low levels of reinnervation. The regeneration success rate was higher with PLC than with POS tubes. Morphometrical analysis of cross-sectional nerves under light microscopy showed the highest number of regenerated myelinated fibers at mid tube and distal nerve in high-permeable PLC guides. Impermeable PLC guides allowed slightly worse levels of regeneration, while low-permeable PLC guides promoted neuroma and limited distal regeneration. The lowest number of regenerated fibers were found in POS tubes. In summary, highly permeable bioresorbable PLC guides offer a suitable alternative for repairing long gaps in injured nerves, approaching the success of autologous nerve grafts.
... 3,13 The growth of axons (myelinated and unmyelinated) across the gap may be enhanced when compared with the use of conventional tubes, as the biodegradable conduits allow better nutrition supply to the regenerating nerve. 172 This type of conduit provides a better structure for the initial matrix and subsequent regenerated nerve. The flexibility of the conduit increases with degradation, resulting in less damage to the maturing regenerated nerve. ...
... The flexibility of the conduit increases with degradation, resulting in less damage to the maturing regenerated nerve. 170,172 Details of some of the materials used for nerve guides are summarized in Table 2. ...
... 157 It was noted that only during the early stage of nerve regeneration, the initial connective regenerating nerve cable, increased in wall permeability, which was an important factor. 172 This was due to a fibrous cover that formed around the regenerated nerve, limiting the exchange between the inside and the outside environment of the conduit. 84,121,172,176 Surface Structure. ...
Peripheral nerve injuries (PNI) are the most common injury types to affect the nervous system. Restoration of nerve function after PNI remains a challenge. Extended gaps in transected peripheral nerves are only repaired using autologous nerve grafting. This technique however, in which nerve tissue is harvested from a donor site and grafted onto a recipient site in the same body, has many limitations and disadvantages. Recent studies have revealed artificial nerve conduits as a promising alternative technique to substitute autologous nerves. This review summarizes different types of artificial nerve grafts used to repair peripheral nerve injuries. These include synthetic and natural polymers, and biological factors. Desirable properties of nerve guides are discussed based on their functionality and effectiveness. In the final part of this review, fabrication methods and commercially available nerve guides are described.
... Thus, an ideal scaffold for biomedical applications must supply a sufficient permeability, which also influences the ability to form fibrin matrices, useful during tissue regeneration processes to guide axons regeneration (She et al., 2008;Gu et al., 2011). Indeed, it has been shown that during the early stage of nerve regeneration, nerve wall permeability increases, and a similar behavior should be mimicked by the conduit (Rodríguez et al., 1999). ...
... A semipermeable nerve conduit is more effective to promote nerve regeneration when compared with low permeable or impermeable conduits (Rodríguez et al., 1999;Vleggeert-Lankamp et al., 2007;Dodla and Bellamkonda, 2008;Chiono and Tonda-Turo, 2015). Moreover, permeability is influenced by the hydrophilic properties of the material; moderate hydrophilicity of the material allows a better cell adhesion, compared to very high hydrophilicity or hydrophobicity (Liu et al., 2007). ...
Peripheral nerve injury treatment is a relevant problem because of nerve lesion high incidence and because of unsatisfactory regeneration after severe injuries, thus resulting in a reduced patient’s life quality. To repair severe nerve injuries characterized by substance loss and to improve the regeneration outcome at both motor and sensory level, different strategies have been investigated. Although autograft remains the gold standard technique, a growing number of research articles concerning nerve conduit use has been reported in the last years. Nerve conduits aim to overcome autograft disadvantages, but they must satisfy some requirements to be suitable for nerve repair. A universal ideal conduit does not exist, since conduit properties have to be evaluated case by case; nevertheless, because of their high biocompatibility and biodegradability, natural-based biomaterials have great potentiality to be used to produce nerve guides. Although they share many characteristics with synthetic biomaterials, natural-based biomaterials should also be preferable because of their extraction sources; indeed, these biomaterials are obtained from different renewable sources or food waste, thus reducing environmental impact and enhancing sustainability in comparison to synthetic ones. This review reports the strengths and weaknesses of natural-based biomaterials used for manufacturing peripheral nerve conduits, analyzing the interactions between natural-based biomaterials and biological environment. Particular attention was paid to the description of the preclinical outcome of nerve regeneration in injury repaired with the different natural-based conduits.
... I. Jeong et al. 2005;S. I. Jeong, Kim, Kim, et al. 2004), cartilage (Jun Xie et al. 2006;Kroeze et al. 2010), and peripheral nerves (De Groot et al. 1997;Rodrıǵuez et al. 1999). It has also been pointed out to support tendon cell proliferation and also been widely applied in ligament/tendon tissue engineering (Kim et al. 2012). ...
... I. Jeong et al. 2005;S. I. Jeong, Kim, Kim, et al. 2004), cartilage (Jun Xie et al. 2006;Kroeze et al. 2010), and peripheral nerves (De Groot et al. 1997;Rodrıǵuez et al. 1999 (Vuornos et al. 2016). Braiding is indeed one of the well-established medical textile technology adapted to the development of different medical constructs, especially like suture and stents, and also ligament and tendon tissues (Aibibu, Hild, and Cherif 2016). ...
Ligament tissue engineering offers a potential approach to recover or replace injured ligament. The three essential elements that have been investigated towards ligament regeneration consist in a suitable scaffold, an adapted cell source, and the supply of biomechanical/biochemical stimulations. In the current study, synthetic polymer poly (L-lactide-co-ε-caprolactone) (PLCL) and silk have been evaluated as suitable candidates to constitute an adapted scaffold. A series of multilayer braided scaffolds based on PLCL and silk, as well as an original silk/PLCL composite scaffold, have been developed and compared. The conducted physicochemical and biological characterizations have demonstrated that both PLCL and silk constitute adapted candidate material to form ligament scaffolds from the mechanical and biological points of view. Moreover, it has been observed that silk/PLCL composite scaffold resulted in adequate mechanical properties and biocompatibility, and therefore could constitute suitable candidate scaffolds for ligament tissue engineering. Both Wharton’s Jelly mesenchymal stem cells (WJ-MSCs) and Bone marrow mesenchymal stem cells (BM-MSCs) have been evaluated to be cell source for ligament regeneration. MSCs behaviors including cell attachment, proliferation, migration and extracellular matrix synthesis have been investigated. In the present study, both MSCS showed a good biocompatibility to interact with PLCL and silk scaffolds. No significant differences have been detected between WJ-MSCs and BM-MSCs. Finally, the effect of biomechanical stimulation on MSCs differentiation towards ligament tissue has been carried out with a tension-torsion bioreactor. Although few cells were detected on scaffold after 7 days of stimulation, MSCs were observed to exhibit an elongated shape along the longitudinal direction of fibers, which may indicate that an adapted mechanical stimulation could promote MSC-scaffold constructs differentiation towards ligamentous tissue. As a conclusion, this study demonstrates the potential of WJ-MSCs and BM-MSCs combined with a new silk/PLCL composite scaffold towards ligament regeneration.
... Non-toxicity, sterilizability, effectiveness and biocompatibility are the minimum requirements of biomaterials for their end applications in human body. Various biomedical applications of synthetic aliphatic polyesters are listed in Figure 5 [256][257][258][259][260][261][262][263][264]. ...
... Biomedical applications of synthetic aliphatic polyesters[256][257][258][259][260][261][262][263][264]. ...
Biodegradable polymers are important as an alternative to conventional non-degradable polymers for sustainable eco-system. The recent trends indicate that the new developments in biodegradable polymers focus on novel polymer systems that can cater the need of biomedical and packaging applications in-terms of performance and economics. The new interest is rapidly moving toward reducing carbon footprint through utilization of carbon dioxide and developing new methods of manufacturing such as 3D printing for specific purposes. This review focus on the present state-of-art and recent developments in biodegradable polymers covering their sources, synthetic methodologies, salient properties, degradation patterns, polymer blends and nanocomposites. As well as biodegradable polymers as a 3D printing material and the use of carbon dioxide as a renewable raw material for biomedical and packaging applications.
... Tube wall pores should be interconnected and with suitable size to allow small molecule permeation; on the other hand pore size greater than 20 mm causes fibroblast infiltration as reported by Sarazin et al. (2004) and should be avoided. Therefore, optimal pore size for external guide should be comprised in 5-30 mm range and preferable at 10-20 mm range (Dodla and Bellamkonda, 2008;Jiang et al., 2010;Pfister et al., 2007a;Rodriguez et al., 1999;Vleggeert-Lankamp et al., 2007), avoiding growth factors outflow and fibroblast infiltration, although allowing nutrient and catabolyte exchange and endothelial cell migration (vascularization). ...
... In addition, electrospinning allows the obtainment of porous polymer substrates with varying porosity degree and mechanical properties, having a high surface to volume ratio, enhancing cellsubstrate interactions. Porous guides have been fabricated by electrospinning, having pores with 1-10 mm size, which avoid the infiltration of fibrous tissue into the NGCs, but allow the ingrowth of endothelial cells (vascularization) when pores have higher size than 5 mm (Dodla and Bellamkonda, 2008;Pfister et al., 2007a;Rodriguez et al., 1999). By changing the collector geometry, substrates with different structural and morphological features can be prepared, such as flat membranes (which can be wrapped into tubular guides or cut into stripes used as NGC fillers), mono-layered or bi-layered hollow tubular structures and fiber mats used as inner fillers. ...
... As a result of this research several polymer implants in the form of tubes have been put into clinical practice. They can be used in the treatment of short gaps of less than 3 cm in length in skin nerves [14][15][16][17], but longer nerve gaps require nerve grafting for reconstruction. ...
... This alginates migration process from the tube's inner space into surroundings was enhanced by PLDL tubes porosity. The lack of porosity in polyurethane tubes blocked fast alginate diffusion from the inner area of tubes and it hampered tissue ingrowth [17,30]. Neural tissue growth inside the tube was also found, but during the longest observation period and after a great part of the alginate gel had been evacuated from the tube into its surroundings. ...
This preliminary research project has been conducted to evaluate different elastic polymer materials in terms of their applicability in peripheral nerve regeneration. Poly(tetrafluoroetylene-co-difluorovinylidene-co-propylene), poly(L-lactide-coD ,L-lactide), and polyurethane were used for the manufacture of tubular implants. Alginate sodium gel and fibers were used as a scaffold to fill in tube nerve grafts and enhance nerve regeneration. The tubes were implanted to reconstruct a 10 mm gap in the sciatic nerve in rats. After 3, 7, 14, 28 days the tubes were retrieved for histological examination. Among tested tubes polyurethane implants were found to be the most suitable because of their mechanical and surgical properties. Other tested implants were found to be unfavorable due to their inappropriate rigidity, elasticity or surgical convenience. Alginate transformation into dense gel form was observed that hindered inner tube space cellular colonization. In consequence of this transformation nerve regeneration was inhibited inside tube nerve grafts. His-tological examination showed massive colonization of the implants with Schwann cells, and growth of new axons was found within Schwann cells growing on tubes external surface. Appropriate time rates for alginate gelation and dis-solveing must be determined to allow undisturbed tissue growth and maturation.
... Rodriguez et al. investigated the rate of regeneration and reinnervation between several PLC and POS tubes with variable permeabilities. They demonstrated that tubes with PLC type and high permeability rates promote reinnervation for long gaps much faster and better [26]. Pfister et al. considered a hydrogel for biodegradable NGC and made rheological measurements [27]. ...
Nerve guidance conduits, or NGCs, used as structures in tissue engineering, play an important role in recon-struction and the growth of axons after nerve injuries. To produce an appropriate scaffold for nerve growth andtransfer the suitable amount of growth factors, these channels should be designed and built with topological,mechanical, and biological properties and functional mass transfer requirements. This study deals with acomputational design approach of a nerve guidance conduit which, after validating the geometry, simulates theblood flow utilizing the Carreau-Yasuda model and optimizes physical properties. The physical properties arehydraulic pressure, porosity, diffusivity and water absorption. The optimization process consists of three distinctsteps: initially, a single objective function is implemented using a genetic algorithm (GA) to determine theoptimal hydraulic pressure for the nerve guidance conduit, resulting in a final value of 0.039 Pa. Subsequently,the GA is employed again to optimize porosity, leading to a final value of 0.6473. Finally, a multi-objectivefunction is utilized to consider porosity, diffusivity, and water absorption, simultaneously.
... PCL has a relatively low degradation rate and high mechanical strength, which make PCL potentially suitable for not only cell-based therapies, but also fabrication of bone substitutes for rapid incorporation and eventually treatment of large defects (as end stage healing). Rodriguez et al. synthesized highly permeable poly(L-lactide-co-ε-caprolactone) for the regeneration of large gaps [37]. In addition to PCL, poly (lactic acid) (PLA), polyether ether ketone (PEEK), and poly vinyl alcohol (PVA) have emerged as potential biocompatible materials for regenerative engineering applications, which allow for FDM-based fabrication of biological tissues and constructs. ...
The repair of orthopedic and maxillofacial defects in modern medicine currently relies heavily on the use of autograft, allograft, void fillers, or other structural material composites. This study examines the in vitro osteo regenerative potential of polycaprolactone (PCL) tissue scaffolding, fabricated via a three-dimensional (3D) additive manufacturing technology, i.e., a pneumatic micro extrusion (PME) process. The objectives of this study were: (i) To examine the innate osteoinductive and osteoconductive potential of 3D-printed PCL tissue scaffolding and (ii) To perform a direct in vitro comparison of 3D-printed PCL scaffolding with allograft Allowash® cancellous bone cubes with regards to cell-scaffold interactions and biocompatibility with three primary human bone marrow (hBM) stem cell lines. This study specifically examined cell survival, cell integration, intra-scaffold cell proliferation, and differentiation of progenitor cells to investigate the potential of 3D-printed PCL scaffolds as an alternative to allograft bone material for the repair of orthopedic injuries. We found that mechanically robust PCL bone scaffolds can be fabricated via the PME process and the resulting material did not elicit detectable cytotoxicity. When the widely used osteogenic model SAOS-2 was cultured in PCL extract medium, no detectable effect was observed on cell viability or proliferation with multiple test groups showing viability ranges of 92.2% to 100% relative to a control group with a standard deviation of ±10%. In addition, we found that the honeycomb infill pattern of the 3D-printed PCL scaffold allowed for superior mesenchymal stem-cell integration, proliferation, and biomass increase. When healthy and active primary hBM cell lines, having documented in vitro growth rates with doubling times of 23.9, 24.67, and 30.94 h, were cultured directly into 3D-printed PCL scaffolds, impressive biomass increase values were observed. It was found that the PCL scaffolding material allowed for biomass increase values of 17.17%, 17.14%, and 18.18%, compared to values of 4.29% for allograph material cultured under identical parameters. It was also found that the honeycomb scaffold infill pattern was superior to the cubic and rectangular matrix structures, and provided a superior microenvironment for osteogenic and hematopoietic progenitor cell activity and auto-differentiation of primary hBM stem cells. Histological and immunohistochemical studies performed in this work confirmed the regenerative potential of PCL matrices in the orthopedic setting by displaying the integration, self-organization, and auto-differentiation of hBM progenitor cells within the matrix. Differentiation products including mineralization, self-organizing “proto-osteon” structures, and in vitro erythropoiesis were observed in conjunction with the documented expression of expected bone marrow differentiative markers including CD-99 (>70%), CD-71 (>60%), and CD-61 (>5%). All of the studies were conducted without the addition of any exogenous chemical or hormonal stimulation and exclusively utilized the abiotic and inert material polycaprolactone; setting this work apart from the vast majority of contemporary investigations into synthetic bone scaffold fabrication In summary, this study demonstrates the unique clinical potential of 3D-printed PCL scaffolds for stem cell expansion and incorporation into advanced microstructures created via PME manufacturing to generate a physiologically inert temporary bony defect graft with significant autograft features for enhanced end-stage healing.
... NGC holds the two nerve stumps coaxially at a certain distance between them, providing a biomimetic environment for nerve regeneration and avoids the formation of fibrotic tissue that would prevent axons from regrowing [10]. Regarding this, permeability must allow the passage of nutrients but at the same limit the infiltration of fibroblasts [11]. NGC should be biodegradable, biocompatible, and it should have similar mechanical properties of native tissue. ...
When a traumatic event causes complete denervation, muscle functional recovery is highly compromised. A possible solution to this issue is the implantation of a biodegradable polymeric tubular scaffold, providing a biomimetic environment to support the nerve regeneration process. However, in the case of consistent peripheral nerve damage, the regeneration capabilities are poor. Hence, a crucial challenge in this field is the development of biodegradable micro- nanostructured polymeric carriers for controlled and sustained release of molecules to enhance nerve regeneration. The aim of these systems is to favor the cellular processes that support nerve regeneration to increase the functional recovery outcome. Drug delivery systems (DDSs) are interesting solutions in the nerve regeneration framework, due to the possibility of specifically targeting the active principle within the site of interest, maximizing its therapeutical efficacy. The scope of this review is to highlight the recent advances regarding the study of biodegradable polymeric DDS for nerve regeneration and to discuss their potential to enhance regenerative performance in those clinical scenarios characterized by severe nerve damage.
... It has shown to alter the proliferation rate and the state of differentiation of SC while preserving their morphology (Yoshino et al 1990 (Eiselt et al 2000) and retains extracellular matrix (Burgi-Savilie et al 1998). The high porosity of alginate hydrogel may result in high permeability of the structure, which has shown to be a beneficial factor Chapter 5: SC compatibility studies in nerve regeneration (Rodriguez et al 1999). This may be further helped by the preservation of SC morphology in alginate hydrogel. ...
There is a clinical need to improve functional outcome after peripheral nerve reconstruction, and also to find a replacement for autologous nerve grafts currently used for this purpose. Schwann cells (SC) are essential for adequate axonal regeneration and the aim of this work was to develop a methodology to enable investigation and characterisation of the effects of SC transplantation and genetic manipulation on peripheral nerve regeneration. Initially an identifiable and pure population of cultured SC was obtained. In vitro evaluation showed that chemical labelling adversely affected SC properties. Transduction of lacZ genetic label was carried out and a stable population of genetically modified SC was obtained. Transduced SC properties and lacZ expression were preserved in vitro for 6 months of continuous culture. Suspension matrix is required for SC transplantation and the suitability of alginate hydrogel was confirmed by in vitro tests to support SC proliferation and neurite sprouting in a neuron-glial co-culture. Defects in the rat sciatic nerve were bridged using resorbable poly-3-hydroxybutyrate (PHB) conduits containing SC. The results showed that the optimal number of SC required to enhance axonal regeneration was 80×l0⁶/ml. Following transplantation of transduced SC, syngeneic SC could be identified for up to 6 weeks and allogeneic SC for up to 3 weeks, as identified by X-gal staining. Immunohistochemistry was used to characterise SC, immune response, and axonal regeneration and the staining quantified by image analysis. Transplantation of both syngeneic and allogeneic SC improved axonal regeneration distance, the quantity of regeneration was better and more sustained with syngeneic SC. Furthermore, addition of liquid fibronectin to alginate improved regeneration which was further enhanced when SC were present. Finally a new technique for nonviral gene delivery of insulin-like growth factor I and mechano growth factor gene with alginate hydrogel matrix showed promising results in improving peripheral nerve regeneration.
... The lactide to caprolactone proportion of the PLCL can be varied to match the aimed tissue properties for different clinical applications [25]. In particular, the PLCL with 70/30 L-lactide/ε-caprolactone proportion enhances the proliferation of Schwann cells and is commonly used for neural guide conduit fabrication for peripheral nerve healing [26][27][28]. Particle leaching is the most common technique to create porous scaffolds [29][30][31][32][33][34][35][36][37][38][39][40][41]. This technique allows for the production of scaffolds with well interconnected pores, whose characteristics are controlled by particle proportion, size, and distribution. ...
Porous scaffolds made of elastomeric materials are of great interest for soft tissue engineering. Poly(L-lactide-co-ε-caprolactone) (PLCL) is a bio-resorbable elastomeric copolymer with tailorable properties, which make this material an appropriate candidate to be used as scaffold for vascular, tendon, and nerve healing applications. Here, extrusion was applied to produce porous scaffolds of PLCL, using NaCl particles as a leachable agent. The effects of the particle proportion and size on leaching performance, dimensional stability, mechanical properties, and ageing of the scaffolds were analyzed. The efficiency of the particle leaching and scaffold swelling when wet were observed to be dependent on the porogenerator proportion, while the secant moduli and ultimate tensile strengths were dependent on the pore size. Porosity, swelling, and mechanical properties of the extruded scaffolds were tailorable, varying with the proportion and size of porogenerator particles and showed similar values to human soft tissues like nerves and veins (E = 7–15 MPa, σu = 7 MPa). Up to 300-mm length micro-porous PLCL tube with 400-µm thickness wall was extruded, proving extrusion as a high-throughput manufacturing process to produce tubular elastomeric bio-resorbable porous scaffolds of unrestricted length with tunable mechanical properties.
... The lactide to caprolactone proportion of the PLCL can be varied to match the aimed tissue properties for different clinical applications [25]. In particular, the PLCL with 70/30 L-lactide/ε-caprolactone proportion enhances the proliferation of Schwann cells and is commonly used for neural guide conduit fabrication for peripheral nerve healing [26][27][28]. Particle leaching is the most common technique to create porous scaffolds [29][30][31][32][33][34][35][36][37][38][39][40][41]. This technique allows for the production of scaffolds with well interconnected pores, whose characteristics are controlled by particle proportion, size, and distribution. ...
Porous scaffolds made of elastomeric materials are of great interest for soft tissue engineering. Poly(L-lactide-co-ε-caprolactone) (PLCL) is a bio-resorbable elastomeric copolymer with tailorable properties, which make this material an appropriate candidate to be used as scaffold for vascular, tendon, and nerve healing applications. Here, extrusion was applied to produce porous scaffolds of PLCL, using NaCl particles as a leachable agent. The effects of the particle proportion and size on leaching performance, dimensional stability, mechanical properties, and ageing of the scaffolds were analyzed. The efficiency of the particle leaching and scaffold swelling when wet were observed to be dependent on the porogenerator proportion, while the secant moduli and ultimate tensile strengths were dependent on the pore size. Porosity, swelling, and mechanical properties of the extruded scaffolds were tailorable, varying with the proportion and size of porogenerator particles and showed similar values to human soft tissues like nerves and veins (E = 7–15 MPa, σu = 7 MPa). Up to 300-mm length micro-porous PLCL tube with 400-µm thickness wall was extruded, proving extrusion as a high-throughput manufacturing process to produce tubular elastomeric bio-resorbable porous scaffolds of unrestricted length with tunable mechanical properties.
... Due to processing difficulties, seven samples were not included in the histology analysis: two autograft at the conduit site, one autograft distal to the graft site, two collagen-filled at the conduit site, and two hollow conduit distal to the conduit site. Color differences are due to staining/drying of toluidine blue regeneration and reorganization of the nerve is effective, but slow enough to deter scar tissue accumulation inhibiting longitudinal nerve growth [32,33]. ...
Background
We compared different surgical techniques for nerve regeneration in a rabbit sciatic nerve gap model using magnetic resonance diffusion tensor imaging (DTI), electrophysiology, limb function, and histology.
Methods
A total of 24 male New Zealand white rabbits were randomized into three groups: autograft (n = 8), hollow conduit (n = 8), and collagen-filled conduit (n = 8). A 10-mm segment of the rabbit proximal sciatic nerve was cut, and autograft or collagen conduit was used to bridge the gap. DTI on a 3-T system was performed preoperatively and 13 weeks after surgery using the contralateral, nonoperated nerve as a control.
Results
Overall, autograft performed better compared with both conduit groups. Differences in axonal diameter were significant (autograft > hollow conduit > collagen-filled conduit) at 13 weeks (autograft vs. hollow conduit, p = 0.001, and hollow conduit vs. collagen-filled conduit, p < 0.001). Significant group differences were found for axial diffusivity but not for any of the other DTI metrics (autograft > hollow conduit > collagen-filled conduit) (autograft vs. hollow conduit, p = 0.001 and hollow conduit vs. collagen-filled conduit, p = 0.021). As compared with hollow conduit (autograft > collagen-filled conduit > hollow conduit), collagen-filled conduit animals demonstrated a nonsignificant increased maximum tetanic force.
Conclusions
Autograft-treated rabbits demonstrated improved sciatic nerve regeneration compared with collagen-filled and hollow conduits as assessed by histologic, functional, and DTI parameters at 13 weeks.
... Nerve guides should be tough and flexible that is capable of bending without kinking. Sufficient permeability of nerve supporting devices is needed for the exchange of fluids between the regeneration environment and the surrounding tissue, avoiding the build-up of pressure due to fluid retention [6]. Among materials that have been suggested for the production of artificial devices for nerve repair biodegradable materials are of particular interest [7]. ...
... Biodegradable nerve tubes are advantageous over their non-degradable ones, obviating the need for their removal when regeneration is completed. These temporary conduits have been mostly based on aliphatic polyesters including poli(glycolid acid) [10][11][12], poly(lactid-co-glicolid acid) [13][14][15][16][17], poly(DL-lactic acid) [18][19][20] and poly(lactic acid-co-ε-caprolactone) [5,21]. The aforementionned group have disadvantage of not only being stiff and brittle materials but also of possessing a relatively high rate of degradation. ...
Strategies to improve healing of damaged nerves include the application of specialized nerve guides, which hold the promise for allowing reanastomosis of the severed or damaged fibers. Studies have demonstrated that the use of a slowly degradable polymeric nerve guide can improve the nature and rate of nerve regeneration across a short gap in small nerves. The objective of this study was to characterize a biodegradable nerve guide based on poly(trimethylene carbonate-co-lactide) for peripheral nerve regeneration and to evaluate its cytotoxicity. The obtained copolymer films were incubated in two different media (distilled water and simulated body fluid), and while the degradation process appeared, pH and ion conductivity changes of solutions were monitored as well as mass loss of the samples. Additionally, mechanical tests (tensile strength, elongation at break and Young’s modulus parameters) before and after different time points were carried out. To evaluate cytotoxicity biological test were done on fibroblasts cells (NIH 3T3). Cell metabolic activity was determined using Alamar Blue reagent and their morphology was observed under fluorescence microscopy. The growth of pH in both media were mostly caused by steadily degradation of carbonate units into alkaline diols. The growth of ion conductivity value at the beginning of the incubation process was associated with the releasing of free ions to the solution. The mechanical parameters decreased with the progress of degradation process. Ringer’s fluid, as more aggressive, caused higher decrease in mechanical properties. The measured contact angles showed good surface wettability. Both surfaces, the top and the bottom, had similar hydrophilicity. Moreover, activity of fibroblasts cells were similar on both sides as well as on the reference TCPS. Good adhesion of NIH 3T3 cells to the surface suggests that the hydrophilic polymers promote colonization of fibroblasts cells on their surface. Biological studies have shown that used cells are very sensitive to surface topography which they colonize and cell viability was higher at the bottom surface, which has a slightly higher average roughness Ra. Thus, fibroblasts cell preferred colonizing rougher than smoother surfaces. Fabricated films does not affect negatively, namely, toxic on cell cultures and forms substrate with favourable surface properties. This was confirmed by the Alamar Blue tests and microscopic observations.
... These data should be quantified in relationship to each other and then determine differentiation among different substrates. PLLA by itself has been used to fabricate tubular nerve guides for implantation after PNS injury [54, 55, 56]. The PLLA fibers used for fabrication for this study have fine, flexible and strong properties. ...
Differentiation of HNPC on Organized Electrospun PLLA Fibers
... The relevance of the nanosize scale fibrous architecture has been widely demonstrated in the case of polycaprolactone (PCL) scaffolds in terms of adhesion/proliferation [8] and cell metabolism [9] and, in the case of poly(lactic acid) (PLA) scaffolds for their ability to promote bone [10,11] or nerve regeneration [12]. Porous scaffolds made of PLC copolymers of different compositions have been used for blood vessels [14,15], bone [16], cartilage [17,18] and nerve regeneration [19] among others. This is because PLC chemical structure combines the high elasticity and low glass transition temperature of PCL with the stiffness of PLA at body temperature into a single biocompatible and biodegradable material [15]. ...
... This could be due to either an actual advantage of smaller cells in successfully reinnervating the distal stump, or to a shift from A to Bϩ morphology due to cell body shrinkage. While a contribution from the latter can never be excluded, in previous studies with different types of sciatic nerve injuries we have consistently found that small nerve fibers (nociceptive and sympathetic) are able to achieve slightly earlier and significantly higher levels of functional reinnervation of targets at the distal paw than large motor and sensory myelinated fibers (Navarro et al., 1994;Rodríguez et al., 1999;Verdú and Navarro, 1997). These differences may be explained by a lesser difficulty of crossing the lesion site, and by a faster rate of regeneration for thinner than for thicker fibers. ...
... In addition, semi-permeable barriers may also facilitate the formation of a supportive fibrin by allowing inward diffusion of local or systemic healing factors [22]. Importantly, semi-permeable barriers have better effects on nerve regeneration [23][24][25][26]. ...
Regeneration and functional recovery of nerves after peripheral nerve injury is the key to peripheral nerve repair. One of the putative therapeutic strategies is to use anti-adhesion polymer films, made of polymeric biomaterials. Recently, a novel biodegradable poly (DL-lactic acid) (PDLLA) film has been prepared using a method of phase transformation with biodegradable polylactic acid polymer as the substrate. This novel, anti-adhesion film has a porous structure, which provides better mechanical properties, better flexibility, more complete diffusion through the polymer of tissue biologic factors like growth factors, and more controllable degradation compared to traditional non-porous films. Little is known, however, about the in vitro and in vivo biocompatibility and cytotoxicity of this type of PDLLA film. Therefore, our aim was to evaluate the biocompatibility and cytotoxicity of this novel PDLLA film using various experimental methods, including a skin irritation test, MTT analysis, and the mouse bone marrow cell micronucleus test, as well as hematology or clinical chemistry measurements in rats after receiving sciatic nerve transection and anastomosis with wrapping of the anastomosis with DLLA films. We demonstrated that exposure to PDLLA film extracts did not generate apparent erythema or edema in rabbit skin and had no effect on the proliferation of Vero cells. Additionally, treatment with PDLLA film extracts did not alter the incidence of micronucleated polychromatic erythrocytes as compared with saline Treated group. Furthermore, implantation of PDLLA film did not alter liver or renal function as measured by serum levels of ALT, AST, TP, A/G, Cr, and BUN, and pathologic examinations showed that implantation of PDLLA film did not cause pathologic changes to the rat liver, kidney, pancreas, or spleen. Taken together, these results suggest that PDLLA films have excellent biocompatibility and no obvious toxicity in vivo, and may be used to prevent nerve adhesion, thereby promoting nerve regeneration.
... The conduits guide the axial growth of neuronal axons. The conduit structure with the appropriate porosity and the size of pores allows free exchange of tissue fluids and nutrients, as well as accumulation of neurotrophic factors that are required for nerve regeneration [3,5]. Over the years, the nerve conduits have evolved from silicone based materials, non-absorbable artificial materials, or biological based material (intravenous, amniotic membrane, etc.) to the modern biodegradable polymers [2,6]. ...
Regeneration and repair of peripheral nerve injury has always been a major problem in the clinic. The conventional technique based on suturing the nerve ends to each other coupled with the implantation of nerve conduits outside is associated with postoperative adhesions and scar problems. Recently, a novel biodegradable poly (DL-lactic acid) (PDLLA) film has been introduced. This novel anti-adhesion film has a porous structure with better mechanical properties, better flexibility, and more controllable degradation as compared to traditional non-porous nerve conduits. However, little is known about the effects of such PDLLA films on regeneration and repair of peripheral nerve injury in vivo. In this study, we evaluated the effects of PDLLA films implantation after sciatic nerve transection and anastomosis on subsequent sciatic nerve regeneration in vivo, using a rat sciatic nerve injury model. Sciatic nerve transection surgery coupled with direct suturing only, suturing and wrapping with traditional nerve conduits, or suturing and wrapping with PDLLA films was performed on adult Wistar rats. The additional wrapping with PDLLA films inhibited the nerve adhesion after 12 weeks recovery from surgery. It also increased the compound muscle action potentials and tibialis and gastrocnemius muscle wet weight ratio following 8 weeks recovery from surgery. Regenerated nerve fibers were relatively straight and the aligned structure was complete in rats with implantations of PDLLA films. The results suggested that PDLLA films can improve the nutritional status in the muscles innervated by the damaged nerves and promote nerve regeneration in vivo.
... PCL conduits do not degrade as quickly or produce toxic degradation products as PGA, while PCL displayed improved functional recovery in rats when compared to PGA, collagen conduits and autografts [70]. All bioabsorbable conduits have illustrated superior functional results compared to their nonreabsorbable counterparts [58], and are comparable to autografts [70,76]. ...
Peripheral nerve repair for complete section injuries employ reconstructive techniques that invariably require sutures in their application. Sutures are unable to seal the nerve, thus incapable of preventing leakage of important intraneural fluids from the regenerating nerve. Furthermore, sutures are technically demanding to apply for direct repairs and often induce detrimental scarring that impedes healing and functional recovery. To overcome these limitations, biocompatible and biodegradable glues have been used to seal and repair peripheral nerves. Although creating a sufficient seal, they can lack flexibility and present infection risks or cytotoxicity. Other adhesive biomaterials have recently emerged into practice that are usually based on proteins such as albumin and collagen or polysaccharides like chitosan. These adhesives form their union to nerve tissue by either photothermal (tissue welding) or photochemical (tissue bonding) activation with laser light. These biomaterial adhesives offer significant advantages over sutures, such as their capacity to unite and seal the epineurium, ease of application, reduced invasiveness and add the potential for drug delivery in situ to facilitate regeneration. This paper reviews a number of different peripheral nerve repair (or reconstructive) techniques currently used clinically and in experimental procedures for nerve injuries with or without tissue deficit.
... Conduit wall permeability is important for effective removal of waste, promotion of new vessel infiltration, and nutrient diffusion into the lumen. Nerves repaired with permeable conduits show more effective nerve regeneration compared to impermeable ones (45). The size and morphology of pores on the nerve conduit wall have an important effect in determining nerve regrowth. ...
Nerve injury secondary to trauma, neurological disease or tumor excision presents a challenge for surgical reconstruction. Current practice for nerve repair involves autologous nerve transplantation, which is associated with significant donor-site morbidity and other complications. Previously artificial nerve conduits made from polycaprolactone, polyglycolic acid and collagen were approved by the FDA (USA) for nerve repair. More recently, there have been significant advances in nerve conduit design that better address the requirements of nerve regrowth. Innovations in materials science, nanotechnology, and biology open the way for the synthesis of new generation nerve repair conduits that address issues currently faced in nerve repair and regeneration. This review discusses recent innovations in this area, including the use of nanotechnology to improve the design of nerve conduits and to enhance nerve regeneration.
Peripheral nerve damages cause loss of sensorimotor and autonomic functions, resulting in a significant burden for the patients. Nerve injuries above a limiting gap length require surgical repair. Although autograft...
Engineered grafts constitute an alternative to autologous transplant for repairing severe peripheral nerve injuries. However, current clinically available solutions have substantial limitations and are not suited for the repair of long nerve defects. A novel design of nerve conduit is presented here, which consists of a chitosan porous matrix embedding a 3D‐printed poly‐ε‐caprolactone mesh. These materials are selected due to their high biocompatibility, safe degradability, and ability to support the nerve regeneration process. The proposed design allows high control over geometrical features, pores morphology, compression resistance, and bending stiffness, yielding tunable and easy‐to‐manipulate grafts. The conduits are tested in chronic animal experiments, aiming to repair a 15‐mm long gap in the sciatic nerve of rats, and the results are compared with an autograft. Electrophysiological and nociception tests performed monthly during a 4‐month follow‐up show that these conduits allow a good degree of muscle functional recovery. Histological analyses show abundant cellularization in the wall and in the lumen of the conduits and regenerated axons within all rats treated with these grafts. It is suggested that the proposed conduits have the potential to repair nerves over the limiting gap length and can be proposed as strategy to overcome the limitations of autograft.
The nervous system is the most important system of the body and damaging this system could be lethal for humans. Restoring the function of a damaged nervous system has always been a challenge due to the complexity of this system and its limited ability of regeneration. Furthermore, several obstacles exist in the repair process of the nervous system. In the central nervous system (CNS) limited clearance of myelin and formation of inhibitory glial scars make regeneration difficult. There is no effective clinical treatment for damages in the CNS while current treatments focus on stabilization and prevention of further damage and consequently on rehabilitation and preparation of prosthetics and mechanical aids. In peripheral nervous system (PNS) damages, the management may be a nerve autograft or allograft while shortage of donors for nerves makes the situation difficult. Size inequality between the donor nerve and the recipient, danger of neuroma formation, and occurrence of infectious diseases are other problems associated with PNS, while indeed complete recovery of function is still not common. Several studies have illustrated that implying tissue engineering strategies for neural repair may lead to considerable improvements in damaged nervous tissues.
Introduction
We hypothesized that hybrid artificial nerves might overcome the limitations of a nerve conduit by isolating nerve fascicles from autologous nerves. Nerve sacrifice during harvest, a drawback of conventional autologous nerve transplantation, may be reduced by the hot dog method. The hot dog method (based on the morphology of hybrid artificial nerves) adds nerve conduits to autologous nerve fascicles.
Methods
Forty-eight rats with a 10-mm sciatic nerve defect were divided into six groups ( n = 8 per group) according to the neural reconstruction method: autologous nerve transplantation, the hot dog method, nerve conduit, nerve fascicle transplantation, sham control, and nerve fascicle isolation were classified as Groups I, II, III, IV, V, and VI, respectively. The sciatic nerve function was assessed in these groups, a histological evaluation was performed, and statistical analyses were conducted based on these data.
Results
Group III (nerve conduit) and Group IV (nerve fascicle transplantation) showed the lowest functional and axonal regenerative effects, followed by Group II (hot dog method) and Group I (autologous nerve transplantation). Group VI (nerve fascicle isolation) tended to achieve better recovery in motor function and axonal regeneration than Group I (autologous nerve transplantation).
Conclusions
The hot dog method is simple, safe, and easy to execute. This method can serve as a new neural reconstruction method that uses artificial nerves.
Peripheral nerve injuries remain among the most challenging medical issues despite numerous efforts to devise methods in fabrication of nerve conduits to functionally regenerate axonal defects. In this regard, the current study offers a holistic perspective in design by considering the mechanical, topographical and structural aspects which are crucial for a successful nerve guide conduit. Poly(e‐caprolactone) and gelatin were employed to serve this purpose in the form of dual‐electrospun films which were rolled and later shaped the assembly of a multichannel conduit. Polyaniline/graphene (PAG) nanocomposite was incorporated to endow the conduit with conductive properties. FTIR analysis, water contact angle measurements, and SEM observations as well as mechanical and conductivity tests were used to evaluate the properties of the conduits. In addition, MTT assay was conducted to assess the proliferation of rat bone marrow‐derived mesenchymal stem cells cultured on the films. Incorporating 2% PAG proved to have superior cell support and proliferation, while guaranteeing electrical conductivity of 10.8 × 10⁻⁵ S/cm and remarkable tensile strength of 3.52 ± 1.3 MPa and 14.12 ± 3.1 MPa for wet and dry conditions, respectively. Overall, the observed results highlight the great potential of the fabricated conduit to be used as a candidate for peripheral nerve defects.
Implantable tubular devices known as nerve guidance conduits (NGCs) have drawn considerable interest as an alternative to autografting in the repair of peripheral nerve injuries. At present, there exists a lack of biodegradable, biocompatible materials for the fabrication of NGCs with physical properties which suitably match the native nerve tissue. Most of the existing reports have been confined to the traditional synthetic aliphatic polyesters due to their naturally-occuring degradation by-products, suitably slow in vivo resoprtion timeframes and relatively diverse and tailorable range of material properties. Moreover, these thermoplastic polymers can be processed into NGCs from various methods and further tweaking of physical properties can be acheved during fabrication. Although there have been many successful reports of nerve gap repair using NGCs made from these materials, the majority have been confined to basic tubular designs across short to medium nerve gaps with at best equivalent outcomes to autografts. This article reviews the performance of poly-α-hydroxyester tubes to date (including modifications to basic hollow conduits) and is intended to aid researchers as they aim to create biomimetic NGCs capable of bridging larger nerve gaps with superior results to autografting. Based on the existing reports, a next-generation bioresorbable NGC should involve a highly flexible poly-a-hydroxyester outer tube, most suitably from a lactide-caprolactone co-polymer, with some combination of internal lumen contact guidance and bioactive neurotrophic factors. However, detailed further experimentation and an interdisciplninary approach will be required to arrive at an ideal final configuration.
Purpose:
To compare recovery in a rat model of sciatic nerve injury using a novel polyglycolic acid (PGA) conduit, which contains collagen fibers within the tube, as compared with both a hollow collagen conduit and nerve autograft. We hypothesize that a conduit with a scaffold will provide improved nerve regeneration over hollow conduits and demonstrate no significant differences when compared with autograft.
Methods:
A total of 72 Sprague-Dawley rats were randomized into 3 experimental groups, in which a unilateral 10-mm sciatic defect was repaired using either nerve autograft, a hollow collagen conduit, or a PGA collagen-filled conduit. Outcomes were measured at 12 and 16 weeks after surgery, and included bilateral tibialis anterior muscle weight, voltage and force maximal contractility, assessment of ankle contracture, and nerve histology.
Results:
In all groups, outcomes improved between 12 and 16 weeks. On average, the autograft group outperformed both conduit groups, and the hollow conduit demonstrated improved outcomes when compared with the PGA collagen-filled conduit. Differences in contractile force, however, were significant only at 12 weeks (autograft > hollow collagen conduit > PGA collagen-filled conduit). At 16 weeks, contractile force demonstrated no significant difference but corroborated the same absolute results (autograft > hollow collagen conduit > PGA collagen-filled conduit).
Conclusions:
Nerve repair using autograft provided superior motor nerve recovery over the 2 conduits for a 10-mm nerve gap in a murine acute transection injury model. The hollow collagen conduit demonstrated superior results when compared with the PGA collagen-filled conduit.
Clinical relevance:
The use of a hollow collagen conduit provides superior motor nerve recovery as compared with a PGA collagen-filled conduit.
Peripheral nerve injury is a large‐scale problem and it is a great challenge to repair the long lesion in a thick nerve. The design of a multi‐tubular conduit with a honeycomb structure by mimicking the anatomy of a peripheral nerve for the potential repair of large defects in thick nerves has been reported. A bilayer mat of electrospun nanofibers is rolled up to form a single tube, with the inner and outer layers comprised aligned and random nanofibers, respectively. Seven such tubes are then assembled into a hexagonal array and encased within the lumen of a larger tube to form the multi‐tubular conduit. By introducing an adhesive to the regions between the tubes, the conduit is robust enough for handling during surgery. The seeded bone marrow stem cells (BMSCs) are able to proliferate in all the tubes with even circumferential and longitudinal distributions. Under chemical induction, the BMSCs are transdifferentiated into Schwann‐like cells in all the tubes. While the cellular version holds great promise for peripheral nerve repair, the multi‐tubular conduit can also be used to investigate the fundamental aspects involved in the development of peripheral nervous system and migration of cells. A multi‐tubular conduit with a honeycomb structure is fabricated by assembling seven small tubes based on electrospun nanofibers into a hexagonal array and wrapped within a larger tube. Bone marrow stem cells are then seeded and transdifferentiated into Schwann‐like cells in all the tubes, generating a cellular conduit for potential use in repairing long lesions in thick nerves.
Peripheral nerve conduits were fabricated from biodegradable polyurethane (PU) which was synthesized by a waterborne process. The biodegradable PU was based on poly(ε-caprolactone) diol and polyethylene butylene adipate diol (2:3 molar ratio) as the soft segment. Conduits formed by the freeze-drying process had asymmetric microporous structure. The PU nerve conduits were used to bridge a 10 mm gap in rat sciatic nerve. Nerve regeneration was evaluated by walking track analysis, magnetic resonance imaging (MRI), electrophysiological, and histological analyses. Results demonstrated that after 6 weeks, walking function was recovered by 40%. MR images showed that the transected nerve was reconnected after 3 weeks and the diameter of the regenerated nerve increased from 3 to 6 weeks. The nerve conduction velocity of the regenerated nerve reached 50% of the normal value after 6 weeks. Histological examination revealed that the cross-sectional area of the regenerated nerve at the midconduit was 0.24 mm2 after 6 weeks. The efficacy of PU nerve conduits based on functional recovery and histology was superior to that of commercial conduits (Neurotube). The PU nerve conduit developed in this study may be a potential candidate for clinical peripheral nerve tissue engineering. This article is protected by copyright. All rights reserved.
Development of new nerve guides is required for replacing autologous nerve grafts for the repair of long gap defects after nerve injury. A nerve guide comprised only of electrospun fibers able to bridge a critical (15 mm) nerve gap in a rat animal model is reported for the first time. The nerve conduits are made of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) (PEOT/PBT), a biocompatible copolymer composed of alternating amorphous, hydrophilic poly(ethylene oxide terephthalate), and crystalline, hydrophobic poly(butylene terephthalate) segments. These guides show suitable mechanical properties, high porosity, and fibers aligned in the longitudinal axis of the guide. In vitro studies show that both neurites and Schwann cells exhibit growth alignment with PA fibers. In vivo studies reveal that, after rat sciatic nerve transection and repair with PEOT/PBT guides, axons grow occupying a larger area compared to silicone tubes. Moreover, after repair of limiting (10 mm) and critical (15 mm) nerve gaps, PEOT/PBT guides significantly increase the percentage of regenerated nerves, the number of regenerated myelinated axons, and improve motor, sensory, and autonomic reinnervation in both gaps. This nerve conduit design combines the properties of PEOT/PBT with electrospun structure, demonstrating that nerve regeneration through long gaps can be achieved through the design of instructive biomaterial constructs.
Two types of biodegradable block polyurethanes based on polycaprolactone (PCL) and poly(ethylene glycol) (PEG) were prepared by controlling the regularity of block arrangements, that is, alternating block polyurethanes PUCL-alt-PEG. Traditional random block polyurethanes PUCL-ran-PEG were also prepared for comparison. Nerve repair scaffolds were prepared from these block polyurethanes. The nerve repair scaffolds were tested in a Sprague–Dawley rat peripheral nerve defect model. The nerve regeneration was evaluated by sciatic function index analysis and histological analyses. Results revealed that polyurethane nerve-guided scaffolds exhibited much greater regeneration capabilities than PCL. Particularly, PUCL-alt-PEG exhibited better nerve regeneration than the autograft. These studies warrant further studies toward optimization of the block polyurethane nerve-guided scaffolds for peripheral nerve regeneration.
The nerval regeneration process was analyzed in poly-caprolactone (PCL)-conduits filled with Schwann cells, fibrin matrix and the neurotrophic factor LIF (Leukemia Inhibitory Factor). Therefore, primary Schwann cells were isolated from newborn rat sciatic nerves. 10 mm gaps in the buccal branch of the facial nerve in rats were bridged with PCL conduits. After 4 weeks the implants were examined histologically and morphometrically within 4 sections (SI = 2,5 mm, S2 = 5 mm, S3 = 7,5 mm, S4 = 10 mm). In order to detect the regeneration process immunhisto-chemistry against neurofilament (S200) and choline acetyl transferase (ChAT), toluidine blue staining and retrograde tracing with FAST BLUE was performed. Successful regeneration throughout all sections was found in the PCL conduits filled with the fibrin/Schwann cell matrix with and without LIF. In the three-dimensional fibrin matrix the Schwann cells were homogenously distributed and showed a high vitality rate.
This chapter will focus primarily on scaffolds for regeneration in the central nervous system (CNS) since there is little endogenous regeneration in the CNS following injury in higher order mammals, whereas, there is significant regeneration in the peripheral nervous system (PNS) following injury. The CNS consists of the brain, spinal cord, and retina. The PNS consists of the sensory neurons which carry impulses from the receptors to the CNS and the motor neurons which carry impulses to the muscles and glands. There is a plethora of texts on the anatomy of the nervous system.
Peripheral nerve injuries lead to variable levels of functional loss depending on the extent of the injury. Despite the modern treatment methods, peripheral nerve regeneration is still a time-consuming process mainly because of the limited regeneration capacity of the nervous system. Unfortunately, attempts to increase the regeneration potential of the peripheral nervous system yielded a limited improvement. However, Tissue engineering emerged as a more promising tool to ease the traditionally laborious process of peripheral nerve regeneration. A tissue-engineered nerve is a combination of a biodegradable scaffold, a neurogenic cell line, and growth factors. The main focus of current research is to improve the cell-scaffold and scaffold-tissue interactions. Engineering a fully biocompatible and natural nerve-like nerve segment should be possible in the future with the improved understanding of biological mechanisms of nerve healing. This chapter provides a detailed look into the components of tissue-engineered nerve grafts along with a review of clinically relevant studies.
This chapter evaluates biological scaffold materials in comparison to conventional synthetic scaffold materials, with a focus on intact acellular extracellular matrix (ECM) scaffold materials. Collagens, glycosaminoglycans, chitosans, and other components of the extracellular matrix are used as implantable scaffold materials. Collagen is the most common and abundant naturally occurring scaffold material that can be extracted from various tissues such as tendons, ligaments, and other connective tissues, solubilized, and reconstituted into fibers of various geometries that could, in turn, be transformed into a variety of shapes and sizes to mimic body structures such as heart valves, blood vessels, and skin. Allogeneic and xenogeneic collagen is generally recognized as "self" tissue when used as a biological scaffold material regardless of its species of origin, and it is subjected to the fundamental biological processes of degradation and integration into adjacent host tissues when left in its native ultrastructure. Intact ECM could be isolated from a large variety of different tissues, including heart valves, blood vessels, skin, nerves, skeletal muscle, tendons, ligaments, small intestine, urinary bladder, and liver. These biological scaffolds could be harvested from several different species including tissues from human, porcine, bovine, and equine or from cells grown in vitro. ECM degradation leads to an initial decrease in overall strength during the early phase of in vivo remodeling, followed by an increase in strength due to the deposition of site-specific ECM and the formation of functional site-appropriate neotissue by infiltrating cells in response to their experienced mechanical stresses.
Despite an intrinsic regenerative capacity of the peripheral nervous system, in most cases of peripheral nerve injury, especially those involving substantial nerve gaps, therapeutic interventions are always required to support peripheral nerve regeneration and functional recovery. With the advancement of tissue engineering and translational medicine, extensive research has focused on the development of tissue-engineered nerve grafts, as a promising alternative to autologous nerve grafts, for bridging nerve gaps in peripheral nerve repair. The typical design of tissue-engineered nerve grafts is composed of a nerve scaffold combined with cellular and/or molecular components. This chapter separately describes a diverse array of physical and biochemical components of tissue-engineered nerve grafts with regard to their features, functions, structure or composition, and fabrication or procurement, and further discusses many representative examples of tissue-engineered nerve grafts and their pros and cons. We also highlight the current clinical applications of tissue-engineered nerve grafts and outline the future prospects for tissue engineering in peripheral nerve regeneration.
Some poly(p-dioxanone) (PDO) homopolymers were first synthesized and the selected PDO was conjugated onto chitosan using a group-protecting method to produce chitosan-poly(p-dioxanone) (CH-PDO) copolymers with various PDO percentages changing from around 30 to 60wt%. The CH-PDO with the PDO content of around 42wt% was used to blend with prescribed amounts of silk fibroin (SF) to build porous single-lumen conduits that are intended to be used for long-gap peripheral nerve repair. Some genipin-crosslinked CH-PDO/SF conduits were endowed with an average porosity of around 60% in their porous wall, and with changed pore-sizes varying from around 10 to ca. 70μm using optimized processing conditions. After being degraded in a PBS medium containing a certain amount of lysozyme for various periods up to 8 weeks, some optimal CH-PDO/SF conduits were able to retain their compressive load and deformation recovery at around 59N/m and 73% in wet state, respectively. In addition, the achieved CH-PDO/SF conduits allowed the permeation of nutritional molecules with various molecular weights while showing a certain ability to prevent cells from infiltrating through the conduit wall. Cell culture confirmed that the optimized CH-PDO/SF conduits were able well supported the growth of rat glioma C6 cells. These results suggest that presently developed CH-PDO/SF conduits have promising potential for long-gap peripheral nerve repair.
Copyright © 2015 Elsevier Ltd. All rights reserved.
This chapter provides a structural overview of the synthetic polymers playing a key role in many applications of regenerative medicine, including implants, tissue engineering scaffolds, and orthopedic fixation devices. It also discusses their physicochemical characteristics, structure-property relationships, applications, and limitations. Nondegradable synthetic polymers such as poly(methyl methacrylate) and polyethers are biologically inert and these materials are developed to reduce to a minimum the host response to the biomaterial. These polymers provide the basis for a plethora of medical devices as diverse as suture materials, orthopedic implants, fracture fixation devices, and catheters and dialysis tubing. These materials are also applied as implantable carriers for the long-term delivery of drugs such as contraceptive hormones. Biodegradable synthetic polymers such as polyesters, poly(α-hydroxy acids), and polylactones offer a number of advantages over nondegradable materials for applications in regenerative medicine. Like all synthetic polymers, they can be synthesized at reproducible quality and purity and fabricated into various shapes with desired bulk and surface properties. Specific advantages include the ability to tailor mechanical properties and degradation kinetics to suit various applications. A wide variety of biodegradable polymers is required, ranging from pliable, elastic materials for soft tissue regeneration to stiff materials that can be used in load-bearing tissues such as bone in order to engineer scaffolds suitable for different applications.
Autologous nerve grafts are the current criterion standard for repair of peripheral nerve injuries when the transected nerve ends are not amenable to primary end-to-end tensionless neurorrhaphy. However, donor-site morbidities such as neuroma formation and permanent loss of function have led to tremendous interest in developing an alternative to this technique. Artificial nerve conduits have therefore emerged as an alternative to autologous nerve grafting for the repair of short peripheral nerve defects of less than 30 mm; however, they do not yet surpass autologous nerve grafts clinically. A thorough understanding of the complex biological reactions that take place during peripheral nerve regeneration will allow researchers to develop a nerve conduit with physical and biological properties similar to those of an autologous nerve graft that supports regeneration over long nerve gaps and in large-diameter nerves. In this article, the authors assess the currently available nerve conduits, summarize research in the field of developing these conduits, and establish areas within this field in which further research would prove most beneficial.
Nerve guide scaffolds from block polyurethanes without any additional growth factors or protein were prepared using a particle leaching method. The scaffolds of block polyurethanes (abbreviated as PUCL-ran-EG) based on poly(ɛ-caprolactone) (PCL-diol) and poly(ethylene glycol) (PEG) possess highly surface-area porous for cell attachment, and can provide biochemical and topographic cues to enhance tissue regeneration. The nerve guide scaffolds have pore size 1-5 μm and porosity 88%. Mechanical tests showed that the polyurethane nerve guide scaffolds have maximum loads of 4.98 ± 0.35 N and maximum stresses of 6.372 ± 0.5 MPa. The histocompatibility efficacy of these nerve guide scaffolds was tested in a rat model for peripheral nerve injury treatment. Four types of guides including PUCL-ran-EG scaffolds, autograft, PCL scaffolds and silicone tubes were compared in the rat model. After 14 weeks, bridging of a 10 mm defect gap by the regenerated nerve was observed in all rats. The nerve regeneration was systematically characterized by sciatic function index (SFI), histological assessment including HE staining, immunohistochemistry, ammonia silver staining, Masson's trichrome staining and TEM observation. Results revealed that polyurethane nerve guide scaffolds exhibit much better regeneration behavior than PCL, silicone tube groups and comparable to autograft. Electrophysiological recovery was also seen in 36%, 76%, and 87% of rats in the PCL, PUCL-ran-EG, and autograft groups respectively, whilst 29.8% was observed in the silicone tube groups. Biodegradation in vitro and in vivo show proper degradation of the PUCL-ran-EG nerve guide scaffolds. This study has demonstrated that without further modification, plain PUCL-ran-EG nerve guide scaffolds can help peripheral nerve regeneration excellently.
Synthetic nerve guidance channels are used to better understand the cellular and molecular events controlling peripheral nerve regeneration. In the present study, the contribution of wound-healing molecules to peripheral nerve regeneration was assessed by varying the molecular weight cutoff of the tubular membrane. Nerve regeneration through polysulfone tubular membranes with molecular weight (Mw) cutoffs of 10(5) and 10(6) Da was analyzed in a transected hamster sciatic nerve model. Cohorts of 6 animals received tubes of either type for 4 or 8 weeks with the distal end of the polymer tube capped. Other cohorts of 6 animals received tubes of either type for 4 weeks with the distal nerve stump secured within the guidance channel so as to create a 4 or 8 mm gap between both nerve stumps. Both types of channels contained regenerated tissue cables extending to the distal end of the guidance channel at both 4 and 8 weeks in the absence of a distal nerve stump. The cables regenerated in the 10(5) Da channels were composed of nerve fascicles surrounded by a loose epineurial sheath, whereas those regenerated in the 10(6) Da channels were composed mainly of granulation tissue. The numbers of myelinated and unmyelinated axons were significantly greater in the 10(5) Da than in the 10(6) Da channels at both 4 and 8 weeks. Both types of channel contained regenerated tissue cables with numerous nerve fascicles when the distal nerve stump was present with either gap length. However, when the gap distance was 8 mm, the 10(6) Da channels contained significantly fewer myelinated axons than the 10(5) Da channels. The present study reveals that the Mw cutoff of a semipermeable guidance channel strongly influences the outcome of peripheral nerve regeneration, possibly by controlling the exchange of molecules between the channel's lumen and the external wound-healing environment. These results suggest that the wound-healing environment secretes humoral factors that can either promote or inhibit the nerve-regeneration process.
One of the ways to reconstruct a nerve defect is to use a biodegradable nerve guide. The aim of this study was to establish a nerve guide constructed of an amorphous copolymer of lactic acid-caprolactone. A pilot study was set up to elucidate the effect of the tube dimensions on nerve regeneration. Four types of nerve guides, with internal diameters ranging from 1.12-1.23 mm and wall thicknesses ranging from 0.34-0.68, were tested for this purpose. We evaluated the biodegradation, foreign body reaction and nerve regeneration by light microscopy, after three different implantation times (1, 2, and 3 months). After 2 months, we observed that all types of nerve guides had changed from a transparent to an opaque and swollen state, and that they had lost their strength. The foreign body reaction was characterized by the presence of giant cells and fibroblasts surrounding the degrading nerve guide. From this pilot study, we conclude that nerve guide type 1, with an internal diameter of 1.23 mm and a wall thickness of 0.34 mm, can ensure nerve regeneration in the case of a 1-cm gap in the sciatic nerve of the rat. Nerve guides types 3 and 4, with relatively small lumens, show nerve compression due to a more pronounced swelling of the degrading tube.
Nerve regeneration was followed in 15 median and 1 ulnar nerve of eight Macaca fascicularis monkeys by serial electrophysiological assessments over a period of three and a half years. Nerve gaps of 5 mm at the wrist were bridged by collagen-based nerve guides, nerve autografts, or direct suture repairs. Thenar muscle reinnervation occurred between 50 and 70 d for all groups, indicating axonal elongation rates of approximately 1 mm/d. The recovery rates of the compound muscle action potential (CMAP) and the compound sensory action potential (CSAP) amplitudes were significantly slower after direct suture repair compared to the other two procedures, although the final levels of recovery were all comparable. Similar results were achieved in one median and one ulnar nerve following nerve guide repair of a 15 mm nerve gap. The functional reinnervation of Pacinian corpuscles was detected in all cases following either nerve graft or nerve guide repair, with similar amplitudes and latencies of the tactile evoked CSAP for both types of repair. Histological analysis demonstrated a significant increase in the number of myelinated axons in the median nerve distal to the nerve lesions following both nerve graft and nerve guide repairs compared to proximal and normal controls, with significant reductions of fiber diameter and corresponding increases in g-ratio. The return of a bimodal frequency distribution of myelinated axon fiber diameter was confirmed by three-dimensional surface plots which illustrate the frequency distribution of the relationship between fiber diameter and g-ratio. These combined results demonstrate that nerve regeneration after repair of a 5 mm nerve gap with a collagen nerve guide in the nonhuman primate is similar to that after graft repair, and the final level of physiological recovery for both repair procedures is comparable to direct suture repair of the median nerve.
Nerve regeneration using artificial biodegradable conduits is of increasing interest. The aim of this study is to evaluate the regeneration and maturation of a nerve after long-term implantation (2 years) of a biodegradable poly-L-lactide/poly-epsilon-caprolactone (PLLA/PCL) copolymeric nerve guide in the sciatic nerve of the rat. After harvesting, we evaluated both the regenerated nerves and the controls, using light microscopy, transmission electron microscopy, and morphometric techniques. Remnants of biomaterial were still present after 2 years of implantation, but the foreign body reaction was very mild at this stage, due to the rounded shapes of the polymer debris. Morphometric analysis showed significant differences between the regenerated nerve and the normal sciatic nerve: the number of myelinated fibers is higher, and the mean fiber diameter of the myelinated fibers in the regenerated nerve is smaller. In conclusion, the results demonstrate that the new PLLA/PCL nerve guide can provide optimal conditions for regeneration and maturation of damaged nerves.
We compared reinnervation of target organs after sciatic nerve section leaving gaps of 2, 4, 6, or 8 mm or gaps repaired with silicone tubes in different groups of mice. Functional reinnervation was assessed by noninvasive methods to determine recovery of sweating, nociceptive, and muscular functions in the hindpaw repeatedly during 3 months postoperation. The increase of gap length between nerve stumps delayed the beginning and reduced the degree of functional recovery achieved either with or without repair. When lesions were left unrepaired, functional reinnervation was only noticeable with a 2-mm gap and practically absent with longer gaps. With tube repair, reinnervation started earlier and achieved higher values than in the corresponding unrepaired groups. Tubulization was most effective with 4-mm gaps and comparatively less with shorter and longer gaps. With 4-mm gaps, recovery was higher when the silicone tube had a cross-sectional area 2.5 times that of the sciatic nerve than with narrower or wider tubes and when the wall was the thinnest available. In all cases muscle reinnervation showed a lower progression than sweating and nociceptive recovery.
Clinical and experimental investigations have shown that recovery of motor and sensory functions after a nerve lesion varies with the type of lesion and the repair technique. We report a study on regeneration of sudomotor axons to sweat glands (SGs) in the mouse paw following different sciatic nerve injury procedures: freezing, crushing, and sectioning. After sectioning, repair was by primary suture or by grafting with the resected section or with conduits of plastic or collagen. Successful regeneration was marked by the reappearance of SG reactivity to pilocarpine. After a freezing or crushing lesion, SGs activated by pilocarpine reappeared by 17 days postoperation and then increased progressively in number until the preoperative number of SGs were again active at 41 days. Cutting the nerve followed by primary repair resulted in a short delay and a significant deficiency in reinnervation. Results obtained with a graft of a segment of resected nerve were similar to primary repair after nerve section and better than when grafts of plastic or collagen tubes were used. This suggests that substances provided by the grafted nerve segment and the type of conduit provided play important roles in the regeneration of unmyelinated axons.
This study evaluates the degree of autotomy induced by anesthesia dolorosa after transection of the sciatic and saphenous nerves in four different strains of mice, and the effectiveness of amitriptyline administration in two of them. The self-mutilating lesions were assessed by means of an autotomy score for one month after denervation. The onset of lesions generally occurred during the first week, starting in the nails and progressing proximally. Autotomy behavior developed differently in the mouse strains studied, involving 88% of the paw areas in OF1 mice, 61% in Balb-C, 35% in NMRI, and 15% in B6CBAF1. Two selected strains, OF1 and NMRI, were treated with amitriptyline (8 mg/kg/day, p.o.) from different intervals pre-operation. Administration starting 14 days before nerve lesion was the most effective treatment schedule for reducing autotomy in both strains.
The present study determines the number and morphology of myelinated fibers that regenerate after resection of the mouse sciatic nerve. In different groups of mice, a resection of 4 or 6 mm of the sciatic nerve was left unrepaired, repaired with silicone or collagen guides or by an autologous nerve graft of the same or smaller calibre. Regeneration was examined, under light microscopy, 3 months after operation and quantified by morphometric analysis of light micrographs of cross-sectional nerve fibers. The results show that, without repair, few nerve fibers reach the distal nerve stump, while tubulization or autografts allowed better regeneration. Tube repair allowed a comparable degree of regeneration to that of an autograft with 4 mm gaps, but lower with 6 mm gaps. Regeneration was limited with a gap of 6 mm in silicone tubes, but was successful in half the mice with collagen tubes. The size and myelination of regenerated fibers were below normal values in all experimental groups, although they were closer to normal with sciatic autografts than after smaller grafts and tubulization. There were no signs of secondary degeneration in the nerve regenerates within silicone and collagen tubes. © 1996 Elsevier Science Ireland Ltd. All rights reserved.
A study was undertaken to compare the regeneration of rat peroneal nerves across a 0.5-cm gap repaired with either a permanent, porous or a resorbable, non-porous artificial nerve graft. The resorbable, impermeable artificial nerve graft was a synthetic passive conduit made from polyglycolic acid (PGA). The permanent, porous artificial nerve graft conduit was manufactured from a hydrophilic elastomeric biopolymer (HEB), and four variations were tested. Qualitative histology on short-term animals revealed similar inflammatory reactions to HEB and PGA. Axonal regeneration was evaluated in longer-term animals after three, four, and six months by qualitative and quantitative histology. Qualitative histology on longer-term animals demonstrated both artificial nerve grafts to be anti-immunogenic. All PGA-artificial nerve graft repairs among three-, four-, and six-month rats contained myelinated axons, as did all HEB-1 repairs. However, three other HEB-graft varieties accounted for a 25 percent failed regeneration rate. Quantitative histologic comparison of repair-site cross-sections in viable PGA and HEB matched pairs demonstrated statistically equivalent myelinated axon counts but larger average myelinated fiber diameters in HEB repairs, with p = .001.
The passage of molecules into the endoneurial environment of the axons of normal peripheral nerve is regulated by two permeability barriers, the perineurial-nerve barrier and the endoneurial blood-nerve barrier. These barriers exist because of the presence of tight junctions between adjacent perineurial cells and adjacent endothelial cells. In the present study we investigated whether permeability barriers form in nerve cables, which develop inside silicone chambers. The sciatic nerves of adult rats were cut, and the proximal and distal ends sutured into opposite ends of silicone chambers that were filled with dialyzed plasma. The presence of barriers was determined with the tracer horseradish peroxidase (HRP), which was injected intravenously and detected histochemically in tissues by light and electron microscopy. At four weeks, a regenerated nerve cable extended across the 10 mm length of each chamber. However, no permeability barriers were present since the reaction product for HRP was visible throughout the cable. At twenty-six weeks, all the axons in cables were gathered into minifascicles. Each minifascicle of axons was surrounded by perineurial cells. Blood vessels were excluded from the minifascicles by the perineurial cells and the vessels were permeable to HRP, thus indicating that their endothelial cells had not formed tight junctions. Despite the leakage of HRP from the excluded vessels, the tracer did not reach the axons because the perineurial cells encircling the minifascicles developed tight junctions. In some animals, the chambers were removed at four weeks to determine whether the chamber influenced barrier development. This manipulation had no effect since cables, with or without chambers, exhibited similar findings at twenty-six weeks. Our results indicate that nerve cables regenerate a perineurial but not an endoneurial permeability barrier. We conclude that axons in long-term cables are protected by only a perineurial permeability barrier.
Rat sciatic nerve regeneration through three synthetic neural prostheses was compared with regeneration through nerve allografts. The synthetic prostheses were either nonpermeable nonabsorbable (Silastic), permeable absorbable (polyglactin mesh), or permeable nonabsorbable (polypropylene mesh). Animals were evaluated at 10, 24, and 90 days. Functional analysis of nerve regeneration was performed by noninvasive methods: electromyography and walking tracks. Nerve tissue was examined with routine histologic and immunofluorescent techniques. A compressive neuropathy developed with the use of the Silastic implant. A neutrophilic inflammatory infiltrate was consistently associated with implantation of the polyglactin mesh. A strong connective tissue response was noted around the polypropylene mesh. Early recovery of nerve function was seen with the Silastic implants, however, overall nerve function was best in the nerve allograft and polypropylene mesh groups. Polyglactin implantation increases the local inflammatory response and should not be used for nerve anastomoses. If Silastic entubulation is used, it should be removed between 24 and 90 days.
A nerve guide made of a benzyl ester of hyaluronic acid (HYAFF11p75) was used to bridge 8 mm gaps in rat tibial nerves. Histologic observations indicated that this biomaterial provoked only a transient, modest inflammatory response, and the resorption rate was compatible with the nerve regeneration processes. Phagocytosis of the biomaterial began after neoangiogenesis and cell migration had taken place from both stumps into the nerve guide material. For comparison, the regeneration achieved was evaluated in nerve guides made of either HYAFF11p75 or Silastic, and in nerves repaired with the autograft technique. Recovery was assessed in vivo 90 days after implantation by measuring the nerve compound action potential (CAP) and conduction velocity (NCV) of the regenerated tibial nerve. The results demonstrate that the nerve guide tubes made of HYAFF11p75 were able to support and direct axonal growth, thereby suggesting a possible use for such biomaterial in the management of short nerve gaps in human pathology.
The present study was designed to evaluate the influence of synthetic guidance channel surface microgeometry on morphological patterns of neural regeneration. Tubes with smooth (S), rough (R), or alternating smooth-rough (S/R) or rough-smooth (R/S) inner surfaces but with identical chemical composition and permeability characteristics were used to bridge a 4-mm nerve gap in a transected mouse sciatic nerve. Animals received S and R channels for 1, 2 and 4 weeks and both S/R and R/S channels for 2 and 4 weeks. At 1 week, the S tubes contained a longitudinally oriented fibrin matrix not contacting the channel's smooth inner wall, whereas R tubes featured an unorganized fibrin matrix which, together with fibroblasts and macrophages, had invaded the channel's rough trabecular network. After 4 weeks, S tubes contained discrete, free-floating nerve cables with numerous myelinated and unmyelinated axons surrounded by a thin, continuous epineurial-like layer, whereas R tubes were completely filled with a loose connective tissue stroma with only a few axons. In combined S/R or R/S channels, the general morphological patterns in individual S or R segments were similar to those observed in pure S or R channels, regardless of whether the tube segment was positioned at the proximal or distal nerve end. Proximal smooth channel segments contained discrete cables which abruptly fanned out to completely fill the lumen in distal rough segments. The opposite pattern was observed with proximal rough and distal smooth segments. At 4 weeks, myelinated axons were observed along the entire length of S/R and R/S tubes. These results suggest that the surface microgeometry of guidance channels influences the outcome of peripheral nerve regeneration, potentially by affecting the early arrangement of the fibrin matrix and/or inducing different cellular responses.
In a primate model, nerve regeneration was evaluated across 2- and 5-cm nerve gaps using a synthetic nerve conduit (glycolide trimethylene carbonate, Maxon) and a biologic conduit (collagen). Two types of conduits from glycolide trimethylene carbonate (Maxon) were evaluated. The first was fashioned into a tube from a single flat piece of Maxon mesh. The second was prefabricated in a crimped form. The other material evaluated was a biologic collagen tube. At 14 months, excellent regeneration was noted across the 2-cm nerve gap through both the collagen and Maxon conduits. At 5 cm, the regeneration, as determined by morphometric analysis, was significantly better across the Maxon prefabricated, crimped conduit, although regeneration at this longer gap in all experimental groups was significantly less than across the shorter nerve gap. At the longer gap, there was no statistical difference between the groups by electrophysiologic assessment.
Sectioned rat sciatic nerves were placed in tubes to study the regrowth of injured nerve processes. First, we characterized quantitatively the regeneration of myelinated fibers at different levels of an acrylic semipermeable tube, from two to 27 weeks postoperatively. From the fifth week, myelinated fiber counts at mid-tube level were equal to the value of an intact nerve, but at the distal part the number of fibers exceeded both mid-tube level and unsectioned nerve values. At the proximal part of the tube an important fiber disorganization was observed. Second, we have compared acrylic semipermeable and totally impermeable silicone tubes at four and 27 weeks postoperatively. In terms of the number of myelinated fibers and the surface of the endoneurium at the mid-tube level, the most effective tube was the impermeable one. This study points out the importance of the tube wall permeability in nerve regeneration.
Reconstruction of a short nerve gap by a nerve graft produces donor-site scarring, loss of donor nerve function, and neuroma formation. This study compared the regeneration achieved after 1 year in 16 monkeys across a 3-cm upper arm ulnar nerve gap with a bioabsorbable polyglycolic acid nerve conduit with the regeneration achieved with a classical interfascicular interpositional sural nerve graft. The results demonstrated electrophysiologic and histologic evidence of neural regeneration across the gaps in all experimental groups. The bioabsorbable nerve conduit groups and the sural nerve graft group had mean fiber diameters, amplitudes, and conduction velocities each significantly less than those of normal control ulnar nerves. There was, however, no significant difference between any of the experimental groups. Electromyography demonstrated recovery of 19 of the 28 (68 percent) intrinsic muscles studied. These results demonstrate that the primate peripheral nerve can regenerate across short nerve gaps when guided by an appropriate nerve conduit, suggesting that a single-stage biodegradable polyglycolic acid conduit may be used as an alternative to a short interfascicular nerve graft.
Light and electron microscopy were used to investigate long-term regeneration in peripheral nerves regenerating across a 10 mm gap through silicone tubes. Schwann cells and axons co-migrated behind an advancing front of fibroblasts, bridging the 10 mm gap between 28 and 35 days following nerve transection. Myelination of regenerated fibres started between 14 and 21 days after transection and occurred in a manner similar to that reported during development. Although these early events were successful in producing morphologically normal-appearing regenerated fibres, complete maturation of many of these fibres was never achieved. Axonal distortion by neurofilaments, axonal degeneration and secondary demyelination were seen at 56 days following nerve transection. These changes progressed in severity with time as more axons advanced through the distal stump towards their peripheral target. Since regeneration occurs in the absence of endoneurial tubes, and because constrictive forces act on the nerve during regeneration, we suggest that these extrinsic factors limit the successful advancement of axons through the distal stump to their target organ.
The presence of a distal nerve segment is considered to be essential for peripheral nerve regeneration through impermeable synthetic guidance channels. The use of a perm-selective material may provide a more appropriate regenerating environment by allowing solute exchange across the wall of the channel. We compared perm-selective acrylic copolymer (AC) channels with impermeable silicone elastomer (SE) channels in terms of regeneration in the absence of a distal nerve stump. Cohorts of 6 animals received AC and SE channels for either 4 or 8 weeks, with the distal end of the polymer tube left open in half of the animals, and plugged with the same polymer ('capped') in the other half. Capped and uncapped AC channels contained regenerated nerve cables which extended fully to the distal end of the channel, whereas capped SE channels contained only 1 mm long granulomatous tissue cables, and uncapped SE channels showed small cables with only a few myelinated axons. The nerve cables regenerated in uncapped AC channels were smaller and contained fewer myelinated axons than those observed in capped AC channels. Capped AC channels sleeved with a tight-fitting silicone tube to render them impermeable, showed no regenerated tissue within their lumen. The use of a perm-selective channel may have allowed the influx of nutrients and growth factors from the external environment while concentrating factors released by the proximal nerve stump.
After placement of stumps of transected rat sciatic nerve in an impermeable tube, the maximum gap the axons can span is 10 mm. The present study shows that the regenerating axons cross much longer gaps if the tube is made permeable. This improvement does not require another nerve as a transplant nor the preplacement of extracellular materials in the tube. Possible mechanisms for this improvement are discussed.
An experimental reason for placing stumps of a transected nerve in an impermeable tube is that factors and soluble substances from the nerve stumps are pooled and separated from cells and soluble substances in the body in general. Previous work showed that certain parameters of regeneration were improved, however, when the impermeable tube was made completely permeable by cutting macroscopic holes in its side. To begin exploring the reasons for these improvements, we covered the holes in the permeable tubes with filters of two different pore sizes, and found that the improvements resulted when the pore size was large enough to allow both fluid and cells to exchange but not when the pore size allowed only fluid to exchange. These findings suggest that cells from the general connective tissue should be given consideration when designing experimental procedures to maximize the regeneration potential of regenerating axons.
These experiments present quantitative data concerning peripheral nerve regeneration in vivo. We used entubulation repair as a model to compare two different types of tubular prostheses, one nonbiodegradable and the other biodegradable. We modified the microenvironment of the regenerating axons within the tubular prostheses by adding a laminin-containing gel to the interior of the tube at the time of initial implantation. The data demonstrate that specific manipulations to the microenvironment of regenerating peripheral axons have quantitative effects on the rate and extent of nerve regeneration. Such effects were dependent on the composition of the tubular prosthesis and varied according to the survival time of the animals. For instance, the laminin gel within the biodegradable tubes enhanced nerve regeneration at 2 weeks but was inhibitory at 6 weeks. Furthermore, such manipulations may have different effects on the number of myelinated axons found within the regenerating nerve cable versus the number of primary motor and sensory neurons giving rise to such axons. We concluded that: the presence of a laminin-containing gel significantly increased the initial rate at which axons from primary sensory and motor neurons cross a transection site; an initial delay in axonal outgrowth at early time points did not necessarily predict diminished outgrowth at later times; and because of the potential for axonal branching the number of myelinated axons found in the midportion of a tubular prosthesis did not always correlate with the number of primary motor and sensory neurons which gave rise to those axons.
One approach to repair of transected nerves is to attempt extrinsic guidance of axons across the gaps. We inserted the proximal and distal stumps of severed mouse sciatic nerves into opposite ends of biodegradable polyester tubes. The nerves and ensheathing tubes were examined after postoperative survival times of as long as 2 years. Myelinated fiber number in each successfully regenerated nerve was measured and correlated with the tube's residual lumen size. In selected regenerated nerves axonal sizes and myelin sheath widths were sampled and compared with control values. Swelling and deformation of tube walls occurred in nearly all tubes. Successful regeneration was obtained through more than half of the implants, and was more probable in tubes with larger initial lumens. Myelinated fiber number in regenerated nerves ranged from 231 to 3561 (normally 3900 to 4200); larger values again were found in tubes with larger initial lumens. Mean axonal areas in regenerated nerves were roughly half of normal, though myelin sheaths became appropriately thick. We concluded that the more biodegradable a tube, the more likely it was to incur distortion and luminal narrowing. Tube composition per se seemed of importance mainly as it related to maintenance of adequate luminal size over the length of the degrading tubes; luminal adequacy, not tube composition, seemed paramount in determining the extent of nerve regeneration.
Recent studies focus on regeneration where nerve stumps are placed in a silicone tube. Since the tube is impermeable, the fluid and cells that collect from the stumps bath the axons. This is presumably beneficial. Making the tube permeable by making holes in its walls should change the patterns of regeneration. If this is done, the major cytologic change is an increase in the fascicular perineurium. There are more individual fascicles, more cells line each fascicle and the lining cells are coated by more prominent external laminae than after similar regeneration in a regular silicone tube or in the normal untransected nerve. For axonal numbers, there are more myelinated and unmyelinated axons in the gap and more unmyelinated axons in the distal stump than after regeneration in a regular silicone tube. The numbers in the holey tube regenerate are statistically different from normal but they are closer to normal than after similar regeneration in a regular silicone tube. There are significantly fewer myelinated and unmyelinated axons than in the normal sural nerve after regeneration through a holey tube, but there are more than after regeneration through a regular tube. The numbers of axons in the nerve to the medial gastrocnemius muscle are not significantly different from normal or from the other regeneration paradigms. These data allow the suggestion that regeneration through a silicone tube with macroscopic holes in its walls may be superior in certain respects to regeneration through a regular impermeable silicone tube.
The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)
The spatial-temporal progress of peripheral nerve regeneration across a 10-mm gap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7-14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1-5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.
The collateral reinnervation of mouse sweat glands has been studied by a method that allows serial evaluation of the course of reinnervation in intact animals. The method is based on the finding that the activation of secretion from newly denervated sweat glands by pilocarpine or nerve stimulation is completely absent seven days after nerve section but returns with reinnervation. These characteristics allowed serial detection of footpad sweat glands newly reinnervated by collateral sprouting of the remaining intact saphenous nerve after section of the sciatic nerve. The number of saphenous innervated glands increased five- to sevenfold and the total saphenous sweat territory was greatly expanded across the volar surface of the hind paw. There was less enlargement when the sciatic nerve was allowed to regenerate and participate in reinnervation of sweat glands. None of the reinnervated glands were dually innervated by saphenous and sciatic nerves, as is the case in normal glands. When the saphenous nerve was sectioned after saphenous collateral sprouting was complete and after sciatic regeneration had innervated an apparent maximal number of glands, the sciatic nerve reacted by advancing farther into the formerly enlarged saphenous territory and re-reinnervated many of the glands. Collateral sprouting by sudomotor axons is more abundant and more widely dispersed than reported for larger nerve fibers to skeletal muscle and to low-threshold mechanoreceptors. It more closely resembles sprouting of nociceptive axons.
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.
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.
Functional reinnervation of sweat glands (SGs), skin, and muscle in the mouse paw after sciatic nerve lesions was evaluated to allow comparisons of the regeneration efficiency of different functional types of nerve fibers. In four groups of mice the sciatic nerve was crushed, sectioned, and left unrepaired or repaired by suture or tubulization. Reappearance of SG secretion and pinprick responses occurred slightly earlier than recordings of compound muscle and nerve action potentials in all groups. The degree of reinnervation, with respect to preoperative control values, of SGs and skin nociceptors was higher than the amplitude of the action potentials, mainly when the nerve injury was severe. The chances for recovery progressively decreased with the severity of the lesion, affecting the larger nerve fibers most. These results indicate that, after injuries of peripheral nerves, all types of nerve fibers are able to regenerate in the mouse, although small size fibers (sudomotor and nociceptive) allow for a higher degree of functional recovery than large myelinated fibers (skeletomotor and sensory).
Transected median nerves in the forearm of two male patients, 12 and 21 years of age, were treated with a chamber technique leaving a 3 to 5 mm gap between the nerve ends. The nerve ends were enclosed in a silicone tube of such a dimension that would not cause compression of the nerve. Post-operative examination including sensory evaluation and assessment of muscle contraction force was carried out after 3 years. In both cases there was excellent motor recovery of the thenar muscles. Outgrowth of sensory fibres was remarkably fast, resulting ultimately in functional sensibility allowing almost normal hand function. 2PD was < or = 6 mm (12-year-old patient) and 8 to 10 mm (21-year-old patient) respectively. In one case the silicone tube was re-explored because of minor local discomfort 2 years after the repair. The former gap was bridged by a smooth continuous nerve-like structure of the same diameter as the adjacent nerve trunk and with no signs of neuroma formation or compression of the nerve.
A copolymer of L-lactide and 6-caprolactone (50:50, w/w) was synthesized and characterized. The thermal behaviour of this material did not show any crystallinity for several months; only after more than 1 yr of aging at room temperature and, particularly, in the in vitro degradation tests did it partially crystallize. The values of tensile strength, percent elongation at break and elastic modulus were, respectively, 25 MPa, 490% and 3 MPa. Transparent, elastic nerve guides having inner diameter of 1.3 mm and wall thickness of 175 microns were prepared.
The rat sciatic nerve does not possess a high potential for regeneration through silastic tubes when the interstump nerve gap is greater than 10 mm. In this study, the effect of NGF treatment on regeneration of the rat sciatic nerve in 10- and 15-mm silastic chambers was compared. In addition, regeneration in 15-mm silastic chambers was compared to regeneration in 15-mm semipermeable chambers. Sections of tubing were implanted and filled with NGF or a control solution of cytochrome C (Cyt. C). Tube implants were removed at various postoperative times and regeneration was assessed histologically and behaviorally. NGF treatment promoted regeneration success rate. It enhanced the initial outgrowth of nonneuronal cells and neuronal fibers into the chamber producing more cellular, organized regenerates. At 2 weeks, in 10-mm chambers, NGF-treated regenerates had fourfold more unmyelinated fibers than controls. At 3 weeks, NGF-treated regenerates possessed threefold more myelinated fibers than controls. After 4 weeks all regenerates had similar numbers of myelinated nerves at the chamber's midpoint. This initial "head start" was sustained peripherally as indicated by the earlier return of sensory function (response to a noxious temperature stimulus) in NGF-treated animals. Finally, regeneration success rate in 15-mm semipermeable tubes is greater than that in 15-mm silastic chambers (NGF and Cyt. C). However, regenerates in silastic chambers possessed twofold more myelinated fibers than regenerates in semipermeable chambers. The positive effects of NGF on neural regeneration and recovery of sensory function provide support for the potential use of NGF in treating peripheral nerve injuries.
Injury to a peripheral nerve is followed by local synthesis and release of neurotrophic factors of importance for the regeneration process. This concept was adopted for repair of transected human median and ulnar nerves in the forearm. As an alternative to conventional microsurgical repair of the nerve trunk, silicone tubes of appropriate size were used to enclose the injury zone, intentionally leaving a gap measuring 3-4 mm between the nerve ends inside the tube. The early results from a prospective, randomized, clinical study comparing this principle with conventional microsurgical technique for repair of human median and ulnar nerves, is presented. Eighteen patients (14 men and 4 women), aged 12-72 (mean, 29.5) years, were randomized to either tubulization (11 cases) or conventional microsurgical repair (7 cases). A battery of tests for sensory and motor functions of the hand were carried out at regular intervals for up to 1 year after surgery. The results show no difference between the both techniques, with the exception of perception of touch, which showed a significant difference (p < .05) at the 3-month checkup in favor of the tubulization technique. At re-exploration 11 months after the initial procedure (1 case), the former gap was replaced by regenerated nerve tissue in direct continuity with the proximal and distal parts of the nerve trunk, the exact level of the former injury being impossible to identify. Study data demonstrate an intrinsic capacity of human major nerve trunks to reconstruct themselves in a preformed space when an optimal environment is offered and the surgical trauma is minimized.
We compared reinnervation of target organs after sciatic nerve resection and repair by tubulization with biodurable tubes of silicone and teflon, or bioresorbable nerve guides of collagen and poly(L-lactide-co-6-caprolactone) (PLC) leaving a 6 mm gap in different groups of mice. All tubes were of 1 mm inside diameter and thin-walled (50 to 250 microm). Functional reinnervation was assessed by noninvasive methods to determine recovery of sweating, sensory and motor functions in the hindpaw repeatedly during 5 months postoperation. PLC guides allowed faster and higher levels of reinnervation for the four functions tested than collagen and silicone tubes, while teflon tubes gave the lowest levels of recovery. Regenerative reinnervation by thin nociceptive and sudomotor fibers was higher than by large sensory and alphamotor fibers in all groups. Resorbable tubes promoted regeneration in a higher proportion of mice than durable tubes. In cases with effective regeneration the nerve cable was multifascicular, with mild to moderate mononuclear cell infiltrates and a thin newly formed perineurium. The number of myelinated fibers was higher in PLC and silicone tubes than in collagen and teflon tubes. There was only minimal inflammatory reaction within the remnants of collagen tubes, but not in the other materials. PLC tubes of slow reabsorption rate seem useful for repairing long gaps in injured nerves.
Complication from silicon-polymer intubulation of nerves
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E!ectiveness of a bioab-sorbable conduit in the repair of peripheral nerves
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Aldini NN, Perego G, Cella GD, et al. E!ectiveness of a bioab-sorbable conduit in the repair of peripheral nerves. Biomaterials 1996;17:959}62.