Peripheral nerves form an extensive network that links the brain and spinal cord to all other parts of the body.1–3 A nerve injury can interfere with communication between the brain and the muscles controlled by a nerve, affecting ability of a person to move certain muscles or have normal sensations.4 Reconstruction of damaged nerves results from different factors that have been investigated by different methods. A number of methods, such as utilizing allograft techniques; cell therapy including Schwann cells (SCs), stem cells, fibroblasts, and olfactory cells; or drug therapy using biologic tubes, designed scaffolds with synthetic and natural materials and oriented channels, and absorbable and nonabsorbable synthetic and natural polymers with unique features benefiting from new nanotechnology were studied for improving the performance of strategies to repair damaged nerve tissue.5–7 Common grafting in surgery was autograft or nerve removal from elsewhere of the body. Unfortunately, autografts had limitations such body injury, repeated surgery and disproportion of grafted nerve tissue in terms of size and structure of nerve tissue8,9 also transplantation of allograft or xenograft had similar problems in addition to the stimulation of the immune system.10–14 Therefore, the studies were conducted on the application of artificial neural tubes to form neural cords.15–17 Clinical investigations were showed functional improvement and regeneration of peripheral nerve tissue with a gap of 3–5 mm by silicone tube.18 One of the scaffolds for nerve regeneration was poly L-lactic acid hollow tubes that was successful for rebuilding of neural fibers with gaps of 14 and 18 mm. Biodegradable sutures composed of polyamide fibers were showed similar results for nerve regeneration with gaps of 7 and 15 mm, but did not suitable for longer gaps.18 Among the synthetic polymers, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microbial polyester can be noted as biocompatible and biodegradable copolymer. PHBV has suitable properties for cellular growth and adhesion and benefits from controllable degradation.19,20 The ability of being prepared as nanofibers has improved the performance of biomaterials. Electrospinning is one of the most important methods for fabrication of nanofibrous scaffolds.21-27 Although the presented nanofibers may mimic the morphologies of extracellular matrix to some extent, some modifications are still required to create a friendly environment for the cells attachment, proliferation, and functions such as communications. Some natural materials such as collagen, chitosan, alginate, some carbohydrates and peptides have been reported as scaffold modifiers.28,29 Chitosan has been proved and regarded as biodegradable noncytotoxic material, which has some interesting biological activities.30–32In vitro studies have shown that chitosan is biocompatible to nerve cells and can be used as nerve conduit material to facilitate nerve cell attachment, differentiation, and growth.33,34 Controlling surface properties is very important for the high performance of adhesion. Biomaterials wettability is an important factor in the surface modification of materials. Surface modification of hydrophobic polymer surfaces can be achieved by wet (acid, alkali), dry (plasma), and radiation treatments (ultraviolet radiation and laser).35–38
In this study, nanofibrous PHBV conduit was fabricated by electrospinning method and cross-linked using chitosan by chemical method. The polymeric film or tube was evaluated by scanning electron microscope (SEM), physical and mechanical analyses, and cellular assays. These conduits were implanted into a 10 mm gap after resection of the sciatic nerve in a rat model, evaluated by macroscopic assessments and histology after 4 months of surgery.
Materials and Methods
Fabrication of Nanofibrous Scaffold
PHBV (molecular weight of 680 KDa) was purchased from Sigma-Aldrich (St. Louis, MO). 2, 2, 2-trifluoroethanol (TFE) was also purchased from Sigma-Aldrich and was used as solvent to prepare PHBV solutions. Both polymer and solvent were used without further purification. Electrospinning apparatus used in this study was prepared from Fanavaran Nano-Meghyas Company (Tehran, Iran). The PHBV was dissolved in TFE at a concentration of 2%w/v and loaded into a glass syringe controlled by syringe pump. A positive high voltage source through a wire was applied at the tip of a syringe needle, and a strong electric field (20 kV) was applied between the PHBV solution and the collector. As soon as the electric field reached a critical value with increasing voltage, mutual charge repulsion overcame the surface tension of the polymer solution and an electrically charged jet was ejected from the tip of a conical shape as the Taylor cone. Ultrafine fibers are formed by narrowing the ejected jet fluid as it undergoes increasing surface charge density due to the evaporation of the solvent. The nanofibers fabricated with a predetermined variables of electrospinning (syringe size: 17 mm, collector speed: 1,000 rpm, injected speed: 2 ml/min, syringe tip distance to collector: 75 mm, voltage: 20 kV, temperature: 30°C, time: 7 h). The electrospinning set has been shown in Figure 1. The electrospun PHBV nanofibrous mat was carefully detached from the collector and dried in vacuum for 2 days at room temperature to remove the solvent molecules completely.
Cross-Linking of Nanofibrous Mat
Chitosan (deacetylation: 75–85%; medium molecular weight: Sigma-Aldrich) was immobilized onto the nanofiber surfaces based on the following protocol. Chitosan was rinsed in acetic acid buffer solution (50 mM, pH = 5.0). Then, the nanofibrous mat was submerged into the 6 M NaOH solution for 15 min. The hydrolyzed nanofibrous mats were rinsed into 2-(N-morpholino)ethanesulfonic acid buffer (pH = 6.0) containing 10 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 10 mM sulfo-N-hydroxysuccinimide to activate the carboxyl groups on the surfaces. The nanofibrous mats were rinsed into chitosan solution (15 mg/ml in acetic acid buffer solution, 50 mM, pH = 5.0) and were shaken gently for 24 h at 4°C. The obtained samples were placed inside a vacuum oven to fully lose the humidity. The chitosan–cross-linked electrospun mat (Figure 1C) was rolled around the cylindrical rod to form a 3D tubular structure and was maintained in this form using a thermal agent (Figure 1D).
The surface characteristics of cross-linked and un–cross-linked nanofibers were studied by a SEM (Cambridge Stereo-scan, S-360, Wetzlar, Germany) to analyze the changes in the surface morphology. The films were first gold sputtered for 2 hours (Joel fine coat) to provide surface conduction before scanning. The sample surfaces static contact angles were investigated by a contact angle measuring apparatus (Krüss G10, Matthews, NC) according to the sessile drop method. For mechanical investigations, the nanofibers were subjected to stress–strain analysis using a universal testing machine under an extension rate of 5 mm/min and 100 N load cell. The specific surface area of nanofibrous mats was determined by surface area and pore size analyzer.
Schwann cells were obtained from sciatic nerves of 8-day-old rats according to earlier described methods.39 In brief, the nerve segments were washed in Dulbecco’s Modified Eagle’s Medium (MDEM)/F-12 twice, then resuspended in 0.3% collagenase type II solution (100 μl per segment) and incubated for 30 min at 37°C. After incubation, the enzymatic solution was carefully removed, and an equal volume of 0.25% trypsin–ethylene diamine tetra-acetic acid (EDTA) was added. The nerve segments were incubated for another 5 min at 37°C and then mechanically dissociated until they formed a homogeneous suspension. Schwann cell basal medium was then added to the suspension at a ratio of 4:1 in order to terminate the activity of the trypsin. The mixture was centrifuged at 800–1,000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in SC basal medium. The SCs were proliferated in the flask and for subculturing were washed using the phosphate-buffered saline (PBS). Then the trypsin enzyme/EDTA was added to the flask (37°C), and the flask was incubated for 90 seconds. The culture media (fetal bovine serum/DMEM) was added to the flask, and the cells were gently pipetted. Then the cell suspension was transferred to a tube (15 ml) (BD Falcon) and centrifuged (400 g) (Germany 5702) for 5 minutes. The solution was removed, and the precipitation was transferred to a new flask (75 cm) for reculturing. Pieces of cell culture (1 cm ×1 cm) from the Petri dish (Control) and the main sample were placed individually in one of the Petri dish wells by using a sterilized pincer. 200,000 cells/well were seeded into a 24 well culture plate and then removed by a pipette and poured onto the control and the main samples. Afterwards, all samples were placed in binder incubator at 37°C for 48 hours and studied using an Invert microscope (Wolf Laboratories, UK). Cell proliferation and viability in vitro were analyzed with the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. Briefly, 5,000 SCs were seeded on nanofibrous PHBV scaffolds. For analysis, 20 μl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (sigma) substrate (2.5 mg/ml stock solution in PBS) was added to each well, and the plates were returned to standard tissue incubator conditions for an additional 4 hours. Medium was then removed, the cells were solubilized in 100 μl of dimethyl sulfoxide, and colorimetric analysis was performed. For scanning electron microscopy study, the scaffolds and cultured SCs were washed by PBS, and then fixed by glutheraldehyde (2.5 %) at 4°C for 2 hours. The samples were dehydrated by alcohols and then kept with tetraoxide osmium vapors at 4°C for 2 hours. The samples were kept in disicator for 48 hours, then coated with gold, and investigated by a SEM (Cambridge Stereo-scan, S-360, Wetzlar, Germany).
Animals and Surgical Procedure
From 15 male wistar white rats aged approximately 4–8 weeks at the beginning of the experiment and weighing 180–220 g, five defects each were grafted with the chitosan–cross-linked nanofibrous conduit, five defects each were grafted with the autograft group as the positive control, five defects with the nongrafted group as the negative control. The protocol for the experiment was approved by the Institutional animal care and use committee of Shahid Beheshti University of Medical Sciences (Iran). Animals were handled according to the guidelines established for animal care at the center. A long segment of sciatic nerve was then resected, leaving a gap of about 10 mm caused by the retraction of nerve ends. For the autograft group, sciatic nerve was resected, and two ends of the nerve were displaced 180 degrees then were sutured. Both the proximal and distal cut ends of the sciatic nerve were telescoped into the tubes and fixed with a single 10-0 nylon suture. After surgery, the animals were placed in separate cages. All animals had free access to standard rat food and water.
Four months after operation, the rats were scarified, then regenerated nerve in the control, the autograft and the grafted with polymeric tubes were removed and prepared for macroscopic analyses.
Histological and Morphological Studies
For histological study, the rats were killed, then the sciatic nerve cords were removed and fixed in 10% formalin at 4°C for 5 days, dehydrated and then paraffin-embedded. Serial 2 μm paraffin sections were cut with a rotating microtome (Microtom) and stained with toluidine blue according to the routine histology protocol and were examined by an optical microscope (Nikon, Japan). Myelin thickness was calculated based on Image J software. Expressing of glial and SCs in regenerated nerves were investigated by immunohistochemical analyses (glial fibrillary acidic protein [GFAP] and S-100). Experimental results were expressed as means ± standard deviation. All data were analyzed by one-way analysis of variance (ANOVA) with Duncan’s multiple range tests (ANOVA; Duncan multiple range test, p < 0.05 and 0.01).
The nanofibrous scaffolds were prepared using the electrospinning method. Figure 2 shows the SEM images of the electrospun nanofibers and the chitosan–cross-linked nanofibers at different magnifications. The smooth and homologous nanofibers have clearly been shown in this figure. The average sizes for the un–cross-linked and the chitosan–cross-linked nanofibers were about 100 and 500 nm, respectively. Figure 2 shows SEM images of the un–cross-linked nanofibrous PHBV mat (A: ×5,000; B: ×20,000) and the chitosan–cross-linked nanofibrous PHBV mat (C: ×5,000; D: ×20,000). Figure 3 illustrated the designed tubular structure and diameter of tube wall.
Mechanical and physical properties of the nanofibrous PHBV scaffolds have been presented in Table 1. The contact angles of 105° and 57° were measured for the electrospun and the chitosan–cross-linked nanofibers, respectively. The 48° difference in the contact angle shows a better hydrophilicity of the cross-linked nanofibers compared to the un–cross-linked samples. The porosity of nanofibrous PHBV and the chitosan–cross-linked scaffolds were calculated 91.62% and 79.58%, and their pore size also measured as 0.45 ± 0.25 μm and 0.28 ± 0.33 μm, respectively. The specific surface area for the electrospun nanofibrous scaffolds was about 138 m2/g and for the cross-linked nanofibrous ones was about 105 m2/g. It is discernible that the electrospun nanofibrous mats have higher porosity and higher level of specific surface area, as well.
Table 2 gives the results of the MTT assay for the control (tissue culture poly styrene [TCPS]), the unmodified nanofibrous PHBV mat and the chitosan–cross-linked nanofibrous mat. These results indicated that there is a high degree of cell viability for the nanofibrous samples. On the other hands, more numbers of SCs were adhered to both the nanofibrous surfaces and could proliferate. Figure 4 shows the attached cells on the unmodified and modified nanofibrous samples and the control ones. Cellular images show a good cell growth in the vicinity of nanofibrous mat cross-linked with chitosan. Figure 5 shows SEM images of cultured SCs on the nanofibrous samples. Figures show good adhesion of the SC on both the nanofibrous surfaces especially for the chitosan–cross-linked nanofibrous mat.
Figures 6 shows the regenerated sciatic nerves after 4 months of surgery. Figure 6A shows that there has occurred no restructuring in the control group, but the Figure 6, B and C, shows the regeneration of the sciatic nerve and the formation of the neural cord. The sciatic nerve in the autograft sample (Figure 6B) shows a uniform and homogenous shape, and its size is proportional to both the distal and proximal ends, but for the chitosan–cross-linked nanofibrous conduit, the neural cord was disproportionate, not uniform, and had less thickness in both the distal and proximal ends.
Histological and Morphological Results
In this research, there was no inflammation in the autograft and the grafted samples with nanofibrous polymeric tube. Microscopic images along the middle section of stained sciatic nerve with toluidine blue are shown in Figure 7. The autograft samples show suitable regenerated nerve fibers with myelin sheet after 4 months of surgery. Also, the grafted samples with nanofibrous conduits show the formation of nerve fibers with myelination. Figure 8 shows the thickness of myelinated nerve fibers after 4 months. Myelin thickness of 0.85 ± 0.04 and 0.72 ± 0.05 micrometers were obtained for the autograft and the cross-linked nanofibrous conduit, respectively.
Staining of tissue with cellular markers (S-100 and GFAP) shows those conduits can increase the expression of markers or in another sense, reproductive increasing of Schwann and glial cells in response to sciatic nerve injury. Figure 9, A and B, shows good expression of S-100 marker for the autograft and the grafted nerves with tubes. Also Figure 9, C and D, shows the expression of glial marker in the sciatic nerve for the all grafted groups, and obtained results were similar to the S-100 analysis. The chitosan–cross-linked nanofibrous conduit expressed good GFAP marker. Expression of this protein shows accelerate process in nerve regeneration.
Discussion and Conclusion
The attempt to replace the lack of certain damaged or entirely destroyed anatomic structures is a challenge and a problem of continuous present interest in many medical fields. In this respect, the research concerning nerve regeneration can not overlook the vast field of biocompatible materials, as some of them may be suitable to be used as nerve conduits. The aim is to produce a structure that promotes nerve regeneration to a level at least comparable to that found in nerve grafts and to eliminate the need of sacrificing healthy nerves in order to repair a damaged one. For peripheral nerve repair, much effort has been devoted to developing artificial nerve grafts to replace traditional autograft techniques, which exhibit some drawbacks. Although artificial nerve grafts constructed from nonresorbable materials (e.g., conduits made from silicone or polyethylene) have yielded some degree of functional recovery, long-term complications often mean that a second surgical procedure is necessary to remove the conduits. These may actually become detrimental by virtue of toxicity or tendency to constrict the nerve. A nerve graft made of bioresorbable (gelatin,35 hyaluron,36 lactosorb,37 fibronectin,38 and poly [DL-lactide-co-caprolactone])39 is thus a promising alternative for promoting successful nerve regeneration. From that perspective, the PHBV tube is a candidate that might replace the nerve graft, at least for the repair of long nerve defects. The tube is very easy to be handled; it can be easily placed and sutured on the nerve, thus eliminating the need of a very accurate nerve suture technique.40,41 The electrospinning technique is widely recognized as a straightforward way to fabricate nanoscale fibrous structures. Because this technique can produce nano- or submicron fibrous scaffolds, which mimic the structure of natural extracellular matrix, it has elicited extensive research interest.42 Another material is chitosan, which has been evaluated extensively to bridge sciatic nerve gaps after injury.43–46 A recent study in dogs evaluated the immunological response to a chitosan prosthesis, which was used for sciatic nerve repair after resection of a 26 mm nerve segment. They found that the material did not alter the cellular or humoral immune response of the organism during sciatic nerve repair. They also concluded that nerve repair, evaluated both morphologically and functionally, were similar to the nonoperated intact control group.47 More recently, groups have demonstrated that bridging resected sciatic nerves with chitosan nerve guides can increase functional recovery.48 These studies indicate that chitosan does have the potential to provide an ideal environment to promote nerve repair. In this study, the nanofibrous conduit cross-linked with chitosan showed suitable physical, mechanical, and structural and cellular properties as the nerve graft. In this study, a 3D, biodegradable, porous, polymeric nerve guidance conduit was described for use in the restoration of the function of injured nerve tissues. The nanofibrous PHBV nerve conduit fabricated by electrospinning and cross-linked with chitosan. The cross-linked nanofibrous conduit showed suitable physical, mechanical, and structural properties as nerve graft, also SCs well adhered on the nanofibrous surfaces especially on the chitosan cross-linked nanofibrous surface. After 4 months of surgery, it was observed that the sciatic nerve truck had been reconstructed with restoration of nerve continuity, formation of nerve fibers with to myelination. Furthermore, it is possible that this study will allow improvements to meet clinical trial requirements in the future.
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