Structure and Properties of Composite Fibers Based on Chitosan and Single-Walled Carbon Nanotubes for Peripheral Nerve Regeneration

This study focused on a potential application of electrically conductive, biocompatible, bioresorbable fibers for tubular conduits aimed at the regeneration of peripheral nerves. The conducting, mechanical, and biological properties of composite fibers based on chitosan and single-walled carbon nanotubes were investigated in this paper. It was shown that introducing 0.5 wt.% of SWCNT into the composite fibers facilitated the formation of a denser fiber structure, resulting in improved strength (σ = 260 MPa) and elastic (E = 14 GPa) characteristics. Additionally, the composite fibers were found to be biocompatible and did not cause significant inflammation or deformation during in vivo studies. A thin layer of connective tissue formed around the fiber.


Introduction
One of the main tasks of tissue engineering is the development of scaffolds based on biocompatible and bioresorbable polymers that can be implanted into a living organism; after implantation, these scaffolds (surrounded by biological media) stimulate cell proliferative activity, which, in turn, promotes the regeneration of damaged tissue. As a result, a new tissue identical to that of the recipient is formed [1][2][3][4][5]. The ability of stem cells to proliferate, grow, and differentiate is determined by electrical signals that take part in cell communications and behavior and, correspondingly, in tissue regeneration processes [6][7][8]. This role of electrical signals is especially significant in the regeneration of peripheral nerves [6,[8][9][10], bone, cartilage, and muscular tissues [1][2][3]11]. Therefore, the scaffolds should possess a certain level of electrical conductivity (close to the electrical characteristics of living tissues and cells [4,12]) to enhance their biocompatibility, modulate cell/tissue responses, and facilitate cell stimulation [6,7,13]. The main factor that should be considered in the development of conducting scaffolds (which are intended to contact tissue responses consists of the preparation of composite materials containing an electrically conductive polymer or filler, specifically, carbon nanoparticles. It has been demonstrated [47] that the introduction of single-walled carbon nanotubes (SWCNTs) into chitosan scaffolds resulted in the ordering of the polymer structures and increases in the elastic moduli and conductivities of the composite films. The chitosanbased films containing up to 2.5 wt.% of single-walled nanotubes were compatible with a culture of human dermal fibroblasts. The data on the ionic and electronic components of the conductivities of the composite films were reported [47].
Although numerous publications devoted to the preparation and study of the structures and properties of biocompatible conducting polymers and composite materials have appeared in recent years, the knowledge of the preparation techniques and electrophysical properties of these materials (and especially fibers) is not sufficient. On the other hand, it is known that the strength and elastic properties of polymers are fully realized in the oriented state, particularly in fibers.
The aims of the present work included the preparation of electrically conductive composite fibers based on bioresorbable polymer chitosan and single-walled carbon nanotubes, studies of their structure, electrical, strength, and deformation characteristics, and an investigation of their biocompatibility. It is expected that the obtained results will help solve an important practical problem: the preparation of electrically conductive, biocompatible, bioresorbable fibers for tubular conduits, which will be used in the regeneration of peripheral nerves.

Materials and Methods
Nanofiber-based tubes can be obtained via electrospinning and microbraiding [48][49][50][51]. The porous structures of tubular implants intended for the regeneration of peripheral nerves should prevent the diffusion of connective and muscular tissue into the diastasis area and promote the vascularization of implant walls. Their lumens should contain anisodiametric pores with diameters comparable to those of the neurites of the damaged nerve.
Poly(L-lactide) (PLA) (Purasorb PL10 (Corbion Purac, Amsterdam, The Netherlands)) was used in the preparation of tubular scaffolds. Using an injection pump, a PLA solution in trichloromethane (15%) was fed through a die into an electric field with a strength of E = 1.5 × 10 4 − 4.0 × 10 5 V/m. Microfibers precipitated on a cylindrical electrode 1.5 mm in diameter; its rotation speed was equal to 1500 rpm, and the distance between electrodes was 0.15 m. The scaffolds fixed on the cylindrical electrode were then heated at 90 • C for 1 h to achieve partial crystallization of the polymer and improve its mechanical properties. The scaffold wall thickness after thermal treatment was equal to 350 µm.
The aqueous suspension containing 0.05-2.5 wt.% of SWCNTs was subjected to ultrasound treatment for 15 min with an IL10-0.63 dispergator (25 kHz, 630 W) [47,52]. Chitosan powder was added to the aqueous suspension in the amount that provided a final concentration in the solution equal to 4 wt.%. The mixture was stirred for 1 h, and concentrated acetic acid was then added to the solution until its concentration in the chitosan/SWCNT mixture was equal to 2%. The composite solution was stirred for 6 h at room temperature. Solutions containing 0.05, 0.5, and 2.5 wt.% of SWCNT to chitosan mass were prepared. The solutions were filtered and deaerated in a vacuum chamber for 24 h at a pressure of 10 kPa. This method of processing makes it possible to obtain a composite with a uniform distribution of particles in the bulk of the polymer [47].
An ethanol/alkali mixture containing C 2 H 5 OH and a 10% aqueous solution of NaOH at a ratio of 1:1 was used as a precipitant in the preparation of the fibers. The solution was Polymers 2023, 15, 2860 4 of 18 extruded using an injection pump through a die 0.6 mm in diameter into the precipitation bath; the feeding rate was 0.2 mL/min, the precipitation time was 150 s, and the values of orientational drawing (λ) varied from 0 to 100%. The prepared fibers were washed with distilled water and dried in air at 50 • C. The scheme of the setup for fiber preparation is presented in Figure 1. mass were prepared. The solutions were filtered and deaerated in a vacuum chamber for 24 h at a pressure of 10 kPa. This method of processing makes it possible to obtain a composite with a uniform distribution of particles in the bulk of the polymer [47].
An ethanol/alkali mixture containing C2H5OH and a 10% aqueous solution of NaOH at a ratio of 1:1 was used as a precipitant in the preparation of the fibers. The solution was extruded using an injection pump through a die 0.6 mm in diameter into the precipitation bath; the feeding rate was 0.2 mL/min, the precipitation time was 150 s, and the values of orientational drawing (λ) varied from 0 to 100%. The prepared fibers were washed with distilled water and dried in air at 50 °C. The scheme of the setup for fiber preparation is presented in Figure 1. The introduction of guiding fibers into the tubular PLA scaffold was performed immediately before surgery. The guiding fibers were oriented along the direction of neurite growth; the filling of the tubular channel lumen did not exceed 25 vol.%.
The structures of the composite fibers were studied using a JSM-7001F scanning electron microscope (Jeol, Tokyo, Japan). Before measurements were taken, the samples were coated with a gold layer 20 nm thick.
The mechanical properties of the composite fibers were studied with the aid of an Instron 5943 universal testing machine; the loading rate was 10 mm/min, and the base length was 100 mm. Before testing, the fibers were kept in a desiccator for 24 h at a relative air humidity of 66%.
The specific volume resistivities of the SWCNT-containing chitosan fibers were measured in a constant electric field at voltage U = 100 V with a Keithley-6487 Picoammeter/voltage source under normal pressure, at room temperature, and with an air humidity of 40%. To improve the contact between the samples and electrodes, a silver paint conductive silver paste (SPI, West Chester, PA, USA) was used. The distance between electrodes was 2 mm. The scheme of the setup is presented in Figure 2. The introduction of guiding fibers into the tubular PLA scaffold was performed immediately before surgery. The guiding fibers were oriented along the direction of neurite growth; the filling of the tubular channel lumen did not exceed 25 vol.%.
The structures of the composite fibers were studied using a JSM-7001F scanning electron microscope (Jeol, Tokyo, Japan). Before measurements were taken, the samples were coated with a gold layer 20 nm thick.
The mechanical properties of the composite fibers were studied with the aid of an Instron 5943 universal testing machine; the loading rate was 10 mm/min, and the base length was 100 mm. Before testing, the fibers were kept in a desiccator for 24 h at a relative air humidity of 66%.
The specific volume resistivities of the SWCNT-containing chitosan fibers were measured in a constant electric field at voltage U = 100 V with a Keithley-6487 Picoammeter/voltage source under normal pressure, at room temperature, and with an air humidity of 40%. To improve the contact between the samples and electrodes, a silver paint conductive silver paste (SPI, West Chester, PA, USA) was used. The distance between electrodes was 2 mm. The scheme of the setup is presented in Figure 2.
The resistivity measurements of three types of fibers were taken: the initial fibers, the samples kept in a desiccator for 24 h at a relative air humidity of 98%, and the samples exposed to the lysozyme-containing phosphate-buffered saline.
Chitosan fibers are bioresorbable; therefore, the in vitro studies of the resorption of the SWCNT-containing composite fibers were performed to reveal changes in their structures and electrical conductivities.
The sterilization of the chitosan samples was carried out under the influence of ozone for 90 min. The resistivity measurements of three types of fibers were taken: the initial fibers, the samples kept in a desiccator for 24 h at a relative air humidity of 98%, and the samples exposed to the lysozyme-containing phosphate-buffered saline.
Chitosan fibers are bioresorbable; therefore, the in vitro studies of the resorption of the SWCNT-containing composite fibers were performed to reveal changes in their structures and electrical conductivities.
The sterilization of the chitosan samples was carried out under the influence of ozone for 90 min.
The in vitro degradation of the fibers was studied in phosphate-buffered saline (PBS, pH 7.4) containing 1.5 mg/mL of lysozyme (human lysozyme, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. The lysozyme concentration was chosen according to [53]. The lysozyme solution was refreshed every third day to ensure continuous enzyme activity [54]. After 7, 14, 21, and 28 days, the samples were removed from the media, rinsed with distilled water, dried at 37 °C until a constant weight was reached [55], and weighed. The extent of in vitro degradation was expressed as the percentage of mass lost from the dried fiber after the PBS and lysozyme treatments.
The conductivities of the samples were measured after 14, 21, and 28 days of exposure to the lysozyme-containing phosphate-buffered saline.
The cytotoxicity studies involved human mesenchymal stromal cell lines (FetMSCs). The cell lines were obtained from the Vertebrate Cell Culture Collection (Institute of Cytology RAS, St Petersburg, Russia). The cells were cultivated in a СО2 incubator at 37 °С in a humidified atmosphere containing air and 5% CO2 in a DMEM nutrient medium (Dulbecco's modified Eagle's medium; Gibco) containing 10% (v/v) of thermally inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA), 1% of L-glutamine, penicillin (50 units/mL), and 50 µg/mL of streptomycin.
The fiber samples were 2 cm long and were immersed in 2 mL of complete nutrient medium and incubated for 4 days. For the evaluation of cytotoxicity, cells (5.0 × 10 3 cells/100 µL/well) were seeded into 96-well plates and cultivated for 24 h for attachment. Then, 100 µL of the medium taken after the incubation of the samples was added. The cells cultivated in the standard nutrient medium were used as control samples.
The cells were incubated for 72 h. After the completion of the incubation period, the medium was removed, and the DMEM/F12 medium (50 µL/well) with MTT (0.1 mg/mL) was introduced. The cells were incubated in a CO2 incubator for 2 h at 37 °С. After the removal of the supernatant, the formazan crystals formed by metabolically active viable The in vitro degradation of the fibers was studied in phosphate-buffered saline (PBS, pH 7.4) containing 1.5 mg/mL of lysozyme (human lysozyme, Sigma-Aldrich, St. Louis, MO, USA) at 37 • C. The lysozyme concentration was chosen according to [53]. The lysozyme solution was refreshed every third day to ensure continuous enzyme activity [54]. After 7, 14, 21, and 28 days, the samples were removed from the media, rinsed with distilled water, dried at 37 • C until a constant weight was reached [55], and weighed. The extent of in vitro degradation was expressed as the percentage of mass lost from the dried fiber after the PBS and lysozyme treatments.
The conductivities of the samples were measured after 14, 21, and 28 days of exposure to the lysozyme-containing phosphate-buffered saline.
The cytotoxicity studies involved human mesenchymal stromal cell lines (FetMSCs). The cell lines were obtained from the Vertebrate Cell Culture Collection (Institute of Cytology RAS, St Petersburg, Russia). The cells were cultivated in a CO 2 incubator at 37 • C in a humidified atmosphere containing air and 5% CO 2 in a DMEM nutrient medium (Dulbecco's modified Eagle's medium; Gibco) containing 10% (v/v) of thermally inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA), 1% of L-glutamine, penicillin (50 units/mL), and 50 µg/mL of streptomycin.
The fiber samples were 2 cm long and were immersed in 2 mL of complete nutrient medium and incubated for 4 days. For the evaluation of cytotoxicity, cells (5.0 × 10 3 cells/ 100 µL/well) were seeded into 96-well plates and cultivated for 24 h for attachment. Then, 100 µL of the medium taken after the incubation of the samples was added. The cells cultivated in the standard nutrient medium were used as control samples.
The cells were incubated for 72 h. After the completion of the incubation period, the medium was removed, and the DMEM/F12 medium (50 µL/well) with MTT (0.1 mg/mL) was introduced. The cells were incubated in a CO 2 incubator for 2 h at 37 • C. After the removal of the supernatant, the formazan crystals formed by metabolically active viable cells were dissolved in dimethylsulfoxide (50 µL/well), and the optical density of the resulting solution was measured at a wavelength of 570 nm with a plate spectrophotometer. The calculations involved a polynomial regression analysis in Microsoft Excel.
In vivo experiments were carried out on male Wistar albino rats, following the rules for working with experimental animals (the principles of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, Strasbourg, 1986, and the Declaration of Helsinki of the World Medical Association on Humane animal welfare 1996). The experimental animals weighed between 180 and 200 g, they were 3 months of age, and there were 10 rats in each group. The animals were operated on under general anesthesia (solution containing Zoletil 100, 0.1 mL, and Rometarum, 20 mg/mL: 0.0125 mL per 0.1 kg of animal weight, administered intraperitoneally).
The rat sciatic nerve (the largest nerve in the animal) was selected for nerve defect replacement with polylactide conduits filled with SWCNT-containing chitosan monofibers. The introduction of guiding fibers into the tubular scaffold was performed immediately before surgery. The diastasis (the distance between the ends of a damaged nerve) was equal to 0.5 cm.
The animals were sacrificed 16 weeks after surgery with CO 2 . For histological studies, fragments of the sciatic nerve with polylactide conduits and chitosan monofibers with different contents of SWCNT were fixed in 10% neutral formalin in phosphate-buffered saline (pH 7.4) for 24 h, dehydrated using a series of ethanol/water solutions with increasing ethanol concentrations, and enclosed in paraffin blocks according to the standard histological technique. To obtain comparable results, the samples were treated simultaneously and under similar conditions. Then, longitudinal cuts of proximal fragments of the conduits and transverse cuts of distal fragments of the nerves (conduit) were obtained. The cuts were 5 µm thick and were prepared with the use of an Accu-Cut SRT 200 microtome (Sakura, Tokyo, Japan), followed by staining with hematoxylin and eosin, using the Picro-Mallory staining technique (BioVitrum, St. Petersburg, Russia). A microscopic analysis of the samples was performed using a Nikon Eclipse E200 light microscope (Nikon, Tokyo, Japan) with 10× ocular and lenses with magnifications of 4, 10, 20, and 40×. Digital images were recorded using a Nikon DS-Fi3 camera (Nikon, Japan).

Scanning Electron Microscopy
It has been noted [43] that chitosan fibers have smooth surfaces and homogeneous internal structures. The microphotographs of composite fibers presented in Figure 3 indicate that their structures contain lamellar elements, which agrees with the results reported in [45,47]. The addition of extremely small amounts of SWCNTs (0.05 wt.%) and the formation of composites causes the appearance of more pronounced surface reliefs. Introducing 0.5 wt.% of SWCNTs (Figure 3b) facilitates the formation of fibers with smooth surfaces. It should be noted that the addition of precisely this amount of SWCNTs (0.5 wt.%) improves the mechanical characteristics of the composite fibers as compared to those of other samples containing 0.05 and 2.5 wt.% of nanotubes ( Figure 4). The microphotograph of the fiber containing 2.5 wt.% of SWCNTs clearly shows the presence of oriented structural elements (appearing due to the increased content of isometrical SWCNT particles). The fibers exposed to the lysozyme solution for 4 weeks retained their sizes and shapes. However, the surface reliefs of the fibers became more pronounced, and fibrillation of the surfaces occurred. The fibers modified with 0.05 wt.% of SWCNTs contain well-oriented structural elements on their surfaces. It should be noted that an increase in SWCNT content led to surface relief sharpening upon exposure to the biologically active medium; the formation of longitudinal pores and cracks is observed (Figure 3e-g). These changes in the surface structures of the composite fibers after exposure to the lysozyme solution are indicative of chitosan resorption that began from the fiber surfaces.

IR Spectroscopy
The prepared pure chitosan and chitosan-based composite fibers were characterized via FTIR spectroscopy. The spectra obtained contain several characteristic bands of different intensities. The wide band in the 3500-3200 cm −1 region is assigned to O-H stretching vibrations (hydroxyl groups involved in the formation of hydrogen bonds); the bands at 3360 and 3290 cm −1 are related to N-H stretching vibrations of amino groups; the bands at 2920 and 2865 cm −1 are assigned to C-H stretching vibrations; the band at 1650 cm −1 is assigned to stretching vibrations of C=O in the N-acyl fragment (Amide I); the peak at 1585 cm  The fibers exposed to the lysozyme solution for 4 weeks retained their sizes and shapes. However, the surface reliefs of the fibers became more pronounced, and fibrillation of the surfaces occurred. The fibers modified with 0.05 wt.% of SWCNTs contain welloriented structural elements on their surfaces. It should be noted that an increase in SWCNT content led to surface relief sharpening upon exposure to the biologically active medium; the formation of longitudinal pores and cracks is observed (Figure 3e-g). These changes in the surface structures of the composite fibers after exposure to the lysozyme solution are indicative of chitosan resorption that began from the fiber surfaces.

IR Spectroscopy
The prepared pure chitosan and chitosan-based composite fibers were characterized via FTIR spectroscopy. The spectra obtained contain several characteristic bands of differ-    The fibers exposed to the lysozyme solution for 4 weeks retained their sizes and shapes. However, the surface reliefs of the fibers became more pronounced, and fibrillation of the surfaces occurred. The fibers modified with 0.05 wt.% of SWCNTs contain welloriented structural elements on their surfaces. It should be noted that an increase in SWCNT content led to surface relief sharpening upon exposure to the biologically active medium; the formation of longitudinal pores and cracks is observed (Figure 3e-g). These changes in the surface structures of the composite fibers after exposure to the lysozyme solution are indicative of chitosan resorption that began from the fiber surfaces.

IR Spectroscopy
The prepared pure chitosan and chitosan-based composite fibers were characterized via FTIR spectroscopy. The spectra obtained contain several characteristic bands of different intensities. The wide band in the 3500-3200 сm −1 region is assigned to O-H stretching vibrations (hydroxyl groups involved in the formation of hydrogen bonds); the bands at  The treatment of the chitosan and composite fibers with a solution of lysozyme (an enzyme of glycosidase family (EC 3.2.1.17)) resulted in the hydrolytic cleavage of the O-glycoside bond in chitosan, which was confirmed via FTIR spectroscopy (a significant decrease in the relative intensity of the band related to the C-O vibrations of the O-glycoside bond). It should be noted that prolonged exposure to the lysozyme solution ( Figure 4) and an increase in the SWCNT content in the composite fiber ( Figure 5) led to the amplification of this effect and an increase in the degree of hydrolysis of the chitosan. It can be assumed that the presence of carbon tubes in the fibers led to the orientation of chitosan macromolecules on their surfaces, as shown in [56], for chitosan macromolecules and chitin nanofibrils. glycoside bond in chitosan, which was confirmed via FTIR spectroscopy (a significant decrease in the relative intensity of the band related to the C-O vibrations of the O-glycoside bond). It should be noted that prolonged exposure to the lysozyme solution ( Figure 4) and an increase in the SWCNT content in the composite fiber ( Figure 5) led to the amplification of this effect and an increase in the degree of hydrolysis of the chitosan. It can be assumed that the presence of carbon tubes in the fibers led to the orientation of chitosan macromolecules on their surfaces, as shown in [56], for chitosan macromolecules and chitin nanofibrils. Also of note is the appearance of a new band at 1737 сm −1 with an increasing degree of chitosan hydrolysis. This band may be attributed to the vibrations of C=O bonds in the aldehyde groups that appear upon the opening of semi-acetal rings of terminal glucosamine units (whose concentrations increase with increasing degrees of hydrolysis). Also of note is the appearance of a new band at 1737 cm −1 with an increasing degree of chitosan hydrolysis. This band may be attributed to the vibrations of C=O bonds in the aldehyde groups that appear upon the opening of semi-acetal rings of terminal glucosamine units (whose concentrations increase with increasing degrees of hydrolysis).

Measurements of Strength and Deformation Characteristics
The dependences of the mechanical characteristics of the composite fibers containing 0.05, 0.5, and 2.5 wt. % of SWCNTs on the degree of orientational drawing are provided in Figure 6a-c.
The data presented in Figure 6 imply that the introduction of nanotubes into the chitosan scaffold exerts a significant influence on the strength, elasticity, and deformation properties of the composite fibers.
The strength of the composite fibers increases by 50%, and their elastic modulus increases by 50% when compared to the corresponding parameters of the pure chitosan samples. For all composites (SWCNT contents 0.05-0.5 wt.%), the tensile strength and elastic modulus values increase monotonically with increasing degrees of orientational drawing; the dependences of the tensile strength and modulus values of the composite fibers on the degree of drawing are similar to those of pure chitosan fibers. Introducing SWCNTs into the chitosan scaffold facilitates the additional orientation of chitosan macromolecules, which increases the strength. A similar effect was observed upon the addition of chitin nanofibrils into chitosan fiber [56]. When a higher amount (2.5 wt.%) of filler is introduced, the behavior of the fiber strength changes: this parameter virtually does not depend on the degree of orientational drawing. This is related to the high content of single-walled carbon nanotubes in the composite solution and the formation of a rigid SWCNT framework. These fibers have high elastic modulus values (17.2 ± 1.2 GPa). The maximum orientational drawing of the fibers was equal to 75%.
The introduction of single-walled carbon nanotubes causes significant deteriorations in the deformation characteristics of the composite fibers; the tensile strain virtually does not change with increasing degrees of fiber drawing, unlike the corresponding characteristics of pure chitosan fibers. The tensile strain of the pure chitosan fiber obtained at λ = 100% is almost similar to that of the fibers containing 0.05-0.5 wt.% SWCNTs, although the tensile strength and elastic modulus values of the composite fibers are greater than those of pure chitosan fibers by 30%. glycoside bond in chitosan, which was confirmed via FTIR spectroscopy (a significant decrease in the relative intensity of the band related to the C-O vibrations of the O-glycoside bond). It should be noted that prolonged exposure to the lysozyme solution ( Figure 4) and an increase in the SWCNT content in the composite fiber ( Figure 5) led to the amplification of this effect and an increase in the degree of hydrolysis of the chitosan. It can be assumed that the presence of carbon tubes in the fibers led to the orientation of chitosan macromolecules on their surfaces, as shown in [56], for chitosan macromolecules and chitin nanofibrils. Also of note is the appearance of a new band at 1737 сm −1 with an increasing degree of chitosan hydrolysis. This band may be attributed to the vibrations of C=O bonds in the aldehyde groups that appear upon the opening of semi-acetal rings of terminal glucosamine units (whose concentrations increase with increasing degrees of hydrolysis).

Measurements of Strength and Deformation Characteristics
The dependences of the mechanical characteristics of the composite fibers containing 0.05, 0.5, and 2.5 wt. % of SWCNTs on the degree of orientational drawing are provided in Figure 6a  The data presented in Figure 6 imply that the introduction of nanotubes into the chitosan scaffold exerts a significant influence on the strength, elasticity, and deformation properties of the composite fibers.
The strength of the composite fibers increases by 50%, and their elastic modulus increases by 50% when compared to the corresponding parameters of the pure chitosan samples. For all composites (SWCNT contents 0.05-0.5 wt.%), the tensile strength and elastic modulus values increase monotonically with increasing degrees of orientational drawing; the dependences of the tensile strength and modulus values of the composite fibers on the degree of drawing are similar to those of pure chitosan fibers. Introducing SWCNTs into the chitosan scaffold facilitates the additional orientation of chitosan mac-  Figure 7 presents the dependences of the specific resistivities of the composite fibers containing 0.05, 0.1, 0.5, 1,0, and 2.5 wt. % of SWCNTs on the filler concentration. The experiments involved the initial fibers and the fibers kept in a desiccator at a relative humidity of 98% for 24 h.

Measurements of Electrical Resistivity of Fibers
As can be seen from Figure 7, introducing SWCNTs into chitosan fiber results in a significant (by six orders of magnitude) decrease in its resistivity.
Pure chitosan fiber possesses ionic conductivity, and its resistivity is equal to 108-109 Ohm·m, which coincides with the data reported in [57]. When the chitosan scaffold contains carbon nanotubes, the composite demonstrates both ionic and electronic conductivities, which cause a decrease in the specific resistivity value. The presence of isometric conducting SWCNT particles among uniaxially oriented chitosan macromolecules promotes the formation of a conducting cluster even at small (lower than 2.5 wt.%) contents of SWCNTs [58,59]. The formation of conducting clusters also enhances the conductivity of the composite fibers.
those of pure chitosan fibers by 30%. Figure 7 presents the dependences of the specific resistivities of the composite fibers containing 0.05, 0.1, 0.5, 1,0, and 2.5 wt. % of SWCNTs on the filler concentration. The experiments involved the initial fibers and the fibers kept in a desiccator at a relative humidity of 98% for 24 h. Figure 7. Dependences of specific volume resistivities of chitosan-based composite fibers on the SWCNT content: the initial chitosan and composite fibers (curve 1) and the fibers kept for 24 h at a relative humidity of 98% (curve 2).

Measurements of Electrical Resistivity of Fibers
As can be seen from Figure 7, introducing SWCNTs into chitosan fiber results in a significant (by six orders of magnitude) decrease in its resistivity.
Pure chitosan fiber possesses ionic conductivity, and its resistivity is equal to 108-109 Ohm·m, which coincides with the data reported in [57]. When the chitosan scaffold contains carbon nanotubes, the composite demonstrates both ionic and electronic conductivities, which cause a decrease in the specific resistivity value. The presence of isometric After the exposure of pure chitosan fibers in a desiccator at a humidity of 98%, sharp decreases in their electrical resistivity values are observed (Figure 7, curve 2). It is known that chitosan is a hydrophilic polymer [60], and an increase in the content of conducting elements (adsorbed dissociated water molecules) results in an increase in the ionic conductivity of a fiber. Introducing SWCNTs causes a decrease in resistivity due to the formation of the conducting cluster and an increase in the electronic component of conductivity.
The dependences of the electrical conductivities of the composite fibers on the time of exposure to the lysozyme-containing buffer solution were obtained (Figure 8). It can be seen that the resistivities of the composite fibers decrease with increasing exposure time. conducting SWCNT particles among uniaxially oriented chitosan macromolecules promotes the formation of a conducting cluster even at small (lower than 2.5 wt.%) contents of SWCNTs [58,59]. The formation of conducting clusters also enhances the conductivity of the composite fibers.
After the exposure of pure chitosan fibers in a desiccator at a humidity of 98%, sharp decreases in their electrical resistivity values are observed (Figure 7, curve 2). It is known that chitosan is a hydrophilic polymer [60], and an increase in the content of conducting elements (adsorbed dissociated water molecules) results in an increase in the ionic conductivity of a fiber. Introducing SWCNTs causes a decrease in resistivity due to the formation of the conducting cluster and an increase in the electronic component of conductivity.
The dependences of the electrical conductivities of the composite fibers on the time of exposure to the lysozyme-containing buffer solution were obtained ( Figure 8). It can be seen that the resistivities of the composite fibers decrease with increasing exposure time. This is explained by the fact that chitosan fibers are hydrophilic [60] and become saturated with electrolyte ions. Additionally, chitosan contains ionogenic -NH3+ groups. In an electrolyte solution, ionogenic groups become hydrated, and chitosan becomes an ionic type of conductor. The higher the SWCNT content, the higher the contribution of the electronic current compared to that of the ionic current. Thus, the conductivity of the compo- This is explained by the fact that chitosan fibers are hydrophilic [60] and become saturated with electrolyte ions. Additionally, chitosan contains ionogenic-NH3+ groups.
In an electrolyte solution, ionogenic groups become hydrated, and chitosan becomes an ionic type of conductor. The higher the SWCNT content, the higher the contribution of the electronic current compared to that of the ionic current. Thus, the conductivity of the composite fiber containing 2.5 wt.% of SWCNTs changes insignificantly when increasing the time of exposure to the lysozyme-containing buffer solution because the fiber contains a dense network of conducting carbon nanotubes. The changes in electrical conductivity are determined by the amount of SWCNTs and their distribution in the scaffold. Thus, the saturation of composite fibers with electrolyte ions during their exposure to a lysozyme solution led to the formation of an ionic-electronic type of conductivity and a decrease in volume resistivity. The mechanical tests and scanning electron microscopy results show that the optimal SWCNT concentration is equal to 0.5 wt.%, which is confirmed by the high values of electrical conductivity (Figure 8). According to the SEM data (Figure 3), the homogeneous structure of the fiber facilitates an increase in electrical conductivity.

Investigation of the Biocompatibility of the Fibers via the MTT Technique
When designing composite materials with improved physical and chemical properties, one must bear in mind that any additional component can influence the biocompatibility of the resulting material. Carbon nanotubes are promising additives for the preparation of materials with improved properties. Soon after their discovery in 1991 [61], these nanosized materials received significant attention from experts in various fields of science, including materials science and biomedical engineering. Although carbon nanotubes possess unique properties and thus impart improved mechanical, electrical, and thermal properties to composite materials [62][63][64], the use of these materials for medicinal purposes is still open to question. On one hand, the carbon nanotubes in the initial state in the cultural medium showed cytotoxicity to certain cell lines, which may be related to the use of metal catalysts (such as nickel) in the preparation of the carbon nanotubes [65]. On the other hand, the carbon nanotubes introduced into composite materials showed high levels of biocompatibility [66][67][68]. The number of publications focused on the investigation of the interactions between carbon nanotubes and cell lines increases every year [52,69,70].
As previously mentioned, the presence of SWCNTs in chitosan fibers significantly improves their structural, mechanical, and conducting properties, but during exposure to a lysozyme solution, the emergence of SWCNTs on the fibers' surfaces is possible. Since the literature contains contradictory data [65], it is necessary to study the cytotoxicity of the obtained composite fibers containing SWCNTs. Our studies have shown that the nutrient medium obtained after 4 days of incubating of the initial composite fiber has no toxic effect on human MSC cells (Figure 9). The number of viable cells in these samples is virtually similar to that in the control sample (the cells cultivated in the standard nutrient medium). An insignificant decrease in the number of viable cells in the case of the sample containing 0.05 wt.% of SWCNTs may be related to a partial release of nanotubes into the nutrient medium. The viability parameters of the cells in the nutrient medium samples in which the fibers with 0.5 and 2.5 wt.% of SWCNTs were kept are similar to the viability parameters of the control sample. As previously noted, it is possible that upon reaching a certain concentration, SWCNTs form a network that prevents the release of individual nanotubes into the nutrient medium. medium). An insignificant decrease in the number of viable cells in the case of the sample containing 0.05 wt.% of SWCNTs may be related to a partial release of nanotubes into the nutrient medium. The viability parameters of the cells in the nutrient medium samples in which the fibers with 0.5 and 2.5 wt.% of SWCNTs were kept are similar to the viability parameters of the control sample. As previously noted, it is possible that upon reaching a certain concentration, SWCNTs form a network that prevents the release of individual nanotubes into the nutrient medium. Figure 9. МТТ test of FetMSC cells cultivated in the nutrient medium after incubation of the following fiber samples: Chit + 0.05%SWCNT-chitosan fibers containing 0.05% SWCNT; Chit + 0.5%SWCNT-chitosan fibers containing 0.5% SWCNTs; Chit + 2.5%SWCNT-chitosan fibers containing 2.5% SWCNTs; Chit + 0.05%SWCNT/lysozyme-chitosan fibers containing 0.05% SWCNTs after exposure to lysozyme solution for 4 weeks; Chit + 0.5%SWCNT/lysozyme-chitosan fibers containing 0.5% SWCNTs after exposure to lysozyme solution for 4 weeks; Chit + 2.5%SWCNT/lysozyme-chitosan fibers containing 2.5% SWCNTs after exposure to lysozyme solution for 4 weeks. . MTT test of FetMSC cells cultivated in the nutrient medium after incubation of the following fiber samples: Chit + 0.05%SWCNT-chitosan fibers containing 0.05% SWCNT; Chit + 0.5%SWCNTchitosan fibers containing 0.5% SWCNTs; Chit + 2.5%SWCNT-chitosan fibers containing 2.5% SWCNTs; Chit + 0.05%SWCNT/lysozyme-chitosan fibers containing 0.05% SWCNTs after exposure to lysozyme solution for 4 weeks; Chit + 0.5%SWCNT/lysozyme-chitosan fibers containing 0.5% SWCNTs after exposure to lysozyme solution for 4 weeks; Chit + 2.5%SWCNT/lysozyme-chitosan fibers containing 2.5% SWCNTs after exposure to lysozyme solution for 4 weeks. Figure 10 presents the results of the morphological analysis of the nerve fibers with various artificial guides. Figure 10 clearly shows the formation of multinucleated foreign body giant cells (FBGC) (arrow 7) in all experiments. These cells form a dense ring along the internal wall of the polylactide scaffold (arrow 2). Additionally, fibroblasts (arrow 8) and a moderate amount of collagen fibers (arrow 10) are localized in the bulk of the scaffold between the chitosan monofibers (arrow 1). This indicates the occurrence of bioresorption processes. Connective tissue fills the lumen of the polylactide scaffold, and growing nerve fibers (arrow 3) are located between connective tissue fibers. Furthermore, the outer surface of the scaffold is coated with connective tissue (arrow 4).

Morphological Analysis
All monofilaments (arrow 1) become encapsulated, localized inside the scaffold eccentrically, and surrounded by connective tissue (arrow 6). This connective tissue is penetrated with numerous thin-walled vessels (arrows 5). It is noteworthy that no active bioresorption of monofibers is observed.
Upon implantation, chitosan monofibers containing 0.05 wt.% of SWCNTs (Figure 10a,b) become deformed due to compressive forces from the surrounding tissues. However, no signs of acute inflammatory reaction were observed after the implantation of chitosan monofibers containing 0.5 wt.% of SWCNTs (Figure 10c,d). These monofibers are surrounded by connective tissue (arrow 6) with moderate amounts of leukocytes (arrow 9).
In contrast, the implantation of chitosan monofibers containing 2.5 wt.% of SWCNTs (Figure 10e,f) results in much greater amounts of leukocytes (arrow 9) and multinucleated foreign body giant cells (FBGC) (arrow 7), indicating the presence of pronounced chronic aseptic inflammation.

Conclusions
Conducting, biocompatible, composite fibers based on chitosan and SWCNTs were developed. It was demonstrated that the specific volume resistivity of the composite samples decreased from 10 7 to 1 Ohm*m when increasing the SWCNT content from 0.05 to 2.5 wt.%.
It was established that the presence of high amounts (2.5 wt.%) of carbon nanotubes (sources of electrical signals) in the composite fibers caused pronounced chronic, aseptic inflammation, although it did not result in a considerable increase in the number of growing nerve fibers.
It was found that the implanted chitosan monofibers containing 0.05 wt.% of SWCNTs became deformed under the action of compressive forces from the surrounding tissues, which led to the formation of a dense connective tissue capsule around the fiber.
The experiments showed that a conduit's best variant of guiding fibers is the composite fiber containing 0.5 wt.% of SWCNTs. The addition of 0.5 wt.% of SWCNTs into composite fibers facilitates the formation of a denser fiber structure, which results in improved strength (σ = 260 MPa) and elastic (E = 14 GPa) characteristics. According to the results of the cytological studies, this composite fiber is biocompatible, it is not deformed in the process of in vivo studies, and it does not cause a pronounced inflammatory reaction. A thin connective tissue capsule is formed around the fiber.