Chitosan Woven Meshes for Use as Biomaterial: From the Wet Spinning Process and Filament Obtention to Mesh Properties

Abstract

This study aimed to obtain wet-spun chitosan (CS) filaments with mechanical properties suitable for the biomaterial weaving process, in addition to evaluating the physical-mechanical and biological behavior of the CS woven meshes obtained. CS filament coagulation rate, drying methodology, and morphology were evaluated. The filament coagulation reaction was completed in 65 seconds, with a logarithmic profile, in compliance with Fick's second law. The proposed drying methodology does not cause dehydration of the chitosan saccharide rings and the stretching condition during drying modified the crystallinity index, mechanical properties, morphology, and diameter of CS filaments. The mechanical, morphological, and biological behaviors of CS woven meshes were investigated. Were observed a uniform pore size, mechanical properties in the wet state similar to those of the human dermis, controlled swelling degree, and degradation of 80% in phosphate buffered saline/lysozyme medium under 5 weeks. In addition, the CS woven meshes were not cytotoxic to L929 Mouse Fibroblast Cell Line, being promising for use as biomaterials.

Keywords:
Chitosan Filaments; Wet spinning; Woven Meshes; Biotextiles; Biomaterials

1. Introduction

Chitosan (CS), a derivative of chitin, is a linear, semi-crystalline polysaccharide known for its non-toxic, biodegradable, and bioabsorbable nature. Notably, the degradation products of chitosan are non-carcinogenic and non-immunogenic, with minimal inflammatory reactions¹-³; these exceptional properties and their capacity to form films and filaments⁴ justify the various studies for biomedical applications such as drug delivery systems⁵-⁸, dressings⁹, surgical suture yarns⁶ tissue engineering¹⁰,¹¹, antimicrobial agents¹², among others.

CS filaments are usually obtained by the wet spinning technique, which allows fibers to be received by extruding polymer solutions in a non-solvent, involving an acid-base neutralization reaction or solvent evaporation¹³-¹⁶. Viscous CS solutions can be made into monofilaments by different coagulation solutions, such as sodium hydroxide¹⁷-¹⁹, potassium hydroxide¹⁷,²⁰,²¹, alcohol (methyl or ethyl), calcium chloride²², sodium hydroxide-sodium sulfate²³, and sodium hydroxide-methanol⁶,²⁴. However, studies on the process steps of CS filaments wet spinning are scarce and poorly detailed, especially regarding the coagulation rate and the influence of stretching on the characteristics of CS filaments.

Textile biomaterials (Biotextiles) may be obtained by conventional textile techniques such as weaving, knitting, and braiding²⁵-²⁹. Compared to knit meshes, woven meshes may be designed to achieve higher tensile strength and dimensional stability²⁵-²⁷,³⁰. Most of the time, weaving techniques are used when high mechanical properties are required, such as in vascular grafts³¹-³³, tendon regeneration³⁴, drug delivery³⁵, hernia repair, and tissue engineering³³,³⁶. CS filaments are also used in various fibrous biomaterials, such as textile manufacturing³⁷-⁴⁰, surgical suture material⁶, dressings⁴¹, and hearing valves⁴².

In biotextile processing, the filaments must meet some criteria for correct textile manufacturing, such as mechanical strength, elasticity, diameter, and length²⁸-³⁰,⁴⁰. In contrast to their biological properties, CS filaments are reported as having low to moderate mechanical properties, limiting their use in load-bearing applications⁴²-⁴⁵. In this sense, different approaches have been proposed to improve the mechanical properties of CS filaments using physical treatments or chemical modifications. Chemical modifications are relatively simple due to the high availability of reactive groups in chitosan chains (amino and hydroxyl). Approaches adopted include: cross-linking with epichlorohydrin⁴⁶, immersion in solutions containing phosphate and phthalate ions¹⁷, modification with formic acid⁴⁴, fiber acetylation²⁰, annealing⁴⁷, chitin nanocrystal reinforcement⁴⁵, and variations in solvent concentrations and coagulation bath composition⁴³. However, these chemical modifications can cause cytotoxic effects²³,⁴³.

In a previous study⁴⁸, cross-linking conditions were demonstrated, which did not affect the biocompatibility of woven chitosan meshes, but introduced other chemicals into the system. To resolve this drawback, in this study, we aimed to improve the mechanical and morphological properties of wet-spun CS filaments through evaluations of the coagulation rate, drying methodology, and effect of elongation during drying. Subsequently, woven meshes were obtained from CS filaments and evaluated for morphology, mechanical properties, degree of swelling, degradation, and cytotoxicity. To the best of our knowledge, this is the first study involving the approach from filament coagulation to the final properties of a chitosan-based textile biomaterial.

2. Materials and Methods

2.1. Materials

Chitosan with a molecular weight of 310 kDa, determined via viscometry using a PSL Rheotek instrument (São Paulo, Brazil) following the method outlined by Il’Ina and Varlamov⁴⁹, was produced at the Northeastern Biomaterials Evaluation and Development Laboratory – CERTBIO (Campina Grande, PB, Brazil). The degree of deacetylation, measured at 86%, was determined using the infrared spectroscopy method with a Perkin Elmer instrument (Beaconsfield, U.K.) following the protocol described by Brugnerotto et al.⁵⁰. Sodium hydroxide (NaOH) was sourced from Neon® (São Paulo, SP, Brazil). Lactic acid 85% and methanol were procured from Anidrol® (Diadema, SP, Brazil). Phosphate buffered saline (PBS, pH 7.4) and lysozyme were acquired from Sigma Aldrich® (Darmstadt, Germany).

2.2. Obtaining chitosan filaments by wet spinning

The preparation of the CS solution (4% w/v) followed previously established methods⁶,⁸,⁵¹. To summarize, the polymer was dissolved in a 0.206 M lactic acid aqueous solution, maintaining a stoichiometric balance concerning the amine groups of chitosan. This dissolution process was carried out under constant mechanical agitation at 25 ± 1 °C for 2 hours. Subsequently, the CS solution was transferred into a syringe with a 20 mL capacity and a 1 mm diameter outlet tip. For the wet spinning process, a coagulation bath consisting of a 70% solution of 0.5 M sodium hydroxide (NaOH) in an aqueous solution and 30% methanol was employed. The CS solution was extruded into the coagulation bath at a constant rate of 45 mL/h using an infusion pump (Pump 11 Pico Plus Elite, Harvard Apparatus, Holliston, MA, USA), as illustrated in Figures 1a and 1b. Subsequently, the CS filaments were carefully removed, subjected to washing with distilled water until the wash water reached a pH of 7, and then underwent stretching (with a deformation, ϵ, of 10% of the initial length), as depicted in Figures 1c and 1d. Finally, the filaments were oven-dried at 60 °C for 1.5 hours.

Figure 1
Steps of the CS filaments wet spinning process. a) arrangement between the infusion pump, the syringe containing the CS solution and the coagulation bath container. b) CS filament extrusion in coagulation bath. c) CS filament stretching (10% of initial length) scheme for drying. d) arrangement of the stretched CS filaments for drying.

In the coagulation step (Figure 1b), the filament coagulation times and profiles, were determined using optical microscopy (OM). Meanwhile, the processes of drying and methanol removal, both carried out in the oven, were assessed through thermogravimetric analysis (TGA). The crystallinity profile of the stretched fibers during the drying process, as well as their mechanical properties and morphology, were assessed using X-ray diffraction (XRD), tensile mechanical testing, and scanning electron microscopy (SEM), respectively.

2.3. Fabrication of chitosan woven meshes

The CS woven meshes were obtained following a previously published methodology. In summary⁴⁸,⁵². A loom constructed from glycol-modified poly(ethylene terephthalate) (PET-G) using 3D printing technology (3DCloner, model DH PLUS, PR, Brazil) was employed in the fabrication of chitosan woven meshes, as illustrated in Figures 2a and 2b. The loom has pins with a diameter and spacing of 3 mm, and edges of 6 mm.

Figure 2
Fabrication of Chitosan (CS) woven meshes. a) plain weft (1/1) obtained by the interweaving of the CS filaments. b) CS woven mesh coated with 1.5% CS solution. c) demolded CS woven mesh d) dimensions of the resulting CS woven mesh.

The initial stage of the weaving process involved aligning the CS filaments in parallel to establish the textile warp, as depicted in Figure 2a. Subsequently, a second CS filament plane was introduced orthogonally into the warp, creating a plain weft pattern (1/1), as indicated in Figure 2b. The dimensions of the resulting CS woven mesh were determined by the filament diameter, and loom geometry (edge length, spacing and diameter of the pins), as shown in Figure 2c. After the weaving process, the woven CS meshes were dip coated for 30 seconds in a 1.5% CS solution (with a molecular weight of 310 kDa; containing 0.077 M lactic acid), neutralized in a 0.5 M NaOH solution for 30 minutes, and then subjected to drying at 50°C for 6 hours to prepare the samples for subsequent characterization, as presented in Figure 2d. The purpose of applying the woven mesh coating is to maintain the orthogonal alignment and weave structure of the filaments.

2.4. Optical Microscopy (OM)

Optical microscopy analysis was employed to assess both the coagulation rate of CS filaments and the morphology of CS woven meshes. The coagulation process of the fibers was captured on video using an optical microscope (Hirox Digital Optical Microscope - Kh 1,300 M, Tokyo, Japan) at a magnification of 50X. CS filaments were extruded into a Petri dish made of polystyrene containing the coagulation bath, and the entire coagulation reaction was recorded over time. Measurements were subsequently conducted using ImageJ software (Java 1.8.0.112 Version, National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, Wisconsin, WI, USA), directly on the filament images acquired during the experiment. The evaluation of the woven mesh morphology also employed the same equipment, with observations made under transmission and reflection modes at magnifications ranging from 20X to 160X. To determine pore sizes in the woven meshes, measurements were carried out using ImageJ software, and a total of 10 measurements were taken for analysis.

2.5. Scanning Electron Microscopy (SEM)

The surface and cross-section morphology of both stretched and unstretched CS filaments were examined using an SEM (Scanning Electron Microscope) from Phenom World, specifically the Pro-X800-07334 model (Eindhoven, The Netherlands). To prepare the samples, cryogenic fracture techniques were employed, followed by a thin gold coating. Imaging was performed with an electron beam accelerated at 15 kV, utilizing a depth of focus of 1 mm and achieving a resolution of 30 nm. For further analysis, measurements of the CS filament diameter were taken from a sample size of n = 10. These measurements, along with pore size determinations, were conducted using ImageJ software.

2.6. Mechanical properties analysis

Uniaxial tensile testing was employed to assess the mechanical properties of both stretched and unstretched CS filaments (with a sample size of n=10 for each condition) as well as dry and hydrated CS woven meshes (n=5). The filaments, measuring 100 mm in length, underwent tensile tests using a 500 kN load cell, a displacement speed of 120 mm/min, and a 100 mm claw distance. Conversely, the dry and hydrated CS woven mesh samples were subjected to testing with a 500 kN load cell, a speed of 100 mm/min, and a 40 mm distance between the claws. To prepare the hydrated woven mesh samples, they were immersed in PBS at 37 ± 0.5 °C for 24 hours. These analyses were carried out at 25 ± 1 °C and under a relative humidity of 60 ± 2%. The testing equipment utilized was a universal testing machine, specifically the Instron Model 6633 (Norwood, MA, USA).

2.7. Thermogravimetric Analysis (TGA)

Following the determination of the optimal coagulation time, the washed specimens underwent dynamic and isothermal thermogravimetric analysis to assess the proposed CS filament drying methodology. Specimens weighing 6 ± 1 mg, housed in aluminum crucibles, were utilized. Dynamic analysis entailed a heating rate of 10 °C/min up to 600 °C within a nitrogen environment boasting a flow rate of 50 mL/min. Subsequently, a constant temperature analysis transpired at a heating rate of 10 °C/min, spanning from 25 to 60 °C, with a temperature hold at 60 °C for 90 minutes. The instrument employed was the TGA Pyris-1 by Perkin Elmer (Waltham, MA, USA).

2.8. X-ray Diffractometry (XRD)

The drying conditions for both the stretched and unstretched CS filaments were assessed using XRD (X-ray Diffraction) analysis conducted with a Shimadzu XRD-7000 diffractometer, equipped with Ni-filtered Cu-Kα radiation. XRD profiles were acquired within the scattering range of 5° < 2θ < 40°, employing a resolution of 0.02° and a scanning rate of 1°/min. The analyses were carried out under the application of 40 kV voltage and 30 mA current. Samples were aligned in parallel and subsequently subjected to the assessment. To gauge alterations in the crystallinity of CS powder, as observed in stretched and unstretched CS filaments, the crystallinity index (CI) was calculated following the equation outlined in our previous research⁶:

$\text{CI (\%)=}\left(\frac{{\text{A}}_{\text{Cr}}}{{\text{A}}_{\text{sample}}}\right)\text{x100}$ (1)

where A_Cr is the area of the crystalline peaks and A_sample is the area under the sample intensity curve.

2.9. Swelling test

The swelling test was conducted on dried CS woven mesh samples (n = 3), each measuring 10 x 10 mm with a thickness of approximately 0.25 ± 0.03 mm. These samples were immersed in a phosphate buffered saline (PBS) solution with a pH of 7.34 at 37 ± 0.5 °C, to simulate a physiological environment. Prior to the immersion, the samples were dried for 6 hours at 50°C, and their initial weights (W₀) were recorded. Subsequently, they were placed in the buffer solution at 37 ± 0.5 °C. At predefined intervals (0.5, 1, 5, 10, 20, and 24 hours), the samples were removed from the solution, gently dried with filter paper, and their weights at the respective times (W_t) were determined. The swelling degree (SD) was calculated using the following equation:

$\text{SD(\%)=}\left(\frac{{\text{W}}_{\text{t}}{\text{-W}}_{\text{0}}}{{\text{W}}_{\text{0}}}\right)\text{x100}$ (2)

where W_t and W₀ represent the weights of swollen and dried state samples, respectively.

2.10. Enzymatic biodegradation

The criteria used to evaluate the in vitro biodegradation of CS woven meshes were the mass loss by gravimetry and morphological changes observed by SEM. CS woven meshes (n=3 for each biodegradation period; dimensions: 10 x 10 mm²) was assessed within a 10 mL phosphate-buffered solution (PBS, pH 7.34) at a controlled temperature of 37 ± 0.5 °C, supplemented with 1.5 µg/mL lysozyme, following the procedure as outlined in the work of Silva et al.⁶. Prior to immersion, the samples were subjected to a drying process at 50 °C for 6 hours and weighed to determine their initial weight (W₀). They were then placed in the PBS/Lysozyme solution at 37 ± 0.5 °C. Periodically, at biodegradation intervals of 7, 14, 21, 28, and 35 days, the samples were retrieved from the solution. They were subsequently rinsed with distilled water, gently dried using filter paper, subjected to another 6-hour drying cycle at 50 °C, and re-weighed (W_t). For differentiation of the enzymatic influence, control samples were subjected to the same conditions as described above, except for lysozyme supplementation. The percentage of mass loss was determined using Equation 3.

$\text{Mass Loss}\left(\text{\%}\right)\text{=}\left(\frac{{\text{W}}_{\text{0}}{\text{-W}}_{\text{t}}}{{\text{W}}_{\text{0}}}\right)\text{x100}$ (3)

where W₀ is the initial mass of the sample, and W_t is the mass of the samples degraded at time t.

After degradation, the morphology of CS woven meshes is assessed through SEM analysis to evaluate any morphological changes resulting from degradation.

2.11. Cytotoxicity test

The cytotoxicity of the chitosan woven meshes was assessed using the L929 Mouse Fibroblast Cell Line (ATCC NCTC clone 929, Rio de Janeiro Cell Bank, Brazil) through the agar diffusion method following ISO 10993-5 (2009)⁵³. In summary, the L929 cells were cultured in RPMI 1640 medium (Gibco - Invitrogen Corporation, Grand Island, USA) until reaching confluence. They were then trypsinized with 0.25% trypsin (Gibco®, Life Technologies) and adjusted to a concentration of 1.0x10⁵ cells/mL. The cell suspension was evenly distributed in 6-well plates (4 ml per well) and incubated for 48 hours. Subsequently, the culture medium was replaced with 1 ml of freshly prepared 2X Eagle MEM agar medium (Gibco®-Invitrogen Corporation, Grand Island, USA) containing 0.01% neutral red solution (Sigma-Aldrich, USA). After allowing the agar to solidify, the samples, along with positive controls (latex sheet) and negative controls (high-density polyethylene – HDPE), all with an area of 100 mm², were positioned in the center of the agar surfaces. The plates were then incubated for 24 hours in a humidified environment at 37 °C ± 1 °C, with 5% ± 1% CO₂. In assessing the degree of cytotoxicity, the extent of the discolored area (dead cells) was measured from the ends of the sample in the quadrants, and the degree of cytotoxicity was related to ISO 10993-5.

The cytotoxicity results were evaluated using a Nikon Eclipse TS100 inverted digital microscope (Minato, Tokyo, Japan), where the discoloration zone and cell lysis were measured. The assay was performed in duplicate.

2.12. Statistical evaluation

T-tests were conducted using Minitab v19.1. The selected confidence level was 95%, with a significance level (α) set at 0.05. When the p-values were less than or equal to α, the distinction between means was regarded as statistically significant. Conversely, when the p-values exceeded α, the distinction between means was considered statistically non-significant.

3. Results and Discussion

3.1. Chitosan filament coagulation rate study

The coagulation rate of the CS filaments was investigated based on images of the filament every 10 s of reaction until complete coagulation at 70 s (Figure 3a). The position of the boundary between the phases (CS solution and CS precipitated), the external radius (R), and the internal radius (r) were verified, and the ratio between the precipitated phase and total sample area was determined according to the relationships illustrated in Figure 3b.

Figure 3
Chitosan Filament Coagulation Rate Study. (a) Images obtained by OM every 10 s of CS filament coagulation reaction and (b) schematic illustration of the parameters R and r evaluated in the cross and longitudinal section of the CS filament during the coagulation reaction.

There is a distinct moving boundary between the coagulated polymer and the uncoagulated chitosan solution in the filament nucleus, associated with the diffusion phenomenon of the chemical specimens involved in the coagulation of the CS solution⁵⁴. In this case, it involves a process based on an acid-base reaction, with the diffusion of chemical species through the solidified CS and CS solution. In the coagulation process, CS (Glc-NH₂), dissolved in lactic acid solution (0.206 M), comes into contact with a basic solution (coagulation bath) and the proton exchange between the solutions causes the precipitation of the CS solution.

The ratio of the precipitated polymer area to the total sample area is the measure of the CS filament coagulation percentage, ranging from 0 (CS solution immediately before contact with the coagulation bath) to 1 (CS fully regenerated by acid-base reaction). Thus, the behavior of the coagulation percentage with the reaction time is shown in Figure 4.

Figure 4
CS filament coagulation profile.

The reaction responsible for the formation of CS filaments is completed in about 65 seconds for 0.5 M of sodium hydroxide in the coagulation bath. Coagulation occurs at higher speeds initially, and is then slowed down by the growth of the barrier to the diffusion process. Coagulation of CS filaments involves the diffusion of coagulant (OH^-) species through newly solidified CS and CS solution, respectively⁵⁴. Therefore, as precipitation of the CS occurs, the thickness of the coagulated wall increases and the diffusion process becomes more difficult. These occur due to the increase in the distance by which OH^- ions need to diffuse for the reaction to take place⁵⁵,⁵⁶.

According to Fick’s second law, the coagulated thickness (measured in mm) should exhibit a linear relationship with the square root of the time (expressed in s^1/2)⁵⁴,⁵⁷,⁵⁸. Moreover, Paul⁵⁹ established the quotient between coagulated thickness and the square root of the time (expressed in mm/s1/2) as a parameter of the wet-spinning process, styled “coagulation rate”. Here the coagulated thickness was expressed as a percentage (%) relative to the total cross-sectional area of the filament.

As observed in Figure 5, the coagulation percentage shows a proportionality relationship with the root of the time, implying suitability for Fick's second law. These results are in line with those reported by⁵⁴-⁵⁶,⁶⁰. The coagulation rate is understood to be the slope of a linear regression. For the system under study, the coagulation rate was 11,95 ± 0,145%/s^1/2.

Figure 5
CS filament coagulation rate.

3.2. Evaluation of chitosan filaments drying methodology

After coagulation, the CS filaments were washed and subjected to thermogravimetric analysis. The results of the dynamic and isothermal analyses are presented in Figures 6a and 6b, respectively.

Figure 6
Thermogravimetric analysis of CS filaments: (a) Dynamic TG and DTG curves of swollen CS filament, at 10°C/ min heating rate, under the N₂ atmosphere (50mL/min) and the initial mass of 6.017 mg and (b) isothermal TG and DTG curves of the swollen CS filaments at 60°C, under the N₂ atmosphere (50mL/min) and the initial mass of 5.383 mg.

The total mass loss in the dynamic analysis (Figure 6a) was 98.44%. In the temperature range between 30 and 150 °C, a first weight loss occurred, about 93.05% in mass, associated with water desorption (weakly bound) and methanol from the coagulant bath⁶¹,⁶². In the range of 180 to 600 °C, three decomposition events occurred, totaling a loss of 5.39% by mass. These events are attributed, respectively, to dehydration of the saccharide rings (strongly bound), depolymerization, and decomposition of the acetylated and deacetylated polymer units, resulting in the release of H₂O, NH₃, CO, CO₂, CH₃COOH and CH₄⁶¹,⁶³.

In the isothermal analysis (Figure 6b), which simulates the proposed drying methodology (90 minutes, 60° C), a single mass loss, approximately 91.57%, in the range of 0 to 20 minutes, related to the loss of loosely bound water and methanol from the coagulation bath⁶⁰,⁶¹. Moreover, up to approximately 100 minutes, no further mass loss steps occur, showing that the drying process under these conditions was effective, without losses in the most strongly bound water, that act in the maintenance of the mechanical properties of CS⁶¹,⁶⁴,⁶⁵. Therefore, we conclude by TG that the drying process was effective in completely removing the utilized methanol, thus dispelling any concerns regarding the persistence of this toxic reagent in the final product⁶⁶.

3.3. Effects of Stretching of Chitosan Filaments

CS filaments were subjected to a 10% deformation in relation to their initial length during the drying process (Figure 1c). The effects of this stretching on the crystallinity profile, mechanical properties, and morphology of the filaments were evaluated.

3.3.1. Crystallinity profile

The effects of dissolution, stretching and drying processes were observed in the XRD patterns of the CS powder, stretched and unstretched CS filaments, as shown in Figure 7.

Figure 7
XRD patterns of CS powder, stretched and unstretched CS filaments, and comparison of the Crystallinity Index (IC) values.

In the XRD patterns of stretched and unstretched CS filaments, a discrete peak shift at 2θ ≈ 20° can be observed for larger angles compared to the CS powder. The peak at 2θ ≈ 10° is shifted to smaller angles, accompanied by a loss of intensity compared to the CS powder XRD pattern. These events occur due to the effects of the CS dilution in the lactic acid solution (0.206 M), which probably interfered with the crystalline regions, hindering an orderly arrangement of the CS chains during polymer precipitation⁶⁷. This effect was confirmed by the calculations of the Crystallinity Index (IC)⁶⁸. CS powder showed higher crystallinity (33%), while for filaments, it was observed that stretching treatment during drying resulted in a higher crystallinity of the filament (24%) compared to unstretched (20%). The stretching promotes alignment and approximation of CS chains, facilitating the crystallization process⁶⁹ and improving the mechanical properties of the filaments⁴³, making it possible to weave the CS filaments.

3.3.2. Mechanical Properties

The mechanical properties of the stretched and unstretched CS filaments were evaluated. Table 1 shows that the stretching condition was statistically significant (p-value < 0.05), caused an increase in Young's modulus (107.12%), maximum load (44.50%), and tensile strength (43.25%), as well as a reduction in sample strain (-10.24%).

Table 1
Mechanical properties of the stretched and unstretched CS filaments.

	Unstretched filament	Stretched filament
Maximum load (N)	4.27 ± 0.3	6.17 ± 0.42
Tensile strength (MPa)	173.04 ± 8.63	247.88 ± 12.61
Strain (%)	2.54 ± 0.05	2.28 ± 0.18
Young's modulus (GPa)	14.07 ± 1.93	29.14 ± 3.0

The increase in Young's modulus, maximum load, and tensile strength occurs because, with stretching, the CS chains are oriented longitudinally in relation to the filaments, facilitating their approximation, resulting in higher crystallinity indices, as observed in the XRD patterns, which directly influence the fibers mechanical properties⁷⁰. Similar results were obtained by Copeland et al.⁶⁹.

The reduction in CS filaments deformation capacity is due to the increase in the crystallinity index, which implies a greater Young's modulus, and greater restriction on the slip of the polymeric chains, which reduces the ability to deform under tension of the filaments⁷¹,⁷².

The mechanical properties of the stretched CS filaments made it possible to easily handle them during the weaving process, as will be seen below, as evidenced by the tensile strength of 248 MPa.

3.3.3. Morphology (SEM)

The surface and cross-section morphology of the stretched and unstretched CS filaments were evaluated by scanning electron microscopy (SEM). Figures 8a and 8b show the surface of the unstretched CS filament, there is a smooth, compact, cylindrical-shaped monofilament surface. In Figures 8e and 8f the same morphology is observed for the CS filaments stretched during drying. The presence of grooves and diameter reduction (from 301 ± 6.4 µm to 212 ± 2.4 µm; p-value < 0.05) induced by stretching is observed. The cross-section images of the unstretched (Figure 8c) and stretched (Figure 8g) CS filaments show the typical morphology of filaments obtained by the wet spinning process. This structure is well-defined as “skin-core” morphology⁷³. As seen in the results of the coagulation rate (Figure 4), during the coagulation stage in the wet spinning process, the skin of the formed fibers coagulates rapidly at the initial moment when the fibers come into contact with the coagulation bath, while the core has time to relax. This phenomenon results in a microstructure where the surface polymer chains are uniformly axially oriented while the core chains are imperfectly packaged and ordered, giving rise to core pores (Figures 8c, 8d, 8g, and 8h)⁷³,⁷⁴.

Figure 8
SEM micrographs of: surface of unstretched (a and b) and stretched (e and f) CS filaments; and cross-section of unstretched (c and d) and stretched (g and h) CS filaments.

The average pore size was decreased by filament stretching from 0.954 ± 0.102 µm to 0.466 ± 0.153 µm (p-value < 0.05). These results are consistent with those observed in the mechanical and XRD analyses of CS filaments, where large pore sizes imply lower mechanical properties and less orderly arrangements (lower CI) of CS filaments⁷².

3.4. Evaluation of Chitosan Woven Meshes

The stretched CS filaments were used for hand weaving, due to their better mechanical properties and minor pore sizes. Thus, the results of the CS woven meshes are shown below.

3.4.1. Morphology (OM and SEM)

The morphology of woven meshes was evaluated by optical microscopy (OM) and scanning electron microscopy (SEM), and the results are presented in Figure 9.

Figure 9
Micrographs of CS woven meshes samples prepared by CS filaments coated by 1.5% CS solution: OM analysis magnifications: of a) 20x and b) 40x; SEM analysis magnifications: c) 100x and d) 500x.

Figures 9a and 9b show the OM micrographs of the CS woven meshes coated with 1.5% CS solution. It can be observed that woven mesh has average pore sizes of 4.87 ± 0.30 mm². The mesh is composed of a continuous monofilament, and has a uniform pore-size end coating through the woven structure. In Figure 9c, we can see an SEM image of the plain weft of the CS filaments in the woven mesh. Figure 9d (SEM images) shows a detail of the CS filament crossings coated with a 1.5% CS solution coating.

3.4.2. Mechanical Properties (Wet and Dry)

The Mechanical properties of the CS woven meshes were evaluated by tensile tests under dry and wet conditions, and the results are shown in Figure 10.

Figure 10
Mechanical properties of the CS woven meshes. a) Stress-Deformation curves of the dry and hydrated CS woven meshes: b) Young’s modulus, c) Tensile Strength, d) Maximum load and e) Strain.

Figure 10a shows representative stress-strain curves (average, n = 5) for the dry and wet CS woven meshes. Under tension, dry CS woven meshes exhibited a short elastic region and a high slope of the curve, followed by failure at approximately 1.25% strain. On the other hand, the hydrated meshes presented a moderate slope and large region under the curve, followed by plastic deformation and breaking at 29.6% strain.

Young’s modulus, tensile strength, maximum load, and strain of both dry and wet CS woven meshes were calculated from the test stress-strain data. Dry CS woven meshes had a high Young’s modulus, 368 ± 75.4 MPa, in comparison with 9 ± 2.1 MPa for the wet samples (Figure 10b). Tensile strength (Figure 10c) and strain (Figure 10d) also decreased in the wet state, from 3.7 ± 0.36 MPa to 2.4 ± 0.26 MPa and from 12.2 ± 0.41 N to 6.3 ± 1.10 N, respectively. The Elongation of CS woven meshes in a wet state (36.6 ± 5.70%) was significantly higher than that of dry CS woven meshes (1.2 ± 0.12%) (Figure 10e). These results are according to those reported in the literature³⁶,⁷⁵-⁷⁷. All of the results are statistically significant (p-value < 0.05).

The variations observed in the mechanical properties of CS woven meshes occurred because the absorbed water molecules interfered with the intermolecular hydrogen bonds of the CS chains, causing an effect similar to that of a plasticizer additive, where the interactions between the CS chains were reduced, facilitating the sliding of the chains during the tractive efforts; reducing the elastic modulus and improving the CS ability to deform⁷⁸-⁸⁰.

The wet mechanical behavior of fibrous biomaterials is reported to be more important than the dry state⁸¹. In this sense, the developed CS woven meshes presented wet mechanical properties similar to those of the human dermis⁸², making them promising for application with dermal grafts or dressings for wound care.

3.4.3. Swelling degree

The swelling test results are presented in Figure 11. It is possible to observe that in the period of 1 h, woven meshes reach their maximum swelling degree, 220 ± 49%, after this period, the swelling degree will fall, oscillating around an average value, of 180 ± 13%. This equilibrium swelling is the result of osmotic force equilibrium, determined by the hydrophilicity and elasticity of the CS polymeric network⁴. The overall performance of a material is affected by its swelling behavior. The maximum swelling value reflects the material's fluid absorption capacity, which is essential for applications such as dressings that need to absorb wound exudates. The equilibrium swelling value suggests the stability of the material over time, ensuring that the biomaterial maintains its integrity and functionality during prolonged use without excessive expansion⁵²,⁸³,⁸⁴. The swelling of CS-based materials in aqueous solution is due to the formation of hydrogen bonds between the absorbed water molecules and the CS hydroxyl (-OH) and free amino (-NH₂) groups⁸⁵,⁸⁶. The results obtained here are in agreement with the reports in the literature⁴,⁸⁶,⁸⁷.

Figure 11
Swelling degree of CS woven meshes in PBS buffer solution.

3.4.4. Enzymatic biodegradation

The biodegradation behavior of CS woven mesh was evaluated in PBS solution buffer (pH 7.4) and PBS containing 1.5 µg/mL of lysozyme, for 5 weeks at 37 ± 0.5°C. Figures 12a, 12b, and 12c, respectively, show mass loss and morphology by SEM of the CS woven mesh.

Figure 12
Biodegradation behavior of CS woven meshes: a) Mass loss with degradation time. SEM images of the degraded CS woven meshes in: b) PBS-lysozyme solution and c) PBS, at 37 °C for 5 weeks (35 days).

In Figure 12a it is possible to observe the biodegradation behavior of CS woven meshes. In up to two weeks, only mass fluctuations occurred due to osmotic swings occurring in the swelling process and interactions between the degradation fluids and the amorphous and crystalline regions of CS woven meshes⁴. In this period, the mass variations of the degraded samples with and without the presence of lysozyme do not differ statistically (p-value > 0.05).

After three weeks, the mass loss increases for both degradation media. However, the mass loss was always higher for CS woven meshes incubated in PBS/Lysozyme solution, differing statistically (p-value < 0.05). In 5 weeks, final mass loss was 58.3 ± 4.23% for CS filaments in PBS solution and 80.3 ± 14.76% for samples in PBS/Lysozyme solution. It is noted that the enzyme accelerates the degradation of CS filaments⁶,⁸⁸-⁹¹.

The surface of CS woven mesh after 5 weeks (35 days) in both degradation media, PBS/Lysozyme, and PBS solutions are presented in Figures 12b and 12c, respectively. It can be observed a breaking in the integrity of the coating of the CS woven meshes, especially stricter for incubated samples in PBS/Lysozyme solution, due to the greater mass loss. Grooves and fissures in the CS filaments in the mesh are also observed, with the highest roughness demonstrated by the sample incubated in PBS/Lysozyme.

An important focus on the development and applications of biodegradable biomaterials is their metabolic fate⁹². Lysozyme is the major responsible for the degradation of CS in the human body, since it cleaves it into its constituent subunits, N-Acetyl-Glucosamine, and glucosamine, facilitating its absorption and/or elimination through normal metabolic pathways²¹,⁹³-⁹⁶.

The mechanisms by which lysozyme accelerates the degradation of chitosan filaments (CS) involve the cleavage of glycosidic bonds in the chitosan backbone, particularly between the N-acetylglucosamine units. This process is facilitated by the affinity of lysozyme for acetylated units⁹⁷,⁹⁸. Compared to the natural degradation process observed in biological systems, lysozyme-mediated degradation is more specific and controlled, whereas, in biological environments, chitosan can be degraded by a broader set of enzymes and hydrolytic processes, resulting in a more gradual and multifaceted degradation⁹³.

3.4.5. Cytotoxicity assay

The cytotoxicity of the CS woven mesh was evaluated by the agar diffusion method using L929. This method values the effects that the material causes on the cells through the agar layer, which allows the diffusion of chemicals from the sample to the cell layer⁵³.

The results obtained from the cytotoxicity assay are shown in Table 2 and Figure 13. The positive control presented a high decolorization index and cell lysis, being classified as severely cytotoxic (Table 2); moreover, it was observed in Figure 13a that cell decolorization, cell lysis, and a light red color in the agar were caused by the deposition of neutral red on the plate after the cell lysis and halo formation. In the negative control, it was verified the presence of the neutral red in the cell cytoplasm (Figure 13b), indicated no cell lysis and no halo formation, and was classified as noncytotoxic (Table 2). Halo formation was not observed for CS woven mesh, like this cell lysis, or decolorization (Figure 13c), as well as the negative control, and the CS woven mesh was classified as noncytotoxic (Table 2). The results found for CS woven meshes in this work are in agreement with other works found in the literature, where several CS-based structures were evaluated, including scaffolds⁹⁹ electroplated nanofibers¹⁰⁰,¹⁰¹, microspheres¹⁰², nanoparticles¹⁰³,¹⁰⁴, porous scaffolds¹⁰⁵, nanocomposite films¹⁰⁶, and sutures for surgical sutures⁶.

Table 2
Results of agar diffusion test.

Tested Materials	Reading the Plates Average (cm)	Decolorization	Lysis Index	Interpretation
Positive control	10.17	4	5	Severely cytotoxic
Negative control	0	0	0	Noncytotoxic
CS woven meshes	0	0	0	Noncytotoxic

Figure 13
Phase contrast microscopy images of L929 cells in the agar diffusion cytotoxicity test: a) C+ positive control, b) C– negative control, c) sample (chitosan woven mesh). All of the images were taken on the same magnification (100x).

Considering the promising biocompatibility results of CS woven meshes, additional in vitro and in vivo studies to evaluate the material's response in more complex biological environments are underway. Aiming to explore potential applications in tissue engineering and regenerative medicine, such as the development of dermal grafts, advanced dressings, and controlled release systems.

4. Conclusions

CS filaments were successfully obtained with improvements in mechanical properties by wet-spinning process step control. The coagulation profile of the CS filaments was determined, and the drying methodology was improved and efficient. The combination of the mechanical properties and morphology of CS filaments obtained by the improved methodology, together with the biological properties of CS was appropriate for the production of woven biotextiles for biomedical applications by the hand-weaving process. Obtained CS woven meshes exhibited controlled swelling, biodegradability, and biocompatibility, without of cytotoxic reactions to rat L929 fibroblasts, and their wet mechanical properties were compatible with the human dermis. Showing potential for application as dermal grafts or dressings for wound treatment. Thus, this study contributes significantly to the literature on CS-based biomaterials by demonstrating a methodology for obtaining CS filaments with mechanical properties adequate to the weaving process, for the manufacture of products for biomedical applications.

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Publication Dates

• Publication in this collection
  20 Dec 2024
• Date of issue
  2024

History

• Received
  04 Mar 2024
• Reviewed
  20 Aug 2024
• Accepted
  20 Sept 2024