Evaluation of Crosslinking Conditions on the Properties of Chitosan Woven Meshes

Abstract

Crosslinking of chitosan (CS) woven meshes is reported in this study. CS filaments were obtained by wet spinning and hand-waved. The weft was coated with CS solutions (3 and 4% w/v) and crosslinked with sodium tripolyphosphate or genipin. CS filaments were free from superficial pores, and their mechanical properties were suitable for the weaving process. CS concentration in the coating, type, and the crosslinking time affected the mesh morphology obtained. Crosslinking by both agents was confirmed by spectroscopy, corroborating the reduction of the hydrophilicity of the crosslinked samples in the swelling and contact angle tests. Crosslinking conditions were effective in reducing the mechanical properties variation in the wet state, as well as regulating the degradation rate of the samples. Furthermore, it was observed that crosslinking did not significantly affect the cell viability of CS woven meshes, making them promising materials for application as stress material in a physiological environment.

Keywords:
Chitosan; filaments; weaving; woven mesh; crosslinking

1. Introduction

The medical textile industry has grown considerably, driving research interests for different solutions in the health field, such as infection control, dressings, wound care products, and medical devices. Thus, there is a significant increase in the development of new fibers and new technologies for manufacturing yarns and fabrics. Many modern medical textile products are made from fiber components based on synthetic polymer, including polylactic acid (PLA), polycaprolactone (PCL), and polypropylene (PP); and natural ones such as collagen, chitin, and chitosan (CS)^1-4.

Chitosan is a natural polysaccharide obtained by chemical or enzymatic deacetylation of chitin. Its chemical structure consists of glucosamine and N-acetyl glucosamine units linked by β- glycosidic bonds (1 → 4). When the degree of deacetylation of CS reaches about 50%, it becomes soluble in an aqueous acidic solution^5,6. It has non-toxicity, biocompatibility, biodegradability, and hemostatic properties, and is easily processed when in weak acidic solutions, allowing the design of different configurations for the development of medical textile products^6-8, such as scaffolds^9,10, films^11-13, and filaments^14-16.

A common technique for developing CS filaments is wet spinning, which consists of extruding a polymeric solution into a non-solvent, promoting an acid-base neutralization^17,18. These filaments can be processed to result in fibrous textile structures, including fabrics, meshes, braids, and non-wovens, which can have an architecture designed to meet specific requirements for applications in the biomedical field^19-21. Neves et al.²¹ developed a fiber-mesh scaffold based on CS/PCL blends for cartilage repair from fiber production via wet spinning. Kim et al.²² produced fabric composites based on CS/hydroxyapatite (HAp) by wet spinning with potential properties for application in bone tissue engineering. Nowotny et al.⁹, by the wet spinning of CS fibers, produced braided scaffolds, achieving a simple approach to design tendon analogs. Yu et al.²³ developed a warp-knitted mesh from CS filaments for stress application in hernia repair.

In a previous work by our research group²⁴, mono and multifilament CS woven meshes were developed. It was observed that the mechanical and morphological properties are modulable based on the configuration of the yarn used in weaving. The authors observed that after hydration, the tensile strength of the monofilament mesh was reduced by 46.8%, while the deformation was increased by 30 times compared to the dry state and its degradation was 80.3% in 5 weeks, suggesting potential applications in the biomedical field, as dressings, or controlled drug release systems.

Mechanical properties of CS-based materials are reduced in the wet state due to the high hydrophilicity and, consequently, the swelling behavior¹⁴. These characteristics can be regulated through crosslinking processes²⁵. In this sense, the use of crosslinking agents such as sodium tripolyphosphate (TPP)^25-28 and genipin^29-31 is widely investigated. In addition, the adjustment of crosslinking process also promotes modulation of the degradation rate for specific applications^32-34.

To the best of our knowledge, the effects of crosslinking on the properties of CS woven meshes have not been investigated yet. In this sense, this work aims to develop crosslinked chitosan woven meshes, to evaluate the effect of crosslinking with TPP and genipin on their properties.

2. Experimental

2.1. Materials

Chitosan (medical degree) 96% deacetylated and molar weight of 310 KDa was produced by CERTBIO (Laboratory of Biomaterials Assessment and Development from Northeast). Phosphate buffer saline, lysozyme, Genipin, and sodium tripolyphosphate (TPP) were bought from Sigma Aldrich. Lactic acid and sodium hydroxide were acquired from Vetec.

2.2. Samples preparations

2.2.1. Filaments

CS filaments were produced according to the methodology reported by Silva et al.³⁵. CS solution with 4% (m/v) was prepared by dissolution of the polysaccharide in lactic acidic solution (1% v/v) under mechanical agitation (400 rpm) for 2 hours at 25 ± 1 °C. Then the CS solution was centrifuged to remove air bubbles and extruded with an infusion pump (Pump 11 Pico Plus Elite, Harvard Apparatus) through a die (45 mL/h, 1 mm diameter outlet tip) immersed in a coagulation bath containing 1 M sodium hydroxide solution. The solidified filaments were submerged in the coagulation bath for 1 h. After total coagulation, the threads were washed in distilled water to pH 7 and dried in a kiln with forced air circulation at 40 °C for 24 h.

2.2.2. Woven meshes

CS threads were used in a handmade weaving process, aiming the obtention of woven mesh materials according to the methodology reported in our other work²⁴. PET-G (glycol-modified poly (ethylene terephthalate) 3D-printed mold used for the weaving process is shown in Figure 1. The diameter and spacing between mold pins were 3 mm, and the edge was 6 mm (Figure 1b,c). After the weaving process, the CS woven meshes were impregnated with 5 mL of 3 and 4% CS solutions to keep the weft between the threads. The coated woven meshes were submerged in 1 M sodium hydroxide solution for 1 h to request the coating, then proceeded with washing for neutralization (pH 7) with distilled water and kiln drying (50 °C for 6 h). The CS solutions for the coating were prepared according to the methodology described above for the threads, varying only the CS concentration.

Figure 1
Views of the mold made by 3D printing for weaving of the CS meshes.

2.2.3. Crosslinked woven meshes

Coated dried CS woven meshes were crosslinked with 100 mL of aqueous solutions at 0.5% (m/v) for both crosslinking agents (genipin and sodium tripolyphosphate) at 15 and 30 minutes. Then, it was proceeded with washing with distilled water and kiln drying at 35 °C for 24 h. The solutions were prepared by adding the crosslinking agents to deionized water under magnetic stirring for 30 minutes. The samples were coded as shown in Table 1.

Table 1
Coding of prepared samples.

Woven mesh samples	CS coating concentration (%)	Crosslinking agent	Crosslinking Time (min)
3CS	3	-	-
3CST15	3	TPP	15
3CST30	3	TPP	30
3CSG15	3	Genipin	15
3CSG30	3	Genipin	30
4CS	4	-	-
4CST15	4	TPP	15
4CST30	4	TPP	30
4CSG15	4	Genipin	15
4CSG30	4	Genipin	30

2.3. Characterizations

CS filaments, non-crosslinked woven meshes, and all crosslinked samples were characterized by optical microscopy (OM) and mechanical tensile test. Some crosslinked samples were evaluated by FT-IR spectroscopy, contact angle, in vitro biodegradation, swelling, and in vitro cytotoxicity tests.

An optical microscope (Hirox Digital Microscope, Kh 1,300 M, Tokyo, Japan) was used to obtain OM images of the CS filaments and woven meshes. The openings of the meshes were evaluated using ImageJ software.

Uniaxial tensile tests were used to characterize the mechanical properties of CS filaments (n = 10) and woven meshes (n = 5). Experiments were conducted on universal mechanical properties testing apparatus (Instron Model 6633), equipped with a load cell of 500 kN, at 24 ± 2 °C and relative humidity of 60% ± 2%. The traction velocity was 120 mm/min for filaments and 100 mm/min for woven meshes samples. The specimens were 100 mm for filaments and 60 mm x 10 mm. FT-IR analyzes were performed on a Perkin Elmer spectrophotometer Spectrum 400 (4000 – 650 cm^-1).

For contact angle tests, was used a goniometer developed by the Mechanical Engineering Academic Unit at UFCG (Federal University of Campina Grande). Deionized water was utilized as a contact liquid. Photographs were taken after 10 seconds of liquid contact with the sample surface (n = 5) and the measurements were performed with Angle Calculator software.

The swelling behavior of the CS woven meshes was evaluated after 24 h of contact with phosphate buffer solution (PBS, pH = 7.34) at 37 ± 0.5 °C, besides that, mechanical properties were too evaluated in the wet state, according to the conditions described above for uniaxial tensile tests. The samples were weighed (n = 5) in dry (W_d) and wet conditions (W_w) for swelling degree calculation according to Equation 1.

$Swellingdegreein24h\left(\text{\%}\right)=\frac{W_w-W_d}{W_d}x100\text{\%}$ (1)

Biodegradation of the CS woven meshes was evaluated through weight loss in phosphate buffer solution (pH = 7.34) at 37 ± 0.5 °C containing lysozyme at 1.5 µg/mL^24,36. The samples were dried at 50 °C for 6 h, weighed (W₀), and conditioned at degradation medium. At each biodegradation period (1, 2, 3, 4, and 5 weeks), the CS woven meshes samples were taken from the solution, washed in distilled water, pre-dried with absorbent paper, and then weighed (W_t). The biodegradation results were obtained from Equation 2.

$Weightloss\left(\%\right)=\frac{W_0-W_t}{W_0}x100\%$ (2)

In vitro cytotoxicity of the CS woven meshes was evaluated using MTT cell availability evaluation test according to ISO 10993 - 5: 2009, with direct contact between samples and L929 fibroblasts cells. The Grubbs test was performed for outliers. The calculation of the standard deviation was performed by Graph Pad Prism 6. The spectrophotometer used to read the viability of the cells was the Victor X3 device, Perkin Elmer, with the measurement wavelength at 570 nm and the reference wavelength at 650 nm.

3. Results and Discussions

3.1. CS filaments preparation

3.1.1. Morphology

Figure 2 presents OM images of CS filaments. A relatively smooth and compact cylindrical monofilament morphology is observed, without surface pores and an average diameter of 0.180 ± 0.013 mm (n = 5). This morphology provides a uniform distribution of stresses along the wire³⁷. There is also the presence of longitudinal grooves, probably due to the outlet tip or orientation of the polymer chains of chitosan caused by the wet spinning process³⁸. In the image of the cross-section of the filament, it is observed an almost circular geometry with a rough surface resulting from the fracture and the presence of pores inside the filament.

Figure 2
OM images of 4% CS filament obtained by wet spinning. Increases of a) 350x the longitudinal section, b) 700x the longitudinal section and c) 1050x the cross-section of the filament.

3.1.2. Mechanical properties

Table 2 shows the mechanical properties of CS filaments obtained by wet spinning. These properties are essential to determine the performance of the treads in the weaving process and, consequently, of the final product. The results obtained here were similar to those reported by Silva et al.^36,39 in his work with chitosan threads for application as surgical suture material.

Table 2
Mechanical properties of CS filament obtained by wet spinning.

Maximum load (N)	6.2 ± 0.42
Tensile strength (MPa)	248 ± 12.61
Strain (%)	2.3 ± 0.18
Youngs Module (GPa)	29.1 ± 3.00

3.2. CS woven meshes preparation

Figures 3a and 3b show OM microscopy of the CS woven meshes coated with 3 and 4% CS solutions, respectively.

Figure 3
OM images of CS woven meshes. a) coated with 3% CS solution, b) coated with 4% CS solution.

Uniform coatings throughout the length of the samples can be observed, showing that both CS solutions, 3 and 4%, were efficient in producing homogeneous coatings. It is also possible to observe marks on the coating at higher magnifications, probably due to the accumulation of solution near the wires. There is a typical morphology of woven structures in the 1/1 ratio (flat fabric)^40-42. In addition to the appearance and uniformity of the coating, the openings of CS woven meshes were 7.5 ± 0.38 and 7.3 ± 0.55 mm² for meshes coated with the 3 and 4% CS solutions, respectively.

3.3. Crosslinked CS woven meshes preparation

3.3.1. Morphology

Figure 4 shows the CS woven meshes after crosslinking with different agents (Genipin and sodium tripolyphosphate) and times (15 and 30 minutes). Sodium tripolyphosphate crosslinked samples showed more uniform morphology than those crosslinked with Genipin, and only the 3CSG15 sample did not show retraction. The retraction and the increase in surface roughness are in agreement with that reported by Hillberg et al.⁴³ for materials crosslinked with genipin. Furthermore, it is notable that the genipin crosslinked samples were dark, in bluish-green tones, characteristic of this reagent⁴⁴.

Figure 4
OM images of CS woven meshes crosslinked with TPP and genipin at different times.

In images magnified by OM, it is possible to observe that the samples obtained with 3% CS coating had fewer rough surfaces compared to those obtained with 4% CS coating, for both crosslinking agents, in particular for the longer crosslinking time (30 minutes). This is probably due to the high viscosity of the more concentrated chitosan solution, which prevented the formation of a more homogeneous film on the filaments⁴⁵.

3.3.2. Mechanical properties

Results of mechanical tests are present in Figure 5. It is possible to notice a growing tendency in the capacity of the woven meshes to support the load with the increase of the CS concentration in the coating. This effect is due to the greater entanglement of polymer chains when more materials are deposited on the filaments^46-48. Crosslinked samples showed a drop of maximum load compared to those uncrosslinked. The strain and tensile strength of CS woven meshes decrease with crosslinking for all tested conditions due to the restriction of the polymeric chains by the introduction of crosslinks^48,49. These results corroborate with the studies reported by Butler et al.⁴⁴, Hwang and Shin⁴⁷, Wyatt et al.⁴⁸, Mi et al.⁵⁰, and Cui et al.⁵¹.

Figure 5
Mechanical properties of crosslinked and uncrosslinked CS woven meshes a) maximum load; b) tensile strength and c) strain.

The samples 3CS, 3CST15, 3CST30, 3CSG15, 4CS, and 4CST30 were the ones that showed higher tensile strength in the dry state, and therefore were selected for the subsequent characterizations.

3.3.3. FT-IR spectroscopy

Analysis of CS woven meshes by FT-IR spectroscopy was performed to evaluate its crosslinking with TPP and genipin. FT-IR spectra are shown in Figure 6a. It is observed that there were interactions between CS and both crosslinking agents, evidenced by the intensity reduction of the hydroxyl and amine bands at 3330 cm^-1 and protonated amine in 1078 cm^-1, indicating a decrease in hydrogen bonds⁵².

Figure 6
FT-IR spectra of crosslinked and uncrosslinked CS woven meshes a) from 4000 to 650 cm^-1; b) from 1800 to 1500 cm^-1 and c) from 1200 to 800 cm^-1.

The peaks intensity reductions around 1577 and 1028 cm^-1 are due to the bonds formed between amino groups of CS and carboxymethyl units of genipin, forming secondary amide^53-55. The effect occurs similarly for the bands located between 3361.42 and 2876.76 cm^-1 since the bonds formed by hydroxyls are also preferential points of chemical crosslinking⁵⁶.

The spectra of the samples with and without crosslinking showed the characteristic absorption bands of CS. At 3330 cm^-1 related to the superposition of the elongation vibrations of the amine and hydroxyl group, peaks around 1575 cm^-1 associated with the protonated amino group, and in 1078 and 1025 cm^-1 resulting from the elongation of the C-N and C-O bonds, respectively⁵¹.

Through the detail presented in Figure 6b, the reduction in band intensity at 1575 cm^-1 can be interpreted as the formation of secondary amide after the protonated amine is consumed by reaction with the genipin ester groups⁴⁴. The increase in the intensity of the vibration band of the C-N bond at 1070 cm^-1 suggests the formation of crosslinked compounds⁵¹.

In Figure 6c, the complexation of chitosan with TPP molecules is suggested by the higher peak intensities at 1150 cm^-1 attributed to the P-O bond and at 892 cm^-1 related to the asymmetric stretching of the P-O-P bond⁵⁷.

3.3.4. Swelling behavior

The Swelling degree (%) of CS woven meshes are shown in Figure 7. The uncrosslinked samples exhibited the highest swelling degrees, followed by those crosslinked with sodium tripolyphosphate and genipin, respectively. On the other hand, the sample 3CS presented the lowest swelling degree, probably due to its higher degree of crosslinking, as suggested by the FT-IR results.

Figure 7
Swelling degree of CS woven meshes crosslinked with TPP and genipin at different times.

CS-based materials swell through hydrophilic interactions (hydrogen bridges) between water molecules and the chitosan amine and hydroxyl groups. As the crosslinking degree increases, the functional groups available for interactions decreases. Furthermore, the crosslinks limit the expansion of the polymeric network, hindering the absorption of fluids^58-60. Thus, the amount of fluid absorbed (degree of swelling) must be inversely related to the crosslinking degree of the samples, as reported by Klein et al.⁶¹.

The effect of swelling on the mechanical properties of CS woven meshes was also investigated. The results are shown in Table 3. After swelling, the uncrosslinked samples presented the lowest maximum loads, which shows the improvement of this property by the crosslinking process. Among the crosslinked samples, those that received the sodium tripolyphosphate treatment performed better than those treated with genipin. The swelling promoted a reduction in tensile strength and an expressive gain in deformation of all samples. However, in the wet state, the sample crosslinked with genipin (3CSG15) showed greater tensile strength than the sample without crosslinking (3CS) and lower deformation capacity than the sodium tripolyphosphate crosslinked samples.

Table 3
Mechanical properties of the CS woven meshes crosslinked with TPP and genipin at different times in the dry and wet state.

Sample	State	Maximum load (N)	Tensile strength (MPa)	Strain (%)
3CS	Dry	21.1 ± 1.38	8.6 ± 0.94	3.4 ± 0.95
	Wet	8.1 ± 0.73	4.1 ± 0.68	32.2 ± 0.38
4CS	Dry	25.2 ± 1.25	6.3 ± 0.41	1.8 ± 0.43
	Wet	9.2 ± 0.16	4.2 ± 0.71	29.3 ± 0.73
3CST15	Dry	21.3 ± 1.04	9.1 ± 2.16	1.6 ± 0.12
	Wet	16.3 ± 0.06	3.9 ± 0.03	16.1 ± 1.6
3CST30	Dry	17.5 ± 1.96	8.1 ± 1.07	1.0 ± 0.20
	Wet	12.8 ± 0.16	3.6 ± 0.06	26.2 ± 2.71
3CSG15	Dry	15.9 ± 1.82	6.5 ± 0.66	1.3 ± 0.14
	Wet	9.4 ± 0.82	4.4 ± 0.06	6.2 ± 0.96
4CST30	Dry	22.8 ± 1.14	6.6 ± 0.79	0.8 ± 0.21
	Wet	15.3 ± 0.25	3.1 ± 0.03	22.7 ± 1.5

• Mean ± Standard deviation.

Table 4 presents the variations (%) of the mechanical properties when going from the dry to the wet state. Crosslinking with sodium tripolyphosphate and genipin reduced the decrease in maximum load by swelling the samples. Variations in tensile strength and deformation appear to have been unaffected by crosslinking.

Table 4
Variation of the mechanical properties when going from dry to a hydrated state.

Sample	Maximum load	Tensile strength	Strain
3CS	-62	-52	847
4CS	-96	-33	1528
3CST15	-23	-57	906
3CST30	-27	-56	2520
3CSG15	-41	-32	377
4CST30	-33	-53	2738

According to Wu et al.⁶², the wet mechanical behavior of biomaterials is more important than the dry state. The water molecules absorbed by the samples interfere with the intermolecular hydrogen bonds of the CS chains. The intermolecular interactions are reduced, facilitating the sliding of the chains and improving the polymers ability to deform^63-65. This behavior in the wet state is reported in the literature by other authors^66-68.

3.3.5. Contact angle

Figure 8 shows the result of the contact angle test. According to Sengupta and Pittman⁶⁹, the wettability of a solid surface decreases as the contact angle with the water drop increases. Angles smaller than 90° mean that the liquid tends to wet the sample, therefore, the surface is hydrophilic. In this case, the investigated samples have a hydrophilic character, as they presented contact angles below 90°.

Figure 8
Contact angle of the CS woven meshes crosslinked with TPP and genipin at different times.

Uncrosslinked samples had the smallest contact angles. That is, they were more hydrophilic than crosslinked samples. There was a decrease in the availability of polar groups in the crosslinked samples, decreasing their polarity and wettability. These results are in accord with those reported by Jin et al.⁷⁰, Karbasi et al.⁷¹, and Tabriz et al.⁷².

3.3.6. Biodegradation

The enzymatic biodegradation profiles of the samples with and without crosslinking are shown in Figure 9. Up to two weeks, all samples showed only mass fluctuations related to swelling and interactions between the degradation fluid and the amorphous and crystalline regions of the samples²⁴. After two weeks, the mass loss starts for most of them. The 3CS and 3CST15 samples had the highest biodegradation rates, followed by the 3CST30 one. The 3CSG15 sample showed a lower degradation rate, with practically constant mass for 4 weeks, showing that crosslinking with genipin was efficient in delaying the biodegradation process of the CS woven meshes.

Figure 9
Mass variation of the CS woven meshes crosslinked with TPP and genipin at different times.

3.3.7. In vitro cytotoxicity

Figure 10 shows the cell viability of the uncrosslinked and crosslinked CS woven meshes. Following ISO 10993-5, all samples demonstrated to be biocompatible, with cell viability higher than 70%. The biocompatibility of chitosan-based materials is widely reported in the literature. Several structures were evaluated, including electrospun nanofibers^73,74, nanoparticles^75,76, microspheres⁷⁷, and porous scaffolds⁷⁸. The crosslinking process did not significantly affect the biocompatibility of the samples, corroborating the works reported by Mi et al.⁷⁹, Bi et al.⁸⁰, Zhang et al.³³, and Dimida et al.³².

Figure 10
Cell viability of the CS woven meshes crosslinked with TPP and genipin at different times.

4. Conclusions

Crosslinked CS woven meshes were obtained from the manual weaving of CS filaments. The crosslinking conditions affected the morphology and mechanical properties of the woven meshes. The effective crosslinking by genipin and TPP was proven by spectroscopy. Crosslinking increased the mechanical stability in the wet state and promoted the modulation of swelling and biodegradation. In addition, it did not significantly affect the hydrophilicity and biocompatibility of CS woven meshes. Thus, the materials developed in this work are promising for stress application in a physiological environment.

5. Acknowledgments

The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Brazil) for financial support, Federal University of Campina Grande - UFCG (PB, Brazil) and Northeast Biomaterials Evaluation and Development Laboratory - CERTBIO for supporting the execution of this work.

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Publication Dates

• Publication in this collection
  18 May 2022
• Date of issue
  2022

History

• Received
  17 Sept 2021
• Reviewed
  07 Dec 2021
• Accepted
  13 Jan 2022