Wound healing properties of triple cross-linked poly (vinyl alcohol)/ methacrylate kappa-carrageenan/chitooligosaccharide hydrogel

Pathum Chandika a, Min-Sung Kim a, Fazlurrahman Khan b, Young-Mog Kim b,c, Seong-Yeong Heo b, Gun-Woo Oh b, Nam Gyun Kim a, Won-Kyo Jung a,b,*

Abstract

To develop an effective and mechanically robust wound dressing, a poly (vinyl alcohol) (PVA)/methacrylate kappa-carrageenan (κ-CaMA) composite hydrogel encapsulated with a chitooligosaccharide (COS) was prepared in a cassette via repeated freeze/thaw cycles, photo-crosslinking, and chemical cross-linking. The chemical, physical, mechanical, in vitro biocompatibility, in vivo wound-healing properties, and antibacterial activity of triple-crosslinked hydrogel were subsequently characterized. The results showed that the PVA/κ-CaMA/COS (PκCaC) hydrogel had a uniformly thick, highly porous three-dimensional architecture with uniformly distributed pores, a high fluid absorption, and retention capacity without disturbing its mechanical stability, and good in vitro biocompatibility. Macroscopic images from the full-thickness skin wound model revealed that the wounds dressed with the proposed Pκ-CaC hydrogel were completely healed by day 14, while the histomorphological results confirmed full re-epithelization and rapid skin-tissue remodeling. This study thus indicates that the composite Pκ-CaC hydrogel has significant potential for use as a wound dressing.

Keywords:
PVA
Methacrylate kappa-carrageenan
Chitooligosaccharide
Hydrogel wound dressing
Wound healing

1. Introduction

The largest organ of the human body is the skin, which acts as the first line of defense but is vulnerable to various types of damage that can potentially compromise the protective barrier against microbial invasion and lead to a loss of body fluids and electrolytes (Zhao et al., 2017). The various metabolites that appear during the early stages of wound healing, such as tissue fluid, proteins, and dead cells, increase the chances of microbial infection and further complicate the healing process. An ideal wound dressing thus acts as a protective barrier against further injury and microbial invasion while also absorbing wound exudate, cleaning up metabolites, and providing a gas-exchange system. It also needs to exhibit appropriate anti-inflammatory, antioxidant, and antibacterial activity in addition to flexibility and mechanical stability (Chen et al., 2019; Zheng et al., 2019).
Hydrogels have excellent properties for wet wound healing, such as favorable fluid absorption and extreme water retention ability due to their highly interconnected porous framework. As a result, the development of new natural-synthetic composite hydrogels paves the way for tunable porous architecture, mechanical and physicochemical properties, and excellent biocompatibility and bioactivity attracted by natural polymers.
Hydrogel wound dressings are generally prepared using simple and non-toxic physical and chemical cross-linking methods, such as ultraviolet (UV) radiation and repeated freezing and thawing. In particular, a technique that has become widespread is photo-crosslinking, which is a form of chemical cross-linking that creates stable covalent bonds between polymer chains in the presence of a photoinitiator and UV radiation (Ou et al., 2017; Yue et al., 2015).
Kappa-carrageenan (κ-Ca) is a water-soluble natural linear sulfated polysaccharide isolated from red seaweed species of the Rhodophyta class, specifically Kappaphycus alvarezii. The relatively low cost and attractive biomimetic properties such as excellent gel-forming ability, high fluid absorption, and retention capacity, biocompatibility, biodegradability, ability in drug delivery (Popa et al., 2011; Yegappan et al., glycosaminoglycan, which is the main component of natural extracellular matrices (Mokhtari et al., 2019) promoted the use of κ-Ca in the fabrication of hydrogels using alone or in the form of composite with different natural and synthetic polymers. κ-Ca consists of one sulphate group per disaccharide unit and gelates upon cooling in the presence of potassium salt as a result of ionic interactions and hydrogen bonds, leading to coil–helix conformational changes and the formation of a uniform hydrogel that is unstable at high temperatures (Chronakis et al., 2000; Tanusorn et al., 2018). However, it has been suggested that methacrylate kappa-carrageenan (κ-CaMA) can be used to fabricate hydrogels with increased mechanical stability by combining ionic and photochemical cross-linking (Tytgat et al., 2019), but it is rigid in design, limiting direct use as a wound dressing. To resolve this issue, the incorporation of a synthetic polymer such as PVA could be employed to develop appropriate hydrogels (Kalantari et al., 2020; Zhang, Jiang, et al., 2019).
PVA hydrogels are typically physically cross-linked and flexible and are produced by creating cross-links between crystalline clusters via repeated freeze/thaw cycles. PVA is currently one of the most frequently employed synthetic polymers for wound management, drug delivery, and other tissue-engineering approaches due to its advantageous characteristics, such as biocompatibility, biodegradability, appropriate mechanical properties, formation of hydrogen bonds, and semi-crystalline nature. However, it’s inadequate swelling, low bioactivity, hydrophilicity, and resistance to protein adsorption compared to natural polymers has limited the use of PVA as a wound-dressing hydrogel on its own. To overcome these inherent disadvantages of PVA and to enhance the mechanical characteristics of natural polymers, blending the two has been proposed by many researchers (Kamoun et al., 2017; Zhang, Liu, et al., 2019).
Water-soluble and low-molecular-weight chitooligosaccharides (COSs) exhibit favorable biocompatibility for the wound-healing process, with a diverse range of antibacterial, anti-inflammation, anti- oxidant, and anti-tumor activity. In a moist environment, COSs are more efficient for wound healing than chitosans due to their rapid interaction with the wound with the right cells (Jafari et al., 2020; Luo et al., 2017). As a wound-healing substitute, COSs enhance the expression of transforming growth factor-beta (TGF-β) by triggering the TGF-β/Smad signal transduction pathway, leading to the transformation of pro- inflammatory macrophages into anti-inflammatory macrophages. This results in the stimulation of TGF-β and enhances fibroblast and keratinocyte proliferation and migration, facilitating rapid re-epithelization and wound healing. In addition, COSs increase the formation of blood vessels by increasing the production of vascular endothelial growth factors (VEGF) (Jafari et al., 2020). Hence, when used as a component of a wound-dressing hydrogel, COSs can play a significant role in accelerating wound healing and preventing bacterial invasion from the surrounding environment while acting as an anti-inflammatory agent.
In the present study, we report modified biopolymer (k-CaMA) and synthetic polymer (PVA) based composite wound dressing hydrogel encapsulated with COS (denoted as Pκ-CaC hereafter) prepared using non-toxic physical, photochemical and ionic cross-linking due to their high biological acceptance. The proposed hydrogel has a better pore structure, better fluid absorption, retention capability, and adequate mechanical stability. Furthermore, direct and indirect cytotoxicity, antibacterial activity, in vivo wound closing, and histomorphological assessment were carried out to confirm the suitability of the hydrogel in wound healing applications.

2. Materials and methods

2.1. Materials

Low molecular weight (MW) COS (1-3 kDa) was obtained from Kitto life Co. (Seoul, Korea). κ-Ca (sulfated linear plant polysaccharide, composed of repeated 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-anhydro-D-galactopyranose units), PVA (MW 89,000-98,000; 99 + % hydrolyzed), methacrylic anhydride (MA), 2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone photo-initiator Irgacure 2959 (PI), fluorescein diacetate (FDA), 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), deuterium oxide (D2O), 2,2- diphenyl-1-picrylhydrazyl (DPPH), Lipopolysaccharide (LPS), were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Hoechst 33, 342 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS), trypsin (250 U/mg), penicillin/streptomycin, Dulbecco Modified Eagle Medium (DMEM), and other materials used in cell culture were purchased from GIBCO™, Invitrogen Corporation, USA. Water used for the research was deionized, and all other chemicals and solvents used for the study were of analytical grade.

2.2. Preparation of triple cross-linked PVA/κ-CaMA/COS hydrogel

5% PVA and freeze-dried κ-CaMA (synthesis and characterization of κ-CaMA can be found in supplementary information S1.1) were prepared in DW at 80 ◦C and 50 ◦C respectively. Both solutions in different ratios were mixed with 1% (w/v) COS and 0.25% (w/v) PI using a magnetic stirrer at room temperature (Table 1). The homogeneous Pκ- CaC solutions were then injected into a 1 mm thick gel cassette and freeze in − 20 ◦C for 18 h and subsequently thawed under UV (365 nm, 4nW/cm2) for 30 min. κ-CaMA monomers in hydrogel cassettes where photo cross-linked by UV exposure at 365 nm, 4nW/cm2 for both sides of cassettes for 30 min at room temperature, and only Pκ-CaC cassettes were subsequently thawed for 6 h. Then hydrogels were subjected to three more freeze-thaw cycles (18: 6 h), excluding the photo cross- linking step. To obtain triple cross-linked hydrogels, the duel cross- linked hydrogels were de-cassette and cross-linked using 5% (w/v) KCl solution.

2.3. Physico-chemical characterization of the hydrogel

2.3.1. Microstructural characterization

The cross-sectional architecture of freeze-dried hydrogels was examined and micro-graphed using a scanning electron microscope (FE- SEM, Hitachi S-2700, Japan) at 5 kV after the sputter-coated with a thin layer of silver using Emi-Tech K500X. The images were analyzed by ImageJ (ImageJ Wayne, Rsband), and pore diameters were calculated from the given formula assuming that the pores are circular, where D and A represent the diameter and area of the pore, respectively.

2.3.2. Porosity

The ethanol infiltration method was used to measure the porosities of fabricated hydrogels. Freeze-dried hydrogels were immersed in absolute ethanol at room temperature under reduced pressure to remove air bubbles and allow to fill ethanol for 5 h, removed, and wipedout surface ethanol. The porosities were calculated by the equation where W0 represents freeze-dried weight, W1 represents ethanol infiltrated hydrogels weight, ρ represents the density of ethanol (0.789 mg/mL), and VS represents geometric volume.

2.3.3. Fourier transform infrared spectroscopy (FTIR) analysis

FTIR spectroscopy was used for chemical characterization of pure materials, κ-CaMA, and developed hydrogels. All spectra were recorded at a resolution of 4 cm− 1 in the frequency of 4000–650 cm− 1 at room temperature using (FTIR–Perkin–Elmer Spectrum, GX Washington DC, USA) spectrometer.

2.3.4. X-ray diffraction analysis (XRD)

Pure biomaterials and composite Pκ-CaCs were characterized by X- ray diffraction (Philips X’pert MPD diffractometer, Eindhoven, Netherland) with CuKα radiation generated at 36 kV and 200 mA; operated with the range of the diffraction angle (2θ) was 10 to 60◦ at a scanning rate of 6◦/min.

2.3.5. Thermal properties

Thermal properties of the PVA and composite hydrogels were measured by the Thermogravimetric analysis (TGA) Pyris 1 TGA analyzer (Perkin Elmer TGA-7, Waltham, MA, USA) within the temperature range of 50 to 700 ◦C at a constant heating rate of 10 ◦C/min under nitrogen gas. The melting behavior of the hydrogels was measured using differential scanning calorimetry (DSC) (Perkin Elmer, Diamond, Waltham, MA, USA) under a healing rate of 10 ◦C/min and argon gas flow rate of 50 mL/min within temperature range from room temperature to 250 ◦C.

2.3.6. Gel fraction

Lyophilized hydrogels (0.8 mm in diameter) were weighted (Wi) and subsequently submerged in 1× phosphate buffer saline (PBS) (pH 7.4) during 24 h at 37 ◦C. After equilibrium swelling, hydrogels were lyophilized and weighted (Wf). The gel fraction was calculated using the following formula. Gel fraction (%) = Wi Wf ×100

2.3.7. Swelling properties
L
yophilized hydrogels were immersed in 1× PBS (pH 7.4) at 37 ◦C until they reached equilibrium. The surface wetness was removed using blotting paper and kept 1 min to further remove surface moisture and measure the weight. The equilibrium-swelling percentages were calculated by the following formula, where Wd and Ws represent the weight of the lyophilized and swollen state of the scaffolds.

2.3.8. Water retention properties

Lyophilized hydrogel reaching its equilibrium swelling in 1× PBS (pH 7.4), kept under open air (relative humidity ≈ 40%) at 37 ◦C for different times. The water retention ratios were calculated according to the equation, where Ws refers to the weight of swollen hydrogel at a specific time, Wd is the weight of the dried hydrogel, and Wi refers to the weight of the fully swelled hydrogel.

2.3.9. Releasing behavior

The releasing behavior of hydrogels was carried out by phenol- Sulfuric assay. Hydrogels were immersed in 1× PBS at 37 ◦C, and dynamics of release were recorded at 490 nm using a microplate reader (Gen 5™ ELISA BioTek, USA) and calculated based on the absorbance of a standard curve.

2.4. Mechanical behavior

2.4.1. Rheological measurements

The dynamic rheological experiments were performed using Discovery HR-2 hybrid Rheometer with 8 mm diameter parallel plate geometry (TA instrument, USA). Hydrogels were loaded with a static load (0.05 N) and deformed at constant amplitude over the dynamic frequency range of 0.1 Hz to 10 Hz and obtained storage and loss modules at a proper strain (5%) at 25 ◦C.

2.4.2. Compressive strength

The tompressive strength of hydrogels was carried out using a universal tensile machine (UTM) (LR5K Plus, Lloyd Instruments). Cylindrical hydrogels with 5 mm height and 8 mm diameter were mounted and preloaded with 0.1 N and subsequently subjected to 20 mm/min crosshead speed at room temperature.

2.5. Indirect and direct cell cytotoxicity

In direct cell cytotoxicity of hydrogels was performed using HDF and HaCaT cells in DMEM supplemented with 10% FBS and 1% penicillin/ streptomycin in an incubator with a humidified atmosphere of 5% CO2 at 37 ◦C according to the International Organization for Standardization (ISO) 10993-5 (Iso, 2009). The detailed procedure can be found in the Supplementary information S1.2.

2.6. Antibacterial activity

Staphylococcus aureus (KCTC 1916), obtained from the Korean Collection for Type Cultures (KCTC, Daejeon, Korea), and Staphylococcus epidermidis (KCCM 40003), obtained from the Korean Culture Center of Microorganisms (KCCM, Sudaemun Seoul, Korea), were cultivated in tryptic soy broth (TSB; Difco Laboratory Inc., Detroit, MI, USA) overnight. The bacterial cell cultures were diluted (1:100) in TSB to achieve an OD of 0.05 at 600 nm. The diluted cell culture was placed in a tube containing the hydrogel specimens and incubated at 37 ◦C for 24 h. After incubation, the cell culture was serially diluted in sterile TSB to a dilution of 10− 8 and spread (100 μl) plated on a tryptone soya agar (TSA) plate. The plates were subsequently incubated at 37 ◦C to allow colonies to form, which were then counted as colony-forming units (CFUs).

2.7. In vivo animal experiment

In vivo wound healing studies were conducted on the 8-week old normal male ICR mice weighing 30 g after acclimatizing for 7 days under a controlled environment (relative humidity: 40–70%, temperature: 20–24 ◦C), according to the protocol approved by the Institutional Animal Care and Use Committee using 6 mice per group. The dorsal surface hair was shaved, and povidone‑iodine and 70% ethanol was used to disinfect the skin surface. Full-thickness skin excision wounds were created at the dorsal surface using 5 mm biopsy punch. Created wounds were dresses with epiderm, PVA, Pκ-CaC 8:2, Pκ-CaC 6:4, and secondary all groups were secured with transparent tegaderm and gauze. All experimental procedures were performed with the mice under general anesthesia with inhaled 20% isoflurane in isopropanol. Wound closure of all experimental groups was photographed using a digital camera at 0, 2, 4, 5, 7, and 14 days post-surgery, and wound closure was plotted as a relative percentage of initial wound size. The wound site and surrounding tissues were collected, and a histomorphological evaluation of sectioned tissue samples were also conducted flowing the method described in Supplementary information S1.3.

2.8. Statistical analysis

All quantitative data are presented as the mean ± standard deviation (SD), with at least three individual experiments conducted using fresh reagents. Significant differences between the groups were assessed using one-way ANOVA followed by Duncan’s test using PASW Statistics 21.0 (SPSS Inc., Chicago, IL, USA). The differences were considered statistically significant at p < 0.05.

3. Results and discussion

3.1. Physicochemical characterization of the hydrogel specimens

3.1.1. Microstructural characterization

SEM micrographs were used to investigate the microstructural morphology of the PVA and triple cross-linked composite hydrogel specimens. As shown in Fig. 1A, under optimal hydrogel-processing conditions, slight and uniform shrinkage was observed for the freeze- dried hydrogel specimens compared to their initial loading thickness in ascending order from PVA to Pκ-CaC 2:8. The magnified SEM micrographs presented in Fig. 1B illustrate the highly interconnected, uniformly porous architecture of all of the hydrogel specimens. PVA base hydrogel demonstrates deformation of polymer network and irregular arrangements after freeze dry and does not revert back to the initial state after re-absorption of water (Bahadoran et al., 2020). However, the present study has shown a highly uniform architecture after freeze- drying because of the casting methods. The pure PVA hydrogel exhibited a denser and more homogeneous porous structure than the other specimens, with a pore size of 1002.5 ± 762.7 μm2, while the average pore size was 1308.9 ± 968.5, 1486.4 ± 1016.8, 1607.3 ± 1240.4, and 1935.1 ± 1476.8 μm2 for the composites PCOS, Pκ-CaC 8:2, Pκ-CaC 6:4, and Pκ-CaC 4:6, respectively. Pκ-CaC 2:8 exhibited an extremely large pore size of 15,517.9 ± 11,976.9 μm2. This increase in pore size can be due to a substantial reduction in physical cross-linking between PVA while maintaining photo- and ionic cross-linking within the κ-CaMA. As a result, the polymer solution's loading thickness remained constant after cross-linking. The pore size distribution also illustrates the shift towards a larger pore size with an increase in κ-CaMA in the composite hydrogel specimens (Fig. 1C).
The porous structure of a hydrogel uptakes the exudate leaking from the wound surface and helps to diffuse nutrients and healing promoters to the wound site while maintaining a suitably moist environment (Oh et al., 2020). Fig. 2 shows that adding κ-CaMA to the PVA hydrogel increased the porosity of the PVA (65.7 ± 2.1) to 68.1 ± 2.5, 71.3 ± 1.3, 75.4 ± 7.3, and 88.5 ± 2.0 for the Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ-CaC 4:6, and Pκ-CaC 2:8 hydrogel specimens, respectively. Overall, this shows that the presence of κ-CaMA loosens the hydrogel structure due to an increase in photo- and ionic cross-linking with an increase in κ-CaMA, suggesting that high levels of κ-CaMA could reduce the structural and mechanical stability of the resulting hydrogel, making it unsuitable for use in wound dressing.

3.1.2. NMR and FTIR spectroscopy

The κ-Ca was modified by substituting the hydroxyl groups with methacrylate from MA in order to facilitate subsequent UV-mediated cross-linking. 1H NMR spectroscopic analysis (Fig. 3A) of the κ-CaMA confirmed the modification of κ-Ca via the presence of methacrylate moieties with a characteristic double peak at 6.3 and 5.9 ppm corresponding to the vinyl protons of the introduced methacrylate. The single peak at 2 ppm corresponded to the methyl protons of the methacrylate group, indicating the successful modification of κ-Ca, which is in agreement with a previous report (Mihaila et al., 2013). The functional groups of pure PVA, κ-Ca, COS, synthesized κ-CaMA, and the composite hydrogel specimens PCOS, Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ-CaC 4:6, and Pκ- CaC 2:8 were then characterized by FTIR (Fig. 3B). The detailed results and discussion were presented in the Supplementary information (S2.1).

3.1.3. X-ray diffraction and thermal properties

The diffraction patterns of neat PVA, κ-Ca, COS, synthesized κ-CaMA, and the composite hydrogel specimens PCOS, Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ-CaC 4:6, and Pκ-CaC 2:8 are displayed in Fig. 4A. The XRD profile for PVA shows a characteristic semi-crystalline diffraction peak at 2θ = 19.5◦ due to the diffraction of the lattice planes as a result of the strong intermolecular and intramolecular hydrogen bonding between the PVA chains. The diffractograms for κ-Ca, COS, and modified κ-CaMA exhibit a weak broad peak near 2θ ≈ 20◦, indicating that they are all amorphous in nature (Pas¸calau et al., 2012˘ ). In the composite specimens, the peak at 2θ = 19.5◦ attributable to the diffraction of the PVA crystals had a lower intensity with an increase in κ-CaMA, indicating a weakening of the semi-crystalline structure and an increase in their amorphous nature. The results also indicated that intermolecular hydrogen bonding between PVA and κ-CaMA dominated, while the chemical cross-linking of the κ-CaMA led to a decrease in physical cross-linking within the PVA and a decrease in the crystallinity of the hydrogel.
DSC thermograms for the pure materials and the cross-linked composite hydrogel specimens PCOS, Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ-CaC 4:6, and Pκ-CaC 2:8 are presented in Fig. 4B. The exothermic peaks for the main thermal transition, the glass transition temperature (Tg), and the melting temperature (Tm) for PVA were compared with the composite dressings. The relative thermal stability of the hydrogel specimens was assessed by comparing the loss of weight over a temperature range of 50–700 ◦C. Fig. 4C presents the resulting thermal degradation profiles for pure PVA, COS, κ-CaMA, and the composite hydrogels. The detailed thermal analysis was given in the Supplementary information S2.2.

3.1.4. Gel fraction, releasing, swelling, and water retention properties

Gel fraction experiments were performed to quantify the cross- linking efficiency of the hydrogel specimens indirectly, and determine the insoluble materials present (Oh et al., 2020). To support the gel fraction results and as an indicator of the release of COS, the cumulative release of carbohydrates from the hydrogel specimens was assessed. Fig. 5B shows an increase in the release ratio with an increase in κ-CaMA from PCOS to Pκ-CaC 2:8 within the first 24 h and a slight increase over the following 24 h. There was a difference of 3.3, 7.0, 9.6, and 11.8% in the release ratio for Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ- CaC 4:6, and Pκ-CaC 2:8,respectively, compared to that of PCOS, which was in agreement with the gel fraction results and indirectly validated the cross-linking efficiency of the specimens.
The swelling behavior of a hydrogel is one of the most important characteristics for potential use as a moist wound-healing material because it is directly related to the ability to absorb the wound exudate and prevent infection, while also affecting its mechanical stability when fully swollen (Fang et al., 2019). As illustrated in Fig. 5C, all of the hydrogel wound dressings exhibited a high water uptake of over 400% within the first hour after contact with PBS, with saturation reached within 24 h and a swelling equilibrium maintained over the following 24 h. After 24 h, the swelling ratio of the PVA, PCOS, Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ-CaC 4:6, and Pκ-CaC 2:8 was 498.6 ± 21.7, 649.2 ± 28.3, 797.1 ± 11.9, 964.8 ± 55.5, 1213.3 ± 79.6, and 3690.1 ± 268.7, respectively, illustrating that the swelling ratio was influenced by the composition of the hydrogel specimens. In addition, the hydrogel swelling rate was directly correlated with the structural density of the hydrogel, with smaller pore sizes exhibiting a lower swelling rate compared to larger pore sizes, which was in agreement with the SEM micrographs.
When considering the practical use of hydrogels in wound dressings, their water retention capability is important for maintaining a moist environment at the site of the wound. The water retention of the composite hydrogel specimens is illustrated in Fig. 5D. The results reveal a decrease in the retention ratio with an increase in the PVA mass ratio of the hydrogel, which also corresponds with the pore size. In addition, a higher retention rate was observed in the hydrogel specimen with high κ-CaMA content, indicating that water may strongly interact with κ-CaMA.

3.2. Mechanical stability

3.2.1. Rheological behavior and compressive strength

Oscillatory shear experiments were conducted to determine the viscoelastic properties of the composite hydrogel specimens. The effect of differences in the biomaterial composition and the degree of cross- linking on the viscoelastic behavior of the hydrogel specimens were assessed using the dynamic shear storage (G′) and shear loss (G′′) modulus, with G′ describing the elastic behavior and G′′ describing the viscous behavior. As shown in Fig. 6A and B, all of the specimens had a high G′ and a low G′′, representing more solid and elastic behavior than fluid-like behavior and indicating they were all likely to recover completely with minimal deformation. The G′ values were independent and linear to the frequency, meaning that all of the hydrogels were strongly cross-linked and had a strong network. Moreover, within the sweep frequency range, the G′ of the hydrogel specimens exhibited a characteristic reduction with the introduction of COS and κ-CaMA (i.e., PVA, PCOS, Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ-CaC 4:6, and Pκ-CaC 2:8), indicating a reduction in the strength of inter- and intra-molecular hydrogen bonding within the PVA. However, the κ-CaMA hydrogel had a high G′ because the κ-CaMA was cross-linked using UV irradiation and K+ ions. In addition, the ratio between the loss and storage moduli, defined as the loss tangent (tan δ = G′′/G′), was employed to describe the overall viscoelastic behavior of the hydrogel specimens, with a tan δ of less than 1 representing more solid- or elastic-like behavior than viscous behavior. Fig. 6C revealed that all the hydrogels were more elastic than viscous, while the elastic properties slightly decreased following the order of PVA, PCOS, Pκ-CaC 8:2, Pκ-CaC 6:4, Pκ-CaC 4:6, and Pκ-CaC 2:8.
To determine the load-bearing capacity of the hydrogel specimens, we conducted uniaxial compressive testing to hydrogel failure. All specimens exhibited an exponential relationship between compression stress and stain with no plateau observed (Fig. 6D). Fig. 6E presents the maximum compressive stress of the hydrogel specimens, showing that incorporating COS into the PVA did not significantly affect the compressive stress or maximum strain. However, the compressive stress was influenced by the inclusion of κ-CaMA, while the Pκ-CaC 8:2 hydrogel did not significantly differ from the PVA or PCOS, which clearly indicates that the triple cross-linking technique was effective. However, a high concentration of κ-CaMA significantly reduced the compressive stress and strain of hydrogels.

3.3. Biological activities

3.3.1. Indirect and direct cell cytotoxicity

When treating a wound, keratinocytes, and fibroblasts in the skin will directly come into contact with the wound dressing. For this reason, the cytotoxicity of hydrogels was evaluated with HDF and HaCaT cells using indirect and direct contact testing in compliance with ISO 10993-5 standards (Iso, 2009). Fig. 7A shows the effect of the degradable elute of each hydrogel specimen on cell viability after 1 or 3 days of incubation was tested using MTT assays, with the results showing no cytotoxic effect and around 100% cell viability for both the HDF and HaCaT groups. In addition, direct contact testing between the HDF and HaCaT cells and hydrogel specimens (Fig. 7B) revealed that the hydrogels were not toxic, with a higher number of cells observed following treatment with the composite hydrogel specimens compared to the control and PVA groups when the cells were observed under a bright-field with FDA fluorescence for live cells and Hoechst 33342 for cell nuclei. Overall, the cytotoxicity results indicate that the proposed hydrogels were biocompatible and promoted cell growth.

3.3.2. Antibacterial activity

Antibacterial activity of hydrogels against S. aureus and S. epidermidis was investigated, and the results are showed in Fig. S1. Results illustrated that the S. aureus and S. epidermidis colonies significantly decreased following treatment with the composite Pk-CaC8:2 hydrogel. Detailed results and discussion are given in S2.3.

3.4. In vivo animal testing

3.4.1. Excisional wound-healing model

The wound-healing properties of Pκ-CaC 8:2 and Pκ-CaC 6:4 hydrogel wound dressings were evaluated with a full-thickness circular excision wound created on the dorsal skin of 8-week-old ICR mice. The wound sites were photographed regularly following the injury, and changes in the size of the wound and scar were evaluated in relation to the original wound size (Fig. 8A and B). Although re-epithelialization of the wounds occurred in all groups, wound healing was more rapid in the hydrogel-treated groups than in the control group, while the remaining scar was more prominent in the control group. The control group exhibited a wound size of 77.0% on day 4, 42.1% on day 7, and 8.9% on day 14, compared to 65.0, 27.1, and 5.2% for Pκ-CaC 8:2 and 58.6, 21.4, and 4.9% for the epiderm group, respectively. The Pκ-CaC 6:4 hydrogel wound dressing also promoted a more rapid wound closure than the control and PVA group, reaching 68.4, 29.4, and 5.6% on days 4, 7, and 14, respectively, indicating that composite hydrogel wound dressings accelerate wound closure, as previously reported in lignin-chitosan-PVA (LCP) composite hydrogel. In comparison with the control group, LCP reached complete wound closure in 15 days while chitosan-PVA dressed group showed moderate healing, indicating chitosan as a natural polymer enhances the wound healing attributed to its attractive biological activities and high fluid absorption and holding capacity (Zhang, Jiang, et al., 2019). The results of the present study suggest that the various biological activities of COS, including wound healing, antibacterial, and antioxidant (Jafari et al., 2020) and high fluid absorption capacity of κ-Ca (Oun & Rhim, 2017) in composite, Pκ-CaC hydrogel enhance the rate of wound closure and reached complete healing by day 14.

3.4.2. Histological characterization

Wound healing is a complex physiological process that consists of several overlapping stages: hemostasis and inflammation, granulation, wound contraction, collagen formation, epithelization, and scar remodeling (Bakr et al., 2021; Chandika et al., 2015). To assess the effect of the proposed hydrogels on skin tissue regeneration, a histomorphological evaluation of the tissue at the center of the wound 14 days after the injury was incurred was conducted using hematoxylin and eosin (H&E) staining (Fig. 8C). Irrespective of the treatment, all of the groups exhibited completed re-epithelization, but with differences in the scar and remodeled tissue. In the control group, the wounded region had full epithelization, with thin and wider scar tissue and dense blood capillaries in the scar tissue. In contrast, the PVA group had comparatively thick scar tissue with fewer blood capillaries, indicating that the treatment has advanced the healing process. Greater re-epithelization, less scar tissue, and more obvious remodeling of the surrounding tissue were prominent in the epiderm, Pκ-CaC 8:2, and Pκ-CaC 6:4 groups. Unlike the control and PVA groups, the Pκ-CaC 8:2 group exhibited a dense, wide, and fully differentiated dermis, with appropriately distributed sebaceous glands, sweat glands, hair follicles, and blood vessels, indicating that the composite dressing enhanced the healing process. Overall, the more rapid wound closure, full re-epithelization, and remodeling of the full-thickness wound illustrate that the proposed composite hydrogel wound dressing provides a suitable environment for effective wound healing.

4. Conclusion

In this study, a composite Pκ-CaC wound-dressing hydrogel was successfully prepared using triple cross-linking based on repeated freeze/thaw cycles and photo-crosslinking. The Pκ-CaC hydrogel exhibited a uniformly thick structure with a uniformly distributed porous microstructure, good fluid absorption and retention, suitable release rates, high mechanical stability, thermal stability, a semi- crystalline structure, and excellent cytocompatibility. An in vivo full- thickness excisional wound-healing mouse model illustrated that the proposed composite Pκ-CaC hydrogel dressing significantly improved wound closure by day 14, and histomorphological evaluation of the tissue in the wounded area identified rapid re-epithelization, differentiated dermis, and epidermis with remained minimal scar tissues. Moreover, the composite Pκ-CaC hydrogel dressing exhibited good antibacterial activity, suggesting that it can be used in wound-healing applications.

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