One-pot method to prepare the guar gum hydrogel dressing and its application in wound repair

Abstract

Background

The skin serves as a critical barrier, safeguarding the body against external threats including bacteria, viruses, and ultraviolet (UV) radiation. Compromised skin integrity can result in pain, hinder daily activities, and elevate the risk of infections. Clinically, dressings are the conventional treatment for skin injuries. However, these often necessitate frequent replacements and may exacerbate wound trauma during removal. Therefore, there is growing interest in developing innovative dressings such as hydrogels, which are celebrated for their softness, adaptability, permeability, and capacity to sustain a moist wound environment. Guar gum, a galactomannan polysaccharide extensively utilized in the food and biomedical sectors, forms highly viscous, biocompatible hydrogels that are promising for medical applications including capsules and wound dressings. Nonetheless, the mechanical strength and antimicrobial properties of guar gum hydrogels require enhancements for optimal medical efficacy.

Objective

This study explores the fortification of guar gum (GG) hydrogels with tannic acid (TA) and citric acid (CA), which are known for their antibacterial, anti-inflammatory, and antioxidant properties, to develop injectable, antimicrobial hydrogel dressings.

Methods

Employing a one-pot synthesis method, this research aimed to create dressings for treating skin injuries in murine models. The hydrogels were characterized using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR), assessed for antibacterial efficacy against Staphylococcus aureus, and evaluated for biocompatibility and therapeutic effectiveness in mice with full-thickness skin injuries.

Results

The results demonstrated successful cross-linking, structural stability, and significant enhancement in wound healing, indicating the potential of these GG-CA-TA hydrogel dressings to broaden the scope of guar gum applications in clinical skin restoration.

Conclusion

In this study, a kind of Guar gum hydrogel was successfully synthesized by one-pot method, which has great potential in clinical skin repair.

Keywords

Guar gum citric acid tannic acid hydrogel wound healing

Introduction

The skin acts as an essential barrier for the human body, defending against external pathogens, regulating temperature, and maintaining homeostasis.¹ Damage to the skin not only results in pain and disrupts daily activities, but also renders the wound prone to bacterial infections from organisms such as Staphylococcus aureus and Streptococcus hemolyticus. Such infections can cause prolonged bleeding, persistent inflammation, and delayed wound healing. Traditional wound dressings used in clinical settings, including gauze and bandages, often require frequent replacement and can allow bacterial ingress once saturated. Critically, they are prone to adhering to the wound bed, which can cause secondary mechanical damage during changes,² diminishing treatment efficacy. In response, the development of innovative dressing types, such as foam, hydrocolloid, and film dressings has been pursued. Notably, hydrogels have gained prominence due to their excellent bioadhesion, biocompatibility, and ability to release drugs in a controlled manner.

Guar gum, a water-soluble galactomannan polysaccharide, is extracted from the endosperm of the leguminous plant guar beans. It is characterized by its non-toxicity, renewability, biocompatibility, and biodegradability. These attributes facilitate the widespread use of guar gum across various sectors including environmental management, food, and petrochemicals, and notably, in the biopharmaceutical industry. Hydroxypropyl guar gum, a derivative of guar gum,³ significantly improves the solubility, dissolution time, and clarity of guar gum solutions through the introduction of new functional groups, while largely retaining the intrinsic properties of guar gum. This derivative has found extensive applications in drug delivery system research and exhibits substantial potential for further applications. Nevertheless,⁴ the synthesis of hydroxypropyl guar gum powder is labor-intensive and time-consuming, which hampers the rapid production of guar gum-based hydrogels. Moreover, the hydrogels produced from hydroxypropyl guar gum suffer from inadequate mechanical strength and suboptimal antibacterial properties, posing challenges to their quick preparation and broad utilization. Hydroxypropyl guar gum hydrogels, when used directly in clinical treatments, do not yield optimal therapeutic outcomes. Consequently, recent research has pivoted towards enhancing these hydrogels through crosslinking with substances such as chitosan, sodium alginate, and carboxymethyl cellulose to improve pharmacological effectiveness.⁵ Citric acid, notable for its bioactivity and the presence of carboxyl and hydroxyl groups in its structure, can form crosslinks with hydroxypropyl guar gum via hydrogen bonding or electrostatic interactions, thereby enhancing the hydrogel's mechanical properties. This crosslinking not only significantly improves the hydrogel's solubility—thus reducing preparation time—but also increases its strength. Despite these improvements, the mechanical properties of the hydrogel remain suboptimal, and the therapeutic effects are not significantly enhanced. Therefore, further modifications are necessary to augment the hydrogel's bioactivity and pharmacological efficacy.⁶

At the same time, tannic acid, a high molecular weight, water-soluble polyphenolic compound,⁷ is derived predominantly from various plants such as gallnuts and pomegranates. Its structure, characterized by numerous phenolic hydroxyl groups, facilitates oxygen scavenging from the environment, leading to oxidation into quinone substances that provide protection to organisms.⁸ Owing to its excellent biocompatibility, antioxidant properties, and antibacterial and anti-inflammatory effects, tannic acid has been extensively utilized in the biomedical sector. Applications include the development of hydrogels and nanomaterials. In particular, the incorporation of tannic acid into guar gum-citric acid hydrogels not only boosts their mechanical strength but also imparts antibacterial and antioxidant properties.⁹ This modification substantially enhances their bioavailability and pharmacological efficacy.

Wound infections are prevalent following dermal injuries, potentially leading to extended periods of bleeding, persistent bacterial colonization, inflammation, and delayed healing processes. These complications can significantly disrupt a patient's quality of life and overall well-being. Traditional dressings used in clinical settings often necessitate frequent replacements and exhibit suboptimal antibacterial properties, highlighting the imperative for innovative dressing technologies.¹⁰ Cationic guar gum, a natural, water-soluble polysaccharide, offers several advantages including non-toxicity, renewability, and biocompatibility, rendering it an excellent candidate for hydrogel dressing formulations. Nonetheless, hydrogels solely based on cationic guar gum pose significant challenges: they are labor-intensive to prepare, exhibit inadequate mechanical strength, and demonstrate minimal pharmacological activity, which hinders their rapid production and broad application.¹¹ The incorporation of citric acid, leveraging its ability to form hydrogen bonds with the abundant hydroxyl groups on guar gum, enhances the solubility and mechanical properties of the hydrogels. However, the pharmacological efficacy remains modest. Tannic acid are rich in phenolic hydroxyl groups, which facilitates the modification of guar gum-citric acid hydroxyl groups via electrostatic hydrogen bonding. This modification not only enhances the mechanical properties of the hydrogel but also imparts antimicrobial and antioxidant functionalities, thereby augmenting the pharmacological efficacy of hydrogel dressings. Such enhancements significantly broaden the potential for the practical use of guar gum gel dressings in skin repair, presenting considerable practical implications.¹²

In summary, this study is designed to develop a tannic acid-modified guar gum gel dressing, with an emphasis on conducting comprehensive physical characterization and in vitro performance evaluations. The effectiveness of this gel dressing in promoting skin repair will be assessed using a full-thickness skin wound model in mice. The outcomes will contribute to validating the therapeutic potential of the gel dressing for clinical applications in dermatology.

Materials and methods

Materials

Hydroxypropyl trimethyl ammonium chloride guar gum (analytical pure) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd Citric acid (analytical pure) was obtained from Shandong Keyuan Biochemical Co., Ltd Tannic acid (analytical pure) was sourced from Shanghai Macklin Biochemical Technology Co., Ltd Disposable sterile syringes of various specifications were acquired from Yueyang Min Kang Medical Materials Co., Ltd 2,2'-Biphenyl-1-picrylhydrazyl (BPH, 96%) was obtained from Shanghai Macklin Biochemical Co., Ltd The culture medium (90 mm Petri dishes) was purchased from Shanghai May 1st Glass Instruments Factory. Plate Count Agar Medium and LB Broth, both classified as biological reagents, were supplied by Beijing Aoboxing Biotech Co., Ltd and Qingdao Hope Bio-Technology Co., Ltd, respectively. Dimethyl Sulfoxide (DMSO), analytical grade, was acquired from Tianjin Fuyu Fine Chemical Co., Ltd Disposable sterile syringes of various specifications were sourced from Yueyang Minkang Medical Materials Co., Ltd PBS Buffer Solid Dry Powder, also analytical grade, was procured from Xi'an Heer Biotechnology Co., Ltd The pet hair clipper of analytical grade was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd Hair removal cream of analytical grade was acquired from Shandong Keyuan Biochemical Co., Ltd Surgical scissors of various sizes were obtained from Shanghai McClintock Biochemical Tech Co. Transparent dressing (product number 1624 W) was sourced from Minnesota Shanghai International Trade Co. Isoflurane, labeled as a biological reagent, was supplied by Rewood Life Science Co., Ltd Disposable sterile syringes of various sizes were procured from Yueyang Min Kang Medical Materials Co., Ltd Needled suture with thread (size 3–0) was purchased from Ningbo Chenghe Microsurgical Instrument Factory.

Kunming mice (KM) (5∼7 weeks old, 25 ± 5 g) were purchased from the Laboratory Animal Center of Xi’an Jiaotong University. They were fed at an animal facility of IVC class (License no. SCXK (Shaanxi) 2015-002) with ad libitum access to food and water under controlled conditions (temperature of 20∼22 °C, relative humidity of 50∼60% and 12-h day/night cycles). All animal experiments complied with the ARRIVE guidelines and were carried out by the UK Animals (Scientific Procedures) Act, 1986.

Methods

Preparation and process optimization of guar glue hydrogel

Under the condition of 25 °C, 40 mg hydroxypropyl guar gum solid powder was dissolved in 1 mL ultra-pure water (pH = 7.0), and the Guar gum solution was prepared by ultrasonography for 5 min after swirling for 30 s. At the same time, the mixture containing 10 mg citric acid and 30 mg tannic acid powder was also dissolved in 1 mL of ultra-pure water, and the citric acid/tannic acid solution was formed after ultrasonic treatment for 5 min. Then, the two solutions were mixed in equal volume, and the stability of Guar glue gel was synthesized by resting for 2 min after vorticity for 30 s.

Fourier transform infrared spectroscopy

To analyze the infrared spectra of hydrogel samples, the potassium bromide disc method was utilized. Initially, guar gum hydrogel was prepared using the optimal ratio determined and subsequently freeze-dried. The resulting freeze-dried guar gum hydrogel powder, along with potassium bromide powder, was desiccated under an infrared lamp to eliminate moisture. These powders were then finely ground together in an agate mortar until a uniform mixture was achieved. A precise quantity of this mixture was then compressed into tablets using a tablet press. Prior to sample analysis, a background scan was conducted using the Fourier Transform Infrared Spectrometer to minimize interference. The guar gum hydrogel samples were scanned over a wavenumber range of 400 cm⁻¹ to 4000 cm⁻¹ to obtain their infrared spectra. This procedure was repeated identically for additional samples including hydroxypropyl guar gum, citric acid, and tannic acid, to ensure consistency in comparison of the spectral data.

Rheological performance test

The rheological properties of guar gum hydrogel were investigated utilizing a rheometer. The prepared guar gum hydrogel sample was injected onto the sample stage of the rheometer at room temperature, forming a disc approximately 2 mm in thickness and 15 mm in diameter. The gap between the rotor and the sample was precisely adjusted to 1 mm. A time sweep test was conducted, during which the data for the storage modulus (G') and loss modulus (G'') of the hydrogel were systematically recorded.

Self-healing performance test

Hydrogel samples were injected onto the rheometer stage to form discs approximately 2 mm thick and 15 mm in diameter. The gap between the rotor and the sample was adjusted to 1 mm. Stress was applied to the hydrogel, and the strain was alternated between 0% and 1000% every 40 s. Changes in the hydrogel's storage modulus (G') and loss modulus (G'') were recorded to evaluate its self-healing properties.

Scanning electron microscopy test

Guar gum hydrogels were prepared using the optimal ratios identified. These hydrogel samples were subsequently subjected to freeze-drying in a lyophilizer for 24 h to ensure complete dehydration. Following freeze-drying, the guar gum hydrogel samples were mounted on a sample plate and coated with gold via sputtering to enhance conductivity. Scanning electron microscopy (SEM) was then employed to capture high-resolution images of the hydrogel's microstructure.

Antioxidant performance test

Following skin injury, the wound environment is characterized by the continuous production of reactive oxygen species (ROS). Hydrogels that exhibit antioxidative properties are increasingly recognized for their potential to facilitate wound healing. The antioxidative capacity of hydrogels is typically evaluated using standardized assays involving the solutions of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). In these assays, the ethanol solution of DPPH, which is initially purple, turns from yellow to transparent upon antioxidant addition, indicating the scavenging of DPPH radicals. Similarly, the blue-green ABTS solution fades to transparent in the presence of antioxidants, reflecting the reduction in ABTS radicals. These color changes quantitatively indicate the extent of radical scavenging and serve as a reliable metric for assessing the antioxidative performance of hydrogels. Detailed methodologies for preparing these solutions are as follows:

DPPH solution: A precise quantity of DPPH was weighed and dissolved in ethanol. The solution was then subjected to ultrasonication for 5 min to ensure thorough mixing. To preserve the integrity of the solution, it was stored in a dark environment;

ABTS solution: A precise quantity of ABTS and potassium persulfate was weighed and dissolved in deionized water. The two solutions were combined in a 1:1 ratio and the mixture was stored in darkness for 12 h to allow the reaction to occur, resulting in the preparation of the ABTS working solution.

Freeze-dried hydrogel, DPPH ethanol solution and ABTS solution were mixed and incubated in the dark for 15 min, and measure the absorbance at 517 nm with a UV-visible spectrophotometer, denoted as A_sample. The blank control group consists of an equal volume of DPPH solution without the sample, with the absorbance denoted as A_control. Calculate the scavenging rate of the hydrogel using the following formula.

c l e a r a n c e r a t e (%) = \frac{A_{control} - A_{s a m p l e}}{A_{c o n t r o l}} * 100 %

(1)

Antimicrobial performance test

LB broth (0.25 g) was mixed with 10 mL of deionized water, shaken, and in another flask, 5.64 g of PCA was dissolved in 240 mL of water. A PBS buffer (pH 7.4) was prepared using 2 L of water. These solutions, along with centrifuge tubes and petri dishes, were sterilized at 121°C for 1 h. After cooling, PCA solution was poured into petri dishes. Staphylococcus aureus was grown in LB broth at room temperature and then incubated at 37°C for 24 h. The culture was centrifuged, supernatant discarded, and the pellet resuspended in PBS, adjusting the optical density to 0.10 at 600 nm. For antibacterial testing, tubes with PBS received the bacterial suspension and incubated at 37°C. Agar plates were inoculated with this mixture, spread, and incubated overnight at 37°C for colony counting of Staphylococcus aureus. This procedure ensures accurate preparation, sterilization, and analysis in microbiological studies.

Cytotoxicity test

Hydrogels are evaluated for biocompatibility using cytotoxicity assays. The MTT assay, a widely acknowledged method, is employed to assess the viability of L929 mouse fibroblasts (3T3) when cultured with hydrogel samples. The experimental protocol is delineated as follows: A quantified amount of freeze-dried hydrogel is first sterilized under ultraviolet light for 30 min and subsequently immersed in a culture medium. This preparation is incubated at 37°C for 24 h; thereafter, the resulting extract is collected and filtered for analysis. Concurrently, L929-3T3 cells are seeded into a 96-well plate and incubated at 37°C in a 5% CO2 atmosphere for 24 h to ensure optimal adhesion. After the initial culture period, the medium was removed and replaced with various concentrations of freeze-dried hydrogel extract; the cultures were then incubated for an additional 24 h. For controls, the blank control wells received only the culture medium, while the negative control wells were supplemented with regular medium and co-cultured with cells. Subsequently, the medium in the wells was discarded, and each well was treated with MTT solution, followed by a further 4-h incubation period. Post incubation, 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, taking care to protect the reaction from light and ensuring thorough mixing by shaking. The absorbance of the solution was measured at 570 nm using an ELISA plate reader. The optical densities recorded as OD0, ODC, and ODS were used to calculate the cell survival rate according to the following formula:

C e l l s u r v i v a l r a t e (%) = \frac{O D_{S} - O D_{0}}{O D_{C} - O D_{0}} \times 100 %

(2)

Guar gum hydrogel dressing promotes skin wound healing in mice test

Mice were anesthetized using isoflurane gas and subsequently secured on the operating table. The dorsal fur was initially trimmed with a pet hair clipper, followed by the application of depilatory cream to ensure complete hair removal. Residual cream was then removed from the skin using a paper towel.¹³ The disinfected area on the mice's back was treated with 75% alcohol. A circular full-thickness skin injury with a diameter of 10 mm was created using surgical scissors. Guar gum gel, with and without tannic acid, was administered onto the wound surface using a 1 mL syringe. The treatment was secured with a 3 M transparent dressing, which was replaced regularly for each group. The experiment is divided into four groups, including the model group (no treatment after model establishment), 3 M dressing group, guar gum hydrogel without tannic acid group, and guar gum hydrogel group, each with three male SPF mice. On days 1, 3, 7, and 14 after surgery, take pictures of the mice with a camera to record their recovery. Finally, use ImageJ software to measure and calculate the wound area of the mice.¹⁴ The initial wound area is denoted as S0, and Si (i = 1, 3, 7, 14) represents the wound area on each day, and calculate the healing rate W (%) using the following formula:

W (%) = (S_{0} - S_{i}) / S_{0} \times 100 %

(3)

Skin wound histopathological test in mice

Wound specimens and adjacent normal skin tissues were collected completely from mice on the 7th and 14th days post-surgery for histological analysis using Hematoxylin and Eosin (H&E) and Masson's trichrome staining.

Statistical analysis

All data were presented as means ± standard deviation (SD). Oneway analysis of variance (ANOVA) was used to analyze the significance among three or more groups using GraphPad Prism version 8.0 software (GraphPad, San Diego, CA). The statistical significance was set as P < 0.05. The statistical analysis of adhesion scores that exhibited a non-parametric distribution was performed using Fisher's exact test.

Results and discussion

Preparation and characterization of guar gum hydrogels

In order to construct a guar gum hydrogel dressing for wound repair, citric acid, tannic acid and guar gum were fully mixed in a one-pot method to obtain an injectable gel dressing. Hydroxypropyl guar gum, citric acid, and tannic acid, all of which are natural water-soluble compounds, were selected to be dissolved in deionized water, the solvent chosen for facilitating the reactions.¹⁵ Hydrogel formation process and results are shown in Figure 1A. Both the guar gum solutions and the citric acid tannin solutions independently maintain a liquid state. However, upon mixing the guar gum solution with the citric acid tannin solution, the resulting system undergoes a transition to a gel state.

Figure 1.

Preparation and characterization of guar gum hydrogels. (A) Preparation of guar gum hydrogel: guar gum solution, citric acid-tannic acid mixture, Guar glue hydrogel. (B) Infrared spectra of GG, CA, TA, and GG-CA-TA. (C) Rheological tests of hydrogels (citric acid as variable): Time scan test of hydrogels, Self-healing tests of hydrogels. (D) Rheological test of hydrogel (hydroxypropyl guar gum as variable): time scan test of hydrogel, self-healing test of hydrogel. (E) SEM of guar gum hydrogel surface (left) and resultant 40 μm pores (right).

Fourier Transform Infrared Spectroscopy (FTIR) was utilized to analyze the infrared spectra of hydroxypropyl guar gum, citric acid, tannic acid, and guar gum hydrogel, employing the potassium bromide tablet method.¹⁶ The analysis covered a wavenumber range from 4000 cm⁻¹ to 400 cm⁻¹. The acquired data were processed to generate the infrared spectra, which are illustrated in Figure 1B. The infrared spectroscopic analysis reveals distinct characteristics of hydroxypropyl guar gum and citric acid. Hydroxypropyl guar gum exhibits a broad absorption peak at 3408 cm⁻¹, attributable to the stretching vibrations of the hydroxyl (OH) groups. Additionally, a weak absorption peak is observed at 2913 cm⁻¹, corresponding to the stretching vibrations of the C-H bonds, and a notable peak at 1050 cm⁻¹, indicative of the C-O-C stretching vibrations in the side-chain sugar ring. In the case of citric acid, the infrared spectrum displays a broad peak near 3500 cm⁻¹, associated with O-H stretching vibrations. Moreover, the spectrum shows a distinct absorption peak at 1709 cm⁻¹, which is characteristic of the C = O stretching vibration in the carboxyl groups. Tannic acid exhibits absorption peaks at 1715 cm⁻¹ and within the range of 1610 to 1480 cm⁻¹. These peaks likely originate from the stretching vibrations of the ester group's C-O and the aromatic ring's C-C bonds in the phenolic structure. Additionally, in the infrared spectrum of the hydrogel, a pronounced and broad absorption peak at 3480 cm⁻¹ is observed, indicative of hydrogen-bonded cross-linking that occurs during the reaction process and results in a blue shift of the peak. Additionally, the presence of hydroxypropyl guar gum within the hydrogel is confirmed by an absorption peak at 2901 cm⁻¹. Furthermore, the peaks at 1723 cm⁻¹, along with those in the range of 1615 cm⁻¹ to 1480 cm⁻¹, substantiate the successful cross-linking of tannic acid with hydroxypropyl guar gum and citric acid, thereby forming a stable guar gum hydrogel.¹⁷

To optimize the synthesis process of guar gum hydrogel, we employed a single-factor experimental approach, examining how variations in the concentration of each monomer—including hydroxypropyl guar gum, citric acid, and tannic acid—affect the rheological properties of the hydrogel.

Citric acid content: Prepare guar gum hydrogels at room temperature to initially produce a hydrogel with established stability. Maintain consistent levels of hydroxypropyl guar gum and tannic acid while exclusively varying the citric acid concentration. This approach aims to investigate the impact of citric acid variations on the gelation characteristics and overall properties of the guar gum hydrogel (Table 1). The composition of each component within the hydrogel is detailed in Table 1.

Table 1.

Content of each component in hydrogel (only the dosage of citric acid was changed).

Guar gum（wt%）	Citric acid（wt%）	Tannin（wt%）
4%	1%	3%
4%	3%	3%
4%	5%	3%

Rheometers were utilized to assess the storage modulus (G') and loss modulus (G'') of the synthesized guar gum hydrogel. The storage modulus G' is indicative of the hydrogel's stiffness and resistance to deformation, whereas the loss modulus G'' characterizes its viscoelastic properties and liquid-like behavior. According to the time sweep rheology results (Figure 1C), the storage modulus consistently exceeds the loss modulus by approximately an order of magnitude throughout the scanning process. This suggests that the samples maintain a predominantly solid state, reflecting robust shape stability of the hydrogel. Further observations reveal a decrease in both the storage and loss moduli with increasing concentrations of citric acid. This phenomenon can be attributed to the molecular structure of citric acid, which includes hydroxyl and carboxyl groups capable of forming hydrogen bonds with guar gum and tannic acid. At higher citric acid concentrations, the limited availability of hydrogen bonding sites restricts the extent of cross-linking. Consequently, excess citric acid molecules remain unincorporated within the three-dimensional network of the hydrogel, compromising its structural integrity. Figure 1C presents the rheological self-healing characteristics of the hydrogel. In the absence of strain, the storage modulus surpasses the loss modulus, confirming the integrity of the hydrogel's three-dimensional network. Upon application of a 1000% strain, the loss modulus exceeds the storage modulus, disrupting the network and transitioning the hydrogel to a liquid-like state. Removal of the stress allows the storage modulus to again dominate, indicating recovery of the network structure. This recovery is repeatable across multiple cycles, demonstrating the hydrogel's robust self-healing properties. Furthermore, increasing concentrations of citric acid correlate with prolonged recovery times after each strain cycle, implying a reduction in self-healing efficiency. Notably, hydrogels with 1wt% citric acid content achieve optimal balance, showcasing superior structural stability and self-healing capabilities. This study suggests that higher citric acid concentrations compromise the mechanical integrity and recovery potential of the hydrogel network.

Tannic acid content: guar gum hydrogels were prepared at room temperature following. Hydroxypropyl guar gum was used as a constant control variable, with the concentrations of hydroxypropyl guar gum and citric acid maintained. The variable under investigation was the amount of tannic acid, which was altered to assess its impact on the physical and chemical properties of the guar gum hydrogel (Table 2). This experimental setup was designed to identify the optimal tannic acid concentration for the hydrogel formulation. The composition of each component within the hydrogel is detailed in Table 2.

Table 2.

The content of each component in guar gum gel (only the amount of tannic acid is changed).

Guar gum（wt%）	Citric acid（wt%）	Tannin（wt%）
8%	1%	3%
8%	1%	5%
8%	1%	7%

Rheological assessments were performed on the synthesized guar gum hydrogel. The time-scan rheology results, depicted in Figure 1D, reveal that throughout the duration of the test, the storage modulus (G') consistently exceeded the loss modulus (G''), confirming the hydrogel's predominately solid state and absence of phase transitions, indicative of robust stability. Moreover, an increase in tannic acid concentration corresponded with a rise in both the storage and loss moduli, implying that tannic acid contributes to the enhancement of hydrogel crosslinking, thereby augmenting its structural integrity. The rheological analysis of the self-healing properties of the hydrogel, as depicted in Figure 1D, demonstrates that throughout the experiments, the storage modulus (G') consistently exceeded the loss modulus (G''). Importantly, the hydrogel was capable of regaining its original storage modulus following multiple strain applications. A noteworthy observation is the influence of tannic acid concentration on the recovery of the storage modulus; with increasing tannic acid levels, the hydrogel's recovered storage modulus progressively approached its initial value. This phenomenon suggests that tannic acid significantly enhances the self-healing capabilities of the guar gum-based hydrogel. Conclusively, the data indicate that increments in tannic acid content not only augment the structural integrity but also improve the self-healing properties of the hydrogel. Optimal performance is observed when the tannic acid concentration reaches 7 wt%. From the outcomes of the single-factor experiments described previously, the optimal composition for synthesizing guar gum hydrogel was established. The most effective formulation consists of 8 wt% hydroxypropyl guar gum, 1 wt% citric acid, and 7 wt% tannic acid.

Using a scanning electron microscope (SEM) at an accelerating voltage of 3 kV, the morphology of guar gum hydrogel was investigated. Figure 1E presents the SEM results. Examination of the SEM images at various magnifications revealed a stable three-dimensional network structure with pore sizes approximately 40 μm. These pores are crucial for facilitating the storage and exchange of external substances, which supports nutrient transport and waste removal during the skin regeneration process. Consequently, guar gum hydrogel exhibits the typical structural characteristics of hydrogels and promotes effective skin repair.

In vitro performance test of guar gum hydrogel

The hydrogel incorporates tannic acid, known for its antioxidant properties. Consequently, the ABTS and DPPH free radical scavenging assays were employed to evaluate these antioxidant capabilities. The guar gum hydrogels were incubated with ABTS and DPPH solutions for 15 min, following which the absorbance was recorded.¹⁸ As illustrated in the corresponding Figure 2A, the free radical scavenging rates for all guar gum hydrogel groups exceeded 95%. This high efficiency suggests that the hydrogels are capable of rapidly releasing tannic acid upon contact with wound environments, thereby facilitating effective free radical clearance. The results clearly indicate that the integration of tannic acid significantly enhances the antioxidant properties of guar gum hydrogels.¹⁹

Figure 2.

(A) Test results of antioxidant properties of guar gum hydrogels ( $\bar{x} \pm SD$ , n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (B) Cytotoxicity test results of guar gum hydrogels ( $\bar{x} \pm SD$ , n = 3, *P < 0.05). (C) Antibacterial effect of hydrogels on Staphylococcus aureus. (D) Antibacterial rate (n = 3, $\bar{x} \pm SD$ , ****P < 0.0001).

An ideal skin dressing must exhibit excellent biocompatibility, which is essential for treating skin injuries.^20,21 This property also serves as a critical metric for assessing the safety of skin dressings. The corresponding results are depicted in Figure 2B. Hydroxypropyl guar gum, citric acid, and tannic acid, known for their excellent biocompatibility, have been extensively utilized in biomedical research, particularly in the fabrication of hydrogels and the exploration of drug delivery systems.²² To validate the biocompatibility of the guar gum-based hydrogel, cytotoxicity assays were conducted using L929-3T3 mouse fibroblast cells. Figure 2B illustrates that the cell viability was maintained above 90% at guar gum hydrogel leachate concentrations of 0.02 mg/mL and 1 mg/mL. Notably, even at a concentration of 0.08 mg/mL, the viability remained above 85%, confirming the hydrogel's high biocompatibility.²³

To evaluate the enhanced antibacterial properties of guar gum hydrogels upon the incorporation of tannic acid, we conducted antibacterial assays using hydrogels in co-culture with bacterial populations. The experimental design included several distinct groups: a control group, a hydroxypropyl guar gum group, a citric acid group, a tannic acid group, and a guar gum hydrogel group. As illustrated in Figure 2C, hydroxypropyl guar gum alone exhibited minimal inhibitory effects on Staphylococcus aureus, likely due to its limited solubility in water which restricts its antibacterial capability.²⁴ Notably, the addition of citric acid and tannic acid significantly reduced bacterial colony formation, with tannic acid showing the most pronounced effect. The results depicted in Figure 2D clearly show minimal colony growth on the medium, which underscores the potent antibacterial properties of tannic acid. Furthermore, the absence of significant bacterial growth on the medium following co-culture with Staphylococcus aureus in the presence of guar gum hydrogel is noteworthy. This observation confirms that the incorporation of citric acid and tannic acid substantially augments the antibacterial efficacy of the guar gum hydrogel.²⁰

Evaluation of skin repair effect of guar gum hydrogel dressing

To investigate the enhancement of wound healing by guar gum hydrogel dressings in mice, a full-thickness skin wound model was established. Survival was noted for all mice throughout the duration of the experiment. Photographic documentation of the wound healing process was conducted on days 1, 3, 7, and 14 post-operation.²⁵ As observed, the wound closure in all mice demonstrated a progressive healing over time (Figure 3A). Notably, during the early to mid-postoperative period, the wounds treated with both the 3 M dressings and the guar gum hydrogel dressings exhibited smaller scab sizes compared to those in the control group and the tannin-free guar gum hydrogel group, indicating an accelerated healing process. By day 14, the wounds in the guar gum hydrogel-treated group had nearly completely healed, with the most rapid hair regrowth observed, suggesting superior recovery performance among all groups tested.²⁶ Subsequently, the wound areas of the photographed mice were subsequently measured, and the wound healing rates were calculated using Image J software to assess their healing status.²⁷ As indicated by the Figure 3B, the healing efficacy of the guar gum hydrogel (GG-CA-TA) was significantly superior to that of the other groups. Specifically, the wound healing rates for the model group (Model), 3 M dressing group (3 M), tannin-free guar gum hydrogel group (GG-CA), and guar gum hydrogel group (GG-CA-TA) on day 3 were 6%, 28%, 22%, and 53%, respectively. By day 7, these rates had increased to 69%, 72%, 75%, and 84%. This demonstrates that guar gum hydrogel (GG-CA-TA) significantly enhances wound healing compared to the other tested dressings, suggesting its potential utility in promoting wound recovery. By the 14th day post-surgery, the wound healing rates were 87% in the guar gum hydrogel group (GG-CA-TA), compared to 79% in the control group (Control). The rates observed in the 3 M dressing group (3 M) and the tannin-free guar gum hydrogel group (GG-CA) were 84% and 78%, respectively. Notably, all these values were lower than those recorded for the guar gum hydrogel group (GG-CA-TA). These findings suggest that guar gum hydrogel facilitates exudate absorption, thereby creating an optimal environment for wound recovery in mice and significantly enhancing wound healing.

Figure 3.

(A) Recovery of full-thickness skin wounds in mice with different treatments. (B) Healing rate. (C) H&E stained sections of mouse wound tissue. Scale bar = 500 μm. (D) Masson stained sections of mouse wound tissue. Scale bar = 500 μm.

On days 7 and 14 post-operatively, wound tissues were harvested from each experimental group of mice. Following harvesting, tissues were embedded in paraffin, sectioned, and subsequently stained using Hematoxylin & Eosin (H&E) and Masson's trichrome techniques. Microscopic evaluation of the stained sections was conducted to assess wound healing processes.²⁸ Representative images from each group are depicted in Figure 3B and Figure 3C. Histological analysis of the H&E-stained sections revealed persistent inflammatory cell presence in the dermal layers of all groups on day 7 post-operation. Notably, the group treated with guar gum hydrogel exhibited the lowest count of inflammatory cells. By day 14, all groups demonstrated the formation of a continuous epidermal basement layer, with distinct stratification of newly formed skin.²⁹ Comparative measurements indicated that the skin regenerated in the guar gum hydrogel group was markedly thicker and more structurally intact than that observed in the other groups. These findings suggest that guar gum hydrogel significantly enhances the regeneration of skin following surgical wounding. Collagen fiber deposition in the regenerated skin of mice was assessed using Masson's trichrome staining method at two time points: seventh and fourteen days post-treatment. The histological analysis of sections from day seven revealed a marked increase in collagen deposition in the treatment groups compared to the control. Further examination of the sections from day fourteen demonstrated that the mice treated with guar gum hydrogel exhibited significantly enhanced collagen deposition relative to other groups. These findings suggest that guar gum hydrogel effectively promotes collagen synthesis in the dermal matrix of mice.³⁰

Conclusions

Hydrogels based on guar gum were synthesized using citric acid and tannic acid as crosslinking agents through a single-factor experimental design to optimize the synthesis process. This methodology not only improved the stability and antibacterial properties of the hydrogels but also streamlined the synthesis process via the “one-pot” method employing deionized water as the solvent, thereby reducing the preparation time. The incorporation of citric and tannic acids promoted crosslinking through hydrogen bonding and electrostatic interactions, which enhanced the mechanical and pharmacological properties of the hydrogels, including their anti-inflammatory activities. Comprehensive evaluations, including rheological analysis and infrared spectroscopy, were conducted to determine the optimal composition of the hydrogel. Subsequent in vitro assessments confirmed its antioxidant and biocompatible properties, underscoring its potential for medical applications, notably in wound healing dressings.

Footnotes

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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