A strategy for fabricating a multifunctional hydrogel with enhanced adhesion and ROS-scavenging capabilities

The cornea is a delicate tissue that is particularly vulnerable to injury, which can lead to blurred vision or even irreversible blindness. It is vital to maintain the cornea’s integrity and transparency throughout the healing process, alongside achieving exceptional biocompatibility. Unfortunately, corneal injuries often result in the excessive production of reactive oxygen species (ROS), which can significantly hinder the healing process. To meet the increasing demand for advanced biomaterials that integrate high transparency, strong adhesion, and effective ROS-scavenging properties for corneal regeneration, this study presents an innovative multifunctional composite hydrogel named GOH-S. This hydrogel is formulated using a careful sequential crosslinking strategy that combines gelatin methacrylate (GelMA), oxidised dextran (ODEX), and thiolated hyaluronic acid (HA-SH). The GOH-S hydrogel features an interconnected porous microstructure and impressive mechanical properties. Its exceptional adhesive strength allows it to securely bond to the wound site, effectively resisting blinking or tear flow and natural eye movements. In testing, GOH-S demonstrated over 85% transparency, minimising visual obstruction for patients while facilitating clinical observation and monitoring. The remarkable potential of the GOH-S hydrogel for corneal repair and regeneration is attributed to its dual functionality: efficient scavenging of harmful ROS and outstanding biocompatibility. This advancement presents a significant opportunity to enhance corneal treatment and improve patient outcomes.

Introduction

Effective wound management continues to present a significant challenge in clinical practice, especially in complex cases that are susceptible to bacterial infections and oxidative stress (Frykberg and Banks, 2015; Millan-Reyes et al., 2025; Wang et al., 2025a). To create an optimal healing environment by isolating the wound, various dressings have been developed, with hydrogels emerging as particularly suitable options (Cheng et al., 2021). Hydrogels are ideal wound dressings due to their high water content, porous structure, biocompatibility, biodegradability, and flexible elasticity. They maintain a moist environment for wounds, promote gas exchange, and easily conform to the wound surface (Negut et al., 2020; Solanki et al., 2023). The cornea is a transparent, avascular tissue that provides a structural barrier for the eyeball. Composed of the epithelium, stroma, and endothelium, the outermost epithelial layer is thin and susceptible to various injuries and trauma (Khosravimelal et al., 2021). The corneal tissue’s vulnerability to injury makes it a primary target for hydrogel therapeutic interventions. Surveys reveal that over 10 million patients experience corneal diseases each year (Ambrožič et al., 2024). During the inflammatory phase of corneal healing, excessive reactive oxygen species (ROS) cause oxidative stress, damaging cellular components and perpetuating inflammation, which severely hinders the healing process (Dunnill et al., 2017; Lopes et al., 2024; Ukaegbu et al., 2025). There is a significant need to develop functional hydrogels that can efficiently scavenge ROS. In the context of corneal applications, the transparency and adhesive properties of these hydrogels are of paramount importance (Wang et al., 2025b). However, existing hydrogel systems demonstrate significant limitations in meeting the essential requirements for biomedical applications. Many of these systems often fail to achieve a simultaneous combination of robust tissue adhesion, high mechanical stability, and intrinsic therapeutic bioactivity within a single formulation (Annabi et al., 2014). Consequently, there is a pressing clinical need for innovative hydrogel-based materials that can effectively eliminate ROS while also demonstrating robust adhesive properties and high transparency.

Natural polymer derivatives, such as gelatin, hyaluronic acid (HA), and dextran (DEX), are increasingly recognised as promising materials for hydrogel construction due to their intrinsic bioactivity (Yang et al., 2018). Gelatin methacryloyl (GelMA) is a modified form of gelatin that retains the arginine-glycine-aspartic acid (RGD) sequence to promote cell adhesion (Augustine et al., 2021). However, single GelMA hydrogels often exhibit limitations such as inadequate mechanical strength, slow cross-linking rates, and insufficient stability at physiological temperatures (Liu et al., 2022). To mitigate these challenges, we sought to integrate DEX into the hydrogel formulation. DEX is a natural biomolecule noted for its exceptional biocompatibility and biodegradability, making it suitable for various biomedical applications (World Stroke et al., 2018). Oxidised dextran (ODEX), a versatile polysaccharide derivative, is obtained by oxidising the hydroxyl groups of DEX. ODEX has abundant aldehyde groups (-CHO) on its molecular chain that can undergo rapid and reversible Schiff base reactions with primary amine groups (-NH₂), forming dynamic covalent bonds (Hudson et al., 2010). HA, a vital component of the extracellular matrix (ECM), plays a significant role in cell signalling by interacting with cell surface receptors to modulate cellular behaviour (Balbino et al., 2015). Cysteine, which can provide ROS clearance, was grafted onto HA in this study to prepare a hyaluronic acid-cysteine conjugate (HA-SH) (Zhang et al., 2024).

Hydrogels that utilise a single crosslinking mechanism frequently encounter challenges in simultaneously fulfilling the opposing demands of injectability and long-term mechanical stability post-implantation (Wang et al., 2019; Bertsch et al., 2023; Tanga et al., 2023). Thus, this study presents the design and fabrication of a novel multi-component, multi-stage crosslinked hydrogel system based on ODEX, GelMA, and HA-SH (Figure 1). The initial formation of a dynamic primary network through a Schiff base reaction from ODEX aldehydes and GelMA amines imparts excellent shear-thinning properties to the hydrogel (Yang et al., 2025b). Subsequently, the integration of HA-SH induces the establishment of a secondary network through Michael addition reactions. The inclusion of HA-SH serves a dual function: it not only confers ROS scavenging capabilities but also enhances the adhesive properties of the hydrogel (Chen et al., 2018; Li et al., 2020). To strengthen the mechanical support further, UV-initiated polymerisation of the methacrylate groups present in GelMA leads to the formation of a robust, covalently crosslinked tertiary network, thus significantly augmenting the mechanical strength and structural stability of the hydrogel (Pamplona et al., 2023; Smits et al., 2024; Yu et al., 2024). This fabrication strategy effectively improves the mechanical properties and ROS scavenging capability of the hydrogel, while achieving an optimal balance between injectability and stability. We systematically evaluated the mechanical properties, rheological behaviour, swelling characteristics, and biocompatibility of the hydrogel. These comprehensive assessments demonstrate their value as a versatile platform for biomedical applications, particularly in the repair of corneal injuries.

Figure 1

Figure 1. Schematic illustration of the GOH-S hydrogel fabrication and multi-stage crosslinking mechanism. The synthesis utilizes GelMA, ODEX, and HA-SH in a sequential strategy. The network is formed through an initial Schiff base reaction, followed by Michael addition, and is finally stabilized by UV-initiated photopolymerization, resulting in a multifunctional hydrogel for corneal repair.

Materials and methods

Material

Gelatin (Type A, from porcine skin, ∼300 g Bloom) was purchased from Sigma-Aldrich. Hyaluronic acid (Mw 800,000–1,500,000), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and 2-Hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Methacrylic anhydride (MA) and L-(+)-cysteine were purchased from Sigma-Aldrich. Dextran (Mw 40,000), sodium periodate, and N-hydroxysuccinimide (NHS) were purchased from Beijing Inotech Technology Co., Ltd. All experiments utilised deionised water.

Synthesis of hydrogel precursors

To construct the desired hydrogel system, three key functionalized polymers—GelMA, ODEX, and HA-SH—were prepared as follows.

GelMA Preparation: 5 g of gelatin was dissolved in 50 mL of deionised water at 60 °C. After the gelatin was completely dissolved, the temperature was lowered to 50 °C, and 3 mL of MA was added to the gelatin solution. Subsequently, the mixture was cooled to room temperature. The resulting solution was dialysed against deionised water for 3 days using a dialysis membrane (MWCO: 12–14 kDa) to remove unreacted MA and other impurities (Lee et al., 2016). Finally, the solution was freeze-dried for 3 days.

ODEX Preparation: ODEX was prepared via sodium periodate oxidation (Sun et al., 2024). In brief, 5 g of DEX was reacted in 200 mL of a 5% (w/v) sodium periodate solution under light-protected conditions for 6 h. Following this, 20 mL of ethylene glycol was added to terminate the reaction. The final product was purified using a dialysis bag (MWCO: 8–14 kDa) and then obtained by freeze-drying.

HA-SH Preparation: The third component, HA-SH, was prepared using EDC/NHS chemistry (Yang et al., 2025a). Initially, 0.5 g of HA was dissolved in water, and 0.96 g EDC and 0.58 g NHS were added at pH 4.8. Next, 0.60 g of L-cysteine was introduced, and the coupling reaction was carried out for 24 h at pH 4.8. The final product was purified using a dialysis bag (MWCO: 8–14 kDa) and then obtained by freeze-drying.

Preparation of GOH-S hydrogels

GelMA, ODEX, and HA-SH were dissolved in phosphate-buffered saline (PBS, pH 7.4) to prepare stock solutions (Zhao et al., 2022). The GOH-S hydrogels were fabricated by first mixing the GelMA and ODEX stock solutions. Subsequently, 0, 5, 10, and 20 mg/mL HA-SH stock solutions were added to the mixture, which were designated as the GO, GOH-S₅, GOH-S₁₀, and GOH-S₂₀ groups, respectively (Table 1). The precursor solutions were cast into moulds and exposed to UV light (365 nm, 10 mW/cm²) for 60 s to initiate photo-crosslinking, thereby forming the stable hydrogel network.

Table 1

Table 1. Experimental groups and composition of the hydrogels.

Structural and morphological characterisation of GOH-S

The chemical structures of the hydrogels were analysed using a Vertex 70 FTIR spectrometer (Germany) fitted with a diamond ATR accessory. A small amount of the lyophilised sample was placed directly onto the ATR crystal, and spectra were recorded from 4,000–400 cm^-1. A spectrum of the empty ATR crystal was used for background correction (Samyn et al., 2012). ¹H NMR (400 MHz, Bruker, Switzerland) was employed to characterise the structures of GelMA, ODEX, and HA-SH. Each sample (10 mg) was dissolved in 0.55 mL of D₂O, and spectra were recorded with 64 scans (Fulmer et al., 2010).

The surface and internal microstructure of the hydrogels were observed using scanning electron microscopy (SEM). To prepare the samples, the hydrogels were flash-frozen in liquid nitrogen to preserve their network structure rapidly. Observations were conducted at different magnifications under an accelerating voltage of 5 kV (Thermo Scientific/Apreo 2S HiVac, Thermo Fisher Scientific Inc., America). The elemental composition of carbon (C), oxygen (O), and sulphur (S) on the hydrogel surfaces was analysed using an integrated Energy-Dispersive X-ray Spectroscopy (EDS) detector.

The transparency of GOH-S hydrogels was assessed by visual inspection using a white background. Quantitative measurements were performed using ultraviolet-visible spectrophotometry (Lambda1050+, PerkinElmer, America), with samples placed in quartz cuvettes and transmittance recorded in the wavelength range of 400–800 nm.

Mechanical properties of GOH-S

The mechanical properties of the hydrogels, including tensile, compressive, and adhesive properties, were evaluated using a universal testing machine (Model CMT6104, Meters Industrial Systems Co., Ltd., China). For tensile testing, dumbbell-shaped specimens were prepared (20 mm × 4 mm × 1 mm). The specimens were stretched at a constant speed of 5 mm/min until fracture, and the stress-strain curve was recorded. For compression testing, cylindrical hydrogel samples (10 mm in diameter and 10 mm in thickness) were compressed at a loading rate of 5 mm/min until rupture, and the compression curve was recorded. The adhesive strength was evaluated using the following method: processed porcine cornea was used as the substrate, with two rectangular hydrogel samples (40 mm × 20 mm × 2 mm) adhered to each side and sandwiched between two glass slides to form a test assembly. The assembly was then stretched at a rate of 5 mm/min using a universal testing machine until sample detachment occurred, with relevant data recorded simultaneously.

The rheological properties of GOH-S hydrogels were measured using a rheometer (MCR 302e, Anton Paar, Austria) to determine the transition between the storage modulus (G′) and loss modulus (G″) in the critical strain region and linear viscoelastic region. The rheological properties of the hydrogels were systematically characterised through a series of dynamic tests. First, the gelation kinetics of the hydrogel were monitored via a time sweep. The changes in the G′ and G″ were continuously recorded for 1,000 s at a constant strain of 1% and an angular frequency of 1 rad/s. Following the stabilisation of the gel network, a dynamic frequency sweep (0.1–100 rad/s) was performed at 1% strain to investigate its frequency-dependent viscoelastic behaviour. To evaluate the stability of its network structure, an amplitude sweep was conducted at a constant frequency of 1 rad/s over a strain range of 1%–2000%.

Swelling and degradation performance of GOH-S₁₀

The swelling behaviour of GOH-S₁₀ hydrogels was evaluated in deionised water at room temperature. The pre-dried and weighed hydrogel samples (W₀) were immersed in an excess of deionised water until swelling equilibrium was reached. At predetermined time intervals, the samples were taken out, and the surface water was carefully blotted away with filter paper before measuring the weight (W_t). The swelling ratio (SR) was calculated according to the equation:

$\text{SR\hspace{0.17em}}\left(\%\right)=\frac{W_t-W_0}{W_0}\times 100$

Where W_t is the weight of the swollen hydrogel at time t, and W₀ is the initial dry weight of the hydrogel.

To evaluate the in vitro degradation behavior of the GOH-S₁₀ hydrogel, the prepared cylindrical hydrogel samples (10 mm in diameter and 2 mm in thickness) were precisely weighed to record their initial dry weight (W₀). Each sample was then individually immersed in PBS containing 1 U/mL collagenase and incubated at 37 °C. At predetermined time points (days 1, 3, 5, and 7), the samples were retrieved from the solution, and subsequently lyophilized. The dry weight at each time point (W_t) was recorded, and the weight loss was calculated using the equation:

$\text{Weightloss\hspace{0.17em}}\left(\%\right)=\frac{W_t-W_0}{W_0}\times 100$

Where W_t is the weight of the swollen hydrogel at time t, and W₀ is the initial dry weight of the hydrogel.

Preparation of hydrogel extracts

The hydrogel extracts for in vitro biocompatibility evaluation were prepared in strict accordance with ISO 10993-12:2021. Prior to extraction, the hydrogel samples were fabricated into specific geometries (10 mm diameter, 2 mm thick discs) and sterilized under UV light for 30 min. The extraction was performed at a surface area/volume ratio of 3 cm²/mL, which aligns with the standard’s recommendation for solid devices with a thickness >0.5 mm. To account for the hydrogel’s absorption, a pre-swelling test was conducted, revealing that the samples absorbed 0.29 ± 0.02 mL of the extraction medium (n ≥ 3). Consequently, the sterilized samples were immersed in DMEM medium, with the total volume being the sum of the base volume required for the 3 cm²/mL ratio and the predetermined absorption volume. The extraction process was carried out in a humidified incubator at 37 °C for 24 h. Following incubation, the supernatant was collected, centrifuged to remove particulates, and subsequently sterilized by filtration through a 0.22 µm syringe filter. The resulting hydrogel extracts were then used for subsequent experiments.

Antioxidant activity assay of GOH-S

The antioxidant activity of GOH-S hydrogels was evaluated using the DPPH (Nanjing Jiancheng Bioengineering Institute, China) and ABTS (Nanjing Jiancheng Bioengineering Institute, China) radical scavenging assays.

DPPH assay: DPPH was dissolved in absolute ethanol to prepare a 0.1 mM DPPH solution, and this solution was stored in the dark at 4 °C before use. Pre-dried and weighed hydrogel samples were immersed in 2 mL of the DPPH solution and incubated at room temperature in the dark for 30 min (Blois, 1958). After incubation, the supernatant was collected, and the absorbance at 517 nm was measured using a microplate reader (SuperMax2800, Shanghai Flash Biotechnology Co., Ltd., China). Ethanol was used as the blank control, and the DPPH solution without hydrogel was used as the control.

ABTS assay: ABTS free radical cations (ABTS⁺) were generated by mixing 7 mM ABTS solution with 2.45 mM potassium persulfate and incubating at room temperature in the dark for 12–16 h (Re et al., 1999). The resulting solution was diluted with ethanol to an absorbance of approximately 0.70 ± 0.02 at 734 nm. Hydrogel samples were immersed in 2 mL of ABTS⁺ solution and incubated at room temperature for 10 min. The absorbance of the supernatant was then measured at 734 nm. ABTS⁺ solution without samples served as the control, and ethanol served as the blank control. The scavenging activity was calculated according to the following formula:

$\text{Scavenging\hspace{0.17em}activity\hspace{0.17em}}\left(\%\right)=\frac{A_c-\left(A_s-A_b\right)}{A_c}\times 100$

Where A_s is the absorbance of the test sample supernatant, A_b is the absorbance of the blank control, and A_c is the absorbance of the free radical solution without the sample.

DCFH-DA Assay: The intracellular ROS scavenging capacity (S0033S, Shanghai Beyotime Biotechnology Co., Ltd., China) following hydrogel treatment was evaluated using a ROS assay kit. Human Corneal Epithelial Cells (HCECs; Hefei Wanwu Biotechnology Co., Ltd., China) were seeded in 24-well plates at a density of 2 × 10⁵ cells per well and cultured for 24 h to allow for complete adhesion. After removing the original culture medium, the cells were incubated with a serum-free medium containing the fluorescent probe DCFH-DA (diluted at 1:1,000) at 37 °C for 30 min. Following incubation, the probe solution was discarded, and the cells were washed three times with serum-free medium. Subsequently, a treatment solution containing 400 μM H₂O₂ was added, and the hydrogel was co-cultured with the cells. The experimental groups were designed as follows: (1) Negative control: cells treated with serum-free medium only; (2) Positive control: cells treated with H₂O₂ without hydrogel; (3) GO hydrogel group: cells treated with H₂O₂ and GO hydrogel; (4) GOH-S₁₀ hydrogel group. Cells treated with H₂O₂ and GOH-S₁₀ hydrogel. After 1 h of treatment, the medium was removed, and the cells were washed with PBS. Finally, both bright-field and fluorescence microscopy images were captured to analyze the intracellular ROS levels.

CCK-8 assay

The cytocompatibility of the hydrogels was assessed using a Cell Counting Kit-8 (CCK-8, Abbkine Scientific Co., Ltd., China). HCECs were seeded into 96-well plates at a density of 5 × 10³ cells/well and incubated overnight to allow for adhesion. Subsequently, the culture medium was replaced with the prepared hydrogel extracts at various concentrations, which constituted the experimental groups. Cells cultured in fresh medium served as the control group. Additionally, wells containing only serum-free DMEM and CCK-8 reagent, but no cells, were set up as the blank group. After incubation for 24 h, the cells were rinsed with PBS, and 100 µL of serum-free DMEM containing 10 µL of CCK-8 reagent was added to each well. The plates were then incubated at 37 °C in the dark for 1 h. The absorbance was measured at 450 nm using a microplate reader (Victor Nivo, PerkinElmer, United Staes). Cell viability was calculated using the following formula:

$\text{Cell\hspace{0.17em}viability\hspace{0.17em}}\left(\%\right)=\frac{A_s-A_b}{A_c-A_b}\times 100$

where A_S is the absorbance of cells in the hydrogel extract-treated group, A_c is the absorbance of the control group, and A_b is the absorbance of the blank group.

Live/dead cell staining

The viability of HCECs cultured in direct contact with the hydrogel samples was evaluated using a Live/Dead staining kit (Calcein-AM/PI). Sterilised hydrogel samples were placed into 24-well plates, and HCECs were seeded onto them at a density of 2 × 10⁴ cells/well. After culturing for 24, 48 and 72 h, the medium was removed, and the cells were gently rinsed with PBS. A working solution containing 2 µM Calcein-AM and 4 µM propidium iodide in serum-free medium was added to each well, followed by incubation at 37 °C for 20 min in the dark. The cells were then washed twice with PBS, and fluorescence images were acquired using an inverted fluorescence microscope (Victor Nivo, PerkinElmer, United Staes).

Proliferation and migration test

The sterile pre-gel solution was injected into 48-well plates and allowed to undergo gelation. Corneal epithelial cells were then seeded onto the surface of the hydrogels at a density of 4 × 10³ cells per well. Cells plated on tissue culture plates at the same density served as the control group. Cell viability was assessed using a Live/Dead Assay Kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. Live cells were stained with green fluorescence. Images were acquired using an inverted fluorescence microscope and cell proliferation was assessed using the CCK-8 assay. Briefly, at the designated time point (1, 4, and 7 days), the culture medium was replaced with a fresh medium containing 10% CCK-8 reagent. The cells were then incubated for 1 h in a humidified incubator at 37 °C with 5% CO₂. Subsequently, the absorbance of the solution was measured at 450 nm using a microplate reader.

A scratch wound assay was performed to evaluate the effect of hydrogels on the migration of HCECs. Cells were seeded in 6-well plates at a density of 1 × 10⁶ cells per well and cultured until reaching 100% confluence. A uniform scratch was created in the center of each well using a 200 μL sterile pipette tip, followed by gentle washing with sterile PBS to remove detached cells. Images of the scratched areas were captured at 0, 24, and 48 h using an inverted microscope (Victor Nivo, PerkinElmer, United Staes) to monitor cell migration.

$\text{Cell\hspace{0.17em}mobility}\left(\%\right)=\frac{A_0-A_t}{A_0}\times 100$

where A₀ is the initial wound area and A_t is the wound area at time.

Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9 software. Differences between groups were analysed using a one-way analysis of variance followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Results

Structure of GOH-S

To confirm the successful synthesis of the hydrogels and the formation of the crosslinked network, we conducted structural characterisation of the key precursors—GelMA, ODEX, and HA-SH—as well as the final GOH-S hydrogel.

The chemical structures of the three key precursors were first verified by ¹H NMR spectroscopy (Figure 2A). The spectrum of GelMA exhibited new resonance peaks at chemical shifts of 5.4 and 5.7 ppm, which are assigned to the vinyl protons of the methacryloyl moiety, confirming successful methacrylation. In contrast to DEX, the ODEX spectrum showed a sharp new signal at approximately 9.4 ppm, characteristic of the -CHO, which provides definitive evidence for the successful oxidative generation of aldehyde groups (Xue et al., 2021). The ¹H NMR spectrum of HA-SH confirmed the successful grafting of cysteine, as evidenced by the emergence of a distinctive multiplet in the range of 2.9–3.1 ppm, corresponding to the β-methylene protons of the cysteine residue. Quantitative analysis of the functional group modification efficiency yielded the following results: The degree of substitution (DS) of GelMA was 35.6% (Shirahama et al., 2016); the oxidation degree of ODEX was 14.9%; and the thiol substitution degree of the HA-SH conjugate was 37.2%. These quantitative results collectively demonstrate that all precursor materials underwent efficient functional group modification, establishing a reliable chemical foundation for the subsequent construction of the hydrogel network.

Figure 2

Figure 2. Structural characterization of hydrogel precursors and the final GOH-S hydrogel. (A) ¹H NMR spectra of the GelMA, ODEX, and HA-SH precursors confirming successful functionalization; (B) ¹H NMR spectrum of the GOH-S hydrogel showing the consumption of reactive groups post-crosslinking; and (C) FTIR spectra of the hydrogel precursors (GelMA, ODEX, HA-SH) and the final GOH-S hydrogel, confirming the consumption of reactive groups and the formation of the crosslinked network.

The ¹H NMR spectrum of the final GOH-S hydrogel (Figure 2B) displayed significantly broadened signals, a typical phenomenon due to restricted molecular motion within the crosslinked network. Crucially, several key changes were observed compared to the precursor spectra. The most notable was the complete disappearance of the aldehyde proton signal from ODEX at ∼9.4 ppm. Concurrently, a dramatic reduction in the signal intensity of the methacryloyl vinyl protons from GelMA (at ∼5.4 and 5.7 ppm) and the cysteine β-methylene protons from HA-SH (at ∼2.9–3.1 ppm) was observed. The disappearance and attenuation of these characteristic peaks of the reactive groups conclusively demonstrate the consumption of these functionalities during the crosslinking reaction, confirming the successful formation of the GOH-S hydrogel.

Complementary evidence was obtained by FTIR spectroscopy. The FTIR spectrum of the GOH-S hydrogel showed the expected characteristic features (Figure 2C). The peak at approximately 1,635 cm^-1 is associated with the amide I band (C=O stretch) of the protein backbone and the residual C=C stretch from GelMA. The characteristic peak of the aldehyde carbonyl group C=O stretch of ODEX, which would appear between 1720 and 1740 cm^-1 (Yan et al., 2024), was absent. This disappearance is attributed to the consumption of aldehyde groups in the formation of Schiff base linkages with amino groups from GelMA. Furthermore, the characteristic thiol (-SH) stretching vibration of HA-SH, which appears as a weak band at 2,550–2,570 cm^-1 (Yang et al., 2025), was significantly weakened, indicating the consumption of free thiols through their participation in crosslinking reactions. Collectively, these spectral changes in the FTIR analysis provide strong supporting evidence for the successful synthesis of the GOH-S hydrogel.

Morphological characterisations of GOH-S

The SEM results showed apparent structural differences among GO, GOH-S₅, GOH-S₁₀, and GOH-S₂₀ hydrogels. The GO hydrogel (Figure 3A) displayed a porous and uniform network, with an average pore area of 6,312.33 ± 3,045.89 µm². The GOH-S₅ group (Figure 3B) exhibited a broader pore size distribution, characterised by a predominance of pores around 3,000 μm², but also included a significant number of pores exceeding 9,000 μm². The GOH-S₁₀ group (Figure 3C) presented markedly smaller and denser pores, with an average pore area of 3,749.61 ± 2,883.91 µm². The GOH-S₂₀ group (Figure 3D) exhibited the smallest and most compact network, with the average pore area further reduced to 1,509.39 ± 763.45 µm².

Figure 3

Figure 3. Microstructural and elemental analysis of the hydrogels. (A–D) Representative SEM images and corresponding pore area distribution histograms for GO, GOH-S₅, GOH-S₁₀, and GOH-S₂₀ hydrogels, respectively; (E) elemental mapping of the GOH-S₁₀ hydrogel demonstrating uniform distribution of carbon (C), oxygen (O), and sulfur (S); and (F) the corresponding EDS spectrum confirming sulfur incorporation. Scale bars in (A–D) represent 200 µm.

To further confirm the successful incorporation and uniform distribution of sulfur-containing functional groups in the hydrogel network, EDS analysis was performed on the hydrogel samples. As shown in Figure 3E, the elemental mapping images of C, O, and S clearly reveal their spatial distributions. It can be observed that S (cyan signals) is uniformly dispersed throughout the porous matrix without noticeable aggregation, indicating a highly homogeneous distribution of sulfur-containing components within the three-dimensional network. Moreover, the distinct characteristic peak of S in the EDS spectrum (Figure 3F) further confirms the successful incorporation of S.

Mechanical properties of GOH-S

Hydrogels intended for ocular applications must possess excellent mechanical properties to maintain their shape and structural integrity in the intraocular environment. Therefore, tensile, compression, and adhesion tests, were conducted on GO, GOH-S₅, GOH-S₁₀, and GOH-S₂₀ hydrogels. In the tensile test (Figure 4A), GOH-S₁₀ exhibited the best performance, with a tensile strength of 34.38 ± 0.57 kPa and a maximum deformation of 69.60%. Compared to the GO hydrogel, GOH-S₅ also showed significant improvements in tensile stress and strain (32.79 ± 0.54 kPa, 60.48%). However, the performance of GOH-S₂₀ declined, with a tensile strength of only 6.40 ± 0.40 kPa. This phenomenon is attributed to the excessive concentration of HA-SH long-chain molecules, which become entangled and create significant steric hindrance, leading to a reduction in the crosslinking density of the hydrogel. A decrease in crosslinking density results in diminished mechanical properties, a conclusion supported by the compression test (Figure 4B). Among the four experimental groups, the GOH-S₁₀ hydrogel withstood the highest compressive stress of 46.96 ± 2.91 kPa. It achieved a maximum deformation of 40.20%, while the maximum compressive stresses for GOH-S₅ and GO were 37.16 ± 0.38 kPa and 24.95 ± 1.44 kPa, respectively. In contrast, the fracture stress of GOH-S₂₀ was only 17.59 ± 0.63 kPa. Representative images of the tensile and compression testing processes are shown in Figures 4G,H.

Figure 4

Figure 4. Characterization of the mechanical and rheological properties of hydrogels. (A) Tensile and (B) compressive stress-strain curves for all hydrogel groups; (C) Adhesion strength of hydrogels to isolated porcine corneas; (D–F) Rheological characterization of hydrogel groups, showing (D) time-sweep, (E) frequency-sweep, and (F) strain-sweep profiles; (G) representative photographs of the hydrogels undergoing tensile (H) compression and (I,J) adhesion testing.

The effectiveness of hydrogels in corneal applications largely depends on their long-term retention capability, making adhesive performance a critical evaluation metric. The adhesive strength between the hydrogels and corneal tissue was quantitatively assessed using a universal testing machine (Figure 4I), with the working principle illustrated schematically in Figure 4J. Specifically, hydrogel samples were adhered to both the anterior and posterior surfaces of fresh porcine corneal tissue to simulate practical application conditions. The test results indicated a positive correlation between the adhesive strength and the concentration of the HA-SH component. As the HA-SH concentration increased from 0 (GO group) to 5, 10, and 20 mg/ml, the corresponding hydrogels (GOH-S₅, GOH-S₁₀, and GOH-S₂₀) exhibited adhesive strengths of 23.08 ± 2.86 kPa, 45.39 ± 3.32 kPa, and 71.43 ± 3.16 kPa, respectively, significantly higher than the 12.81 ± 2.06 kPa observed for the GO group (Figure 4C). In studies on corneal repair hydrogels, adhesive corneal patches with adhesion strengths as high as 98.00 ± 5.60 kPa have been reported (Ghosh et al., 2025). Although the maximum adhesion strength of the GOH-S hydrogels developed in this study reaches 71 kPa, which is lower than the reported value, it is fully adequate to meet the adhesion requirements for corneal patches. This enhancement can be attributed to the formation of disulfide bonds between the thiol groups in HA-SH molecules and cysteine residues in corneal tissue proteins, which significantly strengthens the hydrogel-tissue interfacial binding. The obtained adhesive strength falls within a range that ensures stable adhesion to the corneal surface, preventing detachment from blinking or tear flow. At the same time, adhesive strength level is appropriately controlled to avoid damage to the fragile corneal epithelium and stroma during application or removal, indicating good clinical safety.

Rheological properties of GOH-S

The mechanical properties of the GO, GOH-S₅, GOH-S₁₀ and GOH-S₂₀ hydrogels were systematically characterised via rheological analysis, performing time, frequency, and amplitude sweep tests. Time sweep measurements (Figure 4D) revealed that the G′ of four groups remained consistently and significantly higher than G″) over the entire 600-s duration, confirming the successful formation of a stable three-dimensional network structure.

Frequency sweep tests (Figure 4E) further confirmed the excellent structural stability of the four hydrogels. Over the angular frequency range of 0.1–100 rad/s, G′ consistently remained higher than G″ for all hydrogels, with both moduli exhibiting frequency-dependent strengthening. The dominant behavior of G′ across the entire frequency spectrum verified the characteristic elastic solid-like nature of these hydrogels, demonstrating outstanding structural integrity and mechanical stability under dynamic deformation.

The amplitude sweep results (Figure 4F) elucidated the structural response and yielding behaviour of the material under varying strain. Within the low-strain region, G′ markedly exceeded G″, indicating that the hydrogel remained in the linear viscoelastic regime where the microstructural network remained intact and fully recoverable. With increasing strain, the material transitioned into the nonlinear region, which was accompanied by a rapid decline in G′, suggesting molecular chain disentanglement or disruption of cross-linking sites within the gel network. At approximately 300% strain, the intersection of G′ and G″ curves of GOH-S₅ and GOH-S₁₀ hydrogels indicated a transition in mechanical behavior from an elastic solid to a viscous fluid, demonstrating favorable toughness and recoverability. In contrast, the yield point of the GOH-S₂₀ hydrogel occurred prematurely at about 60% strain, suggesting potential over-crosslinking that led to increased network brittleness, which may compromise structural integrity in practical applications.

Transmittance of the GOH-S

Alongside mechanical integrity, the optical transparency of the hydrogels was quantitatively analysed to ensure unobstructed vision post-application. The GO, GOH-S₅, GOH-S₁₀, and GOH-S₂₀ hydrogels were first examined macroscopically, and their transparency was quantitatively analysed using UV-Vis spectrophotometry. All samples formed stable solid gels at room temperature. GO, GOH-S₅, and GOH-S₁₀ (Figures 5A–D) appeared colourless, uniform, and highly transparent. The letter “A” placed behind them could be clearly seen, which indicates a homogeneous network. In contrast, GOH-S₂₀ (Figure 5D) appeared turbid and translucent, and the letter beneath it looked blurred, suggesting a less uniform structure. To further support these observations, light transmittance was quantified across the 400–800 nm wavelength range (Figure 5E). The results showed that GOH-S₅ and GOH-S₁₀ exhibited the highest transmittance, remaining above 85% throughout the visible spectrum and approaching 100% at 800 nm. This value closely matches the light transmission of the natural cornea and is considerably higher than that of many hydrogels reported in the literature (Borouman et al., 2024), highlighting its significant potential for corneal tissue engineering (Beems and Van Best, 1990). The GO hydrogel also showed good transparency, with a transmittance of approximately 78% at 400 nm that increased steadily with wavelength. In contrast, GOH-S₂₀ demonstrated significantly lower transmittance, measuring only about 65% at 400 nm. This reduced transparency is consistent with the hypothesis of polymer chain entanglement and aggregation at excessive HA-SH concentrations, which also explains its diminished mechanical properties.

Figure 5

Figure 5. Characterisation of the macroscopic morphology, optical and physicochemical properties of GOH-S hydrogels. (A–D) macroscopic images demonstrating the appearance and transparency of (A) GO, (B) GOH-S₅, (C) GOH-S₁₀, and (D) GOH-S₂₀ hydrogels; and (E) UV-Vis transmittance spectra across the 400–800 nm wavelength range. (F) The letters ‘NCU’ formed by extruding GOH-S₁₀ hydrogel through a syringe. (G) Swelling rate curve of GOH-S₁₀ hydrogel over 48 h. (H) In vitro degradation curve of GOH-S₁₀ hydrogel over 7 days. Data in Figures G and H are presented as mean ± standard deviation (n = 3).

Based on the above results, the GOH-S₁₀ hydrogel demonstrated the most well-rounded overall performance, making it particularly suitable for biomedical applications such as corneal patches. Rheological tests revealed that it formed a stable three-dimensional network structure in time sweeps, exhibited excellent structural integrity and dynamic stability in frequency sweeps, confirming the mechanical strength required for long-term retention. Furthermore, amplitude sweeps indicated that it maintained an optimal balance between strength and flexibility even under high strain. Combined with its outstanding tensile, compressive, and adhesive properties, these results suggest that the material achieved an ideal compromise between crosslinking density and structural stability. Meanwhile, optical performance tests showed that GOH-S₁₀ had the high transmittance in the visible region (>85%), meeting the stringent transparency requirements for corneal repair materials without obstructing patient vision. In summary, owing to its excellent mechanical properties and optical transparency, GOH-S₁₀ was selected as the representative sample for further investigation in subsequent studies.

The injectability, swelling and degradation behaviour of GOH-S₁₀

The injectability of the hydrogel was evaluated, with GOH-S₁₀ selected as the representative sample for the injection test. As shown in Figure 5F, GOH-S₁₀ hydrogel was loaded into a syringe and extruded to continuously form a complete “NCU” pattern, demonstrating its favorable injectability and shape-retention capability. The water absorption capacity of the hydrogel was assessed via an in vitro swelling test in PBS (Figure 5G). During the initial swelling stage (0–8 h), the hydrogel absorbed water rapidly, reaching swelling ratios of 175.15% ± 8.00% and 212.25% ± 8.28% at 4 h and 8 h, respectively. The swelling rate then gradually decreased as the hydrogel approached equilibrium, with swelling ratios of 217.65% ± 3.63%, 232.20% ± 4.48%, and 239.40% ± 9.87% at 12 h, 24 h, and 36 h, respectively. After 48 h, the swelling ratio stabilized at 239.40% ± 9.87%, indicating that the hydrogel network had reached its maximum water absorption capacity. To further investigate the in vitro degradation behavior, the freeze-dried GOH-S₁₀ hydrogel was incubated in PBS containing collagenase (Figure 5H). After 7 days of degradation, the hydrogel retained 20% of its initial mass, confirming its controllable degradation characteristics.

Antioxidant activity of hydrogels

To quantitatively evaluate the ROS-scavenging potential of the GO, GOH-S₅, GOH-S₁₀, and GOH-S₂₀ hydrogels, we assessed their antioxidant activity using DPPH and ABTS free radical scavenging assays. The DPPH assay results (Figure 6A) revealed a significant positive correlation between the radical scavenging efficiency and the concentration of HA-SH. The measured DPPH scavenging rates were 63.86% ± 2.55% for GO, 71.81% ± 3.62% for GOH-S₅, 89.64% ± 5.40% for GOH-S₁₀, and 88.79% ± 3.99% for GOH-S₂₀. An apparent concentration-dependent increase was observed from GO to GOH-S₁₀. However, increasing the HA-SH concentration from 1% (w/v) to 2% (w/v) did not lead to a statistically significant improvement in scavenging activity, suggesting that the antioxidant capacity had reached a saturation point.

Figure 6

Figure 6. In vitro antioxidant activity of the hydrogels. Quantitative analysis of free radical scavenging activity using (A) DPPH and (B) ABTS assays; (C) DCFH-DA fluorescence probe assay demonstrating the scavenging effect of GOH-S₁₀ hydrogel on H₂O₂. Green fluorescence indicates intracellular H₂O₂ levels. Scale bar = 100 µm.

To corroborate these findings, ABTS⁺ radical scavenging assays were conducted, which showed a highly consistent trend (Figure 6B). The ABTS⁺ scavenging rates were 31.13% ± 7.23% for GO, 37.75% ± 4.79% for GOH-S₅, 60.91% ± 6.71% for GOH-S₁₀, and 61.41% ± 2.55% for GOH-S₂₀. These results further supported that the antioxidant activity plateaued at 1% w/v HA-SH (GOH-S₁₀), with no significant difference between GOH-S₁₀ and GOH-S₂₀. Collectively, both assays demonstrated that although higher HA-SH concentrations provided more antioxidant sites, beyond 1% (w/v) HA-SH, densification of the hydrogel network may hinder mass transport of free radicals to the active sites, offsetting the theoretical chemical advantage. Thus, GOH-S₁₀, with 1% (w/v) HA-SH, was identified as the optimal formulation, offering strong radical scavenging performance.

To further validate the antioxidant performance of the GOH-S hydrogels at the cellular level, intracellular ROS scavenging capacity was assessed using the DCFH-DA fluorescent probe. As shown in Figure 6C, untreated HCECs (negative control group) exhibited only weak background fluorescence. In contrast, cells treated with 400 μM H₂O₂ for 2 h (positive control group) showed intense green fluorescence, indicating a substantial increase in intracellular ROS levels and severe oxidative stress. Compared with the positive control, cells pretreated with GO hydrogel demonstrated significantly reduced fluorescence intensity, suggesting a certain ROS scavenging ability. Previous studies have reported that L-cysteine can effectively eliminate H₂O₂ under high oxidative stress conditions, which is consistent with the findings of this study (Liu et al., 2020). The GOH-S₁₀ treatment group exhibited a further decrease in fluorescence intensity, nearly returning to the level of the negative control, demonstrating excellent ROS scavenging efficacy and confirming the remarkable antioxidant properties of this hydrogel at the cellular level.

Cytotoxicity test

The excellent biocompatibility of hydrogels is conducive to maintaining a low-cytotoxicity microenvironment, which is crucial for corneal epithelial regeneration and barrier function repair. To evaluate the biosafety of GO and GOH-S₁₀ hydrogels as potential corneal repair materials, we conducted a systematic in vitro assessment of their extracts, including CCK-8 and live/dead cell staining assays.

The CCK-8 assay was employed to evaluate the cytotoxicity of the hydrogel extracts on HCECs. A concentration gradient ranging from 0.50 to 10.00 mg/mL was tested to simulate potential in vivo conditions. As shown in Figure 7A, the GO group exhibited no significant cytotoxicity at any concentration. Even at the highest concentration of 10.00 mg/mL, the cell viability remained high (94.71% ± 1.58%), demonstrating its excellent biocompatibility. Similarly, the GOH-S₁₀ hydrogel (Figure 7B) showed no toxic effects at concentrations below 5.00 mg/mL, with cell viability consistently above 90%. At 10.00 mg/mL, a slight decrease in viability was observed; however, the rate remained above 80%, indicating acceptable biosafety even at this elevated concentration.

Figure 7

Figure 7. In vitro cytotoxicity assessment of hydrogels. (A,B) viability of HCECs cultured with hydrogel extracts for 24 h, assessed by CCK-8 assay for (A) GO and (B) GOH-S₁₀ hydrogels; (C–E) representative fluorescence micrographs of HCECs after direct contact with hydrogels for 24, 48, and 72 h, respectively; and (F–H) quantification of live cell percentages at the corresponding time points. Live cells are stained green (Calcein-AM) and dead cells are stained red (propidium iodide). Scale bars in (C–E) represent 50 µm. Statistical significance is denoted as p < 0.05, *p < 0.01, and **p < 0.0001; ns indicates no significant difference.

To further evaluate the impact of the material on direct cell contact, we performed live/dead staining on the Control group, GO group and GOH-S₁₀ group (Figures 7C–E), and quantitatively analysed the cell survival rates at different time points using ImageJ software (Figures 7F–H). Results indicated that all hydrogel groups supported high cell viability throughout the 72-h culture period. At 24 h, the Control group showed the highest viability (99.69% ± 0.17%), followed closely by the GO hydrogel (99.05% ± 0.59%) and the GOH-S₁₀ hydrogel (98.59% ± 0.36%), with all groups demonstrating viability rates exceeding 98% (Figure 7F). By 48 h, the viability rates across groups became highly comparable, with values of 99.38% ± 0.52% (Control), 99.13% ± 0.70% (GO), and 99.24% ± 0.66% (GOH-S₁₀) (Figure 7G). This excellent viability was sustained through 72 h, where the GO hydrogel remained stable (99.11% ± 0.77%) and the GOH-S₁₀ hydrogel’s viability was similarly high at 99.07% ± 0.41% (Figure 7H).

Assessment of in vitro wound healing capabilities

The cellular compatibility of the hydrogels was systematically evaluated through cell proliferation and migration assays, with the corresponding results presented in Figure 8. During the 7-day culture period, cells grown on both GO and GOH-S₁₀ hydrogels exhibited significantly greater proliferative capacity than those in the control group (Figure 8A). Cell proliferation activity was quantified using the CCK-8 assay (Figure 8B). From day 4 onwards, the proliferation rate of the GOH-S₁₀ group surpassed that of both the control and GO groups (p < 0.05). By day 7, the GOH-S₁₀ group showed the highest proliferation, with an optical density value markedly greater than that of the control group. These results indicated that the GOH-S₁₀ hydrogel markedly enhances corneal epithelial cell proliferation, a key factor in accelerating corneal repair.

Figure 8

Figure 8. Effects of Hydrogels on HCECs Proliferation and Migration. (A) Cell proliferation was assessed via Calcein-AM staining at days 1, 4, and 7 post-culture. Green fluorescence indicates viable cells. Scale bar = 100 µm. (B) Cell proliferation activity was quantified using the CCK-8 assay at days 1, 4, and 7. (C) Representative images of cell scratch healing assays at different time points (0 h, 24 h, 48 h). Dashed lines indicate the initial scratch edges. Scale bar = 100 µm. (D) Semi-quantitative analysis of cell migration area. All data are presented as mean ± standard deviation (n = 3). ns: no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001.

In the cell migration assay (Figure 8C), the control group displayed the slowest migration rate within 48 h, whereas the GOH-S₁₀ group exhibited the most rapid migration. Quantitative analysis (Figure 8D) further confirmed that the wound closure rates in the GO group reached 69.14% ± 1.00% and in the GOH-S₁₀ group reached 78.17% ± 4.31% at 48 h, both of which were significantly higher than the corresponding values in the control group (56.70% ± 2.87% at 48 h). Collectively, these findings demonstrated that the GOH-S10 hydrogel not only possesses excellent cytocompatibility but also promotes the proliferation and migration of human corneal epithelial cells. Its pronounced ability to enhance cell migration suggests that, when applied as a corneal patch, the hydrogel can effectively accelerate epithelial wound repair and facilitate the restoration of the ocular surface barrier. In summary, while fulfilling essential biosafety requirements, the GOH-S₁₀ hydrogel also exhibited functional bioactivity in directing tissue regeneration and promoting wound healing, thereby providing compelling evidence for its potential clinical application in corneal repair.

Discussion

The development of biomaterials for corneal repair represents a significant challenge within the realm of ophthalmic tissue engineering. Despite advances in biomaterials, there remains a critical unmet need for a single material that can simultaneously achieve high transparency, robust tissue adhesion, appropriate mechanical strength, and enduring antioxidant properties (Shirzaei Sani et al., 2019; Wu et al., 2024). This study addresses a specific problem by employing a sophisticated sequential crosslinking strategy to develop the GOH-S₁₀ hydrogel. This innovative composite is based on modified biological macromolecules, including GelMA, ODEX, and HA-SH. Unlike traditional single-network hydrogels, the fabrication process of GOH-S₁₀ involves multiple stages. It begins with a dynamic Schiff base reaction, followed by an efficient Michael addition, and concludes with stabilising photopolymerization (Arkenberg et al., 2020).

A paramount requirement for biomaterials used in the cornea is optical transparency, ensuring unobstructed vision (Meek and Knupp, 2015; Mahdavi et al., 2020; Li and Wang, 2025). The GOH-S₁₀ hydrogel, obtained by incorporating 1% (w/v) HA-SH, exhibited a light transmittance of over 85% across the visible spectrum, a value comparable to that of the native cornea, which is attributed to its homogeneous network structure. Several studies have indicated that increasing the HA content or modifying certain aspects of HA leads to increased light scattering and reduced transmittance in HA-based hydrogels (Lee et al., 2018; Park et al., 2022). Therefore, reactant concentrations must be precisely optimised to ensure efficient crosslinking without causing detrimental physical aggregation (Summonte et al., 2021). Unlike the non-monotonic trend observed in overall mechanical properties, the tissue adhesion strength of the GOH-S hydrogels consistently increased with higher HA-SH concentration. This enhancement is likely attributable to the higher density of thiol groups at the interface, which facilitates the formation of covalent disulfide bonds with corneal tissue (Wirostko et al., 2014; Griesser et al., 2018).

The inflammatory response following corneal trauma generates excessive ROS, a key pathological factor leading to delayed healing and corneal opacity (Zhang et al., 2024). Both DPPH and ABTS assays confirmed the potent free-radical scavenging capabilities of the GOH-S₁₀ hydrogel. Subsequent DCFH-DA probe tests demonstrated that GOH-S₁₀ could effectively remove hydrogen peroxide. In contrast to rapidly degradable polyphenolic antioxidants or nano-antioxidants with potential bioaccumulation concerns, the covalent integration of biocompatible HA-SH into the hydrogel network in GOH-S₁₀ circumvents these safety issues, thereby offering a sustainable and localised antioxidant therapeutic strategy for corneal applications (Brglez Mojzer et al., 2016; Cai et al., 2018; Meng et al., 2023; Chen et al., 2024).

Safety remains a paramount consideration for any biomaterial intended for corneal applications, as it must demonstrate non-toxicity to resident cells and complete biocompatibility upon integration with host tissues (Agrawal et al., 2024; Li and Wang, 2025). To systematically evaluate the cytocompatibility, the material extracts were assessed using the CCK-8 assay and live/dead staining, followed by proliferation and migration experiments with HCECs. The results indicated that the GOH-S₁₀ hydrogel exhibited low cytotoxicity and significantly promoted cell proliferation and migration, demonstrating excellent biocompatibility. In summary, the GOH-S₁₀ hydrogel combines outstanding biocompatibility, favorable mechanical properties, reliable tissue adhesion, high transparency, and sustained antioxidant activity, making it a potential candidate for advanced corneal repair therapies.

Conclusion

This study successfully constructed a multi-component, crosslinked GOH-S₁₀ hydrogel. Initial characterization confirmed that this platform integrates several functionalities—including biocompatibility, mechanical robustness, tissue adhesion, optical transparency, and ROS-scavenging activity—which are desirable for corneal repair. While these in vitro results are promising, this work represents a preliminary proof-of-concept, and its significant limitations must be acknowledged. The primary constraint is that all findings are derived solely from in vitro models, which cannot replicate the complex ocular environment. Consequently, critical translational aspects—such as the hydrogel’s long-term biocompatibility, the durability of its antioxidant effects, and ultimately, its therapeutic efficacy—remain entirely unverified. Future work must therefore prioritize rigorous evaluation in preclinical animal models to comprehensively assess the hydrogel’s safety, functionality, and mechanism of action in vivo. Only through these essential studies can the true translational potential of the GOH-S₁₀ hydrogel be determined.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Author contributions

YiP: Software, Investigation, Visualization, Methodology, Writing – original draft. TY: Conceptualization, Supervision, Data curation, Writing – original draft, Project administration, Software, Visualization. WY: Resources, Methodology, Project administration, Validation, Writing – original draft, Funding acquisition. JX: Writing – review and editing, Supervision, Methodology, Project administration, Data curation. YaP: Project administration, Methodology, Validation, Supervision, Writing – review and editing. CC: Project administration, Methodology, Validation, Conceptualization, Writing – review and editing. YL: Validation, Methodology, Writing – review and editing, Supervision. JL: Software, Writing – original draft, Validation, Project administration, Data curation, Visualization. DW: Resources, Methodology, Writing – review and editing, Project administration. YY: Writing – review and editing, Supervision, Project administration, Writing – original draft, Resources, Data curation, Funding acquisition.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the Natural Science Foundation of Jiangxi Province (20212ACB206022) and the Science and Technology Program of Jiangxi Provincial Health Commission (202110043).

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

Agrawal, P., Tiwari, A., Chowdhury, S. K., Vohra, M., Gour, A., Waghmare, N., et al. (2024). Kuragel: a biomimetic hydrogel scaffold designed to promote corneal regeneration. iScience 27 (5), 109641. doi:10.1016/j.isci.2024.109641

PubMed Abstract | CrossRef Full Text | Google Scholar

Ambrožič, R., Krühne, U., and Plazl, I. (2024). Microfluidics with redox-responsive hydrogels for on-demand BPA degradation. Chem. Eng. J. 485, 149542. doi:10.1016/j.cej.2024.149542

CrossRef Full Text | Google Scholar

Annabi, N., Tamayol, A., Uquillas, J. A., Akbari, M., Bertassoni, L. E., Cha, C., et al. (2014). 25th anniversary article: rational design and applications of hydrogels in regenerative medicine. Adv. Mater 26 (1), 85–123. doi:10.1002/adma.201303233

PubMed Abstract | CrossRef Full Text | Google Scholar

Arkenberg, M. R., Nguyen, H. D., and Lin, C. C. (2020). Recent advances in bio-orthogonal and dynamic crosslinking of biomimetic hydrogels. J. Mater Chem. B 8 (35), 7835–7855. doi:10.1039/d0tb01429j

PubMed Abstract | CrossRef Full Text | Google Scholar

Augustine, R., Zahid, A. A., Hasan, A., Dalvi, Y. B., and Jacob, J. (2021). Cerium oxide nanoparticle-loaded gelatin methacryloyl hydrogel wound-healing patch with free radical scavenging activity. Acs Biomaterials Sci. and Eng. 7 (1), 279–290. doi:10.1021/acsbiomaterials.0c01138

PubMed Abstract | CrossRef Full Text | Google Scholar

Balbino, T. A., Correa, G. S. C., Favaro, M. T. P., Toledo, M. A. S., Azzoni, A. R., and de la Torre, L. G. (2015). Physicochemical and evaluation of cationic liposome, hyaluronic acid and plasmid DNA as pseudo-ternary complexes for gene delivery. Colloids Surfaces A-Physicochemical Eng. Aspects 484, 262–270. doi:10.1016/j.colsurfa.2015.08.005

CrossRef Full Text | Google Scholar

Beems, E. M., and Van Best, J. A. (1990). Light transmission of the cornea in whole human eyes. Exp. Eye Res. 50(4), 393–395. doi:10.1016/0014-4835(90)90140

PubMed Abstract | CrossRef Full Text | Google Scholar

Bertsch, P., Diba, M., Mooney, D. J., and Leeuwenburgh, S. C. G. (2023). Self-healing injectable hydrogels for tissue regeneration. Chem. Rev. 123 (2), 834–873. doi:10.1021/acs.chemrev.2c00179

PubMed Abstract | CrossRef Full Text | Google Scholar

Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature 181 (4617), 1199–1200. doi:10.1038/1811199a0

CrossRef Full Text | Google Scholar

Borouman, S., Sigaroodi, F., Ahmadi Tafti, S. M., Khoshmaram, K., Soleimani, M., and Khani, M. M. (2024). ECM-based bioadhesive hydrogel for sutureless repair of deep anterior corneal defects. Biomater. Sci. 12 (9), 2356–2368. doi:10.1039/d4bm00129j

PubMed Abstract | CrossRef Full Text | Google Scholar

Brglez Mojzer, E., Knez Hrncic, M., Skerget, M., Knez, Z., and Bren, U. (2016). Polyphenols: extraction methods, antioxidative action, bioavailability and anticarcinogenic effects. Molecules 21 (7), 901. doi:10.3390/molecules21070901

PubMed Abstract | CrossRef Full Text | Google Scholar

Cai, Z. Y., Li, X. M., Liang, J. P., Xiang, L. P., Wang, K. R., Shi, Y. L., et al. (2018). Bioavailability of tea catechins and its improvement. Molecules 23 (9), 2346. doi:10.3390/molecules23092346

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, Y., Sui, J., Wang, Q., Yin, Y., Liu, J., Wang, Q., et al. (2018). Injectable self-crosslinking HA-SH/Col I blend hydrogels for in vitro construction of engineered cartilage. Carbohydr. Polym. 190, 57–66. doi:10.1016/j.carbpol.2018.02.057

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, S., Wang, Y., Bao, S., Yao, L., Fu, X., Yu, Y., et al. (2024). Cerium oxide nanoparticles in wound care: a review of mechanisms and therapeutic applications. Front. Bioeng. Biotechnol. 12, 1404651. doi:10.3389/fbioe.2024.1404651

PubMed Abstract | CrossRef Full Text | Google Scholar

Cheng, H., Shi, Z., Yue, K., Huang, X., Xu, Y., Gao, C., et al. (2021). Sprayable hydrogel dressing accelerates wound healing with combined reactive oxygen species-scavenging and antibacterial abilities. Acta Biomater. 124, 219–232. doi:10.1016/j.actbio.2021.02.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Dunnill, C., Patton, T., Brennan, J., Barrett, J., Dryden, M., Cooke, J., et al. (2017). Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process. Int. Wound J. 14 (1), 89–96. doi:10.1111/iwj.12557

PubMed Abstract | CrossRef Full Text | Google Scholar

Frykberg, R. G., and Banks, J. (2015). Challenges in the treatment of chronic wounds. Adv. Wound Care (New Rochelle) 4 (9), 560–582. doi:10.1089/wound.2015.0635

PubMed Abstract | CrossRef Full Text | Google Scholar

Fulmer, G. R., Miller, A. J. M., Sherden, N. H., Gottlieb, H. E., Nudelman, A., Stoltz, B. M., et al. (2010). NMR chemical shifts of trace impurities: common laboratory solvents, organics, and gases in deuterated solvents relevant to the organometallic chemist. Organometallics 29 (9), 2176–2179. doi:10.1021/om100106e

CrossRef Full Text | Google Scholar

Ghosh, A., Bera, A. K., Adhikari, J., Ghosh, S., Singh, V., Basu, S., et al. (2025). Bioprinting of transparent and adhesive corneal patches: integrating photo-crosslinkable dopamine-conjugated silk fibroin and decellularized cornea matrix for sutureless tissue integration and regeneration. Int. J. Biol. Macromol. 306 (Pt 4), 141761. doi:10.1016/j.ijbiomac.2025.141761

PubMed Abstract | CrossRef Full Text | Google Scholar

Griesser, J., Hetenyi, G., and Bernkop-Schnurch, A. (2018). Thiolated hyaluronic acid as versatile mucoadhesive polymer: from the chemistry behind to product developments-what are the capabilities? Polym. (Basel) 10 (3), 243. doi:10.3390/polym10030243

PubMed Abstract | CrossRef Full Text | Google Scholar

Hudson, S. P., Langer, R., Fink, G. R., and Kohane, D. S. (2010). Injectable in situ cross-linking hydrogels for local antifungal therapy. Biomaterials 31 (6), 1444–1452. doi:10.1016/j.biomaterials.2009.11.016

PubMed Abstract | CrossRef Full Text | Google Scholar

Khosravimelal, S., Mobaraki, M., Eftekhari, S., Ahearne, M., Seifalian, A. M., and Gholipourmalekabadi, M. (2021). Hydrogels as emerging materials for cornea wound healing. Small 17 (30), e2006335. doi:10.1002/smll.202006335

PubMed Abstract | CrossRef Full Text | Google Scholar

Lee, B. H., Lum, N., Seow, L. Y., Lim, P. Q., and Tan, L. P. (2016). Synthesis and characterization of types A and B gelatin methacryloyl for bioink applications. Mater. (Basel) 9 (10), 797. doi:10.3390/ma9100797

PubMed Abstract | CrossRef Full Text | Google Scholar

Lee, H. J., Fernandes-Cunha, G. M., and Myung, D. (2018). In situ-forming hyaluronic acid hydrogel through visible light-induced thiol-ene reaction. React. Funct. Polym. 131, 29–35. doi:10.1016/j.reactfunctpolym.2018.06.010

PubMed Abstract | CrossRef Full Text | Google Scholar

Li, Y., and Wang, Z. (2025). Biomaterials for corneal regeneration. Adv. Sci. (Weinh) 12 (6), e2408021. doi:10.1002/advs.202408021

PubMed Abstract | CrossRef Full Text | Google Scholar

Li, X., Chen, M., Wang, P., Yao, Y., Han, X., Liang, J., et al. (2020). A highly interweaved HA-SS-nHAp/collagen hybrid fibering hydrogel enhances osteoinductivity and mineralization. Nanoscale 12 (24), 12869–12882. doi:10.1039/d0nr01824d

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, F., Zang, S., Jing, J., and Zhang, X. (2020). A fluorescent probe based on reversible Michael addition-elimination reaction for the cycle between cysteine and H(2)O(2). Anal. Methods 12 (30), 3797–3801. doi:10.1039/d0ay00904k

PubMed Abstract | CrossRef Full Text | Google Scholar

Liu, H. W., Su, W. T., Liu, C. Y., and Huang, C. C. (2022). Highly organized porous Gelatin-based scaffold by microfluidic 3D-Foaming technology and dynamic culture for cartilage tissue engineering. Int. J. Mol. Sci. 23 (15), 8449. doi:10.3390/ijms23158449

PubMed Abstract | CrossRef Full Text | Google Scholar

Lopes, F. B., Sarandy, M. M., Novaes, R. D., Valacchi, G., and Goncalves, R. V. (2024). OxInflammatory responses in the wound healing process: a systematic review. Antioxidants (Basel) 13 (7), 823. doi:10.3390/antiox13070823

PubMed Abstract | CrossRef Full Text | Google Scholar

Mahdavi, S. S., Abdekhodaie, M. J., Mashayekhan, S., Baradaran-Rafii, A., and Djalilian, A. R. (2020). Bioengineering approaches for corneal regenerative medicine. Tissue Eng. Regen. Med. 17 (5), 567–593. doi:10.1007/s13770-020-00262-8

PubMed Abstract | CrossRef Full Text | Google Scholar

Meek, K. M., and Knupp, C. (2015). Corneal structure and transparency. Prog. Retin Eye Res. 49, 1–16. doi:10.1016/j.preteyeres.2015.07.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Meng, C. Y., Ma, X. Y., Xu, M. Y., Pei, S. F., Liu, Y., Hao, Z. L., et al. (2023). Transcriptomics-based investigation of manganese dioxide nanoparticle toxicity in rats' choroid plexus. Sci. Rep. 13 (1), 8510. doi:10.1038/s41598-023-35341-y

PubMed Abstract | CrossRef Full Text | Google Scholar

Millan-Reyes, M. J., Afanador-Restrepo, D. F., Carcelen-Fraile, M. D. C., Aibar-Almazan, A., Sanchez-Alcala, M., Cano-Sanchez, J., et al. (2025). Reducing infections and improving healing in complex wounds: a systematic review and meta-analysis. J. Clin. Med. 14 (9), 3237. doi:10.3390/jcm14093237

PubMed Abstract | CrossRef Full Text | Google Scholar

Negut, I., Dorcioman, G., and Grumezescu, V. (2020). Scaffolds for wound healing applications. Polym. (Basel) 12 (9), 2010. doi:10.3390/polym12092010

PubMed Abstract | CrossRef Full Text | Google Scholar

Pamplona, R., González-Lana, S., Romero, P., Ochoa, I., Martín-Rapún, R., and Sánchez-Somolinos, C. (2023). Tuning of mechanical properties in photopolymerizable Gelatin-based hydrogels for in vitro cell culture systems. ACS Appl. Polym. Mater. 5 (2), 1487–1498. doi:10.1021/acsapm.2c01980

PubMed Abstract | CrossRef Full Text | Google Scholar

Park, S. K., Ha, M., Kim, E. J., Seo, Y. A., Lee, H. J., Myung, D., et al. (2022). Hyaluronic acid hydrogels crosslinked via blue light-induced thiol-ene reaction for the treatment of rat corneal alkali burn. Regen. Ther. 20, 51–60. doi:10.1016/j.reth.2022.03.005

PubMed Abstract | CrossRef Full Text | Google Scholar

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26 (9-10), 1231–1237. doi:10.1016/s0891-5849(98)00315-3

PubMed Abstract | CrossRef Full Text | Google Scholar

Samyn, P., Van Nieuwkerke, D., Schoukens, G., Vonck, L., Stanssens, D., and Van Den Abbeele, H. (2012). Quality and statistical classification of Brazilian vegetable oils using mid-infrared and Raman spectroscopy. Appl. Spectrosc. 66 (5), 552–565. doi:10.1366/11-06484

PubMed Abstract | CrossRef Full Text | Google Scholar

Shirahama, H., Lee, B. H., Tan, L. P., and Cho, N. J. (2016). Precise tuning of facile one-pot gelatin methacryloyl (GelMA) synthesis. Sci. Rep. 6, 31036. doi:10.1038/srep31036

PubMed Abstract | CrossRef Full Text | Google Scholar

Shirzaei Sani, E., Kheirkhah, A., Rana, D., Sun, Z., Foulsham, W., Sheikhi, A., et al. (2019). Sutureless repair of corneal injuries using naturally derived bioadhesive hydrogels. Sci. Adv. 5 (3), eaav1281. doi:10.1126/sciadv.aav1281

PubMed Abstract | CrossRef Full Text | Google Scholar

Smits, J., van der Pol, A., Goumans, M. J., Bouten, C. V. C., and Jorba, I. (2024). GelMA hydrogel dual photo-crosslinking to dynamically modulate ECM stiffness. Front. Bioeng. Biotechnol. 12, 1363525. doi:10.3389/fbioe.2024.1363525

PubMed Abstract | CrossRef Full Text | Google Scholar

Solanki, D., Vinchhi, P., and Patel, M. M. (2023). Design considerations, formulation approaches, and strategic advances of hydrogel dressings for chronic wound management. ACS Omega 8 (9), 8172–8189. doi:10.1021/acsomega.2c06806

PubMed Abstract | CrossRef Full Text | Google Scholar

Summonte, S., Racaniello, G. F., Lopedota, A., Denora, N., and Bernkop-Schnürch, A. (2021). Thiolated polymeric hydrogels for biomedical application: cross-linking mechanisms. J. Control. Release 330, 470–482. doi:10.1016/j.jconrel.2020.12.037

PubMed Abstract | CrossRef Full Text | Google Scholar

Sun, L., Zhou, J., Lai, J., Zheng, X., Wang, H., Lu, B., et al. (2024). Novel natural polymer-based hydrogel patches with janus asymmetric-adhesion for emergency hemostasis and wound healing. Adv. Funct. Mater. 34 (36), 2401030. doi:10.1002/adfm.202401030

CrossRef Full Text | Google Scholar

Tanga, S., Aucamp, M., and Ramburrun, P. (2023). Injectable thermoresponsive hydrogels for cancer therapy: challenges and prospects. Gels 9 (5), 418. doi:10.3390/gels9050418

PubMed Abstract | CrossRef Full Text | Google Scholar

Ukaegbu, K., Allen, E., and Svoboda, K. K. H. (2025). Reactive oxygen species and antioxidants in wound healing: mechanisms and therapeutic potential. Int. Wound J. 22 (5), e70330. doi:10.1111/iwj.70330

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, Z. J., Xiang, C. P., Yao, X., Le Floch, P., Mendez, J., and Suo, Z. G. (2019). Stretchable materials of high toughness and low hysteresis. Proc. Natl. Acad. Sci. U. S. A. 116 (13), 5967–5972. doi:10.1073/pnas.1821420116

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, J., Lin, Y., Fan, H., Cui, J., Wang, Y., and Wang, Z. (2025a). ROS/pH dual-responsive hydrogel dressings loaded with amphiphilic structured nano micelles for the repair of infected wounds. Int. J. Nanomedicine 20, 8119–8142. doi:10.2147/IJN.S522589

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, R., Wang, Z., and Jiang, N. (2025b). The preparation, properties, and applications of polyphenol hydrogels for the treatment of oral diseases: a comprehensive overview. Int. J. Biol. Macromol. 321 (Pt 3), 146310. doi:10.1016/j.ijbiomac.2025.146310

PubMed Abstract | CrossRef Full Text | Google Scholar

Wirostko, B., Mann, B. K., Williams, D. L., and Prestwich, G. D. (2014). Ophthalmic uses of a thiol-modified hyaluronan-based hydrogel. Adv. Wound Care (New Rochelle) 3 (11), 708–716. doi:10.1089/wound.2014.0572

PubMed Abstract | CrossRef Full Text | Google Scholar

World Stroke, O., Sacks, D., Baxter, B., Campbell, B. C. V., Carpenter, J. S., Cognard, C., et al. (2018). Multisociety consensus quality improvement revised consensus statement for endovascular therapy of acute ischemic stroke. Int. J. Stroke 13 (6), 612–632. doi:10.1177/1747493018778713

PubMed Abstract | CrossRef Full Text | Google Scholar

Wu, K. Y., Qian, S. Y., Faucher, A., and Tran, S. D. (2024). Advancements in hydrogels for corneal healing and tissue engineering. Gels 10 (10), 662. doi:10.3390/gels10100662

PubMed Abstract | CrossRef Full Text | Google Scholar

Xue, X., Wu, Y., Xu, X., Xu, B., Chen, Z., and Li, T. (2021). pH and reduction dual-responsive Bi-Drugs conjugated dextran assemblies for combination chemotherapy and in vitro evaluation. Polym. (Basel) 13 (9), 1515. doi:10.3390/polym13091515

PubMed Abstract | CrossRef Full Text | Google Scholar

Yan, S., Wang, Q., Zhang, S., Huang, Y., Zhu, H., Qi, B., et al. (2024). Oxidized dextran improves the stability and effectively controls the release of curcumin loaded in soybean protein nanocomplexes. Food Chem. 431, 137089. doi:10.1016/j.foodchem.2023.137089

PubMed Abstract | CrossRef Full Text | Google Scholar

Yang, E., Miao, S. D., Zhong, J., Zhang, Z. Y., Mills, D. K., and Zhang, L. G. (2018). Bio-based polymers for 3D printing of bioscaffolds. Polym. Rev. 58 (4), 668–687. doi:10.1080/15583724.2018.1484761

PubMed Abstract | CrossRef Full Text | Google Scholar

Yang, N., Jike, X., Zhang, M., Jiang, T., and Lei, H. (2025a). Synthesis, characterization of thiolated hyaluronic acid and evaluation of its encapsulation effects on Limosilactobacillus reuteri HR7. Int. J. Biol. Macromol. 310 (Pt 4), 143486. doi:10.1016/j.ijbiomac.2025.143486

PubMed Abstract | CrossRef Full Text | Google Scholar

Yang, Y., Liu, Y., Wang, S., Feng, X., Lan, J., and Dong, Z. (2025b). Injectable dual network hydrogel containing Mg2+-Gallate-Based MOF and bone growth polypeptide for extraction socket site preservation. ACS Biomaterials Sci. and Eng. 11, 5600–5615. doi:10.1021/acsbiomaterials.5c01060

PubMed Abstract | CrossRef Full Text | Google Scholar

Yu, H., Luo, X., Li, Y., Shao, L., Yang, F., Pang, Q., et al. (2024). Advanced hybrid strategies of GelMA composite hydrogels in bone defect repair. Polym. (Basel) 16 (21), 3039. doi:10.3390/polym16213039

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhang, W., Wang, H., Pang, J., Huang, Y., Li, H., and Tang, S. (2024). Self-crosslinking hyaluronic acid-based hydrogel with promoting vascularization and ROS scavenging for wound healing. Int. J. Biol. Macromol. 278 (Pt 1), 134570. doi:10.1016/j.ijbiomac.2024.134570

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhao, X., Li, S., Du, X., Li, W., Wang, Q., He, D., et al. (2022). Natural polymer-derived photocurable bioadhesive hydrogels for sutureless keratoplasty. Bioact. Mater 8, 196–209. doi:10.1016/j.bioactmat.2021.07.001

PubMed Abstract | CrossRef Full Text | Google Scholar