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. 2026 Jun 12;11(25):36767–36779. doi: 10.1021/acsomega.5c13610

Research on the Improvement of Oxidative Stress Regulation in Periodontal Ligament Stem Cells and the Promotion of Periodontal Bone Tissue Regeneration by GDF5/ROS-Responsive Degradable Hydrogel

Jing Zhou 1, Yu Zhan 1, He Zhang 1, Jie Yang 1, Wenlei Wu 1,*, Yu Liu 1,*
PMCID: PMC13325396  PMID: 42395953

Abstract

Periodontitis is a chronic inflammatory disorder characterized by a sustained inflammatory stimulus that creates an oxidative stress microenvironment within periodontal tissues. Under such conditions, elevated levels of reactive oxygen species (ROS) disrupt the structural and functional integrity of periodontal ligament stem cells (PDLSCs), inhibit osteogenic differentiation, and impair the regeneration of periodontal bone defects. Herein, a ROS-responsive degradable hydrogel loaded with growth differentiation factor 5 (GDF5) was developed to target this pathological microenvironment. By incorporating a thioketal (TK) linker that can be cleaved by ROS, the hydrogel reacts with high concentrations of ROS, improving the oxidative stress environment. As the reaction occurs, the hydrogel is degraded, and during this process, GDF5 is successfully released into the microenvironment, then exerts a sustained osteogenic-promoting effect. This study does not simply expose GDF5 to a harmful environment. Instead, our hydrogel first rapidly scavenges high levels of ROS upon sensing them, creating a redox-balanced microenvironment for the cells; subsequently, it releases GDF5 through an ROS-triggered mechanism. This sequential logic of “clearing the environment first, then treating” is designed to ensure that the released GDF5 can function in an optimized and functional microenvironment, thereby maximizing its protective effects and promoting its osteogenic activity.

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1. Introduction

Periodontal disease is a chronic inflammatory disease caused by dental plaque biofilm, which results in progressive destruction of the periodontal supporting tissues. In recent years, oxidative stress has been widely regarded as the core mechanism of the key link in the destruction of the connection between microorganisms and host tissues in periodontal disease. When periodontal pathogenic bacteria and their toxins invade the periodontal tissues, the host immune system responds rapidly. To eliminate the pathogens, cells will initiate intense metabolic processes, generating ROS far beyond the normal level. This leads to an explosive growth of ROS in the local periodontal environment. Excessively produced reactive oxygen species induce oxidative stress, which plays a role in bone homeostasis and diseases. , Reactive oxygen species can promote the differentiation and proliferation of osteoclasts through signal pathways such as RANK and mitogen-activated protein kinase, thereby aggravating the absorption of periodontal tissues. Or they can induce the secretion of inflammatory factors by periodontal membrane fibroblasts, causing the differentiation of osteoclasts and aggravating the absorption of periodontal bone tissues.

In recent years, the relationship between mitochondrial homeostasis and periodontal disease has also gradually attracted the attention of scholars. Under disease conditions, the mitochondrial structure and function of periodontal tissue stem cells, odontoblast cells, etc., in the periodontal tissues undergo significant changes, and are related to the progression of periodontal disease. Accordingly, therapeutic modulation of oxidative stress represents an emerging strategy in periodontal therapy. Recent advances in engineered in vitro systemsparticularly those modeling oxidative stress, recapitulating tissue regeneration dynamics, and enabling spatiotemporally controlled growth factor deliveryhave deepened mechanistic insights into periodontal pathogenesis and accelerated the development of personalized interventions, highlighting their significant translational potential in regenerative dentistry. This approach aims to disrupt the self-perpetuating pathological cycle underlying periodontitis, providing potential strategies to halt disease progression and preserve the integrity of periodontal tissues. − The restoration of both the structural morphology and functional integrity of damaged periodontal bone remains a significant challenge in periodontal regenerative therapy.

In recent years, the emergence of bone tissue engineering (BTE) has provided promising new strategies for alveolar bone repair. Advances in BTE are contingent upon innovations in materials science and a deeper understanding of bone biology. This interdisciplinary field is fundamentally built upon three key components: seed cells, growth factors, and scaffolds. , Of the numerous growth factors identified to date, bone morphogenetic proteins (BMPs) have attracted extensive research interest owing to their remarkable osteoinductive capacity and multiple biological activities. Mounting experimental data, together with our prior investigations, demonstrate that growth and differentiation factor 5 (GDF5), belonging to the BMP superfamily, is capable of prominently promoting osteogenic differentiation in periodontal ligament stem cells (PDLSCs). − Despite these favorable effects, clinical application of GDF5 remains greatly restricted on account of its brief biological half-life as well as its spontaneous rapid and unregulated release upon direct in vivo delivery. Consequently, the development of controlled delivery systems for GDF5 during guided tissue regeneration is crucial to maximizing its bioactivity and clinical efficacy.

Whether growth factors can be successfully delivered to bone defect areas and fully exert their effects has always been a key focus for many researchers, especially in the context of periodontitis, which involves oxidative stress. Intelligent hydrogels that can respond precisely and controllably to internal and external cues have aroused extensive research interest over recent decades owing to their versatile prospects within the field of regenerative therapy. For example, Zhao and colleagues developed a hydrogel that scavenges reactive oxygen species (ROS) by cross-linking poly­(vinyl alcohol) (PVA) with ROS-responsive linkers. This hydrogel can effectively reduce ROS levels at wound sites and promote wound healing by upregulating M2 macrophages, thereby facilitating tissue repair. In another study, Xia’s team designed an injectable hydrogel system based on carboxymethyl chitosan and oxidized dextran (CMCS-OD), which forms through dynamic chemical bonds and exhibits self-healing and pH-responsive properties, enabling the controlled release of embelin. Furthermore, it modulates macrophage polarization by regulating the M1/M2 phenotypic balance and suppresses the expression of pro-inflammatory cytokines, thus creating a favorable microenvironment for bone regeneration and mitigating inflammation-induced alveolar bone loss. , These smart hydrogels exhibit programmable and tunable responses under specific stimuli, encompassing tunable breakdown behavior, hydrogel formation, reactive oxygen species elimination, nanostructure conversion, and spatiotemporally controlled drug delivery, offering great potential for precise regenerative therapy.

Herein, we present a reactive oxygen species (ROS)-responsive degradable hydrogel integrated with growth differentiation factor 5 (GDF5), designed to actively target the pathological microenvironment of periodontal bone defects. The hydrogel incorporates a ROS-cleavable thioketal (TK) linker, enabling its controlled degradation in response to elevated ROS concentrations typical of inflammatory conditions. This degradation not only modulates local ROS levels and alleviates inflammation but also gradually improves the oxidative stress landscape. Following the initial ROS-scavenging phase, GDF5 is released in a sustained manner, exerting continuous osteogenic induction within the restored microenvironment. Rather than merely delivering GDF5 into a hostile inflammatory environment, this system operates through a sequential therapeutic logic: it first mitigates oxidative stress to establish a redox-balanced cellular niche, and then initiates the controlled release of bioactive GDF5 via ROS-triggered hydrogel degradation. This “clear-then-treat” strategy is designed to maximize the regenerative efficacy of GDF5 by ensuring its action within a conducive and functionally optimized microenvironment.

2. Materials and Methods

2.1. Preparation of GDF5/ROS-Responsive Degradable Hydrogels

  • (1)

    Synthesis of TK: A mixture of 11.4 g 3-mercaptopropionic acid, 3.7 mL acetone, a proper dosage of trifluoroacetic acid was incorporated into the system, followed by constant stirring at ambient temperature over a 6 h period. Afterward, the resultant mixture was cooled in an ice bath to generate white solid precipitates. After successive triple rinses, the acquired precipitate was subjected to lyophilization and conserved at −20 °C.

  • (2)

    Polymerization of 8-arm PEG-TK-NB and 8-arm PEG-NB: A composite consisting of 378.0 mg TK, 288.0 mg EDC, and 172.5 mg NHS was dissolved in dichloromethane, with continuous agitation under ambient conditions for 3 h. Subsequently, 200.0 mg of 8-arm PEG-NH2 was introduced into the above solution, and the reaction was sustained for another 72 h. Gradient dialysis was conducted via a dialysis membrane with 20%, 15%, 10%, and 5% THF solutions, with each dialysis stage lasting 6 h. This was succeeded by 72 h of dialysis against deionized water, and 8-arm PEG-TK was finally obtained after freeze-drying treatment. For the fabrication of 8-arm PEG-TK-NB, a mixture containing 163.5 mg 8-arm PEG-TK, 115.2 mg EDC, and 69.0 mg NHS was dissolved in DCM and agitated at room temperature for 3 h. Thereafter, 73.8 mg of NB-NH2 was added into the reaction system, which was continuously reacted for 72 h. The purification protocol adopted for 8-arm PEG-TK-NB was identical to that of 8-arm PEG-TK. For control specimen preparation, 8-arm PEG-NB without TK moieties was synthesized. The purification approach for 8-arm PEG-NB was consistent with the aforementioned procedure of 8-arm PEG-TK.

  • (3)

    Synthesis of GDF5/ROS-responsive hydrogel: a precursor solution for the ROS-responsive degradable hydrogel was prepared by mixing 10.0% (w/w) 8-arm PEG-TK-NB with HS-PEG-SH at an 8:5 molar ratio, supplemented with 2 mM RGD and 0.05% (w/w) LAP in deionized water. Aliquot-frozen GDF5 was completely thawed at 37 °C and centrifuged at 1000 rpm for 10 s. Under a sterile laminar flow hood, GDF5 was added to the prepared hydrogel precursor solution. The mixture was exposed to ultraviolet light (365 nm, 30 mW cm–2) for approximately 20 s to form the GDF5/ROS-responsive degradable hydrogel,thereby encapsulating growth factor 5 within the gel network during the curing process, designated as the GDF5/TK­(+) hydrogel. The TK-free hydrogel, designated as the GDF5/TK(−) hydrogel, was prepared using 8-arm PEG-NB following the same procedure as for the GDF5/TK­(+) hydrogel.

2.2. Scanning Electron Microscopy (SEM) Analysis of Hydrogel Microstructure

The prepared hydrogel samples were immersed in deionized water for complete swelling over 24 h, followed by rapid quenching in liquid nitrogen for 1 min. Subsequently, the samples were stored in a −80 °C freezer for 24 h and then subjected to freeze-drying under vacuum for 48 h. A tiny amount of freeze-dried specimen was affixed onto conductive tape, followed by 45 s of gold sputtering at 10 mA via a Quorum SC7620 sputtering device. Surface microstructures were observed by employing a Hitachi Regulus 8100 scanning electron microscope under an accelerating voltage of 3 kV. The porosity and pore size distribution of the hydrogels were quantified using ImageJ analysis software.

2.3. Rheological Characterization of GDF5/ROS Hydrogel

The rheological behavior of the as-prepared hydrogel was analyzed with a Haake Mars 40 rheometer manufactured in Germany. Initially, time-scanning detection was carried out at 1% strain amplitude and 1 Hz frequency, which dynamically traced the changes in storage modulus (G′) and loss modulus (G″) against irradiation duration to realize in situ observation of gel formation. After that, frequency-scanning measurement was implemented over a frequency interval from 1 to 100 rad/s under fixed strain conditions, so as to analyze the frequency-dependent alterations of G′ and G″.

2.4. Evaluation of Hydrogel Degradation Performance

The in vitro degradation behavior of the hydrogels was assessed gravimetrically based on changes in dry mass. Hydrogel samples were prepared by UV curing of a defined volume of precursor solution, followed by freeze-drying for 12 h to obtain a constant initial dry weight, recorded as W 0. For degradation testing, samples in the control group were incubated in phosphate-buffered saline (PBS), while those in the experimental group were immersed in hydrogen peroxide (H2O2) solutions at varying concentrations to simulate oxidative conditions. All specimens were incubated at 37 °C with constant orbital agitation. At preset time points, each specimen was extracted, fully washed with deionized water, and softly dried via filter paper to eliminate superficial free water. Afterward, the specimens were weighed to record the residual dry mass (W 1). The mass retention ratio was computed according to the equation presented below: Remaining mass (%) = (W 1/W 0) × 100. Each time point included three replicate samples to ensure data reliability.

2.5. Analysis of GDF5 Drug Loading Capacity and Determination of In Vitro Release Rate

2.5.1. Determination of GDF5 Encapsulation Efficiency

One gram of solidified GDF5/ROS hydrogel containing varying concentrations of GDF5 was weighed and immersed in 10 mL of PBS solution. The mixture was centrifuged at 1000 rpm for 5 min, and 1 mL of supernatant was collected. The concentration of GDF5 in the supernatant was quantified using a GDF5 ELISA kit according to the manufacturer’s instructions. Based on the standard curve, the GDF5 concentration in the supernatant was determined and applied to the encapsulation efficiency (EE) formula to calculate the EE of GDF5/ROS-responsive degradable hydrogel under different drug-loading conditions. EE (%) = (Ct – Cf)/Ct × 100, where EE denotes encapsulation efficiency, Cf represents the mass of free GDF5 in the supernatant, and Ct signifies the total actual mass of GDF5.

2.5.2. Determination of GDF5 Release Rate

To evaluate the sustained release profile of growth and differentiation factor 5 (GDF5), hydrogel precursor solutions containing 200 ng/mL GDF5 were prepared and dispensed into 6-well plates at a volume of 1 mL per well. The solutions were cross-linked under UV irradiation (365 nm, 30 mW cm–2) to form hydrogels. Each experimental group included three technical replicates. Following gelation, 4 mL of 1× phosphate-buffered saline (PBS) was added to each well, while 2 mL of 1× PBS was placed in the surrounding wells (without hydrogel) to maintain humidity and prevent evaporation. The plates were incubated at 37 °C under continuous shaking at 50 rpm. At predetermined time points, 200 μL of the release medium was collected and transferred to microcentrifuge (EP) tubes. The concentration of released GDF5 was quantified using a commercially available GDF5 ELISA kit (Cloud-Clone Corp., China), following the manufacturer’s instructions. Absorbance values were measured at 450 nm with a microplate reader. Standard curves were generated for each assay, and sample concentrations were determined using the corresponding regression equations. The cumulative release rate of GDF5 was calculated according to the specified formula.

2.6. In Vitro Biocompatibility Assessment of Hydrogels

Periodontal ligament stem cells (PDLSCs) (Procell, China) were seeded in 12-well plates at a density of 3 × 104 cells per well and incubated at 37 °C in a humidified atmosphere with 5% CO2. After 4 h of initial attachment, the culture medium was replaced with hydrogel extract supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS). Cells cultured in standard growth medium without exposure to hydrogel extract served as the control group. Following a 48-h incubation period, the medium and hydrogel extracts were removed, and cells were stained with Calcein-AM/propidium iodide (PI) solution for 30 min at 37 °C. Fluorescence images were acquired using a fluorescence microscope (CKX53, Olympus, Japan). Live cells exhibited green fluorescence (Calcein-AM), while dead cells displayed red fluorescence (PI). Cell viability was further quantitatively evaluated using the CCK-8 assay. Briefly, PDLSCs were seeded in 96-well plates at a density of 5 × 103 cells per well and treated under the same conditions. At predetermined time points, 10 μL of CCK-8 reagent was added to each well, followed by incubation for 1 h. The absorbance was measured at 450 nm using a microplate reader to determine metabolic activity. Relative growth rate (RGR) = [(OD of experimental group – OD of blank group)/(OD of control group – OD of blank group)] × 100%.

2.7. Evaluation of Hydrogel Antioxidant Activity

In our study, oxidative stress modeling was induced by exogenous hydrogen peroxide exposure. The optimal concentration of hydrogen peroxide was determined through CCK-8 assays and flow cytometry analysis. A concentration of 200 μM hydrogen peroxide was subsequently employed for all further cellular experiments (Figure S1).

  • (1)

    In vitro ROS scavenging activity: the antioxidant capacity to eliminate reactive oxygen species (ROS) of synthesized hydrogels was determined via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging detection method. TK­(+) and TK(−) hydrogel samples (20 μL) were mixed with 100 μM DPPH working solution and cultured in 24-well microplates at 37 °C for a continuous 24 h reaction. As a standard antioxidant substance, TEMPOL solution (1 mg/mL) was adopted as the positive control group. After reaction completion, the absorbance value of the collected supernatant was detected at 517 nm wavelength with a microplate spectrophotometer. The inhibition percentage of DPPH radicals was calculated by the following equation: (ADPPH – Asample)/ADPPH × 100, where ADPPH represents the absorbance of the DPPH solution alone and Asample denotes the absorbance of the sample-containing solution. This value reflects the ROS scavenging efficacy of the hydrogels.

  • (2)

    Intracellular ROS detection: DCFH-DA fluorescent probe assay was conducted to assess the capacity of hydrogels to reduce excessive intracellular ROS generation under oxidative stress microenvironments. Periodontal ligament stem cells (PDLSCs) were inoculated into 12-well plates at an inoculation density of 4 × 104 cells/well, and precultured at 37 °C for 12 h to achieve stable cell adhesion. Afterward, PDLSCs were cocultured with TK­(+) or TK(−) hydrogels under 200 μM hydrogen peroxide (H2O2) stimulation, which was utilized to construct the cellular oxidative stress model. Two control groups were set simultaneously: sole H2O2-induced oxidative stress cells without hydrogel treatment, and normal untreated cells without any stimulation. Following 24 h incubation, 15 μM DCFH-DA probe was added into each well, and cells were incubated at 37 °C for another 30 min. Subsequently, cells were collected, and flow cytometry detection was performed to quantitatively analyze the intracellular ROS content. Meanwhile, fluorescence microscopy imaging (CKX53, Olympus, Japan) was performed in parallel tests to directly observe and visualize intracellular ROS fluorescence distribution at the single-cell level.

2.8. Mitochondrial Function Assessment

  • (1)

    Mitochondrial membrane potential analysis: PDLSCs were inoculated into confocal culture dishes at a density of 2 × 104 cells/mL, followed by incubation at 37 °C until steady and complete cellular adhesion was accomplished. The prior culture medium was discarded completely, and adherent cells were gently washed with phosphate-buffered saline (PBS) for 1–2 times. After that, each dish was supplemented with 200 nmol/L JC-1 fluorescent staining reagent, and staining reaction was conducted at 37 °C for 15 min under dark conditions. Upon completion of staining procedure, cells were rinsed twice with PBS, and fluorescent signals were visualized and recorded via laser scanning confocal microscopy. Intact mitochondria possessing high mitochondrial membrane potential can form JC-1 aggregates and emit red fluorescence, whereas depolarized mitochondria exist as JC-1 monomers and display green fluorescence. ImageJ software was adopted for quantitative calculation of the red/green fluorescence intensity ratio, which was utilized to evaluate the alteration level of mitochondrial membrane potential.

  • (2)

    Mitochondrial ATP Content Measurement: PDLSCs were planted into 6-well plates at a density of 2 × 105 cells/mL and cultivated under identical culture environments until full cell attachment. An ATP detection kit (Beyotime, China) was employed to quantify intracellular ATP concentrations, and all operations were strictly performed in line with the official protocol provided by the manufacturer. To eliminate experimental deviations caused by differences in cell quantity and total protein expression, the BCA protein quantification method was utilized to detect total protein content of each sample. Normalization analysis was subsequently carried out by calibrating ATP content against total protein mass. Final ATP data were presented as nmol ATP per milligram protein (nmol/mg), which guarantees precise, reliable and intergroup comparable quantitative experimental outcomes.

2.9. In Vitro Evaluation of the Osteogenic Differentiation Potential of PDLSCs in Response to GDF5/ROS-Responsive Degradable Hydrogel

Upon completion of a 7-day osteogenic differentiation induction, alkaline phosphatase (ALP) activity within PDLSCs was detected via qualitative staining assay with the BCIP/NBT ALP staining kit (Beyotime, China), and all experimental procedures were implemented strictly in compliance with the manufacturer’s operational protocols. After 14 days of osteogenic induction, extracellular calcium mineralized nodules were observed and identified through alizarin red S staining (Beyotime, China). For quantitative evaluation of mineralization degree, stained calcium deposits were dissolved in 10% cetylpyridinium chloride solution (Sigma-Aldrich). The absorbance value of the lysate supernatant was detected at 570 nm wavelength, so as to quantify the relative content of deposited calcium minerals. Total RNA isolation from cells was conducted by using Trizol reagent (Vazyme, China). Subsequently, isolated RNA was reversely transcribed into complementary DNA (cDNA) by employing the HiScript III RT SuperMix kit (Vazyme, China). Transcription levels of osteogenic-related marker genes were evaluated by quantitative real-time PCR (RT-qPCR) technology combined with SYBR Green Mix (Vazyme, China). Relative gene expression data were normalized against the endogenous reference gene GAPDH.

For Western blotting detection, total intracellular proteins were extracted with RIPA lysis buffer (Yoche, China). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electro-transferred onto polyvinylidene fluoride (PVDF) membranes. After membrane blocking with 5% skimmed milk solution, immunoblotting was performed by incubating membranes with primary antibodies targeting RUNX2, osteocalcin (OCN) and β-actin at 4 °C overnight. After sufficient washing steps with TBST buffer, membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies. Immunoreactive protein bands were visualized by enhanced chemiluminescence (ECL) reagent system, and gray intensity analysis of each band was accomplished via densitometric quantification software.

2.10. Statistical Analysis

Data were analyzed using SPSS software (version 27.0). One-way analysis of variance (ANOVA) was employed for multigroup comparisons, while Student’s t-test was used for pairwise comparisons between two groups. Statistical significance was defined as p < 0.05. All results are expressed as mean ± standard deviation (SD). Levels of significance are indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. Each experiment was repeated at least 3 times and 3 replicates were set up.

3. Results and Discussion

3.1. Characterization of GDF5/ROS-Responsive Degradable Hydrogels

3.1.1. Physical and Chemical Properties of GDF5/ROS-Responsive Degradable Hydrogels

The synthesized thioketal (TK) structure was characterized by proton nuclear magnetic resonance (1H NMR), revealing three characteristic peaks at 2.87 ppm (t, 4H), 2.67 ppm (t, 4H), and 1.59 ppm (s, 6H) (Figure A), which are consistent with the previously reported spectral features of the TK moiety. Upon exposure to ultraviolet light, the hydrogels exhibited rapid gelation within approximately 20 s (Figure B). Scanning electron microscopy (SEM) analysis demonstrated that both TK(−) and TK­(+) hydrogels possessed interconnected, sponge-like porous architectures. The average pore size of TK­(+) hydrogels was measured as 167.8 ± 5.1 μm (Figure C). Compressive mechanical properties were evaluated for GDF5/ROS-TK(−) and GDF5/ROS-TK­(+) hydrogels. As illustrated in Figure D, the stress–strain curves of both formulations displayed a “J”-shaped profile, indicative of elastic behavior under compression. The maximum compressive stress reached 114.18 kPa at a strain of 52.81% for GDF5/TK­(+) hydrogels, compared to 108.59 kPa at 50.38% strain for GDF5/TK(−) hydrogels. Rheological analysis confirmed the rapid photo-cross-linking kinetics of both hydrogel systems: the storage modulus (G′) surpassed the loss modulus (G″) within 9.7 s for TK(−) and within 7.4 s for TK­(+), respectively (Figure E).

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(A) 1H NMR analysis confirmed the successful synthesis of the TK linker; (B) schematic illustration of the preparation process for GDF5/ROS-responsive degradable hydrogels; photograph of the hydrogel precursor solution prior to photopolymerization ; photograph of the photopolymerized hydrogel (C) scanning electron microscopy images and quantitative pore size analysis of the hydrogel structures; (D) compressive stress–strain curves of the hydrogels; (E) rheological measurements demonstrating the rapid photo-cross-linking behavior of TK(−) and TK­(+) hydrogels.

Compared with the commonly used maleimide-functionalized polyethylene glycol, which typically requires approximately 10 min for gelation, the norbornene (NB)-functionalized polyethylene glycol synthesized in this study exhibited a significantly faster gelation kinetics. Both hydrogel groups underwent porous polymerization, a process in which the incorporation of cross-linkers generates an interconnected network containing intrinsic pores or voids between polymer chains. The size and uniformity of these pores are governed by factors such as cross-linking density and polymerization kinetics. Both TK(−) and TK­(+) hydrogels displayed high porosity, indicating excellent capacity for fluid retention and permeability. Compressive deformation tests revealed that both hydrogel formulations could withstand strains exceeding 50%, demonstrating sufficient elasticity to endure mechanical compression without fracture. Furthermore, rheological analyses confirmed the rapid gelation capability of the hydrogels, supporting their suitability for applications requiring swift network formation.

3.1.2. Cytotoxicity of GDF5/ROS-Responsive Degradable Hydrogels

As shown in Figure A, Calcein-AM/PI staining combined with fluorescence microscopy revealed uniform cellular distribution on the hydrogel surface, with minimal presence of red-stained dead cells. These findings demonstrate that both GDF5/ROS-TK(−) and GDF5/ROS-TK­(+) hydrogels exhibit favorable cytocompatibility when cocultured with PDLSCs. Therefore, both hydrogel formulations are considered suitable for potential biomedical applications.In the cell proliferation assay (Figure B), the relative proliferation rate of periodontal ligament stem cells (PDLSCs) exceeded 70%, with no evident cytotoxic effects observed.

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Swelling and degradation behavior of GDF5/ROS-responsive degradable hydrogels and sustained release analysis of GDF5 (A) Calcein AM/PI staining of PDLSCs. Scale bar = 100 μm; (B) CCK-8 cell proliferation assay; (C) degradation rates of TK(−) and TK­(+) hydrogels under varying concentrations of H2O2; (D) swelling kinetics of the hydrogels; (E) encapsulation efficiency of GDF5 in GDF5/ROS hydrogel; (F) in vitro cumulative release profile of GDF5 All data are expressed as the mean ± SD * means p < 0.05, ** means p < 0.01, and *** means p < 0.001 relative to the control group. “ns” means not significant. n = 3.

3.1.3. Responsive Degradation and Swelling Properties of GDF5/ROS-Responsive Degradable Hydrogels

Quantitative analysis of the degradation rate (Figure C) demonstrated that TK­(+) hydrogels underwent reactive oxygen species (ROS)-mediated, dose-dependent degradation in vitro. With increasing hydrogen peroxide (H2O2) concentration, the degradation rate of TK­(+) hydrogels progressively accelerated. Upon exposure to 2.5 mM H2O2, the hydrogels exhibited approximately 60% degradation over 20 days; under 12.5 mM H2O2, degradation reached nearly 100% within 10 days. In contrast, H2O2 stimulation had negligible effect on the degradation of TK(−) hydrogels. Concurrently, the swelling behavior of the hydrogels was evaluated (Figure D). TK­(+) hydrogels attained swelling equilibrium after 32 h, whereas TK(−) hydrogels reached equilibrium after 28 h.

Hydrogels provide mechanical support during the early stages of tissue regeneration, and their subsequent degradation is critical for effective tissue formation. Excessively slow degradation may impair cell infiltration, hinder tissue remodeling, and increase the risk of infection; conversely, overly rapid degradation compromises the sustained release of encapsulated therapeutics and undermines structural stability required for tissue regeneration. The concentration-dependent degradation behavior of GDF5/TK­(+) hydrogelsaccelerating with increasing hydrogen peroxide (H2O2) concentrationdemonstrates that TK­(+) hydrogels containing the thioketal (TK) linkage exhibit reactive oxygen species (ROS)-responsive degradability. This feature enables the hydrogels to modulate ROS levels in oxidative stress environments and facilitate the sustained release of loaded drugs. Swelling behavior tests revealed that both hydrogel groups reached certain equilibrium swelling ratios, which are generally associated with high cross-linking densities in polymeric networks.

3.1.4. Encapsulation Efficiency of GDF5 in GDF5/ROS Hydrogel

The encapsulation efficiency of GDF5 in the GDF5/ROS hydrogel is shown in Figure E: as the GDF5 drug loading increases from 1% to 5%, the encapsulation efficiency of GDF5 spheres in the GDF5/ROS hydrogel also increases. When the GDF5 loading rate is 7% to 12%, the encapsulation efficiency of GDF5 spheres in the GDF5/ROS hydrogel can reach 98%. This indicates that the “capture” efficiency of GDF5 by the GDF5/ROS hydrogel is relatively high.

3.1.5. GDF5 Release Profile

The GDF5 release kinetics of both hydrogel groups loaded with GDF5 were evaluated over 1, 3, 5, 7, 14, 21, and 28 days (Figure F). Both formulations exhibited an initial burst release within the first 2 days, followed by a gradual decline in release rate over time. The cumulative release of GDF5 reached 55.78 ± 3.23% for TK­(+) hydrogels and 53.28 ± 1.20% for TK(−) hydrogels by day 28. These results indicate that the GDF5/ROS-responsive degradable hydrogels demonstrate a sustained-release capability for GDF5.

Our TK-based hydrogel for reactive oxygen species (ROS)-responsive delivery of GDF5 aligns with the emerging paradigm of pathophysiologically responsive biomaterials. In contrast to traditional sustained-release systems that provide long-term yet nonspecific factor release, our design enables targeted and on-demand delivery via pathological ROS bursts at the injury site. Among ROS-responsive carriers, phenylboronate ester-based systems exhibit rapid responsiveness to H2O2, while our thioketal (TK) component demonstrates distinct selectivity toward highly reactive hydroxyl radicals. Furthermore, GDF5 was selected as the osteoinductive growth factor, and research indicates that its osteogenic efficacy is comparable to that of advanced BMP-2 delivery systems. Through a series of characterization experiments, we have confirmed the ROS dose-dependent degradation behavior of the GDF5/ROS-responsive hydrogel, preliminarily validating its ROS-responsive functionality. Additionally, the hydrogel demonstrated favorable cytocompatibility, and the loaded GDF5 maintained sustained release, laying the groundwork for subsequent cellular studies and osteoinductive evaluation.

3.2. In Vitro Antioxidant Effect of GDF5/ROS-Responsive Degradable Hydrogels

3.2.1. Analysis of In Vitro ROS Scavenging Capacity

The in vitro reactive oxygen species (ROS) scavenging capacity of the hydrogels was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The TK­(+) hydrogel exhibited a higher ROS inhibition rate (66.52% ± 3.533%) compared to the TK(−) hydrogel (43.70% ± 8.922%), and was slightly lower than that of the antioxidant agent TEMPOL (83.02% ± 5.801%) (Figure A). These findings demonstrate that incorporation of the thioketal (TK) moiety into PEG-based hydrogels significantly enhances their antioxidant activity through effective ROS scavenging. Notably, the TK(−) hydrogel displayed the lowest scavenging efficiency, which may be attributed to the sulfur atoms in the thiol groups of the cross-linker HS-PEG-SH that can donate or transfer electrons and participate in redox processes. However, the DPPH assay possesses limited demonstrative validity. Hence, we further evaluated its antioxidant efficacy through antioxidant gene expression analysis, DCFH-DA fluorescence staining, and flow cytometry.

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Analysis of the antioxidant effects of hydrogels and assessment of mitochondrial function (A) quantitative analysis of ROS inhibition by GDF5/ROS-responsive hydrogel and the antioxidant agent TEMPOL; (B) expression levels of the antioxidant genes catalase (Cat) and superoxide dismutase 1 (Sod1) in periodontal ligament stem cells (PDLSCs); (C) DCFH-DA fluorescence staining for intracellular ROS detection; Scale bar = 100 μm (D) flow cytometry analysis of intracellular ROS levels in PDLSCs; (E) JC-1 staining to assess mitochondrial membrane potential; Scale bar = 500 μm (F) determination of ATP content. All data are expressed as the mean ± SD * means p < 0.05, ** means p < 0.01, *** means p < 0.001 and **** means p < 0.0001 relative to the control group. “ns” means not significant. n = 3.

The expression levels of the antioxidant genes catalase (Cat) and superoxide dismutase 1 (Sod1) in periodontal ligament stem cells (PDLSCs) were analyzed. Downregulation of both genes was observed in the H2O2-exposed control group, whereas upregulation was detected in the hydrogel-treated groups. Moreover, the TK­(+) hydrogel group showed higher expression levels of Cat and Sod1 than the TK(−) hydrogel group (Figure B). These enzymes play essential roles in decomposing and neutralizing ROS, thereby maintaining cellular redox homeostasis and protecting cells from oxidative damage. Collectively, these results underscore the capacity of the TK­(+) hydrogel to modulate the intracellular redox environment and prevent pathological accumulation of ROS.

In addition, cells were stained with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) for fluorescent detection of intracellular ROS (Figure C). Fluorescence microscopy revealed that the ROS level in periodontal ligament stem cells was highest following H2O2 stimulation, whereas fluorescence intensity decreased in the hydrogel-treated groups, with a markedly reduced signal observed in the TK­(+) group. Subsequently, DCFH-DA-stained cells were subjected to quantitative analysis by flow cytometry. Under normal physiological conditions, the basal ROS level in periodontal ligament stem cells was as low as 0.15%. In contrast, H2O2 stimulation induced a substantial increase in intracellular ROS levels, reaching 69.1% (Figure D). Both hydrogels effectively suppressed intracellular ROS accumulation; notably, the TK­(+) hydrogel group exhibited a significantly lower ROS level (13.4%) compared to the TK(−) hydrogel group. These results collectively confirm that the TK­(+) hydrogel exerts a protective effect against excessive intracellular ROS production under oxidative stress.

3.2.2. Mitochondrial Function Assessment

In this study, JC-1 dye was used to assess mitochondrial membrane potential in PDLSCs under CLSM imaging, showing an increased mitochondrial membrane potential in the TK­(+) hydrogel group (Figure E). Mitochondrial ATP production in PDLSCs was quantified using an ATP detection kit, which demonstrated that hydrogen peroxide stimulation markedly reduced ATP generation compared to the control group, whereas the TK­(+) hydrogel treatment group exhibited a significant increase in ATP levels (Figure F). These findings collectively indicate that oxidative stress impairs mitochondrial function, leading to elevated mitochondrial ROS production and decreased membrane potential and ATP synthesis. In contrast, hydrogel treatmentparticularly with TK­(+) hydrogelseffectively mitigated these effects, as evidenced by reduced ROS levels and improved mitochondrial membrane potential and ATP production.

The overproduction of reactive oxygen species (ROS) serves as a central trigger for oxidative stress. − Mitochondria, functioning as the cellular powerhouses, play a vital role in maintaining normal physiological functions of cells. Under oxidative stress conditions, mitochondrial homeostasis is disrupted. Impaired mitochondria not only fail to supply sufficient energy to periodontal ligament stem cells (PDLSCs), but also inhibit their osteogenic differentiation, promote apoptosis, diminish the self-repair capacity of periodontal tissues, and accelerate alveolar bone resorption along with loss of periodontal attachment. , Therefore, maintaining ROS homeostasis, mitigating periodontal oxidative stress, and preserving mitochondrial stability in PDLSCs are essential for protecting their structural functionality and preventing further progression of periodontal pathologies.

In this study, it was demonstrated that the GDF/ROS hydrogel possesses notable antioxidant activity in vitro. Compared with the control group treated with TK-free hydrogel, cells treated with GDF5/TK­(+) hydrogel exhibited reduced intracellular ROS levels, increased expression of antioxidant genes, restored mitochondrial membrane potential, and significantly elevated ATP content. These findings indicate that the hydrogel, through the responsive cleavage of the TK component, alleviates the stimulation of PDLSCs by high ROS levels under periodontal oxidative stress, thereby maintaining mitochondrial homeostasis and protecting cells from oxidative stress-induced damage.

3.3. Osteogenic Effect of the GDF5/ROS-Responsive Degradable Hydrogel

Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were employed to evaluate the effects of the hydrogel on early osteogenic differentiation and late-stage calcium nodule formation in periodontal ligament stem cells (PDLSCs) (Figure A). After 7 days, ALP staining revealed significantly stronger signals in PDLSCs cultured under H2O2-induced oxidative stress and treated with hydrogels, compared to the untreated oxidative stress group. Notably, the TK­(+) hydrogel group exhibited markedly higher ALP activity than the TK(−) hydrogel group, reaching levels comparable to those observed in the nonstressed control group. After 14 days, ARS staining showed evident calcium nodule formation in the control group; however, H2O2 exposure substantially reduced mineralization. Both hydrogelsparticularly the TK­(+) formulationsignificantly enhanced extracellular matrix calcification, indicating effective promotion of osteogenic differentiation and mineralization in PDLSCs. These findings demonstrate that the GDF5/ROS-responsive hydrogel not only promotes the expression of early osteogenic markers such as ALP but also facilitates extracellular matrix mineralization, thereby supporting robust osteogenic differentiation. In addition, given the absence of significant differences in cell viability during the first 5 days, along with the observation of strong ALP activity and mineralization nodule formation after 14 days of osteogenic induction (Figure A), it is indicated that cells in both experimental and control groups maintained favorable viability and functional status throughout the induction period.

4.

4

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Effects of hydrogels on osteogenesis under in vitro oxidative stress conditions (A) representative images of alkaline phosphatase (ALP) and alizarin red S (ARS) staining following treatments with different hydrogels; (B) RT-qPCR analysis of osteogenic gene expression levels in periodontal ligament stem cells (PDLSCs); (C) Western blot assessment of osteogenic protein expression in PDLSCs. All data are expressed as the mean ± SD * means p < 0.05, ** means p < 0.01, *** means p < 0.001 and **** means p < 0.0001 relative to the control group. “ns” means not significant. n = 3.

Real-time quantitative PCR was used to assess the expression levels of key osteogenic genesalkaline phosphatase (Alp), Runt-related transcription factor 2 (Runx2), and osteocalcin (Ocn)in PDLSCs under oxidative stress conditions (Figure B). Downregulation of all three genes was observed in H2O2-exposed cells, whereas their expression was upregulated following hydrogel treatment. Importantly, the TK­(+) hydrogel group displayed significantly higher gene expression levelsapproximately 1.5-fold higher than those in the TK(−) groupconsistent with the histological staining results. Western blot analysis (Figure C) further confirmed that GDF5 treatment increased protein expression of OCN, ALP, and RUNX2, with the most pronounced upregulation observed in the TK­(+) hydrogel group. Taken together, these data indicate that the TK­(+) hydrogel significantly enhances osteogenic gene and protein expression in PDLSCs under oxidative stress, likely by scavenging ROS and establishing a favorable microenvironment for osteogenesis, thereby potentiating the osteoinductive activity of GDF5.

Periodontal bone regeneration represents a central objective in the treatment of periodontitis. PDLSCs, as multipotent mesenchymal stem cells derived from periodontal tissues, play a pivotal role in this process. They are capable of differentiating into osteoblasts and other periodontal supporting cells, migrating to sites of bone defects, secreting osteogenic matrix, promoting mineralization, and releasing paracrine factors that modulate the local microenvironment and stimulate angiogenesis. Their clinical application holds promise for improving the efficacy and predictability of bone regeneration outcomes.

Under standard physiological environments, intracellular ROS are maintained at relatively low concentrations participate in essential cellular processes such as signal transduction and cell signaling. In periodontitis, however, inflammatory cell infiltration and mitochondrial dysfunction lead to excessive ROS accumulation, resulting in oxidative stress. Elevated ROS levels damage key biological macromolecules within periodontal ligament stem cells (PDLSCs), impair their proliferation and osteogenic differentiation, promote apoptosis, and reduce the pool of functional stem cells. This not only directly diminishes osteoblast numbers and activity and suppresses bone matrix synthesis but also enhances osteoclast activation, disrupts bone metabolic homeostasis, and severely compromises periodontal bone regeneration. Our previous studies have demonstrated that modulating intracellular ROS levels can protect cells from oxidative damage, block RANKL-mediated signaling in osteoclast precursor cells, inhibit osteoclast formation, induce osteoclast apoptosis, and regulate the periodontal immune and inflammatory responses, thereby restoring the balance between osteoblasts and osteoclasts. Therefore, maintaining equilibrium between ROS production and antioxidant defense mechanisms is essential for promoting alveolar bone regeneration at periodontal defect sites. GDF5, a member of the BMP family, plays a critical role in the development of bone, cartilage, and tendons. It also promotes periodontal tissue formation and facilitates the directional growth of periodontal ligament fibers toward alveolar bone and cementum during root development, highlighting its significant potential in periodontal regenerative therapy.

This study employed ALP and ARS staining, alongside Western blot and PCR analysis, to investigate the osteogenic potential of GDF5/ROS hydrogels under oxidative stress conditions in periodontal tissues. The results indicate that both GDF5-loaded hydrogel formulations enhance the osteogenic differentiation of PDLSCs to a certain extent, with the GDF5/TK­(+) hydrogel group exhibiting more pronounced effects. Furthermore, it was observed that despite the TK­(+) hydrogel’s ability to moderately regulate ROS levels and thereby ameliorate oxidative stressan inhibitory factor for osteogenesisthe expression levels of ALP, OCN, and RUNX2 induced by GDF5 were markedly elevated. These findings indicate that under the applied experimental conditions, the potent osteoinductive effect of exogenous GDF5 likely serves as the primary driver of cellular differentiation, thereby potentially obscuring the more nuanced regulatory roles that may arise from alterations in the redox microenvironment. ,

4. Conclusion

The present study successfully developed a hydrogel system that not only responds to high levels of ROS within the oxidative stress microenvironment of periodontitis but also exhibits integrated ROS-scavenging and controlled release of GDF5. Through in vitro models, the dual therapeutic effects of this systemmitigating oxidative stress and promoting alveolar bone regenerationwere validated. This work offers a promising strategy to address the challenge of impaired bone regeneration under oxidative stress conditions in periodontal diseases. However, the interaction between the hydrogel system and immune cells within the periodontal inflammatory milieu, as well as its impact on inflammatory phenotype modulation, remains unexplored. Additionally, validation at the in vivo level is currently lacking. Future studies will aim to supplement in vivo evaluations and further elucidate the comprehensive effects of this hydrogel system within an immune–cellular interaction framework.

Supplementary Material

ao5c13610_si_001.pdf (208KB, pdf)

Acknowledgments

This study was supported by the Key Projects of Social Development of the Jiangsu Department of Science and Technology (No. BJ21006; No. BJ2409), Nanjing Medical Science and Technology Development Foundation, Nanjing Department of Health (YKK23180; YKK 22176; GAX24300), and High-Level Hospital Construction Project of Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Institute of Stomatology, Nanjing University (0224C024).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c13610.

  • 1H NMR hydrogen spectrum of the TK structure, hydrogen peroxide-induced model establishment, RT-qPCR primer sequence and GDF5 release under oxidative conditions used in this study (PDF)

Y.L. designed the study. J.Z. and Y.L. conducted the study. J.Z. and Y.Z. collected the data. J.Z. drafted the manuscript. H.Z. and J.Y. revised the manuscript content. Y.L. were devoted to project administration. W.W. contributed to funding acquisition. J.Z., Y.L., and W.W. take responsibility for the integrity of the data analysis. All authors agreed to the published version of the manuscript.

The authors declare no competing financial interest.

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Supplementary Materials

ao5c13610_si_001.pdf (208KB, pdf)

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