Polyphenol-mediated redox-active hydrogel with H₂S gaseous-bioelectric coupling for periodontal bone healing in diabetes

Periodontitis, an infection-driven inflammatory disease, causes irreversible damage to periodontal tissue and tooth loss. Diabetes mellitus exacerbates periodontitis, even with minimal plaque accumulation. Both innate and adaptive immune systems, crucial for periodontium health, are negatively impacted by diabetes. Furthermore, periodontitis-derived inflammatory factors entering the bloodstream worsen glycemic control and increase diabetic complications. Therefore, treating periodontitis in diabetic patients is vital not only for preserving teeth but also for minimizing systemic inflammatory effects on glycemic control and diabetic complications. Current periodontitis and alveolar bone regeneration strategies include subgingival instrumentation, regenerative periodontal surgery, and local antibiotic/bioactive molecule administration. However, these treatments are limited by the over-activated oxidative stress response, excessive inflammation, unbalanced immunomodulation, and impaired mesenchymal stem cell (MSC) function in diabetic conditions. Insufficient alveolar bone formation, secondary bone resorption, and recurrent infection frequently occur during diabetic periodontitis treatment. Guided bone/tissue regeneration (GBR/GTR) techniques, such as collagen-based matrices and pericardium membranes, are widely used but limited by the diabetes-aggravated inflammatory environment. Their effects are often hindered by the limitations of traditional methods. Endogenous signaling, including bioelectric, mechanical, and gaseous signals, dramatically alters cell and tissue composition and microenvironments, orchestrating healing. Hydrogen sulfide (H₂S), an endogenous gasotransmitter, mitigates alveolar bone resorption in periodontitis by attenuating inflammation, apoptosis, and reactive oxygen species (ROS). H₂S also exhibits osteogenic function in periodontal tissue; however, its biosynthesis is impaired in diabetes, adversely affecting MSC migration, angiogenesis, inflammation control, and tissue regeneration. Existing exogenous H₂S delivery methods using H₂S donors have limitations due to their rapid H₂S generation and poor water solubility. Bioelectricity, a key physiological activity, is mediated by endogenous electric fields such as membrane potential and nerve potential. These signals modulate biological processes, from cell cycles and migration to tissue regeneration. Electrical signals from mechanical stress stimulate bone growth and remodeling, and improve cell arrangement essential for functional periodontium regeneration. Traditional GBR/GTR membranes lack conductivity, hindering endogenous electrical signal transmission. Therefore, functional, conductive GBR/GTR membranes coupling endogenous bioelectric signals have significant potential for diabetic periodontal tissue regeneration. Hydrogels, with their extracellular matrix-like structure, are increasingly used in periodontal tissue regeneration. Hydrogels with immunomodulatory capabilities can attenuate inflammatory responses in diabetic periodontal environments. However, many focus on growth factor or stem cell delivery, neglecting the biological effects of hydrogel components. Conductive hydrogel designs often employ conductive fillers with biosafety concerns or limited biofunctionality. Polyphenols, with their biocompatibility, antioxidant properties, and immunomodulatory activity, offer a promising alternative.

The literature extensively documents the challenges in treating periodontitis in diabetic patients due to the complex interplay of hyperglycemia, inflammation, and impaired tissue regeneration. Studies have highlighted the detrimental effects of hyperglycemia on immune cell function, leading to an imbalance in macrophage polarization and impaired mesenchymal stem cell activity. The role of oxidative stress in exacerbating periodontal disease in diabetes has been well-established, with elevated reactive oxygen species contributing to tissue damage and hindering healing. Existing treatment modalities, such as scaling and root planing, along with surgical interventions and local drug delivery, have shown limited success in addressing the complex pathophysiology of diabetic periodontitis. The exploration of novel biomaterials that can address multiple aspects of this disease, such as inflammation, oxidative stress, and impaired stem cell function, has become a crucial area of research. Previous studies have individually investigated the beneficial effects of hydrogen sulfide (H2S) and bioelectric stimulation in promoting tissue regeneration. However, the combined approach of delivering both H2S and harnessing bioelectricity for periodontal regeneration in the context of diabetic periodontitis remains largely unexplored. The use of polyphenols as bioactive components in biomaterials has also been explored, but integrating them with conductive polymers and H2S delivery systems for this specific application remains a novel contribution.

This study developed a polyphenol-mediated redox-active algin/gelatin (AG) hydrogel incorporating a hydrogen sulfide (H₂S) sustained-release system and a conductive poly(3,4-ethylenedioxythiopene)-assembled polydopamine-mediated silk microfiber (PEDOT-PSF) network. The H₂S release system used NaHS-encapsulated bovine serum albumin nanoparticles (BNPs). The PEDOT-PSF network was fabricated using a polyphenol-mediated protection-extraction strategy, ensuring uniform distribution throughout the hydrogel and enhancing mechanical and conductive properties. **Fabrication of PEDOT-PSF:** A polydopamine (PDA)-assisted protection-extraction process was used to fabricate silk microfibers (PSF) from silk cocoons. The resulting PSF with surface catechol groups served as anchor sites for in situ EDOT self-assembly, forming the PEDOT-PSF. **Syntheses of NaHS@BNP:** NaHS-loaded bovine serum albumin nanoparticles (BNPs) were synthesized using a desolvation method. BSA was dissolved in water, and NaHS was dissolved in ethanol. The ethanol solution was added dropwise to the BSA solution while stirring, followed by the addition of EDC for crosslinking. **Preparation of the BNP-PEDOT-PSF-AG hydrogel:** Gelatin was dissolved in a sodium alginate solution, followed by the addition of PEDOT-PSF and BNPs. PEGDE was used for crosslinking. After crosslinking, the hydrogel was soaked in CaCl2 solution for further crosslinking. **Characterization:** The morphology and structure of the materials were analyzed using SEM. H₂S release profiles were measured using an H₂S assay kit. X-ray photoelectron spectroscopy (XPS) was used to analyze the redox activity of the hydrogel. Mechanical properties (compressive strength, recovery properties, degradation rate) were evaluated using a universal testing machine. Electrical conductivity was measured using a two-probe method and verified with an LED test. **In vitro studies:** Human periodontal ligament stem cells (PDLSCs) and THP-1 cells were cultured on various hydrogels under high glucose and inflammatory conditions. Cell proliferation, elongation, and osteogenesis were evaluated using MTT assay, immunofluorescence staining, and quantitative PCR (qPCR). Intracellular ROS levels were measured using DCFH-DA assay. Macrophage polarization was evaluated using flow cytometry. Transcriptome sequencing and gene expression analysis were performed to uncover the underlying molecular mechanisms. High-throughput electrical stimulation (ES) was applied to evaluate the effect of bioelectricity. **In vivo studies:** A diabetic periodontitis rat model was established using streptozotocin (STZ) injection and ligature-induced periodontitis. Hydrogels were implanted into the defect areas, and tissue samples were harvested for micro-CT analysis, histology (HE and Masson staining, TRAP staining), and immunofluorescence (CD68, iNOS, CD163, CD90, CD31, α-SMA, RUNX2, OCN) to assess bone regeneration, angiogenesis, macrophage polarization, and stem cell recruitment. GSH/GSSG ratio and Gpx activity were measured to assess the antioxidative effect.

The developed polyphenol-mediated redox-active hydrogel with H₂S and conductive PEDOT-PSF exhibited significant improvements in periodontal regeneration in a diabetic periodontitis model. **Material characterization:** The hydrogel displayed a uniform porous structure with well-distributed BNPs and PEDOT-PSF. It demonstrated sustained H₂S release for over 21 days and excellent mechanical properties with improved compressive strength and rapid recovery. Importantly, the hydrogel exhibited good electrical conductivity (13.12 ± 1.66 S/m with 2 wt% PEDOT-PSF). **In vitro studies:** The hydrogel promoted PDLSC proliferation, elongation (aspect ratio increased to 4.93±0.45 at 600 mV from 3.04±0.16 without ES), and osteogenesis (significantly higher OCN and ALP expression and activity) under electrical stimulation. Transcriptome analysis revealed upregulation of autophagy-related genes (ATG2B, ATG12P1) and downregulation of inflammatory pathways. The hydrogel showed excellent ROS scavenging activity (significantly lower intracellular ROS levels in THP-1 cells) and immunomodulatory effects, shifting macrophage polarization from M1 to M2 by regulating lipid metabolism (downregulation of lipid biosynthesis and upregulation of lipid catabolism). **In vivo studies:** In the diabetic periodontitis rat model, the hydrogel significantly improved periodontal bone regeneration (BV/TV ratio increased from 57.54% in the blank group to 90.35% in the BNP-PEDOT-PSF-AG group). It also promoted angiogenesis (higher CD31 and α-SMA expression) and significantly reduced osteoclast activity. Immunofluorescence analysis showed a decrease in M1 macrophages and an increase in M2 macrophages. The recruitment of PDLSCs (CD90+ cells) was significantly enhanced. The hydrogel demonstrated excellent antioxidant effects (increased GSH/GSSG ratio and Gpx activity). Histological analysis confirmed enhanced bone formation and improved periodontal ligament fiber arrangement.

This study demonstrates the efficacy of a novel hydrogel in promoting periodontal bone regeneration in a challenging diabetic periodontitis environment. The synergistic effects of H₂S, PEDOT-PSF conductivity, and polyphenol-mediated redox activity create a pro-regenerative microenvironment by addressing multiple pathogenic mechanisms. The sustained release of H₂S promotes stem cell recruitment and angiogenesis, while the conductivity enhances cell alignment and calcium influx, leading to improved osteogenesis. The polyphenol component’s antioxidant and anti-inflammatory properties effectively combat oxidative stress and modulate macrophage polarization, further enhancing tissue regeneration. These results suggest that the combination of gaseous (H₂S) and bioelectric signaling modulation offers a promising therapeutic strategy for treating periodontitis in the context of diabetes. The superior performance of this hydrogel compared to existing therapies highlights the potential for translating this technology to clinical applications.

This study successfully developed a novel polyphenol-mediated redox-active hydrogel that effectively couples endogenous bioelectricity and H₂S to promote periodontal bone regeneration in diabetic periodontitis. The hydrogel's synergistic actions in reducing oxidative stress, modulating inflammation, recruiting stem cells, and promoting osteogenesis offer a promising therapeutic strategy. Future studies could focus on optimizing the hydrogel composition and exploring its potential in combination with other clinical treatments for enhanced therapeutic efficacy.

The study used a rat model of diabetic periodontitis, and the results may not be directly translatable to humans. Long-term in vivo studies are needed to assess the durability and long-term effects of the hydrogel. Further research could also explore the optimal concentration of H₂S and the precise mechanisms of bioelectric signaling in this context. The study's relatively small sample size could also be improved upon.