Polyphenol mediated zinc-oxygen synergistic hydrogel remodels senescent microenvironment for periodontal tissue regeneration

Domain: biology
Published: 2026-03-10
Editorial outcome: minor_revision

Abstract
Senescent mesenchymal stem cells residing in an inflammatory, dysbiotic, and hypoxic microenvironment pose a barrier to periodontal regeneration. Here we introduce a hydrocaffeic acid (HCA)-mediated silk fibroin hydrogel incorporating Mn/HCA-modified calcium peroxide (Mn-hCaO₂) and HCA-modified zeolitic imidazolate framework-8 (hZIF8) to rejuvenate this environment. The strategy imbues the hydrogel with enhanced adhesion and adaptability in periodontal pockets. The Mn-HCA complex acts catalytically to reduce oxidative stress and correct hypoxia. Simultaneously, Zn²⁺ and HCA restore microbial balance and mitigate inflammation. This multifunctional hydrogel targets the senescent periodontal niche by normalizing microbiota homeostasis and promoting a regenerative immune profile. Concurrently, it directly alleviates telomere shortening, DNA damage and oxidative stress in stem cells, thereby rejuvenating cellular function. By specifically addressing hypoxia and zinc deficiency, this polyphenol-mediated synergistic strategy offers a pathway to rejuvenate the senescent periodontal microenvironment, thereby overcoming tissue regeneration barriers and offering a translatable pathway for treating periodontitis and other aging-associated inflammatory diseases.

Introduction
Periodontitis (PD) is an inflammatory disease with a multifactorial etiology, leading to irreversible damage to periodontal support tissue 1. The stress-induced premature senescence of mesenchymal stem cells, characterized by cell cycle arrest and diminished self-renewal and differentiation capacity, may pose a significant barrier to periodontal regeneration 2, 3. In the course of periodontitis, virulent microbial pathogens and the ensuing inflammatory cascade can induce premature senescence of stem cells by causing stress-induced mitochondrial dysfunction and DNA damage 4, 5. Senescent cells emit various intercellular signals into the niche that aggregate local inflammation and impair tissue function 6. The senescent niche, which houses the senescent stem cells, drives a vicious circle of periodontal destruction 7, 8. Owing to the imbalance between limited oxygen availability and soaring energy demand, periodontitis lesions are frequently characterized by hypoxic conditions, which can lead to a cascade of biological responses that hinder the regenerative process 9. Oxygen deprivation can interrupt energy metabolism and stimulate the generation of reactive oxygen species (ROS), which results in oxidative DNA damage, subsequently halting cell cycle progression 10. Countering these effects, oxygen supplementation can contribute to the improvement of bone metabolism and facilitate the regeneration of bone defects 11. Oxygen concentration also plays an indispensable role in modulating the balance of microbiota by impacting the composition and function of microbial communities. Coupled with aberrant oxygen levels, zinc deficiency is also a risk factor in intractable inflammation and failure of tissue repair in periodontitis 12, 13. Zinc is an essential element in many biological processes, including cell growth, immune function and antioxidant defense reaction 14. Low zinc bioavailability can be found in various pathological conditions linked to premature aging, while short-term zinc supplementation has been shown to effectively counteract the adverse effects of zinc deficiency in the elderly 15. Current anti-aging therapies primarily focus on eliminating senescent cells or antagonizing their cellular responses, with relatively limited exploration of how these or related strategies might apply to the complex and diverse periodontal senescent microenvironment 16-18. Reshaping the senescent periodontal niche is a novel approach to rejuvenating stem cells and enhancing tissue regeneration 7, 19, 20.

Hydrogels with biocompatible structures that mimic the extracellular matrix of periodontal cells are widely utilized in periodontal tissue regeneration and functional reconstruction 21. Currently, most hydrogel development focuses on locally sustained and controlled release of drugs that exhibit antibacterial, anti-inflammatory, or pro-regenerative properties 22-24. However, significant challenges remain, as no current hydrogels can effectively modulate the senescent periodontal microenvironment. To remain anchored in this challenging environment, a hydrogel should possess strong wet adhesion and appropriate mechanical properties, ensuring long-term efficacy in the oral cavity. To modulate the senescent periodontal microenvironment, it should simultaneously target multiple aging-related processes, such as hypoxia and zinc ion deficiency. Traditional oxygenating materials like CaO₂ have shortcomings, including insufficient oxygen release duration and excessive H₂O₂ residue, potentially exacerbating oxidative damage to cells. Zeolite imidazolate framework-8 (ZIF-8) is a biocompatible zinc ion donor that has been widely used thanks to its antibacterial activity alongside promotion of angiogenesis and bone regeneration 25, 26. However, the hydrophobic characteristics of ZIF-8 make it difficult to achieve uniform dispersion within a hydrogel network.

Polyphenols, such as tannic acids (TA), epigallocatechin-3-gallate (EGCG) and hydrocaffeic acid (HCA), exhibit immunomodulatory effects due to their unique phenolic hydroxyl groups, which can be beneficial for inflammatory diseases 27. Moreover, polyphenols are metabolized by local microbiota, then serve to regulate their homeostasis, suggesting potential in periodontitis treatment 28. Additionally, the hydrophilicity of polyphenol can alter the wettability of a material's surface through modification. Similar to mussel adhesive proteins, polyphenol-modified hydrogels exhibit strengthened tissue adhesion while retaining the biological properties inherent to polyphenols. Therefore, the construction of polyphenol-mediated hydrogels with anti-aging properties could effectively address the challenges in the senescent periodontal microenvironment.

In this study, we engineered an injectable polyphenol-mediated oxygenating hydrogel based on HCA-mediated manganese-modified CaO nanoparticles (Mn-hCaO₂), HCA-mediated ZIF8 nanoparticles (hZIF8), and silk fibroin methacrylate (SMA) for targeted modulation of the senescent periodontal microenvironment. The abundance of phenolic hydroxyl groups in HCA enhances the antioxidant, adhesive and mechanical properties of the hydrogel. HCA modification can not only improve the dispersibility and stability and stability of improve the dispersibility and stability of nanoparticles in the hydrogel matrix, but also form a cascade enzyme-like catalytic unit together with Mn. This modification offers a simple and safe strategy for constructing an H₂O₂-eliminating oxygenating system. hZIF8 can release a sustained supply of Zn²⁺ to ameliorate zinc ion deficiency. By specifically addressing both hypoxia and zinc ion deficiency, the hydrogel restores the essential micronutrients and oxygen levels, thereby rejuvenating the senescent periodontal microenvironment and enabling senescent cells to re-enter the cell cycle. In this manner, the polyphenol-mediated hydrogel simultaneously targets multiple aging-related processes, such as telomere shortening, DNA damage, cellular antioxidant systems, and oral microbiota homeostasis, which promotes robust periodontal tissue regeneration.

Results
Design Strategy
CaO₂ and ZIF8 were modified with HCA. After modification with HCA, Mnⁿ⁺ was anchored onto the surface of CaO₂ through the chelation effect of phenolic hydroxyl groups (Fig. 1A). CaO₂ reacts with water to release oxygen, alleviating hypoxia, but its hydrolysis produces cytotoxic H₂O₂. Fortunately, the Mnⁿ⁺-HCA shell can catalyze this H₂O₂ into O₂ via catalase (CAT)-like activity. Therefore, the Mnⁿ⁺-HCA chelate complex constitutes a cascade enzyme-like catalytic system, which not only reduces the risk of H₂O₂-induced oxidative stress, but also further promotes the production of O₂ (Fig. 1B). In addition, after modification by HCA, hZIF8 can be uniformly dispersed in SMA hydrogel matrix with Mn-hCaO₂. The hydrogel can be injected into the periodontal pocket and polymerized using UV light irradiation (395 nm) (Fig. 1C). The abundance of phenolic hydroxyl groups enhances the wet adhesive and mechanical properties of the hydrogel, benefitting its application in a dynamic periodontal pocket. The pathological periodontal niche sustains a vicious cycle of hypoxia, microbiota dysbiosis, and pro-inflammatory immune patterns, collectively driving stem cell senescence and impairing tissue regeneration (Fig. 1D). The Mn-hCaO₂/hZIF8/SMA hydrogel disrupts this circuit via synergistic actions of HCA, Zn²⁺ and O₂ release, which mitigate senescence through hypoxia alleviation, oxidative damage protection, telomere maintenance and enhanced DNA repair. Concurrently, it restores microbiotic homeostasis and reprograms immune responses, establishing a rejuvenated microenvironment that supports periodontal tissue repair.

Characterization of Mn-hCaO₂/hZIF8/SMA hydrogel
The spheroid morphology of the Mn-hCaO₂ nanoparticles (NPs), which preserved the characteristic shape of the CaO₂ core, was primarily characterized by scanning electron microscopy (SEM) (Supplementary Fig. 1A, B) and transmission electron microscopy (TEM) (Fig. 2A, B). The successful incorporation of Mn and HCA was confirmed by the uniform distribution of Ca, O, C and Mn in elemental mapping and the presence of C═O and C─O bonds in the high-resolution O 1s spectrum (Fig. 2C). The crystal phase of Mn-hCaO₂ was in good agreement with CaO₂, which indicated that the structural integrity of CaO₂ was not affected (Supplementary Fig. 1C). The zeta potential decreased in hCaO₂ owing to the negative charge of HCA and it slightly increased in Mn-CaO₂ when Mn²⁺ was chelated to the electronegative hydroxyl and carboxyl groups of HCA (Fig. 2D). The concentration of Mn²⁺ was tuned to maximize the oxygen-release performance of the composite (Supplementary Fig. 1D). The oxygen-generating kinetics of Mn-hCaO₂ was measured by a dissolved oxygen (DO) meter when dispersed in phosphate buffered saline (PBS) at 37 ℃, mimicking the in vivo environment (Fig. 2E). The oxygen release curves of CaO₂ and hCaO₂ both reached peaks at 24h with a maximum DO concentrations of 14.75 mg L⁻¹ and 15.92 mg L⁻¹, respectively. Mn-hCaO₂ reached a DO concentration of 20.72 mg L⁻¹ at the second hour, and the DO concentration was maintained higher than 10 mg L⁻¹ to the 60th hour. This sustained concentration is possible because during the oxygen generation reaction of CaO₂, a large amount of H₂O₂ is produced as an intermediate product. Mn-hCaO₂ rapidly decomposed H₂O₂ and the H₂O₂ concentration approached 0 mg L⁻¹ from the 12th hour (Fig. 2F). H₂O₂ rapidly decomposed into oxygen due to the strong catalytic activity of Mnⁿ⁺-HCA shell of the Mn-hCaO₂, which also boosted the production of oxygen. Considering the excessive oxidative stress in the periodontitis microenvironment, the capacity of the NPs to handle external H₂O₂ and •OH was also tested (Fig. 2G, H). The results revealed that Mn-hCaO₂ was efficient in clearing external H₂O₂ and •OH, confirming its ROS-scavenging property.

The hZIF8 was found to form a typical dodecahedral crystal structure similar to that of ZIF8 with a shrink size of around 80 nm, as shown in SEM (Supplementary Fig. 1E, F) and TEM images (Fig. 2I, J). The successful incorporation of HCA was confirmed by the presence of O element on the surface and a decline in zeta potential (Supplementary Fig. 1G). The crystal phase of ZIF8 was maintained after HCA modification (Supplementary Fig. 1H). The hZIF8 had good dispersity in aqueous solution, as was demonstrated by the Tyndall effect (Supplementary Fig. 1I).

The Mn-hCaO₂/hZIF8/SMA hydrogel exhibited good plasticity, tissue adhesion and compression resistance, meeting the requirements of the dynamic and wet conditions of the oral cavity. The compositional ratios of the hydrogel were systematically optimized for functional performance and structural stability. The proportion of hZIF8 to Mn-hCaO₂ was specifically determined through mechanical testing to enhance compressive strength (Supplementary Fig. 2A, B). At the micro level, the hydrogel displayed porous reticular structures with an average pore size of 300 μm and a uniform distribution of C, N, O, Ca and Zn elements on the surface of the hydrogel (Supplementary Fig. 2C-F). The hydrogel is injectable and can adapt to irregular shapes (Supplementary Fig. 2G). The Mn-hCaO₂/hZIF8/SMA pre-gel solution exhibited pronounced shear-thinning behavior with low viscosity, facilitating injection (Fig. 2K). Photo-gelation occurred within 12 seconds under UV irradiation (395 nm, 10 W) (Fig. 2L). Frequency sweep measurements (0.1-10 Hz) confirmed the structural stability of the hydrogel (Supplementary Fig. 2H).

HCA-modified NPs not only endowed the hydrogel with good tissue adhesiveness, but also improved its mechanical properties. The hydrogel is capable of adhering to both soft and hard oral tissues, including alveolar bone, cementum, enamel, and gingiva, demonstrating its suitability for the challenging oral environment (Fig. 2M). We compared the Mn-hCaO₂/hZIF8/SMA hydrogel to pure SMA hydrogel by testing its in situ adhesion to porcine skin, and the results showed the adhesion strength of the hydrogel with HCA-modified NPs was around 15 kPa, three times that of pure SMA hydrogel (Fig. 2N). The compressive modulus of Mn-hCaO₂/hZIF8/SMA hydrogel increased to around 150 kPa, compared to 67 kPa for pure SMA hydrogel, and was able to withstand 80% compressive deformation (Fig. 2O). To mimic the repetitive forces of mastication, cyclic compression to 60% compressive deformation was applied (Fig. 2P). The compression stress remained after 30 cycles, demonstrating the resistance of the material to cyclic compression. These improvements in the modified hydrogel’s mechanical properties could be attributed to the nano-reinforcement effect of Mn-hCaO₂ and hZIF8.

The swelling and degradation properties of a hydrogel material are also crucial for its suitability in tissue regeneration applications. Incorporating hZIF8 and Mn-hCaO₂ increased the swelling capacity of the SMA hydrogel, exhibiting a 155% equilibrium swelling ratio within 10 hours. This enables conformal adaptation to complex periodontal defect geometries, promoting defect filling and retention (Supplementary Fig. 2I). In vitro, the pure and modified hydrogels both exhibited over 90% degradation within 30 days in artificial saliva at 37 °C, which indicated that ZIF8 and Mn-hCaO₂ incorporation did not have significant effects on the degradation rate (Supplementary Fig. 2J). In vivo studies confirmed near complete degradation by day 30 (Supplementary Fig. 2K). The hydrogel degradation is mediated by hydrolysis of methacrylate-derived ester bonds under physiological conditions and aligns with the typical timeline required for new bone regeneration.

Mn-hCaO₂/hZIF8/SMA hydrogel suppresses stem cell senescence and modulates macrophage polarization in vitro
An inflammatory microenvironment induced by microbial virulence was established by applying liposaccharide (LPS) stimuli to human-derived periodontal ligament stem cells (hPDLSCs). The proportion of senescent cells, labeled by a senescence-associated β-galactosidase (SA-β-gal) assay, significantly increased in proportion after 48 hours of inflammatory stimuli (Fig. 3A). Expression of cellular senescence-related genes P16 (Fig. 3B), P21 (Fig. 3C) and P53 (Supplementary Fig. 3A) also increased dramatically, indicating that the hPDLSCs entered a senescent state. Intracellular ROS labeling with a DCFH-DA probe showed increased intracellular ROS levels in the hPDLSCs, suggesting a stress-induced mechanism of cellular senescence (Supplementary Fig. 3B, C). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), two key inflammation-related cytokines of SASP, were significantly increased in mRNA expression (Fig. 3D, E). Treatment with Mn-hCaO₂ significantly suppressed senescence, as demonstrated by a reduction in senescence-related biomarkers and intracellular ROS together with downregulation of pro-inflammatory cytokines. Similarly, hZIF8 supplementation also suppressed these indicators effectively. The combination of both Mn-hCaO₂ and hZIF8 produced the optimum anti-senescent ability in this pro-inflammatory microenvironment. The free radical scavenging ability of Mn-hCaO₂/hZIF8/SMA hydrogel was further confirmed using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, which revealed that phenolic hydroxyl groups of HCA neutralized radical species through electron transfer (Supplementary Fig. 3D). These results demonstrate that the Mn-hCaO₂/hZIF8/SMA hydrogel synergistically alleviates inflammation-induced cellular senescence in hPDLSCs by reducing oxidative stress and suppressing SASP-associated cytokine expression.

Arrested proliferation and differentiation are characteristic of aging hPDLSCs. Initially, we confirmed that light exposure did not impair hPDLSC proliferation using a Cell Count Kit-8 assay (Supplementary Fig. 3E). Under inflammatory challenge, hPDLSC proliferation was significantly suppressed after 1, 3, and 5 days, whereas the LPS+Mn-hCaO₂/hZIF8/SMA group exhibited enhanced proliferation from day 1, suggesting synergistic roles of Mn-hCaO₂ and hZIF8 in promoting cell viability (Fig. 3F). To assess the osteo-inducive property of the hydrogel, a 14-day osteogenic induction was performed in hPDLSCs in an inflammatory microenvironment. The LPS+Mn-hCaO₂/hZIF8/SMA group showed the highest relative mRNA expression of osteoblast markers, including alkaline phosphatase (ALP) (Supplementary Fig. 3F), osteocalcin (OCN) (Fig. 3G) and specificity protein 7 (SP7) (Fig. 3H). The SMA matrix itself could elevate osteogenic activity to some extent by providing a suitable mechanical environment for cell adhesion and stretching; the incorporation of hZIF8 and Mn-hCaO₂ NPs further promoted the ALP activity of hPDLSCs. The results of ALP staining were consistent with the mRNA expression results (Fig. 3I). Taken together, these findings demonstrate that Mn-hCaO₂/hZIF8/SMA hydrogel can effectively restore the proliferation and osteogenic differentiation capability of hPDLSCs compromised by inflammatory stimuli.

Macrophages are a group of pioneering immune cells important to the host response against microbial infection. Different macrophage subtypes can secrete cytokines and chemokines that either promote bone resorption or regeneration, and thus play crucial modulatory roles in the periodontal niche 29, 30. THP-1 cells were induced to generate M0 macrophages, which were then stimulated with LPS for polarization assessment. After 48 h of inflammatory stimuli, the macrophages presented an inclination towards M1 polarization with upregulated expression of pro-inflammatory cytokines including IL-1β and TNF-α, as well as the M1 surface marker CD80 (Fig. 3J). The LPS+Mn-hCaO₂/SMA group and LPS+Mn-hCaO₂/hZIF8/SMA hydrogel successfully reversed this effect and inclined the macrophages toward M2 polarization, characterized by upregulated expression of M2-related markers including arginase-1 (ARG-1), Mer tyrosine kinase (MerTK) and CD206 (Fig. 3K). These results revealed that the Mn-hCaO₂/hZIF8/SMA hydrogel can modulate the M1/M2 ratio, which in turn can produce highly significant changes in the cytokine network in the niche and steer the environment towards a pro-regenerative state.

Molecular pathways underlying the anti-aging activity of Mn-hCaO₂/hZIF8/SMA hydrogel in vitro
To elucidate the mechanisms underlying the anti-aging activity of the Mn-hCaO₂/hZIF8/SMA hydrogel, transcriptome analyses were conducted on hPDLSCs 48 hours after inflammatory stimuli, comparing them to cells treated with four different hydrogels. Venn and volcano plots illustrated the overall distribution of differentially expressed genes (DEGs) across the four groups, with the SMA group serving as the reference. When comparing the Mn-hCaO₂/hZIF8/SMA group to the SMA group, 635 DEGs were identified, including 477 upregulated and 158 downregulated genes. This number significantly exceeded the sum of DEGs observed in the hZIF8/SMA and Mn-hCaO₂/SMA groups compared to the SMA group, indicating a polyphenol-mediated synergistic effect of hZIF8 and Mn-hCaO₂ (Fig. 4A, B, C, D).

Gene ontology (GO) analysis of the DEGs between the Mn-hCaO₂/hZIF8/SMA and SMA groups revealed that the Mn-hCaO₂/hZIF8/SMA hydrogel had a significant impact on several biological processes, including cell cycle, DNA repair, hypoxic response, cellular zinc ion homeostasis, telomere maintenance, aging, cell junctions, collagen biosynthesis, NF-kappaB transcription factor activity, and antimicrobial humoral immune responses mediated by antimicrobial peptides (Fig. 4E).

Gene set enrichment analysis (GSEA) and heatmap analysis confirmed the Mn-hCaO₂/hZIF8/SMA hydrogel's positive regulation of cellular zinc homeostasis, DNA repair, cell cycle, telomere maintenance and antibacterial activity (Fig. 4F, G). ROS-induced DNA damage and telomere dysfunction constitute one of the molecular hallmarks of aging and can amplify inflammatory reactions. The Mn-hCaO₂/hZIF8/SMA hydrogel battled intracellular ROS, endonuclear telomere shortening and DNA damage via multiple mechanisms (Fig. 4H). The release of zinc from the hydrogel upregulated metallothionein (MT), forming a non-enzymatic system that protects stem cells from oxidative damage 31. Additionally, the polyphenol component of the hydrogel upregulated Kelch-like ECH-associated protein 1 (KEAP1), which initiates a robust cytoprotective system mediated by antioxidant response elements (AREs) 32. Sirtuins, important antioxidant targets, were also regulated by the Mn-hCaO₂/hZIF8/SMA hydrogel, modulating aging-associated signaling pathways 33. Regarding telomere maintenance, the Mn-hCaO₂/hZIF8/SMA hydrogel upregulated several key genes: ataxia-telangiectasia-mutated (ATM), which facilitates the recruitment of telomerase; protection of telomere 1 (POT1) and tankyrase 2 (TNKS2), which are essential for telomerase activation and telomere extension 34, 35. The Mn-hCaO₂/hZIF8/SMA hydrogel also altered the ROS-accumulating and hypoxic environment. These processes collectively promote the re-entry of senescent stem cells into the cell cycle. Taken together, these findings suggest that the Mn-hCaO₂/hZIF8/SMA hydrogel revitalizes senescent stem cells by interfering with telomere shortening, DNA damage, and cellular antioxidant systems, attributed to the polyphenol-mediated synergistic effect of hZIF8 and Mn-hCaO₂.

Mn-hCaO₂/hZIF8/SMA hydrogel promotes periodontal regeneration in rat periodontitis
The therapeutic effect of Mn-hCaO₂/hZIF8/SMA hydrogel on periodontitis was evaluated in a rat ligature-induced periodontitis model (Fig. 5A). After 4 weeks of ligation, the rats displayed typical periodontitis symptoms, including gingival inflammation, deep periodontal pockets, attachment loss and root exposure, which confirmed the success of periodontitis model construction (Supplementary Fig. 4). Then, the ligature was removed and different hydrogels were implanted into the periodontal pockets by in situ injection. At the end of a 4-week repair period, the untreated periodontitis control (PD) group still displayed prominent periodontitis symptoms, reflecting unresolved inflammation and insufficient regenerative capacity (Fig. 5B). The Mn-hCaO₂/SMA and Mn-hCaO₂/hZIF8/SMA groups both showed significant improvements upon inspection: the gingiva recovered to a wavy and thin appearance, attaching close to the dental neck, while root exposure was also decreased, indicating periodontal regeneration.

Micro-computed tomography (micro-CT) images and quantitative analysis of alveolar bone crest height (alveolar bone crest-cementoenamel junction distance, ABC-CEJ), bone mineral density (BMD) and bone volume/tissue volume ratio (BV/TV) showed that the SMA matrix, with its fibrous structure and cellular affinity, slightly promoted bone density by providing a suitable mechanical microenvironment for osteogenesis (Fig. 5C, D). Introduction of hZIF8 or Mn-hCaO₂ into the SMA matrix could further improve its osteo-inducive property, resulting in a significant recovery in alveolar bone height. The Mn-hCaO₂/hZIF8/SMA hydrogel exerted a larger effect on alveolar bone height and bone density, indicating a subtle synergistic effect between hZIF8 and Mn-hCaO₂.

The therapeutic effect was further verified via histological and genetic evaluation. As shown by hematoxylin-eosin (H&E) (Fig. 5E) and Masson’s trichrome staining (Supplementary Fig. 5A), in the PD group the periodontitis-affected root furcation area exhibited widened periodontium space, which was filled with disorganized, fibrous connective tissue instead of newly formed bone tissue. The PD group also exhibited destructed gingival papilla and decreased interdental alveolar crest height. Alveolar bone volume was greatly improved in the hZIF8/SMA group and Mn-hCaO₂/SMA group, while the arrangement of periodontal ligament fibers was still unsatisfactory. The Mn-hCaO₂/hZIF8/SMA hydrogel efficiently promoted new bone formation and maturation in the periodontal lesions and displayed a narrower fibrous layer with aligned periodontal ligament fibers. The expression of osteoblast markers was examined by performing immunohistochemical staining and RT-qPCR on the tissue total RNA extracted from rat alveolar bone one week after hydrogel implantation. In healthy tissues, OCN+ osteoblasts were present at the boundary of periodontal ligament and alveolar bone, indicating the active remodeling of alveolar bone (Fig. 5F and Supplementary Fig. 5B). The differences in the OCN+ osteoblast proportions were consistent with the bone repair efficiency of each group. The Mn-hCaO₂/hZIF8/SMA group had the highest proportion of OCN+ osteoblasts, suggesting a strong pro-osteogenic effect. Consistent with this, the Mn-hCaO₂/hZIF8/SMA group also showed early and significant transcriptional upregulation of Ocn, which underscored its strong effect on boosting bone regeneration (Supplementary Fig. 5C, D). Biocompatibility of the hydrogel was confirmed by collecting vital metabolizing organs (liver and kidney) and immune organs (spleen and cervical lymph node) (Supplementary Fig. 5E, F). H&E staining revealed no apparent pathological changes, indicating the hydrogel possessed good biocompatibility.

Mn-hCaO₂/hZIF8/SMA hydrogel modulates senescent periodontal niche via regulating local inflammation and hypoxia in vivo
Immunofluorescence (IF) staining of P16 was performed to label senescent cells in periodontal tissue (Fig. 6A). Only a small percentage of cells were labeled in healthy tissue. In periodontitis, senescent cells were primarily distributed in the periodontal ligament area, where hPDLSCs were situated. A significant decrease in the proportion of senescent cells could be seen in the Mn-hCaO₂/SMA and Mn-hCaO₂/hZIF8/SMA groups (Fig. 6B). This decrease in cellular senescence was further supported by downregulated mRNA expression of P16 and P21 in alveolar bone (Supplementary Fig. 6A, B). SA‑β‑gal staining confirmed extensive inflammation-induced senescence in periodontitis, which was reversed by Mn-hCaO₂/hZIF8/SMA treatment, restoring levels close to those of healthy controls (Fig. 6C). The redox status in periodontal bone tissue was assessed using the reduced glutathione to oxidized glutathione (GSH/GSSG) ratio. Treatment with the Mn-hCaO₂/hZIF8/SMA hydrogel significantly increased the GSH/GSSG ratio, indicating a protective effect against oxidative injury (Fig. 6D). IF staining of HIF-1α and the evaluation of its corresponding mRNA expression showed that the periodontitis-affected tissue displayed a hypoxic state with high Hif-1α expression (Fig. 6E, F and Supplementary Fig. 6C). Hif-1α expression was sharply downregulated with the addition of oxygen-generating Mn-hCaO₂ NPs, confirming that the oxygenating hydrogels can alleviate hypoxia in the periodontal niche.

The severity of local inflammation is a crucial factor in stem cell fate determination. Local inflammation was markedly exacerbated in the rat model of periodontitis, as evidenced by elevated TNF-α expression (Fig. 6 G, H and Supplementary Fig. 6D, E) and neutrophil infiltration indicated by Ly6G⁺ cells (Fig. 6 I, J). Both Mn-hCaO₂/SMA and hZIF8/SMA treatment reduced inflammatory markers, with the composite hydrogel exhibiting the most pronounced anti-inflammatory effects. Collectively, these results confirm that the Mn-hCaO₂/hZIF8/SMA hydrogel can mitigate cellular senescence, oxidative stress, hypoxia, and inflammation, thereby ameliorating the pathological niche that can drive periodontitis.

Mn-hCaO₂/hZIF8/SMA hydrogel modulates microbiotic homeostasis in the senescent periodontal niche
The microbial communities in the subgingival biofilm 1 week after hydrogel application in the rat periodontitis model were catalogued by 16s ribosomal RNA sequencing. Community histograms at the family level demonstrated that Pasteurellaceae, Fusobacteriaceae, Bacteroidaceae, Streptococcaceae and Gemellaceae were the most abundant across the different groups (Fig. 7A). A ternary diagram depicted the differentially abundant taxa in the normal, PD and PD+Mn-hCaO₂/hZIF8/SMA groups, which indicated that the abundance of Fusobacterium and Bacteroides was increased in the PD group (Fig. 7B). At the species level, the abundance of s_Facklamia Tabacinasalis was increased in the Mn-hCaO₂/hZIF8/SMA group, while the abundance of g_Fusobacterium, p_Fusobacteriota, c_Fusobacteriia, f_Fusobacteriaceae and o_Fusobacteriales declined (Fig. 7C). Fusobacterium and Bacteroides are two representative genera of anaerobic bacteria involved in the pathology of periodontitis 36, 37. The PD group had a significant increase in the abundance of these two pathogens, while the PD+Mn-hCaO₂/hZIF8/SMA group returned their abundance to a near-normal level (Fig. 7D, E). This effect might be attributed to the altered oxygen environment, which would be expected to directly inhibit the activities of anaerobes. The relative abundance of periodontal probiotics was also compared. Lactobacillus, which exerts a probiotic effect in periodontitis through the competitive rejection of pathogenic bacteria and has a positive immunomodulatory effect 38, increased in the PD+Mn-hCaO₂/hZIF8/SMA group, though this effect did not reach statistical significance (Fig. 7F). Bacillus, represented by Bacillus subtilis, has been reported to have a protective effect against PD in human and rodents 39, 40. Interestingly, Bacillus was not detected in the PD group, but the hZIF8 and Mn-hCaO₂ containing groups showed considerable abundance of Bacillus (Fig. 7G).

Given the distinct compositional differences between human and rodent periodontal microbiota, we evaluated the hydrogel’s efficacy against Porphyromonas gingivalis, a key human periodontopathogen, in vitro. The Mn-hCaO₂/hZIF8/SMA hydrogel reduced bacterial viability by 90% within 24 hours, as measured by colony-forming unit (CFU) counts (Supplementary Fig. 7 A). Analysis of SEM images revealed extensive cellular damage, including shrinkage and membrane rupture, confirming potent antibacterial activity against this pathogen (Supplementary Fig. 7B). These findings verified that the Mn-hCaO₂/hZIF8/SMA hydrogel was effective in modulating the homeostasis of subgingival microbial communities, thus creating a favorable microenvironment with restored microbiotic homeostasis and a favorable host-microbe relationship for periodontal regeneration.

Overall, our data show that the anti-aging effect of the Mn-hCaO₂/hZIF8/SMA hydrogel was realized by the synergistic effects of multiple mechanisms (Fig.7H). First, the hydrogel restored the homeostasis of periodontal microbiota, eliminating the initiating factor of periodontitis. Second, the hydrogel alleviated the inflammatory and hypoxic environment, providing a favorable microenvironment for tissue regeneration. Third, the hydrogel directly attenuated cellular senescence by maintaining telomeres and defending against oxidative injury, as well as boosting the proliferation and osteogenic differentiation of hPDLSCs, which contributed to successful periodontal regeneration.

Methods
Ethical Statement
The study involving isolation of hPDLSCs from healthy periodontal ligament tissues of human premolars was reviewed and approved by the Research Ethics Committee of West China Hospital of Stomatology (No. WCHSIRB-CT-2023-006). Collection and use of human genetic resources in this study were conducted in compliance with the administrative regulations of the People’s Republic of China on human genetic resources and were approved by the Ministry of Science and Technology of China (No. 2025BAT00592). All animal experiments were approved by the Research Ethics Committee of West China Hospital of Stomatology (WCHSIRB-D-2022-374-R1).

Materials
Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and hydrocaffeic acid (HCA) were purchased from Sigma-Aldrich, USA. Zn(NO₃)₂·6H₂O, MnCl₂·4H₂O, NH₃·H₂O and H₂O₂ were purchased from Kelong, China. CaCl₂, 2-methylimidazole (2-MIM), N,N-dimethylformamide (DMF), glycidyl methacrylate (GMA), poly(ethylene glycol) diacrylate (PEGDA) and other chemical reagents were all purchased from Aladdin, China.

Preparation of CaO₂, hCaO₂ and Mn-hCaO₂ NPs
Typically, 5 g of CaCl₂ was dissolved in 25 mL of deionized (DI) water. The mixture was then poured into 300 mL of ethanol under vigorous stirring. After 10 min, the pH was adjusted to 10 using NH₃·H₂O to create an alkaline environment. Subsequently, 25 mL of 30% H₂O₂ was added dropwise to initiate the reaction. Stirring was continued for 30 min. The product was collected by centrifugation at 5000g for 5 min, washed twice with ethanol, and dried at 60 °C to obtain CaO₂ nanoparticles. For hCaO₂, the same steps were followed with the inclusion of 0.05 g HCA in the initial solution. Mn-hCaO₂ was prepared based on the hCaO₂ procedure with an additional step. After the addition of H₂O₂ and 10 min of reaction, 30 mg of MnCl₂·4H₂O dissolved in 10 mL of DI water was added dropwise to the mixture. Stirring continued for another 30 min. The resulting product was centrifuged at 5000 g for 5 min, washed twice with ethanol, and dried at 60 °C, yielding Mn-hCaO₂ nanoparticles.

Preparation of ZIF8 and hZIF8 NPs
ZIF8 and hZIF8 nanoparticles were synthesized via a one-pot method, with hZIF8 being derived from the basic ZIF8 procedure through the addition of HCA. In a typical synthesis, 4.516 g 2-MIM and 1 mL DMF were dissolved in 17 mL DI water. Separately, 0.232 g Zn(NO₃)₂·6H₂O (Zn: 2-MIM=1:70 in molar ratio) was dissolved in 2 mL DI water and added dropwise to the vigorously stirring 2-MIM solution. After the 3-hour reaction, the product was collected by centrifugation at 10,000 g for 8 min, washed twice with DI water, and dried at 60 °C to obtain ZIF8 nanoparticles. For the preparation of hZIF8 nanoparticles, the 2-MIM/DMF mixture was initially dissolved in 15 mL of DI water. One minute after the addition of the zinc precursor, 5 mg of HCA dissolved in 2 mL of DI water was added dropwise to the reaction mixture. After the 3-hour reaction, the light pink hZIF8 product was collected following the same centrifugation, washing, and drying steps.

Characterizations of NPs
The morphology of the nanoparticles (CaO₂, hCaO₂, Mn-hCaO₂, ZIF8 and hZIF8) were observed by scanning electron microscopy (Gemini 300, Zeiss, Germany). The microstructure and elemental composition of the nanomaterials were obtained using transmission electron microscopy (TEM; Tecnai-F30, FEI, USA) equipped with energy dispersive spectrometer (super-X detector, FEI, USA). The crystal phases of the nanoparticles were assessed by X-ray diffraction (Rigaku Ultima IV, Rigaku, Japan). The chemical states of the nanoparticles were assessed by X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Scientific, USA). The Zeta potential of the nanoparticles was assessed by a particle size & zeta potential analyzer (ZETA-AIZER, Malvern, UK).

Oxygen releasing profiles of Mn-hCaO₂ NPs
For the oxygen releasing curve, 20 mg CaO₂, hCaO₂ or Mn-hCaO₂ were dispersed with 10 mL PBS placed in a sealed container at 37℃, respectively. The dissolved oxygen (DO) concentration of each sample was measured with a dissolved oxygen analyzer (JPBJ608, INESA, China) at the scheduled time (0, 0.5, 2, 5, 12, 24, 36, 48, 60, 78 and 96 hours). The oxygen releasing curve was obtained with the DO concentration versus time. At the same scheduled time, the H₂O₂ concentration of the solution was measured with a H₂O₂ content assay kit (BC3595, Solarbio, China) according to the manufacturer’s instructions at the scheduled time. Absorbance at 415 nm was acquired using a microplate reader (Varioskan PLUX, Thermo Scientific, USA). The H₂O₂ concentration of the samples was calculated referring to a standard curve. The DO and H₂O₂ concentration in PBS were also tested as control under same conditions.

Catalytic activity of Mn-hCaO₂ NPs
First, 20 mg CaO₂, hCaO₂ or Mn-hCaO₂ were dispersed with 10 mL PBS placed in a sealed container at 37 ℃ until the oxygen release was completed, respectively. Then, the samples were placed in an anerobic chamber filled with N₂ gas and 120 μL 30% H₂O₂ solution was added into each sample. The catalytic activity was evaluated with the DO concentration of each sample with a dissolved oxygen analyzer (JPBJ608, INESA, China) at the scheduled time (2, 15, 30, 60, 90, 150 min).

Antioxidant activity of Mn-hCaO₂ NPs
The antioxidant acidities were evaluated with a tetramethylbenzidine (TMB)-based colorimetric method. 20 mg of CaO₂, hCaO₂ or Mn-hCaO₂ was dissolved in 2.85 mL DI water and 0.15 mL 10 g L⁻¹ TMB working solution (T9230, Solarbio, China), respectively. Then, the samples were incubated for 4 h at 37 ℃ in dark environment. The radical scavenging capability was evaluated with the absorbance change at 652 nm using a microplate reader (Varioskan LUX, Thermo Scientific, USA).

Preparation of Mn-hCaO₂/hZIF8/SMA hydrogel
The preparation of the SMA matrix was based on the method first described by Kim et al 52. To degum the silk fibers, silk cocoons were boiled in a 0.25 wt% Na₂CO₃ solution for 30 min and washed with DI water, which was repeated for twice. The degummed silk fibers were dried at 60 ℃ overnight. Afterwards, 20 g degummed silk fibers were dissolved in 200 mL of 9.3 M LiBr solution in a 70 ℃ water bath for the extraction of SF. For the synthesis of SMA, after the SF was extracted with the LiBr solution, the mixture was transferred to a 50 ℃ water bath. Then, 16 mL GMA was added dropwise to start the methacryloyl modification reaction. The stirring continued for 3 h and then 200 mL DI water was added to end the reaction. After that, the mixture was transferred into a dialysis bag (molecular weight cutoff of 14000) for 7 days. The deionized water was refreshed every 8 hours. SMA was stored under dry and photophobic conditions. A 15% (w/v) SMA solution was first prepared by dissolving 0.45 g of SMA in 3 mL of deionized water. Subsequently, 50 μL PEGDA, 8 mg LAP, 20 mg Mn-hCaO₂ NPs and 10 mg hZIF8 NPs were added and thoroughly mixed. This solution was injected into the desired mold or locations and exposed to 395 nm light (10 W) for 15 seconds to induce gelation. The detailed composition and concentrations of each hydrogel were provided in Supplementary Table 1.

Characterization of hydrogels
The morphology and elemental composition of different hydrogels were examined with SEM equipped with an energy dispersive spectrometer (Gemini 300, Zeiss, Germany). For sample preparation, the hydrogels were freeze-dried at -70℃ and sectioned to expose the internal structures. Then, the hydrogel sections were treated with gold sputtering by a sputter coater (JFC-1200, JEOL, Japan) before SEM observation.

Rheology test of hydrogels
The dynamic rheological tests of the hydrogels were characterized using a rheometer (MC2302, Anton Paar, Austria) at room temperature. The viscosity of the initial solution was measured with changed rotate speed (rad/s). To track the crosslinking process, the storage modulus G' (Pa) and loss modulus G" (Pa) changed with time was measured under 10% strain. Then, the hydrogels were prepared in a cylindrical shape with a height of 15 mm and a diameter of 1 mm. Strain amplitude sweeps (0.01-100%) were first performed to determine the linear viscoelasticity region. Dynamic frequency sweeps were performed at angular velocities ranging from 0.1 to 10 Hz at 1.0% strain amplitude.

Compression test of hydrogels
The compressive tests were carried out with a universal test machine (INSTRON 5567, Instron, USA) equipped with a 100 N load cell. The hydrogels were prepared in a cylindrical shape with a height of 11 mm and a diameter of 15 mm. For compressive test, the hydrogels were compressed at a loading rate of 5 mm/min and maximum strain was 80%. For cyclic compressive test, the hydrogels were compressed to a fixed strain of 60% at a loading rate of 5 mm/min and then relaxed for recovery. A total of 30 cycles were conducted. The compressive strength (P) was calculated with the following formula:
P = F/S (Equation 1)
where F is the compressive load and S is the cross-sectional area.

In situ adhesion of hydrogels
The in situ adhesive strength of the different hydrogels were measured by a tensile adhesive test using a universal test machine (INSTRON 5567, Instron, USA) equipped with a 100 N load cell. The hydrogel was in situ formed on fresh porcine skin with a bonding area of 25 mm × 25 mm. Then, the hydrogels were preloaded with 20 N for 10 seconds and pulled with a crosshead speed of 2 mm/min until the samples were displaced from the porcine skin. The adhesion strength (𝜎) was calculated with the following formula:
𝜎=F/A (Equation 2)
where F is the maximum load and A is the bonding area.

Swelling test of hydrogels
The swelling behaviors of the different hydrogels were measured to the evaluate their liquid uptake capacity. The hydrogels were prepared with a height of 5 mm and a diameter of 15 mm. Then, the hydrogels were soaked in 2 mL PBS. At the scheduled time intervals, the samples were taken out and weighed. The mass swelling ratio (SR) was calculated by the following equation:
SR= (W₁-W₀)/W₀ ×100% (Equation 3)
where W₀ is the initial weight of the sample and W₁ is the weight of the sample after PBS absorption.

In vitro degradation profile of hydrogels
The hydrogels were prepared into a cylindrical shape with a height of 20 mm and a diameter of 8 mm and then freeze-dried. The initial weight (M₀) of hydrogel samples was recorded. Then, the hydrogels were soaked into artificial saliva (Yuanye, R30090, China) and the weight (Mᵢ) were recorded at different time intervals. The remaining mass percentage of hydrogel was calculated with the following formula:
Mass remaining (%) = (Mᵢ/M₀) × 100% (Equation 4)

In vivo degradation profile of hydrogels
The hydrogels were prepared into a cylindrical shape with a height of 5 mm and a diameter of 15 mm and freeze-dried. The initial weight (M₁) of hydrogel samples was recorded. 8-week Sprague-Dawley were anesthetized and the dorsal fur is shaved and disinfected. An incision of 18 mm was made and then a subcutaneous pocket is created by blunt dissection. The hydrogels were inserted into the pockets and the incisions were closed with 3-0# suture. The weights of the hydrogels (M₂) were recorded at different time intervals. The remaining mass percentage of hydrogel was calculated with the following formula:
Mass remaining (%) = (M₂/M₁) × 100% (Equation 5)

Isolation, culture and stimulation of hPDLSCs
The hPDLSCs were isolated from healthy periodontal ligament tissues of human premolars obtained following therapeutic extraction for orthodontic reasons. All donor teeth were free of dental diseases, and the patients provided informed consent and had no history of systemic diseases. Periodontal ligament tissue was gently scraped from the middle third of the tooth root using a sterile scalpel and then digested with 3 g L⁻¹ type I collagenase (Gibco, USA) in minimum essential medium α (α-MEM) (Gibco, USA) at 37 °C for 30 min. Digestion was stopped by adding an equal volume of complete α-MEM medium, followed by another centrifugation step. The pellet was resuspended in complete α‑MEM culture medium containing 10% fetal bovine serum (Gemini, USA), 1% penicillin/streptomycin (Gibco, USA) and 1% glutamine (Gibco, USA). The cells were maintained at 37 °C in a 5% CO₂ incubator, with medium changes every 2 days. Cells were passaged upon reaching 80% confluence using 0.25% trypsin, and passages 3-5 were used for subsequent experiments. For cell-hydrogel co-culture experiments, the cells were seeded with a density of 5×10³ well⁻¹ for 96-well plate or 5×10⁴ well⁻¹ for 24-well plate and co-cultured with respective hydrogels (1 mm height, diameter in line with the well). Except the normal group, 1 mg L⁻¹ LPS (Sigma-Aldrich, USA) stimuli was added to the cultural medium after cell attachment to mimic an inflammatory microenvironment.

Cellular senescence and intracellular ROS labeling of hPDLSCs
After 48 hours of LPS stimulation, cellular senescence of hPDLSCs were evaluated with β-Galactosidase staining using a Senescence-associated β-Galactosidase Staining Kit (C0602, Beyotime, China) according to the manufacturer’s protocol. Intracellular ROS of hPDLSCs was labeled by DCFH-DA probe (2′,7′-dichloro fluorescein diacetate) using a reactive oxygen species assay kit (S0033S, Beyotime, China) according to the manufacturer’s protocol.

DPPH free radical scavenging assay of the hydrogels
The free radical scavenging of the hydrogels was tested with a DPPH free radical scavenging assay (SD9360, Solarbio, China). The hydrogels were prepared in a cylindrical shape with a height of 9 mm and a diameter of 2 mm. Then, the hydrogel was added to 3 mL DPPH solution prepared at a concentration of 0.04 g L⁻¹ with methanol. These samples were then placed in an oven at 37°C in the dark for 120 min. Subsequently, the DPPH solution was extracted and its absorbance was measured using a UV-Vis spectrophotometer at a wavelength of 516 nm.

Proliferation of hPDLSCs
Cell proliferation on 1st, 3rd and 5th day was evaluated by CCK8 assay (K1018, APExBIO, USA). After washing with PBS, each well was incubated with a mixture of CCK8 reagent and α-MEM (1:10) at 37°C for 2 h. Cell proliferation was evaluated by the absorbance at 450 nm using a microplate reader (Varioskan LUX, Thermo Scientific, USA).

Osteogenic differentiation of hPDLSCs
The osteogenic differentiation medium was prepared with 10 mM β-sodium glycerophosphate (Sigma, USA), 50 mg L⁻¹ L-ascorbic acid (Sigma, USA) and 100 nM dexamethasone (Sigma, USA). After 14 days of induction, cells were fixed with 4% PFA and ALP staining was performed with a BCIP/NBT Alkaline Phosphatase Color Development Kit according to the manufacturer’s protocol (C3206, Beyotime, China).

Macrophages Polarization
Human myeloid leukemia mononuclear cells, THP-1, were generously offered by the State Key Laboratory of Oral Diseases, West China Hospital of Stomatology. The THP-1 were cultured in Roswell Park Memorial Institute (RPMI) Medium 1640 (Gibco, USA) containing 10 % FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) in a humidified 5% CO₂ incubator at 37 °C. The cells were seeded with a density of 5×10⁴ well⁻¹ for 24-well plate and co-cultured with respective hydrogels (1 mm height, diameter in line with the well). 100 μg L⁻¹ Phorbol 12-Myristate13-Acetate (PMA, Sigma-Aldrich, USA) was applied to induce THP-1 to form macrophages. Except the normal group, 1 mg L⁻¹ LPS (Sigma-Aldrich, USA) stimuli was added to the cultural medium after cell attachment to mimic an inflammatory microenvironment. Macrophages polarization was examined on the 48 hours after stimulation.

Transcriptome sequencing
hPDLSCs treated with different group of hydrogels were collected at the 48th hour after 1 mg L⁻¹ LPS stimuli. The experiment was conducted with 4 independent replicates. The cell samples were treated with TRIzol reagent (Invitrogen, USA) and frozen in liquid nitrogen. RNA sequencing was performed using Illumina HiSeq X10 (Illumina). Bioinformatic analysis was performed using the OmicStudio tools (https://www.omicstudio.cn/tool).

Animal Experiment
A total of 60 eight-weeks-old male Sprague Dawley rats were used for construction of ligament-induced periodontitis model. Sex was not considered as a biological variable in this study. Only male rats were used, primarily to maintain consistency with the established protocols of prior foundational work in this model, while also reducing animal use and focusing resources on the core therapeutic question. The rats were randomly divided into six groups, including 1) normal control, 2) periodontitis, 3) PD+SMA, 4) PD+hZIF8/SMA, 5) PD+Mn-hCaO₂/SMA and 6) PD+Mn-hCaO₂/hZIF8/SMA. The investigators were blinded to the group allocation during data collection and analysis. For periodontitis model construction, the rats were anesthetized with sodium pentobarbital (30 mg kg⁻¹). The 3-0# non-absorbable surgical suture was ligated to the dental cervix of the maxillary first molar and then pressed into the gingival sulcus for four weeks until experimental periodontitis was formed. For hydrogel application, 20 μL premixed precursor solution was injected into the periodontal pocket and crosslinked with blue-violet light (395 nm, 10W) for 15 seconds. Samples were collected four weeks after treatment. The maxilla was collected and fixed with 4% PFA for observation and histology. Fresh alveolar bone tissue surrounding the first molar was collected in liquid nitrogen for RNA extraction. Liver, kidney, spleen and cervical lymph node tissues were collected and fixed with 4% PFA for biotoxicity analysis.

Micro-computed tomography (micro-CT) analysis
Harvested maxilla samples were scanned by a micro-CT scanner (μCT 50, SCANCO, Switzerland). Scanning parameters was set as 70 kV for voltage, 200 μA for current and 10 μm for voxel size. Micro-CT reconstruction and quantitative analysis was performed with the equipped analyzing system. Alveolar bone height (ABC-CEJ) was measured by the distance from the top of alveolar ridge to the cementum-enamel junction. Bone mineral density (BMD) and bone volume to tissue volume (BV/TV) of the furcation area were also analyzed.

Realtime quantitative polymerase chain reaction (RT-qPCR)
Total RNA of alveolar bone tissue or cultured cells was extracted with RNAiso reagent (9108, Takara, Japan) according to the manufacturer’s instructions. The total RNA concentration was measured with a micro-spectrophotometer (NanoDrop One C, Thermo Fisher Scientific, USA). Then, the total RNA was reverse-transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser (RR047A, Takara, Japan). RT-qPCR was performed with QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific, USA) with SYBR Premix Ex Taq II (RR420L, Takara, Japan). Relative gene quantitation was calculated with the 2-ΔΔCT formula. The primers are listed in Supplementary Table 2 and Supplementary Table 3.

Histology
Harvested maxilla samples were fixed with 4% PFA and decalcified in 14% EDTA (pH 7.4) for 8 weeks. The tissues were dehydrated and embedded in paraffin and sectioned by 5-μm thickness with a microtome (HistoCore Biocut R, Leica, Germany). H&E staining (C0105, Beyotime, China), Masson’s trichrome staining (G1340, Solarbio, China) was conducted using paraffin sections according to the manufacturers’ instructions. For immunohistochemical staining, after antigen retrieval with pepsin at 37°C and peroxidase blocking (MXB, China), sections were incubated with primary antibody at 4°C overnight. Detection was performed using the EnVision two‑step system (GK500705, Dako, Denmark), followed by 3,3’-Diaminobenzidine (DAB) development (SW1040, Solarbio, China). Sections were counterstained with hematoxylin, dehydrated, and mounted. Immunofluorescence was performed on frozen sections. The tissues were imbedded in optimal cutting temperature compound (OCT, Sakura, USA) and sectioned by 5-μm thickness with or a cryostat (CM 3050, Leica, Germany). After rewarming and washing, sections were blocked with 3% bovine serum albumin (Sigma-Aldrich, USA) for 2 h and then incubated overnight at 4°C with primary antibodies. Following PBS washes, sections were incubated with fluorophore‑conjugated secondary antibody for 2 h. Nuclei were counterstained with 1 mg L⁻¹ 4',6-diamidino-2-phenylindole (DAPI) (28718-90-3, Sigma-Aldrich, USA). Sections were dehydrated and mounted with an anti‑fade mounting medium (Solarbio, China). Details for used antibodies in immunohistochemistry and immunofluorescence were provided in Supplementary Table 4. β-Galactosidase staining was performed on frozen sections using a Senescence β-Galactosidase Staining Kit (C0602, Beyotime, China) according to the manufacturer’s protocol. Images were acquired with an optimal microscope (Ni-E, Nikon, Japan) or a laser scanning confocal microscope (N-STORM Ti2 A1+, Nikon, Japan).

GSH/GSSG assay
Fresh alveolar bone tissue surrounding the first molar was collected and grounded in liquid nitrogen. Then, GSH/GSSG ratio was measured by a commercial GSH and GSSG Assay Kit (Beyotime, S0053, China) based on the enzymatic recycling method with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

16s ribosomal RNA sequencing
Subgingival biofilm samples were collected after 7 days after hydrogel application. Silk sutures were pressed to gingival sulcus 24 hours in prior for accumulating the microorganisms. The genomic DNA was extracted and the V4 region hypervariable regions of the 16S rDNA were amplified with PCR. The PCR products were quantified with PicoGreen dsDNA Assay Kit (Invitrogen, USA) and then sequenced by NovaSeq PE250 platform (Novogene, China). Data possession was performed based on QIIME2 platform. Bioinformatic analysis was performed using the Novomagic platform (https://magic.novogene.com).

In vitro Antibacterial Assay
Porphyromonas gingivalis (ATCC 33277) was routinely cultured under anaerobic conditions (85% N₂, 10% H₂, 5% CO₂) at 37°C in brain heart infusion (BHI) broth supplemented with 5 mg L⁻¹ hemin and 1 mg L⁻¹ vitamin K. The bacteria were suspended with a density 1×10⁸ CFU mL⁻¹ and co-cultured with different groups of respective hydrogels (1 mm height, diameter in line with the 24-well plate). After 24 hours of anaerobic incubation, the bacteria were harvested by centrifugation at 6000 g for 10 min and washed twice with PBS. Bacterial viability was confirmed by colony-forming unit (CFU) measured with optical density at 600 nm. For SEM imaging, bacterial pellets were fixed in 2.5% glutaraldehyde over night at 4°C. Samples were then dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%, 10 min each). Then they were mounted on silicon wafers, sputter-coated with gold, and observed under a scanning electron microscope.

Statistical analysis
All results were analyzed by a one-way analysis of variance (ANOVA) with a Tukey’s post hoc test. The data are expressed as mean ± standard deviation with n ≥ 3. Differences were considered statistically significant for P < 0.05. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, USA) and Origin 2021 (OriginLab, US).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled \"Polyphenol-Mediated Zinc-Oxygen Synergistic Hydrogel Remodels Senescent Microenvironment for Periodontal Tissue Regeneration\" presents an innovative and well-conceived approach to addressing the challenges of senescent cell accumulation and impaired regeneration in periodontitis. The integration of hydrocaffeic acid-modified CaO₂ and ZIF8 nanoparticles into a silk fibroin hydrogel matrix is scientifically sound and well-supported by comprehensive in vitro and in vivo data. The manuscript is timely and highly-relevant to the fields of tissue engineering, biomaterials, and regenerative medicine.\nTo further strengthen the manuscript and improve its clarity, impact, and accessibility to a broader readership, I have outlined several constructive comments and suggestions below. These are intended to enhance the overall quality of the manuscript and support its eventual publication in this journal.\nHere are my General Comments\n1) Clarify novelty and differentiation:\nThe study is innovative in combining polyphenol-mediated zinc and oxygen delivery; however, the novelty compared to existing oxygenating hydrogels (e.g., ZIF8-based or CaO₂-based) could be further clarified. Please emphasize how your approach substantially outperforms or uniquely modifies current technologies.\n2)Improve language clarity and grammar:\nWhile the scientific content is compelling, the manuscript would benefit from thorough language polishing. For example, awkward phrasing like \"The senescent cells mainly distributed in...\" should be revised to \"Senescent cells were primarily distributed in...\".\n3) Define abbreviations on first use:\nSome abbreviations like SMA, ABC-CEJ, and hPDLSCs are used before being properly defined. Ensure all abbreviations are spelled out at first mention for accessibility to a broader audience.\nHere are my Specific Comments\n1) Mechanistic clarity for synergistic action:\nThe manuscript describes a synergistic effect of Zn²⁺, O₂, and HCA, but it would benefit from a clearer molecular explanation of how these components work together mechanistically. Consider including a simplified mechanistic diagram or expanding the schematic in Figure 1.\n2) Quantitative synergy analysis needed:\nThe manuscript claims a synergistic effect between Mn-hCaO₂ and hZIF8, yet the synergy is not quantitatively assessed. A synergy index or Bliss independence model analysis could help support this claim.\n3) Expand on microbiome modulation implications:\nThe study presents compelling data on microbiota rebalancing but does not discuss long-term microbiome stability or potential off-target effects. Consider adding a brief discussion on the safety and selectivity of microbiota modulation.\n4) Elaborate on in vivo degradation and clearance:\nThere is no information on how long the hydrogel remains in the tissue or its degradation rate. Please include a degradation profile or discuss expected in vivo longevity and any potential for accumulation or toxicity.\n5) Statistics and sample size reporting:\nWhile some graphs include statistical annotations, a clearer summary of biological replicates (n values), animal numbers per group, and statistical tests used (e.g., ANOVA details) should be included in figure legends and methods.\nHere are my comments on formatting and presentation of the manuscript\n1) Improve figure legends for standalone clarity:\nSome figure legends (e.g., Figures 3 and 5) are densely written and assume familiarity with experimental groups. Make figure captions more self-explanatory by briefly restating treatment group compositions.\n2) Supplementary data cross-referencing:\nSeveral references to \"Fig. S1\" or \"Table S1\" lack context. Provide clearer descriptions in the main text when referring to supplementary materials to guide the reader more effectively.", "reviewer_2": "This study presents an innovative and multidisciplinary approach to addressing the critical challenge of cellular senescence in chronic inflammatory microenvironments, particularly in periodontitis. The authors have developed a polyphenol-mediated zinc-oxygen synergistic hydrogel (Mn-hCaO2/hZIF8) that integrates advanced biomaterial engineering with nuanced biological insights to achieve multifactorial modulation of the senescent niche. By combining a cascade oxygenating system with sustained Zn²⁺ delivery, the hydrogel simultaneously alleviates hypoxia, corrects micronutrient deficiencies, and mitigates oxidative stress—key barriers to tissue regeneration. The work stands out for its strategic convergence of nanomedicine, antioxidant chemistry, and microbiome regulation. The hydrogel’s ability to reverse telomere attrition, DNA damage, and microbial dysbiosis while reactivating senescent stem cells underscores its translational potential. The authors transcend conventional single-target anti-senescence strategies by offering a holistic solution that addresses the heterogeneity of senescent microenvironments.\nThis study not only advances regenerative medicine but also provides a blueprint for designing multifunctional hydrogel capable of reprogramming complex pathological niches. The novelty of the therapeutic platform, and the demonstrated efficacy in rejuvenating periodontal tissues make this work a compelling candidate for publication in Nature Communications after addressing the following comments.\n(1) The study demonstrates that the incorporation of Mn-HCA into CaO₂ significantly enhances the antioxidant properties of nanoparticle. It would be valuable to include additional experimental data for the hydrogels as well, for instance, DPPH radical scavenging assays may be a choice.\n(2) The injectability of the multifunctional hydrogel demonstrates considerable clinical appeal for periodontal therapy. The swelling property is fundamental for a hydrogel to conformally fill anatomically complex periodontal defects. Therefore, it is necessary to provide data on the hydrogel’s swelling dynamics in aqua solution (such as PBS or culture medium).\n(3) The glutathione (GSH) to glutathione disulfide (GSSG) ratio is a well-established and critical marker of cellular redox homeostasis, reflecting the balance between oxidative stress and antioxidant capacity within biological systems. Incorporating measurements of the GSH/GSSG ratio into the animal experiments would provide more comprehensive assessment of the hydrogel’s ability in modulating redox balance in vivo.\n(4) The demonstrated dual efficacy of the hydrogel in suppressing anaerobic pathogens and enriching probiotic species represents a significant and encouraging advancement in this field. However, Porphyromonas gingivalis (P.g.), a well-established pathogen in human periodontitis, exhibited notably low abundance in the microbiome sequencing analysis. Considering the inherent differences in periodontal microbiota composition between humans and rodents, the findings remain biologically plausible. Nonetheless, to strengthen the translational relevance of the study, I recommend that the authors conduct in vitro experiments to directly assess the antimicrobial efficacy of the hydrogel against P. g.\n(5) Some minor mistakes in figures should be checked and corrects. E.g., In Fig.7H, ‘jZIF8’ should be corrected with ‘hZIF’. In Fig.S1 B and E, the proportion of C should be added to the graph.", "reviewer_3": "The authors describe an injectable polyphenol-mediated oxygenating hydrogel based on Mn-hCaO2, HCA-mediated ZIF8 nanoparticles, and silk fibroin methacrylate for targeted modulation of senescent periodontal microenvironment. By specifically addressing hypoxia and zinc ion deficiency, the hydrogel could restore the essential micronutrients and oxygen levels, and also rejuvenate the senescent periodontal microenvironment. The polyphenol-mediated hydrogel simultaneously targeted multiple aging-related processes and favored robust periodontal tissue regeneration. However, the following concerns need to be addressed before it can be considered for publication.\n1. How was the content ratio of HCA, Mn2+, Mn-hCaO2, hZIF8 NPs, and SMA screened and determined? The authors did not mention the basis for screening.\n2. In Figure 2M and N, the authors adhered the hydrogel to hard tissue (rat femur) and soft tissue (rat muscle). As the hydrogel was used in periodontal tissue, isn’t alveolar bone, tooth cementum, oral mucosal tissue, and gingival tissue more appropriate?\n3. In Figure 2O and P, the compressive stress of the hydrogels was evaluated. But for an injectable hydrogel, comprehensive rheological properties testing is more important. The authors only conducted rheological characterization of the Mn-hCaO2/hZIF8/SMA hydrogel under frequency from 0.1-10 Hz, which is not enough. Shear thinning properties should be tested especially.\n4. The adhesion strength of hydrogels to porcine skin was around 15 kPa, “strong tissue adhesiveness” might be exaggerated.\n5. Inflammatory microenvironment induced by microbial virulence was established by applying liposaccharide (LPS) stimuli to hPDLSCs. Why did the authors select LPS rather than LPS-PG (LPS from P. gingivalis)? Isn’t it more consistent with the periodontitis condition?\n6. During the rat periodontitis model construction, the hydrogels were irradiated by blue-violet light (395 nm, 10W) for 15-30 seconds. As far as we are concerned, blue-violet light with 395 nm direct exposure to living tissue is harmful. Is the study design reasonable? Moreover, the authors only applied the hydrogel once and conducted the therapeutic evaluation after 4 weeks. Why didn’t apply the hydrogel more times like the clinical situation? The effect of a single dose is definitely limited.\n7. H&E staining in the root furcation area was shown. Since the hydrogel was injected into the periodontal pocket. Isn’t the periodontal pocket area more rational? It is doubted that the hydrogel applied in the periodontal pocket could affect the root furcation area more significantly.\n8. Since the authors have already conducted animal experiments. Evaluation of relative mRNA expression of osteogenesis-related genes in alveolar bone tissue was unnecessary. Immunohistochemistry staining or the ELISA tests of the protein should be more meaningful. The same problems are also reflected in Fig. 6.\n9. Relative abundance of periodontitis-related pathogens and probiotics was shown in Fig. 7. Only Fusobacterium nucleatum was involved. It is suggested to add the results of other periodontitis-related pathogens, especially P.g. and A.a."}, "author_response": "Responses to Reviews’ Comments\nWe sincerely thank all three reviewers for taking time and providing insightful and constructive\nfeedback for our manuscript. We have carefully addressed each of the suggestions and have\nsubstantially revised the manuscript accordingly. The manuscript has undergone thorough\nlanguage polishing. All modifications have been highlighted in the revised manuscript. Point-\nby-point responses to each comment are provided below.\nReviewer #1\nThe manuscript titled \"Polyphenol-Mediated Zinc-Oxygen Synergistic Hydrogel Remodels\nSenescent Microenvironment for Periodontal Tissue Regeneration\" presents an innovative and\nwell-conceived approach to addressing the challenges of senescent cell accumulation and\nimpaired regeneration in periodontitis. The integration of hydrocaffeic acid-modified CaO₂ and\nZIF8 nanoparticles into a silk fibroin hydrogel matrix is scientifically sound and well-supported\nby comprehensive in vitro and in vivo data. The manuscript is timely and highly-relevant to\nthe fields of tissue engineering, biomaterials, and regenerative medicine.\nTo further strengthen the manuscript and improve its clarity, impact, and accessibility to a\nbroader readership, I have outlined several constructive comments and suggestions below.\nThese are intended to enhance the overall quality of the manuscript and support its eventual\npublication in this journal.\nResponse: We sincerely appreciate your encouraging feedback and valuable comments, which\nhave greatly contributed to improving our manuscript. We have carefully addressed all the\nsuggestions provided, as detailed in our responses below.\nHere are my General Comment.\n1) Clarify novelty and differentiation:\nThe study is innovative in combining polyphenol-mediated zinc and oxygen delivery; however,\nthe novelty compared to existing oxygenating hydrogels (e.g., ZIF8-based or CaO₂-based)\ncould be further clarified. Please emphasize how your approach substantially outperforms or\nuniquely modifies current technologies.\nResponse: We sincerely appreciate your insightful comment regarding the novelty of our\npolyphenol-mediated oxygenating system. The novelty compared to existing oxygenating\nhydrogels includes:\nFirst, a key limitation of conventional CaO₂-based oxygenating systems is the production of\ncytotoxic H₂O₂ as a byproduct, which compromises biosafety. CaO₂ generates oxygen via two\nprimary pathways: (1) hydrolysis with water, yielding Ca(OH)₂ and O₂; and (2) a more rapid\nreaction with H⁺ in acidic environments, producing a calcium salt and H₂O₂. Subsequently,\nH₂O₂ decomposes to H₂O and O₂. The acidic conditions prevalent in inflammatory\nmicroenvironments, arising from processes like glycolysis and lipid peroxidation, favor the\nlatter reaction. However, the resulting H₂O₂ byproducts can exacerbate oxidative stress and\nimpede tissue regeneration. To address this limitation, our Mn-HCA modified CaO₂ system\nintegrates enzyme-mimetic catalytic units. The Mn-based catalytic unit enables controlled\noxygen generation from H₂O₂ by leveraging Mn2+/Mn3+ redox cycling, achieving high oxygen-\nyield production while minimizing cytotoxic radical formation for therapeutic applications.\nHCA, as a typical polyphenol compound, mitigates oxidative stress by directly scavenging free\nradicals through hydrogen atom transfer or electron donation from phenolic hydroxyl groups,\nwhile concomitantly upregulating endogenous antioxidant enzymes (e.g., SOD) via activation\nof the anti-inflammatory signaling pathway (e.g., Nrf2 and Sirt). These units concurrently\nenhance oxygen release and scavenge both endogenous and environmental H₂O₂, thereby\nsignificantly improving biosafety.\nSecond, tissue affinity, robust adhesion, and enhanced compressive resistance are critical for\nhydrogels within the periodontal microenvironment to ensure the long-term efficacy. Current\noxygen-releasing materials, however, face significant challenges in achieving these properties.\nConventional CaO₂ or ZIF8 based hydrogels typically exhibit poor aqueous dispersibility and\ninadequate tissue adhesion. To address these limitations, we implemented a polyphenol-\nmediated strategy in our oxygenating system. Surface-functionalized polyphenols on CaO₂ or\nZIF8 nanoparticles leverage their unique phenolic hydroxyl groups to form versatile bonds with\ndiverse functional groups. This approach constructs a dense, dynamic network of chemical\nbonds between polymer chains, substantially enhancing the compressive resistance of the Mn-\nhCaO2/hZIF8/SMA hydrogel. Simultaneously, the polyphenols enable strong interfacial\nadhesion to both soft and mineralized tissues through dynamic hydrogen bonding with soft\ntissues and metal-coordination chelation with calcium ions in hard tissues. Consequently, our\nhydrogel system demonstrates superior mechanical properties compared to conventional\noxygen-releasing platforms, rendering it more suitable for periodontal applications.\nThird, current CaO₂-based oxygenating materials suffer from single-functionality and limited\nefficacy in promoting tissue regeneration, thereby restricting their clinical applicability.\nAlthough numerous reported oxygenating systems demonstrate significantly enhanced oxygen\ngeneration capabilities, demanding regenerative scenarios require multifunctional properties\nbeyond mere oxygenation. Our study has proved that the Mn-HCA component contributes to\nmicrobiota homeostasis restoration and cellular senescence attenuation through telomere\nmaintenance and oxidative injury defense. Concurrently, zinc supplied by hZIF8 further\npromotes tissue regeneration by combating oxidative stress while enhancing the proliferation\nand osteogenic differentiation of stem cells. In conclusion, this integrated platform delivers\nmultifunctional therapeutic effects, including pro-osteogenic, anti-inflammatory, anti-\nsenescence, and microbiota-regulating capabilities, thus enabling targeted treatment within\ncomplex inflammatory microenvironments.\nThe following statements were added to Discussion section.\nLine 564-593 “Current oxygenating materials face significant challenges that limit their\nbiomedical application. First, efforts to augment oxygen generation frequently yield cytotoxic\nH O byproducts, with acidic inflammatory microenvironments arising from glycolytic\n2 2\nmetabolism and lipid peroxidation further amplifying this detrimental cascade. To address this\nlimitation, we constituted Mn-HCA enzyme-mimetic catalytic units. The Mn-based catalysis\nconverts detrimental H O into O through efficient disproportionation, simultaneously\n2 2 2\nmaximizing oxygen yield while suppressing cytotoxic radical formation. HCA, as a typical\npolyphenol compound, mitigates oxidative stress by directly scavenging free radicals through\nhydrogen atom transfer or electron donation from phenolic hydroxyl groups, while\nconcomitantly upregulating endogenous antioxidant enzymes (e.g., SOD) via activation of the\nanti-inflammatory signaling pathway (e.g., Nrf2 and Sirt). These units concurrently enhance\noxygen release and scavenge both endogenous and environmental H₂O₂, thereby significantly\nimproving biosafety. Second, current oxygen-releasing materials exhibit inadequate\nmechanical integration characterized by poor tissue adhesion and compressive resistance in\nperiodontal contexts. To overcome this limitation, we implemented surface-functionalized\npolyphenols on CaO₂ and ZIF8 nanoparticles. This strategy constructed dense, dynamic\nchemical bond networks between polymer chains, substantially enhancing the compressive\nresistance of our Mn-hCaO₂/hZIF8/SMA hydrogel. The polyphenols enabled robust interfacial\nadhesion to both soft and mineralized tissues through dynamic hydrogen bonding with soft\ntissues and metal-coordination chelation with calcium ions in hard tissues. Consequently, our\nhydrogel demonstrates superior mechanical properties, rendering it more suitable for\nperiodontal applications. Furthermore, existing CaO₂-based materials suffer from single-\nfunctionality and limited regenerative efficacy. Our study has shown that the Mn-HCA\ncomponent in our hydrogel contributes to microbiotic homeostasis restoration and cellular\nsenescence attenuation through telomere maintenance and oxidative injury defense.\nConcurrently, zinc supplied by hZIF8 further promotes tissue regeneration by combating\noxidative stress while enhancing the proliferation and osteogenic differentiation of stem cells.\nIn conclusion, this integrated platform delivers multifunctional therapeutic effects, including\npro-osteogenic, anti-inflammatory, anti-senescence, and microbiota-regulating capabilities,\nthus enabling targeted treatment within complex inflammatory microenvironments.”\n2)Improve language clarity and grammar:\nWhile the scientific content is compelling, the manuscript would benefit from thorough\nlanguage polishing. For example, awkward phrasing like \"The senescent cells mainly\ndistributed in...\" should be revised to \"Senescent cells were primarily distributed in...\".\nResponse: We sincerely appreciate your careful reading and valuable suggestion regarding\nlanguage refinement. We have now engaged a professional editing service to thoroughly polish\nthe manuscript, correcting grammatical issues and enhancing overall clarity. All revisions have\nbeen highlighted in the revised manuscript for ease of review. We believe these revisions\nsignificantly improve the readability and precision of the manuscript.\n3) Define abbreviations on first use:\nSome abbreviations like SMA, ABC-CEJ, and hPDLSCs are used before being properly\ndefined. Ensure all abbreviations are spelled out at first mention for accessibility to a broader\naudience.\nResponse: We sincerely apologize for the oversight in defining key abbreviations and\nappreciate you identifying this important accessibility issue. We have now spelled out all\nabbreviations at their first mention in the main text and ensured consistency throughout the\nmanuscript.\nThe following was the first mention of the abbreviations of SMA:\nLine 108-111 “In this study, we engineered an injectable polyphenol-mediated oxygenating\nhydrogel based on HCA-mediated manganese-modified CaO nanoparticles (Mn-hCaO ),\n2 2\nHCA-mediated ZIF8 nanoparticles (hZIF8), and silk fibroin methacrylate (SMA) for targeted\nmodulation of the senescent periodontal microenvironment.”\nThe following was the first mention of the abbreviations of ABC-CEJ:\nLine 390-394 “Micro-computed tomography (micro-CT) images and quantitative analysis of\nalveolar bone crest height (alveolar bone crest-cementoenamel junction distance, ABC-CEJ),\nbone mineral density (BMD) and bone volume/tissue volume ratio (BV/TV) showed that the\nSMA matrix, with its fibrous structure and cellular affinity, slightly promoted bone density by\nproviding a suitable mechanical microenvironment for osteogenesis.”\nThe following was the first mention of the abbreviations of HCA:\nLine 97-99 “Polyphenols, such as tannic acids (TA), epigallocatechin-3-gallate (EGCG) and\nhydrocaffeic acid (HCA), exhibit immunomodulatory effects due to their unique phenolic\nhydroxyl groups, which can be beneficial for inflammatory diseases.”\nHere are my Specific Comments\n1) Mechanistic clarity for synergistic action:\nThe manuscript describes a synergistic effect of Zn²⁺, O₂, and HCA, but it would benefit from\na clearer molecular explanation of how these components work together mechanistically.\nConsider including a simplified mechanistic diagram or expanding the schematic in Figure 1.\nResponse: We sincerely thank the reviewer for this constructive suggestion. In accordance with\nyour recommendation, we have expanded Figure 1D to incorporate a mechanistic diagram\nelucidating the molecular synergy of Zn²⁺, O₂, and HCA. The anti-senescence efficacy of Mn-\nhCaO₂/hZIF-8/SMA hydrogel from coordinated molecular actions: 1) HCA and Zn²⁺\nsynergistically enhance oxidative defense. HCA scavenges ROS via phenolic hydroxyl groups\nwhile activating KEAP1/Nrf2 and sirtuin pathways, whereas Zn²⁺ upregulates metallothionein\nto establish non-enzymatic protection. 2) Zn²⁺ and O₂ co-regulate telomere maintenance\nthrough upregulation of telomerase components. 3) O₂ and HCA jointly mitigate hypoxic stress\nand ROS accumulation, facilitating DNA damage repair. 4) O₂ selectively suppresses anaerobic\npathogens predominant in periodontitis, while Zn²⁺ disrupts microbial metabolism and induces\nmembrane rupture. Notably, the polyphenolic component HCA promotes probiotic\nproliferation, collectively rebalancing microbial homeostasis. These integrated mechanisms\nestablish a regenerative microenvironment that potentiates periodontal tissue repair.\nFigure 1 and corresponding the main text was updated in Design Strategy section.\nLine 135-142 “The pathological periodontal niche sustains a vicious cycle of hypoxia,\nmicrobiota dysbiosis, and pro-inflammatory immune patterns, collectively driving stem cell\nsenescence and impairing tissue regeneration (Fig. 1D). The Mn-hCaO₂/hZIF8/SMA hydrogel\ndisrupts this circuit via synergistic actions of HCA, Zn²⁺ and O₂ release, which mitigate\nsenescence through hypoxia alleviation, oxidative damage protection, telomere maintenance\nand enhanced DNA repair. Concurrently, it restores microbiotic homeostasis and reprograms\nimmune responses, establishing a rejuvenated microenvironment that supports periodontal\ntissue repair.”\nFigure 1 (D) Biological mechanism diagram of Mn-hCaO₂/hZIF-8/SMA hydrogel in\nalleviating senescence in periodontitis. The Mn-hCaO₂/hZIF8/SMA hydrogel alleviated\ncellular senescence and established a ‘rejuvenated’ periodontal niche with the synergistic effect\nof HCA, Zn²⁺ and O₂, which finally supported robust periodontal tissue repair.\n2) Quantitative synergy analysis needed:\nThe manuscript claims a synergistic effect between Mn-hCaO₂ and hZIF8, yet the synergy is\nnot quantitatively assessed. A synergy index or Bliss independence model analysis could help\nsupport this claim.\nResponse: We appreciate the reviewer's valuable suggestion regarding quantitative synergy\nassessment. While classical synergy models like synergy index and Bliss independence provide\nreliable frameworks for conventional pharmacokinetics, their application to our composite Mn-\nhCaO₂/hZIF8/SMA hydrogel system faces inherent limitations due to the material's\nmultifunctional design. Both Mn-hCaO and hZIF8 nanoparticles share HCA functionalization\n2\nthat enables antioxidant and tissue affinitive activities. Direct physical separation of HCA may\nfundamentally influence their biological efficacy. Furthermore, the dynamic\nmicroenvironmental factors following the dose change may introduce confounding factors.\nChanges in component ratios influence hydrogel degradation profiles and mechanical\nproperties, which subsequently regulate cellular responses. These interdependent variables\nobstruct static dose-response analysis required for traditional quantitative synergy assessment.\nWe have acknowledged this limitation in the revised Discussion section. In place of formal\nquantitative modeling, we have instead included additional mechanistic evidence and\ndiscussion to better explain the synergistic interactions observed in our system.\nLine 618-648 “In this study, the positive outcomes from the combined supply of oxygen and\nzinc exceeded the additive effects of each one alone, which indicates synergy between oxygen\nand zinc ions in alleviating hPDLSC senescence and boosting periodontal regeneration. The\noxygen level, as a pivotal environmental component of the niche, can govern fundamental\nprocesses in tissue regeneration 1, 2. Consistent with prior findings, we found that oxygen\nsupply is critical for reversing cellular senescence, facilitating cell cycle re-entry, and\nmodulating periodontal microbiotic homeostasis. Transcriptomic sequencing of hPDLSCs\nfurther revealed that oxygenation significantly upregulated DNA repair processes. The DNA\nrepair pathways, including nucleotide excision repair (NER), base excision repair (BER) and\nhigh-fidelity homologous recombination (HR), are highly reliant on aerobic ATP production3.\nIn inflammatory conditions, energy deficiency and upregulation of HIF-1α cause conversion\nto a transcriptional program that suppresses HR while promoting error-prone repair\nmechanisms like non-homologous end joining (NHEJ), increasing cells’ susceptibility to\nsenescence4. Thus, oxygen availability directly influences DNA repair pathway choice and\nsenescence progression. However, oxygen supplementation can be a double-edged sword:\nexcess oxygen may induce ROS generation and cause oxidative organelle damage. Zinc\ncounteracts this risk by serving as an essential cofactor for multiple enzymes and transcription\nfactors and contributing to the regulation of antioxidant defenses by supporting the activity of\nantioxidant enzymes 5, 6. Meanwhile, zinc is an essential cofactor required for the structural\nintegrity and catalytic function of telomerase 7. Our data also reflected that zinc supply can\nbenefit telomere stability by influencing telomerase-related pathways. When supplied\ntogether, oxygen and zinc exhibit complementary functions: oxygen enables efficient zinc-\ndependent metabolic and synthetic reactions, while zinc mitigates potential oxidative stress.\nBesides these direct anti-senescent activities, our study also indicated that oxygen and zinc\nsynergistically restore microbiotic homeostasis and promote immune modulation, establishing\na pro-regenerative niche for stem cells. These mechanisms underlie the rejuvenating strategy\ndemonstrated here for periodontitis treatment. One limitation of the study is the inapplicability\nof classical synergy models in our multifunctional system owing to the functional inseparability\nof the HCA-modified components and composition-dependent mechanical changes of the\nhydrogels. Future work using dose-response analyses with defined elemental stimuli would\nfacilitate quantitative synergy assessment of oxygen and zinc.”\nReferences\n1. Sammarco, M.C. et al. Endogenous bone regeneration is dependent upon a dynamic\noxygen event. J. Bone Miner. Res. 29, 2336-2345 (2014).\n2. Loeffler, J., Duda, G.N., Sass, F.A. & Dienelt, A. The Metabolic Microenvironment Steers\nBone Tissue Regeneration. Trends Endocrinol. Metab. 29, 99-110 (2018).\n3. Eustermann, S., Patel, A.B., Hopfner, K.-P., He, Y. & Korber, P. Energy-driven genome\nregulation by ATP-dependent chromatin remodellers. Nature Reviews. Molecular Cell Biology\n25, 309-332 (2024).\n4. Peng, L. et al. The hepatic clock synergizes with HIF-1α to regulate nucleotide availability\nduring liver damage repair. Nat. Metab. 7, 148-165 (2025).\n5. Prasad, A.S. & Bao, B. Molecular Mechanisms of Zinc as a Pro-Antioxidant Mediator:\nClinical Therapeutic Implications. Antioxidants 8, 164 (2019).\n6. Maywald, M. & Rink, L. Zinc in Human Health and Infectious Diseases. Biomolecules 12,\n1748 (2022).\n7. Braun, H. et al. ZNF524 directly interacts with telomeric DNA and supports telomere\nintegrity. Nat. Commun. 14, 8252 (2023).\n3) Expand on microbiome modulation implications:\nThe study presents compelling data on microbiota rebalancing but does not discuss long-term\nmicrobiome stability or potential off-target effects. Consider adding a brief discussion on the\nsafety and selectivity of microbiota modulation.\nResponse: We thank the reviewer for raising this important point regarding long-term stability\nand potential off-target effects of microbiome modulation. In response to this comment, we\nhave expanded the Discussion section addressing these aspects.\nLine 595-616 “Long-term stability and off-target effects remain central challenges in the\ndesign of microbiome-modulating biomaterials. To address these issues, we developed a\nhydrogel-based delivery strategy enabling sustained release of oxygen, zinc, and polyphenol,\naimed at remodeling the dysbiotic microenvironment in periodontitis. Oxygen supplementation\nenables selective targeting of aerobic pathogens without introducing exogenous microbes or\nbroad-spectrum antimicrobials. Some polyphenol extracts have been shown to enrich gut\nprobiotics including Bifidobacterium and Lactobacillus, while their application within the oral\nmicrobiota has remained unexplored8, 9. Here, we demonstrated that HCA contributes to\nfavorable rebalancing of the periodontal microbial community by enriching probiotics.\nTogether, these strategies minimize ecological disruption and support a gradual transition\ntoward a stable and healthy microbiome. Microbiome off-target effects, or unintended\nconsequences that occur in the microbial communities of non-targeted regions elsewhere in the\nbody, can pose a concern in strategies that shift microbial balance10. In the present study, the\nconfined application within periodontal pockets, ensured by tissue adhesion, was designed to\nminimize leakage into the oral cavity or gastrointestinal tract. Furthermore, the small applied\nvolume and localized action also diminish risks of off-target impacts on non-targeted microbial\ncommunities. These findings highlight the potential of an injectable polyphenol-mediated\noxygenating hydrogel system to restore ecological balance in situ. Nevertheless, our study is\nlimited by its inability to fully recapitulate the impact of dietary variations on oral microbiome\ndynamics in humans, which may influence treatment outcomes. Future studies under clinically\nrelevant conditions are warranted to validate this strategy as a precise and sustainable\ntherapeutic intervention for periodontitis.”\nReferences\n8. Marchesi, J.R. et al. The gut microbiota and host health: a new clinical frontier. Gut 65,\n330-339 (2016).\n9. Sanders, M.E., Merenstein, D.J., Reid, G., Gibson, G.R. & Rastall, R.A. Probiotics and\nprebiotics in intestinal health and disease: from biology to the clinic. Nature Reviews.\nGastroenterology & Hepatology 16, 605-616 (2019).\n10. DeLeon, O. et al. Microbiome mismatches from microbiota transplants lead to persistent\noff-target metabolic and immunomodulatory effects. Cell 188 (2025).\n4) Elaborate on in vivo degradation and clearance:\nThere is no information on how long the hydrogel remains in the tissue or its degradation rate.\nPlease include a degradation profile or discuss expected in vivo longevity and any potential for\naccumulation or toxicity.\nResponse: We appreciate the reviewer's suggestion regarding hydrogel degradation kinetics\nand potential toxicity accumulation concerns. We have now included comprehensive\ndegradation profiles from both in vitro (Fig. S2 J) and in vivo (Fig. S2 K) studies. In vitro,\ndegradation was assessed in artificial saliva at 37°C, revealing that incorporating ZIF-8 and\nMn-hCaO₂ did not significantly alter the degradation timeline of the hydrogel matrix (SMA),\nwhich exhibited >90% mass loss within 30 days. In vivo, subcutaneous implantation\nexperiments in the back of rat models confirmed complete hydrogel degradation and clearance\nby Day 30, with <5% residual accumulation observed. Mechanistically, hydrogel degradation\noccurs primarily through hydrolysis of the ester bonds introduced via methacrylation of silk\nfibroin. The hydrolysis occurs in physiological pH and temperature conditions, thereby\nexhibiting controlled degradation kinetics that are highly suitable for biomedical applications.\nThe evaluation of potential toxicity was through the H&E staining of kidney, liver, spleen and\ncervical nymph nodes of the rats at 1 month after hydrogel application (Fig. S5E, F). The\nresults showed that no apparent pathological changes were observed, indicating the hydrogel\npossessed good biocompatibility.\nFigure S2 and the corresponding main text was updated in Characterization of Mn-\nhCaO /hZIF8/SMA hydrogel section.\nfor its suitability in tissue regeneration applications.…In vitro, the pure and modified hydrogels\nboth exhibited over 90% degradation within 30 days in artificial saliva at 37 °C, which\nindicated that ZIF-8 and Mn-hCaO₂ incorporation did not have significant effects on the\ndegradation rate (Fig. S2J). In vivo studies confirmed near complete degradation by day 30\n(Fig. S2K). The hydrogel degradation is mediated by hydrolysis of methacrylate-derived ester\nbonds under physiological conditions and aligns with the typical timeline required for new\nbone regeneration.”\n100\n80\n60\n40\n20\n0\n0 5 10 15 20 25 30 35\nTime (d)\n)%(\nnoitagarged\nortiv\nnI\nSMA\nZIF8/SMA\nMn-hCaO /SMA\n100\n)\n%\n(\n80\nn\no\nit\na\n60\nd\na\nr\ng\n40\ne\nd\no\nv\n20\ni\nv\nn\nI\n0\n0 5 10 15 20 25 30\nTime (d)\nFig. S2 (K) In vivo degradation profile of the Mn-hCaO /hZIF8/SMA hydrogel\nLine 800-818\n“In vitro degradation profile of hydrogels\nThe hydrogels were prepared into a cylindrical shape with a height of 20 mm and a diameter\nof 8 mm and freeze-dried. The initial weight (M0) of hydrogel samples was recorded. Then,\nthe hydrogels were soaked into artificial saliva (Yuanye, R30090, China) and the weight (Mi)\nwere recorded at different time intervals. The remaining mass percentage of hydrogel was\ncalculated with the following formula:\nMass remaining (%) = (Mi/M0) × 100% (Equation 4)”\nIn vivo degradation profile of hydrogels\nThe hydrogels were prepared into a cylindrical shape with a height of 5 mm and a diameter of\n15 mm and freeze-dried. The initial weight (M1) of hydrogel samples was recorded. 8-week\nSprague-Dawley were anesthetized and the dorsal fur is shaved and disinfected. An incision of\n18 mm was made and then a subcutaneous pocket is created by blunt dissection. The hydrogels\nwere inserted into the pockets and the incisions were closed with 3-0 suture. The weights of\nthe hydrogels (M2) were recorded at different time intervals. The remaining mass percentage\nof hydrogel was calculated with the following formula:\nMass remaining (%) = (M2/M1) × 100% (Equation 4)”\n5) Statistics and sample size reporting:\nWhile some graphs include statistical annotations, a clearer summary of biological replicates\n(n values), animal numbers per group, and statistical tests used (e.g., ANOVA details) should\nbe included in figure legends and methods.\nResponse: We appreciate your suggestion for statistics reporting. We have now explicitly\nstated the number of biological replicates (n values), animal cohort sizes per experimental\ngroup, and specific statistical tests employed in all relevant figure legends and the Methods\nsection.\nFig. 3 Stem cells senescence attenuation and immunomodulation by Mn-\nhCaO /hZIF8/SMA hydrogel in an inflammatory microenvironment. hPDLSCs and\n2\nmacrophages were treated under the following conditions: NC (standard culture), LPS+NC\n(inflammatory stimulus only), LPS+SMA hydrogel, LPS+hZIF8/SMA hydrogel, LPS+Mn-\nhCaO / SMA hydrogel and LPS+Mn-hCaO /hZIF8/SMA hydrogel. (A) Senescence-associated\n2 2\nβ-galactosidase (SA-β-gal) staining of hPDLSCs from each group. (B, C) Relative mRNA\nexpression of senescence-related genes (P16 and P21) in hPDLSCs. (D, E) Relative mRNA\nexpression of senescence-associated secretory phenotype (SASP)-related genes (IL-1β and\nTNF-α) in hPDLSCs. (F) Proliferative vitality of hPDLSCs across treatment groups measured\non days 1, 3, and 5. (G, H) Relative mRNA expression of osteogenesis-related genes (OCN and\nSP7) in hPDLSCs after 14 days of osteogenic induction. (I) Alkaline phosphatase (ALP)\nstaining of hPDLSCs after 14 days of osteogenic induction. (J, K) Heatmaps showing the\nrelative expression of (J) M1 and (K) M2 macrophage polarization markers in THP-1-derived\nmacrophages across the treatment groups. Data are mean ± SD. N = 3. Significance was\ndetermined by ANOVA with a Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).\nFig. 4 Molecular mechanism of the anti-aging activity of Mn-hCaO /hZIF8/SMA\n2\nhydrogel on hPDLSCs. hPDLSCs, stimulated with LPS, were treated with different hydrogel\ncomponents. The experimental groups are abbreviated by their respective hydrogel names as\nfollows: SMA, hZIF8/SMA, Mn-hCaO /SMA hydrogel and Mn-hCaO /hZIF8/SMA hydrogel.\n2 2\n(A) Venn plot of differentially expressed genes (DEGs) identified in each treatment group\ncompared to the SMA control. (B-D) Volcano plots showing the overall distribution of DEGs\nfor the (B) hZIF8/SMA, (C) Mn-hCaO₂/SMA, and (D) Mn-hCaO₂/hZIF8/SMA groups versus\nthe SMA group. (E) Enriched chord plot based on Gene Ontology (GO) term analysis of the\ntypical DEGs. Red dots: upregulated genes; blue dots: downregulated genes; grey dots: non-\nsignificant genes. (F) Ridge plots showing Gene Set Enrichment Analysis (GSEA) results of\npathways differentially expressed in the Mn-hCaO₂/hZIF8/SMA group compared to the SMA\ngroup. (G) Heatmap of the DEGs across all comparative groups. (H) Schematic illustration\nproposing the molecular mechanism of the hydrogel-cell interaction, integrating the\ntranscriptomic findings. N = 4. Significance was determined by adjusted P < 0.05.\nFig. 5 Therapeutic performance of Mn-hCaO /hZIF8/SMA hydrogel in rat periodontitis.\nperiodontitis control), PD+SMA hydrogel, PD+hZIF8/SMA hydrogel, PD+Mn-hCaO / SMA\n2\nhydrogel and PD+Mn-hCaO2/hZIF8/SMA hydrogel. (A) Schematic illustration of the\nexperimental timeline, including periodontitis induction, hydrogel administration, and\ntherapeutic evaluation. (B) Macroscopic examination of periodontal tissues after 4 weeks of\nhealing. (C, D) Micro-CT analysis of alveolar bone regeneration, quantifying alveolar bone\ncrest height (alveolar bone crest-cementoenamel junction distance, ABC-CEJ distance),\nbone mineral density (BMD), and bone volume/tissue volume ratio (BV/TV). (E) Histological\nevaluation via H&E staining of periodontal tissues, including the root furcation, gingiva and\ninterdental regions. (F) Immunohistochemical (IHC) staining for OCN in the root furcation\nregion. Data are mean ± SD. N = 4. Significance was determined by ANOVA with a Tukey’s\npost hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).\nFig. 6 Mn-hCaO /hZIF8/SMA hydrogel modulates senescent periodontal niche in vivo.\nperiodontitis control), PD+SMA hydrogel, PD+hZIF8/SMA hydrogel, PD+Mn-hCaO / SMA\n2\nhydrogel and PD+Mn-hCaO /hZIF8/SMA hydrogel. (A) Immunofluorescence (IF) staining of\n2\nthe senescence marker P16INK4A in the root furcation area across treatment groups. (B)\nQuantification of P16+ cells. (C) SA-β-Gal staining in the root furcation area. (D) reduced\nglutathione to oxidized glutathione (GSH/GSSG) ratio. (E) IF staining of HIF-1α in the root\nfurcation area. (F) Quantification of HIF1-α+ cells. (G) Immunohistochemical (IHC) staining\nof TNF-α in the root furcation area. (H) Quantification of TNF-α+ cells. (I) IF staining of\nneutrophil marker Ly6G in the root furcation area. (J) Quantification of Ly6G+ cells. Data are\nmean ± SD. N = 5. Significance was determined by ANOVA with a Tukey’s post hoc test (*P\n< 0.05, **P < 0.01, ***P < 0.001).\nFig. 7 The Mn-hCaO /hZIF8/SMA hydrogel modulates on microbiota homeostasis in rat\n2\nperiodontitis. Rats were divided into four experimental groups: NC (healthy control), PD\n(untreated periodontitis control), PD+SMA hydrogel, PD+hZIF8/SMA hydrogel, PD+Mn-\nhCaO / SMA hydrogel and PD+Mn-hCaO /hZIF8/SMA hydrogel. (A) Microbial community\n2 2\nhistogram showing relative abundance at the family level across different treatment groups. (B)\nTernary diagram illustrating differentially abundant taxa among NC, PD, and PD+Mn-\nhCaO₂/hZIF8/SMA groups. (C) LEfSe analysis (LDA score>4, P<0.05) identifying\ndifferentially abundant species between PD and PD+Mn-hCaO₂/hZIF8/SMA groups. (D-G)\nRelative abundance of periodontitis-related pathogens (Fusobacterium and Bacteroides) and\nprobiotics (Lactobacillus and Bacillus) across experimental groups. (H) Proposed mechanism\nof Mn-hCaO₂/hZIF8/SMA hydrogel in periodontitis treatment, illustrating how synergistic\neffects of O₂, polyphenols, and Zn²⁺ promote periodontal regeneration through rejuvenation of\nthe periodontal niche. Data are mean ± SD. N = 3. Significance was determined by ANOVA\nwith a Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).\nHere are my comments on formatting and presentation of the manuscript\n1) Improve figure legends for standalone clarity:\nSome figure legends (e.g., Figures 3 and 5) are densely written and assume familiarity with\nexperimental groups. Make figure captions more self-explanatory by briefly restating treatment\ngroup compositions.\nResponse: We sincerely appreciate this suggestion. We have restructured figure legends and\nadding introductions on treatment group compositions. The revised figure legends are\npresented as the above question.\n2) Supplementary data cross-referencing:\nSeveral references to \"Fig. S1\" or \"Table S1\" lack context. Provide clearer descriptions in the\nmain text when referring to supplementary materials to guide the reader more effectively.\nResponse: We thank you for identifying the insufficient contextualization of supplementary\nmaterial references. We have now revised all mentions of supplementary figures/tables in the\nmain text to explicitly state their analytical purpose.\nLine 159-161 “The spheroid morphology of the Mn-hCaO₂ nanoparticles (NPs), which\npreserved the characteristic shape of the CaO₂ core, was primarily characterized by scanning\nelectron microscopy (SEM) (Fig. S1A, B) and transmission electron microscopy (TEM) (Fig.\n2A, B).”\nLine 185-187 “The hZIF8 was found to form a typical dodecahedral crystal structure similar\nto that of ZIF8 with a shrink size of around 80 nm, as shown in SEM (Fig. S1E, F) and TEM\nimages (Fig. 2I, J).”\nLine 743-744 “The detailed composition and concentrations of each hydrogel were provided\nin Table S1.”\nReviewer #2\nThis study presents an innovative and multidisciplinary approach to addressing the critical\nchallenge of cellular senescence in chronic inflammatory microenvironments, particularly in\nperiodontitis. The authors have developed a polyphenol-mediated zinc-oxygen synergistic\nhydrogel (Mn-hCaO /hZIF8) that integrates advanced biomaterial engineering with nuanced\n2\nbiological insights to achieve multifactorial modulation of the senescent niche. By combining\na cascade oxygenating system with sustained Zn²⁺ delivery, the hydrogel simultaneously\nalleviates hypoxia, corrects micronutrient deficiencies, and mitigates oxidative stress—key\nbarriers to tissue regeneration. The work stands out for its strategic convergence of\nnanomedicine, antioxidant chemistry, and microbiome regulation. The hydrogel’s ability to\nreverse telomere attrition, DNA damage, and microbial dysbiosis while reactivating senescent\nstem cells underscores its translational potential. The authors transcend conventional single-\ntarget anti-senescence strategies by offering a holistic solution that addresses the heterogeneity\nof senescent microenvironments.\nThis study not only advances regenerative medicine but also provides a blueprint for designing\nmultifunctional hydrogel capable of reprogramming complex pathological niches. The novelty\nof the therapeutic platform, and the demonstrated efficacy in rejuvenating periodontal tissues\nmake this work a compelling candidate for publication in Nature Communications after\naddressing the following comments.\nResponse: We sincerely appreciate your encouraging and insightful comments on our work.\nWe have carefully considered all of the comments provided and have performed additional\nexperiments and revisions to address each point in detail. Below, we provide a point-by-point\nresponse to the specific recommendations.\n(1) The study demonstrates that the incorporation of Mn-HCA into CaO₂ significantly enhances\nthe antioxidant properties of nanoparticle. It would be valuable to include additional\nexperimental data for the hydrogels as well, for instance, DPPH radical scavenging assays\nmay be a choice.\nResponse: We sincerely appreciate your insightful suggestion regarding the assessment of\nantioxidant activity. In response to this suggestion, we have conducted additional DPPH radical\nscavenging assays on the hydrogels. The results consistently demonstrated a significantly\nenhanced free-radical scavenging capacity in the Mn-hCaO₂/hZIF8/SMA hydrogel compared\nto control groups, confirming the enhanced antioxidant efficacy mediated by HCA via electron\ntransfer. These findings further supported the proposed mechanism through which the hydrogel\nalleviates oxidative damage.\nFigure S3 and corresponding the main text was updated in Mn-hCaO /hZIF8/SMA hydrogel\n2\nsuppresses stem cell senescence and modulates macrophage polarization in vitro section.\nLine 261-264 “The free radical scavenging ability of Mn-hCaO₂/hZIF-8/SMA hydrogel was\nfurther confirmed using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, which revealed that\nphenolic hydroxyl groups of HCA neutralized radical species through electron transfer (Fig.\nS3 D).”\necnabrosbA\n1\n0\n0\n0\n0\n0\n.\n.\n.\n.\n.\n.\n0\n8\n6\n4\n2\n0\n4 5 0\nW a v\n5e 0l\ne\n0n\ng t h ( n\n5 5m\nD\nS\nh\nM\nM\n0)\nP P H\nM A\nZ IF 8 /S\nn -h C a\nn -h C a\nMO\nO\nA/S\n2/Z\nIF\n8 /S M\n0 0\nA\nFig. S3 (D) UV spectra of the different hydrogels after 120-min reaction with DPPH free\nradicals\nExperimental sections\nLine 840-846\n“DPPH free radical scavenging assay of the hydrogels\nThe free radical scavenging of the hydrogels was tested with a DPPH free radical scavenging\nassay (Solarbio, SD9360, China). The hydrogels were prepared in a cylindrical shape with a\nheight of 9 mm and a diameter of 2 mm.Then, the hydrogel was added to 3 mL DPPH solution\nprepared at a concentration of 0.04 mg/mL with methanol. These samples were then placed in\nan oven at 37°C in the dark for 120 min. Subsequently, the DPPH solution was extracted and\nits absorbance was measured using a UV-Vis spectrophotometer at a wavelength of 516 nm.”\n(2) The injectability of the multifunctional hydrogel demonstrates considerable clinical appeal\nfor periodontal therapy. The swelling property is fundamental for a hydrogel to conformally\nfill anatomically complex periodontal defects. Therefore, it is necessary to provide data on the\nhydrogel’s swelling dynamics in aqua solution (such as PBS or culture medium).\nResponse: We sincerely appreciate your suggestion. As recommended, we have now\nincorporated swelling dynamics data for the hydrogel in PBS at 37 °C. The incorporation of\nZIF8 and Mn-hCaO₂ significantly enhanced the swelling capacity of the SMA hydrogel. The\ncomposite formulation reached an equilibrium swelling ratio of 155% within 10 hours. This\npronounced swelling behavior facilitates conformal adaptation to complex periodontal defect\ngeometries, promoting improved defect filling and retention.\nFigure S2 and corresponding the main text was updated in Characterization of Mn-\nhCaO /hZIF8/SMA hydrogel section.\nfor its suitability in tissue regeneration applications. Incorporating ZIF-8 and Mn–hCaO₂\nincreased the swelling capacity of the SMA hydrogel, exhibiting a 155% equilibrium swelling\nratio within 10 hours. This enables conformal adaptation to complex periodontal defect\ngeometries, promoting defect filling and retention (Fig. S2I).”\n)%(\noitar\ngnillewS\n1\n1\n1\n1\n1\n8\n6\n4\n2\n0\n0\n0\n0\n0\n0\n0 5 1 0 1 5\nT i m\n2\ne\nSZMM\n0\n( h\nM A\nIF 8 /S\nn -h C\nn -h C\n2 5\n)\nMaa AOO\n/S\n2\n/Z 2\n/S M\nExperimental sections\nLine 788-798\n“Swelling behavior of hydrogels\nThe swelling behaviors of the different hydrogels were measured to the evaluate their liquid\nuptake capacity. The hydrogels were prepared with a height of 5 mm and a diameter of 15 mm.\nThen, the hydrogels were soaked in 2mL PBS. At the scheduled time intervals, the samples\nwere taken out and weighed. The mass swelling ratio (SR) was calculated by the following\nequation:\nSR= (W -W )/W ×100% (Equation 3)\n1 0 0\nwhere W is the initial weight of the sample and W is the weight of the sample after PBS\n0 1\nabsorption.”\n(3) The glutathione (GSH) to glutathione disulfide (GSSG) ratio is a well-established and\ncritical marker of cellular redox homeostasis, reflecting the balance between oxidative stress\nand antioxidant capacity within biological systems. Incorporating measurements of the\nGSH/GSSG ratio into the animal experiments would provide more comprehensive assessment\nof the hydrogel’s ability in modulating redox balance in vivo.\nResponse: We thank the reviewer for highlighting the importance of the glutathione redox ratio\n(GSH/GSSG) as a key indicator of cellular redox homeostasis. In response to this suggestion,\nwe have evaluated both the GSH/GSSG ratio and GPX activity following treatment with the\nhydrogel. Consistent with the hydrogel’s free-radical scavenging properties, the hydrogel\nsignificantly increased the GSH/GSSG ratio, demonstrating effective alleviation of oxidative\nstress and enhanced redox balance in vivo.\nFigure 6 and corresponding the main text was updated in Mn-hCaO /hZIF8/SMA hydrogel\n2\nmodulates senescent periodontal niche via regulating local inflammation and hypoxia in\nvivo section.\nLine 447-450 “The redox status in periodontal bone tissue was assessed using the reduced\nglutathione to oxidized glutathione (GSH/GSSG) ratio. Treatment with the Mn-hCaO₂/hZIF-\n8/SMA hydrogel significantly increased the GSH/GSSG ratio, indicating a protective effect\nagainst oxidative injury (Fig. 6D).”\n**\n*\n*\nNormal\nLPS\nLPS+SMA\nLPS+hZIF8/SMA\nLPS+Mn-hCaO2/SMA\nLPS+Mn-hCaO2/hZIF8/SMA\nFig. 6 (D) reduced glutathione to oxidized glutathione (GSH/GSSG) ratio (n=3, ANOVA with\nTukey’s post hoc test, *P < 0.05, **P < 0.01)\nExperimental sections\nLine 940-944\n“GSH/GSSG assay\nFresh alveolar bone tissue surrounding the first molar was collected and grounded in liquid\nnitrogen. Then, GSH/GSSG ratio was measured by a commercial GSH and GSSG Assay Kit\n(Beyotime, S0053, China) based on the enzymatic recycling method with 5,5'-dithiobis(2-\nnitrobenzoic acid) (DTNB).”\n(4) The demonstrated dual efficacy of the hydrogel in suppressing anaerobic pathogens and\nenriching probiotic species represents a significant and encouraging advancement in this field.\nHowever, Porphyromonas gingivalis (P.g.), a well-established pathogen in human periodontitis,\nexhibited notably low abundance in the microbiome sequencing analysis. Considering the\ninherent differences in periodontal microbiota composition between humans and rodents, the\nfindings remain biologically plausible. Nonetheless, to strengthen the translational relevance\nof the study, I recommend that the authors conduct in vitro experiments to directly assess the\nantimicrobial efficacy of the hydrogel against P. g.\nResponse: We thank the reviewer for their positive assessment of the hydrogel’s dual\nfunctionality in modulating the periodontal microbiota and for their constructive suggestion\nregarding P. gingivalis. In response, we conducted additional in vitro antimicrobial assays\nagainst P. gingivalis (ATCC 33277). Treatment with the hydrogel resulted in a 90% reduction\nin bacterial viability, as quantified by colony-forming unit counts after 24 hours. Parallel\nscanning electron microscopy imaging revealed substantial morphological damage, including\ncell shrinkage and membrane rupture. These findings support the antimicrobial efficacy of the\nhydrogel against P. gingivalis.\nFigure S7 and corresponding the main text was updated in Mn-hCaO /hZIF8/SMA hydrogel\n2\nmodulates microbiota homeostasis in the senescent periodontal niche section.\nLine 505-514 “Given the distinct compositional differences between human and rodent\nperiodontal microbiota, we evaluated the hydrogel’s efficacy against Porphyromonas\ngingivalis, a key human periodontopathogen, in vitro. The Mn-hCaO /hZIF8/SMA hydrogel\n2\nreduced bacterial viability by 90% within 24 hours, as measured by colony-forming unit counts\n(Fig. S7 A). Analysis of SEM images revealed extensive cellular damage, including shrinkage\nand membrane rupture, confirming potent antibacterial activity against this pathogen (Fig. S7\nB). These findings verified that the Mn-hCaO /hZIF8/SMA hydrogel was effective in\n2\nmodulating the homeostasis of subgingival microbial communities, thus creating a favorable\nmicroenvironment with restored microbiotic homeostasis and a favorable host-microbe\nrelationship for periodontal regeneration.”\nFig. S7. In vitro anti-microbial property of Mn-hCaO /hZIF8/SMA hydrogel against\nagainst Porphyromonas gingivalis. (B) SEM imaging of bacterial structural deformation. N=\n3, ANOVA with a Tukey’s post hoc test. **P < 0.01, ***P < 0.001.\nExperimental sections\nLine 954-965\n“In vitro Antibacterial Assay\nPorphyromonas gingivalis (ATCC 33277) was routinely cultured under anaerobic conditions\n(85% N₂, 10% H₂, 5% CO₂) at 37°C in brain heart infusion (BHI) broth supplemented with 5\nmg L-1 hemin and 1 mg L-1 vitamin K. The bacteria were suspended with a density 1×108 CFU\nmL−1 and co-cultured with different groups of respective hydrogels (1mm height, diameter in\nline with the 24-well plate). After 24 hours of anaerobic incubation, the bacteria were harvested\nby centrifugation at 6000 g for 10 min and washed twice with PBS. Bacterial viability was\nconfirmed by colony-forming unit (CFU) measured with optical density at 600 nm. For SEM\nimaging, bacterial pellets were fixed in 2.5% glutaraldehyde over night at 4°C. Samples were\nthen dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%, 10 min\neach). Then they were mounted on silicon wafers, sputter-coated with gold, and observed under\na scanning electron microscope.”\n(5) Some minor mistakes in figures should be checked and corrects. E.g., In Fig.7H, ‘jZIF8’\nshould be corrected with ‘hZIF’. In Fig.S1 B and E, the proportion of C should be added to the\ngraph.\nResponse: We sincerely appreciate your meticulous review in identifying these figure\ninaccuracies. The noted errors have been corrected as follows.\nFig.7 (H) Proposed mechanism of Mn-hCaO₂/hZIF8/SMA hydrogel in periodontitis treatment,\nillustrating how synergistic effects of O₂, polyphenols, and Zn²⁺ promote periodontal\nregeneration through rejuvenation of the periodontal niche.\nCOMC a : 1 . 7\n: 4 . 0 9\nn : 0 . 0\n: 9 4 . 0\n8%76 %\n%%\nFig. S1 (B) Element compositions of Mn-hCaO NPs.\nN: 3.38%\nO: 0.49%\nC: 95.41%\nFig. S1 (F) Element compositions of hZIF8 NPs.\nReviewer #3\nThe authors describe an injectable polyphenol-mediated oxygenating hydrogel based on Mn-\nhCaO2, HCA-mediated ZIF8 nanoparticles, and silk fibroin methacrylate for targeted\nmodulation of senescent periodontal microenvironment. By specifically addressing hypoxia\nand zinc ion deficiency, the hydrogel could restore the essential micronutrients and oxygen\nlevels, and also rejuvenate the senescent periodontal microenvironment. The polyphenol-\nmediated hydrogel simultaneously targeted multiple aging-related processes and favored robust\nperiodontal tissue regeneration. However, the following concerns need to be addressed before\nit can be considered for publication.\nResponse: We are grateful for your positive feedback and constructive comments. We have\nrevised the manuscripts by addressing the suggestions as detailed below. We believe these\nrevisions have improved the precision and fluency of our manuscript.\n1. How was the content ratio of HCA, Mn2+, Mn-hCaO2, hZIF8 NPs, and SMA screened and\ndetermined? The authors did not mention the basis for screening.\nResponse: We apologize for lack of clarity in the original manuscript and have included this\nrationalization. The selection of each component was guided by specific functional and\nstructural considerations. The ratio of HCA was adopted based on our previous work\noptimizing polyphenol-based functionalization of hydrogels11. The concentration of Mn²⁺ was\ntuned to maximize the oxygen-release performance of the composite. We found that\nsupplementing 3 mg of MnCl₂ per 5 g of CaCl₂ precursor resulted in optimal oxygen generation\nkinetics. Higher Mn²⁺ levels led to a rapid burst release of oxygen with short duration, whereas\nlower amounts yielded insufficient catalytic activity. The SMA concentration of 15% was\nchosen in accordance with established protocols from prior literature12. The relative proportion\nof hZIF-8 and Mn-hCaO₂ was optimized through mechanical testing. Excessive Mn-hCaO₂\nimpeded the photo-crosslinking process due to light absorption by the dark-colored particles.\nMeanwhile, incorporation of hZIF-8 improved compressive strength through\nnanoreinforcement, although excess amounts adversely affected hydrogel integrity. Thus, the\npresent hydrogel formulation was systematically optimized and rationally designed.\nFigure S1, S2 and corresponding the main text was updated in Characterization of Mn-\nhCaO /hZIF8/SMA hydrogel section.\nperformance of the composite (Fig. S1D).”\n80 5mg\n) 70 10mg\nL\n/g 60 30mg\nm 50mg\n(\nn 5\n6\n0\n0 100mg\ne\ng 40\ny\nx O 30\nd 24\ne 18\nv\nlo 12\ns\ns 6\niD\n0\n0 12 24 36 48 60\nTime (h)\nFig. S1 (D) The oxygen-generating kinetics of Mn-hCaO with different concentration of\nfunctional performance and structural stability. The proportion of hZIF-8 to Mn–hCaO₂ was\nspecifically determined through mechanical testing to enhance compressive strength (Fig. S2A,\nB).”\nFig. S2 (A) Compressive stress of SMA hydrogels with different proportions of Mn-hCaO per\n2\n0.45g SMA. (B) Compressive stress of Mn-hCaO /SMA hydrogels with different proportions\n2\nof hZIF8 per 0.45g SMA.\nReferences\n11. Jiang, Y.A. et al. Bioinspired adhesive and tumor microenvironment responsive\nnanoMOFs assembled 3D-printed scaffold for anti-tumor therapy and bone regeneration. Nano\nToday 39, 101182 (2021).\n12. Wang, B. et al. Biomimetic Microgrooved Methacrylated Silk Fibroin Cartilage Scaffold\nfor Tracheal Injury Repair. Adv. Funct. Mater. (2025).\n2. In Figure 2M and N, the authors adhered the hydrogel to hard tissue (rat femur) and soft\ntissue (rat muscle). As the hydrogel was used in periodontal tissue, isn’t alveolar bone, tooth\ncementum, oral mucosal tissue, and gingival tissue more appropriate?\nResponse: We thank the reviewer for this insightful suggestion. In response, we have\nperformed additional adhesion tests on rat alveolar bone, tooth cementum, tooth enamel and\noral mucosa/gingival tissue. The results demonstrate strong adhesive performance of the\nhydrogel across these dentoalveolar structures, supporting its applicability in periodontal\nsettings.\nFigure 2 and corresponding the main text was updated in Characterization of Mn-\nhCaO2/hZIF8/SMA hydrogel section.\nLine 206-208 “The hydrogel is capable of adhering to both soft and hard oral tissues, including\nalveolar bone, cementum, enamel, and gingiva, demonstrating its suitability for the challenging\noral environment (Fig. 2M).”\nFig.2 (M) The Mn-hCaO2/hZIF8/SMA hydrogel adhering to different soft and hard tissues.\n3. In Figure 2O and P, the compressive stress of the hydrogels was evaluated. But for an\ninjectable hydrogel, comprehensive rheological properties testing is more important. The\nauthors only conducted rheological characterization of the Mn-hCaO /hZIF8/SMA hydrogel\n2\nunder frequency from 0.1-10 Hz, which is not enough. Shear thinning properties should be\ntested especially.\nResponse: We thank the reviewer for highlighting the importance of comprehensive\nrheological characterization for injectable hydrogels. In response to this comment, we have\nperformed additional rheological analyses, including shear-thinning behavior under varying\nshear rates, to more fully capture the hydrogel’s performance under clinically relevant injection\nconditions.\nFigure 2 and corresponding the main text was updated in Characterization of Mn-\nhCaO /hZIF8/SMA hydrogel section.\nthinning behavior with low viscosity, facilitating injection (Fig. 2K). Photo-gelation occurred\nwithin 12 seconds under UV irradiation (395 nm, 10 W) (Fig. 2L).”\n)s·aPm(\nytisocsiV\n2\n1\n1\n0\n5\n0\n5\n0\n0\n0\n0\n0\n0 2 0\nS h\n4e 0a\nr r a\n6t 0e\n( 1 / s\n8) 0 1 0 0\nFig. 2 (K) Shear thinning property of Mn-hCaO₂/hZIF8/SMA hydrogel.\n104\n\"\nG\n,'G\ns\n102\nu\nlu\nd\no 100\nm\ns\ns\no10-2\nl,e\ng G'\na\nr o10-4\nG''\ntS\n10-6\n-10 0 10 20 30 40 50 60\nTime (s)\nFig. 2 (L) UV crosslinking property of Mn-hCaO₂/hZIF8/SMA hydrogel.\nExperimental sections\nLine 753-761\n“Rheology test of hydrogels\nThe dynamic rheological tests of the hydrogels were characterized using a rheometer (MC2302,\nAnton Paar, Austria) at room temperature. The viscosity of the initial solution was measured\nwith changed rotate speed (rad/s). To track the crosslinking process, the storage modulus G'\n(Pa) and loss modulus G\" (Pa) changed with time was measured under 10% strain. Then, the\nhydrogels were prepared in a cylindrical shape with a height of 15 mm and a diameter of 1 mm.\nStrain amplitude sweeps (0.01-100%) were first performed to determine the linear\nviscoelasticity region. Dynamic frequency sweeps were performed at angular velocities\nranging from 0.1 to 10 Hz at 1.0% strain amplitude.”\n4. The adhesion strength of hydrogels to porcine skin was around 15 kPa, “strong tissue\nadhesiveness” might be exaggerated.\nResponse: Thank you for pointing out the exaggerated expression and we apologize for the\nmistake. To ensure precision, we have revised the phrasing to “good tissue adhesiveness\". We\nhave now engaged a professional editing service to thoroughly polish the manuscript,\ncorrecting grammatical issues and enhancing overall clarity. All revisions have been\nhighlighted in the revised manuscript for ease of review. We believe these revisions\nsignificantly improve the readability and precision of the manuscript.\nThe mentioned statements were changed in Characterization of Mn-hCaO /hZIF8/SMA\n2\nhydrogel section.\nLine 205-206 “HCA-modified NPs not only endowed the hydrogel with good tissue\nadhesiveness, but also improved its mechanical properties.”\n5. Inflammatory microenvironment induced by microbial virulence was established by\napplying liposaccharide (LPS) stimuli to hPDLSCs. Why did the authors select LPS rather than\nLPS-PG (LPS from P. gingivalis)? Isn’t it more consistent with the periodontitis condition?\nResponse: We appreciate your insightful inquiry regarding LPS selection. Both standard LPS\nand P. gingivalis-derived LPS-PG are established inflammagens in periodontitis research. The\nPG-LPS can better mimic the inflammatory stimuli as in P.g. dominant periodontitis, while E.\nColi LPS is a broadly-used tool. Previous studies have showed both forms of LPS are similar\nin stimulating local inflammatory responses13. Therefore, the choice of either LPS might not\ninfluence the conclusions of our studies. Further, this approach can facilitate direct\nbenchmarking against established experimental paradigms while revealing the material’s\ntherapeutic versatility across diverse inflammatory pathologies.\nReferences\n13. Liu, R., Desta, T., Raptis, M., Darveau, R.P. & Graves, D.T. P. gingivalis and E. coli\nlipopolysaccharides exhibit different systemic but similar local induction of inflammatory\nmarkers. J. Periodontol. 79, 1241-1247 (2008).\n6. During the rat periodontitis model construction, the hydrogels were irradiated by blue-violet\nlight (395 nm, 10W) for 15-30 seconds. As far as we are concerned, blue-violet light with 395\nnm direct exposure to living tissue is harmful. Is the study design reasonable? Moreover, the\nauthors only applied the hydrogel once and conducted the therapeutic evaluation after 4\nweeks. Why didn’t apply the hydrogel more times like the clinical situation? The effect of a\nsingle dose is definitely limited.\nResponse: We thank the reviewer for raising these important methodological points. Regarding\nlight exposure, 395 nm blue-violet light has been established as safe in multiple biomedical\ncontexts. To directly assess biocompatibility, we exposed human periodontal ligament stem\ncells (hPDLSCs) to 30 seconds of irradiation and evaluated viability via CCK-8 assay over 48\nhours. No significant cytotoxicity was observed, supporting the safety of this photo-\ncrosslinking protocol.\nConcerning treatment frequency, a single application was chosen to align with principles of\nminimal intervention. Repeated injections into periodontal pockets could impede natural\nhealing processes due to physical obstruction and increase patient discomfort, thereby\ncompromising clinical feasibility. The sustained bioactive effects of the hydrogel over four\nweeks further support the rationale for a single-dose regimen.\nFigure S3 and corresponding the main text was updated in Mn-hCaO /hZIF8/SMA\n2\nhydrogel suppressed stem cell senescence and modulated macrophage polarization in\nvitro section.\nLine269-271 “Initially, we confirmed that light exposure did not impair hPDLSC proliferation\nusing a Cell Count Kit-8 assay (Fig. S3E).”\n1.0\n0.8\n0.6\n0.4\n0.2\n0.0\n6h 12h 1d 3d\nmn054DO\necnabrosbA\nns\nControl\nUV\nns\nns\nns\nFig. S3 (E) Cell vitality of hPDLSCs after UV radiation\n7. H&E staining in the root furcation area was shown. Since the hydrogel was injected into the\nperiodontal pocket. Isn’t the periodontal pocket area more rational? It is doubted that the\nhydrogel applied in the periodontal pocket could affect the root furcation area more\nsignificantly.\nResponse: We thank the reviewer for raising this pertinent point regarding the site selection\nfor histological analysis. The periodontal pocket represents the direct application site of the\nhydrogel and more closely mimics horizontal bone loss commonly observed in human\nperiodontitis. However, the choice of the root furcation area in this rat model was based on\nexperimental considerations. The anatomy of the rat first molar, which usually has 4-5 roots,\nintroduces notable variability in interdental bone structure, which may compromise the\nreproducibility of quantitative assessments. In the contrast, the furcation region exhibits\nconsistent and pronounced susceptibility to inflammatory bone resorption in rat periodontitis\nmodel, and its well-defined anatomical boundaries allow for reliable and comparable\nhistological measurements. Therefore, the root furcation region was widely employed in\nperiodontitis studies using SD rats. To provide a comprehensive perspective, we have now\nsupplemented the revised manuscript with H&E staining images of the periodontal pocket area,\nincluding gingival and interdental bone tissues.\nFigure 5 and corresponding the main text was updated in Mn-hCaO /hZIF8/SMA hydrogel\n2\npromotes periodontal regeneration in rat periodontitis section.\nLine399-408 “As shown by hematoxylin-eosin (H&E) (Fig. 5E) and Masson’s trichrome\nstaining (Fig. S5A), in the PD group the periodontitis-affected root furcation area exhibited\nwidened periodontium space, which was filled with disorganized, fibrous connective tissue\ninstead of newly formed bone tissue. The PD group also exhibited destructed gingival papilla\nand decreased interdental alveolar crest height. Alveolar bone volume was greatly improved in\nthe hZIF8/SMA group and Mn-hCaO /SMA group, while the arrangement of periodontal\n2\nligament fibers was still unsatisfactory. The Mn-hCaO /hZIF8/SMA hydrogel efficiently\n2\npromoted new bone formation and maturation in the periodontal lesions and displayed a\nnarrower fibrous layer with aligned periodontal ligament fibers.”\nFig.5 (E) Histological evaluation via H&E staining.\n8. Since the authors have already conducted animal experiments. Evaluation of relative mRNA\nexpression of osteogenesis-related genes in alveolar bone tissue was unnecessary.\nImmunohistochemistry staining or the ELISA tests of the protein should be more meaningful.\nThe same problems are also reflected in Fig. 6.\nResponse: We appreciate your valuable suggestion. We have performed immunohistochemical\nstaining for key protein markers, including TNF-α and OCN (reflecting inflammatory and\nosteogenic responses), as well as P16INK4A (P16), HIF-1α, and Ly6G (reflecting senescence,\nhypoxia, and neutrophil infiltration), to evaluate therapeutic effects at the protein level.\nQuantitative analysis of positively stained cells has been provided to evaluate the efficacy of\nthe hydrogel treatment. Accordingly, we have reorganized Figures 5 and 6 to emphasize these\nprotein-level results. The original mRNA expression data have been moved to the\nSupplementary Materials (Fig. S6) for optional reference.\nFigure 5 and 6 was updated as follows.\nFig. 5 Therapeutic performance of Mn-hCaO /hZIF8/SMA hydrogel in rat periodontitis.\nperiodontitis control), PD+SMA hydrogel, PD+hZIF8/SMA hydrogel, PD+Mn-hCaO / SMA\n2\nhydrogel and PD+Mn-hCaO2/hZIF8/SMA hydrogel. (A) Schematic illustration of the\nexperimental timeline, including periodontitis induction, hydrogel administration, and\ntherapeutic evaluation. (B) Macroscopic examination of periodontal tissues after 4 weeks of\nhealing. (C, D) Micro-CT analysis of alveolar bone regeneration, quantifying alveolar bone\ncrest height (alveolar bone crest-cementoenamel junction distance, ABC-CEJ distance),\nbone mineral density (BMD), and bone volume/tissue volume ratio (BV/TV). (E) Histological\nevaluation via H&E staining of periodontal tissues, including the root furcation, gingiva and\ninterdental regions. (F) Immunohistochemical (IHC) staining for OCN in the root furcation\nregion. Data are mean ± SD. N = 4. Significance was determined by ANOVA with a Tukey’s\npost hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).\nFig. 6 Mn-hCaO /hZIF8/SMA hydrogel modulates senescent periodontal niche in vivo.\nperiodontitis control), PD+SMA hydrogel, PD+hZIF8/SMA hydrogel, PD+Mn-hCaO / SMA\n2\nhydrogel and PD+Mn-hCaO /hZIF8/SMA hydrogel. (A) Immunofluorescence (IF) staining of\n2\nthe senescence marker P16INK4A in the root furcation area across treatment groups. (B)\nQuantification of P16+ cells. (C) SA-β-Gal staining in the root furcation area. (D) reduced\nglutathione to oxidized glutathione (GSH/GSSG) ratio. (E) IF staining of HIF-1α in the root\nfurcation area. (F) Quantification of HIF1-α+ cells. (G) Immunohistochemical (IHC) staining\nof TNF-α in the root furcation area. (H) Quantification of TNF-α+ cells. (I) IF staining of\nneutrophil marker Ly6G in the root furcation area. (J) Quantification of Ly6G+ cells. Data are\nmean ± SD. N = 5. Significance was determined by ANOVA with a Tukey’s post hoc test (*P\n< 0.05, **P < 0.01, ***P < 0.001).\n9. Relative abundance of periodontitis-related pathogens and probiotics was shown in Fig. 7.\nOnly Fusobacterium nucleatum was involved. It is suggested to add the results of other\nperiodontitis-related pathogens, especially P.g. and A.a.\nResponse: We appreciate for this suggestion. We acknowledge that P. g. and A. a. were\nminimally detected in the rat microbiome sequencing data, which likely reflects known\ndifferences in microbial colonization between humans and rodents, particularly the poor\ncolonization capacity of P. g. in rats. To directly evaluate antimicrobial efficacy against this key\npathogen, we conducted additional in vitro assays using P. g (ATCC 33277). Treatment with\nthe hydrogel resulted in a 90% reduction in bacterial viability within 24 hours, as measured by\nCFU counts. Corresponding SEM imaging revealed clear morphological damage, including\ncell shrinkage and membrane rupture, confirming potent antibacterial activity against P.\ngingivalis.\nFigure S7 and corresponding the main text was updated in Mn-hCaO /hZIF8/SMA hydrogel\n2\nmodulates microbiota homeostasis in the senescent periodontal niche section.\nLine 505-514 “Given the distinct compositional differences between human and rodent\nperiodontal microbiota, we evaluated the hydrogel’s efficacy against Porphyromonas\ngingivalis, a key human periodontopathogen, in vitro. The Mn-hCaO /hZIF8/SMA hydrogel\n2\nreduced bacterial viability by 90% within 24 hours, as measured by colony-forming unit counts\n(Fig. S7 A). Analysis of SEM images revealed extensive cellular damage, including shrinkage\nand membrane rupture, confirming potent antibacterial activity against this pathogen (Fig. S7\nB). These findings verified that the Mn-hCaO /hZIF8/SMA hydrogel was effective in\n2\nmodulating the homeostasis of subgingival microbial communities, thus creating a favorable\nmicroenvironment with restored microbiotic homeostasis and a favorable host-microbe\nrelationship for periodontal regeneration.”\nFig. S7. In vitro anti-microbial property of Mn-hCaO /hZIF8/SMA hydrogel against\nagainst Porphyromonas gingivalis. (B) SEM imaging of bacterial structural deformation. N=\n3, ANOVA with a Tukey’s post hoc test. **P < 0.01, ***P < 0.001.\nExperimental sections\nLine 954-965\n“In vitro Antibacterial Assay\nPorphyromonas gingivalis (ATCC 33277) was routinely cultured under anaerobic conditions\n(85% N₂, 10% H₂, 5% CO₂) at 37°C in brain heart infusion (BHI) broth supplemented with 5\nmg L-1 hemin and 1 mg L-1 vitamin K. The bacteria were suspended with a density 1×108 CFU\nmL−1 and co-cultured with different groups of respective hydrogels (1mm height, diameter in\nline with the 24-well plate). After 24 hours of anaerobic incubation, the bacteria were harvested\nby centrifugation at 6000 g for 10 min and washed twice with PBS. Bacterial viability was\nconfirmed by colony-forming unit (CFU) measured with optical density at 600 nm. For SEM\nimaging, bacterial pellets were fixed in 2.5% glutaraldehyde over night at 4°C. Samples were\nthen dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%, 10 min\neach). Then they were mounted on silicon wafers, sputter-coated with gold, and observed under\na scanning electron microscope.”"}, {"round": 2, "reviewer_comments": {"reviewer_2": "After a careful evaluation of the revised manuscript and the response to comments, it is confirmed that the authors have comprehensively addressed all key concerns from the review. The revised manuscript now features a coherent, well-supported narrative with complete experimental data, clear logical connections.\nIn conclusion, the revised manuscript fully addresses initial review comments, so I would recommend acceptance.", "reviewer_3": "The authors have address all the issues that I raised. I have no further question. Therefore, I think the manuscript in the present form is ready to be published."}, "author_response": ""}]

Decision letter