Wet-adhesive metabolic hydrogel for osteoimmune-guided extraction socket healing

Abstract

Dynamic wet interfaces, exemplified by tooth extraction sockets, demand dressings that can maintain firm adhesion despite constant salivary flushing, suction, and mastication while actively guiding tissue regeneration. Conventional hemostatic materials detach easily under these forces, destabilizing clots and impairing healing. Here, we develop a photocurable wet-adhesive metabolic hydrogel (WAM-Gel) that integrates interfacial stability with bioinstructive metabolic signaling. The hydrogel is formed from N-acryloyl glycinamide (NAGA) and acryloyl-6-aminocaproic acid N-hydroxysuccinimide ester (AANHS), which in situ photo-crosslink into a hydrogen-bond-rich, covalently anchored dual network. This architecture provides high compressive strength, considerable burst pressure, and exceptional shear adhesion, thereby ensuring stable sealing under physiologic suction, chewing, and brushing in daily oral activities. Controlled swelling further ensures conformal socket filling and reliable clot stabilization. Beyond adhesion, the hydrogel incorporates β-hydroxybutyrate (BHB) to endow the construct with metabolic immunoregulatory functionality. As a metabolic regulator, BHB orchestrates osteoimmune programming by coupling macrophage phenotype remodeling with enhanced osteogenic differentiation, thereby fostering a pro-regenerative microenvironment. This dual action suppresses inflammatory cytokines, promotes M2 polarization, augments angiogenesis, and accelerates osteogenesis. In vivo, WAM-Gel enhanced early neovascularization, collagen maturation, and bone formation in rat extraction models, and preserved ridge contour with improved trabecular microarchitecture in beagle sockets compared with standard care. Together, this work supports a bioactive sealing strategy that integrates wet stable adhesion and metabolic regulation for osteoimmune guided regeneration at dynamic oral interfaces.

Graphical abstract

Highlights

• •A photocurable wet-adhesive hydrogel enables rapid in situ sealing of extraction sockets under dynamic oral conditions.
• •A hydrogen-bond–rich network with covalent tissue anchoring provides wet-state stability and mechanical resilience.
• •β-hydroxybutyrate (BHB) delivery biases the early osteoimmune niche toward inflammation resolution and regeneration.
• •Rat and beagle models show accelerated healing with improved trabecular microarchitecture and ridge preservation.

1. Introduction

Dynamic wet interfaces represent one of the most persistent obstacles for wound management, as constant fluid flow, fluctuating pressure, and mechanical disturbance undermine barrier stability and compromise regeneration [1,2]. A representative example is the tooth extraction socket, where unstable sealing exposes the wound to saliva and oral microbiota and mechanical forces, disrupts the blood clot that serves as the initial scaffold for healing [3,4].

Loss of this clot predisposes patients to infection, alveolar osteitis (dry socket), persistent pain, and delayed bone regeneration, which severely limit subsequent implant placement and functional recovery [5,6]. Current approaches, including cotton balls, absorbable gelatin sponges, provide only transient hemostasis and rapidly detach under intraoral stresses [7]. Even suturing, which demands advanced surgical skill, extends operation time, may cause additional tissue injury, and often entails a follow-up visit for suture removal [8]. Beyond clinical shortcomings, these materials fail to establish a protective barrier that is mechanically robust, durably adhesive under wet conditions, and biologically active. As a result, they cannot orchestrate the complex interplay of inflammation resolution, angiogenesis, and osteogenesis required for predictable socket regeneration [6]. Overcoming this long-standing barrier requires materials that integrate robust wet adhesion with the ability to program the osteoimmune microenvironment, thereby enabling both reliable physical sealing and active guidance of socket regeneration [9,10].

Hydrogels are widely regarded as promising candidates for post-extraction wound management due to their biocompatibility, favorable shear-thinning behavior and ease of in situ application [9,11]. Their injectability and conformability allow adaptation to irregular socket geometries, while their soft, tissue-like properties minimize secondary trauma [6]. Yet their clinical adoption has been restricted because of three interlinked gaps that are magnified at dynamic wet interfaces. First, their mechanical strength is insufficient: most conventional hydrogels exhibit compressive moduli below 10 kPa, making them unable to withstand the physiological stresses generated by intraoral suction, mastication, and tooth brushing [12,13]. As a result, this mismatch often leads to irreversible deformation or collapse within the first 24-72 h after surgery, disrupting early wound stability and impairing the mechanobiological cues required for bone regeneration [14]. Second, their wet adhesion is weak and unstable: interfacial toughness on hydrated oral tissues is typically below 10 J/m², and excessive swelling further disrupts the tissue-gel interface. Under intraoral negative pressure fluctuations (ranging from −20 to −300 mmHg), such weak and unstable adhesion readily de-bond, often within hours, resulting in loss of barrier function and failure to maintain localized therapeutic delivery [2]. Third, most clinical hydrogel dressings are inherently bioinert, and they act only as passive physical barriers but lack the ability to modulate the immune microenvironment or promote bone formation.

Yet predictable socket healing requires precise coordination of inflammation resolution, angiogenesis, and osteogenesis. Increasing evidence indicates that these regenerative processes are tightly regulated by immunometabolic crosstalk, where metabolites function as signaling mediators that reprogram immune responses and influence stem cell fate. Emerging evidence underscores that bone regeneration is not merely an immunomodulatory or osteogenic process but an immunometabolic event. The transition from inflammation to repair is governed by profound metabolic reprogramming of immune and skeletal cells, where shifts in their energy metabolism dictate functional phenotypes and cell fate decisions [15]. Within this paradigm, metabolites function as potent signaling molecules that couple immune responses with anabolic processes, thereby orchestrating the pace and quality of tissue regeneration [16,17]. Among them, β-hydroxybutyrate (BHB), also known as 3-hydroxybutyrate (3HB), has emerged as a particularly attractive candidate since, as an endogenous ketone body, it suppresses NLRP3 inflammasome activation, promotes M2 macrophage polarization, and enhances osteogenic differentiation of mesenchymal stem cells [18,19]. However, the inability of current hydrogel dressings to integrate such metabolic regulation represents a key limitation, leaving them unable to orchestrate the essential osteoimmune crosstalk for regeneration. Together, these limitations, including inadequate mechanical strength, poor wet adhesion, and lack of metabolic bioactivity, synergistically prevent hydrogels from exerting spatiotemporal control over healing and limit their capacity to restore alveolar ridge function [20].

To address these interconnected challenges, we engineered a photo-crosslinkable hydrogel that not only achieves robust wet adhesion and mechanical resilience but also serves as a sustained delivery platform for BHB, leveraging its immunometabolic role to actively guide the healing process. (Fig. 1). Specifically, we developed a photo-crosslinkable hydrogel composed of NAGA and AANHS. This architecture is engineered to withstand intraoral negative pressures and mechanical agitation, while maintaining conformal sealing with controlled swelling. This performance is attributed to the robust hydrogen bonding of NAGA after curing. The succinimide group present in AANHS enables strong adhesion to oral tissues, endowing the hydrogel with excellent bonding properties. Unlike conventional skin-applied bioadhesives, this combination of strong adhesion and mechanical resilience is particularly suited to the challenging intraoral environment, which is characterized by frequent movement, high negative pressure, and a moist environment [21]. To move beyond passive physical barrier, we incorporated BHB as an immunometabolic cue, converting the hydrogel into an active scaffold capable of synchronizing inflammation resolution, angiogenesis, and osteogenesis. We hypothesized that sustained BHB release would downregulate excessive inflammation, promote pro-healing macrophage polarization, stimulate vascular ingrowth, and accelerate osteogenic differentiation. In this study, we systematically investigated the physicochemical properties, mechanical performance, and wet adhesion of this hydrogel, as well as its immunoregulatory and osteogenic effects in vitro and in vivo. Finally, we validated its efficacy in both rat and beagle extraction models, demonstrating its potential as a multifunctional scaffold paradigm for guiding tissue regeneration across dynamic wet interfaces.

Fig. 1.

Schematic illustration of the design, application and biological functions of WAM-Gel for Tooth extraction socket healing.

2. Results & discussion

2.1. Design and mechanism of WAM-Gel

The rationally design of WAM-Gel was guided by the clinical requirements of tooth extraction sockets, where dynamic wet conditions demand stable sealing and coordinated tissue regeneration [6]. To fulfill these demands, we developed an in-situ photo-crosslink WAM-Gel with a hierarchical design logic, linking molecular interactions, macroscopic stability, and metabolic immunoregulation.

At the molecular level, two complementary monomers were selected, with NAGA forming a reversible hydrogen-bonded network that provides toughness and elasticity, and AANHS forming covalent bonds with tissue-surface amines to ensure durable anchoring in wet and dynamic environments. Upon UV irradiation, these components rapidly polymerize to yield a dual-crosslinked architecture that integrates reversible energy dissipating bonds with permanent covalent anchors. At the macroscopic level, this dual-network architecture translates into robust mechanical performance and reliable sealing. The hydrogel precursor is injectable, readily adapts to irregular socket geometries and gels within minutes. The resulting hydrogel withstands several fold higher negative pressures than those generated during oral suction, resists dislodgement under mastication and brushing, and remains structurally stable for up to five days in a salivary environment [2]. These properties ensure conformal sealing of the extraction socket and effective stabilization of the blood clot, a prerequisite for orderly healing [22].

To extend beyond physical stabilization, the hydrogel was further integrated with BHB, an endogenous metabolic mediator with immunomodulatory and osteogenic potential [23]. Sustained BHB release reprograms the osteoimmune microenvironment by suppressing inflammasome-driven inflammation, promoting M2 macrophage polarization, enhancing angiogenesis, and accelerating osteogenic differentiation [24]. Through this integration, WAM-Gel is transformed from a passive barrier into a bioactive scaffold capable of programming the osteoimmune microenvironment.

In summary, the design of WAM Gel establishes a direct link from molecular interactions (NAGA/AANHS) to macroscopic stability (wet adhesion and mechanical resilience) and extends to functional immunoregulation (BHB mediated macrophage polarization and osteogenesis). This hierarchical design enables predictable socket sealing and provides a generalizable strategy for regeneration across dynamic wet interfaces (Fig. 1).

The design of WAM-Gel represents a strategic advance from passive biomaterial barriers to actively orchestrated regenerative platforms. While conventional socket management relies on mechanically inadequate materials or single factor biological delivery, our methodology simultaneously overcomes the physical and biological challenges of wound healing. The synergistic combination of covalent tissue anchoring and energy dissipating hydrogen bonds creates a dual-network architecture that demonstrates superior wet-adhesion stability compared to clinical standards like gelatin sponges and fibrin glues, while its sustained release profile addresses the critical limitation of burst-release drug delivery systems [25,26].

More significantly, the incorporation of BHB positions WAM-Gel at the forefront of immunometabolic reprogramming strategies. By employing an endogenous metabolite to concurrently resolve inflammation and promote anabolic processes, this system circumvents the pleiotropic risks and cost inefficiencies associated with recombinant growth factors or synthetic drugs [24,27]. This metabolic therapy approach via engineered biomaterials offers a novel strategy for modulating the hostile microenvironments characteristic of chronic and complex wounds [23,27].

Beyond tooth extraction sockets, the fundamental design principle established here, integrating robust wet-tissue adhesion with targeted metabolic signaling, presents a generalizable platform for regenerative medicine. This strategy shows immediate translational potential for managing diabetic ulcers, sealing gastrointestinal defects, and enhancing the integration of implantable devices, where reliable interfacial stability in dynamic fluid environments and precise immunomodulation are equally critical for successful outcomes.

2.2. Network formation and properties of WA-Gel

To evaluate whether the molecular design of WAM-Gel translates into rapid in situ curing and a stable dual-network suited for immediate socket sealing, the physicochemical and rheological properties of WA-Gel (WAM-Gel without BHB) were investigated (Fig. 2). A schematic synthesis route is shown in Fig. 2A, which illustrated that NAGA contributes a reversible hydrogen-bonded network for energy dissipation, while AANHS participates polymerization and provides covalent anchoring to tissue amines. Rheological analysis revealed that a fast gelation upon UV exposure, with the sol-gel transition occurring at approximately 20 s and complete curing within 2 min, as evidenced by the crossover and continued rise of the storage modulus (G′, solid curve) over the loss modulus (G″, hollow curve) (Fig. 2B). Frequency sweep measurements further confirmed the stable solid like behavior across a wide range of frequencies (G′>G″), supporting intraoperative, time efficient handling and early mechanical stabilization (Fig. 2C).

Fig. 2.

Physicochemical properties of WA-Gel. (A) Schematic illustration of the synthesis and physicochemical characterization of WA-Gel. (B) Real-time modulus curves during gelation under UV light. (C) Rheological frequency sweep curves of hydrogels. (D) Swelling curves of hydrogels (n = 3). (E) Tensile strength. (F) Tensile modulus. (G) Elongation. (H) Compressive strength. (I) Compressive modulus. (J) Compressive strength curves. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

We next examined swelling as a proxy for conformal sealing and interface stability under wet conditions (Fig. 2D). Increasing AANHS content led to higher equilibrium swelling. The formulation containing 2% AANHS exhibited a moderate swelling ratio (≈25%), which facilitates intimate adaptation to irregular socket walls without undermining adhesion or clot integrity [28,29], whereas 5% AANHS caused excessive swelling that is unfavorable for extraction site applications [30,31]. These results identify AANHS content as a controllable molecular parameter governing the balance between conformability and interfacial stability.

Uniaxial tensile tests were used to assess resistance to traction and displacement relevant to oral motion. With increasing AANHS content, tensile strength and tensile modulus decreased (Fig. 2E–F), while the elongation at break increased (Fig. 2G), indicating enhanced ductility at the expense of stiffness. Similarly, under compression, both compressive strength and modulus declined with higher AANHS ratio (Fig. 2H–I). The full-range compressive strain-stress curves (Fig. 2J) indicated that the 2% AANHS hydrogel achieved a well-balanced mechanical performance, combining sufficient deformability to accommodate physiological perturbations while maintaining resistance to intraoral stresses, which consistent with requirements during the critical 24-72 h stabilization window.

Collectively, these results indicate that AANHS content co-modulates interfacial anchoring strength and bulk mechanical integrity. While increasing covalent sites may strengthen potential adhesion, excessive AANHS appears to introduce network heterogeneity and swelling-driven internal stresses that diminish cohesive strength. In practice, the 2% AANHS formulation provided the most clinically relevant window by coupling moderate swelling with adequate toughness and manageable stiffness, and by sustaining structural and adhesive stability in saliva mimicking conditions for multiple days [9,32]. This calibrated mapping from molecular properties to macroscopic behavior supports subsequent biological evaluation and underscores a general design principle for dynamic wet interfaces, namely optimizing rather than maximizing crosslinker density to jointly satisfy adhesion, mechanical resilience, and swelling control.

2.3. Adhesion properties of WA-Gel

To determine whether the molecular design translates into stable tissue sealing, we quantified adhesion across formulations with varied AANHS content (Fig. 3). On porcine skin, the shear adhesion strength increased significantly with higher AANHS concentration, demonstrating the role of covalent anchoring in strengthening the interface (Fig. 3A). To simulate the challenging wet oral environment, samples were immersed underwater for 24 h before testing. Notably, the 2% AANHS formulation retained a shear strength exceeding 100 kPa with minimal loss, while 0% and 5% AANHS hydrogels showed a dramatic decline in adhesion strength (<50 kPa) (Fig. 3B). This non-monotonic trend is consistent with our design map: insufficient AANHS yields too few anchors, while excessive AANHS increases swelling/heterogeneity that disrupts intimate contact at the interface.

Fig. 3.

Adhesion properties of WA-Gel. (A) Shear strength of WA-Gel to pig isolated epidermis, n = 6. (B) Shear strength after 24-h immersion in water, demonstrating the retention of adhesion under wet conditions, n = 4. (C) Burst pressure measurement of the WA-Gel on porcine intestine, compared to physiological intraoral negative pressure and blood pressure thresholds, n = 4. (D) Long-term shear adhesion stability of the 2% AANHS hydrogel over 28 days, n = 6. (E) Images showing the hydrogel adhered to porcine skin under mechanical challenges: stretching, bending, and twisting. (F) Adhesion persistence after underwater rinsing followed by mechanical stress. (G) Shear and tensile adhesion strength of the hydrogel on gingival tissue, n = 5. (H) Comparison of adhesive performance with other reported wet-adhesive hydrogels [[33], [34], [35], [36], [37], [38], [39], [40], [41]]. (I, J) Adhesion test in a clinically relevant model: hydrogel applied to extracted porcine mandibular socket and subjected to tensile testing (I), with quantitative results shown in (J), n = 4. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Sealing capacity was further assessed by burst pressure measurements on porcine small intestine (Fig. 3C). the 2% AANHS hydrogel withstood pressures that exceed reported intraoral negative pressure maxima and typical blood pressures [12,42,43], establishing a practical safety margin for events such as coughing, sneezing, or vigorous suction. Beyond peak values, the hydrogel preserved adhesion integrity throughout mechanical perturbations that mimic oral activity, supporting its suitability for dynamic wet interfaces.

Long-term adhesion stability was assessed over 28 days. The shear strength of the 2% AANHS hydrogel slightly increased during days 1-3 and maintained ≥200 kPa thereafter, demonstrating durable bonding under storage conditions (Fig. 3D). We next visualized robustness via sequential challenges on porcine skin. The hydrogel was firmly attached during stretching, bending, and twisting (Fig. 3E), and retained strong adhesion even after underwater rinsing for 1 min followed by the same mechanical tests (Fig. 3F). Quantitative adhesion tests on gingival tissue yielded consistently high shear and tensile strength (Fig. 3G), supporting translational relevance to periodontal settings.

Comparative analysis with representative wet-adhesive hydrogels from the literature shows that the 2% AANHS hydrogel occupies the upper range of adhesion with soft tissue while simultaneously offering underwater retention and long-term stability (Fig. 3H) [[33], [34], [35], [36], [37], [38], [39], [40], [41]]. Finally, an in vitro socket model was used to simulate clinical handling, the precursor was injected into freshly extracted porcine mandibular sockets and photo-cured in situ, after which tensile testing confirmed strong adhesion and effective adaptation to the irregular socket anatomy (Fig. 3I–J).

Together, these data demonstrate that the dual anchoring interface, including rapid physical interactions reinforced by covalent bonding, confers high, water resistant, and durable adhesion. The 2% AANHS window provides the best balance between anchoring density and cohesive integrity, enabling reliable sealing under hydrated, mechanically perturbed conditions that are characteristic of post-extraction wounds.

2.4. From WA-Gel to WAM-Gel: Loading of BHB with desirable biological effects

To complement mechanical sealing with biological programming, we evaluated whether BHB, an endogenous ketone metabolite with reported anti-inflammatory and pro-osteogenic activities, which holds promising potential for modulating the inflammatory microenvironment of a healing tooth socket.

We first sought to delineate its impact at the transcriptomic level in human gingival fibroblasts (HGFs) challenged with Porphyromonas gingivalis (p.g.). Principal component analysis (PCA) of RNA-seq data separated the four groups, indicating distinct transcriptional states under BHB and/or p.g. treatment (Fig. 4A). The differential expression analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment suggested that BHB pretreatment attenuated pathways related to inflammation and apoptosis, while promoting those associated with extracellular matrix organization, cell cycle progression, and tissue regeneration (Fig. 4B–D, Fig. S10-11). These results provide mechanistic evidence that BHB exerts a dual action via mitigating inflammatory damage and activating intrinsic repair programs, which consistent with the needs of early socket healing.

Fig. 4.

Biological effect of the BHB and WAM-Gel. (A-D) RNA-Seq analysis of HGF. (A) PCA plot illustrating sample clusters of blank, BHB, p.g., p.g.-BHB group. (B) Top 15 of GO enrichment analysis of p.g.-BHB vs P.g. group. (C) Top 15 of GO Biological Process (BP) enrichment analysis of p.g.-BHB vs P.g. group. (D) Top 20 of KEGG enrichment analysis of p.g.-BHB vs p.g. (E) Representative ALP staining of mineralization induction BMSCs at day 7. Scale bar = 200 μm. (F) Flow cytometry of macrophage polarization in RAW264.7 cells after stimulation for 48 h. (G, H) Percentage of (G) CD86⁺CD206⁻cells and (H) CD206⁺CD86⁻cells in different treatments. n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Guided by these findings, BHB was incorporated into the optimized WA-Gel (2% AANHS) to obtain the bioactive WAM-Gel. HPLC confirmed successful loading, with characteristic BHB peaks detected in WAM-Gel extracts, and a calibration curve enabled quantification of loading and release (Fig. S8A–B). In vitro release in PBS (pH 7.4, 37 °C) displayed an initial burst over the first 6 h followed by sustained release to 24 h (Fig. S9). This profile is aligned with the temporal demands of post-extraction healing, the initial burst release provides immediate immunomodulatory signaling during the acute inflammatory phase, while the sustained release maintains local exposure as the wound transitions toward proliferation and matrix deposition. Thus, this release kinetics mirrors the natural progression of wound healing, positioning WAM-Gel as a temporally intelligent delivery system rather than a passive carrier [44].

Functionally, the combination of stable wet adhesion and time staged BHB exposure is designed to secure the blood clot and, in parallel, bias the osteoimmune milieu toward regeneration [45]. In the context of known BHB activities, including suppression of NLRP3 inflammasome signaling, promotion of M2 macrophage polarization, and support of osteogenic differentiation [18,24,27], the observed transcriptomic changes in HGFs provide mechanistic plausibility for elevating BHB from a mere anti-inflammatory agent to a master regulator of the wound healing transcriptome (Fig. 4A–D, Fig. S10-S11). Its ability to simultaneously suppress destructive inflammatory pathways while activating regenerative programs in human gingival fibroblasts represents a paradigm distinct from conventional single target therapeutic approaches [46,47]. This dual action mechanism is particularly valuable in the complex socket microenvironment, where unchecked inflammation and inadequate tissue formation often coexist and mutually reinforce healing impairment [48,49]. Beyond its bioactivity, employing the endogenous metabolite BHB as the therapeutic agent confers distinct practical benefits relative to recombinant growth factors such as BMP-2 or FGF, or to synthetic drugs. As a naturally occurring small molecule, BHB exhibits a favorable safety profile characterized by low immunogenicity and minimal toxicity, thereby circumventing potential complications arising from the administration of foreign proteins. Furthermore, its high chemical stability supports long-term storage and simplifies material handling and distribution key considerations for clinical translation. From a manufacturing standpoint, BHB can be produced cost-effectively at scale, in contrast to the complex and expensive processes required for recombinant proteins. Collectively, these attributes superior safety, stability, and cost-effectiveness establish BHB as a translationally viable candidate that simultaneously mitigates risks associated with supraphysiological dosing of exogenous proteins [50,51].

Taken together, the mechanistic evidence and the delivery performance form a coherent design to function link, that is BHB engages gene programs relevant to dampening inflammation and supporting repair in gingival stromal cells, and the WAM-Gel delivers BHB locally with an early to sustained profile suited to dynamic wet tissues. This integration of transcriptomic validation with controlled local delivery establishes a modular framework for metabolic cue-enabled hydrogels and sets the stage for downstream evaluation of macrophage polarization, angiogenesis, and osteogenesis in vitro and in vivo.

2.5. Biocompatibility of WAM-Gel

To support clinical translation, we first established a tiered biosafety profile aligned with the gel's intended intraoral use and time scale. Hemocompatibility analysis confirmed negligible hemolysis, well below the 5% acceptance threshold (Fig. S12), indicating a low risk of interfering early coagulation in the fresh extraction socket [52]. In vitro cytocompatibility was then assessed using BMSCs and HGFs. CCK-8 assays revealed that the WAM-Gel extracts had no inhibition for cell proliferation in both BMSCs and HGFs cultures exposed to the gel extracts (Fig. S13). Further, live/dead staining (Calcein-AM/PI) showed predominantly viable cells (green fluorescence) with minimal cell death (red fluorescence) (Fig. S14). These results confirmed that WA-Gel and its extracts do not impose cytotoxic burden on stromal or soft-tissue cells, thereby preserving the cellular substrate required for downstream regeneration and ensuring that the therapeutic effects of BHB are not undermined by material-related toxicity [53].

In vivo biocompatibility was evaluated by subcutaneous implantation of photo-cured WAM-Gel in SD rats. After 7 days, the peri-implant tissue displayed no abnormal reaction, and the histology of major organs (heart, liver, spleen, lungs, and kidneys) showed no pathological changes (Fig. S15). The absence of local irritation and distal organ findings supports the systemic safety of the cured network, including the initiator system and early degradation products [54]. Collectively, these data provide convergent evidence that WAM-Gel satisfies the key biocompatibility constraints for an intraoral sealant that simultaneously releases an active metabolic cue, justifying progression to functional efficacy studies.

2.6. In vitro osteoimmune modulation of WAM-Gel

We next tested the central functional premise of WAM-Gel, which is whether stable wet sealing can be paired with a metabolic cue to bias the socket microenvironment toward regeneration. Under osteogenic induction, both free BHB and BHB-loading WAM-Gel enhanced early osteogenesis of BMSCs, evidenced by intensified ALP staining compared to controls (Fig. 4E). Preservation of this effect after incorporation confirmed that the fabrication process and matrix do not compromise mineralization promoting capacity of BHB and that the release achieved in vitro is sufficient to trigger early differentiation.

In parallel, we quantified immunomodulation using RAW264.7 macrophages. Flow cytometry analysis demonstrated a marked shift toward an M2-like phenotype after treatment with BHB or WAM-Gel, with reduced expression of the M1 marker CD86 and a concurrent upregulation of the M2 marker CD206 (Fig. 4F). Quantitative results further confirmed that both BHB and WAM-Gel treatment groups exhibited a higher ratio of CD206⁺ cells and a lower ratio of CD86⁺ cells relative to the control (Fig. 4G–H), indicating effective polarization toward a pro-healing state known to support inflammation resolution, angiogenesis, and osteogenesis [55,56]. The fact that WAM-Gel recapitulates the effect of free BHB indicates not only successful delivery but therapeutically relevant exposure to immune cells, consistent with the 0-24 h release profile (Fig. S9), and the early inflammatory window of socket healing.

Collectively, these findings demonstrate that WAM-Gel possesses dual functionality, simultaneously promoting bone formation and directing the macrophage polarization, consistent with a coupled osteoimmune mechanism rather than isolated endpoints [57]. In this framework, BHB-driven M2 macrophage polarization is expected to lower inflammatory noise and enhance vascular/osteogenic cues, creating a favorable niche in which stem cell-mediated bone formation proceeds more efficiently [58]. The in vitro data thus provide a mechanistic bridge between the hydrogel's time-staged release and the coordinated biological processes required for regeneration, motivating subsequent in vivo evaluation of socket sealing, inflammation resolution, and bone microarchitecture.

2.7. WAM-Gel accelerates alveolar bone healing in rat extraction model

We tested whether wet-stable sealing together with a metabolic cue improves socket repair in vivo. Maxillary first molars were extracted and sockets received Blank (untreated socket), Gelatin Sponge, or WAM-Gel (Fig. 5A–B). Histological analysis via Hematoxylin and Eosin (H&E) staining showed more organized granulation and fewer necrotic areas in the WAM-Gel group at days 3 and 7. By day 21, the mucosal layer was more continuous than in both controls (Fig. 5C). Masson's trichrome staining further demonstrated denser collagen deposition and osteoid matrix formation across the healing period, indicating faster matrix maturation (Fig. 5D). These patterns are consistent with an earlier transition from the inflammation to repair and are in line with known immunomodulatory and angiogenic potential of BHB [59].

Fig. 5.

In vivo evaluation of alveolar bone regeneration in rat M1 tooth extraction model treated with WAM-Gel. (A) Schematic of the experimental timeline and surgical procedure of maxillary first molar extraction. (B) Representative image showing the application of WAM-Gel into a fresh extraction socket. (C) H&E staining and (D) Masson staining of healing sockets at days 3, 7, and 21 post-operations. Scale bar = 200 μm. (E) Representative micro-CT image of a longitudinal section through the extraction socket axis. Scale bar = 1 mm. (F) Representative 3D reconstructed micro-CT image of the healed alveolar ridge. Scale bar = 1 mm. (G-K) Quantitative micro-CT analysis of bone regeneration parameters: (G) bone volume fraction (BV/TV), (H) bone surface density (BS/TV), (I) trabecular thickness (Tb.Th), (J) trabecular number (Tb.N), and (K) trabecular separation (Tb.Sp). n = 4, ∗p < 0.05, ∗∗p < 0.01.

Micro-CT analysis at day 21 supported the histological improvements. Three-dimensional (3D) reconstructions showed greater mineralized tissue formation and better preservation of the alveolar ridge morphology with WAM-Gel (Fig. 5E and F). Quantitative morphometric analysis confirmed a higher bone volume fraction (BV/TV) compared to Blank and Gelatin Sponge groups (Fig. 5G). Furthermore, the bone surface density (BS/TV) and trabecular number (Tb.N) were increased, while trabecular separation (Tb.Sp) was decreased (Fig. 5H–K), yielding a trabecular network that is denser and more interconnected. And these features associated with improved biomechanical competence. Trabecular thickness (Tb.Th) showed an upward trend without reaching statistical significance (Fig. 5I). The resulting trabecular network was denser and more interconnected, a feature associated with improved biomechanical competence [60]. Relative to natural healing and a standard clinical product, WAM-Gel orchestrated a stronger healing response [61,62]. Together, these data indicate that WAM-Gel improves both the rate and the quality of socket bone regeneration, moving beyond volume fill toward structurally meaningful restoration.

2.8. In vivo immunomodulation and osteogenesis with WAM-Gel

To mechanistically link the regenerative outcomes and local biological response, we performed a temporal immunohistochemical (IHC) analysis of the socket microenvironment, tracking key markers of inflammation, vascularization, and osteogenesis within the healing socket at days 3, 7, and 21 post-operations. IHC staining for TNF-α and CD68 showed reduced inflammatory response and macrophage burden in WAM-Gel-treated sockets at all time points compared with both controls (Fig. 6A–B, E-F). These findings are consistent with early in vitro observations and with BHB-associated promotion of M2 polarization and inhibition of NLRP3 inflammasome activaty [19,63]. In parallel, CD31 staining revealed an enlarged endothelial area as early as day 3 that remained elevated versus Blank at days 7 and 21 (Fig. 6C–G), suggesting that timely angiogenesis that supports oxygen and nutrients delivery to the hypoxic wound site. The temporal coupling with lower inflammation and enhanced angiogenesis agrees with a permissive niche for anabolic processes [64].

Fig. 6.

Immunohistochemical analysis of socket healing mechanisms modulated by WAM-Gel. (A-D) IHC staining of (A)TNFα, (B) CD68, (C) CD31, (D) COL1A1 at day 3, 7 and 21. Scale bar = 50 μm. (E-H) Quantitative analysis of IHC staining: (E) TNF-α positive area, (F) CD68 positive cells, (G) CD31 high positive area, and (H) Means of COL1A1⁺ in socket. n = 4, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Crucially, this low-inflammation and enhanced angiogenesis state coincided with an early rise in osteogenic activity [65,66]. COL1A1 staining increased markedly in the WAM-Gel-treated sockets beginning day 3 (Fig. 6D–H), implying prompt initiation of collagenous matrix synthesis. These findings demonstrate that the temporal alignment of reduced inflammation, increased vessel formation, and activated matrix programs supports a coordinated mechanism, in which WAM-Gel's wet-stable seal secures the clot while time-staged BHB exposure biases macrophages toward pro-healing phenotypes, permits vessel ingrowth, and unlocks osteogenic activities. This coordination shortens the time to the reparative and remodeling phases and aligns with the micro-CT gains observed in Fig. 5. Rather than acting through a single pathway, WAM-Gel does not merely passively allow healing to occur but active the anabolic phase by synchronously resolving inflammation and angiogenic potential, thereby unlocking the innate regenerative capacity of the tissue to compress the healing timeline and improve tissue quality [57,67].

2.9. WAM-Gel accelerates alveolar bone healing in beagle extraction model

To evaluate translational performance in a large animal model, we tested WAM-Gel in beagle dogs, whose oral anatomy and healing kinetics approximate human conditions (Fig. 7A–B) [68]. After 28 days, Micro-CT results, including representative sagittal/coronal/axial views and full-socket reconstructions, showed that more substantial mineralized tissue formation and superior maintenance of the original ridge contour with WAM-Gel compared with Blank and Gelatin Sponge groups (Fig. 7C). Quantitative morphometric analysis confirmed the WAM-Gel group exhibited a higher BV/TV compared to both the Blank and Gelatin Sponge groups (Fig. 7D). Furthermore, BS/TV, Tb.Th, and Tb.N were all greater than Blank, while Tb.Sp was reduced (Fig. 7E–H), indicating the trabecular network was denser, thicker, and more connected. These features that indicate a more mature regenerative state and support ridge preservation as a clinical endpoint that affects implant feasibility [69]. These results demonstrate that WAM-Gel significantly promotes extraction socket healing and alveolar ridge preservation in a clinically relevant large animal model.

Fig. 7.

Treatment effect of WAM-Gel promotes socket healing in Beagle P1 tooth extraction model. (A) Schematic diagram of tooth-extraction and treatment in beagles. (B) Representative image showing the application of WAM-Gel into a beagle fresh extraction socket. (C) Representative micro-CT images showing three orthogonal views (sagittal, coronal, and axial) of longitudinal sections through the extraction socket axis. Scale bar: 10 mm. (D-H) Quantitative micro-CT analysis of bone regeneration: (D) bone volume fraction (BV/TV), (E) bone surface density (BS/TV), (F) trabecular thickness (Tb.Th), (G) trabecular number (Tb.N), and (H) trabecular separation (Tb.Sp). Blank group n = 6, Gs and WAM-Gel group n = 8, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

The concordance between rat and beagle outcomes strengthens confidence in the mechanism and in scalability under clinically relevant mechanical and microbial challenges. By uniting wet-stable adhesion with metabolic immunoregulation, WAM-Gel consistently preserved ridge form and improved bone formation across species. These results de-risk translation by showing that the same integrated design with secure clot sealing plus time-staged BHB signaling, which remains effective in a complex oral environment, positioning WAM-Gel as a candidate for socket preservation and implant-ready regeneration.

To bridge the gap toward clinical translation, several critical steps must be undertaken. Long-term studies in large animal models are required to evaluate complete bone remodeling, maturation, and the restoration of load-bearing function under physiological masticatory forces. Validation in clinically relevant disease models, such as periodontitis or diabetes, is essential to assess the material's efficacy under conditions of chronic inflammation and a dysregulated immune microenvironment. Furthermore, the transition toward clinical application necessitates the development of standardized manufacturing processes aligned with Good Manufacturing Practice guidelines, including the establishment of reliable terminal sterilization methods and formal stability studies. Finally, further pharmacokinetic investigations could help refine the release kinetics of the active molecule to better match the spatiotemporal demands of the complex healing process. Addressing these aspects systematically will be indispensable for translating the current preclinical findings into future clinical use.

3. Conclusion

This study establishes WAM-Gel that couples stable interfacial sealing with coordinated osteoimmune repair in tooth extraction sockets. A hydrogen-bond enhanced, covalently anchored dual network enables rapid in situ curing, wet-state adhesion, and mechanical resilience, while controlled delivery of BHB provides a metabolic cue to bias early healing. This integrated design sustained clot stability under intraoral stresses, tempered early inflammation, supported timely angiogenesis, and advanced osteogenic activity. Taken together, the results support WAM-Gel as a credible path toward implant-ready socket preservation and outline a general materials framework for dynamic wet interfaces that is to optimize a dual-mode adhesive network for wet stability, and embed an endogenous metabolic cue for time-aligned immunoregulation and tissue anabolism.

CRediT authorship contribution statement

Zifan Zhao: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft. Jing Zhang: Formal analysis, Investigation, Methodology, Visualization. Hu Chen: Investigation, Validation. Xu Zhang: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing – review & editing. Yuchun Sun: Funding acquisition, Project administration, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.