Injectable living hydrogel as engineered biotherapeutic to promote tooth-extraction wound healing and alveolar bone regeneration

Abstract

Effective wound healing and functional bone regeneration following tooth extraction remain clinical challenges, underscoring the significant need for multifunctional strategies that address the complex, multistage and translational demands of socket management. Herein, an injectable live biotherapeutic hydrogel was fabricated by integrating probiotic Lactobacillus rhamnosus GG (LGG) and calcium phosphate nanoparticles (CP NPs) into a photopolymerizable poly (ethylene glycol) (PEG) matrix. Upon in situ injection and activation with dental blue light, the hydrogel rapidly forms a conformal, protective and bioactive scaffold to the extraction socket. Therapeutically, LGG probiotics remodel the wound microenvironment through antibacterial and immunomodulatory bioactivities to promote early-stage healing. Simultaneously, LGG can facilitate the release of calcium and phosphate ions from CP NPs, which synergizes with microbe-assisted biomineralization to promote osteogenic differentiation and bone regeneration. In vitro and in vivo validations confirmed that this probiotic-mineral biotherapeutic hydrogel concurrently integrates infection control, immune regulation, and osteoinduction within a single clinically deployable platform, representing not only a transformative strategy for post-extraction socket management but also a paradigm for the development of living biomaterials in dynamic tissue engineering applications.

Graphical abstract

A. Engineering the Hydrogel: An injectable, live biotherapeutic hydrogel is fabricated by embedding probiotic LGG and calcium phosphate nanoparticles (CP NPs) within a photopolymerizable PEG matrix.B. Precise Clinical Application: The hydrogel is injected into the extraction socket and rapidly crosslinked in situ using dental blue light, forming a perfect fit within the wound.C. Spatiotemporally Orchestrated Healing: The construct dynamically regulates the socket microenvironment, sequentially combating pathogens, modulating immunity, and ultimately boosting bone regeneration.

Highlights

• •Probiotic LGG orchestrates early wound healing by suppressing pathogens, modulating inflammation, and guiding macrophage polarization.
• •LGG-derived acids promote Ca/P release and, with microbe-assisted mineralization, synergistically enhance bone regeneration.
• •An injectable, light-activated living hydrogel integrates probiotics and minerals into a clinically viable, chairside therapy for extraction sockets.

1. Introduction

Teeth play an essential role in mastication, phonation, and psychosocial well-being. However, tooth loss remains a prevalent clinical challenge with profound functional and aesthetic consequences. While dental implants provide an effective solution for tooth replacement, their application and success critically rely on adequate alveolar bone volume, which is a prerequisite often compromised by post-extraction bone defects or impaired socket healing [1]. Various interventions have been explored to promote extraction socket healing [[2], [3], [4]] and bone regeneration [5,6] through hemostatic control, biofilm inhibition, or regeneration enhancement. Nonetheless, alveolar socket repair is a highly orchestrated and temporally regulated process involving clot stabilization, inflammatory modulation, granulation tissue formation, bone generation and remodeling [7,8]. This multifaceted cascade is particularly susceptible to disruption by bacterial infection [9,10], especially within the warm, humid, and microbe-rich oral environment [11]. These challenges underscore the urgent need for multifunctional and clinically practical therapeutics capable of coordinating infection control, immune regulation, and bone regeneration during post-extraction wound management.

Nature-inspired living biomaterials are emerging as a paradigm shift in the development of versatile therapeutics. Through millennia of evolution, microorganisms have refined biosynthetic systems that enable self-replication, metabolic activity, and environmental responsiveness. From a materials science perspective, these living microorganisms can be conceptualized as powerful nanoscale bioreactors, forming the foundation of engineered living biomaterials [12]. Their integration with synthetic matrices has ushered in a transformative concept of “Materials Come Alive” in biomaterials science [13], offering unprecedented therapeutic archetypes with autonomous adaptation, cascade biosynthesis, and dynamic host interactions that greatly surpass conventional therapies [14,15]. These advantages make living biomaterials particularly suited for the dynamic environments of post-extraction sockets.

Probiotics are microorganisms that confer health benefits to the host. Building on their long history of safe use, probiotics and their dynamic interactions with host physiology have spurred the development of live biotherapeutics, which are defined by the FDA as “therapeutic strategies comprising live microorganisms designed to prevent or treat diseases” [16]. These probiotic-based biotherapeutics have been explored for gastrointestinal disorders, diabetic wound healing, and tissue regeneration [[16], [17], [18]], with multifaceted mechanisms encompassing pathogen suppression, immune modulation, and stimulation of tissue repair [14,17]. Furthermore, probiotic metabolites such as lactic acid and butyrate have been shown to support neuroplasticity and bone remodeling, highlighting their pleiotropic regenerative potential [19].

The oral cavity, as the gateway to the digestive tract, provides a warm, humid, and nutrient-rich environment that is particularly well-suited for the development of probiotic-based biotherapeutics. Lactobacillus rhamnosus GG (LGG), a clinically validated probiotic strain, exhibits multimodal bioactivities that are ideally conducive to oral wound healing: competitive inhibition of pathogens [20]; secretion of antimicrobial compounds such as bacteriocins, organic acids, hydrogen peroxide to preserve microbial homeostasis [20,21]; modulation of macrophages for immune regulation [22,23]; promotion of tissue-repair cytokines to accelerate healing [24,25]; and enhancement of osteogenesis while inhibiting osteoclastogenesis to support bone regeneration [19,[26], [27], [28]]. These versatile bioactivities render LGG a promising candidate for engineered live biotherapeutics targeting the complex pathophysiology of pathogen suppression, immune modulation, and regenerative stimulation during post-extraction wound repair. However, conventional administration routes, such as oral intake or gavage, encounter challenges in maintaining therapeutic concentrations and vitality of probiotics at wound sites. Moreover, the irregular anatomy and dynamic physiology of the oral cavity impose additional barriers to precise and durable biotherapeutics delivery.

To enhance the therapeutic efficacy and clinical applicability of live biotherapeutics, advanced delivery systems are essential. Hydrogel encapsulation provides an ideal platform since these “living hydrogels” not only localize probiotic viability at the application site, but also facilitate the gas and nutrient exchange, as well as controlled release of bioactive factors through the hydrated porous networks [29]. Furthermore, their facile integration with functional augmentation, such as inorganic additives, can further enhance the biological response and osteoconductivity for bone regeneration [30,31]. Calcium phosphate nanoparticles (CP NPs) have been widely employed in scaffold fabrication to synergistically enhance stem cell osteogenic differentiation, mineralization, and bone regeneration [[31], [32], [33]]. Moreover, interactions between mineral ions and microbial cell walls can facilitate the nucleation and growth of mineral crystals [34,35], suggesting that integration of living hydrogel with CP NPs may harness the dynamic reciprocity of natural biomineralization processes to provide both structural support and bioactive signaling cues for enhanced bone regeneration.

Based on these rationales, an injectable, photo-crosslinkable, poly (ethylene glycol) (PEG)-based hydrogel integrating LGG and CP NPs was engineered as a multifunctional live biotherapeutic for tooth-extraction wound healing and alveolar bone regeneration (Scheme 1A). It is hypothesized that this composite hydrogel can be injected and rapidly photo-crosslinked in situ using dental blue light to precisely conform the extraction sockets anatomy (Scheme 1B). Its porous and hydrophilic network supports LGG viability and bioactivity, thereby imparting antibacterial, immunomodulatory, and pro-regenerative capacities to accelerate wound healing during initial stages. In addition, lactic acid produced by LGG may facilitate the release of calcium and phosphate ions from CP NPs, synergizing with microbe-assisted biomineralization to promote subsequent mineral deposition and bone formation. This strategy transcends prior probiotic-based approaches, which typically rely on oral administration with limited site-specific retention, by establishing a spatially confined, on-demand activated living biomaterial capable of orchestrating multiple stages of socket repair. By integrating antibacterial, immunoregulatory, and osteogenic functions into a single, minimally invasive platform (Scheme 1C), this live biotherapeutic hydrogel introduces a previously unexplored probiotic–mineral synergy and offers a comprehensive and clinically translatable approach for coordinating the multistage processes of socket repair, representing an innovative paradigm for engineered biotherapeutics to address complex regenerative challenges.

Scheme 1.

Schematic illustration of the engineered live biotherapeutic hydrogel for enhancing post-extraction wound healing and bone regeneration. (A) Fabrication of the injectable, light-responsive live biotherapeutic hydrogel by incorporating Lactobacillus rhamnosus GG (LGG) and calcium phosphate nanoparticles (CP NPs) into a photopolymerizable poly (ethylene glycol) (PEG) matrix. (B) Biomedical application of the live biotherapeutic hydrogel, which can be injected into tooth extraction sockets and rapidly crosslinked in situ under dental blue light to conform precisely to the socket anatomy. (C) The live biotherapeutic hydrogel spatiotemporally modulates the local microenvironment by recapitulating key phases of socket repair, including pathogen inhibition, immune modulation, and bone regeneration.

2. Results and discussion

2.1. Synthesis and characterization of the live biotherapeutic hydrogel

The oral cavity hosts one of the most diverse and densely populated microbial ecosystems in the human body, making effective closure of the extraction site with a protective scaffold critical for mitigating pathogen invasion and post-extraction infection risks. Given the irregular anatomical architecture of the extraction socket and the need for clinical feasibility, an injectable, dental blue light-responsive hydrogel was developed as a chairside-applicable biotherapeutic scaffold. The hydrogel matrix was constructed using 4-arm poly (ethylene glycol) diacrylate (PEG-DA) as the photopolymerizable backbone and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as the photoinitiator (Fig. 1A). A human dental model was used to mimic clinical application (Fig. 1B), and the LAP, LGG-containing PEG-DA solutions were preloaded separately in a dual-barrel syringe. Upon mixing, the resulting formulation was precisely injected into the tooth extraction socket and photopolymerized using a standard dental blue light for 30 s, forming an in situ crosslinked, milky-white biotherapeutic hydrogel that conformed tightly to the socket's geometry.

Fig. 1.

Fabrication and characterization of the live biotherapeutic hydrogel. (A) Schematic illustration of the synthesis of the live biotherapeutic hydrogel. The system employs poly (ethylene glycol) diacrylate (PEG-DA) as the photocrosslinkable polymer matrix and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as the photoinitiator to encapsulate Lactobacillus rhamnosus GG (LGG) and calcium phosphate nanoparticles (CP NPs). (B) Digital images depicting the clinical application of the live biotherapeutic hydrogel. Following tooth extraction, the precursor solutions containing LGG and CP NPs are mixed, injected into the socket, and photopolymerized in situ using a dental blue light. (C) Confocal microscopy images showing the distribution and viability of LGG within the hydrogels after 24 h of incubation in PBS, saliva, or MRS medium. Scale bar: 200 μm. Quantitative analysis of (D) fluorescence intensity and (E) colony-forming unit (CFU) counts of encapsulated LGG under different culture conditions. (F) SEM images of lyophilized hydrogel showing LGG (pseudo-colored green) and CP NPs (blue). Scale bar: 2 μm. (G) Rheological analysis of the hydrogels.

The high hydrophilicity of hydrogels makes them ideal carriers for probiotics by providing a moist and protective microenvironment that preserves probiotic viability and biotherapeutic efficacy while minimizing premature release [29]. To evaluate the viability and spatial distribution of LGG within the hydrogel under physiologically relevant conditions, the hydrogels were incubated in phosphate buffer saline (PBS), saliva, and DeMan–Rogosa–Sharpe (MRS) medium and analyzed by laser scanning confocal microscopy (LSCM) (Fig. 1C). The homogeneous, sparsely distributed green fluorescence throughout the matrix (Figs. S1–3) indicated the excellent biocompatibility and suitability of the light-responsive hydrogels for probiotic encapsulation and delivery. The progressive increase in both fluorescence intensity (Fig. 1D) and colony-forming unit (CFU) counting (Fig. 1E) further confirmed the sustained viability of LGG within the hydrogel. Notably, LGG exposed to saliva exhibited moderate growth, demonstrating that the hydrogel matrix could support LGG survival and preserve its potential therapeutic functionality in the oral environment.

Unlike other superficial wounds, successful tooth-extraction socket healing necessitates bone regeneration to ensure structural and mechanical support for future prosthetic restoration. To enhance the osteoinductive properties of the scaffold, CP NPs were incorporated into the hydrogel to fabricate a composite living hydrogel (CP-LGG@Gel). Scanning electron microscopy (SEM) images of the lyophilized hydrogels revealed a porous, interconnected microstructure, with pore sizes estimated to be 10–20 μm (Fig. 1F, Fig. S4), which is conducive to cell infiltration and nutrient exchange. The integration of LGG and CP NPs did not alter the porous morphology of the hydrogels, and LGG cells retained their structural integrity and uniform distribution within the network, demonstrating that the encapsulation did not compromise the structure of probiotics or the architecture of the scaffold.

For clinical translation, an ideal injectable hydrogel should exhibit appropriate viscosity to ensure precise delivery and rapid gelation following administration. To evaluate the sol–gel transition kinetics, the hydrogels were monitored by rheological assessments (Fig. 1G). Before light exposure, all formulations displayed higher loss modulus (G″) than storage modulus (G′), confirming their fluid state suitable for injection. Upon dental light irradiation, a sharp G′ increase surpassed G″, indicating rapid photopolymerization. All hydrogels demonstrated stabilized G′ and G″ values post-irradiation, signifying that the incorporation of LGG and CP NPs had no adverse effect on gelation kinetics or mechanical stability. The compressive stress–strain curve was shown in Fig. S5, and the calculated compressive modulus (Ec) was approximately 40 kPa. These results collectively indicate the developed light-responsive composite hydrogel exhibits excellent injectability, rapid in situ gelation, robust structural integrity, and favorable biocompatibility, underscoring its potential as a clinically translatable platform for local administration of a live biotherapeutic in the management of tooth-extraction wounds.

2.2. Efficacy of the live biotherapeutic hydrogel against pathogenic bacteria

Leveraging the well-documented antimicrobial properties of LGG, the engineered living hydrogel was hypothesized to inhibit pathogen colonization during post-extraction wound healing. To evaluate its antibacterial efficacy, the hydrogel was challenged with three wound-relevant pathogens: Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Enterococcus faecalis (E. faecalis). After 48 h of co-culture, visual inspection and optical density (OD) measurements (Fig. 2A–D,G) revealed that bacterial suspensions incubated with blank hydrogels (Gel groups) exhibited characteristic turbidity indicative of bacterial growth, whereas LGG-loaded hydrogels (LGG@Gel groups) maintained optical clarity throughout the incubation period. OD analyses confirmed that CP-LGG@Gel hydrogels significantly suppressed bacterial growth compared to blank gels, with complete inhibition observed in LGG@Gel groups.

Fig. 2.

Antibacterial ability of the live biotherapeutic hydrogels and probiotic-facilitated biomineralization. Representative photographs and corresponding optical density (OD) values of (A) S. aureus, (D) E. coli and (G) E. faecalis suspensions after 48 h co-culture with the hydrogels. Quantification of CFUs demonstrating the antibacterial efficacy of the hydrogels against (B) S. aureus, (E) E. coli, and (H) E. faecalis. Agar diffusion assay showing inhibition zones formed by the hydrogels against (C) S. aureus, (F) E. coli, and (I) E. faecalis. (J) Schematic illustration of the proposed mechanism of LGG-facilitated biomineralization. (K) TEM images showing mineral deposition (indicated by red arrows) on the surface of LGG. Scale bars: 200 nm. (L) SEM images showing time-dependent mineral formation on LGG surface after incubation in PBS or CP NPs suspension for 7 and 14 days. Scale bars: 400 nm. (M) SEM images of the hydrogels after immersion in PBS for 7 days. Scale bar: 2 μm. LGG cells (green), CP NPs (blue), and biomineral deposits (violet) are pseudo-colored for visualization. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

These observations were corroborated by CFU assays (Fig. 2B–E,H). In LGG@Gel groups, no detectable colonies confirmed the complete eradication of pathogens. The CP-LGG@Gel groups demonstrated a significant reduction in viable pathogens, indicating the reliable antibacterial efficacy of the composite formulation. Further assessments via agar disk diffusion assays (Fig. 2C–F,I) revealed that LGG-containing hydrogels generated substantial inhibition zones against all tested pathogens, whereas control hydrogels showed no antimicrobial activity. Notably, the incorporation of CP NPs moderately attenuated the antibacterial activity, likely due to progressive surface mineralization partially shielding LGG and buffering local acidity. This reflects a programmed functional transition, balancing early antimicrobial protection with subsequent biomineralization and biosafety.

The oral cavity hosts a diverse microbial ecosystem, wherein pathogen growth can significantly compromise post-extraction wound healing. As a well-established human-derived probiotic, LGG has been proven to modulate the local microenvironment through the secretion of bioactive substances, such as bacteriocins, hydrogen peroxide, and organic acids, to inhibit pathogenic proliferation and promote microecological homeostasis [20,21]. The results of antibacterial measurements collectively demonstrate that the engineered live biotherapeutic hydrogel exhibits robust and sustained antibacterial activity, underscoring its potential as a dual-functional wound dressing for infection control and regenerative support in post-extraction socket management.

2.3. Probiotic LGG facilitates biomineralization

Microorganisms, including lactic acid bacteria, have evolved sophisticated mechanisms for biologically regulated mineralization, offering significant potential for biotechnological applications in the biosynthesis of biogenic minerals [[35], [36], [37]]. The cell surface-associated proteins (CSPs) of LGG have been proposed to serve as templates for mineralization [34,38]. Moreover, the cell envelope of Gram-positive bacteria like LGG is enriched with metal-chelating functional groups (e.g., COO⁻ and PO₄³⁻), which provide abundant nucleation sites by anchoring metal cations (e.g., Ca²⁺), thereby facilitating the accumulation of counteranions (e.g., HPO₄²⁻) and subsequent nucleation of mineral phases [34,35]. To investigate the biomineralization potential of LGG, the Transwell-assisted exposure to CP NPs suspension was used to eliminate direct physical contact while permitting ionic diffusion. The mineral deposition on bacterial surfaces was analyzed, and the schematic rationale is envisioned in Fig. 2J.

Transmission electron microscopy (TEM) analysis revealed dense mineral aggregates adhering to LGG cell surfaces following incubation with CP NPs suspension (Fig. 2K), confirming that the CSPs of LGG act as effective templates for mineral formation. A time-dependent reduction in surface negativity (Fig. S6), together with accumulation of minerals on LGG surfaces (Fig. 2L), further demonstrated progressive calcium phosphate deposition. Consistently, LGG-containing systems exhibited an early, transient decrease in pH followed by a gradual return toward physiological levels (Fig. S7), reflecting attenuation of bacterial metabolic activity and the establishment of a self-regulated microenvironment favorable for biomineralization and biosafety. Notably, mineral deposition persisted even after LGG became metabolically inactive (Day 14 in Fig. 2L), indicating that the mineralization process is predominantly governed by structural features of the bacterial surface rather than active metabolism. This progressive loss of metabolic activity represents a deliberate biosafety advantage, as it restricts uncontrolled bacterial proliferation while preserving the mineralization-directing function of LGG. Collectively, this programmed transition from early biological activity to late-stage structural templating enables localized, self-limiting therapeutic action and provides stable structural cues to support osteogenic differentiation. The large surface area of bacterial cells enables a high-density display of functional groups that promote mineral nucleation, and the cell envelope of Gram-positive bacteria presents numerous negatively charged ligands capable of chelating metallic ions, thereby facilitating the initiation and growth of biomineral deposits [34,35].

Extracellular mineralization typically requires three core elements [35]: adequate supply of precursor materials (e.g., dissolved inorganic phosphate or carbonate), availability of nucleation sites, and a conducive local microenvironment. In this platform, these requirements are satisfied through the synergistic interactions of the functional components. The CP NPs embedded in the hydrogel serve as reservoirs of inorganic minerals, which can be gradually solubilized under the mild acidification induced by LGG metabolic by-products (e.g., lactic acid). This promotes localized supersaturation of calcium and phosphate ions, leading to mineral precipitation onto the nucleation sites provided by the LGG surface proteins. These proteins, through their negatively charged residues, sequester Ca²⁺ and facilitate the nucleation, growth, and organization of minerals. Moreover, microbial metabolism of LGG maintains dynamic pH gradients, enabling local supersaturation and controlled precipitation.

The introduction of inorganic CP NPs into hydrogel matrix could not only enhance the mechanical properties of the scaffold but also reinforce its biological functionality for bone regeneration. After immersion of CP-LGG@Gel in PBS for 7 days (Fig. 2M), the observed mineral deposits on LGG surface confirmed the dual role of LGG as both a biotherapeutic agent with intrinsic health-promoting effects and a bio-catalyst facilitating biomineralization. Notably, mineralized LGG surfaces function as distributed biomineralization templates, creating osteoconductive microdomains that promote mineral deposition. In the early phase, viable LGG acts as a biotherapeutic agent, exerting antibacterial and immunomodulatory effects while facilitating local calcium and phosphate release from CP NPs (Fig. S8). As mineralization progresses, LGG gradually loses metabolic activity and transitions into a bio-catalytic mineralization template, wherein surface-associated proteins and negatively charged functional groups guide calcium phosphate nucleation and growth independent of bacterial viability, thereby enabling localized, self-limiting osteogenic stimulation with reduced long-term biosafety concerns. The microbially mediated biominerals synthesis via mimicking natural mineralization processes has demonstrated distinct advantages in supporting cell adhesion, proliferation, and osteogenic differentiation, offering a compelling strategy for promoting bone regeneration [36,37].

2.4. Macrophage activation induced by live biotherapeutic hydrogel

Probiotic-based biotherapeutics have emerged as promising immunomodulatory agents for managing inflammation and immune-related disorders [39,40]. To investigate the immunoregulatory capacity of the biotherapeutic hydrogel, the polarization of RAW264.7 macrophages was examined using a Transwell co-culture system (Fig. 3A). After 24 h of co-culture, quantitative RT-PCR analysis revealed concurrent upregulation of both M1-associated markers (CD86, TNF-α, iNOS) and M2-associated anti-inflammatory IL-10 in LGG@Gel and CP-LGG@Gel groups (Fig. 3B). This biphasic response suggests that LGG initiates transient pro-inflammatory signaling while simultaneously priming anti-inflammatory pathways in the early phase, which is a characteristic immunomodulatory feature of probiotics [41,42]. By 48 h, TNF-α levels declined, whereas M2 markers (CD206, IL-10, Arg-1) showed substantial upregulation (Fig. 3C), suggesting a timely shift from M1 to M2 phenotype. This temporal progression reflects the resolution of inflammation, in which early immune responses focused on pathogen clearance are succeeded by reparative processes.

Fig. 3.

In vitro immunomodulatory effects of the live biotherapeutic hydrogel. (A) Schematic representation of the Transwell co-culture system used to evaluate macrophage polarization using RAW264.7 cells. Quantitative RT-PCR analysis of macrophage polarization markers after (B) 24 h and (C) 48 h of co-culture, with gene expression levels normalized to the control group. (D) Flow cytometry analysis, quantification of (E) CD80⁺ (M1 macrophage marker), and (F) CD206⁺ (M2 macrophage marker) cell populations after 48 h of co-culture. (G) Representative immunofluorescence images of macrophages stained for CD86 (red, M1 marker), CD206 (green, M2 marker), and nuclei (blue, DAPI) after 48 h of co-culture. Scale bars: 30 μm. Quantification of fluorescence intensities for (H) CD86 and (I) CD206. (J) Principal component analysis (PCA) demonstrating distinct transcriptomic profiles between the control and CP-LGG@Gel-treated groups. (K) Volcano plot of RNA-sequencing results highlighting significantly upregulated (red) and downregulated (green) genes. (L) Gene Ontology (GO) enrichment analysis of differentially expressed genes in macrophages treated with CP-LGG@Gel. (M) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identifying significantly enriched signaling pathways affected by the treatment. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Flow cytometry analysis further confirmed this trend (Fig. 3D–F), which revealed increased populations of CD206⁺ (M2) macrophages in LGG-containing groups by 48 h. Immunofluorescence staining and quantification (Fig. 3G–I) further demonstrated the predominant CD206 (M2) signal in LGG-containing groups after 48 h. These findings demonstrate that both LGG@Gel and CP-LGG@Gel effectively shift macrophages from a pro-inflammatory state to an anti-inflammatory, regenerative phenotype.

Transcriptomic analysis was performed to provide deeper insight into the immunoregulation mechanisms. Principal component analysis (PCA) showed distinct clustering of CP-LGG@Gel-treated macrophages (Fig. 3J), with 2007 differentially expressed genes (1796 upregulated and 211 downregulated) compared to controls (Fig. 3K). Gene Ontology (GO) enrichment analysis (Fig. 3L) highlighted significant involvement in immune-regulatory processes such as positive regulation of adaptive immune responses, leukocyte chemotaxis, regulation of leukocyte mediated cytotoxicity, response to tumor necrosis factor, and positive regulation of T cell-mediated immunity. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed CP-LGG@Gel treatment primarily activated pathways including cytokine–cytokine receptor interaction, Epstein–Barr virus infection, and TNF signaling pathways (Fig. 3M), suggesting that a broad range of immune-modulatory pathways are involved in the immunoregulatory effects mediated by the live biotherapeutic hydrogel.

These findings align with documented properties of LGG, which can modestly stimulate pro-inflammatory cytokine production in the absence of pre-existing inflammation while suppressing excessive immune responses under inflammatory conditions [42,43]. This context-dependent dual regulation is particularly valuable for maintaining immune homeostasis: it boosts host defense when needed while preventing prolonged inflammation, and confers a significant advantage over conventional single-mode strategies. Collectively, these results highlight that LGG creates a dynamic and balanced immunoregulatory microenvironment, making it particularly suitable for oral tissue repair, where early inflammatory control and timely transition from pro-inflammatory to anti-inflammatory phenotypes are critical for preventing chronic inflammation and promoting regeneration.

2.5. Biocompatibility and osteoinductivity of the biotherapeutic hydrogel

The biocompatibility and osteogenic potential of the hydrogels were evaluated using rat bone marrow mesenchymal stem cells (BMSCs) cultured with hydrogel leachates (Fig. 4A). Cell viability assessment via MTT assay revealed no significant differences across all groups (Fig. 4B), confirming the cytocompatibility of the hydrogels. Morphological analysis demonstrated that BMSCs maintained a healthy, well-spread morphology under all conditions (Fig. 4C), indicating the favorable potential of these hydrogels to serve as scaffolds for wound healing and tissue regeneration.

Fig. 4.

Proliferation and osteogenic differentiation of BMSCs induced by the live biotherapeutic hydrogels. (A) Schematic illustration of the co-culture setup between hydrogel extracts and BMSCs. (B) MTT assay showing BMSCs viability after 24 and 48 h of culture with hydrogel leachates compared to PBS control. (C) Representative immunofluorescence images showing the morphology of BMSCs. Scale bar: 50 μm. (D) Alkaline phosphatase (ALP) staining and (E) quantitative ALP activity of BMSCs after 7 and 14 days of culture. (F) Alizarin Red S staining and (G) quantification of calcium nodule deposition at 14 and 21 days. (H, I) RT-PCR analysis of osteogenic gene expression at 7 and 14 days. (J, K) Western blot analysis of key osteogenic marker proteins at 7 and 14 days. (L, M) Densitometric quantification of protein expression levels from Western blots. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Subsequently, the osteoinductive capacity of the platform was then evaluated. Alkaline phosphatase (ALP) staining demonstrated intense enzymatic activity in CP@Gel and CP-LGG@Gel groups at days 7 and 14 (Fig. 4D), with quantitative analysis confirming the highest ALP expression in CP-LGG@Gel (Fig. 4E), suggesting its superior capacity to promote osteogenic differentiation. Mineralization assessment via Alizarin Red S (ARS) staining revealed substantially enhanced calcium nodule deposition in CP@Gel and CP-LGG@Gel groups at days 14 and 21 (Fig. 4F and G), demonstrating that the incorporation of CP NPs effectively promoted matrix mineralization of BMSCs.

To further explore the osteogenic potential of the scaffolds, the expression of osteogenesis-related genes, including bone morphogenetic protein 2 (Bmp2), runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alpl), type I collagen (Col1a1), Osterix (Osx), bone sialoprotein (Ibsp), Osteopontin (Opn), and osteocalcin (Ocn), was quantified by RT-PCR analysis. As shown in Fig. 4H and I, BMSCs in the CP-LGG@Gel group exhibited significantly upregulated expression of osteogenesis-related genes on day 7 and day 14. Western blot analysis confirmed the enhanced protein expression of key osteogenic markers including BMP-2, Runx2, ALPL and OPN in the CP-LGG@Gel group (Fig. 4J and K), collectively demonstrating the strong osteoinductive potential of the platform for bone tissue engineering.

The enhanced osteoinductive effects likely arise from synergistic interactions between the scaffold components. The incorporated CP NPs function as mineral reservoirs and their progressive degradation elevates extracellular calcium and phosphate concentrations, which can activate calcium-sensing receptors and phosphate transporters, stimulating osteogenic signaling pathways to promote the osteogenic differentiation of stem cells and mineral deposition in the extracellular matrix [33,[44], [45], [46]]. The LGG encapsulation in CP-LGG@Gel could increase the calcium release from CP NPs, augmenting the phosphate metabolism and osteogenic commitment of BMSCs.

Beyond its role in calcium and phosphate regulation, LGG also promotes osteogenesis through diverse biological pathways. Clinical studies have shown that LGG supplementation benefits skeletal health by preventing pathological bone loss and promoting bone formation via improving intestinal barrier function, stimulating mesenchymal stem cell migration, and regulating the immune responses [19,27,47]. Furthermore, LGG has been reported to improve periodontal bone regeneration by modulating gut microbiota, increasing butyrate production, and stimulating pro-osteogenic metabolites [26]. Its culture supernatant has also been shown to rescue the proliferation and osteogenesis of mesenchymal stem cells, thereby mitigating alveolar bone loss [27]. By leveraging the synergistic interactions between inorganic CP NPs and biological probiotic LGG, the composite biotherapeutic hydrogel achieved enhanced osteoinductivity. This multifaceted osteogenic stimulation—combining ion-mediated signaling with probiotic-derived bioactivity—represents a significant advancement over conventional mono-functional scaffolds, offering a promising platform for advanced bone regeneration applications.

2.6. In vivo therapeutic efficacy of the biotherapeutic hydrogel in tooth extraction socket regeneration

Living hydrogels serve not only as physical barriers that shield open wounds from bacterial invasion but also as bioactive matrices that orchestrate a favorable local microenvironment for tissue regeneration. To evaluate the therapeutic efficacy of the platform, an in vivo tooth extraction model was established by removing the maxillary first molars of rats (Fig. 5A). Hydrogel precursors were injected into fresh extraction sockets and photopolymerized in situ immediately using a dental LED light, forming a conformal protective layer. Wound healing and regenerative outcomes were then assessed (Fig. 5B). Quantification of serum cytokines (TNF-α, IL-1β, IL-6, IL-10) revealed minimal intergroup variation (Fig. 5C), confirming good in vivo biocompatibility of the live biotherapeutic hydrogels without eliciting systemic inflammatory responses.

Fig. 5.

Therapeutic efficacy of the live biotherapeutic hydrogels in promoting socket healing following tooth extraction in rats. (A) Experimental rat tooth extraction model. Following bilateral extraction of maxillary first molars, hydrogels were applied into the sockets at the time of surgery. Representative images show in situ-injected and light-polymerized hydrogel (arrows) conforming precisely to the socket anatomy. (B) Schematic timeline of the experimental procedure. Rats were euthanized at postoperative days 1, 3, 7, and 14 for tissue harvesting and analysis. (C) Quantification of systemic cytokine levels (TNF-α, IL-1β, IL-6, and IL-10) in serum at designated time points. (D) Representative macroscopic images, and (E) quantification of wound closure ratios, illustrating healing progression at extraction sites. M2: second molar. (F) Histological analysis of extraction sockets by H&E and Masson's trichrome staining. Black dashed lines indicate defect margins; black stars denote inflammatory layers; yellow stars mark blood clots. (G) Histomorphometric analysis quantifying tissue composition within the healing sockets. (H) Immunofluorescence staining of iNOS (red, M1 macrophage marker) and CD206 (green, M2 macrophage marker), with DAPI (blue) nuclear counterstaining. (I) Quantitative analysis of positive immunofluorescence areas for iNOS and CD206 within the defect region. (J) Immunofluorescence staining for osteogenic markers OCN (red), Runx2 (green), and DAPI (blue). (K) Quantification of OCN⁺ and Runx2⁺ areas indicating osteoblast activity. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Wound healing assessment (Fig. 5D and E) demonstrated that the control group exhibited incomplete epithelial coverage by day 3, while the living hydrogel-treated sites showed faster closure. Complete re-epithelialization was observed by day 7 in the CP-LGG@Gel group, whereas epithelial defects persisted in the controls. This accelerated mucosal barrier restoration is clinically significant for mitigating infection risks in the microbe-rich oral environment, highlighting the efficacy of the live biotherapeutic hydrogel as a safe and effective dressing for oral wound management.

Tooth socket healing involves sequential stages, including clot formation, inflammatory cell infiltration, granulation tissue development, bone formation and remodeling [7,8]. Histological analysis at day 1 (Fig. 5F) showed that all sockets were predominantly occupied by well-formed blood clots (indicated by yellow stars), providing essential structural and biochemical cues for wound stabilization and activation of the repair cascade [48]. No noticeable differences in tissue composition among groups (Fig. 5G) confirmed the in situ-formed hydrogels were biocompatible and non-disruptive, effectively protecting clots during the initial healing stages and supporting the subsequent regenerative processes.

Given the high infection risk in oral wounds, immunofluorescence staining of iNOS (M1 macrophages marker) and CD206 (M2 marker) was performed to investigate the immune modulation during the healing process (Fig. 5H and I). Both LGG@Gel and CP-LGG@Gel groups exhibited enhanced macrophage recruitment in the early stages, suggesting the immune activation to combat pathogens and orchestrate tissue repair. Double immunofluorescence staining of Runx2 and osteocalcin (OCN) was conducted to assess osteogenic activity, which confirmed that bone metabolism remained inactive on day 1 (Fig. 5J and K).

By day 3 (Fig. 6A and B), the hydrogel-treated groups exhibited tissue organization progressing from the socket walls, along with abundant neutrophil infiltration and the formation of nascent connective tissue. By day 7 (Fig. 6C and D), the CP-LGG@Gel group exhibited newly formed woven bone and mineralizing matrix filling the socket centripetally, forming interconnected trabeculae (yellow arrows). In contrast, sockets in the control group remained filled with granulation tissue (green stars) and showed limited bone formation confined to the socket walls. Increased CD206⁺ cell infiltration was seen in CP-LGG@Gel group on day 3 (Fig. 6E and F), and the M2 dominance became more pronounced by day 7 (Fig. 6G and H), demonstrating the immunomodulatory capability of the biotherapeutic hydrogel to support an anti-inflammatory and pro-regenerative environment. Concurrently, Runx2⁺ cells were widely distributed in the CP@Gel, LGG@Gel, and CP-LGG@Gel groups on days 3 and 7 (Fig. 6I–L), with CP-LGG@Gel exhibiting the highest OCN expression on day 7, indicating enhanced osteoblast differentiation and matrix maturation.

Fig. 6.

Histological evaluation of socket healing at days 3 and 7 post-tooth extraction. (A, C) H&E staining and Masson's trichrome staining of extraction sockets at days 3 and day 7 post-surgery. Black dashed lines indicate the boundaries of the extraction defects; black stars denote inflammatory layers; green stars mark granulation tissue, and yellow arrows highlight the newly formed woven bone. (B, D) Quantitative histomorphometric analysis showing the proportion of different tissues within extraction sockets. (E, G) Representative immunofluorescence images showing M1 (iNOS, red) and M2 (CD206, green) macrophage markers, with DAPI (blue) nuclear staining, in the defect regions at days 3 and 7 post-surgery. (F, H) Quantification of the iNOS⁺ and CD206⁺ areas in the defect regions at days 3 and 7 post-surgery. (I, K) Immunofluorescence staining for osteogenic markers OCN (red) and Runx2 (green), with DAPI (blue) counterstaining. (J, L) Quantitative analysis of OCN⁺ and Runx2⁺ areas reflecting osteogenic activity. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Histological analysis on day 14 (Fig. 7A and B) revealed sparse trabeculae and large interspaces in the control group, while hydrogel-treated groups exhibited denser, continuous bone structures with higher osteoblast density. The CP-LGG@Gel group showed the greatest extent of mature bone and the least remaining connective tissue, demonstrating its superior regenerative outcomes. Immunofluorescence analysis confirmed the dominant CD206⁺ macrophages widely distributed (Fig. 7C and D), and significantly elevated Runx2 and OCN expression in CP-LGG@Gel-treated sockets on day 14 (Fig. 7E and F), demonstrating the robust osteogenesis and matrix mineralization of the composite scaffold.

Fig. 7.

Histological and radiographic evaluation of socket healing and bone regeneration on day 14 post-extraction. (A) H&E and Masson's trichrome staining of extraction sockets on day 14. (B) Quantification of the proportions of mineralized bone within the extraction sockets. (C) Representative immunofluorescence images showing M1 macrophage marker (iNOS, red) and M2 macrophage marker (CD206, green), with nuclei counterstained by DAPI (blue). (D) Quantification of iNOS⁺ and CD206⁺ areas within the defect regions. (E) Immunofluorescence staining of osteogenic markers OCN (red) and Runx2 (green), with DAPI (blue) for nuclear counterstaining. (F) Quantitative analysis of OCN⁺ and Runx2⁺ areas within the regenerated tissue. (G, H) Sagittal and 3D-reconstructed micro-CT images showing the progression of bone healing in the extraction sockets on days 7 and 14. (I–L) Quantitative micro-CT analysis of bone regeneration, including the total tissue volume (TV), bone volume (BV), bone volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb. Sp.). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Micro-CT images of the maxillae at day 7 (Fig. 7G) revealed the initial bone formation progressing centripetally from the socket walls. Advanced bone regeneration reaching the center of socket in CP-LGG@Gel group at day 7, and more hyperdense areas with trabecular bone by day 14 demonstrated greater bone generation (Fig. 7H). Quantitative analysis of micro-CT reconstructions, including measurements of total tissue volume (TV), bone volume (BV), bone volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb. Sp.), further confirmed that the CP-LGG@Gel group exhibited the highest levels of bone formation and maturation.

These findings demonstrate the biotherapeutic hydrogel platform significantly improved wound healing and bone regeneration in tooth extraction sockets, which might be attributed to the synergistic mechanisms. Upon loading CP NPs and LGG, the composite hydrogel was injected and rapidly formed within the tooth extraction sockets, serving not only as a physical barrier to prevent bacterial contamination but also as a protective matrix to provide a favorable microenvironment for tissue repair. During the early phase, LGG contributes to pathogen suppression, immune modulation, mucosal barrier re-establishment, and stem cell differentiation to accelerate wound healing and bone regeneration. The osteogenic potential was augmented by the CP NPs, which could offer a local mineral reservoir, directly promoting osteogenic differentiation and minerals deposition. LGG could also secrete acids to increase local calcium and phosphate ion availability and offer nucleation sites for biomineralization, fostering bone formation.

Though the live biotherapeutic hydrogel in this study was locally applied in oral cavity, the possibility of limited LGG dissemination to the gastrointestinal tract cannot be completely excluded. Such exposure is unlikely to raise safety concerns, as LGG is a clinically approved probiotic known to modulate immune responses and enhance bone metabolism via the gut-bone axis [19,26]. These systemic interactions may even contribute synergistically to the observed osteoregenerative outcomes. LGG has been used for decades in oral supplementation, gavage, and clinical probiotic formulations, where its safety profile relies on controlled metabolic activity, transient mucosal colonization, and immune-mediated clearance [49]. Building upon this established clinical foundation, the present strategy further enhances biosafety and local efficacy by encapsulating LGG within a light-responsive, PEG-based hydrogel matrix, confining LGG within the extraction socket and minimizing uncontrolled dissemination, rapid systemic clearance, and immediate immune elimination. Collectively, these features underscore the translational promise of integrating clinically validated probiotics with inorganic bioactive components to synergistically promote biomineralization for bone tissue engineering.

3. Conclusion

To precisely align with the distinct and highly orchestrated physiological processes involved in post-extraction socket healing, while satisfying clinical demands for procedural efficiency, a light-responsive hybrid living hydrogel was tailored in the present study by integrating probiotic LGG and CP NPs into a PEG-based matrix. This injectable platform enables rapid in situ photopolymerization, ensuring excellent anatomical conformity to the irregular geometry of extraction sockets and facilitating seamless chairside application. Therapeutically, LGG confers multifunctional bioactivity, including broad-spectrum antimicrobial effects and dynamic immunomodulation via spatiotemporal macrophage polarization, thereby optimizing the local microenvironment and accelerating wound healing. Simultaneously, LGG promotes biomineralization by facilitating the dissolution of CP NPs and microbe-assisted mineral deposition. These coordinated mechanisms synergistically create a regenerative niche that supports both soft tissue healing and alveolar bone regeneration.

By leveraging the synergistic capabilities of living probiotics, bioactive inorganic components, and a light-responsive injectable hydrogel scaffold, this study establishes a comprehensive engineered biotherapeutic strategy that simultaneously addresses infection control, immune modulation, and bone regeneration within a single clinically translatable system. Beyond demonstrating robust therapeutic efficacy, biocompatibility, and procedural practicality in extraction socket management, this minimally invasive living hydrogel offers a versatile and adaptable solution with broader regenerative potential, including ridge preservation, peri-implantitis intervention, and periodontal regeneration. More broadly, the design principle of coupling probiotics with inorganic bioactive cues within a supportive matrix offers a generalizable framework for engineering living biomaterials capable of modulating complex pathological microenvironments, extending its relevance across regenerative medicine applications.

Despite the favorable biosafety and therapeutic performance demonstrated in this study, further optimization and systematic evaluation remain warranted to facilitate clinical translation. Future investigations will focus on elucidating the mechanistic basis of probiotic-mediated biomineralization and osteogenesis, as well as comprehensively assessing long-term biodegradation behavior, systemic biosafety, and ecological impacts on the oral and gut microbiome. In addition, optimization of scaffold degradability, dosage parameters, and validation in large-animal models will be pursued to further refine this platform and accelerate its translational feasibility.

CRediT authorship contribution statement

Jingmei Guo: Writing – original draft, Validation, Software, Project administration, Methodology, Investigation, Conceptualization. Wenlong Lei: Visualization, Software, Methodology, Investigation. Boyi Li: Formal analysis, Data curation. Kaifeng Li: Software. Jiyun Li: Visualization. Zhuoran Wang: Methodology. He Liu: Writing – review & editing. Cui Huang: Supervision, Project administration, Funding acquisition, Conceptualization. Ya Shen: Supervision, Project administration, Investigation.

Ethics approval and consent to participate

All experimental procedures were approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (Approval No. S07923040G). All animal experiments were conducted in accordance with institutional guidelines and ethical standards.

Declaration of competing interest

Ya Shen is an associate editor for Bioactive Materials and was not involved in the editorial review or the decision to publish this article. He Liu is an editorial board member for Bioactive Materials and was not involved in the editorial review or the decision to publish this article. 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: