Human periodontal ligament stem cells-derived small extracellular vesicles mitigate periodontitis by regulating macrophage pyroptosis

Abstract

Background

This study aimed to investigate whether human periodontal ligament stem cells (hPDLSCs)-derived small extracellular vesicles (P-sEVs) can improve the periodontal inflammatory microenvironment and promote periodontal tissue regeneration by regulating macrophage pyroptosis.

Methods

The expression of the GSDMD and CD68 were examined using HE and IHC in healthy and periodontitis gingival tissues. Pyroptotic levels in macrophages co-cultured with hPDLSCs or P-sEVs were assessed using various methods in vitro. The hydroxybutyl chitosan hydrogel (HBCH)/P-sEVs repair system was implanted into a rat periodontitis bone defect model in vivo.

Results

GSDMD and CD68 were significantly elevated in inflamed gingival tissues. Treatment with P. gingivalis-LPS and ATP significantly upregulated the expression of NF-κB, NLRP3, caspase-1, GSDMD-N, IL-1β, and so on at both mRNA and protein levels, and significantly enhancing lactate dehydrogenase release, the percentage of cells with damaged membranes and so on. However, these pathological effects were mitigated by the paracrine effects of hPDLSCs or direct action of P-sEVs. The HBCH/P-sEVs repair system enhanced hPDLSCs proliferation and osteogenesis, while simultaneously reducing pyroptosis and promoting alveolar bone regeneration in rats.

Conclusions

P-sEVs significantly reduced macrophages pyroptosis induced by P. gingivalis-LPS/ATP by inhibiting the NF-κB/NLRP3/GSDMD signalling pathway in vitro. Furthermore, P-sEVs could be integrated with temperature-sensitive HBCH to establish a repair and regeneration system with potential clinical applications in the treatment of periodontitis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-07533-9.

Introduction

Periodontitis is an inflammatory condition affecting the supporting tissues of the teeth and represents a significant public health challenge [1]. Although its underlying mechanisms remain incompletely understood, disruption of the homeostatic interaction between the oral microbiome and host immune response is recognised as a critical driver of periodontitis [2]. This disruption leads to persistent inflammation, destruction of periodontal tissues, and even systemic diseases [3]. Therefore, a deeper investigation into the pathogenesis of periodontitis is essential to enable early diagnosis and advance precision treatment strategies.

Innate immune responses serve as the first line of defence in the pathogenesis of periodontitis [4]. Pyroptosis, a form of programmed cell death closely associated with innate immunity [5]. This process recruits and activates additional immune cells, thereby amplifying the innate immune response. Macrophages are key effector cells in the innate immune system that may play a critical role in both the occurrence and function of pyroptosis [6, 7]. However, the role of macrophage-driven pyroptosis as a fundamental mechanism in the pathogenesis of periodontitis remains unclear. In a previous study, our group identified that macrophage pyroptosis participates in the initiation and progression of periodontitis [8]. Therefore, reversing macrophage pyroptosis has a significant potential for controlling and treating periodontitis.

Recent studies have highlighted that small extracellular vesicles (sEVs) carrying specific bioactive molecules establish crucial signalling networks with human periodontal ligament stem cells (hPDLSCs) under both healthy and diseased periodontal conditions [9]. hPDLSCs play a pivotal role in the regulation of inflammation, osteogenic differentiation, and cell communication, and their functionality is primarily dependent on the periodontal sEVs (P-sEVs) they secrete [10, 11]. However, the direct application of P-sEVs faces considerable challenges, such as difficulties in targeting specific sites, difficulties in controlled release, and poor stability, which limit their therapeutic efficacy and clinical application in precision and targeted treatments [12]. Hydrogels [13], particularly hydroxybutyl chitosan hydrogels (HBCHs), offer superior water solubility and temperature sensitivity, making them ideal carriers for P-sEVs [14]. When combined with P-sEVs, HBCH not only addresses these challenges but also provides an effective, targeted, and precise therapeutic strategy for clinical applications in periodontitis and tissue regeneration.

The potential mechanisms by which P-sEVs act as immunomodulators by targeting macrophages to alleviate periodontal inflammation and promote periodontal bone regeneration remain unclear. Therefore, this study aimed to investigate the impact and underlying mechanisms of hPDLSCs-derived sEVs on macrophage pyroptosis, with the goal of improving the periodontal inflammatory microenvironment and promoting periodontal tissue regeneration. To achieve this, a co-culture model of hPDLSCs or P-sEVs with macrophages was established to assess their effects on macrophage pyroptosis. Additionally, a composite hydrogel incorporating P-sEVs into phase-reversible temperature-sensitive HBCH was developed and implanted into a rat model of periodontitis-associated bone defects. This allowed for the evaluation of its regulatory effects on cell pyroptosis and tissue regeneration, providing novel therapeutic approaches and potential targets for the treatment of periodontitis.

Results

Overexpression of canonical pyroptosis-related factors in periodontitis

Gingival tissues were obtained from patients with severe periodontitis and from age- and sex-matched healthy individuals. HE staining revealed a substantial infiltration of inflammatory cells into the gingival tissue of patients with periodontitis (Fig. 1A). IHC (Fig. 1B) demonstrated an elevated expression of GSDMD and the macrophage marker CD68 in patients with periodontitis. Moreover, IHC double staining demonstrated increased GSDMD expression, specifically in macrophages.

Fig. 1.

Macrophage pyroptosis is elevated in the periodontal tissues of patients with periodontitis. (A) HE and (B) immunohistochemical (IHC) staining of GSDMD and CD68 in human gingival tissue sections from healthy individuals and patients with periodontitis. Signals for GSDMD and CD68 appear brown in sections counterstained with haematoxylin (blue). In double IHC staining, GSDMD and CD68 appear pink and brown, respectively. White arrows indicate inflammatory regions in HE staining, black arrows represent expression of GSDMD or CD68 in immunohistochemistry, and red arrows denote regions where GSDMD and CD68 are co-localised. Quantitative analysis was performed on the immunohistochemical staining of GSDMD and CD68 in gingival tissues. (C–F) mRNA levels of NFKB1 (F), NLRP3 (G), GSDMD-N (H), and IL1B (I) measured using qRT-PCR and normalised to the expression of the housekeeping gene GAPDH. (G) Uniform Manifold Approximation and Projection (UMAP) plot of periodontal tissue cells from patients with periodontitis (PD, n = 5). (H) Proportions of different cell subtypes in PD tissues. (I) Bubble plot showing the relative expression levels of GSDMD across cell subtypes. Data represent the mean ± standard deviation (SD), n = 3; *P < 0.05, **P < 0.01, ***P < 0.001

To further confirm the occurrence of pyroptosis in periodontitis, the expression of NF-κB, NLRP3, GSDMD-N, and IL-1β were assessed using qRT-PCR. The results demonstrated a significant upregulation of these target genes in gingival tissues affected by periodontitis (Fig. 1C, D, E, and F).

Building on these findings, publicly available single-cell RNA sequencing datasets from patients with periodontitis were analyzed to investigate the role of macrophages. Uniform Manifold Approximation and Projection (UMAP) clustering based on gene expression profiles identified distinct cellular populations within periodontitis tissues, with T lymphocytes, macrophages, and B lymphocytes showing notable enrichment compared to other cell types (Fig. 1G, H). Subsequent cell annotation demonstrated that GSDMD was expressed across multiple cell types, with comparatively higher expression in macrophages (Fig. 1I).

Identification and characterization of hPDLSCs and P-sEVs

hPDLSCs were isolated using the tissue block method (Fig. 2A), followed by cell sub-culturing (Fig. 2B). The primary culture exhibited spindle-shaped fibroblast-like morphology (Fig. 2C). After 10‒14 days of culture at low density, single cells efficiently formed evenly distributed colonies on the culture dish (Fig. 2D). Lipogenic differentiation was induced for 28 days, and the presence of lipid droplets was evident upon Oil Red O staining (Fig. 2E). Osteogenic differentiation was induced for 26 days, and calcified nodules were observed through alizarin red S staining (Fig. 2F). Flow cytometric analysis confirmed the expression of the cell surface markers CD29 (Fig. 2H), CD73 (Fig. 2I), CD90 (Fig. 2J), and CD105 (Fig. 2K), whereas CD45 expression was absent (Fig. 2G).

Fig. 2.

Identification of hPDLSCs and P-sEVs. (A, B) Cultured primary cells derived from the human periodontal ligament using the tissue block method. (C) Cells exhibiting a typical fibroblast-like morphology. (D) hPDLSCs were plated at a low density and stained with 0.1% toluidine blue after 14 days. (E) Oil Red O staining of adipogenically stimulated hPDLSCs. (F) Alizarin Red staining of osteogenically stimulated hPDLSCs. (G–K) Flow cytometry analysis showing the expression of surface markers CD45 (G), CD29 (H), CD73 (I), CD90 (J), and CD105 (K). (L) Morphology of P-sEVs assessed using TEM. (M) P-sEVs particle size. (N) Western blot analysis of P-sEVs markers (Alix, CD63, and TSG101) and a cytosolic marker (β-actin)

The P-sEVs were collected through differential centrifugation and displayed a disk-shaped membrane structure under TEM (Fig. 2L). Particle size analysis indicated an average diameter of 132.1 nm (Fig. 2M), and the P-sEVs markers CD63, Alix, and TSG101 were all positively expressed (Fig. 2N).

P. gingivalis-LPS triggers canonical pyroptosis in macrophages in vitro

Macrophages were initially treated with varying concentrations (0, 0.1, 1, or 10 µg/mL) of P. gingivalis-LPS for 11 h, followed by a 1-h exposure to 5 mM ATP. qRT-PCR analysis revealed a concentration-dependent increase in the expressions of NF-κB, NLRP3, caspase-1, GSDMD-N, and IL-1β following treatment with P. gingivalis-LPS. The most pronounced effect was observed at a concentration of 10 µg/mL of P. gingivalis-LPS combined with ATP (Fig. 3A, E). Furthermore, total cellular proteins were extracted from each treated and control group to determine the protein levels of the target genes through western blot analysis, and these results were consistent with the qRT-PCR findings (Fig. 3B, F). Under fluorescence microscopy, combinations of 1 µg/mL and 10 µg/mL concentrations of P. gingivalis-LPS with ATP induced notable alterations in cellular membrane integrity at 12 h post-exposure. Hoechst 33,324 staining was used for nuclear visualisation, and PI staining was employed to identify dying cells with damaged membranes (Fig. 3C, G). The CCK-8 assay results indicated a comparable cell survival rate of approximately 85% for both concentrations of P. gingivalis-LPS (Fig. 3D).

Fig. 3.

P. gingivalis-LPS combined with ATP effectively induces GSDMD-mediated pyroptosis in macrophages in vitro. (A, E) mRNA expression levels of NFKB1, NLRP3, CASP1, GSDMD-N, and IL1B measured by qRT-PCR in THP-1 (A) and rat primary macrophages (E) after 11 h of stimulation with varying concentrations of P. gingivalis-LPS followed by 1 h of treatment with 5 mM ATP, normalized to GAPDH. (B, F) Protein expression levels of NF-κB, NLRP3, and GSDMD-N measured by Western blot in THP-1 (B) and rat primary macrophages (F) under the same conditions, normalized to GAPDH. (C, G) Representative images of Hoechst 33,342 (blue) /PI (red) double-staining captured by fluorescence microscopy in THP-1 (C) and rat primary macrophages (G), with merged channels showing co-localization of nuclear and pyroptotic signals. (D) Cell viability assessed by CCK-8 assay in THP-1 macrophages following pyroptosis induction with varying concentrations of P. gingivalis-LPS. Data are presented as mean ± standard deviation (SD), n = 3. *P < 0.05, **P < 0.01, ***P < 0.001

THP-1 macrophages silenced NF-κB expression through lentiviral transfection at an MOI of 30 and induced pyroptosis in vitro following silencing. After 24 h of lentiviral transfection, the receptor cells were observed under a fluorescence microscope to confirm successful transfection (Fig. S1A). The qRT-PCR results indicated minimal expression of NF-κB mRNA, and the expression of NLRP3 mRNA and GSDMD mRNA was significantly reduced in transfected cells after induction with 10 µg/mL P. gingivalis-LPS combined with 5 mM ATP (Fig. S1B). Western blot results mirrored the trends observed in mRNA expression for NF-κB, NLRP3, and GSDMD proteins (Fig. S1C). Under conditions of NF-κB silencing and pyroptosis induction in THP-1 macrophages, combined treatment with MCC950 did not significantly reduce the expression of NLRP3 and GSDMD, whereas disulfiram treatment further decreased GSDMD expression (Fig. S1D, E).

Macrophage pyroptosis was attenuated by hPDLSCs and P-sEVs

As previously demonstrated, the combination of P. gingivalis-LPS and ATP activates the canonical pyroptosis pathway in macrophages for 24 h. Notably, hPDLSCs significantly suppressed the expressions of related factors, including NF-κB (Fig. S2A, G), NLRP3 (Fig. S2B, H), caspase-1 (Fig. S2C, I), GSDMD (Fig. S2D, J), IL-1β (Fig. S2E, K), and IL-18 (Fig. S2F, L), in macrophages undergoing pyroptosis.

We assessed the protein levels of NF-κB, NLRP3, caspase-1, ASC, GSDMD, GSDMD-N, IL-1β, and cleaved-IL-1β through western blotting (Fig. 4A, G). Protein expressions was markedly higher in the single-culture pyroptosis group than in the single-culture control and co-culture control groups. However, their expression was significantly lower in the co-culture pyroptosis group than in the single-culture pyroptosis group, which was consistent with the qRT-PCR results. ELISA revealed decreased expression of IL-1β (Fig. 4B) and IL-18 (Fig. 4C) in the culture supernatants of the treatment group compared with the pyroptosis group. Caspase-1 activity measurements revealed that the combination of P. gingivalis-LPS and ATP markedly enhanced caspase-1 activity in THP-1 macrophages. However, co-culture with hPDLSCs significantly reversed this effect (Fig. 4D).

Fig. 4.

hPDLSCs and P-sEVs inhibit macrophage pyroptosis at the protein level. THP-1 and rat primary macrophages were stimulated with 10 µg/mL P. gingivalis-LPS for 11 h, followed by 5 mM ATP for 1 h, and then co-cultured with hPDLSCs (24 h) or P-sEVs (24 h and 48 h). (A, G) Western blot analysis of NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-GSDMD, GSDMD-N, and so on in THP-1 (A) and rat primary macrophages (G) co-cultured with hPDLSCs, respectively. (B, C) Levels of IL-1β (B) and IL-18 (C) in THP-1 macrophage supernatants measured by ELISA. (D) Caspase-1 activity in THP-1 macrophages assessed using a caspase-1 activity assay kit. (E, H) Uptake of PKH67-labeled P-sEVs (green) by THP-1 (E) and rat primary macrophages (H) after 24 h of co-culture with P-sEVs; nuclei were stained with DAPI (blue). (F, I) Western blot analysis of pro-caspase-1, cleaved caspase-1, pro-GSDMD, and GSDMD-N in THP-1 (F) and rat primary macrophages (I) co-cultured with P-sEVs, respectively. GAPDH was used as the loading control. Values are presented as mean ± standard deviation (SD) from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Higher intracellular PI signal intensity indicated compromised membrane integrity under stressed conditions (Fig. S3A, C, F, H). Compared with the negative control, exposure to the P. gingivalis-LPS/ATP regimen significantly increased the proportion of PI-permeable cells (red) with damaged membranes. The percentage of PI-positive cells significantly decreased in the presence of hPDLSCs. P. gingivalis-LPS/ATP combination significantly increased ROS levels in macrophages, whereas hPDLSCs co-culture attenuated ROS-mediated oxidative stress through paracrine secretion (Fig. S3B, D, G, I). Cytotoxicity assay kits were employed to determine the amount of LDH released (Fig. S3E), yielding results consistent with those for ROS.

P-sEVs were fully internalised by macrophages after 24 h of co-culture (Fig. 4E, H). Total proteins were extracted from normal or induced macrophages after 24 and 48 h of co-culture with P-sEVs. Western blot analysis demonstrated that a high concentration of P-sEVs significantly inhibited the activation of caspase-1 at 24 h and GSDMD at 48 h in THP-1 macrophages, whereas a low concentration did not exert a significant inhibitory effect on pyroptosis during these periods (Fig. 4F). In contrast, in rat primary macrophages, both low and high concentrations significantly suppressed the activation of caspase-1 and GSDMD at 24 h and 48 h (Fig. 4I).

Characterization of HBCH/P-sEVs repair system

The HBCH and HBCH/P-sEVs repair systems comprise thermo sensitive hydrogels that exhibit biphasic reversible properties (Fig. 5A). SEM revealed a reticular structure of HBCH, with P-sEVs adhering to this network (Fig. 5B). FTIR analysis demonstrated a modification in the degree of substitution of HBCH compared with that of CS, whereas the incorporation of P-sEVs did not affect the characteristic peaks of HBCH (Fig. 5C). In the sustained release experiment, the release profiles of HBCH/P-sEVs-l and HBCH/P-sEVs-h were approximately 20% per day during the first 7 days, followed by a plateau after day 10, reaching a release rate of 80–90% by day 14 and 95% by week 4 (Fig. 5D). Rheological analysis revealed that the phase transition temperature was approximately 17 °C, with a notable decrease corresponding to an increased volume ratio of P-sEVs (Fig. 5E).

Fig. 5.

Characterisation of the HBCH/P-sEVs repair system. (A) HBCH and the HBCH/P-sEVs repair system are thermosensitive hydrogels exhibiting biphasic reversible properties. (B) SEM images showing the attachment of P-sEVs to the HBCH mesh. (C) FTIR analysis of CSH, HBCH, and HBCH/P-sEVs. (D) Release profile of the HBCH/P-sEVs repair system over 4 weeks. (E) Rheological analysis of HBCH, HBCH/P-sEVs-l, and HBCH/P-sEVs-h

Extract of HBCH/P-sEVs repair system significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs

The results demonstrated that hPDLSCs completely internalised the P-sEVs within 24 h (Fig. 6A). The CCK-8 assay revealed that the extract from HBCH/P-sEVs significantly promoted hPDLSCs proliferation over a 5-day period, with the highest concentration of P-sEVs producing the most pronounced effect (Fig. 6B). In addition, western blot analysis (Fig. 6C) and ALP staining (Fig. 6D) revealed that the extract from HBCH/P-sEVs significantly promoted the osteogenic differentiation of hPDLSCs.

Fig. 6.

Effects of the HBCH/P-sEVs repair system on proliferation and osteogenesis of PDLSCs. (A) Cellular internalisation of P-sEVs by PDLSCs. (B) Effects of HBCH and varying concentrations of HBCH/P-sEVs on PDLSC proliferation from days 1 to 7 assessed using the CCK-8 assay. (C) Western blot analysis of ALP, RUNX2, and OPN protein levels in PDLSCs treated with HBCH and different concentrations of HBCH/P-sEVs after 7 or 14 days of culture in osteogenic medium. GAPDH served as an internal control. (D) ALP staining of PDLSCs treated with HBCH and different concentrations of HBCH/P-sEVs after 14 days of osteogenic induction. Values are presented as mean ± SD from three independent experiments (*P < 0.05, ** P < 0.01, *** P < 0.001)

HBCH/P-sEVs repair system reduced pyroptosis and facilitated alveolar bone regeneration in rats

IHC results demonstrated that the positive expression of Caspase-1 (indicated by brown staining) in the HBCH/P-sEVs-h group was significantly lower than that in the other groups at the 4-week time point. At 8 weeks, although Caspase-1 expression was reduced in the treatment groups compared to the untreated group, no significant differences were observed among the treatment groups. (Fig. 7A). At 4 weeks, both the HBCH/P-sEVs-l and HBCH/P-sEVs-h groups exhibited a significant reduction in GSDMD-positive expression (brown) compared to the other groups. After 8 weeks, GSDMD-positive expression was markedly lower in the HBCH, HBCH/P-sEVs-l, and HBCH/P-sEVs-h groups compared to the remaining groups (Fig. 7B). After 4 and 8 weeks of either no implantation or implantation of various repair systems in a rat model of periodontitis with bone defects, micro-CT imaging and mandibular analyses were conducted across different treatment groups. The images revealed that the HBCH/P-sEVs-l and HBCH/P-sEVs-h groups demonstrated superior efficacy in regenerating the alveolar bone compared to the other groups at both time points. Quantitative analysis of the bone volume fraction and bone mineral density confirmed these findings, showing that both HBCH/P-sEVs-l and HBCH/P-sEVs-h significantly enhanced bone tissue regeneration compared to the untreated and HBCH groups at 4 and 8 weeks (Fig. 7C). To further evaluate the effects on bone tissue regeneration, we performed HE and Masson’s trichrome staining on mandibular specimens. HE staining revealed significant healing of the alveolar bone margins in the HBCH/P-sEVs-l and HBCH/P-sEVs-h groups at 4 and 8 weeks, with new collagen fibre formation being more pronounced at 8 weeks (Fig. 7D). Masson’s trichrome staining demonstrated increased expression of type I collagen fibres (blue) within the bone-defect areas in both HBCH/P-sEVs-l and HBCH/P-sEVs-h groups at 4 and 8 weeks (Fig. 7E).

Fig. 7.

HBCH/P-sEVs repair system reduced pyroptosis and promoted alveolar bone regeneration in rats. (A, B) IHC staining for caspase-1 (A) and GSDMD (B) in rat mandibular specimens. Brown signals indicate caspase-1 and GSDMD positivity. (C) Micro-computed tomography image of the alveolar bone on the buccal side of the left mandibular first molar. Quantitative analysis of bone volume fraction and bone mineral density at 4 and 8 weeks. * P < 0.05, ** P < 0.01, *** P < 0.001. (D, E) HE staining (D) and Masson staining (E) of rat mandibular specimen sections. Red, green, and blue arrows denote teeth, gingiva, and alveolar bone, respectively; pink/orange arrows highlight newly formed tissues. Bone defect areas and HBCH/P-sEVs are outlined by black dashed lines

Discussion

GSDMD-mediated macrophage pyroptosis plays a key role in the onset and progression of periodontitis. However, its specific involvement remains underexplored [15]. Previous studies have indicated elevated levels of the pyroptosis marker GSDME and the macrophage marker CD68 in the gingival tissues of patients with periodontitis, suggesting a link between GSDME-mediated pyroptosis and periodontitis [8]. This study further investigates the role of macrophage pyroptosis in periodontitis. H&E staining demonstrated significant macrophage infiltration in inflamed periodontal tissues compared with normal controls, accompanied by increased expression and co-localization of GSDMD and CD68, which was further confirmed by immunohistochemistry and qRT-PCR. Re-analysis of published single-cell RNA sequencing data revealed that T lymphocytes, macrophages, and B lymphocytes were enriched relative to other cell types in the periodontal tissues of patients with periodontitis, with GSDMD being highly expressed in macrophages. These findings highlight the critical role of GSDMD-mediated macrophage pyroptosis in the progression of periodontitis, offering insights into the molecular mechanisms underlying this disease.

P. gingivalis-LPS/ATP induces pyroptosis in macrophages via the NF-κB/NLRP3/GSDMD signalling pathway. To further elucidate the molecular mechanisms underlying macrophage-associated pyroptosis in periodontitis, we investigated the NF-κB/NLRP3/GSDMD signaling pathway in both THP-1 macrophages and rat primary macrophage [16–20]. Previous studies have demonstrated that ATP combined with Escherichia coli-LPS can induce macrophage pyroptosis [21].

To simulate the pathogenic process of periodontitis, we used P. gingivalis-LPS at a concentration of 10 µg/mL combined with ATP, which produced the most pronounced effects in macrophages. These included characteristic pyroptotic morphology, elevated NLRP3 expression, cleavage of the GSDMD N-terminal domain, and activation of IL-1β and caspase-1. Since NF-κB is the primary responder to pathogenic and/or inflammatory stimuli, such as LPS exposure, it plays a critical role in initiating downstream signals, including the activation of NLRP3 [22]. To further validate this, NF-κB was silenced in THP-1 macrophages, resulting in a marked reduction in NLRP3 and GSDMD expression during subsequent pyroptosis induction. Under NF-κB silencing, the lack of further suppression of NLRP3 and GSDMD-N expression by combined MCC950 treatment indicates that the regulatory effect of NF-κB is primarily mediated through NLRP3; moreover, the use of disulfiram confirmed that this pathway indeed executes pyroptosis via the canonical GSDMD-dependent mechanism. This study elucidates the specific molecular mechanisms by which P. gingivalis-LPS and ATP contribute to periodontitis progression, emphasising the NF-κB/NLRP3/GSDMD axis as a key pathway in macrophage pyroptosis. These findings highlight the potential of this signalling axis as a therapeutic target for the treatment of periodontitis.

The paracrine effects of hPDLSCs and the direct actions of P-sEVs suppress pyroptosis in macrophages via the NF-κB/NLRP3/GSDMD signalling axis. P-sEVs, which are produced through the paracrine activity of hPDLSCs, carry various bioactive molecules that facilitate intercellular communication between hPDLSCs and other cell types [11]. The application of P-sEVs as a therapeutic strategy to promote periodontal tissue regeneration has become a significant research focus in the context of periodontitis treatment [23, 24]. However, there are limited studies on the immunoregulatory mechanisms of P-sEVs in periodontitis. In this study, macrophage pyroptosis was successfully induced and subsequently assessed in vitro using co-culture experiments with hPDLSCs and P-sEVs. The results demonstrated a significant downregulation of NF-κB, NLRP3, caspase-1, and GSDMD at both the gene and protein levels in the co-culture groups. These findings indicate that hPDLSCs, through paracrine signaling, and P-sEVs, through direct interaction, modulate the NF-κB/NLRP3/GSDMD signaling axis to inhibit macrophage pyroptosis. Moreover, P-sEVs were internalized by both THP-1 and rat primary macrophages. Both high (50 µg/mL) and low (10 µg/mL) concentrations of P-sEVs effectively inhibited macrophage pyroptosis, suggesting that P-sEVs may function as potent “immunomodulators” capable of attenuating excessive inflammatory responses. hPDLSC-derived sEVs have become a focal point in contemporary medical research due to their favorable safety profile, scalability, and potential for targeted delivery. Nevertheless, further investigations are needed to elucidate their underlying mechanisms and to optimize their therapeutic efficacy for clinical applications.

HBCH/P-sEVs composite therapy effectively promoted periodontal osteogenesis and inhibited pyroptosis. Recently, hydrogels have gained prominence in tissue engineering because of their ability to provide a controlled and sustained release of encapsulated agents [25, 26]. Among these, HBCH has been extensively developed and applied in various fields, including stem cell culture and drug delivery [27, 28]. To enhance the application of P-sEVs in the local periodontal environment and optimise them for clinical use, we incorporated P-sEVs into HBCH to synthesise an HBCH/P-sEVs composite therapy system. This therapeutic system exhibits excellent properties, including temperature sensitivity, biocompatibility, reversible phase transition, and sustained release, which significantly improves the clinical applicability of P-sEVs. In vitro studies of the HBCH/P-sEVs composite system validated its biological functionality, demonstrating that it not only promoted the proliferation of hPDLSCs but also enhanced their osteogenic differentiation. Furthermore, when implanted into a rat model of periodontitis-induced bone defects, this material system effectively promoted osteogenesis and inhibited pyroptosis in the inflammatory periodontal microenvironment. These findings highlight the advantages of the HBCH/P-sEVs composite system in terms of immunoregulation, safety, and tissue regeneration, establishing a strong foundation for its clinical application in periodontitis treatment.

Overall, this study demonstrates that P-sEVs can attenuate P. gingivalis-LPS/ATP-induced macrophage pyroptosis by inhibiting the NF-κB/NLRP3/GSDMD pathway and, when combined with HBCH, offer a potential strategy for periodontal repair and regeneration (Fig. 8). However, the precise underlying mechanisms remain to be fully elucidated and warrant further investigation.

Fig. 8.

Schematic diagram showing that P-sEVs suppress P. gingivalis-LPS/ATP-induced pyroptosis in macrophages via the NF-κB/NLRP3/GSDMD pathway, and can be incorporated with thermosensitive HBCH to construct a potential repair and regeneration system for periodontitis therapy

Conclusions

In summary, macrophage pyroptosis plays a crucial role in the onset and progression of periodontitis. In vitro experiments utilizing 10 µg/mL of P. gingivalis-LPS for 11 h, combined with 1 h of 5 mM ATP, effectively mimicked the periodontal inflammatory microenvironment and activated GSDMD-mediated pyroptosis in macrophages. The paracrine effect of hPDLSCs and P-sEVs efficiently inhibited the activation of the NF-κB/NLRP3/GSDMD-mediated classical pyroptosis pathway. In vivo, a combination of P-sEVs and phase-reversible temperature-sensitive HBCH was implanted into an inflammatory bone defect model in rats, where it promoted osteogenesis and exerted antipyroptotic effects in the inflammatory periodontal microenvironment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Caixiu Yang: conceptualization, formal analysis, writing–original draft. Wenxuan Wang: methodology, writing–original draft. Chao Feng and Shuo Xu: writing–review and editing Li Zhang: data curation. Yanwen Wang: validation, writing–review and editing. Xiaoming Xing: Supervision, visualization. Qiuxia Ji: supervision, project administration, funding acquisition.

Data availability

The data used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval

This study was approved by the Ethics Committee of the Qingdao University Affiliated Hospital (QYFY WZLL 27126). The research was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki.

Patient consent

All patients involved in this study provided written informed consent. Prior to agreeing to participate, all patients were fully informed about the purpose, scope, potential risks, and benefits of the study. They clearly understood that participation was entirely voluntary.

Competing interests

The authors declare no competing interests.