hPDLSC-ApoEVs attenuate periodontitis and enhance bone regeneration via NF-κB/S100A9-Mediated M2 macrophage polarization

Abstract

Background

Periodontitis is the most prevalent oral disease worldwide, leading to inflammation, alveolar bone loss, and tooth loss. Mesenchymal stem cells (MSCs) transplantation has the potential to alleviate periodontitis and restore alveolar bone, but numerous studies have found that transplanted MSCs undergo apoptosis, releasing apoptotic extracellular vesicles (ApoEVs). Human periodontal ligament stem cells (hPDLSCs) ApoEVs have immunomodulatory and tissue regenerative potential. Still, their role in periodontitis treatment and alveolar bone restoration has not been investigated yet.

Methods

The study isolated hPDLSCs-ApoEVs and investigated the modulatory effect on macrophaghe. We explored the specific molecular mechanisms of hPDLSC-ApoEVs in regulating macrophage polarization in vitro through mRNA-seq and inhibitors. Co-culture experiment demonstrated that hPDLSC-ApoEVs promoted hPDLSC osteogenic differentiation by regulating macrophage polarization. Finally, we verified that hPDLSC-ApoEVs inhibited periodontitis-induced periodontal tissue defects and promoted periodontal bone tissue regeneration in vivo.

Results

This study found that hPDLSCs-ApoEVs regulate M0 to anti-inflammatory M2 macrophage polarization, as indicated by the upregulation of CD163, IL-10, and ARG1. hPDLSCs-ApoEVs activated NF-κB signaling that upregulated S100A9 in macrophages. hPDLSCs-ApoEVs failed M2 macrophage polarization during inhibition of NF-κB or S100A9. Furthermore, hPDLSCs-ApoEVs-treated macrophage-conditioned medium robustly promoted osteogenic differentiation of hPDLSCs in vitro. Finally, we verified these results in the periodontitis model of male C57BL/6 mice. Local injection of hPDLSCs-ApoEVs facilitated macrophage M2 polarization, alleviated periodontitis, and restored alveolar bone.

Conclusion

In conclusion, these findings provide new evidence for the clinical application of hPDLSCs-ApoEVs in periodontitis treatment via macrophage immune modulation-mediated alveolar bone restoration.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04617-7.

Introduction

Periodontitis is a chronic inflammatory disease that damages periodontal tissues, often resulting in persistent pain, gingival swelling, alveolar bone loss, and eventual tooth loss [1]. The complex anatomy and composition of the periodontium pose significant challenges to tissue regeneration [2]. Various treatment modalities, including cell-based therapies, have been explored to address these issues. Mesenchymal stem cells (MSCs) have been widely studied for decades due to their potential in disease treatment and tissue regeneration [3]. Among these, human periodontal ligament stem cells (hPDLSCs) are particularly promising because of their plasticity, ease of harvesting, and ability to promote periodontal tissue repair and regeneration [4, 5]. However, the clinical application of stem cell therapies faces several limitations. The inflammatory environment in periodontitis induces oxidative stress, cell death, and impaired differentiation of transplanted cells [6]. Additionally, ethical concerns, limited cell yield, and the loss of “stemness” further restrict the widespread adoption of traditional stem cell therapies for periodontitis [7]. These challenges underscore the urgent need for alternative therapeutic strategies to treat periodontitis effectively.

Research has demonstrated that transplanted stem cells often undergo apoptosis and rapidly disappear after transplantation [8]. The byproducts of this apoptosis, along with their paracrine effects, play a key role in regulating macrophage immunomodulation, which in turn influences disease progression and severity [8]. These findings highlight the potential of active substances derived from apoptotic mesenchymal stem cells (MSCs) as a basis for developing non-cellular therapeutic approaches [9]. Consequently, identifying the specific components of apoptotic MSC byproducts that contribute to disease treatment and tissue regeneration is crucial for advancing innovative therapies.

Apoptotic cell-derived extracellular vesicles (ApoEVs) are key byproducts of apoptotic cells, playing essential roles in maintaining stem cell homeostasis, regulating macrophage activity, promoting tissue regeneration, and inhibiting tumor growth [10–14]. Recognized as critical therapeutic agents in stem cell transplantation, ApoEVs offer a non-cellular approach with lower immunogenicity than traditional stem cell therapies, alongside demonstrated anti-inflammatory and tissue regenerative properties^15,16. During apoptosis, cells encapsulate themselves into ApoEVs, which attract macrophages for recognition and phagocytosis. These vesicles modulate macrophage activity and activate anti-inflammatory or pro-inflammatory signaling pathways through paracrine effects [15]. Additionally, ApoEVs can naturally enter macrophages via immune recognition, making them powerful regulators of macrophage functions [16].

Macrophages display diverse functional phenotypes in response to various stimuli, which are closely linked to the progression and resolution of periodontitis [17–20]. M1 macrophages are associated with tissue damage and bone resorption, while M2 macrophages contribute to tissue repair and regeneration [21–23]. Previous studies have demonstrated that ApoEVs derived from platelets and mesenchymal stem cells (MSCs) can either suppress M1 macrophage polarization or promote M2 macrophage polarization [17, 18]. Although the regenerative potential of human periodontal ligament stem cells (hPDLSCs) in periodontal tissues is well-documented [4], the specific role of hPDLSCs-derived ApoEVs in treating periodontitis and restoring alveolar bone remains poorly understood.

This study aimed to investigate the role of hPDLSCs-derived apoptotic extracellular vesicles (hPDLSCs-ApoEVs) in macrophage immunomodulation for periodontitis treatment and alveolar bone restoration, while elucidating the underlying molecular mechanisms. Using a combination of in vitro experiments and in vivo validation in a mouse periodontitis model, we explored the effects of hPDLSCs-ApoEVs on macrophage polarization and osteogenic differentiation of precursor cells. Our findings demonstrate that hPDLSCs-ApoEVs promote M2 macrophage polarization through the NF-κB/S100A9 signaling axis, alleviating periodontitis and restoring alveolar bone. These results provide a foundation for developing innovative, minimally invasive strategies to treat periodontal diseases, addressing the critical need for effective tissue regeneration approaches in periodontology.

Materials and methods

Particular materials and methods are shown in Appendix A (Supplementary Material).

Isolation and culture of hPDLSCs

hPDLSCs were obtained from healthy human premolars extracted for orthodontic purposes with informed consent at the Preventive Dentistry Department of the School and Hospital of Stomatology, Guangzhou Medical University. The experiments were conducted with the approval of the Medical Ethical Committee of the School and Hospital of Stomatology, Guangzhou Medical University (Approval Number: JCYJ2023001). Periodontal ligament (PDL) tissues were obtained from the middle part of the premolar root surface. The PDL tissue was minced into small pieces and digested with collagenase I (4 mg/mL; Merck, USA) and dispase II (4 mg/mL; Merck, USA) for 2 h at 37 °C. After digestion, suspensions were filtered using a 70 μm cell strainer and cultured in α-minimal essential medium (α-MEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 100 U/mL penicillin/streptomycin (P/S; Gibco, USA) at 37 °C with 5% CO₂. The medium was changed every 3 days. These cells were recognized as P0 cells. Upon reaching 80–90% confluence, cells were trypsinized and further passaged. Cells (passages 3 to 5) were utilized for subsequent experiments.

Isolation of hPDLSC-ApoEVs

The hPDLSCs were exposed to 0.5 µM staurosporine (MedChemExpress, USA) for 12 h at 37 °C to induce apoptosis. Subsequently, the supernatant was first centrifuged at 1,000 g for 10 min at 4 °C to remove cell debris. The resulting supernatant was centrifugated at 16,000 g for 30 min at 4 °C to obtain ApoEVs in the pellet. The isolated ApoEVs were washed twice with PBS, resuspended in PBS, and quantified by a BCA Protein Assay Kit (Beyotime, Hunan, China).

mRNA-seq analysis

The total RNA of hPDLSCs treated for 12 h with or without hPDLSC-ApoEVs was isolated by the TRIzol reagent (Invitrogen, USA). mRNA Sequencing was performed by Guangzhou Huayin Medical Laboratory Center Co., Ltd. (Guangzhou, China). The raw sequencing data underwent quality control and the high-quality sequencing data were compared with the designated reference genome. The datasets of mRNA-seq are available in the Sequence Read Archive (SRA) repository (PRJNA1269638), (https://www.ncbi.nlm.nih.gov/sra/PRJNA1269638).

Macrophage conditioned media experiment

THP-1 cells were plated in six-well plates at a density of 2 × 10⁶ cells/well and differentiated into macrophages by treatment with 100 ng/mL PMA for 24 h. Subsequently, THP-1 macrophages were cultured in RPMI-1640 supplemented with 10% FBS and 100 U/mL P/S, with or without 5 µg/mL ApoEVs for 48 h at 37 °C. After incubation, the medium was discarded, cells were washed twice with PBS and then incubated in α-MEM medium (without FBS and P/S) for 24 h. The supernatants were filtered using 0.22 μm pore-size filters (Millipore, Burlington, MA, USA) to obtain conditioned medium (CM), prepared by mixing with α‐MEM at a 1:1 ratio. The medium from unstimulated THP-1 macrophages was designated the CM-M0 group, and the medium from biomaterial-stimulated macrophages was designated the CM-ApoEVs group. hPDLSCs were seeded in six-well or forty-eight-well plates, and after reaching 80–90% confluence, the culture medium was replaced with CM for 7 days. hPDLSCs cultured in an OM served as a control.

Periodontitis models

Male wild-type (WT) C57BL/6 mice aged 6–8 weeks (18–20 g) purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd. were selected for establishing the periodontitis model. The Ethics Committee for Animal Experiments at Guangzhou Medical University approved the animal studies used in this study (Approval Number: G2023-153). Mice were housed in specific pathogen-free conditions (24 °C, 12 h light/dark cycle, and 50% humidity) with ad libitum access to food and water. They were randomly assigned to the following groups (n = 6, total = 12): (1) the PBS group and (2) the hPDLSC-ApoEVs group.

To establish the mouse periodontitis model, 5 − 0 silk ligatures were tied around the second maxillary molars on the left side of the mice under the anesthesia of 0.15 mL/10 g 1.5% tribromoethanol. After ligating for 7 days, the ligatures were removed. PBS containing or not containing apoptotic cell-derived extracellular vesicles was injected into the palatal periodontal pocket of the second maxillary molars using a microliter syringe (20 µL). The mice that the silk ligatures fell off after 7 days were excluded. The contralateral molars without ligation served as randomly selected controls for bone defect measurement. Throughout the experiment, the ligatures remained intact in each mouse. After 7 days, the mice were euthanized using CO₂, and the heart, liver, spleen, lungs, kidneys, and maxillary bones were harvested for further analysis. The work has been reported in line with the ARRIVE guidelines 2.0.

Statistical analysis

All experimental data were analyzed using GraphPad Prism 9.0 statistical software, and quantitative data were expressed as mean ± standard deviation (x ± s). A p-value < 0.05 was considered statistically significant. For comparisons between two groups, the independent samples t-test was employed when the data satisfied the assumptions of normal distribution and homogeneity of variances; otherwise, the Mann-Whitney U test was used. A one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used for statistical comparisons among multiple groups (> 2).

Results

Isolation and identification of hPDLSCs

Initially, hPDLSCs were carefully isolated from the human periodontal ligament. Multilineage differentiation assays confirmed their ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages (Figure. 1A-C). Additionally, hPDLSCs exhibited strong proliferation potential (Figure. 1D). Colony-forming assays further demonstrated that the isolated primary cells possessed clonal proliferation and differentiation capabilities (Figure. 1D). Flow cytometry analysis revealed that over 98% of hPDLSCs expressed MSC surface markers CD73, CD105, CD29, CD44, and CD90, while hematopoietic markers CD34 and CD45 were expressed in less than 2% of cells (Figure. 1E). These findings confirm the stem cell characteristics of the isolated hPDLSCs.

Fig. 1.

Identification and functional characterization of human periodontal ligament stem cells (hPDLSCs). (A) Oil Red O staining demonstrating lipid droplet accumulation in hPDLSCs following adipogenic differentiation. (B) Alizarin Red staining revealing calcium-rich mineralized matrix deposition after osteogenic induction. (C) Alcian Blue staining detecting sulfated glycosaminoglycans, indicative of chondrogenic differentiation. (D) Colony-forming unit (CFU) assay illustrating the clonogenic potential of hPDLSCs. (E) Flow cytometry analysis confirming the expression of mesenchymal stem cell (MSC) surface markers (e.g., CD73, CD90, CD105) in hPDLSCs

Characterization of hPDLSCs-ApoEVs

Flow cytometry analysis was employed to assess the identity and purity of the isolated hPDLSC-derived apoptotic extracellular vesicles (hPDLSC-ApoEVs). The results showed high expression of Annexin V, a well-established marker for ApoEVs (Figure. 2A). Dynamic light scattering (DLS) analysis revealed that the size of these vesicles ranged from 200 to 400 nm (Figure. 2B). Transmission electron microscopy (TEM) images further confirmed their characteristic cup-shaped morphology and distinct bilayer membrane structure, with diameters approximately between 200 and 300 nm (Figure. 2C). Western blot analysis demonstrated the presence of cleaved-caspase 3, a specific marker for ApoEVs, within the vesicles. Notably, caspase 3 expression was significantly lower in hPDLSCs-ApoEVs compared to the parent hPDLSCs (Figure. 2D). These findings collectively validate the successful isolation and characterization of hPDLSC-ApoEVs.

Fig. 2.

Characterization of human periodontal ligament stem cell-derived apoptotic extracellular vesicles (hPDLSCs-ApoEVs). (A) Flow cytometry analysis of Annexin V and propidium iodide (PI) staining to confirm the apoptotic origin of hPDLSCs-ApoEVs. (B) Dynamic light scattering (DLS) analysis showing the particle size distribution profile of hPDLSCs-ApoEVs. (C) Representative transmission electron microscopy (TEM) images depicting the morphology and ultrastructure of hPDLSCs-ApoEVs. (D) Western blot analysis detecting protein levels of caspase-3 and its activated form, cleaved-caspase-3, in hPDLSCs-ApoEVs. Full-length blots/gels are presented in Appendix Fig. 6

Internalization of hPDLSCs-ApoEVs by THP-1 macrophages

To explore the interaction between hPDLSCs-ApoEVs and macrophages, THP-1 cells were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA). Confocal fluorescence microscopy analysis confirmed the differentiation, as PMA-treated THP-1 cells exhibited significantly higher expression of the macrophage marker CD68 compared to untreated THP-1 cells (Figure. 3A, B). To assess ApoEV uptake, PKH-67-labeled hPDLSCs-ApoEVs (green) were incubated with macrophages. Confocal imaging revealed successful internalization of the labeled ApoEVs by the macrophages (Figure. 3C, Appendix Fig. 3A-B). The co-localization of green (ApoEVs) and red (macrophages) fluorescence confirmed that the ApoEVs were actively taken up by the macrophages, rather than merely adhering to their surface. This demonstrates a direct interaction between hPDLSCs-ApoEVs and macrophages.

Fig. 3.

Internalization of human periodontal ligament stem cell-derived apoptotic extracellular vesicles (hPDLSC-ApoEVs) by THP-1 macrophages. (A, B) Immunofluorescence analysis of CD68 expression in THP-1 macrophages, comparing phorbol 12-myristate 13-acetate (PMA)-treated and untreated conditions. (C) Confocal microscopy images showing cellular uptake of PKH67-labeled hPDLSC-ApoEVs (green) by THP-1 macrophages, with actin cytoskeleton stained using phalloidin (red). Data are expressed as mean ± standard deviation (SD) from three independent experiments (n = 3). Statistical significance between groups is indicated as ***p < 0.001

hPDLSCs-ApoEVs promoted M2 macrophage polarization

hPDLSCs-ApoEVs demonstrated cytocompatibility with macrophages at concentrations of 2.5, 5, 10, and 20 µg/mL (Figure. 4A). Compared to the untreated group, hPDLSCs-ApoEVs treatment significantly upregulated the expression of M2 macrophage markers at both mRNA (ARG-1, IL-10, and CD206) and protein levels (CD163 and ARG-1), as confirmed by Western blot analysis (Figure. 4B-F) and RT-qPCR (Figure. 4G-I). Notably, only the 10 µg/mL concentration of hPDLSCs-ApoEVs significantly suppressed the M1 macrophage marker INOS. Immunofluorescence analysis further validated these results (Figure. 4J-O). These findings suggest that hPDLSCs-ApoEVs promote macrophage M2 polarization, which is closely associated with anti-inflammatory and tissue regenerative processes, highlighting their potential role in immunomodulation and periodontal tissue repair.

Fig. 4.

Modulation of THP-1 macrophage polarization by human periodontal ligament stem cell-derived apoptotic extracellular vesicles (hPDLSCs-ApoEVs). (A) Cell Counting Kit-8 (CCK-8) assay assessing the viability of THP-1 macrophages. (B–F) Western blot analysis of M1 macrophage markers (CD80 and inducible nitric oxide synthase, iNOS) and M2 macrophage markers (CD163 and arginase-1, ARG-1). Full-length blots/gels are presented in Appendix Figs. 7, 8, 9 and 10. (G–I) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of M2 macrophage markers (ARG-1, interleukin-10 [IL-10], and CD206). (J–L) Immunofluorescence staining of CD163 and IL-10 in THP-1 macrophages. (M–O) Immunofluorescence staining of CD80 and iNOS in THP-1 macrophages. Data are presented as mean ± standard deviation (SD) from three independent experiments (n = 3). Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001 (ns: not significant)

hPDLSCs-ApoEVs activated NF-κB/S100A9 signaling in macrophages to promote M2 polarization

To further explore the mechanisms underlying M2 macrophage polarization, mRNA sequencing (mRNA-seq) was performed. Gene Ontology (GO) analysis revealed that inflammation-related signaling pathways were significantly enriched in macrophages treated with hPDLSCs-ApoEVs (5 µg/mL) (Figure. 5A). Additionally, mRNA-seq results showed a marked upregulation of S100A9 expression in the hPDLSCs-ApoEVs-treated group compared to the control (Figure. 5B). These findings suggest that the anti-inflammatory effects induced by hPDLSCs-ApoEVs may drive M2 macrophage polarization, potentially mediated by the upregulation of S100A9 and activation of inflammation-related pathways. This highlights a key molecular mechanism by which hPDLSCs-ApoEVs promote macrophage immunomodulation and tissue repair.

Fig. 5.

hPDLSCs-ApoEVs promote M2 polarization of THP-1 macrophages via the NF-κB/S100A9 signaling pathway. (A) Gene Ontology (GO) analysis categorizing enriched terms in biological processes, cellular components, and molecular functions. (B) Volcano plot illustrating the distribution of differentially expressed genes. (C–E) Western blot analysis of CD163, phosphorylated NF-κB p65 (p-p65), total NF-κB p65 (t-p65), and S100A9 protein expression in THP-1 macrophages. Full-length blots/gels are presented in Appendix Figs. 11, 12, 13, 14 and 15. (F–H) Immunofluorescence staining of CD163 and interleukin-10 (IL-10) in THP-1 macrophages. Data are presented as mean ± standard deviation (SD) from three independent experiments (n = 3). Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001 (ns: not significant). Bay: BAY 11-7082 (NF- κB inhibitor), Paq: Paquinimod (S100A9 inhibitor)

Given the critical role of NF-κB in immune regulation and its upregulated signaling in response to hPDLSCs-ApoEVs, we investigated the involvement of the NF-κB/S100A9 axis in hPDLSCs-ApoEVs-mediated M2 macrophage polarization. After 24 h of hPDLSCs-ApoEVs treatment, the ratio of phosphorylated NF-κB p65 (p-p65) to total NF-κB p65 (t-p65) significantly increased, accompanied by upregulated S100A9 expression. Inhibition of NF-κB signaling downregulated hPDLSCs-ApoEVs-induced S100A9 and CD163 expression in macrophages (Figure. 5C, E). Similarly, inhibition of S100A9 abolished ApoEVs-induced M2 macrophage polarization but did not affect NF-κB phosphorylation (Figure. 5D), indicating that NF-κB acts upstream of S100A9 in ApoEVs-treated macrophages. Immunofluorescence analysis further confirmed that inhibition of NF-κB or S100A9 reduced CD163 (green) and IL-10 (red) expression in ApoEVs-treated macrophages (Figure. 5F-H). To further elucidate the activation status of NF-κB, we detect P65 nuclear translocation and phosphorylation of IκB-α. Immunofluorescence analysis indicated that ApoEVs-treated macrophage exhibited no significant nuclear translocation (Appendix Fig. 4A-B.). Western blot analysis showed that ApoEVs-stimulated macrophages showed no significant difference in IκB-α phosphorylation (Appendix Fig. 4C.). These results demonstrate that hPDLSCs-ApoEVs promote M2 macrophage polarization, likely through the NF-κB/S100A9 axis, highlighting a key molecular mechanism underlying their immunomodulatory effects.

hPDLSCs-ApoEVs-Treated macrophages promoted osteogenic differentiation of hPDLSCs

To explore the impact of hPDLSCs-ApoEVs-treated macrophages on the osteogenic potential of hPDLSCs, we assessed the effect of conditioned medium (CM) from macrophages on hPDLSCs. CM was prepared using THP-1 macrophages, as previously described [24]. Alkaline phosphatase (ALP) staining revealed that the CM-ApoEVs group significantly enhanced ALP expression at day 7 compared to other groups (Figure. 6A, B). Similarly, Alizarin Red staining demonstrated increased calcium deposition at day 21 in the CM-ApoEVs group compared to the osteogenic medium (OM) and CM-M0 groups (Figure. 6C, D).

Fig. 6.

hPDLSC-ApoEVs enhance osteogenic differentiation of hPDLSCs by promoting macrophage M2 polarization. (A, B) Alkaline phosphatase (ALP) staining of hPDLSCs. (C, D) Alizarin Red staining of hPDLSCs to assess mineralized matrix deposition. (E) Immunofluorescence analysis of osteogenic markers osteopontin (OPN) and collagen type I (COL-1) in hPDLSCs, with nuclei counterstained using DAPI. (F–I) Western blot analysis of OPN, COL-1, and runt-related transcription factor 2 (RUNX2) protein expression in hPDLSCs. Full-length blots/gels are presented in Appendix Figs. 16, 17 and 18. Data are presented as mean ± standard deviation (SD) from three independent experiments (n = 3). Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001 (ns: not significant). ALP: Alkaline phosphatase; DAPI: 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; OPN: Osteopontin; COL-1: Collagen Type I; RUNX2: Runt-related transcription factor 2. OM: Osteogenic medium, CM-M0:M0 macrophage-derived conditioned medium, CM-ApoEVs: ApoEVs-treated macrophage-derived conditioned medium

Immunofluorescence (Figure. 6E, Appendix Figure. 1A-B) and Western blot analysis (Figure. 6F-I) further supported these findings, showing robust expression of osteogenic markers, including osteopontin (OPN), runt-related transcription factor 2 (RUNX2), and collagen type I (COL-1), in the CM-ApoEVs group compared to the CM-M0 group. To exclude the direct effect of ApoEVs on hPDLSCs, we detected mRNA expression of osteogenic marker. QRT-PCR analysis indicated that CM-ApoEVs significantly increased mRNA expression of osteogenic marker including bone morphogenetic protein 4 (BMP4), osteocalcin (OCN), bone morphogenetic protein 2 (BMP2) and bone sialoprotein (BSP) of hPDLSCs (Appendix Fig. 5A-D). Compared to CM, ApoEVs exerted a weaker effect on osteogenic gene expression. While OPN and BSP showed no significant alterations, BMP2 and BMP4 exhibited modest enhancement—though the magnitude remained lower than that induced by CM. These results collectively indicate that hPDLSCs-ApoEVs enhance the osteogenic differentiation of hPDLSCs through macrophage immunomodulation, underscoring their potential efficacy in promoting alveolar bone regeneration.

hPDLSCs-ApoEVs alleviated periodontitis and restored alveolar bone in mice

To further evaluate the potential of hPDLSCs-ApoEVs in periodontal bone regeneration, hPDLSCs-ApoEVs were locally injected into the periodontal region of mice with periodontitis. Micro-CT and histological analysis revealed improved alveolar bone quality in hPDLSCs-ApoEVs-treated mice compared to PBS-treated controls (Figure. 7A-C). Additionally, periodontal tissues in ApoEVs-treated mice exhibited reduced immune cell infiltration. To investigate the role of the NF-κB p65/S100A9 signaling pathway in M2 macrophage polarization during periodontitis, immunohistochemical analysis was performed, showing increased expression of p-p65 and S100A9 in the hPDLSCs-ApoEVs group compared to the control group (Figure. 7D-F).

Fig. 7.

hPDLSCs-ApoEVs alleviate periodontitis and restore alveolar bone resorption in a mouse model. (A) Micro-computed tomography (Micro-CT) images of alveolar bone (n = 6). (B) Quantitative analysis of micro-CT data. (C) Hematoxylin and eosin (H&E) staining of periodontal tissue (n = 3). (D) Immunohistochemical staining of periodontal tissue (n = 3). (E–H) Quantitative analysis of immunohistochemical staining results. (I) Masson’s trichrome staining of periodontal tissue (n = 3). (J) Immunofluorescence staining analysis of periodontal tissue (n = 3). (K) Schematic representation of the proposed mechanism: hPDLSCs-ApoEVs induce macrophage M2 polarization via the NF-κB/S100A9 signaling axis, promoting periodontal tissue regeneration (n = 3). Data are presented as mean ± standard deviation (SD). Statistical significance is indicated as *p < 0.05 and **p < 0.01

Immunohistochemical and Masson staining further demonstrated that hPDLSCs-ApoEVs enhanced the expression of osteogenic markers, including osteopontin (OPN), runt-related transcription factor 2 (RUNX2), and collagen, indicating improved bone regeneration potential in the inflamed periodontal environment (Figure. 7D, G-I). Immunofluorescence analysis confirmed elevated levels of S100A9 and the M2 macrophage marker CD163 in the periodontal tissues of hPDLSCs-ApoEVs-treated mice (Figure. 7J). Importantly, serum biochemistry and histology of vital organs showed no adverse effects on liver or kidney function, confirming the biosafety of hPDLSCs-ApoEVs treatment in vivo (Appendix Figure. 2A-K).

These findings demonstrate that hPDLSCs-ApoEVs alleviate periodontitis and promote alveolar bone restoration in mice through NF-κB/S100A9 axis-mediated M2 macrophage polarization, highlighting their therapeutic potential for periodontal tissue regeneration.

Discussion

The balance between osteoblast and osteoclast activity is crucial for maintaining periodontal bone homeostasis and overall periodontal health [25]. Mesenchymal stem cell-derived apoptotic extracellular vesicles (MSCs-ApoEVs) have demonstrated significant potential in bone tissue regeneration by regulating osteogenesis and immunomodulation [26]. Among MSCs, periodontal ligament stem cells (PDLSCs) are particularly promising for tissue regeneration and the treatment of diseases such as periodontitis. In this study, we found that hPDLSCs-ApoEVs promote M2 macrophage polarization primarily through the NF-κB/S100A9 signaling pathway. This M2 polarization further enhanced the osteogenic differentiation of hPDLSCs. Our in vivo experiments confirmed that hPDLSCs-ApoEVs alleviate periodontitis and restore alveolar bone in a mouse periodontitis model. We observed M2 macrophage polarization, activation of NF-κB signaling, and upregulation of S100A9 in the periodontal tissues of hPDLSCs-ApoEVs-treated mice. These findings demonstrate that hPDLSCs-ApoEVs mitigate periodontitis and promote alveolar bone regeneration through macrophage immunomodulation. This highlights the potential clinical application of hPDLSCs-ApoEVs as a novel therapeutic strategy for periodontitis treatment.

A higher ratio of M1 to M2 macrophages in periodontal tissue is strongly associated with the progression and severity of periodontitis [27]. Previous studies have shown that hPDLSCs-induced M2 macrophage polarization promotes periodontal tissue regeneration [28], and apoptotic bodies from adipose-derived MSCs have demonstrated the ability to polarize macrophages toward the M2 phenotype during diabetic wound healing [29]. In our in vitro study, hPDLSCs-ApoEVs exhibited significant potential to induce M2 macrophage polarization. Transcriptome analysis, supported by RT-qPCR and Western blot, revealed upregulation of NF-κB and S100A9 in hPDLSCs-ApoEVs-treated macrophages. This aligns with previous findings that the TLR4/NF-κB/S100A9 cascade drives M2 polarization [30]. Notably, inhibition of NF-κB reversed hPDLSCs-ApoEVs-induced M2 macrophage polarization and downregulated S100A9 expression, while inhibition of S100A9 also reversed M2 polarization without affecting NF-κB activation. These results indicate that NF-κB acts upstream of S100A9 and that hPDLSCs-ApoEVs promote M2 macrophage polarization through the NF-κB/S100A9 signaling axis. The precise biological mechanism by which ApoEVs activated NF-κB remains to be elucidated. Park SJ et al. reported that ApoEVs could act as damage-associated molecular patterns (DAMPs) and activated NF-κB through sphingosine-1-phosphate receptors 1 (S1PR1) [31]. Whether hPDLSC-ApoEVs activated NF-κB through analogous pathways remains further exploration. Curiously, NF-κB and S100A9 are traditionally linked to pro-inflammatory M1 polarization in diseases but not M2 polarization. NF-κB pathway, a multi-functional pathway, consists of canonical and non-canonical pathways [32]. El-Shinawi M er al. reported that activation of NF-κB induced secretion of M2 cytokine. Our result showed that hPDLSC-ApoEVs induced P65 phosphorylation, yet exerted no significant effects on IκBα phosphorylation and nuclear translocation of P65 [33]. Hu et al. reported that Fusobacterium nucleatum facilitated M2 Macrophage Polarization by activating TLR4/NF-κB/S100A9 cascade [30]. Our result indicated that hPDLSC-ApoEVs elicit a non-canonical NF-κB activation state characterized by P65 phosphorylation that selectively engaged M2-polarizing pathways while. The exact mechanisms underlying this subunit-specific regulation remained to be elucidated. S100A9, a calcium-binding protein, serves as a key DAMPs released by activated phagocytes. It acts as an endogenous ligand for the TLR4 complex, promoting inflammatory progression, suppressing M2 macrophage differentiation, and inducing pro-inflammatory functions [34, 35]. Although S100A9 is widely recognized as a pro-inflammatory mediator, emerging evidence suggests its involvement in M2 macrophage polarization and tissue repair. Kwak T et al. reported that upregulation of S100A9 in macrophages increases PGE2 production and upregulates C/EBP-β which induced M2 polarization [36]. Hu et al. demonstrate that activation of the TLR4/NF-κB/S100A9 cascade facilitates M2 polarization [30]. Additionally, S100A9 deficiency during the repair phase following acute kidney injury impairs M2 polarization, leading to renal fibrosis and persistent damage [37]. Thus, S100A9 may regulate inflammatory processes and restore homeostasis, with appropriate expression levels potentially enhancing host defense and immune equilibrium [38].

This mechanism underscores the therapeutic potential of hPDLSCs-ApoEVs in modulating macrophage behavior to support tissue repair and regeneration in periodontitis.

M2 macrophages play a critical role in exerting anti-inflammatory effects and promoting the osteogenic differentiation of precursor cells [39]. Periodontal ligament stem cells (PDLSCs) are vital precursor cells for periodontal tissue regeneration, as they can differentiate into cementum, alveolar bone, and periodontal ligament-like tissues [4]. In this study, we found that macrophages treated with hPDLSCs-ApoEVs significantly enhance the osteogenic differentiation of hPDLSCs. These results demonstrate that hPDLSCs-ApoEVs-induced M2 macrophage polarization exerts anti-inflammatory effects and promotes periodontal tissue regeneration. This highlights the dual therapeutic potential of hPDLSCs-ApoEVs in modulating inflammation and supporting tissue repair in periodontitis.

Pathogenic microorganisms, virulence factors, and local inflammatory responses contribute to bone resorption in periodontitis [25]. In mesenchymal stem cell (MSC)-based therapies, tissue regeneration is closely linked to the immunomodulatory effects mediated by the apoptosis of transplanted MSCs. For instance, a previous study demonstrated that apoptosis of PDLSCs induced by orthodontic forces promotes osteogenesis [40]. In this study, we treated periodontitis mice with local injections of hPDLSCs-ApoEVs and found that hPDLSCs-ApoEVs alleviated periodontitis and restored alveolar bone. The periodontal region of hPDLSCs-ApoEVs-treated mice exhibited more M2 macrophages, along with increased expression of p-p65 (NF-κB) and S100A9. These findings further confirm that hPDLSCs-ApoEVs mitigate periodontitis and promote alveolar bone regeneration through NF-κB/S100A9-regulated M2 macrophage polarization, highlighting their therapeutic potential for periodontal tissue repair.

Recent studies have highlighted the immunomodulatory and tissue regenerative potential of mesenchymal stem cell-derived apoptotic extracellular vesicles (MSCs-ApoEVs) [11, 41, 42]. Our findings emphasize the potential of hPDLSCs-ApoEVs as a promising alternative to hPDLSCs for periodontitis treatment. While we demonstrated that hPDLSCs-ApoEVs induce M2 macrophage polarization, the specific cargo components responsible for this effect remain unidentified. Additionally, although local administration of hPDLSCs-ApoEVs in a mouse periodontitis model showed no systemic adverse effects, further studies using hPDLSCs-ApoEVs derived from the same species are necessary to validate their safety and efficacy for clinical applications. These steps are critical to advancing hPDLSCs-ApoEVs as a viable therapeutic strategy for periodontitis.

Conclusion

This study demonstrates that hPDLSCs-ApoEVs promote M2 macrophage polarization via the NF-κB/S100A9 axis, alleviating periodontitis and enhancing alveolar bone regeneration. hPDLSCs-ApoEVs restored alveolar bone, increased M2 macrophages, and upregulated NF-κB/S100A9 in a mouse periodontitis model. Inhibition studies confirmed NF-κB as an upstream regulator of S100A9, clarifying the mechanism behind hPDLSCs-ApoEVs-mediated immunomodulation. This study highlights hPDLSCs-ApoEVs as a minimally invasive, cell-free strategy for periodontal regeneration and periodontitis treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ApoEV

Apoptotic extracellular vesicle

ALP

Alkaline phosphatase

Arg-1

Arginase 1

COL-1

Collagen type I

CD80

T-lymphocyte activation antigen CD80

CD163

Scavenger receptor cysteine-rich type 1 protein M130

CD206

Macrophage mannose receptor 1

CM

Conditioned medium

DAPI

2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride

hPDLSC

Human periodontal ligament stem cell

IL-10

Interleukin 10

iNOS

Nitric oxide synthase 2, inducible

MSC

Mesenchymal stem cell

OM

Osteogenic medium

OPN

Osteopontin

PDL

Periodontal ligament

RUNX2

Runt-related transcription factor 2

TEM

Transmission electron microscopy

THP-1 cell

Tohoku Hospital Pediatrics-1 cell

WT

Wild-type

Author contributions

ZZ, ZX, LY, JP, and LL contributed to the conception, design, data acquisition, analysis, and interpretation, and drafted and critically revised the manuscript; YY, LZ and LZ contributed to conception, design, data acquisition, analysis, interpretation and critically revised the manuscript; GZ, JW and JX contributed to data acquisition, analysis and interpretation and critically revised the manuscript; WP, QD and YL contributed to data acquisition and analysis and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

Funding

This study was supported by the National Key Research and Development Program of China (2021YFE0108000) and the Department of Education of Guangdong Province of China (No. 2023ZDZX2034) and the Research Capability Improvement Program of Guangzhou Medical University (No. 2024SRP157).

Data availability

The datasets of mRNA-seq are available in the SRA repository (PRJNA1269638), (https://www.ncbi.nlm.nih.gov/sra/PRJNA1269638). The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The experimental protocol of human samples ”Mechanism of apoptotic extracellular vesicles of human periodontal ligament stem cells promoting periodontal tissue regeneration and repair by regulating macrophage polarization” was approved by the Medical Research Ethics Association of Affiliated Stomatology Hospital of Guangzhou Medical University (JCYJ2023001) on February 28th, 2023. Animal experiments ”Mechanism of apoptotic extracellular vesicles of human periodontal ligament stem cells promoting periodontal tissue regeneration and repair by regulating macrophage polarization” were conducted by the Institutional Animal Care and Use Committee of Guangzhou Medical University (G2023-153) on March 2nd, 2023.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.