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. 2026 Oct 1;21(4):2152–2161. doi: 10.1016/j.jds.2025.07.3580

Avian mesenchymal stem cell-derived extracellular vesicle alleviates the Porphyromonas gingivalis lipopolysaccharide induced gingival fibroblast senescence

Han-Chieh Yang a,†, Earl Fu a,b,†, Yu-Tang Chin a, Ju-Sheng Shieh a, Wei-Ni Lin c, Cheng-Yang Chiang a,d,*
PMCID: PMC13689649  PMID: 42924215

Abstract

Background/purpose

We have shown that extracellular vesicles (EVs) derived from Polygonum multiflorum extract bio-pulsed avian mesenchymal stem cells exhibit anti-inflammatory and proliferation-promoting properties. A significant association has been identified between periodontitis, higher biological age, and accelerated biological aging. This study investigated whether the bio-pulsed EVs could alleviate Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-induced senescence in human gingival fibroblasts (hGFs).

Materials and methods

Pg-LPS was used to induce cellular inflammation and senescence in hGFs. The cytokines IL-1β, IL-6, and TNF-α were assessed by quantitative polymerase chain reaction (qPCR), and cytotoxicity was assessed with MTS. Senescence after repeated stimulations was evaluated by flow cytometry, SA-β-gal staining, and p16, p21, and p53 mRNA and protein (qPCR and Western blot), and telomere length (qPCR). Cell proliferation and apoptosis markers of Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) were evaluated by colorimetric assay.

Results

Cytotoxicity was noted in hGFs only at 5.0 μg/mL LPS. LPS at 0.5 μg/mL significantly upregulated IL-1β, IL-6, and TNF-α mRNA expressions. Repeated LPS significantly increased cell size (1.0 μg/mL), the number of SA-β-gal-staining cells (0.5 and 1.0 μg/mL), and mRNA and protein expressions for p16, p21, and p53 (1.0 μg/mL), indicating senescence. Bio-pulsed EVs significantly increased CCK-8 but reduced LDH releases, indicating improved hGFs viability. Furthermore, the bio-pulsed EVs suppressed the LPS-induced upregulation of pro-inflammatory cytokines and senescence markers, but reversed telomere shortening.

Conclusion

Single Pg LPS triggered inflammatory responses in hGFs, whereas repeated exposures induced senescence. The bio-pulsed EVs effectively mitigated Pg LPS-induced inflammation and senescence, highlighting the preventive/therapeutic potential.

Keywords: Senescence, Lipopolysaccharides, Inflammation, Stem cells, Extracellular vesicles

Introduction

The volume of global publications on aging associated with periodontitis has increased dramatically and is expected to continue rising.1 A significant association has been identified between higher biological age, accelerated biological aging, and periodontitis.2 Accumulating evidence indicates that cellular senescence plays a causal role in age-related dysfunctions and pathologies.3,4 Senescent cells contribute to disease progression by perpetuating inflammation through the secretion of senescence-associated secretory phenotype (SASP) factors, which recruit immune cells and exacerbate tissue destruction.5 Senescence-associated changes have been observed in the gingival tissues of aged mice as well as in cultured human gingival fibroblasts.6 A recent study further demonstrated that senescent cell accumulation increases with the progression of periodontitis; the inhibition of cellular senescence via metformin administration successfully alleviated inflammation and bone loss.7 These findings emphasize that inflammation is both a cause and a consequence of senescence. Chronic inflammation drives senescence, while persistent inflammatory stimuli, such as those in osteoarthritis, can induce senescence in adjacent non-senescent cells.8 A deeper understanding of these mechanisms offers promising new opportunities for managing periodontitis, particularly in aging populations.

Recently, stem cells and their derived extracellular vesicles (EVs) have been investigated for their anti-aging properties.9,10 These vesicles contain various bioactive molecules, including proteins, lipids, RNAs, and DNAs.9–13 These allow them to transfer signals to recipient cells, regulate immune responses, suppress excessive inflammation, and establish a favorable environment for tissue repair, regeneration, and anti-aging processes. In human dermal fibroblasts, EVs secreted by human adipose-derived stem cells have been shown to mitigate aging by inhibiting aging-related proteins such as p53, p21, and p16, and by reducing senescence-associated β-galactosidase (SA-β-gal) activity.9 Additionally, implanting dental pulp stem cells combined with extracts from Polygonum multiflorum Thunb, a traditional Chinese medicinal herb known for its anti-aging effects, has been reported to significantly accelerate periodontal tissue regeneration by enhancing cell recruitment and potentially promoting angiogenesis.14 Furthermore, EVs derived from Polygonum multiflorum extract bio-pulsed avian mesenchymal stem cell have been found not only to reduce lipopolysaccharide (LPS)-induced expressions of pro-inflammatory cytokines, but also to promote cell proliferation and wound healing in human dermal or gingival fibroblasts.15 However, whether the bio-pulsed EVs can prevent chronic inflammation–induced cellular senescence in the periodontium remains unknown. Therefore, the aims of this study were: (1) to determine whether Porphyromonas gingivalis (Pg) LPS can induce cellular inflammation and senescence in human gingival fibroblasts; and (2) to evaluate the effects of the bio-pulsed EVs on LPS-induced inflammation and senescence in gingival fibroblasts.

Materials and methods

Culture of gingival fibroblast cell and treatment of Porphyromonas gingivalis lipopolysaccharide or extracellular vesicles

Clinically healthy gingival tissues were obtained from patients undergoing crown lengthening or wisdom tooth extraction at the Department of Periodontology, Tri-Service General Hospital (August 16, 2023–August 15, 2024) with IRB approval (No. A202305147 and B202605046, TSGH). Written informed consent was obtained from all participants. Connective tissue fragments were digested in 50 μl collagenase (Sigma, St. Louis, MO, USA) and 5 ml DMEM-F12 (Gibco, Gaithersburg, MD, USA) at 37 °C, 5% CO2 for 24 h, rinsed with PBS, and cut into ~1 mm3 pieces. After centrifugation at 1500 rpm for 5 min, the fragments were plated in a 10 cm dish with DMEM-F12 (89%), antibiotics (1%) (Gibco), and FBS (10%) (Hyclone, Logan, UT, USA) and incubated for seven days without a medium change. Migrating gingival fibroblasts were observed under an inverted fluorescence microscope, and tissue fragments were subsequently removed. The medium was replaced every 48 h. Upon confluence, cells were passaged, washed with PBS, trypsinized (Gibco), centrifuged, resuspended, and replated. Pg LPS (InvivoGen, San Diego, CA, USA) was used to induce cellular inflammation and senescence via single or repeated stimulations. Single-dose LPS treatment was used to induce inflammation, whereas repeated LPS stimulations across passages were applied to model cellular senescence, as modified from previous studies.16–18 Biopulsed avian mesenchymal stem cell-derived extracellular vesicles (Bio-pulsed ScEVs; ExoGiov, AM BioTech, Taipei, Taiwan) were commercially obtained as a standardized product source. The underlying bio-pulsed avian MSC-EV production platform had been previously established and characterized by our group in an earlier study, in which avian mesenchymal stem cells were bio-pulsed prior to EV isolation, purification, lyophilization, and characterization. 15 In the present study, the commercially sourced Biopulsed ScEV preparation was used for functional testing in human gingival fibroblasts.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay

To assess the cytotoxicity of Pg LPS on hGFs, the fifth-passaged fibroblasts were seeded into a 96-well plate (1000 viable cells/well, 100 μl medium) and incubated for 6 h. Cells were treated with LPS at 0.0, 0.5, or 1.0 μg/mL for 72 h (30 wells per dosage group). Subsequently, 20 μl of MTS reagent (CellTiter96 AQ) (Promega, Madison, WI, USA) was added per well and incubated for 2 h before measuring absorbance at 490 nm (Thermo Scientific, Waltham, MA, USA).19

Quantitative polymerase chain reaction analysis of inflammatory cytokines and senescence markers

Total RNA was extracted using a NucleoSpin RNA kit (Macherey–Nagel, Bethlehem, PA, USA) following the manufacturer’s instructions. RNA purity and concentration were determined using a Nanodrop spectrophotometer. Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s protocol. Gene expression of the inflammatory cytokines of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), and the senescence markers of p16 (p16INK4a, cyclin-dependent kinase inhibitor 2A), p21 (Cyclin-Dependent Kinase Inhibitor 1A), and tumor protein p53 (p53) was quantified using the Rotor-Gene SYBR Green PCR Kit (Qiagen, Hilden, Germany) on a LightCycler 480 system (Roche, Vienna, Austria). The cycling conditions included denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Primer sequences were as follows: IL-1β, forward primer: 5′-ATGATGGCTTATTACAGTGGCAA-3′ and reverse primer: 5′-GTCGGAGATTCGTAGCTGGA-3’. IL-6, forward primer: 5′-GGTACATCCTCGACGGCATCT-3′ and reverse primer: 5′-GTGCCTCTTTGCTGCTTTCAC-3’. TNF-α, forward primer: 5′-CCTCTCTCTAATCAGCCCTCTG-3′ and reverse primer: 5′-GAGGACCTGGGAGTAGATGAG-3’. p16, forward primer: 5′-CAGTGGACCTGGCTGAGGAG-3′ and reverse primer: 5′-CTCAGGGTACGGGGTCCTC-3’. p21, forward primer: 5′-CCTGTCACTGTCTTGTACCCT-3′ and reverse primer: 5′-GCGTTTGGAGTGGTAGAAATCTG-3’. p53, forward primer: 5′-CAGCACATGACGGAGGTTGT-3′ and reverse primer: 5′-TCATCCAAATACTCCACACGC-3’.

Flow cytometry analysis

hGFs were treated with LPS (0.0, 0.001, 0.01, 0.1, 1.0 μg/mL) on day 1, with subsequent medium changes containing fresh LPS on days 3, 5, and 7. On day 9, cells were analyzed using flow cytometry (Thermo Scientific) to assess morphological changes.20

Senescence-associated β-galactosidase staining

Cells were further identified using the SA-β Gal Staining Kit (Abcam, Cambridge, United Kingdom). After nine days of culture, cells were fixed and stained with X-gal solution and incubated at 37 °C for 24 h. Stained cells were observed under an inverted fluorescence microscope (IX71, OLYMPUS, Tokyo, Japan), and the percentage of senescent cells was quantified.

Western blot for senescence marker

Protein levels of p16, p21, and p53 were assessed using Western blotting. Protein lysates were quantified using the Bradford assay (Bio-Rad, Pleasanton, CA, USA), denatured in sample buffer, and separated by SDS-PAGE. Proteins were transferred to PVDF membranes, blocked, and probed with primary antibodies against p16, p21, and p53, followed by HRP-conjugated secondary antibodies. Bands were visualized using chemiluminescence (Bio-Rad).

Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays

The effects of EVs on LPS-induced hGFs cell proliferation and apoptosis were determined by CCK-8 and LDH assays (Dojindo, Tokyo, Japan), respectively.21 Fifth-passage hGFs were treated with EVs (0–1 × 106 EV/ml) for 72 h. LDH release was measured by transferring supernatant to a new plate, mixing with LDH reagent, and reading absorbance at 490 nm. For CCK-8, fresh medium with 10 μl CCK-8 reagent was added, incubated for 2 h, and measured at 450 nm.

Telomere length measurement

Genomic DNA was extracted using the Abcam genomic DNA extraction kit (Abcam). Telomere length was quantified using the Relative Human Telomere Length Quantification qPCR Assay Kit (ScienCell, Carlsbad, CA, USA). The T/S ratio was calculated using the ΔΔCt method.22

Statistical analysis

Data were expressed as mean ± SD. Statistical analyses were performed using Prism 6 (GraphPad Software Inc, San Diego, CA, USA). Group differences were analyzed using independent t-tests, with significance set at P < 0.05.

Results

Effect of Porphyromonas gingivalis lipopolysaccharide on cellular inflammation in human gingival fibroblasts

Cell viability showed no significant differences among groups treated with various concentrations of LPS, except at 5.0 μg/mL, where a significant decrease was observed (Fig. 1A). Treatment with 0.5 μg/mL LPS significantly increased the mRNA expression levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α compared to the phosphate-buffered saline controls (Fig. 1B).

Figure 1.

Figure 1

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Effect of Porphyromonas gingivalis lipopolysaccharide on cellular inflammation in gingival fibroblasts.

The cell viabilities of human gingival fibroblasts (hGFs) after lipopolysaccharide (LPS) treatments, determined by MTS assay, were shown (A). The expressions of pro-inflammatory cytokines of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α after LPS treatment for 3 days were exhibited (B). (Data presented mean and standard deviation, NS: no statistical difference; *: P < 0.05, n = 5, by the independent t-test).

Effect of Porphyromonas gingivalis lipopolysaccharide on cellular senescence in human gingival fibroblasts

Cell size in hGFs was evaluated following repeated LPS treatments (0–1.0 μg/mL), and the size was significantly increased only at 1.0 μg/mL (Fig. 2A). SA-β-gal staining revealed a significant increase in positively stained cells after repeated LPS treatments at 0.5 and 1.0 μg/mL compared to untreated controls (Fig. 2B). Furthermore, repeated LPS treatments at 1.0 μg/mL significantly upregulated the mRNA and protein expression levels of senescence markers p16, p21, and p53 (Fig. 2C and D).

Figure 2.

Figure 2

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Effect of Porphyromonas gingivalis lipopolysaccharide repeated treatments on cellular senescence in human gingival fibroblasts.

Cell sizes of fibroblast cultures were examined by flow cytometry after 4-time stimulations with lipopolysaccharide (LPS) (A). The distributions of senescence-associated β-galactosidase (SA-β-gal) positively stained gingival fibroblasts were observed under the microscope after repeated LPS (the left histographies, 200× magnification), while the quantitative analysis was shown on the right plot (B). The mRNA and protein expressions for p16, p21, and p53 in fibroblasts after the repeated LPS (1.0 μg/mL) were compared in C and D. (Data presented mean and standard deviation, NS: no statistical difference, Cont.: control; *: P < 0.05, n = 5, by independent t-test).

Effect of extracellular vesicles on lipopolysaccharide-induced inflammation and senescence in human gingival fibroblasts

Effect of extracellular vesicles on fibroblast proliferation and apoptosis

Treatment with EVs (101 - 106 particles/ml) significantly increased the proliferation marker (CCK-8 assay) and decreased the apoptotic marker (LDH release) in hGFs compared to the group without EV (Fig. 3A).

Figure 3.

Figure 3

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Effect of extracellular vesicles on repeated lipopolysaccharide-induced pro-inflammatory cytokine expression.

Effects of extracellular vesicles (EVs) on cell proliferation and apoptosis in gingival fibroblasts were examined using Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays, respectively (A). The effects of EVs on the mRNA expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in fibroblasts after lipopolysaccharide (LPS) treatment were shown (B, C, and D, respectively). (Data presented mean and standard deviation, NS: no statistical difference; *: significant difference at P < 0.05 vs 0 μg/mL LPS, #: significant difference at P < 0.05 vs those with LPS treatment with 0.5 or 1.0 μg/mL, and NS: no statistical difference, by independent t-test) (n = 5) (White bar: without EV, and black bar: with EVs).

Effect of extracellular vesicles on lipopolysaccharide-induced pro-inflammatory cytokine expressions

Repeated Pg LPS treatments significantly increased IL-1β mRNA levels compared to those of no LPS controls, despite 0.5 and 1.0 μg/mL; however, the co-treatment with EVs (104 particles/ml) significantly reduced the enhanced LPS-induced IL-1β expression (Fig. 3B). Similar attenuation patterns were observed for IL-6 and TNF-α (Fig. 3C and D).

Effect of extracellular vesicles on lipopolysaccharide-induced cellular senescence

As shown in Fig. 4A, repeated LPS exposures (0.5 and 1.0 μg/mL) significantly increased the mRNA levels of senescence markers p16, p21, and p53 compared to controls, and the co-treatment with EVs significantly decreased LPS-induced mRNA levels of p16, p21, and p53 (no statistical significance for p53 at 1.0 μg/mL LPS). Additionally, the repeated stimulation led to telomere shortening in hGFs (Fig. 4B), whereas co-treatment with EVs preserved telomere length, with significant protection observed at 0.5 μg/mL LPS, but not at 1.0 μg/mL. A similar finding of EV treatment significantly reduced LPS-induced SA-β-gal expression in hGFs was also observed (Fig. 4C).

Figure 4.

Figure 4

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Effect of extracellular vesicles on the repeated lipopolysaccharide-induced senescence in human gingival fibroblasts.

The effects of extracellular vesicles (EVs) on the expression of p16, p21, and p53 mRNA in fibroblasts with and without lipopolysaccharide (LPS) treatment were presented (A), on telomere length were exhibited (B), and on the senescence-associated β-galactosidase (SA-β-gal) cytochemical staining was observed (C). (Data presented mean and standard deviation; *: P < 0.05, NS: no statistical difference, n = 5, by independent t-test).

Discussion

The present in vitro study investigated whether the EVs derived from Polygonum multiflorum extract bio-pulsed avian mesenchymal stem cells could attenuate cellular inflammation and senescence in hGFs. Initially, cellular inflammation was assessed by measuring the mRNA expression levels of pro-inflammatory cytokines following a single stimulation with Pg LPS (Fig. 1). To evaluate cellular senescence, hGFs was repeatedly exposed to LPS, and senescence-associated characteristics, including changes in cell size, SA-β-galactosidase (SA-β-gal) activity, the expression of p16, p21, and p53, and telomere length, were examined (Figs. 2 and 4). As shown in Fig. 2A, a significant increase in cell size was observed only at the highest LPS concentration (1.0 μg/mL), suggesting that the critical threshold for LPS-induced senescence lies between 0.1 and 1.0 μg/mL. Senescence was further confirmed by SA-β-gal staining, which revealed a significant increase in the proportion of positively stained cells at 0.5 and 1.0 μg/mL LPS concentrations (Fig. 2B). Additionally, repeated LPS treatments (1.0 μg/mL) significantly upregulated the mRNA and protein expression levels of the senescence markers p16, p21, and p53 (Fig. 2C and D), further supporting the induction of senescence in hGFs.

Studies have shown that senescent cells secrete SASP factors, including pro-inflammatory cytokines, proteases, and growth factors, which can exacerbate local inflammatory responses and compromise the structural integrity of tissues.5,23 Multiple components of the SASP can also induce senescence in neighboring non-senescent cells through a process known as paracrine senescence.24 The periodontal pathogen Pg can activate the immune system through virulence factors, such as LPS, thereby triggering a cascade of inflammatory responses.25 In gingival fibroblasts, LPS stimulation has been shown to increase reactive oxygen species (ROS) accumulation; however, within 24 h, the resulting oxidative stress does not appear to cause significant DNA damage or apoptosis.26 However, prolonged exposure to inflammation for 48 h leads to a marked increase in oxidative stress, as indicated by elevated 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and a higher number of apoptotic cells.26 Furthermore, Pg-derived LPS has been shown to induce senescent morphological changes and the expression of biochemical senescence markers, leading to premature senescence of alveolar osteocytes.27 In the context of periodontitis, inflammaging, a chronic, sterile, low-grade inflammatory state associated with aging, has also been proposed as a contributing factor.16 It has been reported that the associations between lower telomerase activity and/or shorter telomeres in immune cells and elevated systemic cytokine levels in patients with chronic disorders.28 For dental pulp stem cells, LPS stimulation upregulated the expression of SASP markers, such as p53 and p21.16 Despite the complexity and multifactorial nature of aging, healthy aging or increased longevity is generally associated with longer telomeres and a lower inflammatory profile.29,30 In the present study, LPS stimulation induced pro-inflammatory cytokine expressions in hGFs, regardless of whether cells were exposed once or repeatedly (Figs. 1 and 3). However, after repeated stimulations, aging could be noted, which could be identified through additional assessments such as the expression of senescence markers (p16, p21, p53), telomere shortening, or morphological changes.29,31

In the present study, we further investigated whether the bio-pulsed EVs could prevent LPS-induced cellular senescence in hGFs. Treatment with EVs enhanced hGFs proliferation while reducing apoptosis (Fig. 3A) and significantly attenuated LPS-induced expression of pro-inflammatory cytokines (Fig. 3B–D). Although the responses did not exhibit a strictly dose-dependent pattern, it may be attributable to a saturation effect of EV uptake by recipient cells.32,33 More importantly, EV treatment mitigated LPS-induced cellular senescence, as evidenced by downregulation of p16, p21, and p53 mRNA expression levels and the preservation of telomere length (Fig. 4). Previous studies have reported that conditioned media and exosomes derived from human induced pluripotent stem cells ameliorate both genotypic and phenotypic changes associated with UV-induced photoaging and natural senescence in human dermal fibroblasts.34,35 Furthermore, when highly purified EVs derived from the induced pluripotent stem cells were applied, senescent mesenchymal stem cells exhibited reduced ROS levels and alleviated aging-related phenotypes.34,35 Similarly, exosomes derived from human adipose-derived stem cells have been shown to significantly suppress the expression of senescence-related proteins, including p53, p21, and p16, thereby demonstrating potent anti-senescence effects in human dermal fibroblasts.9 Exosomes from human umbilical cord-derived mesenchymal stem cells have also been reported to promote axonal and Schwann cell regeneration while modulating the inflammatory response by downregulating pro-inflammatory and upregulating anti-inflammatory cytokines.36 Collectively, these findings suggest that EVs, particularly those derived from varied mesenchymal stem cells, may serve as promising therapeutic agents for tissue repair and wound healing, or even the mitigation of cellular senescence.37,38 In this study, EV and LPS were supplied simultaneously in starting the experiment, indicating a prevention of LPS-induced telomere attrition by EV. Similarly, previous studies have suggested that stem cell-derived EVs may alleviate ROS accumulation, reduce DNA damage responses, and preserve telomere integrity under inflammatory or stress conditions.10,34

In the present study, the exact biomolecular composition of the EVs was not analyzed; however, it is well recognized that the secretome profile of stem cells is highly dependent on the microenvironment in which they are cultured, and can be modified through preconditioning strategies during in vitro culture.39 Polygonum multiflorum Thunb (he-shou-wu), a well-known traditional Chinese medicinal herb, has long been utilized for its anti-aging properties.40 In that study, tetrahydroxystilbene glucoside, a major bioactive component extracted from Polygonum multiflorum Thunb, was used to bio-pulse avian mesenchymal stem cells before EV isolation. A previous study demonstrated that glucoside can reduce nitric oxide production and suppress inducible nitric oxide synthase expression, via downregulation of the NF-κB signaling pathway in LPS-stimulated mouse peritoneal macrophages. 41 It can also attenuate inflammatory cytokine expression in LPS-induced gingival fibroblasts and ligature-induced periodontitis models.42 The glucoside also exhibits various biological activities associated with anti-aging and preventing age-related diseases.

More recently, derivatives of dental pulp stem cells stimulated with the Polygonum multiflorum extracts have been reported to enhance cellular activity and display anti-inflammatory effects.43 In addition, the glucoside bio-pulsing of avian mesenchymal stem cells has been shown to yield EVs with higher contents of exosomal proteins and RNAs compared to non-pulsed cells, resulting in enhanced proliferation, wound healing, and upregulated gene expression in human dermal fibroblasts and hair follicle dermal papilla cells.15 Consistent with these findings, our present results demonstrated that the bio-pulsed EVs effectively alleviated LPS-induced cellular inflammation and senescence in hGFs. Our results in EVs significantly enhance CCK-8 activity (a marker of proliferation), but decrease LDH release (a marker of apoptosis) across a range of EV concentrations (101 to 106 particles/ml) (Fig. 3A), further suggesting the protective effects may involve regulation of cellular proliferation and apoptosis. Although the exact molecular components responsible for the anti-senescent effects were not investigated in this study, previous studies have shown that EV cargos, including micro-RNAs, proteins, and regulatory molecules,12,13 may modulate inflammatory and senescence-associated pathways such as ROS generation, NF-κB activation, and p53/p21 signaling. In particular, RAB27A, a well-known regulator of exosome secretion and vesicle trafficking, has been implicated in intercellular communication during inflammation and senescence.44

In conclusion, this in vitro study demonstrated that Pg LPS triggers cellular inflammation in hGFs and that repeated LPS stimulation leads to cellular senescence. Moreover, the bio-pulsed EV treatment mitigated both LPS-induced inflammation and senescence, suggesting its potential application in improving periodontal health.

Acknowledgments

This study was supported by a grant from the Tri-Service General Hospital (TSGH-D112-054, TSGH-D115-072) and from Cardinal Tien Hospital (CTH112A-NDMC-2223), Taiwan.

Funding Statement

This study was supported by a grant from the Tri-Service General Hospital (TSGH-D112-054, TSGH-D115-072) and from Cardinal Tien Hospital (CTH112A-NDMC-2223), Taiwan.

Footnotes

Declaration of competing interest: The authors have no conflicts of interest to report.

References

  • 1. Liu X, Li H. Global trends in research on aging associated with periodontitis from 2002 to 2023: a bibliometric analysis. Front Endocrinol (Lausanne) 2024;15:1374027. doi: 10.3389/fendo.2024.1374027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Li S, Wen C, Bai X, Yang D. Association between biological aging and periodontitis using NHANES 2009–2014 and mendelian randomization. Sci Rep. 2024;14:10089. doi: 10.1038/s41598-024-61002-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Correia-Melo C, Passos JF. Mitochondria: are they causal players in cellular senescence? Biochim Biophys Acta. 2015;1847:1373–9. doi: 10.1016/j.bbabio.2015.05.017. [DOI] [PubMed] [Google Scholar]
  • 4. Davalli P, Mitic T, Caporali A, Lauriola A, D’Arca D. ROS, cell senescence, and novel molecular mechanisms in aging and agerelated diseases. Oxid Med Cell Longev. 2016;2016:3565127. doi: 10.1155/2016/3565127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Rodier F, Coppe JP, Patil CK, et al. Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nat Cell Biol. 2009;11:973–9. doi: 10.1038/ncb1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Furukawa M, Matsuda K, Aoki Y, et al. Analysis of senescence in gingival tissues and gingival fibroblast cultures. Clin Exp Dent Res. 2022;8:939–49. doi: 10.1002/cre2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Fu L, Yin C, Zhao Q, et al. CD81+ fibroblasts, a unique subpopulation with accelerated cellular senescence, exaggerate inflammation and activate neutrophils via C3/C3aR1 axis in periodontitis. eLife. 2024;13:RP96908. [Google Scholar]
  • 8. Zhang XX, He SH, Liang X, Li W, Li TF, Li DF. Aging, cell senescence, the pathogenesis and targeted therapies of osteoarthritis. Front Pharmacol. 2021;12:728100. doi: 10.3389/fphar.2021.728100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Guo JA, Yu PJ, Yang DQ, Chen W. The antisenescence effect of exosomes from human adipose-derived stem cells on skin fibroblasts. Biomed Res Int. 2022;2022:1034316. doi: 10.1155/2022/1034316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Liao CM, Luo T, von der Ohe J, de Juan Mora B, Schmitt R, Hass R. Human MSC-derived exosomes reduce cellular senescence in renal epithelial cells. Int J Mol Sci. 2021;22:13562. doi: 10.3390/ijms222413562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Teng X, Chen L, Chen W, Yang J, Yang Z, Shen Z. Mesenchymal stem cell-derived exosomes improve the microenvironment of infarcted myocardium contributing to angiogenesis and anti-inflammation. Cell Physiol Biochem. 2015;37:2415–24. doi: 10.1159/000438594. [DOI] [PubMed] [Google Scholar]
  • 12. Cha H, Hong S, Park JH, Park HH. Stem cell-derived exosomes and nanovesicles: promotion of cell proliferation, migration, and anti-senescence for treatment of wound damage and skin ageing. Pharmaceutics. 2020;12:1135. doi: 10.3390/pharmaceutics12121135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Xu D, Tahara H. The role of exosomes and microRNAs in senescence and aging. Adv Drug Deliv Rev. 2013;65:368–75. doi: 10.1016/j.addr.2012.07.010. [DOI] [PubMed] [Google Scholar]
  • 14. Lin CY, Tsai MS, Kuo PJ, et al. 2,3,5,4’-Tetrahydroxystilbene-2-O-beta-d-glucoside promotes the effects of dental pulp stem cells on rebuilding periodontal tissues in experimental periodontal defects. J Periodontol. 2021;92:306–16. doi: 10.1002/JPER.19-0563. [DOI] [PubMed] [Google Scholar]
  • 15. Shieh JS, Chin YT, Chiu HC, et al. Bio-pulsed stimulation effectively improves the production of Aian mesenchymal stem cell-derived extracellular vesicles that enhance the bioactivity of skin fibroblasts and hair follicle cells. Int J Mol Sci. 2022;23:15010. doi: 10.3390/ijms232315010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Feng G, Zheng K, Cao T, et al. Repeated stimulation by LPS promotes the senescence of DPSCs via TLR4/MyD88-NF-κBp53/p21 signaling. Cytotechnology. 2018;70:1023–35. doi: 10.1007/s10616-017-0180-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Ricciarelli R, Azzi A, Zingg JM. Reduction of senescence-associated beta-galactosidase activity by vitamin E in human fibroblasts depends on subjects’ age and cell passage number. Biofactors. 2020;46:665–74. doi: 10.1002/biof.1636. [DOI] [PubMed] [Google Scholar]
  • 18. Kim CO, Huh AJ, Han SH, Kim JM. Analysis of cellular senescence induced by lipopolysaccharide in pulmonary alveolar epithelial cells. Arch Gerontol Geriatr. 2012;54:e35–41. doi: 10.1016/j.archger.2011.07.016. [DOI] [PubMed] [Google Scholar]
  • 19. Gray DH, Tull D, Ueno T, et al. A unique thymic fibroblast population revealed by the monoclonal antibody MTS-15. J Immunol. 2007;178:4956–65. doi: 10.4049/jimmunol.178.8.4956. [DOI] [PubMed] [Google Scholar]
  • 20. Pollizzi KN, Waickman AT, Patel CH, Sun IH, Powell JD. Cellular size as a means of tracking mTOR activity and cell fate of CD4+ T cells upon antigen recognition. PLoS One. 2015;10:e0121710. doi: 10.1371/journal.pone.0121710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Song SJ, Raja IS, Lee YB, et al. Comparison of cytotoxicity of black phosphorus nanosheets in different types of fibroblasts. Biomater Res. 2019;23:23. doi: 10.1186/s40824-019-0174-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Dahlgren PN, Bishop K, Dey S, Herbert BS, Tanaka H. Development of a new monochrome multiplex qPCR method for relative telomere length measurement in cancer. Neoplasia. 2018;20:425–31. doi: 10.1016/j.neo.2018.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Lopes-Paciencia S, Saint-Germain E, Rowell M-C, Ruiz AF, Kalegari P, Ferbeyre G. The senescence-associated secretory phenotype and its regulation. Cytokine. 2019;117:15–22. doi: 10.1016/j.cyto.2019.01.013. [DOI] [PubMed] [Google Scholar]
  • 24. Acosta JC, Banito A, Wuestefeld T, et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol. 2013;15:978–90. doi: 10.1038/ncb2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Nativel B, Couret D, Giraud P, et al. Porphyromonas gingivalis lipopolysaccharides act exclusively through TLR4 with a resilience between mouse and human. Sci Rep. 2017;7:15789. doi: 10.1038/s41598-017-16190-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Cheng R, Choudhury D, Liu C, Billet S, Hu T, Bhowmick NA. Gingival fibroblasts resist apoptosis in response to oxidative stress in a model of periodontal diseases. Cell Death Discov. 2015;1:15046. doi: 10.1038/cddiscovery.2015.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Aquino-Martinez R, Rowsey JL, Fraser DG, et al. LPS-induced premature osteocyte senescence: implications in inflammatory alveolar bone loss and periodontal disease pathogenesis. Bone. 2020;132:115220. doi: 10.1016/j.bone.2019.115220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Kordinas V, Ioannidis A, Chatzipanagiotou S. The telomere/telomerase system in chronic inflammatory diseases. Cause or effect? Genes. 2016;7:60. doi: 10.3390/genes7090060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Djojosubroto MW, Choi YS, Lee HW, Rudolph KL. Telomeres and telomerase in aging, regeneration and cancer. Mol Cells. 2003;15:164–75. [PubMed] [Google Scholar]
  • 30. Arai Y, Martin-Ruiz CM, Takayama M, et al. Inflammation, but not telomere length, predicts successful ageing at extreme old age: a longitudinal study of semi-supercentenarians. EBioMedicine. 2015;2:1549–58. doi: 10.1016/j.ebiom.2015.07.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Lanz MC, Zatulovskiy E, Swaffer MP, et al. Increasing cell size remodels the proteome and promotes senescence. Mol Cell. 2022;82:3255–32569e8. doi: 10.1016/j.molcel.2022.07.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Mulcahy LA, Pink RC, Carter DR. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles. 2014;3:24641. doi: 10.3402/jev.v3.24641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Horibe S, Tanahashi T, Kawauchi S, Murakami Y, Rikitake Y. Mechanism of recipient cell-dependent differences in exosome uptake. BMC Cancer. 2018;18:47. doi: 10.1186/s12885-017-3958-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Oh M, Lee J, Kim YJ, Rhee WJ, Park JH. Exosomes derived from human induced pluripotent stem cells ameliorate the aging of skin fibroblasts. Int J Mol Sci. 2018;19:1715. doi: 10.3390/ijms19061715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Oh M, Kim Y, Son Y, Yoo H, Park JH. Promotive effects of human induced pluripotent stem cell-conditioned medium on the proliferation and migration of dermal fibroblasts. Biotechnol Bioproc E. 2017;22:561–8. [Google Scholar]
  • 36. Ma Y, Dong L, Zhou D, et al. Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats. J Cell Mol Med. 2019;23:2822–35. doi: 10.1111/jcmm.14190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Chen B, Li Q, Zhao B, Wang Y. Stem cell-derived extracellular vesicles as a novel potential therapeutic tool for tissue repair. Stem Cells Transl Med. 2017;6:1753–8. doi: 10.1002/sctm.16-0477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med. 2015;13:49. doi: 10.1186/s12967-015-0417-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Pendse S, Kale V, Vaidya A. Extracellular vesicles isolated from mesenchymal stromal cells primed with hypoxia: novel strategy in regenerative medicine. Curr Stem Cell Res Ther. 2021;16:243–61. doi: 10.2174/1574888X15999200918110638. [DOI] [PubMed] [Google Scholar]
  • 40. Ling S, Xu JW. Biological activities of 2,3,5,4’-tetrahydroxystilbene-2-O-beta-D-glucoside in antiaging and antiagingrelated disease treatments. Oxid Med Cell Longev. 2016;2016:4973239. doi: 10.1155/2016/4973239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Cha DS, Jeon H. Anti-inflammatory effect of MeOH extracts of the stem of Polygonum multiflorum in LPS-stimulated mouse peritoneal macrophages. Nat Prod Sci. 2009;15:83–9. [Google Scholar]
  • 42. Chin YT, Hsieh MT, Lin CY, et al. 2, 3, 5, 4′-tetrahydroxystilbene-2-O-β-glucoside isolated from polygoni multiflori ameliorates the development of periodontitis. Mediat Inflamm. 2016;2016:6953459. doi: 10.1155/2016/6953459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Chin YT, Liu CM, Chen TY, et al. 2,3,5,4’-tetrahydroxystilbene-2-O-beta-D-glucoside-stimulated dental pulp stem cellsderived conditioned medium enhances cell activity and antiinflammation. J Dent Sci. 2021;16:586–98. doi: 10.1016/j.jds.2020.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Takasugi M. Emerging roles of extracellular vesicles in cellular senescence and aging. Aging Cell. 2018;17:e12734. doi: 10.1111/acel.12734. [DOI] [PMC free article] [PubMed] [Google Scholar]

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