Skip to main content

Advertisement

SpringerLink
Log in

Saliva biofilm-derived outer membrane vesicles regulate biofilm formation and immune response of oral epithelial cells on titanium surfaces

  • Research
  • Published:
Clinical Oral Investigations Aims and scope Submit manuscript

Abstract

Objectives

While the significant roles of outer membrane vesicles (OMVs) from individual oral bacterial species in bacterial-host interactions are known, the involvement of saliva biofilm-derived OMVs in peri-implant disease pathogenesis remains unclear. This study aimed to investigate the effect of saliva biofilm-derived OMVs on regulating saliva biofilm formation and modulating the immune response of the epithelial cells on titanium surfaces.

Materials and methods

Saliva derived biofilms were cultured on tissue culture plates (TCP) for 4 days using pooled saliva from four healthy donors. OMVs secreted from the TCP bound biofilm (referred to as OMVs or healthy saliva biofilm OMVs) were enriched using the size-exclusion chromatography method. We then evaluated the effects of these OMVs on the viability, metabolic activity, and the presence of oral pathogens in saliva biofilm grown on titanium discs for 24 h and 72 h. Furthermore, the impact of OMVs on the mRNA expression and inflammatory cytokines [interleukin (IL)-6, IL-1α, and monocyte chemoattractant protein-1 (MCP-1)] in human oral epithelial cells (OKF6/TERT-2) was investigated using RT-qPCR and enzyme-linked immunosorbent assay (ELISA), respectively.

Results

Healthy saliva biofilm OMVs improved the biomass and activity of saliva biofilm cultured on the titanium surfaces, with inhibited Porphyromonas gingivalis and Fusobacterium nucleatum, and enhanced Streptococcus mutans expression. Additionally, OMVs increased pro-inflammatory cytokine IL-6 mRNA and IL-6 cytokine expression in human oral epithelial cells. However, IL-1α and MCP-1 cytokines were inhibited 24-hour post-incubation with OMVs.

Conclusion

Healthy saliva biofilm derived OMVs regulate the activity and pathogen composition of biofilms formed on titanium, while modulating the secretion of pro-inflammation factors of oral epithelial cells grown on titanium surfaces.

Clinical relevance

Healthy saliva biofilm OMVs may regulate the early biofilm formation on abutment surfaces and modulate epithelial cell immune response, which may alter the peri-implant niche and participate in the pathogenesis of peri-implant disease.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

All data collected and analysed within this study are available from the corresponding author on request.

References

  1. Renvert S et al (2018) Peri-implant health, peri-implant mucositis, and peri-implantitis: case definitions and diagnostic considerations. J Clin Periodontol 45(Suppl 20):S278–s285

    PubMed  Google Scholar 

  2. Salvi GE, Monje A, Tomasi C (2018) Long-term biological complications of dental implants placed either in pristine or in augmented sites: a systematic review and meta-analysis. Clin Oral Implants Res 29(Suppl 16):294–310

    PubMed  Google Scholar 

  3. Salvi GE et al (2022) Physiopathology of peri-implant diseases. Clin Implant Dent Relat Res 25(4):629–639. https://doi.org/10.1111/cid.13167

    Article  PubMed  Google Scholar 

  4. Lasserre JF, Brecx MC, Toma S (2018) Oral microbes, biofilms and their role in periodontal and peri-implant diseases. Materials (Basel) 11(10):1802

    PubMed  ADS  Google Scholar 

  5. Körtvélyessy G et al (2021) Bioactive coatings for dental implants: a review of alternative strategies to prevent peri-implantitis induced by anaerobic bacteria. Anaerobe 70:102404

    PubMed  Google Scholar 

  6. Zheng H et al (2015) Subgingival microbiome in patients with healthy and ailing dental implants. Sci Rep 5:10948

    PubMed  PubMed Central  ADS  CAS  Google Scholar 

  7. Belibasakis GN, Manoil D (2021) Microbial community-driven etiopathogenesis of peri-implantitis. J Dent Res 100(1):21–28

    PubMed  CAS  Google Scholar 

  8. Schwechheimer C, Kuehn MJ (2015) Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13(10):605–619

    PubMed  PubMed Central  CAS  Google Scholar 

  9. Chen H et al (2022) Recent advances in biomedical applications of bacterial outer membrane vesicles. J Mater Chem B 10(37):7384–7396

    PubMed  CAS  Google Scholar 

  10. Han P, Bartold PM, Ivanovski S (2022) The emerging role of small extracellular vesicles in saliva and gingival crevicular fluid as diagnostics for periodontitis. J Periodontal Res 57(1):219–231

    PubMed  CAS  Google Scholar 

  11. Ma L, Cao Z (2021) Membrane vesicles from periodontal pathogens and their potential roles in periodontal disease and systemic illnesses. J Periodontal Res 56(4):646–655

    PubMed  CAS  Google Scholar 

  12. Okamura H et al (2021) Outer membrane vesicles of Porphyromonas gingivalis: novel communication tool and strategy. Jpn Dent Sci Rev 57:138–146

    PubMed  PubMed Central  Google Scholar 

  13. Zhang Z et al (2022) Porphyromonas gingivalis outer membrane vesicles inhibit the invasion of Fusobacterium nucleatum into oral epithelial cells by downregulating FadA and FomA. J Periodontol 93(4):515–525

    PubMed  CAS  Google Scholar 

  14. Uemura Y et al (2022) Porphyromonas gingivalis outer membrane vesicles stimulate gingival epithelial cells to induce pro-inflammatory cytokines via the MAPK and STING pathways. Biomedicines 10(10):2643

    PubMed  PubMed Central  CAS  Google Scholar 

  15. Oscarsson J et al (2019) Tools of Aggregatibacter actinomycetemcomitans to evade the host response. J Clin Med 8(7):1079

    PubMed  PubMed Central  CAS  Google Scholar 

  16. Duan XB et al (2017) Marginal bone loss around non-submerged implants is associated with salivary microbiome during bone healing. Int J Oral Sci 9(2):95–103

    PubMed  PubMed Central  CAS  Google Scholar 

  17. Pallos D et al (2021) Salivary microbial dysbiosis is associated with peri-implantitis: a case-control study in a Brazilian population. Front Cell Infect Microbiol 11:696432

    PubMed  Google Scholar 

  18. Han P et al (2021) Salivary outer membrane vesicles and DNA methylation of small extracellular vesicles as biomarkers for periodontal status: a pilot study. Int J Mol Sci 22(5):2423

    PubMed  PubMed Central  CAS  Google Scholar 

  19. Ramachandra SS et al (2023) Fabrication and characterization of a 3D polymicrobial microcosm biofilm model using melt electrowritten scaffolds. Biomater Adv 145:213251

    PubMed  CAS  Google Scholar 

  20. Han P et al (2023) TNF-α and OSX mRNA of salivary small extracellular vesicles in periodontitis: a pilot study. Tissue Eng Part C Methods 29(7):298–306

    PubMed  CAS  Google Scholar 

  21. Liaw A et al (2023) Salivary histone deacetylase in periodontal disease: a cross-sectional pilot study. J Periodontal Res 58(2):433–443

    PubMed  CAS  Google Scholar 

  22. Tonetti MS, Greenwell H, Kornman KS (2018) Staging and grading of periodontitis: framework and proposal of a new classification and case definition. J Clin Periodontol 45(Suppl 20):S149–s161

    PubMed  Google Scholar 

  23. Ramachandra SS et al (2023) An in vitro dynamic bioreactor model for evaluating antimicrobial effectiveness on periodontal polymicrobial biofilms: a proof-of-concept study. J Periodontol. https://doi.org/10.1002/JPER.23-0086. Epub ahead of print

  24. Han PA-O et al (2022) Iron accumulation is associated with periodontal destruction in a mouse model of HFE-related haemochromatosis. J Periodontal Res 57(2) (1600-0765 (Electronic)):294–304

    PubMed  CAS  Google Scholar 

  25. Han P et al (2023) Saliva diagnosis using small extracellular vesicles and salivaomics. Methods Mol Biol 2588:25–39

    PubMed  CAS  Google Scholar 

  26. Han P et al (2023) TNF-αand OSX mRNA of salivary small extracellular vesicles in periodontitis: a pilot study. Tissue Eng 29(7):298–306. https://doi.org/10.1089/ten.TEC.2023.0051

    Article  CAS  Google Scholar 

  27. Han P et al (2023) Effects of periodontal cells-derived extracellular vesicles on mesenchymal stromal cell function. J Periodontal Res 58(6):1188–1200. https://doi.org/10.1111/jre.13171

    Article  PubMed  CAS  Google Scholar 

  28. Théry C et al (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7(1):1535750

    MathSciNet  PubMed  PubMed Central  Google Scholar 

  29. Walker SA et al (2022) Sucrose-based cryoprotective storage of extracellular vesicles. Extracell Vesicle 1:100016

    Google Scholar 

  30. Guo T et al (2021) Influence of sterilization on the performance of anodized nanoporous titanium implants. Mater Sci Eng: C 130:112429

    ADS  CAS  Google Scholar 

  31. Jayasree A et al (2022) Electrochemically nano-engineered titanium: influence of dual micro-nanotopography of anisotropic nanopores on bioactivity and antimicrobial activity. Mater Today Adv 15:100256

    CAS  Google Scholar 

  32. Chen X et al (2020) Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro. BMC Microbiol 20(1):1–11

    MathSciNet  CAS  Google Scholar 

  33. Sauer K et al (2022) The biofilm life cycle: expanding the conceptual model of biofilm formation. Nat Rev Microbiol 20(10):608–620

    PubMed  PubMed Central  CAS  Google Scholar 

  34. Jayasree A et al (2022) Gallium-doped dual micro-nano titanium dental implants towards soft-tissue integration and bactericidal functions. Mater Today Adv 16:100297

    CAS  Google Scholar 

  35. Han P et al (2020) Detection of salivary small extracellular vesicles associated inflammatory cytokines gene methylation in gingivitis. Int J Mol Sci 21(15):5273

    PubMed  PubMed Central  CAS  Google Scholar 

  36. Rajasekar A, Varghese SS (2022) Microbiological profile in periodontitis and peri-implantitis: a systematic review. J Long Term Eff Med Implants 32(4):83–94

    PubMed  Google Scholar 

  37. Quirynen M et al (2006) Dynamics of initial subgingival colonization of ‘pristine’ peri-implant pockets. Clin Oral Implants Res 17(1):25–37

    PubMed  Google Scholar 

  38. Fürst MM et al (2007) Bacterial colonization immediately after installation on oral titanium implants. Clin Oral Implants Res 18(4):501–508

    PubMed  ADS  Google Scholar 

  39. Nice JB et al (2018) Aggregatibacter actinomycetemcomitans leukotoxin is delivered to host cells in an LFA-1-indepdendent manner when associated with outer membrane vesicles. Toxins (Basel) 10(10):414

    PubMed  CAS  Google Scholar 

  40. Lim Y et al (2022) Activation of bone marrow-derived dendritic cells and CD4(+) T cell differentiation by outer membrane vesicles of periodontal pathogens. J Oral Microbiol 14(1):2123550

    MathSciNet  PubMed  PubMed Central  Google Scholar 

  41. Lynch JB, Alegado RA (2017) Spheres of hope, packets of doom: the good and bad of outer membrane vesicles in interspecies and ecological dynamics. J Bacteriol 199(15). https://doi.org/10.1128/jb.00012-17

  42. Kamaguchi A et al (2003) Effect of Porphyromonas gingivalis vesicles on coaggregation of Staphylococcus aureus to oral microorganisms. Curr Microbiol 47(6):485–491

    PubMed  CAS  Google Scholar 

  43. Yonezawa H et al (2009) Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation. BMC Microbiol 9:197

    PubMed  PubMed Central  Google Scholar 

  44. Seike S et al (2020) Outer membrane vesicles released from Aeromonas strains are involved in the biofilm formation. Front Microbiol 11:613650

    PubMed  Google Scholar 

  45. Zhao Z et al (2022) Regulation of the formation and structure of biofilms by quorum sensing signal molecules packaged in outer membrane vesicles. Sci Total Environ 806(Pt 4):151403

    PubMed  ADS  CAS  Google Scholar 

  46. da Silva Barreira D et al (2023) Membrane vesicles released by Lacticaseibacillus casei BL23 inhibit the biofilm formation of Salmonella enteritidis. Sci Rep 13(1):1163

    PubMed  PubMed Central  ADS  Google Scholar 

  47. Wang Y et al (2021) Inhibition of Streptococcus mutans biofilms with bacterial-derived outer membrane vesicles. BMC Microbiol 21(1):234

    PubMed  PubMed Central  CAS  Google Scholar 

  48. Furuta N et al (2009) Porphyromonas gingivalis outer membrane vesicles enter human epithelial cells via an endocytic pathway and are sorted to lysosomal compartments. Infect Immun 77(10):4187–4196

    PubMed  PubMed Central  CAS  Google Scholar 

  49. Cañas MA et al (2018) Outer membrane vesicles from probiotic and commensal Escherichia coli activate NOD1-mediated immune responses in intestinal epithelial cells. Front Microbiol 9:498

    PubMed  PubMed Central  Google Scholar 

  50. Ahmadi Badi S et al (2019) Induction effects of Bacteroides fragilis derived outer membrane vesicles on toll like receptor 2, toll like receptor 4 genes expression and cytokines concentration in human intestinal epithelial cells. Cell J 21(1):57–61

    PubMed  Google Scholar 

  51. Fábrega MJ et al (2017) Intestinal anti-inflammatory effects of outer membrane vesicles from Escherichia coli Nissle 1917 in DSS-experimental colitis in mice. Front Microbiol 8:1274

    PubMed  PubMed Central  Google Scholar 

  52. Caruana JC, Walper SA (2020) Bacterial membrane vesicles as mediators of microbe-microbe and microbe-host community interactions. Front Microbiol 11:432

    PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by the National Natural Science Foundation of China (No. 82271005), the Guangdong Basic and Applied Basic Research Foundation, China (No. 2021A1515010821), the International Team for Implantology (ITI, grant number 1586_2021), Osteology Foundation (Grant No. 20-162), and grant from the China Scholarship Council to BH.

Author information

Author notes

  1. Baoxin Huang and Chun Liu have contributed equally to the work and are co-first authors.

Authors and Affiliations

  1. Hospital of Stomatology, Sun Yat-Sen University, Guangzhou, People’s Republic of China

    Baoxin Huang & Jieting Yang

  2. Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat-Sen University, Guangzhou, People’s Republic of China

    Baoxin Huang & Jieting Yang

  3. The University of Queensland, School of Dentistry, QLD, Brisbane, 4006, Australia

    Baoxin Huang, Chun Liu, Enmao Xiang, Sašo Ivanovski & Pingping Han

  4. The University of Queensland, School of Dentistry, Centre for Oral-Facial Regeneration, Rehabilitation and Reconstruction (COR3), Brisbane, Queensland, Australia

    Baoxin Huang, Chun Liu, Enmao Xiang, Sašo Ivanovski & Pingping Han

Authors
  1. Baoxin Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jieting Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Enmao Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sašo Ivanovski
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pingping Han
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.H., P.H., and S.I. conceptualized this study. C.L. collected the samples. J.Y. prepared the titanium disc. B.H., C.L., and E.X. performed the experiments, and data analysis and drafted the manuscript. P.H. and S.I. guided B.H. and C.L. on data interpretation, and critically reviewed and revised the manuscript. All authors approved the article submission.

Corresponding authors

Correspondence to Sašo Ivanovski or Pingping Han.

Ethics declarations

Ethics approval

The current study was conducted in accordance with the World Medical Association Declaration of Helsinki (version, 2013). The ethical approval was obtained from The University of Queensland, institutional human ethics committee, approval number HREC2019/HE001113.

Consent to participate

The written informed consent of all the participating subjects was obtained prior to enrolling in this study.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

ESM 1

(DOCX 750 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huang, B., Liu, C., Yang, J. et al. Saliva biofilm-derived outer membrane vesicles regulate biofilm formation and immune response of oral epithelial cells on titanium surfaces. Clin Oral Invest 28, 75 (2024). https://doi.org/10.1007/s00784-023-05454-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00784-023-05454-9

Keywords

Search

Navigation