Skip to main content
Log in

Biofilms Developed on Dental Implant Titanium Surfaces with Different Roughness: Comparison Between In Vitro and In Vivo Studies

  • Published:
Current Microbiology Aims and scope Submit manuscript


Microbial biofilms developed on dental implants play a major role in perimplantitis’ pathogenesis. Many studies have indicated that surface roughness is the main feature favoring biofilm development in vitro, but its actual influence in vivo has still to be confirmed. In this study, the amount of biofilm formed on differently treated titanium surfaces, showing distinct roughness, has been examined both in vivo and in vitro by Confocal Laser Scanning Microscopy. In vitro studies availed of biofilm developed by Pseudomonas aeruginosa or by salivary bacteria from volunteer donors. In vivo biofilm production was obtained by exposing titanium discs to the oral cavity of healthy volunteers. In vitro experiments showed that P. aeruginosa and, to a lesser extent, salivary bacteria produce more biomass and develop thicker biofilms on laser-treated and sandblasted titanium surfaces with respect to machined ones. In vivo experiments confirmed that bacterial colonization starts on sites of surface unevenness, but failed to disclose biomass differences among biofilms formed on surfaces with different roughness. Our study revealed that biofilm developed in vitro is more easily influenced by surface features than biofilm formed by complex communities in the mouth, where the cooperation of a variety of bacterial species and the presence of a wide range of nutrients and conditions allow bacteria to optimize substrate colonization. Therefore, quantitative differences observed in vitro among surfaces with different characteristics may not be predictive of different colonization rates in vivo.

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

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others


  1. Al-Ahmad A, Wiedmann-Al-Ahmad M, Faust J, Bächle M, Follo M, Wolkewitz M, Hannig C, Hellwig E, Carvalho C, Kohal R (2010) Biofilm formation and composition on different implant materials in vivo. J Biomed Mater Res B 95(1):101–109

    Article  CAS  Google Scholar 

  2. Albertini M, López-Cerero L, O’Sullivan MG, Chereguini CF, Ballesta S, Ríos V, Herrero-Climent M, Bullón P (2015) Assessment of periodontal and opportunistic flora in patients with peri-implantitis. Clin Oral Implants Res 26(8):937–941

    Article  PubMed  Google Scholar 

  3. Bürgers R, Gerlach T, Hahnel S, Schwarz F, Handel G, Gosau M (2010) In vivo and in vitro biofilm formation on two different titanium implant surfaces. Clin Oral Implants Res 21(2):156–164

    Article  PubMed  Google Scholar 

  4. Cavalcanti IM, Ricomini Filho AP, Lucena-Ferreira SC, da Silva WJ, Paes Leme AF, Senna PM, Del Bel Cury AA (2014) Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma. Arch Oral Biol 59(7):695–703

    Article  CAS  PubMed  Google Scholar 

  5. Drago L, Bortolin M, De Vecchi E, Agrappi S, Weinstein RL, Mattina R, Francetti L (2016) Antibiofilm activity of sandblasted and laser-modified titanium against microorganisms isolated from peri-implantitis lesions. J Chemother 28(5):383–389

    Article  CAS  PubMed  Google Scholar 

  6. Grössner-Schreiber B, Griepentrog M, Haustein I, Müller WD, Lange KP, Briedigkeit H, Göbel UB (2001) Plaque formation on surface modified dental implants. An in vitro study. Clin Oral Implants Res 12(6):543–551

    Article  PubMed  Google Scholar 

  7. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S (2000) Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146:2395–2407

    Article  CAS  PubMed  Google Scholar 

  8. Huang MS, Chen LK, Ou KL, Cheng HY, Wang CS (2015) Rapid osseointegration of titanium implant with innovative nanoporous surface modification: animal model and clinical trial. Implant Dent 24(4):441–447

    PubMed  Google Scholar 

  9. John G, Becker J, Schwarz F (2015) Modified implant surface with slower and less initial biofilm formation. Clin Implant Dent Relat Res 17:461–468

    Article  PubMed  Google Scholar 

  10. Kirmanidou Y, Sidira M, Drosou M-E, Bennani V, Bakopoulou A, Tsouknidas A et al (2016) New Ti-alloys and surface modifications to improve the mechanical properties and the biological response to orthopedic and dental implants: a review. BioMed Res Int 2016:1–21.

    Article  Google Scholar 

  11. Le Guéhennec L, Soueidan A, Layrolle P, Amouriq Y (2007) Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater 23(7):844–854

    Article  PubMed  Google Scholar 

  12. Lindhe J, Meyle J. Group D of European Workshop on Periodontology (2008) Peri-implant diseases: consensus report of the Sixth European Workshop on Periodontology. J Clin Periodontol. 35: 282–285

    Article  PubMed  Google Scholar 

  13. Mabboux F, Ponsonnet L, Morrier JJ, Jaffrezic N, Barsotti O (2004) Surface free energy and bacterial retention to saliva-coated dental implant materials. An in vitro study. Colloids Surf B 39:199–205

    Article  CAS  Google Scholar 

  14. Okshevsky M, Meyer RL (2015) The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Crit Rev Microbiol 41(3):341–352

    Article  CAS  PubMed  Google Scholar 

  15. Palmer RJ Jr, Wu R, Gordon S, Bloomquist CG, Liljemark WF, Kilian M, Kolenbrander PE (2001) Retrieval of biofilms from the oral cavity. Methods Enzymol 337:393–403

    Article  PubMed  Google Scholar 

  16. Quirynen M, Van der Mei HC, Bollen CM et al (1994) The influence of surface-free energy on supra and subgingival plaque microbiology. An in vivo study on implants. J Periodontol 65:162–167

    Article  CAS  PubMed  Google Scholar 

  17. Quirynen M, van Steenberghe D (1993) Bacterial colonization of the internal part of two-stage implants. An in vivo study. Clin Oral Implant Res 4:158–161

    Article  CAS  Google Scholar 

  18. Rosan B, Lamont RJ (2000) Dental plaque formation. Microbes Infect 2:1599–1607

    Article  CAS  PubMed  Google Scholar 

  19. Rybtke M, Hultqvist LD, Givskov M, Tolker-Nielsen T (2015) Pseudomonas aeruginosa biofilm infections: community structure, antimicrobial tolerance and immune response. J Mol Biol 427(23):3628–3645

    Article  CAS  PubMed  Google Scholar 

  20. Sánchez MC, Llama-Palacios A, Fernández E, Figuero E, Marín MJ, León R, Blanc V, Herrera D, Sanz M (2014) An in vitro biofilm model associated to dental implants: structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces. Dent Mater 30:1161–1171

    Article  PubMed  Google Scholar 

  21. Shalabi MM, Gortemaker A, Van’t Hof MA, Jansen JA, Creugers NHJ (2006) Implant surface roughness and bone healing: a systematic review. J Dent Res 85:496–500

    Article  CAS  PubMed  Google Scholar 

  22. Shibata Y, Tanimoto Y (2015) A review of improved fixation methods for dental implants. Part I: surface optimization for rapid osseointegration. J Prosthodont Res 59:20–33

    Article  PubMed  Google Scholar 

  23. Subramani K, Jung RE, Molenberg A, Hammerle CH (2009) Biofilm on dental implants: a review of the literature. Int J Oral Maxillofac Implants 24:616–626

    PubMed  Google Scholar 

  24. Teughels W, Van Assche N, Sliepen I, Quirynen M (2006) Effect of material characteristics and/or surface topography on biofilm development. Clin Oral Implants Res 17:68–81

    Article  PubMed  Google Scholar 

  25. van Dijk J (1987) Surface-free energy and bacterial adhesion. An in vivo study in beagle dogs. J Clin Periodontol 14:300–304

    Article  PubMed  Google Scholar 

  26. Wennerberg A, Albrektsson T (2009) Effects of titanium surface topography on bone integration: a systematic review. Clin Oral Implants Res 20:172–184

    Article  PubMed  Google Scholar 

  27. Zitzmann NU, Berglundh T (2008) Definition and prevalence of peri-implant diseases. J Clin Periodontol 35:286–291

    Article  PubMed  Google Scholar 

Download references


The authors would like to thank GEASS s.r.l (Pozzuolo del Friuli, Udine) for donating some of the materials used in this study. CLSM images were generated in the Optical Microscopy Center of the University of Trieste at the Life Sciences Department, funded as detailed at “” Special thanks are due to the facility manager, Dr. Gabriele Baj, for his expert advice.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Lucilla Dolzani.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1 Acrylic splint carrying titanium discs (TIF 1938 KB)


Supplementary Figure 2 CLSM image of a LT titanium surface covered with biofilm developed after 1-day exposure to the oral environment (magnification: 60x) (TIF 12400 KB)


Supplementary Figure 3 Biofilm developed on glass slides by P. aeruginosa (a) and Mixed Salivary bacteria (b). Staining: Hucker crystal violet; magnification: 600x. Method: sterile standard microscopy glass slides with self-adhesive 12 wells silicone gaskets (Ibidi) were used. 200 µl of BHI containing either 5x105 bacteria from a pure P. aeruginosa culture or 5 µl of MSB were dispensed into each well. Slides were covered with the supplied lid and incubated for 48 hours at 37°C to allow biofilm development. Each well was then washed 3 times with sterile saline, the gasket was removed and biofilm was fixed by exposure at 60° for 1 hour. The slide was stained for 15 min with Hucker crystal violet and observed under the light microscope (TIF 8274 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bevilacqua, L., Milan, A., Del Lupo, V. et al. Biofilms Developed on Dental Implant Titanium Surfaces with Different Roughness: Comparison Between In Vitro and In Vivo Studies. Curr Microbiol 75, 766–772 (2018).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: