Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ



The aim of the present study was to investigate the efficacy of a new preparation in dental prophylaxis containing zinc-carbonate hydroxyapatite microclusters (Biorepair) for oral biofilm management.

Methods and materials

Initial biofilm formation was carried out in situ with bovine enamel slabs fixed to individual upper jaw splints worn by six subjects. Rinses with the customary preparation as well as with subfractions (hydroxyapatite microclusters in saline solution; liquid phase without particles) were adopted for 1 min in situ after 1 min of pellicle formation, and the bacterial colonization was recorded after 6 h and 12 h, respectively. Rinses with chlorhexidine served as a reference. The adherent microorganisms were quantified and visualized using DAPI staining and live–dead staining (BacLight). Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight).


Application of the customary preparation and of the separate components distinctly reduced the initial bacterial colonization of the enamel surface in situ as visualized and quantified with all techniques. After 12 h, 1.3 × 107 ± 2.0 × 107 bacteria/cm² were detected on unrinsed control samples with DAPI staining; 2.4 × 106 ± 3.3 × 106 after application of Biorepair (12 h after CHX-rinse; 1.3 × 105 ± 9.2 × 104). Also, pure hydroxyapatite microclusters in saline solution (2.1 × 106 ± 3.0 × 106) as well as the liquid phase without particles (5.1 × 105 ± 3.3 × 105) reduced the amount of adherent bacteria. Furthermore, antimicrobial effects on S. mutans were observed in vitro.


The preparation is an effective compound for biofilm management in the oral cavity due to antiadherent and antibacterial effects.

Clinical relevance

The tested mouthrinse seems to be a reasonable amendment for dental prophylaxis.

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  1. 1.

    Ismail AI, Hasson H (2008) Fluoride supplements, dental caries and fluorosis: a systematic review. J Am Dent Assoc 139:1457–1468

    PubMed  Google Scholar 

  2. 2.

    Griffin SO, Regnier E, Griffin PM et al (2007) Effectiveness of fluoride in preventing caries in adults. J Dent Res 86:410–415

    PubMed  Article  Google Scholar 

  3. 3.

    Hellwig E, Lennon AM (2004) Systemic versus topical fluoride. Caries Res 38:258–262

    PubMed  Article  Google Scholar 

  4. 4.

    Marsh PD (2004) Dental plaque as a microbial biofilm. Caries Res 38:204–211

    PubMed  Article  Google Scholar 

  5. 5.

    Hannig C, Spies B, Spitzmuller B et al (2010) Efficacy of enzymatic mouth rinses for immobilisation of protective enzymes in the in situ pellicle. Arch Oral Biol 55:1–6

    PubMed  Article  Google Scholar 

  6. 6.

    Cross KJ, Huq NL, Reynolds EC (2007) Casein phosphopeptides in oral health—chemistry and clinical applications. Curr Pharm Des 13:793–800

    PubMed  Article  Google Scholar 

  7. 7.

    Hannig M, Hannig C (2010) Nanomaterials in preventive dentistry. Nat Nanotechnol 5:565–569

    PubMed  Article  Google Scholar 

  8. 8.

    Hannig C, Hannig M (2010) Natural enamel wear—a physiological source of hydroxylapatite nanoparticles for biofilm management and tooth repair? Med Hypotheses 74:670–672

    PubMed  Article  Google Scholar 

  9. 9.

    Roveri N, Palazzo B, Iafisco M (2008) The role of biomimetism in developing nanostructured inorganic matrices for drug delivery. Expert Opin Drug Deliv 5:861–877

    PubMed  Article  Google Scholar 

  10. 10.

    Battistella E, Lelli M, Palazzo B et al. (2009) Surface enamel remineralisation: biomimetic apatite nanocrystals and fluoride ions different effects. J Nanomaterials

  11. 11.

    Tschoppe P, Zandim DL, Martus P et al (2011) Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 39:430–437

    PubMed  Article  Google Scholar 

  12. 12.

    Poggio C, Lombardini M, Colombo M et al (2010) Impact of two toothpastes on repairing enamel erosion produced by a soft drink: an AFM in vitro study. J Dent 38:868–874

    PubMed  Article  Google Scholar 

  13. 13.

    Hannig C, Hannig M, Rehmer O et al (2007) Fluorescence microscopic visualization and quantification of initial bacterial colonization on enamel in situ. Arch Oral Biol 52:1048–1056

    PubMed  Article  Google Scholar 

  14. 14.

    Hannig C, Follo M, Hellwig E et al (2010) Visualization of adherent microorganisms using different techniques. J Med Microbiol 59:1–7

    PubMed  Article  Google Scholar 

  15. 15.

    Hannig C, Kirsch J, Al-Ahmad A et al. (2012) Do edible oils reduce bacterial colonization of enamel in situ? Clinical oral investigations:in press

  16. 16.

    Jung DJ, Al-Ahmad A, Follo M et al (2010) Visualization of initial bacterial colonization on dentine and enamel in situ. J Microbiol Methods 81:166–174

    PubMed  Article  Google Scholar 

  17. 17.

    Al-Ahmad A, Follo M, Selzer AC et al (2009) Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization. J Med Microbiol 58:1359–1366

    PubMed  Article  Google Scholar 

  18. 18.

    Schwartz T, Hoffmann S, Obst U (2003) Formation of natural biofilms during chlorine dioxide and UV disinfection in a public drinking water distribution system. J Appl Microbiol 95:591–601

    PubMed  Article  Google Scholar 

  19. 19.

    Faul F, Erdfelder E, Buchner A et al (2009) Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav Res Methods 41:1149–1160

    PubMed  Article  Google Scholar 

  20. 20.

    Faul F, Erdfelder E, Lang AG et al (2007) G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 39:175–191

    PubMed  Article  Google Scholar 

  21. 21.

    Hannig C, Hannig M (2009) The oral cavity—a key system to understand substratum-dependent bioadhesion on solid surfaces in man. Clin Oral Investig 13:123–139

    PubMed  Article  Google Scholar 

  22. 22.

    Hannig M (1999) Transmission electron microscopy of early plaque formation on dental materials in vivo. Eur J Oral Sci 107:55–64

    PubMed  Article  Google Scholar 

  23. 23.

    Hannig C, Sorg J, Spitzmuller B et al (2009) Polyphenolic beverages reduce initial bacterial adherence to enamel in situ. J Dent 37:560–566

    PubMed  Article  Google Scholar 

  24. 24.

    Hannig C, Spitzmuller B, Al-Ahmad A et al (2008) Effects of Cistus-tea on bacterial colonization and enzyme activities of the in situ pellicle. J Dent 36:540–545

    PubMed  Article  Google Scholar 

  25. 25.

    Gjermo P, Saxton CA (1991) Antibacterial dentifrices. Clinical data and relevance with emphasis on zinc/triclosan. J Clin Periodontol 18:468–473

    PubMed  Article  Google Scholar 

  26. 26.

    Soderberg TA (1990) Effects of zinc oxide, rosin and resin acids and their combinations on bacterial growth and inflammatory cells. Scand J Plast Reconstr Surg Hand Surg Suppl 22:1–87

    PubMed  Google Scholar 

  27. 27.

    Raghupathi KR, Koodali RT, Manna AC (2011) Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir 27:4020–4028

    PubMed  Article  Google Scholar 

  28. 28.

    Bowen WH (1994) Food components and caries. Adv Dent Res 8:215–220

    PubMed  Google Scholar 

  29. 29.

    Mandel ID (1994) Antimicrobial mouthrinses: overview and update. J Am Dent Assoc 125(Suppl 2):2S–10S

    PubMed  Google Scholar 

  30. 30.

    Sarikaya M, Tamerler C, Jen AK et al (2003) Molecular biomimetics: nanotechnology through biology. Nat Mater 2:577–585

    PubMed  Article  Google Scholar 

  31. 31.

    Venegas SC, Palacios JM, Apella MC et al (2006) Calcium modulates interactions between bacteria and hydroxyapatite. J Dent Res 85:1124–1128

    PubMed  Article  Google Scholar 

  32. 32.

    Kezic S, Kammeyer A, Calkoen F et al (2009) Natural moisturizing factor components in the stratum corneum as biomarkers of filaggrin genotype: evaluation of minimally invasive methods. Br J Dermatol 161:1098–1104

    PubMed  Article  Google Scholar 

  33. 33.

    Rose RK (2000) Binding characteristics of Streptococcus mutans for calcium and casein phosphopeptide. Caries Res 34:427–431

    PubMed  Article  Google Scholar 

  34. 34.

    Rose RK (2000) The role of calcium in oral streptococcal aggregation and the implications for biofilm formation and retention. Biochim Biophys Acta 1475:76–82

    PubMed  Article  Google Scholar 

  35. 35.

    Rose RK (2000) Effects of an anticariogenic casein phosphopeptide on calcium diffusion in streptococcal model dental plaques. Arch Oral Biol 45:569–575

    PubMed  Article  Google Scholar 

  36. 36.

    Boverhof DR, David RM (2010) Nanomaterial characterization: considerations and needs for hazard assessment and safety evaluation. Anal Bioanal Chem 396:953–961

    PubMed  Article  Google Scholar 

  37. 37.

    Holgate ST (2010) Exposure, uptake, distribution and toxicity of nanomaterials in humans. J Biomed Nanotechnol 6:1–19

    PubMed  Article  Google Scholar 

  38. 38.

    Buzea C, Pacheco I, Robbie K (2007) Nanomaterials and nanoparticles: sources and toxicity. Biointerphases 2:MR17–71

    PubMed  Article  Google Scholar 

  39. 39.

    Frohlich E, Roblegg E (2012) Models for oral uptake of nanoparticles in consumer products. Toxicology 291:10–17

    PubMed  Article  Google Scholar 

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We would like to thank Bettina Spitzmüller, University of Freiburg, for the excellent support in the laboratory.

Conflict of interest

The authors declare that they have no conflict of interest.

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Correspondence to C. Hannig.

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Hannig, C., Basche, S., Burghardt, T. et al. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Invest 17, 805–814 (2013).

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  • Nanomaterial
  • Mouthwash
  • Hydroxyapatite
  • Biofilm
  • Pellicle
  • Bacterial adherence