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Plasma Medicine
SJR: 0.278 SNIP: 0.183 CiteScore™: 0.57

ISSN Print: 1947-5764
ISSN Online: 1947-5772

Plasma Medicine

DOI: 10.1615/PlasmaMed.2018027314
pages 1-14

Ex Vivo Study Comparing Three Cold Atmospheric Plasma (CAP) Sources for Biofilm Removal on Microstructured Titanium

Saskia Preissner
Department of Operative and Preventive Dentistry, Charité-Universitätsmedizin Berlin, Berlin, Germany
Ann Cathrin Poehlmann
Charité Universitätsmedizin Berlin, Berlin, Germany
Andreas Schubert
IZI Fraunhofer, Leipzig, Germany
Antje Lehmann
IOM Leipzig, Leipzig, Germany
Thomas Arnold
cIOM Leipzig, Leipzig, Germany
Olaf Nell
University Homburg, Homburg, Germany
Stefan Rupf
University Homburg, Homburg, Germany


The aim of the present experimental study was to test three different plasma sources on the removal of 72 h oral biofilms. It was hypothesized that cold atmospheric plasma (CAP) lowers biofilm coverage significantly. In vivo biofilms were formed on sand-blasted/acid-etched titanium discs (n = 40) mounted on splints worn for 72 h by eight volunteers. Specimens were randomly divided into five groups: CAP I received indirect plasma application, CAP II received direct plasma application, CAP III received microwave-driven pulsed plasma application (90 s each). The chlorhexidine (CHX) group was cleaned with a curette and rinsed with CHX. Biofilms of a control group received no treatment. After treatment, all specimens were rinsed for 10 s using a dental air/water spray (2 bar). The vitality of microorganisms was detected by cultivation on agar plates for 24 h and 48 h. The presence of biofilms and their quantity on the titanium samples was investigated by fluorescence microscopy (FM) using live/dead staining. A biofilm's quality was analyzed by scanning electron microscopy (SEM). All treated samples showed a reduced growth on agar plates compared with the control group. FM analysis showed significantly lowered biofilm coverage in all treatment groups compared with the negative control group (t test, p < 0.05). Within the plasma treatment groups, there was a significant difference between CAP II and CAP III ( p = 0.032). SEM showed disintegrated biofilms in all test groups. CAP reduces and disintegrates oral biofilms. Adjuvant application plasma could lead to more effective antimicrobial therapies for peri-implantitis.


  1. Haertel B, von Woedtke T, Weltmann KD, Lindequist U. , Non-thermal atmospheric-pressure plasma possible application in wound healing. Biomol Ther (Seoul). 2014;22(6):477-90.

  2. Metelmann HR, von Woedtke T, Weltmann KD. , Plasmamedizin: Kaltplasma in der medizinischen Anwendung. 2016.

  3. Arjunan KP, Sharma VK, Ptasinska S. , Effects of atmospheric pressure plasmas on isolated and cellular DNA-a review. Int J Mol Sci. 2015;16(2):2971-3016.

  4. Laroussi M, Kong MG, Morfill G, Stolz W. , Plasma medicine: Applications of low-temperature gas plasmas in medicine and biology. Cambridge: Cambridge University Press; 2012.

  5. Von Woedtke TRS, Masur K, Weltmann KD. , Plasmas for medicine. Phys Rep. 2013; 530:291-320.

  6. Babington P, Rajjoub K, Canady J, Siu A, Keidar M, Sherman JH. , Use of cold atmospheric plasma in the treatment of cancer. Biointerphases. 2015;10(2):029403.

  7. Isbary G, Morfill G, Schmidt HU, Georgi M, Ramrath K, Heinlin J, Karrer S, Landthaler M, Shimizu T, Steffes B, Bunk W, Monetti R, Zimmermann JL, Pompl R, Stolz W., A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients. Br J Dermatol. 2010;163(1):78-82.

  8. Rupf S, Lehmann A, Hannig M, Schafer B, Schubert A, Feldmann U, Schindler A. , Killing of adherent oral microbes by a non-thermal atmospheric plasmajet. J Med Microbiol. 2010;59:206-12.

  9. Sharkey MA, Chebbi A, McDonnell KA, Staunton C, Dowling DP. , Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments. Biointerphases. 2015;10(2):029507.

  10. Southwood LL, Baxter GM. , Instrument sterilization, skin preparation, and wound management. Vet Clin North Am Equine Pract. 1996;12(2):173-94.

  11. Aboubakr HA, Williams P, Gangal U, Youssef MM, El-Sohaimy SA, Bruggeman PJ, Goyal SM, Virucidal effect of cold atmospheric gaseous plasma on feline calicivirus, a surrogate for human norovirus. Appl Environ Microbiol. 2015;81(ll):3612-22.

  12. Alkawareek MY, Algwari QT, Gorman SP, Graham WG, O'Connell D, Gilmore BE, Application of atmospheric pressure nonthermal plasma for the in vitro eradication of bacterial biofilms. FEMS Immunol Med Microbiol. 2012;65(2):381-4.

  13. Klampfl TG, Isbary G, Shimizu T, Li YF, Zimmermann JL, Stolz W, Schlegel J, Morfill GE, Schmidt H-U, Cold atmospheric air plasma sterilization against spores and other microorganisms of clinical interest. Appl Environ Microbiol. 2012;78(15):5077-82.

  14. Kvam E, Davis B, Mondello F, Garner AL. , Nonthermal atmospheric plasma rapidly disinfects multidrug-resistant microbes by inducing cell surface damage. Antimicrob Agents Chemother. 2012;56(4):2028-36.

  15. Maisch T, Shimizu T, Isbary G, Heinlin J, Karrer S, Klampfl TG, Li YF, Morfill G, Zimmermann JL. , Contact-free inactivation of Candida albicans biofilms by cold atmospheric air plasma. Appl Environ Microbiol. 2012;78(12):4242-7.

  16. Maisch T, Shimizu T, Li YF, Heinlin J, Karrer S, Morfill G, Zimmermann JL. , Decolonisation of MRSA, S. aureus and E. coli by cold-atmospheric plasma using a porcine skin model in vitro. PLoS One. 2012;7(4):e34610.

  17. Yoo EM, Uhm SH, Kwon JS, Choi HS, Choi EH, Kim KM, Kim KN. , The study on inhibition of planktonic bacterial growth by non-thermal atmospheric pressure plasmajet treated surfaces for dental application. J Biomed Nanotechnol. 2015;11(2):334-41.

  18. Mai-Prochnow A, Clauson M, Hong J, Murphy AB. , Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma. Sci Rep. 2016;6:38610.

  19. Napp M, Daeschlein G, von Podewils S, Hinz P, Emmert S, Haase H, Spitzmueller R, Gumbel D, Kasch R, Junger M., In vitro susceptibility of methicillin-resistant and methicillin-susceptible strains of Staphylococcus aureus to two different cold atmospheric plasma sources. Infection. 2016;44(4):531-7.

  20. Smeets R, Henningsen A, Jung O, Heiland M, Hammacher C, Stein JM. , Definition, etiology, prevention and treatment of peri-implantitis—a review. Head Face Med. 2014;10:34.

  21. Schwarz F, Becker J, Bach J, Bartsch K, Beck J, Blume M, Ighaut G, Kebschull M, Ritter L, Schlee M, Steisch M, Stiller M, Wolf T. , S3-Leitlinie Die Behandlung peri-implantarer Infektionen an Zahn-implantaten. AWMF Online. 2016.

  22. Lee CT, Huang YW, Zhu L, Weltman R. , Prevalences of peri-implantitis and peri-implant mucositis: systematic review and meta-analysis. J Dent. 2017;62:1-12.

  23. Lang NP, Berglundh T, Working Group 4 of Seventh European Workshop on Periodontology. Peri-implant diseases: Where are we now?—Consensus of the Seventh European Workshop on Periodontology. J Clin Periodontal. 2011;38 Suppl 11:178-81.

  24. Pesce P, Canullo L, Grusovin MG, de Bruyn H, Cosyn J, Pera P. , Systematic review of some prosthetic risk factors for peri-implantitis. J Prosthet Dent. 2015;114(3):346-50.

  25. Lindhe Jl, Meyle J, Group D of European Workshop on Periodontology. Peri-implant diseases: Consensus report of the Sixth European Workshop on Periodontology. J Clin Periodontol. 2008;35(8 Suppl):282-5.

  26. Hannig C, Hannig M. , The oral cavity—a key system to understand substratum-dependent bioadhe-sion on solid surfaces in man. Clin Oral Investig. 2009;13(2):123-39.

  27. Marsh PD. , Dental plaque as a biofilm and a microbial community: Implications for health and disease. BMC Oral Health. 2006;6 Suppl 1:S14.

  28. Subramani K, Jung RE, Molenberg A, Hammerle CH. , Biofilm on dental implants: a review of the literature. Int J Oral Maxillofac Implants. 2009;24(4):616-26.

  29. Horikawa T, Odatsu T, Itoh T, Soejima Y, Morinaga H, Abe N, Tsuchiya N, Iijima T, Sawase T. , Retrospective cohort study of rough-surface titanium implants with at least 25 years' function. Int J Implant Dent. 2017;3(1):42.

  30. de Araujo Nobre M, Malo P, Prevalence of periodontitis, dental caries, and peri-implant pathology and their relation with systemic status and smoking habits: Results of an open-cohort study with 22009 patients in a private rehabilitation center. J Dent. 2017;67:36-42.

  31. Muthukuru M, Zainvi A, Esplugues EO, Flemmig TF. , Non-surgical therapy for the management of peri-implantitis: a systematic review. Clin Oral Implants Res. 2012;23 Suppl 6:77-83.

  32. Rupf S, Idlibi AN, Marrawi FA, Hannig M, Schubert A, von Mueller L, Spitzer W, Holtmann H, Lehmann A, Rueppell A, Schindler A. , Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology. PLoS One. 2011;6(10):e25893.

  33. Idlibi AN, Al-Marrawi F, Hannig M, Lehmann A, Rueppell A, Schindler A, Jentsch H, Rupf S., Destruction of oral biofilms formed in situ on machined titanium (Ti) surfaces by cold atmospheric plasma. Biofouling. 2013;29(4):369-79.

  34. Lehmann A, Pietag F, Arnold T. , Human health risk evaluation of a microwave-driven atmospheric plasmajet as medical device. Clin Plasma Med. 2017;7-8:16-23.

  35. Preissner S, Wirtz HC, Tietz AK, Abu-Sirhan S, Herbst SR, Hartwig S, Pierdzioch P, Schmidt-West-hausen AM, Dommisch H, Hertel M, Bactericidal efficacy of tissue tolerable plasma on microrough titanium dental implants: An in-vitro-study. J Biophotonics. 2016;9(6):637-44.

  36. Jablonowski L, Fricke K, Matthes R, Holtfreter B, Schluter R, von Woedtke T, Weltmann KD, Ko-cher T., Removal of naturally grown human biofilm with an atmospheric pressure plasma jet: An in-vitro study. J Biophotonics. 2017;10(5):718-26.

  37. Matthes R, Hubner NO, Bender C, Koban I, Horn S, Bekeschus S, Weltmann KD, Kocher T, Kramer A, Assadian O., Efficacy of different carrier gases for barrier discharge plasma generation compared with chlorhexidine on the survival of Pseudomonas aeruginosa embedded in biofilm in vitro. Skin Pharmacol Physiol. 2014;27(3):148-57.

  38. Duske K, Jablonowski L, Koban I, Matthes R, Holtfreter B, Sckell A, Nebe JB, von Woedtke T, Weltmann KD, Kocher T. , Cold atmospheric plasma in combination with mechanical treatment improves osteoblast growth on biofilm covered titanium discs. Biomaterials. 2015;52: 327-34.

  39. Lee JH, Kim YH, Choi EH, Kim KM, Kim KN. , Air atmospheric-pressure plasma-jet treatment enhances the attachment of human gingival fibroblasts for early peri-implant soft tissue seals on titanium dental implant abutments. Acta Odontol Scand. 2015;73(1):67-75.

  40. Danna NR, Beutel BG, Tovar N, Witek L, Marin C, Bonfante EA, Granato R, Suzuki M, Coelho PG., Assessment of atmospheric pressure plasma treatment for implant osseointegration. Biomed Res Int. 2015;2015:761718.

  41. Daeschlein G, von Woedtke T, Kindel E, Brandenburg R, Weltmann K-D, Junger M., Antibacterial activity of an atmospheric pressure plasma jet against relevant wound pathogens in vitro on a simulated wound environment. Plasma Processes and Polymers. 2010;7(3-4):224-30.

  42. Han L, Patil S, Keener KM, Cullen PJ, Bourke P. , Bacterial inactivation by high-voltage atmospheric cold plasma: influence of process parameters and effects on cell leakage and DNA. J Appl Microbiol. 2014;116(4):784-94.

  43. Heinlin J, Zimmermann JL, Zeman F, Bunk W, Isbary G, Landthaler M, Maisch T, Monetti R, Morfill G, Shimizu T, Steinbauer J, Stolz W, Karrer S., Randomized placebo-controlled human pilot study of cold atmospheric argon plasma on skin graft donor sites. Wound Repair Regen. 2013;21(6):800-7.

  44. Matthes R, Assadian O, Kramer A. , Repeated applications of cold atmospheric pressure plasma does not induce resistance in Staphylococcus aureus embedded in biofilms. GMS Hyg Infect Control. 2014;9(3):Doc17.