Original study - ZZI 02/2012

Disinfection and removal of biofilms on microstructured titanium by cold atmospheric plasma

S. Rupf1, A. N. Idlibi1, N. Umanskaya1, M. Hannig1, F. Nothdurft2, A. Lehmann3, A. Schindler3, L. v. Müller4, W. Spitzer5

Introduction: Biofilms on dental implants play an important role in the genesis of inflammatory periimplant disease. Decontamination of microstructured titanium (mTi) is still a challenge for the dental practitioner. Cold atmospheric plasma jets offer disinfecting capabilities at biologically acceptable temperatures. This experimental study investigated disinfective and destructive effects of cold atmospheric plasma on oral biofilms formed in situ on mTi surfaces.

Material and Methods: mTi discs (sandblasted/etched, N = 120) were exposed to the oral environment of two healthy volunteers for 24 hours to produce biofilms. Plasma treatment was carried out by means of a meander like CC line by line scanning with a pulsed microwave-driven (2.45 GHz) plasma jet (2.5 s/mm², 2.0 l/min He, 3 W or 5 W microwave mean power). Following plasma treatment some specimens were air/water sprayed and subsequently subjected to a second plasma treatment. Non-irradiated biofilms, biofilms treated with chlorhexidine and mTi discs without biofilms served as controls. Disinfection of biofilms was assessed by contact agar samples (RODAC technique) and fluorescence microscopy (vital/dead staining). Biofilm morphology was visualized by scanning electron microscopy; biofilm coverage was measured by fluorescence microscopy. Total protein was quantified colorimetrically.

Results: Depending on the plasma jet power the mTi surface temperature at the plasma contact point varied between 39 and 43°C. After plasma treatment only, disintegration as well as reduction of biofilm viability and of total protein were observed. The additional application of air/water spray resulted in a further reduction of biofilm viability and of total protein. Fluorescence and protein amounts were reduced comparable to control specimens without biofilms after a second plasma treatment. The microstructured surface of the samples was not altered by plasma treatment.

Conclusions: In this experimental study cold atmospheric plasma technology combined with air/water spray enabled complete elimination of oral biofilms from mTi. This new approach may enable new routes for the therapy of inflammatory periimplant disease, while preserving microstructured surfaces.

Keywords: cold atmospheric plasma; biofilm removal; experimental study; oral biofilm; microstructured titanium; SEM; fluorescence microscopy; cultivation

Introduction

Microbial biofilms are the most important factor in the pathogenesis of inflammatory processes on dental implants [14]. When inflammation spreads to the bone, clinically established perimucositis can progress to periimplantitis, which is the most frequent cause of implant failure. The incidence of periimplantitis is reported in the literature as 12–43 % [27]. The outstanding biotolerability of modern implant materials, such as titanium or zirconium oxide, promotes deposition of endogenous cells but also adsorption of biomolecular pellicles and microorganisms [2]. Biofilms impede the colonization of implant surfaces by osteoblasts and other endogenous cells and can interfere with both osseointegration and re-osseointegration after exposure of the implant surface to the oral environment [21, 22]. For these reasons, implant surface decontamination methods are the subject of intensive research. Techniques currently employed for surface decontamination of dental implants include mechanical scaling, smoothing and polishing using Teflon, plastic or metal curettes or rotaries. Ultrasonically activated curettes are also used in combination with antimicrobial rinses, air abrasion polishing and laser decontamination [1, 4, 5, 17, 21–23]. However, since modern implant surfaces often have a microstructured surface [25], biofilm removal continues to be a technical challenge. Mechanical processing leads to destruction of the surface texture while biofilm is often incompletely removed, laser decontamination can damage the surrounding tissue due by heating, and chemical disinfectant methods leave behind biofilms that are morphologically intact. Methods that enable complete biofilm removal while preserving the surface texture of dental implants have so far been available for clinical use only to a small extent [20].

An alternative disinfection method for biofilms is the use of physical plasmas. In physics, plasmas constitute the fourth state, in addition to solids, liquids and gases. Physical plasmas are ionized gases that consist largely or completely of free charge carriers. These include electrons, ions, free radicals and charged molecules. In recent times, it has become possible to miniaturize plasma sources and keep plasmas stable for use under environmental conditions with very little effort [10, 18]. The delivery of excitation energy can also be pulsed. The plasma jet is ignited for a few microseconds and cooled by the gas flow between pulses. This makes it possible to produce plasma with biologically acceptable temperatures below 40°C. The noble gases helium and argon are used as carrier for the plasma. Addition of chemically active gases such as oxygen or nitrogen produces reactive oxygen species (NO-, O- and OH-radicals), which are capable of reacting with surfaces. This allows mild etching and cleaning processes to be performed. Plasmas have the further advantage that they can penetrate hollow spaces and clefts, which they also clean or disinfect. Previous investigations have shown that adherent microorganisms can be reduced by several orders of magnitude with cold plasmas at biologically acceptable temperatures. It has also been shown that biofilms are disinfected effectively [6–10, 18, 19, 24].

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