Eva-Maria Decker

Multifunctional nature of UV-irradiated nanocrystalline anatase thin films for biomedical applications

Anatase is known to decompose organic material by photocatalysis and to enhance surface wettability once irradiated by ultraviolet (UV) light. In this study, pulse magnetron-sputtered anatase thin films were investigated for their suitability with respect to specific biomedical applications, namely superhydrophilic and biofilm degrading implant surfaces. UV-induced hydrophilicity was quantified by static and dynamic contact angle analysis. Photocatalytic protein decomposition was analyzed by quartz crystal microbalance with dissipation. The surfaces were characterized by X-ray diffraction, atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. The radical formation on anatase, responsible for photocatalytic effects, was analyzed by electron spin resonance spectroscopy. Results have shown that the nanocrystalline anatase films, in contrast to reference titanium surfaces, were sensitive to UV irradiation and showed rapid switching towards superhydrophilicity. The observed decrease in carbon adsorbents and the increase in the fraction of surface hydroxyl groups upon UV irradiation might contribute to this hydrophilic behavior. UV irradiation of anatase pre-conditioned with albumin protein layers induces the photocatalytic decomposition of these model biofilms. The observed degradation is mainly caused by hydroxyl radicals. It is concluded that nanocrystalline anatase films offer different functions at implant interfaces, e.g. bedside hydrophilization of anatase-coated implants for improved osseointegration or the in situ decomposition of conditioning films forming the basal layer of biofilms in the oral cavity.

DEPOSITION AND RETENTION OF VITAL AND DEAD STREPTOCOCCUS SANGUINIS CELLS ON GLASS SURFACES IN A FLOW-CHAMBER SYSTEM

The proportion of vital as compared with dead Streptococcus sanguinis cells attached to glass surfaces was monitored and related to varying proportions of planktonic vital as compared with dead Strep. sanguinis cells. In a flow chamber with six parallel-mounted glass plates, Strep. sanguinis was suspended in pretreated sterile human saliva. Deposition of Strep. sanguinis took place, with a proportion of vital sanguinis streptococci in saliva (%VSs) of 90%, 45% or 22.5%. After exposure times of 30, 60, 90, 120 and 240 min, adherent microorganisms were labelled with two fluorescence stains to differentiate between vital and dead bacteria. Proportions of vital attached streptococci (%VSa) were determined microscopically. Dead bacteria were detected on all glass plates. The %VSa at 30 min and 60 min was significantly lower than the baseline %VSs. During the course of a single run the %VSa frequently increased after either 30, 60 or 90 min without exceeding the %VSs at 4 h. %VSs was the only variable exerting a significant effect on %VSa at 30 and 60 min. It is suggested that during the initial events of microbial attachment the dead rather than vital Strep. sanguinis cells attach preferably to solid surfaces.

Formation and Photocatalytic Decomposition of a Pellicle on Anatase Surfaces

The acquired dental pellicle plays a critical role in the adhesion and detachment of dental plaque bacteria. It has been reported that titanium dioxide biomaterials decompose single-protein films by photocatalysis. However, it is not known whether this can also be achieved with complex structured pellicle films. This in vitro study investigated in real-time the formation and photocatalytic decomposition of human pellicle at anatase-saliva interfaces. Nanostructured polycrystalline anatase layers were deposited on titanium-coated quartz crystals by magnetron-sputtering, serving as a model for titanium implant surfaces. The quartz crystals were used as acoustic sensors in a quartz-crystal microbalance (QCM) system with dissipation. In situ UV irradiation of pellicle-covered anatase caused a statistically significant decrease of the adsorbed salivary mass. In contrast, photocatalytic decomposition of pellicle could not be observed on reference titanium surfaces. Wettability characterization revealed superhydrophilicity of anatase upon UV irradiation, whereas titanium was unaffected. XPS measurements provide further information concerning the decomposition of the salivary films. The results suggest that the photocatalytic activity of polycrystalline anatase-modified biomaterial surfaces is able to decompose complex structured macromolecular pellicle films. Therefore, this study opens the way to surface modifications supporting therapeutic approaches of biofilm removal.

The efficacy of a single pocket irrigation on subgingival microbial vitality

Abstract The object of this study was to monitor the proportion of vital bacteria (microbial vitality: VF in %) present in subgingival dental plaque following one single subgingival irrigation with saline (S), chlorhexidine (CHX) or povidone iodine (I2), but without any subgingival instrumentation. Its effect on the main composition of the microflora was also assessed. Seventeen patients with adult periodontitis took part in this investigation. In each patient four initially untreated pockets (pocket depth 5–11 mm) associated with bleeding were selected for the standardised pocket irrigation and plaque sampling at baseline (0 h) and after the following 1 h, 24 h, 7 days and 31 days. The subgingival irrigation was only performed once (0 h). One pocket per quadrant was irrigated using 0.9% prereduced S, 0.2% CHX or 0.05% I2 (Iso-Betadine Buccale). The remaining untreated pocket without any irrigation served as an additional control (C). Using an acrylic splint as a guide, paperpoints were inserted into the pocket precisely at the same site to collect subgingival plaque. The bleeding on sampling (BOS) was thereafter noted. The proportions of bacterial morphotypes were examined by darkfield microscopy. VF was evaluated using a vital fluorescence staining. The undisturbed subgingival dental plaque was composed of 86% (median value) vital bacteria. The sampling procedure alone and the saline irrigation led to a decrease in the number of spirochetes but had no influence on the vitality of the flora. Large variations in VF could be observed in the short-term (1 h, 24 h) irrigation effect of CHX and I2. The reduction of VF was still significant after 7 days (VFCHX 30–80%, VFI2 35–80%) but persisted up to 31 days only after I2 irrigation (VFI2 12–90%). The findings indicated that all single subgingival irrigations resulted in a temporary change of the subgingival microflora while povidone iodine produced the longest lasting antimicrobial effect. Any clinical advantage of this situation should be further investigated.

Dynamic Production of Soluble Extracellular Polysaccharides by Streptococcus mutans

Caries development in the presence of Streptococcus mutans is associated not only with the production of extracellular water-insoluble polymers but also is based on water-soluble polysaccharides. The aim of this study was the evaluation of a novel glucan-specific Lectin assay for monitoring water-soluble EPS produced by S. mutans during several growth periods in different media. S. mutans cultures were grown for 24 h, 48 h, and 144 h in medium deficient of sucrose (A) and medium supplemented with 5% sucrose (B). Microtiter well plates were coated with cell-free supernatants followed by the addition of labeled Concanavalin-A and enzyme substrate. The substrate reactions were kinetically detected at 405 nm. The validation of the assay was performed using carbohydrates dextran, xanthan, and sucrose as reference. This new Concanavalin-A-based assay showed the highest sensitivity for dextran and revealed that the glucan production of S. mutans reached its maximum at 144 h in medium B according to bacterial maturation.

Antimicrobial efficiency of mouthrinses versus and in combination with different photodynamic therapies on periodontal pathogens in an experimental study

BACKGROUND AND OBJECTIVE In the therapy of destructive periodontal disease, chemical antimicrobial agents and increasingly photodynamic therapy (PDT) play an important adjunctive role to standard mechanical anti-infective treatment procedures. However, both antiseptic methods have their shortcomings in terms of eliminating periodontal pathogens. The aim of the study was to compare the antibacterial efficacy of different antiseptic mouthrinses, of a conventional and a new, modified PDTplus as well as of the different antiseptic mouthrinses combined with either the conventional or the modified PDTplus against periopathogens. MATERIAL AND METHODS Six representative periodontitis-associated bacterial strains were grown for 24 h under anaerobic conditions. After mixing the individual cell pellets they were exposed to 10 different antiseptic mouthrinse formulations: chlorhexidine (0.2%, 0.06%, CHX); CHX + cetylpyridinium chloride (each 0.05%); sodium hypochlorite (0.05%); polyhexanide (0.04%, PHMB1; 0.1%, PHMB2); octenidine dihydrochloride (0.1%); fluoride (250 ppm); essential oils; povidone iodine (10%); and saline (0.9%, NaCl) as control. Furthermore, the bacteria were treated with conventional PDT based on light-emitting diodes and a new modified photodisinfection combining photosensitizer with hydrogen peroxide to PDTplus also based on light-emitting diodes. In addition to the single treatments, a combined application of antiseptic exposure followed by use of PDT or PDTplus was performed. The microbial viability was characterized by analyzing colony growth and fluorescence-based vitality proportions. RESULTS Nearly all mouthrinses caused a statistically significant growth inhibition. The most effective antiseptics, CHX (0.2%), CHX/cetylpyridinium chloride and octenidine dihydrochloride, inhibited bacterial growth completely. Conventional PDT resulted in moderate reduction of colony growth. The modified PDTplus achieved maximum antimicrobial effect. The combination of antiseptic exposure and PDT against periopathogens predominantly increased antibacterial efficacy compared to the single applications. The mouthrinse containing essential oil seemed to interfere with PDT. CONCLUSION A combination therapy of preceding chemotherapeutical exposure and subsequent photodisinfection may be a more effective and promising antibacterial treatment than single applications of the antiseptic methods. The modified PDTplus using oxygen-enriched toluidine showed a superior antibacterial effect on periodontal pathogens to conventional PDT and to the majority of the investigated mouthrinses.

Initiale Biofilmbildungen durch dynamische Proteinwechselwirkungen an oberflächenmodfizierten Titanimplantaten

Osseointegrated dental titanium implants simultaneously contact subgingivally hard and transgingivally soft tissue as well as saliva in the oral cavity. Biofilm (dental plaque) formation is discussed as a main reason for reduced longterm functionality of the implants by inducing inflammatory processes. Early stages of plaque formation are characterized by macromolecular interactions during which conditioning films are formed which modulate following cell adhesion processes. However, there is less knowledge about time-dependent changes of biomaterial/biosystem interfaces during these conditioning processes. In our study, we investigated macromolecular film formation of human serum albumin (HSA) and whole human sterile saliva at the interface of pure titanium, of polyphosphazene Polyzene®-F and of the amorphous fluoropolymer Teflon® AF by means of tensiometric multiloop Wilhelmy-technique and dynamic contact angle analysis (DCA). This technique allows to detect changes in wettability parameters such as advancing and receding contact angles and contact angle hysteresis during macromolecular driven changes at the interface. Since roughness is a main factor influencing contact angle measurements, the surfaces under research have been prepared to have identical roughness on the micrometer scale as proven by profilometric roughness measurements. By this, roughness induced wetting parameter shifts should be excluded and first of all be caused by adsorption processes from the aqueous bioliquids. Having strong differences in hydrophilicity and contact angle hysteresis, all materials showed shifts towards very similar wetting properties during HSA and saliva contact. During adsorption, hydrophilization and homogenisation of all surfaces could be stated according to the analyzed hysteresis shifts. A remarkable exception could be found in the system Teflon AF/HSA. There, the initially very homogeneous fluoropolymer became heterogenous by the contact with the protein, indicated by large equilibrium hysteresis after adsorption. Among other online-methods, e.g. mass and viscoelasticity sensitive quartz crystal microbalances, DCA yield real-time data of interfacial processes during the initial contact of a biomaterial and host bioliquids such as saliva. The importance of tensiometric methods is based on the easy possibility to detect not only contact angles and contact angle hysteresis but the wetting tension, directly considering the surface tension of the aqueous bioliquid. Wetting tension has been discussed to be a main factor in modulating interfacial processes in aqueous biosystems rather than surface free energy. Danksagung Unser Dank gilt der Polyzenix®-GmbH (Ulm, Germany) für die Unterstützung dieser Studien. Ebenso danken wir B. Herzog (Angewandte Physik, Universität Tübingen) für die Herstellung der Titan-Sputterschichten. Diese Studie wurde im Rahmen der TZB-lnitiative (Tübinger Zentrum für Biofilmforschung, http://www.medical-biofilms.net) des Schwerpunktinitiierungsprogramms des Universitätsklinikums Tübingen durchgeführt. Literatur [1] Andrade J. D., Smith L. M., Gregonis D. E.: In: Andrade J. D. (Hrsg.)rSurface and Interfacial Aspects of Biomedical Polymers, Chapter 5, Plenum Press, New York 1985 [2] Bico J., Thiele M., Quere D.: Wetting of textured surfaces. Colloids Surfaces A 206: 41-46, 2002 [3] Bico J., Tordeux C., Quere D.: Rough wetting. Europhys Lett 55: 214-220, 2001 [4] Bränemark P. I., Breine U„ Adell R., Hansson B. 0., Lindstrom H., Ohlssen Α.: Intraosseous anchorage of dental prostheses. Scand J Plast Reconstr Surg 3: 81-100, 1969 [5] Cassie A. B. D.: Discuss Faraday Soc 3(1): 11-16, 1948 [6] Cooper L. F.: A role for surface topography in creating and maintaining bone at titanium endosseous implants. J Prosthet Dent 84: 522-534, 2000 [7] Decker E. M„ Weiger R., von Ohle C„ Wiech I., Brecx M.: Susceptibility of planktonic versus attached Streptococcus sanguinis cells to Chlorhexidine. Clin Oral Invest 7(2): 98102, 2003 [8] Decker E. L., Frank B., Suo Y., Garoff S.: Physics of contact angle measurement. Colloids Surfaces A 156: 177189, 1998 [9] Dettre R. H., Johnson R. E. : Contact angle hysteresis. IV. Contact angle measurements on heterogeneous surfaces. J Phys Chem 69(5): 1507-1516, 1965 2 9 0 B O m a t e r i a l i e n 4 (4) , 2003 Frank Rupp: Initiale Biofilmbildungen durch dynamische Proteinwechselwirkungen ORIGINAL· ARBEITEN