Biofilm formation on metal alloys, zirconia and polyetherketoneketone as implant materials in vivo.

OBJECTIVES: This study investigated biofilm formation on discs of metal alloys, zirconia and polyetherketoneketone in vivo. MATERIAL AND METHODS: Sixteen healthy volunteers conducted two runs of 24 hr each wearing an intraoral splint with 15 discs representing five different materials (gold-based [EL] and silver-based [PA] noble metal alloys; zirconia [ZR]; polyetherketoneketone [PEKK]; titanium zirconium alloy [TiZr]). Safranin staining assays and colony-forming unit (CFU) counts were conducted. Linear mixed-effects models were used to compare materials, and geometric mean ratios with 95% confidence interval were calculated with the level of significance set at α = 0.05. RESULTS: Less biofilm mass and lower CFU counts were found on PA and EL, while ZR and PEKK developed similar levels as the reference material TiZr alloy. Compared with PA, biofilm mass was 1.5 times higher for EL (p = .004), 1.7 times higher for PEKK (p < .001), 2.2 times higher for TiZr (p < .001) and 2.4 times higher for ZR (p < .001). The culturing method confirmed these results for EL and PA with lower CFU compared to TiZr. The biomass staining technique and cell culturing correlated for EL and PA. CONCLUSION: Silver-based noble alloy and gold-based high noble alloy demonstrated the least biofilm formation indicating a potential clinical use as material for implant components in the transmucosal compartment. Zirconia and Polyetherketoneketone revealed similar results as the reference material titanium zirconium alloy used in commercially available titanium dental implant.

Antimicrobial efficacy of copper-doped titanium surfaces for dental implants.

The aim of this in vitro study was to quantify the antibacterial effect of a copper-deposited titanium surface as a model for dental implants on the peri-implantitis-associated strain Porphyromonas gingivalis (DSM 20709). A spark-assisted anodization method in a combined deposition-anodization process was applied to deposit copper on discs made of titanium. This method allows the deposition of different concentrations of copper on the surface by varying the process time. Conventional culturing was used to investigate the adhesion of P. gingivalis onto the discs over 2, 4, and 6 h as well as to study the antibacterial effect of copper released in solution. The viability of the bacterial cells is strongly inhibited on copper-deposited discs and reaches a CFU reduction of 3 log-units after 6 h in comparison to the reference. The copper released in solution causes a reduction of 4 log-units after a 6 h incubation time. With a 6 h incubation time, the CFU count decreases with increasing copper concentrations on the disc (by 2% for the 1.3 µg/disc; 32% for the 5.6 µg/disc; and 34% for the 9.5 µg/disc). However, at a higher copper concentration of 17.7 µg/disc, after 6 h, the decrease in the CFU count is less pronounced than that observed in solution, where a further decrease is observed. In conclusion, copper-functionalized titanium significantly reduces the survival of adhered bacteria and decreases the viable bacterial count in the environment surrounding the titanium. Thus, the area surrounding implants is being protected by copper released from the surface, forming a "safe zone" for improved implant healing.

Beneficial Oral Biofilms as Smart Bioactive Interfaces.

Periodontitis is a very common health problem caused by formation of pathogenic bacterial biofilm that triggers inflammation resulting in either reversible gingivitis or irreversible periodontal hard and soft tissue damages, leading to loss of teeth when left untreated. Commensal bacteria play an important role in oral health in many aspects. Mainly by colonizing oral tissues, they (i) contribute to maturation of immune response, and (ii) foreclose attachment of pathobiont and, therefore, prevent from infection. The main goal of the study was to investigate if blocking of receptors on a commensal biofilm can prevent or reduce the attachment of pathogenic strains. To do so, biofilm produced by commensal Streptococcus sanguinis was treated with whole cell lysate of pathobionts Fusobacterium nucleatum or Porphyromonas gingivalis, followed by incubation with respective strain(s). The study revealed significant reduction in pathobiont adhesion to lysate-treated commensal biofilm. Therefore, adhesion of pathobionts onto the lysate-blocked biofilm was hindered; however, not completely eliminated supporting the idea that such approach in the oral cavity would benefit the production of a well-balanced and healthy bioactive interface.

Determinants of biofilm formation and cleanability of titanium surfaces.

OBJECTIVE: The aim of the present study was to analyze biofilm formation on four different titanium-based surfaces (machined titanium zirconium (TiZr) alloy, M; machined, acid-etched TiZr alloy, modMA; machined, sandblasted, acid-etched TiZr alloy, modSLA; and micro-grooved titanium aluminum vanadium alloy, TAV MG) in an experimental human model. MATERIAL AND METHODS: Custom-made discs were mounted in individual intraoral splint housings and worn by 16 volunteers for 24 h. The safranin staining assay, isothermal microcalorimetry (IMC), and SEM were applied before and after surface cleaning. RESULTS: The hydrophilic surfaces modMA and modSLA with greater surface micro-roughness exhibited significantly more biofilm than the hydrophobic surfaces TAV MG and M. The standardized cleaning procedure substantially reduced the biofilm mass on all surfaces. After cleaning, the IMC analyses demonstrated a longer lag time of the growth curve on TAV MG compared to modSLA. Inter- and intraindividual variations in biofilm formation on the titanium discs were evident throughout the study. CONCLUSIONS: Surface hydrophilicity and roughness enhanced biofilm formation in vivo, whereas surface topography was the most influential factor that determined surface cleanability. While the grooved surface retained larger amounts of initial biofilm, the machined surface was easier to clean, but proliferation indicated by increased metabolic activity (growth rate) in IMC occurred despite mechanical biofilm removal.

In Vitro Biofilm Formation on Titanium and Zirconia Implant Surfaces.

BACKGROUND: It has been hypothesized that zirconia might have a reduced bacterial adhesion compared with titanium; however, results from experimental studies are rather controversial. The aim of the present study is to compare biofilm formation on zirconia and titanium implant surfaces using an in vitro three-species biofilm and human plaque samples. METHODS: Experimental disks made of titanium (Ti) or zirconia (ZrO2) with a machined (M) or a sandblasted (SLA) and acid-etched (ZLA) surface topography were produced. An in vitro three-species biofilm or human plaque samples were applied for bacterial adhesion to each type of disk, which after 72 hours of incubation was assessed using an anaerobic flow chamber model. RESULTS: Zirconia showed a statistically significant reduction in three-species biofilm thickness compared with titanium (ZrO2-M: 8.41 µm; ZrO2-ZLA: 17.47 µm; Ti-M: 13.12 µm; Ti-SLA: 21.97 µm); however, no differences were found regarding three-species-biofilm mass and metabolism. Human plaque analysis showed optical density values of 0.06 and 0.08 for ZrO2-M and ZrO2-ZLA, and values of 0.1 and 0.13 for Ti-M and Ti-SLA, respectively; indicating a statistically significant reduction in human biofilm mass on zirconia compared with titanium. Additionally, zirconia revealed a statistically significant reduction in human plaque thickness (ZrO2-M: 9.04 µm; ZrO2-ZLA: 13.83 µm; Ti-M: 13.42 µm; Ti-SLA: 21.3 µm) but a similar human plaque metabolism compared with titanium. CONCLUSION: Zirconia implant surfaces showed a statistically significant reduction in human plaque biofilm formation after 72 hours of incubation in an experimental anaerobic flow chamber model compared with titanium implant surfaces.

[Not Available]. / Bedeutung der Hepatitis C-Virusinfektion in der zahnärztlichen Praxis.

This article gives a short overview about the epidemiology, transmission, clinical appearance, testing and therapy of hepatitis C virus infection and its importance in the healthcare setting. Standard infection control measures in dental practice remain essential for the prevention of HCV transmission.

Microcalorimetric determination of the effects of amoxicillin, metronidazole, and their combination on in vitro biofilm.

BACKGROUND: The mechanism of action of adjuvant antibiotic therapy in the treatment of peri-implantitis is not well understood. The aim of this study is to investigate antibiotic susceptibility of an in vitro biofilm by isothermal microcalorimetry (IMC). METHODS: Titanium disks containing a 72-hour three-species biofilm (Streptococcus sanguinis DSM20068, Fusobacterium nucleatum ATCC10953, and Porphyromonas gingivalis DSM20709) were placed in a series of IMC ampoules with nutrient agar supplemented with increasing concentrations of amoxicillin, metronidazole, or their combination and incubated anaerobically for 10 days. Lag time and maximum growth rate were determined from continuous heat-flow recordings of metabolic activity. To validate the IMC biofilm results, adherent S. sanguinis and P. gingivalis were incubated anaerobically in medium supplemented with antibiotics at 37°C for 24 hours, and their vitality was determined by live/dead staining, conventional culturing, and IMC. RESULTS: In all biofilm samples incubated with antibiotics, a prolonged lag phase was observed compared with controls (P <0.05). Maximum growth rate was significantly lower for samples treated with either amoxicillin or metronidazole compared with controls (P <0.05). Combining the antibiotics did not improve this effect. Concentrations exceeding 10 times the minimum inhibitory concentration completely inhibited the growth of adherent S. sanguinis and P. gingivalis, whereas lower concentrations resulted in only a delay in the lag phase. A poor correlation was observed between live/dead staining and conventional culturing. CONCLUSIONS: IMC gives new evidence about antibiotic effects on oral biofilms and is more informative than conventional culture and live/dead assays. The combination of antibiotics was found to be more efficient than metronidazole alone; however, only minor differences in growth inhibition were detected compared with amoxicillin alone.

Antimicrobial activity of short- and medium-term applications of polyhexamethylene biguanide, chlorhexidine digluconate and calcium hydroxide in infected immature bovine teeth in vitro.

AIM: To compare the antimicrobial activity of polyhexamethylene biguanide (Prontosan wound gel, Pr) and chlorhexidine digluconate (CHX) after short- and medium-term application with the disinfection ability of calcium hydroxide (Ca) in a model using immature bovine teeth. METHODS: Sixty immature bovine roots were infected with Enterococcus faecalis and randomly assigned to six groups (n = 10). Disinfectants were applied into the root canal for 10 min (CHX-10 min and Pr-10 min) or 7 days (CHX-7d, Pr-7d and Ca-7d(g) ). In the negative control group (Co-n), no disinfectant was used. Dentine samples were collected, and the total count of bacteria and colony-forming units were determined. The log10 -transformed Colony-forming units (CFU) data were analysed using a Kruskal-Wallis test with post hoc Wilcoxon multiple-comparison tests. RESULTS: The application of disinfectants led to a significant reduction in CFUs in all groups compared with group Co-n. When compared to Ca-7d(g) , CHX-7d (P = 0.290), CHX-10 min (P = 0.963) and Pr-7d (P = 0.095) revealed no significant differences. Pr-10 min had a significantly higher CFU value than Ca-7d(g) (P = 0.0004), CHX-10 min (P = 0.0009) and Pr-7d (P = 0.0006). CONCLUSIONS: Within the limitations of this study, sufficient antimicrobial effect may be reached by a short-term application of CHX. For the application of 1% Prontosan wound gel, a medium-term use (7 day) is required, while short-term use (10 min) is less effective.

Effects of Er:YAG laser on bacteria associated with titanium surfaces and cellular response in vitro.

This in vitro study examined (a) the anti-bacterial efficacy of a pulsed erbium-doped yttrium aluminum garnet (Er:YAG) laser applied to Streptococcus sanguinis or Porphyromonas gingivalis adhered to either polished or microstructured titanium implant surfaces, (b) the response of osteoblast-like cells and (c) adhesion of oral bacteria to titanium surfaces after laser irradiation. Thereto, (a) bacteria adhered to titanium disks were irradiated with a pulsed Er:YAG laser (λ = 2,940 nm) at two different power settings: a lower mode (12.74 J/cm(2) calculated energy density) and a higher mode (63.69 J/cm(2)). (b) After laser irradiation with both settings of sterile titanium, disks were seeded with 10(4) MG-63 cells/cm(2). Adhesion and proliferation were determined after 1, 4, and 24 h by fluorescence microscopy and scanning electron microscopy. (c) Bacterial adhesion was also studied on irradiated (test) and non-irradiated (control) surfaces. Adhered P. gingivalis were effectively killed, even at the lower laser setting, independent of the material's surface. S. sanguinis cells adhered were effectively killed only at the higher setting of 63.69 J/cm(2). Laser irradiation of titanium surfaces had no significant effects on (b) adhesion or proliferation of osteoblast-like MG-63 cells or (c) adhesion of both oral bacterial species in comparison to untreated surfaces. An effective decontamination of polished and rough titanium implant surfaces with a Er:YAG laser could only be achieved with a fluence of 63.69 J/cm(2). Even though this setting may lead to certain surface alterations, no significant adverse effect on subsequent colonization and proliferation of MG-63 cells or increased bacterial adhesion was found in comparison to untreated control surfaces.

Efficacy of various side-to-side toothbrushes for noncontact biofilm removal.

OBJECTIVES: The aim of this study was to evaluate the efficacy of four different powered toothbrushes with side-to-side action for noncontact biofilm removal in vitro. MATERIALS AND METHODS: A three-species biofilm was formed in vitro on protein-coated titanium disks using a flow chamber combined with a static biofilm growth model. Subsequently, the biofilm-coated substrates were exposed to four different side-to-side toothbrushes (A, B, C, and D) with various brushing times (2, 4, and 6 s) and brushing (bristle-to-disk) distances (0, 2, and 4 mm). The biofilm volumes were measured using volumetric analyses with confocal laser scanning microscope images and Imaris version 7.5.2 software. RESULTS: The median percentages of biofilm reduction by the analyzed toothbrushes ranged from 9 % to 80 %. The abilities of the tested toothbrushes to remove the in vitro biofilm differed significantly (p < 0.05). Two of the tested toothbrushes (C and D) were capable of significant biofilm reduction by noncontact brushing. CONCLUSIONS: It was possible to reduce a three-species in vitro biofilm by noncontact brushing with two out of four side-to-side toothbrushes. CLINICAL RELEVANCE: Toothbrushes C and D show in vitro a high efficacy in biofilm removal without bristle contact.

Isothermal microcalorimetry provides new insights into biofilm variability and dynamics.

The purpose of this study was to investigate a three-species in vitro biofilm with peri-implantitis-related bacteria for its variability and metabolic activity. Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis were suspended in simulated body fluid containing 0.2% glucose to form biofilms on polished, protein-coated implant-grade titanium disks over 72 h using a flow chamber system. Thereafter, biofilm-coated disks were characterized by scanning electron microscopy and fluorescence in situ hybridization/confocal laser scanning microscopy. To assess metabolic activity within the biofilms, their heat flow was recorded for 480 h at 37 °C by IMC. The microscopic methods revealed that the total number of bacteria in the biofilms varied slightly among specimens (2.59 × 10(4)  ± 0.67 × 10(4)  cells mm(-2) ), whereas all three species were found constantly with unchanged proportions (S. sanguinis 41.3 ± 4.8%, F. nucleatum 17.7 ± 2.1%, and P. gingivalis 41.0 ± 4.9%). IMC revealed minor differences in time-to-peak heat flow (20.6 ± 4.5 h), a trend consistent with the small variation in bacterial species proportions as shown by microscopy. Peak heat flow (35.8 ± 42.6 µW), mean heat flow (13.1 ± 22.0 µW), and total heat over 480 h (23.5 ± 37.2 J) showed very high variation. These IMC results may be attributed to differences in the initial cell counts and relative proportions of the three species, their distribution and embedment in exopolysaccharide matrix on the test specimens. The present results provide new insights into variability and dynamics of biofilms on titanium disks, aspects that should be explored in future studies of dental surfaces.

Influence of gaseous ozone in peri-implantitis: bactericidal efficacy and cellular response. An in vitro study using titanium and zirconia.

Dental implants are prone to bacterial colonization which may result in bone destruction and implant loss. Treatments of peri-implant disease aim to reduce bacterial adherence while leaving the implant surface intact for attachment of bone-regenerating host cells. The aims of this study were to investigate the antimicrobial efficacy of gaseous ozone on bacteria adhered to various titanium and zirconia surfaces and to evaluate adhesion of osteoblast-like MG-63 cells to ozone-treated surfaces. Saliva-coated titanium (SLA and polished) and zirconia (acid etched and polished) disks served as substrates for the adherence of Streptococcus sanguinis DSM20068 and Porphyromonas gingivalis ATCC33277. The test specimens were treated with gaseous ozone (140 ppm; 33 mL/s) for 6 and 24 s. Bacteria were resuspended using ultrasonication, serially diluted and cultured. MG-63 cell adhesion was analyzed with reference to cell attachment, morphology, spreading, and proliferation. Surface topography as well as cell morphology of the test specimens were inspected by SEM. The highest bacterial adherence was found on titanium SLA whereas the other surfaces revealed 50-75% less adherent bacteria. P. gingivalis was eliminated by ozone from all surfaces within 24 s to below the detection limit (≥99.94% reduction). S. sanguinis was more resistant and showed the highest reduction on zirconia substrates (>90% reduction). Ozone treatment did not affect the surface structures of the test specimens and did not influence osteoblastic cell adhesion and proliferation negatively. Titanium (polished) and zirconia (acid etched and polished) had a lower colonization potential and may be suitable material for implant abutments. Gaseous ozone showed selective efficacy to reduce adherent bacteria on titanium and zirconia without affecting adhesion and proliferation of osteoblastic cells. This in vitro study may provide a solid basis for clinical studies on gaseous ozone treatment of peri-implantitis and revealed an essential base for sufficient tissue regeneration.

Quantification of vital adherent Streptococcus sanguinis cells on protein-coated titanium after disinfectant treatment.

The quantification of vital adherent bacteria is challenging, especially when efficacy of antimicrobial agents is to be evaluated. In this study three different methods were compared in order to quantify vital adherent Streptococcus sanguinis cells after exposure to disinfectants. An anaerobic flow chamber model accomplished initial adhesion of S. sanguinis on protein-coated titanium. Effects of chlorhexidine, Betadine®, Octenidol®, and ProntOral® were assessed by quantifying vital cells using Live/Dead BacLight™, conventional culturing and isothermal microcalorimetry (IMC). Results were analysed by Kruskal-Wallis one-way analysis of variance. Live/dead staining revealed highest vital cell counts (P < 0.05) and demonstrated dose-dependent effect for all disinfectants. Microcalorimetry showed time-delayed heat flow peaks that were proportioned to the remaining number of viable cells. Over 48 h there was no difference in total heat between treated and untreated samples (P > 0.05), indicating equivalent numbers of bacteria were created and disinfectants delayed growth but did not eliminate it. In conclusion, contrary to culturing, live/dead staining enables detection of cells that may be viable but non-cultivable. Microcalorimetry allows unique evaluation of relative disinfectant effects by quantifying differences in time delay of regrowth of remaining vital cells.

Bactericidal effects of different laser systems on bacteria adhered to dental implant surfaces: an in vitro study comparing zirconia with titanium.

OBJECTIVES: The purpose of this study was to examine in vitro the anti-bacterial efficacy of two different laser systems (CO(2) and diode) applied to Streptococcus sanguinis or Porphyromonas gingivalis cells in suspensions or adhered to zirconia or titanium dental implant materials, with two different surfaces each. MATERIALS AND METHODS: Bacteria were irradiated at two different power settings with either a CO(2) (lambda=10,600 nm) or a diode laser (lambda=810 nm). The lower mode is used clinically (for CO(2) 100 J/cm(2), diode 50 J/cm(2)) and the higher may alter the materials' surface (for CO(2) 1200 J/cm(2), diode 150 J/cm(2)). After irradiation, the number of viable bacteria was determined by culture. RESULTS: Planktonic cells of both species were more resistant to the laser irradiations than bacteria that adhered to surfaces. Adhered P. gingivalis were effectively killed at both wavelengths lambda=10,600 and 810 nm even at the lower settings, independent of the material. S. sanguinis cells that adhered to either zirconia surface were effectively killed by the CO(2) laser at the lower setting of 100 J/cm(2). However, the higher settings of both lasers were needed to reduce S. sanguinis that adhered to titanium surfaces. The CO(2) laser at the lower setting and the diode laser at the higher setting effectively reduced the viability of S. sanguinis or P. gingivalis that adhered to zirconia surfaces. CONCLUSIONS: Under irradiation conditions known not to alter zirconia implant surfaces in vitro, both CO(2) laser (100 J/cm(2)) and the diode laser (150 J/cm(2)) effectively reduced the viability of adhered S. sanguinis or P. gingivalis.

Comparison of the immediate effects of gaseous ozone and chlorhexidine gel on bacteria in cavitated carious lesions in children in vivo.

Clinical application of ozone gas has been shown to arrest the progression of dentinal caries in children. In this study, we compare the immediate effects of gaseous ozone and chlorhexidine gel on bacteria in cavitated carious lesions in children. Forty children, each with at least two open occlusal carious lesions, were enrolled in the study. Two teeth were chosen randomly. In one lesion, overlying soft biological material was removed, whilst the other lesion was not excavated. Cavities were rinsed with sterile water and dried with air. A standardised sample was taken from the mesial part of each lesion. Then, gaseous ozone (HealOzone) or 1% chlorhexidine gel (Corsodyl) was applied for 30 s on both lesions of 20 children each, and a second sample was taken from the distal part of each lesion. The anaerobic microbiota was cultivated; the number of colony forming units was calculated per milligram sample. The two-sided paired t test showed no significant (P > 0.05) differences in the reduction of total bacterial counts per milligram comparing samples before and after ozone or chlorhexidine application. The tests also showed no statistically significant difference whether the superficial decayed dentine had been removed before ozone or with chlorhexidine treatment or not. It can be concluded that gaseous ozone or chlorhexidine gel application for 30 s to deep occlusal carious cavities had no significant immediate antimicrobial effects whether the superficial decayed layers dentine were removed or not.

Adhesion of Streptococcus sanguinis to glass surfaces measured by isothermal microcalorimetry (IMC).

Bacterial adhesion is the first step in the development of the oral biofilm, called dental plaque. Plaque is the cause of caries, periodontal diseases, and periimplantitis. Investigations of dental plaque, including bacterial adhesion, employ various in vivo and in vitro models using microscopic methods. Microcalorimetry offers another direct approach. The model organism Streptococcus sanguinis is one of the first colonizers adhering to the saliva-coated human tooth surfaces or dental materials within minutes after tooth cleaning. TAM III thermostats, equipped with microcalorimeters, were used for isothermal microcalorimetric (IMC) measurements of heat production as a function of time, expressed by power-time (p-t) curves. Continuous measurements of heat production of growing S. sanguinis cells showed their overall metabolic activity and were highly reproducible. For the adhesion experiments the bacteria were allowed to adhere to different amounts of glass beads. Growing S. sanguinis cells produced a characteristic p-t curve with a maximum of 500 microW at 4.5 h when reaching 10(9) cells ml(-1). The same number of stationary S. sanguinis cells, suspended in PBS produced only approximately 30 microW at 0.5 h due to adhesion. But the amount of heat increased with available glass surface area, indicating that a portion of the heat of adhesion was measured. Similar results were obtained with stationary S. sanguinis cells suspended in human saliva. This study shows that microcalorimetric evaluation of initial bacterial adhesion is indeed possible and may become a rapid, reproducible screening method to study adhesion of different bacteria to different dental materials or to modified surfaces.

Adhesion of Streptococcus sanguinis to dental implant and restorative materials in vitro.

Bacterial adhesion to tooth surfaces or dental materials starts immediately upon exposure to the oral environment. The aim of this study, therefore, was to compare the adhesion of Streptococcus sanguinis to saliva-coated human enamel and dental materials - during a one-hour period - using an in vitro flow chamber system which mimicked the oral cavity. After fluorescent staining, the number of adhered cells and their vitality were recorded. The dental materials used were: titanium (Rematitan M), gold (Neocast 3), ceramic (Vita Omega 900), and composite (Tetric Ceram). The number of adherent bacterial cells was higher on titanium, gold, and ceramic surfaces and lower on composite as compared to enamel. As for the percentage of adherent vital cells, it was higher on enamel than on the restorative materials tested. These results suggested that variations in the number and vitality of the adherent pioneer oral bacteria, S. sanguinis, in the in vitro system depended on the surface characteristics of the substratum and the acquired salivary pellicle. The in vitro adhesion model used herein provided a simple and reproducible approach to investigate the impact of surface-modified dental materials on bacterial adhesion and vitality.