Adhesion of oral streptococci to enamel and dental materials : studies using a flow chamber and microcalorimetry

Dental plaque formation on human teeth is the crucial etiologic factor in the pathogenesis of oral diseases: caries, periodontal diseases, and peri-implantitis. Dental plaque has been defined as a diverse community of microorganisms found on teeth as a biofilm, embedded in an extracellular polymer matrix of bacterial origin and including host components. Bacterial colonization starts with the adhesion of early colonizers, called pioneer bacteria, to the salivary pellicle on teeth as well as on dental materials within minutes after tooth cleaning. The early colonizers, mostly streptococcoci, contribute to plaque development and ultimately to oral diseases. Investigations of dental plaque, including bacterial adhesion, employ various in vivo and in vitro models and use microscopic methods to assess surface phenomena. The complexity of the oral environment makes it difficult to generate an in vitro system including all relevant aspects. The studies presented (paper I-III) were aimed to adapt two in vitro models, a flow chamber system and a microcalorimetric technique, for investigating adhesion of oral streptococci to human enamel, glass and different dental materials. The dental materials used for the flow chamber experiments (paper I + II) were titanium (Rematitan®M), gold (Neocast 3), ceramic (Vita Omega 900), composite (Tetric Ceram), and four different all-ceramics. The early colonizing Streptococcus sanguinis, S. oralis, and the caries-inducing S. mutans, and S. sobrinus were used as the model organisms. The saliva-coated materials were incubated with the bacteria in the flow chamber during one hour. Number and vitality of adhering bacteria were determined microscopically after staining. The results suggested that variations in the number and vitality of the adherent oral streptococci depend on the surface characteristics of the substrata and the acquired salivary pellicle. It also depended on the bacterial species, as S. mutans and S. sobrinus adhered about 10 x less than S. sanguinis and S. oralis. Isothermal Microcalorimetry (paper III) indeed allowed evaluation of initial bacterial adhesion of S. sanguinis to glass. Maximum heat flow measurements during adhesion were about 10 fold lower than during bacterial growth. Experiments showed that increased surface areas, provided by increased amounts of glass beads, were associated with higher energy release. Heat flow was higher when cells were suspended in human saliva than in PBS. Based on these results both methods appear to be applicable to study bacterial adhesion to new dental restorative or implant materials. Advantages and disadvantages of the methods are discussed

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 30s 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 30s to deep occlusal carious cavities had no significant immediate antimicrobial effects whether the superficial decayed layers dentine were removed or no

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 × 104 ± 0.67 × 104 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 surface

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 = 2,940nm) at two different power settings: a lower mode (12.74J/cm2 calculated energy density) and a higher mode (63.69J/cm2). (b) After laser irradiation with both settings of sterile titanium, disks were seeded with 104 MG-63 cells/cm2. Adhesion and proliferation were determined after 1, 4, and 24h 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 63.69J/cm2. 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 63.69J/cm2. 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

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 (P0.05), 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 cell

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 (140ppm; 33mL/s) for 6 and 24s. 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 24s 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 regeneratio

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 6s) and brushing (bristle-to-disk) distances (0, 2, and 4mm). 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

Silver coordination compounds as light-stable, nano-structured and anti-bacterial coatings for dental implant and restorative materials

Silver coordination polymer chains were deposited on Au(111) as a model surface, as well as on gold alloy and titanium as dental implant and restorative materials. The topography of the surface was analysed on the model substrate and it was found to be a nano-structured crystalline material. In vitro investigations in a flow chamber imitating the oral environment prove the anti-bacterial properties of the silver compound

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