Effects of nanomodified titanium surfaces considering bacterial colonization and viability of osteoblasts and fibroblasts.

This study investigates nanostructured titanium surfaces (Ti2 spikes) that promote the viability of osteoblasts and fibroblasts and prevent bacterial colonisation. Helium ion irradiation was adopted to produce nanometric-sized cones on titanium. Human osteoblasts (hFOB) and human gingiva fibroblasts (hGF) were used for analysis. A viability and a cytotoxicity assay were conducted to evaluate the lactate dehydrogenase (LDH) activity and assess cell damage in Ti2 spikes compared to titanium discs with a sandblasted and acid-etched (Ti2 SLA) surface. The antibacterial activity was investigated against Escherichia coli, Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis. In the course of the cultivation, both hGF and hFOB demonstrated significantly reduced viability on the Ti2 spikes surface. hGF cells exhibited a slight but significant increase in LDH release. In contrast, hFOB showed reduced cytotoxicity on this surface. On the Ti2 spikes surface, hGF cells exhibited a significant reduction in gene expression of VCL, Src-1, and ITGα5. However, the integrin subunits ITGα1 and ITGα3 showed upregulation on the Ti2 spikes surface. The Ti2 spikes surface significantly increased the expression of almost all osteogenic markers. The results of conventional culturing demonstrated a statistically significant decrease in the number of viable cells for S. mutans, F. nucleaum, and greater quantities of P. gingivalis on Ti2 spikes surface compared to control. However, no such reduction was detected for E. coli. The long-term success of implants relies on establishing and maintaining hard and soft peri-implant tissues. Ti2 spikes represent a novel and promising approach to enhance osseointegration and optimize biocompatibility.

Effect of Dentifrice Ingredients on Volume and Vitality of a Simulated Periodontal Multispecies Biofilm.

The aim of this in vitro study was to investigate the effect of different toothpaste ingredients on biofilm volume and vitality in an established non-contact biofilm removal model. A multi-species biofilm comprising Porphyromonas gingivalis, Streptococcus sanguinis, and Fusobacterium nucleatum was grown on protein-coated titanium disks. Six disks per group were exposed to 4 seconds non-contact brushing using a sonic toothbrush. Four groups assessed slurries containing different ingredients, i.e., dexpanthenol (DP), peppermint oil (PO), cocamidopropyl betaine (CB), and sodium hydroxide (NaOH), one positive control group with the slurry of a toothpaste (POS), and a negative control group with physiological saline (NEG). Biofilm volume and vitality were measured using live-dead staining and confocal laser scanning microscopy. Statistical analysis comprised descriptive statistics and inter-group differences. In the test groups, lowest vitality and volume were found for CB (50.2 ± 11.9%) and PO (3.6 × 105 ± 1.8 × 105 µm3), respectively. Significant differences regarding biofilm vitality were found comparing CB and PO (p = 0.033), CB and NEG (p = 0.014), NaOH and NEG (p = 0.033), and POS and NEG (p = 0.037). However, no significant inter-group differences for biofilm volume were observed. These findings suggest that CB as a toothpaste ingredient had a considerable impact on biofilm vitality even in a non-contact brushing setting, while no considerable impact on biofilm volume was found.

Stannous Source in Toothpastes Leads to Differences in Their Antimicrobial Efficacy.

PURPOSE: The aim of this in-vitro study was to investigate the antimicrobial efficacy of identical experimental toothpastes with different stannous sources. MATERIALS AND METHODS: Streptococcus mutans biofilms were grown on protein-coated glass disks in static conditions for 24 h and thereafter exposed to toothpaste slurries or physiological saline (negative control; n = 15) for 30 s. Four experimental toothpastes were applied in this study, containing either stannous chloride (SnCl2; B: 3500 ppm Sn2+, and D: 3600 ppm Sn2+) or stannous fluoride (SnF2; C: 3500 ppm Sn2+, and E: 3600 ppm Sn2+). Marketed toothpaste meridol® (A: 3300 ppm SnF2) served as control. All five toothpastes contained amine fluoride (AmF). The biofilms were placed on agar surface and their metabolic activity was assessed by isothermal microcalorimetry over 96 h. The heat flow data was analysed for growth rate and lag time using grofit package in software R. Additionally, reduction of active biofilm compared to untreated control was calculated. RESULTS: All toothpastes significantly prolong the lag time of treated biofilms in comparison to negative control (p < 0.05). Toothpastes containing SnF2 (C and E) prolonged the lag time statistically significantly compared to toothpastes containing SnCl2 (B and D) (p < 0.05). The maximum growth rate was statistically significantly reduced by all tested toothpastes compared to the untreated control group (p < 0.05). Toothpastes containing SnF2 (A, C and E) reached 59.9 ± 7.8, 61.9 ± 7.7, and 55.6 ± 7.0% reduction of active biofilm, respectively. Thus, they exhibit statistically significantly better results than toothpastes B (52.9 ± 9.9%) and D (44.7 ± 7.6%). Toothpaste D, which contains a slightly higher concentration of Sn2+, was the least effective in reducing active biofilm. CONCLUSION: The toothpastes containing SnF2 combined with AmF had the highest antimicrobial efficacy in this study.

Piranha-etched titanium nanostructure reduces biofilm formation in vitro.

OBJECTIVES: Nano-modified surfaces for dental implants may improve gingival fibroblast adhesion and antibacterial characteristics through cell-surface interactions. The present study investigated how a nanocavity titanium surface impacts the viability and adhesion of human gingival fibroblasts (HGF-1) and compared its response to Porphyromonas gingivalis with those of marketed implant surfaces. MATERIAL AND METHODS: Commercial titanium and zirconia disks, namely, sandblasted and acid-etched titanium (SLA), sandblasted and acid-etched zirconia (ZLA), polished titanium (PT) and polished zirconia (ZrP), and nanostructured disks (NTDs) were tested. Polished titanium disks were etched with a 1:1 combination of 98% H2SO4 and 30% H2O2 (piranha etching) for 5 h at room temperature to produce the NTDs. Atomic force microscopy was used to measure the surface topography, roughness, adhesion force, and work of adhesion. MTT assays and immunofluorescence staining were used to examine cell viability and adhesion after incubation of HGF-1 cells on the disk surfaces. After incubation with P. gingivalis, conventional culture, live/dead staining, and SEM were used to determine the antibacterial properties of NTD, SLA, ZLA, PT, and ZrP. RESULTS: Etching created nanocavities with 10-20-nm edge-to-edge diameters. Chemical etching increased the average surface roughness and decreased the surface adherence, while polishing and flattening of ZrP increased adhesion. However, only the NTDs inhibited biofilm formation and bacterial adherence. The NTDs showed antibacterial effects and P. gingivalis vitality reductions. The HGF-1 cells demonstrated greater viability on the NTDs compared to the controls. CONCLUSION: Nanocavities with 10-20-nm edge-to-edge diameters on titanium disks hindered P. gingivalis adhesion and supported the adhesion of gingival fibroblasts when compared to the surfaces of currently marketed titanium or zirconia dental implants. CLINICAL RELEVANCE: This study prepared an effective antibacterial nanoporous surface, assessed its effects against oral pathogens, and demonstrated that surface characteristics on a nanoscale level influenced oral pathogens and gingival fibroblasts. CLINICAL TRIAL REGISTRATION: not applicable.

Antimicrobial and mechanical assessment of cellulose-based thermoformable material for invisible dental braces with natural essential oils protecting from biofilm formation.

Controlling biofilm formation in the oral cavity during orthodontic treatments is crucial. Therefore, antimicrobial surfaces for invisible dental appliances are of interest to both therapists and patients. Here we present a cellulose-based thermoformable material used for invisible braces that can be loaded with essential oils (EOs) having antibacterial and antifungal properties. We hypothesize that this material can absorb and release EOs, thus providing an antimicrobial effect without compromising the safety and mechanical properties necessary for dental invisible braces. Conventional microbiology and isothermal microcalorimetry analyses revealed that the thermoformable material loaded with essential oils significantly delayed the biofilm formation of oral streptococci (S. mutans and S. mitis) under static conditions (p < 0.05) and while simulating saliva flow (p < 0.05). In addition, cytotoxicity tests (ISO 10993-5), revealed that the loaded material is well tolerated by human gingival fibroblasts. Finally, the loading with antibacterial agents did not significantly alter the mechanical properties and stability of the material (initial force (p = 0.916); initial stress (p = 0.465)). Compared to gold-standard clear aligner materials, this material offers a reliable transmission of forces for orthodontic treatments. Moreover, this approach exhibits the potential for acting as an oral drug delivery platform for multiple compounds.

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

OBJECTIVES: Composition of implant material and its surface structure is decisive for oral biofilm accumulation. This study investigated biofilm formation on eight different materials. MATERIALS AND METHODS: Eighteen healthy subjects wore intraoral splints fitted with two sets of eight materials for 24 h: zirconia [ZrO2 ]; silver-gold-palladium [AgAuPd]; titanium zirconium [TiZr]; Pagalinor [PA]; hydroxyapatite [HA]; silver-platinum [AgPt]; titanium aluminum niobium [TAN]; titanium grade4 [TiGr4]. Total biomass was stained by safranin to assess plaque accumulation while conventional culturing (CFU) was conducted to investigate viable parts of the biofilm. Cell viability of human gingival fibroblasts (HGF-1) was assessed in vitro. Statistical evaluation was performed with linear mixed-effects models to compare materials (geometric mean ratios, 95% CI), with the level of significance set at ɑ = .05. RESULTS: Less biofilm mass and CFU were found on noble metal alloys (AgPt, AgAuPd, and PA). Compared to AgPt, PA had 2.7-times higher biofilm mass value, AgAuPd was 3.9-times, TiGr4 was 4.1-times, TiZr was 5.9-times, TAN was 7.7-times, HA was 7.8-times, and ZrO2 was 9.1-times higher (each p < .001). Similarly, CFU data were significantly lower on AgPt, AgAuPd had 4.1-times higher CFU values, PA was 8.9-times, TiGr4 was 11.2-times, HA was 12.5-times, TiZr was 13.3-times, TAN was 16.9-times, and ZrO2 was 18.5-times higher (each p < .001). HGF-1 viability varied between 47 ± 24.5% (HA) and 94.4 ± 24.6% (PA). CONCLUSION: Noble alloys are considered as beneficial materials for the transmucosal part of oral implants, as less biofilm mass, lower bacterial counts, and greater cell viability were detected than on titanium- or zirconia-based materials.

Evaluation of antibacterial properties of fluoride-containing mouth rinses differing in their acidic compound using a Streptococcus mutans biofilm.

This in vitro study assessed the antibacterial effect on Streptococcus mutans biofilms of mouth rinses with 700 ppm F- (derived from NaF) that differed only in their acid compounds (malic (A), citric (B), tartaric (C), fumaric (D), hydrochloric (E), phosphoric (F), and lactic (G) acid) used to adjust pH. S. mutans (ATCC 25175) was grown for 22 h at 37°C, harvested, resuspended in simulated body fluid and biofilm formation followed for 24 h at 37°C. Thereafter, biofilms were treated with experimental rinses for 30 s, and placed in TAM48 isothermal microcalorimeter at 37°C for 72 h. Applying Gompertz growth model parameters lag time and growth rate were determined from heatflow curves; additionally, reduction of active biofilms was calculated. Moreover, samples were live/dead stained and analyzed by confocal scanning microscopy. All mouth rinses were showing statistically significant lag time and reduction of active biofilm (p<0.05, A 19.1+/-2.3h and 58.5+/-7.7%, B 15.5+/-1.1h and 41.9+/-5.3%, C 17.6+/-1.9h and 53.1+/-7.5%, D 18.4+/-2.4h and 55.8+/-8.8%, E 20.2+/-3.3h and 61.5+/-10.0%, F 20.2+/-3.0h and 61.6+/-9.3%, and G 18.3+/-2.5h and 55.3+/-8.9%). Interestingly, there were no differences found between the treated groups (p>0.05, A 0.064+/-0.004 1/h, B 0.063+/-0.005 1/h, C 0.065+/-0.004 1/h, D 0.067+/-0.004 1/h, E 0.066+/-0.006 1/h, F 0.067+/-0.004 1/h, G 0.066+/-0.006 1/h) for the maximum growth rate. Vitality staining supported these findings.. The present investigation demonstrates that the type of acid compounds used to produce the rinses did not show any negative effect on the antimicrobial properties of the tested products as all of them exhibited a similar efficacy against S. mutans biofilms.

Saliva profiling with differential scanning calorimetry: A feasibility study with ex vivo samples.

Differential scanning calorimetry (DSC) has been used widely to study various biomarkers from blood, less is known about the protein profiles from saliva. The aim of the study was to investigate the use DSC in order to detect saliva thermal profiles and determine the most appropriate sampling procedure to collect and process saliva. Saliva was collected from 25 healthy young individuals and processed using different protocols based on centrifugation and filtering. The most effective protocol was centrifugation at 5000g for 10 min at 4°C followed by filtration through Millex 0.45 µm filter. Prepared samples were transferred to 3 mL calorimetric ampoules and then loaded into TAM48 calibrated to 30°C until analysis. DSC scans were recorded from 30°C to 90°C at a scan rate of 1°C/h with a pre-conditioning the samples to starting temperature for 1 h. The results show that the peak distribution of protein melting points was clearly bimodal, and the majority of peaks appeared between 40-50°C. Another set of peaks is visible between 65°C- 75°C. Additionally, the peak amplitude and area under the peak are less affected by the concentration of protein in the sample than by the individual differences between people. In conclusion, the study shows that with right preparation of the samples, there is a possibility to have thermograms of salivary proteins that show peaks in similar temperature regions between different healthy volunteers.

Novel Titanium Nanospike Structure Using Low-Energy Helium Ion Bombardment for the Transgingival Part of a Dental Implant.

AIM(S): The aim of the study was to fabricate a nanospike surface on a titanium alloy surface using a newly established method of low-energy helium ion bombardment. Various methods to achieve nanospike formation on titanium have been introduced recently, and their antibacterial properties have been mainly investigated with respect to Escherichia coli and Staphylococcus aureus. Oral pathogens such as Porphyromonas gingivalis play an important role in the development of peri-implantitis. For that reason, the antibacterial properties of the novel, nanostructured titanium surface against P. gingivalis were assessed, and a possible effect on the viability of gingival fibroblasts was evaluated. MATERIALS AND METHODS: Helium sputtering was employed for developing titanium surfaces with nanospikes of 500 nm (ND) in height; commercially available smooth-machined (MD) and sandblasted and acid-etched titanium disks (SLA) were used as controls. Surface structure characterization was performed through scanning electron microscopy (SEM) and atomic force microscopy (AFM). Following incubation with P. gingivalis, antibacterial properties were determined via conventional culturing and SEM. Additionally, the viability of human gingival fibroblasts (HGFs) was tested through MTT assay, and cell morphology was assessed through SEM. RESULTS: SEM images confirmed the successful establishment of a nanospike surface with required heights, albeit with heterogeneity. AFM images of the 500 nm nanospike surface revealed that the roughness is dominated by large-scale hills and valleys. For frame sizes of 5 × 5 µm and smaller, the average roughness is dominated by the height of the titanium spikes. ND successfully induces dysmorphisms within P. gingivalis cultures following the incubation period, while conventional culturing reveals a 17% and 20% reduction for ND compared to MD and SLA, respectively. Moreover, the nanospike surfaces did not affect the viability of human growth fibroblasts despite their sharp surface. CONCLUSION(S): This study successfully developed a novel titanium-nanospike-based structuration technique for titanium surfaces. In addition, the nanospikes did not hinder gingival fibroblast viability. Enhanced antimicrobial effects for such a novel nanospike-based resurfacing technique can be achieved through further optimizations for nanospike spacing and height parameters.

Bacterial extracellular polymeric substances as potential saliva substitute.

This proof-of-principle study aims to find commensal oral bacteria that can produce extracellular polymeric substances (EPS), which have similar lubrication properties to saliva and could serve as saliva substitutes. Saliva and plaque samples were collected from 21 generally healthy individuals. Primary screening was done by conventional culturing and Gram-staining; all species selected for further analysis were identified by MALDI-TOF and deposited in DSMZ. Lactobacillus gasseri (DSM32453 and DSM32455), Lactobacillus rhamnosus (DSM32452), Lactobacillus paracasei (DSM32454), and Streptococcus sanguinis (DSM32456) produced 413.6, 415.7, 431.1, 426.8, and 877.6 µg/ml of EPS, respectively. At the same time calcium dissolution could not be detected for both L. gasseri strains, minimal dissolution for the other three: S. sanguinis 0.3 mm, and 3.7 mm for L. rhamnosus and L. paracasei. There were no differences found between the EPS samples and the saliva for the effect of shear rate on the viscosity and for the effect of sliding speed on lubrication properties. In conclusion, five commensal bacterial strains have been isolated, all able to produce EPS and lead to no or to low calcium dissolution. EPS produced exhibits rheological and tribological properties comparable to human saliva. A total of four out of five selected strains are probiotic and, therefore, may exhibit additional beneficial influence within the oral cavity.

Influence of different zirconia surface treatments on biofilm formation in vitro and in situ.

OBJECTIVES: To determine whether the surface treatment of zirconia affects biofilm formation in an in vitro three-species biofilm model and in situ. MATERIAL AND METHODS: Zirconia surfaces considered for the transmucosal portion of a zirconia implant were compared with polished pure titanium grade 4 (Tp). Disks 13 mm in diameter of either polished (Zp), polished and heat-treated (Zpt), machined (Zm), machined and heat-treated (Zmt) and sandblasted, etched and heat-treated (Z14) zirconia were fabricated. Surface roughness and wettability of specimens was measured. Biofilm formation was evaluated by safranin staining and scanning electron microscopy (SEM) using a three-species model, and intraorally with 16 volunteers carrying oral splints in two independent experiments. Relative biofilm formation was compared with Kruskal-Wallis followed by Bonferroni post hoc test (α = 0.05). RESULTS: In vitro biofilm formation with optical density values on Zp (0.14 ± 0.01), Zpt (0.14 ± 0.02), Zm (0.13 ± 0.01) and Zmt (0.13 ± 0.01) was significantly lower than on Tp (0.21 ± 0.05) and Z14 (0.20 ± 0.04) (p < .05). In situ biofilm formation was significantly higher on Z14 (0.56 ± 0.45) (p < .05), while no significant differences in optical density were observed among Zp (0.25 ± 0.20), Zm (0.36 ± 0.34) and Tp (0.28 ± 0.22). SEM analysis supported quantitative findings. CONCLUSIONS: In the in vitro, three-species biofilm model differences in material and surface roughness affected biofilm formation. In situ biofilm formation was mainly affected by the surface roughness of the specimens. Polishing of zirconia is recommended to reduce biofilm formation, while heat treatment has no significant effect.

Cinnamaldehyde as antimicrobial in cellulose-based dental appliances.

AIMS: In the context of minor orthodontic intervention using clear aligner technologies, we determined antimicrobial properties of a cellulose-based material loaded with essential oils such as cinnamaldehyde. METHODS AND RESULTS: Isothermal microcalorimetry was used to assess the growth of bacterial biofilms at the interface between the tested material and the solid growth medium. The calorimetric data were analyzed using conventional growth models (Gompertz and Richards), and inhibition at 12 and 24 h was calculated. CONCLUSIONS: The tested material showed antimicrobial properties against Staphylococcus epidermidis as well as Streptococcus mutans and Streptococcus mitis clinical isolates. The inhibition was more pronounced against S. epidermidis, for which growth rate was reduced by 70% and lag phase was extended by 12 h. For S. mutans and S. mitis, the decrease in growth rate was 20% and 10%, and the lag phase increased by 2 and 6 h, respectively. SIGNIFICANCE AND IMPACT: Clear aligners for minor teeth alignment are becoming very popular. As they must be worn for at least 22 h per day for up to 40 weeks, it is important that they remain clean and do not promote caries formation or other oral infections. Therefore, introducing material with antimicrobial properties is expected to maintain oral hygiene during the aligner therapy. Here, we demonstrate the use of cinnamaldehyde for reducing microbial growth and biofilm formation on cellulose-based dental clear aligners.

Effect of a Nanostructured Titanium Surface on Gingival Cell Adhesion, Viability and Properties against P. gingivalis.

OBJECTIVES: The transgingival part of titanium implants is either machined or polished. Cell-surface interactions as a result of nano-modified surfaces could help gingival fibroblast adhesion and support antibacterial properties by means of the physico-mechanical aspects of the surfaces. The aim of the present study was to determine how a nanocavity titanium surface affects the viability and adhesion of human gingival fibroblasts (HGF-1). Additionally, its properties against Porphyromonas gingivalis were tested. MATERIAL AND METHODS: Two different specimens were evaluated: commercially available machined titanium discs (MD) and nanostructured discs (ND). To obtain ND, machined titanium discs with a diameter of 15 mm were etched with a 1:1 mixture of 98% H2SO4 and 30% H2O2 (piranha etching) for 5 h at room temperature. Surface topography characterization was performed via scanning electron microscopy (SEM) and atomic force microscopy (AFM). Samples were exposed to HGF-1 to assess the effect on cell viability and adhesion, which were compared between the two groups by means of MTT assay, immunofluorescence and flow cytometry. After incubation with P. gingivalis, antibacterial properties of MD and ND were determined by conventional culturing, live/dead staining and SEM. Results: The present study successfully created a nanostructured surface on commercially available machined titanium discs. The etching process created cavities with a 10-20 nm edge-to-edge diameter. MD and ND show similar adhesion forces equal to about 10-30 nN. The achieved nanostructuration reduced the cell alignment along machining structures and did not negatively affect the proliferation of gingival fibroblasts when compared to MD. No differences in the expression levels of both actin and vinculin proteins, after incubation on MD or ND, were observed. However, the novel ND surface failed to show antibacterial effects against P. gingivalis. CONCLUSION: Antibacterial effects against P. gingivalis cannot be achieved with nanocavities within a range of 10-20 nm and based on the piranha etching procedure. The proliferation of HGF-1 and the expression levels and localization of the structural proteins actin and vinculin were not influenced by the surface nanostructuration. Further studies on the strength of the gingival cell adhesion should be performed in the future. CLINICAL RELEVANCE: Since osseointegration is well investigated, mucointegration is an important part of future research and developments. Little is known about how nanostructures on the machined transgingival part of an implant could possibly influence the surrounding tissue. Targeting titanium surfaces with improved antimicrobial properties requires extensive preclinical basic research to gain clinical relevance.

Cariogenic Biofilms and Caries from Birth to Old Age.

Caries is a complex microbial disease characterized by a multifactorial etiology. The disease is driven by cariogenic microbiota that metabolize dietary carbohydrates into acids, creating prolonged periods of low pH on the biofilm surrounding the teeth, which will result in loss of calcium from the teeth leading to carious lesions. Caries remains a major public health problem globally, ranking first for the decay of permanent teeth (2.3 billion people) and 12th for deciduous teeth (560 million children) according to the Global Burden of Disease study by the WHO in 2015. Different factors play a role in the development of the disease: (i) individual factors such as tooth morphology, saliva, and the oral microbiome, (ii) behavioral factors such as frequency and amount of fermentable carbohydrates in the host's diet and overall oral hygiene, and (iii) socioeconomic status and host genetics as well as modifying factors such as fluoride. Various models exist which explain the transition from a health-compatible oral microbiota to a cariogenic microbiota. Longitudinal studies may increase our knowledge of the oral microbial compositions in different age groups by analyzing the temporal sequence leading to carious lesions. Understanding the factors which control microbial colonization early in life as well as the keystone species that should be present or absent may provide us with strategies for the acquisition and maintenance of a health-promoting oral microbiome. Thus, the importance lies in understanding caries etiology to improve strategies for diagnosis, risk assessment, prevention, and (operative) treatment.

Efficacy of Experimental Mouth Rinses on Caries-Related Biofilms in vitro.

This study assessed the efficacy of tin and Polyethylenglycol (PEG-3) tallow aminopropylamine in different concentrations on Streptococcus mutans (S. mutans) biofilms to establish a new screening process for different antimicrobial agents and to gain more information on the antibacterial effects of these agents on cariogenic biofilms. Isothermal microcalorimetry (IMC) was used to determine differences in two growth parameters: lag time and growth rate; additionally, reduction in active biofilms was calculated. Experimental mouth rinses with 400 and 800 ppm tin derived from stannous fluoride (SnF2) revealed results (43.4 and 49.9% active biofilm reduction, respectively) similar to meridol mouth rinse (400 ppm tin combined with 1,567 ppm PEG-3 tallow aminopropylamine; 55.3% active biofilm reduction) (p > 0.05), while no growth of S. mutans biofilms was detected during 72 h for samples treated with an experimental rinse containing 1,600 ppm tin (100% active biofilm reduction). Only the highest concentration (12,536 ppm) of rinses containing PEG-3 tallow aminopropylamine derived from amine fluoride (AmF) revealed comparable results to meridol (57.5% reduction in active biofilm). Lower concentrations of PEG-3 tallow aminopropylamine showed reductions of 16.9% for 3,134 ppm and 33.5% for 6,268 ppm. Maximum growth rate was significantly lower for all the samples containing SnF2 than for the samples containing control biofilms (p < 0.05); no differences were found between the control and all the PEG-3 tallow aminopropylamine (p > 0.05). The growth parameters showed high reproducibility rates within the treated groups of biofilms and for the controls; thus, the screening method provided reliable results.

A Review of Methods to Determine Viability, Vitality, and Metabolic Rates in Microbiology.

Viability and metabolic assays are commonly used as proxies to assess the overall metabolism of microorganisms. The variety of these assays combined with little information provided by some assay kits or online protocols often leads to mistakes or poor interpretation of the results. In addition, the use of some of these assays is restricted to simple systems (mostly pure cultures), and care must be taken in their application to environmental samples. In this review, the necessary data are compiled to understand the reactions or measurements performed in many of the assays commonly used in various aspects of microbiology. Also, their relationships to each other, as metabolism links many of these assays, resulting in correlations between measured values and parameters, are discussed. Finally, the limitations of these assays are discussed.

Effect of divalent ions on cariogenic biofilm formation.

BACKGROUND: Divalent cations are able to interact with exopolysaccharides (EPS) and thus are capable to modify the structure and composition of dental biofilm. At the moment, little is known about the adsorption of metals by cariogenic EPS; thus, the aim of the present study was to evaluate the effect of divalent ions (calcium, magnesium, and zinc) on the growth and biofilm formation of mutans streptococci and on the dissolution of hydroxyapatite as well as to investigate their binding to the bacterial EPS. RESULTS: S. mutans strains used in this study show the highest tolerance towards calcium of the ions tested. Growth parameters showed no differences to control condition for both strains up to 100 mM; revealing natural tolerance to higher concentration of calcium in the surroundings. Although excessive levels of calcium did not impair the growth parameters, it also did not have a positive effect on biofilm formation or its binding affinity to EPS. Magnesium-saturated environment proved to be counterproductive as strains were able to dissolve more Ca2+ from the tooth surface in the presence of magnesium, therefore releasing excessive amounts of Ca2+ in the environment and leading to the progression of the disease. Thus, this supports the idea of self-regulation, when more Ca2+ is released, more calcium is bound to the biofilm strengthening its structure and however, also less is left for remineralization. Zinc inhibited bacterial adhesion already at low concentrations and had a strong antibacterial effect on the strains as well as on calcium dissolution; leading to less biofilm and less EPS. Additionally, Zn2+ had almost always the lowest affinity to all EPS; thus, the unbound zinc could also still remain in the surrounding environment and keep its antimicrobial properties. CONCLUSION: It is important to maintain a stable relationship between calcium, magnesium and zinc as excessive concentrations of one can easily destroy the balance between the three in cariogenic environment and lead to progression of the disease.

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.

Tolerance and Persister Formation in Oral Streptococci.

The aim of this study was to analyze the potential influence of long-term exposure in subinhibitory concentrations of chlorhexidine on the emergence of tolerant and/or persistent cells in oral streptococci. The two oral streptococcal isolates S. mutans ATCC25175 and S. sobrinus ATCC33402 were incubated, after long-term subinhibitory exposure to chlorhexidine, in liquid growth media containing high concentrations of chlorhexidine. A distinct subpopulation of more chlorhexidine-tolerant cells could be detected in streptococci that had been previously exposed to subinhibitory concentrations of chlorhexidine but not in the control strains. These more biocide-tolerant and persisting microbial subpopulations might also arise in vivo. Therefore, the rational and proper use of antimicrobials in dentistry, especially when used over a long period of time, is crucial.

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.