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.

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.

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.

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.

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.

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.

Influence of the amplitude of different side-to-side toothbrushes on noncontact biofilm removal.

OBJECTIVES: To investigate the impact of the lateral deflection of toothbrush bristles (amplitude) of three side-to-side toothbrushes for noncontact biofilm removal in an artificial interdental space model. MATERIALS AND METHODS: A three-species biofilm (Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus sanguinis) was formed in vitro on protein-coated titanium disks. A flow chamber system was combined with a static biofilm growth model. The amplitudes of three commercial side-to-side toothbrushes were evaluated by means of a dose response analysis. The amplitudes were decreased in steps (100%, 85%, 70%, 55%, and 40%). Subsequently, the biofilm-coated substrates were exposed to the toothbrushes. The biofilms were analyzed with confocal laser scanning microscope images and measured using volumetric analyses. RESULTS: The predictability of interdental biofilm reduction differed among the toothbrushes. A lower variety in the results of repeated experiments occurred in toothbrush C compared to toothbrushes A and B. Toothbrush C obtained highest percentage of biofilm reduction by 85% of amplitude power setting (median biofilm reduction 76%). Decreasing the amplitude from 85 to 40% resulted in reduced biofilm reduction (p = 0.029). In contrast, no significance could be observed for the differences of the tested amplitudes within toothbrushes A and B (p > 0.05). Between the toothbrushes, a significant difference in interdental biofilm reduction was found between C-A (p = 0.029) and C-B (p = 0.029) with amplitude of 85%. CONCLUSIONS: The amplitude of one of the investigated side-to-side toothbrushes affected the biofilm reduction predictably in an interdental space model. CLINICAL RELEVANCE: Within certain toothbrushes, a specific amplitude power setting may demonstrate beneficial effects on noncontact biofilm removal.

Influence of the oscillation frequency of different side-to-side toothbrushes on noncontact biofilm removal.

OBJECTIVES: The objective of this study was to investigate the influence of different oscillation frequencies of three powered toothbrushes with side-to-side action for noncontact biofilm removal in an artificial interdental space model. MATERIALS AND METHODS: A three-species biofilm (Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus sanguinis) was formed in vitro on protein-coated titanium disks using a flow chamber system combined with a static biofilm growth model. The oscillation frequencies of three commercial side-to-side toothbrushes were evaluated by means of a dose response. The frequency was decreased in steps (100, 85, 70, 55, and 40%). Subsequently, the biofilm-coated substrates were exposed to the side-to-side toothbrushes. The biofilm volumes were measured using volumetric analyses (Imaris 8.1.2) with confocal laser scanning microscope images (Zeiss LSM700). RESULTS: Compared to maximum oscillation frequency (100%), lower oscillation frequencies (up to 40%) resulted in reduced median percentages of biofilm reduction (median biofilm reduction up to 53% for maximum oscillation frequency, and up to 13% for 40% oscillation frequency) (p ≥ 0.03). In addition, decreasing the oscillation frequencies of the side-to-side toothbrushes showed an enhanced variety in the results of repeated experiments. CONCLUSIONS: The oscillation frequency of the tested side-to-side toothbrushes affected the biofilm reduction in an interdental space model. CLINICAL RELEVANCE: Within a toothbrush, higher oscillation frequencies may lead to beneficial effects on interdental biofilm removal by noncontact brushing.

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.

Bacterial colonization of resin composite cements: influence of material composition and surface roughness.

So-called secondary caries may develop in the cement gap between the tooth and the bonded restoration. Cement materials with a low susceptibility to biofilm formation are therefore desirable. In the present study, the adhesion of Strepococcus mutans onto three adhesive (Multilink Automix, RelyX Ultimate, and Panavia V5) and three self-adhesive (Multilink Speed Cem, RelyX Unicem 2 Automix, and Panavia SA plus) resin composite cements was evaluated. Previous studies have failed to evaluate concomitantly the effect of both the composition of the cements and their surface roughness on biofilm formation. The presence of S. mutans on cement surfaces with differing degrees of roughness was therefore recorded using fluorescence microscopy and crystal violet staining, and the composition of the cements was analyzed using energy-dispersive X-ray spectroscopy mapping. Biofilm formation on resin composite cements was found to be higher on rougher surfaces, implying that adequate polishing of the cement gap is essential. The use of copper-containing cements (Multilink Automix, Panavia V5, and Panavia SA plus) significantly reduced biofilm formation.

Efficacy of various side-to-side toothbrushes and impact of brushing parameters on noncontact biofilm removal in an interdental space model.

OBJECTIVES: The objective of this study was to evaluate the efficacy of four different side-to-side toothbrushes and the impact of various brushing parameters on noncontact biofilm removal in an adjustable interdental space model. MATERIALS AND METHODS: A three-species biofilm, consisting of Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus sanguinis, was formed in vitro on protein-coated titanium disks using a flow chamber combined with a static biofilm growth model. Subsequently, the biofilm-coated disks were exposed to four different powered toothbrushes (A, B, C, D). The parameters distance (0 and 1 mm), brushing time (2, 4, and 6 s), interdental space width (1, 2, and 3 mm), and toothbrush angulation (45° and 90°) were tested. The biofilm volumes were determined using volumetric analyses with confocal laser scanning microscope (Zeiss LSM700) images and Imaris version 7.7.2 software. RESULTS: The median percentages of simulated interdental biofilm reduction by the tested toothbrushes ranged from 7 to 64 %. The abilities of the analyzed toothbrushes to reduce the in vitro biofilm differed significantly (p < 0.05). Three of the tested toothbrushes (A, B, C) were able to significantly reduce a simulated interdental biofilm by noncontact brushing (p ≤ 0.005). The brushing parameters and their combinations tested in the experiments revealed only minor effects on in vitro interdental biofilm reduction (p > 0.05). CONCLUSIONS: A three-species in vitro biofilm could be altered by noncontact brushing with toothbrushes A, B, and C in an artificial interdental space model. CLINICAL RELEVANCE: Certain side-to-side toothbrushes demonstrate in vitro a high efficacy in interdental biofilm removal without bristle-to-biofilm contact.

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.

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.

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.

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.

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.

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.

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.

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.