Glycolysis and Automated Plaque Regrowth Method for Evaluation of Antimicrobial Performance.

PURPOSE: This study explored the potential of a new in vitro method in evaluating antiplaque benefits from five sets of antimicrobial systems including cetylpyridinium chloride (CPC), stannous fluoride (SnF2), Listerine essential oil mouthwashes (+/- alcohol), zinc chloride (ZnCl2), and sodium fluoride. (NaF). METHODS: Gingival dental plaque was collected and propagated using sterilized tryptic soy broth and sucrose, and then allocated into separate glycolysis and regrowth recipes for antiplaque evaluations. Glycolysis measurements (in duplicate) were recorded via pH microelectrode on plaque-treatment samples thermomixed (1200 rpm, 37 °C) for 4 h. For plaque regrowth, optical densities (in duplicate) were automatically collected on plaque-treatment samples using a microplate reader (linear shaking, 37 °C) from baseline to 4 h. RESULTS: Calculations of percent change in pH and optical density were performed and analyzed for each set of antimicrobial treatment groups. Statistical analysis (one-way ANOVA, Student-Newman-Keuls stepwise comparison tests) revealed dose responses and significant differences (p < 0.05) among treatment groups, including between negative and clinically relevant positive controls. CONCLUSIONS: This lab method produces results consistent with published clinical observations. This glycolysis and plaque growth method is sensitive to antimicrobial mechanisms of action, and may offer a convenient and clinically relevant screening tool in the evaluation of putative antimicrobial agents and formulations.

In situ remineralization of white-spot enamel lesions by 500 and 1,100 ppm F dentifrices.

The aim of this study was to evaluate the remineralization potential of three silica-containing NaF dentifrice systems in an intraoral model. Subjects (N = 30) in this randomized, three-phase, 28-day, crossover study served as their own control. Each participant wore a customized orthodontic appliance attached to a mandibular molar and contained one tooth block with caries-like lesion. For each phase, participants engaged in twice-daily brushing for 2 min with one of the following dentifrices: 500 ppm F, 500 ppm F plus functionalized ß-tricalcium phosphate (fTCP), and a clinically proven 1,100 ppm F. After each phase, appliances were removed, and specimens were analyzed using surface microhardness (SMH), transverse microradiography (TMR), and cross-sectional microhardness (CSMH). Statistically significant (p < 0.05) remineralization of white-spot lesions relative to baseline occurred for each dentifrice as determined with SMH and TMR. No significant differences (p > 0.05) in SMH were found among the three groups, but trending revealed the 500 ppm F plus fTCP produced 26% and 27% greater SMH recovery relative to 500 and 1,100 ppm F, respectively. Similarly, no significant differences (p > 0.05) in TMR were found among the groups. However, the 500 ppm F plus fTCP dentifrice produced 10% and 38% greater mineral recovery relative to 500 and 1,100 ppm F, respectively, while reducing the lesion depth 30% and 52%, respectively. Significant differences (p < 0.05) in CSMH existed among the three dentifice groups at different enamel depths, but statistical differences (p < 0.05) in relative lesion size were only found between 500 ppm F plus fTCP and 500 ppm F. The combination of fTCP and fluoride in a single-compartment, water-based dentifrice can cooperate with fluoride to produce significant remineralization. These results suggest that the combination of 500 ppm F with fTCP may provide comparable anticaries benefits relative to a 1,100 ppm F dentifrice.

SEM evaluation of demineralized dentin treated with professional-strength NaF topical pastes.

PURPOSE: To qualitatively explore the effects of prescription-strength topical NaF pastes on patent tubules in a pH cycling model comprising remineralization and demineralization phases. METHODS: 3 mm diameter dentin cores were extracted from bovine teeth, mounted in acrylic rods, ground and polished, and initially demineralized in 10 mL unstirred 50% citric acid (pH = 1.2) for 2 minutes to remove the smear layer and expose the dentin tubules. 72 acceptable specimens were then divided into six groups (12 per group) and subjected to a two-phase pH cycling study. The six pastes evaluated were as follows: fluoride-free Silly Strawberry, Clinpro 5000 and Clinpro Tooth Crème, MI Paste Plus, Sensodyne NUPRO 5000 and Topex Renew. First, the specimens were subjected to a remineralization regimen (Phase A), with each day comprising inverted immersion of specimens in three paste treatments (10 g paste: 20 mL distilled water) performed for 1 minute apiece with the immersion in artificial saliva (pH = 7.0) for 2 hours in between each treatment as well as overnight. After 7 days, the specimens were imaged using field emission scanning electron microscopy (FESEM). The remaining six specimens from each group were then cycled through a demineralization regimen (Phase B), with each day comprising inverted immersion of specimens in three rounds of a 1-minute treatment (10 g paste: 20 ml distilled water) followed immediately by a 3-minute static acid challenge (0.3% citric acid, pH = 3.8) with 2 hours in artificial saliva in between each round as well as overnight. After 3 days, the specimens were again imaged using FESEM. RESULTS: After the Phase A regimen (i.e. remineralization phase), treatment with Silly Strawberry (fluoride-free), Sensodyne NUPRO 5000 and Topex Renew yielded qualitatively similar tubule pore appearance, whereby deposits appeared to have formed around the tubule openings. Clinpro 5000, MI Paste Plus and Clinpro Tooth Crème appeared to occlude dentin tubules as shown by the relatively smaller diameter tubule openings. Relative comparisons among these pastes revealed Clinpro 5000 appeared especially effective in narrowing and incorporating mineral within the tubule, while the MI Paste Plus and Clinpro Tooth Crème appear to produce comparable tubule occlusion. After the Phase B regimen (i.e. resistance to demineralization), qualitative comparisons revealed the fluoride-free, MI Paste Plus, NUPRO and Renew pastes did not appear to occlude dentin tubules as well as Clinpro 5000 and Clinpro Tooth Crème.

Surfactant-modified beta-TCP: structure, properties, and in vitro remineralization of subsurface enamel lesions.

A hybrid material comprised of beta-tricalcium phosphate (beta-TCP) and sodium lauryl sulfate (SLS) was prepared using a mechanochemical process, examined using particle size analysis, IR spectroscopy, (31)P, (23)Na, and (13)C solid-state NMR spectroscopy, and calcium dissolution experiments, and probed for in vitro remineralization of subsurface enamel lesions. Our results suggest that while the (31)P environments of beta-TCP remain unchanged during solid-state processing, there is noticeable shifting among the SLS (23)Na and (13)C environments. Therefore, given the structure of beta-TCP, along with our IR examinations and calcium dissolution isotherms, SLS appears to interface strongly with the cation deficient C(3) symmetry site of the beta-TCP hexagonal crystal lattice with probable emphasis placed on the underbonded CaO(3) polyhedra. To demonstrate the utility of the surface-active TCP material in dental applications, we combined the TCP-SLS with 5,000 ppm F (NaF) and evaluated the remineralization potential of subsurface enamel lesions via an in vitro remineralization/demineralization pH cycling dental model. Using surface and longitudinal microhardness measurements, the TCP-SLS plus 5,000 ppm F system was found to significantly boost remineralization of subsurface enamel lesions, with microhardness values increasing up to 30% greater than fluoride alone.

In vitro remineralization efficacy of NaF systems containing unique forms of calcium.

PURPOSE: To determine the in vitro remineralization potential of a new calcium phosphate technology in a 1000 ppm F system. METHODS: 3 mm diameter bovine enamel specimens were mounted, ground and polished, and softened in a carbopol-lactic acid solution (pH = 5.0) for 36 hours at 37 degrees C. Specimens were then measured for baseline Vickers microhardness and stratified (N = 10, mean VHN = 35) into the following groups: Group A: distilled water (negative control); Group B: MI Paste Plus (900 ppm F); Group C: Theramed SOS (1450 ppm F); Group D: "control" dentifrice (1000 ppm F); and, Group E: "test" dentifrice (1000 ppm F) admixed with a new functionalized calcium phosphate system. The groups were then cycled for 10 days in a pH cycling model consisting of four 2-minute treatment periods (diluted 1:3 with distilled water) and one 4-hour acid challenge (carbopol-lactic acid, pH = 5.0) per day. Between these events, specimens were immersed in artificial saliva (pH = 7.0). After 10 days of cycling, the specimens were measured for Vickers surface microhardness and were subsequently microdrilled, with the powder measured for fluoride content using a calibrated fluoride-sensitive electrode. RESULTS: Significant differences resulted between the distilled water and fluoride-containing groups. Among the fluoride-containing groups, Group B demonstrated statistically low levels of enamel fluoride deposition and deltaVHN, while Group E statistically outperformed Group D. Among the groups with different calcium systems (Groups B, C, and E), Groups C and E were found to be statistically equivalent and superior to Group B with respect to both bioavailable fluoride and deltaVHN. Based on our results, these data demonstrated the combination of a new calcium phosphate technology plus 1000 ppm F, produced significantly greater remineralization relative to both the 1000 ppm F test dentifrice and MI Paste Plus, and was statistically equivalent to Theramed SOS.

In vitro assessments of experimental NaF dentifrices containing a prospective calcium phosphate technology.

PURPOSE: To determine the fluoride dose response of experimental NaF dentifrices containing a prospective calcium phosphate technology, along with the corresponding relative enamel and dentin abrasion values. METHODS: 3 mm diameter bovine enamel specimens were mounted, ground and polished, and softened in a carbopol-lactic acid solution (pH = 5.0) for 36 hours at 37 degrees C. Specimens were then measured for baseline Vickers microhardness and stratified (N = 18, mean VHN = 33) into eight groups. These groups consisted of a placebo paste, four test dentifrices (A, B, C, D) with three of the four (A, B, C) containing a promising calcium phosphate ingredient, Crest Cavity Protection, MI Paste Plus, and PreviDent Booster 5000. The groups were cycled in a lesion reversal pH cycling model consisting of four 2-minute treatment periods (diluted 1:3 with DI water) and one 4-hour acid challenge (carbopol-lactic acid, pH = 5.0) per day. Between these events, specimens were immersed in artificial saliva (pH = 7.0). After 20 days of cycling, the specimens were microdrilled and analyzed for fluoride content, and also measured for Vickers surface microhardness after 10 and 20 days of cycling and after a 2-hour and 16-hour post-cycle acid challenge (carbopol-lactic acid, pH = 5.0). Separately, relative dentin and enamel abrasion (RDA and REA) were performed using the ADA recommended radiotracer method. RESULTS: A fluoride dose response was observed for the test dentifrices after 10 and 20 days of pH cycling, with test dentifrice C promoting the highest remineralization among the groups while both the MI Paste Plus and PreviDent systems provide the least remineralization (one-way ANOVA, SNK, P < 0.05). With respect to enamel fluoride uptake, the group facilitating the highest incorporation of fluoride into the enamel lesion was test dentifrice C, while the least effective NaF system was the MI Paste Plus (one-way ANOVA, SNK, P < 0.05). In terms of formulation abrasion, the REA scores were similar among the test dentifrices, MI Paste Plus, and PreviDent and compared favorably to the ADA reference material score (one-way ANOVA, SNK, P < 0.05); relative to the ADA reference material RDA score, the data indicate that MI Paste Plus was essentially non-abrasive, while PreviDent was significantly more abrasive to dentin (one-way ANOVA, SNK, P < 0.05). Altogether, these data show the developmental test dentifrices demonstrate a fluoride dose response and show great promise in remineralizing white-spot enamel lesions relative to MI Paste Plus and PreviDent.

Noncontact remineralization of incipient lesions treated with a 5% sodium fluoride varnish in vitro.

PURPOSE: Fluoride varnishes are appealing topical fluoride preparations that may provide anticaries benefits. The purpose of this in vitro study was to assess the noncontact remineralization effects of a commercial 5% sodium fluoride varnish on white spot lesions (WSLs). METHODS: Three-millimeter diameter enamel cores were extracted from bovine teeth, mounted in acrylic rods, ground and polished, and initially demineralized to create WSLs. Specimens were evaluated for surface microhardness and divided (N=6) into two groups (water control or noncontact 5% sodium fluoride white varnish with tricalcium phosphate, where one 0.50 ml unit dose was applied to acrylic rods instead of directly on WSLs). Groups were cycled in a three-day regimen consisting of two rounds of one-hour treatments and one-hour static immersions in demineralization solution. Between these events, WSLs were immersed in artificial saliva. Remineralization was evaluated using surface and cross-sectional microhardness and high-resolution scanning electron microscopy (SEM). RESULTS: The noncontact varnish treatment produced significantly greater percent surface microhardness recoveries (P<.05) and smaller subsurface lesions compared to the control group (P<.05). SEM revealed comparatively greater WSL porosity reduction for noncontact varnish. CONCLUSIONS: Noncontact application of a commercial 5% sodium fluoride varnish reduced white spot lesion porosity and produced significant acid-resistant white spot lesion remineralization.

Effects on dentin treated with eluted multi-mineral varnish in vitro.

PURPOSE: The purpose of this pilot study was to assess the non-contact effects of a NaF varnish on the morphology and elemental compositions of sound and demineralized dentin in an in vitro pH cycling model. METHODS: 3 mm diameter dentin cores were extracted from bovine teeth, mounted in acrylic rods, and ground and polished. Sound and demineralized (in 10 mL unstirred 50% citric acid (pH = 1.2) for two minutes) specimens were divided into four groups (N=6) corresponding to treatments with either water (control group) or non-contact Vanish® 5% NaF White Varnish with tri-calcium phosphate (3M ESPE). For the non-contact varnish group, one 0.50 ml unit-dose was applied to acrylic rods and positioned in the same stopper as three dentin specimens. The four groups were then cycled in a regimen consisting of three rounds of one-hour treatments with each round followed by immersion in a three-minute static acid challenge (0.3% citric acid, pH = 3.8). For each one-hour treatment immersion the test groups were immersed in distilled water. The specimens were thoroughly rinsed with distilled water after each treatment and after each acid challenge. After the last challenge, specimens were rinsed with distilled water and immersed in artificial saliva (AS) overnight. All treatments, acid challenges and immersion in AS solution were performed in an incubator set at 37°C. After three days, the specimens were assessed using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). RESULTS: SEM and EDS results demonstrated the non-contact varnish group can provide mineralization benefits relative to the control group for both sound and demineralized dentin substrates. Depositions from mineral eluting from the varnish developed on the intertubular and intratubular regions alike, with the most prominent depositions appearing on initially demineralized dentin. CONCLUSIONS: In this observational study, acid-resistant mineral depositions were produced on the sound and dentin surfaces without direct application from a multi-mineral varnish containing fluoride, calcium and phosphate.

Remineralization of eroded enamel lesions by simulated saliva in vitro.

PURPOSE: The purpose of this study was to evaluate the effects of two simulated saliva (SS) remineralization solutions comprising different calcium-inorganic phosphate (Ca/P(i)) ratios on eroded enamel. METHODS: 3 mm diameter enamel cores were extracted from bovine teeth, mounted in acrylic rods, ground and polished,and initially demineralized with either 0.3% (120 minutes) or 1.0% (30 minutes) citric acid solutions (pH 3.8). Both sets of initially eroded specimens were evaluated for surface microhardness (N=10) and treated with either 0.3 or 1.6 Ca/P(i) ratio SS. Groups were first exposed to a seven-day remineralization period and then were cycled in a three-day regimen consisting daily of three rounds of two-hour plus overnight SS treatments and three 10-minute static immersions in demineralization solution. Specimens were assessed using surface microhardness and scanning electron microscopy. RESULTS: Initial erosion from 0.3% citric acid led to elliptical-shaped pore openings several microns in length and in depth and contrasted significantly with respect to 1% citric acid. The greatest remineralization was observed from the 0.3 Ca/P(i) SS, while the 1.6 Ca/P(i) SS produced the least. CONCLUSIONS: This study demonstrated the nature of remineralization of eroded enamel depends on both initial erosive conditions and the Ca/P(i) ratio of simulated saliva.

In situ remineralisation of eroded enamel lesions by NaF rinses.

OBJECTIVE: The purpose of this pilot study was to evaluate the remineralisation of eroded enamel by NaF rinses in an intra-oral model. METHODS: Serving as their own control, subjects (N=80) participated in a randomised, four-leg (20 subjects/leg), 28-day, parallel design study. In each leg, each participant wore a customised orthodontic bracket attached to a mandibular molar that contained one tooth block having an initial erosive lesion (0.3% citric acid, pH 3.75, 2 h). Within the 28-day period, participants engaged in twice-daily brushing for 1 min with a fluoride-free dentifrice followed by 1-min rinsing with one of the following aqueous rinses: fluoride-free (0 ppm F), 225 ppm F, 225 ppm F plus functionalised ß-tricalcium phosphate (fTCP), and 450 ppm F. Following intra-oral exposure, appliances were removed and specimens were analysed using surface microhardness (SMH) and transverse microradiography (TMR). RESULTS: Statistically significant (p<0.05) remineralisation, as determined by SMH and TMR, of the eroded enamel relative to baseline occurred for each fluoride system. No significant differences in SMH were observed amongst the fluoride groups (p>0.05), however, 225 ppm plus fTCP produced 27% and 7% SMH indent length reduction relative to 225 ppm F and 450 ppm F, respectively. No significant differences in TMR were observed amongst the fluoride groups (p>0.05), however, 225 ppm F plus fTCP and 450 ppm F produced significant (p<0.05) mineral gains relative to the fluoride-free control, whilst 225 ppm F did not (p>0.05). Relative to the 225 ppm F group, the 450 ppm F and 225 ppm F plus fTCP groups produced 65% and 61% greater mineral change, respectively. CONCLUSIONS: These pilot results demonstrate this model is sensitive to fluoride and that addition of fTCP to an aqueous rinse containing 225 ppm F may provide significant remineralisation benefits. Therefore, the combination of relatively low levels of fluoride and fTCP might be an effective alternative to a high fluoride treatment for anti-erosion benefits.

Preparation, characterization and in vitro efficacy of an acid-modified beta-TCP material for dental hard-tissue remineralization.

A blended material composed of beta-tricalcium phosphate (beta-TCP) and fumaric acid (FA) was prepared using a mechanochemical process. The structure and properties of the TCP-FA material was probed using particle size analysis, infrared, (31)P and (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy, powder X-ray diffraction and calcium bioavailability. NMR studies showed that orthophosphate environments within beta-TCP remain largely unaffected in the presence of FA during mechanochemical processing; alternately, (13)C data indicated the carboxylic groups of FA are strongly affected during processing with beta-TCP. X-ray results reveal beta-TCP diffraction plane shifting with lattice contractions likely arising at the C(3) symmetry site. While milled beta-TCP (mTCP) produces a higher flux of bioavailable calcium relative to native beta-TCP, the mechanochemical conditioning of TCP-FA generates more than seven times the level of ionic calcium relative to mTCP. Collectively, the results from these studies indicate FA interfaces with calcium oxide polyhedra of the beta-TCP hexagonal crystal lattice, especially with the underbonded CaO(3) cluster manifested within the C(3) symmetry site of the beta-TCP motif. An in vitro remineralization/demineralization pH cycling dental model was then used to assess the potential of the TCP-FA material in reversing early stage non-cavitated enamel lesions. Characterization of the remineralization via surface and longitudinal microhardness measurements demonstrated that the TCP-FA material provides statistically superior remineralization relative to milled and native beta-TCP.

Remineralization of eroded enamel by a NaF rinse containing a novel calcium phosphate agent in an in situ model: a pilot study.

PURPOSE: An in situ study evaluated the remineralization potential of 225 ppm fluoride (F) rinses with and without a calcium phosphate agent (TCP-Si-Ur) on eroded enamel. METHODS: 20 human patients participated in this IRB approved study. Enamel blocks extracted from 20 human molars were assigned to each of the three study phases (G1, G2, G3). Each block was eroded using 1% citric acid (pH = 2.5), with a slice cut from each block to establish baseline lesion parameters (ie, integrated mineral loss ΔZ, and lesion depth LD) using transverse microradiography (TMR). Participants and assigned blocks were randomly divided into three 28-day phases. The blocks were mounted into modified orthodontic brackets and bonded to the buccal surface of one of the subject's mandibular molars. The appliance remained in the subject's mouth for 28 days. Prior to each study phase, participants observed a one-week-washout period using a fluoride-free dentifrice. In each phase, participants brushed with the fluoride-free dentifrice for 1 min, followed by one of the following coded treatments: G1: 225 ppm F + 40 ppm TCP-Si-Ur rinse (1 min); G2: 225 ppm F rinse (1 min); G3: no rinse (saliva-only). After each phase, appliances were removed and specimens were analyzed using TMR. RESULTS: TMR data (ie, ΔZ and LD) revealed all three groups significantly remineralized eroded enamel (paired t-tests, P < 0.001). Net mineralization (% change in ΔZ, LD) were as follows (mean (std.dev): G1: 44.1 (22.6), 30.5 (27.0); G2: 30.0 (7.4), 29.4 (10.5); G3: 23.8 (16.4), 25.7 (15.5). Furthermore, G1 was found to cause significantly more remineralization than G2 (P = 0.039) and G3, (P = 0.002). CONCLUSION: Mouthrinse containing 225 ppm F plus TCP-Si-Ur provided significantly greater remineralization relative to 225 ppm F only or saliva alone.

Bioactivity of novel self-assembled crystalline Nb2O5 microstructures in simulated and human salivas.

Crystalline, self-assembled niobium oxide microstructures formed in situ via potentiostatic anodization of niobium foil in an HF(aq) electrolyte solution are proposed as exceptional nucleators of Ca-P minerals, including hydroxyapatite. This material was tested for bioactivity through immersion in simulated and pooled human salivas. The simulated saliva formulation was based on mineral content found in stimulated human saliva and has a molar Ca/P ratio of 1:3.7. Oxide microstructures and mineral morphologies were examined using scanning electron microscopy. Differences in the mineral phase and morphology were attributed to the contrasting complexities of the two supersaturated solutions, with proteins and enzymes in human saliva most likely imparting a significant role. Dimensions of the niobium oxide microstructures and mineral deposits were characterized using profilometry. Energy dispersive spectroscopy, x-ray diffraction, Raman spectroscopy and electron microprobe analysis were utilized in identifying the nucleated mineral phases. Nucleation from human saliva resulted in mixed-phase mineral formations including amorphous calcium phosphate and poorly crystalline apatites. On the other hand, mineral nucleation from simulated saliva was more specific to hydroxyapatite. Based on these results, we demonstrate that a crystalline, self-assembled metal oxide is a unique and efficient nucleator of hydroxyapatite and other Ca-P minerals in supersaturated salivary solutions.

Structure and ionic interactions of organic-inorganic composite polymer electrolytes studied by solid-state NMR and Raman spectroscopy.

Solid-state NMR studies of composite polymer electrolytes are reported. The materials consist of polyethylene oxide and an organic inorganic composite, together with a lithium salt, and are candidates for electrolytes in solid-state lithium ion batteries. Silicon and aluminum MAS and multiple quantum MAS are used to characterize the network character of the organic-inorganic composite, and spin diffusion measurements are used to determine the nanostructure of the polymer/composite blending. Multiple quantum spin counting is used to measure the ion aggregation. The NMR results are supported by Raman spectra, calorimetry, and impedance spectroscopy. From these experiments it is concluded that the composite suppresses polymer crystallization without suppressing its local mobility, and also suppresses the tendency for the ions to aggregate. This polymer composite thus appears very promising for application in lithium ion batteries.