Abstract
Objectives
To compare and explore the dose-response of phytate-containing 1150 ppm fluoride toothpastes on model caries lesions and to determine the impact of zinc ions.
Methods
This was a single-centre, randomised, blinded (examiner/laboratory analyst), six-treatment, four-period crossover, in situ study in adults with a removable bilateral maxillary partial denture. Study treatments were toothpastes containing: 0.425% phytate/F; 0.85% phytate/F; 0.85% phytate/Zn/F; F-only; Zn/F and a 0% F placebo. Where present, F was 1150 ppm as NaF; Zn was 0.3% as ZnCl 2 . Human enamel specimens containing early-stage, surface-softened (A-lesions) or more advanced, subsurface (B-lesions) caries lesions were placed into the buccal flanges of participants’ modified partial denture (one of each lesion type per side). A-lesions were removed after 14 days of twice-daily treatment use; B-lesions were removed after a further 14 days. A-lesions were analysed for surface microhardness recovery. Both lesion types were analysed by transverse microradiography and for enamel fluoride uptake, with B-lesions additionally analysed by quantitative light-induced fluorescence. Comparison was carried out using an analysis of covariance model.
Results
Statistically significant differences between 1150 ppm F and the placebo toothpastes (p < 0.05) were shown for all measures, validating the model. No differences between fluoride toothpastes were observed for any measure with little evidence of a dose-response for phytate. Study treatments were generally well-tolerated.
Conclusions
Results suggest phytate has little impact on fluoride’s ability to promote early-stage lesion remineralisation or prevent more advanced lesion demineralisation in this in situ caries model. Similarly, results suggest zinc ions do not impair fluoride efficacy.
Clinical significance
Toothpastes may contain therapeutic or cosmetic agents that could interfere with fluoride’s caries prevention efficacy. The present in situ caries study has demonstrated that phytate, added to provide enhanced extrinsic stain removal/prevention, and zinc, added to inhibit malodour, do not impair fluoride efficacy.
1
Introduction
Modern toothpastes are multifunctional, delivering therapeutic benefits to combat a variety of oral conditions (e.g., caries, gingivitis, dentine hypersensitivity, erosive tooth wear) while also offering cosmetic benefits such as control of extrinsic enamel staining or inhibition of oral malodour. The incorporation of ‘cosmetic’ ingredients allows toothpastes to be tailored to an individual’s requirements; however, consideration must be given to ensure that the positive actions of therapeutic ingredients are not negated by the addition of such ingredients.
An important cosmetic function of a toothpaste is to control extrinsic dental stain. Common approaches include the use of physical abrasives, such as particulate silica or alumina, or soluble chemical agents with affinity for calcium ions, such as condensed phosphates (e.g., pyrophosphate, tripolyphosphate or hexametaphosphate) that interfere with attachment of stain molecules to enamel surfaces [ ]. Chemical cleaning agents are an attractive addition to a conventional toothpaste to augment the abrasive-based stain control system without increasing toothpaste abrasivity [ ]. However, condensed polyphosphate agents have been reported to interfere with the bioactivity of fluoride by inhibiting the exchange of minerals at the enamel surface, thereby interfering with the ability of fluoride to remineralise enamel [ ]. Few long-term clinical studies exist to clarify whether condensed phosphates impair the anticaries properties of fluoride, those that do, have not indicated an effect [ ]. However, such observations may not necessarily be extrapolated to all phosphate classes and a polyphosphate that provides the tooth whitening benefits typical of the class, without impact on fluoride-promoted remineralisation, is therefore of great interest.
Phytate is an organic polyphosphate ion naturally found in cereals and seeds where it is the principal storage molecule of phosphorous in plant tissues [ ]. Chemically, phytate is similar to condensed phosphates by virtue of multiple phosphate groups, but in contrast to condensed (linear) phosphates, it is cyclic in nature with six phosphates groups bound to a cyclohexane ring (and no direct phosphate–phosphate bonds). Recently, a 12-week study has indicated enhanced stain removal ability for a toothpaste containing 0.85% w/w sodium phytate compared to a regular toothpaste [ ]. Phytate may also offer caries protective benefits of its own. In vitro , phytate has been shown to modify the transport of ions across enamel and dentine surfaces, with potential benefits in terms of caries progression [ ] In animal caries studies, phytate has been shown to exhibit anticaries effects in the absence of fluoride [ ].
A second important cosmetic function of a toothpaste is control of oral malodour. Zinc ions are commonly added to toothpaste formulations to reduce malodour by chemically binding to volatile odour molecules [ ] and by inhibiting malodour-generating plaque bacteria [ ]. Zinc has an affinity for enamel surfaces via binding to surface phosphate groups and so also has the potential to interfere with fluoride’s action against caries [ ]. Since a further benefit of use of zinc toothpastes is a reduction in calculus formation [ ] via inhibition of formation of apatites and their precursors [ ], it has the potential to interfere with enamel remineralisation. In vitro mechanistic studies have demonstrated the potential for this to occur in both a positive and negative fashion [ ]. Furthermore, like phytate, zinc may offer its own protection against demineralisation. Data from in vitro and in situ studies support this proposition [ ]. Overall, the effect of zinc on caries development is proposed to be neutral [ ].
It is important to understand the interaction of these ‘cosmetic’ agents on fluoride efficacy, separately and together, when formulated in a toothpaste. A program was undertaken to investigate the remineralisation/demineralisation effects of phytate and zinc on fluoride efficacy from toothpastes. Studies have already been performed to understand their effects on enamel remineralisation/demineralisation using a relatively simple, single-treatment ‘erosion’ in situ protocol [ ]. These studies showed significant inhibition of fluoride-induced remineralisation by phytate and by zinc. In the current study, a protocol based on an established longer-term in situ caries model [ ] was employed to examine the effects of phytate (at two concentrations, the primary focus of the study) and zinc (the secondary focus) separately and together in a regular fluoride toothpaste on remineralisation and prevention of further demineralisation of enamel.
2
Materials and methods
This was a single-centre, randomised, blinded (examiner and laboratory analyst), six-treatment, four-period crossover, in situ study conducted at the Oral Health Research Institute of the Indiana University School of Dentistry, Indianapolis, Indiana, USA. The protocol was approved by the Indiana University Institutional Review Board (IRB #1512088822) and the study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. The study was registered at clinicaltrials.gov (NCT02751320). There were minor amendments to the protocol that did not affect study flow or outcomes.
2.1
Participants
All participants provided written informed consent prior to screening. Participants aged 18–85 years were eligible for inclusion if they had good oral health and a removable bilateral maxillary partial denture with sufficient room in the posterior buccal flange areas to accommodate two enamel specimens and an unstimulated and stimulated salivary flow rate of at least 0.2 and 0.8 mL/min, respectively. Exclusion criteria included: pregnancy; breastfeeding; taken antibiotics or had received professional fluoride application in the 2 weeks prior to screening; use of fluoride supplements or mouth rinse; dental restorations in poor state of repair; active caries or moderate/severe periodontal disease; using a denture adhesive to secure the denture other than non-zinc Poligrip ® (GSK Consumer Healthcare, Weybridge, UK).
2.2
In situ remineralisation model
This study used the in situ caries remineralisation model developed by Zero et al. [ ]. Human permanent teeth were cut into 4 × 4 mm enamel specimens, which were polished to create flat surfaces as described previously. Surface-softened lesions (A-lesions) were created according to a modified method of White [ ] by immersing enamel specimens in 40 mL 0.05 mol/dm 3 lactate 50% saturated with respect to hydroxyapatite and containing 0.2% (wt/vol) Carbopol ® 907 (BF Goodrich, Cleveland, OH, USA) adjusted to pH 5.0 with KOH at 37 °C for 24 h. Subsurface lesions (B-lesions) were prepared by demineralising enamel specimens at 37 °C for 9 days in a solution containing 0.1 M lactate, 4.1 mM CaCl 2 , 8.0 mM KH 2 PO 4 and 0.2% w/v Carbopol ® 907, pH adjusted to pH 5.0 using KOH [ ]. Lesions were deemed acceptable if lesioned areas displayed uniform opacity and surface shine on exposure to overhead light. Specimens were stored in a moist environment to prevent dehydration and were sterilised by ethylene oxide gas prior to insertion into dentures.
At each treatment start visit, one A-lesion and one B-lesion enamel specimen was placed flush in each buccal flange area of the bilateral mandibular denture for a total of two of each specimen type per denture. Each enamel specimen was covered with Polyester Knit Fabric (Item P01628, Bard Peripheral Vascular, Tempe, AZ, USA) to encourage plaque formation [ ]. Specimens were mounted such that the surface was flush with the buccal flange surface and were luted in place using a light cured dental composite (Triad VLC material, Dentsply Int., York, PA, USA). Upon study completion, the denture was permanently repaired with acrylic.
2.3
Study design and treatments
At the screening visit, participants provided written informed consent and their demographic details and medical and current medication history was recorded. They underwent an oral soft tissue (OST) and oral hard tissue (OHT) examination and their stimulated and unstimulated salivary flow rate was assessed. An OST examination was also performed before and after each treatment period; an OHT examination was performed following dental prophylaxis and at the end of each treatment period. Prior to each treatment period, participants received a dental prophylaxis using a non-fluoride prophylaxis and floss. They were issued with a washout toothpaste (Negative Control 0 ppm fluoride, as below) and study toothbrush (Aquafresh ® Clean Control Everyday Clean™ toothbrush, GSK Consumer Healthcare, supplied new for each washout and treatment period).
At the first treatment visit, eligible participants were randomised to a treatment sequence according to a schedule provided by the Biostatistics Department of GSK Consumer Healthcare. Given the number of treatment arms, an incomplete block design was employed to reduce the length of participation for each subject and so that all possible pairs of treatments were observed, ideally an equal number of times. Details of study treatments are provided in Table 1 .
| Name | Phytate (as sodium phytate, w/w) | Zinc chloride, (w/w) ** | Fluoride (ppm F, as NaF) |
|---|---|---|---|
| 0.425% Phy/F | 0.425% | – | 1150 |
| 0.85% Phy/F | 0.85% | – | 1150 |
| 0.85% Phy/Zn/F | 0.85% | 0.3% | 1150 |
| Placebo | – | – | – |
| F-only | – | – | 1150 |
| Zn/F | – | 0.3% | 1150 |
* All toothpastes were formulated in a silica abrasive base that contained sodium lauryl sulphate as the primary surfactant.
** Zinc-containing toothpastes included sodium citrate to stabilise the ionic form of zinc.
Toothpastes were supplied in plain white tubes with a label containing protocol number, product code, directions for use/storage and emergency contact number. The examiner, laboratory analysts, study participants, study statistician, data management staff and other employees of the sponsor who could influence study outcomes were blinded to treatment allocation and order of treatment.
Participants used 1.5 g (±0.1 g) of their assigned toothpaste twice a day, applied to a wet toothbrush, and brushed only their natural teeth for one timed minute, ensuring the enamel specimens were not brushed. They then expectorated and rinsed with 15 mL tap water for approximately 10 s. The first brushing for each participant for each treatment period was under study site supervision with their brushing technique re-assessed at each treatment visit. Participants wore their mandibular partial denture for 24 h a day but could remove it for water-rinsing following eating and for cleaning, taking care to avoid brushing the specimens. Participants could not eat or drink anything for at least 30 min following brushing and were instructed not to eat canned sardines during the study as they could contain high fluoride levels. Participants followed the brushing schedule for 28 days per treatment period and returned to the study centre at days 14 and 28 when the two A-lesion specimens and two B-lesion specimens were removed, respectively.
There was a washout-period of approximately 1 week between each treatment period during which participants followed their usual dental hygiene practices for at least 4 days. They then returned to the study centre for a dental prophylaxis. For the 2–3 days prior to the treatment start visits, participants discontinued all usual oral hygiene practices (with the exception of inter-dental cleaners) and used only the study washout fluoride-free toothpaste and toothbrush. For the study duration, participants were instructed not to use any other denture cleaning products or denture adhesive on the lower partial denture or a denture adhesive, with the exception of supplied, zinc free, Polygrip, on the upper partial or full denture.
2.4
Outcome measures and their assessment
The primary endpoint for analysis was percentage surface microhardness recovery (%SMHR) [ ] of the A-lesions with the primary objective being to evaluate if %SMHR was related to sodium phytate dose. SMH was measured by assessing indentation lengths using a Wilson 2100 Hardness Tester (Norwood, MA, USA) equipped with a Knoop diamond, before (B) and after (D) in vitro demineralisation of the enamel specimens and after intraoral toothpaste treatment (R). Values were averaged across the two enamel specimens per participant to give a single observation. %SMHR was calculated as = [(D-R)/(D-B)] × 100 [ ].
Additional endpoints for the A-lesions were measurement of the amount of fluoride incorporated into the enamel (EFU) and the transverse microradiography (TMR)-derived parameters of ΔZ (integrated mineral loss in vol%min × μm), L (lesion depth in μm) and SZ max (surface zone mineral density in vol%min). For the B-lesions, secondary endpoints evaluated included EFU, ΔF (lesion fluorescence loss, determined using quantitative light-induced fluorescence [QLF]) and the TMR-derived variables of ΔM (change from baseline integrated mineral loss), ΔL (change from baseline lesion depth) and ΔSZ max (change from baseline surface zone mineral density).
TMR measurements were made by sectioning the enamel specimens into 100 μm plano-parallel thin slices using a Silverstone-Taylor Hard Tissue Microtome (Scientific Fabrications, Lafayette, CO, USA). Specimens were polished, mounted on plates and X-rayed at 20 kV and 30 mA at a distance of 42 cm for 65 min. Micrographs were examined by Zeiss EOM microscope using TMR software v.3.0.0.11 (Inspektor Research Systems BV, Amsterdam, The Netherlands). Mineral content was calculated from the grey intensity levels of the section images [ ]. Lesions were required to have a baseline mineral loss (ΔZ) of 2500 ± 600 vol% min × μm and a maximum mineral density at the surface zone (SZ max ) of at least 40 vol%min. After treatment, a further section was taken from each lesion specimen for radiography assessment. To avoid potentially compromising the primary endpoint, %SMHR of A-lesions, baseline TMR measures were not performed; TMR data for the A-lesions represents the lesion condition at the end of treatment while TMR data for the B-lesions represents change from baseline.
Changes in mineral content (ΔM), lesion depth (ΔL) and maximum mineral density at the surface-zone (ΔSZ max ) were calculated as respective post-treatment values minus baseline values. Positive numbers denote remineralisation, increase in lesion depth and increase in SZ max , respectively; negative numbers denote further demineralisation, decrease in lesion depth and decrease in SZ max , respectively.
For QLF, the B-lesion specimens were air-dried for at least 10 min before QLF measurements were performed using the QLF-D Biluminator 2 (Inspektor Research, the Netherlands). A sound enamel reference area had previously been denoted/protected on each specimen through the use of clear nail varnish. Acquired QLF images were analysed using dedicated QLF Analysis software (C4 Research Software Suite, Inspektor Research Systems BV, Amsterdam, The Netherlands). ΔF base values were recorded at a threshold level of 5%, i.e., a minimum of 5% fluorescence loss between sound and uncovered demineralised enamel. After completion of the 28-day treatment period, QLF images were obtained on all B-lesions again (ΔF post ). The change that occurred in lesion fluorescence loss (ΔΔF) following treatment was calculated as follows: ΔΔF = ΔF post − ΔF base , where positive numbers denote remineralisation, negative numbers denote further demineralisation.
EFU was determined using the microdrill enamel biopsy technique, as previously described [ ]. Four microdrill samples from each enamel specimen were pooled and a value for fluoride content determined. Participant-wise measurements were calculated separately for A- and B-lesions as the mean values from the two respective enamel blocks.
2.5
Safety
Assessments of tolerability were made with respect to OST abnormalities and adverse events (AEs) reported by participants following the first dental prophylaxis until 5 days following last administration of study treatment. Each AE was assessed for intensity (mild, moderate, severe) and whether it was related to study treatment, according to clinical judgement. Safety assessments were based on the safety population, defined as all participants who were randomised and received at least one dose of study toothpaste.
2.6
Statistical analysis
This was an exploratory study to examine the effects of phytate (at two concentrations, the primary focus of the study) and zinc (the secondary focus) separately and together in a regular fluoride toothpaste on remineralisation and prevention of further demineralisation of enamel. Given the exploratory nature of this comparison, no formal sample size calculation was performed. However, to ensure validity of this established model (and permit comment on comparison between the fluoride toothpastes), a formal size calculation was performed on the Placebo and F-only controls. This study aimed to randomize approximately 62 participants to ensure approximately 50 completed the entire study. In this incomplete block design, with 50 evaluable participants this study had >90% power at a 5% significance level (using 2-sided testing) to detect differences between the Placebo and F-only groups for the main comparators (%SMHR for A-lesions, TMR [ΔM] for B-lesions).
The intent-to-treat (ITT) population, on which all efficacy analyses were performed, was defined as all participants who were randomised, received study toothpastes at least once and provided at least one post-baseline efficacy assessment. The per-protocol (PP) population was defined as all participants in the ITT population who had at least one efficacy assessment considered unaffected by protocol violations.
Validity of the study assessment measures was predefined as acceptable if there was a statistically significant difference (p < 0.05) between the F-only and Placebo toothpastes, favouring the former. If model analysis method validity was not achieved, no further analysis of any endpoint was performed using that technique. In all analyses, if a participant was missing an enamel specimen, the mean was computed over the available enamel specimens. All pairwise treatment comparisons were performed using 2-sided testing at the 5% significance level. The assumption of normality of residuals was investigated and met for all endpoints. No adjustment for multiple comparisons was employed as the primary comparison had been defined.
%SMHR was analysed using an analysis of covariance (ANCOVA) model with participant as a random effect and fixed effects for study period and treatment. The covariates were based on participant-level (mean across treatment periods) and period level (period-level minus participant-level) baseline and pre-treatment acid challenge enamel SMH/indentation lengths. Primary comparisons were: F-only vs Placebo (validity of model, fluoride effect); 0.425% Phy/F vs F-only; 0.85% Phy/F vs F-only; 0.425% Phy/F vs 0.85% Phy/F. Linear and quadratic contrasts were fitted to establish whether there was a dose-response relationship of phytate (0%, 0.425%, and 0.85%, all containing 1150 ppm fluoride). Secondary comparisons were: 0.85% Phy/Zn/F vs Zn/F; Zn/F vs F-only; 0.85% Phy/F vs 0.85% Phy/Zn/F.
Participant-wise change in mineral content (ΔM) of TMR values at week 4 and participant-wise EFU at 2 and 4 weeks were secondary efficacy variables. Participant-wise measurements were calculated as the mean values from the two enamel blocks for A- and B-lesions. Treatment comparisons were performed using a mixed model ANCOVA including participant as a random effect and fixed effects for study period and treatment. It also included the participant-level (mean across treatment periods) baseline and period-level (period-level minus participant-level) baseline ΔZ measurement as covariates. EFU was analysed using analysis of variance (ANOVA) with participant as a random effect and fixed effects for study period and treatment for both A and B-lesions. Linear and quadratic contrasts were fitted to establish whether there was a dose-response relationship of phytate (0%, 0.425%, and 0.85% all containing 1150 ppm fluoride). Secondary comparisons were: 0.85% Phy/Zn/F vs F-only; Zn/F vs F-only; 0.85% Phy/F vs 0.85% Phy/Zn/F.
All TMR parameters (change in mineral content [ΔM], change in the lesion depth [ΔL] and maximum mineral density at the surface-zone) for A-lesions at week 2, change in the lesion depth (ΔL), maximum mineral density at the surface-zone of TMR and change in lesion fluorescence loss (ΔΔF) of the B-lesions at week 4 were exploratory efficacy variables. All TMR parameters at week 2 for A-lesions were analysed the same way as EFU. Change in the lesion depth (ΔL), maximum mineral density at the surface-zone and change in lesion fluorescence loss (ΔΔF) of the B-lesions were analysed in the same way as ΔM of TMR at week 4. Baseline (participant and period level) lesion depth, SZmax and ΔF were used as covariates, respectively.
2
Materials and methods
This was a single-centre, randomised, blinded (examiner and laboratory analyst), six-treatment, four-period crossover, in situ study conducted at the Oral Health Research Institute of the Indiana University School of Dentistry, Indianapolis, Indiana, USA. The protocol was approved by the Indiana University Institutional Review Board (IRB #1512088822) and the study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. The study was registered at clinicaltrials.gov (NCT02751320). There were minor amendments to the protocol that did not affect study flow or outcomes.
2.1
Participants
All participants provided written informed consent prior to screening. Participants aged 18–85 years were eligible for inclusion if they had good oral health and a removable bilateral maxillary partial denture with sufficient room in the posterior buccal flange areas to accommodate two enamel specimens and an unstimulated and stimulated salivary flow rate of at least 0.2 and 0.8 mL/min, respectively. Exclusion criteria included: pregnancy; breastfeeding; taken antibiotics or had received professional fluoride application in the 2 weeks prior to screening; use of fluoride supplements or mouth rinse; dental restorations in poor state of repair; active caries or moderate/severe periodontal disease; using a denture adhesive to secure the denture other than non-zinc Poligrip ® (GSK Consumer Healthcare, Weybridge, UK).
2.2
In situ remineralisation model
This study used the in situ caries remineralisation model developed by Zero et al. [ ]. Human permanent teeth were cut into 4 × 4 mm enamel specimens, which were polished to create flat surfaces as described previously. Surface-softened lesions (A-lesions) were created according to a modified method of White [ ] by immersing enamel specimens in 40 mL 0.05 mol/dm 3 lactate 50% saturated with respect to hydroxyapatite and containing 0.2% (wt/vol) Carbopol ® 907 (BF Goodrich, Cleveland, OH, USA) adjusted to pH 5.0 with KOH at 37 °C for 24 h. Subsurface lesions (B-lesions) were prepared by demineralising enamel specimens at 37 °C for 9 days in a solution containing 0.1 M lactate, 4.1 mM CaCl 2 , 8.0 mM KH 2 PO 4 and 0.2% w/v Carbopol ® 907, pH adjusted to pH 5.0 using KOH [ ]. Lesions were deemed acceptable if lesioned areas displayed uniform opacity and surface shine on exposure to overhead light. Specimens were stored in a moist environment to prevent dehydration and were sterilised by ethylene oxide gas prior to insertion into dentures.
At each treatment start visit, one A-lesion and one B-lesion enamel specimen was placed flush in each buccal flange area of the bilateral mandibular denture for a total of two of each specimen type per denture. Each enamel specimen was covered with Polyester Knit Fabric (Item P01628, Bard Peripheral Vascular, Tempe, AZ, USA) to encourage plaque formation [ ]. Specimens were mounted such that the surface was flush with the buccal flange surface and were luted in place using a light cured dental composite (Triad VLC material, Dentsply Int., York, PA, USA). Upon study completion, the denture was permanently repaired with acrylic.
2.3
Study design and treatments
At the screening visit, participants provided written informed consent and their demographic details and medical and current medication history was recorded. They underwent an oral soft tissue (OST) and oral hard tissue (OHT) examination and their stimulated and unstimulated salivary flow rate was assessed. An OST examination was also performed before and after each treatment period; an OHT examination was performed following dental prophylaxis and at the end of each treatment period. Prior to each treatment period, participants received a dental prophylaxis using a non-fluoride prophylaxis and floss. They were issued with a washout toothpaste (Negative Control 0 ppm fluoride, as below) and study toothbrush (Aquafresh ® Clean Control Everyday Clean™ toothbrush, GSK Consumer Healthcare, supplied new for each washout and treatment period).
At the first treatment visit, eligible participants were randomised to a treatment sequence according to a schedule provided by the Biostatistics Department of GSK Consumer Healthcare. Given the number of treatment arms, an incomplete block design was employed to reduce the length of participation for each subject and so that all possible pairs of treatments were observed, ideally an equal number of times. Details of study treatments are provided in Table 1 .
