Enhanced enamel benefits from a novel fluoride toothpaste

Transcription

1 International Dental Journal (2009) 59, Enhanced enamel benefits from a novel fluoride toothpaste Andrew Joiner, Fred Schäfer, Kate Hornby, Mark Long, Margaret Evans, Tim Beasley and Pam Abraham Bebington, UK Objectives: Through the understanding of tooth enamel science and insights into the beneficial role calcium can play in the caries process, a novel fluoride toothpaste has been developed containing micro-calcium. This paper describes a series of in vitro studies to assess delivery of the micro-calcium to a plaque biofilm, delivery of radiolabelled micro-calcium to subsurface enamel lesions and the rehardening of acid softened enamel by this novel toothpaste. Two clinical studies evaluated the delivery of calcium to the mouth. Methods: Uptake of micro-calcium to a plaque biofilm was assessed using a Calgary Biofilm Device and measuring the calcium levels delivered to the biofilm from the micro-calcium containing toothpaste, a calcium carbonate toothpaste, a silica toothpaste or water controls. Sound and subsurface enamel lesions were treated with 45 Ca labelled micro-calcium toothpaste in an in vitro ph cycling study and the uptake of labelled calcium determined. Acid softened enamel specimens were treated with either the micro-calcium containing toothpaste, a calcium carbonate toothpaste or a non-fluoride silica toothpaste in an in vitro remineralisation protocol and the changes in surface microhardness measured. Calcium delivery in vivo was determined in two double-blind, randomised cross-over studies. Subjects brushed their teeth for one minute with either the micro-calcium containing toothpaste or a silica toothpaste. Immediately after brushing and at fixed time intervals up to one hour, unstimulated saliva samples were taken and the total calcium concentration determined. Results: Significantly (p<0.05) more calcium was delivered to a plaque biofilm from the micro-calcium containing toothpaste than the controls. The radiolabelled micro-calcium study demonstrated the uptake of calcium to subsurface enamel lesions. In the remineralisation study, acid softened enamel became significantly harder (p<0.05) following treatment with the micro-calcium containing toothpaste than compared to the control toothpastes. It was shown in the two clinical studies that more calcium was delivered to the mouth following the use of the micro-calcium containing toothpaste than compared to the silica toothpaste. The product differences were in excess of 50% and were of statistical significance (p<0.001). Conclusions: The studies show that the new toothpaste containing micro-calcium delivered elevated levels of calcium to the mouth, promoted enhanced remineralisation of demineralised enamel lesions and thus can help repair early signs of tooth decay. Key words: Calcium, remineralisation, caries, enamel, fluoride FDI/World Dental Press /09/ (Supplement 1) It is well established that regular brushing of the teeth with a fluoride containing toothpaste can protect enamel from cariogenic attacks and can help to repair demineralised enamel, thus reducing the dental caries incidence and conveying an essential oral health benefit 1-3. In general, this is accomplished by the toothpaste, in combination with a toothbrush, cleaning away dental plaque from the tooth surfaces via primarily a mechanical action and concomitantly delivering an efficacious level of a fluoride into the oral cavity 1,4. It is widely agreed that the presence of low, but slightly elevated levels of fluoride in plaque and saliva is the predominant means by which fluoride exerts its anti-caries effect 5,6. For example, it has been shown that the low levels of fluoride typically found in saliva hours after the use of fluoride containing toothpaste can significantly doi: /idj_2412joiner10 IDJ 2412-Joiner.indd /08/ :42:00

2 245 reduce the dissolution of sound enamel and significantly increase the uptake of calcium to demineralised enamel 7,8. In addition, clinical studies have demonstrated an inverse relationship between fluoride concentration in saliva and/or dental plaque and the prevalence of caries levels 9,10. Calcium and phosphate are necessary components for the maintenance of strong and healthy teeth. These are found in saliva and form part of the natural defence against caries by influencing the de- and re-mineralisation processes that can occur in teeth. The ionic activities of calcium and phosphate in plaque fluid and its ph are also important in determining the stability of the tooth surface under a cariogenic challenge 11. Under normal circumstances, plaque fluid is supersaturated with respect to enamel minerals and its ph is close to neutral 12. However, periods of undersaturation can occur, usually following meals, where the plaque bacteria metabolise sugars and carbohydrates to produce organic acids which in turn lower the ph at or near the tooth surface 13. Under these conditions an instability in the enamel mineral phases can occur and this results in a net flow of calcium and phosphate ions from the tooth to the surrounding fluid. The dissolution will continue until the ph and calcium and phosphate activities reach levels where the enamel minerals are stable again. The extent of dissolution depends on a number of factors including ph, the types and amounts of acids produced, and the ionic activities of calcium and phosphate When conditions return to supersaturation, it is possible for new minerals to be precipitated, replenishing the minerals that were lost during periods of undersaturation 17. The levels of calcium and phosphate in saliva and plaque have been shown to be inversely associated with caries incidence and low levels of plaque calcium as a predictor of future caries Similar findings have been reported from epidemiological studies which provide strong evidence that caries incidence is inversely related to calcium concentration in saliva 21. One reason put forward for this correlation is that increased calcium concentration in saliva will reduce solubility of enamel on the one hand and also increase the driving force for remineralisation of the early stages of enamel demineralisation. Margolis and Moreno 22 found that the degree of saturation with respect to enamel (DS En ) was lower in the plaque samples of caries-positive subjects as compared to plaque samples from caries-free subjects following a cariogenic challenge. Similar observations were made in plaque fluid samples of caries prone subjects 23,24. The magnitude of DS En is dependent on the available calcium and phosphate ions and theoretically small changes in calcium levels will have a greater effect than similar changes in phosphate levels 25. This has been confirmed via in vitro mechanistic studies with calcium being approximately twenty times as potent as phosphate in inhibiting enamel dissolution through increased DS En 26,27. Thus extended elevation of plaque calcium levels would be an effective means of reducing undersaturation during a cariogenic challenge 25. The relative importance of calcium and phosphate and the rate at which these can be supplied by saliva may also be an important factor in enamel remineralisation 28. Laboratory experiments have indicated that, at equal degrees of supersaturation, an optimal rate of enamel remineralisation can be obtained with a calcium/phosphate ratio of However in plaque fluid and saliva, there is an excess of phosphate present, and the Ca/P ratio is approximately Therefore, it has been suggested that the calcium ion concentration may be the main rate-limiting mineral constituent 28. Calcium delivery to plaque and saliva A number of in vitro and clinical studies have investigated the delivery of additional calcium to plaque from oral care products and its potential benefits to oral health. For example, Pearce 31 has demonstrated the ability to raise calcium concentrations in plaque using a mouthrinse containing urea and calcium chloride. Further, Pearce and Nelson 32 have shown that such increased concentrations were associated with a reduction in softening and porosity of enamel surfaces covered with plaque in situ. Pearce et al. 33 investigated a calcium and phosphate containing mouthrinse designed to precipitate HAP in plaque and showed that plaque fluid levels of calcium were elevated in subjects using the calcium/phosphate mouthrinse compared to a placebo mouthrinse following exposure to a sucrose solution. Duke et al. 34 investigated the addition of calcium glycerophosphate (CaGP) to a sodium monofluorophosphate (SMFP) containing calcium carbonate/silica toothpaste. Plaque collected from subjects using this toothpaste was shown to contain more calcium than plaque collected from subjects using the control toothpaste containing no CaGP 34. Similar results were found in approximal plaque collected after 1h following the use of CaGP containing toothpaste versus control toothpaste, and that the elevated levels were maintained for at least 8h after use 35. In an in vitro biofilm flow cell model grown on bovine enamel, CaGP was pulsed in 1h before, during or 1h after a sucrose challenge and it was shown that there was a significant dose response of decreasing demineralisation as CaGP concentration increased, with pulsing before the sucrose challenge giving the greatest protection from demineralisation 36. In a literature review on the role of CaGP and caries, the reduction of caries by CaGP containing toothpastes in a number of clinical trials is described and it was concluded that the elevation of calcium levels in plaque is the most likely explanation for the clinical effect 25. A dicalcium phosphate dihydrate (DCPD) containing toothpaste indicated an increase in the calcium activity and DS En in plaque fluid 12h after use compared to a silica control 37,38. Using 45 Ca radiolabelled DCPD, it Joiner et al.: Enhanced enamel benefits from a novel fluoride toothpaste IDJ 2412-Joiner.indd /08/ :42:01

3 246 was shown that the calcium from the DCPD was incorporated into enamel with a concomitant reduction in enamel solubility in a rat model 37. In addition, an intra-oral brushing model with a fluoride/ 44 Ca labelled DCPD toothpaste showed that 44 Ca from DCPD could be detected in demineralised enamel after six days of treatment and in whole plaque 18h after the last treatment 38. The incorporation of casein-phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP- ACP) in a mouthwash have been demonstrated to increase the levels of calcium and phosphate ions in supragingival plaque, and to promote the remineralisation of enamel subsurface lesions in situ 39,40. It has been shown that the CPP-ACP binds to model plaque in vitro with twice the affinity of free calcium ions, providing a large calcium reservoir 41. Chewing gums containing either monocalcium phosphate monohydrate or a mixture of dicalcium phosphate anhydrous and tetracalcium phosphate produced a very pronounced and persistent (16 minutes) elevation in the saturation of saliva with respect to tooth mineral 42. Following a sucrose rinse, the use of a chewing gum containing α-tricalcium phosphate significantly increased the calcium concentrations in saliva, plaque and plaque fluid, and increased the ph, compared to the use of a control chewing gum 43. Laboratory experiments have shown that tooth mineral itself has the ability to buffer the ph and hence restrict the impact of cariogenic challenges. For example, Zaura et al. 44 have shown that the ph at the bottom of in situ plaque formed in grooves cut of different materials and treated with glucose, was dependent on the solubility of the material, with dentine having a higher ph than enamel, which in turn was higher than polyacrylate. In another in vitro study where a dentine block was placed next to an enamel block, the dentine block acted as a sacrificial source of calcium and phosphate and inhibited the enamel dissolution in solutions initially undersaturated with respect to both minerals 45. Relationship between plaque calcium and fluoride There is considerable evidence in the literature that there is a linear relationship between levels of calcium and fluoride in plaque It has been hypothesised that the ability of plaque to retain fluoride for longer periods is determined mainly by the calcium concentration in plaque 48. Further, it has been suggested that efforts to increase the cariostatic potential of fluoride could be based on methods to increase the concentration of calcium in plaque 48. To this end, Vogel et al. 52 demonstrated that a calcium lactate pre-rinse before the use of a sodium fluoride mouthrinse significantly increased the one hour salivary fluoride concentration versus the sodium fluoride mouthrinse only. However, in a second experiment where a water rinse was used between the two rinses to reduce calcium carryover, salivary fluoride concentration was reduced, suggesting that the water rinse removed free calcium from oral tissues. The combination of the calcium lactate pre-rinse followed by a fluoride rinse also gave a significantly elevated overnight salivary fluoride concentration versus the fluoride rinse alone 53. The combination of a 150mM calcium lactate pre-rinse and a fluoride rinse was also shown to increase plaque and plaque fluid fluoride concentrations after one hour compared to the fluoride rinse alone 54. Elevated salivary fluoride levels after one hour were also obtained when using a 150mM calcium lactate pre-rinse, or a CaGP containing toothpaste followed by a sodium fluoride rinse, and when using the calcium lactate pre-rinse followed by a sodium fluoride toothpaste, suggesting that an increase in the concentration of calcium given shortly before a fluoride rinse or toothpaste may increase the cariostatic effect of the fluoride product 55. However, in contrast, when a 20mM calcium chloride pre-rinse was used in conjunction with a fluoride toothpaste, this combination had only a minor effect on salivary calcium and fluoride concentrations and none on plaque concentrations after 1 and 12 hours 51. In a further study with 150mM calcium lactate pre-rinse followed by a fluoride toothpaste, Pessan et al. 50 confirmed significant elevated fluoride salivary concentrations and no significant elevation of plaque fluoride concentrations after 1h. The elevation of calcium levels in plaque has the potential benefit of enhancing the remineralisation of enamel and aiding the potential of fluoride in this remineralisation process. For example, the remineralisation of artificially demineralised bovine enamel specimens was shown to be significantly improved by the use of a 150mM calcium lactate pre-rinse followed by the use of either a fluoride or non-fluoride toothpaste over 14 days in an in situ model 56. Blake-Haskins et al. 57 demonstrated in vitro an increased remineralisation of subsurface enamel lesions with a combination of a calcium prerinse followed by a fluoride treatment versus fluoride treatment alone. The impact of calcium levels on enamel remineralisation is further confirmed by in vitro experiments using conditions found during a typical acid challenge in terms of ph, fluoride, calcium and phosphate concentrations, where it was found that significantly enhanced remineralisation of sub-surface enamel lesions was achieved by increasing the calcium concentration whilst maintaining fluoride concentration 58. Plaque-acid neutralisation and buffering by calcium carbonate It is known, from the classic studies of Stephan 59 that following rinsing with a glucose solution, the ph of plaque can drop sharply and be maintained at a low ph for some considerable time due to the generation International Dental Journal (2009) Vol. 59/No.4 (Supplement 1) IDJ 2412-Joiner.indd /08/ :42:01

4 247 of plaque acids. Further studies have demonstrated that this plaque ph drop is related to the susceptibility of an individual to be caries-prone or caries-free, with larger drops being exhibited by caries-prone subjects 60. Thus, routes that can limit the drop in plaque ph have the potential to reduce the impact of plaque acids on enamel demineralisation. Calcium carbonate is an alkaline, buffering agent commonly used in toothpaste as an abrasive agent for cleaning and stain removal 4,61,62. Duke 63 has described the deposition of calcium carbonate abrasive particles, with a mean diameter in the range 6-8 microns, into dental plaque for several hours after pre-treatment with a calcium carbonate-based toothpaste. The solubility of calcium carbonate particles increase in the presence of acid and have the potential to be partially dissolved when dental plaque ph drops after sugar consumption, releasing calcium ions. Thus, calcium carbonate has the potential not only of reducing hydrogen ion concentration through neutralisation but also simultaneously increasing calcium ion concentration in plaque fluid. The overall effect is to reduce the cariogenicity of plaque when most needed under sugar challenges and the associated plaque ph drop. Indeed, calcium carbonate-based toothpastes have been shown to reduce the ph drop of plaque using a number of in vitro and in vivo protocols The impact of calcium carbonate on enamel re- and demineralisation processes has been investigated using in situ protocols, where a calcium carbonate/smfp toothpaste was shown to be significantly more effective at reducing enamel demineralisation and enhancing enamel remineralisation than a silica/smfp toothpaste 66,67. In addition, it was recorded that more fluoride was found in the test plaque treated with the calcium carbonate toothpaste. The authors conclude that these results suggest that calcium carbonate abrasive may enhance the effect of fluoride present in toothpaste on dental caries control 66. Tooth erosion and calcium Erosion is defined as the loss of hard tissue by chemical means not derived from bacteria, that is, the dissolution of hard tissue by acid where the acid source is not derived from oral bacteria 68. Erosion may be caused by either intrinsic sources, such as stomach acid reflux, or extrinsic sources, which are often associated with the consumption of acidic foods and beverages such as orange juice and cola. The calcium and phosphate contents of an acidic foodstuff or beverage are important factors for determining their erosive potential as they will influence the concentration gradient within the local environment of the tooth surface and hence DS En 69. Indeed, the addition of calcium to acidic beverages such as fruit juice, soft drinks, carbonated beverages and sports drinks, and to acidic candies has been shown to significantly reduce their erosive potential Calcium carbonate toothpaste containing micro-calcium Continuing efforts are being made by manufacturers to improve fluoride toothpaste by, for example, improving fluoride delivery/retention or by adding other beneficial agents. From the discussion above, the advantages of delivering a calcium source to the mouth which can give elevated calcium levels in the mouth has the potential to limit acid challenges by reducing enamel demineralisation whilst promoting enamel remineralisation. To this end, a combination of calcium sources (calcium glycerophosphate and micro-calcium carbonate, termed micro-calcium) was added to a calcium carbonate/smfp toothpaste, in order to enhance the overall delivery of calcium to plaque and saliva with a concomitant improvement in tooth enamel benefits. This paper describes a series of in vitro and in vivo studies investigating the properties of this new toothpaste formulation including delivery of calcium to a biofilm, delivery of calcium to demineralised enamel and its impact on enamel hardness and the extent to which it can increase the concentration of calcium in saliva. Delivery of calcium to a biofilm in vitro Materials and methods In order to evaluate the delivery of calcium from the new toothpaste containing micro-calcium to a plaque biofilm, the in vitro Calgary Biofilm Device (CBD) was used 81. In outline, the CBD allows a bacterial biofilm to be established on pegs attached to the lid of a 96-well plate. Biofilms can be treated and evaluated by incubation in 96-well plates containing various solutions. The Guggenheim consortium was used to represent the plaque biofilm and consisted of the following organisms: Streptococcus oralis, Streptococcus sobrinus, Actinomyces naeslundii, Fusobaterium nucleatum, Veilonalla dispar and Candida albicans 82. The Guggenheim consortium was cultured as described by Shapiro et al. 83 and then allowed to form a biofilm on whole human saliva pellicle coated pegs. The biofilms were treated for 1min with either the toothpaste containing micro-calcium, a commercially available calcium carbonate/smfp toothpaste, a commercial silica-based toothpaste or water, with stirring at room temperature (n=16). The test toothpastes were prepared as slurries (1:3) in deionised sterile water. After product treatment, the pegs were washed three times with sterile water. The QuantiChrom calcium kit (BioAssay Systems, CA, USA) was used to measure the amount of calcium delivered to the biofilm 84. For measurement of total calcium delivered to the plaque biofilm, the CBD (n=8) were treated with 1M hydrochloric acid for 60mins. An aliquot of the solution was removed (5µl) and to this was added the QuantiChrom reagent (200µl), followed Joiner et al.: Enhanced enamel benefits from a novel fluoride toothpaste IDJ 2412-Joiner.indd /08/ :42:01

5 248 Table 1 Mean values of total and soluble calcium concentrations in an in vitro biofilm following various treatments Treatment Total [Ca] (s.d.) (mg/ml) Soluble [Ca] (s.d.) (mg/ml) Micro-calcium toothpaste (0.390) a (0.019) a Calcium carbonate toothpaste (0.151) b (0.003) b Silica toothpaste (0.186) b (0.002) b Water (0.119) b (0.001) b Superscripts in columns with different letters are statistically different (p<0.05, Tukey Kramer) Table 2 ph cycling protocol Time Treatment Duration Early a.m. Mid-late p.m. Toothpaste slurry Acid buffer Neutral buffer Toothpaste slurry Acid buffer Neutral buffer 5 min 15 min 6-7 hours 5 min 15 min Overnight Table 3 45 Ca uptake to sound and subsurface lesion enamel specimens from a radiolabelled micro-calcium carbonate containing toothpaste (n=4) Specimen Type Mean Calcium Uptake (s.d.) (mg/mm 2 ) Sound enamel 3.15 (1.50) Subsurface lesion 6.65 (1.07) by 1M sodium hydroxide (5µl) and the absorbance read at 612nm. In order to measure the amount of soluble calcium present, the CBD (n=8) was incubated directly in the QuantiChrom reagent at room temperature. A standard curve was generated using known concentrations of calcium and this was used to calculate the soluble and total calcium concentrations. Results The total and soluble amounts of calcium measured in the in vitro biofilm following treatment with various toothpastes are shown in Table 1. Statistical analysis (ANOVA, Tukey-Kramer) showed that the biofilm treated with the toothpaste containing micro-calcium had a significantly (p<0.05) higher level of both soluble and total calcium when compared to the other three treatments. All other treatment comparisons were non significant for both soluble and total calcium. Calcium uptake by enamel from 45 Ca radiolabelled micro-calcium carbonate in vitro Materials and methods Paired bovine enamel blocks were cut using a diamond saw to give specimens of approximately 5x3mm. One set of the specimens was mounted in dental wax and the edges of the specimens painted using acid resistant nail varnish. Sub-surface lesions were created using an acidified 8% methyl cellulose gel (0.1M lactic acid, ph 4.6) with incubation at 37 C for 10 days. The specimens were analysed by Transverse Microradiography to ensure a balance in lesion depth and mineral loss. The other paired set of specimens was used as sound enamel. Four specimens were mounted on each perspex rod using dental wax and the edges were painted with the nail varnish. In total, there were eight sound and eight sub surface lesions used in the experiment. The surface area of the specimens exposed to the toothpaste treatments was measured by optical methods. A sample of the micro-calcium carbonate was 45 Ca labelled using a neutron irradiation source. This isotope is a beta emitter with a half-life of 156 days. The radiolabelled micro-calcium carbonate was added to a 3:1 water:toothpaste slurry at an incorporation level of 2% w/w in the original toothpaste. An acidic buffer (50 mm acetic acid, 1.50 mm calcium chloride dihydrate, 0.90 mm potassium dihydrogen orthophosphate, 130 mm potassium chloride, ph 5.0) and neutral buffer (20 mm HEPES, 1.50 mm calcium chloride dihydrate, 0.90 mm potassium dihydrogen orthophosphate, 130 mm potassium chloride, ph 7.0) were also prepared. The bovine enamel specimens were cycled daily for 4 days through toothpaste slurry, acid buffer and neutral buffer with deionised water washings (15ml) between treatments according to Table 2. The specimens were then individually mounted onto a glass slide using dental wax. To each specimen was added 1M perchloric acid (50µl) for 3mins. This solution was removed with a pipette and placed into a labelled eppendorf tube. Each specimen was washed three times with 1M sodium acetate (50µl) and the washings combined with the acid treatment in the eppendorf tube. The perchloric acid and sodium acetate washings were repeated a further four times. The International Dental Journal (2009) Vol. 59/No.4 (Supplement 1) IDJ 2412-Joiner.indd /08/ :42:01

6 249 Table 4 Remineralisation of enamel following various toothpaste treatments Treatment Mean change in SMH s.d. Statistical analysis a Micro-calcium toothpaste A CaCO 3 /SMFP B Non-F/Silica C a Statistical analysis of variance means with different letters are significantly different (p<0.05, Tukey-Kramer) combined perchloric acid and sodium acetate washings were analysed for 45 Ca and the mass of calcium uptake determined. This value was divided by the surface area of the specimen to give calcium uptake expressed as mg/mm 2 and the mean calcium uptake values for sound and subsurface lesions calculated. Results The mean calcium uptake values for sound and subsurface lesions are shown in Table 3. Statistical analysis (t-test, two sample two tailed) shows there to be a statistical difference (p<0.05) between the two types of specimens. Enamel remineralisation in vitro Materials and methods Bovine enamel blocks (3x3mm) were treated with 1% citric acid, ph 3.8 for 20mins at room temperature. The surface microhardness (SMH) of the demineralised enamel blocks was then measured using a Mitutoyo HM122 Microhardness Tester (Mitutoyo Corp., Japan) and Knoop diamond under a load of 50g for 10s and stratified into three treatment groups (n=8). The enamel blocks were incubated with whole human saliva for 2h and rinsed with deionised water. They were then treated with either the micro-calcium containing toothpaste, a calcium carbonate/smfp toothpaste or a non-fluoride silica-based toothpaste for 3mins. Toothpaste treatments were prepared as 1:2 (toothpaste:water) slurry in deionised water. The enamel blocks were then rinsed with deionised water for 30s and placed in a neutral buffer (ph 7.0) containing 20mM HEPES, 1mM CaCl 2, 12.7mM KH 2 PO 4 and 130mM KCl, for 24 hours. After water rinsing, the SMH of the enamel blocks was remeasured and the change in SMH calculated. Results The change in SMH following various toothpaste treatments and a remineralising solution are shown in Table 4. Statistical analyses (ANOVA) showed an overall difference between treatments and paired comparisons (Tukey-Kramer) showed there were significant differences between all treatments (p<0.05). Evaluation of calcium delivery in vivo Material and Methods Two double-blind, randomised, cross-over studies of identical design and study conditions were carried out. The objective of both studies was to compare total calcium concentration in saliva before and after brushing with toothpaste containing calcium carbonate abrasive plus micro-calcium (test) or brushing with toothpaste containing silica abrasive (control). The study population (of each study) consisted of male and female adults who were of general good health. They had to have a minimum of 20 natural teeth, free of untreated caries or periodontal disease. Pregnant women and nursing mothers were excluded as were subjects with allergies to normal toothpaste ingredients. At the start of each study subjects were required to give written informed consent, complete a medical history form, and undergo a dental screening to establish whether they were suitable. Subjects were given a calcium-free toothpaste to use throughout each study. In each study, subjects brushed their teeth at the study location for 1min with the test toothpaste (micro-calcium containing toothpaste) or the control toothpaste (silica abrasive). Both toothpastes contained fluoride either as 1.1% SMFP (test) or 0.32%NaF (control). Immediately before brushing and at fixed time intervals after brushing (5, 15, 30, 60min) unstimulated whole mouth saliva samples were taken. The total calcium concentration in the saliva samples was determined by an Inductively Coupled Plasma (ICP) Atomic Emission Spectrometer (Varian Vista ICP, Varian Instruments, Walnut Creek CA, USA). The protocols of both studies were reviewed and approved by an independent Research Ethics Committee. The outcome variable was the total calcium concentration in saliva at each sampling time. In addition an integrated calcium delivery (ICD) value was determined. This was defined as the sum of the areas of the trapezoids formed by linear interpolation of neighbouring sampling times from 5-60min. The level of significance for all statistical analyses was set at p < Joiner et al.: Enhanced enamel benefits from a novel fluoride toothpaste IDJ 2412-Joiner.indd /08/ :42:01

7 250 Table 5 Mean (s.d.) total calcium concentration (ppm) in saliva before and 15 minutes after brushing: Study 1 Results Pre 15 min n Mean (s.d.) Mean (s.d.) Control (24.6) 56.5 (13.6) Test (25.5) (125.1) Table 6 Mean (s.d.) total calcium concentration (ppm) in saliva before and 15 minutes after brushing: Study 2 Pre 15 min n Mean (s.d.) Mean (s.d.) Control (26.4) 53.3 (11.6) Test (26.9) (141.3) Study 1 A total of 37 subjects were enrolled and 36 subjects completed the study. The mean total calcium concentration (ppm) in saliva samples taken before (pre) and 15min after brushing are shown in Table 5. The difference between the two groups in pre-brushing calcium concentrations was not statistically significant. In contrast, the calcium concentrations in saliva after brushing were significantly higher in the test group than in then control group (p<0.001). The integrated calcium delivery (ICD) value between 5-60min was 10,158ppm*min for the test paste and 3,439 ppm*min for the control paste. The difference between the two groups was statistically significant (p<0.001). The ICD value in the test group was 5,583ppm*min higher than the corresponding ICD value based on the normal, pre-brushing calcium concentration in saliva. This increase was statistically significant (p<0.001). The ICD value in the control group was 1,236ppm*min lower than the corresponding ICD value based on the normal, pre-brushing calcium concentration in saliva (p<0.001). Study 2 A total of 38 subjects were enrolled and 37 subjects completed the study. The mean total calcium concentration (ppm) in saliva samples taken before (pre) and at 15min after brushing are shown in Table 6. The difference between the two groups in pre-brushing calcium concentrations was not statistically significant. In contrast, the calcium concentrations in saliva after brushing was significantly higher in the test group than in then control group (p<0.001). The integrated calcium delivery (ICD) value between 5-60min was 10,562ppm*min for the test paste and 3,224ppm*min for the control paste. The difference between the two groups was statistically significant (p<0.001). The ICD value in the test group was 6,091ppm*min higher than the corresponding ICD value based on the normal, pre-brushing calcium concentration in saliva. This increase was statistically significant (p<0.001). In contrast, the ICD value in the control group was 1,071ppm*min lower than the corresponding ICD value based on the normal, pre-brushing calcium concentration in saliva (p<0.001). Discussion A number of in vitro biofilm models have been described in the literature for the evaluation of the effects of oral care products on various parameters such as biofilm removal and biocidal activity 4,85,86. The biofilm used in the current study is based on a consortium of orally relevant microbes and designed as a simple in vitro system for modelling supragingival plaque 82,83. The model has been further developed to test toothpaste formulations where it has been shown not to be significantly affected by major formulation components such as sodium lauryl sulphate 87. Further, the calcium delivery experiment in the current paper is a non-brushing protocol and so would tend to mimic delivery to a relatively undisturbed biofilm present in occlusal fissures, interproximal regions or other difficult to clean locations i.e. areas where caries incidence may be greater. Under the conditions described in the current work, the in vitro biofilm had relatively low levels of total (acid extractable) and soluble calcium. Upon treatment with the silica or calcium carbonate toothpaste, there was a numerical increase in both total and soluble calcium but the difference from the water treatment was not of statistical significance. However, following treatment with the micro-calcium containing toothpaste, the total calcium increased by over eleven-fold and the free calcium by over six-fold, versus the calcium carbonate control toothpaste, demonstrating the delivery of additional calcium to the biofilm by the micro-calcium containing toothpaste. The significant increase in calcium concentration in the soluble form suggests that the micro-calcium containing toothpaste can increase the activity of calcium ions in plaque fluid, which might increase the driving force for remineralisation. Further, with the added delivery and retention of the calcium sources within plaque, this may give further protection by reducing plaque ph drop and/or acting as a calcium source to reduce undersaturation during a cariogenic challenge and serving as a new source of mineral ions between cariogenic challenges to promote remineralisation. Nakashima et al. 88 investigated a nano-sized calcium carbonate described as several tens to hundreds of nanometres in size. This was shown to exhibit better retention properties on oral surfaces than calcium carbonate abrasive particles of approximately 10 microns International Dental Journal (2009) Vol. 59/No.4 (Supplement 1) IDJ 2412-Joiner.indd /08/ :42:01

8 251 in diameter. Thus, the additional calcium delivery to the plaque from the micro-calcium containing toothpaste versus the calcium carbonate-based toothpaste in the current study is most likely due to the relative smaller particle size of the micro-calcium carbonate versus the regular calcium carbonate particles. In the 45 Ca labelled micro-calcium carbonate experiment ph cycling conditions were used as this approach has been described as one means of bringing in vitro models much closer to reality 89. Under these conditions, 45 Ca was detected in both sound and subsurface lesion enamel specimens. This indicates that calcium ions from the micro-calcium carbonate has become available to be involved in the remineralisation process and is subsequently incorporated into the enamel specimens. In the remineralisation study, the initial acid softening of the enamel with 1% citric acid (ph 3.8) is considered typical of the acid challenge from orange juice 90 and has been used in a number of in vitro studies to evaluate erosion processes 90,91. The remineralisation solution in the current study was chosen to mimic concentrations found in plaque fluid, where the calcium:phosphate ratio is typically less than 0.5 i.e. relatively phosphate rich 30,92. Since caries is initiated and progressed beneath a layer of plaque, it therefore may be more important to study de- and remineralisation processes of enamel using experimental solutions representative of plaque fluid. Under such conditions, the micro-calcium containing toothpaste showed significantly more remineralisation than the calcium carbonate toothpaste or the non-fluoride toothpaste. The two in vivo calcium delivery studies provided very consistent measures of total calcium concentration in saliva, both before and after brushing. Before brushing, the mean total saliva calcium concentrations were very similar in the test and control group in both studies ranging from 78-85ppm (equivalent to about 2mmol/l calcium ion). These concentrations of calcium in saliva measured before brushing agree reasonable well with data reported in the literature. In this study baseline values were about 2mmol/l while data published by Sewon and co-workers 93,94 showed mmol/l and Poureslami et al. found 1.4mmol/l. Normal ranges of 1-2 mmol/l are reported by Dawes in Edgar and O Mullane 95. After brushing with the test toothpaste, an increase in saliva calcium concentration was found in both studies which indicated delivery and deposition of calcium in the mouth. Again, very similar values were observed in the two studies, with at least a 2-fold increase in saliva calcium concentration measured 15min after brushing. In contrast, no increase in saliva calcium was apparent after brushing with the control toothpaste. In fact, 15min after brushing the calcium concentration in saliva was significantly lower than before brushing. In addition to single time-point measurements of calcium concentration in saliva, an integrated measure of calcium delivery (ICD) was calculated, analogous to the methodology applied in pharmacokinetic studies and fluoride oral delivery studies 96,97. The results showed that the ICD value, calculated over 1h, was nearly three times higher after brushing with the test toothpaste than after brushing with the control toothpaste and twice as high compared to baseline. Conclusions Through the understanding of tooth enamel science and insights into the beneficial role calcium can play in the caries process a new fluoride toothpaste has been developed containing micro-calcium. It was shown in two clinical studies that more calcium was delivered to the mouth following the use of the micro-calcium containing toothpaste than compared to a silica toothpaste. The product differences were in excess of 50% and were of statistical significance (p<0.001). An in vitro study has shown significantly (p<0.05) greater delivery of calcium to a plaque biofilm from the micro-calcium containing toothpaste than control toothpastes. A radiolabelled micro-calcium carbonate study has demonstrated the uptake of calcium to subsurface enamel lesions. In a remineralisation study, acid softened enamel became significantly harder (p<0.05) following treatment with the micro-calcium containing toothpaste than compared to a calcium carbonate toothpaste and non-fluoride silica toothpaste. Therefore, the new toothpaste containing microcalcium is able to deliver elevated levels of calcium to the mouth, promotes enhanced remineralisation of demineralised enamel lesions and thus can help repair early signs of tooth decay. References 1. Stamm JW. Multi-function toothpastes for better oral health: a behavioural perspective. Int Dent J : Stephens KW. Fluoride toothpastes, rinses, and tablets. Adv Dent Res : Twetman S, Axelsson S, Dahlgren H et al. Caries-preventative effect of fluoride toothpaste: a systematic review. Acta Odontol Scand : Joiner A. The cleaning of teeth. In: Handbook for Cleaning/Decontamination of Surfaces. Eds. Johansson I, Somasundaran P. Elsevier, Amsterdam. 1 st Ed. 2007, p ten Cate JM. Current concepts on the theories of the mechanism of action of fluoride. Acta Odontol Scand : Featherstone JD. Prevention and reversal of dental caries: role of low level fluoride. Community Dent Oral Epidemiol : Page DJ. A study of the effect of fluoride delivered from solution and dentifrices on enamel demineralization. Caries Res : Gibbs CD, Atherton SE, Huntington E et al. Effect of low levels of fluoride on calcium-uptake by demineralised human enamel. Arch Oral Biol : Gaugler RW, Bruton WF. Fluoride concentration in dental plaque of naval recruits with and without caries. Arch Oral Biol : Joiner et al.: Enhanced enamel benefits from a novel fluoride toothpaste IDJ 2412-Joiner.indd /08/ :42:01

9 Nobre dos Santos M, Melo dos Santos L, Francisco SB et al. Relationship among dental plaque composition, daily sugar exposure and caries in the primary dentition. Caries Res : Margolis HC, Murphy BJ, Moreno EC. Development of carious-like lesions in partially saturated lactate buffers. Caries Res : Moreno EC, Margolis HC. Composition of human plaque fluid. J Dent Res : Dodds MWJ, Edgar WM. The relationship between plaque ph, plaque acid anion profiles, and oral carbohydrate retention after ingestion of several reference foods by human subjects. J Dent Res : Theuns HM, Van Dijk JWE, Driessens FCM et al. Effect of time and degree of saturation of buffer solutions on artificial carious lesion formation in human tooth enamel. Caries Res : Theuns HM, Van Dijk JWE, Driessens FCM et al. Effect of ph of buffer solution on artificial carious lesion formation in human tooth enamel. Caries Res : Margolis HC, Moreno EC. Kinetic and thermodynamic aspects of enamel demineralization. Caries Res : Chow LC, Vogel GL. Enhancing remineralisation. Oper Dent Supp Duckworth RM, Huntington E. On the relationship between calculus and caries. In: The Teeth and Their Environment Duckworth RM (ed), Monogr Oral Sci Basel, Karger, : Shaw L, Murray JJ, Burchell CK et al. Calcium and phosphorus content of plaque and saliva in relation to dental caries. Caries Res : Pearce EI, Dong YM, Yue L et al. Plaque minerals in the prediction of caries activity. Community Dent Oral Epidemiol : Sewon L, Mäkelä M. A study of the possible correlation of high salivary calcium levels with periodontal and dental conditions in young adults. Arch Oral Biol : S211-S Margolis HC, Moreno EC. Composition of pooled plaque fluid from caries-free and caries-positive individuals following sucrose exposure. J Dent Res : Margolis HC, Zhang YP, Gewirtz A et al. Cariogenic potential of pooled plaque fluid from exposed root surfaces in humans. Arch Oral Biol : Gao XJ, Fan Y, Kent RL et al. Association of caries activity with the composition of dental plaque fluid. J Dent Res : Lynch RJM. Calcium glycerophosphate and caries: a literature review. Int Dent J : Tanaka M, Kadoma Y. Comparative reduction of enamel demineralization by calcium and phosphate in vitro. Caries Res : Lynch RJM, ten Cate JM. The anti-caries efficacy of calcium carbonate-based fluoride toothpastes. Int Dent J : Ten Cate JM. In situ models, physico-chemical aspects. Adv Dent Res 1994, 8: Exterkate RAM, Damen JJ, ten Cate JM. A single-section model for enamel de- and remineralization studies. 1. The effects of different Ca/P ratios in remineralisation solutions. J Dent Res : Margolis HC. An assessment of recent advances in the study of the chemistry and biochemistry of dental plaque. J Dent Res : Pearce EIF. Effect of plaque mineralization on experimental dental caries. Caries Res : Pearce EIF, Nelson DGA. In vivo comparison of caries inhibition by a plaque mineral enriching mouthrinse and fluoride dentifrice. Caries Res : Pearce EIF, Margolis HC, Kent Jr RL. Effect of in situ plaque mineral supplementation on the state of saturation of plaque fluid during sugar induced acidogenesis. Eur J Oral Sci : Duke SA, Rees DA, Forward GC. Increased plaque calcium and phosphorus concentrations using a calcium carbonate toothpaste containing calcium glycerophosphate and sodium monofluorophosphate. Caries Res : International Dental Journal (2009) Vol. 59/No.4 (Supplement 1) 35. Sidi AD, Wilson RF. Fluoride, calcium and inorganic phosphorus concentrations in approximal plaque collected from young adults 1 and 24 h after toothbrushing with fluoride toothpastes. Caries Res : Lynch RJM, ten Cate JM. Effect of calcium glycerophosphate on demineralization in an in vitro biofilm model. Caries Res : Gaffar A, Blake-Haskins J, Mellberg J. In vivo studies with dicalcium phosphate dihydrate/mfp system for caries prevention. Int Dent J : Sullivan RJ, Charig A, Blake-Haskins J et al. In vivo detection of calcium from dicalcium phosphate dihydrate dentifrices in demineralised human enamel and plaque. Adv Dent Res : Reynolds EC, Cai F, Shen P et al. Retention in plaque and remineralisation of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res : Reynolds EC, Cai F, Cochrane NJ et al. Fluoride and casein phosphopeptide-amorphous calcium phosphate. J Dent Res : Rose RK. Binding characteristics of Streptococcus mutans for calcium and casein phosphopeptide. Caries Res : Chow LC, Takagi S, Shern RJ et al. Effects on whole saliva of chewing gums containing calcium phosphates. J Dent Res : Vogel GL, Zhang Z, Carey CM et al. Composition of plaque and saliva following a sucrose challenge and use of an α-tricalciumphosphate-containing chewing gum. J Dent Res : Zaura E, Buijs MJ, ten Cate JM. The effects of solubility of artificial fissures on plaque ph. J Dent Res : Lynch RJM, ten Cate JM. The effect of adjacent dentine blocks on the demineralisation and remineralisation of enamel in vitro. Caries Res : Kato K, Nakagaki H, Takami Y et al. A method for determining the distribution of fluoride, calcium and phosphorus in human dental plaque and the effect of a single in vivo fluoride rinse. Arch Oral Biol : Schamschula RG, Bunzel M, Agus HM et al. Plaque minerals and caries experience: Associations and inter-relationships. J Dent Res : Whitford GM, Wasdin JL, Schafer TE et al. Plaque fluoride concentrations are dependent on plaque calcium concentrations. Caries Res : Pessan JP, Silva SMB, Lauris JRP et al. Fluoride uptake by plaque from water and from dentifrice. J Dent Res : Pessan JP, Sicca CM, de Souza TS et al. Fluoride concentrations in dental plaque and saliva after the use of a fluoride dentifrice preceded by a calcium lactate rinse. Eur J Oral Sci : Whitford GM, Buzalaf MAR, Bijella MFB et al. Plaque fluoride concentrations in a community without water fluoridation: effects of calcium and use of a fluoride or placebo dentifrice. Caries Res : Vogel GL, Chow LC, Carey CM et al. Effect of a calcium prerinse on salivary fluoride after a 228 ppm fluoride rinse. Caries Res : Vogel GL, Chow LC, Carey CM. Calcium pre-rinse greatly increases overnight salivary fluoride after 228 ppm fluoride rinse. Caries Res : Vogel GL, Schumacher GE, Chow LC et al. Ca pre-rinse greatly increases plaque and plaque fluid F. J Dent Res : Vogel GL, Shim D, Schumacher GE et al. Salivary fluoride from fluoride dentifrices or rinses after use of a calcium pre-rinse or calcium dentifrice. Caries Res : Magalhaes AC, de Almeida Furlani T, de Moraes Italiani F et al. Effect of calcium pre-rinse and fluoride dentifrice on remineralisation of artificially demineralised enamel and on the composition of the dental biofilm formed in situ. Arch Oral Biol : IDJ 2412-Joiner.indd /08/ :42:01

10 Blake-Haskins JC, Mellberg JR, Snyder C. Effect of calcium in model plaque on the anticaries activity of fluoride in vitro. J Dent Res : Lynch RJM, Mony U, ten Cate JM. De- and remineralisation of enamel lesions at low ph under simulated plaque-fluid conditions. Caries Res : Stephan RM. Changes in hydrogen-ion concentrations on tooth surfaces and carious lesions. J Amer Dent Assoc : Stephan RM. Intra-oral hydrogen ion concentration associated with dental caries activity. J Dent Res : Matheson JR, Cox TF, Baylor N et al. Effect of toothpaste with natural calcium carbonate/perlite on extrinsic tooth stain. Int Dent J : Duke SA. Effect of a chalk-based toothpaste on ph changes in dental plaque in vivo. Caries Res : Duke SA. Effect induced by a chalk-based toothpaste on the ph changes of plaque challenged by a high sugar diet over an 8-hour period. Caries Res : Brading MG, Cromwell VJ, Green AK et al. The role of triclosan in dentifrice formulations, with particular reference to a new 0.3% triclosan calcium carbonate-based system. Int Dent J : Tahmassebi J, Duggal MS, Curzon ME. Effect of a calcium carbonate-based toothpaste with 0.3% triclosan on ph changes in dental plaque in vivo. Caries Res : Cury JA, Francisco SB, Simoes GS et al. Effect of calcium carbonate-based dentifrice on enamel demineralization in situ. Caries Res : Cury JA, Simoes GS, Del Bel Cury AA et al. Effect of a calcium carbonate-based dentifrice on inbsitu enamel remineralization. Caries Res : Imfeld T. Dental erosion. Definition, classification and links. Eur J Oral Sci : Lussi A, Jaeggi T. Chemical factors. In: Dental Erosion Lussi A (Ed). Monogr Oral Sci 2006, 20: Larsen MJ, Nyvad B. Enamel erosion by some soft drinks and orange juice relative to their ph, buffering effect and contents of calcium phosphate. Caries Res : Davis RE, Marshall TA, Qian F et al. In vitro protection against dental erosion afforded by commercially available, calcium-fortified 100 percent juices. J Amer Dent Assoc : Attin T, Weiss K, Becker K et al. Impact of modified acidic soft drinks on enamel erosion. Oral Dis : Hughes JA, West NX, Parker DM et al. Development and evaluation of a low erosive blackcurrant juice drink. 3. Final drink and concentrate, formulae comparison in situ and overview of the concept. J Dent : Hara AT, Zero DT. Analysis of the erosive potential of calciumcontaining acidic beverages. Eur J Oral Sci : West NX, Hughes JA, Parker DM et al. Development of low erosive carbonated blackcurrant drink compared to a conventional carbonated drink. J Dent : Magalhaes AC, Moraes SM, Rios D et al. Effect of ion supplementation of a commercial soft drink on tooth enamel erosion. Food Add Contam Part A : Venables MC, Shaw L, Jeukendrup AE et al. Erosive effect of a new sports drink on dental enamel during exercise. Med Sci Sports Exerc : Hooper S, West NX, Sharif N et al. A comparison of enamel erosion by a new sports drink compared to two proprietary products: a controlled, crossover study in situ. J Dent : Ramalingam L, Messer LB, Reynolds EC. Adding casein phosphopeptide-amorphous calcium phosphate to sports drinks to eliminate in vitro erosion. Pediatr Dent : Jensdottir T, Nauntofte B, Buchwald C et al. Effects of calcium on the erosive potential of acidic candies in saliva. Caries Res : Ceri H, Olson ME, Stremick C et al. The Calgary biofilm device: new technology for rapid determination of antibiotic susceptibility of bacterial biofilms. J Clin Microbiol : Guggenheim B, Giertsen E, Schupbach P et al. Validation of an in vitro biofilm model of supragingival plaque. J Dent Res : Shapiro S, Giertsen E, Guggenheim B. An in vitro oral biofilm model for comparing the efficacy of antimicrobial mouthrinses. Caries Res : Gohr CM, Fahey M, Rosenthal AK. Calcific tendonitis: a model. Conn Tiss Res : Sissons CH. Artificial dental plaque biofilm model systems. Adv Dent Res : Baehni PC, Takeuchi Y. Anti-plaque agents in the prevention of biofilm-associated oral diseases. Oral Dis : Brading MG, Cromwell VJ, Jones NM et al. Anti-microbial efficacy and mode of action studies on a new zinc/triclosan formulation. Int Dent J : Nakashima S, Yoshie M, Sano H et al. Effect of a test dentifrice containing nano-sized calcium carbonate on remineralization of enamel lesions in vitro. J Oral Sci : Pickles MJ, Evans M, Philpotts CJ et al. In vitro efficacy of a whitening toothpaste containing calcium carbonate and perlite. Int Dent J : Fowler C, Willson R, Rees GD. In vitro microhardness studies on a new anti-erosion desensitizing toothpaste. J Clin Dent [Spec Iss]: Lussi A, Megert B, Eggenberger D et al. Impact of different toothpastes on the prevention of erosion. Caries Res : Margolis HC, Zhang YP, Lee CY et al. Kinetics of enamel demineralization in vitro. J Dent Res : Sewon LA, Karjalainen SM, Sainio M et al. Calcium and other salivary factors in periodontitis-affected subjects prior to treatment. J Clin Periodontol : Sewon LA, Karjalainen SM, Soderling E et al. Associations between salivary calcium and oral health. J Clin Periodontol : Dawes C. Factors influencing salivary flow rate and composition. In: Saliva and Oral Health (Eds. Edgar WM, O Mullane DM) 2 nd Ed., British Dental Association, London, 1996, Duckworth RM, Morgan SN. Oral fluoride retention after use of fluoride dentifrices. Caries Res : Duckworth RM, Knoop DTM, Stephen KW. Effect of mouthrinsing after toothbrushing with a fluoride dentifrice on human salivary fluoride levels. Caries Res : Correspondence to: A. Joiner, Unilever Oral Care, Quarry Road East, Bebington, Wirral, CH63 3JW, UK. Andrew.Joiner@Unilever.com Joiner et al.: Enhanced enamel benefits from a novel fluoride toothpaste IDJ 2412-Joiner.indd /08/ :42:02