A novel electron-microscopic method for measurement of mineral content in enamel lesions.

OBJECTIVE: To assess Scanning Electron Microscopy in Back-Scattered Emission mode (BSE-SEM) for measurement of lesion mineral content as a function of depth. Direct comparison is made with Transverse Micro-Radiography (TMR) and Surface Micro-Hardness (SMH) on carious and erosive lesions. DESIGN: Caries lesions prepared from sound bovine enamel at 37 °C and pH 4.6 in unsaturated (7d) or part-saturated (8d, 4.1 mM Ca2+, 8 mM Pi) lactic acid /methyl cellulose gel system, followed by TMR analysis. Erosive lesions prepared from sound bovine enamel (1% citric acid, pH3.8, room temperature) for 5, 10, 15 or 20 min at n = 10 per treatment group. SMH readings (Vickers diamond, 1.9 N, 20 s) were taken from acid-treated and reference areas of each sample. BSE-SEM performed on polished cross-sections of lesioned samples (Jeol JSM6490LV SEM; high vacuum, 10 keV beam voltage, magnification x500 with constant working distance of 10 mm). Under identical SEM conditions, polished standards i.e. MgF2, alumina, Mg, Al and Si provided a calibration plot of BSE-SEM signal vs. atomic number (z¯). Mineral content vs. depth plots were derived from the cross-sectional BSE-SEM data. RESULTS: Cross-sectional BSE-SEM images clearly differentiate between caries and erosive lesions. Comparison of caries lesion mineral loss from BSE-SEM with TMR data showed good correlation (R2 = 0.98). Similarly, comparison of BSE-SEM data from erosive lesions showed good correlation (R2 = 0.99) with hardness loss data from SMH. CONCLUSION: BSE-SEM provides a relatively rapid and cost-effective method for the assessment of mineral content in demineralised tooth enamel and is applicable to both caries and erosive lesions.

Effect of toothbrushing duration and dentifrice quantity on enamel remineralisation: An in situ randomized clinical trial.

OBJECTIVES: The influence of toothbrushing duration and dentifrice quantity on fluoride efficacy against dental caries is poorly understood. This study investigated effects of these two oral hygiene factors on enamel remineralisation (measured as surface microhardness recovery [SMHR]), enamel fluoride uptake (EFU), and net acid resistance (NAR) post-remineralisation in a randomized clinical study using an in situ caries model. METHODS: Subjects (n=63) wore their partial dentures holding partially demineralised human enamel specimens and brushed twice-daily for two weeks, following each of five regimens: brushing for 120 or 45s with 1.5g of 1150ppm F (as NaF) dentifrice; for 120 or 45s with 0.5g of this dentifrice; and for 120s with 1.5g of 250ppm F (NaF) dentifrice. RESULTS: Comparing brushing for 120s against brushing for 45s, SMHR and EFU increased by 20.0% and 26.9% respectively when 1.5g dentifrice was used; and by 22.8% and 19.9% respectively when 0.5g dentifrice was used. Comparing brushing with 1.5g against brushing with 0.5g dentifrice, SMHR and EFU increased by 35.3% and 51.3% respectively when brushing for 120s, and by 38.4% and 43.0% respectively when brushing for 45s. Increasing brushing duration and dentifrice quantity also increased the NAR value. The effects of these two oral hygiene factors on SMHR, EFU, and NAR were statistically significant (p<0.05 in all cases). CONCLUSION: Brushing duration and dentifrice quantity have the potential to influence the anti-caries effectiveness of fluoride dentifrices. Study NCT01563172 on ClinicalTrials.gov. CLINICAL SIGNIFICANCE: The effect of two key oral hygiene regimen factors - toothbrushing duration and dentifrice quantity - on fluoride's anticaries effectiveness is unclear. This 2-week home-use in situ remineralisation clinical study showed both these factors can influence fluoride bioactivity, and so can potentially affect fluoride's ability to protect against caries.

Dose-response effect of fluoride dentifrice on remineralisation and further demineralisation of erosive lesions: A randomised in situ clinical study.

OBJECTIVE: The objective was to evaluate the ability of fluoride in a conventional, non-specialised sodium fluoride-silica dentifrice to promote tooth remineralisation and enamel fluoride uptake (EFU), and assess the resistance of the newly formed mineral to attack by dietary acid, across the concentration range used in mass-market dentifrices. METHODS: Subjects wore a palatal appliance containing eight polished bovine enamel specimens, each including an early erosive lesion. In a randomised full-crossover sequence, 62 healthy subjects were treated with dentifrices containing four different fluoride concentrations: no fluoride; 250ppm, 1150ppm and 1426ppm fluoride. At each treatment visit, under supervision, subjects brushed with 1.5g dentifrice and rinsed once while wearing the appliance; the appliance was removed after a 4-h remineralisation period and effects on the enamel specimens determined. The primary efficacy variable was surface microhardness recovery (SMHR); others included EFU, relative erosion resistance (RER) and comparative erosion resistance. RESULTS: Highly significant linear and, with the exception of SMHR, quadratic dose-response relationships were observed between all efficacy variables and fluoride concentration. For SMHR, EFU and RER, values for the different fluoride concentrations were statistically resolved from one another, with the exception of the two highest fluoride concentrations. The degree of remineralisation and the acid resistance of enamel after treatment were closely related to EFU. CONCLUSION: After a single brushing, conventional non-specialised sodium fluoride-silica dentifrices promoted remineralisation of early enamel lesions, and imparted increased acid-resistance to the enamel surface, in a dose-dependent manner at least up to 1500ppm fluoride. CLINICAL SIGNIFICANCE: Enamel erosive tissue loss is an increasing concern, associated with modern diets. This study demonstrated that sodium fluoride, in a conventional non-specialised dentifrice formulation, can promote repair of the earliest stages of enamel erosion after a single application, in a dose-dependent fashion across the fluoride concentration range used in mass-market dentifrices. This study is registered in the GlaxoSmithKline Study Register (ID RH01299), available at: www.gsk-clinicalstudyregister.com/study/RH01299.

Comparison of Knoop and Vickers surface microhardness and transverse microradiography for the study of early caries lesion formation in human and bovine enamel.

OBJECTIVE: The aims of the present laboratory study were twofold: a) to investigate the suitability of Knoop and Vickers surface microhardness (SMH) in comparison to transverse microradiography (TMR) to investigate early enamel caries lesion formation; b) to compare the kinetics of caries lesion initiation and progression between human and bovine enamel. DESIGN: Specimens (90×bovine and 90×human enamel) were divided into six groups (demineralization times of 8/16/24/32/40/48h) of 15 per enamel type and demineralized using a partially saturated lactic acid solution. SMH was measured before and after demineralization and changes in indentation length (ΔIL) calculated. Lesions were characterized using TMR. Data were analyzed (two-way ANOVA) and Pearson correlation coefficients calculated. RESULTS: ΔIL increased with increasing demineralization times but plateaued after 40h, whereas lesion depth (L) and integrated mineral loss (ΔZ) increased almost linearly throughout. No differences between Knoop and Vickers SMH in their ability to measure enamel demineralization were observed as both correlated strongly. Overall, ΔIL correlated strongly with ΔZ and L but only moderately with the degree of surface zone mineralization, whereas ΔZ and L correlated strongly. Bovine demineralized faster than human enamel (all techniques). CONCLUSIONS: Lesions in bovine formed faster than in human enamel, although the resulting lesions were almost indistinguishable in their mineral distribution characteristics. Early caries lesion demineralization can be sufficiently studied by SMH, but its limitations on the assessment of the mineral status of more demineralized lesions must be considered. Ideally, complementary techniques to assess changes in both physical and chemical lesion characteristics would be employed.

Physical chemical effects of zinc on in vitro enamel demineralization.

OBJECTIVES: Zinc salts are formulated into oral health products as antibacterial agents, yet their interaction with enamel is not clearly understood. The aim was to investigate the effect of zinc concentration [Zn(2+)] on the in vitro demineralization of enamel during exposure to caries-simulating conditions. Furthermore, the possible mechanism of zinc's action for reducing demineralization was determined. METHODS: Enamel blocks and synthetic hydroxyapatite (HAp) were demineralized in a range of zinc-containing acidic solutions (0-3565ppm [Zn(2+)]) at pH 4.0 and 37°C. Inductively coupled-plasma optical emission spectroscopy (ICP-OES) was used to measure ion release into solution. Enamel blocks were analysed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and HAp by X-ray diffraction (XRD) and neutron diffraction (ND). RESULTS: ICP-OES analysis of the acidic solutions showed a decrease in [Ca(2+)] and [PO4(3-)] release with increasing [Zn(2+)]. FTIR revealed a α-hopeite (α-Zn3(PO4)2.4H2O)-like phase on the enamel surfaces at >107ppm [Zn(2+)]. XRD and ND analysis confirmed a zinc-phosphate phase present alongside the HAp. CONCLUSIONS: This study confirms that zinc reduces enamel demineralization. Under the conditions studied, zinc acts predominantly on enamel surfaces at PO4(3-) sites in the HAp lattice to possibly form an α-hopeite-like phase. CLINICAL SIGNIFICANCE: These results have a significant implication on the understanding of the fundamental chemistry of zinc in toothpastes and demonstrate its therapeutic potential in preventing tooth mineral loss.

Effect of gallium on growth of Streptococcus mutans NCTC 10449 and dental tissues.

Gallium-doped phosphate-based glasses (Ga-PBG) were assessed for their impact on Streptococcus mutans and dental mineralisation, firstly by disc diffusion assays followed by biofilms grown on nitrocellulose filter membrane (NFM) and constant-depth film fermentor (CDFF). Short-time exposure (10 min) effects of Ga-PBG on S. mutans biofilm were compared with that of 0.2% chlorhexidine. The effects of Ga-PBG on bovine enamel (which was investigated under pH-cycling condition) and dentine were analysed using transverse microradiography (TMR), profilometry and inductively coupled plasma optical-emission spectrometry (ICP-OES). The disc diffusion assays showed inhibition zones of 24.5 ± 0.5 mm for Ga-PBG compared with controls (C-PBG). Ga-PBG showed statistically significant growth inhibition of S. mutans biofilms on NFM (p = 0.001) and CDFF (p < 0.046) compared with hydroxyapatite (HA) and C-PBG. The CDFF assay revealed a maximum of 2.11 log colony-forming unit (CFU) reduction at 48 h, but short-time exposure effects were comparable with that of 0.2% chlorhexidine only on older biofilms (maximum of 0.59 vs. 0.69 log CFU reduction at 120 h). TMR analyses of the enamel revealed non-significant mineral loss (p = 0.37) only in the case of Ga-PBG samples compared with controls including sodium fluoride. ICP-OES analyses indicated transient gallium adsorption into dentine by calcium displacement. The results confirmed that gallium inhibited S. mutans growth and appears to have the potential to protect the enamel surface under conditions representative of the oral environment. Further work is needed to establish whether it has an application in daily oral hygiene procedures to prevent or reduce caries.

Effects of fluoride on in vitro enamel demineralization analyzed by ¹9F MAS-NMR.

The mechanistic action of fluoride on inhibition of enamel demineralization was investigated using (19)F magic angle spinning nuclear magnetic resonance (MAS-NMR). The aim of this study was to monitor the fluoride-mineral phase formed on the enamel as a function of the concentration of fluoride ions [F(-)] in the demineralizing medium. The secondary aim was to investigate fluorapatite formation on enamel in the mechanism of fluoride anti-caries efficacy. Enamel blocks were immersed into demineralization solutions of 0.1 M acetic acid (pH 4) with increasing concentrations of fluoride up to 2,262 ppm. At and below 45 ppm [F(-)] in the solution, (19)F MAS-NMR showed fluoride-substituted apatite formation, and above 45 ppm, calcium fluoride (CaF2) formed in increasing proportions. Further increases in [F(-)] caused no further reduction in demineralization, but increased the proportion of CaF2 formed. Additionally, the combined effect of strontium and fluoride on enamel demineralization was also investigated using (19)F MAS-NMR. The presence of 43 ppm [Sr(2+)] in addition to 45 ppm [F(-)] increases the fraction of fluoride-substituted apatite, but delays formation of CaF2 when compared to the demineralization of enamel in fluoride-only solution.

Characteristics of methylcellulose acid gel lesions created in human and bovine enamel.

Acid gel caries lesions were created in 3,100 human and bovine enamel specimens and studied with transverse microradiography. Small, significant differences were found. Human enamel lesions were found to be less demineralized, shallower, had a higher ratio of integrated mineral loss (ΔZ) to lesion depth (L), a lower degree of surface zone mineralization (SZ(max)) and showed less variability than those in bovine enamel. SZ(max) showed the highest variability. Between tissues, L differed the most, ΔZ the least. Biological variation within bovine enamel is perhaps not only bigger than previously assumed, it may also overshadow any structural and chemical differences between tissues.

Remineralization agents - new and effective or just marketing hype?

This is a review of the need for better remineralization and of the status of calcium-based remineralizing agents for use in anti-caries toothpastes. Use of fluoride toothpastes has markedly reduced caries. However, the decline may be over or in reverse. There is a limit to what fluoride alone can do; complementary agents are needed. Using plaque as a reservoir for calcium-based agents holds promise. Plaque fluid is already supersaturated with respect to relevant calcium phosphates at neutral pH; extra calcium may lead to surface-blocking and sub-optimal lesion consolidation. However, at cariogenic pH, lesions may be more porous to the ingress of mineral, leading to fuller consolidation, and controlled release of calcium should reduce undersaturation with respect to enamel and accelerate deposition of fluorhydroxyapatite. Clinical data to validate in vitro screening models are scarce. Direct progression to in situ models may often be appropriate. The spectrum of lesion types, from softening to relatively advanced subsurface, and lesion activity should be considered. Far from being 'marketing hype', progress with calcium-based remineralizing agents is both encouraging and scientifically sound. Clinical evidence exists for the efficacy of some agents, but further unequivocal clinical data are needed before these agents might be considered 'effective' when delivered from toothpaste.

Effect of fluoride, lesion baseline severity and mineral distribution on lesion progression.

The present study investigated the effects of fluoride (F) concentration, lesion baseline severity (ΔZ(base)) and mineral distribution on lesion progression. Artificial caries lesions were created using three protocols [methylcellulose acid gel (MeC), hydroxyethylcellulose acid gel (HEC), carboxymethylcellulose acid solution (CMC)] and with low and high ΔZ(base) groups by varying demineralization times within protocols. Subsequently, lesions were immersed in a demineralizing solution for 24 h in the presence of 0, 1, 2 or 5 ppm F. Changes in mineral distribution characteristics of caries lesions were studied using transverse microradiography. At baseline, the protocols yielded lesions with three distinctly different mineral distributions. Secondary demineralization revealed differences in F response between and within lesion types. In general, lowΔZ lesions were more responsive to F than highΔZ lesions. LowΔZ MeC lesions showed the greatest range of response among all lesions, whereas highΔZ HEC lesions were almost unaffected by F. Laminations were observed in the presence of F in all but highΔZ HEC and CMC lesions. Changes in mineral distribution effected by F were most pronounced in MeC lesions, with remineralization/mineral redeposition in the original lesion body at the expense of sound enamel beyond the original lesion in a dose-response manner. Both ΔZ(base) and lesion mineral distribution directly impact the F response and the extent of secondary demineralization of caries lesions. Further studies - in situ and on natural white spot lesions - are required to better mimic in vivo caries under laboratory conditions.

Effects of zinc and fluoride on the remineralisation of artificial carious lesions under simulated plaque fluid conditions.

The aim was to study the effects of zinc (Zn) and fluoride (F) on remineralisation at plaque fluid concentrations. Artificial carious lesions were created in 2 acid-gel demineralising systems (initially infinitely undersaturated and partially saturated with respect to enamel) giving lesions with different mineral distribution characteristics (high and low R values, respectively) but similar integrated mineral loss values. Lesions of both types were assigned to 1 of 4 groups and remineralised for 5 days at 37°C. Zn and F were added, based on plaque fluid concentrations 1 h after application, to give 4 treatments: 231 µmol/l Zn, 10.5 µmol/l F, Zn/F combined and an unmodified control solution (non-F/non-Zn). Subsequently remineralisation was measured using microradiography. High-R lesions were analysed for calcium, phosphorus, F and Zn using electron probe micro-analysis. All lesions underwent statistically significant remineralisation. For low-R lesions, remineralisation was in the order F(a) < non-F/non-Zn(a) < Zn(a, b) < Zn/F(b), and for high-R lesions F(a) < non-F/non-Zn(b) < Zn(b) < Zn/F(c) (treatments with the same superscript letter not significantly different, at p < 0.05). Qualitatively, remineralisation occurred throughout non-F/non-Zn and Zn groups, predominantly at the surface zone (F) and within the lesion body (Zn/F). Electron probe micro-analysis revealed Zn in relatively large amounts in the outer regions (Zn, Zn/F). F was abundant not only at the surface (F), but also in the lesion body (Zn/F). Calcium:phosphate ratios were similar to hydroxyapatite (all). To conclude, under static remineralising conditions simulating plaque fluid, Zn/F treatment gave significantly greater remineralisation than did F treatment, possibly because Zn in the Zn/F group maintained greater surface zone porosity compared with F, facilitating greater lesion body remineralisation.

Enamel demineralization and remineralization under plaque fluid-like conditions: a quantitative light-induced fluorescence study.

The present study investigated de- and remineralization in enamel lesions under plaque fluid (PF)-like conditions using quantitative light-induced fluorescence (QLF). Preformed lesions were exposed to partially saturated lactic acid solutions, varying in pH and fluoride concentration ([F]) based on a 5 × 3 factorial study design (0/0.1/0.5/1.5/4 ppm F; pH 4.9/5.2/5.5). Average fluorescence loss (ΔF) was monitored for 11 days. Subsequently, lesions were demineralized in a partially saturated acetic acid solution for two 24-hour periods. Data were analyzed using repeated measures analysis of covariance. Lesions exposed to PF at 4 ppm F and pH 5.5 showed not only the most remineralization (ΔΔF = 28.2 ± 14.0%) for all groups after 11 days, but also the most demineralization (ΔΔF = -19.3 ± 13.5%) after subsequent acetic acid exposure. Increased [F] resulted in more remineralization, regardless of pH. Higher pH values resulted in more remineralization. No remineralization was observed in lesions exposed to F-free solutions, regardless of pH. Remineralization was noticeable under the following conditions: pH 4.9 - [F] = 4 ppm, pH 5.2 - [F] ≥ 1.5 ppm, and pH 5.5 - [F] ≥ 0.5 ppm. Overall, [F] had a stronger effect on remineralization than pH. Subsequent demineralization showed that little protection was offered by PF-like solutions, and further demineralization compared with baseline was observed on lesions not remineralized initially. [F] had a stronger effect on net mineral change than pH. The present study has shown that QLF is a valuable tool in studying lesion de- and remineralization under PF-like conditions, where [F] was shown to be more important than pH.

A review of the structure of human and bovine dental hard tissues and their physicochemical behaviour in relation to erosive challenge and remineralisation.

OBJECTIVES: This review sets out to examine the suitability of bovine hard dental material in lieu of human material when investigating dental erosion, to review the evidence for the major factors popularly attributed to dental erosion: pH, pKa, acid type, erosion duration, temperature and stirring rate as well as examine the case for the use of fluoride in an anti-erosion capacity. DATA SOURCES: Published works were selected using online search software ICI Web of Knowledge and Pubmed, with key terms such as "enamel", "erosion" and "bovine AND human" and cross referenced with relevant papers cited in the indices. RESULTS: The growing trend of dental erosion, coupled to legislative changes has precipitated a recent shortage of human enamel and dentine for experimental work. This in turn has resulted in the increasing use of cheap and readily available alternate supplies being sourced. This alternate supply principally originates from beef cattle under 20 months of age, under the assumption that bovine enamel and dentine will behave in a manner similar to human material. Recent experiments attempting to compare the physicochemical properties of these two species have shown that erosion is not simply a matter of bulk tissue loss resulting from acid exposure, but a multi-factorial event encompassing ever increasing and varied complexity of the inter-relationship between solvent and substrate. CONCLUSIONS: Accurate data from the published literature regarding the comparative properties of human and bovine hard dental tissue remains scarce but consensus appears to accept the continuing use of bovine enamel as a substitute for human enamel. This lack of comparative data is further hampered by the lack of an established, standardised protocol with which to evaluate the two species. In addition, much debate remains regarding the significant principal factors responsible for dental erosion and ways to minimise the pathological manifestation.

In situ fluoride response of caries lesions with different mineral distributions at baseline.

The present in situ study investigated the fluoride response of caries lesions with similar mineral loss but two distinct mineral distributions (low- and high-'R', calculated as the ratio of mineral loss to lesion depth). Sixteen subjects wore eight gauze-covered enamel specimens with preformed lesions placed buccally on their mandibular partial dentures for periods up to 4 weeks. The participants brushed twice daily for 1 min with an 1,100 ppm F (as NaF) dentifrice. After 3 and 4 weeks, specimens were retrieved and analyzed microradiographically (TMR) and by quantitative light fluorescence (QLF). TMR results revealed that low- and high-R lesions showed opposite behaviors - low-R lesions further demineralized, whereas high-R lesions exhibited some remineralization. In comparison, lesion depth increased in low-R, but remained unchanged in high-R lesions; R decreased in both, but more in high-R lesions; mineral density at the lesion surface remained unchanged in low-R, but increased in high-R lesions. Differences in mineral loss between lesion types increased further between 3 and 4 weeks. QLF did not mirror TMR results as low-R lesions were found to remineralize, whereas high-R lesions remained unchanged. It is likely that low-R lesions differ from high-R lesions chemically and microstructurally; therefore rendering low-R lesion more susceptible to further dissolution. During lesion formation, low-R in contrast to high-R lesions may not lose all of the solubility-determining impurities such as magnesium and carbonate, which can reprecipitate again in different mineral phases within the lesion. In conclusion, mineral distribution at baseline directly impacts in situ lesion response to fluoride.

The effect of brushing time and dentifrice quantity on fluoride delivery in vivo and enamel surface microhardness in situ.

While the clinical anticaries efficacy of fluoride toothpaste is now without question, our understanding of the relation of fluoride efficacy to brushing time and dentifrice quantity is limited. The aim of this in situ study was to determine how differences in brushing time and dentifrice quantity influence (i) fluoride distribution immediately after brushing, (ii) clearance of fluoride in saliva, (iii) enamel fluoride uptake (EFU) and (iv) enamel strengthening, via the increase in surface microhardness. The study compared brushing times of 30, 45, 60, 120 and 180 s with 1.5 g of dentifrice containing 1,100 microg/g fluoride as sodium fluoride. In addition, 60 s of brushing with 0.5 g dentifrice was evaluated. A longer brushing time progressively reduced retention of dentifrice in the brush, thereby increasing the amount delivered into the mouth. A longer brushing time also increased fluoride concentrations in saliva for at least 2 h after the conclusion of brushing, showing that increased contact time promoted fluoride retention in the oral cavity. There was a statistically significant positive linear relationship between brushing time and both enamel strengthening and EFU. Compared to 0.5 g dentifrice, brushing with 1.5 g dentifrice more than doubled the fluoride recovered in saliva after brushing and increased EFU. In conclusion, the results of this preliminary, short-term usage study suggest for the first time that both brushing time and dentifrice quantity may be important determinants both of fluoride retention in the oral cavity and consequent enamel remineralization.

Effect of lesion characteristics and mineralizing solution type on enamel remineralization in vitro.

The aim was to study the effect of lesion preparation technique and solution composition on remineralization of artificial lesions in vitro. Lesions were prepared with similar total mineral loss, but different mineral distribution, i.e., low (14.0) or high R (34.8) values. Lesions from both groups were remineralized (10 days, 37 degrees C) in two different solutions, with similar supersaturation with respect to hydroxyapatite (St), but calcium:phosphate ratios representing either hydroxyapatite stoichiometry or plaque fluid (PF). Remineralization was quantified microradiographically, mineral distribution was compared with natural white-spot lesions. Mineral loss and depth decreased significantly, and surface-zone mineral content (Zmax) increased significantly, in all lesions. Overall there was a significant relationship of decreasing remineralization with increasing Zmax, but not within either lesion type. PF was significantly more efficient than St in high-R lesions, with lesions remineralizing almost completely in PF. Remineralization was not significantly different in PF or St for low-R lesions but in high-R lesions, PF was more efficient than St, possibly through differences in relative saturations with respect to different calcium phosphates. Differences in area:solution ratios and baseline Zmax values may also have explained the different response to PF. Low-R lesions were similar to natural white-spot lesions in terms of mineral distribution, whereas high-R were not. Concluding, both lesion and remineralizing solution type had a marked influence on remineralization. It is proposed that use of low-R lesions would be more appropriate where more physiologically relevant mineral distribution is required, whereas high-R lesions would be appropriate for studying inherent remineralizing efficiency.

The effect of fluoride at plaque fluid concentrations on enamel de- and remineralisation at low pH.

The aim was to study the effect of fluoride, at concentrations typical of plaque fluid, on de- and remineralisation of subsurface lesions at low pH. Artificial lesions in human enamel were microradiographed to quantify mineral loss and placed in acid-gel systems at pH 4.8, 5.0 and 5.2. Calcium and phosphate were added to give initial Ca and Pi concentrations of either 4.1 and 8.0 mM, or 4.7 and 9.7 mM, at each pH value. Further, at each pH and combination of Ca and Pi, fluoride was added to the gels to give initial concentrations of 1, 2 or 5 ppm, with a non-fluoride control group. The lesions were removed after 10 days and change in mineral content quantified. Those in the non-fluoride control groups had demineralised further. Those exposed to fluoride had remineralised, the amount increasing with increasing fluoride concentration, up to a maximum value of approximately 75%. Calcium activity in the gels was reduced significantly, to levels similar to those reported for plaque fluid at low pH. Fluoride activity was also reduced, though to a lesser extent. These findings contrast with those from studies which have simulated conditions on smooth surface sites and which used experimental solutions composed to reflect salivary fluoride concentrations, where net demineralisation was observed at low pH. This reflects the need for further study of de- and remineralisation under plaque-fluid conditions. In conclusion, subsurface lesions were remineralised at low pH by fluoride at concentrations found in plaque fluid during a cariogenic challenge.

The effect of lesion characteristics at baseline on subsequent de- and remineralisation behaviour.

The aim was to study the effect of lesion characteristics at baseline on subsequent de- and remineralisation behaviour. Artificial lesions used during in vitro and intra-oral studies exhibit an increasing tendency toward net remineralisation with increasing integrated mineral loss at baseline (DeltaZ(base)). Proposed explanations include: (i) small lesions may be more vulnerable to demineralisation, and (ii) large lesions may be more difficult to remineralise. To evaluate these hypotheses, subsurface lesions were created in numerous blocks of human and bovine enamel, with a range of depths and DeltaZ(base), measured by microradiography. One group of lesions was further demineralised, a second group remineralised, and subsequently, both groups re-analysed. Under demineralising conditions, there was a marked decrease in further mineral loss with increasing DeltaZ(base). Under remineralising conditions lesions gained mineral in proportion to DeltaZ(base). The decrease in demineralisation of lesions with larger DeltaZ(base) may be partially a result of decreased intrinsic solubility through modified chemical composition, e.g. loss of magnesium, carbonate etc. The results may explain the tendency toward net remineralisation with increasing DeltaZ(base) in pH-cycling regimes.

Effect of calcium glycerophosphate on demineralization in an in vitro biofilm model.

The aim was to investigate the anti-caries properties of calcium glycerophosphate (CaGP) using an in vitro bacterial flow cell model. Four flow cells, inoculated from a chemostat containing a seven-organism bacterial consortium, were pulsed with sucrose twice daily, to provide an acidic challenge and pH-cycling conditions. Blocks of enamel and dentine were mounted in each flow cell. In a study on the effect of CaGP concentration, CaGP was pulsed into three of the flow cells, at the same time as the sucrose, to give concentrations of 0.10, 0.25 and 0.50%. Water was pulsed into the fourth flow cell with the sucrose. Microradiography revealed a significant dose response of decreasing demineralization as CaGP concentration increased. Reductions at 0.25 and 0.5% were significant when compared to the control. A second study investigated the effect of timing of CaGP pulsing, relative to sucrose, on enamel and dentine demineralization. CaGP (flow cell concentration 0.2%), was pulsed 1 h before, during or 1 h after the sucrose pulse; a water control was employed. In enamel, pulsing CaGP before the sucrose reduced demineralization significantly compared to concurrent pulsing, which in turn gave a significant reduction compared to pulsing after sucrose, which did not reduce demineralization significantly compared to the water control. In dentine, CaGP reduced demineralization significantly only when pulsed before the sucrose. The findings suggest that in vivo, the anti-caries potential of CaGP may be greater if it is applied before a cariogenic challenge.

The effect of adjacent dentine blocks on the demineralisation and remineralisation of enamel in vitro.

The aim was to investigate interactions between enamel and dentine at low pH under conditions simulating those at the enamel-dentine junction. Sound enamel blocks were demineralised in acid-gel systems, at pH 4.6, either in isolation, next to one, or in the middle of two, abutting dentine blocks. The gels were initially infinitely undersaturated with respect to enamel. In a second study, enamel blocks containing pre-formed lesions were placed in acid-gel systems, at pH 5.0, either in isolation or next to dentine blocks. The systems were initially either partially or infinitely undersaturated. In the partially saturated systems, calcium and phosphate concentrations were representative of plaque fluid. In the first study, demineralisation of enamel next to one dentine block was reduced in inverse proportion to the distance from the dentine. Demineralisation of enamel between two dentine blocks was retarded markedly across the whole block. In the second study, in the partially saturated systems, enamel lesions next to dentine blocks remineralised, whereas those in isolation demineralised further. We suggest that diffusion of dissolved dentine mineral over the enamel in the infinitely undersaturated system was sufficient to reduce undersaturation, thus retarding demineralisation, and that in the partially saturated systems, dentine dissolution together with the added calcium phosphate caused remineralisation of enamel lesions. Fluoride released from dissolving dentine may have augmented these effects. Different rates of demineralisation in enamel and dentine, or enamel remineralisation with concurrent dentine demineralisation, enabled by differences in their solubilities, could help explain the progression of so-called 'hidden caries'.