R.J.M. Lynch

Effect of Lesion Characteristics and Mineralising Solution Type on Enamel Remineralisation in vitro

The aim was to study the effect of lesion preparation technique and solution composition on remineralisation 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 remineralised (10 days, 37°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). Remineralisation 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 remineralisation with increasing Zmax, but not within either lesion type. PF was significantly more efficient than St in high-R lesions, with lesions remineralising almost completely in PF. Remineralisation 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 remineralising solution type had a marked influence on remineralisation. 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 remineralising efficiency.

A comparison of terahertz pulsed imaging with transmission microradiography for depth measurement of enamel demineralisation in vitro.

Terahertz pulsed imaging (TPI) is a relatively new, non-ionising and non-destructive imaging technique for studying hard tissues which does not require tooth section preparation, unlike transmission microradiography (TMR). If TPI can measure the depths of caries/demineralisation lesions accurately the same tooth samples could be reused and remeasured during in vitro and in situ studies on de- and/or remineralisation. The aim of this study was to compare TPI and TMR for measuring the depths of a range of artificially induced bovine enamel demineralised lesions in vitro. Bovine slabs with artificial caries, induced to different levels of demineralisation by two different but standard demineralisation techniques (‘acid gel’ and ‘carbopol’) were measured by TPI and TMR and the readings compared. The set of TPI/TMR measurements obtained on the gel-demineralised slabs showed an extremely high coefficient of determination (r2 = 0.995). Detailed analysis of the results and theoretical considerations (involving the relationship between refractive index profiling and mineral loss profile) are used to explain the findings and show that for acid gel lesions TPI is measuring demineralisation in the range of 47% of that of TMR depth plus an intercept of 16 µm, with further calculations allowing the TMR depths to be determined to within 5% using TPI.

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 µg/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.

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 (ΔZbase). 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 ΔZbase, 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 ΔZbase. Under remineralising conditions lesions gained mineral in proportion to ΔZbase. The decrease in demineralisation of lesions with larger ΔZbase 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 ΔZbase in pH-cycling regimes.

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.

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 Fa < non-F/non-Zna < Zna, b < Zn/Fb, and for high-R lesions Fa < non-F/non-Znb < Znb < Zn/Fc (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.

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.

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.

A continuous culture biofilm model of cariogenic responses

Aims: To validate an in vitro model for the analysis of physiological and ecological responses to sugar challenge in bacterial populations, and subsequent changes in enamel mineralization.

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.