Chemical and structural challenges in remineralization of dental enamel lesions

Abstract – The aim of the present paper was to identify some chemical and structural factors which may prevent a full remineralization of caries lesions and to study whether it is possible to overcome such obstacles. Samples of powdered enamel apatite were equilibrated with solutions metastably supersaturated with respect to enamel hydroxyapatite and fluorapatite. After 10 min and 60 min of equilibration at 20°C the suspensions were centrifuged and the calcium and phosphate concentrations and the pH were determined in the supernatant. In parallel studies, 50–75-μm-thick sections of 27 fluorotic teeth of a severity of 5–7 according to Thylstrup and Fejerskov's classification were examined by microradiography and in polarized light using distilled water, and Thoulett's media or seen dry in air. Five obstacles inhibiting remineralization were identified: 1) Although remineralizing solutions or saliva are supersaturated with respect to enamel apatite the total amount of calcium and phosphate dissolved in it is small, so that after precipitation of the dissolved mineral only 1/20 000-1/30 000 of the volume of the mineralizing solution is occupied by mineral. 2) The concentration gradients from the mineralizing solution into the enamel is small, which indicates a slow diffusion into and out of the lesion. 3) The uptake of calcium and phosphate by the enamel apatite crystals is so rapid that the aqueous phase within the pores can be presumed to be only marginally supersaturated in the deeper parts of the lesion. 4) The surface layer of the enamel lesions was found to be a serious obstacle to remineralization so that a subsurface area remains hypomineralized after exposure to salivary remineralization even for a lifetime. 5) Nucleation of new apatite crystals to substitute lost crystals is an unsolved problem.     

Enhanced enamel remineralization under acidic conditions in vitro

We conducted this study to test the hypothesis that acidic solutions undersaturated with respect to enamel and supersaturated with respect to fluorapatite can enhance enamel remineralization by reducing preferential remineralization of the outer lesion and promoting mineral ion penetration. We used quantitative microradiography to assess mineral changes in artificial surface-softened and subsurface lesions in human enamel in vitro, induced by such an acidic solution and by a neutral remineralizing solution. For surface-softened lesions, the extent of remineralization was similar for both solutions, although preferential remineralization of the outer lesion was observed with the neutral solution. For subsurface lesions, preferential remineralization of the outer lesion was not observed with either solution. However, the extent of subsurface lesion remineralization by the acidic solution was significantly greater than that observed with the neutral solution. Results obtained are noted to reflect inherent differences in lesion type and the properties of the solutions studied.     

On the chemical and physical nature of erosions and caries lesions in dental enamel.

The aim of the present paper was to study the relation between the development of enamel erosion and caries lesion. Intact teeth were exposed to a gently agitated 50 mM acetate buffer, with or without fluoride for various periods of time. Further, intact teeth were exposed to an agitated 0.2 M HCl solution. It was found that irrespective of experimental conditions the aqueous phase was initially unsaturated with respect to both hydroxyapatite and fluorapatite during which period fluorhydroxyapatite was dissolved. Histological examination showed that the dissolving apatite originated from the enamel surface and the resulting lesion exhibited the signs of a dental erosion. The depth of the erosive lesion was found to be a direct function of the amount of mineral dissolved before the aqueous phase became saturated with respect to fluorapatite. The calcium, phosphate and fluoride dissolved made the aqueous phase first saturated and later supersaturated with respect to fluorapatite and a reuptake of fluoride in the enamel began, now under development of a caries-like lesion with preservation of the surface layer over a subsurface demineralizing zone. The demineralization depended on the volume of the aqueous phase. In conclusion, in a closed system with a limited amount of unsaturated solution a double lesion, an erosion over a caries lesion, will develop according to the changing saturation with respect to fluorapatite.     

Effect of monofluorophosphate on calcium phosphate formation in supersaturated solutions

The effect of purified monofluorophosphate (MFP) on the formation of hydroxyapatite (HA) in supersaturated calcium phosphate solutions was determined. In solutions initially at pH 7.4 and seeded with HA crystals, MFP was hydrolysed to a small extent, releasing F-. Once the crystal growth-enhancing property of this F- was compensated for, 4 mmol/l MFP could be shown to inhibit precipitation by 40%. Without compensation for F-, MFP appeared to inhibit precipitation by only 18%. This inhibition was weaker than that caused by pyrophosphate or ethane-1-hydroxy-1,1-diphosphonic acid. Because MFP is hydrolysed on apatite surfaces to F-, use of sodium MFP as an anticaries agent is unlikely to cause significant inhibition of enamel remineralization.     

酪蛋白磷酸肽-磷酸钙溶液体外再矿化作用的化学分析及显微硬度测试

目的研究酪蛋白磷酸肽-磷酸钙溶液在体外对牛牙釉质早期人工龋的再矿化作用。方法用不同浓度的酪蛋白磷酸肽-磷酸钙（CPP—CP）溶液对牛牙釉质早期人工龋进行再矿化处理后，首先采用化学分析法测量处理液中的残余矿物质，得出沉积在龋损区内的矿物质含量；然后采用显微硬度法测量处理前后牙釉质剖面硬度变化。结果CPP—CP溶液可以在体外使龋损牙釉质发生再矿化，再矿化作用与CPP—CP浓度呈剂量-效应关系。再矿化后龋损区矿物沉积量与再矿化液的CPP—CP浓度成正比。结论酪蛋白磷酸肽-磷酸钙溶液能凭借高钙磷浓度梯度促进牛牙釉质早期人工龋的再矿化。     

A kinetic and morphological study of mineralization of bovine tooth enamel surfaces

A new constant composition method was used to study the kinetics of remineralization of enamel surfaces. The labial enamel surfaces were treated with 20 per cent phosphoric acid, 0.05 M HCl, EDTA (2 per cent, pH 4.0) yielding Type I etched surface and with EDTA (1–2 per cent, pH 7.0) to give a Type II etched surface. Remineralization experiments were made in stable supersaturated calcium phosphate solutions undersaturated with respect to dicalcium phosphate dihydrate and octacalcium phosphate phases, at pH 7.40 and 37 °C. Type I-etched surfaces yielded similar remineralization rates, but these were greater than those for Type II surfaces. Confirmation of hydroxyapatite as the exclusive remineralizing phase was made by various physico-chemical methods. Scanning electron microscopy of the remineralized phase, suggested various sites for remineralization.     

Influences of bicarbonate on processes of enamel subsurface remineralization and demineralization: assessment using micro‐Raman spectroscopy and transverse microradiography

In the present study, we investigated, using micro‐Raman spectroscopy (Raman) and transverse microradiography, the influence of bicarbonate [sodium hydrogen carbonate (NaHCO₃)] on the effects of carbonate ions in the mineral phase during demineralization (acid resistance test) of subsurface lesions. Baseline lesions were created by demineralizing bovine enamel, and specimens were then exposed to remineralization solutions containing 0, 5, or 50 mM bicarbonate. Acid resistance tests were performed on remineralized and sound enamel specimens. Raman spectra showed that carbonate and phosphate were incorporated into both surface layers and lesion bodies during remineralization in the presence of bicarbonate. Moreover, the presence of bicarbonate did not affect the rates of remineralization, although the average mineral profiles of remineralized enamel differed from those of sound enamel after acid resistance tests. Raman analyses enabled close evaluation of site‐specific characteristics of carbonate and phosphate in subsurface lesions. In conclusion, incorporation of carbonate and phosphate ions into enamel subsurface lesions during remineralization does not affect the magnitude of remineralization or acid resistance.     

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.     

pH-cycling of enamel and dentin lesions in the presence of low concentrations of fluoride

F-dentifrice usage causes slightly elevated fluoride levels in saliva. Therefore, the effects of permanent low fluoride concentrations versus daily dentifrice treatments were studied on enamel and dentin lesions in a pH-cycling model of alternating demineralization and remineralization. Groups received 1) no fluoride treatment. 2) 3 μM (0.06 ppm) F continuously present during re- and demineralization or 3) daily 5–min F-dentifrice treatments. Solutions were analyzed for changes in calcium and fluoride. Cumulative results (10 d) showed that for the non-fluoride group the dentin lesions increased, while for enamel lesions mineral uptake and loss were balanced. Addition of 3 μM F caused small, non-significant, enhancement of remineralization (1–7%). while demineralization was significantly inhibited for both tissues (9–23%). The daily dentifrice treatments resulted in a balance between mineral uptake and loss of dentin, due to inhibited demineralization (-33%) and enhanced remineralization (+ 79%). For enamel, the F-dentifrice treatments resulted in 43% reduction of demineralization, with no significant effect on remineralization. Fluoride loss from the 3 μM F cycling solutions was significant (up to 50%) and constant during the experimental period. Microradiographic analysis showed remineralization at the lesion front in enamel. In dentin, the lesion depth was increased in all groups, with concomitant mineral deposition in the surface region of the dentifrice group. Results indicate that slightly elevated fluoride levels may be considerably less effective in inhibiting lesion progression in dentin than in enamel, and suggest mineral uptake and loss to occur at similar depths for enamel lesions, while demineralization and remineralization occur at different depths in dentin.     

Enamel remineralization: how to explain it to patients

The term remineralization of initial enamel caries is frequently used, but mainly at research conferences and in lecture rooms. Clinicians avoid discussing remineralization and white-spot enamel lesion formation with patients because details of the processes are complex. Patients, therefore, incorrectly assume that cavitation occurs right at the onset of caries and that a restoration must be placed to halt further progression of the lesion. Although laboratory and clinical studies have shown that initial white-spot enamel lesions can remineralize, patients have little or no access to this information. The schematic diagrams in this paper explain the diffusion of organic acids from plaque between enamel crystals, the partial loss of mineral from enamel crystals, and subsequent demineralization and remineralization that produces the white-spot lesion. Further, these diagrams may help clinicians explain remineralization as simply as possible to patients who present with white-spot enamel lesions.     

Remineralization of caries lesions extending into dentin.

Remineralization is one aspect of the overall process of tooth decay. However, it is primarily studied in shallow lesions. The aim of this study was to explore whether caries lesions in enamel and extending into the dentin can be remineralized. A single-section model was developed for the longitudinal and non-destructive monitoring of changes in enamel and dentin. Lesions at least 200 microm into dentin were formed in undersaturated acetate buffers. Next, the lesions were divided into groups (three treatment and one control) and remineralized. The treatments were: weekly immersion in 1,000 ppm fluoride, single treatment with methanehydroxybisphosphonate, and a constant level of 1 ppm fluoride. De- and remineralization was assessed by transverse microradiography. Remineralization was observed in enamel, but also in dentin, indicating that, deep into dentin, the pores become supersaturated to apatite formation. Treatments affected remineralization only in the outer part of enamel. Both findings are explained by a relatively fast diffusion of mineral ions, with precipitation being rate-limiting. The results suggest that dentin remineralization, underneath enamel, can be achieved and could possibly be used in clinical treatment strategies.     

The effect of fluoride in the remineralization of enamel caries and caries-like lesions in vitro

Remineralization occurs naturally during the formation of a carious lesion in human dental enamel and is seen in the form of two of the four classical histological zones of the lesion. Exposure of small lesions to a synthetic calcifying in vitro results in a significant increase in remineralization of the lesion. The degree of remineralization achieved depends upon the presence of fluoride ions in the calcifying fluid and its degree of supersaturation. It appears that only low levels of fluoride are required to trigger the mechanism of remineralization; raising the fluoride level further does not result in a greater degree of remineralization. The calcium ion concentration of the calcifying fluid is critical with respect to determining which components are supersaturated and which in turn determines the degree of remineralization achieved. With levels of 3.0 mM calcium only the superficial region of the lesion is remineralized whereas with a calcium concentration of 1.0 mM, remineralization occurs in depth. It is hoped that studies on remineralization phenomena will provide further insight into the mechanisms of caries formation and be the basis for a more powerful and effective form of caries prevention.     

Dental caries: a dynamic disease process

Abstract Dental caries is a transmissible bacterial disease process caused by acids from bacterial metabolism diffusing into enamel and dentine and dissolving the mineral. The bacteria responsible produce organic acids as a by-product of their metabolism of fermentable carbohydrates. The caries process is a continuum resulting from many cycles of demineralization and remineralization. Demineralization begins at the atomic level at the crystal surface inside the enamel or dentine and can continue unless halted with the end-point being cavitation. There are many possibilities to intervene in this continuing process to arrest or reverse the progress of the lesion. Remineralization is the natural repair process for non-cavitated lesions, and relies on calcium and phosphate ions assisted by fluoride to rebuild a new surface on existing crystal remnants in subsurface lesions remaining after demineralization. These remineralized crystals are acid resistant, being much less soluble than the original mineral.     

Crystallographic changes in human tooth enamel during in-vitro caries simulation.

Subsurface demineralization of human dental enamel similar to natural caries was produced by the use of lactate buffers containing diphosphonate (MHDP) as a surface dissolution inhibitor. X-ray diffraction was used to follow crystallographic changes in the surface layer to a depth of 40 μm during artificial carious lesion formation. The various lesion zones were dissected and submitted to X-ray diffraction. It was concluded that: the intact surface layer is formed from the original enamel, after partial demineralization, by deposition of calcium and phosphate, dissolved from underlying enamel, into defects of existing crystals; dicalcium phosphate CaHPO₄ is formed in the inner lesion (translucent) zone; carbonate-containing apatite is progressively lost during lesion formation; and that well-formed hydroxyapatite is left during subsurface dissolution.     

Amelogenin control over apatite crystal growth is affected by the pH and degree of ionic saturation

Authors – Habelitz S, DenBesten PK, Marshall SJ, Marshall GW, Li W Objective – To study the mechanisms which promote the interactions of amelogenin proteins with the forming mineral to establish suitable conditions for the biomimetic synthesis of enamel in vitro. Design – Saturated calcium phosphate solutions were used in conjunction with recombinant amelogenin proteins to induce mineral formation on glass‐ceramics substrates containing oriented fluoroapatite crystals (FAP). The height of mineral layers formed on these substrates within 24 h was measured by atomic force microscopy (AFM). Experimental Variables – The effect of protein concentration, pH and degree of saturation (DS) on the growth of apatite mineral was evaluated. Mineralization experiments were performed at 0, 0.4 and 1.6 mg/ml amelogenin concentrations. Mineralization solutions were used at pH values of 6.5, 7.4, 8.0 and 8.8 and DS of calcium and phosphate between 9 and 13. Outcome Measure – Height and morphology of mineralized layer formed on glass‐ceramic substrates as determined from AFM measurements. Results – Homogeneous nucleation and crystal growth of thin layers on the FAP were observed, when calcium and phosphate ions were added. The height of these layers grown on (001) planes of FAP was strongly dependent on the protein concentration and pH. At concentrations of 0 and 0.4 mg/ml crystal grew 5–15 nm on the FAP, while they grew approximately to 200 nm at 1.6 mg/ml. The enhanced crystal growth was observed only at pH 6.5, 7.4 and 8.0, while layers only 20 nm thick were obtained at pH 8.8. An increase in DS resulted in uncontrolled growth of calcium phosphate mineral covering large areas of the substrate. Conclusions – Protein concentration, pH and the saturation of the mineralizing solution need to be considered carefully to provide suitable conditions for amelogenin‐guided growth of apatite crystals.     

Effects of two fluoride gels on fluoride uptake and phosphorus loss during artificial caries formation

Blocks of human enamel were cycled through a demineralization--F-treatment-remineralization procedure and then analyzed for fluoride and the presence of caries-like lesions. Treatments with a sodium fluoride gel (5000 ppm F) increased the enamel fluoride concentration to 6500 ppm F, whereas a stannous fluoride gel (1000 ppm F) increased enamel fluoride to about 1200 ppm F. Although a control treatment (water) allowed caries-like lesions to form, as observed by microradiography, no lesions were found in either of the fluoride-treated groups. When the experiment was repeated with radioactive teeth, mineral loss, as determined by release of 32P, was again greatest in the water-treated control group, but some loss was observed in the fluoride treatment groups. The least loss was found in the sodium fluoride group. It was concluded that the fluoride treatments not only increased enamel resistance but also enhanced remineralization so that calcium phosphate was replaced during the subsequent remineralization phase. Because of the probability that stannous ions were deposited during the stannous fluoride treatments, some of the apparent calcium phosphate re-deposition in this group was probably stannous compounds.     

Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate

The aim of this clinical study was to investigate the acid resistance of enamel lesions remineralized in situ by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP: Recaldent). The study utilized a double-blind, randomized, crossover design with two treatments: (i) sugar-free gum containing 18.8 mg of CPP-ACP, and (ii) sugar-free gum not containing CPP-ACP as control. Subjects wore removable palatal appliances with insets of human enamel containing demineralized subsurface lesions and chewed the gum for 20 min 4 times per day for 14 days. After each treatment the enamel slabs were removed and half of each lesion challenged with acid in vitro for 8 or 16 h. The level of remineralization was determined using microradiography. The gum containing CPP-ACP produced approximately twice the level of remineralization as the control sugar-free gum. The 8- and 16-hour acid challenge of the lesions remineralized with the control gum resulted in 65.4 and 88.0% reductions, respectively, of deposited mineral, while for the CPP-ACP-remineralized lesions the corresponding reductions were 30.5 and 41.8%. The acid challenge after in situ remineralization for both control and CPP-ACP-treated lesions resulted in demineralization underneath the remineralized zone, indicating that the remineralized mineral was more resistant to subsequent acid challenge. The results show that sugar-free gum containing CPP-ACP is superior to an equivalent gum not containing CPP-ACP in remineralization of enamel subsurface lesions in situ with mineral that is more resistant to subsequent acid challenge.     

Enamel subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride

Casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) and amorphous calcium fluoride phosphate (CPP-ACFP) solutions have been shown to remineralise enamel subsurface lesions. The aim of this study was to determine the effect of ion composition of CPP-ACP and CPP-ACFP solutions on enamel subsurface lesion remineralisation in vitro. CPP-bound and free calcium, phosphate and fluoride ion concentrations in the solutions were determined after ultrafiltration. The ion activities of the free ion species present were calculated using an iterative computational program. The mineral deposited in the subsurface lesions was analysed using transverse microradiography and electron microprobe. CPP was found to stabilise high concentrations of calcium, phosphate and fluoride ions at all pH values (7.0-4.5). Remineralisation of the subsurface lesions was observed at all pH values tested with a maximum at pH 5.5. The CPP-ACFP solutions produced greater remineralisation than the CPP-ACP solutions at pH 5.5 and below. The mineral formed in the subsurface lesions was consistent with hydroxyapatite and fluorapatite for remineralisation with CPP-ACP and CPP-ACFP, respectively. The activity gradient of the neutral ion pair CaHPO(4)(0) into the lesion was significantly correlated with remineralisation and together with HF(0) were identified as important species for diffusion.     

Influence of salivary macromolecules and fluoride on enamel lesion remineralization in vitro

The aim of this study was to investigate the influence of salivary macromolecules on enamel lesion remineralization in the presence or absence of fluoride. Paraffin-stimulated whole saliva was centrifuged, and the supernatant was dialyzed in 1,000 molecular-weight cutoff dialysis tubes, first against a phosphate buffer and then against a mineral solution containing Ca and phosphate. Artificial subsurface lesions of human enamel, created in pH 4.5 acetate buffer, were remineralized for 28 days in 4 remineralizing solutions: group C--mineral solution as a control; group S--mineral solution + dialyzed saliva; group F--mineral solution + 1 ppm F; group SF--mineral solution + dialyzed saliva + 1 ppm F. Changes in relative mineral concentration in the lesions were assessed by transverse microradiography. The results showed statistically significant mineral gains in the lesion body in groups C (DeltaZ = 3,254 +/- 1,562% x microm) and SF (DeltaZ = 2,973 +/- 1,349% x microm), but not in groups S (DeltaZ = 5,192 +/- 1,863% x microm) and F (DeltaZ = 4,310 +/- 1,138% x microm) compared with the baseline group (DeltaZ = 5,414 +/- 461% x microm). It was also found that the mineral density at the surface layer in group F (75.0 +/- 15.7%) was greater than that in the baseline group (30.1 +/- 12.3%) with statistical significance, but not in group SF (39.9 +/- 16.5%). It was concluded that the macromolecules inhibited lesion remineralization fundamentally but that these molecules, in the presence of fluoride, seemed to play an important role in the continuation of remineralization by reducing mineral gains at the surface layer.     

The formation and remineralization of artificial white spot lesions: a constant composition approach

Artificial white spot lesions have been prepared on bovine enamel surfaces by the controlled addition of lactate buffer containing methylhydroxydiphosphonate ions. The rates of remineralization have been measured using a constant composition method in solutions of calcium phosphate of low supersaturation. Deposition of new hydroxyapatite solid phase takes place exclusively within the white spot lesion at a rate appreciably slower than that of an acid-etched enamel surface, probably due to the presence of methylhydroxydiphosphonate located within the intact surface region of the lesion.