Energy dispersive x-ray spectroscopy evaluation of demineralized human enamel after titanium tetrafluoride application.

OBJECTIVE: Evaluate the presence of a titanium dioxide layer after application of titanium tetrafluoride on human permanent tooth enamel. STUDY DESIGN: The sample consisted of unerupted third molars. After the removal of the roots, each tooth was mesiodistally divided into 2 fragments, one reserved for the experimental group and the other for the control group. Before the treatments the fragments were artificially demineralized. The experimental group (n=5) received an application of 4% titanium tetrafluoride, for one minute and the control group (n=5) did not receive any treatment. The samples were sputter-coated with a 20-30nm gold layer as the energy dispersive x-ray spectrometer analysis was carried out in a scanning electron microscope and the results were descriptively analyzed. RESULTS: The titanium dioxide layer was present on all experimental samples with a titanium peak varying between 6.82 and 26.37%. This layer was not found in the control group. Fluoride and calcium fluoride precipitates were present in the samples treated with titanium tetrafluoride. CONCLUSION: Titanium dioxide layer was formed after one titanium tetrafluoride application, but it was not uniform. Further studies should be carried out so that both the morphology and thickness of such layers can be better understood.

In vitro remineralizing effect of fluoride varnishes containing sodium trimetaphosphate.

This study analyzed the effects of fluoride varnishes supplemented with sodium trimetaphosphate (TMP) on the remineralization of caries-like lesions in vitro. Bovine enamel discs were selected through surface hardness (SH) and caries-like lesions were induced. SH was again determined and the blocks were divided into 7 experimental groups (n = 24/group): placebo (no fluoride or TMP), 5% TMP, 2.5% NaF, 2.5% NaF/5% TMP, 5% NaF, 5% NaF/5% TMP and commercial formulation (Duraphat™, 5% NaF), following a blind protocol. Discs were treated with the varnishes and kept in a remineralizing solution for 4 h and a demineralizing solution for 2 h. Varnishes were then removed and half of the discs were used for analysis of loosely (CaF2) and firmly bound fluoride. The remaining discs were submitted to a pH-cycling regimen for 6 days. The percentage of surface hardness recovery (%SHR), cross-sectional hardness (ΔKHN) and enamel CaF2 and fluoride were determined. Data were analyzed by ANOVA and by Student-Newman-Keuls' test (p < 0.05). A dose-response relationship was observed between fluoride concentrations in the varnishes and %SHR. The 5% TMP varnish led to %SHR similar to that obtained for the placebo. When TMP was used in association with fluoride, however, significantly higher %SHR were observed in comparison with their counterparts without TMP. Moreover, ΔKHN obtained for the 5% NaF/5% TMP was significantly lower among all groups tested. Higher concentrations of CaF2 and fluoride were observed for Duraphat and 5% NaF, followed by 5% NaF/5% TMP, 2.5% NaF and 2.5% NaF/5% TMP (p < 0.05). It was concluded that the supplementation of fluoride varnishes with TMP leads to enhanced remineralizing effect of artificial caries lesions in vitro.

Stability against brushing abrasion and the erosion-protective effect of different fluoride compounds.

This study aimed to analyse the impact of brushing on the protective effect of different fluoride solutions on enamel and dentin erosion. Bovine enamel and dentin specimens were rinsed once with TiF4, AmF, SnF2 (0.5 M F, 2 min) or water (control). Specimens were either left unbrushed or brushed with 10, 20, 50, 100 or 500 brushing strokes in an automatic brushing machine (2 N, non-fluoridated toothpaste slurry). Ten specimens per group were eroded with hydrochloric acid (HCl) (pH 2.3) for 60 s, and calcium release into the acid was determined by atomic absorption spectroscopy. Additionally, enamel and dentin surfaces were analysed by X-ray energy-dispersive spectroscopy (EDS) (n = 6/group) and scanning electron microscopy (SEM) (n = 2/group) before brushing and after 500 brushing strokes. Statistical analysis (p < 0.05) was performed by three- and one-way ANOVA (calcium release) or repeated measures ANOVA (EDS). TiF4, AmF and SnF2 reduced the erosive calcium loss in unbrushed specimens to 58-67% (enamel) and 23-31% (dentin) of control. Calcium release increased with increasing brushing strokes prior to erosion and amounted to 70-88% (enamel) and 45-78% (dentin) of control after 500 brushing strokes. Brushing reduced the surface concentration of fluoride (AmF), tin (SnF2) and titanium (TiF4). SEM revealed that surface precipitates were affected by long-term brushing. Brushing reduced the protective potential of TiF4, AmF and SnF2 solutions. However, considering a small number of brushing strokes, the protective effect of fluoride solutions is only slightly affected by brushing abrasion.

A calcium prerinse required to form calcium fluoride in plaque from a sodium fluoride rinse.

The purpose of this study is to determine whether a calcium (Ca) prerinse used before a 228 µg/g (ppm) fluoride (F) rinse would induce the formation of 'calcium fluoride-like' (CaF2-like) deposits in human dental plaque. Sixty minutes after the use of the Ca prerinse/F rinse, plaque samples were collected from 10 volunteers, homogenized, and split into 2 aliquots. The plaque mass from one aliquot was then extracted with a 'plaque-like' solution that extracted all the CaF2-like deposits. The total F in both aliquots was then determined and compared. The results demonstrated that, as in previous studies, the Ca prerinse induced large increases in plaque fluid and total plaque F. However, unlike previous results without the Ca prerinse, 30% of the plaque F deposits were CaF2 or CaF2-like. Given that maintaining an elevated F concentration in the vicinity of a developing lesion may play an important role in the cariostatic effect of this ion, and the potential advantages of CaF2-like deposits as an F source, these results suggest that a Ca prerinse may increase the cariostatic effect of topical agents.

In situ evaluation of a low fluoride concentration gel with sodium trimetaphosphate in enamel remineralization.

PURPOSE: To evaluate the capability of gels with low fluoride (F) concentration and supplemented with sodium trimetaphosphate (TMP) to promote in situ enamel remineralization. METHODS: Bovine enamel blocks were selected on the basis of their surface hardness after demineralization, and divided into five groups: gel without F or TMP (placebo); gel with 4,500 ppm F (4,500); gel with 4,500 ppm F + 5% TMP (4,500 5% TMP); gel with 9,000 ppm F (9,000) and gel with 12,300 ppm F (12,300). The study design was blind and cross-over: 12 subjects used palatal devices with four demineralized enamel blocks for 3 days, after topical fluoride application (TFA). Two blocks were removed immediately for analysis of the loosely bound fluoride (CaF2) and firmly bound fluoride (F) after TFA in enamel. In the remaining blocks, the percentage of surface hardness recovery (% SH), cross-sectional hardness (deltaKHN) and CaF2 and F were determined after remineralization. The results were subjected to ANOVA and Bonferroni tests (P < 0.05). RESULTS: The groups 4,500 5% TMP, 9,000, and 12,300 showed the best results with regard to % SH (P < 0.05). Lower deltaKHN values were observed in the 4,500 5% TMP and 12,300 gel groups (P < 0.05). Higher concentrations of CaF2 and F were observed in the 12,300 group, followed by the 4,500 5% TMP and 9,000 groups (P > 0.05). It was concluded that it is possible to promote enamel remineralization using gels with low fluoride concentration supplemented with TMP.

Pilot-scale fluoride-containing wastewater treatment by the ballasted flocculation process.

A pilot-scale ballasted flocculation system was used to remove fluoride from one type of industrial wastewater. The system included the formation of calcium fluoride (CaF2) using calcium hydroxide followed by coagulation sedimentation. Calcium fluoride was recycled as nuclei for enhancing CaF2 precipitation and as a ballasting agent for improving fluoride removal and flocculation efficiency. Factors affecting fluoride and turbidity removal efficiencies, including pH in the CaF2-reacting tank and coagulation-mixing tank, sludge recycling ratio, and dosages of FeCl3 and polyacrylamide (PAM), were investigated in the pilot-scale system. The recycled CaF2 precipitates improved CaF2 formation kinetics, enhanced fluoride removal and flocculation performance. Under the optimized condition, the ballast flocculation process reduced fluoride concentration from 288.9 to 10.67 mg/L and the turbidity from 129.6 NTU to below 2.5 NTU.

Effect of Er,Cr:YSGG laser and professional fluoride application on enamel demineralization and on fluoride retention.

This study evaluated the effect of Er,Cr:YSGG laser irradiation and professional fluoride application on enamel demineralization and on fluoride formation and retention. In a blind in vitro study, 264 human enamel slabs were distributed into 8 groups: G1--untreated; G2--treated with acidulated phosphate fluoride gel (APF gel, 1.23% F) for 4 min; G3, G4 and G5--irradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively; G6, G7 and G8--preirradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively, and subjected to APF gel application. Twenty slabs of each group were submitted to a pH-cycling regimen, and enamel demineralization was evaluated in 10 slabs of each group. In the other 10 slabs, CaF2-like material was determined. To evaluate F formed, 10 additional slabs of each group, not subjected to the pH cycling, were submitted to analysis of CaF2-like material and fluorapatite, while the other 3 slabs of each group were evaluated by scanning electron microscopy. The F content was also measured in all pH-cycling solutions. Laser at 8.5 J/cm2 and APF treatment reduced enamel demineralization compared to the control (p < 0.05), but the combination of these treatments was not more efficient than their isolated effect. A higher concentration of retained CaF2-like material was found in laser groups followed by APF in comparison with the APF gel treatment group. The findings suggest that laser treatment at 8.5 J/cm2 was able to decrease hardness loss, even though no additive effect with APF was observed. In addition, laser treatment increased the formation and retention of CaF2 on dental enamel.

Dose-response effects of zinc and fluoride on caries lesion remineralization.

The present mechanistic in vitro study aimed to investigate dose-response effects of zinc and fluoride on caries lesion remineralization and subsequent protection from demineralization. Artificial caries lesions were created using a methylcellulose acid gel system. Lesions were remineralized for 2 weeks using citrate-containing artificial saliva which was supplemented with zinc (0-153 µmol/l) and fluoride (1.1 or 52.6 µmol/l) in a 7 × 2 factorial design. Lesions were also remineralized in the absence of zinc and citrate, but in the presence of fluoride. After remineralization, all lesions were demineralized for 1 day under identical conditions. Changes in mineral distribution characteristics of caries lesions after remineralization and secondary demineralization were studied using transverse microradiography. At 1.1 µmol/l fluoride, zinc exhibited detrimental effects on remineralization in a dose-response manner and mainly by preventing remineralization near the lesion surface. At 52.6 µmol/l fluoride, zinc retarded remineralization only at the highest concentration tested. Zinc enhanced overall remineralization at 3.8-15.3 µmol/l. At 76.5 and less so at 153 µmol/l, zinc showed extensive remineralization of deeper parts within the lesions at the expense of remineralization near the surface. Citrate did not interfere with remineralization at 1.1 µmol/l fluoride, but enhanced remineralization at 52.6 µmol/l fluoride. Lesions exhibiting preferential remineralization in deeper parts showed higher mineral loss after secondary demineralization, suggesting the formation of more soluble mineral phases during remineralization. In summary, zinc and fluoride showed synergistic effects in enhancing lesion remineralization, however only at elevated fluoride concentrations.

Effect of acidulated phosphate fluoride gel application time on enamel demineralization of deciduous and permanent teeth.

Although the effect of acidulated phosphate fluoride gel (APF gel) on caries reduction in permanent teeth is based on evidence, the relevance of the clinical application time is still under debate. Also, the effect of 4- versus 1-min application has not been evaluated in deciduous enamel. In a blind, crossover, in situ study of 14 days, 16 adult volunteers wore palatal appliances containing slabs of human permanent and deciduous enamel. At the beginning of each phase, the slabs were submitted to one of the following treatments: no APF application (negative control); APF gel (1.23% F) application for 1 or 4 min. Biofilm accumulation on the slab surface was allowed and the slabs were subjected eight times a day to 20% sucrose, simulating a high cariogenic challenge condition. On the 15th day of each phase, fluoride retained as CaF(2) and fluorapatite (FAp) was determined on the enamel of the slabs and demineralization was assessed by cross-sectional microhardness. Fluoride as CaF(2) and FAp, formed by APF gel application on the enamel slabs not subjected to the cariogenic challenge, was also determined. APF gel reduced demineralization in both enamel types (p < 0.05), but the difference between 1 and 4 min was not statistically significant (p > 0.05). CaF(2) and FAp formed and retained on deciduous and permanent enamel was significantly higher in APF gel groups (p < 0.05), but no significant difference was found between 1 and 4 min (p > 0.05). The findings suggest that 1 min of APF gel application provides a similar effect on inhibition of demineralization as 4 min, for both permanent and deciduous enamel.

Prevention of erosive/abrasive enamel wear due to orange juice modified with dietary supplements.

OBJECTIVE: The aim of this study was to evaluate the erosive/abrasive enamel wear after contact with orange juices modified with different dietary supplements. METHODS: A total of 96 bovine enamel samples were prepared and allocated to eight groups (1-8; n = 12). Samples were eroded (120 s) in 200 ml of the following eight solutions: 1: water (control), 2: orange juice, 3: water + calcium effervescent tablet, 4: orange juice + calcium effervescent tablet, 5: water + 0.75 g acid/base regulating powder (Probase), 6: water + 0.375 g Probase, 7: orange juice + 0.75 g Probase and 8: orange juice + 0.375 g Probase. After erosion, the samples were brushed with 40 brushing strokes (load 2.5 N). Enamel wear was measured using surface profilometry after 20 and 40 cycles of erosion/abrasion respectively. RESULTS: Highest mean enamel wear (± SD) after 20 and 40 cycles of erosion/abrasion was observed for the unmodified orange juice (group 2) (0.605 ± 0.240 µm; 1.375 ± 0.496 µm respectively). The enamel wear in all other groups (3-8) was significantly lower (P < 0.0001 respectively) with no significant difference within these groups and compared with water (control). CONCLUSION: Erosive/abrasive enamel wear induces by orange juice and tooth brushing could be reduced significantly by modification with free available dietary supplements.

In vivo effects of fluoride on enamel permeability.

This in vivo study evaluated the effects of topical fluoride application on enamel by repeated scanning electron microscopy analysis of replicas. Baseline fluid droplets were employed as qualitative indication of enamel permeability. CaF(2)-like globules were detected in vivo after fluoride application and were not found after professional brushing, ultrasound action, or chemical extraction. Absence of water permeability of enamel was demonstrated even after removal of CaF(2)-like globules. Droplets reappeared within 1 h in sodium fluoride-treated teeth, but they did not reappear even after 1 week following topical enamel treatment with acidulated phosphate fluoride. Teeth treated with an acidulate fluoride-free solution showed lack of CaF(2)-like globules and no droplets for at least 1 week as detected in acidulate phosphate fluoride-treated teeth. The caries-preventing action of fluoride may be due to its ability to decrease permeability and diffusion pathways. CaF(2)-like globules seem to be indirectly involved in enamel protection over time maintaining an impermeable barrier, and phosphoric acid seemed to play an unexpected fluoride-independent preventive role.

No calcium-fluoride-like deposits detected in plaque shortly after a sodium fluoride mouthrinse.

Plaque 'calcium-fluoride-like' (CaF(2)-like) and fluoride deposits held by biological/bacterial calcium fluoride (Ca-F) bonds appear to be the source of cariostatic concentrations of fluoride in plaque fluid. The aim of this study was to quantify the amounts of plaque fluoride held in these reservoirs after a sodium fluoride rinse. 30 and 60 min after a 228 microg/g fluoride rinse, plaque samples were collected from 11 volunteers. Each sample was homogenized, split into 2 aliquots (aliquots 1 and 2), centrifuged, and the recovered plaque fluid combined and analyzed using microelectrodes. The plaque mass from aliquot 1 was retained. The plaque mass from aliquot 2 was extracted several times with a solution having the same fluoride, calcium and pH as the plaque fluid in order to extract the plaque CaF(2)-like deposits. The total fluoride in both aliquots was then determined. In a second experiment, the extraction completeness was examined by applying the above procedure to in vitro precipitates containing known amounts of CaF(2)-like deposits. Nearly identical fluoride concentrations were found in both plaque aliquots. The extraction of the CaF(2)-like precipitates formed in vitro removed more than 80% of these deposits. The results suggest that either CaF(2)-like deposits were not formed in plaque or, if these deposits had been formed, they were rapidly lost. The inability to form persistent amounts of CaF(2)-like deposits in plaque may account for the relatively rapid loss of plaque fluid fluoride after the use of conventional fluoride dentifrices or rinses.

Effects of carboxymethyl cellulose-based saliva substitutes with varying degrees of saturation with respect to calcium phosphates on artificial enamel lesions.

The aim of the present study was to evaluate the effects of experimental saliva substitutes based on carboxymethyl cellulose (CMC) differing in degrees of saturation with respect to calcium phosphates on the mineral loss of enamel in vitro. Demineralized bovine specimens (subsurface lesions) were exposed to one of six experimental CMC-based solutions with theoretical degrees of saturation with respect to octacalcium phosphate (S(OCP)) of S0, S0.5, S1, S2, S4, and S8 for 10 weeks. A previously studied saliva substitute (Glandosane) and two aqueous solutions (C0 and C1) served as controls. Mineral losses and lesion depths before and after storage were evaluated from microradiographs. Free and bound calcium as well as phosphate and fluoride concentrations were determined. According to these measurements, S(OCP) of S2, S4, and S8 was 0.3, 1.1, and 3.4, respectively. Storage in Glandosane and both negative controls resulted in significant demineralization (p < 0.05). Only S2 significantly remineralized the specimens (p < 0.05). All other solutions showed neutral effects. No significant differences in mineralization between S0 and C0 as well as between S1 and C1 could be observed (p > 0.05). It can be concluded that a CMC-based solution actually unsaturated with respect to octacalcium phosphate (S2) shows most pronounced remineralization capability under the conditions chosen. This might be explained by a more favorable balance between calcium bound to CMC in an adsorbed layer at the enamel-liquid interface and heterogeneous nucleation of calcium phosphates within a solution compared to solutions either supersaturated or having lower levels of saturation.

Expression of osteopontin and type I collagen of hFOB 1.19 cells on sintered fluoridated hydroxyapatite composite bone graft materials.

PURPOSE: The biological effect of fluoridated hydroxyapatite (FHA) graft materials has been attributed to their fluoride ion content; but, only few studies have been conducted to explore the osteoblastic cellular response to physicochemical characteristics of them. We hypothesized that the effect of varied sintered FHA composites on osteoblastic behavior would attribute certain specified physicochemical characteristics of apatites. MATERIALS: Sintered FHA composites were prepared by sintering method with varied gravity percentages of calcium fluoride and hydroxyapatite. Scanning electron microscopic, x-ray diffraction, and Fourier-transform infrared analysis were recorded. The human fetal-osteoblast (hFOB 1.19) cells were seeded on the apatites and tissue culture plates. Responses to the apatites were assessed in terms of osteopontin (OPN) and type I collagen, COL I, gene differentiation. RESULTS: We observed the calcined hydroxyapatite (OHAp), sintered F- OHAps, and hydroxy fluorapatites (OH-FAps) with different physicochemical characteristics. The x-ray diffraction analysis showed sintered apatites to be fluorapatites. Otherwise, Fourier-transform infrared spectral patterns could differentiate the sintered F-OHAps from OH-FAps by the existence of OH, OH···F, or OH···F···OH bands. With ≤ 1 wt% CaF2 added, sintered F-OHAp composites expressed both OH and OH···F bands. With >1 wt% CaF2 added, sintered OH-FAp composites expressed both OH···F and OH···F···OH bands. Sintered F-OHAp composites could enhance OPN and COL I gene expression after 6-day culture (P ≤ 0.05). Otherwise, sintered OH-FAp composites inhibited the expression. CONCLUSION: The results revealed that sintered F-OHAp composites with both OH and OH···F bands were bioactive bone graft materials.

Biologic precipitation of fluorite.

X-ray diffraction patterns show that the statoliths of marine mysid crustaceans are composed of fluorite, and that this mineral is also a principal phase of the gizzard plates of some tectibranch gastropods. A phosphatic phase is also indicated by chemical analyses in the gizzard plates, but its crystallochemical characterization has not been feasible by x-ray diffraction. The occurrence of fluorite in mysid statoliths confirms the earlier interpretations based on insufficient documentation. Fixation of fluorine in hard tissues of marine invertebrates is extensive in the shelf seawaters and minor in the bathyal zone of the oceans.

Mechanism of fluoride dentifrice effect on enamel demineralization.

Although the anticaries effect of fluoride (F) dentifrices is clearly established, the relative importance of F taken up by dental plaque not removed by brushing and of F products (CaF(2)-like) formed on totally cleaned enamel for the subsequent inhibition of demineralization is not known. Both effects were evaluated using conventional (1,100 microg F/g) and low-F concentration (500 microg F/g) dentifrices in a randomized, crossover, double-blind in situ study. Enamel blocks not treated or pretreated with the dentifrices to form CaF(2)-like deposits were mounted in palatal appliances in contact with a Streptococcus mutans test plaque. Volunteers brushed with non-F (negative control), low-F or conventional dentifrices and inserted the appliance in the mouth. F concentration in the fluid and solid phases of the test plaque was determined after 30 min, and a rinse with 20% sucrose solution was performed. After additional 45 min, plaque was collected and the loss of surface hardness at different test-plaque depths was measured. CaF(2)-like deposition on enamel and F taken up by plaque due to the use of F dentifrices were able to significantly increase F concentration in the fluid phase of the test plaque, but only the latter significantly reduced the loss of hardness because of the 20-30 times higher F concentration. Also, significant differences between the low-F and conventional dentifrices were observed for F on enamel, in plaque and on the subsequent loss of hardness. The results suggest that uptake of F by dental plaque not removed by brushing may be the main cause of the anticaries effect of F dentifrices.

Effect of iontophoresis on fluoride uptake in enamel with artificial caries lesion.

Iontophoresis is a noninvasive technique, based on the application of a constant low-intensity electric current to facilitate the release of a variety of drugs, whether ionized or not, through biological membranes. The objective of this research was to evaluate the effect of iontophoresis using different electric current intensities on the uptake of fluoride in dental enamel with artificial caries lesions. In this in vitro operator-blind experiment, bovine enamel blocks (n = 10/group) with caries-like lesions and predetermined surface hardness were randomized into 6 groups: placebo gel without fluoride applied with a current of 0.8 mA (negative control), 2% NaF gel without application of any current, and 2% NaF gel applied with currents of 0.1, 0.2, 0.4 and 0.8 mA. Cathodic iontophoresis was applied for 4 min. The concentration of loosely bound fluoride (calcium fluoride) and firmly bound fluoride (fluorapatite) was determined. The results were analyzed by the nonparametric Kruskal-Wallis and Dunn tests. Iontophoresis at 0.8 mA, combined with the application of fluoridated gel (2% NaF), increased fluoride uptake in enamel with caries-like lesions, as either calcium fluoride or fluorapatite.

The effect of saliva on the formation of KOH-soluble fluoride after topical application of amine fluoride solutions of varying fluoride concentration and pH.

The effect of saliva on the amount of KOH-soluble fluoride formed on a sound enamel surface after application of amine fluoride solution of varying fluoride concentrations (1, 0.5 and 0.25% F) and pH (5.3, 4.5 and 4.0) was examined in an in vitro study. The saliva pretreatment increased the amount of KOH-soluble fluoride at the highest pH value. For any given fluoride concentration, the presence of saliva did not influence the amount of KOH-soluble fluoride. These data suggest that saliva could enhance the amount of KOH-soluble fluoride if the topical fluoride preparations are not highly acid.

Solid sampling determination of total fluorine in baby food samples by high-resolution continuum source graphite furnace molecular absorption spectrometry.

This study describes the applicability of solid sampling technique for the determination of fluorine in various baby foods via molecular absorption of calcium monofluoride generated in a graphite furnace of high-resolution continuum source atomic absorption spectrometry. Fluorine was determined at CaF wavelength, 606.440nm in a graphite tube applying a pyrolysis temperature of 1000°C and a molecule forming temperature of 2200°C. The limit of detection and characteristic mass of the method were 0.20ng and 0.17ng of fluorine, respectively. The fluorine concentrations determined in standard reference sample (bush branches and leaves) were in good agreement with the certified values. By applying the optimized parameters, the concentration of fluorine in various baby foods were determined. The fluorine concentrations were ranged from

Ion probe study of fluorine gradients in outermost layers of human enamel.

F concentrations in the outermost layers of human tooth enamel were studied with the aid of a secondary ion microanalyzer. Concentration profiles were recorded in continuous sputtering analysis from the surface down to a depth of about 0.3 micrometer. Samples previously subjected to topical fluoride treatment were compared with reference specimens. In some samples, the results were compared with those obtained at greater depths by macroscopic etching analysis.