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Interactions of dentine desensitisers with human dentine: Morphology and composition
j o u r n a l o f d e n t i s t ry 4 1 s 4 ( 2 0 1 3 ) s 2 8 – s 3 9
Available online at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Interactions of dentine desensitisers with human dentine: Morphology and composition George Eliadesa,*, Maria Mantzouranib, Roberto Labellab, Bruna Muttic, Deepak Sharmad a
Department of Biomaterials, University of Athens, School of Dentistry, Greece Oral Care Scientific & Professional Affairs, Johnson & Johnson Consumer Services EAME Ltd, Maidenhead, UK c Oral Care Research and Development, Johnson and Johnson GmbH, Neuss, Germany d Oral Care Research and Development, Johnson & Johnson Consumer & Personal Products Worldwide, Skillman, NJ, USA b
keywords
abstract
Dentinal tubule occlusion Potassium oxalate mouthrinse FTIR spectroscopy Raman microscopy EDX spectroscopy SEM
Objective: To evaluate the effect of desensitising agents on human dentine morphology and composition. Methods: Randomly assigned human coronal-dentine specimens were subjected to: (a) no treatment (smear-layer control, n=4); (b) acid etching with 6% citric acid (demineralised control, n=4); (c) treatment with desensitising agents (12 cycles of 60 s treatment with 60 s between-treatment rinsing, n=6 per agent ); and (d) exposure to acidic challenge (pH 5.0 for 90 s, n=6 per agent). The tested products were: Listerine® Advanced Defence Sensitive (LADS; 1.4% potassium oxalate) mouthrinse, Colgate® Sensitive Pro-Relief™ mouthrinse, and toothpaste slurries (paste/water 1:2 wt/wt ratio) of Colgate® Sensitive Pro-Relief™ paste, Crest® Sensitive paste and Sensodyne® Repair and Protect paste. All dentine surfaces were studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Raman microscopy and high vacuum scanning electron microscopy with energy-dispersive X-ray microanalysis (HV-SEM/EDX). Results: Desensitising slurry treatments occluded tubule orifices of acid-etched dentine, creating a randomly distributed surface pattern of particle aggregates. The greatest intratubular penetration of occluding particles was found in dentine treated with LADS. The atomic ratios of Ca/N and Ca/P, and the mineral/matrix ratios increased after toothpasteslurry treatments compared with the acid-etched dentine. However, the acidic challenge removed most surface precipitates and further demineralised these substrates. Before the acidic challenge, the surface features were least affected in specimens treated with Sensodyne® Repair and Protect. After the acidic challenge, the sub-surface occlusion features were least affected in specimens treated with LADS. Clinical significance: Although most tested products achieved occlusion of dentinal tubules and provided evidence of mineral deposits, the deposit formed by LADS demonstrated the greatest resistance to acidic challenge, which simulates intra-oral demineralisation phases. © 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The hydrodynamic theory of dentine sensitivity hypothesises that minute fluid shifts within exposed, patent dentinal
tubules in response to hydrodynamic stimuli activate intrapulpal nerves to cause brief, sharp, well-localised tooth pain.1–3 A corollary to that theory is that anything that can partially occlude dentinal tubules will reduce dentine sensitivity.4 Another corollary is that agents that can reduce
* Corresponding author at: Department of Biomaterials, University of Athens, School of Dentistry, 2 Thivon Str, Goudi,115 27 Athens, Greece. Tel.: +30 210 7461101; fax: +30 210 7461306 E-mail address: [email protected] (G. Eliades) 0300-5712/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
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nerve excitability will reduce dentine sensitivity. Hodosh discovered that 5–30% potassium nitrate applied topically to sensitive dentine could reduce dentine sensitivity,5 while others have shown that raising potassium ion concentrations in tissue fluids reduced nerve excitability.6–8 In vivo studies have confirmed the hydrodynamic theory9 and support the inclusion of 5% potassium nitrate in many desensitising dentifrices. Many currently available desensitising dentifrices and mouthrinses contain “tubule-occluding agents” and have been marketed based on different technologies, with several mechanisms proposed for their interaction with dentine. The purpose of this in vitro study was to evaluate the surface interactions with human dentine of four commercially available dentine-desensitising products and a new 1.4% potassium oxalate mouthrinse (Listerine® Advanced Defence Sensitive; LADS). The null hypotheses were: (1) there is no between-treatment difference in acidetched dentine morphology and composition after the desensitising treatments have been applied; and (2) acidic erosion challenge does not alter the morphology or elemental and molecular composition of dentine surfaces treated with these products.
2.
Materials and methods
2.1
Products tested
2.3
2.4
Desensitising treatment
One dentine sample was placed in each well of a 12-well plate (treatment plate) and 2 mL of test solution (toothpaste/ water slurry or mouthrinse) was applied. One plate was used for each product and solutions were changed after each treatment. The treatment plate was agitated on an orbital shaker at 100 rpm for 60 s to simulate salivary flow. Each dentine sample was transferred to a second 12-well plate (rinse plate) with 2 mL diH2O per well and the plate was agitated as above. The diH2O was removed from the wells with a pipette and the dentine specimens kept undisturbed for 5 min. The dentine specimens were replaced in their respective wells in the treatment plate and the treatment/ rinse cycle repeated a total of 12 times. Half of the specimens (n=6) for each treatment were used for the erosion challenge procedure and the remaining specimens (n=6) were retained for microscopic and spectroscopic analysis.
2.5
2.2
2.6.1
Disk-shaped coronal human dentine specimens (diameter 7–9 mm, thickness 0.7–0.9 mm, n=68; provided by J&J) were placed in individual wells of 12-well plates. The dentine disks were randomly assigned to Groups A–G. Group A comprised the reference specimens, which were untreated and retained a smear-layer (n=4). Group B comprised control acid-etched specimens that were not subjected to any product treatment (n=4). Specimens in Groups C–G (n=12 per group) were acid etched and then treated as described below. Groups C, D and E were treated with toothpaste slurries (Group C: SDRP, Group D: CRSN, Group E: CSRT); Groups F and G were treated with mouthrinses (Group F: CSRM, Group G: LADS). The margins of the top surfaces of the dentine specimens to be exposed to the desensitising and erosion-challenge treatments were marked with permanent ink dots.
Etching procedure
Dentine specimens in Groups B–G were acid etched by immersion in 6% citric acid and sonication at 37 kHz for 180 s, followed by sonication in diH2O for 90 s. The acidetched specimens were stored individually in vials with moist paper in the cap of the vial to keep specimens hydrated. Before applying desensitising treatments, specimens were hydrated in diH2O for 30 min.
The products tested were three toothpastes [Crest® Sensitivity (CRSN), Procter & Gamble, Cincinnati, OH, USA; Sensodyne® Repair and Protect (SDRP), GlaxoSmithKline, Philadelphia, PA, USA; Colgate® Sensitive Pro-Relief (CSRT), Colgate-Palmolive Company, New York, NY, USA] and two mouthrinses [Colgate® Sensitive Pro-Relief (CSRM), Colgate-Palmolive Company, New York, NY, USA; 1.4% potassium oxalate-containing mouthrinse (LADS), Johnson & Johnson Consumer & Personal Products Worldwide (J&J), Skillman, NJ, USA]. All the test products were provided to the investigators by J&J without commercial names and in a blinded fashion. Toothpastes were used as water slurries at 1:2 wt/wt ratios with distilled water (diH2O) after thorough hand and vortex mixing. Slurries were used within 30 min after preparation and new slurries were prepared every 30 min. Mouthrinses were used without modification.
Dentine specimen preparation
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Erosion challenge
Half of the dentine specimens subjected to the desensitising products were exposed to the following erosion challenge. The dentine specimens were individually immersed in 10 mL of demineralisation solution (0.1 M lactic acid saturated with hydroxyapatite; pH 5.0) and sonicated in a water bath at 37 kHz for 90 s. Each dentine specimen was then immersed in 10 mL diH2O for 30 s without sonication.
2.6
Analytical procedures
The top surfaces of specimens (n=2) from Groups A and B and from Groups C–G [after desensitising alone (n=4) and after desensitising and erosion challenge treatments (n=4)] were assessed as follows.
Attenuated total reflectance Fourier transform infrared spectroscopy
An attenuated total reflectance (ATR) cell was used (Golden Gate, Specac, Augusta, USA) attached to a Fourier transform infrared (FTIR) spectrometer (Spectrum GX, Perkin-Elmer, Bacon, UK) operated under the following conditions: deuterated triglycerine sulphate detector; 2000–650 cm-1 wavenumber range; 4 cm-1 resolution; single-reflection diamond ATR element with zinc selenide focusing lenses; 2 mm diameter sampling area; ~2 μm depth of analysis at 1000 cm-1 and 40 scans co-addition. Spectra were subjected to baseline and ATR corrections.
2.6.2
Raman microscopy
The same specimens were studied using a Raman microscope (InVia, Renihaw, Gloucestershire, UK) employing a charge-
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coupled device detector and a 785 nm diode laser. Spectra were recorded under the following conditions: 2000–0 cm-1 Raman shift (wavenumber range); 1200 l/mm grading, 4 cm-1 resolution; 50× magnification; 3 mW laser power at sample; 10 s acquisition time; and ~5–6 μm depth of analysis.
2.6.3
High vacuum scanning electron microscopy and energy-dispersive X-ray microanalysis
All the specimens were subjected to controlled dehydration in a desiccator coated with a ~20 nm thick carbon film in a sputter coater (Bal Tec SCD 004 Sputter Coater with OCD 030 attachment, Balzers, Vaduz, USA) and examined by high vacuum scanning electron microscopy (HV-SEM) under the following conditions: solid state compositional backscattered detector for phase identification; 15 kV accelerating voltage; 10-6 Torr pressure; 90 μA beam current; and 1300× magnification. For energy-dispersive X-ray (EDX) analysis a liquid nitrogencooled Si(Li) detector with a super-ultrathin beryllium window (EDAX Sapphire CDU, EDAX Int, Mahwah, USA) and Genesis software (version 5.1) were used with the following acquisition parameters: 110 μA beam current; 1300× magnification (100×100 μm2 analysis area); 34% detector dead time; 0.4° tilt angle; 35.75° take-off angle; 130.44 eV resolution; and ~3 μm depth of analysis. Qualitative analysis was performed in standardless mode after ZAF and C-coating corrections. Separate specimens (n=2) from Groups A and B, and from Groups C–G [after desensitising alone (n=2) and after desensitising and erosion challenge treatments (n=2)] were sectioned with a thin diamond disk (from the bottom side upwards to a depth of 0.5 mm) and then fractured with a sharp chisel. The fractured surfaces were coated with 20 nm of gold in a sputter coater and studied by HV-SEM as detailed above at magnifications of 2000×, 2500× and 8000×.
The SEM backscattered electron (BE) image demonstrated tubule orifices closed with smear plugs, with well-defined peritubular dentine collars due to their higher mineral content (Fig. 1C). The EDX analysis showed the presence of strong Ca and P peaks, along with C, O, Na, Mg, Si, S and Cl, which is consistent with a highly mineralised dentine surface (atomic ratios: Ca/N = 1.9; Ca/P = 1.5; Fig. 1G). The BE image of the longitudinally fractured specimen exhibited highly mineralised peritubular dentine walls, extending up to the cut surface, with tubule orifices closed by smear-layer plugs (Fig. 1E).
3.2
The ATR-FTIR spectrum of the acid-etched specimens showed a demineralised surface (Fig. 1A, red) as evidenced by the strong reduction in -PO43- v1,v3 and -CO32- (mostly of the b-type at 1405 and 871 cm-1) and an increase in dentine organic matrix (Amide I, II, III, CH2, CH3) peak intensities, resulting in an RIR of 0.2. The residual mineral was a mixture of highly (1023 cm-1) and poorly crystalline apatite (1080 cm-1). The demineralisation was also confirmed by the reduction in the intensity of the -PO43- v1 (958 cm-1) peak and the increase in the intensity of Amide I and III peaks in the Raman spectrum (Fig. 1B, bottom), leading to an RRM of 3.7. The BE image revealed a dentine surface free of smear layer, with open tubule orifices and loss of peritubular dentine, all typical findings of acid-etched dentine (Fig. 1D). The EDX analysis showed a mineral-deficient dentine surface with a strong reduction in all mineral peaks and an increase in C, N and O (atomic ratios: Ca/N = 0.05, Ca/P = 2; Fig. 1H). The BE images of fractured dentine specimens (Fig. 1F) clearly showed a superficial zone of demineralised collagen collapsed onto subsurface mineralised dentine. The mean thickness of this zone was estimated to be 3 μm.
3.3
3. 3.1
Acid-etched dentine specimens (Group B)
Acid-etched dentine specimens subjected to SDRP (Group C)
Results Smear-layer covered dentine specimens (Group A)
The ATR-FTIR spectrum of smear-layer covered dentine specimens (Fig. 1A, blue) demonstrated peaks of -PO43- (v1,v3 at 1180–900 cm-1, v4 at 600–550 cm-1) and -CO32- (1450 cm-1 of a-type + CH; 1405 and 871 cm-1 of b-type), assigned to dentine mineral and peaks of Amide I (1655–1640 cm-1), Amide II (1550–1530 cm-1), Amide III (1250 cm-1) and CH2/CH (1400–1350 cm-1) assigned to the organic components of dentine (mainly collagen type I). The mineral (-PO43- v1,v3 at 1180–900 cm-1 range) to organic matrix (Amide I at 1712–1596 cm-1 range) net peak absorbance area ratio from ATR-FTIR measurements (RIR) was calculated as 3.7. The complex peak of the -PO43- v1,v3 band (~1040 cm-1) exhibited high- and low-frequency shoulders attributed to the various states of the apatite mineral. The Raman spectrum (Fig. 1B, top) showed the characteristic peaks of -PO43- ( v1 at 958 cm-1, v2 at 434 cm-1, v4 at 590 cm-1), -CO32- (v1 at 1083 cm-1, v2 at 865 cm-1, v3 at 1415 cm-1), Amide I (1665–1650 cm-1), Amide III (1270 cm-1), CH2, CH3 groups (G at 1455–1320 cm-1) and lattice vibrations (350–0 cm-1). The mineral (-PO43- v1 at 958 cm-1) to organic matrix (Amide I at 1662 cm-1) net peak absorbance height ratio from Raman measurements (RRM) was calculated as 11.4.
ATR-FTIR spectra of SDRP-treated surfaces before and after acidic challenge are depicted in Fig. 2A. In comparison with the acid-etched control (Fig. 1A, red), SDRP produced a minor increase in the complex -PO43- v1,v3 peak at ~1080 cm-1, with a low-frequency shoulder at 1030 cm-1, which was reduced following the acidic challenge although it retained the original peak shape. The RIR ratios before and after the acidic challenge were 0.3 and 0.2, respectively. The results of Raman analysis (Fig. 2B) confirmed the mineral changes (RRM before, 6.3; RRM after, 4.3). A representative BE image from an area fully covered by the SDRP components is shown in Fig. 2C. A granular layer covered the acid-etched dentine structure, with a great number of particles attached, which consisted of elements with a high mean atomic number. The EDX analysis (Fig. 2G) showed the presence of Si and Ti along with the other dentine elements (atomic ratios: Ca/N = 0.1, Ca/P = 0.8). However, after acidic challenge this layer was largely removed (Fig. 2D). No material deposition was found within dentinal tubules before or after the acidic challenge (Figs. 2E and F, respectively).
3.4
Acid-etched dentine specimens subjected to CRSN (Group D)
ATR-FTIR spectra showed that treatment with CRSN increased the complex -PO43- v1,v3 peak, with a predominant component
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Fig. 1 – Dentine specimens of the control groups. (A) Attenuated total reflectance Fourier transform infrared spectra of smear layer (blue) and acid-etched dentine (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra of smear layer (top) and acid-etched dentine (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image of smear-layer covered dentine surface (1300×, bar 20 μm); (D) BE image of acid-etched dentine surface (1300×, bar 20 μm); (E) BE image of fractured specimen with smear layer (2000×, bar 20 μm); (F) BE image of fractured specimen of acid-etched dentine (2000×, bar 20 μm); (G) energy-dispersive X-ray (EDX) spectrum of smear-layer covered dentine; (H) EDX spectrum of acid-etched dentine.
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Fig. 2 – Dentine specimens treated with Sensodyne Repair and Protect toothpaste (SDRP) slurry. (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm); (G) energy-dispersive X-ray spectrum of treated dentine.
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Fig. 3 – Dentine specimens treated with Crest Sensitivity toothpaste (CRSN) slurry. (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm).
at ~1080 cm-1 that was slightly altered after the acidic challenge (Fig. 3A). The RIR ratios were 0.4 before and 0.3 after the acidic challenge. The Raman spectra (Fig. 3B) confirmed the additional demineralisation after the acidic challenge, showing a reduction of the intensity of the central apatite peak and an increase of the contribution of amides, with minor changes in the spectral profile attributed to increased CH3/CH2 group vibrations. The RRM ratios were 6.3 and 3.5 after the CRSN treatment and following the acidic challenge, respectively. The BE image of the dentine surface after treatment (Fig. 3C) shows the presence of sparsely distributed particles with higher mean atomic number than observed in
demineralised dentine after CRSN treatment; these particles were not observed following the acidic challenge (Fig. 3D). The BE images of the fractured surfaces showed tubular structures free of any precipitants after CRSN treatment (Fig. 3E). The acidic challenge further exposed demineralised collagen, opening more lateral tubule orifices within tubules (Fig. 3F).
3.5
Acid-etched dentine specimens subjected to CSRT (Group E)
The ATR-FTIR spectrum following CSRT treatment (Fig. 4A) demonstrated chemical groups found in the native toothpaste
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(Fig. 4B) with an RIR of 0.1. However, after the acidic challenge a typical spectrum of demineralised dentine was recorded, with traces of residual mineral (-PO43- v1,v3 peak at ~1080 cm-1; RIR 0.1). The Raman spectra demonstrated increased intensities of the -CO32-, CH3/CH2 and TiO2 (140 cm-1) peaks after CSRT treatment, but the contribution of the -CO32- peak was reduced after the acidic challenge, and the TiO2 peak disappeared (Fig. 4C). The RRM values measured before and after acidic challenge were 6.7 and 5.1, respectively. The BE images (Fig. 4D) clearly showed that a granular structure remained on the dentine surface after CSRT treatment, leading to atomic ratios for Ca/N of 0.2 and for Ca/P of 2.2, as determined by EDX analysis (Fig. 4H). Nevertheless, this structure was completely removed from the dentine surface following the acidic challenge (Fig. 4E). The BE images of the fractured specimens after CSRT treatment showed a zone of low mean atomic number material at the dentine surface, with evidence of micro-granular inclusions (Fig. 4F); however, most tubules were free of granular material. The acidic challenge removed this zone (Fig. 4G).
3.6
Acid-etched dentine specimens subjected to CSRM (Group F)
No differences were observed between the ATR-FTIR spectra for untreated acid-etched dentine control (Group B), dentine treated with CSRM and dentine treated with CSRM followed by acidic challenge, apart from minor changes in the -PO43- v1,v3 peak at ~1080 cm-1. The RIR ratios were 0.2 before and 0.1 after the acidic challenge (Fig. 5A). The Raman spectra documented the additional demineralisation after the acidic challenge, with increases in the dentine organic matrix peaks (Amide I, CH3/CH2, Amide III relative to the -PO43- v1 (958 cm-1) peak, leading to an RRM of 6.2 before and 5.4 after the acidic challenge (Fig. 5B). The BE images were similar to those for the acid-etched dentine control, with no evidence of surface precipitates (Figs. 5C and D). The BE images of the fractured specimens showed few crystalline particles located at tubular openings, before and after the acidic challenge (Figs. 5E and F).
3.7
Acid-etched dentine specimens subjected to LADS (1.4% potassium oxalate) (Group G)
The ATR-FTIR spectra of LADS-treated dentine before and after the acidic challenge were identical, and similar to that of the demineralised dentine control (RIR 0.2 before and 0.1 after the acidic challenge; Fig. 6A). The complex -PO43- v1,v3 peak was clearly resolved into two components (1080 and 1030 cm-1). The Raman spectra (Fig. 6B) documented an additional demineralisation after the acidic challenge (seen as a reduction in the intensity of the central apatite peak and an increase of the amide contribution), with no changes in the molecular composition (RRM 3.8 before; RRM 2.2 after). The BE image of the LADS–treated dentine surface (Fig. 6C) showed open tubules with scarce crystals in tubule orifices with atomic ratios for Ca/N of 0.1 and for Ca/P of 2.6, as provided by the EDX analysis (Fig. 6I). After the acidic challenge, the dentine surface (Fig. 6D) was similar to that of the acid-etched control. However, the BE image of the fractured surfaces showed many crystalline structures inside tubule orifices after LADS treatment, some extending to a depth of more than 20 μm (Figs. 6E and G). After the acidic challenge, crystalline structures were still detected on and within tubules (Figs. 6F and H).
4.
Discussion
The present study was designed to investigate the effects of desensitising toothpastes and mouthrinses on the morphology and elemental/molecular composition of aciddemineralised dentine surfaces. The results showed that there were differences in acid-etched dentine morphology and composition after desensitising treatments, requiring rejection of the study’s first null hypothesis. The fact that changes were observed following acid challenges to treated dentine surfaces for three of the five desensitising products requires partial rejection of the second null hypothesis. Occlusion of the patent dentinal tubules in dentine with acid-etched surfaces has been extensively used to study the efficacy of desensitising products with putative occluding technologies.10,11 In the present study, dentine specimens were acid etched with 6% citric acid (pH 1.5) for 3 min and then rinsed in water, with sonication, to avoid extensive demineralisation and formation of a residual smear layer or mineral-salt deposition on the dentine surface. The validation of the method is supported by the SEM images of the acidetched dentine control specimens, in which opened tubules were observed with a 3–5 μm superficially demineralised zone. Dentine surfaces covered with a smear-layer, as produced by sectioning, were used as a fully mineralised dentine control, as they combine the highest mineral content with closedtubule morphology. The desensitising toothpaste agents were tested as slurries with water (2:1 wt/wt ratio) to simulate the slurry used during toothbrushing. Nevertheless, no toothbrushing was performed, to avoid mineral removal from dentine surfaces due to abrasion.11 Instead, an orbital shaker was used to simulate the effect of saliva flow on the precipitated minerals, which also facilitated comparison with the mouthrinses. For the acidic challenge, an organic acid (lactic acid, a by-product of bacterial plaque metabolism) saturated with hydroxyapatite was used at a mild pH, but with sonication, to remove the loosely bound or weakly attached desensitising compounds from dentine, without excessive in-depth demineralisation. ATR-FTIR, Raman microscopy and HV-SEM/EDX were the main analytical techniques used in the study. Although complementary information is given by these techniques, there are differences in their mean sampling depth (2 μm for ATR-FTIR, 5–6 μm for Raman microscopy and 3 μm for EDX in the conditions of the present study), which should be taken into account when interpreting the results. For imaging, only a compositional backscattered detector was employed. This detector minimises the edge effects observed on very rough surfaces (such as fractured specimens) by secondary electron detectors, provides atomic number contrast for easy phase identification, and better matches to the depth of EDX analysis.12 In the present study, the assessment of the SEM images was mainly qualitative, in contrast to the other techniques. Therefore, the SEM results for the extent of tubule occlusion should be carefully interpreted, as applies for most relevant studies. The toothpastes tested were based on bioglass (7.5% Ca-Naphosphosilicate; SDRP), 0.454% SnF2/Na-hexametaphosphate (CRSN) and 8.0% arginine/CaCO3 (CSRT) technologies, whereas the mouthrinses were based on 0.8% arginine, methyl vinyl ether and maleic anhydride copolymer, pyrophosphates and sodium fluoride (CSRM) and 1.4% potassium oxalate (LADS) technologies.
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Fig. 4 – Dentine specimens treated with Colgate Sensitive Pro Relief toothpaste (CSRT) slurry. (A) Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) ATR-FTIR spectra of native material after solution in water and dehydration. Absorbance scale 2000–600 cm-1 region; (C) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (D) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (E) BE image after acidic challenge (1300×, bar 20 μm); (F) BE image of fractured specimen after treatment (2000×, bar 20 μm); (G) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm); (H) energy-dispersive X-ray spectrum of treated dentine.
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Fig. 5 – Dentine specimens treated with Colgate Sensitive Pro Relief mouthrinse (CSRM). (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm).
Treatment of acid-etched dentine surfaces with the desensitising toothpaste slurries resulted in the development of precipitates (mainly granular material) covering tubule orifices to various extents. More organised integuments were found after SDRP and CSRT treatments, with the mouthrinses being the least effective. The particles identified on dentine surfaces were mainly composed of C, N, O, Na, Mg, Si, P, S, Cl and Ca. After desensitisation treatments, dentine surface Ca was increased in the CSRT-treated group and Si in the SDRPtreated group, apparently due to adsorption or precipitation of Ca and Si, respectively, from the desensitising agents.
Ti was also found in SDRP and CSRT groups as TiO2, as confirmed by Raman microscopy. The surface specificity of ATR-FTIR demonstrated limited evidence of mineralisation as monitored by the -PO43- v1,v3 band region. The mineral/ matrix ratios measured on desensitised specimens ranged from 0.4 to 0.2, with 0.2 calculated for the acid-etched control and 3.7 for the smear-layer control. However, care should be taken when analysing the complex -PO43- v1,v3 dentine band (1180–900 cm−1) after desensitisation, because many native components of the toothpastes used demonstrated peaks in the same region. The characteristic splitting of the complex
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Fig. 6 – Dentine specimens treated with Listerine Advanced Defence Sensitive (LADS) mouthrinse. (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm); (G) BE image of fractured specimen after treatment (8000×, bar 5 μm); (H) BE image of fractured specimen after acidic challenge (8000×, bar 5 μm); (I) energy-dispersive X-ray spectrum of treated dentine.
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-PO43- v1,v3 band into two well-resolved peaks (~1080 cm-1 and ~1030 cm-1) after CSRT, CSRM, and especially after LADS treatments, may imply the presence of more crystalline apatite on the dentine surface.13 The Raman analysis showed more intense -PO43- v1 peaks on acid-etched dentine than seen by ATR-FTIR, apparently due to the greater depth of analysis despite the low laser power used, which resulted in subsurface interferences from intact tissue. Nevertheless, the calculated mineral/matrix ratios were consistent with the ranking determined by ATR-FTIR (6.5–3.6 on treated surfaces vs 4.1 on acid-etched control surfaces and 11.4 on the smear-layer control surface). The images of the fractured specimens demonstrated distinct differences between the toothpaste slurries and the mouthrinses. Although the dentine surface morphology was almost free of precipitates with mouthrinses, crystals were identified within dental tubule canals and orifices. This contrasts with the toothpaste slurries, for which no intratubular extension of the surface-precipitated phases was observed. This was very characteristic after LADS treatment, with the crystals extending up to a depth of 20 μm. It is likely that the rheological characteristics of this potassium oxalate mouthrinse increased its capacity for penetration into tubules to mediate sealing by reacting with ionised Ca and forming insoluble calcium oxalate crystals, known to reduce the hydraulic conductance of dentine.14 The shape of the crystals formed after LADS treatment is consistent with the formation of calcium oxalate monohydrate.15 The crystals found after CSRM treatment were located more superficially. After the acidic challenge all surface-precipitated phases were removed and the morphology of the treated dentine surfaces resembled that of the acid-etched control, except for some sporadic crystals located in tubule orifices after SDRP. This suggests that the retention of the precipitated phases with dentine is rather weak. The mineral/matrix ratios determined both by ATR-FTIR and Raman microscopy were reduced owing to the additional demineralisation. On fractured specimens, more crystals were identified in dentine specimens treated with LADS than in other treated specimens. The development of crystals deep in the tubules following LADS treatment, along with their relatively low solubility, probably maintained the tubular occlusion despite the acidic challenge. The professional application of acidic oxalate solutions to desensitise dentine has long been established, but the clinical effects have not been considered permanent.16 However, in the form of a mouthrinse, a positive effect may be maintained due to the repeated applications.
with significant intratubular subsurface crystalline formation, which resisted the acidic challenge to a greater extent than the deposits formed by the other mouthrinse (CSRM) or any of the toothpaste slurries tested.
Conflict of interest George Eliades received funding from Johnson & Johnson. Maria Mantzourani and Roberto Labella are employees of Johnson & Johnson Consumer Services EAME Ltd. Bruna Mutti is an employee of Johnson & Johnson GmbH. Deepak Sharma is an employee of Johnson & Johnson Consumer & Personal Products Worldwide, Division of Johnson & Johnson Consumer Companies Inc.
Acknowledgements This work was supported by Johnson & Johnson Consumer & Personal Products Worldwide, Division of Johnson & Johnson Consumer Companies, Inc. Editorial assistance was provided by Dr Julie Ponting of Anthemis Consulting Ltd, funded by Johnson & Johnson Consumer Services EAME Ltd.
references 1.
2.
3.
4.
5. 6.
7.
8.
9.
5.
Conclusions 10.
The desensitising treatments performed with the toothpaste slurries induced significant morphological and compositional changes on the surface of acid-etched dentine. These changes were mostly related to surface occlusion by precipitation of amorphous material. Nevertheless, the acidic challenge removed all these artificial surface layers, creating a dentine surface that resembled that of the acid-etched control. The mouthrinses showed a different reaction pattern. After use of these desensitising treatments, dentine surfaces mostly had opened tubules. One mouthrinse (LADS) was associated
11.
12.
13.
Brännström M, Aström A. Study on mechanisms of pain elicited from dentin. Journal of Dental Research 1964; 43: 61925. Brännström M, Aström A. The hydrodynamics of dentine – its possible relationship to dentinal pain. International Dental Journal 1972; 22: 219-27. Pashley DH. Dentin permeability, dentin sensitivity and treatment through tubule occlusion. Journal of Endodontics 1986; 12: 465-74. Pashley DH, Tay FR, Haywood VB, Collins MA, Drisko CL. Consensus-based recommendations for the diagnosis and management of dentin hypersensitivity. Compendium of Continuing Education in Dentistry 2008; 29: 1-37. Hodosh M. A superior desensitizer – potassium nitrate. Journal of the American Dental Association 1974; 88: 831-2. Orchardson R, Peacock JM. Factors affecting nerve excitability and conduction as a basis for desensitizing dentine. Archives of Oral Biology 1994; 39(Suppl): 81S-6S. Peacock J, Orchardson R. Effect of potassium ions on action potential conduction in A- and C-fibers in rat spinal nerves. Journal of Dental Research 1995; 74: 634-41. Peacock JM, Orchardson R. Action potential conduction block of nerves in vitro by potassium citrate, potassium tartrate and potassium oxalate. Journal of Clinical Periodontology 1999; 26: 33-7. Ajcharanukul O, Kraivaphan P, Wanachantararak S, Vongsavan N, Matthews B. Effects of potassium ions on dentine sensitivity in man. Archives of Oral Biology 2007; 152: 632-9. Wang Z, Jiang T, Sauro S, Wang Y, Thompson I, Watson TF, et al. Dentine remineralization induced by two bioactive glasses developed for air abrasion purposes. Journal of Dentistry 2011; 39: 746-56. Wang Z, Jiang T, Sauro S, Pashley DH, Toledano M, Osorio R, et al. The dentine remineralizing activity of a desensitizing bioactive glass-containing toothpastes: an in vitro study. Australian Dentistry Journal 2011; 58: 1-10. O’Connor DJ, Sexton BA, Smart RCS. Surface analysis methods in materials science. Springer Series in Surface Sciences 23. Berlin (Heidelberg): Springer Verlag, 1992. Gadaleta SJ, Camacho NP, Mendelsohn R, Boskey AL. Fourier transform infrared microscopy of calcified turkey leg tendon.
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Calcified Tissue International 1996; 58: 17-23. 14. Greenhill JD, Pashley DH. The effects of desensitizing agents on the hydraulic conductance of human dentin in vitro. Journal of Dental Research 1981; 60: 686-98. 15. Shen Y, Yue W, Xie A, Li S, Qian Z. Effects of amino acids on
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crystal growth of CaC2O4 in reverse microemulsion. Colloids and Surfaces. B, Biointerfaces 2005; 10: 120-4. 16. Kolker JL, Vargas MA, Armstrong SR, Dawson DV. Effect of desensitizing agents on dentin permeability and dentin tubule occlusion. Journal of Adhesive Dentistry 2002; 4: 211-21.
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Interactions of dentine desensitisers with human dentine: Morphology and composition George Eliadesa,*, Maria Mantzouranib, Roberto Labellab, Bruna Muttic, Deepak Sharmad a
Department of Biomaterials, University of Athens, School of Dentistry, Greece Oral Care Scientific & Professional Affairs, Johnson & Johnson Consumer Services EAME Ltd, Maidenhead, UK c Oral Care Research and Development, Johnson and Johnson GmbH, Neuss, Germany d Oral Care Research and Development, Johnson & Johnson Consumer & Personal Products Worldwide, Skillman, NJ, USA b
keywords
abstract
Dentinal tubule occlusion Potassium oxalate mouthrinse FTIR spectroscopy Raman microscopy EDX spectroscopy SEM
Objective: To evaluate the effect of desensitising agents on human dentine morphology and composition. Methods: Randomly assigned human coronal-dentine specimens were subjected to: (a) no treatment (smear-layer control, n=4); (b) acid etching with 6% citric acid (demineralised control, n=4); (c) treatment with desensitising agents (12 cycles of 60 s treatment with 60 s between-treatment rinsing, n=6 per agent ); and (d) exposure to acidic challenge (pH 5.0 for 90 s, n=6 per agent). The tested products were: Listerine® Advanced Defence Sensitive (LADS; 1.4% potassium oxalate) mouthrinse, Colgate® Sensitive Pro-Relief™ mouthrinse, and toothpaste slurries (paste/water 1:2 wt/wt ratio) of Colgate® Sensitive Pro-Relief™ paste, Crest® Sensitive paste and Sensodyne® Repair and Protect paste. All dentine surfaces were studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Raman microscopy and high vacuum scanning electron microscopy with energy-dispersive X-ray microanalysis (HV-SEM/EDX). Results: Desensitising slurry treatments occluded tubule orifices of acid-etched dentine, creating a randomly distributed surface pattern of particle aggregates. The greatest intratubular penetration of occluding particles was found in dentine treated with LADS. The atomic ratios of Ca/N and Ca/P, and the mineral/matrix ratios increased after toothpasteslurry treatments compared with the acid-etched dentine. However, the acidic challenge removed most surface precipitates and further demineralised these substrates. Before the acidic challenge, the surface features were least affected in specimens treated with Sensodyne® Repair and Protect. After the acidic challenge, the sub-surface occlusion features were least affected in specimens treated with LADS. Clinical significance: Although most tested products achieved occlusion of dentinal tubules and provided evidence of mineral deposits, the deposit formed by LADS demonstrated the greatest resistance to acidic challenge, which simulates intra-oral demineralisation phases. © 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The hydrodynamic theory of dentine sensitivity hypothesises that minute fluid shifts within exposed, patent dentinal
tubules in response to hydrodynamic stimuli activate intrapulpal nerves to cause brief, sharp, well-localised tooth pain.1–3 A corollary to that theory is that anything that can partially occlude dentinal tubules will reduce dentine sensitivity.4 Another corollary is that agents that can reduce
* Corresponding author at: Department of Biomaterials, University of Athens, School of Dentistry, 2 Thivon Str, Goudi,115 27 Athens, Greece. Tel.: +30 210 7461101; fax: +30 210 7461306 E-mail address: [email protected] (G. Eliades) 0300-5712/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
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nerve excitability will reduce dentine sensitivity. Hodosh discovered that 5–30% potassium nitrate applied topically to sensitive dentine could reduce dentine sensitivity,5 while others have shown that raising potassium ion concentrations in tissue fluids reduced nerve excitability.6–8 In vivo studies have confirmed the hydrodynamic theory9 and support the inclusion of 5% potassium nitrate in many desensitising dentifrices. Many currently available desensitising dentifrices and mouthrinses contain “tubule-occluding agents” and have been marketed based on different technologies, with several mechanisms proposed for their interaction with dentine. The purpose of this in vitro study was to evaluate the surface interactions with human dentine of four commercially available dentine-desensitising products and a new 1.4% potassium oxalate mouthrinse (Listerine® Advanced Defence Sensitive; LADS). The null hypotheses were: (1) there is no between-treatment difference in acidetched dentine morphology and composition after the desensitising treatments have been applied; and (2) acidic erosion challenge does not alter the morphology or elemental and molecular composition of dentine surfaces treated with these products.
2.
Materials and methods
2.1
Products tested
2.3
2.4
Desensitising treatment
One dentine sample was placed in each well of a 12-well plate (treatment plate) and 2 mL of test solution (toothpaste/ water slurry or mouthrinse) was applied. One plate was used for each product and solutions were changed after each treatment. The treatment plate was agitated on an orbital shaker at 100 rpm for 60 s to simulate salivary flow. Each dentine sample was transferred to a second 12-well plate (rinse plate) with 2 mL diH2O per well and the plate was agitated as above. The diH2O was removed from the wells with a pipette and the dentine specimens kept undisturbed for 5 min. The dentine specimens were replaced in their respective wells in the treatment plate and the treatment/ rinse cycle repeated a total of 12 times. Half of the specimens (n=6) for each treatment were used for the erosion challenge procedure and the remaining specimens (n=6) were retained for microscopic and spectroscopic analysis.
2.5
2.2
2.6.1
Disk-shaped coronal human dentine specimens (diameter 7–9 mm, thickness 0.7–0.9 mm, n=68; provided by J&J) were placed in individual wells of 12-well plates. The dentine disks were randomly assigned to Groups A–G. Group A comprised the reference specimens, which were untreated and retained a smear-layer (n=4). Group B comprised control acid-etched specimens that were not subjected to any product treatment (n=4). Specimens in Groups C–G (n=12 per group) were acid etched and then treated as described below. Groups C, D and E were treated with toothpaste slurries (Group C: SDRP, Group D: CRSN, Group E: CSRT); Groups F and G were treated with mouthrinses (Group F: CSRM, Group G: LADS). The margins of the top surfaces of the dentine specimens to be exposed to the desensitising and erosion-challenge treatments were marked with permanent ink dots.
Etching procedure
Dentine specimens in Groups B–G were acid etched by immersion in 6% citric acid and sonication at 37 kHz for 180 s, followed by sonication in diH2O for 90 s. The acidetched specimens were stored individually in vials with moist paper in the cap of the vial to keep specimens hydrated. Before applying desensitising treatments, specimens were hydrated in diH2O for 30 min.
The products tested were three toothpastes [Crest® Sensitivity (CRSN), Procter & Gamble, Cincinnati, OH, USA; Sensodyne® Repair and Protect (SDRP), GlaxoSmithKline, Philadelphia, PA, USA; Colgate® Sensitive Pro-Relief (CSRT), Colgate-Palmolive Company, New York, NY, USA] and two mouthrinses [Colgate® Sensitive Pro-Relief (CSRM), Colgate-Palmolive Company, New York, NY, USA; 1.4% potassium oxalate-containing mouthrinse (LADS), Johnson & Johnson Consumer & Personal Products Worldwide (J&J), Skillman, NJ, USA]. All the test products were provided to the investigators by J&J without commercial names and in a blinded fashion. Toothpastes were used as water slurries at 1:2 wt/wt ratios with distilled water (diH2O) after thorough hand and vortex mixing. Slurries were used within 30 min after preparation and new slurries were prepared every 30 min. Mouthrinses were used without modification.
Dentine specimen preparation
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Erosion challenge
Half of the dentine specimens subjected to the desensitising products were exposed to the following erosion challenge. The dentine specimens were individually immersed in 10 mL of demineralisation solution (0.1 M lactic acid saturated with hydroxyapatite; pH 5.0) and sonicated in a water bath at 37 kHz for 90 s. Each dentine specimen was then immersed in 10 mL diH2O for 30 s without sonication.
2.6
Analytical procedures
The top surfaces of specimens (n=2) from Groups A and B and from Groups C–G [after desensitising alone (n=4) and after desensitising and erosion challenge treatments (n=4)] were assessed as follows.
Attenuated total reflectance Fourier transform infrared spectroscopy
An attenuated total reflectance (ATR) cell was used (Golden Gate, Specac, Augusta, USA) attached to a Fourier transform infrared (FTIR) spectrometer (Spectrum GX, Perkin-Elmer, Bacon, UK) operated under the following conditions: deuterated triglycerine sulphate detector; 2000–650 cm-1 wavenumber range; 4 cm-1 resolution; single-reflection diamond ATR element with zinc selenide focusing lenses; 2 mm diameter sampling area; ~2 μm depth of analysis at 1000 cm-1 and 40 scans co-addition. Spectra were subjected to baseline and ATR corrections.
2.6.2
Raman microscopy
The same specimens were studied using a Raman microscope (InVia, Renihaw, Gloucestershire, UK) employing a charge-
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coupled device detector and a 785 nm diode laser. Spectra were recorded under the following conditions: 2000–0 cm-1 Raman shift (wavenumber range); 1200 l/mm grading, 4 cm-1 resolution; 50× magnification; 3 mW laser power at sample; 10 s acquisition time; and ~5–6 μm depth of analysis.
2.6.3
High vacuum scanning electron microscopy and energy-dispersive X-ray microanalysis
All the specimens were subjected to controlled dehydration in a desiccator coated with a ~20 nm thick carbon film in a sputter coater (Bal Tec SCD 004 Sputter Coater with OCD 030 attachment, Balzers, Vaduz, USA) and examined by high vacuum scanning electron microscopy (HV-SEM) under the following conditions: solid state compositional backscattered detector for phase identification; 15 kV accelerating voltage; 10-6 Torr pressure; 90 μA beam current; and 1300× magnification. For energy-dispersive X-ray (EDX) analysis a liquid nitrogencooled Si(Li) detector with a super-ultrathin beryllium window (EDAX Sapphire CDU, EDAX Int, Mahwah, USA) and Genesis software (version 5.1) were used with the following acquisition parameters: 110 μA beam current; 1300× magnification (100×100 μm2 analysis area); 34% detector dead time; 0.4° tilt angle; 35.75° take-off angle; 130.44 eV resolution; and ~3 μm depth of analysis. Qualitative analysis was performed in standardless mode after ZAF and C-coating corrections. Separate specimens (n=2) from Groups A and B, and from Groups C–G [after desensitising alone (n=2) and after desensitising and erosion challenge treatments (n=2)] were sectioned with a thin diamond disk (from the bottom side upwards to a depth of 0.5 mm) and then fractured with a sharp chisel. The fractured surfaces were coated with 20 nm of gold in a sputter coater and studied by HV-SEM as detailed above at magnifications of 2000×, 2500× and 8000×.
The SEM backscattered electron (BE) image demonstrated tubule orifices closed with smear plugs, with well-defined peritubular dentine collars due to their higher mineral content (Fig. 1C). The EDX analysis showed the presence of strong Ca and P peaks, along with C, O, Na, Mg, Si, S and Cl, which is consistent with a highly mineralised dentine surface (atomic ratios: Ca/N = 1.9; Ca/P = 1.5; Fig. 1G). The BE image of the longitudinally fractured specimen exhibited highly mineralised peritubular dentine walls, extending up to the cut surface, with tubule orifices closed by smear-layer plugs (Fig. 1E).
3.2
The ATR-FTIR spectrum of the acid-etched specimens showed a demineralised surface (Fig. 1A, red) as evidenced by the strong reduction in -PO43- v1,v3 and -CO32- (mostly of the b-type at 1405 and 871 cm-1) and an increase in dentine organic matrix (Amide I, II, III, CH2, CH3) peak intensities, resulting in an RIR of 0.2. The residual mineral was a mixture of highly (1023 cm-1) and poorly crystalline apatite (1080 cm-1). The demineralisation was also confirmed by the reduction in the intensity of the -PO43- v1 (958 cm-1) peak and the increase in the intensity of Amide I and III peaks in the Raman spectrum (Fig. 1B, bottom), leading to an RRM of 3.7. The BE image revealed a dentine surface free of smear layer, with open tubule orifices and loss of peritubular dentine, all typical findings of acid-etched dentine (Fig. 1D). The EDX analysis showed a mineral-deficient dentine surface with a strong reduction in all mineral peaks and an increase in C, N and O (atomic ratios: Ca/N = 0.05, Ca/P = 2; Fig. 1H). The BE images of fractured dentine specimens (Fig. 1F) clearly showed a superficial zone of demineralised collagen collapsed onto subsurface mineralised dentine. The mean thickness of this zone was estimated to be 3 μm.
3.3
3. 3.1
Acid-etched dentine specimens (Group B)
Acid-etched dentine specimens subjected to SDRP (Group C)
Results Smear-layer covered dentine specimens (Group A)
The ATR-FTIR spectrum of smear-layer covered dentine specimens (Fig. 1A, blue) demonstrated peaks of -PO43- (v1,v3 at 1180–900 cm-1, v4 at 600–550 cm-1) and -CO32- (1450 cm-1 of a-type + CH; 1405 and 871 cm-1 of b-type), assigned to dentine mineral and peaks of Amide I (1655–1640 cm-1), Amide II (1550–1530 cm-1), Amide III (1250 cm-1) and CH2/CH (1400–1350 cm-1) assigned to the organic components of dentine (mainly collagen type I). The mineral (-PO43- v1,v3 at 1180–900 cm-1 range) to organic matrix (Amide I at 1712–1596 cm-1 range) net peak absorbance area ratio from ATR-FTIR measurements (RIR) was calculated as 3.7. The complex peak of the -PO43- v1,v3 band (~1040 cm-1) exhibited high- and low-frequency shoulders attributed to the various states of the apatite mineral. The Raman spectrum (Fig. 1B, top) showed the characteristic peaks of -PO43- ( v1 at 958 cm-1, v2 at 434 cm-1, v4 at 590 cm-1), -CO32- (v1 at 1083 cm-1, v2 at 865 cm-1, v3 at 1415 cm-1), Amide I (1665–1650 cm-1), Amide III (1270 cm-1), CH2, CH3 groups (G at 1455–1320 cm-1) and lattice vibrations (350–0 cm-1). The mineral (-PO43- v1 at 958 cm-1) to organic matrix (Amide I at 1662 cm-1) net peak absorbance height ratio from Raman measurements (RRM) was calculated as 11.4.
ATR-FTIR spectra of SDRP-treated surfaces before and after acidic challenge are depicted in Fig. 2A. In comparison with the acid-etched control (Fig. 1A, red), SDRP produced a minor increase in the complex -PO43- v1,v3 peak at ~1080 cm-1, with a low-frequency shoulder at 1030 cm-1, which was reduced following the acidic challenge although it retained the original peak shape. The RIR ratios before and after the acidic challenge were 0.3 and 0.2, respectively. The results of Raman analysis (Fig. 2B) confirmed the mineral changes (RRM before, 6.3; RRM after, 4.3). A representative BE image from an area fully covered by the SDRP components is shown in Fig. 2C. A granular layer covered the acid-etched dentine structure, with a great number of particles attached, which consisted of elements with a high mean atomic number. The EDX analysis (Fig. 2G) showed the presence of Si and Ti along with the other dentine elements (atomic ratios: Ca/N = 0.1, Ca/P = 0.8). However, after acidic challenge this layer was largely removed (Fig. 2D). No material deposition was found within dentinal tubules before or after the acidic challenge (Figs. 2E and F, respectively).
3.4
Acid-etched dentine specimens subjected to CRSN (Group D)
ATR-FTIR spectra showed that treatment with CRSN increased the complex -PO43- v1,v3 peak, with a predominant component
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Fig. 1 – Dentine specimens of the control groups. (A) Attenuated total reflectance Fourier transform infrared spectra of smear layer (blue) and acid-etched dentine (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra of smear layer (top) and acid-etched dentine (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image of smear-layer covered dentine surface (1300×, bar 20 μm); (D) BE image of acid-etched dentine surface (1300×, bar 20 μm); (E) BE image of fractured specimen with smear layer (2000×, bar 20 μm); (F) BE image of fractured specimen of acid-etched dentine (2000×, bar 20 μm); (G) energy-dispersive X-ray (EDX) spectrum of smear-layer covered dentine; (H) EDX spectrum of acid-etched dentine.
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Fig. 2 – Dentine specimens treated with Sensodyne Repair and Protect toothpaste (SDRP) slurry. (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm); (G) energy-dispersive X-ray spectrum of treated dentine.
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Fig. 3 – Dentine specimens treated with Crest Sensitivity toothpaste (CRSN) slurry. (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm).
at ~1080 cm-1 that was slightly altered after the acidic challenge (Fig. 3A). The RIR ratios were 0.4 before and 0.3 after the acidic challenge. The Raman spectra (Fig. 3B) confirmed the additional demineralisation after the acidic challenge, showing a reduction of the intensity of the central apatite peak and an increase of the contribution of amides, with minor changes in the spectral profile attributed to increased CH3/CH2 group vibrations. The RRM ratios were 6.3 and 3.5 after the CRSN treatment and following the acidic challenge, respectively. The BE image of the dentine surface after treatment (Fig. 3C) shows the presence of sparsely distributed particles with higher mean atomic number than observed in
demineralised dentine after CRSN treatment; these particles were not observed following the acidic challenge (Fig. 3D). The BE images of the fractured surfaces showed tubular structures free of any precipitants after CRSN treatment (Fig. 3E). The acidic challenge further exposed demineralised collagen, opening more lateral tubule orifices within tubules (Fig. 3F).
3.5
Acid-etched dentine specimens subjected to CSRT (Group E)
The ATR-FTIR spectrum following CSRT treatment (Fig. 4A) demonstrated chemical groups found in the native toothpaste
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(Fig. 4B) with an RIR of 0.1. However, after the acidic challenge a typical spectrum of demineralised dentine was recorded, with traces of residual mineral (-PO43- v1,v3 peak at ~1080 cm-1; RIR 0.1). The Raman spectra demonstrated increased intensities of the -CO32-, CH3/CH2 and TiO2 (140 cm-1) peaks after CSRT treatment, but the contribution of the -CO32- peak was reduced after the acidic challenge, and the TiO2 peak disappeared (Fig. 4C). The RRM values measured before and after acidic challenge were 6.7 and 5.1, respectively. The BE images (Fig. 4D) clearly showed that a granular structure remained on the dentine surface after CSRT treatment, leading to atomic ratios for Ca/N of 0.2 and for Ca/P of 2.2, as determined by EDX analysis (Fig. 4H). Nevertheless, this structure was completely removed from the dentine surface following the acidic challenge (Fig. 4E). The BE images of the fractured specimens after CSRT treatment showed a zone of low mean atomic number material at the dentine surface, with evidence of micro-granular inclusions (Fig. 4F); however, most tubules were free of granular material. The acidic challenge removed this zone (Fig. 4G).
3.6
Acid-etched dentine specimens subjected to CSRM (Group F)
No differences were observed between the ATR-FTIR spectra for untreated acid-etched dentine control (Group B), dentine treated with CSRM and dentine treated with CSRM followed by acidic challenge, apart from minor changes in the -PO43- v1,v3 peak at ~1080 cm-1. The RIR ratios were 0.2 before and 0.1 after the acidic challenge (Fig. 5A). The Raman spectra documented the additional demineralisation after the acidic challenge, with increases in the dentine organic matrix peaks (Amide I, CH3/CH2, Amide III relative to the -PO43- v1 (958 cm-1) peak, leading to an RRM of 6.2 before and 5.4 after the acidic challenge (Fig. 5B). The BE images were similar to those for the acid-etched dentine control, with no evidence of surface precipitates (Figs. 5C and D). The BE images of the fractured specimens showed few crystalline particles located at tubular openings, before and after the acidic challenge (Figs. 5E and F).
3.7
Acid-etched dentine specimens subjected to LADS (1.4% potassium oxalate) (Group G)
The ATR-FTIR spectra of LADS-treated dentine before and after the acidic challenge were identical, and similar to that of the demineralised dentine control (RIR 0.2 before and 0.1 after the acidic challenge; Fig. 6A). The complex -PO43- v1,v3 peak was clearly resolved into two components (1080 and 1030 cm-1). The Raman spectra (Fig. 6B) documented an additional demineralisation after the acidic challenge (seen as a reduction in the intensity of the central apatite peak and an increase of the amide contribution), with no changes in the molecular composition (RRM 3.8 before; RRM 2.2 after). The BE image of the LADS–treated dentine surface (Fig. 6C) showed open tubules with scarce crystals in tubule orifices with atomic ratios for Ca/N of 0.1 and for Ca/P of 2.6, as provided by the EDX analysis (Fig. 6I). After the acidic challenge, the dentine surface (Fig. 6D) was similar to that of the acid-etched control. However, the BE image of the fractured surfaces showed many crystalline structures inside tubule orifices after LADS treatment, some extending to a depth of more than 20 μm (Figs. 6E and G). After the acidic challenge, crystalline structures were still detected on and within tubules (Figs. 6F and H).
4.
Discussion
The present study was designed to investigate the effects of desensitising toothpastes and mouthrinses on the morphology and elemental/molecular composition of aciddemineralised dentine surfaces. The results showed that there were differences in acid-etched dentine morphology and composition after desensitising treatments, requiring rejection of the study’s first null hypothesis. The fact that changes were observed following acid challenges to treated dentine surfaces for three of the five desensitising products requires partial rejection of the second null hypothesis. Occlusion of the patent dentinal tubules in dentine with acid-etched surfaces has been extensively used to study the efficacy of desensitising products with putative occluding technologies.10,11 In the present study, dentine specimens were acid etched with 6% citric acid (pH 1.5) for 3 min and then rinsed in water, with sonication, to avoid extensive demineralisation and formation of a residual smear layer or mineral-salt deposition on the dentine surface. The validation of the method is supported by the SEM images of the acidetched dentine control specimens, in which opened tubules were observed with a 3–5 μm superficially demineralised zone. Dentine surfaces covered with a smear-layer, as produced by sectioning, were used as a fully mineralised dentine control, as they combine the highest mineral content with closedtubule morphology. The desensitising toothpaste agents were tested as slurries with water (2:1 wt/wt ratio) to simulate the slurry used during toothbrushing. Nevertheless, no toothbrushing was performed, to avoid mineral removal from dentine surfaces due to abrasion.11 Instead, an orbital shaker was used to simulate the effect of saliva flow on the precipitated minerals, which also facilitated comparison with the mouthrinses. For the acidic challenge, an organic acid (lactic acid, a by-product of bacterial plaque metabolism) saturated with hydroxyapatite was used at a mild pH, but with sonication, to remove the loosely bound or weakly attached desensitising compounds from dentine, without excessive in-depth demineralisation. ATR-FTIR, Raman microscopy and HV-SEM/EDX were the main analytical techniques used in the study. Although complementary information is given by these techniques, there are differences in their mean sampling depth (2 μm for ATR-FTIR, 5–6 μm for Raman microscopy and 3 μm for EDX in the conditions of the present study), which should be taken into account when interpreting the results. For imaging, only a compositional backscattered detector was employed. This detector minimises the edge effects observed on very rough surfaces (such as fractured specimens) by secondary electron detectors, provides atomic number contrast for easy phase identification, and better matches to the depth of EDX analysis.12 In the present study, the assessment of the SEM images was mainly qualitative, in contrast to the other techniques. Therefore, the SEM results for the extent of tubule occlusion should be carefully interpreted, as applies for most relevant studies. The toothpastes tested were based on bioglass (7.5% Ca-Naphosphosilicate; SDRP), 0.454% SnF2/Na-hexametaphosphate (CRSN) and 8.0% arginine/CaCO3 (CSRT) technologies, whereas the mouthrinses were based on 0.8% arginine, methyl vinyl ether and maleic anhydride copolymer, pyrophosphates and sodium fluoride (CSRM) and 1.4% potassium oxalate (LADS) technologies.
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Fig. 4 – Dentine specimens treated with Colgate Sensitive Pro Relief toothpaste (CSRT) slurry. (A) Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) ATR-FTIR spectra of native material after solution in water and dehydration. Absorbance scale 2000–600 cm-1 region; (C) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (D) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (E) BE image after acidic challenge (1300×, bar 20 μm); (F) BE image of fractured specimen after treatment (2000×, bar 20 μm); (G) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm); (H) energy-dispersive X-ray spectrum of treated dentine.
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j o u r n a l o f d e n t i s t ry 4 1 s 4 ( 2 0 1 3 ) s 2 8 – s 3 9
Fig. 5 – Dentine specimens treated with Colgate Sensitive Pro Relief mouthrinse (CSRM). (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm).
Treatment of acid-etched dentine surfaces with the desensitising toothpaste slurries resulted in the development of precipitates (mainly granular material) covering tubule orifices to various extents. More organised integuments were found after SDRP and CSRT treatments, with the mouthrinses being the least effective. The particles identified on dentine surfaces were mainly composed of C, N, O, Na, Mg, Si, P, S, Cl and Ca. After desensitisation treatments, dentine surface Ca was increased in the CSRT-treated group and Si in the SDRPtreated group, apparently due to adsorption or precipitation of Ca and Si, respectively, from the desensitising agents.
Ti was also found in SDRP and CSRT groups as TiO2, as confirmed by Raman microscopy. The surface specificity of ATR-FTIR demonstrated limited evidence of mineralisation as monitored by the -PO43- v1,v3 band region. The mineral/ matrix ratios measured on desensitised specimens ranged from 0.4 to 0.2, with 0.2 calculated for the acid-etched control and 3.7 for the smear-layer control. However, care should be taken when analysing the complex -PO43- v1,v3 dentine band (1180–900 cm−1) after desensitisation, because many native components of the toothpastes used demonstrated peaks in the same region. The characteristic splitting of the complex
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Fig. 6 – Dentine specimens treated with Listerine Advanced Defence Sensitive (LADS) mouthrinse. (A) Attenuated total reflectance Fourier transform infrared spectra after treatment (blue) and acidic challenge (red). Absorbance scale, 2000–600 cm-1 region; (B) Raman spectra after treatment (top) and acidic challenge (bottom). Linear intensity scale, 2000–0 cm-1 region; (C) backscattered electron (BE) image after treatment (1300×, bar 20 μm); (D) BE image after acidic challenge (1300×, bar 20 μm); (E) BE image of fractured specimen after treatment (2000×, bar 20 μm); (F) BE image of fractured specimen after acidic challenge (2000×, bar 20 μm); (G) BE image of fractured specimen after treatment (8000×, bar 5 μm); (H) BE image of fractured specimen after acidic challenge (8000×, bar 5 μm); (I) energy-dispersive X-ray spectrum of treated dentine.
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-PO43- v1,v3 band into two well-resolved peaks (~1080 cm-1 and ~1030 cm-1) after CSRT, CSRM, and especially after LADS treatments, may imply the presence of more crystalline apatite on the dentine surface.13 The Raman analysis showed more intense -PO43- v1 peaks on acid-etched dentine than seen by ATR-FTIR, apparently due to the greater depth of analysis despite the low laser power used, which resulted in subsurface interferences from intact tissue. Nevertheless, the calculated mineral/matrix ratios were consistent with the ranking determined by ATR-FTIR (6.5–3.6 on treated surfaces vs 4.1 on acid-etched control surfaces and 11.4 on the smear-layer control surface). The images of the fractured specimens demonstrated distinct differences between the toothpaste slurries and the mouthrinses. Although the dentine surface morphology was almost free of precipitates with mouthrinses, crystals were identified within dental tubule canals and orifices. This contrasts with the toothpaste slurries, for which no intratubular extension of the surface-precipitated phases was observed. This was very characteristic after LADS treatment, with the crystals extending up to a depth of 20 μm. It is likely that the rheological characteristics of this potassium oxalate mouthrinse increased its capacity for penetration into tubules to mediate sealing by reacting with ionised Ca and forming insoluble calcium oxalate crystals, known to reduce the hydraulic conductance of dentine.14 The shape of the crystals formed after LADS treatment is consistent with the formation of calcium oxalate monohydrate.15 The crystals found after CSRM treatment were located more superficially. After the acidic challenge all surface-precipitated phases were removed and the morphology of the treated dentine surfaces resembled that of the acid-etched control, except for some sporadic crystals located in tubule orifices after SDRP. This suggests that the retention of the precipitated phases with dentine is rather weak. The mineral/matrix ratios determined both by ATR-FTIR and Raman microscopy were reduced owing to the additional demineralisation. On fractured specimens, more crystals were identified in dentine specimens treated with LADS than in other treated specimens. The development of crystals deep in the tubules following LADS treatment, along with their relatively low solubility, probably maintained the tubular occlusion despite the acidic challenge. The professional application of acidic oxalate solutions to desensitise dentine has long been established, but the clinical effects have not been considered permanent.16 However, in the form of a mouthrinse, a positive effect may be maintained due to the repeated applications.
with significant intratubular subsurface crystalline formation, which resisted the acidic challenge to a greater extent than the deposits formed by the other mouthrinse (CSRM) or any of the toothpaste slurries tested.
Conflict of interest George Eliades received funding from Johnson & Johnson. Maria Mantzourani and Roberto Labella are employees of Johnson & Johnson Consumer Services EAME Ltd. Bruna Mutti is an employee of Johnson & Johnson GmbH. Deepak Sharma is an employee of Johnson & Johnson Consumer & Personal Products Worldwide, Division of Johnson & Johnson Consumer Companies Inc.
Acknowledgements This work was supported by Johnson & Johnson Consumer & Personal Products Worldwide, Division of Johnson & Johnson Consumer Companies, Inc. Editorial assistance was provided by Dr Julie Ponting of Anthemis Consulting Ltd, funded by Johnson & Johnson Consumer Services EAME Ltd.
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The desensitising treatments performed with the toothpaste slurries induced significant morphological and compositional changes on the surface of acid-etched dentine. These changes were mostly related to surface occlusion by precipitation of amorphous material. Nevertheless, the acidic challenge removed all these artificial surface layers, creating a dentine surface that resembled that of the acid-etched control. The mouthrinses showed a different reaction pattern. After use of these desensitising treatments, dentine surfaces mostly had opened tubules. One mouthrinse (LADS) was associated
11.
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