SEARCH

SEARCH BY CITATION

Keywords:

  • Bioactive glass;
  • dentine hypersensitivity;
  • toothpaste;
  • attenuated total reflection Fourier transformed infrared spectroscopy;
  • scanning electron microscopy;
  • energy dispersive X-ray

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background:  Dentine hypersensitivity occurs easily on exposed dentine. This study aimed to evaluate the effectiveness of a novel bioactive glass-containing toothpaste on dentine permeability and remineralization.

Methods:  Thirty dentine discs were divided into three groups of 10 specimens each. The groups corresponded to the following brushing treatments: no brush, distilled water, and bioactive glass-containing toothpaste (Novamin). The toothpaste was applied twice a day for 7 days. Dentine permeability was measured after ethylene-diamine-tetraacetic acid (EDTA) etching, initial application, 3-day application, 7-day application and citric acid challenge, respectively. The dentine morphology was investigated using scanning electron microscopy (SEM). Attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy was performed to monitor the mineral variation on demineralized dentine. Qualitative information of elemental variation before and after treatments on completely demineralized dentine was detected by energy dispersive X-ray (EDX) analysis.

Results:  The bioactive glass-containing toothpaste significantly reduced dentine permeability after the 7-day treatment and showed, under SEM, excellent resistance to acid challenge compared to the other groups. ATR/FTIR and EDX revealed increased mineral content after treatment with Novamin.

Conclusions:  As the innovative bioactive glass-containing toothpaste occlude dentinal tubules and resist acid challenge, it may be useful for the treatment of dentine hypersensitivity and dentine remineralization.


Abbreviations and acronyms:
AS

artificial saliva

ATR

attenuated total reflection

ATR/FTIR

attenuated total reflection Fourier transform infrared spectroscopy

CEJ

cemento-enamel junction

CHA

carbonated hydroxyapatite

EDTA

ethylene-diamine-tetraacetic acid

EDX

energy dispersive X-ray

HA

hydroxyapatite

SEM

scanning electron microscopy

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dentine hypersensitivity remains an oral health problem in adults on a global scale. It is characterized by a high prevalence of exposed cervical dentine.1 Many strategies have been evoked for its treatment, resulting in generally effective and immediate reduction of pain.2 However, based on the hydrodynamic theory,3 the occlusion of dentinal tubules is the main method to treat dentine hypersensitivity by decreasing stimuli-evoked fluid shifts inside the dentinal tubules, and thereby relieving symptoms of sensitivity.4,5

Several studies have shown that desensitizing products including varnishes, resins and remineralizing agents are able to provide tubule obturation.6,7 However, toothbrushing using desensitizing toothpastes could also be an acceptable at-home everyday treatment of dentine hypersensitivity. As a result, in vitro8 and in vivo9,10 studies have claimed success in relieving dentine hypersensitivity by using various desensitizing toothpastes. Nevertheless, most of the previous studies focused on the immediate occlusion effect of dentinal tubules with little evidence of whether they could resist pulpal pressure, daily brushing procedures or acidic foods over time.

There is also little evidence of the bioactivity of toothpastes in apatite formation, which may be extremely important in preventing the recurrence of dentine hypersensitivity by remineralization effects. In this regard, a recent bioactive glass-containing toothpaste has been developed because of the excellent remineralizing characteristic of its active principle.11,12 Although the capability of a bioactive glass-containing dentifrice in occluding dentinal tubules has been shown,13,14 these in vitro studies have only provided evidence for immediate dentine hypersensitivity treatment. Further studies are needed to compare its bioactivity to common desensitizing toothpastes and prove its effect over time following daily brushing and citric acid challenge.

The present study was undertaken to evaluate the ability of desensitizing toothpastes in forming apatite and/or mineral complexes on demineralized dentine, monitored by a non-destructive approach known as attenuated total reflection Fourier transformed infrared (ATR/FTIR) spectroscopy. Dentine permeability was also quantitatively evaluated over 7 days to estimate the ability of desensitizing toothpastes to reduce the dentinal fluid flow following daily brushing and citric acid challenge.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sample preparation for dentine permeability evaluation

Caries-free extracted human third molars were obtained from healthy adults under a protocol approved by the local Ethics Committee of the School and Hospital of Stomatology, Wuhan University, China. The teeth were stored in 0.5% thymol at 4 °C for no longer than a month prior to use.

Thirty dentine discs with a thickness of 1.0 ± 0.1 mm were prepared by sectioning each tooth over the cemento-enamel (CEJ) junction using a low-speed water cooled diamond saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA). The occlusal enamel was removed from each tooth exposing the middle dentine. A standard smear layer was created on both sides of the dentine specimens using 600-grit silicon carbide paper for 30 seconds under constant water irrigation.15

Experimental design for dentine permeability evaluation

The dentine discs were randomly divided into three groups, containing 10 specimens each, which received the following treatments: Group 1 (Control) – EDTA-etched specimens; Group 2 – EDTA-etched specimens brushed with distilled water for 2 minutes twice a day for 7 days; and Group 3 – EDTA-etched specimens brushed with 1.0 g bioactive glass-containing toothpaste (Novamin) (Glaxo Smith Kline, Weybridge, Surrey, UK) for 2 minutes twice a day (at 8 am and 5 pm) for a period of 7 days between brushing.

All specimens from each group were kept in artificial saliva (AS) at 37 °C (pH 7.4) which was replaced every 24 hours.16 The composition of the AS is listed in Table 1. The active components of the desensitizing toothpaste used in this study were calcium sodium phosphosilicate and silicon dioxide. A toothbrush (SpinBrushPro, Procter & Gamble Co., Cincinnati, OH, USA) with extra soft bristles was applied to the occlusal surface at an inclination of about 90° under a constant loading (2 N) for 150 strokes/minute for 2 minutes. The brushing load was measured with a top loading balance during brushing. After all measurements of dentine permeability had been completed over the 7-day period, all specimens were immersed in 6 wt% citric acid (pH 1.5) for 1 minute, followed by rinsing in distilled water for 2 minutes to determine how acid resistant were the reductions in permeability.

Table 1.   Composition of artificial saliva storage solution
CompositionConcentration (mmol/L)
CaCl21.5
KCl50
KH2PO40.9
Tris20

Dentine permeability measurement

Thirty dentine discs were used for the dentine permeability measurement. The smear layers on both sides of the dentine discs were removed by immersion in 0.5 M EDTA solution (pH 7.4) for 2 minutes under constant stirring (75 rpm). The permeability values obtained after EDTA etching were arbitrarily assigned a value of 100%. The permeability of dentine was measured by a fluid filtration system in a split-chamber unit at a simulated pulpal pressure of 20 cm H2O. Each dentine disc was held tightly between a pair of rubber ‘O’ rings, each connected to a plexiglass slab (2 cm × 2 cm × 0.5 cm) providing an exposed dentine surface area of 0.38 cm2 for filtration of deionized water. Briefly, fluid flow was measured by following the movement of an air bubble trapped within a 25 μL capacity microcapillary tube (Microcaps, Fisher Scientific, Atlanta, GA, USA) that was horizontally positioned between the pressure reservoir and the dentine disc. Fluid flow across the dentine discs were then calculated as hydraulic conductance (Lp) (μLmin−1 cm−2 cm H2O−1), by dividing the fluid flow (μL min−1) by the exposed dentine surface area (cm2) and the water pressure (cm H2O).17,18 The permeability of each specimen was expressed as a percentage (Lp%) of the fluid flow through the EDTA-etched (2 minutes) dentine disc of the same specimen. Dentine permeability was measured at five time points: EDTA-etching, initial brush, 3-day brush, 7-day brush and after citric acid challenge.

Statistical analysis was performed by SPSS 16.0 (SPSS Inc. Chicago, IL, USA) for Windows. Means and standard deviations of Lp% values were calculated, and the experimental data obtained were reported as the percentage of the permeability values created by EDTA etching (2 minutes). Homogeneity of variance was determined by using Levene’s test. Two-way repeated measures ANOVA was applied to evaluate the dentine permeability results, considering treatment as the main effect and treatment time as the repeated measure. Pairwise comparisons procedures for one-way repeated measures ANOVA were made within each group. Post hoc multiple comparisons were used to isolate and compare the significant results using Tukey test at a 5% significance level.

ATR/FTIR spectroscopy

ATR/FTIR spectra of the specimens of the three brushed groups were obtained before and after EDTA etching, 7-day brushing and citric acid challenge. Three additional extracted teeth were used to obtain three mid-coronal dentine discs. The dentine discs were sectioned in two halves and then completely demineralized at 25 °C in 0.5 M EDTA solution (pH 7.4) with continuous stirring for 15 days (Demin). The end point of complete demineralization was monitored using digital radiography.19 Two dentine halves were stored in AS (Demin + AS) without any treatment for 7 days. The remaining dentine specimens (two halves for each group) were treated with the toothpaste by brushing for 2 minutes on the dentine surface (Demin + DW, Demin + Novamin). The specimens were finally rinsed in deionized water and immersed in AS for 7 days. Spectra were obtained before and after treatments using a Nicolet 5700 FTIR spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a diamond attenuated total reflection (ATR) accessory. The pulpal surface of each specimen was marked by means of a water-cooled high-speed handpiece with a fine bur. The specimens were then put onto the diamond crystal of the ATR accessory with the marked surface facing up. Spectra were collected in the range 800–1800 cm−1 at 4 cm−1 resolution using 64 scans. ATR/FTIR spectra were analysed by OMNIC 8 software (Nicolet, Madison, WI, USA), and the spectra of water was obtained and subtracted from each original spectra.20 Subsequently, raw spectra were processed by smoothing, baseline corrected, and normalized to the amide I peak.21 In addition, ATR/FTIR spectra of Novamin toothpaste alone were also recorded.

SEM/EDX analysis

Subsequent to the ATR/FTIR spectroscopy, the completely demineralized specimens were rinsed with deionized water and dried in a desiccator and coated with carbon in a vacuum evaporator (EIKO IB-3 ion coater, Hitachi, Japan). An energy dispersive X-ray (EDX) apparatus attached to a SEM (FEI QUANTA-200, Eindhoven, Netherlands) was used to identify elemental analysis on the dentine surface induced by the different treatments. Novamin toothpaste was also dispersed in distilled water and then dipped onto aluminum paper prior to being air dried. Novamin particle size and dentine surface morphology changes following the 7-day brushing and acid challenge were obtained using the SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dentine permeability measurements

Table 2 shows the dentine permeability data produced by three groups at different time points, expressed as percentages of the permeability after EDTA etching for 2 minutes. The Lp% values of the Novamin group gradually decreased over the 7 days. The two-way repeated ANOVA analysis for brushing treatment revealed a significant main effect regarding the treatment time (p < 0.001), treatment group (p < 0.001), and time × group interaction (p < 0.001). Post hoc Tukey test indicated that the Novamin-treated group reduced dentine permeability 85.1%, down to a residual value of only 14.9%, which was significantly lower than other groups as an overall effect (p < 0.001). Brushing with distilled water also led to significant Lp% decrease instantly and at 3-day time points (p < 0.05), while in the control group no significant increase was observed in the 7-day treatment (p > 0.05). Although all the groups showed significant Lp% recovery after acid challenge, Novamin exhibited a significantly lower increase in Lp% values following acid challenge compared to the other groups (p < 0.001). Pairwise comparisons for one-way repeated ANOVA of Novamin indicated that dentine permeability at 7 days was significantly lower compared to the initial brush or the 3-day brush (p < 0.05).

Table 2.   Dentine permeability after treatment for 7 days
GroupsEDTA applicationInstant brush3-day brush7-day brushAcid challenge
  1. The values (expressed as (%)) were reported as means ± standard deviations. Lp after EDTA treatment represented the maximum permeability (Lp = 100%). Groups with different superscripts in the same row are statistically significant (p < 0.05).

Control (AS immersion)100 ± 0a100.2 ± 7.8a99.9 ± 9.0a98.4 ± 10.7a141.6 ± 10.9a1
Distilled water (DW)100 ± 0b60.3 ± 9.4b164.2 ± 7.7b264.5 ± 9.8b2112.9 ± 12.1b3
Novamin100 ± 0c23.7 ± 9.8c119.1 ± 8.5c214.9 ± 7.6c329.0 ± 9.5c4

ATR/FTIR spectroscopy

The ATR/FTIR spectra of untreated mineralized dentine, toothpaste treated dentine and completely demineralized dentine are shown in Figs 1 and 2. All representative spectra were recorded in the region of 800–1800 cm−1. The phosphate ν1, ν3 stretching (885–1180 cm−1) and CO32−ν2 bands (845–885 cm−1) result from the mineral components of the dentine (i.e. apatite),22 while the amide I, II and III bands at 1180–1725 cm−1 arise from the organic components. The obvious intensity changes of the phosphate ν1, ν3 bands indicated demineralization and remineralization effects (Figs 1 and 2).

image

Figure 1.  A representative series of ATR/FTIR spectra of treated dentine surface were marked in different colours in sequence (1–4) depicting four types of treatments: untreated dentine (1-black); EDTA etching for 2 minutes (2-red); 7-day brush (3-blue); and acid-challenge (4-green). The spectra were normalized to amide I. (a) control, (b) distilled water (DW), (c) Novamin.

Download figure to PowerPoint

image

Figure 2.  ATR/FTIR spectra (a1–c1) and EDX analysis (a2–c2) of dentine surface on completely demineralized dentine after treatments followed by 7-day AS immersion. (a1, a2) Demin + AS, (b1, b2) Demin + DW, (c1, c2) Demin + Novamin.

Download figure to PowerPoint

After baseline correction and normalization of all bands to the amide I band for each specimen, the intensity of peaks ν1, ν3 remained relatively high prior to EDTA etching. However, after immersing in 0.5 M EDTA (pH 7.4) for 2 minutes, the intensity of ν1 and ν3 decreased dramatically (Fig 1). When EDTA-treated dentine were brushed with Novamin for 7 days, apatite peak was at 1080 cm−1, even higher than the peaks observed before EDTA etching (Fig 1c). Nevertheless, remarkable differences were noted among groups after citric acid challenge. Novamin showed the highest phosphate peak after citric acid (Fig 1). Some variations were found in phosphate v1, ν3 bands in the control group, probably induced by the deposition of some compounds of calcium and phosphates induced AS (Fig 1a).23 However, brushing with distilled water showed even lower apatite peaks (ν1, ν3) after 7-day treatment (Fig 1b).

No image could be captured by digital radiography after 15 days of complete demineralization. However, completely demineralized dentine displayed no v1, ν3 bands at 885–1180 cm−1 and small bands after 7 days of immersion in AS (Fig 2a1). A slight increase was also found in v1, ν3 bands in the Demin + AS group, probably induced by the deposition of some compound of calcium and phosphates induced AS (Fig 2a1). Similarly, very small phosphate peaks (ν1, ν3) were found in the Demin + DW group (Fig 2b1). In the Demin + Novamin group, the phosphate peaks (ν1, ν3) were faint before Novamin application but a very strong apatite peak at 1080 cm−1 was detected after AS immersion (Fig 2c1), indicating a clear sign of dentine remineralization bioactivity.

A unique phosphate peak at 1040 cm−1 was only observed in the ATR/FTIR spectra of Novamin paste, which indicated the calcium-sodium-phosphosilicate contained in the toothpaste (Fig 3a). However, no amide peak was detected which indicated organic substance.

image

Figure 3.  (a) ATR/FTIR spectra of original Novamin paste and (b) SEM microscopic image of Novamin particles at 35 000× magnification.

Download figure to PowerPoint

SEM/EDX examination

Novamin particle size was confirmed by SEM which showed agglomerated, nanometric particles approximately 0.1 μm at 35 000× magnification (Fig 3b).

The dentine specimens immersed in AS for 7 days resulted in some irregular deposits on the dentine surface, but most of the dentinal tubules were open (Fig 4a1 and a2), while those brushed with distilled water showed few precipitates on the dentine surface and some were partially obliterated (Fig 4b1 and b2). The application of Novamin toothpaste for 7 days was responsible for the production of a smear layer-like coating that occluded most of the tubule orifices. Very few tubule orifices remained partially open (Fig 4c1 and c2).

image

Figure 4.  SEM micrographs of the dentine surface morphology following different brushing treatments for 7 days prior to acid challenge. (a1, a2) EDTA-etched dentine surface after AS immersion for 7 days. Most of the dentinal tubules were open. Note some irregular deposits on the surface or even in the tubules (a2). (b1, b2) Dentine surface appeared smooth after brushing with distilled water for 7 days but some debris in the tubules could still be noted (b2). (c1, c2) Dentine was covered by a smear layer, leaving very few open tubules following brushing with Novamin for 7 days. Dentinal tubules were occluded by debris which completely obliterated dentinal orifices.

Download figure to PowerPoint

After challenging the dentine surfaces with citric acid solution, little debris could be observed in the groups that did not receive toothpaste treatment, especially for the AS immersion group (Fig 5a3–b4). A more uniform and smooth morphology was detected on the dentine surface with Novamin for 7 days and exposed to citric acid attack. However, the dentine surface remained covered by a homogeneous layer which occluded the dentinal tubules with small acid-resistant granular deposits (Fig 5c3 and c4).

image

Figure 5.  SEM micrographs of 7-day treated dentine surface after exposure to 6% citric acid for 1 minute. (a3, a4) Citric acid immersion enlarged the dentinal tubules by removing subsurface peritubular dentine. Most deposits in the tubules no longer existed in the no brush group. (b3, b4) In the distilled water (DW) brushed group, porous dentine was evident because citric acid dissolved the deposits on the dentine surface and in the tubules. (c3, c4) Partially open dentinal tubules could be observed after acid challenge in the Novamin treated group. However, a thin film with granular deposits was still visible on the top of the dentine surface covering most tubules (c4).

Download figure to PowerPoint

The EDX analysis undertaken on the completely demineralized dentine surface immersed in AS for 7 days showed a large amount of oxygen, potassium and chloride. Calcium and phosphate were rarely observed (Fig 2a2). The most common elements detected in Demin + DW group were carbon and oxygen (Fig 2b2). Demin + Novamin showed high amounts of calcium and phosphate accompanied by a small amount of silicon (Fig 2c2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The evaluation of apatite and/or mineral complex depositions in scaffolds, when immersed in simulated body fluid, is a commonly accepted method to establish the bioactivity of a constituent to remineralization.23 This study was designed to evaluate the bioactive properties of desensitizing toothpastes related to their ability to reduce the dentinal fluid flow over 7 days following daily brushing and citric acid challenge. In order to accomplish the latter, a hydrostatic fluid filtration system was also used to estimate the dentine permeability changes induced by the experimental treatments.

ATR/FTIR spectroscopy has been shown to be an effective non-destructive approach for surface analysis which ensures high comparability of spectra before and after treatments.21 ATR/FTIR and EDX showed important changes in mineral content of the treated dentine. For instance, the ATR/FTIR analysis showed that EDTA etching caused dissolution of the mineral phase of the dentine by decreasing and sharpening the contour of phosphate peaks. The calcium-sodium-phosphosilicate containing toothpaste was the only product used in this study to clearly induce signs of dentine remineralization by the formation of apatite. In most previous in vitro studies,13,14,24 the remineralization property of Novamin toothpaste has not been accurately illustrated due to a lack of appropriate remineralization time for minerals to react and precipitate on the dental tissues. On the contrary, the 7-day immersion in AS was used in the current study to provide a more realistic scenario for dentine remineralization. Moreover, it could be noticed that the intensity phosphate peaks of the DW group was even lower than the AS control group after the 7-day treatment (Fig 1a and b). This may be because the brush strokes with water removed some of the mineral deposition on the dentine surface. Both partially demineralized (EDTA 2 minutes) and completely demineralized dentine (EDTA 15 days) have been applied in this study, showing different remineralization capabilities. The reason for choosing a completely demineralized model was to eliminate the ambiguity in differentiating the remineralized apatite crystallites from remnant apatite existing in dentine.25

It is important to consider that calcium-sodium-phosphosilicate is an inorganic compound in the class of highly biocompatible materials that reacts in aqueous environments to release calcium (Ca2+), sodium (Na+), and phosphate ions (PO43−).26 More specifically, Na+ ions begin to exchange with hydrogen cations (H+ or H3O+), which allows Ca2+ and PO43− to be released from the filler particle structure. Ca2+ and PO43− ions from the Novamin toothpaste, along with mineral ions in saliva, are able to form a calcium phosphate (Ca-P) layer onto dentine surfaces or into tubules,12,27 which may result in the physical occlusion enhanced by silicates.28 Burwell26 also reported that when calcium-sodium-phosphosilicate bioactive glass is introduced into the oral environment, the material could result in the formation of carbonated hydroxyapatite (CHA), a mineral chemically similar to that in natural teeth.

In accordance with the results obtained using the ATR/FTIR in Novamin treated demineralized dentine, the EDX analysis showed large amounts of calcium, phosphate and carbon, confirming that Novamin toothpaste may have remineralizing properties.

The bioactive glass particles in Novamin toothpaste may also induce hydroxyapatite (HA) precipitation from their interaction with AS and offer acid resistance to the dentine surfaces and smear layer.24 The results of this study showed that the dentine specimens treated with Novamin showed higher content of minerals and apatite compared to the other toothpastes, even after citric acid challenge.

These observations were consistent with attained fluid permeability measurements. Indeed, the tubule occlusion capability and the remineralizing effects of Novamin were quantitatively demonstrated by the dentine permeability test which showed the lowest fluid filtration among all the groups even after the citric acid challenge. A brushing force of around 2 N has been used by most researchers to simulate normal toothbrushing force.29,30

The SEM examination showed that the dentine surfaces treated by Novamin appeared to have few exposed dentinal tubules and less diameter enlargement after 6% citric acid challenge. Also in this case, the resistance to brushing and acid challenge procedures may be explained by the combined effect of the remineralization and mechanical occlusion of Novamin mentioned above.

The dentine demineralized specimens treated with Novamin and immersed in AS for 7 days without brushing showed that smear layer-like deposits could be found on the dentine surface but not in the tubules by SEM. These may be explained by the fact that delicate mineral deposits from AS could not resist the rinsing action of 20 cm pressure in the permeability test and were flushed out of the tubules. It is important to consider that the first brushing procedures led to a neat 40% decrease of dentine permeability. This may have been due to more smear layer creation by bristles and abrasives.8

After citric acid challenge, there were increases in Lp% values in all groups, but Novamin showed the lowest increment (14.1%) (Table 2). The results of the permeability tests and those obtained with the SEM/EDX observations, which showed a Ca-P containing layer on Novamin treated dentine even after acid challenge, support the concept that a desensitizing toothpaste with remineralizing properties may be more appropriate for long-term home treatment of dentine hypersensitivity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by the Key Technologies Research and Development Program of Hubei Province of China (No. 2007AA301B28) and the Youth Chenguang Project of Science and Technology of Wuhan City, China (No. 200950431186).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References