Abstract
Objective
This study aimed to compare the in vitro dentinal tubule occluding efficacy of two different methods using a nano-scaled bioactive glass (BG)-containing desensitising agent.
Methods
Citric acid treated dentine discs were randomly divided into 7 groups (n = 8). Group A1, A2 and A3: dentine discs coated with BG desensitising paste; Group B1, B2 and B3: dentine discs coated with BG desensitising paste and covered with matched transparent trays; and control group (Group C): dentine discs treated with deionised water. Field Emission Scanning Electron Microscopy (FE-SEM) was used to capture topographical images of each dentine discs after they were immersed in artificial saliva for corresponding treatment time and dentinal tubules exposure rates were thus measured. Elemental compositions of dentine discs were identified using Energy-Dispersive X-ray Spectroscopy (EDX).
Results
FE-SEM revealed better tubule occluding effects in Group B. Dentinal tubules in Group B3 were totally occluded with continuous homogeneous minerals to a depth of 20.6-24.7 μm. Dentinal tubule exposure rates in Group B1, Group B2 and Group B3 were lower than that in Group A1, Group A2 and Group A3. EDX indicated that occluding deposits observed in each group were calcium-deficient hydroxyapatite.
Conclusions
The application of transparent trays in combination with nano-scaled BG-containing desensitising paste could increase the dentinal tubule occluding effectiveness of the latter one and shorten the treatment time.
Clinical relevance
Transparent trays could be used in combination with BGs-containing desensitising paste as the containers of the latter one in order to increase the dentinal tubule occluding effectiveness. This could lead to the development of a new therapeutic technique for treating dentine hypersensitivity.
1
Introduction
Dentine hypersensitivity (DH) is a common clinical dental condition. It is characterised by distinctive short, sharp pain arising from exposed dentine in response to external stimuli, which typically are thermal, evaporative, tactile, osmotic, or chemical . The prevalence of DH varies considerably from the differences in populations studied and in methods employed, and would increase significantly in patients with periodontal surgeries . The hydrodynamic theory proposed by Brännström is the most generally accepted theory regarding the mechanism of DH . Such a theory suggests that pain-provoking stimuli would increase the flow of dentinal tubular fluid, or change the flow direction, and consequently, stimulate the nerves around odontoblasts, leading to DH. An understanding of the hydrodynamic mechanism provides occlusion of dentinal tubules (which could help to reduce fluid flow and decrease dentine permeability) and reduction of intradental nerve excitability (eg. by the application of chemical agents containing potassium ions) as the two main DH treatment strategies, and evokes employments of dentine desensitising products containing fluoride, strontium salts, oxalate, glutaraldehyde, dendrimers, and more recently studied and utilised bioactive glasses .
Bioactive glasses (BGs) are highly biocompatible materials containing calcium sodium phosphosilicate as their active ingredient. Due to their abilities in inducing the formation of calcium phosphate precipitates in physiological liquid environments, BGs have been increasingly studied in the field of human hard tissue regeneration and remineralisation . This property, together with their appropriate particle size, provides BGs with potentials in occluding dentinal tubules, as well as promoting dentine remineralisation and inhibiting dentine demineralisation . Desensitising dentifrices and toothpastes containing NovaMin ® (based on the original 45S5 Bioglass) as their major constituents have been reported to achieve dentinal tubule occlusion in vitro and relieve DH symptoms in vivo [17–20]. Du et al. have conducted a double-blind clinical trial to compare the DH relieving effect of a NovaMin ® -containing dentifrice with a strontium chloride-containing dentifrice and found that although DH was alleviated or even disappeared after the application of both products for 6 weeks, visual analogue scale (VAS) measurements indicated that NovaMin ® -containing dentifrice was more effective in reducing dentine sensitivity . Pradeep et al. have compared the effectiveness of a BGs-containing toothpaste with toothpastes containing potassium nitrate or amine fluoride, and reached similar conclusions . In addition, BGs exhibit strong antimicrobial activities in vitro , which might be beneficial to the relief of DH symptoms by inhibiting pulpal response against bacterial .
Although there are advantages such as easy and immediate availability and low cost in the use of over-the-counter (OTC) dentine desensitisers (such as those mentioned above), one of the major drawbacks is that they require at least 2–4 weeks to relieve DH symptoms . Due to the important but time-consuming apatite crystalisation processes, bioactive glass toothpastes or dentifrices might even take up to 6 weeks to show their effects. In-office desensitisers (eg. BGs-containing gels or pastes) are thus considered to overcome this shortcoming. However, in the present clinical practice, most of these BG desensitisers play a role by coating. This might take more than 2 weeks for obvious effectiveness to be observed, due to saliva flow, food and beverage consumption, and tooth brushing . Poor patient compliance will also reduce the desensitising effect.
The aim of this in vitro study was to investigate whether a modified BGs desensitiser applying method could improve the dentinal tubules occluding effects. In this modified method, transparent trays were employed in combination with simple coating of BGs desensitiser. Transparent trays which were made of polyvinylacetatic were commonly used as orthodontic retainers and the container of home-use bleaching agents in dental clinics and in this study they were used as a supporter to ensure sufficient contact time and effective concentration of BGs-containing desensitiser during treatment. The null hypothesis was that no difference would be found between the modified method and the simple coating method.
2
Materials and methods
2.1
Dentine disc preparation
Extracted human third molars without caries, fractures or restorations were selected from patients between 18 and 40 years old with the written informed consent under a protocol approved by the Ethics Committee of Affiliated Stomatology Hospital of Nanjing Medical University. The teeth were cleaned thoroughly and stored in 0.1% thymol aqueous solution at 4 °C for a week prior to use .
Fifty-six dentine discs, free of coronal enamel or pulp tissue, each with a thickness of approximately 1.0 mm, were prepared by making two parallel cuts perpendicular to the long axis of each tooth, above the cement-enamel junction (CEJ), using a low-speed water cooled diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA). The coronal surfaces of these dentine discs were sequentially polished with 400, 800 and 1200 grit carbide polishing papers under running water. The smear layer was removed with an ultrasonic cleaner (FS20, Fisher Scientific Co., Pittsburgh, PA, USA) in deionised water (DW) for 30 s.
2.2
Transparent tray preparation
Columned epoxy resin bases (15 mm in diameter and 15 mm in height) were prepared and then embedded with alginate impression material. Resin discs (1.5 mm in thickness) were made of light-cured composite resin (Filtek Z350 XT, 3 M ESPE, St. Paul, MN, USA) and placed on columned epoxy resin bases to occupy space for dentine discs. Transparent trays matched with the resin discs and embedded columned epoxy resin bases beneath them were fabricated with 1.0-mm polyvinylacetate plates (Erkoflex, Erkodent Erich Kopp GmbH, Pfalzgrafenweiler, Germany) by a vacuum thermalforming unit (Erkoform-3d, Erkodent Erich Kopp GmbH). The resin discs were then removed.
2.3
Experimental procedures
Dentine discs were treated with 6% citric acid for 2 min to establish sensitive dentine model, within which the dentinal tubules were almost totally open . After rinsed with DW for 30 s, these treated dentine discs were equally divided into 7 groups, each containing 8 discs.
Group A1, A2 and A3: dentine discs were coated with BG desensitising paste (DDS™, Datsing Bio-tech Co., LTD, Beijing, China) for 2 min twice a day using cotton swabs.
Group B1, B2 and B3: dentine discs were fixed on top of the columned epoxy resin bases, coated with BG desensitising paste (DDS™, Datsing Bio-tech Co., LTD) for 2 min, and covered with matched transparent trays. This procedure was repeated once a day.
Group C (control group): dentine discs were treated with DW for 2 min, twice a day.
Artificial saliva (AS) containing CaCl 2 (0.7 mM/L), MgCl 2 ·6H 2 O(0.2 mM/L), KH 2 PO 4 (4.0 mM/L), KCl (30 mM/L), NaN 3 (0.3 mM/L) and HEPES buffer (20 mM/L) was used in this study . The pH of AS was adjusted to 7.4. In subsequent experiments, after coated with BG desensitising paste or treated with the desensitising procedure using BG desensitising paste combined with transparent trays, dentine discs were immediately immersed in AS, which was changed every 24 h. Treatments for Group A1 and B1, Group A2 and B2, and Group A3 and B3 lasted for 1 day, 3 days and 7 days, respectively, and Group C for 7 days. Dentine discs were then rinsed with DW for 30 s and dried for 48 h under vacuum. All the dentine discs were split perpendicularly to their treated surfaces and then characterised by Field Emission Scanning Electron Microscopy (FE-SEM) and Energy-Dispersive X-ray Spectroscopy (EDX).
2.4
Dentine discs characterisations
Surface topography of dentine discs from each treatment group was observed using FE-SEM (LEO 1530VP, LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) with a beam voltage of 15 kV after dentine discs were dried for 48 h under vacuum and sputter-coated with gold. Each image was calibrated individually using a 10 μm bar on the negative and analysed using image analysis software ImagePro Plus 6.0 for Windows (Media Cybernetics, Silver Spring, MD, USA). A standard area was marked on each image and only those tubules within this area of interest (AOI) were measured. Tubules that overlapped the edges of the marked area and confused the data by including only portions of tubules were excluded. The grayscale used ranged from 0 to 255 where 0 = black and 255 = white and a level was chosen for the threshold such that only the tubules were highlighted. Eight images captured under this protocol were selected randomly from each dentine disc. The tubule exposure rate of each dentine disc was calculated and the mean diameter of dentine discs was measured. An attached energy-dispersive X-ray spectroscopy (EDX) apparatus was used to identify elemental composition on the dentine surface treated with different methods and the vertical sections were observed with FE-SEM as well.
2.5
Statistical analysis
Statistical analysis was performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). Differences of tubule exposure ratio between Group A and Group B were analysed using a paired t -test. Differences of Ca/P ratio of each group were analysed using one-way ANOVA analysis. P-values less than 0.05 were considered statistically significant.
2
Materials and methods
2.1
Dentine disc preparation
Extracted human third molars without caries, fractures or restorations were selected from patients between 18 and 40 years old with the written informed consent under a protocol approved by the Ethics Committee of Affiliated Stomatology Hospital of Nanjing Medical University. The teeth were cleaned thoroughly and stored in 0.1% thymol aqueous solution at 4 °C for a week prior to use .
Fifty-six dentine discs, free of coronal enamel or pulp tissue, each with a thickness of approximately 1.0 mm, were prepared by making two parallel cuts perpendicular to the long axis of each tooth, above the cement-enamel junction (CEJ), using a low-speed water cooled diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA). The coronal surfaces of these dentine discs were sequentially polished with 400, 800 and 1200 grit carbide polishing papers under running water. The smear layer was removed with an ultrasonic cleaner (FS20, Fisher Scientific Co., Pittsburgh, PA, USA) in deionised water (DW) for 30 s.
2.2
Transparent tray preparation
Columned epoxy resin bases (15 mm in diameter and 15 mm in height) were prepared and then embedded with alginate impression material. Resin discs (1.5 mm in thickness) were made of light-cured composite resin (Filtek Z350 XT, 3 M ESPE, St. Paul, MN, USA) and placed on columned epoxy resin bases to occupy space for dentine discs. Transparent trays matched with the resin discs and embedded columned epoxy resin bases beneath them were fabricated with 1.0-mm polyvinylacetate plates (Erkoflex, Erkodent Erich Kopp GmbH, Pfalzgrafenweiler, Germany) by a vacuum thermalforming unit (Erkoform-3d, Erkodent Erich Kopp GmbH). The resin discs were then removed.
2.3
Experimental procedures
Dentine discs were treated with 6% citric acid for 2 min to establish sensitive dentine model, within which the dentinal tubules were almost totally open . After rinsed with DW for 30 s, these treated dentine discs were equally divided into 7 groups, each containing 8 discs.
Group A1, A2 and A3: dentine discs were coated with BG desensitising paste (DDS™, Datsing Bio-tech Co., LTD, Beijing, China) for 2 min twice a day using cotton swabs.
Group B1, B2 and B3: dentine discs were fixed on top of the columned epoxy resin bases, coated with BG desensitising paste (DDS™, Datsing Bio-tech Co., LTD) for 2 min, and covered with matched transparent trays. This procedure was repeated once a day.
Group C (control group): dentine discs were treated with DW for 2 min, twice a day.
Artificial saliva (AS) containing CaCl 2 (0.7 mM/L), MgCl 2 ·6H 2 O(0.2 mM/L), KH 2 PO 4 (4.0 mM/L), KCl (30 mM/L), NaN 3 (0.3 mM/L) and HEPES buffer (20 mM/L) was used in this study . The pH of AS was adjusted to 7.4. In subsequent experiments, after coated with BG desensitising paste or treated with the desensitising procedure using BG desensitising paste combined with transparent trays, dentine discs were immediately immersed in AS, which was changed every 24 h. Treatments for Group A1 and B1, Group A2 and B2, and Group A3 and B3 lasted for 1 day, 3 days and 7 days, respectively, and Group C for 7 days. Dentine discs were then rinsed with DW for 30 s and dried for 48 h under vacuum. All the dentine discs were split perpendicularly to their treated surfaces and then characterised by Field Emission Scanning Electron Microscopy (FE-SEM) and Energy-Dispersive X-ray Spectroscopy (EDX).
2.4
Dentine discs characterisations
Surface topography of dentine discs from each treatment group was observed using FE-SEM (LEO 1530VP, LEO Elektronenmikroskopie GmbH, Oberkochen, Germany) with a beam voltage of 15 kV after dentine discs were dried for 48 h under vacuum and sputter-coated with gold. Each image was calibrated individually using a 10 μm bar on the negative and analysed using image analysis software ImagePro Plus 6.0 for Windows (Media Cybernetics, Silver Spring, MD, USA). A standard area was marked on each image and only those tubules within this area of interest (AOI) were measured. Tubules that overlapped the edges of the marked area and confused the data by including only portions of tubules were excluded. The grayscale used ranged from 0 to 255 where 0 = black and 255 = white and a level was chosen for the threshold such that only the tubules were highlighted. Eight images captured under this protocol were selected randomly from each dentine disc. The tubule exposure rate of each dentine disc was calculated and the mean diameter of dentine discs was measured. An attached energy-dispersive X-ray spectroscopy (EDX) apparatus was used to identify elemental composition on the dentine surface treated with different methods and the vertical sections were observed with FE-SEM as well.
2.5
Statistical analysis
Statistical analysis was performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). Differences of tubule exposure ratio between Group A and Group B were analysed using a paired t -test. Differences of Ca/P ratio of each group were analysed using one-way ANOVA analysis. P-values less than 0.05 were considered statistically significant.
3
Results
3.1
Scanning electron microscopy
3.1.1
Surface morphology observation and analysis
FE-SEM was used to observe surface morphologies of dentine discs treated in each experimental group. The exposure rates of dentinal tubules in each group was calculated using an image analysis software (ImagePro Plus 6.0). As shown in Fig. 1 , a portion of dentinal tubules in Group A1 were occluded with loose, granular shaped occlusive material particles. The diameter of the tubules was about 2.4 μm ( Fig. 1 A1). In Group A2, the majority of tubules were incompletely occluded with occlusive materials, most of which were combined and only a small amount was isolated and granular shaped. Curving gaps were observed between occlusive materials and tubule walls. The diameter of dentinal tubules in Group A2 decreased to 1.7–2.2 μm ( Fig. 1 A2), compared with which in Group A1. Dentinal tubules in Group A3 were completely occluded and the diameter of the tubules decreased to 1.1–1.4 μm. The central areas of occluded tubules were concave ( Fig. 1 A3).
