Abstract
Objective
Dentin hypersensitivity (DH) is a commonly occurring dental condition, and bioactive glasses (BG) are used in dentifrice formulations for treating DH by forming a surface layer of hydroxycarbonate apatite (HCA) on the tooth, thereby occluding exposed dentinal tubules. Fluoride-containing BG, however, form fluorapatite, which is more stable toward acid attack, and provide a more sustainable option for treating DH.
Methods
Melt-derived multi-component BG (SiO 2 –P 2 O 5 –CaO–CaF 2 –SrO–SrF 2 –ZnO–Na 2 O–K 2 O) with increasing CaF 2 +SrF 2 content (0–32.7 mol%) were prepared. Apatite formation, occlusion of dentinal tubules in dentin discs and ion release in Tris buffer were characterized in vitro over up to 7 days using X-ray diffraction, infrared spectroscopy, scanning electron microscopy and inductively coupled plasma emission spectroscopy.
Results
The fluoride-containing bioactive glasses formed apatite from as early as 6 h, while the fluoride-free control did not form apatite within 7 days. The glasses successfully occluded dentinal tubules by formation of apatite crystals and released ions such as fluoride, strontium and potassium.
Significance
Fluoride significantly improved apatite formation of the BG, allowing for treatment of DH by occlusion of dentinal tubules. The BG also released therapeutically active ions, such as strontium and fluoride for caries prevention, zinc for bactericidal properties and potassium, which is used as a desensitizing agent in dentifrices.
1
Introduction
Dentin hypersensitivity (DH) is one of the most commonly occurring clinical dental conditions, and up to 69% of the UK population have reported experiencing some form of tooth sensitivity . Although the etiology of DH is multi-factorial and not yet fully understood , it is attributed to the general increase in exposed root surfaces of the teeth from periodontal disease, toothbrush abrasion or cyclic loading fatigue of the thin enamel near the cemento-enamel junction . The currently accepted theory for a DH mechanism is the hydrodynamic theory , which proposes that external stimuli such as cold, hot, tactile or osmotic pressure, when applied to exposed dentin, cause fluid movement within the dentinal tubules. This fluid movement stimulates mechanoreceptors near the base of the tubule and may, if certain physiological parameters are met, trigger a pain response. The hydrodynamic theory is based on the understanding that open tubules allow fluid flow through the tubules , which results in pressure changes that excite the nerve endings in the dental pulp, and it is consistent with the observation that when DH is treated with a tubule-occluding agent, this will result in a reduction in DH .
Occlusion of exposed dentinal tubules is therefore a common approach for treating DH, and several over-the-counter (OTC) dentifrices propose tubule occlusion as their mode of action. Strontium chloride is the active ingredient in the original Sensodyne ® dentifrice (GlaxoSmithKline, London, UK) and was the first tubule occluding agent incorporated into a dentifrice; later products also contained strontium acetate . Fluoride was first proposed as a desensitizing agent in 1941 and has subsequently been used in dentifrices, gels, mouth rinses and varnishes. Recently, a bioactive glass (NovaMin ® , developed by NovaMin Technology Inc., Alachua, FL, USA) based on the original 45S5 Bioglass ® (US Biomaterials Corp., Jacksonville, FL, USA) composition has been incorporated as a remineralising ingredient in dentifrice formulations for treating DH by precipitating hydroxycarbonate apatite (HCA) onto the tooth surface and subsequently occluding the dentinal tubules . However, concerns have been expressed over the long-term durability of HCA in the mouth, and formation of fluorapatite (FAp) rather than HCA is preferable, as it is more resistant to acid attack and would therefore dissolve less readily when teeth are exposed to acidic conditions (e.g. during consumption of fruit juice and carbonated beverages).
It was recently shown that fluoride-containing bioactive glasses form FAp rather than HCA in physiological solutions . Here a series of bioactive glasses (SiO 2 –P 2 O 5 –CaO–CaF 2 –SrO–SrF 2 –ZnO–Na 2 O–K 2 O) were produced, which form FAp in physiological solutions, release strontium and fluoride for caries prevention, zinc for bactericidal properties and potassium, which is currently used as a desensitizing agent in dentifrices. The aim was to investigate apatite formation, ion release and occlusion of dentinal tubules in vitro .
2
Experimental procedure
2.1
Glass synthesis and characterization
Fluoride (CaF 2 and SrF 2 ) was added to the glasses rather than substituting it for CaO or SrO, as this was shown to maintain glass solubility, bioactivity and network connectivity . Glasses ( Table 1 ) were prepared using a melt-quench route. Mixtures of analytical grade SiO 2 (Prince Minerals Ltd., Stoke-on-Trent, UK), P 2 O 5 , CaCO 3 , SrCO 3 , Na 2 CO 3 , CaF 2 and SrF 2 (Sigma–Aldrich, Gillingham, UK) were melted in a platinum–rhodium crucible for 1 h at 1420 °C in an electric furnace (EHF 17/3, Lenton, Hope Valley, UK). A batch size of 100 g was used. After melting, the glasses were rapidly quenched into water to prevent crystallization. After drying, the glass frit was ground using a vibratory mill (Gyro mill, Glen Creston, London, UK) for 7 min and sieved using a 38 μm mesh analytical sieve (Endecotts Ltd., London, UK). The amorphous structure of the glasses was confirmed by powder X-ray diffraction (XRD; X’Pert PRO, PANalytical, Cambridge, UK).
| SiO 2 | P 2 O 5 | CaO | CaF 2 | SrO | SrF 2 | Na 2 O | K 2 O | ZnO | CaF 2 + SrF 2 | |
|---|---|---|---|---|---|---|---|---|---|---|
| F0 | 44.00 | 5.00 | 15.00 | 15.00 | 10.00 | 10.00 | 1.00 | |||
| F4 | 41.91 | 4.76 | 14.29 | 2.38 | 14.29 | 2.38 | 9.53 | 9.53 | 0.95 | 4.76 |
| F9 | 39.92 | 4.54 | 13.61 | 4.64 | 13.61 | 4.64 | 9.07 | 9.07 | 0.91 | 9.28 |
| F13 | 38.01 | 4.32 | 12.96 | 6.81 | 12.96 | 6.81 | 8.64 | 8.64 | 0.86 | 13.62 |
| F17 | 36.19 | 4.11 | 12.34 | 8.88 | 12.34 | 8.88 | 8.22 | 8.22 | 0.82 | 17.76 |
| F25 | 32.76 | 3.72 | 11.17 | 12.77 | 11.17 | 12.77 | 7.45 | 7.45 | 0.74 | 25.54 |
| F32 | 29.61 | 3.36 | 10.09 | 16.36 | 10.09 | 16.36 | 6.73 | 6.73 | 0.67 | 32.72 |
The glass transition temperature ( T g ) was determined using differential scanning calorimetry (DSC, Stanton Redcroft DSC1500, Rheometric Scientific, Epsom, UK). 50 mg of glass frit was analyzed in a platinum crucible using analytical grade alumina powder as reference with a heating rate of 10 K/min.
2.2
Tris buffer study
Tris buffer solution was prepared by dissolving 15.090 g tris(hydroxymethyl)aminomethane (Sigma–Aldrich) in 800 mL deionised water, adding 44.2 mL 1 M hydrochloric acid (Sigma–Aldrich), heating to 37 °C over night, adjusting the pH to 7.30 using 1 M hydrochloric acid using a pH meter (Oakton Instruments, Nijkerk, Netherlands) and filling to a total volume of 2000 mL using deionised water. Tris buffer solution was kept at 37 °C.
50 mL of Tris buffer was pipetted into 150 mL PE bottles. pH was measured using a pH meter (Oakton Instruments) and 75 mg of glass powder (<38 μm) was dispersed in the Tris buffer solution. Samples were placed in an orbital shaker at 37 °C at an agitation rate of 60 rpm for 3, 6, 9, 24, 72 and 168 h. Tris buffer from the same batch without glass powder was used as control. After removing the samples from the shaker pH was measured and solutions were filtered through medium porosity filter paper (5 μm particle retention, VWR International, Lutterworth, UK) and kept at 4 °C. The filter paper was dried at 37 °C and the dried powders were analyzed using Fourier-transform infrared spectroscopy (FTIR; Spectrum GX, Perkin-Elmer, Waltham, MA, USA) and XRD.
Fluoride-release into Tris buffer was measured using a fluoride-selective electrode (Orion 9609BNWP with Orion pH/ISE meter 710, both Thermo Scientific, Waltham, MA, USA). Calibration was performed using standard solutions prepared using Tris buffer to account for ionic strength. For elemental analysis, solutions were acidified using 69% nitric acid and quantitatively analyzed by inductively coupled plasma–optical emission spectroscopy (ICP; Varian Vista-PRO, Varian Ltd., Oxford, UK).
2.3
Occlusion of dentinal tubules
Caries-free extracted maxillary and mandibular third molars used in this study were obtained from the tooth bank at the Royal London Dental hospital. Studies were performed in accordance with the guidelines by the Queen Mary Research Ethics Committee. In vitro occlusion of dentinal tubules was investigated using the dentin disc model . The teeth were sectioned mesio-distally into discs approximately 1 mm thick using an internal edge annular diamond blade (Microslice annular blade, Ultratec, USA) mounted on a Microslice 2 saw (Malvern Instruments Ltd., UK) and halved. The discs were stored in sodium hypochlorite until required.
The dentin discs were etched with 6% citric acid for two minutes and rinsed with distilled water, prior to placement in a Tris buffer-filled container together with glass powder as outlined in Section 2.2 . Controls were kept in Tris buffer without glass powder. Following storage in Tris buffer for up to 7 days the discs were allowed to dry in a dessiccator for two days, attached to aluminum stubs with carbon conducting cement, carbon sputter-coated and viewed in a scanning electron microscope (SEM, FEI Inspect F) using back-scattered electron mode at 20 kV. Untreated dentin discs were mounted, sputter coated with gold/palladium and subsequently viewed in a Cambridge stereoscan 90B SEM at a constant working distance of 10 mm. Variable accelerating voltages (10 or 15 kV) were selected to optimize image quality.
2
Experimental procedure
2.1
Glass synthesis and characterization
Fluoride (CaF 2 and SrF 2 ) was added to the glasses rather than substituting it for CaO or SrO, as this was shown to maintain glass solubility, bioactivity and network connectivity . Glasses ( Table 1 ) were prepared using a melt-quench route. Mixtures of analytical grade SiO 2 (Prince Minerals Ltd., Stoke-on-Trent, UK), P 2 O 5 , CaCO 3 , SrCO 3 , Na 2 CO 3 , CaF 2 and SrF 2 (Sigma–Aldrich, Gillingham, UK) were melted in a platinum–rhodium crucible for 1 h at 1420 °C in an electric furnace (EHF 17/3, Lenton, Hope Valley, UK). A batch size of 100 g was used. After melting, the glasses were rapidly quenched into water to prevent crystallization. After drying, the glass frit was ground using a vibratory mill (Gyro mill, Glen Creston, London, UK) for 7 min and sieved using a 38 μm mesh analytical sieve (Endecotts Ltd., London, UK). The amorphous structure of the glasses was confirmed by powder X-ray diffraction (XRD; X’Pert PRO, PANalytical, Cambridge, UK).
| SiO 2 | P 2 O 5 | CaO | CaF 2 | SrO | SrF 2 | Na 2 O | K 2 O | ZnO | CaF 2 + SrF 2 | |
|---|---|---|---|---|---|---|---|---|---|---|
| F0 | 44.00 | 5.00 | 15.00 | 15.00 | 10.00 | 10.00 | 1.00 | |||
| F4 | 41.91 | 4.76 | 14.29 | 2.38 | 14.29 | 2.38 | 9.53 | 9.53 | 0.95 | 4.76 |
| F9 | 39.92 | 4.54 | 13.61 | 4.64 | 13.61 | 4.64 | 9.07 | 9.07 | 0.91 | 9.28 |
| F13 | 38.01 | 4.32 | 12.96 | 6.81 | 12.96 | 6.81 | 8.64 | 8.64 | 0.86 | 13.62 |
| F17 | 36.19 | 4.11 | 12.34 | 8.88 | 12.34 | 8.88 | 8.22 | 8.22 | 0.82 | 17.76 |
| F25 | 32.76 | 3.72 | 11.17 | 12.77 | 11.17 | 12.77 | 7.45 | 7.45 | 0.74 | 25.54 |
| F32 | 29.61 | 3.36 | 10.09 | 16.36 | 10.09 | 16.36 | 6.73 | 6.73 | 0.67 | 32.72 |
The glass transition temperature ( T g ) was determined using differential scanning calorimetry (DSC, Stanton Redcroft DSC1500, Rheometric Scientific, Epsom, UK). 50 mg of glass frit was analyzed in a platinum crucible using analytical grade alumina powder as reference with a heating rate of 10 K/min.
2.2
Tris buffer study
Tris buffer solution was prepared by dissolving 15.090 g tris(hydroxymethyl)aminomethane (Sigma–Aldrich) in 800 mL deionised water, adding 44.2 mL 1 M hydrochloric acid (Sigma–Aldrich), heating to 37 °C over night, adjusting the pH to 7.30 using 1 M hydrochloric acid using a pH meter (Oakton Instruments, Nijkerk, Netherlands) and filling to a total volume of 2000 mL using deionised water. Tris buffer solution was kept at 37 °C.
50 mL of Tris buffer was pipetted into 150 mL PE bottles. pH was measured using a pH meter (Oakton Instruments) and 75 mg of glass powder (<38 μm) was dispersed in the Tris buffer solution. Samples were placed in an orbital shaker at 37 °C at an agitation rate of 60 rpm for 3, 6, 9, 24, 72 and 168 h. Tris buffer from the same batch without glass powder was used as control. After removing the samples from the shaker pH was measured and solutions were filtered through medium porosity filter paper (5 μm particle retention, VWR International, Lutterworth, UK) and kept at 4 °C. The filter paper was dried at 37 °C and the dried powders were analyzed using Fourier-transform infrared spectroscopy (FTIR; Spectrum GX, Perkin-Elmer, Waltham, MA, USA) and XRD.
Fluoride-release into Tris buffer was measured using a fluoride-selective electrode (Orion 9609BNWP with Orion pH/ISE meter 710, both Thermo Scientific, Waltham, MA, USA). Calibration was performed using standard solutions prepared using Tris buffer to account for ionic strength. For elemental analysis, solutions were acidified using 69% nitric acid and quantitatively analyzed by inductively coupled plasma–optical emission spectroscopy (ICP; Varian Vista-PRO, Varian Ltd., Oxford, UK).
2.3
Occlusion of dentinal tubules
Caries-free extracted maxillary and mandibular third molars used in this study were obtained from the tooth bank at the Royal London Dental hospital. Studies were performed in accordance with the guidelines by the Queen Mary Research Ethics Committee. In vitro occlusion of dentinal tubules was investigated using the dentin disc model . The teeth were sectioned mesio-distally into discs approximately 1 mm thick using an internal edge annular diamond blade (Microslice annular blade, Ultratec, USA) mounted on a Microslice 2 saw (Malvern Instruments Ltd., UK) and halved. The discs were stored in sodium hypochlorite until required.
The dentin discs were etched with 6% citric acid for two minutes and rinsed with distilled water, prior to placement in a Tris buffer-filled container together with glass powder as outlined in Section 2.2 . Controls were kept in Tris buffer without glass powder. Following storage in Tris buffer for up to 7 days the discs were allowed to dry in a dessiccator for two days, attached to aluminum stubs with carbon conducting cement, carbon sputter-coated and viewed in a scanning electron microscope (SEM, FEI Inspect F) using back-scattered electron mode at 20 kV. Untreated dentin discs were mounted, sputter coated with gold/palladium and subsequently viewed in a Cambridge stereoscan 90B SEM at a constant working distance of 10 mm. Variable accelerating voltages (10 or 15 kV) were selected to optimize image quality.
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