Dentine remineralization induced by two bioactive glasses developed for air abrasion purposes

Dentine remineralization induced by two bioactive glasses developed for air abrasion purposes

Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 746-756, Copyright © 2011 Elsevier Ltd

Abstract

Objectives

The present study aimed to evaluate dentine remineralization through a 7-day period of artificial saliva (AS) storage induced by bioactive glass 45S5 (BAG) and by bioactive glass modified with soda-lime spherical glass.

Methods

Partially demineralized dentine disks were treated by BAG or the spherical-glass modified bioactive glass (M-BAG) and subsequently immersed in AS for 7 days. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to quantitatively analyse the mineral variation of the dentine surface by calculating the spectra parameters. X-ray diffraction (XRD) and energy-dispersive X-ray analysis (EDX) were performed on completely demineralized dentine to confirm the apatite formation. The roughness of the dentine surface was evaluated by atomic force microscopy (AFM) and the morphology was also examined by scanning electron microscopy (SEM).

Results

ATR-FTIR showed a significant increase of the mineral matrix area ratio in dentine specimens treated with the two bioactive glasses subsequent to 7 days of AS storage. The XRD spectrum exhibited apatite growth and mineral elements could be found on completely demineralized samples analysed by EDX after remineralization treatments. The dentine specimens treated with bioactive glass showed lower roughness, and most of the dentinal tubules appeared completely occluded during the AFM and SEM examination.

Conclusions

Although the concentration of bioactive glass in the M-BAG is 60% of that contained in the original version, both formulations have similar potential in dentine remineralization. These bioactive powders developed for air-abrasive use may be considered as innovative bioactive materials for therapeutic remineralization of dental hard tissues.

Introduction

Dentine is a tubular permeable structure composed of organic matrix embedded in crystalline apatite which accounts for the most part of dental hard tissues. Although a physiological equilibrium exists between remineralization and demineralization of the dental hard tissues in the oral cavity, an increase in organic acids derived from dental plaque or an acidic diet may lead to a change of this equilibrium inducing demineralization of the tooth surface. If the demineralization effect overcomes the self-regulated remineralization, excessive loss of mineral content from the dental hard tissues may occur jeopardising the integrity of the teeth. In particular clinical situations (i.e. abrasion or erosion) and in the proximity of specific morphological areas (i.e. gingival recession), this demineralization may result in the exposure of the dentinal tubules, though the formation of reactionary dentine in the pulp and dentine sclerosis inside the tubules may exist as a compensatory response.

Several studies have been aimed at reincorporating mineral into demineralized dentine to achieve remineralization. Bioactive glass is an extensively studied biomaterial in the field of tissue engineering, bone regeneration and dentine remineralization due to the remarkable bioactive capability in forming hydroxycarbonate apatite (HCA) when submerged in simulated body fluid solutions. It has been recently demonstrated that it is possible to achieve hierarchy remineralization via apatite precipitation using calcium silicates in a biomimetic approach. Although it has been successfully proven that materials based on bioactive substance have the potential to promote dentine remineralization, only a limited number of studies have quantitatively monitored the remineralization process of demineralized dentine. Bioactive glass 45S5 (BAG) is currently used in dentistry for air polishing procedures and dentine desensitization. Moreover, bioactive glass might be used within air-cutting systems in restorative dentistry for minimally invasive cavity preparation. Nevertheless, there is still the need to create alternative bioactive glass formulations which may allow clinicians to perform more selective air-polishing or caries removal procedures offering, at the same time, the possibility for therapeutic remineralization of the dental hard tissues.

The current study was designed to quantitatively compare the apatite forming capacity of the original and spherical-glass modified bioactive glass (M-BAG) in dentine remineralization using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) which is commonly used for the characterization of chemical changes in bone and other mineralized tissues as a non-destructive approach. Qualitative analysis by means of X-ray diffraction (XRD), Scanning electron microscopy–energy-dispersive X-ray analysis (SEM–EDX) and atomic force microscopy (AFM) were also used to analyse the chemical and morphological changes to the dentine surface. The null hypothesis of this study was that the two bioactive glasses (BAG and M-BAG) did not differ significantly in their remineralization ability on demineralized dentine compared to a control.

Materials and methods

Sample preparation

Forty-five caries-free extracted human third molars were obtained under a protocol approved by the local Ethics Committee of the School and Hospital of Stomatology Wuhan University, China. Dentine disks with a thickness of 1.0 ± 0.1 mm, were prepared by sectioning each tooth 1.5 mm above the cemento-enamel junction using a low-speed water cooled diamond saw (Isomet, Buehler, Lake Bluff, IL, USA). The surrounding enamel of the disks was cut off, creating square dentine specimens (5 mm × 5 mm × 1 mm). A standard smear layer was created on both sides of the dentine specimens using 600-grit silicon carbide paper (Yuli Abrasive Belts, Shanghai, China) for 30 s under constant water irrigation.

Bioactive glasses and treatment procedures

The compositions of BAG and M-BAG are listed in Table 1 . Bioactive glass 45S5 (BAG) (particle size: 30–90 μm) was mixed with 40 wt% of soda lime spherical-glass (particle size range: 4–80 μm) to create the spherical-glass-modified bioactive glass (M-BAG). The M-BAG mixture was kept under agitation at 60 rpm for 24 h and subsequently left undisturbed at 37 °C in a desiccator for a further 24 h. Dentine disks were randomly divided into three principal groups ( n = 10): artificial saliva (AS) as a control, BAG and M-BAG and then cut into two halves using a water-cooled high-speed handpiece with a fine carbide bur (AEU-25, Tulsa Dentsply, USA). Half of the specimens were partially demineralized in 0.5 M EDTA solution (pH 7.4) for 5 min, whilst the other halves of the specimens were completely demineralized in a 0.5 M EDTA solution (pH 7.4) at 25 °C for 15 days. The specimens were finally copiously rinsed with distilled water for 2 min and monitored using digital radiography to ensure that a complete demineralization was achieved as previously accomplished.

Table 1
Composition of the materials used for dentine remineralization.
Treatments Maufacturer Components Particle size range (μm)
BAG OSspray Ltd., London, UK 45 wt% SiO 2 , 24.5 wt% CaO, 24.6 wt% Na 2 O, 5.8 wt% P 2 O 5 30–90
M-BAG OSspray Ltd., London, UK 60% BAG + 40% Soda-lime spherical glass a (73 wt% SiO 2 , 14 wt% Na 2 O, 9 wt% CaO, 4 wt% MgO) 4–80

a The Soda-lime spherical glass (Spheriglass Glass Microspheres) was provided by Potters Europe – Engineered Glass Materials Division, Suffolk, United Kingdom.

The dentine surface was then slightly rubbed with 20 mg of BAG or M-BAG using a wet cotton pellet for 1 min, followed by copious rinsing (1 min) with distilled water. No treatment was performed in the control group. All treated specimens were subsequently stored in AS (pH 7.4), containing 1.5 mmol/l CaCl 2 , 50 mmol/l KCl, 0.9 mmol/l KH 2 PO 4 , 20 mmol/l Tris, at 37 °C for 7 days. The AS was replaced every 24 h with 20 ml of fresh AS.

ATR-FTIR spectroscopy

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectra were obtained from partially and totally demineralized dentine specimens subsequent to 1-day remineralization, 3-day remineralization and 7-day remineralization periods in AS using a Nicolet 5700 FTIR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a diamond crystal attenuated total reflection (ATR) accessory. Prior to the spectrophotometric analysis, the specimens were rinsed with distilled water for 30 s and then completely air dried.

Reference points were marked on the area of examination in each specimen in order to perform the analysis at the same position before and after treatments. Spectra were collected in the range of 800–1800 cm −1 at 4 cm −1 resolution for a total of 64 scans, and analysed by OMNIC 8 software (Nicolet, Madison, WI, USA). Each specimen was analysed in 3 different positions before and after treatment. The spectrum acquired at the end of the analyses represented the average of all the three single scans. Spectra of water was obtained and subtracted from each of the original spectra which were then processed by smoothing, baseline corrected, and normalized to the amide I peak. The mineral matrix ratio (the ratio of integrated areas of the phosphate v 1 , v 3 contour to the amide I peak) was measured in all spectra in order to quantitatively calculate the extent of remineralization. In addition, ATR-FTIR spectra from the bioactive glass powders were also obtained to characterize their chemical structures.

XRD analysis

X-ray diffraction (XRD) analysis was carried out before and after the 7-day remineralization for six additional completely demineralized specimens in AS, BAG and M-BAG groups by X’ Pert PRO Dy2198 (Spectric Pte, Singapore) with a CuKα generator working at 40 kV and 40 mA. The range of scanning angles was from 20° to 55°. In addition, an untreated dentine disc was measured to obtain a spectrum of the sound dentine as a control, and bioactive glass powders were also measured for characterization.

SEM–EDX analysis

Six further completely demineralized specimens were remineralized for 7 days, rinsed with distilled water, dried in a desiccator and finally processed for carbon coating. The particle size of the bioactive glasses and changes in dentine surface morphology for partially demineralized samples following the 7-day remineralization were obtained using the SEM (QUANTA-200, FEI, Eindhoven, Netherlands) at 20 kV. An EDX apparatus associated with a SEM was used to identify the content of chemical elements of the dentine surface before and after the treatments. The EDX analysis of the two bioactive glasses powders was also performed.

AFM examination

Three additional dentine disks were prepared as previously described and metallographically polished through silicon carbide papers (Yuli Abrasive Belts, Shanghai, China) starting with a 600-grit down to 4000-grit. A final polishing step was performed using a 0.25 μm diamond paste to provide an ultra-flat surface suitable for AFM scanning procedures. A Digital Instruments Nanoscope IV (Veeco, NY, USA) was used to measure the microstructure and surface roughness changes on the partially demineralized dentine disc after 7-day treatment using a contact mode. Fields of view at 20 μm × 20 μm scan size and 0.5 Hz scan rate were employed.

In each image, five randomized boxes of 2 μm × 2 μm were drawn using the Nanoscope V530b4 (Veeco, NY, USA) at the intertubular dentine, and mean roughness ( R a ) and root mean square roughness ( R rms ) were determined.

Statistical analysis

Statistical analysis was performed by SPSS 16.0 (SPSS, Chicago, IL, USA) for Windows. Means and standard deviations of the mineral matrix peak area ratio from ATR-FTIR measurement analysis were calculated. Homogeneity of variance was determined using Levene’s test. Two-way repeated measures ANOVA was applied to evaluate the ATR-FTIR results, considering the treatment as main effect and treatment time as the repeated measure at a 5% significance level. Pair-wise comparisons for one-way repeated measures ANOVA with LSD adjustment were made within each group. One-way ANOVA was performed to analyse the AFM roughness values ( R a and R rms ).

Results

Material characterization

In accordance with the contents of the bioactive glasses provided by the manufacturer ( Table 1 ), the SEM analysis showed that the morphology of bioactive glasses was characterized by micrometric irregular particles with a clump-like shape (BAG; Fig. 1 A ) or spherical shape (M-BAG; Fig. 1 E) with a dimension of approximately 50 μm, which is consistent with another study showing the micron-sized bioactive glass. SEM–EDX on BAG powder exhibited the presence of silicon, calcium and phosphate, whilst M-BAG powder showed higher silicon peak intensity and lower phosphate content with the presence of magnesium ( Fig. 1 B and F). ATR-FTIR spectra of the two bioactive glasses presented wide vibrational bands at 800–1200 cm −1 ( Fig. 1 C and G). BAG displayed vibrations at 900, 1000 and 1450 cm −1 corresponding to P–O stretch, Si–O–Si stretch and CO 3 2− v 3 band, and M-BAG contained P–O and Si–O–Si stretch at 920 and 1000 cm −1 , respectively. The XRD pattern of the two bioactive glasses showed the presence of a peak at 2 = 20–35° with a broad contour and low intensity, which indicated the amorphous state of both glasses ( Fig. 1 D and H).

Characterization of bioactive glasses measured by SEM–EDX, ATR-FTIR and XRD. (A) BAG particles were clump-like with sharp edges about 50 μm in size. (B) EDX showed silicon, calcium, phosphate, oxygen and sodium peaks for BAG. (C) ATR-FTIR spectra of BAG. (D) XRD spectra of BAG. (E) M-BAG exhibited round-shaped particles together with smaller clump-like shaped ones. (F) Besides a high silicon peak, traces of calcium, sodium, magnesium, oxygen and carbon were displayed. (G) ATR-FTIR spectra of M-BAG. (H) XRD spectra of M-BAG.
Fig. 1
Characterization of bioactive glasses measured by SEM–EDX, ATR-FTIR and XRD. (A) BAG particles were clump-like with sharp edges about 50 μm in size. (B) EDX showed silicon, calcium, phosphate, oxygen and sodium peaks for BAG. (C) ATR-FTIR spectra of BAG. (D) XRD spectra of BAG. (E) M-BAG exhibited round-shaped particles together with smaller clump-like shaped ones. (F) Besides a high silicon peak, traces of calcium, sodium, magnesium, oxygen and carbon were displayed. (G) ATR-FTIR spectra of M-BAG. (H) XRD spectra of M-BAG.

ATR-FTIR spectroscopy

All representative spectra recorded in the region of 800–1800 cm −1 presented phosphate bands at 885–1180 cm −1 , representative of mineral components, assigned to the phosphate v 1 , v 3 stretching mode, and amide bands from 1180 to 1725 cm −1 from organic components.

Phosphate v 1 , v 3 bands at 1010 cm −1 were detected and increased during the 7-day period in BAG and M-BAG groups ( Fig. 2 B and C ). However, a few changes of phosphate peaks could also be observed in the control group during the 7-day incubation ( Fig. 2 A). The mineral matrix area ratio increased significantly from 4.96 (BAG) and 4.64 (M-BAG) up to 13.81 and 15.06, respectively after 7-day AS immersion ( P < 0.05) ( Fig. 2 D). The two-way repeated ANOVA showed a statistically significant main effect for both time and treatment ( P < 0.05), as well as time × treatment interaction ( P < 0.05). However, pairwise comparisons indicated no significant difference in mineral matrix area ratio between these two bioactive glasses ( P > 0.05), but they both showed significantly larger ratio values than the control group ( P < 0.05).

A representative series of ATR-FTIR spectra (A)–(C) recorded on the dentine surface were marked in different colours for different time points at EDTA 5 min (black), 1-day (red), 3-day (blue) and 7-day (green) treatments. The spectra have been normalized to amide I. (A) Artificial saliva (AS), (B) BAG, (C) M-BAG. The mineral matrix area ratio after different time points for each group was present as well (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Fig. 2
A representative series of ATR-FTIR spectra (A)–(C) recorded on the dentine surface were marked in different colours for different time points at EDTA 5 min (black), 1-day (red), 3-day (blue) and 7-day (green) treatments. The spectra have been normalized to amide I. (A) Artificial saliva (AS), (B) BAG, (C) M-BAG. The mineral matrix area ratio after different time points for each group was present as well (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

There was no clearly identifiable phosphate band at 885–1180 cm −1 on the completely demineralized dentine and the mineral matrix area ratio was very small. Only a slight increment of mineral matrix area ratio was detected after 7 days of immersion in AS ( P > 0.05) ( Fig. 3 A and D ). In the BAG group, the phosphate peaks (v 1 , v 3 ) were weak before application but a stronger apatite peak was detected after the 7-day remineralization ( Fig. 3 B). M-BAG induced a similar phosphate v 1 , v 3 band as BAG, indicating a sign of dentine remineralization bioactivity ( Fig. 3 C). However, there was no significant difference in the increase of mineral matrix area ratio between the two bioactive glass groups ( P > 0.05) ( Fig. 3 D).

A representative series of ATR-FTIR spectra (A)–(C) recorded on the completely demineralized dentine surface were marked in different colours for different time points which are complete demineralization (black) and 7-day remineralization (red). The spectra have been normalized to amide I. (A) Artificial saliva (AS), (B) BAG, (C) M-BAG. The mineral matrix area ratio after different time points for each group was present as well (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Fig. 3
A representative series of ATR-FTIR spectra (A)–(C) recorded on the completely demineralized dentine surface were marked in different colours for different time points which are complete demineralization (black) and 7-day remineralization (red). The spectra have been normalized to amide I. (A) Artificial saliva (AS), (B) BAG, (C) M-BAG. The mineral matrix area ratio after different time points for each group was present as well (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

XRD measurement

The X-ray diffraction analyses performed in completely demineralized dentine, 7-day remineralized dentine and untreated dentine are displayed in Fig. 4 A . The untreated dentine generally exhibited a characteristic X-ray diffraction pattern of hydroxyapatite ( Fig. 4 A) (JCPDS 9-432). Completely demineralized specimens showed no obvious diffraction peak. After a 7-day immersion in AS, the diffraction patterns exhibited a more obvious halo ( Fig. 4 A) at 2 = 20–35° even though no sharp diffraction peak was presented. However, after 7-day remineralization, a characteristic reflection was observed at approximately 26° of (0 0 2) reflection in the two bioactive glasses groups. A (2 1 1) reflection diffraction peak at about 31.8° was visible ( Fig. 4 A), indicating the presence of apatite compared to the pattern of untreated dentine.

XRD patterns and EDX on completely demineralized dentine specimens. (A) XRD spectra of untreated dentine (red) and completely demineralized dentine (black) treated by artificial saliva (AS) (yellow), BAG (green) and M-BAG (blue) after setting in AS for 7 days. (B–D) EDX analysis of completely demineralized dentine after AS (B), BAG (C) and M-BAG (D) remineralization for 7 days. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Fig. 4
XRD patterns and EDX on completely demineralized dentine specimens. (A) XRD spectra of untreated dentine (red) and completely demineralized dentine (black) treated by artificial saliva (AS) (yellow), BAG (green) and M-BAG (blue) after setting in AS for 7 days. (B–D) EDX analysis of completely demineralized dentine after AS (B), BAG (C) and M-BAG (D) remineralization for 7 days. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

SEM–EDX analysis

The SEM–EDX analysis commenced on the completely demineralized dentine surface showed traces of calcium and phosphate and relatively large amounts of carbon and oxygen ( Fig. 4 B). After 7-day AS storage, EDX detected no apatite element growth and SEM showed a dentine surface characterized by exposed dentinal tubules ( Fig. 5 A1 ). However, after bioactive glass treatments, calcium and phosphate became the dominant elements including the presence of sodium and silicon ( Fig. 4 C and D). The dentine surface was completely covered with almost all dentinal tubules occluded ( Fig. 5 B1 and C1). Separate micro-sized bioactive glass particles became aggregates of apatite spherulites (BAG) or needle like crystals (M-BAG) of nano-size (about 0.3 μm) in the homogeneous layer.

Dentine surface morphology of the partially demineralized dentine after different treatments. (A1, A2) SEM and AFM images of EDTA-etched (5 min) dentine surface after AS immersion for 7 days. (B1, B2) SEM and AFM images of EDTA-etched (5 min) dentine surface after BAG induced 7-day remineralization. (C1, C2) SEM and AFM images of EDTA-etched (5 min) dentine surface after M-BAG induced 7-day remineralization.
Fig. 5
Dentine surface morphology of the partially demineralized dentine after different treatments. (A1, A2) SEM and AFM images of EDTA-etched (5 min) dentine surface after AS immersion for 7 days. (B1, B2) SEM and AFM images of EDTA-etched (5 min) dentine surface after BAG induced 7-day remineralization. (C1, C2) SEM and AFM images of EDTA-etched (5 min) dentine surface after M-BAG induced 7-day remineralization.

AFM examination

The AFM ultra-morphology analysis resulted in a series of 20 μm × 20 μm three-dimensional images of dentine disks after the different treatments ( Fig. 5 A2, B2 and C2). The microstructure of 5 min EDTA etched dentine displayed open dentinal tubules after 7-day immersion in AS ( Fig. 5 A2). The demineralized specimens treated with both the bioactive glasses presented dentine surfaces characterized by deposits which covered the lumen of the dentinal tubules after the 7-day treatment ( Fig. 5 B2 and C2).

Mean roughness ( R a ) and root mean square roughness ( R rms ) are presented in Table 2 . Baseline roughness values after polishing the dentine surface were significantly lower than those after treatments ( P < 0.05). However, BAG and M-BAG groups showed significant lower R a and R rms values than the AS group after 7-day remineralization ( P < 0.05), indicating that the mineral precipitation from bioactive glass remineralization resulted in a smoother surface. No difference was found between the two bioactive glass groups ( P > 0.05).

Table 2
Means (standard deviations) of the intertubular mean roughness ( R a ) and root mean square roughness ( R rms ) after different treatments.
Treatment R a (nm) R rms (nm)
Baseline (after polishing) 41.16 (14.55) a 55.97 (13.29) d
Artificial saliva (AS) 115.66 (33.34) b 134.56 (30.82) e
BAG 85.71 (14.75) c 101.88 (13.60) f
M-BAG 67.12 (16.29) c 81.62 (18.21) f
Groups with the different superscript letters are statistically significant ( P < 0.05).

Discussion

Bioactive glass 45S5 (BAG), a highly biocompatible calcium sodium phosphosilicate, is currently used in the dental clinic for air polishing procedures, or incorporated into toothpastes, desensitizing pastes, bonding materials and varnishes in order to create hybrid remineralizing materials. Although bioactive glass was originally developed as a bone conductive material, it may also react with saliva inducing a dissolution of Ca 2+ , PO 4 3− and Si 4+ at the glass surface and subsequent precipitation of a polycondensated silica-rich layer (Si-gel) which serves as a template for the formation of a calcium phosphate (Ca/P) which subsequently crystallise into HCA.

The formation of a crystallised layer of HCA has also been recently observed after the application of bioactive glass containing toothpaste. A recent study used BAG in air-polishing procedures achieving dentinal tubule occlusion and dentine permeability reduction of up to 80%. Moreover, BAG was less aggressive than traditional alumina powders in air-polishing treatments. The use of M-BAG might further reduce the eventuality of an excessive dental tissue loss due to the presence of round-shaped soda-lime glass contained within the composition of the powder ( Fig. 1 B and F). Indeed, spherical particles of the powders achieve rounded internal angles which are considered to be ideal for restorative materials ensuring better marginal adaptation and reduction of voids after air abrasion. The null hypothesis that the two bioactive glasses (BAG and M-BAG) did not differ significantly compared to the control in their remineralization ability must be rejected. Indeed, this study has also provided evidences that the M-BAG has preserved its bioactivity compared to the original Bioglass 45S5 (BAG). The use of these two bioactive glasses during air-abrasion procedures might offer an important contribution to the therapeutic remineralization of dental hard tissues.

The two bioactive glass formulations tested in this study were able to form HCA in demineralized dentine. The remineralization process induced by the two bioactive glasses used in this study were probably due to a simultaneous bioactive phenomena characterized by Si 4+ release and a subsequent polycondensation reaction induced by precipitation of calcium and phosphates on the organic matrix creating the template for the formation of a calcium phosphate phase. The reaction of polycondensation forms H 2 O which remains physically bonded with the Si–O–Si surface forming the hydrated silica rich layer. The polycondensated silicon-rich layer (Si-gel) then precipitates into an amorphous CaO–P 2 O 5 due to the incorporation of soluble Ca 2+ and PO 4 3− . The further incorporation of various mineral ions from AS helps the amorphous CaO–P 2 O 5 to convert into HCA. These processes were supported in this study by high calcium and phosphates peaks from EDX and by the increase of the mineral matrix area ratio observed in the ATR-FTIR analysis ( Figs. 2 and 4 ). Moreover, the results obtained during SEM examination also demonstrated that the dimensions of BAG and M-BAG powder particles were micrometrical before application in partially demineralized dentine, whilst after 7-day AS storage they turned into nano-sized particles ( Fig. 5 B1 and C1).

The rationale behind using partially and completely demineralized models in this study was based on the elimination of the ambiguity in differentiating the remineralized apatite crystallites from remnant apatite seed crystallites existing in partially demineralized dentine. The completely demineralized dentine may ascertain that remineralization was able to occur without a contribution from non-collagenous proteins (NCPs) and proteoglycans released from mineralized dentine. However, the two bioglass powders showed a good remineralization effect on both the partially and completely demineralized models. The formation of apatite might improve the sealing of dentinal tubules as evidenced by the SEM and AFM results. The optimal occlusion effect of the bioactive materials should also play an important role if they are applied in the treatment of post-operative dentine hypersensitivity. Moreover, bioactive glasses showed lower intertubular roughness values ( R a and R rms ) in comparison to that observed in the partially demineralized control specimens after 7-day treatment ( Table 2 ). The R rms was reported to take less account of the variations of low scanning frequency in an AFM study. Surface roughness of the dentine affects not only the aesthetic properties but also bacterial adhesion and plaque formation. In the present study, bioactive glass treatment was able to achieve a smoother surface than control group by crystalline phase formation. Such improvement of surface roughness might promote the resistance to bacterial adherence.

Vollenweider et al. assessed quantitatively the remineralization capability of ultrafine bioactive glass using thermogravimetry demonstrating enhanced mechanical properties in dentine. In addition, spectroscopic analyses by Fourier transform infrared spectroscopy (FTIR) have been commonly used to qualitatively support the presence of apatite formation amongst dentine remineralization investigations. However, no experiment has been performed to quantitatively evaluate dentine remineralization in real-time induced by a bioactive substance developed for restorative purposes of dentistry. The advantage of the spectroscopic methods is the non-destructive approach which provides continuous evaluation of the remineralization process. Unlike Fourier transform infrared spectroscopy (FTIR), which only accepts powder samples, ATR-FTIR has been shown to be an effective method for dentine mineral detection. A recent study demonstrated the bleaching effects of hydrogen peroxide on mineral and organic components of dentine before and after treatments by means of ATR-FTIR. Some more recent studies revealed the bioactivity of calcium silicate-based materials by means of ATR-FTIR and micro-Raman. In the present study, we found the mineral matrix area ratio of phosphate contour increased dramatically during the 7-day treatment on partially demineralized specimens in bioactive glass groups. Interestingly, the remineralization effect on completely demineralized dentine was weaker than the partially demineralized dentine, which is possibly due to the absence of dentine mineral contribution and difficulty in crystal nucleation. However, a low mineral matrix area ratio could be found on the completely demineralized dentine before treatments ( Fig. 3 ). This is probably because the digital radiography used for the demineralization detection was not as sensitive as ATR-FTIR. Even though no graph was captured by the digital radiography, ATR-FTIR could still detect a very slight intensity of the phosphate peak on the completely demineralized samples.

Furthermore, it should be pointed out that the contour for phosphate v 1 , v 3 became broader during the remineralization period. The broadened phosphate contour probably indicates that the crystallinity of the apatite layer was decreasing. However, it has been reported that the presence of labile non-apatite environments of phosphate ions is one of the critical characteristics of poorly crystalline apatites in dentine and bone. Such environments were believed to be located in a hydrated layer at the surfaces of the crystals, whereas the relatively ordered hydroxyapatite phase may exist in the core of the crystals.

Unlike the ATR-FTIR technique which is very surface sensitive due to its long-range frequency effect, the measurement of XRD as well as EDX might be influenced by the remnant mineral in the dentine after partial demineralization and thus be unable to detect mineral changes on the surface. Therefore, XRD and EDX were only performed on completely demineralized dentine in this study. With the support of SEM–EDX analysis, it could be noted that the compositions of the two bioactive glasses were consistent with the contents provided by the manufacturer in Table 1 (e.g. the presence of magnesium in M-BAG). High silicon peaks were observed in the bioactive glass powders, whilst lower intensity peaks were detected on the dentine surface. This might be due to the accumulation in the subsurface calcium silicates hydrate phase or release of a small amount of silicon into the AS solution.

XRD of the completely demineralized dentine suffering a 7-day remineralization induced by bioactive glasses yielded major peaks at the (0 0 2) and (2 1 1) planes, which appeared at the same positions as the spectra of natural dentine with higher peak intensity, further supporting the finding of HCA on the completely demineralized dentine surface. Meanwhile, it has been proven that demineralized dentine with phosphoproteins has the ability to induce apatite formation. Therefore, this amorphous phase might be a product of the interaction of the bioactive glass treated dentine surface with the surrounding AS solution. Additionally, even after only AS immersion without bioactive glass treatment, slight changes were observed by XRD. Since the AS is rich in calcium and phosphate ions, it is likely that remineralization was initiated when the specimens were put into the AS solution. The halo with stronger intensity after AS immersion when compared to completely demineralized dentine might indicate an amorphous phase which is possibly still in the early stage of HCA formation. Kielbassa et al. , and Tschoppe et al. revealed the remineralization effects of natural saliva and saliva substitutes on enamel and dentine by means of transversal microradiography (TMR). More recent studies indicated some nano-sized hydroxyapatite containing materials have strong remineralization effects on the subsurface enamel and dentine lesions using different techniques. In comparison to the surface measurements used in this study such as ATR-FTIR, TMR can quantitatively monitor the mineral variation at different lesion depths of the tooth specimens.

Even though the bioactive glasses developed a calcium-phosphate layer on the dentine surface, this phenomenon requires further clarification in future studies focused on the depth of the remineralization. Further in vitro analyses such as nano-indentation are also required to evaluate if the two bioglass powders used in this study may re-establish the biomechanical properties of hardness and elasticity of the dentine.

Conclusions

Within the limits of this study, it may be concluded that the two bioactive glasses possessed high remineralization ability as revealed by real-time spectroscopic techniques and showed surface morphology changes on dentine surface after immersion in AS.

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Dentine remineralization induced by two bioactive glasses developed for air abrasion purposes Zhejun Wang , Tao Jiang , Salvatore Sauro , Yining Wang , Ian Thompson , Timothy F. Watson , Yue Sa , Wenzhong Xing , Ya Shen and Markus Haapasalo Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 746-756, Copyright © 2011 Elsevier Ltd Abstract Objectives The present study aimed to evaluate dentine remineralization through a 7-day period of artificial saliva (AS) storage induced by bioactive glass 45S5 (BAG) and by bioactive glass modified with soda-lime spherical glass. Methods Partially demineralized dentine disks were treated by BAG or the spherical-glass modified bioactive glass (M-BAG) and subsequently immersed in AS for 7 days. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to quantitatively analyse the mineral variation of the dentine surface by calculating the spectra parameters. X-ray diffraction (XRD) and energy-dispersive X-ray analysis (EDX) were performed on completely demineralized dentine to confirm the apatite formation. The roughness of the dentine surface was evaluated by atomic force microscopy (AFM) and the morphology was also examined by scanning electron microscopy (SEM). Results ATR-FTIR showed a significant increase of the mineral matrix area ratio in dentine specimens treated with the two bioactive glasses subsequent to 7 days of AS storage. The XRD spectrum exhibited apatite growth and mineral elements could be found on completely demineralized samples analysed by EDX after remineralization treatments. The dentine specimens treated with bioactive glass showed lower roughness, and most of the dentinal tubules appeared completely occluded during the AFM and SEM examination. Conclusions Although the concentration of bioactive glass in the M-BAG is 60% of that contained in the original version, both formulations have similar potential in dentine remineralization. These bioactive powders developed for air-abrasive use may be considered as innovative bioactive materials for therapeutic remineralization of dental hard tissues. 1 Introduction Dentine is a tubular permeable structure composed of organic matrix embedded in crystalline apatite which accounts for the most part of dental hard tissues. Although a physiological equilibrium exists between remineralization and demineralization of the dental hard tissues in the oral cavity, an increase in organic acids derived from dental plaque or an acidic diet may lead to a change of this equilibrium inducing demineralization of the tooth surface. If the demineralization effect overcomes the self-regulated remineralization, excessive loss of mineral content from the dental hard tissues may occur jeopardising the integrity of the teeth. In particular clinical situations (i.e. abrasion or erosion) and in the proximity of specific morphological areas (i.e. gingival recession), this demineralization may result in the exposure of the dentinal tubules, though the formation of reactionary dentine in the pulp and dentine sclerosis inside the tubules may exist as a compensatory response. Several studies have been aimed at reincorporating mineral into demineralized dentine to achieve remineralization. Bioactive glass is an extensively studied biomaterial in the field of tissue engineering, bone regeneration and dentine remineralization due to the remarkable bioactive capability in forming hydroxycarbonate apatite (HCA) when submerged in simulated body fluid solutions. It has been recently demonstrated that it is possible to achieve hierarchy remineralization via apatite precipitation using calcium silicates in a biomimetic approach. Although it has been successfully proven that materials based on bioactive substance have the potential to promote dentine remineralization, only a limited number of studies have quantitatively monitored the remineralization process of demineralized dentine. Bioactive glass 45S5 (BAG) is currently used in dentistry for air polishing procedures and dentine desensitization. Moreover, bioactive glass might be used within air-cutting systems in restorative dentistry for minimally invasive cavity preparation. Nevertheless, there is still the need to create alternative bioactive glass formulations which may allow clinicians to perform more selective air-polishing or caries removal procedures offering, at the same time, the possibility for therapeutic remineralization of the dental hard tissues. The current study was designed to quantitatively compare the apatite forming capacity of the original and spherical-glass modified bioactive glass (M-BAG) in dentine remineralization using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) which is commonly used for the characterization of chemical changes in bone and other mineralized tissues as a non-destructive approach. Qualitative analysis by means of X-ray diffraction (XRD), Scanning electron microscopy–energy-dispersive X-ray analysis (SEM–EDX) and atomic force microscopy (AFM) were also used to analyse the chemical and morphological changes to the dentine surface. The null hypothesis of this study was that the two bioactive glasses (BAG and M-BAG) did not differ significantly in their remineralization ability on demineralized dentine compared to a control. 2 Materials and methods 2.1 Sample preparation Forty-five caries-free extracted human third molars were obtained under a protocol approved by the local Ethics Committee of the School and Hospital of Stomatology Wuhan University, China. Dentine disks with a thickness of 1.0 ± 0.1 mm, were prepared by sectioning each tooth 1.5 mm above the cemento-enamel junction using a low-speed water cooled diamond saw (Isomet, Buehler, Lake Bluff, IL, USA). The surrounding enamel of the disks was cut off, creating square dentine specimens (5 mm × 5 mm × 1 mm). A standard smear layer was created on both sides of the dentine specimens using 600-grit silicon carbide paper (Yuli Abrasive Belts, Shanghai, China) for 30 s under constant water irrigation. 2.2 Bioactive glasses and treatment procedures The compositions of BAG and M-BAG are listed in Table 1 . Bioactive glass 45S5 (BAG) (particle size: 30–90 μm) was mixed with 40 wt% of soda lime spherical-glass (particle size range: 4–80 μm) to create the spherical-glass-modified bioactive glass (M-BAG). The M-BAG mixture was kept under agitation at 60 rpm for 24 h and subsequently left undisturbed at 37 °C in a desiccator for a further 24 h. Dentine disks were randomly divided into three principal groups ( n = 10): artificial saliva (AS) as a control, BAG and M-BAG and then cut into two halves using a water-cooled high-speed handpiece with a fine carbide bur (AEU-25, Tulsa Dentsply, USA). Half of the specimens were partially demineralized in 0.5 M EDTA solution (pH 7.4) for 5 min, whilst the other halves of the specimens were completely demineralized in a 0.5 M EDTA solution (pH 7.4) at 25 °C for 15 days. The specimens were finally copiously rinsed with distilled water for 2 min and monitored using digital radiography to ensure that a complete demineralization was achieved as previously accomplished. Table 1 Composition of the materials used for dentine remineralization. Treatments Maufacturer Components Particle size range (μm) BAG OSspray Ltd., London, UK 45 wt% SiO 2 , 24.5 wt% CaO, 24.6 wt% Na 2 O, 5.8 wt% P 2 O 5 30–90 M-BAG OSspray Ltd., London, UK 60% BAG + 40% Soda-lime spherical glass a (73 wt% SiO 2 , 14 wt% Na 2 O, 9 wt% CaO, 4 wt% MgO) 4–80 a The Soda-lime spherical glass (Spheriglass Glass Microspheres) was provided by Potters Europe – Engineered Glass Materials Division, Suffolk, United Kingdom. The dentine surface was then slightly rubbed with 20 mg of BAG or M-BAG using a wet cotton pellet for 1 min, followed by copious rinsing (1 min) with distilled water. No treatment was performed in the control group. All treated specimens were subsequently stored in AS (pH 7.4), containing 1.5 mmol/l CaCl 2 , 50 mmol/l KCl, 0.9 mmol/l KH 2 PO 4 , 20 mmol/l Tris, at 37 °C for 7 days. The AS was replaced every 24 h with 20 ml of fresh AS. 2.3 ATR-FTIR spectroscopy Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectra were obtained from partially and totally demineralized dentine specimens subsequent to 1-day remineralization, 3-day remineralization and 7-day remineralization periods in AS using a Nicolet 5700 FTIR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a diamond crystal attenuated total reflection (ATR) accessory. Prior to the spectrophotometric analysis, the specimens were rinsed with distilled water for 30 s and then completely air dried. Reference points were marked on the area of examination in each specimen in order to perform the analysis at the same position before and after treatments. Spectra were collected in the range of 800–1800 cm −1 at 4 cm −1 resolution for a total of 64 scans, and analysed by OMNIC 8 software (Nicolet, Madison, WI, USA). Each specimen was analysed in 3 different positions before and after treatment. The spectrum acquired at the end of the analyses represented the average of all the three single scans. Spectra of water was obtained and subtracted from each of the original spectra which were then processed by smoothing, baseline corrected, and normalized to the amide I peak. The mineral matrix ratio (the ratio of integrated areas of the phosphate v 1 , v 3 contour to the amide I peak) was measured in all spectra in order to quantitatively calculate the extent of remineralization. In addition, ATR-FTIR spectra from the bioactive glass powders were also obtained to characterize their chemical structures. 2.4 XRD analysis X-ray diffraction (XRD) analysis was carried out before and after the 7-day remineralization for six additional completely demineralized specimens in AS, BAG and M-BAG groups by X’ Pert PRO Dy2198 (Spectric Pte, Singapore) with a CuKα generator working at 40 kV and 40 mA. The range of scanning angles was from 20° to 55°. In addition, an untreated dentine disc was measured to obtain a spectrum of the sound dentine as a control, and bioactive glass powders were also measured for characterization. 2.5 SEM–EDX analysis Six further completely demineralized specimens were remineralized for 7 days, rinsed with distilled water, dried in a desiccator and finally processed for carbon coating. The particle size of the bioactive glasses and changes in dentine surface morphology for partially demineralized samples following the 7-day remineralization were obtained using the SEM (QUANTA-200, FEI, Eindhoven, Netherlands) at 20 kV. An EDX apparatus associated with a SEM was used to identify the content of chemical elements of the dentine surface before and after the treatments. The EDX analysis of the two bioactive glasses powders was also performed. 2.6 AFM examination Three additional dentine disks were prepared as previously described and metallographically polished through silicon carbide papers (Yuli Abrasive Belts, Shanghai, China) starting with a 600-grit down to 4000-grit. A final polishing step was performed using a 0.25 μm diamond paste to provide an ultra-flat surface suitable for AFM scanning procedures. A Digital Instruments Nanoscope IV (Veeco, NY, USA) was used to measure the microstructure and surface roughness changes on the partially demineralized dentine disc after 7-day treatment using a contact mode. Fields of view at 20 μm × 20 μm scan size and 0.5 Hz scan rate were employed. In each image, five randomized boxes of 2 μm × 2 μm were drawn using the Nanoscope V530b4 (Veeco, NY, USA) at the intertubular dentine, and mean roughness ( R a ) and root mean square roughness ( R rms ) were determined. 2.7 Statistical analysis Statistical analysis was performed by SPSS 16.0 (SPSS, Chicago, IL, USA) for Windows. Means and standard deviations of the mineral matrix peak area ratio from ATR-FTIR measurement analysis were calculated. Homogeneity of variance was determined using Levene's test. Two-way repeated measures ANOVA was applied to evaluate the ATR-FTIR results, considering the treatment as main effect and treatment time as the repeated measure at a 5% significance level. Pair-wise comparisons for one-way repeated measures ANOVA with LSD adjustment were made within each group. One-way ANOVA was performed to analyse the AFM roughness values ( R a and R rms ). 3 Results 3.1 Material characterization In accordance with the contents of the bioactive glasses provided by the manufacturer ( Table 1 ), the SEM analysis showed that the morphology of bioactive glasses was characterized by micrometric irregular particles with a clump-like shape (BAG; Fig. 1 A ) or spherical shape (M-BAG; Fig. 1 E) with a dimension of approximately 50 μm, which is consistent with another study showing the micron-sized bioactive glass. SEM–EDX on BAG powder exhibited the presence of silicon, calcium and phosphate, whilst M-BAG powder showed higher silicon peak intensity and lower phosphate content with the presence of magnesium ( Fig. 1 B and F). ATR-FTIR spectra of the two bioactive glasses presented wide vibrational bands at 800–1200 cm −1 ( Fig. 1 C and G). BAG displayed vibrations at 900, 1000 and 1450 cm −1 corresponding to P–O stretch, Si–O–Si stretch and CO 3 2− v 3 band, and M-BAG contained P–O and Si–O–Si stretch at 920 and 1000 cm −1 , respectively. The XRD pattern of the two bioactive glasses showed the presence of a peak at 2 = 20–35° with a broad contour and low intensity, which indicated the amorphous state of both glasses ( Fig. 1 D and H). Fig. 1 Characterization of bioactive glasses measured by SEM–EDX, ATR-FTIR and XRD. (A) BAG particles were clump-like with sharp edges about 50 μm in size. (B) EDX showed silicon, calcium, phosphate, oxygen and sodium peaks for BAG. (C) ATR-FTIR spectra of BAG. (D) XRD spectra of BAG. (E) M-BAG exhibited round-shaped particles together with smaller clump-like shaped ones. (F) Besides a high silicon peak, traces of calcium, sodium, magnesium, oxygen and carbon were displayed. (G) ATR-FTIR spectra of M-BAG. (H) XRD spectra of M-BAG. 3.2 ATR-FTIR spectroscopy All representative spectra recorded in the region of 800–1800 cm −1 presented phosphate bands at 885–1180 cm −1 , representative of mineral components, assigned to the phosphate v 1 , v 3 stretching mode, and amide bands from 1180 to 1725 cm −1 from organic components. Phosphate v 1 , v 3 bands at 1010 cm −1 were detected and increased during the 7-day period in BAG and M-BAG groups ( Fig. 2 B and C ). However, a few changes of phosphate peaks could also be observed in the control group during the 7-day incubation ( Fig. 2 A). The mineral matrix area ratio increased significantly from 4.96 (BAG) and 4.64 (M-BAG) up to 13.81 and 15.06, respectively after 7-day AS immersion ( P < 0.05) ( Fig. 2 D). The two-way repeated ANOVA showed a statistically significant main effect for both time and treatment ( P < 0.05), as well as time × treatment interaction ( P < 0.05). However, pairwise comparisons indicated no significant difference in mineral matrix area ratio between these two bioactive glasses ( P > 0.05), but they both showed significantly larger ratio values than the control group ( P < 0.05). Fig. 2 A representative series of ATR-FTIR spectra (A)–(C) recorded on the dentine surface were marked in different colours for different time points at EDTA 5 min (black), 1-day (red), 3-day (blue) and 7-day (green) treatments. The spectra have been normalized to amide I. (A) Artificial saliva (AS), (B) BAG, (C) M-BAG. The mineral matrix area ratio after different time points for each group was present as well (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.) There was no clearly identifiable phosphate band at 885–1180 cm −1 on the completely demineralized dentine and the mineral matrix area ratio was very small. Only a slight increment of mineral matrix area ratio was detected after 7 days of immersion in AS ( P > 0.05) ( Fig. 3 A and D ). In the BAG group, the phosphate peaks (v 1 , v 3 ) were weak before application but a stronger apatite peak was detected after the 7-day remineralization ( Fig. 3 B). M-BAG induced a similar phosphate v 1 , v 3 band as BAG, indicating a sign of dentine remineralization bioactivity ( Fig. 3 C). However, there was no significant difference in the increase of mineral matrix area ratio between the two bioactive glass groups ( P > 0.05) ( Fig. 3 D). Fig. 3 A representative series of ATR-FTIR spectra (A)–(C) recorded on the completely demineralized dentine surface were marked in different colours for different time points which are complete demineralization (black) and 7-day remineralization (red). The spectra have been normalized to amide I. (A) Artificial saliva (AS), (B) BAG, (C) M-BAG. The mineral matrix area ratio after different time points for each group was present as well (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.) 3.3 XRD measurement The X-ray diffraction analyses performed in completely demineralized dentine, 7-day remineralized dentine and untreated dentine are displayed in Fig. 4 A . The untreated dentine generally exhibited a characteristic X-ray diffraction pattern of hydroxyapatite ( Fig. 4 A) (JCPDS 9-432). Completely demineralized specimens showed no obvious diffraction peak. After a 7-day immersion in AS, the diffraction patterns exhibited a more obvious halo ( Fig. 4 A) at 2 = 20–35° even though no sharp diffraction peak was presented. However, after 7-day remineralization, a characteristic reflection was observed at approximately 26° of (0 0 2) reflection in the two bioactive glasses groups. A (2 1 1) reflection diffraction peak at about 31.8° was visible ( Fig. 4 A), indicating the presence of apatite compared to the pattern of untreated dentine. Fig. 4 XRD patterns and EDX on completely demineralized dentine specimens. (A) XRD spectra of untreated dentine (red) and completely demineralized dentine (black) treated by artificial saliva (AS) (yellow), BAG (green) and M-BAG (blue) after setting in AS for 7 days. (B–D) EDX analysis of completely demineralized dentine after AS (B), BAG (C) and M-BAG (D) remineralization for 7 days. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.) 3.4 SEM–EDX analysis The SEM–EDX analysis commenced on the completely demineralized dentine surface showed traces of calcium and phosphate and relatively large amounts of carbon and oxygen ( Fig. 4 B). After 7-day AS storage, EDX detected no apatite element growth and SEM showed a dentine surface characterized by exposed dentinal tubules ( Fig. 5 A1 ). However, after bioactive glass treatments, calcium and phosphate became the dominant elements including the presence of sodium and silicon ( Fig. 4 C and D). The dentine surface was completely covered with almost all dentinal tubules occluded ( Fig. 5 B1 and C1). Separate micro-sized bioactive glass particles became aggregates of apatite spherulites (BAG) or needle like crystals (M-BAG) of nano-size (about 0.3 μm) in the homogeneous layer. Fig. 5 Dentine surface morphology of the partially demineralized dentine after different treatments. (A1, A2) SEM and AFM images of EDTA-etched (5 min) dentine surface after AS immersion for 7 days. (B1, B2) SEM and AFM images of EDTA-etched (5 min) dentine surface after BAG induced 7-day remineralization. (C1, C2) SEM and AFM images of EDTA-etched (5 min) dentine surface after M-BAG induced 7-day remineralization. 3.5 AFM examination The AFM ultra-morphology analysis resulted in a series of 20 μm × 20 μm three-dimensional images of dentine disks after the different treatments ( Fig. 5 A2, B2 and C2). The microstructure of 5 min EDTA etched dentine displayed open dentinal tubules after 7-day immersion in AS ( Fig. 5 A2). The demineralized specimens treated with both the bioactive glasses presented dentine surfaces characterized by deposits which covered the lumen of the dentinal tubules after the 7-day treatment ( Fig. 5 B2 and C2). Mean roughness ( R a ) and root mean square roughness ( R rms ) are presented in Table 2 . Baseline roughness values after polishing the dentine surface were significantly lower than those after treatments ( P < 0.05). However, BAG and M-BAG groups showed significant lower R a and R rms values than the AS group after 7-day remineralization ( P < 0.05), indicating that the mineral precipitation from bioactive glass remineralization resulted in a smoother surface. No difference was found between the two bioactive glass groups ( P > 0.05). Table 2 Means (standard deviations) of the intertubular mean roughness ( R a ) and root mean square roughness ( R rms ) after different treatments. Treatment R a (nm) R rms (nm) Baseline (after polishing) 41.16 (14.55) a 55.97 (13.29) d Artificial saliva (AS) 115.66 (33.34) b 134.56 (30.82) e BAG 85.71 (14.75) c 101.88 (13.60) f M-BAG 67.12 (16.29) c 81.62 (18.21) f Groups with the different superscript letters are statistically significant ( P < 0.05). 4 Discussion Bioactive glass 45S5 (BAG), a highly biocompatible calcium sodium phosphosilicate, is currently used in the dental clinic for air polishing procedures, or incorporated into toothpastes, desensitizing pastes, bonding materials and varnishes in order to create hybrid remineralizing materials. Although bioactive glass was originally developed as a bone conductive material, it may also react with saliva inducing a dissolution of Ca 2+ , PO 4 3− and Si 4+ at the glass surface and subsequent precipitation of a polycondensated silica-rich layer (Si-gel) which serves as a template for the formation of a calcium phosphate (Ca/P) which subsequently crystallise into HCA. The formation of a crystallised layer of HCA has also been recently observed after the application of bioactive glass containing toothpaste. A recent study used BAG in air-polishing procedures achieving dentinal tubule occlusion and dentine permeability reduction of up to 80%. Moreover, BAG was less aggressive than traditional alumina powders in air-polishing treatments. The use of M-BAG might further reduce the eventuality of an excessive dental tissue loss due to the presence of round-shaped soda-lime glass contained within the composition of the powder ( Fig. 1 B and F). Indeed, spherical particles of the powders achieve rounded internal angles which are considered to be ideal for restorative materials ensuring better marginal adaptation and reduction of voids after air abrasion. The null hypothesis that the two bioactive glasses (BAG and M-BAG) did not differ significantly compared to the control in their remineralization ability must be rejected. Indeed, this study has also provided evidences that the M-BAG has preserved its bioactivity compared to the original Bioglass 45S5 (BAG). The use of these two bioactive glasses during air-abrasion procedures might offer an important contribution to the therapeutic remineralization of dental hard tissues. The two bioactive glass formulations tested in this study were able to form HCA in demineralized dentine. The remineralization process induced by the two bioactive glasses used in this study were probably due to a simultaneous bioactive phenomena characterized by Si 4+ release and a subsequent polycondensation reaction induced by precipitation of calcium and phosphates on the organic matrix creating the template for the formation of a calcium phosphate phase. The reaction of polycondensation forms H 2 O which remains physically bonded with the Si–O–Si surface forming the hydrated silica rich layer. The polycondensated silicon-rich layer (Si-gel) then precipitates into an amorphous CaO–P 2 O 5 due to the incorporation of soluble Ca 2+ and PO 4 3− . The further incorporation of various mineral ions from AS helps the amorphous CaO–P 2 O 5 to convert into HCA. These processes were supported in this study by high calcium and phosphates peaks from EDX and by the increase of the mineral matrix area ratio observed in the ATR-FTIR analysis ( Figs. 2 and 4 ). Moreover, the results obtained during SEM examination also demonstrated that the dimensions of BAG and M-BAG powder particles were micrometrical before application in partially demineralized dentine, whilst after 7-day AS storage they turned into nano-sized particles ( Fig. 5 B1 and C1). The rationale behind using partially and completely demineralized models in this study was based on the elimination of the ambiguity in differentiating the remineralized apatite crystallites from remnant apatite seed crystallites existing in partially demineralized dentine. The completely demineralized dentine may ascertain that remineralization was able to occur without a contribution from non-collagenous proteins (NCPs) and proteoglycans released from mineralized dentine. However, the two bioglass powders showed a good remineralization effect on both the partially and completely demineralized models. The formation of apatite might improve the sealing of dentinal tubules as evidenced by the SEM and AFM results. The optimal occlusion effect of the bioactive materials should also play an important role if they are applied in the treatment of post-operative dentine hypersensitivity. Moreover, bioactive glasses showed lower intertubular roughness values ( R a and R rms ) in comparison to that observed in the partially demineralized control specimens after 7-day treatment ( Table 2 ). The R rms was reported to take less account of the variations of low scanning frequency in an AFM study. Surface roughness of the dentine affects not only the aesthetic properties but also bacterial adhesion and plaque formation. In the present study, bioactive glass treatment was able to achieve a smoother surface than control group by crystalline phase formation. Such improvement of surface roughness might promote the resistance to bacterial adherence. Vollenweider et al. assessed quantitatively the remineralization capability of ultrafine bioactive glass using thermogravimetry demonstrating enhanced mechanical properties in dentine. In addition, spectroscopic analyses by Fourier transform infrared spectroscopy (FTIR) have been commonly used to qualitatively support the presence of apatite formation amongst dentine remineralization investigations. However, no experiment has been performed to quantitatively evaluate dentine remineralization in real-time induced by a bioactive substance developed for restorative purposes of dentistry. The advantage of the spectroscopic methods is the non-destructive approach which provides continuous evaluation of the remineralization process. Unlike Fourier transform infrared spectroscopy (FTIR), which only accepts powder samples, ATR-FTIR has been shown to be an effective method for dentine mineral detection. A recent study demonstrated the bleaching effects of hydrogen peroxide on mineral and organic components of dentine before and after treatments by means of ATR-FTIR. Some more recent studies revealed the bioactivity of calcium silicate-based materials by means of ATR-FTIR and micro-Raman. In the present study, we found the mineral matrix area ratio of phosphate contour increased dramatically during the 7-day treatment on partially demineralized specimens in bioactive glass groups. Interestingly, the remineralization effect on completely demineralized dentine was weaker than the partially demineralized dentine, which is possibly due to the absence of dentine mineral contribution and difficulty in crystal nucleation. However, a low mineral matrix area ratio could be found on the completely demineralized dentine before treatments ( Fig. 3 ). This is probably because the digital radiography used for the demineralization detection was not as sensitive as ATR-FTIR. Even though no graph was captured by the digital radiography, ATR-FTIR could still detect a very slight intensity of the phosphate peak on the completely demineralized samples. Furthermore, it should be pointed out that the contour for phosphate v 1 , v 3 became broader during the remineralization period. The broadened phosphate contour probably indicates that the crystallinity of the apatite layer was decreasing. However, it has been reported that the presence of labile non-apatite environments of phosphate ions is one of the critical characteristics of poorly crystalline apatites in dentine and bone. Such environments were believed to be located in a hydrated layer at the surfaces of the crystals, whereas the relatively ordered hydroxyapatite phase may exist in the core of the crystals. Unlike the ATR-FTIR technique which is very surface sensitive due to its long-range frequency effect, the measurement of XRD as well as EDX might be influenced by the remnant mineral in the dentine after partial demineralization and thus be unable to detect mineral changes on the surface. Therefore, XRD and EDX were only performed on completely demineralized dentine in this study. With the support of SEM–EDX analysis, it could be noted that the compositions of the two bioactive glasses were consistent with the contents provided by the manufacturer in Table 1 (e.g. the presence of magnesium in M-BAG). High silicon peaks were observed in the bioactive glass powders, whilst lower intensity peaks were detected on the dentine surface. This might be due to the accumulation in the subsurface calcium silicates hydrate phase or release of a small amount of silicon into the AS solution. XRD of the completely demineralized dentine suffering a 7-day remineralization induced by bioactive glasses yielded major peaks at the (0 0 2) and (2 1 1) planes, which appeared at the same positions as the spectra of natural dentine with higher peak intensity, further supporting the finding of HCA on the completely demineralized dentine surface. Meanwhile, it has been proven that demineralized dentine with phosphoproteins has the ability to induce apatite formation. Therefore, this amorphous phase might be a product of the interaction of the bioactive glass treated dentine surface with the surrounding AS solution. Additionally, even after only AS immersion without bioactive glass treatment, slight changes were observed by XRD. Since the AS is rich in calcium and phosphate ions, it is likely that remineralization was initiated when the specimens were put into the AS solution. The halo with stronger intensity after AS immersion when compared to completely demineralized dentine might indicate an amorphous phase which is possibly still in the early stage of HCA formation. Kielbassa et al. , and Tschoppe et al. revealed the remineralization effects of natural saliva and saliva substitutes on enamel and dentine by means of transversal microradiography (TMR). More recent studies indicated some nano-sized hydroxyapatite containing materials have strong remineralization effects on the subsurface enamel and dentine lesions using different techniques. In comparison to the surface measurements used in this study such as ATR-FTIR, TMR can quantitatively monitor the mineral variation at different lesion depths of the tooth specimens. Even though the bioactive glasses developed a calcium-phosphate layer on the dentine surface, this phenomenon requires further clarification in future studies focused on the depth of the remineralization. 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