# Effects of a chewing gum containing phosphoryl oligosaccharides of calcium (POs-Ca) and fluoride on remineralization and crystallization of enamel subsurface lesions in situ

## Effects of a chewing gum containing phosphoryl oligosaccharides of calcium (POs-Ca) and fluoride on remineralization and crystallization of enamel subsurface lesions in situ

Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 771-779, Copyright © 2011 Elsevier Ltd

## Objectives

Manufacturers are adding fluoride (F) to calcium-containing chewing gums to further promote enamel remineralization. The aim of this study was to assess the effect of a chewing gum containing phosphoryl oligosaccharides of calcium (POs-Ca) and fluoride on remineralization of enamel subsurface lesions, in a double-blind, randomized controlled in situ trial.

## Methods

Thirty-six volunteer subjects wore removable buccal appliances with three different insets of bovine enamel with subsurface demineralized lesions. For 14 days the subjects chewed one of the three chewing gums (placebo, POs-Ca, POs-Ca + F), three times a day. After each treatment period, the insets were removed from the appliance, embedded, sectioned, polished and then subjected to laboratory tests; mineral level was determined by transverse microradiography (TMR; n = 36), and hydroxyapatite (HAp) crystallites were assessed by synchrotron radiation wide-angle X-ray diffraction (WAXRD; n = 13). Data were analysed by t -test or Wilcoxon rank-sum test with Bonferroni corrections at 0.05 significance level.

## Results

Chewing POs-Ca and POs-Ca + F gums resulted in 21.9 ± 10.6 and 26.3 ± 9.4 (mean ± SD) percentage mineral recovery, which was significantly higher than that of placebo gum (15.0 ± 11.4) ( p < 0.05). Chewing POs-Ca + F gum resulted in 24.9 ± 5.4 (mean ± SD) percentage HAp crystallites recovery, which was significantly higher compared to POs-Ca (16.0 ± 4.1%) or placebo (11.1 ± 4.8%) gums ( p < 0.05).

## Conclusions

Addition of POs-Ca to the chewing gum resulted in significant remineralization of enamel subsurface lesions. Although POs-Ca + F gum was not superior in TMR recovery rate when compared with POs-Ca gum, WAXRD results highlighted the importance of fluoride ion bioavailability in the formation of HAp crystallites in enamel subsurface lesions in situ ( NCT01377493 ).

## Introduction

The anticariogenic effects of sugar-free chewing gums have been demonstrated in a number of clinical trials. Recently, manufacturers have been adding calcium in various forms to the sugar-free chewing gums to enhance their potential anticariogenic action. Phosphoryl oligosaccharides of calcium (POs-Ca; Ezaki Glico, Osaka, Japan) is a form of soluble, bioavailable calcium that has been added to sugar-free gum. POs-Ca is produced by enzymatic hydrolysation of potato starch, and the calcium in POs-Ca is delivered as bioavailable ions.

On the other hand, fluoride content from various sources such as dentifrices, fluoridated water and green or black tea has been recognized as the source of beneficial dental effects. The use of fluoride agents is frequently recommended to remineralize the enamel subsurface lesions. Whilst addition of fluoride to chewing gums has been suggested as an effective oral health measure , few clinical reports have investigated the effects of sugar-free gums containing both the newly introduced calcium compounds and fluoride on enamel subsurface lesions.

The use of chewing gum increases the flow rate of saliva. Meanwhile, it has been reported that the solubility of calcium and fluoride compounds in saliva is low ; therefore, it is likely that when a sugar-free gum is chewed, the calcium and fluoride ions would not release into saliva from the gum in a bioavailable form. Under in vitro conditions, both calcium and fluoride compounds at high concentration showed a limited solubility in saliva. However, ‘in-house’ tests showed that both calcium and fluoride ions were released from a gum containing POs-Ca and fluoride, and had high bioavailability in saliva when the gum was chewed for 20 min (Ezaki Glico, personal communication). It was reported that as a calcium compound, POs-Ca had the advantage of a high water solubility ; therefore, in combination with fluoride, it may be desirable for improving the remineralization potential of the sugar-free chewing gums.

Most of the studies focusing on the beneficial effects of chewing gums and other agents on remineralization of enamel subsurface lesions have used transverse microradiography (TMR) to measure the mineral contents. TMR has been recognized as a gold standard for analysing the recovery of mineral content; however, this technique may not clarify the microstructure of the ‘remineralized’ zone in the subsurface lesion, as both crystalline and amorphous calcium phosphate phases are referred to as mineral content in the TMR data. In this regard, detailed structural analysis of the remineralized enamel may require employment of other techniques such as cross-sectional elemental analysis, crystallography and hardness measurements.

Synchrotron crystallography is a powerful technique for the study of the microstructure and crystal lattice of enamel. Wide-angle X-ray diffraction (WAXRD) enables qualitative and quantitative investigation of hydroxyapatite (HAp) crystallites at submicron and atomic levels. In this regard, a microbeam (6 μm in diameter) synchrotron radiation X-ray diffraction from Super Photon ring 8 GeV (SPring-8) was used to evaluate enamel crystallites in 100 μm-deep subsurface lesion. More recently, quantitative comparative in vitro study between TMR and WAXRD results indicated that both the spatial distribution and orientation of the restored HAp crystals in the remineralization process at the subsurface lesion could be simultaneously analysed by the WAXRD.

To date, no study has reported on the effect of chewing gum containing both calcium and fluoride on enamel remineralization, and the relationship between TMR and WAXRD profiles of enamel subsurface lesions in situ . The aim of this double-blind, randomized, controlled in situ study was to assess the effect of chewing gum containing POs-Ca and fluoride on enamel subsurface lesions remineralization and crystallites in comparison with POs-Ca or placebo (control) chewing gums. The null hypothesis was that there were no differences in the degree of enamel remineralization of subsurface lesions between the three chewing gums.

## Study design and protocol

Approval for this study was obtained from the Ethics Committee for Human Research at Tokyo Medical and Dental University (#392). Thirty-six healthy adult subjects (18 males and 18 females) were recruited (age 20–31 years) after obtaining informed consent. The volunteers were all residents of metropolitan Tokyo, where the tap water is not fluoridated (mean fluoride level: 0.07 ppm). The volunteers were examined to confirm that there was no current caries activity, periodontal condition or any other oral disease, and that each subject had 22 or more permanent teeth. The subjects were not taking any antibiotics or other medication which could potentially influence their oral conditions, such as the composition and flow rate of saliva. It was also confirmed that for each subject, the stimulated saliva flow rate, measured whilst chewing a pellet of unflavoured paraffin wax, was higher than 0.3 ml/min. Health history questionnaires at baseline and health update questionnaires during the treatment periods were used to monitor the subjects.

This study was based on a double-blind, randomized, controlled in situ design with three sugar-free gums as described in Table 1 . One of the gums was a placebo sugar-free chewing gum without POs-Ca (Ezaki Glico) and the other two contained 2.5 wt% POs-Ca or 2.5% POs-Ca and 1.2% fluoride-containing green tea extract, namely POs-Ca and POs-Ca + F. The placebo and intervention chewing gums were identical in appearance, taste and smell without any observable differences. The chewing gum slabs were received in code-labelled packages stored at room temperature. The codes were randomly assigned by a controller and remained unknown to the volunteers and operators until all remineralization data had been obtained. The subjects were randomly divided into three groups ( n = 12) according to the design presented in Fig. 1 . The treatment was performed in three periods of 2 weeks each, during which the subjects in each groups received one of the gums randomly. Between each two treatment periods, there was a resting period of 1 week at which treated enamel insets of appliances were exchanged for new demineralized insets according to the cross-over design. Subjects then crossed over to another gum product. This was repeated until each subject had used each of the three gums. The subjects wearing the appliances chewed two slabs of gum three times every day (at 8:00 am, 12:00 pm and 5:00 pm) for 20 min each time. After this and before removing the intraoral appliance, the subjects continued wearing the appliances for 20 more minutes whilst refraining from eating or drinking.

Table 1
Composition of the chewing gums used in the study (wt%).
Ingredients Gum
Placebo POs-Ca POs-Ca + F
POs-Ca 0 2.5 2.5
Green tea extract fluoride 0 0 1.2
Maltitol 41.0 41.0 41.0
Xylitol 27.5 25.0 23.8
Gum base 24.0 24.0 24.0
Flavour, etc. 7.5 7.5 7.5
Participant disposition for the randomized control trial. (A) Placebo gum, (B) POs-Ca gum and (C) POs-Ca + F gum.

## Preparation of enamel subsurface lesions

Fresh bovine incisors were obtained from a local slaughterhouse (Yokohama, Japan). Enamel blocks 7 mm × 10 mm × 2 mm (width × length × depth) were cut from the bovine incisors using a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) under running water, and embedded in acrylic resin (Unifast Trad; GC, Tokyo, Japan). The outer enamel surface was polished to a mirror finish using wet polishing papers (800, 1000, and 2000 grit lapping papers; 3M, St. Paul, MN, USA). Three zones, namely sound, demineralized (DEM), and remineralized (REM), were assigned on the polished enamel surface of each block as follows. First, one third of the surface on each block was covered with a nail varnish (Nail POP; Chamon, Kyonggi, South Korea) ; this part served as the sound portion. Subsurface lesions were then formed on the remaining surface using the two-layer demineralization method with 8% methylcellulose gel (Methocel MC, Fluka, Everett, WA, USA) and 0.1 M lactate buffer (pH 4.6) at 37 °C for 14 days. The ratio of gel to lactate buffer was 3:5. After demineralization, the blocks were sterilized by ethylene oxide gas sterilizer (Steri-Gas; 3M, St. Paul, MN, USA). Another one-third of the surface was then covered by the nail varnish, and the remaining exposed one-third of enamel surface served as remineralization zone. In total, 108 blocks were prepared as insets for insertion into the intraoral appliances.

## Intraoral appliances

Removable lower-buccal acrylic appliances covering premolars to molars were fabricated for each subject ( Fig. 2 ). The appliances were retained by two stainless steel circumferential clasps on canine and first molar, with a trough to house for enamel insets. Subjects were instructed to insert and remove the intraoral appliances according to the treatment design. They were also asked to rinse the appliances briefly after removal with tap water and store them at 37 °C in sealed and humidified containers until the next insertion. After completion of each 2-week treatment period, the enamel blocks were removed from the appliances, rinsed with distilled/deionized water and stored under moist conditions in labelled microcentrifuge tubes until laboratory tests.

Schematic for specimen preparation and the intraoral appliance design for the in situ trial and TMR or WAXRD analyses.

## TMR (microradiography and microdensitometry)

For TMR assessment of treated enamel bocks in each group ( n = 36), approximately 150-μm-thick slabs were obtained by cutting the enamel blocks through the middle and perpendicular to the treated surfaces using a water-cooled diamond-coated wire saw (Well Diamond Wire Saws, Mannheim, Germany). The surfaces of the sections were carefully polished, whilst the thickness of each section was monitored by a micrometre (Mitutoyo, Tokyo, Japan). In this manner, transverse sections, each including three zones (sound, DEM, REM), were obtained. The thin sections were then microradiographed together with high-purity aluminium step wedges (12.5 μm × 21 steps) as mineral density phantoms onto high-resolution plates (Konica Minolta Opto Products, Yamanashi, Japan) by Cu Kα X-ray generated at 20 kV and 20 mA for 13 min (PW-3830; Philips, Eindhoven, Netherlands). The mineral profiles of the sound, DEM and REM zones were obtained from the digital images of the microradiographs. The uniform density at the sound zone was confirmed, indicating the flatness and plano-parallelism of the prepared sections. The digital microradiographs were then analysed using a computerized image-analysis system (TMR v1.25; Inspektor Research System, Amsterdam, Netherlands). The output parameters obtained were the mineral content profile of the lesion, lesion depth and integrated mineral loss (ML). In this analysis, the mineral content of sound enamel was assumed to be 87 vol%. The mineral recovery rate for each specimen was calculated from the ML of DEM (ML DEM ) and REM (ML REM ) as follows: recovery rate (% R ) = (ML DEM − ML REM )/ML DEM × 100.

## Wide-angle X-ray diffraction with an X-ray microbeam

The wide-angle X-ray diffraction measurement for 13 specimens randomly selected for each gum was calculated in the same manner as described by Yagi et al. and Tanaka et al. The experiments were carried out at the BL40XU beamline of the SPring-8 (Japan Synchrotron Radiation Research Institute, Hyogo, Japan) third-generation synchrotron radiation facility with an X-ray energy of 15.0 keV (bandwidth ∼3%). The diameter of the X-ray microbeam at the sample (spot size) was about 6 μm (full width at half-maximum). The X-ray detector consisted of an X-ray image intensifier (V5445P; Hamamatsu Photonics, Hamamatsu, Japan) coupled to a cooled CCD camera (ORAC-II-ER; Hamamatsu Photonics, Hamamatsu, Japan). Enamel sections, which had been examined by TMR, were subjected to the WAXRD measurements. The sections had been kept sealed with water, and carefully inspected under a stereomicroscope (Olympus, Tokyo, Japan) to check for any changes in the sample shape or damage to the edge, before the WAXRD measurements. All X-ray measurements were made at ambient temperature and humidity. Each section was mounted vertically so that its enamel edge was horizontal; the X-ray beam passed perpendicularly through the slice. The sample was moved upwards in 5 μm steps so that the X-ray beam could scan across the enamel, from the surface towards the dentine. At each step a WAXRD pattern was recorded. In the WAXRD patterns, the (1 0 0) equatorial reflection (at a Bragg spacing of 0.815 nm) was used to obtain the intensity profile along the depth up to 150 μm depth in the enamel. The intensity profiles were then normalized by the average intensity between 150 and 200 μm from the surface. In this manner, the subsurface lesion (DEM, REM) and sound zones of each sample were evaluated on a region up to the depth of 200 μm. The intensity profiles were then integrated over 150 μm from the surface to obtain crystal content of this zone. The resulting integrated crystal contents of the 150-μm-thick surface layer of DEM and REM ( C DEM and C REM ) were then used to calculate HAp crystallites recovery rate after each chewing gum treatment as follows:

HAprecoveryrate(%)=CDEMCREMCDEM×100. $\text{HAp}\text{}\text{recovery}\text{}\text{rate}\text{}\left(%\right)=\frac{{C}_{\text{DEM}}-{C}_{\text{REM}}}{{C}_{\text{DEM}}}×100\text{.}$

## Gum analysis

In order to analyse the contents of the chewing gums, 5 pellets (about 1.4 g/pellet) of each chewing gum were pounded with a pestle in 50 ml of the artificial saliva for 20 min at 37 °C. This procedure simulated chewing two pellets of gum with 20-ml saliva that is usually secreted during 20-min chewing. The artificial saliva was prepared containing 100 mM KCl (Wako Pure Chemical Industries, Osaka, Japan), 3.6 mM KH 2 PO 4 (Wako Pure Chemical Industries, Osaka, Japan), 1.5 mM CaCl 2 (Wako Pure Chemical Industries, Osaka, Japan), and 20 mM HEPES-K (Dojindo, Kumamoto, Japan) (pH 7.0); the pH was then adjusted to 6.5, as described previously. The gum extracts were collected every 5 min during pounding. Then, the extracts were centrifuged (10,000 × g × 5 min) to remove gum debris. The calcium and phosphate concentrations of the supernatants were determined by the inductively coupled plasma-atomic emission spectroscopy (ICP-AES SPS7700; Seiko Instruments, Tokyo, Japan) and a p -methylaminophenol reduction method using Phospha C-test kit (Wako Pure Chemical Industries, Osaka, Japan), respectively. The fluoride ion concentrations of the supernatants were analysed with a calibrated fluoride ion electrode (6561-10C; Horiba, Kyoto, Japan) with F detection range from 0.02 to 19,000 ppm.

## Data analysis

The data were first tested for normality. The ML DEM and % R data were examined at the 95% level of significance by t -test with Bonferroni corrections. The data of HAp crystallites recovery rate were analysed by Wilcoxon rank-sum test with Bonferroni correction. To avoid an accumulation of errors due to multiple comparisons, the Bonferroni correction modified the significance level by dividing it ( p < 0.05) between the number of comparisons made. Where p < 0.0017, the comparison was considered to be statistically significant. All statistical analyses were performed using SPSS version 11.0 software (SPSS, Chicago, IL, USA).

## Results

All randomized subjects completed the in situ study without significant protocol violations and were therefore included in the analysis. Health questionnaires indicated no adverse events.

## Enamel remineralization

The mean mineral density profiles of the DEM and REM zones in each gum are shown in Fig. 3 (a–c) , and representative microradiographic images of the subsurface lesions before and after chewing each gum are presented in Fig. 4 . ML data of the enamel subsurface lesion of DEM and REM regions for each gum are summarized in Table 2 . No significant differences were found in the initial lesions used for the in situ study as shown by the initial ML DEM values for the three groups ( p > 0.05). The mean mineral recovery rate (% R ) (mean ± SD) for the different gums (placebo, POs-Ca, POs-Ca + F) was 15.0 ± 11.4, 21.9 ± 10.6 and 26.3 ± 9.4, respectively. Chewing POs-Ca and POs-Ca + F gums resulted in significantly greater average recovery rate than that produced by chewing the control gum ( p < 0.05). However, there was no statistical difference in recovery rate between POs-Ca and POs-Ca + F ( p > 0.05).

Mean TMR (a–c) profiles of all specimens for three chewing gums. (a) The placebo chewing gum, (b) POs-Ca chewing gum and (c) POs-Ca + F chewing gum. DEM: demineralized zone; REM: remineralized zone. Note that whilst all the chewing gums resulted in some mineral gain, (b) and (c) resulted in a significantly higher mineral recovery compared to (a).
Representative TMR microradiographs of the subsurface lesions before and after remineralization for three chewing gums. DEM: demineralized zone; REM: remineralized zone. The placebo gum resulted in a minor remineralization in the subsurface lesion whereas for POs-Ca and POs-Ca + F, decreased radiolucency through the body of the lesions was visible, especially for POs-Ca + F.
Table 2
Remineralization of enamel subsurface lesions assessed by TMR (mean ± SD).
Sample ML DEM (vol% μm) ML REM (vol% μm) ML DEM − ML REM (vol% μm) % R
Placebo 3461 ± 625 2942 ± 664 519.5 ± 368.1 15.0 ± 11.4 b,c
POs-Ca 3471 ± 865 2711 ± 704 760.1 ± 460.3 21.9 ± 10.6 b
POs-Ca + F 3628 ± 594 2675 ± 533 952.7 ± 385.6 26.3 ± 9.4 c
Mean initial lesion depth = 133 ± 32 μm. Values marked by similar lowercase superscript letters are significantly different ( p < 0.05). ML DEM : mineral loss (Δ Z ) of demineralized enamel, ML REM : mineral loss (Δ Z ) of remineralized enamel after chewing each gum, % R : mineral recovery [(ML DEM − ML REM /ML DEM )/100].

## WAXRD

The mean HAp crystallite recovery rates (%) (mean ± SD) for the different gums (placebo, POs-Ca, POs-Ca + F) are 11.1 ± 4.7, 16.0 ± 4.1 and 24.9 ± 5.4, respectively. Chewing POs-Ca and POs-Ca + F gums resulted in significantly greater HAp crystallite recovery rate than that produced by chewing the placebo gum ( p < 0.05). Moreover, there was a statistical difference in the HAp crystallite recovery rate between POs-Ca and POs-Ca + F ( p < 0.05). Representative normalized WAXRD profile plots are presented in Fig. 5 (a–c) .

Representative WAXRD profiles for three chewing gums. (a) The placebo chewing gum, (b) POs-Ca chewing gum and (c) POs-Ca + F chewing gum. DEM: demineralized zone; REM: remineralized zone. The difference between REM and DEM in (c) resulted in a significantly higher crystal recovery compared to (a) and (b). The WAXRD profiles are not as smooth as in TMR, owing to the beam spot size.

## Gum ion analysis

Analyses of ions released into artificial saliva from the three gums with regard to calcium, phosphate and fluoride ions are presented in Table 3 . For the placebo gum, only a small amount of soluble calcium was released from the gum base. The POs-Ca gum showed a higher level of extractable calcium in the artificial saliva (up to 7.1 mM) similar to POs-Ca + F gum (up to 7.3 mM). The highest amount of fluoride ion release (1.25 ppm) was found in POs-Ca + F gum at 20 min.

Table 3
Ion analysis results for calcium, inorganic phosphate and fluoride levels (mean ± SD).
Sample Time (min) Ca (mM) P (mM) F (ppm)
Placebo 5 2.5 ± 0.2 3.1 ± 0.3 0.07 ± 0.03
10 2.6 ± 0.1 3.1 ± 0.3 0.06 ± 0.02
15 2.9 ± 0.9 3.2 ± 0.2 0.06 ± 0.01
20 2.5 ± 0.2 3.2 ± 0.3 0.06 ± 0.01
POs-Ca 5 4.1 ± 0.5 3.2 ± 0.3 0.05 ± 0.03
10 5.7 ± 0.4 3.1 ± 0.3 0.07 ± 0.01
15 6.4 ± 0.9 3.2 ± 0.4 0.08 ± 0.01
20 7.1 ± 0.8 3.3 ± 0.4 0.07 ± 0.01
POs-Ca + F 5 4.5 ± 0.1 3.2 ± 0.2 0.52 ± 0.13
10 5.9 ± 0.1 3.2 ± 0.3 0.84 ± 0.15
15 7.0 ± 0.5 3.3 ± 0.2 1.03 ± 0.21
20 7.3 ± 0.5 3.3 ± 0.3 1.25 ± 0.31

## Discussion

An in situ model was chosen in the present study in order to simulate the everyday situation to test different chewing gum treatments aimed at improving the demineralized enamel. The use of bovine enamel has been verified in a number of in vitro studies to evaluate the effect of anticariogenic agents on enhancing enamel remineralization and inhibiting enamel demineralization. Using the in situ model, human and bovine enamel have shown similar characteristics in subsurface lesion remineralization under irradiation or abrasion influence. However, bovine enamel is not similar to human enamel in some aspects; for example, it may have a higher porosity, potentially leading to a faster demineralization rate. The sound bovine enamel had a lower mineral concentration than human enamel. On the other hand, these differences result in a relatively more consistent response to treatments in the experimental groups for bovine enamel when compared to human enamel. These points should be taken into consideration when interpreting any demineralization and remineralization study using bovine enamel, even if there were no qualitative differences between bovine and human enamel. Nevertheless, an objective comparison amongst groups required a method which took into account the baseline variations amongst the demineralized lesions (shallow vs. deep lesions). In the current study, the lower variability of bovine enamel specimens enabled creation of standardized baseline lesions required for the cross-over study; there were no significant differences in the integrated mineral content of demineralized lesions (ML DEM ) subjected to remineralization in all experimental groups, indicating that a comparison of remineralization amongst the lesions would be valid ( Table 2 ). Furthermore, the recovery rate variables were used for statistical comparisons in this study, rather than direct comparison of remineralization values ( i.e. ML REM and C REM ) amongst different treatments.

Considering the long period (6 weeks in total) of the trial for each volunteer, a small removable lower-buccal acrylic appliance was designed to reduce the discomfort of the subject when wearing them. The appliances were constructed with two circumferential clasps to provide adequate retention and avoid any dislodgment during chewing, whilst allowing frequent insertion and removal of the appliance during the trial period. The enamel slabs were sized to accurately fit the stainless steel housings on the appliances and retained by a small amount of dental wax to minimize the potential hazard of displacement or swallowing of the specimen during chewing. No such events were reported by the subjects in this study. Significant mineral recovery rates resulted after the 2-week periods not only with the test gums but also with the placebo gum. An extended period of chewing could have resulted in higher recovery rates, but it was beyond the scope of this randomized controlled trial.

Two X-ray analysis techniques were used to investigate the microstructure of the subsurface lesion at different depth levels. Microradiographic assessment by TMR provides mineral content profiles, but cannot render the crystal structure of the tissue. In an in vitro study comparing both TMR and WAXRD, both the spatial distribution and orientation of the restored HAp crystals in the remineralization process at the subsurface lesion could be simultaneously analysed by the WAXRD. In order to investigate the superficial structure, specially under the fluoride containing gum, a microbeam design was considered for quantitative measurement of HAp crystals and fluorapatite crystals. It was previously suggested that the intensity of the (1 0 0) equatorial reflections was not greatly affected by the tilt of the c -axis of the HAp crystals, and therefore gave a more reliable measure of the amount of the crystals. Using the 6-μm-diameter microbeam diffracted throughout the thickness of the enamel slice, the WAXRD could provide an average relative quantification of crystallites at the subsurface lesion in this study, as presented in Fig. 5 (a–c). This work may be the first in situ trial to employ a crystallographic method at a third-generation synchrotron radiation facility to compare the recrystallization by different remineralizing agents, besides the conventional mineral recovery data. Further development of the crystallography methodologies may enable their laboratory application, without the need to use the costly synchrotron radiation facilities.

Both tests conducted on enamel in this study confirmed increased remineralization by both POs-Ca gums after 2 weeks, compared to the placebo. It was reported that an effective remineralization of subsurface lesions required the agent to diffuse past the pellicle-covered enamel surface and into the subsurface lesion area. Unless the particles are small enough, calcium will rapidly adsorb onto the surface layer, decrease the pore volume of the layer and obstruct the diffusion pathways. Recent research demonstrated that nano-sized particles had an ability for biomimetic repair of the enamel subsurface lesion. Moreover, considering the current results, it was assumed that POs-Ca was capable of providing calcium ions in a bioavailable form on the enamel surface, driving diffusion of the ions into the subsurface lesion to promote remineralization due to the small size of POs-Ca (average molecular mass: about 800; maximum chain length: about 4 nm).

The gum analysis at a neutral pH in this study demonstrated high solubility of the POs-Ca, with both POs-Ca and POs-Ca + F gums releasing a similar amount of up to 7.3 mM calcium ions into the artificial saliva. This high calcium amount was extractable into both water and saliva without requiring any acid for solubilization. According to the most recent literature, another form of bioavailable calcium such as casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) required acid for the solubilization. These findings highlighted the important role of the POs-Ca, not only in a marked increase in calcium ion solubility, but also in localizing the calcium ions in saliva to facilitate intraoral subsurface enamel lesion remineralization.

HAp crystals are formed in the oral environment from supersaturated calcium and phosphate ions, and the presence of fluoride ions is likely to further promote the remineralization process by forming fluorapatite crystals that are more resistant to future demineralization compared to HAp. In the current study, a low fluoride ion concentration (around 1 ppm) extracted from POs-Ca + F gum was detected in the gum analysis. On the effects of low levels of fluoride on remineralization, it was reported that a fluoride treatment continuously present at 1 ppm was beneficial for repair of deep lesions, at least in the outer enamel. On the other hand, it has been suggested that calcium fluoride (CaF 2 ) deposits on or within the surface that form due to the interaction of fluoride and calcium ions may be protected by salivary constituents and act as a fluoride reservoir ; however, the formation of CaF 2 was considered unlikely in the current study, since the combinational conditions of a low pH and a high fluoride concentration that favour formation of CaF 2 were not a feature in situ . Such a low fluoride level as in the extracted solution did not cause CaF 2 precipitation under the artificial saliva in vitro . Moreover, the effects of high-fluoride levels in the forms of gel or mouthwash may not last as long as previously hypothesized ; therefore, frequent exposure to low but slightly elevated levels of fluoride in saliva and plaque from sources such as water or the chewing gum can perhaps more effectively help to prevent and reverse caries by inhibiting demineralization and enhancing remineralization.

It has been reported that the presence of fluoride in in vitro remineralization, even at low concentrations, may induce a continuous deposition of minerals on the lesion surface and gradually block the pores present on the demineralized surface layer. In this study, any tendency towards some kind of hypermineralization on the top surface of remineralized subsurface lesion was not detected on the TMR profile of POs-Ca + F group. In this regard, apart from aspects discussed earlier, it should be noted that in the in situ trial, formation of an acquired pellicle is inevitable, and therefore the role of macromolecules from natural saliva should not be overlooked. It was reported that in the presence of low fluoride concentrations, these molecules seemed to play an important role in the continuation of remineralization by reducing mineral gains on the surface layer.

According to the results, it was assumed that the fluoridated gum in this study effectively exerted ionized fluoride which enhanced HAp crystallites during remineralization. Whilst there was no significant difference between the two tested gums in TMR profile, an enhanced crystalline structure in POs-Ca + F group could be confirmed by the WAXRD technique. This was most likely due to a chemical change by the substitution of the hydroxyl groups of HAp by fluoride ions and formation of a less soluble fluorinated apatite. Nevertheless, elemental composition analysis and localization of fluoride incorporated into the remineralized subsurface lesions may be required to elucidate the remineralization mechanism of fluoride-containing gum under the in situ model. On top of the X-ray analysis techniques employed in the current study, which showed remarkable similarities around the mid-depth of the lesion for the test gums, it may be suggested that microhardness can provide a direct measure of effective mineralization in in situ trials. Further cross-sectional investigation using both mineral and hardness profiles may shed more light on the healing process of the subsurface lesions. Moreover, other techniques such as optical coherence tomography (OCT), surface pH measurement and atomic force microscopy (AFM) would be useful for evaluation of enamel remineralization.

This study confirmed the advantage of synergistic use of bioavailable calcium (POs-Ca) with fluoride on remineralization and crystal growth within the subsurface lesion in situ . However, clarifying the mechanism of the contribution and possibility of crystal nucleation by the compound warrants further research.

## Conclusions

In conclusion, this in situ study demonstrated that POs-Ca containing gums could significantly contribute to remineralization and recrystallization within enamel subsurface lesions, compared to a placebo gum. Furthermore, POs-Ca + F gum was superior in crystal recovery when compared with POs-Ca gum according to WAXRD analysis. POs-Ca + F gum appeared to be an effective vehicle for fluoride supplementation.

## Acknowledgements

This project was supported by grant #21592413 from the Japan Society for the Promotion of Science , and for Global Center of Excellence Program ; International Research Center for Molecular Science in Tooth and Bone Diseases in Tokyo Medical and Dental University . The authors would like to thank Dr. Naoto Yagi, Dr. Hiroshi Kamasaka, Dr. Hiroshi Takii and Ms. Tomoko Tanaka for their helpful advice towards this manuscript. The experiments at SPring-8 were conducted with the approval of the SPring-8 Project Review Committee (2009A1885, 2010A1278).

1 These authors contributed equally to this work.

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Effects of a chewing gum containing phosphoryl oligosaccharides of calcium (POs-Ca) and fluoride on remineralization and crystallization of enamel subsurface lesions in situ Yuichi Kitasako , Miyuki Tanaka , Alireza Sadr , Hidenori Hamba , Masaomi Ikeda and Junji Tagami Journal of Dentistry, 2011-11-01, Volume 39, Issue 11, Pages 771-779, Copyright © 2011 Elsevier Ltd Abstract Objectives Manufacturers are adding fluoride (F) to calcium-containing chewing gums to further promote enamel remineralization. The aim of this study was to assess the effect of a chewing gum containing phosphoryl oligosaccharides of calcium (POs-Ca) and fluoride on remineralization of enamel subsurface lesions, in a double-blind, randomized controlled in situ trial. Methods Thirty-six volunteer subjects wore removable buccal appliances with three different insets of bovine enamel with subsurface demineralized lesions. For 14 days the subjects chewed one of the three chewing gums (placebo, POs-Ca, POs-Ca + F), three times a day. After each treatment period, the insets were removed from the appliance, embedded, sectioned, polished and then subjected to laboratory tests; mineral level was determined by transverse microradiography (TMR; n = 36), and hydroxyapatite (HAp) crystallites were assessed by synchrotron radiation wide-angle X-ray diffraction (WAXRD; n = 13). Data were analysed by t -test or Wilcoxon rank-sum test with Bonferroni corrections at 0.05 significance level. Results Chewing POs-Ca and POs-Ca + F gums resulted in 21.9 ± 10.6 and 26.3 ± 9.4 (mean ± SD) percentage mineral recovery, which was significantly higher than that of placebo gum (15.0 ± 11.4) ( p < 0.05). Chewing POs-Ca + F gum resulted in 24.9 ± 5.4 (mean ± SD) percentage HAp crystallites recovery, which was significantly higher compared to POs-Ca (16.0 ± 4.1%) or placebo (11.1 ± 4.8%) gums ( p < 0.05). Conclusions Addition of POs-Ca to the chewing gum resulted in significant remineralization of enamel subsurface lesions. Although POs-Ca + F gum was not superior in TMR recovery rate when compared with POs-Ca gum, WAXRD results highlighted the importance of fluoride ion bioavailability in the formation of HAp crystallites in enamel subsurface lesions in situ ( NCT01377493 ). 1 Introduction The anticariogenic effects of sugar-free chewing gums have been demonstrated in a number of clinical trials. Recently, manufacturers have been adding calcium in various forms to the sugar-free chewing gums to enhance their potential anticariogenic action. Phosphoryl oligosaccharides of calcium (POs-Ca; Ezaki Glico, Osaka, Japan) is a form of soluble, bioavailable calcium that has been added to sugar-free gum. POs-Ca is produced by enzymatic hydrolysation of potato starch, and the calcium in POs-Ca is delivered as bioavailable ions. On the other hand, fluoride content from various sources such as dentifrices, fluoridated water and green or black tea has been recognized as the source of beneficial dental effects. The use of fluoride agents is frequently recommended to remineralize the enamel subsurface lesions. Whilst addition of fluoride to chewing gums has been suggested as an effective oral health measure , few clinical reports have investigated the effects of sugar-free gums containing both the newly introduced calcium compounds and fluoride on enamel subsurface lesions. The use of chewing gum increases the flow rate of saliva. Meanwhile, it has been reported that the solubility of calcium and fluoride compounds in saliva is low ; therefore, it is likely that when a sugar-free gum is chewed, the calcium and fluoride ions would not release into saliva from the gum in a bioavailable form. Under in vitro conditions, both calcium and fluoride compounds at high concentration showed a limited solubility in saliva. However, ‘in-house’ tests showed that both calcium and fluoride ions were released from a gum containing POs-Ca and fluoride, and had high bioavailability in saliva when the gum was chewed for 20 min (Ezaki Glico, personal communication). It was reported that as a calcium compound, POs-Ca had the advantage of a high water solubility ; therefore, in combination with fluoride, it may be desirable for improving the remineralization potential of the sugar-free chewing gums. Most of the studies focusing on the beneficial effects of chewing gums and other agents on remineralization of enamel subsurface lesions have used transverse microradiography (TMR) to measure the mineral contents. TMR has been recognized as a gold standard for analysing the recovery of mineral content; however, this technique may not clarify the microstructure of the ‘remineralized’ zone in the subsurface lesion, as both crystalline and amorphous calcium phosphate phases are referred to as mineral content in the TMR data. In this regard, detailed structural analysis of the remineralized enamel may require employment of other techniques such as cross-sectional elemental analysis, crystallography and hardness measurements. Synchrotron crystallography is a powerful technique for the study of the microstructure and crystal lattice of enamel. Wide-angle X-ray diffraction (WAXRD) enables qualitative and quantitative investigation of hydroxyapatite (HAp) crystallites at submicron and atomic levels. In this regard, a microbeam (6 μm in diameter) synchrotron radiation X-ray diffraction from Super Photon ring 8 GeV (SPring-8) was used to evaluate enamel crystallites in 100 μm-deep subsurface lesion. More recently, quantitative comparative in vitro study between TMR and WAXRD results indicated that both the spatial distribution and orientation of the restored HAp crystals in the remineralization process at the subsurface lesion could be simultaneously analysed by the WAXRD. To date, no study has reported on the effect of chewing gum containing both calcium and fluoride on enamel remineralization, and the relationship between TMR and WAXRD profiles of enamel subsurface lesions in situ . The aim of this double-blind, randomized, controlled in situ study was to assess the effect of chewing gum containing POs-Ca and fluoride on enamel subsurface lesions remineralization and crystallites in comparison with POs-Ca or placebo (control) chewing gums. The null hypothesis was that there were no differences in the degree of enamel remineralization of subsurface lesions between the three chewing gums. 2 Materials and methods 2.1 Study design and protocol Approval for this study was obtained from the Ethics Committee for Human Research at Tokyo Medical and Dental University (#392). Thirty-six healthy adult subjects (18 males and 18 females) were recruited (age 20–31 years) after obtaining informed consent. The volunteers were all residents of metropolitan Tokyo, where the tap water is not fluoridated (mean fluoride level: 0.07 ppm). The volunteers were examined to confirm that there was no current caries activity, periodontal condition or any other oral disease, and that each subject had 22 or more permanent teeth. The subjects were not taking any antibiotics or other medication which could potentially influence their oral conditions, such as the composition and flow rate of saliva. It was also confirmed that for each subject, the stimulated saliva flow rate, measured whilst chewing a pellet of unflavoured paraffin wax, was higher than 0.3 ml/min. Health history questionnaires at baseline and health update questionnaires during the treatment periods were used to monitor the subjects. This study was based on a double-blind, randomized, controlled in situ design with three sugar-free gums as described in Table 1 . One of the gums was a placebo sugar-free chewing gum without POs-Ca (Ezaki Glico) and the other two contained 2.5 wt% POs-Ca or 2.5% POs-Ca and 1.2% fluoride-containing green tea extract, namely POs-Ca and POs-Ca + F. The placebo and intervention chewing gums were identical in appearance, taste and smell without any observable differences. The chewing gum slabs were received in code-labelled packages stored at room temperature. The codes were randomly assigned by a controller and remained unknown to the volunteers and operators until all remineralization data had been obtained. The subjects were randomly divided into three groups ( n = 12) according to the design presented in Fig. 1 . The treatment was performed in three periods of 2 weeks each, during which the subjects in each groups received one of the gums randomly. Between each two treatment periods, there was a resting period of 1 week at which treated enamel insets of appliances were exchanged for new demineralized insets according to the cross-over design. Subjects then crossed over to another gum product. This was repeated until each subject had used each of the three gums. The subjects wearing the appliances chewed two slabs of gum three times every day (at 8:00 am, 12:00 pm and 5:00 pm) for 20 min each time. After this and before removing the intraoral appliance, the subjects continued wearing the appliances for 20 more minutes whilst refraining from eating or drinking. Table 1 Composition of the chewing gums used in the study (wt%). Ingredients Gum Placebo POs-Ca POs-Ca + F POs-Ca 0 2.5 2.5 Green tea extract fluoride 0 0 1.2 Maltitol 41.0 41.0 41.0 Xylitol 27.5 25.0 23.8 Gum base 24.0 24.0 24.0 Flavour, etc. 7.5 7.5 7.5 Fig. 1 Participant disposition for the randomized control trial. (A) Placebo gum, (B) POs-Ca gum and (C) POs-Ca + F gum. 2.2 Preparation of enamel subsurface lesions Fresh bovine incisors were obtained from a local slaughterhouse (Yokohama, Japan). Enamel blocks 7 mm × 10 mm × 2 mm (width × length × depth) were cut from the bovine incisors using a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) under running water, and embedded in acrylic resin (Unifast Trad; GC, Tokyo, Japan). The outer enamel surface was polished to a mirror finish using wet polishing papers (800, 1000, and 2000 grit lapping papers; 3M, St. Paul, MN, USA). Three zones, namely sound, demineralized (DEM), and remineralized (REM), were assigned on the polished enamel surface of each block as follows. First, one third of the surface on each block was covered with a nail varnish (Nail POP; Chamon, Kyonggi, South Korea) ; this part served as the sound portion. Subsurface lesions were then formed on the remaining surface using the two-layer demineralization method with 8% methylcellulose gel (Methocel MC, Fluka, Everett, WA, USA) and 0.1 M lactate buffer (pH 4.6) at 37 °C for 14 days. The ratio of gel to lactate buffer was 3:5. After demineralization, the blocks were sterilized by ethylene oxide gas sterilizer (Steri-Gas; 3M, St. Paul, MN, USA). Another one-third of the surface was then covered by the nail varnish, and the remaining exposed one-third of enamel surface served as remineralization zone. In total, 108 blocks were prepared as insets for insertion into the intraoral appliances. 2.3 Intraoral appliances Removable lower-buccal acrylic appliances covering premolars to molars were fabricated for each subject ( Fig. 2 ). The appliances were retained by two stainless steel circumferential clasps on canine and first molar, with a trough to house for enamel insets. Subjects were instructed to insert and remove the intraoral appliances according to the treatment design. They were also asked to rinse the appliances briefly after removal with tap water and store them at 37 °C in sealed and humidified containers until the next insertion. After completion of each 2-week treatment period, the enamel blocks were removed from the appliances, rinsed with distilled/deionized water and stored under moist conditions in labelled microcentrifuge tubes until laboratory tests. Fig. 2 Schematic for specimen preparation and the intraoral appliance design for the in situ trial and TMR or WAXRD analyses. 2.4 TMR (microradiography and microdensitometry) For TMR assessment of treated enamel bocks in each group ( n = 36), approximately 150-μm-thick slabs were obtained by cutting the enamel blocks through the middle and perpendicular to the treated surfaces using a water-cooled diamond-coated wire saw (Well Diamond Wire Saws, Mannheim, Germany). The surfaces of the sections were carefully polished, whilst the thickness of each section was monitored by a micrometre (Mitutoyo, Tokyo, Japan). In this manner, transverse sections, each including three zones (sound, DEM, REM), were obtained. The thin sections were then microradiographed together with high-purity aluminium step wedges (12.5 μm × 21 steps) as mineral density phantoms onto high-resolution plates (Konica Minolta Opto Products, Yamanashi, Japan) by Cu Kα X-ray generated at 20 kV and 20 mA for 13 min (PW-3830; Philips, Eindhoven, Netherlands). The mineral profiles of the sound, DEM and REM zones were obtained from the digital images of the microradiographs. The uniform density at the sound zone was confirmed, indicating the flatness and plano-parallelism of the prepared sections. The digital microradiographs were then analysed using a computerized image-analysis system (TMR v1.25; Inspektor Research System, Amsterdam, Netherlands). The output parameters obtained were the mineral content profile of the lesion, lesion depth and integrated mineral loss (ML). In this analysis, the mineral content of sound enamel was assumed to be 87 vol%. The mineral recovery rate for each specimen was calculated from the ML of DEM (ML DEM ) and REM (ML REM ) as follows: recovery rate (% R ) = (ML DEM − ML REM )/ML DEM × 100. 2.5 Wide-angle X-ray diffraction with an X-ray microbeam The wide-angle X-ray diffraction measurement for 13 specimens randomly selected for each gum was calculated in the same manner as described by Yagi et al. and Tanaka et al. The experiments were carried out at the BL40XU beamline of the SPring-8 (Japan Synchrotron Radiation Research Institute, Hyogo, Japan) third-generation synchrotron radiation facility with an X-ray energy of 15.0 keV (bandwidth ∼3%). The diameter of the X-ray microbeam at the sample (spot size) was about 6 μm (full width at half-maximum). The X-ray detector consisted of an X-ray image intensifier (V5445P; Hamamatsu Photonics, Hamamatsu, Japan) coupled to a cooled CCD camera (ORAC-II-ER; Hamamatsu Photonics, Hamamatsu, Japan). Enamel sections, which had been examined by TMR, were subjected to the WAXRD measurements. The sections had been kept sealed with water, and carefully inspected under a stereomicroscope (Olympus, Tokyo, Japan) to check for any changes in the sample shape or damage to the edge, before the WAXRD measurements. All X-ray measurements were made at ambient temperature and humidity. Each section was mounted vertically so that its enamel edge was horizontal; the X-ray beam passed perpendicularly through the slice. The sample was moved upwards in 5 μm steps so that the X-ray beam could scan across the enamel, from the surface towards the dentine. At each step a WAXRD pattern was recorded. In the WAXRD patterns, the (1 0 0) equatorial reflection (at a Bragg spacing of 0.815 nm) was used to obtain the intensity profile along the depth up to 150 μm depth in the enamel. The intensity profiles were then normalized by the average intensity between 150 and 200 μm from the surface. In this manner, the subsurface lesion (DEM, REM) and sound zones of each sample were evaluated on a region up to the depth of 200 μm. The intensity profiles were then integrated over 150 μm from the surface to obtain crystal content of this zone. The resulting integrated crystal contents of the 150-μm-thick surface layer of DEM and REM ( C DEM and C REM ) were then used to calculate HAp crystallites recovery rate after each chewing gum treatment as follows: HAprecoveryrate(%)=CDEM−CREMCDEM×100. HAp recovery rate ( % ) = C DEM − C REM C DEM × 100 . 2.6 Gum analysis In order to analyse the contents of the chewing gums, 5 pellets (about 1.4 g/pellet) of each chewing gum were pounded with a pestle in 50 ml of the artificial saliva for 20 min at 37 °C. This procedure simulated chewing two pellets of gum with 20-ml saliva that is usually secreted during 20-min chewing. The artificial saliva was prepared containing 100 mM KCl (Wako Pure Chemical Industries, Osaka, Japan), 3.6 mM KH 2 PO 4 (Wako Pure Chemical Industries, Osaka, Japan), 1.5 mM CaCl 2 (Wako Pure Chemical Industries, Osaka, Japan), and 20 mM HEPES-K (Dojindo, Kumamoto, Japan) (pH 7.0); the pH was then adjusted to 6.5, as described previously. The gum extracts were collected every 5 min during pounding. Then, the extracts were centrifuged (10,000 × g × 5 min) to remove gum debris. The calcium and phosphate concentrations of the supernatants were determined by the inductively coupled plasma-atomic emission spectroscopy (ICP-AES SPS7700; Seiko Instruments, Tokyo, Japan) and a p -methylaminophenol reduction method using Phospha C-test kit (Wako Pure Chemical Industries, Osaka, Japan), respectively. The fluoride ion concentrations of the supernatants were analysed with a calibrated fluoride ion electrode (6561-10C; Horiba, Kyoto, Japan) with F detection range from 0.02 to 19,000 ppm. 2.7 Data analysis The data were first tested for normality. The ML DEM and % R data were examined at the 95% level of significance by t -test with Bonferroni corrections. The data of HAp crystallites recovery rate were analysed by Wilcoxon rank-sum test with Bonferroni correction. To avoid an accumulation of errors due to multiple comparisons, the Bonferroni correction modified the significance level by dividing it ( p < 0.05) between the number of comparisons made. Where p < 0.0017, the comparison was considered to be statistically significant. All statistical analyses were performed using SPSS version 11.0 software (SPSS, Chicago, IL, USA). 3 Results All randomized subjects completed the in situ study without significant protocol violations and were therefore included in the analysis. Health questionnaires indicated no adverse events. 3.1 Enamel remineralization The mean mineral density profiles of the DEM and REM zones in each gum are shown in Fig. 3 (a–c) , and representative microradiographic images of the subsurface lesions before and after chewing each gum are presented in Fig. 4 . ML data of the enamel subsurface lesion of DEM and REM regions for each gum are summarized in Table 2 . No significant differences were found in the initial lesions used for the in situ study as shown by the initial ML DEM values for the three groups ( p > 0.05). The mean mineral recovery rate (% R ) (mean ± SD) for the different gums (placebo, POs-Ca, POs-Ca + F) was 15.0 ± 11.4, 21.9 ± 10.6 and 26.3 ± 9.4, respectively. Chewing POs-Ca and POs-Ca + F gums resulted in significantly greater average recovery rate than that produced by chewing the control gum ( p < 0.05). However, there was no statistical difference in recovery rate between POs-Ca and POs-Ca + F ( p > 0.05). Fig. 3 Mean TMR (a–c) profiles of all specimens for three chewing gums. (a) The placebo chewing gum, (b) POs-Ca chewing gum and (c) POs-Ca + F chewing gum. DEM: demineralized zone; REM: remineralized zone. Note that whilst all the chewing gums resulted in some mineral gain, (b) and (c) resulted in a significantly higher mineral recovery compared to (a). Fig. 4 Representative TMR microradiographs of the subsurface lesions before and after remineralization for three chewing gums. DEM: demineralized zone; REM: remineralized zone. The placebo gum resulted in a minor remineralization in the subsurface lesion whereas for POs-Ca and POs-Ca + F, decreased radiolucency through the body of the lesions was visible, especially for POs-Ca + F. Table 2 Remineralization of enamel subsurface lesions assessed by TMR (mean ± SD). Sample ML DEM (vol% μm) ML REM (vol% μm) ML DEM − ML REM (vol% μm) % R Placebo 3461 ± 625 2942 ± 664 519.5 ± 368.1 15.0 ± 11.4 b,c POs-Ca 3471 ± 865 2711 ± 704 760.1 ± 460.3 21.9 ± 10.6 b POs-Ca + F 3628 ± 594 2675 ± 533 952.7 ± 385.6 26.3 ± 9.4 c Mean initial lesion depth = 133 ± 32 μm. Values marked by similar lowercase superscript letters are significantly different ( p < 0.05). ML DEM : mineral loss (Δ Z ) of demineralized enamel, ML REM : mineral loss (Δ Z ) of remineralized enamel after chewing each gum, % R : mineral recovery [(ML DEM − ML REM /ML DEM )/100]. 3.2 WAXRD The mean HAp crystallite recovery rates (%) (mean ± SD) for the different gums (placebo, POs-Ca, POs-Ca + F) are 11.1 ± 4.7, 16.0 ± 4.1 and 24.9 ± 5.4, respectively. Chewing POs-Ca and POs-Ca + F gums resulted in significantly greater HAp crystallite recovery rate than that produced by chewing the placebo gum ( p < 0.05). Moreover, there was a statistical difference in the HAp crystallite recovery rate between POs-Ca and POs-Ca + F ( p < 0.05). Representative normalized WAXRD profile plots are presented in Fig. 5 (a–c) . Fig. 5 Representative WAXRD profiles for three chewing gums. (a) The placebo chewing gum, (b) POs-Ca chewing gum and (c) POs-Ca + F chewing gum. DEM: demineralized zone; REM: remineralized zone. The difference between REM and DEM in (c) resulted in a significantly higher crystal recovery compared to (a) and (b). The WAXRD profiles are not as smooth as in TMR, owing to the beam spot size. 3.3 Gum ion analysis Analyses of ions released into artificial saliva from the three gums with regard to calcium, phosphate and fluoride ions are presented in Table 3 . For the placebo gum, only a small amount of soluble calcium was released from the gum base. The POs-Ca gum showed a higher level of extractable calcium in the artificial saliva (up to 7.1 mM) similar to POs-Ca + F gum (up to 7.3 mM). The highest amount of fluoride ion release (1.25 ppm) was found in POs-Ca + F gum at 20 min. Table 3 Ion analysis results for calcium, inorganic phosphate and fluoride levels (mean ± SD). Sample Time (min) Ca (mM) P (mM) F (ppm) Placebo 5 2.5 ± 0.2 3.1 ± 0.3 0.07 ± 0.03 10 2.6 ± 0.1 3.1 ± 0.3 0.06 ± 0.02 15 2.9 ± 0.9 3.2 ± 0.2 0.06 ± 0.01 20 2.5 ± 0.2 3.2 ± 0.3 0.06 ± 0.01 POs-Ca 5 4.1 ± 0.5 3.2 ± 0.3 0.05 ± 0.03 10 5.7 ± 0.4 3.1 ± 0.3 0.07 ± 0.01 15 6.4 ± 0.9 3.2 ± 0.4 0.08 ± 0.01 20 7.1 ± 0.8 3.3 ± 0.4 0.07 ± 0.01 POs-Ca + F 5 4.5 ± 0.1 3.2 ± 0.2 0.52 ± 0.13 10 5.9 ± 0.1 3.2 ± 0.3 0.84 ± 0.15 15 7.0 ± 0.5 3.3 ± 0.2 1.03 ± 0.21 20 7.3 ± 0.5 3.3 ± 0.3 1.25 ± 0.31 4 Discussion An in situ model was chosen in the present study in order to simulate the everyday situation to test different chewing gum treatments aimed at improving the demineralized enamel. The use of bovine enamel has been verified in a number of in vitro studies to evaluate the effect of anticariogenic agents on enhancing enamel remineralization and inhibiting enamel demineralization. Using the in situ model, human and bovine enamel have shown similar characteristics in subsurface lesion remineralization under irradiation or abrasion influence. However, bovine enamel is not similar to human enamel in some aspects; for example, it may have a higher porosity, potentially leading to a faster demineralization rate. The sound bovine enamel had a lower mineral concentration than human enamel. On the other hand, these differences result in a relatively more consistent response to treatments in the experimental groups for bovine enamel when compared to human enamel. These points should be taken into consideration when interpreting any demineralization and remineralization study using bovine enamel, even if there were no qualitative differences between bovine and human enamel. Nevertheless, an objective comparison amongst groups required a method which took into account the baseline variations amongst the demineralized lesions (shallow vs. deep lesions). In the current study, the lower variability of bovine enamel specimens enabled creation of standardized baseline lesions required for the cross-over study; there were no significant differences in the integrated mineral content of demineralized lesions (ML DEM ) subjected to remineralization in all experimental groups, indicating that a comparison of remineralization amongst the lesions would be valid ( Table 2 ). Furthermore, the recovery rate variables were used for statistical comparisons in this study, rather than direct comparison of remineralization values ( i.e. ML REM and C REM ) amongst different treatments. Considering the long period (6 weeks in total) of the trial for each volunteer, a small removable lower-buccal acrylic appliance was designed to reduce the discomfort of the subject when wearing them. The appliances were constructed with two circumferential clasps to provide adequate retention and avoid any dislodgment during chewing, whilst allowing frequent insertion and removal of the appliance during the trial period. The enamel slabs were sized to accurately fit the stainless steel housings on the appliances and retained by a small amount of dental wax to minimize the potential hazard of displacement or swallowing of the specimen during chewing. No such events were reported by the subjects in this study. Significant mineral recovery rates resulted after the 2-week periods not only with the test gums but also with the placebo gum. An extended period of chewing could have resulted in higher recovery rates, but it was beyond the scope of this randomized controlled trial. Two X-ray analysis techniques were used to investigate the microstructure of the subsurface lesion at different depth levels. Microradiographic assessment by TMR provides mineral content profiles, but cannot render the crystal structure of the tissue. In an in vitro study comparing both TMR and WAXRD, both the spatial distribution and orientation of the restored HAp crystals in the remineralization process at the subsurface lesion could be simultaneously analysed by the WAXRD. In order to investigate the superficial structure, specially under the fluoride containing gum, a microbeam design was considered for quantitative measurement of HAp crystals and fluorapatite crystals. It was previously suggested that the intensity of the (1 0 0) equatorial reflections was not greatly affected by the tilt of the c -axis of the HAp crystals, and therefore gave a more reliable measure of the amount of the crystals. Using the 6-μm-diameter microbeam diffracted throughout the thickness of the enamel slice, the WAXRD could provide an average relative quantification of crystallites at the subsurface lesion in this study, as presented in Fig. 5 (a–c). This work may be the first in situ trial to employ a crystallographic method at a third-generation synchrotron radiation facility to compare the recrystallization by different remineralizing agents, besides the conventional mineral recovery data. Further development of the crystallography methodologies may enable their laboratory application, without the need to use the costly synchrotron radiation facilities. Both tests conducted on enamel in this study confirmed increased remineralization by both POs-Ca gums after 2 weeks, compared to the placebo. It was reported that an effective remineralization of subsurface lesions required the agent to diffuse past the pellicle-covered enamel surface and into the subsurface lesion area. Unless the particles are small enough, calcium will rapidly adsorb onto the surface layer, decrease the pore volume of the layer and obstruct the diffusion pathways. Recent research demonstrated that nano-sized particles had an ability for biomimetic repair of the enamel subsurface lesion. Moreover, considering the current results, it was assumed that POs-Ca was capable of providing calcium ions in a bioavailable form on the enamel surface, driving diffusion of the ions into the subsurface lesion to promote remineralization due to the small size of POs-Ca (average molecular mass: about 800; maximum chain length: about 4 nm). The gum analysis at a neutral pH in this study demonstrated high solubility of the POs-Ca, with both POs-Ca and POs-Ca + F gums releasing a similar amount of up to 7.3 mM calcium ions into the artificial saliva. This high calcium amount was extractable into both water and saliva without requiring any acid for solubilization. According to the most recent literature, another form of bioavailable calcium such as casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) required acid for the solubilization. These findings highlighted the important role of the POs-Ca, not only in a marked increase in calcium ion solubility, but also in localizing the calcium ions in saliva to facilitate intraoral subsurface enamel lesion remineralization. HAp crystals are formed in the oral environment from supersaturated calcium and phosphate ions, and the presence of fluoride ions is likely to further promote the remineralization process by forming fluorapatite crystals that are more resistant to future demineralization compared to HAp. In the current study, a low fluoride ion concentration (around 1 ppm) extracted from POs-Ca + F gum was detected in the gum analysis. On the effects of low levels of fluoride on remineralization, it was reported that a fluoride treatment continuously present at 1 ppm was beneficial for repair of deep lesions, at least in the outer enamel. On the other hand, it has been suggested that calcium fluoride (CaF 2 ) deposits on or within the surface that form due to the interaction of fluoride and calcium ions may be protected by salivary constituents and act as a fluoride reservoir ; however, the formation of CaF 2 was considered unlikely in the current study, since the combinational conditions of a low pH and a high fluoride concentration that favour formation of CaF 2 were not a feature in situ . Such a low fluoride level as in the extracted solution did not cause CaF 2 precipitation under the artificial saliva in vitro . Moreover, the effects of high-fluoride levels in the forms of gel or mouthwash may not last as long as previously hypothesized ; therefore, frequent exposure to low but slightly elevated levels of fluoride in saliva and plaque from sources such as water or the chewing gum can perhaps more effectively help to prevent and reverse caries by inhibiting demineralization and enhancing remineralization. It has been reported that the presence of fluoride in in vitro remineralization, even at low concentrations, may induce a continuous deposition of minerals on the lesion surface and gradually block the pores present on the demineralized surface layer. In this study, any tendency towards some kind of hypermineralization on the top surface of remineralized subsurface lesion was not detected on the TMR profile of POs-Ca + F group. In this regard, apart from aspects discussed earlier, it should be noted that in the in situ trial, formation of an acquired pellicle is inevitable, and therefore the role of macromolecules from natural saliva should not be overlooked. It was reported that in the presence of low fluoride concentrations, these molecules seemed to play an important role in the continuation of remineralization by reducing mineral gains on the surface layer. According to the results, it was assumed that the fluoridated gum in this study effectively exerted ionized fluoride which enhanced HAp crystallites during remineralization. Whilst there was no significant difference between the two tested gums in TMR profile, an enhanced crystalline structure in POs-Ca + F group could be confirmed by the WAXRD technique. This was most likely due to a chemical change by the substitution of the hydroxyl groups of HAp by fluoride ions and formation of a less soluble fluorinated apatite. Nevertheless, elemental composition analysis and localization of fluoride incorporated into the remineralized subsurface lesions may be required to elucidate the remineralization mechanism of fluoride-containing gum under the in situ model. On top of the X-ray analysis techniques employed in the current study, which showed remarkable similarities around the mid-depth of the lesion for the test gums, it may be suggested that microhardness can provide a direct measure of effective mineralization in in situ trials. Further cross-sectional investigation using both mineral and hardness profiles may shed more light on the healing process of the subsurface lesions. Moreover, other techniques such as optical coherence tomography (OCT), surface pH measurement and atomic force microscopy (AFM) would be useful for evaluation of enamel remineralization. This study confirmed the advantage of synergistic use of bioavailable calcium (POs-Ca) with fluoride on remineralization and crystal growth within the subsurface lesion in situ . However, clarifying the mechanism of the contribution and possibility of crystal nucleation by the compound warrants further research. 5 Conclusions In conclusion, this in situ study demonstrated that POs-Ca containing gums could significantly contribute to remineralization and recrystallization within enamel subsurface lesions, compared to a placebo gum. Furthermore, POs-Ca + F gum was superior in crystal recovery when compared with POs-Ca gum according to WAXRD analysis. POs-Ca + F gum appeared to be an effective vehicle for fluoride supplementation. 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