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J Dent Res 85(10):919-923, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Laser-Fluoride Effect on Root Demineralization

X.-L. Gao1, J.-S. Pan2, and C.-Y. Hsu1,*

1 Department of Preventive Dentistry, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, Republic of Singapore 119074; and
2 Materials Science & Characterization Laboratory, Institute of Materials Research and Engineering, Republic of Singapore

* corresponding author, pndhsus{at}nus.edu.sg


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the individual cariostatic effects of laser and fluoride have been shown, the combined effect of CO2 laser and fluoride on root demineralization remains uncertain and was the main aim of this study. By using a pH-cycling system and Polarized Light Microscopy, we demonstrated the synergistic effect of fluoride combined with CO2 laser treatment on reducing root demineralization. The mean lesion depths (in µm) for each group were 160 ± 14 (Control), 113 ± 8 (Laser treatment alone), 111 ± 6 (Fluoride treatment alone), and 25 ± 7 (Fluoride followed by laser treatment). A significant laser-enhanced fluoride uptake, characterized by the ToF-SIMS analysis, was revealed by the 37% and 400% increments in loosely and firmly bound fluorides (both p < 0.002) in laser-irradiated areas, compared with the non-irradiated controls. In conclusion, there is a significant synergistic effect of combined CO2 laser and fluoride treatment on the inhibition of root demineralization, possibly due to laser-enhanced fluoride uptake in the root.

KEY WORDS: CO2 laser • fluoride • root • demineralization • fluoride uptake


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the decline of dental caries in developed countries, a continuous increase in root caries prevalence has been reported and is regarded as a serious threat to oral health for adults in all age groups (Katz, 1995; Blinkhorn and Davies, 1996; NIH, 2000; Griffin et al., 2004). Moreover, a further increase in the prevalence of root caries in the 21st century has been predicted, highlighting the importance of the search for effective methods of root caries prevention (Blinkhorn and Davies, 1996; Griffin et al., 2004).

Accumulated evidence has demonstrated significant effects of lasers in the prevention of both enamel caries (Featherstone et al., 1991; Hsu et al., 2000) and root caries (Westerman et al., 1994). Effects of the combined fluoride and laser treatment in the inhibition of caries have been reported for both enamel and root (Featherstone et al., 1991; Flaitz et al., 1995; Hicks et al., 1995a,b, 1997; Hsu et al., 2001). However, the effect and mechanism of combined CO2 laser and fluoride treatment in inhibiting root caries remain to be elucidated.

In a previous study, we demonstrated the effect of lasers in enhancing fluoride uptake in enamel (Hsu et al., 2004), but their effect on root tissues is not yet clear.

The objectives of this study were: (1) to evaluate the combined effect of CO2 laser and fluoride in inhibiting root demineralization, and (2) to investigate the effect of CO2 laser on loosely and firmly bound fluoride uptake in the root.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tooth Preparation & Grouping
The procedure for tooth collection was approved under the exemption category of the Institutional Review Board of the National University of Singapore (NUS-IRB reference code: 04-153E). After extraction, the teeth were stored at 4°C in 0.1% thymol solution. Before the experiment, all teeth were cleaned by being gently brushed and carefully scaled, for the removal of debris, attached soft tissues, and calculus. The root surfaces were not polished or sonicated.

For objective (1), 15 sound teeth were selected and viewed under a stereomicroscope (Olympus® SZ40, Olympus Optical Co. LTD, Tokyo, Japan). By using acid-resistant varnish (ExpressFinish®, Maybelline Inc., Clark, NJ, USA), we created 4 3 mm x 1 mm windows (2 on the distal surface and 2 on the mesial surface) on each tooth, 1 mm below the cemento-enamel junction, with a 2-mm distance between the 2 windows on the same root surface. The 4 windows on the same tooth were randomly assigned to 4 groups: (1) Control, (2) Laser treatment alone, (3) Fluoride treatment alone, and (4) Fluoride followed by laser treatment.

For objective (2), we selected 12 teeth. Each tooth was cut buccallingually into 2 halves, which were randomly assigned to "non-KOH" and "KOH" groups, respectively. Each tooth half contained 2 windows, created with the above-mentioned varnishing method, and were randomly assigned as the "Laser" and "Non-laser" windows, respectively. Therefore, the 4 windows on the same tooth were assigned into 4 groups: (5) Non-laser/Non-KOH, (6) Non-laser/KOH, (7) Laser/Non-KOH, and (8) Laser/KOH.

Fluoride Treatment
A neutral fluoride gel, Perfect ChoiceTM (Challenge Products, Inc., Louisville, CO, USA), containing 2.0% NaF (sodium fluoride), was applied for 4 min to each of the windows in Groups (3)–(8), and wiped off with tissue papers.

Laser Treatment
On the windows of Groups (2), (4), (7), and (8), a carbon dioxide (CO2) laser treatment (SharplanTM Compact 20C, STI Laser Industries Ltd., Or-Akiva, Israel) was carried out with a "single pulse" tissue exposure mode (i.e., 1 laser shot was emitted with each step on the foot-switch), with a wavelength of 10.6 µm, 1-mm spot size, 0.3-W power setting, and irradiating duration of 0.1 sec. When the tooth was moved under the laser tip, the window surface was irradiated spot by spot, with a single laser shot on each spot. The irradiation was repeated back and forth, for a total of 4 shots on each spot.

With a laser monitor, LASERSTARTM (Model FL 250A-SH, Ophir Optronics Ltd., Wilmington, MA, USA), the mean energy for each shot was measured to be 8.9 mJ. Therefore, the average power output was calculated to be 0.09 W. Based on the external output measurement, the average fluence per shot was calculated to be 1.14 J/cm2. After laser treatment, all the samples were rinsed in running de-ionized and distilled water for 10 min.

Potassium Hydroxide (KOH) Treatment
The tooth halves in the "KOH" groups were treated with 1 M KOH solution at room temperature, with a stirring speed of 120 rpm, for 24 hrs, for removal of the loosely bound fluoride (Caslavska et al., 1991). We thus calculated "loosely bound" fluoride uptake by deducting the firmly bound fluoride uptake from the total fluoride uptake. After KOH treatment, the tooth halves were rinsed in running de-ionized and distilled water for 10 min.

pH-Cycling
Each of the 15 teeth was cut longitudinally into 4 root fragments, representing Groups (1)–(4). After the cut surfaces were varnished, the root fragments were subjected to a two-day pH-cycling scheme. To prevent cross-contamination between fluoride and non-fluoride groups, we immersed the 15 root fragments (each with a 3 mm x 1 mm window) for each group, separately, in a 1000-mL demineralizing solution (pH 4.8, containing 0.05 M acetic acid, 2.2 mM calcium, and 2.2 mM phosphate ions) for 18 hrs, and then in a 1000-mL remineralizing solution (pH 7.0, containing 0.15 M potassium chloride, 1.5 mM calcium, and 0.9 mM phosphate ions) for 6 hrs each day at a temperature of 37°C, with a stirring speed of 132 rpm. The pH-cycling started with the demineralizing phase. A five-minute wash in de-ionized and distilled water was done between each 2 treatment phases and at the end of the cycling.

Tooth-sectioning
All teeth were sectioned longitudinally, perpendicular to the root surfaces through the central part of each window, with a Silverstone-TaylorTM hard-tissue microtome (Series 1000 Deluxe, Sci. Fab., Littleton, CO, USA). From each window, 3 sections were obtained, with a thickness of about 100 µm for Groups (1)–(4) and 600 µm for Groups (5)–(8).

Polarized Light Microscopic (PLM) Characterization
After immersion in water, all the sections of teeth from Groups (1)–(4) were examined at 40X magnification with a Polarized Light Microscope (Model BX51, Olympus®, Tokyo, Japan). We traced and measured a representative area of the central region (500 µm in width) of each lesion, using Micro ImageTM software (Olympus), as previously described (Hsu et al., 1998). The area value was divided by 500 to represent the average lesion depth of the section. The measurements on 3 sections from each window were averaged to provide the depth of each window lesion.

Fluoride Measurement
By using Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS® IV, ION-TOF GmbH, Münster, Germany), we carried out elemental analysis on the cut surfaces of the sections of Groups (5)–(8). For each section, we analyzed 2 correlated spots (50 µm x 50 µm) beneath the root surface. The first spot was selected below the center of the window. The second spot, serving as the control, was 1.5 mm apical to the first spot, in the non-window area (Fig. 1Go).


Figure 1
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  		Figure 1. Two spots on the cut surface of each section for fluoride measurement. Spot 1 was selected below the center of the window. Spot 2, serving as the control, was 1.5 mm apical to Spot 1, in the non-window area.

 
Each selected spot was bombarded with an Ar+ primary ion beam (10 KeV, 50 µm spot, 3.0 pA, and a 200-second acquisition time). With the ToF-SIMS Software (Version 3.14), the elemental intensity data were obtained, and the intensity of ionized 19F was normalized against the intensity of ionized 31P, the reference element for negative ions in the tooth (Duckworth and Gilbert, 1992). For each section, a pair of F/P values was obtained for the window and the control areas. By calculating the difference between these 2 paired values ({Delta}F/P), we obtained the relative amount of fluoride uptake.

Statistical Analysis
For objective (1), "lesion depth (in µm)" was used as the dependent variable. The independent variables included "laser treatment", "fluoride treatment", and "tooth structure". After the homogeneity of variance was revealed by the Levene test, an ANOVA model for the factorial design was constructed to evaluate the main effects of independent variables and their interactions. The Tukey-Kramer test was chosen for multiple pairwise comparisons.

For objective (2), {Delta}F/P was used as the dependent variable. The independent variables included "laser treatment", "fluoride form" (either firmly or loosely bound), and "tooth structure". We constructed an ANOVA model for the factorial design for assessing the main effects of independent variables and their potential interactions. We used paired t tests to evaluate the significance of the differences between "laser" and "non-laser" groups in both firmly and loosely bound fluoride uptakes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Qualitative Assessment
The representative PLM images of artificial root caries lesions for the 4 groups are illustrated, and cementum was found to be intact on root surfaces (Fig. 2Go). In Groups (3) and (4), bandings parallel to the lesion surface were found in most of the lesions, indicative of remineralization (Wefel, 1994). On several sections of Group (4), complete inhibition of root demineralization was observed in some areas. A concave outer lesion on the dried surface, indicating substantial mineral loss and the shrinkage of unsupported collagen in the outer layer of the lesion (Wefel, 1994), was identified in all groups except Group (4).


Figure 2
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  		Figure 2. Representative polarized light microscopic images of artificial root-caries lesions on the sections for Groups (1) Control, (2) Laser treatment alone, (3) Fluoride treatment alone, and (4) Fluoride followed by laser treatment.

 
Quantitative Assessment
The mean (and standard deviation) of lesion depths with the percentage reduction in each treatment group were compared (Table 1Go). Both Laser and Fluoride treatments showed about 30% reduced lesion depth, whereas the combined treatment resulted in an 84.5% decrease in lesion depth.


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  		Table 1. Changes of Lesion Depths
 
The mean and standard deviation of loosely and firmly bound fluoride uptakes with and without laser treatment were assessed (Table 2Go). The fluoride uptake in laser-irradiated root was about 5 times that in the non-irradiated root in the firmly bound form, with a 37% increase in the loosely bound form.


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  		Table 2. Comparison of Fluoride Uptake
 
Statistical Significance
Both laser and fluoride effects on lesion depth, and the interaction between laser and fluoride, were statistically significant (all p < 0.001). There was a significant difference in lesion depth between each pair of groups (all p < 0.001), except between Groups (2) and (3) (p > 0.05). The responses of different teeth to the laser or fluoride treatment were homogeneous (p = 0.243).

The effects of "laser" and "fluoride form" on fluoride uptake were all statistically significant (both p < 0.001). The root structure of sample teeth related to fluoride uptake was homogeneous (p = 0.538). There were significant interactions between "tooth" and "fluoride form" (p = 0.021) and between "laser" and "fluoride form" (p < 0.001). There were significant differences in fluoride uptake between "laser" and "non-laser" groups, in both firmly bound and loosely bound forms (both p < 0.002).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Synergistic Effect of Laser and Fluoride on Root Caries Prevention
In this study, laser treatment alone and fluoride treatment alone resulted in comparable and moderate levels of root caries inhibition (about 30%). When laser was combined with fluoride treatment, a synergistic inhibition (about 85%) was achieved.

The combined treatment effect reported here is striking when compared with the results of 2 previous studies using about 10 times the energy density for a "Fluoride + Argon Laser" treatment on the root, to achieve a 54–66% reduction in lesion depth (Hicks et al., 1995b, 1997).

Although a synergistic effect of laser and chemical inhibitors on the dissolution of enamel has been demonstrated (Fox et al., 1992), it was unclear whether the intensified cariostatic effect on the root is synergistic or just an additive effect of laser and fluoride (Hicks et al., 1995b, 1997). Our results strongly indicate a synergistic rather than an additive effect of the combined laser and fluoride treatments.

A recent study has shown that CO2 laser treatment did not achieve significant caries inhibition in dentin (Featherstone et al., 2003). That study differed from ours in the selection of experimental/control sites on different teeth without a "combined fluoride-laser treatment" group, a stronger acid challenge in the pH-cycling scheme, and different laser parameters.

Laser-induced Fluoride Uptake
It is clear that CO2 laser treatment not only increases the deposition of calcium fluoride on the root surface, but also markedly facilitates the incorporation of fluoride into the crystalline structure, in the form of apatitic fluoride (ten Cate, 1999).

In this study, 32% of the fluoride uptake in the laser-treated root was in the firmly bound form and 68% in the loosely bound form. Our previous study showed that 45% and 55% of the fluoride uptake in irradiated enamel was in the firmly and loosely bound forms, respectively (Hsu et al., 2004). The laser treatment seemed to be more effective in enhancing loosely bound fluoride uptake in the root than in enamel. Having much greater porosity and surface roughness than enamel, the root may have greater propensity for the loosely bound fluoride to deposit; while, for enamel, with a much higher crystalline density than the root, fluoride may be more easily incorporated into the firmly bound form.

Without laser treatment, fluoride uptake has been regarded as a process of diffusion with simultaneous chemical reaction (Duckworth and Braden, 1967). For laser-enhanced fluoride uptake, several potential mechanisms have been postulated. These include laser-induced carbonate loss and the subsequent substitution of fluoride ions for carbonate (Goodman and Kaufman, 1977), enhanced fluoride uptake through laser-induced temperature increase (Goodman and Kaufman, 1977), an increase in micropores or microspaces in dental hard tissues (Oho and Morioka, 1990), and an increase of the root-surface area due to the roughening caused by laser irradiation (Westerman et al., 1998).

Possible Mechanisms of the Synergistic Effect on Caries Prevention
Laser treatment significantly increased fluoride uptake by 400% and 37% in the loosely and firmly bound forms, respectively. During acid attack, the loosely bound calcium fluoride may release the fluoride ions to inhibit demineralization and enhance remineralization. The firmly bound fluoride incorporated into the crystalline structure may increase crystal stability and acid resistance. A micro x-ray diffraction study has revealed a contraction in the a-axis dimension and an improvement in the enamel crystallinity induced by laser irradiation, with a further reduction of the a-axis of hydroxyapatite crystals caused by the combined laser-fluoride treatment (Deng and Hsu, 2005). This may be the case in the root as well. Furthermore, the firmly bound fluoride may serve as a fluoride reservoir, with a greater substantivity than that of the loosely bound fluoride. The amount and depth of fluoride uptake in dentin have been reported to be several times greater than those in enamel (Acuna et al., 1990).

Therefore, the formation of a more acid-resistant root structure and an increased fluoride reservoir may be the main reasons for the substantial reduction of lesion depth demonstrated in the combined laser-fluoride group.

Although fluoride has been regarded as an effective agent in caries prevention and has been widely applied for several decades, it has not led to the elimination of dental caries, which remains an ubiquitous disease affecting all age groups (Winn et al., 1996). Even intensified fluoride programs have a limited cariostatic potential on the tooth root (ten Cate, 1999). As indicated in this study, a combined fluoride and CO2 laser treatment may offer a clinically promising therapy for root caries prevention.


   ACKNOWLEDGMENTS
 
We thank Ms. Doreen Lai for her technical support in ToF-SIMS and Dr. Wang Yougan for his suggestions on statistical analysis. This study was financially supported by the Academic Research Funds (R-222-000-007-112 and R222-000-013-112) of the National University of Singapore.

Received October 24, 2004; Last revision May 10, 2006; Accepted June 21, 2006


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acuna V, von Beetzen M, Caracatsanis M, Sundstrom F (1990). In vitro fluoride uptake by enamel and dentin. A comparative study of two varnishes. Acta Odontol Scand 48:89–92.[ISI][Medline]

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