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J Dent Res 85(7):617-621, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Clinical

In situ Mineral Loss Inhibition by CO2 Laser and Fluoride

L.K.A. Rodrigues1, M. Nobre dos Santos2,*, and J.D.B. Featherstone3

1 Faculty of Pharmacy, Dentistry and Nursing, Federal University of Ceará, Fortaleza, CE, Brazil;
2 Faculty of Dentistry of Piracicaba, State University of Campinas, Av. Limeira 901, Piracicaba, SP, 13414-903, Brazil; and
3 University of California, San Francisco, CA, USA

* corresponding author, nobre{at}fop.unicamp.br


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Laser and fluoride treatments have been shown to inhibit enamel demineralization in the laboratory. However, the intra-oral effects of this association have not been tested. This study assessed in situ the effect of a Transversely Excited Atmospheric CO2 laser ({lambda} = 9.6 µm) and the use of pressure fluoridated dentifrice on enamel demineralization. During two 14-day phases, 17 volunteers wore palatal appliances containing human enamel slabs assigned to treatment groups, as follows: (1) non-fluoride dentifrice, (2) CO2 laser irradiation plus non-fluoride dentifrice, (3) fluoride dentifrice, and (4) CO2 laser irradiation plus fluoride dentifrice. A 20% sucrose solution was dripped onto the slabs 8 times per day. The specimens treated with laser and/or fluoridated dentifrice presented a significantly lower mineral loss when compared with those from the non-fluoride dentifrice group. The results suggested that CO2 laser treatment of enamel inhibits demineralization in the human mouth, being more effective when associated with fluoride.

KEY WORDS: CO2 laser • fluoride • dental enamel • demineralization • dental caries


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In spite of the decline observed in dental caries, it still represents the most prevalent chronic childhood oral disease (Oral Health in America, 2000). In addition, the disease has become polarized, with carious surfaces being mostly in certain groups of children who present high caries activity (Tayanin et al., 2005). Consequently, the use of combined therapies for this population might be a promising method to prevent and control dental caries.

The efficacy of CO2 laser irradiation combined with fluoride in inhibiting enamel demineralization has been demonstrated by several laboratory investigations (Featherstone et al., 1991; Fox et al., 1992; Hsu et al., 1998; Hsu et al., 2001; Nobre dos Santos et al., 2001). However, there is no report about the in situ or in vivo caries-preventive effect of CO2 laser combined with fluoride dentifrice on dental enamel. Furthermore, no study has tested a transversely excited atmospheric pressure (TEA) CO2 laser operating at 9.6- µm wavelength combined with a fluoride dentifrice used under intra-oral conditions. It must be emphasized that the most efficient wavelengths for preventing dental caries are 9.3 and 9.6 µm, due to the high absorption coefficient in dental enamel at these wavelengths (Featherstone et al., 1998).

Thus, the objective of this study was to assess in situ the combined effects of a 9.6-µm TEA CO2 laser and fluoride dentifrice on the inhibition of human dental enamel demineralization.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Design
This study was approved by the Research and Ethics Committee of the Faculty of Dentistry of Piracicaba-UNICAMP (Protocol No. 114/2003), and all volunteers signed an informed consent. It was performed in two phases (Paes Leme et al., 2004), during which 17 volunteers, from 20 to 33 years old, wore acrylic palatal appliances containing two human enamel slabs. In each phase, each slab received one of the following treatments: (1) non-fluoride dentifrice (Control group), (2) laser irradiation plus non-fluoride dentifrice (Laser group), (3) fluoride dentifrice (Fluoride group), and (4) laser irradiation plus fluoride dentifrice (Laser+fluoride group). The volunteers were randomly allocated to treatments, and those who received treatments "Control" and "Laser" in the first phase received "Fluoride" and "Laser+fluoride" in the second one, and vice versa (Fig. 1Go). The use of two treatments (split-mouth) in the same intra-oral palatal appliance was supported by the absence of any possible cross-over effect between irradiated and non-irradiated enamel slabs. It should be emphasized that the split-mouth design was not used for dentifrice treatments, because of the possibility of carry-across effects. Therefore, both treatments that used fluoridated dentifrice were performed in the same phase. On the 14th day of each phase, the slabs were collected and sectioned, and enamel mineral loss was determined by cross-sectional microhardness analyses. For statistical analysis, the volunteer was considered as a unit, and the treatment as an individualized block. This study was triple-blind, since the examiner, the volunteers, as well as the technician who performed the analyses could not identify the dentifrice type or the irradiated slabs.


Figure 1
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  		Figure 1. Experimental design used in the study. Adapted from Pecharki et al.(2005).

 
Enamel Slabs and Palatal Device Preparation
Forty extracted impacted human third molars, which were sterilized by gamma radiation (Amaechi et al., 1999), were used to perform this in situ study. Eighty enamel slabs (5 x 5 x 2 mm) were obtained, and the surfaces to be treated were polished for 30 sec by means of a 5-µm alumina/water suspension to expose fresh enamel. The slabs were coated with an acid-resistant varnish, leaving a window (12.25 mm2) of exposed anatomical surface enamel, and randomly divided into four groups of 17 specimens according to the treatments. Twelve dental enamel slabs, which were not worn in the mouth, were reserved for SEM analyses. For each volunteer, 2 acrylic custom-made palatal devices were made in which 2 cavities (6 x 6 x 3 mm) were prepared on the left and right sides, and into each of which one slab was placed. To allow plaque to accumulate, and to protect it from mechanical disturbance, we positioned a plastic mesh on the acrylic resin, leaving a 1-mm space from the slab surface (Benelli et al., 1993). To avoid carry-across effect, both treatments that used fluoridated dentifrice were performed in the same phase. Within each palatal device, the position of the slab was randomly determined.

Laser Irradiation
The exposed enamel area of 40 slabs was irradiated in the laboratory with a TEA CO2 (Argus Photonics Group, Jupiter, FL, USA) laser at 9.6-µm wavelength, 5-µs pulse duration, 10-Hz repetition rate, 1.5-mm beam diameter, and 1.5 J/cm2 per pulse (Nobre dos Santos et al., 2001). We measured and calibrated the laser energy using a calorimeter, and measured the laser spot size (same as the beam diameter) by scanning the beam with a razor blade. To provide uniform coverage of each window, we used a computer-operated micrometer-driven x-y stage, and each spot was irradiated with 25 pulses. The sample was moved one-third of the beam diameter, and an overlapping spot was irradiated with 25 pulses, and so on, providing an approximately uniform coverage over the entire specimen experimental surface.

Scanning Electron Microscopy
To verify the laser effect on surface enamel morphology, we evaluated 6 irradiated and 6 non-irradiated slabs by scanning electron microscopy (SEM). The specimens were coated with a thin layer of gold (approximately 10–12 nm thick). Observations were then made with a JEOL JSM-5600 LV Scanning Electron Microscope (Jeol Inc., Peabody, MA, USA) at 15 kV and magnifications up to 3000.

Intra-oral Phase
During the lead-in and wash-out periods, the volunteers brushed their teeth with a non-fluoride silica-based dentifrice prepared for this study (FGM Dentistry Product, Joinville/SC, Brazil). Next, all volunteers began wearing palatal devices and using the appropriate dentifrice according to the treatments.

To provide a cariogenic challenge, the volunteers were instructed to remove the appliance and drip one drop of 20% sucrose solution onto each mesh that was above the enamel slab, 8 times per day at pre-determined times (8.00, 9.30, 11.00, 14.00, 15.30, 17.00, 19.00, and 21.00) (Pecharki et al., 2005). Before the palatal appliance was re-placed in the mouth, a five-minute waiting time was standardized for sucrose diffusion into the dental plaque. As stated by Paes Leme et al.(2004), this model is able to simulate a high-caries-risk situation.

Fluoridated dentifrice (silica-based, containing 1100 µg F/g, w:w, as NaF; FGM Dentistry Product, Joinville/SC, Brazil) was used by the volunteers in the phase in which they underwent Fluoride and Laser+fluoride treatments. In the other phase, Control and Laser treatments were carried out, and the previously described non-fluoridated dentifrice was used. The dentifrice treatment was performed only 3 times a day, at pre-determined times, which were after main mealtimes and when volunteers habitually performed oral hygiene. The appliances were extra-orally brushed, except the enamel slabs, and volunteers were asked to brush carefully over the covering meshes, to avoid disturbing the plaque. They were asked to brush their teeth and appliance for up to 5 min. All volunteers consumed fluoridated water (0.70 mg F/L) and received oral and written instructions to wear the appliances all the time, including at night. They were allowed to remove the appliances only during meals and when performing oral hygiene (Cury et al., 2000).

Microhardness Analysis
Each enamel slab was longitudinally sectioned by a cut through the center of the exposed enamel area. The segments were embedded in acrylic resin and serially polished. Cross-sectional microhardness measurements were made with a microhardness tester (Future Tech FM-ARS; Tokyo, Japan) with a Knoop diamond under a 25 g load for 5 sec. Three lanes of 13 indentations each were made in the central region of the slab, and distance between them was set at 100 µm. The indentations were made at the following depths: 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, and 200 µm from the outer enamel (Hara et al., 2003).

The mean Knoop hardness number values at each distance from the surface were obtained and converted into volume percent mineral (Featherstone et al., 1983). Volume percent mineral was plotted against depth for each specimen, and the integrated mineral content of the lesion was calculated relative to underlying sound enamel. The mean sound enamel values for the computation of integrated mineral loss were obtained from inner sound enamel under the lesion in the same tooth. To compute {Delta}Z (integrated mineral loss), we subtracted the integrated mineral content of the lesion from the value obtained for sound enamel (Featherstone et al., 1988). Based on the mean {Delta}Z parameter, the percentage of demineralization inhibition was calculated individually in comparison with the control leg, and the mean was then calculated for the experimental groups.

Statistical Analysis
To assess the effects of the treatments, we analyzed the dependent variable {Delta}Z; the assumptions of equality of variances and normal distribution of errors were checked. The factors included in the analysis were volunteer, first or second phase, slab position in device, and treatment. Equality of variances, normal distribution of errors, and absence of outliers were satisfied, and analysis of variance, followed by the Tukey multiple comparison test, was applied. The software SAS system (version 8.02, SAS Institute Inc., Cary, NC, USA) was used, and the significance limit was set at 5%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The mean {Delta}Z values in the experimental groups were lower, to a statistically significant extent, than the values found for the control (no fluoride, no laser) group (TableGo). However, there was no statistically significant difference between the Laser and Fluoride groups (TableGo). In addition, when laser irradiation was associated with fluoridated dentifrice use, a statistically significant lower mineral loss value was found.


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  		Table. Mineral Loss, {Delta}Z (vol% x µm) in Human Dental Enamel, According to Groups (mean ± SD)
 
The SEM observations showed evidence of melting and fusion in specimens treated with CO2 laser (Fig. 2Go).


Figure 2
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  		Figure 2. Representative scanning electron micrographs of irradiated specimens. Surface morphology of transition area between normal and irradiated enamel at 100x magnification (a) and irradiated enamel at 3000x magnification (b).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Enamel specimens exposed to CO2 laser irradiation, whether or not associated with fluoridated dentifrice, presented significantly less mineral loss than specimens treated with non-fluoridated dentifrice (TableGo). In addition, the combination of CO2 laser irradiation with fluoride dentifrice use provided significantly more protection against demineralization than did laser irradiation or fluoride dentifrice use alone. The decreased mineral loss associated with the combined therapy applied may translate into more protection against caries. Analysis of these in situ data confirms reports from several in vitro studies that found an additional effect when this laser was combined with fluoride (Featherstone et al., 1991; Fox et al., 1992; Hsu et al., 1998; Hsu et al., 2001; Nobre dos Santos et al., 2001, 2002). This effect was attributed not only to enamel changes promoted by the CO2 laser, but also to a possible increase in fluoride uptake in irradiated enamel, as previously reported (Chin-Ying et al., 2004; Tepper et al., 2004). There are two possible mechanisms involved in the laser-induced increase of fluoride uptake. One suggestion is that laser-fluoride treatment produces numerous spherical precipitates that morphologically resemble calcium-fluoride-like deposits on dental surfaces, and which serve as a reservoir to replenish fluoride (Chin-Ying et al., 2004). Another mechanism emphasizes the role of lasers in enhancing fluoride uptake into the tooth crystalline structure in the form of firmly bound fluoride (Meurman et al., 1997).

Since fluoride was not present at the time of irradiation, heat was not responsible for fluoride uptake. As evidenced by SEM analysis, laser-induced surface changes, such as an increase in cracks and roughness, might have played an important role in increasing calcium-fluoride-like material formation (Putt et al., 1978). However, there could be complications from laser-induced enamel changes, such as increased potential for stain uptake, but this has yet to be determined in further clinical studies. This technology may be considered a practical approach when used in specific clinical situations in limited areas, such as in surfaces adjacent to restorations, or in the pits and fissures of occlusal surfaces, where irradiation might act as a prophylactic measure against caries formation, as has been demonstrated in vitro (Konishi et al., 1999; Nobre dos Santos et al., 2002; Klein et al., 2005).

The laser treatment alone produced inhibition of enamel demineralization comparable with that found by three-times-a-day use of fluoridated dentifrice, which was also shown by a previous in situ study (Featherstone et al., 2001). However, these authors did not combine the laser and fluoride treatment, and they found caries inhibition by laser irradiation only in those individuals who were considered demineralizers, that is, those who were exposed to a highly cariogenic diet daily and, consequently, showed higher enamel mineral loss, expressed as the {Delta}Z parameter. Thus, it seems that laser treatment did not enhance remineralization in the absence of fluoride, but only inhibited demineralization. This may explain why fluoride dentifrice use presented a higher percentage of caries inhibition than laser treatment, since fluoride can interfere physicochemically with caries development by reducing demineralization and enhancing remineralization (Dawes and Weatherell, 1990).

As expected, the fluoridated dentifrice inhibited enamel demineralization in the present intra-oral model. This confirms previous in situ studies evaluating the role of fluoridated dentifrice in caries inhibition (Paes Leme et al., 2004). However, no statistically significant difference was found between the Fluoride and Laser groups, showing that laser treatment may be a good alternative in those cases where fluoride is apparently not as effective as it is on smooth surfaces, such as in pit and fissure regions. A previous in vitro study, in occlusal surfaces treated by CO2 laser treatment, showed caries inhibition similar to that achieved for smooth surfaces (Nobre dos Santos et al., 2002). The fusion and melting observed in the present study (Fig. 2Go) are in agreement with previous reports (Nelson et al., 1986; McCormack et al., 1995; Kantorowitz et al., 1998) where higher fluences (energy/surface area) were used than in our study. However, in this study, a short pulse duration of 5 µs, rather than 100 µs, was used to match more closely the thermal relaxation time of enamel at this wavelength.

In conclusion, CO2 laser treatment, with a 9.6-µm wavelength and a pulse duration of 5 µs, whether or not associated with fluoride use, inhibits subsequent enamel mineral loss in an in situ high-caries-challenge situation. In particular, the combined use of this specific laser treatment plus fluoride was more successful than either laser treatment or fluoride alone in the inhibition of mineral loss in the mouth. The results of this in situ study suggest that the combination of this specific laser treatment with fluoride therapy may be effective as a caries inhibition treatment.


   ACKNOWLEDGMENTS
 
We thank the volunteers for their valuable participation. This research was supported by FAPESP, grant 00/09702-8. The study was an extension of work supported by NIH/NIDCR grant RO1-DE09958. The authors especially thank the Oral Biochemistry Laboratory of the Faculty of Dentistry of Piracicaba for the use of their microhardness tester. Dentifrices used in this study were prepared and donated by FGM Dental Products, Joinville-SC, Brazil. The first author received a scholarship from CAPES-PICDT-UFC during her PhD program. Dr. Daniel Fried and Charles Le at UCSF are thanked sincerely for the laser irradiation of the specimens and their input into the study.

Received July 26, 2005; Last revision March 2, 2006; Accepted March 23, 2006


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 ABSTRACT
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 RESULTS
 DISCUSSION
 REFERENCES
 
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