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Effectiveness of carbonate removal and demineralization inhibition in primary teeth using a 9.3-μm carbon dioxide laser

Open AccessPublished:December 05, 2022DOI:https://doi.org/10.1016/j.jfscie.2022.100017

      Abstract

      Background

      Studies on permanent dental enamel have shown that irradiation with a 9.3-μm carbon dioxide (CO2) laser can safely inhibit caries progression in vivo and carieslike lesion formation in vitro. The authors conducted a study to investigate whether 9.3-μm CO2 laser irradiation could safely inhibit carieslike lesion formation in primary teeth in vitro.

      Methods

      Teeth were irradiated with a 9.3-μm CO2 laser at a pulse fluence of 0.8 J/cm2 and scanned automatically over a target area of 5.8 mm2. Two test groups of 15 extracted human molars each were used: (1) laser-irradiated and (2) laser-irradiated plus additional fluoride. Both groups used nonirradiated areas as nonlaser-treated controls. After irradiation, artificial carieslike lesions were generated through a validated pH-cycling protocol. Relative mineral loss (ΔZ) was determined by cross-sectional microhardness testing in depth. Fourier transform infrared spectroscopy was performed on additional 10 irradiated samples to investigate the removal of acid-soluble carbonate groups from the mineral.

      Results

      Inhibition of carieslike lesion formation relative to untreated enamel was (1) laser-irradiated alone: 56.2%, (2) fluoride alone: 55.0%, (3) laser plus fluoride: 76.5% (significantly different from 1 and 2; P < 0.01). Carbonate removal by laser irradiation (SD) was 50.4% (7.7%) at the surface and detectable to a depth of at least 14 μm.

      Conclusion

      Irradiation with 9.3-μm CO2 laser on primary teeth reduced the formation of carieslike lesions and was associated with significant removal of acid-soluble carbonate groups from the enamel mineral. This inhibition was accomplished without an unsafe rise in pulpal temperature and without significant microscopic or visible damage to the enamel surface.

      Graphical abstract

      Key Words

      Caries continues to be a major problem in infants and toddlers. Primary teeth are more susceptible to caries than permanent teeth, and caries progresses more rapidly. Caries preventive methods such as antibacterial mouthrinses and high-concentration fluoride toothpaste cannot be used in children younger than 6 years. Caries management involves sealants, fluoride toothpaste, fluoride varnish and silver diamine fluoride. Fluoride therapy alone, however, is insufficient to control cavity formation in young children at high caries risk. Sealants can allow for bacterial growth through microleakage sites, and sealant retention can be an ongoing problem. The use of a novel 9.3-μm carbon dioxide laser to alter tooth enamel in such a way as to render it more acid-resistant presents a unique approach to improving caries resistance in a safe way. This technique has been well-investigated over the past several decades in permanent teeth and has been shown to be effective in inhibiting demineralization both in laboratory and clinical studies, especially in combination with fluoride. This laboratory study provides novel information regarding the effectiveness and safety of this laser treatment on primary teeth. This treatment has the potential to revolutionize pediatric and preventive dentistry, especially if combined with conventional treatments.

      Introduction

      Caries in primary teeth is a global problem that starts at a young age.
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      Caries management is difficult in this age group as high-concentration fluoride toothpaste (5,000 ppm fluoride) and mouthrinses such as chlorhexidine cannot be used in children younger than 6 years. The American Academy of Pediatric Dentistry publishes best practice guidelines recommending what can be used for low-, moderate-, and high-risk young children.
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      These guidelines include daily brushing with fluoride-containing toothpaste and the use of fluoride varnish. Even when following these guidelines, caries progresses in high-risk young children.
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      Furthermore, fluorosis from fluoride ingestion and staining from silver diamine fluoride are concerns.
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      There is a need for innovative methods beyond the standard of care to prevent or reverse dental caries in primary teeth.
      The use of specifically designed 9.3-μm carbon dioxide (CO2) laser irradiation provides an alternative method for reducing demineralization caused by caries, and this has been shown in laboratory and clinical studies on permanent teeth.
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      In-vivo occlusal caries prevention by pulsed CO2-laser and fluoride varnish treatment: a clinical pilot study.
      The primary effect that 9.3-μm CO2 laser irradiation has on dental enamel is the removal of acid-soluble carbonate groups from the carbonated hydroxyapatite enamel mineral, and subsequent remineralization with fluoride, calcium, and phosphate groups further enhances acid resistance.
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      Fundamental interactions of lasers with dental hard tissues.
      Although other marketed wavelengths, such as erbium lasers, have been studied in the laboratory to show a reduction in carious lesion formation, none have shown a clinically viable inhibition of demineralization.
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      Fundamental interactions of lasers with dental hard tissues.
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      • Matsumoto K.
      Caries-preventive effect of Er:YAG laser irradiation with or without water mist.
      The key to the success of 9.3- or 9.6-μm CO2 laser irradiation in reducing enamel solubility is that these wavelengths are strongly absorbed by the phosphate groups in the carbonated hydroxyapatite mineral of the teeth.
      • Featherstone J.D.B.
      • Fried D.
      Fundamental interactions of lasers with dental hard tissues.
      This allows for a rapid, safe, and controlled superficial heating to the necessary temperatures to remove the carbonate groups without additional damage to the enamel structure or an unsafe rise in pulpal temperature.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      ,
      • Esteves-Oliveira M.
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      • Heussen N.
      • Eduardo C.P.
      • Lampert F.
      • Apel C.
      Rehardening of acid-softened enamel and prevention of enamel softening through CO2 laser irradiation.
      Significant research and clinical studies have focused on permanent teeth to investigate the safety and efficacy of inhibiting demineralization using CO2 lasers at 2 wavelengths, 9.3 and 9.6 μm, both of which have similar preferential absorption in hydroxyapatite.
      • Rechmann P.
      • Charland D.A.
      • Rechmann B.M.T.
      • Le C.Q.
      • Featherstone J.D.B.
      In-vivo occlusal caries prevention by pulsed CO2-laser and fluoride varnish treatment: a clinical pilot study.
      ,
      • Featherstone J.D.B.
      • Fried D.
      Fundamental interactions of lasers with dental hard tissues.
      ,
      • Rechmann P.
      • Fried D.
      • Le C.Q.
      • et al.
      Caries inhibition in vital teeth using 9.6-μm CO2-laser irradiation.
      Studies from 2020 and 2021 have shown effective inhibition of demineralization with a commercial 9.3-μm CO2 laser using irradiation conditions that would be useful in a clinical workflow.
      • Rechmann P.
      • Le C.Q.
      • Kinsel R.
      • Kerbage C.
      • Rechmann B.M.T.
      In vitro CO2 9.3-μm short-pulsed laser caries prevention-effects of a newly developed laser irradiation pattern.
      ,
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      The objective of this study was to evaluate the effectiveness of 9.3-μm CO2 laser irradiation for the inhibition of demineralization of enamel in primary teeth and the associated removal of acid-soluble carbonate groups. In addition, the depth effect of laser irradiation on enamel was investigated by measuring carbonate removal at incremental depths under the enamel surface. The system and energy delivery method were based on previous work on permanent enamel, which was designed to allow for a clinically relevant application in terms of treatment speed and safe energy levels that did not result in significant surface damage or overheating of the pulp.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.

      Methods

      Laser settings

      Laser irradiation was performed using a 9.3-μm CO2 laser (Solea, Convergent Dental). A beam of 1-mm diameter (measured by 1/e2 method) was scanned over a 5.8-mm2 area at a pulse repetition rate of 333 Hz for 0.38 seconds. A pulse duration of 20 μs and fluence of 0.8 J/cm2 were used. The fluence value used was the same as the median fluence used in a previous study with similar parameters for permanent enamel.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      The pulse repetition rate was reduced compared with that of the previous study to lower the average power of irradiation, accounting for the smaller size and less mineralized structure of primary teeth. Preliminary tests showed that this irradiation provided a good balance of safety and effectiveness. The handpiece was held at approximately 10 mm from the irradiation surface while treating the teeth. A continuous airflow (SD) out of the handpiece was maintained at 9 (1) L/min for cooling the tooth.

      Samples for assessment of carbonate removal from irradiation

      Ten sound human primary molars with no evidence of caries or fluorosis (Therametric) were cleaned. The samples had been exposed to 0.1% thymol solution during shipment and were less than 3 months old after extraction.
      The samples were mounted in acrylic with the buccal or lingual surface exposed to investigate the acid-resistant layer created by laser irradiation. A mounted cross-section of the enamel was generated using a diamond saw (Isomet, Buehler), and the enamel samples were serially polished up to 1 μm diamond suspension using an automated grinder polisher (Forcipol 1V with Forcimet, Metkon Instruments).

      Fourier transform infrared spectroscopy for carbonate removal analysis

      Infrared spectra in the range of 4,000 through 500 cm−1 were obtained from the enamel before and after irradiation using Fourier transform infrared spectroscopy (FTIR) with a diamond window in the ATR setup (Nicolet iS5 with iD7 air, ThermoFisher Scientific). Advanced ATR correction was used to correct for the wavelength shift by the material, and the spectra were normalized to the highest peaks, which corresponded to phosphate groups. The resultant spectra matched previous work.
      • Featherstone J.D.B.
      • Fried D.
      Fundamental interactions of lasers with dental hard tissues.
      To calculate the percentage carbonate removed (%CR) by laser irradiation, the area under the carbonate and phosphate peaks was determined using the FTIR spectral analysis software (Omnic 8, Thermo Fisher Scientific). The carbonate peak was chosen to be 1,600 through 1,300 cm−1, and the phosphate peak range was chosen to be 1,200 through 650 cm−1. The percentage of carbonate groups removed was then calculated using the following formula:
      %CR=100(1CO3Area/PO4Area(after)CO3Area/PO4Area(before))


      where CO
      • Chaffee B.W.
      • Featherstone J.D.B.
      • Zhan L.
      Pediatric caries risk assessment as a predictor of caries outcomes.
      Area and PO
      • Chibinski A.C.
      • Wambier L.M.
      • Feltrin J.
      • Loguercio A.D.
      • Wambier D.S.
      • Reis A.
      Silver diamine fluoride has efficacy in controlling caries progression in primary teeth: a systematic review and meta-analysis.
      Area are, respectively, the areas under the CO
      • Chaffee B.W.
      • Featherstone J.D.B.
      • Zhan L.
      Pediatric caries risk assessment as a predictor of caries outcomes.
      peak and PO
      • Chibinski A.C.
      • Wambier L.M.
      • Feltrin J.
      • Loguercio A.D.
      • Wambier D.S.
      • Reis A.
      Silver diamine fluoride has efficacy in controlling caries progression in primary teeth: a systematic review and meta-analysis.
      peak of the FTIR spectrum.

      Microscopy images

      These samples were imaged under a 3-dimensional digital reflection microscope (RH-2000; Hirox-US) before and after irradiation, both with and without cross-polarization at ×700 magnification. These images were used to assess the surface effects of the irradiation.

      Pulpal temperature

      Modeled after previous work,
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      ,
      • Ertuǧrul C.Ç.
      • Ertuǧrul I.F.
      Temperature change in pulp chamber of primary teeth during curing of coloured compomers: an in vitro study using pulpal blood microcirculation model.
      ,
      • Krmek S.J.
      • Miletic I.
      • Simeon P.
      • Mehicić GP
      • Anić I
      • Radisić B
      The temperature changes in the pulp chamber during cavity preparation with the Er:YAG laser using a very short pulse.
      type J thermocouples (5TC-TT-J-36-36, Omega Engineering) were placed through a drilled hole through the root into the roof of the pulp chamber of 10 extracted human primary molars with thermally conductive paste and tape (3M). Radiographic images of the teeth were acquired to confirm that the thermocouple tip was mounted correctly, touching the pulp chamber ceiling. The tooth was held upright in clay on a heating plate, and its baseline temperature (SD) was maintained at 35.0 (3.0) °C. The temperature change during 40 seconds of continuous irradiation over the occlusal surfaces (with 1 second of cooling time between consecutive scans of 0.38 seconds) was recorded. This irradiation duration was more than that required to treat a tooth but was chosen to compare with previously measured pulpal temperature rise on permanent teeth.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      Data were collected using a temperature logger (HH806U, Omega Engineering).

      Specimen used for demineralization reduction

      Thirty human primary molars with no signs of caries or fluorosis were obtained less than 3 months postextraction and stored in thymol solution (Therametrics). The samples were rinsed, air-dried, mounted, and separated into 2 groups of 15 each. The samples underwent pH cycling without (group 1) or with (group 2) additional fluoride. The fluoride used was a sodium fluoride toothpaste slurry, made with a 1:3 ratio of 1,100 ppm fluoride toothpaste (Crest Cavity Protection; P&G) to distilled water. As done previously, samples were laser-irradiated on either side of an area with the least curvature on the buccal or lingual aspect of the crown.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      The remaining nonirradiated areas were maintained as control regions on each sample. An acid-resistant, quick-curing nail polish was used to mask the regional boundaries.

      pH-cycling and artificial lesion formation

      Demineralizing and remineralizing solutions were cycled to simulate aggressive carious lesion formation. A pH-cycling regimen with these solutions, as described by Rechmann et al,
      • Rechmann P.
      • Rechmann B.M.T.
      • Groves Jr., W.H.
      • et al.
      Caries inhibition with a CO2 9.3-μm laser: an in vitro study.
      • Argenta R.M.O.
      • Tabchoury C.P.M.
      • Cury J.A.
      A modified pH-cycling model to evaluate fluoride effect on enamel demineralization.
      • Stookey G.K.
      • Featherstone J.D.B.
      • Rapozo-Hilo M.
      • et al.
      The Featherstone laboratory pH cycling model: a prospective, multi-site validation exercise.
      with steps of 6 hours in demineralization and 18 hours in remineralization over a total of 9 days of cycling, was followed. The demineralizing solution at pH 4.4 was made with 75 mM acetate buffer with 2 mM calcium and phosphate. A simple saliva-mimicking remineralizing solution at pH 7.1 was made from 0.1 M Tris, 0.8 mM calcium, and 2.4 mM phosphate. Group 2 samples were exposed to the fluoride toothpaste slurry for 1 minute after each step in the cycling. After 5 pH cycles, the solutions were replaced with fresh solutions.

      Cross-sectional microhardness measurements

      To determine the relative mineral loss (ΔZ) in the laser-treated area, a cross-section perpendicular to the laser-irradiated surface area was created by cutting the enamel with a diamond saw at the approximate middle of the treated area. Then the samples were serially polished to a 1-μm diamond suspension finish to prepare for the cross-sectional microhardness testing described in previous studies.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      ,
      • Rechmann P.
      • Rechmann B.M.T.
      • Groves Jr., W.H.
      • et al.
      Caries inhibition with a CO2 9.3-μm laser: an in vitro study.
      ,
      • Stookey G.K.
      • Featherstone J.D.B.
      • Rapozo-Hilo M.
      • et al.
      The Featherstone laboratory pH cycling model: a prospective, multi-site validation exercise.
      The samples were serially indented using a microhardness indenter Matsuzawa Seiki DMH-2 (Matsuzawa, Akita Pref) in a straight line under the surface with 25 g of force loads for 10 seconds for each indent with steps of 15 μm starting at 15 μm from the outer surface until a depth of 210 μm was reached, for 14 total indents, as performed previously.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      The length of each indent was measured as the long diagonal generated from the Knoop tip using the digital microscope at ×2,000 magnification. The volume percentage of mineral content was then calculated at each indentation position using the following formula:
      vol%=4.3KHN+11.3


      where KHN is the Knoop hardness number obtained from the length of the intention.
      • Stookey G.K.
      • Featherstone J.D.B.
      • Rapozo-Hilo M.
      • et al.
      The Featherstone laboratory pH cycling model: a prospective, multi-site validation exercise.
      ,
      • Featherstone J.D.
      • ten Cate J.M.
      • Shariati M.
      • Arends J.
      Comparison of artificial caries-like lesions by quantitative microradiography and microhardness profiles.
      ΔZ, a measure of depth mineral loss, was then calculated as the area under the curve according to Stookey et al.
      • Stookey G.K.
      • Featherstone J.D.B.
      • Rapozo-Hilo M.
      • et al.
      The Featherstone laboratory pH cycling model: a prospective, multi-site validation exercise.

      Statistical methods and sample size calculation

      Based on previous work, sample sizes have been shown to be sufficient for these studies.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      ,
      • Rechmann P.
      • Rechmann B.M.T.
      • Groves Jr., W.H.
      • et al.
      Caries inhibition with a CO2 9.3-μm laser: an in vitro study.
      Data were analyzed in Minitab 18 (Minitab). The Shapiro-Wilk test was done on the groups to verify that the data followed normal distribution. One-way analysis of variance was performed on the data set with post hoc Tukey tests for comparison of individual groups.

      Results

      Laser surface effects

      Figure 1 shows microscopy surface images at ×400 magnification for the nonirradiated and irradiated enamel regions. Each cross-polarized image was taken at a location identical to that of the corresponding regular surface image. The images reveal minor superficial changes from laser irradiation, such as crazing, which is obvious in the cross-polarized images because it occurs several micrometers into the structure. The images show that the effect of laser irradiation does not visibly damage the surface and that the treatment is safe.
      Figure thumbnail gr1
      Figure 1Microscopy images of the primary molar enamel surface in irradiated and nonirradiated areas. Cross-polarization microscopy enabled the crazing pattern caused by laser irradiation to be observed.

      Fourier transform infrared spectroscopy

      Infrared spectra of primary molar enamel before and after irradiation are shown in Figure 2. When normalized to the phosphate peaks, the carbonate peaks revealed a significant reduction in response to laser irradiation, with an average (SD) of 50.4% (7.7%).
      Figure thumbnail gr2
      Figure 2A. Normalized Fourier transform infrared spectroscopy spectra before and after irradiation. The carbonate peaks between 1,600-1,300 cm−1 show a reduction in size from laser irradiation relative to the phosphate peak between 1,200-650 cm−1. B. Percentage carbonate removed by laser irradiation as a function of depth (SD). A return to the original carbonate content (gray line at 0%) was found to be at least 14 μm deep.
      Percentage carbonate removed as a function of depth under the enamel surface is shown in Figure 2. Carbonate content was indistinguishable from nonirradiated enamel after a depth of at least 14 μm. The data followed an exponential decay, fitted by the equation:
      %CR=50.4e0.232d


      where %CR is the percentage carbonate removed from the enamel, e is exponential, and d is the depth under the enamel surface.

      ΔZ

      ΔZ values, indicative of the size of the artificial lesion, are shown in Table 1. One-way analysis of variance revealed that significant differences in ΔZ occurred between the treatment groups. The reduction in ΔZ from fluoride alone without laser irradiation was 55.0%, which is similar to that of laser irradiation alone (56.3%). The combination of irradiation and additional fluoride reduced demineralization by 76.5% compared with untreated controls without additional fluoride. Post hoc Tukey tests showed that the combination treatment of laser irradiation and additional fluoride provided the most significant benefit in reducing ΔZ for each laser fluence used (P < .01 for all).
      Table 1Relative mineral loss data
      GroupAdditional FluorideIrradiated ΔZ (SD)
      Groups with the same superscripted letters are not significantly different.
      Nonirradiated, ΔZ (SD)Laser Reduction, % ΔZnP Value (95%)
      1No1,532 (475)a3,503 (823)c56.315< .001
      2Yes822 (405)b1,578 (416)a47.915< .001
      Groups with the same superscripted letters are not significantly different.
      Figure 3 shows the averages of the mineral concentration as a function of depth under the enamel surface for the groups. Overall lesion depth was typically less than 120 μm. The remineralized outer layer is represented by a typically higher mineral concentration at 15 μm than at 30 μm. The fluoride-treated groups showed a deeper remineralization effect on average.
      Figure thumbnail gr3
      Figure 3Mineral volume percentage concentration as a function of depth under the enamel surface after pH cycling presented with standard error bars. The average baseline mineral concentration for primary enamel was 83% (gray dotted line). Irradiation coupled with additional fluoride during pH cycling provided the strongest resistance to demineralization.

      Pulpal temperature

      Forty seconds of laser irradiation cycling on the surfaces of the extracted teeth resulted in an average (SD) temperature increase of 3.4 (1.6) °C in the pulp chamber. As shown in Figure 4, the increase in pulpal temperature on average shows with 95% CI that the rise is below the Arrhenius damage threshold.
      • Zach L.
      • Cohen G.
      Pulp response to externally applied heat.
      • Baldissara P.
      • Catapano S.
      • Scotti R.
      Clinical and histological evaluation of thermal injury thresholds in human teeth: a preliminary study.
      • Dewey W.C.
      Arrhenius relationships from the molecule and cell to the clinic.
      Forty seconds of laser irradiation covered the entire occlusal surface more than 3 times.
      Figure thumbnail gr4
      Figure 4Pulpal temperature rise for primary molars over 40 seconds of continuous irradiation treatment on the occlusal surface. The mean rise (blue line) for the 10 samples is shown with a 95% CI (gray shaded area). The gray dashed line represents the Arrhenius damage threshold, where irreversible tissue damage may occur.

      Discussion

      This study shows that laser irradiation at 9.3-μm wavelength on primary tooth enamel is similar to that on permanent teeth in inhibiting mineral loss. An acid-resistant layer of modified hydroxyapatite is generated in a fraction of a second by heat from this irradiation.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      ,
      • Featherstone J.D.
      • Nelson D.G.
      Laser effects on dental hard tissues.
      Organic components surrounding the enamel rods are removed or shrunk near the surface during irradiation. Together, these surface changes have been described previously as crazing of the surface.
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      ,
      • Kim J.W.
      • Lee R.
      • Chan K.H.
      • Jew J.M.
      • Fried D.
      Influence of a pulsed CO2 laser operating at 9.4 μm on the surface morphology, reflectivity, and acid resistance of dental enamel below the threshold for melting.
      The formation of fluoride-containing hydroxyapatite further reduces the solubility of the acid-resistant layer. Our results are in general agreement with findings reported previously for ΔZ in depth
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      ,
      • Rechmann P.
      • Rechmann B.M.T.
      • Groves Jr., W.H.
      • et al.
      Caries inhibition with a CO2 9.3-μm laser: an in vitro study.
      because of acidity challenges. Our study verified that the computer-assisted method of scanning laser pulses over enamel surfaces at a high repetition rate is effective for clinical application in primary teeth as well as permanent teeth.
      To the best of our knowledge, this study presents the first FTIR data on carbonate removal after irradiation with a 9.3-μm CO2 laser in primary tooth enamel as a function of depth under the surface. An exponential fall in the percentage of CR was found until at least a 14 μm depth. This is consistent with a previous measurement using an acid challenge after similar irradiation at this wavelength,
      • Badreddine A.H.
      • Couitt S.
      • Donovan J.
      • Cantor-Balan R.
      • Kerbage C.
      • Rechmann P.
      Demineralization inhibition by high-speed scanning of 9.3 μm CO2 single laser pulses over enamel.
      which provides evidence that the depth of effect of irradiation delivered in this study is largely a product of pulse fluence and wavelength. When considering the rapid rise of surface temperature at 9.3 and 9.6 μm,
      • Fried D.
      • Seka W.D.
      • Glena R.E.
      • Featherstone J.D.
      Thermal response of hard dental tissues to 9- through 11-μm CO2-laser irradiation.
      it is not surprising that the bulk of the effect may be driven by individual pulse fluence (or pulse irradiance).
      In an earlier study, carbonate removal was measured using 9.6- and 10.6-μm CO2 laser irradiation as a function of depth in bovine enamel.
      • Zuerlein M.J.
      • Fried D.
      • Featherstone J.D.B.
      Modeling the modification depth of carbon dioxide laser-treated enamel.
      The plots showed a similar rapid drop-off in the first 6 μm, followed by a slower return to baseline and a corresponding thermal model to corroborate that data. Our data at 9.3-μm irradiation show an exponential decay that correlates with that thermal model and a drop-off toward the baseline that lies between 9.6 μm and 10.6 μm, which may be explained by their absorption depths in enamel.
      • Featherstone J.D.B.
      • Fried D.
      Fundamental interactions of lasers with dental hard tissues.
      When digitized and fit to an exponential equation, the thermal model data from that previous work
      • Zuerlein M.J.
      • Fried D.
      • Featherstone J.D.B.
      Modeling the modification depth of carbon dioxide laser-treated enamel.
      produce exponential coefficients that fall in the range of −0.18 through −0.28, similar to the −0.232 value from carbonate removal presented in our study. Because carbonate removal without significant structural changes occurs primarily in the range of 400 through 800 °C, this provides evidence that the fitted equation from carbonate removal may be used to estimate enamel temperature as a function of depth.
      The volume percentage of mineral concentration in permanent teeth was reported to be an average of 85%.
      • Nelson D.G.
      • Wefel J.S.
      • Jongebloed W.L.
      • Featherstone J.D.B.
      Morphology, histology and crystallography of human dental enamel treated with pulsed low-energy infrared laser radiation.
      Through microhardness indentation, we found that primary teeth mineral concentration was 83% on average. This concentration in primary teeth was lower than in permanent teeth, which was expected because of the larger amount of organic material and slightly increased porosity.
      • Angmar B.
      • Carlström D.
      • Glas J.E.
      Studies on the ultrastructure of dental enamel, IV: the mineralization of normal human enamel.
      ,
      • Mortimer K.V.
      The relationship of deciduous enamel structure to dental disease.
      Despite the lower mineral content, a similar reduction in demineralization was observed in primary molars compared with permanent molars.
      • Badreddine A.H.
      • Couitt S.
      • Kerbage C.
      Histopathological and biomechanical changes in soft palate in response to non-ablative 9.3-μm CO2 laser irradiation: an in vivo study.
      ,
      • Rechmann P.
      • Rechmann B.M.T.
      • Groves Jr., W.H.
      • et al.
      Caries inhibition with a CO2 9.3-μm laser: an in vitro study.
      No obvious differences were observed on the surface to account for this, but 1 possibility is that the porosity was sealed by irradiation and organic content was removed, providing an opportunity for remineralization of the inorganic matrix components of enamel near the surface. Furthermore, a more extensive form of this has been presented in other studies, in which sealing pores such as dentinal tubules with CO2 lasers has been investigated.
      • Matsumoto K.
      • Kimura Y.
      Laser therapy of dentin hypersensitivity.
      ,
      • Lee E.M.R.
      • Borges R.
      • Marchi J.
      • de Paula Eduardo C.
      • Marques M.M.
      Bioactive glass and high-intensity lasers as a promising treatment for dentin hypersensitivity: an in vitro study.
      The depth of carbonate removal (at least 14 μm) roughly matches the remineralization depth shown on average, corresponding to the decrease in mineral volume concentration less than 15 μm under the surface. This provides a good indication that the effect of irradiation is not just in decreasing the solubility of the enamel but also in enhancing its capability to remineralize. Possibly, this is due to vacancies in the mineral structure and the areas left behind by vaporized organic components.
      The use of fluoride in this pH cycling model provided a major additional benefit to the irradiation treatment, which has been seen before and can be explained by an enhanced remineralization effect accompanying carbonate removal.
      • Esteves-Oliveira M.
      • Pasaporti C.
      • Heussen N.
      • Eduardo C.P.
      • Lampert F.
      • Apel C.
      Rehardening of acid-softened enamel and prevention of enamel softening through CO2 laser irradiation.
      ,
      • Bahrololoomi Z.
      • Fotuhi Ardakani F.
      • Sorouri M.
      In vitro comparison of the effects of diode laser and CO2 laser on topical fluoride uptake in primary teeth.
      One unexpected and interesting result observed in this study and shown in Figure 4 was the observance of a higher mineral concentration at 30 μm under the surface of the samples treated with additional fluoride. In comparison, as expected, samples without additional fluoride showed a decrease in mineral concentration under the remineralized surface (especially < 15 μm). This may indicate that the laser treatment followed by fluoride allows for a deeper remineralization effect or a more controlled rate of enamel dissolution under the surface.
      Because of the smaller size of primary molars, it was important to investigate the pulpal temperature rise during this treatment. The Arrhenius damage threshold for soft tissue can be considered in this case to be 5.5 °C over the starting temperature.
      • Zach L.
      • Cohen G.
      Pulp response to externally applied heat.
      A thorough overtreatment of the occlusal surface with at least 3 full scans over the entire surface revealed that the treatment was safe. Furthermore, no cracking or other damage to the tooth structure was observed. Desiccation occurs, but this can be compensated clinically by simply rehydrating the tooth structure.
      This study used a sodium fluoride toothpaste introduced during the demineralization-remineralization pH cycling, but clinically, any use of fluoride after laser irradiation would be expected to yield a benefit. In particular, using a varnish for prolonged release of fluoride may further enhance the formation of an acid-resistant fluorapatite layer, as shown in an in vivo study.
      • Rechmann P.
      • Charland D.A.
      • Rechmann B.M.T.
      • Le C.Q.
      • Featherstone J.D.B.
      In-vivo occlusal caries prevention by pulsed CO2-laser and fluoride varnish treatment: a clinical pilot study.

      Conclusions

      Irradiation with a 9.3-μm CO2 laser on primary enamel removed 50.4% of carbonate and showed a measurable depth of carbonate removal of at least 14 μm on average. With a combined treatment of laser irradiation and additional fluoride, an additional reduction in demineralization in artificial lesion formation of 47.9% was found relative to fluoride alone, which amounted to a reduction of 76.5% relative to untreated controls. No adverse surface effects were observed, and the rise in pulpal temperature was kept at a safe level. The high-speed scanning of 9.3-μm CO2 short-pulsed laser irradiation presented here enables a clinician to provide a quick and safe method to reduce demineralization in primary teeth.

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