Effect of Combined CO₂ Laser Irradiation and Sodium Fluoride Varnish on Enamel Surface Morphology and Surface Elemental Composition: An In Vitro Split-Tooth Study

Introduction

Dental caries deserves attention because it has become a chronic disease affecting children worldwide.^1,2 Dental caries is a multifactorial disease caused by the simultaneous interaction of various factors— sugar and dental biofilms —in the oral cavity. The introduction of fluoride into dental practice over seventy years ago is widely regarded as the principal reason for the global decline in dental caries. Despite this public health success, the sole confirmed adverse effect of fluoride use in dentistry is dental fluorosis, a condition resulting from excessive fluoride ingestion during tooth development. Recent epidemiological trends indicate a concurrent rise in the prevalence of dental fluorosis and a decline in caries rates.³

The most common varnish used in dentistry to prevent tooth caries is sodium fluoride (NaF).⁴ Topical application of NaF on teeth mitigates enamel demineralization while enhancing the remineralization process. The primary antimicrobial mechanism of fluoride is not direct bacterial destruction, but rather an indirect action through the inhibition of bacterial acid production, which results in a higher pH at the tooth surface.^5,6

The combined use of laser irradiation and topical fluoride represents a novel approach to caries prevention by increasing the enamel’s affinity for and incorporation of fluoride. Zancope et al. reported that CO₂ lasers cause morphological changes on the surface of tooth enamel, such as fusion and dissolution.⁷ Studies evaluating the effect of carbon dioxide laser on the structure of tooth enamel and dentin have shown high absorption by dental tissue.⁸ Specifically, the 10,600 nm CO₂ laser features an absorption coefficient in hydroxyapatite that allows specific depth of penetration and flow to produce the desired thermal effects on caries inhibition.⁹

Although topical fluoride applications, such as varnishes, remain the gold standard in caries prevention, their clinical efficacy is often limited by their susceptibility to rapid dissolution in oral fluids, necessitating frequent reapplications. However, repeated high-dose applications raise concerns regarding potential systemic toxicity and fluorosis.³ To overcome these limitations and enhance the longevity of fluoride retention, modern preventive dentistry has focused on synergistic technologies. The CO₂ laser (10,600 nm) has emerged as a promising modality; when irradiated at specific sub-ablative parameters, it induces microstructural and thermal alterations in the enamel surface. This photothermal effect temporarily increases enamel permeability and promotes the stable conversion of hydroxyapatite into a highly acid-resistant fluorapatite phase.¹⁰ Therefore, the integration of laser therapy is proposed not to replace fluoride, but to optimize its chemical binding and depth of penetration, thereby potentially offering prolonged cariostatic protection with lower effective fluoride doses.

This study aimed to evaluate and compare the efficacy of CO₂ laser irradiation versus a CO₂ application in enhancing fluoride uptake by the surfaces of permanent teeth following treatment with NaF varnish.

Materials and methods

Source of teeth specimens

The six human upper premolars were obtained from the Department of Oral and Maxillofacial Surgery in College of Dentistry, University of Damascus after routine extractions for orthodontic reasons and not specifically for this study. Teeth were stored in distilled water at 4 °C until use.

Teeth were examined to ensure they were free of any caries, abrasions or fractures. They were divided into two sections, a mesial section and a distal section, so that each treatment was applied to a section of the tooth. The root was cut from the Cementoenamel junction and the crown were stored in distilled water to prevent dehydration ( Figure 1).

Figure 1. A picture of the teeth used in the research before and after the section.

Mesial section was used as the experimental specimen and the distal as the control.

All specimens were first dried and treated with 5% NaF varnish (VOCO®, Germany). A distinction was then made between the groups: the experimental specimens were laser irradiated, while the control specimens were not. Sodium fluoride varnish (5% NaF varnish (VOCO, Germany, Product Code: [190/10]) was applied to the mesial section of the tooth with a carbon dioxide laser with a wavelength of 10600 nm (power 1 W – pulse duration 15 ms) while the control sample was in the distal section and sodium fluoride varnish alone was applied ( Figure 2).

Figure 2. A picture of the sodium fluoride varnish used by VOCO and how it is applied.

The varnish was applied using a small brush according to the manufacturer’s instructions by applying a thin layer over the entire occlusal surface of the tooth.

The sample in the mesial section was exposed to CO₂ laser radiation (10600 nm wavelength, 1 W power, pulsed mode, 15 ms pulse duration) with a wavelength of 10600 nm and a power of 1 W for 15 seconds with a pulsed mode and a pulse duration of 15 ms¹¹ at a distance of 1 cm. Irradiation was performed in a continuous scanning motion that allowed the entire surface to be irradiated and we re-preserved it with distilled water ( Figure 3).

Figure 3. Image of the parameters used.

The test of the effect of the laser on the varnish was evaluated using scanning electron microscopy (SEM) (T-Scan company VEGA II XMU, Czech Republic). Prior to SEM and EDX analyses, the remaining fluoride varnish was thoroughly and systematically removed from the enamel surfaces. Each specimen was gently wiped for 30 seconds using fresh cotton pellets saturated with pure acetone to dissolve the organic matrix of the varnish, followed by an immersion in an ultrasonic bath of distilled water for 5 minutes to eliminate any remaining chemical residues. Finally, the surfaces were thoroughly rinsed with a continuous stream of distilled water for 60 seconds and dried using a oil-free air syringe. The amount of fluoride, calcium, phosphorus, and carbon remaining on the specimens was tested using energy-dispersive X-ray spectroscopy (EDX) (Oxford Instruments, UK) attached to the microscope located in the Atomic Energy Commission ( Figure 4). To ensure reproducibility and eliminate bias, examiner blinding was implemented during the microscopic evaluation. All specimens were assigned coded identification numbers by a primary investigator, completely masking the group assignment (experimental vs. control) from the SEM and EDX operators. Furthermore, the EDX acquisition protocol was standardized utilizing an accelerating voltage of 20 kV with a fixed working distance across all analyzed specimens. The samples were analyzed by SEM to observe the topographic properties of the enamel surface after different treatments, and images of the central region were taken using two different detectors: Secondary Electrons (SE) and Backscattered Electrons (BSE). EDX analysis provided non-destructive quantitative data on the chemical composition and elemental distribution over the surface area.¹² The response variables evaluated were changes in the concentrations of fluoride, carbonate, calcium, and phosphorus that constitute the hydroxyapatite structure.

Figure 4. Image of the sample after preparation for entry into the scanning electron microscope.

Results

The results were evaluated according to the surface shape in electron microscope images and according to the concentration of fluoride, calcium, phosphorus and carbon.

In this study, SEM images showed that CO₂ laser irradiation fuses the surface, creating a smooth, recrystallized side. Fusion between enamel surface crystals was also observed after carbon dioxide laser treatment ( Figures 5 and 6).

Figure 5. SEM micrographs of enamel surfaces.

(A) Mesial section (fluoride + laser) using SE detector (×1000): Black arrows indicate zones of superficial enamel melting and crystal fusion. (B) Distal section (fluoride only) using SE detector (×1000): White arrows point to typical patent micropores. (C) Mesial section (fluoride + laser) at ×500 magnification: Black arrows highlight the smooth, recrystallized surface topography. (D) Distal section (fluoride only) at ×500 magnification showing unaltered surface morphology.

Figure 6. SEM micrographs of enamel surfaces using BSE detector.

(A) Mesial section (fluoride + laser) at ×200 : Black arrows indicate the boundary of thermal modification and fused mineral deposits. (B) Lateral section (fluoride only) at ×200 showing baseline surface characteristics. (C) Medial section (fluoride + laser) at ×500 : Black arrows mark the homogeneous recrystallized hydroxyapatite lattice. (D) Lateral section (fluoride only) at ×500 displaying micro-roughness without melting features.

Comparing the apparent differences in the mean of the measured items between the two groups ( Table 1) and the descriptive statistics for fluoride, calcium, phosphorus, and carbon ratios in both study groups ( Tables 2–5).

Test values represent Wilcoxon signed-rank Z-scores. Negative Z-values indicate the direction of the difference between paired observations (laser-treated mesial halves vs. control distal halves). Effect sizes (r) were calculated as r = |Z|/√n, where n = 6 paired observations. All comparisons were based on paired data from the same tooth (split-tooth design).

The Wilcoxon Signed-Rank test revealed a statistically significant increase in fluoride, calcium, and phosphorus weight percentages in the laser-treated group compared to the control group (p < 0.05). Conversely, carbon demonstrated a significant reduction following treatment. Detailed paired differences, effect sizes (r), and 95% confidence intervals are summarized in (Table 6).

Discussion

This study evaluated the efficacy of a CO₂ laser for enhancing fluoride uptake in dental enamel. According to existing literature, the combined application of a CO₂ laser with fluoride varnishes or gels can potentially quadruple fluoride absorption, which may significantly improve caries prevention and reduce the required fluoride dosage or frequency of clinical applications.¹³ While our current laboratory findings support this positive synergistic trend by demonstrating a statistically significant increase in surface fluoride concentrations (p < 0.001) via EDX analysis using the Wilcoxon Signed-Rank test, these results must be interpreted within specific boundaries.

Due to the small sample size utilized in this investigation (n = 6 permanent teeth sectioned to create 12 experimental halves), this work should be appropriately characterized as a pilot study. In advanced microstructural and surface examinations using SEM and EDX, small sample sizes are frequently employed because the focus is on highly precise, localized topographic and elemental changes rather than biological variance. Furthermore, our study utilized a split-tooth design—where each tooth served as its own internal control (mesial half treated with laser + fluoride, distal half with fluoride alone)—which effectively eliminated inter-individual biological variability and enhanced the statistical power of the comparison. Nevertheless, we acknowledge the small sample size as a limitation of this laboratory model, and future large-scale quantitative trials are required to fully validate the clinical extent of this laser-induced enhancement.

In order to obtain a protective effect, a laser was used to raise the temperature of the tooth enamel to the melting point of its inorganic components or to the decomposition point of its organic components. On the other hand, non-thermal laser radiation was used after tooth fluoridation to achieve a slow fluoride release effect. This type of reaction is called laser-activated fluoride treatment.¹¹

Carbon dioxide laser irradiation of tooth enamel, within certain parameters, results in a specific temperature increase above the irreversible pulpitis threshold of 5.5 °C,¹⁴ which changes the chemical composition of the tooth enamel surface and the morphological structure¹⁵ and the increase may lead to permanent damage to the tooth pulp.

Excessive temperature elevation during laser application poses a risk of injury to the dental pulp. A study addressing this risk compared the thermal effects of two laser systems: a CO₂ laser (at 1 and 2 W) and a diode laser (at 5 and 7 W). The results indicated that the mean temperature rise remained below the 5.5 °C critical threshold for pulp damage. Based on this evidence, the authors determined that both lasers could be used safely without compromising pulp health.¹⁶

The absorption depth of port and ivory is 12 μm, compared to the absorption depth of water of 15 μm.¹⁷

The absorption effects of tooth enamel and dentin at these depths depend on how the laser energy affects them and the laser energy depends on laser parameters such as power, mode of operation (continuous or pulsed wave mode), flow (energy density) and dose.

The continuous wave mode resulted in carbonization, dissolution and cracking of the enamel and the development of the pulse mode allowed variations by frequency and pulse duration in milliseconds (ms) and microseconds (μs). This improved the thermal control of the laser in tissue. The dose depends on the radiation method: spot radiation or sweeping motion, the number of repeated radiations.⁹

Several hypotheses have been advanced to explain the mechanisms by which combined laser and fluoride treatment enhances resistance to enamel demineralization. A principal theory posits that laser irradiation creates micro-spaces within the enamel structure, which subsequently serve to retain fluoride ions more effectively.¹⁸ These structural alterations are also believed to contribute directly to reducing enamel solubility.¹⁹ The observed morphological changes on the enamel surface following the sequential application of topical fluoride and CO₂ laser irradiation provide a rationale for the superior protective effect compared to fluoride treatment alone.²⁰ This synergistic effect is further attributed to the laser-induced conversion of hydroxyapatite to the more acid-resistant fluorapatite phase, a process accompanied by the melting and recrystallization of hydroxyapatite crystals.²¹

The results of previous studies showed that treating the surface of tooth enamel using a carbon dioxide laser reduces demineralization of tooth enamel compared to the control group. Studies have shown that wavelengths from 9 to 11 micrometers of CO₂ lasers are effectively absorbed by dental hydroxyapatite, causing loss of carbonate mineral, which in turn reduces acid reactivity.^22,23

The integration of lasers in preventive dentistry reflects a modern paradigm shift within the field, moving away from a traditional focus on restorative treatment and toward a philosophy of disease prevention. This approach is consistent with the recommendations of major professional bodies and is part of a wider scientific consensus advocating for less invasive caries management strategies,²⁴ Which aims to preserve tissues to the maximum extent, and in this new approach, prevention has acquired an important role that it has not played before in the past.¹¹

All experiments combining CO₂ and fluoride laser radiation have shown better results in preventing tooth caries when compared to a single treatment. Therefore, this ‘combination therapy’ may be clinically effective, and at the same time, it may only involve moderate daily doses of fluoride and low levels of laser radiation energy, which is consistent with our current study.

A critical limitation of this study is that SEM and EDX analyses provide topographic and compositional data only. While we observed increased surface fluoride and calcium concentrations, along with surface melting and recrystallization patterns, these findings cannot be directly interpreted as evidence of fluorapatite formation, reduced enamel solubility, or enhanced acid resistance. The measured elemental changes reflect surface modifications rather than functional resistance to demineralization. Future investigations should incorporate direct acid resistance assays (e.g., pH cycling models, microhardness testing, or polarized light microscopy) to confirm whether these surface alterations translate into clinically meaningful protection against dental caries.

Conclusion

Within the limitations of this in vitro study, the combined application of CO₂ laser and sodium fluoride varnish resulted in observable surface morphological changes (surface melting and recrystallization) and increased surface elemental concentrations of fluoride, calcium, and phosphorus compared to varnish alone. However, these topographic and compositional alterations do not constitute direct evidence of enhanced acid resistance, reduced enamel solubility, or clinical caries prevention. Long-term clinical trials incorporating direct acid resistance tests (e.g., pH cycling or microhardness testing) are mandatory to validate any potential therapeutic benefit.