Evaluation of the effect of a nanobioglass varnish and Er,Cr: YSGG laser on remineralization of demineralized deciduous teeth

ABSTRACT

Background:

This in vitro experimental study aimed to evaluate and compare the remineralizing effects of nanobioglass varnish and Er, Cr: YSGG laser on demineralized deciduous teeth as potential alternatives to fluoride therapy.

Materials and Methods:

This in vitro experimental study was conducted to evaluate and compare the remineralizing effects of nanobioglass varnish and Er,Cr: YSGG laser on demineralized deciduous teeth. Eighty-four extracted primary incisors were randomly assigned to seven groups, including positive and negative controls, fluoride varnish, nanobioglass varnish, Er,Cr: YSGG laser, and combined varnish–laser treatments. Bioactive glass nanoparticles were synthesized by the sol-gel method and characterized using X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy. Surface microhardness was measured using the Vickers hardness test. Data were analyzed using the one-way analysis of variance and independent t-tests using the SPSS software version 23 at a significance level of 0.05.

Results:

Significant differences in surface microhardness were observed among the groups (P < 0.0001). The nanobioglass varnish followed by laser irradiation group showed the highest improvement in enamel microhardness compared to other treatments.

Conclusion:

Er,Cr: YSGG laser, either alone or in combination with nanobioglass varnish, significantly enhances the remineralization of demineralized deciduous enamel. This combination may serve as an effective nonfluoride alternative for managing primary caries lesions.

INTRODUCTION

Dental caries is a prevalent chronic infectious disease caused by acid-producing bacteria, primarily Streptococcus mutans, which metabolize dietary sugars and gradually demineralize the tooth surface. When the natural balance between demineralization and remineralization is disrupted, carious lesions develop, leading to significant morphological and chemical alterations in the enamel. The progression of caries in deciduous teeth is typically more rapid than in permanent dentition because of differences in chemical composition and enamel thickness. In primary incisors, early lesions often appear as whitespots along the gingival margin.[1] Untreated dental caries can adversely affect the quality of life (QoL) and dietary intake of preschool children. Those with untreated decay show significantly lower oral health-related QoL compared with their caries-free peers.[2,3]

Conventional restorative treatments for carious deciduous teeth are often challenging, particularly in young or uncooperative patients. Therefore, noninvasive approaches that promote remineralization of early enamel lesions have gained increasing attention. Recently, various remineralizing agents have been developed to preserve enamel integrity and prevent the progression of carious lesions.[4] Among these, bioactive glass (BAG) – an inorganic material composed of sodium, calcium, phosphorus, and silica (sodium–calcium phosphosilicate) – has demonstrated excellent biocompatibility and remineralizing potential in both enamel and dentin. BAG particles adhere to dentin and form a hydroxycarbonate apatite layer chemically similar to the tooth’s mineral phase while exhibiting minimal cytotoxicity to pulp cells. Furthermore, BAG possesses antimicrobial properties against cariogenic bacteria.[5,6]

In addition to chemical agents, erbium lasers such as Er,Cr: YSGG (2.78 μm) and Er: YAG (2.94 μm) have been widely used in dentistry for their photobiomodulatory effects. Laser irradiation induces controlled thermal and structural changes in enamel without causing ablation, thereby enhancing enamel resistance to acid attacks and preventing demineralization.[7] The Er,Cr: YSGG laser, in particular, demonstrates greater absorption by hydroxyapatite – the primary mineral component of enamel – allowing deeper energy penetration and minimal thermal damage to the pulp and surrounding tissues. This makes it more advantageous than the Er: YAG laser for caries prevention.[8]

Despite the proven individual benefits of BAG and Er,Cr: YSGG laser therapy, limited evidence exists regarding their combined effect on enamel remineralization in deciduous teeth. Therefore, this study aimed to evaluate the effectiveness of Er,Cr: YSGG laser (2780 nm) and nanobioglass varnish, used individually and in combination, in preventing and reversing enamel demineralization in primary teeth.

MATERIALS AND METHODS

This in vitro experimental study aimed to evaluate and compare the remineralizing effects of nanobioglass varnish and Er,Cr: YSGG laser on demineralized deciduous teeth. The study was approved by the Ethics Committee of Hamadan University of Medical Sciences, Hamadan, Iran (Ethics approval code: IR. UMSHA. REC.1401.810).

Nanobioglass synthesis

Nanobioglass was synthesized using the sol–gel method. In this process, tetraethyl orthosilicate was mixed with calcium nitrate in a water–ethanol solution (1:2). To adjust the pH to 2, 1 M citric acid was gradually added, and the mixture was stirred until a clear solution (solution A) was obtained. A second solution (solution B) containing 2% polyethylene glycol (molecular weight 2000) and diammonium hydrogen phosphate was then prepared and its pH was adjusted to 10 by adding ammonia. Solutions A and B were combined and stirred continuously for 10 h to form a homogeneous gel. The resulting white gel was washed with deionized water, filtered, and dried. It was subsequently lyophilized and calcined in an oven at 650°C for 10 h to obtain fine nanobioglass powder. Finally, a 5% nanobioglass varnish was prepared by dissolving the synthesized powder in an alcoholic solution-containing natural resin.

Nanobioglass characterization

X-ray diffraction analysis

Phase identification of the synthesized nanobioglass was confirmed using X-ray diffraction (XRD). The powdered nanoparticles were scanned in the 2 θ range of 15°–60°, and the resulting diffraction patterns were analyzed to verify the crystalline phase formation and purity of the synthesized material.

Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy was employed to determine the functional groups and chemical bonds present in the synthesized nanobioglass. For this analysis, the nanoparticles were mixed with potassium bromide powder, pressed into transparent pellets, and analyzed using a PerkinElmer Frontier FTIR spectrometer. The spectra were recorded over a wavenumber range of 4000–400 cm⁻¹ to identify characteristic peaks corresponding to phosphate, silicate, and hydroxyl groups.

Morphology of particles

Transmission electron microscopy (TEM) was used to observe the morphology, particle size, and distribution of the nanobioglass particles. The TEM micrographs provided detailed visualization of particle shape uniformity and nanoscale dimensions, confirming the formation of well-dispersed spherical particles.

Specimen preparation

Eighty-four extracted deciduous teeth with intact and healthy crowns were used. Teeth showing any signs of caries, restorations, cracks, wear, or enamel hypoplasia were excluded. After extraction – performed for orthodontic or other clinical reasons – the teeth were stored in normal saline at the room temperature until analysis. Each tooth was sectioned at the cemento-enamel junction using a diamond disc (D and Z, Berlin, Germany) to separate the crown from the root. The crowns were embedded in self-cure acrylic resin, and their buccal surfaces were sequentially polished with silicon carbide abrasive papers of 800, 1000, and 2000 grit to produce the flat enamel surfaces suitable for microhardness testing. The specimens were then randomly divided into seven groups (n = 12 per group):

1. Nondemineralized group (positive control)

2. Demineralized group (negative control)

3. Fluoride varnish group

4. Nanobioglass varnish group

5. Laser group

6. Laser followed by nanobioglass varnish group

7. Nanobioglass varnish followed by laser group.

Artificial carious lesion creation and demineralization

All groups except the nondemineralized control were subjected to artificial demineralization. The samples were immersed individually in 10 mL of demineralizing solution and incubated at 37°C for 48 h to produce incipient carious lesions. The demineralizing solution consisted of 1.5 mL acetic acid, 2.9 g NaCl, 5 mL NaN₃ (2% w/w), 5 mL NaF (100 ppm), 0.13 g NaH₂PO₄, and 0.12 g CaCl₂, with the pH adjusted to 4.5. To maintain a stable pH and avoid ion accumulation, the solution was replaced daily. After demineralization, the samples were rinsed with artificial saliva of equivalent ionic composition and stored in fresh artificial saliva at the room temperature until surface treatment.

Surface treatment and microhardness evaluation

All samples were air-dried for 30 s before surface treatment. The positive and negative control groups did not receive any intervention. In the nanobioglass varnish group, a 5% nanobioglass varnish was applied to the enamel surface for 1 min. The treated specimens were then immersed in deionized water at 37°C for 24 h, after which any residual varnish was gently removed with a scalpel blade.

For the laser group, an Er, Cr: YSGG laser (2.78 μm) was applied perpendicularly to the enamel surface using the S-mode setting. The laser was operated at a power of 0.25 W, a frequency of 20 Hz, with water and air levels set to 0%. The beam was moved in an unfocused scanning motion at a 1 mm distance for 30 s.

In the “laser+varnish” combination group, the laser was applied first, followed by the varnish. In the “varnish+laser” group, varnish application preceded laser irradiation. After treatment, all samples were rinsed with artificial saliva and subjected to microhardness testing.

Microhardness was measured at three central points on each enamel surface using a Vickers hardness testing machine (load: 200 N; dwell time: 10 s). Indentations were spaced 100 μm apart, and the mean value was recorded as the Vickers Hardness Number for each specimen.

Statistical analysis

All the data were analyzed using the SPSS software (version 23.0; IBM Corp., Armonk, NY, USA). The normality of data distribution was assessed using the Shapiro–Wilk test. Mean Vickers microhardness values were compared among the groups using the one-way analysis of variance and pairwise comparisons were conducted using independent t-tests. Data were expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

RESULTS

Nanoactivebioglass characterization

The XRD pattern presented in Figure 1 illustrates the crystallographic characteristics of the synthesized nanobioglass. Analysis of the diffraction peaks using X’Pert HighScore software (Malvern Panalytical B.V., Almelo, Overijssel, The Netherlands) confirmed that the nanoparticles calcined at 650°C exhibited a well-defined crystalline structure.

Figure 1.

(a) X-ray diffraction (XRD) pattern and (b) Fourier transform infrared (FTIR) spectrum of synthesized nanobioglass nanoparticles. The XRD pattern confirms the crystalline structure of the particles, while the FTIR spectrum shows characteristic bands of phosphate (PO₄³⁻), carbonate (CO₃²⁻), and silicate (Si–O–Si) groups, along with hydroxyl (O–H) stretching at 3413 cm⁻¹, confirming successful synthesis.

The characteristic diffraction peaks and their corresponding 2θ positions closely matched those reported in the JCPDS card no. 33-0302,[9] thereby verifying the crystalline nature of the synthesized material. A distinct and sharp peak was detected at approximately 2θ = 32.69°, corresponding to the Miller index (300), as shown in Figure 1a, further supporting the formation of a crystalline bioglass phase.

The FTIR spectrum [Figure 1b] provided additional confirmation of successful nanobioglass synthesis. The spectrum displayed characteristic absorption bands associated with phosphate (PO₄³⁻), carbonate (CO₃²⁻), and silicate (Si–O–Si) groups at approximately 459, 1086, and 790 cm⁻¹, respectively. The broad absorption peak observed at 3413 cm⁻¹ corresponded to O–H stretching vibrations, indicating the presence of hydroxyl groups and bound water within the material structure.[10]

These spectral features confirm the expected chemical composition of BAG.

TEM analysis was performed to examine the morphological features of the synthesized nanoparticles. As shown in Figure 2a, the particles exhibited a spherical morphology with an average diameter of <50 nm. The corresponding particle size distribution histogram [Figure 2b] demonstrated uniform particle dispersion, indicating a high level of synthesis control and reproducibility. These findings are in agreement with previously reported studies on nanobioglass materials and confirm the high-quality synthesis achieved in the present study.

Figure 2.

(a) Transmission electron microscopy image showing the spherical morphology of nanobioglass nanoparticles. (b) Particle size distribution histogram demonstrating uniformity and narrow dispersion, with an average particle size of <50 nm.

Based on these favorable characterization results, the synthesized nanobioglass was incorporated into a varnish formulation to ensure optimal adhesion, adequate surface penetration, and prolonged retention time on enamel surfaces. This formulation aimed to enhance the potential of nanobioglass as a clinically applicable remineralizing agent.

Microhardness is elevated after surface treatment

Following surface treatments, the Vickers microhardness values of all 84 enamel samples were measured to evaluate remineralization efficacy. A comparison of mean microhardness values among the seven experimental groups demonstrated that the nanobioglass varnish followed by laser irradiation (Varnish B + Laser) group exhibited the highest enamel hardness [Figure 3]. Both the Varnish B + Laser and the fluoride varnish (Varnish A) groups showed significantly higher microhardness compared to the positive control group (P < 0.01 and P < 0.05, respectively). All treated groups exhibited significantly higher hardness compared with the demineralized (negative control) group, with the most pronounced improvements observed in the Varnish A, Laser, and Varnish B + Laser groups (P < 0.001). Moreover, the Varnish B + Laser treatment produced significantly greater microhardness than varnish alone (P = 0.0206) and also outperformed the Laser + Varnish sequence (P = 0.0401), as shown in Table 1.

Figure 3.

Microhardness assessment of deciduous teeth in the experimental groups. Demineralization caused a significant reduction in enamel microhardness, whereas surface treatments using nanobioglass varnish, fluoride varnish, laser, or their combinations led to substantial recovery. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistically significant differences compared with the demineralized (negative control) group. #P < 0.05 and ##P < 0.01 indicate statistically significant differences between the indicated groups.

Table 1.

Comparison of microhardness (Vickers hardness number) differences among experimental groups of deciduous teeth

I Group	J Group	Mean difference (I−J) (VHN)	SE (VHN)	Significance (P)
Varnish A versus	Varnish B	−18.16	17.34	0.3067
	Laser	5.81	17.45	0.7422
	Laser–varnish B	−12.59	16.88	0.4636
	Varnish B–laser	19.83	14.32	0.1801
Varnish B versus	Laser	23.98	18.42	0.2070
	Laser–varnish B	5.58	17.82	0.7575
	Varnish B–laser	37.99	15.18	0.0206*
Laser versus	Laser–varnish B	−18.40	17.89	0.3148
	Varnish B–laser	14.02	15.50	0.3756
Laser–varnish B versus	Varnish B–laser	32.42	14.86	0.0401*

An asterisk (*) indicates a statistically significant mean difference at the 0.05 level (P<0.05). VHN: Vickers hardness number; SE: Standard error

Fluoride is known to enhance enamel resistance by forming a fluorapatite layer on the enamel surface; however, this layer is relatively superficial due to limited ion penetration and may cause adverse effects such as dental or skeletal fluorosis when used excessively. In contrast, BAG materials promote remineralization through ion exchange reactions that release calcium, phosphate, sodium, and silicon ions upon contact with aqueous media such as saliva. These ions lead to the formation of hydroxycarbonate apatite crystals, which chemically bond to enamel and dentin surfaces.[11,12] Consequently, BAG demonstrates strong remineralizing potential with minimal biological risks. Previous studies have shown that BAG-containing toothpastes exhibit superior efficacy in remineralizing early carious lesions compared to fluoride-based formulations.[13] In the present study, both fluoride and nanobioglass varnishes achieved comparable microhardness outcomes, indicating that BAG can serve as an effective fluoride-free remineralizing agent.

DISCUSSION

The Er,Cr: YSGG laser used in this study demonstrated significant potential in enhancing enamel hardness. This laser wavelength is strongly absorbed by water and hydroxyl groups within hydroxyapatite, allowing it to induce photothermal and photomechanical effects that modify enamel structure without causing thermal damage to the pulp. These effects increase enamel crystallinity, reduce permeability, and improve resistance to acid dissolution.[14] Furthermore, when used in conjunction with a remineralizing agent, the Er,Cr: YSGG laser can enhance the diffusion of ions into enamel by creating microchannels or micro-porosities, thereby facilitating deeper agent penetration and stronger mineral deposition.[15,16,17] Although the present study found no statistically significant difference in hardness between laser-only treatment and the combined laser–varnish approach, previous investigations have reported varying outcomes. Kamel et al.[18] demonstrated that applying BAG paste before Er,Cr: YSGG laser irradiation resulted in greater remineralization and superior esthetic outcomes compared to laser treatment alone. Similarly, Moslemi et al. and Poosti et al.[19,20] found that combining remineralizing agents with Er,Cr: YSGG or fractional CO₂ laser treatment improved enamel acid resistance more effectively than either method used individually. Damar et al.[21] also observed comparable remineralization effects between fluoride-free agents used alone and those combined with laser therapy, suggesting that both approaches are effective but may vary based on enamel condition and experimental parameters.

In contrast, Yilmaz et al.[22] reported no significant synergistic effect when combining Er,Cr: YSGG laser with different remineralizing agents, including casein phosphopeptide-amorphous calcium phosphate with fluoride, fluoride varnishes, and ROCS^® medical mineral gel. Their findings, however, still confirmed the independent remineralizing potential of the Er,Cr: YSGG laser, as evidenced by structural changes in enamel hydroxyapatite observed via XRD.

The variability in reported results across different studies can be attributed to differences in experimental methodologies, evaluation techniques (such as scanning electron microscopy vs. microhardness testing), laser parameters, and the types of remineralizing materials used (e.g., pastes, gels, or varnishes). Despite these methodological differences, the collective evidence supports the potential of both BAG and Er,Cr: YSGG laser treatments as effective noninvasive options for enhancing enamel remineralization in primary teeth.

CONCLUSION

The results of this study demonstrated that all treatment groups showed higher enamel microhardness values compared with the control group, confirming the remineralizing potential of the tested interventions. High-power Er,Cr: YSGG laser irradiation, whether applied alone or in combination with nanobioglass varnish, effectively halted or reversed enamel demineralization in deciduous teeth. Given the potential risks associated with excessive fluoride exposure – such as dental fluorosis and systemic toxicity in children – nonfluoride remineralizing materials represent a promising and safer alternative. Among these, BAG nanoparticles offer comparable or even superior efficacy to fluoride in promoting enamel remineralization, particularly when combined with laser therapy. Therefore, the use of nanobioglass, either independently or as an adjunct to Er,Cr: YSGG laser treatment, can be considered a viable noninvasive strategy for preventing and managing early carious lesions in primary teeth.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.

Funding Statement

This research was financially supported by Hamadan University of Medical Sciences, Hamadan, Iran, under Grant Number 1401120210511.