Biomimetic management of orthodontic white spot lesions

Abstract

Objectives: white spot lesions (WSLs) may develop in patients with inadequate oral hygiene during orthodontic treatment. This study aimed to develop a natural remineralization agent by creating artificial WSLs in vitro. Materials/methods: WSLs were created in teeth with orthodontic buttons in vitro. The teeth were divided into the following groups: P11-4 (group 1), Nano-HA solution before sintering (group 2), Nano-HA solution after sintering (group 3), P11-4 and Nano-HA mixture before sintering (group 4), P11-4 and Nano-HA mixture after sintering (group 5), boron-containing Nano-HA mixture before sintering (group 6), boron-containing Nano-HA mixture after sintering (group 7), CPP-ACP (group 8), and artificial saliva (group 9). Measurements were taken before and after demineralization, and at the 7th, 14th, 21st, and 28th days of remineralization. Images were recorded using DIAGNOcam and VistaCamIX, area measurements were made using ImageJ, and SEM was used for remineralization assessment. Results: all groups except group 9 showed a reduction in the WSL area, with statistically significant results. SEM analysis revealed the lowest remineralization in groups 8 and 9, while the other groups exhibited more intense remineralization. Conclusions: the most successful groups for WSL remineralization were those containing pure Nano-HA (groups 2 and 3), with the other groups showing varying levels of remineralization.

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1. Introduction

White spot lesions (WSLs) emerge as a consequence of enamel exposure to acids produced by cariogenic bacteria. These acids penetrate the enamel, leading to the dissolution of hydroxyapatite (HA) minerals. The occurrence of WSLs signifies the initial stage of enamel demineralization.¹ During orthodontic treatment, WSLs can emerge within as little as one month due to alterations in the oral microbiota and a decline in plaque pH, particularly in patients with inadequate oral hygiene.² Aesthetic concerns arise from lesions in the anterior region and, if not addressed promptly and effectively, can progress to cavitated caries. The prevention of these lesions during patient monitoring and treatment, and the prompt management of their occurrence with effective remineralization agents are essential steps.

Despite the growing interest in the use of various nano-sized HA (Nano-HA) or calcium phosphate (CaPO₄) derivatives, with or without fluoride, for the management of WSLs,³ their clinical efficacy remains unproven. The clinical application of calcium phosphates, which exhibit low solubility, in combination with fluoride ions, poses significant challenges.

A number of studies have been conducted with the objective of restoring the mineral content of enamel through biomimetic approaches.^4,5 The aim of these studies has been to return the structure and function of the original tooth to its initial state. These studies have contributed significantly to understanding the enamel remineralization process. Nevertheless, the subsurface remineralization of WSLs using biomimetic methods has yet to be firmly established in clinical applications. Despite extensive research into treating demineralized enamel with biomimetic materials, achieving the desired outcomes remains challenging. Nevertheless, the guidance of remineralization of damaged enamel through biomimetic structures such as peptides offers significant clinical implications.

The biomimetic material P11-4, a self-assembling peptide, is a next-generation product that has shown promising results for WSL remineralization in recent studies.^6,7

Hydroxyapatite, an essential component of enamel's inorganic structure, serves as a source of calcium and phosphate ions that are critical for the remineralization of demineralized enamel areas.⁸ Nano-HA-based toothpaste has demonstrated effectiveness in the remineralization of teeth affected by WSLs.

The objective of the present study was to replicate the organic and inorganic structures of enamel by applying the enamel matrix protein P11-4 and Nano-HA in a simultaneous manner, with a view to investigating their combined effects on WSL remineralization. Furthermore, the regenerative effects of Nano-HA materials synthesized via different methods and with varying nanoparticle sizes were compared. Commercially available casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) was utilized as a positive control in these experiments.

Research indicates that the addition of boron to resin-based sealants can inhibit the progression of carious lesions, prevent bacterial adhesion to tooth surfaces, and enhance remineralization.⁹ Furthermore, boron has been utilized for its antibacterial properties in the production of various healthcare materials.¹⁰ The present study incorporated boron into Nano-HA groups synthesized via different methods and evaluated its regenerative properties and effects on WSLs.

The effectiveness of the applied materials for WSL remineralization was assessed using infrared digital imaging transillumination (DIAGNOcam), fluorescence imaging (VistaCam IX), and scanning electron microscopy (SEM), which is regarded as the gold standard for diagnosing WSLs. The objective of this study was to develop a natural remineralization model and agent for WSLs by creating artificial WSLs on enamel surfaces bonded with orthodontic buttons in vitro. The study's findings are expected to inform the development of novel products, including dental gels and toothpastes, aimed at promoting enamel remineralization.

2. Materials and methods

Ethical approval for the study was obtained from the Hamidiye Scientific Research Ethics Committee of the University of Health Sciences (approval number 20/139).

The extracted human premolars used in this study were obtained as anonymized biological waste following routine dental extractions performed for clinical purposes. Only sound premolars without caries, restorations, visible white spot lesions, or structural defects were included in the study.

The boron-doped and undoped nano-hydroxyapatite powders utilized in the study were synthesized by the research team.^11,12

The sample size was determined by power analysis using G*Power software (version 3.1.9.2). Based on a significance level of α = 0.05, a sample size of 12 per group was calculated to provide a test power of 95%. To account for a potential 20% loss due to experimental errors or unforeseen circumstances, a total of 126 teeth, with 14 premolars in each group, were included in the study. Standard 4 × 4 mm² areas were created on the buccal surfaces of the teeth, and orthodontic buttons were bonded above these areas. The remaining regions were coated with double-layer nail varnish in order to prevent them from being affected by the demineralization solution. The tooth roots were embedded in cold acrylic molds up to one-third of their length and were divided into nine groups (Fig. 1).

Fig. 1 Flowchart.

In the present study, a demineralization solution was utilized to generate WSLs. The solution contained 2.2 mM CaCl₂ (calcium chloride), 2.2 mM NaH₂PO₄ (sodium dihydrogen phosphate dihydrate), and 0.05 M acetic acid. The pH of the solution was then adjusted to 4.4 by the addition of 1 M KOH (potassium hydroxide).¹³

In order to induce the formation of WSLs, the samples were immersed in a demineralization solution for a period of four days, with the solution being refreshed on two occasions on each day. The demineralization process was conducted at room temperature (23 ± 1 °C), with manual agitation performed twice daily. At the end of the fourth day, the teeth were rinsed with distilled water, dried using an air–water spray, and the prepared 4 × 4 mm² areas on the buccal surfaces were examined for the presence of an opaque, chalky appearance. Following demineralization, the orthodontic buttons were removed from the teeth, and the nail polish was removed. The samples with WSLs were stored in distilled water to prevent dehydration. In this study, nine groups were formed, including positive and negative control groups, to develop next-generation dental materials capable of biomimetic remineralization of WSLs (Table 1).

Table 1 Groups and applied remineralization agents

Group number	Applied remineralization agents
Group 1	P11-4 (self-assembling peptide)
Group 2	Nano-HA solution (pre-sintering)
Group 3	Nano-HA solution (post-sintering)
Group 4	P11-4 and Nano-HA mixture (pre-sintering)
Group 5	P11-4 and Nano-HA mixture (post-sintering)
Group 6	2% Boron-doped Nano-HA mixture (pre-sintering)
Group 7	2% Boron-doped Nano-HA mixture (post-sintering)
Group 8	Casein phosphopeptide–amorphous calcium phosphate (CPP–ACP)
Group 9	Artificial saliva

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2.1. A comprehensive overview of the groups included in the study

Group 1: the oligopeptide material P11-4 (Curodont Repair, Credentis AG, Switzerland), a biomimetic material employed for the remineralization of WSLs, was designated as the first experimental group. Its preparation and application procedures were meticulously performed in accordance with the manufacturer's guidelines. The artificial saliva solution containing the samples was replaced with a fresh solution on a twice-daily basis for a period of 28 days.

Group 2: an “acid-based method” was employed for the synthesis of HA using a wet chemical method, owing to its advantages such as the absence of foreign elements in the reaction between calcium hydroxide [Ca(OH)₂] and phosphoric acid [H₃PO₄], and water being the only byproduct aside from HA [Ca₁₀(PO₄)₆(OH)₂].¹⁴ Following a 24-hour ageing period (prior to sintering), the suspension was directly utilized in the “group 2” experiment.

Group 3: HA powders obtained by the method in group 2 were subjected to a heating process in a furnace at a temperature of 1000 °C for a duration of 4 hours. Following this, the powders were ground for a period of 10 minutes in an agate mortar. Subsequently, 7.5 grams of HA powder were introduced into 250 milliliters of deionized water, which was then subjected to a magnetic stirring process for a duration of 10 minutes. Thereafter, the mixture was subjected to a homogenization process using ultrasonic waves for a duration of 2 minutes. The resulting suspension was then utilized in the designated “group 3” experiment.

Group 4: the suspension prepared in the group 2 experiment was applied to the surfaces with WSLs following the P11-4 treatment and utilized in the group 4 experiment.

Group 5: the suspension prepared in the “group 3” experiment was applied to the surfaces with WSLs after P11-4 treatment and used in the “group 5” experiment.

Group 6: in the production of B-doped HA using the acid-based method, the same steps as those used in the synthesis of pure HA were followed. Following a 24-hour ageing period (prior to sintering), the suspension was utilized directly in the “group 6” experiment.

Group 7: HA powders synthesized using the acid-based method in conjunction with boric acid (H₃BO₃) powders were employed. A quantity of 7.5 grams of “B-doped HA” powder, obtained by a solid-state method, was added to 250 milliliters of deionized water on a magnetic stirrer. The mixture was stirred for a period of 10 minutes, followed by 2 minutes of ultrasonic homogenization. The resulting suspension was used in the “group 7” study.

Group 8: GC Tooth Mousse (GC Corporation, GC International, Itabashiku, Tokyo, Japan) is a cream-based product containing water-soluble casein phosphopeptide amorphous calcium phosphate. In this study, CPP-ACP was designated as the positive control group in group 8.

Group 9: the artificial saliva solution used in the study (negative control group) contained 1.5 mM CaCl₂ (calcium chloride), 0.9 mM NaH₂PO₄ (sodium dihydrogen phosphate dehydrate), and 0.15 M KCl (potassium chloride), with a pH of 7.0.¹³

The remineralization agents prepared in groups 2, 3, 6 and 7 were applied to the WSL-formed area twice a day (every 12 hours) for 28 days. After application, the samples were allowed to dry for 5 minutes and then stored in glass bottles filled with artificial saliva.

In groups 4 and 5, the P11-4 material was applied according to the manufacturer's instructions only at the beginning of the study, and the product was allowed to dry for 5 minutes. Group 4 was treated with HA obtained by the method of group 2, while group 5 received HA obtained by the method of group 3.

After the materials were applied and allowed to dry for 5 minutes, the samples were stored in glass bottles filled with artificial saliva. The artificial saliva was changed every 12 hours after each application. The effectiveness of the remineralization materials used in the study, which involved the formation of WSLs on human teeth in an in vitro environment, was evaluated according to the times indicated in Table 2.

Table 2 Evaluation time points of the samples

T0	T1	T2	T3	T4	T5
Baseline	Demineralization	After the application of remineralization agents
		1 week	2 weeks	3 weeks	4 weeks

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The obtained samples were evaluated using an infrared digital imaging transillumination device (DIAGNOcam), a fluorescence imaging device (VistaCam IX) and a scanning electron microscope (SEM). The samples were photographed using a VistaCam IX imaging device and a DIAGNOcam device inside a black box at scheduled time intervals from the intact enamel surface, after WSL formation and after the application of remineralization agents. After the formation of WSLs and the application of remineralization agents, area measurements were made using the ImageJ image analysis program (ImageJ, National Institutes of Health, Bethesda, MD, USA) and the amount of lesion reduction was determined.¹⁵

2.2. Statistical analysis

The Friedman test was employed to compare more than two continuous variables that were dependent and not conforming to a normal distribution. The Wilcoxon test was used to compare two continuous variables that were dependent and not conforming to a normal distribution. The Kruskal–Wallis test was used to compare more than two continuous variables that were independent and not conforming to a normal distribution. The level of statistical significance was set at 0.05. The analyses were performed using MedCalc Statistical Software (version 12.7.7).

3. Results

SEM analysis at 10k× magnification revealed that the materials used in this study exhibited different remineralization characteristics on the WSL surface (Fig. 2).

Fig. 2 SEM images at 10k× magnification showing healthy enamel at T0, demineralized enamel at T1, and enamel after the application of remineralization agents at T5 (4th week).

Group 1 (P11-4): the areas of remineralization appeared darker and fibrous structures became more prominent on the surface. These fibrillar structures formed an organized skeletal structure and the pores were largely closed.

Group 2 (Nano-HA solution, pre-sintering): dark areas of remineralization were visible and Nano-HA crystals were concentrated on the surface, closing the pores.

Group 3 (Nano-HA solution, post-sintering): a homogeneous and regular area of remineralization was observed. Nano-HA crystals formed a layer on the surface, significantly closing the pores.

Group 4 (P11-4 and Nano-HA mixture, pre-sintering): globular crystal structures and dark areas of remineralization were visible. Pores were partially closed.

Group 5 (P11-4 and Nano-HA mixture, post-sintering): globular and crystalline structures were regularly organized, areas of remineralization were more intense, and pores were largely closed.

Group 6 (2% boron modified Nano-HA, pre-sintering): bright crystalline structures largely filled the pores, and the surface appeared smoother.

Group 7 (2% boron modified Nano-HA, post-sintering): the pores were almost completely closed and bright Nano-HA crystals formed dense clusters. The regular crystal structures formed after sintering were remarkable.

Group 8 (positive control, CPP-ACP): dark areas of remineralization and surface accumulation of Ca and PO₄ were prominent. Pores were partially occluded, and remineralization was limited but effective.

Group 9 (negative control, artificial saliva): remineralization was minimal, and pore structures were largely preserved. However, minimal remineralizing effects of saliva were observed on the surface.

The DIAGNOcam images showed that the healthy enamel tissue appeared light grey, whereas after the formation of the WSL, the boundaries became distinctly dark grey. After the application of materials, the remineralized areas changed from dark grey to light grey. In the VistaCam IX images, the healthy enamel tissue appeared light green and after the formation of the WSL, the boundaries became distinctly dark green. After the application of materials, the remineralized areas changed from dark green to light green (Fig. 3).

Fig. 3 Images of G3 and G5 after WSL and remineralization agent application at T5 (4th week): (A) DIAGNOcam and (B) VistaCam IX.

Images of the samples at T0, T1, T2, T3 and T4 taken with the intraoral camera head of the VistaCam IX unit were analyzed using the area calculation module of Image J software. The percentage reduction in the WSL area was calculated based on the T1 period (initial formation of the WSL). The ΔArea values obtained with the remineralization agents are shown in Table 3. In intra-group comparisons, the area reduction values of the WSL achieved by the remineralization agents were measured at weeks 1, 2, 3 and 4 and were found to be statistically significant in all groups except group 9 (negative control, artificial saliva) (p < 0.001). When comparing between groups, a statistically significant difference in WSL area reduction was observed between the nine groups (p < 0.001). Pairwise comparisons of time periods showed significant differences in WSL formation (T1–T2, T1–T3, T1–T4, T2–T3, T2–T4, T2–T5, T3–T4, T3–T5, T4–T5) in all groups except the artificial saliva group (Wilcoxon test, <0.005).

Table 3 Percentage area change of white spot lesions after remineralization

Group	Description	T2, ΔArea 1 (week 1 – WSL)	T3, ΔArea 2 (week 2 – WSL)	T4, ΔArea 3 (week 3 – WSL)	T5, ΔArea 4 (week 4 – WSL)	p 2
p 1: 1–9 (Kruskal–Wallis test), p2: T1–T5 (Friedman test).
Group 1	P11-4	−0.02	−0.03	−0.04	−0.05	<0.001
Group 2	HA before synth.	−0.07	−0.10	−0.13	−0.16	<0.001
Group 3	HA after synth.	−0.02	−0.08	−0.11	−0.13	<0.001
Group 4	P11-4 + HA before synth.	−0.02	−0.03	−0.04	−0.05	<0.001
Group 5	P11-4 + HA after synth.	−0.01	−0.04	−0.05	−0.05	<0.001
Group 6	B-HA before synth.	0.002	−0.01	−0.01	−0.02	<0.001
Group 7	B-HA after synth.	−0.01	−0.01	−0.02	−0.03	<0.001
Group 8	CPP-ACP	−0.01	−0.02	−0.03	−0.03	<0.001
Group 9	Artificial saliva	−0.00	−0.00	−0.00	−0.00	<0.001
p 1		<0.001	<0.001	<0.001	<0.001

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These quantitative findings were also supported by close-up images obtained with the VistaCam IX device; for instance, a noticeable reduction in opacity was observed in the T1 and T5 images of group 3, highlighting the aesthetic impact of remineralization (Fig. 4).

Fig. 4 Close-up VistaCam IX images of group 3: (A) T1 – after WSL formation and (B) T5 – at the fourth week after remineralization. Visible reduction in lesion opacity from A to B indicates remineralization and aesthetic improvement.

Compared to the negative control group (artificial saliva, group 9), the greatest area reduction was observed in group 2 (Nano-HA solution before sintering). This was followed by group 3 (Nano-HA solution after sintering). Groups 1 (P11-4), 4 (P11-4-Nano-HA before sintering) and 5 (P11-4-Nano-HA after sintering) showed similar efficacy in reducing the WSL area. Groups 7 (B-Nano-HA After Sintering) and 8 (CPP-ACP) also showed a smaller reduction in area, while the smallest change was observed in group 6 (B-Nano-HA Before Sintering) (Fig. 5).

Fig. 5 Graphical representation of the percentage change in WSL areas after remineralization.

4. Discussion

In our study, the reduction in the WSL area after the application of remineralization agents was evaluated weekly in all groups. It was found that P11-4 (group 1) produced a significant reduction from the first week. Üstün et al.¹⁶ also reported that P11-4 was effective from the first day and that the highest remineralization was observed at the end of 30 days. Kirkham et al.¹⁷ reported that P11-4 induced hydroxyapatite crystal formation and increased mineralization within 5 days. Similarly, Lo et al.¹⁸ found that P11-4 showed remineralization efficacy by the third day. In our study, P11-4 also started to reduce the lesion area from the first week and showed the highest efficacy at the fourth week.

When comparing the efficacy of P11-4 and CPP-ACP agents, both were found to significantly reduce the WSL area, but P11-4 was more effective. SEM analysis showed that P11-4 produced a more robust and natural structure on the enamel surface compared to CPP-ACP. These results are consistent with previous studies in the literature demonstrating the superior remineralization efficacy of P11-4.¹⁹

The findings of studies evaluating the remineralization efficacy of the P11-4 material and HA-containing agents applied separately to WSL surfaces demonstrate the superiority of biomimetics, materials closely resembling natural structures.^8,19–21 In the present study, Nano-HA solutions synthesized by the research team using different methods were combined with the P11-4 material to mimic both the organic and inorganic structures of enamel. Thus, a novel biomimetic strategy was applied for the remineralization of WSLs, offering a unique contribution to the literature.

Although resin infiltration is widely used clinically in the treatment of white spot lesions, this method requires aggressive surface etching with approximately 15% hydrochloric acid for 90–120 seconds, which irreversibly alters the natural structure of the lesion.²² Moreover, due to the limited penetration of the low-viscosity resin into the porous structure, demineralized areas in the deeper layers of the lesion may remain, leading to irregular morphology.²³ Furthermore, this approach does not support true enamel regeneration; instead, it merely fills the voids with resin to mask the lesion, and therefore it is not considered a biomimetic method.

The contribution of this study lies in its systematic evaluation of the combined use of P11-4 and Nano-HA on the remineralization process, both quantitatively and morphologically. While P11-4 mimics enamel matrix proteins and primarily targets the organic scaffold, Nano-HA provides calcium and phosphate ions to support the reconstruction of the inorganic enamel structure. When applied together, these two agents simultaneously target different phases of the remineralization process, leading to a more efficient healing response. In particular, the area reduction observed in group 5 during weeks 2 and 3 was more pronounced and occurred earlier compared to group 1, indicating enhanced efficacy. These clinical outcomes were further supported by the SEM analyses. In group 1, although initial mineral deposition was detected, it appeared irregular, dispersed, and interspersed with porous regions, suggesting that P11-4 alone provided a limited improvement in surface morphology. In contrast, group 4, which received a combination of P11-4 and pre-synthesized Nano-HA, demonstrated a more homogeneous, organized, and relatively denser crystal formation with a notable reduction in porosity. The most striking morphological results were observed in group 5. This group exhibited a compact, continuous mineral layer with large and tightly bound HA crystals uniformly distributed across the surface. Such a structure indicates that this combination not only reduced lesion size more effectively but also restored surface architecture to a greater extent. These findings demonstrate that the synergistic use of P11-4 and Nano-HA enhances remineralization not only in terms of quantity but also in the structural quality and organization of the mineral layer. To the best of our knowledge, no previous study has provided such a comprehensive assessment of this combination through both quantitative and micromorphological analyses, making this work a novel and valuable contribution to the field.

In this study, Nano-HA solutions were prepared using different synthesis methods and applied to enamel surfaces with WSLs in group 4 (P11-4 and Nano-HA mixture pre-sintering) and group 5 (P11-4 and Nano-HA mixture post-sintering). Images obtained from the VistaCam IX and DIAGNOcam devices revealed that WSL areas began to shrink from the first week and healthy tooth tissue started to form. SEM analyses showed a reduction in the porous structure of the WSL beginning from the first week, with the presence of remineralization layers and HA globular structures identified in the second, third, and fourth weeks.

Despite the fact that groups 1 and 4 exhibited analogous effects in terms of the reduction of WSL areas, group 5 was found to be more efficacious than groups 1 and 4 in the second and third weeks. However, by the fourth week, the values for groups 1, 4, and 5 became equivalent. It is therefore recommended that longer-term studies exceeding one month are conducted in order to more accurately evaluate the combined effects of enamel matrix proteins and Nano-HA apatite, which mimic the structure of enamel.

Another objective of our study is to ascertain the most effective synthesis method for enhancing the effectiveness of Nano-HA on remineralization. In the present study, group 2 (Nano-HA solution before sintering) and group 3 (Nano-HA solution after sintering) were found to promote remineralization in WSLs. It is noteworthy that group 2 was identified as the most effective group in terms of WSL area reduction parameters, as determined by image analysis using the DIAGNOcam and VistaCam IX devices. Group 3 demonstrated the second-highest effectiveness after group 2. These findings indicate that smaller and more amorphous particles based on NaOH (group 2) are more effective in remineralization. The study by Kamath et al.,²⁴ which compared a Nano-HA-containing material with CPP-ACPF, highlights the scientific innovation and originality that our research contributes.

From the SEM analyses, Nano-HA formation was observed in the lesion areas on SEM images, similar to those in previous studies on remineralization using Nano-HA,²⁵ with remineralization areas visible even after the first week. Regarding area reduction, a consistent decrease was detected in group 2 starting from the first week, while the greatest reduction in group 3 was observed in the second week. In light of these findings, it is recommended that future clinical studies consider applying these two materials for a minimum of two weeks.

In previous studies, boron mineral has been shown to be biocompatible, to enhance the mechanical properties of drugs and materials, to inhibit caries progression in resin-based sealants, and to exhibit antibacterial properties.^10,26,27 In addition, when combined with hydroxyapatite (HA) and boron nitride nanotubes, it has been found to enhance osteoblast activity.⁹ The objective of this study was to evaluate the effect of boron on WSLs, based on these findings. A solution containing boron was formulated with the Nano-HA material and administered in group 6 (2% boron-doped Nano-HA mixture before sintering) and group 7 (2% boron-doped Nano-HA mixture after sintering).

In the SEM analysis, group 6 exhibited a near-healthy enamel appearance, particularly in the 4th week, and the fish scale appearance began to fade from the 1st week. In group 7, the formation of HA crystals continued to increase. The evaluation using the DIAGNOcam and VistaCam IX devices revealed that the most significant activity appeared in group 6 from the 1st week onwards, while in group 7, more remineralization images were observed in the 3rd and 4th weeks.

Group 6 was obtained by adding boron to group 2 using an acid-based method, while in group 7, boron was added to the Nano-HA in group 3 using a solid-state method. The measurement of WSL area reduction values revealed that group 7 exhibited a reduction similar to that in group 8 (CPP-ACP), yet demonstrated a greater reduction in the lesion area in comparison to group 9 (artificial saliva). Group 6 demonstrated a smaller reduction compared to group 8 but a greater reduction compared to group 9 (artificial saliva). The effectiveness of the enamel remineralization agent was found to be dependent on the presence of boron, which was observed to reduce the effectiveness of Nano-HA.

Recent studies have highlighted that excessive boron doping may adversely affect the physicochemical and structural properties of hydroxyapatite (HA). For example, Jodati et al.²⁸ reported that increasing B³⁺ concentration during HA synthesis reduces crystallinity and particle size, potentially weakening the material's mechanical strength. Supporting this, Pazarçeviren et al.²⁹ demonstrated that boron incorporation alters HA's surface morphology, increases porosity, and induces lattice distortions, all of which may compromise crystal integrity and biological performance. These findings suggest that while boron may provide beneficial bioactivity, its concentration must be carefully controlled to maintain both remineralization efficacy and mechanical stability, particularly in clinical contexts requiring long-term durability and biocompatibility.

The enhanced remineralization observed in group 7 in comparison to group 6 can be ascribed to the solid-state technique employed for amalgamating Nano-HA and boron, a method that appears to diminish the efficacy of Nano-HA to a lesser extent compared to the acid-based technique. The antibacterial property of boron, which is its strongest feature, was not evaluated in this study. Consequently, it is recommended that future research focus on the combined effects of Nano-HA's remineralization properties and boron's antibacterial activity. It is anticipated that the findings of this study will provide a valuable foundation for future research in this domain.

Teeth with WSLs possess a natural remineralization potential due to the presence of saliva; however, this effect does not reach the desired level.³⁰ In the present study, teeth from group 9, the negative control group, were immersed in artificial saliva at 12-hour intervals in order to evaluate the natural remineralization potential of saliva. Scanning electron microscopy (SEM) analysis revealed the presence of calcium (Ca²⁺) and phosphate (PO₄³⁻) deposits on the surface of the WSL; however, the porous structure on the lesion surface persisted from the first to the fourth week, with the pores remaining open and only a low level of remineralization observed.

While our in vitro model provided valuable insights into the remineralization potential of biomimetic materials under controlled conditions, certain limitations regarding clinical translatability should be acknowledged. The absence of dynamic oral environment factors – including the salivary flow rate (0.8–1.5 mL min⁻¹in vivo), microbial biofilm interactions, and dietary pH fluctuations – may affect the extrapolation of these findings to clinical settings. Specifically, the regulatory roles of salivary proteins (e.g., statherin's calcium-binding capacity and histatin's antimicrobial activity³) were not fully replicated. Furthermore, the complex interplay between cariogenic biofilms (particularly Streptococcus mutans and Lactobacillus spp.).³¹ and remineralization processes remains to be investigated in this model system. Notably, the superior performance of unsintered Nano-HA (Group 2) and P11-4's structural mimicry of natural enamel observed in our study provide compelling preclinical evidence, though future investigations incorporating biofilm-adapted models would strengthen clinical relevance.

The limitations of our study include the short follow-up period (4 weeks) and the inability of in vitro conditions to fully replicate the clinical environment. Additionally, the measurements obtained with the devices used were limited to surface effects, and the use of advanced techniques (FTIR, EDS, XRD, and Raman microscopy) is recommended for evaluating deeper layers. Furthermore, microhardness testing should also be considered to comprehensively assess the mechanical properties of enamel tissue.

4.1. Recommendations for future research

In consideration of the aforementioned findings and limitations, this study can be regarded as a significant advancement in the management of WSLs during orthodontic treatment using biomimetic materials. For future research, it would be advantageous to investigate the effects of different orthodontic treatment methods on WSLs and to adopt prospective study designs to mitigate the limitations of in vitro methods.

In addition, the incorporation of bacterial models into studies to more realistically replicate the oral environment could offer valuable insights, particularly in understanding the antibacterial efficacy of boron-containing materials. A rigorous examination of the effects of varying concentrations, production methodologies, combinations, and application protocols of biomimetic agents, including Nano-HA and P11-4, could facilitate the optimization of the clinical effectiveness of these materials. Optimization of the ratios of material combinations used in the study and the sintering processes is a further research avenue that could be explored. In order to evaluate the antibacterial efficacy of boron materials, in vitro studies simulating the bacterial environment of the oral cavity could be planned, thereby enabling the simultaneous investigation of both the remineralization properties of Nano-HA and the antibacterial activity of boron.

Furthermore, the employment of advanced imaging techniques to investigate the process of remineralization in the deeper layers of lesions has the potential to enhance the accuracy of measurements and facilitate comparisons across studies. These recommendations have the capacity to enhance the understanding of WSL management and the clinical applicability of biomimetic materials, contributing to the development of more effective treatment strategies.

Ethical approval

The ethical approval required for the present study was reviewed and approved by the Ethics Committee of Health Sciences University Hamidiye Scientific Research on 24th April 2020, with the approval number 20/139.

Conflicts of interest

The authors declare that there is no conflict of interest.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

The research was supported by the Health Sciences University Scientific Research Projects Coordination Unit (project number: 2020/046).

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