Effect of immediate and delayed composite restoration on shear bond strength to remineralized initial enamel lesions: an in vitro study

Abstract

Dental caries weakens enamel, and remineralizing agents may help restore its structure; however, their effect on bonding to composite resin and the optimal timing of bonding after remineralization remain unclear. This study evaluated the effect of hydroxyapatite (Karex gel) and fluoride-containing hydroxyapatite (Remin Pro) as well as post-remineralization bonding timing on the shear bond strength (SBS) of composite resin to enamel. Eighty extracted human premolars and molars were divided into four groups: non-demineralized enamel (CI), demineralized enamel (CD), hydroxyapatite-treated enamel (KP), and fluoride-containing hydroxyapatite-treated enamel (RP). Except for the CI group, all teeth were demineralized. The RP and KP groups were treated with remineralizing agents for 28 days, while the CI and CD groups were stored in artificial saliva during this period. Each group was further divided based on the timing of composite bonding after remineralization: immediate (24 h) and delayed (7 days). SBS was measured at a crosshead speed of 0.5 mm/min, and failure modes were evaluated under microscopy. Data were analyzed using two-way robust ANOVA, Shapiro–Wilk test, and Bonferroni post hoc analysis. Failure modes were compared using Fisher–Freeman–Halton and McNemar tests (p < 0.05). The group and time interaction was statistically significant (p = 0.004). Immediate bonding (24 h) showed similar SBS values across all groups (n = 40). At 7 days (n = 40), the fluoride-containing hydroxyapatite group (RP) demonstrated significantly higher SBS than the other groups (9.4 MPa), followed by KP group (7.45 MPa), while the lowest values were observed in the demineralized (CD) and control (CI) groups (3.25 MPa and 4.55 MPa, respectively). Most failures were adhesive, while mixed and cohesive failures were more frequent in remineralized groups. These findings suggest that fluoride-containing hydroxyapatite may enhance enamel composite bond strength after adequate post-remineralization time. The observed SBS values are more suitable for orthodontic/low-stress applications, and further maturation or surface conditioning may be required for load-bearing restorations.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s44445-026-00229-w.

Introduction

Dental caries remains the most prevalent dental disease worldwide (Kassebaum et al. 2017, Schwendicke et al. 2019, Banerjee et al. 2017). It is a multifactorial condition influenced by time, bacteria, fermentable carbohydrates, and host factors (Schwendicke et al. 2019). Caries develops when the intraoral ecological balance between demineralization and remineralization is disrupted (Schwendicke et al. 2019, Banerjee et al. 2017). The demineralization process begins with the dissolution of hydroxyapatite crystals in enamel, leading to early white-spot lesions in which the subsurface is affected while the outer enamel surface often remains intact (Schwendicke et al. 2019, Vieira et al. 2015, Weber et al. 2018). If left untreated, these lesions may progress into cavitated defects involving dentin (Schwendicke et al. 2019, Vieira et al. 2015, Weber et al. 2018). Remineralization, on the other hand, is the natural redeposition of calcium and phosphate, and it plays a central role in hindering lesion progression and forms the basis of minimally invasive dentistry (Schwendicke et al. 2019).

The key to preventing this progression is restoring balance between demineralization and remineralization. Modifying risk factors plays a central role in achieving such balance and shifting the lesions toward remineralization (Schwendicke et al. 2019, Featherstone 2000). Reducing the frequency of sugar consumption lowers acid challenges and helps maintain a healthier pH (Moynihan and Kelly 2014). Adequate saliva flow also acts as a natural protective factor by providing calcium and phosphate ions necessary for enamel repair (Schwendicke et al. 2019, Mejare et al. 2013). Good oral hygiene further reduces bacterial load and acid production (Slayton et al. 2018). Daily use of fluoride toothpaste adds another layer of protection by supporting remineralization (Marinho et al. 2013, Walsh et al. 2010, Wolff and Schenkel 2018).

Once these factors are addressed, remineralizing agents can be used to enhance natural repair by supplying additional bioavailable ions (Schwendicke et al. 2019). Fluoride remains the most established agent, delivered via water fluoridation, toothpaste, mouth rinses, and professional applications (Marinho et al. 2013, Walsh et al. 2010, Wolff and Schenkel 2018, Marinho et al. 2016, Gao et al. 2016, Fung et al. 2018). It promotes the formation of acid-resistant fluorapatite, inhibits bacterial metabolism, and slows caries progression (Walsh et al. 2010, Cate 1999, Marsh 2003). However, excessive fluoride can cause dental fluorosis in developing teeth, and its remineralizing effect is largely limited to the superficial layers of enamel (DenBesten and Li 2011, Cury and Tenuta 2009).

Nanohydroxyapatite (n-HA) has attracted significant interest because its crystal structure closely resembles natural enamel. The nanoparticles can penetrate superficial enamel defects, serve as a scaffold for mineral deposition, and improve surface microhardness (Anil et al. 2022, Wierichs et al. 2022, Huang et al. 2009, El-Haddad et al. 2025). n-HA is considered highly biocompatible and shows strong affinity for demineralized enamel, with the ability to fill surface porosities and potentially reinforce weakened areas; however, robust clinical evidence supporting these effects is still limited (Anil et al. 2022, Wierichs et al. 2022, El-Haddad et al. 2025). A synergistic effect is reported when fluoride is combined with nanohydroxyapatite. The calcium-phosphate nanocrystals enhance fluoride uptake and promote formation of fluoridated apatite, improving both depth and quality of remineralization compared to either agent alone (Erdem et al. 2022, Epasinghe et al. 2017, Ramalingam and Subramanian 2018).

Other available remineralizing materials include casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP), bioactive glass, tricalcium phosphate, amorphous calcium phosphate, and P11-4, in addition to xylitol-based products (Zhang et al. 2023, Taha et al. 2017, Kumar et al. 2022, Philip 2019). Because these agents modify enamel structure and chemistry, they may influence the bonding performance of restorative materials. While composite restorations remain necessary for cavitated lesions, early remineralization preserves tooth structure, reduces cavity size, and may enhance long-term adhesive outcomes. However, certain agents may alter enamel in ways that either enhance or compromise bond strength, underscoring the need to evaluate their effects before restoration.

Although previous studies have investigated the effects of remineralizing agents on enamel characteristics and bonding performance, limited information is available regarding how differences in remineralizing formulations influence subsequent resin bonding. In particular, the effect of fluoride incorporation into hydroxyapatite-based materials and the influence of the time interval between remineralization and restoration remain insufficiently investigated (Erdem et al. 2022, Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025, Alagha 2020). Changes occurring after remineralization may affect the interaction between the enamel surface and adhesive systems (Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025). Therefore, evaluating fluoride-containing and fluoride-free hydroxyapatite-based materials at different restoration intervals may provide further insight into their effects on bonding performance.

Accordingly, this study aims to assess how restoration timing affects the bonding of composite resin to enamel remineralized with different agents: Remin Pro (fluoride-containing hydroxyapatite [HA + F]) and Karex gel (hydroxyapatite [HA]). The specific time points of 24 h and 7 days were selected to evaluate immediate bonding performance versus a delayed approach that allows for initial mineral maturation and stabilization within a simulated oral environment (Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025). This study may provide clinical guidance on the optimal timing for restoration to achieve more durable and stable bonding to remineralized enamel. Shear bond strength testing, stereomicroscopy, and scanning electron microscopy (SEM) were used to characterize bonding performance and failure modes. The null hypothesis of the study is that restoration timing (immediate and delayed) and the type of remineralizing agent will not significantly affect the shear bond strength of composite resin to remineralized enamel.

Materials and methods

Sample selection and preparation

The sample size was determined based on previous studies that demonstrated significant differences in shear bond strength among experimental groups (Arslandaş Dinçtürk and Kedici Alp 2025, Alagha 2020, Rabee et al. 2020). An a priori power analysis was conducted using G*Power software (version 3.1.9.7) for a two-way ANOVA design. A large effect size (f = 0.50) was assumed based on the expected differences among groups, with an α level of 0.05 and a statistical power of 80%. The analysis indicated that a minimum of 80 specimens were required. Accordingly, 80 extracted human premolars and molars with intact enamel were selected and randomly allocated to the experimental groups (10 specimens per subgroup). Teeth with caries, cracks, fractures, demineralized areas, or staining were excluded after examination under a microscope. Selected teeth were disinfected in 0.5% chloramine-T solution for 24 h and stored in distilled water at 37 °C until use. The roots were sectioned 2 mm apical to the cemento-enamel junction (CEJ). Each tooth was embedded in acrylic resin mold with the enamel surface exposed and aligned flush with the mold surface to standardize the bonding substrate. The exposed enamel surfaces were polished using 1000-grit silicon carbide paper, followed by 1200-grit silicon carbide paper under water irrigation to create a flat surface and remove the prismless enamel layer. To define the bonding area and standardize adhesive and composite placement during the bonding procedure, a 4 × 4 mm sticker was applied to the enamel surface of each tooth, and all remaining surfaces were coated with two layers of nail polish to isolate the working area.

Specimen grouping and treatment

The 80 teeth were stratified according to tooth type (premolars and molars) to ensure balanced distribution among groups and were then randomly allocated to the four experimental groups (n = 20 each) using simple randomization:

• KP (Karex Gelée; Dentsply Sirona, Germany): Hydroxyapatite (HA)-based material, the enamel surface was demineralized and subsequently remineralized with Karex for 28 days.

• RP (Remin Pro; VOCO GmbH, Germany): Hydroxyapatite with fluoride (HA + F)-based material, the enamel surface was demineralized and subsequently remineralized with Remin Pro for 28 days.

• CD (negative control): Enamel surfaces were subjected to artificial demineralization without subsequent treatment and maintained in artificial saliva for 28 days.

• CI (positive control): Enamel surfaces were not subjected to any artificial demineralization procedure and maintained in artificial saliva for 28 days. The experimental design and grouping of specimens are summarized in a flowchart (Fig. 1).

Fig. 1.

Flowchart showing specimen grouping, treatment (demineralization/remineralization), restoration timing, and subsequent shear bond testing

Preparation of demineralization and remineralization solutions

A 0.3% citric acid demineralization solution was prepared by dissolving 15 g of citric acid in 5 L of distilled water (pH 2.3) (Üstün and Güven 2022). The pH adjustment was performed using hydrochloric acid (HCl) as described by Nikiforuk and Sreebny (1953) (Nikiforuk and Sreebny 1953). The remineralization solution (artificial saliva without organic components) was prepared in 5 L of distilled water containing 0.550 g CaCl₂, 18.6 g KCl, 13.60 g KH₂PO₄, and 0.05 g NaNO₃ (pH 7.0), with pH adjustment performed using potassium hydroxide (KOH) (Üstün and Güven 2022). The pH of both solutions was monitored throughout the experimental period to ensure stability.

Remineralization and demineralization procedures

The enamel specimens were subjected to four erosive cycles at 8-hour intervals to induce initial enamel lesions. Demineralization was achieved using a 0.3% citric acid solution, alternated with immersion in artificial saliva (Fig. 2). To prevent dehydration after lesion formation, all specimens were stored in distilled water. The solutions were renewed for each cycle. This protocol was intended solely to create standardized initial enamel lesions prior to the remineralization phase. Following lesion induction, the remineralizing materials were applied to the enamel surfaces of the KP and RP groups using a gloved finger for 30 s and left undisturbed for an additional 150 s, according to the manufacturers’ instructions. This remineralization protocol was repeated once daily for 28 days. Specimens in the CI and CD groups were stored in fresh artificial saliva for the same 28-day period without any remineralizing treatment. After each application or storage period, all specimens were rinsed, dried, and stored in fresh artificial saliva in an incubator at 37 °C (Memmert UN 110, Schwabach, Germany) (Fig. 2).

Fig. 2.

The early enamel lesion formation cycle was performed at 8 h-intervals

Restoration protocol

After the 28-day remineralization phase, all specimens were transferred to distilled water for 24 h. Each group (n = 20) was then divided into two equal subgroups of (n = 10) based on the timing of restoration:

1. Immediate restoration: composite was placed 24 h after the remineralization period.

2. Delayed restoration: composite was placed 7 days after the remineralization period.

Composite restorations were bonded to the enamel surfaces using a universal adhesive applied in selective-etch mode, which has been demonstrated to provide superior enamel bonding performance (Rosa et al. 2015). The enamel surface was selectively etched with 37% phosphoric acid gel (ProEtch, Promida, Eskişehir, Türkiye) for 30 s, rinsed thoroughly and gently air-dried. The universal adhesive (G-Premio BOND; GC Corporation, Tokyo, Japan) was applied according to the manufacturer’s instructions and light-cured using an LED curing unit (D-Light Pro) operating at 1400 mW/cm² for 10 s. Composite resin cylinders were prepared using posterior nanohybrid composite (G-ænial Posterior; GC Corporation, Tokyo, Japan) packed into a custom silicone mold measuring 2 mm in height and 1.5 mm in width. The composite was placed in 1-mm increments, with each increment light-cured for 20 s. After removal of the mold, additional light curing was performed to ensure complete polymerization. Details of all materials used in this study are presented in Table 1.

Table 1.

Materials used in this study

Material	Ingredients	Manufacturer
Remin Pro	Hydroxyapatite, sodium fluoride (1450 ppm fluoride), xylitol	VOCO GmbH, Cuxhaven, Germany
Karex Gelée	Hydroxyapatite, calcium, xylitol	Dr. Kurt Wolff, Bielefeld, Germany
G-Premio Bond	10-MDP, 4-META, MDTP, methacrylate acidic ester, distilled water, acetone, photoinitiators, silica	GC, Tokyo, Japan
G-aenial Posterior Composite	UDMA, dimethacrylate co-monomers, Bis-GMA-free resin matrix, strontium and lanthanoid fillers, fluoroaluminosilicate glass, silica	GC, Tokyo, Japan
ProEtch Etching Gel	37% orthophosphoric acid	Promida, Eskişehir, Türkiye

In the first subgroup, specimens were subjected to a pH cycling protocol, bonded to composite resin, and subsequently stored in artificial saliva for 24 h before shear bond strength testing. In the second subgroup, specimens were stored in artificial saliva and exposed to daily pH cycling for 7 days then bonded to composite resin. These specimens were also stored in artificial saliva for 24 h before testing. The pH-cycling procedures involved 15 erosive cycles for each specimen, with each cycle consisting of 6 s of immersion in citric acid solution and 6 s of storage in artificial saliva. In between erosive challenges, the samples were incubated in artificial saliva and cleaned with deionized water (Üstün and Güven 2022). This post-treatment pH-cycling protocol was designed to simulate repeated short-term acidic challenges in the oral environment following remineralization, and it differed from the initial demineralization protocol used for lesion creation. Specimens from both groups (24-hour and 7-day) were subsequently stored in artificial saliva at 37 °C in an incubator (Memmert UN 110, Schwabach, Germany) after bonding. Specimens in the first group were tested after 24 h, whereas specimens in the second group were maintained under the same conditions for 7 days before shear bond strength (SBS) testing. All investigators involved in SBS testing, failure mode analysis, and scanning electron microscopy (SEM) evaluation were blinded to specimen group allocation to minimize assessment bias.

Shear bond strength testing

The specimens were placed in the mounting jigs of the testing apparatus (Bisco Shear Bond Tester; Bisco, Schaumburg, Ill, USA). The loading device was aligned to contact the composite-enamel interface, ensuring that the applied force was directed parallel to the bonded surface. Each specimen was loaded to failure under shear force at a crosshead speed of 0.5 mm/min.

Failure mode analysis

After completion of the shear bond strength (SBS) testing, specimens from each main group were examined under a stereomicroscope (SZX-ILLB100, Olympus Optical Co. Ltd., Tokyo, Japan) at 10× magnification to evaluate the failure mode. Failures were classified as follows:

• Adhesive failure: the resin composite was completely detached from the enamel surface.

• Cohesive failure: the fracture occurred within the resin composite itself.

• Mixed failure: the fracture involved both adhesive and cohesive components.

Additionally, representative specimens exhibiting adhesive and mixed failure modes were examined using a scanning electron microscope (SEM, Zeiss Sigma 300, Germany) to characterize interfacial failure morphology. Images were obtained at ×200, ×500, and ×1000 magnifications (Fig. 4).

Fig. 4.

Scanning electron microscopy (SEM) images at ×200, ×500, and ×1000 magnifications showing the failure modes: (a) adhesive failure, characterized by the presence of only the tooth surface, and (b) mixed failure, showing remnants of composite material on the tooth surface

Statistical analysis

Data were analyzed using IBM SPSS Statistics (Version 23.0; IBM Corp.; Armonk, NY, USA) and the WRS2 package in R (Version 4.4.0). The Shapiro-Wilk test was used to assess the normality of quantitative variables. Since the shear bond strength (SBS) data were not normally distributed, a two-way robust ANOVA was performed to compare the effects of group and restoration time. Multiple comparisons were performed using the Bonferroni adjustment. Categorical variables, such as failure modes, were compared between groups using the Fisher-Freeman-Halton test, and paired categorical comparisons within groups were analysed using the McNemar test. Quantitative data were presented as mean ± standard deviation (SD) and median (minimum–maximum), while categorical data were presented as frequency and percentage. Statistical significance was set at p < 0.05.

Results

Shear bond strength (SBS)

The median SBS values (MPa) for each group at 24 h and 7 days are summarized in Table 2. The main effect of group was not statistically significant (p = 0.171), and the main effect of time was also not statistically significant (p = 0.637). However, a statistically significant group × time interaction was observed (p = 0.004).

Table 2.

Comparison of bond strength values by group and time

Time	Group				Total		Q	P
	KP	RP	CD	CI
After 24 h	7.1 (4.7–12) a	6.7 (4.3–7.8) a	6.65 (5.2–8.9) a	7.95 (6.3–35.3) a	6.9 (4.3–35.3)	Group	1.670	0.171
After 7 days	7.45 (6.3–29.7) a	9.4 (8.3–22) b	3.25 (2.2–5.2) c	4.55 (3.3–6.5) c	6.35 (2.2–29.7)	Time	0.223	0.637
Total	7.3 (4.7–29.7)	8.05 (4.3–22)	5.2 (2.2–8.9)	6.4 (3.3–35.3)	6.85 (2.2–35.3)	Group×Time	13.540	0.004*

*Values are presented as median (minimum–maximum). Different superscript letters indicate significant differences between groups. Q: robust ANOVA test statistic. p < 0.05

At 24 h, no statistically significant differences in SBS were detected among the groups (Table 2). The CI group demonstrated the highest median SBS value (7.95 MPa), while the CD group exhibited the lowest value (6.65 MPa). The CI group included a single high SBS value (35.3 MPa), which was retained in the dataset and accounted for using the robust statistical analysis and median-based reporting approach.

At 7 days, statistically significant differences in SBS were observed among the groups. The RP group demonstrated the highest median SBS (9.4 MPa) and was significantly different from the CD and CI groups, which showed the lowest SBS values (3.25 MPa and 4.55 MPa, respectively). These differences are reflected by the different superscript letters (Fig. 3).

Fig. 3.

Shear bond strength values demonstrating the interaction between experimental groups and time

Failure mode analysis

Failure modes following SBS testing are summarized in Table 3.

Table 3.

Comparison of failure modes across groups over time

Failure Mode	KP	RP	CD	CI	Total	Fisher-Freeman-Halton	p-value
After 24 h						4.854	0.681
Adhesive	8 (80%)	7 (70%)	9 (90%)	9 (90%)	33 (82.5%)
Mixed	1(10%)	3 (30%)	1 (10%)	1 (10%)	6 (15%)
Cohesive	1 (10%)	0 (0%)	0 (0%)	0 (0%)	1 (2.5%)
After 7 days						1.551	1.000
Adhesive	9 (90%)	10 (100%)	9 (90%)	9 (90%)	37 (92.5%)
Mixed	1 (10%)	0 (0%)	1 (10%)	1 (10%)	3 (7.5%)
McNemar test (p-value)	------- -------	------- -------	------ 1.000	------ 1.000	------ ------

*Fisher–Freeman–Halton test for comparison among groups; **McNemar test for within-group comparison over time. Values are presented as frequency (percentage)

• 24-Hours Evaluation: Adhesive failures predominated across all groups (KP: 80%, RP: 70%, CD: 90%, CI: 90%). Cohesive failures were observed only in KP (10%), whereas mixed failures were observed in KP (10%) and RP (30%) groups. No statistically significant differences were detected among the groups (p = 0.681).

• 7-Days Evaluation: In intergroup comparisons, adhesive failures remained predominant (KP: 90%, RP: 100%, CD: 90%, CI: 90%), with minor occurrences of mixed failures in KP (10%), CD (10%), and CI (10%) groups. No significant differences were found among the groups (p = 1.000).

• Failure modes morphology was evaluated by SEM, as shown in (Fig. 4).

• Intragroup Comparisons: Within-group comparisons of failure modes between 24 h and 7 days also revealed no statistically significant differences (p = 1.000).

• SEM analysis revealed distinct surface features among the experimental groups. In the RP group, globular mineral-like deposits were observed on the enamel surface (Fig. 5), whereas these features were less evident or absent in the other groups. Energy-dispersive X-ray (EDX) spectroscopy analysis of RP samples demonstrated the presence of fluorine alongside calcium and phosphorus (Supplementary Files 1 and 2), suggesting the possible presence of fluoride-containing mineral deposits within the analyzed surface layer. In contrast, the KP group exhibited a relatively smooth surface with minor crystal-like formations, suggesting possible hydroxyapatite (HA) deposition (Fig. 5).

Fig. 5.

Representative scanning electron microscopy (SEM) images showing enamel surface morphology following remineralization with (a) hydroxyapatite (KP) and (b) fluoride-containing hydroxyapatite (RP)

Discussion

This study evaluated how the timing of restoration following enamel remineralization with different agents affects the shear bond strength (SBS) of resin composite to enamel, compared with non-demineralized (CI) and demineralized (CD) enamel, at two restoration time intervals: 24 h and 7 days.

SBS values at 24 h were comparable among all groups and did not differ statistically significantly. Theoretically, the bonding strength of the demineralized group (CD) would be expected to be lower; however, this was not observed (Rizvi et al. 2016). This finding could be because the enamel surface was partially restored over the 28-day storage period in artificial saliva following demineralization (Rizvi et al. 2016, Harper et al. 2020). Artificial saliva, being a mineral-rich medium, promotes remineralization through bioavailable calcium and phosphate ions. Furthermore, it should be considered that phosphoric acid etching may partially remove the superficial remineralized layer, potentially masking early differences between groups.

Similarly, the remineralized group (KP and RP) did not demonstrate superior SBS at 24 h. Immediate bonding after remineralization may occur before the enamel surface fully matures and before deposited ions are fully integrated into the hydroxyapatite lattice (Featherstone 2000, Cate 1999). Consequently, composite bonding at this early stage may not fully benefit from enamel recovery. After 7 days, the remineralized groups showed higher SBS compared to both CD and CI, suggesting that additional time after remineralization enhances bond strength (Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025, Agob et al. 2018). Exposure to saliva and pH cycling in our study may have allowed the mineral layer to mature, become denser and more homogeneous, and provide a more stable substrate for adhesive resin penetration. The results of this study agree with previous evidence showing that enamel remineralization may improve bonding performance after sufficient maturation time (Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025, Agob et al. 2018). For example, Rizvi et al. (2016) reported that CPP-ACP-treated enamel shows no early improvement in bond strength at 1 h or 1 day, despite visible microstructural changes, but demonstrates a significant and progressive increase after 2 days, 1 week, and 2 weeks (Rizvi et al. 2016). Similarly, the present study showed no significant differences in SBS among intact, demineralized, and remineralized groups at 24 h, whereas after 7 days, the remineralized groups (RP and KP) exhibited significantly higher SBS compared with both CI and CD groups.

The CD group further illustrates the importance of restoration timing. Although 28-day storage in artificial saliva temporarily improved the enamel surface through superficial mineral deposition, the underlying demineralized enamel remained structurally weak. During subsequent 7 days of pH cycling, this superficial layer was likely dissolved, leading to a sharp decline in SBS. In contrast, enamel treated with remineralizing agents appears to have developed a deeper, more integrated, and chemically stable mineral layer that continued to mature over 7 days, thereby enhancing shear bond strength (Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025, Mielczarek and Michalik 2007).

Consistently, the CD group exhibited the lowest SBS, particularly after 7 days of pH cycling (median 3.25 MPa). Acid-induced demineralization might have disrupted enamel prisms and reduced hardness, limiting the formation of stable resin tags and decreasing the substrate’s ability to support mechanical interlocking (Arslandaş Dinçtürk and Kedici Alp 2025). Previous studies have shown that bonding to demineralized enamel is compromised unless remineralizing agents are applied to partially restore the enamel surface and improve bonding effectiveness (Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025).

The remineralized groups (KP and RP) generally outperformed both non-demineralized enamel (CI) and demineralized enamel (CD) groups, which aligns with previous findings showing that remineralizing agents enhance bonding compared to untreated substrates (Arslandaş Dinçtürk and Kedici Alp 2025, Agob et al. 2018, Mielczarek and Michalik 2007). Although sound enamel is typically considered an ideal bonding substrate, remineralization may have modified the enamel surface through deposition of calcium phosphate minerals, including fluorapatite, thereby improving its response to acid etching and resin infiltration. Increased surface mineral content and microhardness may also have contributed to this enhanced bond strength. However, previous studies have reported inconsistent findings. Some researches have shown that remineralization improves bonding of demineralized enamel but restores bond strength only to levels comparable with sound enamel, rather than exceeding them (Shirani et al. 2025). In contrast, Patel et al. (2021) reported no significant difference in bond strength between remineralized and control enamel groups, possibly because bonding was performed immediately after remineralization without an extended maturation period (Patel et al. 2021). These discrepancies may be related to differences in remineralizing agents, enamel conditions, maturation periods, and bonding protocols. Therefore, the mechanisms underlying the improved bonding observed after remineralization remain speculative and require further investigation (Arslandaş Dinçtürk and Kedici Alp 2025, Agob et al. 2018, Mielczarek and Michalik 2007).

Distinct differences were observed between the two agents. After 7 days, the RP (HA + F) group exhibited the highest SBS (median 9.4 MPa), exceeding the KP (HA) group (7.45 MPa). This superior performance may be attributed to the synergistic effect of hydroxyapatite and fluoride in the RP group (Erdem et al. 2022, Epasinghe et al. 2017). Fluoride promotes the formation of fluorapatite, a denser and more acid-resistant mineral phase than hydroxyapatite, enhancing resin infiltration and micromechanical interlocking while stabilizing the enamel resin interface during pH cycling (Cate 1999, Cury and Tenuta 2009). The resulting microporosities may be more uniform and stable, allowing resin tags to interlock securely within the mineral structure. During pH cycling, the fluorapatite-enriched surface resists further demineralization, preserving the bonded interface (Alagha 2020).

SEM analysis in the present study (Fig. 5) revealed globular mineral deposits on the RP enamel surface, which were morphologically suggestive of fluoride-containing mineral deposits (Alrefai 2024). This observation was further supported by EDX findings demonstrating the presence of fluorine alongside calcium and phosphorus in RP samples (Supplementary Files 1 and 2), suggesting the incorporation of fluoride-containing mineral phases within the analyzed surface layer. In contrast, the KP group, containing fluoride-free hydroxyapatite, showed only a modest increase in SBS (from 7.1 to 7.45 MPa). Although hydroxyapatite supports mineral deposition, the absence of fluoride may make the surface more susceptible to acid challenges and may require longer maturation periods to achieve comparable stability (Cate 1999, Cury and Tenuta 2009, Huang et al. 2009, Rabee et al. 2020).

These findings underscore the importance of enamel surface condition and time-dependent mineral maturation on bonding performance. While freshly remineralized enamel at 24 h provided an adequate substrate for bonding, superior bond strength was achieved after 7 days, indicating that remineralization is a progressive process requiring sufficient time to stabilize. Exposure to a pH-cycling environment, particularly in the presence of fluoride as observed in the RP group, appears to promote continued mineral deposition and enamel maturation, ultimately enhancing adhesion (Featherstone 2000, Cury and Tenuta 2009).

Failure mode analysis supported the SBS findings, with adhesive failures predominating across all groups and time points, suggesting that the enamel-resin interface remained the primary site of stress concentration. Overall, demineralization compromised bond strength, whereas remineralization, especially fluoride-mediated remineralization, restored and enhanced adhesion over time.

Clinically, these findings suggest that the timing of composite resin application following remineralization directly influences enamel composite adhesion performance. Delaying composite bonding for several days may support further mineral maturation and stabilization of the enamel surface, resulting in improved shear bond strength (Rizvi et al. 2016, Arslandaş Dinçtürk and Kedici Alp 2025, Alagha 2020, Rabee et al. 2020, Üstün and Güven 2022, Nikiforuk and Sreebny 1953, Rosa et al. 2015, Harper et al. 2020, Agob et al. 2018, Mielczarek and Michalik 2007). Consequently, these findings may assist clinicians in determining the optimal interval between remineralization therapy and adhesive restorative procedures.

Remineralization therapies are commonly recommended for orthodontic patients with white spot lesions, high-caries-risk individuals, and pediatric or special-needs patients undergoing preventive remineralization treatment prior to restorative procedures (Slayton et al. 2018, Gao et al. 2016, Fung et al. 2018, Anil et al. 2022, Philip 2019). In such cases, optimizing post-remineralization bonding timing may enhance bonding outcomes (Cury and Tenuta 2009, Philip 2019, Rizvi et al. 2016).

The required shear bond strength (SBS) varies according to specific clinical application. In this study, both remineralized groups achieved shear bond strength values within the range reported for orthodontic bracket bonding (6–10 MPa) at both 24 h and 7 days, indicating potential suitability for low-stress adhesive applications (Reynolds 1975). However, the observed values remained below the thresholds typically required for load-bearing direct composite restorations (17–20 MPa), with a maximum median value of 9.4 MPa (Roberson et al. 2006). Therefore, while remineralization improved early bond strength sufficiently for composite in low-stress applications (such as orthodontic bracket bonding) when applied shortly after remineralization treatment, allowing additional post-remineralization maturation time may yield superior enamel-composite adhesion better suited for load-bearing restorations.

To better validate these clinical timelines, future studies should include longer follow-up periods and aging simulation. Additional limitations of the present study include the absence of comprehensive compositional analysis, variability in enamel structure due to non-standardized patient factors, the absence of organic components in the prepared artificial saliva, and the lack of thermocycling before SBS testing. Furthermore, as an in vitro study, the experimental conditions cannot fully reproduce the complexity of the oral environment, including bacterial biofilm activity, salivary variations, and masticatory loading. Additionally, fluorine detection by EDX has higher uncertainty due to the challenges of analyzing light elements at low concentrations.

Future studies should expand upon these findings by incorporating quantitative analyses, such as surface hardness testing and compositional techniques (e.g., Raman spectroscopy, FTIR, XPS, or ToF-SIMS), to correlate the extent of fluorapatite formation with improvements in bond strength. Additionally, long-term evaluation beyond 7 days, including thermocycling and extended pH cycling, would help determine whether the enhanced SBS in the RP group is maintained over time. Such investigations would further validate the clinical significance of applying fluoride-containing and hydroxyapatite-based remineralization protocols prior to adhesive restoration. These findings are clinically relevant for managing white spot lesions, orthodontic patients, and high caries-risk individuals.

Conclusion

Within the limitations of this in vitro study, fluoride-containing hydroxyapatite remineralization appeared to provide a more favourable enamel surface for subsequent adhesive procedures compared with fluoride-free hydroxyapatite. The findings suggest that delaying restoration after remineralization may allow further mineral maturation and improve bonding performance. However, clinical studies with long-term evaluation are required to confirm the optimal timing between remineralization therapy and adhesive restorative procedures.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

M. A: Conceptualization, methodology, laboratory experimentation, data analysis, and writing – original draft.M. M. A: Methodology review, data analysis, writing – original draft, and writing – review and editing.A. D: Supervision, methodology review, and writing – review and editing.All authors have read and approved the final version of the manuscript.

Data availability

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

Declarations

Competing interests

The authors declare no competing interests. This in vitro study was conducted at the Faculty of Dentistry, Van Yüzüncü Yıl University, Department of Restorative Dentistry. Ethical approval was obtained from the Clinical Research Ethics Committee (Decision No. 02/28.08.2024).