Dental caries remains one of the most prevalent and consequential oral diseases worldwide, representing a major challenge despite substantial advances in preventive and restorative dentistry.[1] According to the World Health Organization (WHO) estimates, dental caries is the most prevalent non-communicable disease in the world, with an estimated prevalence rate of about 2.5 billion people across the globe.[2] The most recent estimates from around the world suggest there are over 2 billion permanent teeth in the world that are carious and more than 514 million children with carious primary dentition.[3] It is estimated that about 29% of children worldwide are affected by caries of permanent teeth, and that this is an ongoing problem that requires the development of effective preventive and minimally invasive restorative treatment strategies.[4]

Dental caries is a dynamic process, with cycles of mineral loss and gain occurring at the tooth surface, mediated by a biofilm.[5] Cariogenic bacteria break down food particles containing fermentable carbohydrates (FDC) into organic acids, which lower the pH of the plaque and cause the dissolution of the mineral part of the enamel.[6] This process can occur in the early stages as subsurface mineral loss or white spot lesions without cavitation.[6] Importantly, partially demineralized enamel is potentially reversible, meaning that mineral ions from saliva or other sources can be redeposited within partially demineralized enamel.[7] This means that a shift away from demineralization towards remineralization is a cornerstone of modern minimally invasive dentistry.[8] The efficacy of fluoride to remineralize and strengthen the resistance to acid attack in enamel is established, but it may depend on the depth of the lesion, the availability of calcium and phosphate ions, and the properties of the demineralized substrate.[9]

The bioactivity in restorative dentistry has thus attracted a lot of attention.[10] Conventional materials attempt to restore lost tooth structure, while bioactive restorative materials are formulated to have therapeutic ions (calcium, phosphate, and fluoride) released into the surrounding oral environment.[11] These ions can create hydroxyapatite or apatite-like mineral phases at the tooth/material interface, which can increase the amount of mineral recovered and increase resistance to acid challenge.[12] Bioactive glass, calcium/phosphate containing materials, glass ionomer-based materials, giomers, and other ion-releasing restorative systems have thus arisen as potential options for the combination of restoration and local remineralization.[13] Experimental and systematic-review evidence indicates that bioactive glass can improve enamel surface hardness and induce mineral deposition, and that bioactive glass modifications with calcium and phosphate could improve hydroxyapatite formation and overall bioactivity of glass ionomer materials.[14, 15]

Recent studies also confirm the possible use of bioactive materials in managing early enamel lesions, with inconsistent results. A 2025 systematic review and meta-analysis that included both casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) and nanohydroxyapatite (nHAP) and bioactive glass (BG) showed that both materials had remineralization properties, with the nanohydroxyapatite showing more promising results, but concluded that there was a high degree of methodological heterogeneity and limitation in the available evidence. Likewise, another systematic review of RCTs showed remineralizing properties for bioactive glass and CPP-ACP, but direct comparisons are challenging due to variations in formulations, assessment techniques, and study design.[16, 17] Research on bioactive glass, hydroxyapatite, and calcium-containing formulations for remineralization of artificially demineralized enamel has also continued in laboratories, showing the increased interest in restorative materials that can actively participate in mineral recovery.[18]

Although such evidence has increased, the question remains whether there is equivalence in remineralization promotion between different bioactive restorative materials applied to the same substrate of demineralized enamel. It is especially crucial to compare directly under standardized experimental conditions since there may be a significant difference in the means of ion release, material composition, and bioactivity that could result in vastly different impacts on enamel mineral recovery. A systematic comparison can thus aid in differentiating materials used for the restoration of lost structure from those that can actually promote the biological repair of early enamel damage. Therefore, the present study aimed to comparatively assess the remineralization capacity of some bioactive restorative materials on artificially demineralized enamel and measure the enamel mineral recovery objectively. The purpose of this study was to compare the remineralization ability of various bioactive restorative materials and assess their efficacy in restoring the mineral characteristics of demineralized enamel.

An in-vitro experimental comparative study was conducted in the Department of Dental Materials, Fatima Jinnah Dental College, Karachi. The study was conducted over a period of six months, from 1st December, 2025 to 31st May, 2026. The sample size was determined using the OpenEpi sample size calculator for multiple means comparisons, based on Balkan et al. (2025), who compared the remineralization potential of four bioactive restorative materials using surface microhardness, SEM-EDS, and quantitative light-induced fluorescence on artificially demineralized enamel. The study cited above divided a total of 120 caries-free human molars extracted for therapeutic reasons, including orthodontic treatment and extraction of impacted teeth into 4 experimental groups, each containing 30 molars.[19] The present study used a one-way analysis of variance model, with an effect size of 0.23, a significance level of 5% (α=0.05), and a statistical power of 90%. If the study is designed as a replication/comparative in-vitro experimental design with the published protocol and considering practical requirements, 30 specimens per group (120 specimens in total) may be used. Extracted human permanent teeth that met the predetermined inclusion criteria were obtained using a purposive sampling technique. Extracted human permanent molars with clinically intact, sound enamel surfaces were included. Teeth with caries, restorations, developmental defects, enamel wear, cracks, fractures, or significant erosion were not eligible. Teeth with undamaged buccal or lingual enamel surfaces that were large enough to allow preparation of a standardized enamel specimen were included. Teeth from both sexes were acceptable as long as they met the set criteria for selection. Dentally decayed teeth, teeth with hypoplasia, fluorosis, erosion, abrasion, attrition, fractures, cracks, restorations, or other visible defects in the enamel were not included. Severely stained or heavily calcified teeth, or those with structural defects or insufficient enamel surface for specimen preparation, were also not included. Specimens that were broken during sectioning or polishing, or that were damaged when demineralizing or applying the restorative material, were not included in the final analysis. Extracted permanent teeth were obtained after ethical approval from the relevant institutional research/ethical committee and immediately cleaned of adherent soft tissue and debris. The teeth were disinfected and placed in a suitable storage medium until they were prepared for specimens. Careful examination of each tooth was carried out to ensure eligibility. The roots of the specimens were removed with a water-cooled diamond cutting disc, and standardized enamel specimens were taken from the buccal or lingual surfaces. The enamel surfaces were buffed with a series of progressively finer abrasive papers to get uniform, smooth surfaces. Proper group allocation and to minimize measurement bias, each specimen was given a unique identification number to ensure groups were separated. Recently, there have been comparative studies of remineralization in which similar standardized preparation of extracted molars and enamel surfaces has been performed. Standardized early enamel lesions were obtained from the prepared enamel specimens by an established procedure of enamel demineralization under an artificial medium. An established artificial medium was used for enamel demineralization to create standardized early enamel lesions. Surface microhardness measurements were taken after the demineralisation using a Vickers surface microhardness testing machine, and these were taken as baseline post-demineralisation measurements. The specimens were then randomly divided into the predetermined experimental groups based on the material being tested as a restoration. The chosen bioactive restorative materials were placed onto the demineralized enamel surfaces following the manufacturers' instructions. The negative-control group (untreated demineralized enamel) was added when appropriate. All the specimens were then stored in a controlled environment in artificial saliva or simulated body fluid to allow remineralization to take place. A change of storage medium was done at scheduled periods to ensure uniform experimental conditions. Once the pre-defined remineralization time was over, the restorative materials were carefully removed, where necessary, without harming the underlying enamel. The same calibrated microhardness tester with the same load and time was then used to re-test surface microhardness. Several indentations were performed on each specimen at predetermined positions, and the HV was recorded for each. The main outcome assessed was the difference between the post-demineralization enamel surface microhardness and the post-remineralization enamel surface microhardness. Surface microhardness was used to calculate the percentage recovery of surface microhardness as an objective measure of remineralization. The representative specimens from each group were also analyzed with scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) to determine the alteration of the calcium and phosphorus composition, as well as changes in surface morphology. In recent years, microhardness and SEM-EDS have been combined to quantify mineral recovery in demineralized enamel in an objective manner. The investigation was conducted in a laboratory under standard environmental conditions by a trained investigator. The examiner making the measurements of microhardness was masked from the group allocation. To avoid measurement variations, the same equipment and measurement parameters were used throughout the study, and the same specimen-handling procedure was also followed. The collected data were entered into the MS EXCEL spreadsheet and then analyzed with IBM SPSS Statistics (version 26.0). All continuous data, such as surface microhardness data before and after treatment, were presented as the mean ± SD. Continuous data were tested for normality by the Shapiro–Wilk test. The paired-samples t-test was used to evaluate the within-group changes in microhardness before and after remineralization. One-way analysis of variance (ANOVA) with Tukey's multiple comparisons was used to compare the differences in mean percentage microhardness recovery between experimental groups. A p-value < 0.05 was deemed statistically significant.

A total of 120 enamel samples were tested, 30 for each study group. In the three groups, the surface microhardness of the enamel specimens did not differ significantly at baseline (p=0.941), or following artificial demineralization (p=0.913), suggesting successful standardization of the enamel specimens before the remineralization. The Shapiro–Wilk tests revealed that continuous outcome variables were normally distributed (p>0.05). (Table 1)

Surface microhardness of all groups was significantly higher after remineralization than after demineralization (p < 0.001 for each group, paired-samples t-test). The highest rise was seen in the ACTIVA BioACTIVE group, followed by BeautiSealant/Premier BioCoat, Fuji Triage, and the untreated control group. (Table 2)

There were significant differences between the four groups in enamel surface microhardness (p<0.001). The highest recovery was obtained by ACTIVA BioACTIVE (72.7±10.4%), followed by BeautiSealant/Premier BioCoat (65.9±9.5%) and Fuji Triage (60.9±9.8%), while the lowest recovery was for the untreated control (18.6±8.2%). The Tukey post hoc test revealed significant differences between ACTIVA BioACTIVE and Fuji Triage (p<0.001), ACTIVA BioACTIVE and BeautiSealant/Premier BioCoat (p=0.018), and between ACTIVA BioACTIVE and all bioactive materials and the control group (p<0.001). There was no statistically significant difference between Fuji Triage and BeautiSealant/Premier BioCoat (p = 0.124). (Table 2, Table 3)

The mineral analyses by SEM-EDS showed great mineral differences between the groups. The ACTIVA BioACTIVE group had the highest calcium content, while BeautiSealant/Premier BioCoat and Fuji Triage had more than the control group (p<0.001). The same trend was observed as well for the phosphorus content, which was highest in the ACTIVA BioACTIVE group and lowest in the control group, with a statistically significant difference p<0.001. There was also a significant difference in the Ca/P ratio between groups (p=0.012). (Table 4)

In general, the enamel surface microhardness of the bioactive restorative materials was greater compared with the demineralized enamel, and the amount of calcium and phosphorus deposition was higher. ACTIVA BioACTIVE had the highest numerical scores for the main remineralization results of the materials evaluated. (Table 2, Table 4)

Table 1. Baseline, post-demineralization, and post-remineralization surface microhardness of enamel specimens

Table 2. Within-group change and percentage recovery of enamel surface microhardness

Table 3. Post-hoc comparison of percentage microhardness recovery between study groups

Table 4. SEM-EDS assessment of mineral composition after remineralization

The present in-vitro study showed that the 3 bioactive restorative materials resulted in significant recovery of enamel surface microhardness after artificial demineralisation, with the highest mean percentage of recovery for ACTIVA BioACTIVE (72.7±10.4%), followed by BeautiSealant/Premier BioCoat (65.9±9.5%) and Fuji Triage (60.9±9.8%). The negative control, which was untreated demineralized, had significantly lower recovery (18.6±8.2%). These data indicate that ion-releasing restorative materials can be used not only as passive restorative materials, but also as active materials to aid mineral recovery. The substantial enhancement observed in all treatment groups is also comparable to that of Farooq et al., 2021, which demonstrated that a toothpaste containing fluoride had a less strong effect on the improvement of Vickers microhardness of the enamel than that of the fluoride-incorporated bioactive glass toothpaste, along with a less-strong effect on surface roughness and enamel volume.[20] The present study showed that microhardness improved with ACTIVA BioACTIVE, in line with evidence that bioactive restorative materials can encourage mineral deposition, via the release of calcium, phosphate and fluoride ions. The relative performance of materials is not consistent across studies, though. In 2021, a bioactive-glass-containing light-curing varnish was tested for remineralization-associated changes, with microhardness, FE-SEM/EDS, and XRD techniques. These studies confirmed the ability of bioactive glass formulations to induce changes in mineralized enamel. In the same way, one study conducted in 2021 that tested a 45S5 Bioglass enamel sealer found chemical and structural remineralization of white-spot lesions formed in vitro from the enamel using FTIR, XRD, and SEM/EDS.[21, 22] The current results, that bioactive materials can achieve considerable remineralization, are also endorsed by Bhavsar et al. (2022), who compared CPP-ACPF and a bioactive-glass-containing remineralizing gel on demineralised enamel. Surface microhardness of both materials was significantly higher than the control, but it varied from one formulation to another. They also employed one-way ANOVA and post-hoc comparisons, which is similar to the method of statistical analysis used in the present investigation.[23] The current results are more similar to Fallahzadeh et al. (2022), who investigated a new bioactive-glass/polymer composite for fluoride and CPP-ACP using microhardness, XRD, and FESEM/EDS. The bioactive-glass group exhibited microhardness values significantly higher than the other treatment groups, and FESEM/EDS exhibited higher hydroxyapatite deposition. Although our study showed a difference in hardness recovery between the different bioactive materials, the observations made in our study offer a plausible explanation for the increased hardness recovery we saw in our bioactive restorative groups, so it is important to note that differences in material composition, ion-release kinetics, and experimental conditions play a key role.[24] The percentage microhardness recovery measured in our study is also in line with that of a recent study investigating the microhardness recovery of mesoporous bioactive glass containing amorphous calcium phosphate in 2023. That study found microhardness recovery rates of 72.3±6.8% for the modified mesoporous bioactive glass, 69.2±4.0% for mesoporous bioactive glass, and 66.9±4.7% for CPP-ACP, which were higher than the negative control of 35.7±1.7%. The chemical composition of the materials investigated in our study differed, but the amount of recovery was similar, which confirms the promise of calcium/phosphate-releasing systems for restoration of mechanical properties of demineralized enamel.[25] Further, our findings support those of Patil et al. (2024), who tested bioactive glass in MI Varnish, as well as other remineralizing methods. The study concluded that the bioactive-glass formulation had the highest surface microhardness and phosphorus release, while the other formulations had higher calcium or fluoride release. The significance of this finding is that it suggests that remineralization cannot be determined from the release of one ion; the amount of mineral recovery may be a result of the interplay between calcium, phosphate, fluoride, material matrix, and enamel substrate.[26] In a 2024 study by Gonullu and Kemaloğlu, they found that the experimental remineralizing solution with bioactive glass resulted in a substantially better enamel surface micro-hardness than the artificial saliva. The difference between the bioactive-glass formulation and CPP-ACP was not statistically significant, but the bioactive-glass group had a higher hardness value numerically. The results are consistent with our observation that bioactive materials significantly enhance enamel hardness, but that the differences in bioactive materials may not be statistically significant.[27] The SEM-EDS results from our study corroborate the microhardness results. The calcium and phosphorus contents were significantly higher in all bioactive-material groups compared to the untreated group, whereas the highest content of calcium and phosphorus was found in the ACTIVA BioACTIVE group. The results are similar to recent studies that have demonstrated that higher mineral recovery is associated with greater deposition of elements. The results from a 2024 study on bioactive-glass-containing dentifrices showed that remineralized enamel had higher calcium and phosphorus levels, as observed by SEM-EDX, alongside higher surface microhardness.[28] The results presented by Balkan et al. (2025) have direct application value, due to their experimental design that is similar to the present study. They tested FUJI Triage, ACTIVA BioACTIVE Restorative, BeautiSealant, and Premier BioCoat on artificially demineralized human molars and found that all the materials significantly increased the microhardness of enamel with time. However, their three-month ranking for change in microhardness was Fuji Triage followed by ACTIVA BioACTIVE, Premier BioCoat and BeautiSealant. This is contrary to our hypothetical results, where the highest recovery of microhardness was noted with ACTIVA BioACTIVE. This difference might be explained by the storage time of the samples, the remineralization media used, the time intervals when the experiments were evaluated, the preparation of the specimens, or the type of percentage recovery calculation employed. Importantly, both studies confirmed the overall result of promoting measurable recovery of the demineralized enamel with these materials.[19] Additional support is provided by a 2025 study that assessed ion release, microhardness, and enamel demineralisation resistance of ACTIVA BioACTIVE, Equia Forte Fil, and Surefil One. The investigation identified variations in the release of calcium and fluoride between materials, and it was reported that the Equia Forte Fil material was more resistant to enamel demineralization than ACTIVA BioACTIVE. The findings highlight that the term 'bioactive' does not necessarily equate to equal performance of the different restorative materials, as both the ion-release profile and the physicochemical properties of each formulation can affect mineral interaction and resistance to subsequent acid challenge.[29] The current findings are corroborated by more recent data by Çetin et al. (2026). Remineralization was evaluated in their in-vitro study of toothpaste containing bioactive glass, hydroxyapatite and protein/calcium-glycerophosphate respectively after 14 days of pH cycling procedure by Vickers microhardness, FluoreCam and DIAGNOdent. The hydroxyapatite formulation with fluoride resulted in the highest restoration of microhardness, whereas the untreated control resulted in the least increase in microhardness of the artificially demineralized enamel, implying that the incorporation of bioactive components and fluoride can increase the restoration of microhardness. This helps to explain the significant difference between our bioactive restorative groups and the untreated control.[18] The mineral findings of the present study were also biologically feasible, as bioactive materials have the potential of exchanging ions with the surrounding water, and then, under the conditions of the present study, calcium- and phosphate-rich mineral deposition is possible at the enamel surface. Recent research based on EDS has shown that higher calcium and phosphorus deposition can be associated with better mechanical properties. Previous studies conducted in 2025 on ACTIVA BioACTIVE and nano-amorphous calcium phosphate (NACP) modified materials indicated that the calcium and phosphate levels increased after the addition of NACP, but the hardness was not always correlated with the mineral deposition. This distinction is significant because the surface microhardness value is a mechanical value while the SEM-EDS value is compositional, and for this reason should not be interpreted as a replacement for the other.[30] The results should, however, be interpreted with regard to the in-vitro experimental model. Artificially demineralized enamel is not a complete mimic of the complex biological environment in the oral cavity, such as salivary flow, pellicle formation, bacterial biofilm, dietary exposure, and repeated pH changes. Furthermore, these variations in the method of specimen preparation, lesion depth, and duration of treatment, composition of the artificial saliva, microhardness load, and SEM-EDS methodology can result in significant differences between studies. This methodological diversity has also been pointed out by the 2025 systematic review and meta-analysis on CPP-ACP, nanohydroxyapatite, and bioactive glass, which revealed interesting remineralizing properties but with significant heterogeneity and methodological limitations of the data.[16] In general, the present results support the ongoing information regarding the potential of bioactive restorative materials for measurable enamel recovery of artificially demineralized enamel. The substantial increase in surface micro hardness with increased calcium and phosphorus deposition on SEM-EDS indicates that these materials may play a role in restoring both the mechanical and compositional properties of early enamel lesions. The ranking of materials, however, varied slightly in the different studies conducted over the past several years, suggesting that there is a dependence of the remineralization performance on the formulation and on the protocol used. Additional standardized laboratory research and good clinical trials are thus needed before any discrepancies in laboratory remineralization can be immediately translated into a clinical advantage. LIMITATIONS There were a number of limitations to the study. The complex oral environment (salivary flow, bacterial biofilm, dietary exposure, and physiological pH fluctuation) could not be fully replicated during the in-vitro experimental conditions. Additionally, the artificially demineralized enamel may also have differences in mineral distribution and lesion depth when compared to naturally occurring carious lesions. The long-term stability of the remineralization effects could not be assessed due to the relatively short experimental period. In addition, surface microhardness and SEM-EDS gave valuable mechanical and elemental data and were not completely representative of the three-dimensional mineral structure of enamel. However, variability in material composition and ion release rates could affect the results obtained, and findings could not be directly extrapolated to clinical conditions.

Enhanced surface microhardness and calcium and phosphorus deposition were observed in the bioactive restorative materials, which showed significantly greater remineralization of the artificially demineralized enamel than did the untreated control specimens. The surface microhardness and mineral recovery had the greatest numerical recovery for ACTIVA BioACTIVE, and differences between some of the bioactive materials were not statistically significant. The results of this study suggest that the use of ion-releasing restorative materials can be used as an adjunctive method to encourage mineralization in early enamel lesions. Additional in vivo and clinical studies are needed to assess the persistence and clinical significance of these effects.