INTRODUCTION

In July 2018, a group of 50 key opinion leaders met to develop a consensus statement on the definition of a bioactive restorative dental material.¹ From that consensus meeting, a bioactive restorative material was defined as one that would restore missing tooth structure and additionally “stimulate or direct specific cellular or tissue responses or control interactions with microbiological species”.¹ One example of a bioactive effect was described as having “components that dissolve and can be identified with normal physiologic species involved in a biological process.” During the process of secondary caries, hydroxyapatite is detached from the surface of the tooth surrounding a dental restoration. A restoration that can release the ions present in the mineral content of the tooth would, in theory, be able to assist the process of remineralization in the tooth structure surrounding the restoration and decrease secondary caries.² As calcium and fluoride are components of hydroxyapatite or fluorapatite in tooth structure, materials that release fluoride (such as glass ionomers) and those that release calcium could both be considered bioactive. The recent trend, however, has been to identify calcium-releasing materials as bioactive, since this is the major component of hydroxyapatite.

Glass ionomer materials release fluoride in both an initial burst (first 24 hours) when glass particles react with polyalkenoic acid during the setting reaction and a prolonged long-term release when glass dissolves in the acidified water of the hydrogel matrix.^3,4 The fluoride release from glass ionomer materials plateaus after 10–20 days,⁵ and continued low-level fluoride release has been reported for up to 3 years.⁶ A similar release profile has been reported for resin-modified glass ionomer (RMGI) materials.⁷ Polyacid-modified composites (also called compomers) do not have a burst release of fluoride; however, they have a constant fluoride release at lower level than glass ionomer or RMGI materials.^8,9 Fluoride release is increased from restorative materials when they are placed in an acidic solution, presumably due to dissociation of the material.^10,11 Application of fluoride-containing solutions may provide a so-called recharge of restorative materials that may allow sustained fluoride release by washout of fluoride retained on the surface of a material or from ion-containing solute within pores of the material.¹² As glass ionomer materials are more permeable and have higher water sorption than resin-based materials, they act as a better reservoir for fluoride.^12–15

Fluoride released from dental materials can be incorporated structurally into surrounding tooth structure (fluorapatite) or precipitate onto the tooth surface as a calcium fluoride layer.¹² Structural incorporation of fluoride in teeth produces larger apatite crystals, which increases resistance to carious lesion formation.¹⁶ A deposited fluoride layer may facilitate reprecipitation of minerals, also helping to prevent mineral ion loss caused by the caries process.¹⁷ The amount of fluoride uptake from ion-releasing restorative materials is higher in dentin than enamel.¹⁸ Several laboratory studies have demonstrated the ability of fluoride-releasing materials to inhibit demineralization of surrounding enamel or root dentin.^19–28 A clinical study has also shown less demineralization around extracted primary teeth restored with glass ionomer than with amalgam.²⁹ Clinical trials have reported a higher incidence of secondary caries around resin composite and amalgam restorations than fluoride-releasing materials, particularly in patients who were non-compliant with extrinsic fluoride application.^30–33 The clinical benefit of fluoride-releasing materials is not evident in all clinical trials. A systematic review of 28 clinical trials reported that there was insufficient evidence to support a treatment effect of inhibition of secondary caries by glass ionomer materials. This review reported that there was an equal number of studies with an incidence of caries in the glass ionomer group as those with an incidence of caries in the control group. The magnitude of the difference in incidence of caries for either material was not analyzed.³⁴

Several calcium-releasing restorative materials are commercially available with claims of bioactivity. Some of the bioactive restorative materials currently on the market, such as Activa BioACTIVE-Restorative (Pulpdent Corp, Boston, MA, USA), could be considered a compomer, as its polymer matrix is predominantly composed of a dual-cured resin methacrylate with added polyacid monomers. A recent study identified the filler as fluoroaluminasilicate glass.³⁵ Previous studies have reported small amounts of calcium released from Activa BioACTIVE-Restorative.^35,36 A recent study identified a spherical calcium-containing filler particle within the material that may be responsible for the release.³⁶ A new, modified, single syringe delivery system is now available on the market (Activa Presto, Pulpdent). An experimental premarket version of this material, which can also be considered a compomer, was tested in the present study. Another commercially available calcium-releasing restorative material, Cention N (Ivoclar Vivadent, Lichtenstein, Schaan), which contains methacrylate monomers as well as calcium fluorosilicate glass fillers, was also tested in the present study.³⁵ Cention N has been shown to inhibit caries formation at restoration margins in vitro.³⁷

The purpose of the present study was to determine if two calcium-releasing materials (an experimental material and Cention N) could inhibit demineralization of surrounding root dentin relative to reference RMGI (positive control) and resin composite (negative control) materials. The study methodology was cyclic remineralization and demineralization of restorations placed in extracted human teeth. The null hypothesis was that there would be no difference in the area of demineralization inhibition provided by the two calcium-releasing materials relative to the reference resin composite or RMGI materials.

METHODS AND MATERIALS

Forty freshly extracted human molars (mixture of maxillary and mandibular, and 1st-3rd molars) from unidentified donors were collected from the Oral and Maxillofacial Surgery department. Caries-free molars with relatively flat buccal surfaces were selected. Prior to restoration, the teeth were immersed in distilled water for up to 6 months and randomized into four groups of 10 specimens each. Approximately 4 mm × 3 mm (±0.2 mm) box preparations (2-mm deep) were prepared on the buccal surfaces of the human molars with an enamel occlusal margin, while the gingival margin was placed in root dentin. All preparations were performed with a #557 carbide bur in a high-speed handpiece with copious water irrigation.

One resin composite (Filtek Supreme Ultra, 3M Oral Care, St Paul, MN, USA), two calcium-releasing materials (Experimental material, Pulp-dent; Cention N, Ivoclar Vivadent), and one RMGI material (Fuji II LC, GC America, Alsip, IL, USA) were used to fill the prepared box (Table 1). For the Filtek Supreme Ultra, Cention N and experimental material groups, an adhesive (Scotchbond Universal, 3M Oral Care) used in the self-etch mode was applied with 20 seconds of agitation followed by solvent evaporation with 5 seconds of gentle air. The adhesive was then light cured with an LED curing light (Elipar S10, 3M Oral Care, output > 1000mW/cm²) for 10 seconds. The restorative materials were then placed into the preparations and light cured for 20 seconds using an LED curing light (Elipar S10). After finishing and polishing, the restorations were examined using a digital microscope (VHX-600, Keyence, Osaka, Japan) under 20× magnification to ensure an effective marginal seal was present, and there was no excess material present over the restoration margin. Any resin composite flash present over the restoration margin was removed with a polishing disk (medium Sof-Lex disc, 3M Oral Care).

Table 1: Materials Used in This Study

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Afterwards, the teeth were incubated for 24 hours in distilled water at 37°C to allow complete polymerization of all materials. The surface of the teeth were dried with a paper towel. Acid-resistant varnish with different colors was painted onto the teeth for different groups, leaving a window including the restoration and 2 mm of tooth structure uncoated surrounding the restoration.

An artificial caries model was used based on a previously published protocol.^27,38 A 500 mL demineralization solution composed of 3.7 mL lactic acid (0.1 M), 0.111 g CaCl₂ (2 mM Ca) and 0.136 g KH₂PO₄ (2 mM PO₄), 0.5 g thymol (0.1% to prevent bacterial growth), and NaOH (to adjust pH=4.5) was prepared. A 500 mL remineralization solution composed of 0.083 g CaCl₂ (1.5 mM Ca), 0.061 g KH₂PO₄ (0.9 mM Ca), 0.121 g Tris-(hydroxymethyl) aminomethane (20 mM, pH=7.0), and 0.5 g thymol (0.1% to prevent bacterial growth) was prepared. The specimens were immersed in the demineralization solution for 4 hours, followed by the remineralization solution for 20 hours and cycled daily. A 5-minute wash in running deionized water was performed between each cycle. To ensure that the solution was agitated through the experiment, a magnetic stirrer was utilized in both the demineralization and remineralization solutions.

After 1 month, the specimens were embedded into polymethyl methacrylate (Ortho-Jet, Lang Dental Manufacturing Co, Wheeling, IL, USA) and sectioned into approximately 150 μm sections using a low-speed sectioning saw (IsoMet, Beuhler, Lake Bluff, IL, USA). Two cuts were made through the buccal–lingual plane of the tooth through the mesial–distal center of the restoration to obtain the 150-μm section. The sections were then hand polished with 600 grit silicon carbide paper to achieve a final thickness of 100 μm ± 20 μm. Any sections with artifacts (flash over the margin, voids at the margin) were discarded, and an additional section was taken from the specimen. A small number of specimens separated at the tooth–restoration interface during polishing.

Sections were viewed with polarized light microscopy at 10× (VHX-600, Keyence). The zone of demineralization was defined as the area in which a visual detection in the appearance of the root dentin could be viewed. The zone of demineralization started at the external surface of the tooth and progressed in a pulpal direction. For some specimens, an “area of inhibition” was observed adjacent to the restoration in which the zone of demineralization did not progress as deep in the pulpal direction. For other specimens, a “wall lesion” was formed in which the zone of demineralization progressed deeper in the pulpal direction at the area adjacent to the restoration. A line demarking the front line of the zone of demineralization was defined for each tooth (Figure 1, through point A and point B to point C). An “area of inhibition” was defined as any area of unaltered root dentin adjacent to the restorative material and shallower (away from pulp) than the line (Figure 1, area between points B, C, and D). A “wall lesion” was defined as any area of demineralized root dentin that was adjacent to the restorative material and deeper (towards pulp) than the line (Figure 1, area between points B, C, and E). These areas were hand traced with internal image evaluation software and measured. Areas of inhibition were recorded as positive values, and areas of wall lesions were recorded as negative values.

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Data were compared with one-way analysis of variance (ANOVA) and Tukey honestly significant difference (HSD) analysis. The significance level was set at α=0.05. All analyses were conducted using SAS 9.4 (SAS Institute, Cary, NC, USA).

RESULTS

All specimens in the Filtek Supreme Ultra group (negative control) produced wall lesions; whereas, all specimens in the Fuji II LC, experimental composite, and Cention N groups produced “areas of inhibition.” The mean and standard deviation of the “areas of inhibition” and “wall lesions” were as follows: Fuji II LC (5412±2754), Cention N (2768±1576), experimental composite (1484±1585), and Filtek Supreme Ultra (−1119±1029). The measurements of each specimen are listed in Table 2. Negative or positive values indicate from which side of the front line of demineralization the area is measured. The negative values for Filtek Supreme Ultra denote that the wall lesions were located deeper (towards pulp) than the front line of the zone of demineralization. The oneway ANOVA determined that there were significant differences between materials (p<0.01). The Tukey post hoc analysis determined that the experimental composite and Cention N groups had no statistically significant difference but produced smaller zones of inhibition than the Fuji II LC group. Representative specimens from each group are shown in Figures 2–5.

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Table 2: Areas of “Inhibition” (Positive Values) or “Wall Lesions” (Negative Values) in Root Dentin Adjacent to Different Restorative Materials

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DISCUSSION

In this study, there was significantly more demineralization inhibition adjacent to the Cention N and the experimental material than the reference resin composite but less than that adjacent to the RMGI material. Therefore, we reject the null hypothesis. The ability of this test to discriminate between different materials demonstrates its utility.

Previous studies have shown that fluoride-releasing restorative materials can inhibit demineralization around both enamel and dentin. One previous study reported that materials ranked in their protective ability (from the most to the least): glass ionomer, RMGI, giomer, compomer, and resin composite.²⁰ Several studies have confirmed that glass ionomers and RMGI materials provide more caries inhibition than resin composites.^{19–22,26–28} One study reported that a compomer material provided similar protection as glass ionomer and/or RMGI materials²¹; whereas, others reported that compomers provided less protection.^20,22,26

Both Cention N and the experimental material could be considered to be similar to compomers, as they contain methacrylate functional groups and fluoride-releasing fillers. A previous study reported that Cention N releases more fluoride than Fuji II LC.³⁹ A previous version of Activa BioACTIVE-Restorative showed very little fluoride release, less than Cention N.³⁵ The differentiating characteristic of Cention N and the experimental materials from glass ionomers or RMGIs is that they are formulated with calcium-containing components; whereas, the RMGI used in this study does not release calcium. Cention N releases significantly more calcium than a previous version of Activa BioACTIVE-Restorative.³⁵ Despite the additional calcium release, both Cention N and the experimental material showed less demineralization inhibition than that provided by Fuji II LC—a RMGI material. Our study confirms previous findings by Donly, who also reported that Cention N produced less protection than a RMGI material.³⁷Figures 4 and 5 demonstrate that both Cention N and Fuji II LC contain porosities, so the difference in performance may not be related to the presence of porosity. These porosities did not interfere with measurement of demineralization.

Both Cention N and the experimental material were used with an adhesive. An adhesive was used was due to clinical trials suggesting Cention N and a previous version of Activa BioACTIVE-Restorative show superior clinical performance when an adhesive is used, specifically improved marginal integrity and retention.^40,41In vitro testing has shown that placement of an adhesive prior to application of fluoride-releasing materials can prevent the demineralization inhibition effect of the fluoride-releasing material.^19,25 Perhaps the adhesive used in this study could have prevented the restorative materials from inhibiting demineralization in the surrounding tooth structure. In the previous artificial caries testing performed with Cention N, the authors did not mention if an adhesive was used.³⁷

There is no standardized method for performing artificial caries testing, and the methodology used in different studies has varied. The pH cycling regimen used in the present study was based on a previous published regimen²⁷ originally credited to Featherstone.³⁸ In the present study, lactic acid was chosen for the demineralization solution rather than acetate. In some studies, specimens have been placed in acidic solutions only (pH=4–5) for between 3 days and 10 weeks.^{19,21,22,24,28} Other studies have performed pH cycling between a pH of 4.5 or 5 and 7.^20,27 Other studies produced demineralization through exposure to a bacterial caries model. In these studies, the resin composites were placed in a broth containing Streptococcus mutans and lactobacillus.^22–25,27 The results of some studies suggest that bacterial models produce similar results as pH cycling models; whereas, others show different outcomes. For example, a study determined that glass ionomer and RMGI materials provided protection from demineralization in a pH cycling model; however, glass ionomer or RMGI materials performed similarly to traditional resin composites, if the demineralization was caused by a bacterial caries model.²² In contrast, another study showed that glass ionomer and RMGI materials showed more protection from demineralization than resin composite in both a bacterial caries model and a pH cycling caries model.²⁷

The methodology used to measure demineralization varied between studies. Most studies measured demineralization through polarized light microscopy^19,20,21,24; however, it can also be measured with confocal microscopy²² or microradiography.^23–25 The measurement of demineralization has usually included either outer wall lesions,^21,26,27 areas of wall lesions/inhibition, or both.^{19,20,22,24,25} Outer wall lesions describe the amount of demineralization present from the external surface of the tooth to the internal limit of demineralization. Often the outer wall lesion is measured at a point 100–200 microns away from the restoration margin. Outer wall lesions are meant to describe the amount of protection provided by a restorative material at a location distant to its interface with the tooth. On the other hand, areas of wall lesion or inhibition are the areas immediately next to the restoration that are measured as described in the methodology of this study. Other methods have been used to estimate the area of a wall lesion or inhibition, such as measuring the linear distance (pulpal-external) of demineralization near the restoration margin²² or estimating the thickness (occlusal-gingival) of the wall lesion or area of inhibition.^24,28 Measurement of the exact area of inhibition or wall lesion, as performed in the present study, provides information of both the thickness and the depth of these areas. In the present study, the wall lesions and areas of inhibition were measured relative to the front line of the zone of demineralization rather than the external surface of the tooth. This decision was made because teeth experienced different depths of their zone of demineralization relative to their external surface, which could be related to the properties of the tooth rather than the restorative material.

Several technique variables were determined to be critical toward achieving observable lesions within the specimens. The most critical factor was accurately monitoring the pH of the demineralization solution. Due to neutralization from excess water surrounding specimens or possibly from the specimens themselves, the pH of the solution required frequent adjustment by the addition of lactic acid. Daily calibration of the solution was necessary to maintain a constant pH. Possibly the use of acetate to produce an acidic pH would have helped to better buffer the solution. Another critical factor was the volume of solution used per specimen. Through pilot testing, a solution of 500 mL could produce lesions when 40 specimens were immersed within it. A lesser volume of solution with a similar number of specimens would not create consistent demineralization lesions. Alternatively, specimens from each group could have been stored in separate containers of 125 mL, assuming the specimens would be completely immersed in liquid, while the liquid is being stirred.

Future studies should examine the caries inhibition produced by calcium-releasing materials placed with and without a dental adhesive. Although it would not be prudent to use these materials clinically without adhesive, ^40,41 this test methodology could determine whether calcium-releasing materials offer additional benefit from fluoride-releasing materials without the confounding factor of an intermediary adhesive. Another future study could explore the development of an adhesive that would not prevent any protective effects of ion-releasing materials. The artificial caries model could also be modified such that the remineralization solution contained fluoride in order to simulate the effects of topical fluoride application (ie, fluoridated toothpaste). Previous protocols have incorporated fluoride into the remineralization (0.05 ppm) and demineralization (0.03 ppm) solutions²⁷ or applied topical fluoride as part of the daily cycle.³⁸ Finally, future studies could monitor the progression of demineralization by removing, sectioning, and viewing teeth at various time points.

CONCLUSIONS

In this study, two calcium-releasing materials used with an adhesive provided more protection from root dentin demineralization than a resin composite used with an adhesive; however, these materials provided less protection than a RMGI material used without an adhesive. These findings suggest that calcium-releasing materials, sometimes referred to as bioactive materials, may provide additional utility beyond that of bonded resin composites for prevention of demineralization around restorations. A RMGI material used without an adhesive still provides superior protection from tooth demineralization.