CPP-ACP added to a GIC was distributed throughout the cement.
GIC can be recharged with CPP-ACP/F by topical application to the cement.
Recharging enhanced calcium, phosphate and fluoride ion release from the cement.
Recharging increased the surface hardness (acid resistance) of the cement.
Daily recharging of GIC may increase longevity of the cement and enhance caries protection.
To assess the effect of CPP-ACP/F recharging on ion release and hardness of GIC Fuji-Triage (VII) and Fuji-Triage-EP (VII-EP) containing CPP-ACP/F.
CPP-ACP distribution in Fuji-Triage-EP was determined using immunofluorescence. Thirty blocks of Fuji-Triage and Fuji-Triage-EP with the same surface area were placed individually in 5 mL of 50 mM lactic acid (pH 5) for three days. Every 12 h ten Fuji-Triage and ten Fuji-Triage-EP blocks were treated with 2 mL of either MI Paste Plus (CPP-ACP/F) solution (1 g paste + 4 mL water), Placebo MI paste solution (no CPP-ACP/F), or distilled water for 2 min. After each 2 min treatment the blocks were rinsed with distilled water and placed back into the acid. Calcium, inorganic phosphate and fluoride levels in the acid solution were measured using atomic absorption spectrophotometry, colorimetry and ion specific electrode respectively. Vickers surface hardness of the GIC was also determined. Data were analysed using a two-sample t-test and one-way ANOVA with a Bonferroni-Holm correction for multiple comparisons.
CPP-ACP was distributed throughout Fuji-Triage-EP. Significantly (p < 0.001) higher calcium, inorganic phosphate and fluoride ion release and greater surface hardness (acid resistance) was observed in both GIC’s treated with the CPP-ACP/F paste. Fuji-Triage-EP released higher ion levels and exhibited greater surface hardness (acid resistance) than Fuji-Triage.
Topical application of CPP-ACP/F paste to GIC Fuji-Triage-EP recharged ion release and increased surface hardness (acid resistance) which may help improve properties and resistance to degradation as well as improve ion release for caries control.
Glass-ionomer cements (GICs) exhibit several clinical advantages compared with other dental restorative materials and are widely used in dentistry for a variety of applications including caries stabilisation, fissure sealing and as a surface protecting material for high caries-risk surfaces such as root surfaces. The main advantages of GICs are their ability to strongly adhere to tooth tissue [ ], to slowly release fluoride ions, and their low coefficients of thermal expansion [ ]. GIC’s fluoride ion release properties are extremely important as fluoride ions promote enamel and dentine remineralisation and may also interfere with the metabolism of plaque bacteria, thereby slowing the progression and aiding the regression of early caries lesions [ ]. Fuji-Triage (VII) GIC is routinely used for pit and fissure sealing, root surface protection, and intermediate restoration due to its better fluoride-ion release properties and ease of placement.
The ability of fluoride ion to remineralise early caries lesions has been shown to be calcium ion limited and this has led to considerable interest by oral care and dental materials’ companies to incorporate a source of bioavailable calcium ions into their products [ ]. Dental materials that release calcium and phosphate ions as well as fluoride ions can produce local environments supersaturated with respect to fluorapatite at the margins of the restoration to help prevent caries. Hence these materials can be used as a source of ions to maintain calcium, phosphate and fluoride ion activities which influence the progression and the regression of early caries lesions. Recently GICs and resin composites have been modified to incorporate soluble calcium and phosphate ions in an attempt to improve their anticariogenic properties [ ]. The conventional GIC, Fuji-Triage (VII) has been modified to include casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) which is a saliva biomimetic and an excellent source of bioavailable calcium and inorganic phosphate [ , ]. The CPP contains the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- and stabilises supersaturated solutions of calcium (Ca), inorganic phosphate (Pi) and fluoride (F) ions to produce CPP-amorphous calcium fluoride phosphate nanocomplexes (CPP-ACFP) [ ]. The CPP are biomimetics of the salivary protein statherin which contains the sequence Asp-Ser(P)-Ser(P)-Glu-Glu-, however due to the greater content of Ser(P) residues, particularly in the sequence –Ser(P)-Ile/Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- of the CPP, they are superior to statherin in their ability to inhibit demineralisation and stabilise and deliver Ca, Pi and F ions [ ]. As the CPP can deliver bioavailable Ca, Pi and F ions together they are superior to F alone in remineralising enamel subsurface lesions in situ and in vivo [ , ]. This greater remineralisation potential translates to enhanced clinical efficacy in slowing the progression and enhancing the regression of dental caries, which has been demonstrated in multiple independent clinical trials [ , ].
The anticariogenic potential of CPP-ACP has been attributed to the ability of the CPP to localise amorphous calcium phosphate together with F at the tooth surface, thereby helping to maintain a state of supersaturation with respect to tooth mineral [ , ]. Fuji-Triage (VII) containing 3% CPP-ACP, designated Fuji-Triage-EP (VII-EP), has been shown to be superior to Fuji-Triage and other restorative materials in the release of F, Ca and Pi ions and in protecting adjacent tooth tissue from acid demineralisation [ , , ]. Furthermore, the addition of CPP-ACP to Fuji-Triage-EP results in a decreased bacterial biofilm on the surface of that cement when compared with Fuji-Triage without CPP-ACP [ ]. The mechanism of action of the CPP-ACP in decreasing biofilm development was proposed to be related to the highly charged/polar nature of CPP-ACP changing the surface properties of the GIC. However, in none of these studies was the distribution of the CPP-ACP in the set GIC material investigated. Knowledge of the distribution of CPP-ACP in the GIC may help understand the mechanism by which the CPP-ACP enhanced Ca, Pi and F ion release and decreased bacterial biofilm development.
Furthermore, the release of any soluble component of a GIC in vivo will eventually subside and drop below therapeutic levels when the release has exhausted the component concentration in the nanoporosities and exposed surface of the material. However, various studies have shown that nanoporous GICs can act as rechargeable ion release systems (reservoir effect), which may provide a long-term caries inhibitory effect due to enhanced ion release through recharging [ , ]. Recently it was shown that the fluoride levels of Fuji-Triage GIC could be recharged using mouthwashes or slurries of toothpastes containing fluoride [ ]. This demonstrated recharging of the nanoporosities of Fuji-Triage GIC with F suggested that it may be possible to recharge this GIC with CPP-ACP/F nanocomplexes.
The aims of this study were to assess the distribution of CPP-ACP in Fuji-Triage-EP (VII-EP) using immunofluorescence and to determine the effect of CPP-ACP/F recharging upon MI Paste Plus application on surface microhardness (acid resistance) and ion release. The null hypotheses were that CPP-ACP/F recharging by MI Paste Plus application did not change calcium, phosphate and fluoride ion release, and that there was no difference in the surface hardness between different treatment groups.
Materials and methods
GIC block preparation
Fuji-Triage (VII) and Fuji-Triage with added 3% w/w CPP–ACP (Fuji-Triage-EP, also marketed as Fuji-VII EP) were provided by GC Corporation (Tokyo, Japan) in capsule form. Standardised GIC blocks measuring 3 × 6 × 6 mm 3 were created using polyvinyl siloxane impression material (eliteHD + light body, Zhermack SpA, Badia Polesine, Italy) molds as described previously by Zalizniak et al. [ ]. Briefly, the GIC blocks were prepared by placing the materials in the mold with the top and bottom surfaces covered by plastic strips, which were manually pressed between two glass slides. The GIC was allowed to set inside the molds for 24 h in an incubator (37 °C, ∼100% relative humidity). After cooling to room temperature, the blocks were removed from the molds, and the two major parallel surfaces of the blocks were lapped with 600 grit silicon carbide paper (Norton Tufbak, Saint-Gobain Abrasives Ltd., Auckland, NZ) to provide a standardised surface finish [ ].
Immunofluorescence analysis using confocal microscope
To determine the CPP-ACP distribution in blocks of Fuji-Triage-EP and Fuji-Triage, as a negative control, using immunofluorescence, the GIC exposed surfaces of the blocks as well as fractured cross-sectional surfaces were examined. These exposed surfaces were rinsed with Tris-buffered saline (TBS) and then blocked with 1% normal goat serum in TBS. The surfaces were then exposed to 0.2% anti-CPP antibody in TBS and then thoroughly rinsed with TBS. Following the rinsing, the secondary antibody (FITC-conjugated goat anti-rabbit IgG) was applied, followed by further rinsing with TBS. Images were taken with an Axiovert 200 M inverted microscope (Carl Zeiss, Göttingen, Germany) fitted with a Zeiss LSM 510 META Confocal scan head with the 458/477/488 nm Argon laser and a 10X plan apochromatic objective.
Acid challenge and recharging
Thirty blocks of Fuji-Triage and 30 blocks of Fuji-Triage-EP containing 3% w/w CPP-ACP were suspended in an acidic solution over a three-day period as described previously by Mazzaoui et al. [ ]. Each block was individually placed in 5 mL of 50 mM lactic acid (adjusted to pH 5). Every 12 h, one third of the blocks (10 blocks of Fuji-Triage and 10 blocks of Fuji-Triage-EP) were treated with 2 mL of MI Paste Plus (CPP-ACP/F) solution (1 g MI Paste Plus + 4 mL water) for 2 min (MI Paste Plus Treatment). MI Paste Plus contains 10% CPP-ACP and 900 ppm F as sodium fluoride. Another third was treated with placebo MI paste solution (no CPP-ACP/F) at the same dilution and for the same treatment time (Placebo Treatment). The ingredients listed on the MI Paste Plus were water, glycerol, CPP-ACP, d -sorbitol, sodium carboxymethyl cellulose, propylene glycol, silicon dioxide, titanium dioxide, xylitol, phosphoric acid, sodium fluoride, flavouring, sodium saccharin, ethyl p-hydroxybenzoate, magnesium oxide, guar gum, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate. The placebo MI Paste contained the same ingredients without the CPP-ACP and sodium fluoride. The last third of the GIC blocks was placed in distilled water for 2 min (No Treatment Control). After each 2 min treatment the blocks were gently rinsed for 30 s with distilled water and immediately placed back into the acid solution without drying. Treatments were delivered every 12 h and the three day period was commenced with a 2 min pre-treatment (MI Paste, Placebo MI Paste or water as described above) applied after the blocks were lapped but before being placed into the acid solution for the first time. The acid solutions were changed every 24 h and were analysed for ion release. The surface hardness of the GIC blocks was determined before and after the treatment period.
Ion release measurement
Calcium (Ca), inorganic phosphate (Pi) and fluoride (F) levels in the lactic acid solution were measured using atomic absorption spectrophotometry (Ca), colorimetry (Pi) and ion specific electrode (F) respectively, as described in a previous study [ ]. Briefly, to determine the Ca concentration sample solutions (1 mL) were acidified with 1 M HCl (0.5 mL) and diluted with 2% lanthanum chloride (0.5 mL) and analysed on a Varian AA240 atomic absorption spectroscope (Varian Australia Pty. Ltd.) against a set of seven standards ranging from 0 to 250 mM calcium. Pi concentrations were determined colorimetrically using a spectrophotometer (UV–vis spectrophotometer, Varian Australia, Pty. Ltd.). The samples that were analysed were prepared by taking 100 mL of solution, diluting with 500 mL of 4.2% ammonium molybdate and adding 20 mL 1.5% of Tween 20 (Sigma–Aldrich, St. Louis, MO). The Pi concentration was determined by comparing the spectrophotometer readings of the samples against a set of seven standards ranging in Pi concentrations from 0 to 100 mM. The concentration of F ions was determined using an ion selective electrode (Radiometer analytical, ISE C301 F, Lyon, France) connected to an ion analyser (Radiometer analytical, Ion Check 45, France). Sample solutions (1 mL) were diluted with 1 mL total ionic strength adjustment buffer (Merck Pty. Ltd., Kilsyth, VIC, Australia) and measured against a set of eight F standards ranging from 0 to 1000 μM.
Vickers hardness measurements for the GIC blocks before and after treatments over a three-day period were determined using a Microhardness Tester (MHT-10, Anton Paar GmbH, Graz, Austria) attached to a microscope (Leica DMPL, Leica Microsystems Wetzlar GmbH, Germany) as described previously [ ]. Three indentations separated by a distance of at least three times the indentation size were made on each block (force, 1.0N; dwell, 6 s; rate, 0.99 N/min). Images of the indentations were acquired through a calibrated digital camera (Leica DFC320) mounted on the microscope and distance measurements made using Image Tool software (Version 3.0, UTHSC, San Antonio, TX) which were then converted into Vickers hardness values.
Differences in ion levels released and Vickers surface hardness values between Fuji-Triage and Fuji-Triage-EP were analysed using a two-sample t-test. Differences in mean ion levels and Vickers hardness measurements for each of the two GIC materials across the three treatments were measured using one-way ANOVA with a Bonferroni-Holm correction for multiple comparisons. All analyses were conducted using statistical software (Stata version 14.2; StataCorp LLC., College Station, USA).
Immunofluorescence image analysis using confocal microscopy demonstrated general distribution of the CPP-ACP throughout Fuji-Triage-EP by the presence of intense red fluorescent staining at the exposed GIC surfaces as well as along fractured cross-sectional surfaces ( Fig. 1 ). Fluorescent staining was absent in the negative control GIC Fuji-Triage which did not contain CPP-ACP ( Fig. 1 ).