Evaluation of calcium release and pH value of light-cured cavity liners for pulp-capping materials

Introduction: Cavity liners based calcium hydroxide present low strength and high solubility that is consider a disadvantage. In order to enhance these properties it was developed a light-cured cavity liner based calcium hydroxide containing resin. Objective: The purpose of this study was to evaluate the pH and calcium release of light-cured cavity liners. Material and method: There were prepared specimens (n=10) with the Ultra-Blend  plus, Biocal (light cured cements) and Hydro C (control). The samples were stored in 10 mL of distilled water and maintained at 37 °C. After 24 hours, 7, and 14 days, there were analyzed pH and the release of calcium levels. The data were statistically analyzed by ANOVA and Tukey test (α = 0.05). Result: The results of pH showed Hydro C > Ultra-Blend plus > Biocal. Biocal presented worst calcium release. Conclusion: Light-cured materials present lower calcium release and alteration in pH values. Descriptors: Calcium hydroxide; dental cavity lining; dydrogen-ion concentration.


INTRODUCTION
The use of cavity liners has been recommended in order to prevent the diffusion of residual and/or leached components through the dentin tubules.These materials are also used to overcome the problems related to the potential toxicity of dentin bonding agents when applied in deep cavities [1][2][3] .Cavity liner based calcium hydroxide has been largely used as a powerful cavity liner [4][5][6] .
Calcium hydroxide in cavity liners dissociate into ions calcium and hydroxyl, which are responsible for the antimicrobial properties and induction of mineralization 7,8 .The calcium ions release is essential for the mineralization process, subsequently it promotes cell migration and differentiation 9,10 while the hydroxyl ions provide high pH (about 12), and promotes enzymatic inhibition of microorganisms 6,11 .Those hydroxyl ions also release alkaline phosphatase, which participates in the mineralization process 10,11 .Therefore, the use of calcium hydroxide on the pulp tissue promotes repair 12 and subsequent formation of dentin bridge 4,13 .
Cavity liners must be resistant to solubility in water, to organic solvents of acid etching, exhibit resistance to support the restoration, and resist to occlusal forces during mastication 14 in order to protect the pulp 15  and restorative materials 16,17 , in addition presents difficulty in manipulation and application 18,19 .
With the purpose of improving the properties of conventional calcium hydroxide cavity liners, were developed resin-based cavity liners containing calcium hydroxide.These materials are light-cured 20 , highly resistant to etchants 21 , present superior physical properties, and handling characteristics 14 .Currently, there are no studies showing if resin-based cavity liners with calcium hydroxide present alteration in pH and release of calcium ions when compared to conventional cavity liners.Thus, the aim of the present investigation was to evaluate pH and calcium release of resin-based cavity liners light-cured with calcium hydroxide.The null hypothesis was that all cements showed similar calcium release and pH change.

Analysis of pH and Calcium Release
Ten samples of each material were prepared using polyethylene tubes (n=10).The tubes with dimensions of 10 mm in height and 1 mm in diameter were filled with the cavity liner based in calcium hydroxide (Table 1).Hydro C was evaluated after cured and Ultra-Blend plus and Biocal  were evaluated light-cured and not light-cured.The samples of Ultra-Blend  plus and Biocal  were light-cured with a light-curing unit (1200 mW/cm 2 ) during 20 seconds, as recommended by the manufacturer.The tubes filled with materials were immersed in 10 mL of distilled water and stored in individual flasks at 37 °C at a relative humidity 100% in 24 hours, 7 and 14 days.After each period the tubes were removed and transferred to new flasks with 10 mL of distilled water.The solutions contained in the flask were used after each experimental period to determine pH and release of calcium (Ca 2+ ), based on the methodology used by Santos et al. 22 .The measurement of pH was performed by digital pHmeter (Q-400 Quimis instrument, São Paulo, SP, BR), which was previously calibrated with buffer solutions (pH 4.0 and 7.0).To evaluate the release of calcium was used atomic absorption spectrophotometry (Spectraa 55B -Varian, Inc., Palo Alto, CA, EUA).Lanthanum oxide was added to all samples to eliminate ionic interference specific for phosphate ions.Solutions containing calcium concentrations of 0, 1, 2, 3, 4 and 5 ppm were used for calibration.The data of pH and calcium release were subjected to parametric tests ANOVA and Tukey post-hoc, with 5% of significance level.

RESULT
Table 2 shows the results of the pH test.Hydro C obtained the highest pH values in all experimental periods (p<0.05).Ultra-Blend plus showed higher pH than Biocal (p<0.05).After 24 hours, Ultra-Blend plus which was not light-cured showed higher pH values than the same material photo polymerized (p<0.05).On 7 and 14 days, Ultra-Blend plus light-cured and not light-cured showed no significantly difference (p<0.05).Biocal, which was light-cured, showed acid pH during all experimental periods.Table 3 shows the results obtained in the evaluation of calcium release.Ultra-Blend plus not cured released more calcium when compared to Ultra-Blend plus cured, Hydro C and Biocal (cured and not cured) after 24 hours and 7 days (p<0.05).In 14 days, Hydro C showed higher release of calcium than the others cavity liners (p<0.05).Ultra-Blend plus not cured showed intermediate values when compared with the others.Biocal cured and not cured had the lowest calcium release, with no significant difference from Ultra-Blend plus cured in 14 days (p<0.05).

DISCUSSION
A conventional calcium hydroxide and two resin-based cements (cured and not cured) were used as lining materials and were evaluated in terms of calcium release and pH change.The null hypothesis was rejected, because all cements showed different calcium release and pH values during the evaluated periods.
Calcium hydroxide cements as Dycal  and Hydro C  are the most commonly conventional lining materials used in direct and indirect pulp capping, because they present alkaline pH, biocompatibility, induce pulp-dentin mineralization, and reduce infection 23,24 .
Lining materials should remain on the interface between the tooth and the restoration, some of these conventional materials based with calcium hydroxide has shown high water sorption and solubility 14 the cavity liners containing resin components were created to improve those mechanical properties.
In the present study, Hydro C, showed high pH values (p<0.05) and had an increase of calcium release throughout the evaluation.This data is similar with the literature 25 , where the principal font of ions release is calcium hydroxide, but calcium silicate cements release more hydroxyl and calcium ions, which were not evaluated in this study.
Non-cured Ultra-Blend plus presented superior calcium release than Hydro C, cured Ultra-Blend plus and Biocal groups after 24 h and 7 days, despite the lower pH values compared to Hydro C. Currently, cavity liners containing resin components need to be cured and this causes a reduction in release of calcium ions.Therefore, in comparison with other study 25 , Dycal present lower calcium release than calcium silicate materials, in neutral pH.This could be compared with the superior calcium release of non-cured Ultra-Blend plus in lower pH value.
Cured Ultra-Blend plus presented lower calcium release than Hydro C during the first period (24h), but after 7 days both did not obtain statistically significant difference.Although Duarte et al. 10 has analyzed calcium release of Ultra-Blend and Hydro C with spectrophotometry through atomic absorption and a calcium cathode lamp, Ultra-Blend presented higher value than Hydro C for 24 h and 7 days.Duarte et al. 10 attributed this value to the presence of hydroxyapatite in the material, which could be released and detected by spectrophotometer.The different methodology of this study might have shown distinct results.
Cured Ultra-Blend plus showed neutral pH (around 7.50) without significant difference after 7 and 14 days than the same material non-cured.However, Ultra-blend plus non-cured was better in the first period analyzed (24 h).In addition Ultra-blend plus cured and non-cured were better than Biocal throughout the experiment (p<0.05).Biocal cured had acid pH in this study.
The difference of ions release could be observed in vitro, however that may not occur in vivo.Further studies are being carried out to evaluate the interaction between the calcium release and pH change,

CONCLUSION
Within the limitations of this study, it is concluded that: 1-Hydro C had the highest pH rates; 2-Hydro C had an increase of calcium release during the experiment; 3-Ultra-Blend not cured had better calcium release than others materials for 24h and 7 days; 4-Ultra-Blend not cured had better pH and calcium release than Ultra-Blend cured; 5-Biocal cured had the lowest pH value and the worst calcium release for all experimental period.

Table 1 .
Materials used in this study

Table 2 .
pH values recorded at different time periods (mean and SD)

Table 3 .
Calcium release recorded over different periods of time (mean and SD) ,b,c,d The different letters in the same line indicate significant differences between groups (p<0.05).Pereira, Cruvinel, Dantas et al.Rev Odontol UNESP.2018 July-Aug.;47(4): 205-209 208 208/209 also mechanical tests should be added for a better understand of dental cavity liners. a