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The aim was to validate a novel protocol to measure the cariostatic efficacies of demineralization inhibitors by repeating previous SMR (scanning microradiography) studies investigating the dose response of Zn²⁺ and F^– on demineralization kinetics in vitro using real-time Ca²⁺ ion selective electrodes (ISEs). In this study, Ca²⁺ release was used as a proxy for the extent of demineralization. Forty-eight hydroxyapatite (HAP) discs were allocated into 16 groups (n = 3) and adding either increasing [Zn²⁺], or [F^–], similar to those used in the previous SMR studies. Each HAP disc was immersed in 50 mL, pH 4.0, buffered acetic acid for 1 h, and real-time ISE methodology was used to monitor the rate of increase in [Ca²⁺] in the demineralization solution. Next, either zinc acetate or sodium fluoride was added into each demineralization solution accordingly. Then after each [Zn²⁺] or [F^–] addition, the HAP disc was further demineralized for 1 h, and ISE measurements were continued. The percentage reduction in the rate of calcium loss from hydroxyapatite (PRCL_HAP) at each [Zn²⁺] or [F^–] was calculated from the decrease in Ca²⁺ release rate, similar to that used in the previous SMR studies. A log-linear relationship between mean PRCL_HAP and inhibitor concentration was found for both Zn²⁺ and F^–, similar to that reported for each ion in the previous SMR studies. In conclusion, real-time Ca²⁺ ISE systems can be used to measure the cariostatic efficacies of demineralization inhibitors.

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