The pH-influence on Activation of Endogenous Enzymes From Human Dentin

Objectives: The aim of this in vitro study was to evaluate the pH-influence on the activation of human coronal and root dentin MMPs.
Methods: Coronal (CR) and root dentin (RD) from recently extracted human molars were pulverized to powder and the proteins were extracted with 1% phosphoric acid (10min, 4⁰C). Then, the extract containing proteins and the partially demineralized powder were separately incubated in one of the following solutions: APMA (control) or a buffer solution with different pHs (2.5, 4.5, 5.0, 6.0 and 7.0). After the incubation, the proteins were separated by electrophoresis in a polyacrylamide gel co-polymerized with gelatin to quantify MMPs activities, by means of zymography. To assess the solubilized dentin collagen, the partially demineralized dentin powder treated with different pHs was incubated in an artificial saliva (24h, 37⁰C) and the amount of hydroxyproline (HYP) released in media was measured.
Results: Zymography revealed gelatinolytic activities corresponding to MMP-2, for both CD and RD, in all experimental groups, demonstrating that MMPs activity is pH-dependent. No form of MMP-9 was identified. For both substrates, the lowest pH solutions (2.5, 4.5 and 5.0) yielded the higher gelatinolytic activity than did the highest pH solutions (6.0 and 7.0). For HYP analysis, the groups pH2.5 and pH4.5 showed absorbance values below the detection limit of the equipment. For the other groups, for both coronal and root dentin, statistically significant diferences were found between them (p<0.05). The amount of HYP was higher for pH7.0 than all other groups (p<0.05), except for pH6.0. No statistical difference was found between pH6.0, pH5.0 and control (p>0.05).
Conclusions: Low pH solutions are able to increase the activation of human coronal and root MMP-2. On the other hand, in pHs close to neutral there is a higher degradation of demineralized dentin organic matrix by these endogenous enzymes.