The aim of this study is to investigate how hydrogen peroxide (HP) treatments affect enamel porosity and protein structure, as well as their impact on tooth shade and hardness over time. Fifty healthy teeth were collected from adult patients. Teeth were randomly divided into 2 groups, the first was incubated with 30 % HP while the second, the control group, was incubated with distilled water (DW). Tooth shade and enamel microhardness were evaluated using a digital spectrophotometer and a Vickers tester, respectively, at different time intervals after treatment. The specific surface area of the enamel was measured using a surface area analyzer and the Brunauer-Emmett-Teller (BET) equation. Protein structure was analyzed using circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). Shade analysis revealed that, in the short term, HP treatment significantly increased lightness and Hue and decreased chroma compared to DW (p < 0.05); however, 1 week after treatment some of the initial gains in tooth lightness were partially lost. Hardness analysis revealed significant decreases in microhardness in the bleached group compared to the control group (p < 0.05). BET analysis revealed that HP treatment increased enamel surface area and reduced its average pore size. CD and DLS analyses showed that proteins from HP-treated teeth mostly adopted a non-random conformation and had smaller average protein sizes compared to the control. HP treatment lightened tooth shade and significantly reduced enamel microhardness over time. This could be related to changes in surface area and porosity caused by denaturalization of the enamel proteins. Our findings showed that HP induced denaturation of enamel proteins, resulting in increased enamel porosity and reduced microhardness. These changes in enamel properties could help explain clinical complications observed with these treatments such as increased sensitivity.

Objective: The plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP) – rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore ectopic mineralization (calcification). The mutant mouse strain D2, Ahsg-/- combines fetuin-A deficiency with the mineralization-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue mineralization. Here we studied mechanisms leading to soft tissue mineralization, organ damage and premature aging in these mice. Approach and Results: We analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice. Fetuin-A deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature aging. Importantly, early stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the ectopic mineralization was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis. Conclusions: Collectively, these results demonstrate that pathological mineralization can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of mineralized matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.