Cellular Effects and Medium-Induced Transformations of Non-Apatitic Calcium Phosphates in Relation to Breast Tumor Microcalcifications

Abstract Breast tumor microcalcifications (MCs), primarily composed of calcium phosphates (CaPs), exhibit significant heterogeneity in composition, crystallinity, and ionic substitution. To date, apatite and magnesium-rich whitlockite are the only CaP phases identified in clinical breast tissue samples, while other phases potentially related to pathological biomineralization, such as brushite and monetite, were reported only for other pathologies. Magnesium is frequently incorporated in CaPs formed under physiological or pathological conditions, where it strongly influences crystal structure, crystallinity, solubility, and surface chemistry. Expanding the understanding of magnesium effects beyond apatite-based systems is therefore essential to elucidate phase-dependent cellular behavior. Here, we engineered a panel of synthetic CaP phases, including brushite, low- and high-Mg-doped brushite, whitlockite, monetite, and Mg-doped monetite, which serve as simplified model CaP systems to investigate structure–property–cellular response relationships. Characterization in the cell-free medium revealed Mg/Ca-dependent morphological variations, partial dissolution, reprecipitation, and surface modifications prior to cellular exposure, indicating that cells interact with a dynamic, medium-transformed material system. Upon interaction with non-tumorigenic and precancerous human breast epithelial cells, cellular responses were dependent on both crystal phase and malignancy stage. Under the tested conditions, monetite and high-Mg brushite led to a lower relative proliferation rate in precancerous cells compared to non-malignant cells. Within brushite samples, higher Mg content correlated with reduced proliferation in precancerous cells, whereas this trend was absent for monetite, suggesting that particle effects depend on both chemical composition and crystal phase. These findings demonstrate that magnesium incorporation into non-apatitic CaP phases differentially regulates breast epithelial cell behavior, supporting the view that minerals function as active components of the microenvironment rather than passive deposits.