Sustainable production of osteoinductive Co2+, Mg2+ and Mn2+ -substituted apatites particles by one-pot conversion of biogenic calcium carbonate

Biogenic CaCO₃ microparticles obtained from oyster shells Crassostrea gigas were used as starting material for synthesizing Co²⁺, Mg²⁺ and Mn²⁺-doped apatite nano-submicroparticles, through a one-step hydrothermal conversion. The conversion was completed at 200 °C for 7 days, yielding metal-doped apatite and whitlockite in percentages of 5.3 wt% when adding Co²⁺, 28.7 wt% for Mg²⁺, and 0 wt% for Mn²⁺. Samples were cytocompatible with murine pancreatic endothelial cells (MS1), murine mesenchymal stem cells (m17.ASC), and murine osteoblast's progenitors (mOBPs) cells. The analysis by flow cytometry and TEM-EDX revealed strong particle-cell interactions, sustained internalization across m17.ASC and mOBPs cells, and potential progressive apatite dissolution in the cellular environment. Additionally, incubating these cells with the metal-doped samples promoted their osteogenic differentiation without needing an osteogenic differentiation medium. Indeed, the evaluation of gene expression by quantitative real-time PCR, the detection of alkaline phosphatase activity, and the ability to induce the mineralization in the cellular matrix analyzed by alizarin red staining revealed that all particles (and particularly the carbonated apatite and the Mg-doped sample) encouraged the osteogenic commitment. This approach represents a sustainable way to valorize and transform aquaculture and canning industries' mineral waste (shells) in highly demanded osteoinductive materials.