Synthesis, characterization, and cytotoxicity assay of calcium oxalate dihydrate crystals in various shapes

Abstract

Various morphologies of calcium oxalate dihydrate (COD) crystals containing cross-shaped, flower-like, thin-bipyramid, thick-bipyramid, and elongated-bipyramid structures, with a size of approximately 5 μm, were prepared by varying the reactant concentration, reaction temperature, stirring speed, and additive. X-ray diffraction (XRD) and infrared spectroscopy (FT-IR) showed that the synthesized crystals were pure-phase COD crystals. The factors affecting the morphology of COD and the mechanism of COD formation were discussed. Physicochemical properties such as specific surface area, pore structure, pore size, zeta potential, and conductivity of the crystals were identified. The cytotoxicity of five different shapes of COD crystals in human kidney proximal tubular epithelial (HK-2) cells was investigated using the Cell Counting Kit-8 (CCK-8) assay and propidium iodide (PI) staining assay. The cytotoxicity was ranked in the following order: COD-elongated-bipyramid > COD-thick-bipyramid ≈ COD-cross-shaped > COD-thin-bipyramid > COD-flower-like. The toxicity of tetragonal bipyramid COD crystals was closely correlated with the area of the Ca²⁺ ion-rich (100) face; the crystals with large (100) faces showed a higher toxicity to HK-2 cells. The cytotoxicity of cross-shaped COD and flower-like COD was affected by the sharpness of their crystal edges; COD-CS with sharp corners caused more serious injuries to cell membranes. These findings are helpful in understanding the effects of mineralization conditions and additives on crystal morphology as well as to identify the relationship between crystal morphology and cytotoxicity. The results also provide a reference for the injury difference of various shapes of crystals from normal subjects and stone patients to the renal epithelial cells and the formation mechanism of calcium oxalate stones.