Experimental results were analyzed statistically using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). with high cytotoxicity caused more calcium deposits around the cell surface, higher expression levels of osteogenic protein, and stronger osteogenic transformation abilities. These findings elucidated the relationship between crystal shape and cytotoxicity and provided theoretical recommendations for decreasing the risks of vascular calcification. Subject terms: Bioinorganic chemistry, Cell death, Risk factors Introduction Vascular calcifications (VCs) are actively regulated biological processes associated with hydroxyapatite (HAP) crystallization in the extracellular matrix and in middle and intimal cells of the arterial wall1. VCs are highly regulated cell-mediated processes, which possess many similarities to bone formation. The center cells of calcification process are vascular easy muscle cells (VSMCs)2. During calcification process, when enough SKI-II calcium and phosphorus ions accumulate in the matrix vesicles, it will lead to the deposition of calcium phosphate, which SKI-II will then be converted into octacalcium phosphate and finally converted into insoluble HAP, and HAP repeats nucleation and crystallization in the same approach and expands the deposition area3. Precipitate complexes formed in biological tissues exhibit distinct polymorphic morphology due to different growth environments and different pathological conditions; that is, they appear round, spherical, needle, rod, and laminated particles4C7. Villa-Bellosta et al.6 found that HAP is the only crystalline phase in the SKI-II calcium and phosphate deposition of lysed and living cells. Rounded crystallites (5C10?nm) exhibiting a random orientation were existed in lysed cells, while the deposits in living cells were composed of 10?nm thick long fiber crystals embedded in an amorphous matrix. Liu et al.5 obtained and analyzed pellets isolated from the serum of uremia patients through SEM. The pellets have laminated shapes and crystallized needle-like projections (30C500?nm). EDS analysis has demonstrated that this consist of obtained pellets are similar to those of HAP precursor and indicative of CaP crystals, whereas no detectable particles are found in normal serum. Fully mineralized vesicles in tissues with atherosclerosis are composed of numerous spherical and needle-shaped mineral deposits4. Chiou et al.7 classified calcific HNPCC depositions into arc, fragmented or punctuated, nodular, and cystic shapes based on ultrasonographic findings. Many studies8C14 have confirmed that HAP crystals cause damage to VSMCs and induce cell phenotype transformation, which in turn promote vascular calcification. For example, exogenous calcifying nanoparticles, which are nanosized complexes of CaP mineral and proteins, are endocytosed by aortic clean muscle cells, thereby decreasing cell viability, accumulating apoptotic bodies at mineralization sites, and accelerating vascular calcification11. Ewence et al.14 reported CaP crystals induce cell death in human aortic SMCs SKI-II depending on their size and composition. However, the effects of the morphological characteristics of HAP crystals on cytotoxicity and vascular calcification have not been reported. The size and morphological characteristics of crystals are two important physical parameters that affect cytotoxicity. Sage et al.12 cultured mouse aorta vascular easy muscle cells SKI-II (MASMCs) with different concentrations of nano-HAP for 24?h and found that crystals stimulate the osteogenic transformation of MASMCs in a concentration-dependent manner. Nahar-Gohad et al.10 showed that HAP induces the osteogenic transformation of rat aortic easy muscle cells through CaSR- and bone morphogenetic factor-2 (BMP-2)-mediated pathways, thereby leading to the increased expression of the following osteogenic markers: Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN). The inhibitory mechanisms of diethyl citrate (Et2Cit), sodium citrate (Na3Cit), and phosphonoformic acid in calcification induced by high Pi in mouse aortic easy muscle cells (MOVAS) have been investigated15. The damage mechanism of nanosized HAP on MOVAS and the inhibitory effects of the anticoagulants Et2Cit and Na3Cit on injury have been explored16. Differences in damage to easy muscle cells caused by nano-HAP crystals with different sizes and shapes have rarely been reported. In this study, the effects of.