Vascular smooth muscle cell damage is a key step in inducing vascular calcification that yields hydroxyapatite (HAP) as a major product. cytotoxicity of each crystal was positively correlated with the following factors: large specific surface area, high electrical conductivity and low surface charge. HAP accelerated calcium deposits on the A7R5 cell surface and induced the expression of osteogenic proteins, such as BMP-2, Runx2, OCN, and ALP. The crystals with high cytotoxicity caused more calcium deposits on 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 Marimastat theoretical references for decreasing the risks of vascular calcification. strong class=”kwd-title” 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 smooth muscle cells (VSMCs)2. During calcification process, when enough calcium and phosphorus ions accumulate in the matrix vesicles, it will lead to the deposition of calcium phosphate, which 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 CCND2 morphology due to different growth environments and different pathological conditions; that is, they appear round, spherical, needle, rod, and laminated particles4C7. Villa-Bellosta em et al /em .6 found that HAP is the only crystalline phase in the calcium and phosphate deposition of lysed and living cells. Rounded crystallites (5C10?nm) exhibiting a random orientation were existed in lysed cells, Marimastat while the deposits in living cells were composed of 10?nm thick long fiber crystals embedded in an amorphous matrix. Liu em et al /em .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 the 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 em et al /em .7 classified calcific 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 Marimastat 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 smooth muscle cells, thereby decreasing cell viability, accumulating apoptotic bodies at mineralization sites, and accelerating vascular calcification11. Ewence em et al /em .14 reported CaP crystals induce cell death in human aortic SMCs 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 em et al /em .12 cultured mouse aorta vascular smooth muscle cells (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 em et al /em .10 showed that HAP induces the osteogenic transformation of rat aortic smooth 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 smooth 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 smooth muscle cells caused by nano-HAP crystals with different sizes and shapes have rarely been reported. In this study, the effects of the differences in the morphological characteristics of nano-HAP on rat aortic smooth muscle cell (A7R5) injury and its phenotypic transformation were investigated to provide a basis for determining the effects of the physicochemical properties.