Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells.

Objective: This study aimed to investigate the damage mechanism of nanosized hydroxyapatite (nano-HAp) on mouse aortic smooth muscle cells (MOVASs) and the injury-inhibiting effects of diethyl citrate (EtCit) and sodium citrate (NaCit) to develop new drugs that can simultaneously induce anticoagulation and inhibit vascular calcification.

Methods: The change in cell viability was evaluated using a cell proliferation assay kit, and the amount of lactate dehydrogenase (LDH) released was measured using an LDH kit. Intracellular reactive oxygen species (ROS) and mitochondrial damage were detected by DCFH-DA staining and JC-1 staining. Cell apoptosis and necrosis were detected by Annexin V staining. Intracellular calcium concentration and lysosomal integrity were measured using Fluo-4/AM and acridine orange, respectively.

Results: Nano-HAp decreased cell viability and damaged the cell membrane, resulting in the release of a large amount of LDH. Nano-HAp entered the cells and damaged the mitochondria, and then induced cell apoptosis by producing a large amount of ROS. In addition, nano-HAp increased the intracellular Ca concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant EtCit or NaCit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et Cit and NaCit could also chelate with Ca to inhibit the intracellular Ca elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of EtCit and NaCit exhibited strong inhibitory effects. The inhibitory capacity of NaCit was stronger than that of EtCit at similar concentrations.

Conclusion: Both EtCit and NaCit significantly reduced the cytotoxicity of nano-HAp on MOVASs and inhibited the apoptosis and necrosis induced by nano-HAp crystals. The chelating function of citrate resulted in both anticoagulation and binding to HAp. EtCit and NaCit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp.