Abstract: BACKGROUND:Nano-hydroxyapatite has excellent biocompatibility, biological activity, and modifiability, but the synthesized nano-hydroxyapatite has a high specific surface energy, which makes it agglomerate in solution.
OBJECTIVE: To compare the effects of ultrasound and different dispersants on the aggregation, cytotoxicity and intracellular distribution of nano-hydroxyapatite. 
METHODS: Needle like nano-hydroxyapatite was prepared by chemical precipitation method, and kept for further use after high pressure sterilization. Different concentrations of dispersant sodium hexametaphosphate (0, 0.25, 0.5, 1, 2 mmol/L), sodium citrate (0, 0.25, 0.5, 1, 2 mmol/L), sodium polymethacrylate (0%, 0.0625%, 0.125%, 0.25%, 0.5%) were co-cultured with MC3T3E1 cells. Cell proliferation was detected by CCK-8 assay. The appropriate concentration of dispersant was screened for subsequent experiments. The nano-hydroxyapatite solution was divided into five groups: the control group was not dispersed; and the remaining four groups were subjected to ultrasonic dispersant, 1 mmol/L sodium hexametaphosphate dispersant, 1 mmol/L sodium citrate dispersant, and 0.125% sodium polymethacrylate dispersant. The aggregate size of nano-hydroxyapatite was detected. The hydroxyapatite was treated with ultrasound, 1 mmol/L sodium hexametaphosphate, 1 mmol/L sodium citrate, and 0.125% sodium polyacrylate dispersants and then co-cultured with MC3T3E1 cells. Undispersed nano-hydroxyapatite co-cultured with MC3T3E1 cells was considered as control. Cell proliferation was detected by CCK-8 assay. At 1 day after culture, the distribution of nano-hydroxyapatite was observed by transmission electron microscope.
RESULTS AND CONCLUSION: (1) Sodium hexametaphosphate at a concentration of 1 mmol/L and lower had no obvious cytotoxicity. Sodium citrate at 0.25-2 mmol/L had no obvious cytotoxicity. Sodium polymethacrylate at different concentrations had certain cytotoxicity in time- and concentration-dependent manners. Subsequent experiments selected 1 mmol/L sodium hexametaphosphate, 1 mmol/L sodium citrate, and 0.125% sodium polyacrylate for dispersion treatment. (2) All three dispersants significantly reduced the agglomeration size of nano-hydroxyapatite, and sodium hexametaphosphate had the best dispersion effect. (3) The dispersion method and the addition of dispersant could significantly affect the biological function of nano-hydroxyapatite. Sodium citrate promoted the proliferation of cells co-cultured with nano-hydroxyapatite. Ultrasound and sodium polymethacrylate inhibited proliferation of cells co-cultured with nano-hydroxyapatite. (4) Transmission electron microscopy showed that in the control group and the ultrasound group, large agglomerates of nano-hydroxyapatite existed inside and outside the cell; the nano-hydroxyapatite dispersed by the three dispersants also had agglomeration, but the size of the agglomeration was significantly smaller. Among them, the nano-aggregation size of the sodium hexametaphosphate group was the smallest. Some nanoparticles were wrapped in a double-layer membrane in the cell to form a structure similar to “small vacuoles”. (5) The results show that the commonly used dispersants themselves have certain cytotoxicity, and the addition of dispersants will significantly reduce the agglomeration size of nano-hydroxyapatite and its intracellular distribution, and affect cell proliferation.

Key words: bone, material, nano-hydroxyapatite, agglomerates, disperse, dispersant, ultrasound, bone grafting materials, cytotoxicity, cell distribution, cell proliferation