Oxidized Low-Density Lipoprotein Promotes In Vitro Calcification

Abstract

Calcification plays an important role in the human body in maintaining homeostasis. In the human body, the presence of a high amount of oxidized low-density lipoprotein (ox-LDL) is a consistent feature of the local areas that are common sites of ectopic calcification, namely dental calculus, renal calculus, and the areas affected by arteriosclerosis. Hence, ox-LDL may have some effect on calcification. Scanning electron microscopy (SEM) observation revealed a high amount of amorphous calcium phosphate (ACP) when ox-LDL was included in the solution. In the in vitro experiment, the highest amount of precipitation of calcium phosphate was observed in the solution containing ox-LDL compared to the inclusion of other biomaterials and was 4.2 times higher than that of deionized water for 4.86 mM calcium and 2.71 mM phosphate. The morphology of calcium phosphate precipitates in the solution containing ox-LDL differed from that of the precipitates in solutions containing other biomaterials, as determined by transmission electron microscopy (TEM). Through the time course observation of the sediments using TEM, it was observed that the sediments changed from spherical or oval shape to a thin film shape. These results indicate that sediments acquired a long-range order array, and the phase transitioned from non-crystalline to crystalline with an increased time and density of ACP. Thus, it is concluded that ox-LDL promoted ACP precipitation and it plays an important role in ectopic calcification.

Keywords: amorphous calcium phosphate; calcification; crystallization; dentin; oxidized low-density lipoprotein.

Figure 1

SEM image of the calcium phosphate precipitate with biomaterials. (a) Precipitate from 4.86 mM calcium and 2.71 mM phosphate without biomaterials; (b) 4.86 mM calcium and 2.71 mM phosphate with 0.025% oxidized low-density lipoprotein (ox-LDL); (c) 4.86 mM calcium and 2.71 mM phosphate with 0.025% dextran sulfate; (d) 23.66 mM calcium and 13.18 mM phosphate without biomaterials; (e) 23.66 mM calcium and 13.18 mM phosphate with 0.025% ox-LDL; and (f): 23.66 mM calcium and 13.18 mM phosphate with 0.025% dextran sulfate.

Figure 2

Effect of the biomaterials on calcium and phosphate reaction. Absorbance values were measured at a wavelength of 650 nm. DW: Deionized water, LDL: Low-density lipoprotein, HDL: high-density lipoprotein, DHA: Docosahexaenoic acid, EPA: Eicosapentaenoic acid, LPS: lipopolysaccharides.

Figure 3

Effect of the concentration of biomaterials on the calcium and phosphate reaction. Absorbance values were measured at a wavelength of 650 nm.

Figure 4

Observation of calcium phosphate precipitation by transmission electron microscopy. Calcium and phosphate concentrations were 12.02 mM and 6.70 mM. The concentrations of biomaterials were 0.025%. (e)–(g) are high magnifications of the white squares in (b)–(d); (a) without biomaterials; (b) oxidized low-density lipoprotein (ox-LDL); (c) albumin,; (d) dextran sulfate; (e) ox-LDL; (f) albumin; (g) dextran sulfate. Magnifications: (a) 10,000× (b)–(g): 30,000×.

Figure 5

TEM images of the precipitate with 0.025% ox-LDL: (a) 12.02 mM calcium and 6.70 mM phosphate; (b) 39.33 mM calcium and 21.92 mM phosphate.

Figure 6

Time course analysis of the crystal shape in the solution containing 0.025% ox-LDL by transmission electron microscopy: (a) 5 min; (b) 30 min; (c) 24 h.