Size-Dependent Cytotoxicity of Hydroxyapatite Crystals on Renal Epithelial Cells

Figures & data

Table 1 Basic Physical and Chemical Properties of HAP with Different Sizes

Crystal Type	Crystal Size*	Zeta Potential in Water/mV	Zeta Potential in Culture Medium/mV	Conductivity in Water/mS/cm	Conductivity in Culture Medium mS/cm	Specific Surface Area SBET/m^2/g	Pore Volume /mm^3/g	Pore Size /nm
HAP-40nm	37.94±8.70 nm	−4.00	−3.21	0.53	14.43	76.2	29.2	6.77
HAP-70nm	71.27±5.37 nm	−8.19	−4.14	0.42	14.13	67.0	4.19	35.3
HAP-1µm	0.96±0.19 µm	−15.00	−13.94	0.21	13.60	6.16	0.003	1.37
HAP-2µm	1.92±0.59 µm	−29.28	−24.08	0.16	12.70	1.18	0.001	6.67

Note: *The average crystal size was determined by Nano Measurer Software from the SEM images.

Figure 1 SEM images of HAP with different sizes (A). Cell viability (B) and LDH release (C) of HK-2 cells after injury by HAP crystals with different concentrations and sizes for 24 h. (**P<0.01, *P<0.05 vs control); Crystal concentration: 63, 125, 250, 500 μg/mL, respectively; treatment time: 24 h. Scale bars: 200 nm (HAP-40nm, HAP-70 nm), 1 μm (HAP-1μm, HAP-2μm).

Figure 2 Cell morphology (A) and cytoskeleton (B) of HK-2 cells after injury by HAPs with different sizes. Crystal concentration: 250 μg/mL; treatment time: 24 h. Scale bars of cell morphology: 50 µm (400x). Scale bars of cytoskeleton: 20 μm (Merged images, 630x); 5 μm (Enlarged images).

Figure 3 Intracellular ROS levels of HK-2 cells after injury by HAPs with different sizes. (A) Qualitative observation of ROS; (B) Flow cytometry analysis results of ROS; (C) ROS quantitative histogram. Crystal concentration: 250 μg/mL; treatment time: 24 h. Scale bars: 50 μm (400x).

Figure 4 Flow cytometry analysis of internalized HAP crystals (A) and intracellular Ca²⁺ ion concentration [Ca²⁺]_i (B) in HK-2 cells after injury by HAPs with different sizes. Quantitative analysis results of internalized HAP crystals (C) and [Ca²⁺]_i (D). The linear relationship of cell death rate and the proportion of cells with internalized crystals (E). The linear relationship of [Ca²⁺]_i and cell viability (F). R² is a linear correlation coefficient. Crystal concentration: 250 μg/mL; treatment time: 24 h.

Figure 5 Mitochondrial membrane potential (Δψm) changes of HK-2 cells after injury by HAP with different sizes. (A) Qualitative observation of the changes in Δψm under fluorescence microscope; (B) Scatter plot of Δψm detected by flow cytometry; (C) Histogram for quantitative determination of aggregation degree of JC-1 in mitochondria, JC-1 monomers represent decreased Δψm. Crystal concentration: 250 μg/mL; treatment time: 24 h. Scale bars: 50 μm (200x).

Figure 6 Changes in cell cycle distribution of HK-2 cells after injury by HAPs with different sizes. (A) Cell cycle images detected by Flow cytometry; (B) quantitative histogram of cell cycle distribution; (C) correlation between cell viability and retention capacity at G0/G1 phase. Crystal concentration: 250 μg/mL; treatment time: 24 h.

Figure 7 Changes of apoptosis and necrosis rate of HK-2 cells after injury by HAPs with different sizes. (A) Qualitative observation of apoptosis and necrosis under fluorescence microscope; (B) quantitative scatter plot of apoptosis and necrosis; (C) statistical result of necrosis rate. Crystal concentration: 250 μg/mL; treatment time: 24 h. Scale bars: 50 μm (100x).

Figure 8 Schematic diagram of cytotoxicity mechanism of HK-2 cells induced by HAP with different sizes.