Osteoblast response to zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate

Abstract

Calcium phosphate bioceramics, such as hydroxyapatite, have long been used as bone substitutes because of their proven biocompatibility and bone binding properties in vivo. Recently, a zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized, which is more soluble than hydroxyapatite and allows for controlled release of calcium and phosphate ions. These ions have been postulated to increase osteoblast differentiation and mineralization in vitro. The focus of this work is to elucidate the physicochemical properties of Zr-ACP and to measure cell response to Zr-ACP in vitro using a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line. Cells were cultured in osteogenic medium and mineral was added to culture at different stages in cell maturation. Culture in the presence of Zr-ACP showed significant increases in cell proliferation, alkaline phosphatase activity (ALP), and osteopontin (OPN) synthesis, whereas collagen synthesis was unaffected. In addition, calcium and phosphate ion concentrations and medium pH were found to transiently increase with the addition of Zr-ACP, and are hypothesized to be responsible for the osteogenic effect of Zr-ACP.

Figure 1

X-ray diffractometry. XRD patterns for (a) Zr-ACP and (b) HAP powders.

Figure 2

Ion concentration. (a) Phosphate and (b) calcium ion concentration in growth medium following addition of 5 mg/mL Zr-ACP and HAP. Dotted lines represent concentrations for n = 2 control samples. Bars represent the mean ± standard error for n = 2 samples. All measurements are significantly different from control (p ≤ 0.05) except calcium concentration at 15 min after addition of HAP.

Figure 3

pH as a function of time. Change in pH of growth medium with addition of Zr-ACP or HAP. Bars represent the mean ± standard error for n = 4 samples. An asterisk indicates statistical difference from the control group (p ≤ 0.05).

Figure 4

Cell number. Zr-ACP or HAP (5 mg/mL) was added at day 0 and cells were assayed at days 3, 7, 10, and 14. Each bar represents the mean ± standard error for n = 8 wells. An asterisk indicates statistical difference from the control group (p ≤ 0.05), and a pound symbol indicates statistical difference from the HAP group (p ≤ 0.05).

Figure 5

Cell number. Zr-ACP or HAP (5 mg/mL) was added at (a) day 4 and assayed at days 5, 7, and 14, and (b) added at day 11 and assayed at days 12 and 14. Each bar represents the mean ± standard error for n = 8 wells. An asterisk indicates statistical difference from the control group (p ≤ 0.05), and a pound symbol indicates statistical difference from the HAP group (p ≤ 0.05).

Figure 6

Alkaline phosphatase activity. Zr-ACP or HAP (5 mg/mL) was (a) added at day 4 and assayed at days 5, 7, and 14, and (b) added at day 11 and assayed at days 12 and 14. Each bar represents the mean ± standard error for n = 8 wells. An asterisk indicates statistical difference from the control group (p ≤ 0.05), and a pound symbol indicates statistical difference from the HAP group (p ≤ 0.05).

Figure 7

Osteopontin expression. (a) Representative bands for OPN and G3PDH for cells cultured at 14 days where 5 mg/mL of Zr-ACP or HAP or no mineral was added at day 0. Each lane represents one replicate (well). (b) Relative OPN band densities (normalized by G3PDH) versus addition time of minerals. Each bar represents the mean ± standard error for n = 6 wells. An asterisk indicates statistical difference from the control group (p ≤ 0.05), and a pound symbol indicates statistical difference from the HAP group (p ≤ 0.05).

Figure 8

Collagen synthesis. (a) Counts per minute from collagen protein fractions normalized by mean cell number and (b) collagenous protein as a percentage of the total protein synthesized plotted versus addition time of minerals. Each bar represents the mean ± standard error for n = 4 wells.