The effects of low dose X-irradiation on osteoblastic MC3T3-E1 cells in vitro

Abstract

Background: It has been indicated that moderate or high dose of X-irradiation could delay fracture union and cause osteoradionecrosis, in part, mediated by its effect on proliferation and differentiation of osteoblasts. However, whether low dose irradiation (LDI) has similar roles on osteoblasts is still unknown. In this study, we investigated whether and to what extent LDI could affect the proliferation, differentiation and mineralization of osteoblasts in vitro.

Methods: The MC3T3-E1 cells were exposed to single dose of X-irradiation with 0, 0.1, 0.5, 1.0 Gy respectively. Cell proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization was evaluated by methylthiazoletetrazolium (MTT) and bromodeoxyuridine (BrdU) assay, flow cytometry, ALP viability kit and von Kossa staining, respectively. Osteocalcin (OCN) and core-binding factor α1 (Cbfα1) expressions were measured by real time-PCR and western blot, respectively.

Results: The proliferation of the cells exposed to 2.0 Gy was significantly lower than those exposed to ≤1.0 Gy (p < 0.05) from Day 4 to Day 8, measured by MTT assay and BrdU incorporation. For cells exposed to ≤1.0 Gy, increasing dosages of X-irradiation had no significant effect on cell proliferation and apoptosis. Importantly, LDI of 0.5 and 1 Gy increased ALP activities and mineralized nodules of MC3T3-E1 cells. In addition, mRNA and protein expressions of OCN and Cbfα1 were also markedly increased after treatment with LDI at 0.5 and 1 Gy.

Conclusions: LDI have different effects on proliferation and differentiation of osteoblasts from those of high dose of X-irradiation, which might suggest that LDI could lead to promotion of fracture healing through enhancing the differentiation and mineralization of osteoblasts.

Figure 1

Growth curve of irradiated MC3T3-E1 cells assessed by MTT assay. MC3T3-E1 cells were exposed to 0, 0.1, 0.5, 1.0 and 2.0 Gy of X-ray irradiation respectively. The effect of irradiation on cell growth was assessed by MTT assay at 24-h intervals until day 8 after been seeded. Optical density (OD) was measured at 495 nm and data were expressed as the mean ± SD of triplicate experiments (n = 3). From day 4 to day 8, relative proliferation rate of the cells exposed to 2.0 Gy was significantly lower than those of the control group (0 Gy) (* p < 0.05, ** p < 0.01). On day 8, relative proliferation rates of the cells exposed to 0.5 and 1 Gy were lower than those of the control group and 0.1 Gy group(* p < 0.05).

Figure 2

Proliferation of MC3T3-E1 cells after irradiation assessed by BrdU incorporation assay. MC3T3-E1 cells were exposed to 0, 0.1, 0.5, 1.0 and 2.0 Gy of X-ray irradiation respectively. Cell proliferation was determined by BrdU incorporation assay at 24-h intervals until day 8 after been seeded. Absorbance in each well was measured at 450 nm and data were expressed as the mean ± SD of triplicate experiments (n = 3). From day 3 to day 8, the DNA synthesis of the cells exposed to 2.0 Gy was significantly lower than those of the control group (0 Gy) (* p < 0.05, ** p < 0.01). On day 8, the DNA synthesis of the cells exposed to 0.5 and 1 Gy were lower than those of the control group and 0.1 Gy group(* p < 0.05).

Figure 3

Apoptosis of irradiated MC3T3-E1 cells on day 6–8 after been seeded. MC3T3-E1 cells were exposed to 0, 0.1, 0.5, 1.0 Gy of X-irradiation. Cells on day 6–8 were double stained with annexin-V-FITC and PI and analyzed by flow cytometry. Data were expressed as the mean ± SD of triplicate experiments (n = 3). For each dose, apoptosis cells (including early apoptotic and late apoptotic cells) was gradually increased from day 6 to day 8 and the number of apoptosis cell on day 8 was significantly more than that on day 6 (p < 0.05).

Figure 4

Effects of X-irradiation on ALP activity and staining of MC3T3-E1 cells. MC3T3-E1 cells were exposed to 0, 0.1, 0.5 and 1.0 Gy of X-irradiation respectively and cells were cultured for 7 days after irradiation. (A) ALP activity was measured and the data were expressed as mean ± SD (n = 3). The ALP activity of 0.5 Gy and 1.0 Gy groups was significantly increased than that of the control group (0 Gy) (*p < 0.05, **p < 0.01). However, 0.1 Gy groups had no significant difference compared with the control. (B) represented plate view of ALP staining. There were more positive cells in 0.5 and 1.0 Gy groups compared with the control.

Figure 5

Effects of X-irradiation on mineralized nodules of MC3T3-E1 cells. MC3T3-E1 cells were exposed to 0, 0.1, 0.5 and 1.0 Gy of X-irradiation respectively and von Kossa staining was performed on day 14 after irradiation. A represented microscope images. The number of mineralized nodules of osteoblasts was counted and shown in B. Data were expressed as mean ± SD of 3 wells. Significant difference in 0.5 and 1.0 Gy groups was observed compared with the control group (0 Gy) (**p<0.01).

Figure 6

OCN, Cbfα1 mRNA expression of irradiated MC3T3-E1 cells. MC3T3-E1 cells were exposed to 0, 0.1, 0.5 and 1.0 Gy of X-irradiation respectively and cells were harvested on day 10 after irradiation. Total RNA was collected and real-time PCR was performed, β-actin as an endogenous control. Results were expressed as fold change over the control group (0 Gy). (A) OCN mRNA expression in 0.5 and 1 Gy groups were significantly increased compared with the control group (*p < 0.05, **p < 0.01). (B) Cbfα1 mRNA expression in 0.5 and 1 Gy groups were significantly increased compared with the control group (*p < 0.05).

Figure 7

OCN, Cbfα1 protein expression of irradiated MC3T3-E1 cells. MC3T3-E1 cells were exposed to 0, 0.1, 0.5 and 1.0 Gy of X-irradiation respectively and cells were harvested on day 10 after irradiation. (A) The protein productions of OCN and Cbfα1 were detected by Western blot with β-actin as an endogenous control. (B) The quantitative date of OCN (B1) and Cbfα1 (B2) was band-density ratios compared to the control group. X-irradiation of 0.5 and 1 Gy resulted in obviously increase of protein level of OCN and Cbfα1 (*P < 0.05).