Lower dosage of aspirin promotes cell growth and osteogenic differentiation in murine bone marrow stromal cells

Abstract

Background/purpose: The effect of aspirin on bone regeneration remains controversial. This study aimed to determine the effect of various concentrations of aspirin on cell viability, osteogenic differentiation, cell cycle, and apoptosis on ST2 cells to find an effective range of aspirin for bone regeneration induction.

Materials and methods: Cell viability was measured with MTT assay after being stimulated with aspirin for 1 day, 2 days, 3 days, 5 days, and 7 days. Alkaline phosphatase (ALP) activity was measured after cells were treated for 1 day, 3 days, and 7 days. Expression of runt-related transcription factor 2 (Runx-2) was evaluated using Western-blot analysis at 3 days and 7 days. Flow cytometry was used for cell cycle and apoptosis measurement after cells were treated for 48 hours.

Results: Lower concentrations of aspirin (1μΜ and 10μM) promoted cell growth and increased ALP levels and Runx-2 expression, while higher concentrations (100μΜ and 1000μΜ) inhibited cell growth (P < 0.05), and lost their effect on ALP activity after 3 days, while even showing an inhibitory effect on the expression of Runx-2. Aspirin at a concentration of 100μM promoted cell mitosis from the S phase to the G2/M phase, and 1000μM arrested the cell cycle in the resting phase G0/G1 (P < 0.05). Parallel apoptosis/necrosis studies showed the percentage of cells in apoptosis decreased dramatically at any dose of aspirin.

Conclusion: A lower dosage of aspirin could promote ST2 cell growth, osteogenic differentiation, and inhibit their apoptosis which indicates that aspirin can be used as an alternative for bone regeneration.

Keywords: apoptosis; aspirin; bone marrow stromal cells; cell cycle; osteogenic differentiation; proliferation.

Figure 1

Cell morphology observation after recovery. (A) Twenty-four hours after recovery (×100), cells were in long spindle shapes and large intercellular space was observed; (B) 48h after recovery (×200); (C) 48h after recovery (×100) cells connected each other into a net, the oval nucleus was in the middle.

Figure 2

Effect of different doses of aspirin on viability of ST2 cells (n = 6). ST2 cells were incubated with various concentrations of aspirin for 1 day, 2 days, 3 days, 5 days, and 7 days, respectively. The proliferation of ST2 cells was measured with MTT assay (490 nm wavelength). Data represents means ± standard deviation (n = 6, six replicates per time point for each experimental condition). ** P < 0.01 compared with control (0μM aspirin treatment). OD = optical density.

Figure 3

Effect of different doses of aspirin on (alkaline phosphatase) ALP activity of ST2 cells. ST2 cells were cultured with various concentrations of aspirin for 1 day, 3 days, and 7 days. Data were normalized for total protein content to account for the effects. The ALP activity incubated with 1μM and 10μM aspirin significantly increased. Data represents means ± standard deviation (n = 6, 6 replicates per time-point for each experimental condition). By one-way analysis of variance and Students–Newman–Keuls test, significant differences were shown. * P < 0.05 compared with control. ** P < 0.01 compared with control. *** P < 0.05 versus 100μM and 1000μM.

Figure 4

(A) Effect of aspirin on runt-related transcription factor 2 (Runx-2) expression of ST2 cells. ST2 cells were cultured in an osteogenesis medium with different doses of aspirin for 3 days and 7 days; (B)western-blot analysis showed that on Day 3, all doses of aspirin promoted Runx-2 expression. On Day 7, only 1μM and 10μM of aspirin increased Runx-2 levels compared with the control, the higher ones (100μM and 1000μM) showed an inhibitory effect on the contrary. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as a loading control.

Figure 5

Effect of aspirin on cell cycle distribution of ST2 cells. ST2 cells were cultured in serum-free medium for 24 hours and then treated with the indicated concentrations of aspirin for 48 hours. Flow cytometric analysis showed that 100μM aspirin significantly increased the proportion of cells in the G2/M phase and G0/G1 phase for 1000μM aspirin. Data was shown as means ± standard deviation (n = 4, 4 replicates per time-point for each experimental condition). By one-way analysis of variance and Students–Newman–Keuls test, significant differences were shown. * P < 0.05 compared with the same phase of cell cycle of control.

Figure 6

(A) Control group; (B) typical result for the effect on cell cycle of 1μM; (C) 10μM; (D) 100μM; and (E) 1000μM of aspirin with flow cytometry.

Figure 7

(A) Control group; (B) typical result for the effect on cell apoptosis rate of 1μM; (C) 10μM; (D) 100μM; and (E) 1000μM of aspirin with flow cytometry. The number of viable cells was counted in the lower left quadrant (C3), early apoptosis in lower right quadrant (C4), late apoptosis in upper right quadrant (C2), and necrosis in upper left quadrant (C1).