Mechanical Stretch Promotes the Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Erythropoietin

Abstract

Introduction: The effects of erythropoietin (EPO) on the behaviors of bone marrow mesenchymal stem cells (BMSCs) subjected to mechanical stretch remain unclear. This study was therefore aimed at establishing the dose-response effect of EPO stimulation on rat BMSCs and investigating the effects of mechanical stretch combined with EPO on the proliferation and osteogenic differentiation of BMSCs.

Material and methods: The proliferation and osteogenic differentiation of rat BMSCs were examined and compared using EPO with different concentrations. Thereafter, BMSCs were subjected to 10% elongation using a Flexcell strain unit, combined with 20 IU/ml EPO. The proliferation of BMSCs was detected by Cell Counting Kit-8, colony formation assay, and cell cycle assay; meanwhile, the mRNA expression levels of Ets-1, C-myc, Ccnd1, and C-fos were detected by reverse transcription and real-time quantitative PCR (qPCR). The osteogenic differentiation of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA expression levels of ALP, OCN, COL, and Runx2 were detected by qPCR. The role of the extracellular signal-regulated kinases 1/2 (ERK1/2) in the osteogenesis of BMSCs stimulated by mechanical stretch combined with 20 IU/ml EPO was examined by Western blot.

Results: Our results showed that effects of EPO on BMSCs included a dose-response relationship, with the 20 IU/ml EPO yielding the largest. Mechanical stretch combined with 20 IU/ml EPO promoted proliferation and osteogenic differentiation of BMSCs. The increase in ALP, mineral deposition, and osteoblastic genes induced by the mechanical stretch-EPO combination was inhibited by U0126, an ERK1/2 inhibitor.

Conclusion: EPO was able to promote the proliferation and osteogenic differentiation of BMSCs, and these effects were enhanced when combined with mechanical stretch. The underlying mechanism may be related to the activation of the ERK1/2 signaling pathway.

Figure 1

Representative images of BMSCs before and after mechanical stretch. Before mechanical stretch, the obtained cells were fibroblast-like, spindle-shaped, and randomly oriented (a). After mechanical stretch, the BMSCs were still fibroblast-like and spindle-shaped but became much thinner compared to the nonstretch cells. The cells were oriented perpendicularly to the axis of the external strain (b). In addition, immunophenotypic characterization was analyzed by flow cytometry, indicating that the obtained cells were BMSCs (c). The arrow indicates direction of stretch field. Bar = 50 μm.

Figure 2

The proliferation and ALP staining of BMSCs under the stimulus of different concentrations of EPO. A dose-response relationship was evident among the experiment groups, and the group of 20 IU/ml EPO showed the maximum value, with the cell proliferation significantly increased about 3-fold compared to the control (a). On the other hand, the staining of the cells treated with EPO is much more obvious than that in the control. Furthermore, the EPO dosage of 20 IU/ml yielded the largest effect relative to the control (b). ^∗p < 0.05 and ^∗∗p < 0.001 compared with the control group.

Figure 3

Effects of mechanical stretch combined with 20 IU/ml EPO on the cell proliferation, the distribution of the cell cycles, and the expression of proliferation genes. Under the stimulus of stretch with and without EPO, the cell proliferation increased significantly compared to the control. Among the experiment groups, the STR+EPO group showed the maximum value, with the cell proliferation significantly increased about 4.5-fold compared to the control (a). The number of cell colonies under the intervention of mechanical stretch with and without EPO. One representative well was presented for each group (b). Quantitative results for these assays are also presented as bar graphs. The number of cell colonies that developed was evidently higher under the intervention of mechanical stretch combined with 20 IU/ml EPO compared to the other groups (b). The results showed that mechanical stretch combined with 20 IU/ml EPO stimulated cell proliferation by promoting cell cycle progression from G1 to S-G2/M phases. Besides, qPCR was used to analyze the proliferation genes. BMSCs subjected to mechanical stretch with or without EPO clearly show a change in the mRNA expression levels of Ets-1, C-myc, Ccnd1, and C-fos compared to the control. The mRNA expression of Ets-1 in the group subjected to mechanical stretch combined with 20 IU/ml EPO showed the maximum value, with the expression significantly increased about 5.32-fold compared to the control. The mRNA expressions of C-myc, Ccnd1, and C-fos in the group subjected to mechanical stretch combined with 20 IU/ml EPO increased about 5.59-fold, 5.72-fold, and 5.58-fold, respectively, compared to the control (c). ^∗p < 0.05 and ^∗∗p < 0.001 compared with the control group.

Figure 4

Flow cytometry was used to analyze the distribution of the cell cycles. There was a significantly high S-G2/M phase population with percentages of 27.0 ± 0.74 in the experimental cells (strains combined with 20 IU/ml EPO) compared to the other groups of cells with percentages of 10.7 ± 0.85, 17.9 ± 0.79, and 20.6 ± 0.68 (3B). ^∗p < 0.05 and ^∗∗p < 0.001 compared with the control group.

Figure 5

The ALP staining of the cells subjected to mechanical stretch with or without EPO was more obvious than that of the control (a). The group subjected to mechanical stretch combined with 20 IU/ml EPO yielded the largest ALP activity relative to the control (b). BMSCs subjected to mechanical stretch with or without EPO show a change in the mRNA expression levels of ALP, OCN, COL, and Runx2 compared to the control group. The mRNA expression of ALP in the group of mechanical stretch combined with 20 IU/ml EPO showed the maximum value, with the expression significantly increased about 5.37-fold compared to that of the control. The mRNA expressions of OCN, COL, and Runx2 in the group of mechanical stretch combined with 20 IU/ml EPO increased about 5.85-fold, 5.41-fold, and 5.42-fold, respectively, compared to those of the control (c). ^∗p < 0.05 and ^∗∗p < 0.001 compared with the control group.

Figure 6

The levels of p-ERK1/2 under mechanical stretch combined with 20 IU/ml EPO. The p-ERK1/2 expression of the cells subjected to mechanical stretch with or without EPO was more obvious than the control. The group subjected to mechanical stretch combined with 20 IU/ml EPO yielded the largest effect relative to the control (a, b). Furthermore, the U0126, an inhibitor of ERK1/2, inhibited the ALP activity induced by mechanical stretch combined with 20 IU/ml EPO (c). ^∗p < 0.05 and ^∗∗p < 0.001 compared with the control group.