Mechanical strain plays a critical role in the proliferation, differentiation and maturation of bone cells. As mechanical receptor cells, osteoblasts perceive and respond to stress force, such as those associated with compression, strain and shear stress. However, the underlying molecular mechanisms of this process remain unclear. Using a four-point bending device, mouse MC3T3-E1 cells was exposed to mechanical tensile strain. Cell proliferation was determined to be most efficient when stimulated once a day by mechanical strain at a frequency of 0.5 Hz and intensities of 2500 µε with once a day, and a periodicity of 1 h/day for 3 days. The applied mechanical strain resulted in the altered expression of 1992 genes, 41 of which are involved in the mitogen-activated protein kinase (MAPK) signaling pathway. Activation of ERK by mechanical strain promoted cell proliferation and inactivation of ERK by PD98059 suppressed proliferation, confirming that ERK plays an important role in the response to mechanical strain. Furthermore, the membrane-associated receptors integrin β1 and integrin β5 were determined to regulate ERK activity and the proliferation of mechanical strain-treated MC3T3-E1 cells in opposite ways. The knockdown of integrin β1 led to the inhibition of ERK activity and cell proliferation, whereas the knockdown of integrin β5 led to the enhancement of both processes. This study proposes a novel mechanism by which mechanical strain regulates bone growth and remodeling.

BackgroundThe extracellular matrix (ECM) provides a supportive microenvironment for cells, which is suitable as a tissue engineering scaffold. Mechanical stimulus plays a significant role in the fate of osteoblast, suggesting that it regulates ECM formation. Therefore, we investigated the influence of mechanical stimulus on ECM formation and bioactivity.MethodsMouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with mechanical tensile strain. After removing the cells, the ECMs coated on dishes were prepared. The ECM protein and calcium were assayed and MC3T3-E1 cells were re-seeded on the ECM-coated dishes to assess osteoinductive potential of the ECM.ResultsThe cyclic tensile strain increased collagen, bone morphogenetic protein 2 (BMP-2), BMP-4, and calcium levels in the ECM. Compared with the ECM produced by unstrained osteoblasts, those of mechanically stimulated osteoblasts promoted alkaline phosphatase activity, elevated BMP-2 and osteopontin levels and mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and increased secreted calcium of the re-seeded MC3T3-E1 cells.ConclusionMechanical strain promoted ECM production of osteoblasts in vitro, increased BMP-2/4 levels, and improved osteoinductive potential of the ECM. This study provided a novel method to enhance bioactivity of bone ECM in vitro via mechanical strain to osteoblasts.

As integrins are mechanoresponsive, there exists an intimate relationship between integrins and mechanical strain. Integrin-β1 mediates the impact of mechanical strain on bone. Mechanical strain induces bone formation through the activation of β-catenin pathways, which suggests that integrin-β1 mediates β-catenin signaling in osteoblasts in response to mechanical strain. In the present study, we examined the role of integrin-β1 in Wnt/β-catenin signal transduction in mechanically strained osteoblasts. MC3T3-E1 osteoblastic cells were transfected with integrin-β1 small interfering RNA (si-Itgβ1), and exposed to mechanical tensile strain of 2,500 microstrain (µε) using a four-point bending device. The mechanical strain enhanced the mRNA expression of integrin-β1, the protein levels of phosphorylated (p-) glycogen synthase kinase-3β (GSK‑3β) and β-catenin, simultaneously increased the mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), the protein levels of bone morphogenetic protein (BMP)-2 and -4 and enhanced the alkaline phosphatase (ALP) activity of the ME3T3-E1 cells. The elevations were inhibited by si-Itgβ1. Additionally, the mechanical strain induced the nuclear translocation of β-catenin into the nucleus, which was also inhibited by si-Itgβ1. These findings indicated that mechanical strain promoted osteoblastic differentiation through integrin‑β1‑mediated β-catenin signaling.

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