Orthodontic tooth movement induces alveolar bone resorption and formation via mechanical stimuli. Specifically, the force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators of bone formation in the response of osteoblasts to mechanical stimuli. Fluid shear stress induces ATP release in osteoblastic MC3T3-E1 cells and ATP promotes bone formation via the P2X7 receptor in rat calvarial cells. However, the effect of tension force (TF)-induced ATP on osteogenesis is poorly understood. Therefore, we examined the effect of TF on ATP production, the expression of osteogenesis-related transcription factors, P2X7, extracellular matrix proteins, alkaline phosphatase (ALP) activity, and osteogenesis in osteoblasts.
Methods:
MC3T3-E1 cells were plated on flexible-bottom plates, incubated in the presence or absence of the P2X7 antagonist A438079, and then stimulated with cyclic TF (6 or 18%, at 6 cycles/min of 5 sec strain, 5 sec relaxation) for 24 hours using a Flexercell Strain Unit 3000. ATP release in the culture medium was measured using the luciferin/luciferase assay. The expression of osteogenesis-related transcription factors, P2X7, and extracellular matrix proteins was determined at the mRNA level by real-time PCR and at protein level by Western blotting or ELISA. ALP activity was estimated by ALP staining, indicating the enzyme activity. The calcium content in mineralized nodules was determined using the calcium E-test kit.
Results:
TF at 6% induced ATP production, the expression of P2X7, osteogenesis-related transcription factor, and extracellular matrix protein. It also increased the calcium content in mineralized nodules and ALP activity. Moreover, the P2X7 antagonist A438079 blocked these TF-induced phenomena.
Conclusions:
These results suggest that ATP stimulated by TF promotes osteogenesis via the P2X7 receptor in osteoblasts.