Abstract
Mechanobiological stimuli, such as low-intensity pulsed ultrasound (LIPUS), have been shown to promote bone regeneration and fresh fracture repair, but the fundamental biophysical mechanisms involved remain elusive. Here, we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes. This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment. Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection. MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation, and knockdown of Piezo1 restricted the increase in cell migration and proliferation. After labeling with Fluo-8, MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation. No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells, which was similar to the results in the GsMTx4-treated group. The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased (P < 0.01) after LIPUS stimulation. In addition, Phalloidin-iFluor-labeled F-actin filaments immediately accumulated in the perinuclear region after LIPUS stimulation, continued for 5 min, and then returned to their initial levels at 30 min. These results suggest that Piezo1 can transduce LIPUS-induced mechanical signals into intracellular calcium. The influx of Ca2+ serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization, which regulate the proliferation of MC3T3-E1 cells.
Highlights
Millions of fractures occur in the United States every year, with the average rate of nonunion fractures being roughly between 5% and 10%, which is predicted to increase over time.[1,2] The risk of nonunion fracture is mainly related to several factors, including the severity of the injury and type of treatment
ERK1/2 to ERK1/2 (p-ERK1/2 vs. ERK1/2) in the MC3T3-E1 cell group was significantly increased from 0.483 ± 0.069 before low-intensity pulsed ultrasound (LIPUS) stimulation to 0.975 ± 0.026 after LIPUS stimulation (n = 3, P < 0.01, Student’s t-test)
Piezo[1] expression was recently discovered at the plasma membrane in human dental pulp-derived mesenchymal stem cells,[24] stem cells extracted from human exfoliated deciduous teeth,[25] mouse urothelium cells,[26] human umbilical vein endothelial cells,[19] and HEK293T cells.[13]
Summary
Millions of fractures occur in the United States every year, with the average rate of nonunion fractures being roughly between 5% and 10%, which is predicted to increase over time.[1,2] The risk of nonunion fracture is mainly related to several factors, including the severity of the injury and type of treatment. For the treatment of fracture or bone defects, several treatment modalities can be considered, either alone or in combination, for optimization of the bone healing process.[3] In addition to typical approaches, such as fixation and bone transport, mechanobiological interventions have shown promise in promoting cellular proliferation and tissue adaptation; of these strategies, low-intensity pulsed ultrasound (LIPUS)[4] and pulsed electromagnetic fields[5] have been extensively utilized in the clinical setting to enhance bone regeneration and fresh fracture as noninvasive modalities of biophysical stimulation.
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