Higher-intensity ultrasound accelerates fracture healing via mechanosensitive ion channel Piezo1

Osteoporosis-related fractures are a major public health problem. Mechanobiological stimulation utilizing lowintensity pulsed ultrasound (LIPUS) is the most widely accepted modality for accelerating fracture healing. However, recent evidence has demonstrated the ineffectiveness of LIPUS, and the biophysical mechanisms of ultrasound-induced bone formation also remain elusive. Here, we demonstrate that ultrasound at a higher intensity than LIPUS effectively accelerates fracture healing in a mouse osteoporotic fracture model. Higherintensity ultrasound promoted chondrogenesis and hypertrophic differentiation of chondrocytes in the fracture callus. Higher-intensity ultrasound also increased osteoblasts and newly formed bone in the callus, resulting in accelerated endochondral ossification during fracture healing. In addition, we found that accelerated fracture healing by ultrasound exposure was attenuated when the mechanosensitive ion channel Piezo1 was inhibited by GsMTx4. Ultrasound-induced new bone formation in the callus was attenuated in fractured mice treated with GsMTx4. Similar results were also confirmed in a 3D osteocyte-osteoblast co-culture system, where osteocytic Piezo1 knockdown attenuated the expression of osteoblastic genes after ultrasound exposure. Together these results demonstrate that higher-intensity ultrasound than clinically used LIPUS can accelerate endochondral ossification after fractures. Furthermore, our results suggest that mechanotransduction via Piezo1 mediates ultrasound-stimulated fracture healing and bone formation.