In-vitro stimulation of cells of the musculoskeletal system with focused Low-Intensity Pulsed Ultrasound (FLIPUS): Analyses of cellular activities in response to the optimized acoustic dose

Low-Intensity Pulsed Ultrasound (LIPUS) is a safe treatment for fresh fractures, delayed- and non-union bone. Certain inconsistency is associated with the reported in-vitro data, which could be attributed to difficulties in reproducing similar conditions using unfocused transducers in experimental in-vitro set-ups. In our study we address the above issues by using focused LIPUS (FLIPUS), which avoids the previously described potential sources of physical side-effects and therefore, allows more robust “acoustic dose” definition and an “acoustic dose - observed effect” correlation in an in-vitro setting. In our experiments all relevant sound field parameters were measured and adjusted such that they provide an optimal and even sound transmission. Five days after daily FLIPUS stimulation, gene expression of osteopontin (OPN), Runx2, collagen-1 (Col-1), E11 antigen and dentin matrix protein-1 (DMP-1) was up-regulated in rat mesenchymal stem cells (rMSCs). These cells under reduced nutrient supply also showed higher proliferation rate on days 3 and 4. Enhanced proliferation on days 2, 3 and 4 was observed in murine MC3T3-E1 pre-osteoblasts after the FLIPUS treatment under similar conditions. Increased expressions of early response genes c-jun and cyclooxygenase-2 (COX-2) were observed 30 min post-FLIPUS-exposure in the same cells. FLIPUS induced enhanced phosphorylation of Smad1/5/8 (mothers against decapentaplegic) transcription factors in MC3T3-E1s, when Bone Morphogenetic Protein-2 (BMP-2) and FLIPUS were applied simultaneously. These results indicate that FLIPUS is a convenient set-up to study the mechanism of bone regeneration in response to ultrasound in vitro.