Low Intensity Pulsed Ultrasound Synergistically Modulates Sost and Opg/rankl Ratio in Mlo-y4 Osteocytes in Vitro

Introduction: Bone remodeling is a well coupled biological process of bone resorption and bone formation, which is predominantly regulated by mechanical load. Recent years, osteocytes, the most abundant cell population in bone, have been evidenced to be playing a key role in the modulation of bone remodeling. As a form of mechanical stimulation, low intensity (<30mW/cm2) pulsed ultrasound (LIPUS) has been reported to be effective in enhancing angiogenesis1, growth2 and healing3 of bone. In vitro, the effect of LIPUS on chondrocytes4 and bone cells5 has been studied however the mechanisms are still unclear, especially the effect of LIPUS on the osteocytic modulation of bone remodeling. Sclerostin (SOST) is a negative bone formation regulator, which is exclusively expressed in mature osteocytes. Mechanical loading was shown to be able to significantly reduce the SOST production in vivo6. However, whether SOST can be modulated by LIPUS is unknown. OPG and RANKL are essential paracrine mediators of bone remodeling. Mechanical load (strain) has been shown to modulate bone formation and resorption balance through the OPG/RANKL axis7. Lately, we used fluid shear stress (FSS) to mechanically load MLO-Y4 osteocytes and found that 1 hour of 12 dynes/cm2 FSS drastically decreased the membrane-bound RANKL level by 65%. Reversibly, the RANKL level was returned back to the basal level when FSS ceased8. However, the modulating effect of LIPUS on OPG/RANKL is not known. Based on the data mentioned above, we hypothesize that LIPUS induces anabolic responses in osteocytes, in turn modulates the remodeling of bone. To investigate why and how LIPUS impacts bone remodeling on cellular level, we subjected MLO-Y4 osteocytes to LIPUS in culture. To determine the upstream signaling pathways involved, we tested the activation of ERK1/2. To explore the downstream functional changes, we examined the protein production of metabolic bone markers as cyclooxygenase-2 (COX-2), sclerostin (SOST), osteoprotegerin (OPG) and receptor activator of NFkB ligand (RANKL). Materials and Methods: Cell Culture: An osteocyte cell line-MLO-Y4 cells (from Dr. Lynda Bonewald, UMKC) were cultured in MEM supplemented with 5% FBS and 5% BS. LIPUS Application: MLO-Y4 osteocytes were seeded and grown on Ø35 mm Petri dishes coated with collagen Type I. Upon confluence, the cells were serum starved for 24 hours, then subjected to LIPUS (30mW/cm2, 1.5MHz frequency, 1kHz repetition, EXOGEN 2000+ system, Smith & Nephew, Inc.) for either 10 minutes to test signaling pathway of ERK1/2, or 20 minutes followed by 6 hours of post-incubation to …