Abstract
Objective: NFκB negatively affects bone metabolism through inflammatory pathways, whereas Nrf2 benefits it by regulating antioxidant/detoxifying enzymes through antioxidant response element (ARE) activity. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances osteogenic differentiation in 3D culture and whether it affects osteoblastic differentiation-associated markers such as calcium and alkaline phosphatase (ALP), and the regulation of NFκB and/or ARE.
Methods: Murine MC3T3-E1 preosteoblasts were used to produce 3D-spheroids, which were treated with LIPUS. Time-dependent morphological change of spheroids was evaluated by microscopy, histology, and micro-CT. To analyze changes in ARE levels, a combination of LIPUS with an optimal concentration and timing of the Nrf2-inducer sulforaphane was applied. Osteoblast differentiation and mineralization were achieved through stimulation with differentiation medium for 21-28 days, during which LIPUS was used every day at 24-hour intervals. Differentiation was evaluated using calcium and ALP assay. Finally, cells were transduced with SIN-lenti-NFκB/SIN-lenti-ARE construct, and the effects of single-shot LIPUS on NFκB and ARE were evaluated using Nano-Glo® Luciferase Assay.
Results: Morphologically, in LIPUS group, shrinkage behaviour, which may correlate with cell differentiation, was observed to be more pronounced. NFκB activity with LIPUS was significantly lower than without treatment, whereas ARE activity showed significant increase only immediately after LIPUS treatment. Conversely, LIPUS enhanced ARE activity in combination with sulforaphane administration. After 28 days, the calcium content and ALP activity of LIPUS-treated spheroids increased significantly.
Conclusion: This study suggests that LIPUS may have NFκB-downregulating, limited ARE-raising effects in combination with sulforaphane, and elevating osteoblast mineralization three-dimensionally. Thus, LIPUS could be useful for treating fractures in conditions of oxidative stress and hyper-inflammation.