Effect of low‐intensity pulsed ultrasound on murine calvarial derived periosteal cells

Abstract

Background : The replacement of lost natural teeth using osseointegrated implants require adequate bone volume permitting their anchorage.Many studies have shown potential advantages of stem-cell based therapies in bone tissue engineering. Periosteal cells have been suggested as a promising source for bone tissue engineering, and low-intensity pulsed ultrasound (LIPUS) has shown successful bone healing of delayed unions, non-unions and simulated fracture healing in animal models and in clinical treatments.Aim/Hypothesis : The aim of this study was to investigate the effect of LIPUS on the proliferation and the osteogenic differentiation of murine calvarial derived periosteal cells.Materials and Methods : Four-week-old SLC ICR female mice were euthanized, and then their calvarias were extracted and washed with phosphate-buffered saline (PBS). Six hundred mg gelatin was inserted inside a 60 mm culture dish and immersed in a 6 ml culture medium. The periosteum was derived from the calvarias and put it onto the gelatin. Then the gelatin was inverted upside-down and incubated at 37°C with 5% CO2. The culture medium was changed every 3 days. Cells at the passage of three were treated with LIPUS with an intensity of 30 mW cm2, frequency of 3 MHz, pulse repetition frequency (PRF) of 1 kHz and exposure time of 20 min per day up to 21 days. The cell proliferation and viability were analyzed by cell counting kit-8 (CCK-8) assay, PrestoBlue assay and Live Dead Cell Assay. Osteogenic differentiation was determined by alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red staining and RT-real-time-PCR of osteogenic marker genes.Results : Cell proliferation is significantly accelerated by LIPUS more than that of the control group. Furthermore, more osteogenic differentiation was observed in LIPUS treated groups. Conclusion and Clinical Implications :The results suggested that LIPUS could enhance cell proliferation and osteogenic differentiation of murine calvarial derived periosteal cells and could be part of effective treatment methods for future clinical applications.