Effects of Low-intensity pulsed ultrasound (LIPUS) on osteogenic differentiation of murine periosteum-derived cells

Abstract

The replacement of lost natural teeth using osseointegrated implants require adequate bone volume permitting their anchorage. The periosteum is a specialized fibrous tissue composed of fibroblast, osteoblast, and progenitor cells and optimal source for bone tissue engineering based on its accessibility and rapid proliferation in vivo and in vitro. Low-intensity pulsed ultrasound (LIPUS) has shown successful bone healing of delayed, non-unions and fracture healing in animal models and in clinical treatments. This study aimed to investigate the effects of LIPUS on the proliferation and osteogenic differentiation of murine calvarial periosteum-derived cells (PDCs). PDCs were treated with daily ultrasound stimulation 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 with PrestoBlue Cell Viability assay. Osteogenic differentiation was determined with alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red staining, and real-time reverse transcriptase- polymerase chain reaction. The results indicated that LIPUS stimulation did not significantly affect the viability of PDCs. LIPUS significantly increased ALP activity on day 7 of culture and markedly promoted the formation of mineralized nodules on day 21. The mRNA expression of ALP, RUNX2, OSX, and OCN was significantly up-regulated by LIPUS stimulation. Thus, LIPUS stimulation increases osteogenic differentiation of PDCs and is useful for potential bone regeneration.