Low Intensity Pulsed Ultrasound for bone regeneration therapy: a controlled in vitro study method

Jill Savva, Margaret Lucas, Helen Mulvana, Medical and Industrial Ultrasonics, School of Engineering, University of Glasgow


Low Intensity Pulsed Ultrasound (LIPUS) stimulates healing of fractured bone. Many in vitro studies, including Tang et al. [Molecular Pharmacology, 2006; 69(6): pp2047-2057], investigate the mechanisms involved by measuring cellular markers of bone regrowth under various acoustic conditions.  But comparison between trials is difficult due to inadequate acoustic characterisation. Many set-ups are prone to standing waves and plate resonances, making acoustic conditions difficult to predict. Additionally, spatial-average acoustic intensity (ISATA) is the standard measure of exposure, whereas mechanical bio-effects, considered the most likely mechanism, are more associated with peak-negative pressure (PNP).

This study aims to establish a robust and repeatable protocol for in vitro investigation of LIPUS and proposes the use of PNP to compare results.


Murine osteoblast cells (MC3T3-E1) were grown to confluency in a custom cell-holder, comprising a circular 3D-printed frame (VeroGrayTM) bounded by 6µm Mylar membranes (Goodfellow, UK), providing an acoustically transparent window and cell growth surface. Self-sealing septa allow injection of cells and growth media.

The cell-holder was positioned 100mm from a purpose-built LIPUS transducer in a tank of sterilised water at 37°C and exposed for 20 minutes to LIPUS with frequency 1MHz, pulse width 200µs, repetition rate 1kHz, PNP 0kPa (control) to 500kPa.  The maximum and spatial-averaged PNPs and Intensities were determined prior to exposure by scanning with a 0.5mm needle hydrophone (Precision Acoustics, UK) and correcting for membrane attenuation.

After incubating for 24 hours, the concentration of Prostaglandin E2 (PGE2), a marker of bone regrowth used by Tang et al., was measured using an ELISA kit (Abcam AB133021) and standard microplate reader (Tecan, AT). 


PGE2 up-regulation was assessed against PNP to establish optimal conditions for LIPUS stimulation of bone regrowth. Intensity data allowed comparison with existing studies.


The results will validate the protocol for controlled investigation of the mechanisms involved in LIPUS stimulation.

View the Poster here