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
Background:
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.
Methods:
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).
Results:
PGE2 up-regulation was assessed against PNP to establish optimal conditions for LIPUS stimulation of bone regrowth. Intensity data allowed comparison with existing studies.
Conclusions:
The results will validate the protocol for controlled investigation of the mechanisms involved in LIPUS stimulation.