Low-Intensity Pulsed Ultrasound Stimulates a Bone-Forming Response in UMR-106 Cells Stuart J. Warden,* Jenny M. Favaloro,† Kim L. Bennell,* ,1 Joan M. McMeeken,* Kong-Wah Ng,‡ Jeffery D. Zajac,§ and John D. Wark† ,¶ *Centre for Sports Medicine Research and Education, School of Physiotherapy, †Department of Medicine, Royal Melbourne Hospital, and §Department of Medicine, Austin and Repatriation Medical Centre, University of Melbourne, Melbourne, Victoria, Australia; ‡St. Vincent’s Institute of Medical Research, Australia; and ¶ Bone and Mineral Service, Royal Melbourne Hospital, Melbourne, Victoria, Australia Received July 20, 2001 Low-intensity (< 100 mW/cm 2 ) pulsed ultrasound (US) is an established therapy for fracture repair. In both animal and human trials, such US has been shown to facilitate fresh fracture repair and initiate healing in fractures with repair defects. However, the mechanism by which US achieves these outcomes is not clear. One possible mechanism is the direct stim- ulation of bone formation. To investigate this hypoth- esis, the current study investigated the mRNA re- sponse of isolated bone-forming cells (UMR-106 cells) to a single 20-min dose of low-intensity pulsed US. Using a novel US-cell coupling method, US was found to stimulate expression of the immediate-early re- sponse genes c-fos and COX-2 and elevate mRNA levels for the bone matrix proteins ALP and OC. These find- ings suggest that low-intensity pulsed US has a direct effect on bone formation. This may contribute to the beneficial effect of low-intensity pulsed US on fracture repair. © 2001 Academic Press Key Words: fracture healing; mechanical loading; mechanotransduction; RT-PCR; ultrasound therapy; UMR-106 cells. Low-intensity (100 mW/cm 2 ) pulsed ultrasound (US) is an established therapy for fracture repair (1). In animal fracture models, such US has been shown to facilitate union (2, 3) accelerating mechanical strength return by 30 –38% (4). In humans, low-intensity pulsed US has been shown to induce a 30 –38% reduction in the time to union in fresh fractures (5–7), and reduce the incidence of delayed union (8). When applied to established nonunited fractures, the same US has been shown to stimulate union in over 85% of cases (9). Despite its pronounced effects during fracture re- pair, the underlying mechanisms of action of low- intensity pulsed US remain unclear. One possible mechanism is the direct stimulation of bone formation. Nolte et al. (10) found low-intensity pulsed US to stim- ulate bone rudiment ossification suggesting a direct effect of US on osteogenesis. Investigating the mecha- nism for this apparent bone forming response, Kokubu et al. (11) demonstrated low-intensity pulsed US to induce a threefold increase in prostaglandin E 2 (PGE 2 ) production in murine osteoblasts (MC3T3 cells). This resulted from the upregulation of cyclooxygenase-2 (COX-2). COX-2 and PGE 2 are known mediators in a bone forming response to external stimuli (12). More recently, Naruse et al. (13) using bone-marrow-derived stromal cells (ST2 cells) showed low-intensity pulsed US to induce the transient expression of the immediate-early response gene c-fos and elevate mRNA levels for insulin-like growth factor-I (IGF-I), osteocalcin (OC) and bone sialoprotein (BSP). These changes are consistent with a bone-forming response. Although isolated bone cells have been shown to be responsive to low-intensity pulsed US, the methods of US-cell coupling previously used may have influenced the US dose introduced. Optimal US-cell coupling oc- curs when there is minimal attenuation of US energy between the US treatment head and cells allowing for an accurately known dose of US to be repeatedly intro- duced. In previous studies (11, 13), cells were plated in standard six-well perspex dishes. US was directed at the bottom of the wells and was required to pass through the perspex before influencing the cells. Given the acoustic properties of perspex, the incident dose of Abbreviations used: US, ultrasound; PGE 2 , prostaglandin E 2 ; COX-2, cyclooxygenase-2; IGF-I, insulin-like growth factor-I; OC, osteocalcin; BSP, bone sialoprotein; UEC(s), ultrasound exposure chamber(s); TGF-, transforming growth factor-; ALP, alkaline phosphatase; GAPDH, glyceraldhyde-3-phosphate dehydrogenase. 1 To whom correspondence and reprint requests should be addressed at Centre for Sports Medicine Research and Education, School of Phys- iotherapy, University of Melbourne, Victoria, 3010 Australia. Fax: +61- 3-8344-4188. E-mail: k.bennell@unimelb.edu.au. Biochemical and Biophysical Research Communications 286, 443– 450 (2001) doi:10.1006/bbrc.2001.5412, available online at http://www.idealibrary.com on 443 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.