doi:10.1016/j.ultrasmedbio.2005.01.013 ● Original Contribution EARLY GENE RESPONSE TO LOW-INTENSITY PULSED ULTRASOUND IN RAT OSTEOBLASTIC CELLS KOTARO SENA,ROBERT M. LEVEN,KHURRAM MAZHAR,DALE R. SUMNER, AMARJIT S. VIRDI Department of Anatomy and Cell Biology, Rush University Medical Center, Chicago, IL, USA (Received 01 June 2004; revised 20 January 2005; in final form 27 January 2005) Abstract—The aim of the current research was to quantify the changes in gene expression in rat bone marrow derived stromal cells (BMSC) to low intensity pulsed ultrasound (LIPUS) during early time points after the ultrasound application. LIPUS at 1.5 MHz, 30 mW/cm 2 was applied to BMSC for a single 20 min treatment. Real-time PCR was carried out to quantify the expression of early response genes and bone differentiation marker genes 0.5, 1, 3, 6 and 12 h after the end of the LIPUS treatment. Compared with the controls, LIPUS treatment resulted in elevated transient expression of early response genes (c-jun, c-myc, COX-2, Egr-1, TSC-22) as well as the bone differentiation marker genes, osteonectin and osteopontin, at 3 h. This induction of early response genes as well as extracellular matrix genes associated with cell proliferation and differentiation may represent the effect of LIPUS to cells of osteoblastic lineage. (E-mail: amarjit_virdi@rush.edu) © 2005 World Federation for Ultrasound in Medicine & Biology. Key Words: Low-intensity pulsed ultrasound, Fracture healing, Bone marrow derived stromal cells, Gene expression. INTRODUCTION Bone fracture healing is a complex biological process that involves the spatial and temporal orchestration of various cell types, large numbers of genes and extracel- lular matrix. The process of fracture healing can be divided into several stages such as immediate injury response, repair (intramembranous ossification, endo- chondral ossification) and remodeling. While each stage has distinct histological features, they share several un- derlying events that are common in fracture repair. These events include cell proliferation and differentiation, che- motaxis and the synthesis of extracellular matrix (Bolan- der 1992). Although there are few therapeutic options for the enhancement of local bone repair, the medical appli- cation of low-intensity pulsed ultrasound (LIPUS) is an established therapy for fracture repair. In 1994, LIPUS was approved for the stimulation of fresh bone fracture healing in the United States by the Food and Drug Administration and, in 2000, approval was extended to the treatment of established nonunions (Rubin et al. 2001). In animal fracture models, such ultrasound appli- cation has been shown to facilitate union, accelerating mechanical strength (Duarte 1983; Wang et al. 1994). In humans, double-blind, prospective, placebo-controlled clinical trials demonstrated the reduction of time to union in fresh fractures by up to 40% with the use of LIPUS (Heckman et al. 1994; Kristiansen et al. 1997). When applied in the clinical treatment of delayed unions and nonunions, LIPUS has been shown to stimulate union in a relatively short period of time (Mayr et al. 2000). Despite its pronounced effects during fracture repair, the underlying mechanisms of action of LIPUS remain un- clear. Kokubu et al. (1999) demonstrated LIPUS to in- crease prostaglandin E2 (PGE 2 ) production via the in- duction of cyclooxygenase-2 (COX-2) in a mouse osteo- blastic cell line (MC3T3-E1). Naruse et al. (2000) showed LIPUS to induce the transient expression of the immediate-early response gene c-fos and to elevate gene expression for bone sialoprotein (BSP), insulin-like growth factor-1 (IGF-1) and osteocalcin (OC), using a mouse bone marrow stromal derived cell line (ST2 cell). More recently, these authors also reported that LIPUS elevated gene expression of the same genes in bone marrow derived osteoblastic cells (Naruse et al. 2003). Address correspondence to: Department of Anatomy and Cell Biology, Rush University Medical Center, 600 S. Paulina Street - AF507, Chicago, IL 60612, USA. E-mail: amarjit_virdi@rush.edu Ultrasound in Med. & Biol., Vol. 31, No. 5, pp. 703–708, 2005 Copyright © 2005 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/05/$–see front matter 703