Original contribution
Involvement of adenosine 5′-triphosphate in ultrasound-induced fracture repair

https://doi.org/10.1016/j.ultrasmedbio.2005.04.017Get rights and content

Abstract

Ultrasound (US) accelerates fracture healing; however, the mechanism of this effect remains unclear. Adenosine 5′-triphosphate (ATP) stimulates bone remodeling and is released constitutively from intact osteoblasts; this is a process that is enhanced after mechanical stimulation. We hypothesized that ATP release from osteoblasts is increased after US stimulation and that this leads to accelerated fracture healing. US was applied to SaOS-2 human osteoblasts and the concentration of ATP in the cell culture medium was determined. Cell proliferation and gene expression were subsequently investigated. Increased concentrations of ATP were detected in the culture medium of US-treated cells and both ATP and US stimulation caused increased receptor activator of nuclear factor-kappa B ligand (RANKL), decreased osteoprotegerin expression and increased cell proliferation by SaOS-2 cells. These findings indicate that US causes ATP release by osteoblasts in vitro and that this may contribute to accelerated fracture healing by enhancing osteoblast proliferation and increasing RANKL expression and decreasing osteoprotegerin expression by osteoblasts to promote osteoclastogenesis. (E-mail: [email protected])

Introduction

Ultrasound (US) is the term given to sound waves with a frequency that is above the limit of human audibility. They are propagating pressure waves that transfer mechanical energy to tissues and cells when applied therapeutically. Mechanical strains received by cells may result in biochemical events that, in the skeleton, have been shown to promote bone formation. Numerous studies have revealed that low-intensity (< 100 mW/cm2) pulsed US increases the rate of fracture healing. Double-blind placebo-controlled studies on US-treated human fractures of the distal radius, tibial shaft and scaphoid have reported improvement in healing rates by approximately 30 to 40% (Kristiansen et al 1997, Heckman et al 1994). In addition, US has been shown to reduce the incidence of delayed union (Cook et al 1997, Mayr et al 2000) and to stimulate union in established nonunited fractures in over 85% of cases (Mayr et al. 2000). The mechanism by which US induces these responses, however, remains unclear. Fracture healing is a complex process that involves cell proliferation and differentiation, chemotaxis and the synthesis of extracellular matrix (Bolander 1992). Numerous factors have been implicated in the stimulation of these fracture-repair processes, including platelet-derived growth factor (Ito et al. 2000), basic fibroblast growth factor (Kawaguchi et al. 1994), nitric oxide (Diwan et al. 2000), prostaglandin E2 (Keller et al. 1993) and transforming growth factor beta (Bolander 1992) and increased production of these factors by osteoblasts has been reported after US stimulation (Ito et al 2000, Doan et al 1999, Li et al 2002, Li et al 2003, Reher et al 2002). Interleukin-8 production by osteoblasts is increased by US stimulation (Reher et al. 1999), which may accelerate fracture healing via its angiogenic activity.

Recent data have implicated adenosine 5′-triphosphate (ATP) as an important extracellular signaling molecule in the bone microenvironment (Bowler et al 2001, Hoebertz et al 2003). Although ATP is more commonly known for its role as an intracellular energy source, it is also active extracellularly as an agonist at a group of membrane receptors termed P2 receptors (Burnstock 1978, Abbracchio and Burnstock 1994), multiple subtypes of which are expressed by bone-forming osteoblasts (Hoebertz et al 2000, Maier et al 1997) and bone-resorbing osteoclasts (Hoebertz et al 2000, Naemsch et al 1999, Wiebe et al 1999, Buckley et al 2002). ATP has been shown to increase proliferation and DNA synthesis by osteoblast-like cells after P2 receptor stimulation (Shimegi 1996, Nakamura et al 2000) and, as well as these influences on osteoblasts, ATP stimulates both osteoclast formation and resorptive activity (Buckley et al 2002, Morrison et al 1998; Bowler et al. 1998; Hoebertz et al. 2001). There is, therefore, a growing volume of literature documenting ATP as an important factor in the bone microenvironment, in particular, its strong influence on accelerating remodeling, the highly coordinated process of bone regeneration where osteoclastic resorption is thought to activate osteoblastic bone formation in a process described as coupling.

High concentrations of ATP in the extracellular environment are likely to be found at trauma sites, such as fractures, where cell lysis results in the release of ATP, which is usually at a concentration of 3 to 5 mM inside the cell. It can, therefore, be hypothesized that ATP influences the increased rate of modeling and remodeling that occurs during fracture healing. ATP is released constitutively from healthy osteoblasts (Buckley et al. 2003) and this process is enhanced after mechanical stimulation from fluid shear (Romanello et al. 2001). Similar findings have been reported in other cell types (Burnstock 1999, Homolya et al 2000). Nucleotides have been suggested to play a role in mechanotransduction in bone, where detection of mechanical deformation by skeletal cells results in remodeling (Jorgensen et al. 1997).

These previous findings led us to hypothesize that ATP is released after US stimulation of osteoblasts and that release of this nucleotide could be involved in some of the processes that lead to fracture repair. This study reports the use of the Exogen 2000+/Sonic Accelerated Fracture Healing System (SAFHS) (Smith and Nephew, Cambridge, UK) to apply US stimulation to osteoblast-like SaOS-2 cells and the effects of this stimulation on ATP release, cell proliferation and osteoblast gene expression. In particular, the fundamental factors in controlling osteoclastogenesis, receptor activator for nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG), will be studied because their role in initiating or inhibiting osteoclastogenesis strongly influences the remodeling process. Variations in expression of other genes, including osteocalcin and osteonectin, will be examined to determine changes in osteoblast differentiation after US stimulation.

Section snippets

Materials

Dulbecco’s modified Eagle’s medium (DMEM), F-12 HAM medium, fetal calf serum (FCS), l-glutamine, penicillin and streptomycin, trypsin-ethylenediaminetetraacetic acid, 5X 1st strand reaction buffer, and Superscript™ II reverse transcriptase were purchased from Invitrogen, Paisley, UK. Deoxyribonuclease (Dnase I), ribonuclease (Rnase) inhibitor and deoxynucleotide triphosphates were from Roche Diagnostics, Welwyn Garden City, UK. Nucleotides, TRI-REAGENT™ and diethylpyrocarbonate were purchased

ATP release from SaOS-2 cells after ultrasound stimulation

ATP concentration in cell culture medium samples collected from dishes of confluent SaOS-2 reporter cells exposed to US or from control cells, was measured by light emission after addition of NMR containing luciferin and luciferase in a tube luminometer. ATP concentration before introduction of the US probe was subtracted from subsequent sample measurements. Two samples were taken at each time point and the average was calculated. This procedure was repeated on 10 separate occasions, using

Discussion

After reports of increased ATP release from human osteoblasts in response to mechanical stimulation and publication of numerous studies demonstrating a decrease in time to fracture healing caused by US application, this study has aimed to determine whether or not US induces ATP release from osteoblasts. In addition, we have investigated the effects of US stimulation on osteoblast gene expression and cell proliferation and compared these responses to those induced by ATP, to deduce whether or

Summary

The findings reported in this study reveal that osteoblasts respond to US stimulation by increasing ATP release and that the role of this local mediator in fracture repair can be inferred from its similar effects to US in osteoblast gene expression and cell proliferation. Increased expression of RANKL and decreased expression of OPG by osteoblasts to promote osteoclastogenesis and enhanced proliferation of osteoblasts may be mechanisms via which US-induced ATP release from osteoblasts leads to

Acknowledgements

The authors acknowledge Smith and Nephew for provision of the Exogen 2000+/Sonic accelerated fracture healing system. The authors thank the Arthritis Research Campaign (ARC) for funding of J.P. Dillon and K. Buckley.

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