Original contribution
Skeletal effects of low-intensity pulsed ultrasound on the ovariectomized rodent

https://doi.org/10.1016/S0301-5629(01)00376-3Get rights and content

Abstract

Growing evidence supports low-intensity pulsed ultrasound (US) as an osteogenic mechanical stimulus. Its effects on isolated bone cells and on fractured bone are established. However, its effects on osteoporosis are not clear. This study examined US effects on ovariectomy (OVX) induced bone changes within the rodent hindlimb (distal femur and proximal tibia), and on normal bone in animals following sham-OVX. Animals were exposed to daily unilateral active-US and contralateral inactive-US for 12 weeks. Bone status was assessed using dual energy X-ray absorptiometry and histomorphometry. Ovariectomy resulted in significant bone changes. Low-intensity pulsed US did not influence these changes. These results suggest that the US dose introduced may not be a beneficial treatment for osteoporosis, and that intact bone may be less sensitive to US than fractured bone and isolated bone cells. This may relate to the biophysical mechanisms of action of US, US-bone interactions and tissue level processes taking place. (E-mail: [email protected])

Introduction

Osteoporosis, the preeminent clinical problem resulting from low bone mass and increased bone fragility, represents a major public health concern, causing increased morbidity and mortality in individuals who are often otherwise healthy. The mainstay of osteoporosis management is pharmacological intervention. However, as systemic treatments, these do not specifically target osteoporotic sites prone to fracture. In addition, they can result in a range of undesireable side-effects, limiting their application in various subgroups within the community. Due to the increasing global problem of osteoporosis associated with the aging of the population, there is a need to develop new interventions for this disease. The use of nonpharmacological therapies, either in isolation or in combination with pharmacological intervention, warrants evaluation.

Ultrasound (US) represents a potential nonpharmacological intervention for osteoporosis. Ultrasound refers to high-frequency nonaudible acoustic energy which travels in the form of a mechanical wave (Chivers 1991). A mechanical wave is one in which energy is transmitted by the movement of particles within the medium through which the wave is travelling (ter Haar 1987). As these mechanical waves travel as a relatively focussed beam (typical effective radiating area = 5 cm2), US can be directed onto specific regions to exert a local mechanical stimulus.

There is good biologic rationale to justify the application of US-produced mechanical stimuli to bone for the treatment of osteoporosis. Bone is a dynamic tissue which remodels in response to applied mechanical stimuli. This response has been thoroughly documented both in vitro and in vivo (Duncan and Turner 1995). As a form of mechanical stimulation, US may produce a similar remodeling response. This theory is supported by accumulating evidence demonstrating an osteogenic effect of pulsed-wave US at low (<100 mW/cm2) spatial-averaged temporal-averaged intensities Warden et al 2000a, Warden et al 2000c.

Currently, the most frequent application of low-intensity pulsed US to bone is during fracture repair. In animal fracture models, such US has been shown to facilitate the rate of endochondral bone formation Duarte 1983, Ito et al 1998, Wang et al 1994, increasing the return of bone mineral density (BMD) at the fracture site (Ito et al. 1998). This resulted in the facilitation of mechanical strength return by a factor of 1.4 to 1.6 (Pilla et al. 1990). In humans, low-intensity pulsed US has been shown to facilitate the return of bone mineral content (BMC) during limb lengthening procedures (Sato et al. 1999) and to accelerate fresh fracture repair by 38% Heckman et al 1994, Kristiansen et al 1997. When applied clinically to nonunited fractures, the same US has been shown to stimulate union in over 85% of cases Frankel 1998, Mayr et al 2000.

Additional evidence for an osteogenic effect of low-intensity pulsed US is provided by a growing number of in vitro investigations. These suggest a direct cellular basis for US effects on bone with osteoblastic changes including the transient expression of the immediate-early response genes c-fos (Naruse et al. 2000) and COX-2 (Kokubu et al. 1999), and elevated mRNA levels for insulin-like growth factor-I, osteocalcin and bone sialoprotein (Naruse et al. 2000). Other in vitro findings include an increase in osteoblastic proliferation Doan et al 1999, Reher et al 1998, collagen and noncollagenous protein synthesis Doan et al 1999, Reher et al 1997, calcium uptake (Ryaby et al. 1989) and prostaglandin E2 production (Kokubu et al. 1999). Further, low-intensity pulsed US has been shown to stimulate in vitro endochondral ossification in isolated bone rudiments, resulting in a threefold increase in the length of the calcified diaphysis (Nolte et al. 1999).

Despite this indirect evidence suggesting that low-intensity pulsed US may stimulate osteogenesis in intact bone, there is conflicting research investigating this potential application. In growing rodents, low-intensity pulsed US applied for 4 weeks had no effect on hindlimb BMD (Spadaro and Albanese 1998). Similarly, it had no effect on bone mineral loss associated with wing bandaging in pigeons (Wimsatt et al. 2000). Conversely, in five osteoporotic bedridden patients, daily low-intensity pulsed US applied for 4 weeks to the neck of femur resulted in an 8.9% average increase in BMD compared with baseline (Arai et al. 1993). Bone mineral density on the contralateral nontreated side decreased by 4.0% within the same period. This latter finding suggests an anabolic effect of low-intensity pulsed US on intact bone. It is apparent that further research is necessary to evaluate whether low-intensity pulsed US should be pursued as a possible clinical modality for osteoporosis. The aim of this investigation was to examine the effect of low-intensity pulsed US on hindlimb (distal femoral and proximal tibial) bone loss following OVX in rodents. A second aim was to investigate the effect of the same US on normal rodent bone in animals following sham-OVX.

Section snippets

Animals

Following ethical approval (Pharmacology and Physiology Animal Experimentation and Ethics Sub-Committee, The University of Melbourne, Parkville, VIC, Australia), 70 virgin female Sprague-Dawley rats were obtained at birth from the Biologic Research Facility at the University of Melbourne (Parkville, VIC, Australia). Animals were matured for a period of 26 weeks. Throughout maturation and the duration of the experiment, the housing room was maintained at 20°C with a 12 h light/12 h dark light

Animal weights

There were no significant differences in weight between the groups at baseline (p > .05). Surgery had a significant effect on weight gain, with OVX-C increasing their weight by 28.6% (97.0 ± 26.2 g) over the intervention period compared to a 15.7% (51.8 ± 18.8 g) increase in sham-OVX-C (p < 0.001; OVX-C vs. sham-OVX-C). Weight gain in restrained animals was significantly lower than in cage control animals following both OVX (p = .002; OVX-B vs. OVX-C) and sham-OVX (p = .001; sham-OVX-B vs.

Discussion and summary

To our knowledge, this is the first controlled study investigating the potential of low-intensity pulsed US as a treatment for osteoporosis. Using the OVX rodent as a model of osteoporosis, low-intensity pulsed US at the dose introduced had no effect on bone loss within the distal femur or proximal tibia. Similarly, it had no effect on normal rodent bone following sham-OVX. This nonsignificant finding supports that of Spadaro and Albanese (1998). Using the same dosage, low-intensity pulsed US

Acknowledgements

This work was funded by the Rebecca L. Cooper Medical Research Foundation. The authors gratefully acknowledge the expertise of Dr. A. Richards and Mr. A. Stirling (Ultrasound Standards, National Measurement Laboratory, Division of Telecommunications and Industrial Physics, CSIRO) in providing assistance with ultrasound design and testing, and experimental methodology; Dr K. Lay (Rowville Veterinary Clinic) in performing animal surgery; Ms. E. Wilson in assisting with US interventions; Ms. B.

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