Mitigation of bone loss with ultrasound induced dynamic mechanical signals in an OVX induced rat model of osteopenia

Abstract

This study tests the hypothesis that an ultrasound generated dynamic mechanical signal can attenuate bone loss in an estrogen deficient model of osteopenia. Eighty-four 16-week-old Sprague–Dawley rats were divided into six groups: baseline control, age-matched control, ovariectomy (OVX) control, OVX + 5 mW/cm² ultrasound (US), OVX + 30 mW/cm² US and OVX + 100 mW/cm² US. Low intensity pulsed ultrasound (LIPUS) was delivered transdermally at the L4/L5 vertebrae, using gel-coupled plane wave US transducers. The signal, characterized by 200 μs pulses of 1.5 MHz sine waves repeating at 1 kHz with spatial-averaged temporal-averaged (SATA) intensities of 5, 30 or 100 mW/cm², was applied 20 min/day, 5 days/week for 4 weeks. OVX treatment reduced bone volume fraction 40% and compromised microstructure at 4 weeks. LIPUS treatment, however, significantly increased BV/TV (+33%) compared to OVX controls for the 100 mW/cm² treated group. SMI and Tb.N showed significant improvements compared with OVX for the 100 mW/cm² treated group and Tb.Th was significantly improved in the 30 and 100 mW/cm² treated groups. Improvements in bone's microstructural characteristics with 100 mW/cm² US treatment translated into improved load bearing characteristics, including a significant 42% increase in apparent level elastic modulus compared to OVX controls. Significant improvement of trabecular mechanical strength was also observed in the treated animals, e.g., principal compressive stress (represent bone's ability to resist loads) was significantly higher compared to OVX controls. Histomorphometric analysis also showed that treatment with 100 mW/cm² US resulted in a 76% improvement in MS/BS. In addition, measures of bone quantity and quality at the femoral metaphysis suggest that LIPUS is site specific. This study indicates that localized ultrasound treatment, delivered at specific intensities, has beneficial effects on intact bone and may represent a novel intervention for bone loss.

Introduction

Osteoporosis is a disease characterized by decreased bone mass and progressive erosion of the microstructure. As a result, key skeletal sites such as the hip, spine and wrist are at increased risk of fracture in response to minimal trauma. Current treatments include hormonal, pharmacologic and mechanical strain interventions. Hormonal and pharmacologic interventions are often associated with adverse side effects and target the skeleton as a whole, as opposed to specifically targeting skeletal sites at increased risk for failure. Mechanical strain interventions, however, are noninvasive and have demonstrated promising results. In vivo studies have shown low-magnitude, high-frequency vibrations to be anabolic in both human [1] and animal models [2]. In addition, whole bone accelerations have been shown to be anabolic to bone [3], [4]. A contributing mechanism, by which low-magnitude mechanical stimulations act, could involve bone fluid flow. Previous studies have shown that in the absence of mechanical strain, intramedullary bone fluid flow can drive bone remodeling [5], [6].

It has been suggested that in bone, ultrasound behaves as a mechanical wave, generating local pressure gradients. This may result in the production of anabolic shear forces on cell membranes or changes in local solute concentrations [7], [8]. These gradients could drive local fluid flow, potentially resulting in an anabolic signal. Therefore, low intensity pulsed ultrasound (LIPUS) may offer a noninvasive method for delivery of high frequency, low amplitude and large cycle number, dynamic mechanical signals.

In vitro studies have shown that LIPUS is capable of increasing osteoblast proliferation and stimulating endochondral ossification in excised tissues [9], [10], [11], [12]. It has also been shown that ultrasound signal intensity plays an important role in modulating the response of osteoblasts in vitro [13], [14]. One potential mechanism by which ultrasound acts could involve integrin receptors. An in vitro study has shown that osteoblasts up-regulate inducible nitric-oxide synthase (iNOS) via an integrin receptor in osteoblasts [15]. In addition, ultrasound may act to increase bone morphogenetic protein-2 (BMP-2) [16]. In vivo studies have also shown that LIPUS accelerated fracture healing in both animal [17], [18], [19], [20], [21] and human models [22], [23], [24], [25]. In addition, Yang et al. showed that bone's response to ultrasound during fracture healing was sensitive to ultrasound signal intensity [21]. While LIPUS has demonstrated effects on regulating osteoblastic activities in vitro and promote fracture healing in vivo, one may assume that ultrasound may play a role in modulating osteopenia associated with estrogen deficiency and aging as well. However, there is limited, conflicting evidence with respect to the effectiveness of LIPUS in treating non-fracture related bone diseases in vivo [26], [27], [28], [29], [30]. In one study, 26-week-old rats (~ 332 g) were ovariectomized and LIPUS (30 mW/cm², 1 MHz pulsed at 1 kHz, 20 min/day, 6 days/week, for 12 weeks) was delivered to the proximal tibia. This study found that ultrasound (US) signals had no effect on wet weight or bone formation rate (BFR) [27]. Another study evaluated the effects of LIPUS (30 mW/cm², 1.5 MHz pulsed at 1 kHz, 20 min/day, for 20 days) at the proximal femur of 200 g female Holstman rats subjected to 30 days OVX prior to treatment. Histological analysis using mason trichrome staining showed qualitative improvements in ultrasound treated animals not observed in control groups [29]. In a more recent study, LIPUS (1.5 MHz, 1.0 kHz pulse repetition, 30 mW/cm², with intensity of 200 μs pulse length) was applied to 14-week-old OVX mice for 6-week, and indicated that bone volume of treated limb was significantly enhanced compared to the contralateral control [38]. In light of the differences among US stimulation protocols used in these studies, it is remained unclear what role ultrasound signal parameters, in particular signal intensity, play in bone's response.

The objective of this study was to explore the therapeutic potential of LIPUS for treatment of bone loss associated with estrogen deficient osteopenia using high resolution three-dimensional imaging and computational structural analysis techniques. This study tests the hypothesis that an ultrasound generated dynamic mechanical signal can mediate bone loss and changes to structural integrity in an estrogen deficient model of osteopenia. To this end, we have completed a study in which we tested the effectiveness of various US signal intensities in preserving bone's microarchitecture and mechanical integrity using high resolution imaging, dynamic histomorphometry and computer modeling techniques.