Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound

Abstract

Bone growth and repair are under the control of biochemical and mechanical signals. Low-intensity pulsed ultrasound (LIPUS) stimulation at 30 mW/cm² is an established, widely used and FDA approved intervention for accelerating bone healing in fractures and non-unions. Although this LIPUS signal accelerates mineralization and bone regeneration, the actual intensity experienced by the cells at the target site might be lower, due to the possible attenuation caused by the overlying soft tissue. The aim of this study was to investigate whether LIPUS intensities below 30 mW/cm² are able to provoke phenotypic responses in bone cells. Rat bone marrow stromal cells were cultured under defined conditions and the effect of 2, 15, 30 mW/cm² and sham treatments were studied at early (cell activation), middle (differentiation into osteogenic cells) and late (biological mineralization) stages of osteogenic differentiation. We observed that not only 30 mW/cm² but also 2 and 15 mW/cm², modulated ERK1/2 and p38 intracellular signaling pathways as compared to the sham treatment. After 5 days with daily treatments of 2, 15 and 30 mW/cm², alkaline phosphatase activity, an early indicator of osteoblast differentiation, increased by 79%, 147% and 209%, respectively, compared to sham, indicating that various intensities of LIPUS were able to initiate osteogenic differentiation. While all LIPUS treatments showed higher mineralization, interestingly, the highest increase of 225% was observed in cells treated with 2 mW/cm². As the intensity increased to 15 and 30 mW/cm², the increase in the level of mineralization dropped to 120% and 82%. Our data show that LIPUS intensities lower than the current clinical standard have a positive effect on osteogenic differentiation of rat bone marrow stromal cells. Although Exogen™ at 30 mW/cm² continues to be effective and should be used as a clinical therapy for fracture healing, if confirmed in vivo, the increased mineralization at lower intensities might be the first step towards redefining the most effective LIPUS intensity for clinical use.

Introduction

Bone, a highly specialized form of connective tissue consists of a stiff outer region (cortical bone) which overlies the softer inner structure (cancellous or trabecular bone). Bone formation and regeneration involve chemotaxis, cell proliferation, differentiation and the synthesis of an extracellular matrix; a result of interactions amongst biochemical, biomechanical, cellular and hormonal signals [1]. Bone formation is defined as intramembranous or endochondral depending on whether a membranous tissue or cartilage is the precursor for bone. The fracture healing site however, goes through a cascade of distinct but interdependent stages; such as immediate inflammatory response, soft callus formation, hard callus formation (endochondral ossification) and remodeling phases [2]. Osteogenic stem cell growth and differentiation play an important role in the repair phase of fracture healing. However, a significant percentage of fractured bones experience delayed healing, and in some case, result into a non-union. Low-intensity pulsed ultrasound (LIPUS) stimulation is a clinically established, widely used and FDA approved intervention for accelerating bone growth during healing of fractures, non-unions and other osseous defects, based on in vivo studies [3], [4], [5], [6], [7], [8], [9]. Numerous studies have also shown its direct effect on bone cells in in vitro culture systems [10], [11], [12], [13], [14]. LIPUS has been shown to increase the rate of fracture repair at all stages of the healing process, and is most effective when applied during all stages of the repair [15]. Although the mechanism by which LIPUS induces these responses is unclear, it is known that the increase in the rate of fracture healing is induced by mechanical strains received by cells translated into biochemical events [16], [17], [18].

Therapeutic ultrasound with frequencies varying between 0.5–1.5 MHz and intensities 30–200 mW/cm² are known to promote healing, bone deposition and growth [12], [19], [20], [13], [21]. LIPUS has predominantly been used at an intensity of 30 mW/cm² to enhance bone fracture repair [19], [15], orthopedic implant fixation [9], non-union healing [22], [23] and distraction osteogenesis [24], [25], [26]. But the difficulty in elucidating the effects of LIPUS has been in determining the precise energy delivered to the target cells [19]. LIPUS is a longitudinal wave with compressions and rarefactions, acting as mechanical perturbations on the extra cellular matrix (ECM) of the target cells. The delivered energy depends on the matrix composition of the overlying tissue through which the waves traverse, as well as the coupling between the transducer and sample [27], [28]. It is known that as ultrasound passes through a medium it is exponentially attenuated as described by the equation I = I₀e^−μx, where I is the target intensity of the sound wave, I₀ is the initial intensity, μ is the intensity attenuation coefficient and x is the distance traveled by the sound wave. Thus, it is reasonable to assume that the soft tissues around the fracture will attenuate the ultrasound intensity and the ECM will be exposed to intensities lower than the dose delivered by the transducer. The attenuation caused by the soft tissue around the fracture repair site, exposes the periosteal surface to a much lower intensity than applied. In an in vivo set-up the ultrasound absorption coefficient for soft tissue is in the range of 0.2–0.6 dB/cm MHz whereas it is about 1 dB/cm MHz for collagen fiber rich tissues like skin and tendon [28]. If a homogeneous tissue model is considered with a soft tissue attenuation of 0.5 dB/cm MHz, theoretically, 2 and 15 mW/cm² corresponds to a tissue thickness of 15.68 and 4 cm, respectively.

In vivo studies respond to overall biological effects, but cellular mechanism and actions are better studied in vitro. In the past, the majority of the in vitro studies have used LIPUS at 30 mW/cm² [12], [29], [10], [13], [14], [30], [31], [32]. A handful of studies have attempted to use lower intensities for stimulation considering this evident attenuation and have shown an increase in the expression of type X collagen in chick chondrocytes by 2 mW/cm² LIPUS [33]. Iwashina et al. have shown 15 mW/cm² to stimulate cell proliferation in rabbit intervertebral disc cells [34]. In addition, previous work suggest diagnostic ultrasound with intensities as low as 12 mW/cm² can improve radiographic healing and increase bone density in rat femora [35]. Other evidences present that, ultrasound intensities ranging between 140 and 990 mW/cm² show pronounced and differential effects on cell function directly related to the applied intensity [36]. It was our goal in this study to see the effect of LIPUS intensities lower than the one used clinically in eliciting phenotypic responses in bone cells. Integrins, a family of transmembrane cell adhesion molecules, through their structural connection are proposed to transmit the mechanical signal from the ECM to the cell and initiate intracellular signaling [16], [18]. Mitogen-activated protein kinases (MAPKs) are activated in response to mechanical stress [37], [38] and mediate transduction of the mechanical stimulation into intracellular signals that regulate cell proliferation and differentiation [39], [40], [41].

We hypothesize that LIPUS intensities lower than 30 mW/cm² are able to provoke responses in bone cells that may result in guiding them towards osteogenic differentiation. To test this hypothesis we stimulated rat bone marrow stromal cells (rBMSC) in osteogenic-defined medium with intensities of LIPUS lower than 30 mW/cm² and compared them with sham (0 mW/cm²) treated cells. This in vitro model was used as it serves as an excellent system to study pathways of osteogenic differentiation, alkaline phosphatase (ALP) activity with extracellular matrix and subsequent mineralization as hallmarks of the fully mature osteoblastic phenotype[42], [43] in response to LIPUS stimulation. Early response to LIPUS, 30 min after first stimulation, was studied by observing phosphorylation of extracellular signal-regulated kinases (ERKs) and p38 MAPK known to be activated in response to LIPUS stimulation [44]. The p38 MAPK is a stress activated protein kinase (SAPK) which participates in the response of cells to stress whereas ERK1/2 play a significant role in proliferation and differentiation. Further daily LIPUS stimulation up to 7 days were studied by assaying for alkaline phosphatase (ALP) activity, a known early marker of osteoblast phenotype maturation [45], [46], [47]. Finally, stimulation for up to 24 days was studied by observing in vitro mineral formation, a characteristic feature of biological mineralization.