Prediction of trabecular bone qualitative properties using scanning quantitative ultrasound.

Microgravity induced bone loss represents a critical health problem in astronauts, particularly occurred in weight-supporting skeleton, which leads to osteopenia and increase of fracture risk. Lack of suitable evaluation modality makes it difficult for monitoring skeletal status in long term space mission and increases potential risk of complication. Such disuse osteopenia and osteoporosis compromise trabecular bone density, and architectural and mechanical properties. While X-ray based imaging would not be practical in space, quantitative ultrasound may provide advantages to characterize bone density and strength through wave propagation in complex trabecular structure. This study used a scanning confocal acoustic diagnostic and navigation system (SCAN) to evaluate trabecular bone quality in 60 cubic trabecular samples harvested from adult sheep. Ultrasound image based SCAN measurements in structural and strength properties were validated by µCT and compressive mechanical testing. This result indicated a moderately strong negative correlations observed between broadband ultrasonic attenuation (BUA) and µCT-determined bone volume fraction (BV/TV, R2=0.53). Strong correlations were observed between ultrasound velocity (UV) and bone's mechanical strength and structural parameters, i.e., bulk Young's modulus (R2=0.67) and BV/TV (R2=0.85). The predictions for bone density and mechanical strength were significantly improved by using a linear combination of both BUA and UV, yielding R2=0.92 for BV/TV and R2=0.71 for bulk Young's modulus. These results imply that quantitative ultrasound can characterize trabecular structural and mechanical properties through measurements of particular ultrasound parameters, and potentially provide an excellent estimation for bone's structural integrity.

Extended high-frame rate imaging method with limited-diffraction beams.

Fast three-dimensional (3-D) ultrasound imaging is a technical challenge. Previously, a high-frame rate (HFR) imaging theory was developed in which a pulsed plane wave was used in transmission, and limited-diffraction array beam weightings were applied to received echo signals to produce a spatial Fourier transform of object function for 3-D image reconstruction. In this paper, the theory is extended to include explicitly various transmission schemes such as multiple limited-diffraction array beams and steered plane waves. A relationship between the limited-diffraction array beam weighting of received echo signals and a 2-D Fourier transform of the same signals over a transducer aperture is established. To verify the extended theory, computer simulations, in vitro experiments on phantoms, and in vivo experiments on the human kidney and heart were performed. Results show that image resolution and contrast are increased over a large field of view as more and more limited-diffraction array beams with different parameters or plane waves steered at different angles are used in transmissions. Thus, the method provides a continuous compromise between image quality and image frame rate that is inversely proportional to the number of transmissions used to obtain a single frame of image. From both simulations and experiments, the extended theory holds a great promise for future HFR 3-D imaging.

High frame rate imaging system for limited diffraction array beam imaging with square-wave aperture weightings.

A general-purpose high frame rate (HFR) medical imaging system has been developed. This system has 128 independent linear transmitters, each of which is capable of producing an arbitrary broadband (about 0.05-10 MHz) waveform of up to +/- 144 V peak voltage on a 75-ohm resistive load using a 12-bit/40-MHz digital-to-analog converter. The system also has 128 independent, broadband (about 0.25-10 MHz), and time-variable-gain receiver channels, each of which has a 12-bit/40-MHz analog-to-digital converter and up to 512 MB of memory. The system is controlled by a personal computer (PC), and radio frequency echo data of each channel are transferred to the same PC via a standard USB 2.0 port for image reconstructions. Using the HFR imaging system, we have developed a new limited-diffraction array beam imaging method with square-wave aperture voltage weightings. With this method, in principle, only one or two transmitters are required to excite a fully populated two-dimensional (2-D) array transducer to achieve an equivalent dynamic focusing in both transmission and reception to reconstruct a high-quality three-dimensional image without the need of the time delays of traditional beam focusing and steering, potentially simplifying the transmitter subsystem of an imager. To validate the method, for simplicity, 2-D imaging experiments were performed using the system. In the in vitro experiment, a custom-made, 128-element, 0.32-mm pitch, 3.5-MHz center frequency linear array transducer with about 50% fractional bandwidth was used to reconstruct images of an ATS 539 tissue-mimicking phantom at an axial distance of 130 mm with a field of view of more than 90 degrees. In the in vivo experiment of a human heart, images with a field of view of more than 90 degrees at 120-mm axial distance were obtained with a 128-element, 2.5-MHz center frequency, 0.15-mm pitch Acuson V2 phased array. To ensure that the system was operated under the limits set by the U.S. Food and Drug Administration, the mechanical index, thermal index, and acoustic output were measured. Results show that higher-quality images can be reconstructed with the square-wave aperture weighting method due to an increased penetration depth as compared to the exact weighting method developed previously, and a frame rate of 486 per second was achieved at a pulse repetition frequency of about 5348 Hz for the human heart.

Fourier-Bessel field calculation and tuning of a CW annular array.

A 1-D Fourier-Bessel series method for computing and tuning the linear lossless field of flat continuous wave (CW) annular arrays is given and discussed with both numerical simulation and experimental verification. The technique provides a new method for modelling and manipulating the propagated field by linking the quantized surface pressure profile to a set of limited diffraction Bessel beams propagating into the medium. In the limit, these become a known set of nondiffracting Bessel beams satisfying the lossless linear wave equation, which allow us to derive a linear matrix formulation for the field in terms of the ring pressures on the transducer surface. Tuning (beamforming) of the field then follows by formulating a least squares design with respect to the transducer ring pressures. Results are presented in the context of a 10-ring annular array operating at 2.5 MHz in water.

Interrelation between external oscillatory muscle coupling amplitude and in vivo intramedullary pressure related bone adaptation.

Interstitial bone fluid flow (IBFF) is suggested as a communication medium that bridges external physical signals and internal cellular activities in the bone, which thus regulates bone remodeling. Intramedullary pressure (ImP) is one main regulatory factor of IBFF and bone adaptation related mechanotransduction. Our group has recently observed that dynamic hydraulic stimulation (DHS), as an external oscillatory muscle coupling, was able to induce local ImP with minimal bone strain as well as to mitigate disuse bone loss. The current study aimed to evaluate the dose dependent relationship between DHS's amplitude, i.e., 15 and 30mmHg, and in vivo ImP induction, as well as this correlation on bone's phenotypic change. Simultaneous measurements of ImP and DHS cuff pressures were obtained from rats under DHS with various magnitudes and a constant frequency of 2Hz. ImP inductions and cuff pressures upon DHS loading showed a positively proportional response over the amplitude sweep. The relationship between ImP and DHS cuff pressure was evaluated and shown to be proportional, in which ImP was raised with increases of DHS cuff pressure amplitudes (R(2)=0.98). A 4-week in vivo experiment using a rat hindlimb suspension model demonstrated that the mitigation effect of DHS on disuse trabecular bone was highly dose dependent and related to DHS's amplitude, where a higher ImP led to a higher bone volume. This study suggested that sufficient physiological DHS is needed to generate ImP. Oscillatory DHS, potentially induces local fluid flow, has shown dose dependence in attenuation of disuse osteopenia.

Reversal of the detrimental effects of simulated microgravity on human osteoblasts by modified low intensity pulsed ultrasound.

Microgravity (MG) is known to induce bone loss in astronauts during long-duration space mission because of a lack of sufficient mechanical stimulation under MG. It has been demonstrated that mechanical signals are essential for maintaining cell viability and motility, and they possibly serve as a countermeasure to the catabolic effects of MG. The objective of this study was to examine the effects of high-frequency acoustic wave signals on osteoblasts in a simulated microgravity (SMG) environment (created using 1-D clinostat bioreactor) using a modified low-intensity pulsed ultrasound (mLIPUS). Specifically, we evaluated the hypothesis that osteoblasts (human fetal osteoblastic cell line) exposure to mLIPUS for 20 min/d at 30 mW/cm(2) will significantly reduce the detrimental effects of SMG. Effects of SMG with mLIPUS were analyzed using the MTS proliferation assay for proliferation, phalloidin for F-actin staining, Sirius red stain for collagen, and Alizarin red for mineralization. Our data showed that osteoblast exposure to SMG results in significant decreases in proliferation (∼ -38% and ∼ -44% on days 4 and 6, respectively; p < 0.01), collagen content (∼ -22%; p < 0.05) and mineralization (∼ -37%; p < 0.05) and actin stress fibers. In contrast, mLIPUS stimulation in SMG condition significantly increases the rate of proliferation (∼24% by day 6; p < 0.05), collagen content (∼52%; p < 0.05) and matrix mineralization (∼25%; p < 0.001) along with restoring formation of actin stress fibers in the SMG-exposed osteoblasts. These data suggest that the acoustic wave can potentially be used as a countermeasure for disuse osteopenia.

Dynamic hydraulic fluid stimulation regulated intramedullary pressure.

Physical signals within the bone, i.e. generated from mechanical loading, have the potential to initiate skeletal adaptation. Strong evidence has pointed to bone fluid flow (BFF) as a media between an external load and the bone cells, in which altered velocity and pressure can ultimately initiate the mechanotransduction and the remodeling process within the bone. Load-induced BFF can be altered by factors such as intramedullary pressure (ImP) and/or bone matrix strain, mediating bone adaptation. Previous studies have shown that BFF induced by ImP alone, with minimum bone strain, can initiate bone remodeling. However, identifying induced ImP dynamics and bone strain factor in vivo using a non-invasive method still remains challenging. To apply ImP as a means for alteration of BFF, it was hypothesized that non-invasive dynamic hydraulic stimulation (DHS) can induce local ImP with minimal bone strain to potentially elicit osteogenic adaptive responses via bone-muscle coupling. The goal of this study was to evaluate the immediate effects on local and distant ImP and strain in response to a range of loading frequencies using DHS. Simultaneous femoral and tibial ImP and bone strain values were measured in three 15-month-old female Sprague Dawley rats during DHS loading on the tibia with frequencies of 1Hz to 10Hz. DHS showed noticeable effects on ImP induction in the stimulated tibia in a nonlinear fashion in response to DHS over the range of loading frequencies, where they peaked at 2Hz. DHS at various loading frequencies generated minimal bone strain in the tibiae. Maximal bone strain measured at all loading frequencies was less than 8µÎµ. No detectable induction of ImP or bone strain was observed in the femur. This study suggested that oscillatory DHS may regulate the local fluid dynamics with minimal mechanical strain in the bone, which serves critically in bone adaptation. These results clearly implied DHS's potential as an effective, non-invasive intervention for osteopenia and osteoporosis treatments.

Dynamic hydraulic flow stimulation on mitigation of trabecular bone loss in a rat functional disuse model.

Bone fluid flow (BFF) has been demonstrated as a critical regulator in mechanotransductive signaling and bone adaptation. Intramedullary pressure (ImP) and matrix strain have been identified as potential generators to regulate BFF. To elevate in vivo oscillatory BFF using ImP, a dynamic hydraulic stimulation (DHS) approach was developed. The objective of this study was to evaluate the effects of DHS on mitigation of bone loss and structural alteration in a rat hindlimb suspension (HLS) functional disuse model. Sixty-one 5-month old female Sprague-Dawley rats were divided into five groups: 1) baseline control, 2) age-matched control, 3) HLS, 4) HLS+static loading, and 5) HLS+DHS. Hydraulic flow stimulation was carried out daily on a "10 min on-5 min off-10 min on" loading regime, 5 days/week, for a total of 4 weeks in the tibial region. The metaphyseal trabecular regions of the proximal tibiae were analyzed using µCT and histomorphometry. Four weeks of HLS resulted in a significant loss of trabecular bone, leading to structural deterioration. HLS with static loading alone was not sufficient to attenuate the bone loss. Bone quantity and microarchitecture were significantly improved by applying DHS loading, resulting increase of 83% in bone volume fraction, 25% in trabecular number and mitigation of 26% in trabecular separation compared to HLS control. Histomorphometry analysis on trabecular mineralization coincided with the µCT analysis, in which DHS loading yielded increases of 34% in histomorphometric BV/TV, 121% in MS/BS, 190% in BFR/BS and 146% in BFR/BV, compared to the HLS control. Overall, the data demonstrated that dynamic hydraulic flow loading has potentials to provide regulatory signals for mitigating bone loss induced by functional disuse. This approach may provide a new alternative mechanical intervention for future clinical treatment for osteoporosis.

Mechanobiological modulation of cytoskeleton and calcium influx in osteoblastic cells by short-term focused acoustic radiation force.

Mechanotransduction has demonstrated potential for regulating tissue adaptation in vivo and cellular activities in vitro. It is well documented that ultrasound can produce a wide variety of biological effects in biological systems. For example, pulsed ultrasound can be used to noninvasively accelerate the rate of bone fracture healing. Although a wide range of studies has been performed, mechanism for this therapeutic effect on bone healing is currently unknown. To elucidate the mechanism of cellular response to mechanical stimuli induced by pulsed ultrasound radiation, we developed a method to apply focused acoustic radiation force (ARF) (duration, one minute) on osteoblastic MC3T3-E1 cells and observed cellular responses to ARF using a spinning disk confocal microscope. This study demonstrates that the focused ARF induced F-actin cytoskeletal rearrangement in MC3T3-E1 cells. In addition, these cells showed an increase in intracellular calcium concentration following the application of focused ARF. Furthermore, passive bending movement was noted in primary cilium that were treated with focused ARF. Cell viability was not affected. Application of pulsed ultrasound radiation generated only a minimal temperature rise of 0.1°C, and induced a streaming resulting fluid shear stress of 0.186 dyne/cm(2), suggesting that hyperthermia and acoustic streaming might not be the main causes of the observed cell responses. In conclusion, these data provide more insight in the interactions between acoustic mechanical stress and osteoblastic cells. This experimental system could serve as basis for further exploration of the mechanosensing mechanism of osteoblasts triggered by ultrasound.

Frequency specific ultrasound attenuation is sensitive to trabecular bone structure.

This study investigated the efficacy of frequency modulated ultrasound attenuation in the assessment of the trabecular structural properties. Four frequency modulated signals were created to represent four frequency bands centered at 500 kHz, 900 kHz, 1.3 MHz and 1.7 MHz with the bandwidth of 400 kHz. Five 1-cm trabecular cubes were harvested from fresh bovine distal femur. The cubes underwent four steps of demineralization process to expand the sample size to 25 with the greater variations of the structural properties for the better correlation study. Pearson correlation study was performed between the ultrasound attenuation in four frequency bands and the trabecular structural properties. The results showed that correlations of frequency modulated ultrasound attenuation to the trabecular structural properties are dependent on frequency bands. The attenuation in proximal-distal orientation had the highest correlation to BV/TV (R(2) = 0.73, p < 0.001) and trabecular thickness (R(2) = 0.50, p < 0.001) at the frequency band centered at 1.7 MHz. It was equivalent in the four frequency bands in correlation to the trabecular number (average R(2) = 0.80, p < 0.001) and to the trabecular separation (average R(2) = 0.83, p < 0.001). The attenuation in anterio-posterial orientation had the highest correlation to BV/TV (R(2) = 0.80, p < 0.001) and trabecular thickness (R(2) = 0.71, p < 0.001) at the frequency band centered at 1.3 MHz. The attenuation in the first frequency band was the most sensitive to the trabecular number (R(2) = 0.71, p < 0.001) and trabecular separation (R(2) = 0.80, p < 0.001). No significant correlation was observed for the attenuation in medial-lateral orientation across the four frequency bands.

Prediction of trabecular bone principal structural orientation using quantitative ultrasound scanning.

Bone has the ability to adapt its structure in response to the mechanical environment as defined as Wolff's Law. The alignment of trabecular structure is intended to adapt to the particular mechanical milieu applied to it. Due to the absence of normal mechanical loading, it will be extremely important to assess the anisotropic deterioration of bone during the extreme conditions, i.e., long term space mission and disease orientated disuse, to predict risk of fractures. The propagation of ultrasound wave in trabecular bone is substantially influenced by the anisotropy of the trabecular structure. Previous studies have shown that both ultrasound velocity and amplitude is dependent on the incident angle of the ultrasound signal into the bone sample. In this work, seven bovine trabecular bone balls were used for rotational ultrasound measurement around three anatomical axes to elucidate the ability of ultrasound to identify trabecular orientation. Both ultrasound attenuation (ATT) and fast wave velocity (UV) were used to calculate the principal orientation of the trabecular bone. By comparing to the mean intercept length (MIL) tensor obtained from µCT, the angle difference of the prediction by UV was 4.45°, while it resulted in 11.67° angle difference between direction predicted by µCT and the prediction by ATT. This result demonstrates the ability of ultrasound as a non-invasive measurement tool for the principal structural orientation of the trabecular bone.

Extension of the distributed point source method for ultrasonic field modeling.

The distributed point source method (DPSM) was recently proposed for ultrasonic field modeling and other applications. This method uses distributed point sources, placed slightly behind transducer surface, to model the ultrasound field. The acoustic strength of each point source is obtained through matrix inversion that requires the number of target points on the transducer surface to be equal to the number of point sources. In this work, DPSM was extended and further developed to overcome the limitations of the original method and provide a solid mathematical explanation of the physical principle behind the method. With the extension, the acoustic strength of the point sources was calculated as the solution to the least squares minimization problem instead of using direct matrix inversion. As numerical examples, the ultrasound fields of circular and rectangular transducers were calculated using the extended and original DPSMs which were then systematically compared with the results calculated using the theoretical solution and the exact spatial impulse response method. The numerical results showed the extended method can model ultrasonic fields accurately without the scaling step required by the original method. The extended method has potential applications in ultrasonic field modeling, tissue characterization, nondestructive testing, and ultrasound system optimization.

A new algorithm for spatial impulse response of rectangular planar transducers.

Previous solutions for spatial impulse responses of rectangular planar transducers require either approximations or complex geometrical considerations. This paper describes a new, simplified and exact solution using only trigonometric functions and simple set operations. This solution, which can be numerically implemented with a straightforward algorithm, is an exact implementation of the Rayleigh integral without any far field or paraxial approximation. Additionally, a nonlinear relationship was also established for spatial impulse responses from two field points which share the same projection point on the transducer surface plane. By incorporating this relationship in the algorithm, the computational efficiency of spatial impulse responses and continuous fields is improved about 20-folds and 14-folds, respectively. This algorithm has practical applications in designing l-D linear/phased arrays, 1.5-D arrays and 2-D arrays, as demonstrated through numerical simulations with array transducers. Experiments were also conducted to verify the new solution and results show that the algorithm is both accurate and efficient. The application of this method may include development of ultrasound imaging system for hard and soft tissue nondestructive assessment.

Evaluation of a pulsed phase-locked loop system for noninvasive tracking of bone deformation under loading with finite element and strain analysis.

Ultrasound has been widely used to nondestructively evaluate various materials, including biological tissues. Quantitative ultrasound has been used to assess bone quality and fracture risk. A pulsed phase-locked loop (PPLL) method has been proven for very sensitive tracking of ultrasound time-of-flight (TOF) changes. The objective of this work was to determine if the PPLL TOF tracking is sensitive to bone deformation changes during loading. The ability to noninvasively detect bone deformations has many implications, including assessment of bone strength and more accurate osteoporosis diagnostics and fracture risk prediction using a measure of bone mechanical quality. Fresh sheep femur cortical bone shell samples were instrumented with three 3-element rosette strain gauges and then tested under mechanical compression with eight loading levels using an MTS machine. Samples were divided into two groups based on internal marrow cavity content: with original marrow, or replaced with water. During compressive loading ultrasound waves were measured through acoustic transmission across the mid-diaphysis of bone. Finite element analysis (FEA) was used to describe ultrasound propagation path length changes under loading based on µCT-determined bone geometry. The results indicated that PPLL output correlates well to measured axial strain, with R(2) values of 0.70 ± 0.27 and 0.62 ± 0.29 for the marrow and water groups, respectively. The PPLL output correlates better with the ultrasound path length changes extracted from FEA. For the two validated FEA tests, correlation was improved to R(2) = 0.993 and R(2) = 0.879 through cortical path, from 0.815 and 0.794 via marrow path, respectively. This study shows that PPLL readings are sensitive to displacement changes during external bone loading, which may have potential to noninvasively assess bone strain and tissue mechanical properties.

Low-Intensity Amplitude Modulated Ultrasound Increases Osteoblastic Mineralization.

The purpose of this study was to assess the effect of pulsed amplitude modulated ultrasound (pAMUS) on the level of mineralization in osteoblast cell in comparison to cells stimulated with low-intensity pulsed ultrasound (LIPUS). To make the ultrasound effects more enhanced and targeted at region of interest, this study uses a novel approach of applying pulsed amplitude modulated ultrasound to osteoblast cells. The pAMUS signal was generated using two signal generators. Pulsed signal was amplified through a power amplifier and drove two identical focused ultrasound probes, focusing at the same point in the culture dish. The effects of pAMUS were evaluated using a pAMUS signal of 45 kHz and 100 kHz with 20% duty cycle. The hydrophone verified the formation of a focal point at equal distances (16 mm) from the surface of both transducers. Intensity profile using computer controlled 2D scanner showed circular focal point with a diameter of approximately 10 mm. The effect of the signal was studied using MC3T3-E1 cells cultured in osteogenic medium at time points Day 7, 12 and 18. The cells were analyzed for ALP activity and calcium mineralization. The pAMUS significantly increased the ALP activity and matrix calcification in comparison with LIPUS stimulated cultures.

Effects of phase cancellation and receiver aperture size on broadband ultrasonic attenuation for trabecular bone in vitro.

Phase cancellation in ultrasound due to large receiver size has been proposed as a contributing factor to the inaccuracy of estimating broadband ultrasound attenuation (BUA), which is used to characterize bone quality. Transducers with aperture size ranging from 2 to 5 mm have been used in previous attempts to study the effect of phase cancellation. However, these receivers themselves are susceptible to phase cancellation because aperture size is close to one center wavelength (about 3 mm at 500 KHz in water). This study uses an ultra small receiver (aperture size: 0.2 mm) in conjunction with a newly developed two-dimensional (2-D) synthetic array system to investigate the effects of phase cancellation and receiver aperture size on BUA estimations of bone tissue. In vitro ultrasound measurements were conducted on 54 trabecular bone samples (harvested from sheep femurs) in a confocal configuration with a focused transmitter and synthesized focused receivers of different aperture sizes. Phase sensitive (PS) and phase insensitive (PI) detections were performed. The results show that phase cancellation does have a significant effect on BUA. The normalized BUA (nBUA) with PS is 8.1% higher than PI nBUA while PI BUA is well correlated with PS BUA. Receiver aperture size also influences the BUA reading for both PI and PS detection and smaller receiver aperture tends to result in higher BUA readings. The results also indicate that the receiver aperture size used in the confocal configuration with PI detection should at least equal the aperture of the transmitter to capture most of the energy redistributed by the interference and diffraction from the trabecular bone.

Field computation for two-dimensional array transducers with limited diffraction array beams.

A method is developed for calculating fields produced with a two-dimensional (2D) array transducer. This method decomposes an arbitrary 2D aperture weighting function into a set of limited diffraction array beams. Using the analytical expressions of limited diffraction beams, arbitrary continuous wave (cw) or pulse wave (pw) fields of 2D arrays can be obtained with a simple superposition of these beams. In addition, this method can be simplified and applied to a 1D array transducer of a finite or infinite elevation height. For beams produced with axially symmetric aperture weighting functions, this method can be reduced to the Fourier-Bessel method studied previously where an annular array transducer can be used. The advantage of the method is that it is accurate and computationally efficient, especially in regions that are not far from the surface of the transducer (near field), where it is important for medical imaging. Both computer simulations and a synthetic array experiment are carried out to verify the method. Results (Bessel beam, focused Gaussian beam, X wave and asymmetric array beams) show that the method is accurate as compared to that using the Rayleigh-Sommerfeld diffraction formula and agrees well with the experiment.

Theory and experiment of Fourier-Bessel field calculation and tuning of a pulsed wave annular array.

A one-dimensional (1D) Fourier-Bessel series method for computing and tuning (beamforming) the linear lossless field of flat pulsed wave annular arrays is developed and supported with both numerical simulation and experimental verification. The technique represents a new method for modeling and tuning the propagated field by linking the quantized surface pressure profile to a known set of limited diffraction Bessel beams propagating into the medium. This enables derivation of an analytic expression for the field at any point in space and time in terms of the transducer surface pressure profile. Tuning of the field then also follows by formulating a least-squares design for the transducer surface pressure with respect to a given desired field in space and time. Simulated and experimental results for both field computation and tuning are presented in the context of a 10-ring annular array operating at a central frequency of 2.5 MHz in water.