Chiaki Miyasaka

Metastatic breast cancer cells suppress osteoblast adhesion and differentiation.

Bone is a primary target for colonization of metastatic breast cancer cells. Once present, the breast cancer cells activate osteoclasts, thereby stimulating bone loss. Bone degradation is accompanied by pain and increased susceptibility to fractures. However, targeted inhibition of osteoclasts does not completely prevent lesion progression, nor does it heal the lesions. This suggests that breast cancer cells may also affect osteoblasts, cells that build bone. The focus of this study was to determine the ability of breast cancer cells to alter osteoblast function. MC3T3-E1 osteoblasts were cultured with conditioned medium from MDA-MB-231 breast cancer cells and subsequently assayed for changes in differentiation. Osteoblast differentiation was monitored by expression of osteocalcin, bone sialoprotein and alkaline phosphatase, and by mineralization. Osteoblasts cultured with MDA-MB-231 conditioned medium did not express these mature bone proteins, nor did they mineralize a matrix. Inhibition of osteoblast differentiation was found to be due to transforming growth factor β present in MDA-MB-231 conditioned medium. Interestingly, breast cancer conditioned medium also altered cell adhesion. When osteoblasts were assayed for adhesion properties using interference reflection microscopy and scanning acoustic microscopy, there was a reduction in focal adhesion plaques and sites of detachment were clearly visible. F-actin was disassembled and punctate in osteoblasts cultured with MDA-MB-231 conditioned medium rather than organized in long stress fibers. Taken together, these observations suggest that metastatic breast cancer cells alter osteoblast adhesion and prevent differentiation. These affects could account for the continued loss of bone after osteoclast inhibition in patients with bone-metastatic breast cancer.

Acoustic imaging of thick biological tissue

Up to now, biomedical imaging with ultrasound for observing a cellular tissue structure has been limited to very thinly sliced tissue at very high ultrasonic frequencies, i.e., 1 GHz. In this paper, we present the results of a systematic study to use a 150 to 200 MHz frequency range for thickly sliced biological tissue. A mechanical scanning reflection acoustic microscope (SAM) was used for obtaining horizontal cross-sectional images (C-scans) showing cellular structures. In the study, sectioned specimens of human breast cancer and tissues from the small intestine were prepared and examined. Some accessories for biomedical application were integrated into our SAM (Sonix HS-1000 and Olympus UH-3), which operated in pulse-wave and tone-burst wave modes, respectively. We found that the frequency 100 to 200 MHz provides optimal balance between resolution and penetration depth for examining the thickly sliced specimens. The images obtained with the lens focused at different depths revealed cellular structures whose morphology was very similar to that seen in the thinly sectioned specimens with optical and scanning acoustic microscopy. The SAM operation in the pulse-echo mode permits the imaging of tissue structure at the surface, and it also opens up the potential for attenuation imaging representing reflection from the substrate behind the thick specimen. We present such images of breast cancer proving the methods applicability to overall tumor detection. SAM with a high-frequency tone-burst ultrasonic wave reveals details of tissue structure, and both methods may serve as additional diagnostic tools in a hospital environment.

Study of Cellular Adhesion with Scanning Acoustic Microscopy

A mechanical scanning acoustic reflection microscope was applied to living cells (e.g., osteoblasts) to observe their undisguised shapes and to evaluate their adhesive conditions at a substrate interface. A conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. To characterize the cellular adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for 2 days, then assayed with the scanning acoustic reflection microscope. At 600 MHz the scanning acoustic reflection microscope clearly indicated that MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium had both an abnormal shape and poor adhesion at the substrate interface. The results arc compared with those obtained with laser scanning con- focal microscopy and are supported by a simple multilayer model.

Characterization of Stress at a Ceramic/Metal Joined Interface by the V(z) Technique of Scanning Acoustic Microscopy

It is well known that the process of heating and then cooling dissimilar materials introduces considerable stress at and near the interface. In this article, first, the surface wave velocity distributions obtained with the V(z) curve technique were found to compare well with residual stress distribution measured by the finely collimated X-ray diffraction technique. Second, a delamination was introduced at the interface. The V(z) curve technique was then used again to measure the surface acoustic wave velocity along the interface. The defective specimens showed significantly different patterns of surface acoustic wave velocities. Thus, this study presents useful guidelines in discriminating between sound and defective ceramic/metal joints by scanning acoustic microscopy.

Pupil function splitting method in calculating acoustic microscopic signals for elastic discontinuities

This paper deals with the output signals of scanning acoustic microscopes observing the elastic discontinuities on sample surface, and presents a new method of calculation. The formulation is based on the angular-spectrum method together with a ray-optical approximation. The key treatment is splitting of the pupil function into two parts. This method clarifies the image-forming factors in acoustic microscopy including large and fine fringes parallel to discontinuities. Calculation for an edge and a metal/ceramic joint showed good agreements with the experimental results.

Non-destructive evaluation (NDE) of aerospace composites: acoustic microscopy

Abstract: This chapter presents two case studies on the use of acoustic microscopy for the characterization of carbon fiber-reinforced epoxy composites (CFRP) and aramid fiber composites (AFRP). The emphasis is on impact damage ranging from hard impacts (bullets, micrometereorites, hail, etc.) to soft impacts (bird strike) typically encountered in aerospace applications. The chapter ends with challenges and future trends, such as the embedding of sensors in composite panels.

Opto-acoustic technique to evaluate adhesion strength of thin-film systems

An opto-acoustic technique is proposed to evaluate the adhesion strength of thin film systems at the film-substrate interface. The thin-film system to be examined is configured as an end-mirror of a Michelson interferometer, and driven from the rear with an acoustic transducer at audible frequencies. The amplitude of the resultant oscillation of the film is quantified as the variation in the contrast of the interferometric fringe pattern observed with a digital camera at 30 frames/s. As a proof of concept, experiment has been conducted with the use of a pair of strongly and weakly adhered Au-coated Si-wafer specimens. The technique successfully differentiates the adhesion strength of the specimens.

NDE of friction stir welds of Al alloys using high-frequency acoustic microscopy

This paper presents results on high-frequency ultrasonic imaging of bonds between different materials with application to friction stir welding (FSW). The unique feature of this paper is the use of high numerical aperture lenses that allow the excitation of leaky surface acoustic waves. These interact with weak bonds to produce wave interference of incident and reflected waves at weak bond interfaces. The interference provides a vivid contrast marking the weak interface bond as an easily recognisable feature. Presented are a description of the high-frequency lenses, simulation of the contrast phenomenon and the application to friction stir welds. The welds were between cast A356 and wrought 6061 Al alloys obtained under four different processing conditions. The selected sections of these were imaged at high frequencies in the GHz range by bringing the acoustic lenses towards the specimens surface far enough so that the peripheral lens rays were incident at large enough angles to exceed the first and second critical angles and to excite leaky surface acoustic waves. The interference of these waves allowed the imaging of the bond details with a resolution of about 1.5–3 μm. The features and flaws observed in the images were correlated with those reported on in the literature as well as those obtained from scanning electron microscopy and tensile property evaluation. The quality of weld bonds inferred from the images was correlated to the tensile fracture experiments, which showed that improperly bonded specimens showed a relatively larger number of flaws. The results suggest that high-frequency acoustic microscopy is indeed a useful method for the diagnostic evaluation of FSW, especially since acoustic microscopes are now available as portable instruments commercially.

Fine mapping of tissue properties on excised samples of melanoma and skin without the need for histological staining

This paper develops a novel two-frequency approach for noninvasive evaluation of cancerous tissue with optimum depth and resolution. Frequencies of about 50 MHz are used in thickly sliced tissue to detect differences of the relative attenuation (C-scan mode scanning) with relatively limited resolution. Thus, suspect zones can be identified according to a quantitative criterion. These suspect zones are then selected for preparation of thin, transversal slices from within the original thick slices. Very-high-resolution (1-μm) visualization of cells is obtained at around 600 MHz on these transversal sections and adjacent sections are prepared for histological study in parallel. The techniques feasibility and potential are demonstrated on both normal and cancerous (melanoma) skin tissue. Isotropy of the specimens is experimentally verified to ensure that conditions were coherent for use of a 5-layer, angular spectrum model made to simulate longitudinal velocity, allowing estimation of longitudinal velocity from semiquantitative V(z) data.

Multilayer transfer matrix characterization of complex materials with scanning acoustic microscopy

A multilayer structured thin film system, such as a biomedical thin film, MEMS (Micro Electric Mechanical System)/NEMS (Nano Electric Mechanical System) devices, and semiconductors, is widely used in various fields of industries. To non-destructively evaluate the multilayer structured thin film system, a mechanical scanning acoustic reflection microscope has been well recognized as a useful tool in recent years. Especially, the V(z) curve method with the scanning acoustic microscope is used to characterize the very small area of the system. In this study, V(z) curve simulation software for simulating transducer output when we transmit an ultrasound wave into the specimen has been developed. In the software, the Thompson-Haskell transfer matrix method is applied to solve for the reflectance function. All input and output interfaces incorporated in a GUI interface for users’ convenience. Surface acoustic wave velocities are calculated from the simulated V(z) curves. For the precise calculation advanced signal processing techniques are utilized. The surface acoustic wave velocity is compared to that from an experiment with a bulk solid. We also tested the simulation’s thickness sensitivity by simulating models with different thickness in nanoscale. A series of experiments with multilayered solids are carried out and the results are compared with the simulation results. It was the first time a comparison of analytical versus experimental for V(z) curves for multilayered system were performed. For the multilayered specimen, silicon (100) is used as a substrate. Titanium (thickness: 10 nanometer) and platinum (thickness: 100 nanometer) are deposited respectively.