Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels.

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrates increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels.

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrate increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

Sensitivity improvements of a resonance-based tactile sensor.

Resonance-based contact-impedance measurement refers to the application of resonance sensors based on the measurement of the changes in the resonance curve of an ultrasonic resonator in contact with a surface. The advantage of the resonance sensor is that it is very sensitive to small changes in the contact impedance. A sensitive micro tactile sensor (MTS) was developed, which measured the elasticity of soft living tissues at the single-cell level. In the present paper, we studied the method of improving the touch and stiffness sensitivity of the MTS. First, the dependence of touch sensitivity in relation to the resonator length was studied by calculating the sensitivity coefficient at each length ranging from 9 to 40 mm. The highest touch sensitivity was obtained with a 30-mm-long glass needle driven at a resonance frequency of 100 kHz. Next, the numerical calculation of contact impedance showed that the highest stiffness sensitivity was achieved when the driving frequency was 100 kHz and the contact-tip diameter of the MTS was 10 µm. The theoretical model was then confirmed experimentally using a phase-locked-loop-based digital feedback oscillation circuit. It was found that the developed MTS, whose resonant frequency was 97.030 kHz, performed with the highest sensitivity of 53.2 × 106 Hz/N at the driving frequency of 97.986 kHz, i.e. the highest sensitivity was achieved at 956 Hz above the resonant frequency.

Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application.

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment.

Emerging roles of hypoxia-inducible factors and reactive oxygen species in cancer and pluripotent stem cells.

Eukaryotic organisms require oxygen homeostasis to maintain proper cellular function for survival. During conditions of low oxygen tension (hypoxia), cells activate the transcription of genes that induce an adaptive response, which supplies oxygen to tissues. Hypoxia and hypoxia-inducible factors (HIFs) may contribute to the maintenance of putative cancer stem cells, which can continue self-renewal indefinitely and express stemness genes in hypoxic stress environments (stem cell niches). Reactive oxygen species (ROS) have long been recognized as toxic by-products of aerobic metabolism that are harmful to living cells, leading to DNA damage, senescence, or cell death. HIFs may promote a cancer stem cell state, whereas the loss of HIFs induces the production of cellular ROS and activation of proteins p53 and p16(Ink4a), which lead to tumor cell death and senescence. ROS seem to inhibit HIF regulation in cancer cells. By contrast, controversial data have suggested that hypoxia increases the generation of ROS, which prevents hydroxylation of HIF proteins by inducing their transcription as negative feedback. Moreover, hypoxic conditions enhance the generation of induced pluripotent stem cells (iPSCs). During reprogramming of somatic cells into a PSC state, cells attain a metabolic state typically observed in embryonic stem cells (ESCs). ESCs and iPSCs share similar bioenergetic metabolisms, including decreased mitochondrial number and activity, and induced anaerobic glycolysis. This review discusses the current knowledge regarding the emerging roles of ROS homeostasis in cellular reprogramming and the implications of hypoxic regulation in cancer development.

Role of tumor suppressor genes in the cancer-associated reprogramming of human induced pluripotent stem cells.

Because of their pluripotent characteristics, human induced pluripotent stem cells (iPSCs) possess great potential for therapeutic application and for the study of degenerative disorders. These cells are generated from normal somatic cells, multipotent stem cells, or cancer cells. They express embryonic stem cell markers, such as OCT4, SOX2, NANOG, SSEA-3, SSEA-4, and REX1, and can differentiate into all adult tissue types, both in vitro and in vivo. However, some of the pluripotency-promoting factors have been implicated in tumorigenesis. Here, we describe the merits of tumor suppresser genes as reprogramming factors for the generation of iPSCs without tumorigenic activity. The initial step of reprogramming is induction of the exogenous pluripotent factors to generate the oxidative stress that leads to senescence by DNA damage and metabolic stresses, thus inducing the expression of tumor suppressor genes such as p21CIP1 and p16INK4a through the activation of p53 to be the pre-induced pluripotent stem cells (pre-iPSCs). The later stage includes overcoming the barrier of reprogramming-induced senescence or cell-cycle arrest by shutting off the function of these tumor suppressor genes, followed by the induction of endogenous stemness genes for the full commitment of iPSCs (full-iPSCs). Thus, the reactive oxygen species (ROS) produced by oxidative stress might be critical for the induction of endogenous reprogramming-factor genes via epigenetic changes or antioxidant reactions. We also discuss the critical role of tumor suppressor genes in the evaluation of the tumorigenicity of human cancer cell-derived pluripotent stem cells, and describe how to overcome their tumorigenic properties for application in stem cell therapy in the field of regenerative medicine.

Softening of the mouse zona pellucida during oocyte maturation.

A change in the elasticity and the resistance to dissolution of the mouse zona pellucida (ZP) was quantitatively evaluated at immature germinal vesicle (GV), mature metaphase II (MII) and fertilized pronuclear (PN) stages. Young's modulus of the ZP was measured using a micro tactile sensor (MTS), a highly sensitive resonator-based sensor for a micro scale elasticity measurement. 0.25% α-chymotrypsin was used for the ZP dissolution assay. The results of measuring the ZP elasticity and the dissolution time clearly showed that the ZP softened during oocyte maturation and the ZP hardened after fertilization. The results indicate that the amount of the zona softening can be a criterion to evaluate oocyte quality for the selection of top quality mature oocyte before in vitro fertilization (IVF) treatment.

Surface density mapping of natural tissue by a scanning haptic microscope (SHM).

To expand the performance capacity of the scanning haptic microscope (SHM) beyond surface mapping microscopy of elastic modulus or topography, surface density mapping of a natural tissue was performed by applying a measurement theory of SHM, in which a frequency change occurs upon contact of the sample surface with the SHM sensor - a microtactile sensor (MTS) that vibrates at a pre-determined constant oscillation frequency. This change was mainly stiffness-dependent at a low oscillation frequency and density-dependent at a high oscillation frequency. Two paragon examples with extremely different densities but similar macroscopic elastic moduli in the range of natural soft tissues were selected: one was agar hydrogels and the other silicon organogels with extremely low (less than 25 mg/cm(3)) and high densities (ca. 1300 mg/cm(3)), respectively. Measurements were performed in saline solution near the second-order resonance frequency, which led to the elastic modulus, and near the third-order resonance frequency. There was little difference in the frequency changes between the two resonance frequencies in agar gels. In contrast, in silicone gels, a large frequency change by MTS contact was observed near the third-order resonance frequency, indicating that the frequency change near the third-order resonance frequency reflected changes in both density and elastic modulus. Therefore, a density image of the canine aortic wall was subsequently obtained by subtracting the image observed near the second-order resonance frequency from that near the third-order resonance frequency. The elastin-rich region had a higher density than the collagen-rich region.

Observation of local elastic distribution in aortic tissues under static strain condition by use of a scanning haptic microscope.

The purpose of this study was to observe variation in the local elastic distribution in aortic tissue walls under different static strain conditions, including physiological strain, by use of a scanning haptic microscope (SHM). Strain was applied by stretching aortic tissues in the circumferential direction by the simple tensile method or by the rod-insertion method to mimic in vivo internal pressure loading. SHM measurements in a saline solution at room temperature were performed on canine thoracic aorta using a glass needle probe with a diameter of ca 5 µm and a scanning area and point pitch of 160 × 80 µm and 2 µm, respectively. Under strain of 0-0.23, corresponding to internal pressure of 0-150 mmHg, wavy-shaped elastin fibers stretched until they were almost straightened, and the average elastic modulus increased almost linearly. Although there was little difference between the images obtained for the two different stretching methods, under high strain (>0.36; 250 mmHg) significant circumferential orientation of the collagen fibrils occurred with an increase in the average elastic modulus. It was concluded that the pressure resistance of the aorta under physiological strain was mainly afforded by elastin fibers; collagen fibrils contributed little except under much higher pressures.

Pyrrole-imidazole polyamide targeting transforming growth factor ß1 ameliorates encapsulating peritoneal sclerosis.

OBJECTIVE: Encapsulating peritoneal sclerosis (EPS) is a devastating fibrotic complication in patients treated with peritoneal dialysis (PD). Transforming growth factor ß1 (TGF-ß1) is a pivotal factor in the induction of EPS. METHODS: To develop pyrrole-imidazole (PI) polyamide, a novel gene silencer, targeted to the TGF-ß1 promoter (Polyamide) for EPS, we examined the effects of Polyamide on messenger RNA (mRNA) expression of TGF-ß1, vascular endothelial growth factor (VEGF), and extracellular matrix (ECM) in mesothelial cells in vitro, and on the thickness of injured peritoneum evaluated by histology and high-resolution regional elasticity mapping in rats in vivo. RESULTS: Polyamide significantly lowered mRNA expression of TGF-ß1 and ECM in vitro. Polyamide labeled with fluorescein isothiocyanate was taken up into the injured peritoneum and was strongly localized in the nuclei of most cells. Polyamide 1 mg was injected intraperitoneally 1 or 3 times in rats receiving a daily intraperitoneal injection of chlorhexidine gluconate and ethanol (CHX) for 14 days. Polyamide significantly suppressed peritoneal thickening and the abundance of TGF-ß1 and fibronectin mRNA, but did not affect expression of VEGF mRNA in the injured peritoneum. Elasticity distribution mapping showed that average elasticity was significantly lower in Polyamide-treated rats than in rats treated solely with CHX. CONCLUSIONS: Polyamide suppressed the stiffness, ECM formation, and thickening of the injured peritoneum that occurs during EPS pathogenesis. These data suggest that PI polyamide targeted to the TGF-ß1 promoter will be a specific and feasible therapeutic strategy for patients with EPS.

Variations in local elastic modulus along the length of the aorta as observed by use of a scanning haptic microscope (SHM).

Variations in microscopic elastic structures along the entire length of canine aorta were evaluated by use of a scanning haptic microscope (SHM). The total aorta from the aortic arch to the abdominal aorta was divided into 6 approximately equal segments. After embedding the aorta in agar, it was cut into horizontal circumferential segments to obtain disk-like agar portions containing ring-like samples of aorta with flat surfaces (thickness, approximately 1 mm). The elastic modulus and topography of the samples under no-load conditions were simultaneously measured along the entire thickness of the wall by SHM by using a probe with a diameter of 5 µm and a spatial resolution of 2 µm at a rate of 0.3 s/point. The elastic modulus of the wall was the highest on the side of the luminal surface and decreased gradually toward the adventitial side. This tendency was similar to that of the change in the elastin fiber content. During the evaluation of the mid-portion of each tunica media segment, the highest elastic modulus (40.8 ± 3.5 kPa) was identified at the thoracic section of the aorta that had the highest density of elastic fibers. Under no-load conditions, portions of the aorta with high elastin density have a high elastic modulus.

Surface elasticity imaging of vascular tissues in a liquid environment by a scanning haptic microscope.

The objective of this study was to make an elasticity distribution image of natural arteries in a liquid environment at high resolution at the micrometer level and at a wide area at the sub-square millimeter level by improving the scanning haptic microscope (SHM), developed previously for characterization of the stiffness of natural tissues. The circumferential sections (thickness, 1.0 mm) of small-caliber porcine arteries (approximately 3-mm diameter) were used as a sample. Measurement was performed by soaking a probe (diameter, 5 microm; spatial resolution, less than 2 microm) in saline solution at an appropriate depth. The vascular tissues were segregated by multi-layering a high elasticity region with mainly elastin (50.8 +/- 13.8 kPa) and a low one with mainly collagen and smooth muscle cells (17.0 +/- 9.0 kPa), as observed previously in high humidity conditions. The elasticity was measured repeatedly with little change for over 4 h in a liquid environment, which enabled observation with maintenance of high precision of a large area of at least 1,200 x 100 microm, whereas the elasticity was increased with time by the dehydration of samples with shrinkage in the air, in which an averaged elasticity in the overall area was approximately doubled within 2 h. This simple, inexpensive system allows observation of the distribution of the surface elasticity at the extracellular matrix level of vascular tissues in a liquid environment close to the natural one.

Improvement of hydrogelation abilities and handling of photocurable gelatin-based crosslinking materials.

Three types of eosin-derivatized gelatins (eosin-gelatins) with different molecular weights (M(w)) of ca. 15 kDa (low-molecular-weight eosin-gelatin, LEG), ca. 30 kDa (medium-molecular-weight eosin-gelatin, MEG), and ca. 95 kDa (high-molecular-weight eosin-gelatin, HEG) were prepared. All the eosin-gelatins except for HEG dissolved completely in water at 37 degrees C within several hours even at high concentrations of 35 or 40 wt % along with polyamine (poly(N,N-dimethylaminopropylacrylamide)) to produce photo-crosslinkable materials. The materials had appropriate viscosity for in situ molding at 37 degrees C and could be handled as a liquid at low temperatures of up to 25 degrees C. Upon photoirradiation for several tens of seconds, the materials were converted almost completely to hydrogels in the desired form with a microporous network structure by the radical coupling reaction. The mechanical strength of the produced hydrogels could be controlled by selecting a particular molecular weight or concentration of eosin-gelatins. The hydrogels obtained from LEG (40 wt %) or MEG (35 wt %) had elasticity similar to that of goat periodontal tissue. The handling of the photo-crosslinkable materials at room temperature and their photogelation ability were drastically improved by reducing the M(w) of eosin-gelatin. The potential usefulness of the photo-crosslinkable materials to periodontal regeneration has been discussed.

Local elasticity imaging of vascular tissues using a tactile mapping system.

This study aimed to map the elasticity of a natural artery at the micron level by using a tactile mapping system (TMS) that was recently developed for characterization of the stiffness of tissue slices. The sample used was a circumferential section (thickness, approximately 1 mm) of a small-caliber porcine artery (diameter, approximately 3 mm). Elasticity was measured with a probe of diameter 1 microm and a spatial resolution of 2 microm at a rate of 0.3 s per point, without significant sample invasion. Topographical measurements were also performed simultaneously. Wavy regions of high elasticity, layered in the circumferential direction, were measured at the tunica media, which was identified as an elastin-rich region. The Young's modulus of the elastin-rich region in the media was 50.8 +/- 13.8 kPa, and that of the elastin-rich region of the lamina elastica interna was 69.0 +/- 12.8 kPa. Both these values were higher than the Young's modulus of the other regions in the media, including smooth muscle cells and collagen fibrils (17.0 +/- 9.0 kPa). TMS is simple and inexpensive to perform and allows observation of the distribution of the surface elastic modulus at the extracellular matrix level in vascular tissue. TMS is expected to be a powerful tool in evaluation of the maturation and degree of reconstruction in the development of tissue-engineered or artificial tissues and organs.

Tactile mapping system: a novel imaging technology for surface topography and elasticity of tissues or organs.

OBJECTIVE: : We demonstrated that the tactile mapping system (TMS) has a high degree of spatial precision in the distribution mapping of surface elasticity of tissues or organs. METHODS: : Samples used were a circumferential section of a small-caliber porcine artery (diameter: ∼3 mm) and an elasticity test pattern with a line and space configuration for the distribution mapping of elasticity, prepared by regional micropatterning of a 14-µm thick gelatin hydrogel coating on a polyurethane sheet. Surface topography and elasticity in normal saline were simultaneously investigated by TMS using a probe with a diameter of 5 or 12 µm, a spatial interval of 1 to 5 µm, and an indentation depth of 4 µm. RESULTS: : In the test pattern, a spatial resolution in TMS of <5 µm was acquired under water with a minimal probe diameter and spatial interval of the probe movement. TMS was used for the distribution mapping of surface elasticity in a flat, circumferential section (thickness: ∼0.5 mm) of a porcine artery, and the concentric layers of the vascular wall, including the collagen-rich and elastin-rich layers, could be clearly differentiated in terms of surface elasticity at the spatial resolution of <2 µm. CONCLUSIONS: : TMS is a simple and inexpensive technique for the distribution mapping of the surface elasticity in vascular tissues at the spatial resolution <2 µm. TMS has the ability to analyze a complex structure of the tissue samples under normal saline.

Nuclear transfer to study the nuclear reprogramming of human stem cells.

Research of stem cells will enable us to understand the development and function of tissues and organs in mammals. The ability to induce regeneration of new tissues from embryonic stem (ES) cells derived from cloned blastocysts via nuclear transfer can be expected in the not-too-distant future. The fact that there is no way except nuclear cloning for the return of differentiated cells to undifferentiated cells remains an interesting problem to be solved. We describe protocols for the production of cloned calves from bovine ES cells to study nuclear reprogramming ability of stem cells. The frequency of term pregnancies for blastocysts from ES cells is higher than those of early pregnancies and maintained pregnancies after nuclear transfer with bovine somatic cells. We also describe protocols for gene introduction into bovine ES cells in vitro, particularly the human leukocyte antigens (HLA). Bovine ES cells provide a powerful tool for the generation of transgenic clonal offspring. This technique, when perfected for humans, may be critical for neural stem cell transplantation.

High resolution regional elasticity mapping of the human prostate.

What is it that the clinician "feels" during a digital rectal examination? To answer this question, it is necessary to measure the elastic properties of the prostate and verify the stiffness values with histological examination. Therefore, we devised an Elasticity Mapping System to evaluate the elastic properties of various histopathological grades of prostate cancer in relation to benign prostatic hyperplasia (BPH) and normal tissue. The system consists of a micro tactile sensor, a three-axis (XYZ) with one (fine Z) micromanipulation stage, a stereoscope camera and a measurement chamber. Using this methodology we mapped the elasticity of human prostate cancer (CaP) and it was obviously observed that the node was significantly harder than surrounding normal tissues and had some textures.

Mouse zona pellucida dynamically changes its elasticity during oocyte maturation, fertilization and early embryo development.

A change in the elasticity of mouse zona pellucida was quantitatively evaluated during oocyte maturation, fertilization and early embryo development. Young's modulus of zona pellucida of germinal vesicle (GV), metaphase-II (MII), pronuclear (PN), 2cell, 4cell, 8cell, morulae (M) and early blastocyst (EB) stages was measured using a micro tactile sensor (MTS) and a chamber exclusively designed for the measurement. The MTS has very high sensitivity and a deformation of only 5 microm was sufficient to calculate the Young's modulus and the oocyte/embryo maintained its original spherical shape during the measurement. The Young's modulus of GV, MII, PN, 2cell, 4cell, 8cell, M and EB was 22.8+/-10.4 kPa (n=30), 8.26+/-5.22 kPa (n=74), 22.3+/-10.5 kPa (n=66), 13.8+/-3.54 kPa (n=41), 12.6+/-3.34 kPa (n=19), 5.97+/-4.97 kPa (n=6), 1.88+/-1.34 kPa (n=8) and 3.39+/-1.86 kPa (n=4), respectively. Experimental results clearly demonstrated that the mouse zona pellucida hardened following fertilization. Interestingly, once the zona pellucida hardened at the PN stage, it gradually softened as the embryo developed (i.e. it was found that the zona hardening is a transient phenomenon). Furthermore, the zona pellucida of the GV oocyte was as hard as that of the PN embryo and became soft as it matured to the MII stage. In addition, the safety of the MTS measurement for oocytes and embryos was discussed both theoretically and experimentally.

Considerations in the design and sensitivity optimization of the micro tactile sensor.

Although miniaturization has been considered the only technology with which to increase sensitivity of tactile sensors, we recently developed the micro tactile sensor (MTS) that performs with high sensitivity without microfabrication. In this study, we examined design and sensitivity optimization of the MTS using theory based upon Mason's equivalent circuit. The touch probe, which is attached to the lead zirconate titanate (PZT) element, was expressed as a purely inductive circuit component. Resonance frequency was calculated as a function of the length of the touch probe, and sensitivity was predicted to be dependent on the length. Furthermore, many kinds of MTS were fabricated with different touch probe lengths, and actual sensitivity was measured as phase shift between nonloaded and loaded conditions. And, from the consideration of theory and experimental data, a sensitivity coefficient was proposed and found to be useful.

Remote sensing of mechanical properties of materials using a novel ultrasound transducer and signal processing.

An ultrasound-based remote sensing method to evaluate the mechanical properties of materials is presented. This method consists of a disk-shaped, piezoelectric transducer, operating at its resonance frequency, and a phase-shifted, feedback circuit. Mechanical parameters are derived by analyzing the signal contained in the phase-shifted values of the reflected signal. It is concluded that, using this novel transducer system and signal processing, remote mechanical measurements can be made. Such measurements obviate the need to apply the force-deformation approach and may be used to enable stiffness imaging.