Madhu Sudhan Reddy Gudur

Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0-20 mg ml(-1)), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml(-1) HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml(-1) HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 2 0mg ml(-1) HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.

High-frequency rapid B-mode ultrasound imaging for real-time monitoring of lesion formation and gas body activity during high-intensity focused ultrasound ablation

The goal of this study was to examine the ability of high-frame-rate, high-resolution imaging to monitor tissue necrosis and gas-body activities formed during high-intensity focused ultrasound (HIFU) application. Ex vivo porcine cardiac tissue specimens (n = 24) were treated with HIFU exposure (4.33 MHz, 77 to 130 Hz pulse repetition frequency (PRF), 25 to 50% duty cycle, 0.2 to 1 s, 2600 W/cm2). RF data from Bmode ultrasound imaging were obtained before, during, and after HIFU exposure at a frame rate ranging from 77 to 130 Hz using an ultrasound imaging system with a center frequency of 55 MHz. The time history of changes in the integrated backscatter (IBS), calibrated spectral parameters, and echo-decorrelation parameters of the RF data were assessed for lesion identification by comparison against gross sections. Temporal maximum IBS with +12 dB threshold achieved the best identification with a receiver-operating characteristic (ROC) curve area of 0.96. Frame-to-frame echo decorrelation identified and tracked transient gas-body activities. Macroscopic (millimetersized) cavities formed when the estimated initial expansion rate of gas bodies (rate of expansion in lateral-to-beam direction) crossed 0.8 mm/s. Together, these assessments provide a method for monitoring spatiotemporal evolution of lesion and gas-body activity and for predicting macroscopic cavity formation.

Noninvasive quantification of in vitro osteoblastic differentiation in 3D engineered tissue constructs using spectral ultrasound imaging.

Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5–15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×104 at day 1 to 0.9±0.2×104 at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.

High-Frequency Ultrasound M-Mode Imaging for Identifying Lesion and Bubble Activity During High-Intensity Focused Ultrasound Ablation

Effective real-time monitoring of high-intensity focused ultrasound (HIFU) ablation is important for application of HIFU technology in interventional electrophysiology. This study investigated rapid, high-frequency M-mode ultrasound imaging for monitoring spatiotemporal changes during HIFU application. HIFU (4.33 MHz, 1 kHz PRF, 50% duty cycle, 1 s, 2600‒6100 W/cm²) was applied to ex vivo porcine cardiac tissue specimens with a confocally and perpendicularly aligned high-frequency imaging system (Visualsonics Vevo 770, 55 MHz center frequency). Radio-frequency (RF) data from M-mode imaging (1 kHz PRF, 2 s × 7 mm) was acquired before, during and after HIFU treatment (n = 12). Among several strategies, the temporal maximum integrated backscatter with a threshold of +12 dB change showed the best results for identifying final lesion width (receiver-operating characteristic curve area 0.91 ± 0.04, accuracy 85 ± 8%, compared with macroscopic images of lesions). A criterion based on a line-to-line decorrelation coefficient is proposed for identification of transient gas bodies.

Two-Bubble Acoustic Tweezing Cytometry for Biomechanical Probing and Stimulation of Cells

The study of mechanotransduction relies on tools that are capable of applying mechanical forces to elicit and assess cellular responses. Here we report a new (to our knowledge) technique, called two-bubble acoustic tweezing cytometry (TB-ATC), for generating spatiotemporally controlled subcellular mechanical forces on live cells by acoustic actuation of paired microbubbles targeted to the cell adhesion receptor integrin. By measuring the ultrasound-induced activities of cell-bound microbubbles and the actin cytoskeleton contractile force responses, we determine that TB-ATC elicits mechanoresponsive cellular changes via cyclic, paired displacements of integrin-bound microbubbles driven by the attractive secondary acoustic radiation force (sARF) between the bubbles in an ultrasound field. We demonstrate the feasibility of dual-mode TB-ATC for both subcellular probing and mechanical stimulation. By exploiting the robust and unique interaction of ultrasound with microbubbles, TB-ATC provides distinct advantages for experimentation and quantification of applied forces and cellular responses for biomechanical probing and stimulation of cells.

Characterization of the pancreas in vivo using EUS spectrum analysis with electronic array echoendoscopes

BACKGROUND Spectral analysis of the radiofrequency (RF) signals that underlie grayscale EUS images has been used to provide quantitative, objective information about tissue histology. OBJECTIVE Our purpose was to validate RF spectral analysis as a method to distinguish between chronic pancreatitis (CP) and pancreatic cancer (PC). DESIGN AND SETTING A prospective study of eligible patients was conducted to analyze the RF data obtained by using electronic array echoendoscopes. PATIENTS Pancreatic images were obtained by using electronic array echoendoscopes from 41 patients in a prospective study, including 15 patients with PC, 15 with CP, and 11 with a normal pancreas. MAIN OUTCOME MEASUREMENTS Midband fit, slope, intercept, correlation coefficient, and root mean square deviation from a linear regression of the calibrated power spectra were determined and compared among the groups. RESULTS Statistical analysis showed that significant differences were observable between groups for mean midband fit, intercept, and root mean square deviation (t test, P < .05). Discriminant analysis of these parameters was then performed to classify the data. For CP (n = 15) versus PC (n = 15), the same parameters provided 83% accuracy and an area under the curve of 0.83. LIMITATIONS Moderate sample size and spatial averaging inherent in the technique. CONCLUSIONS This study shows that mean spectral parameters of the backscattered signals obtained by using electronic array echoendoscopes can provide a noninvasive method to quantitatively discriminate between CP and PC.

Transmural ultrasound imaging of thermal lesion and action potential changes in perfused canine cardiac wedge preparations by high intensity focused ultrasound ablation

Intra-procedural imaging is important for guiding cardiac arrhythmia ablation. It is difficult to obtain intra-procedural correlation of thermal lesion formation with action potential (AP) changes in the transmural plane during ablation. This study tested parametric ultrasound imaging for transmural imaging of lesion and AP changes in high intensity focused ultrasound (HIFU) ablation using coronary perfused canine ventricular wedge preparations (n = 13). The preparations were paced from epi/endocardial surfaces and subjected to HIFU application (3.5 MHz, 11 Hz pulse-repetition-frequency, 70% duty cycle, duration 4 s, 3500 W/cm2), during which simultaneous optical mapping (1 kframes/s) using di-4-ANEPPS and ultrasound imaging (30 MHz) of the same transmural surface of the wedge were performed. Spatiotemporally correlated AP measurements and ultrasound imaging allowed quantification of the reduction of AP amplitude (APA), shortening of AP duration at 50% repolarization, AP triangulation, decrease of optical AP rise, and change of conduction velocity along tissue depth direction within and surrounding HIFU lesions. The threshold of irreversible change in APA correlating to lesions was determined to be 43±1% with a receiver operating characteristic (ROC) area under curve (AUC) of 0.96±0.01 (n = 13). Ultrasound imaging parameters such as integrated backscatter, Rayleigh (α) and log-normal (σ) parameters, cumulative extrema of σ were tested, with the cumulative extrema of σ performing the best in detecting lesion (ROC AUC 0.89±0.01, n = 13) and change of APA (ROC AUC 0.79±0.03, n = 13). In conclusion, characteristic tissue and AP changes in HIFU ablation were identified and spatiotemporally correlated using optical mapping and ultrasound imaging. Parametric ultrasound imaging using cumulative extrema of σ can detect HIFU lesion and APA reduction.

TOMOGRAPHIC RECONSTRUCTION OF TISSUE PROPERTIES AND TEMPERATURE INCREASE FOR HIGH-INTENSITY FOCUSED ULTRASOUND APPLICATIONS

The acoustic and thermal properties as well as the temperature change within a tissue volume during high-intensity focused ultrasound ablation are critically important for treatment planning and monitoring. Described in this article is a tomographic reconstruction method used to determine the tissue properties and increase in temperature in a 3-D volume. On the basis of the iterative finite-element solution to the bioheat equation coupled with Tikhonov regularization techniques, our reconstruction algorithm solves the inverse problem of bioheat transfer and uses the time-dependent temperature measured on a tissue surface to obtain the acoustic absorption coefficient, thermal diffusivity and temperature increase within the subsurface volume. Numerical simulations were performed to validate the reconstruction algorithm. The method was initially conducted in ex vivo experiments in which time-dependent temperature on a tissue surface was measured using high-resolution, non-invasive infrared thermography.

Lymph node characterization in vivo using endoscopic ultrasound spectrum analysis with electronic array echo endoscopes

Our purpose was to demonstrate the use of radiofrequency spectral analysis to distinguish between benign and malignant lymph nodes with data obtained using electronic array echo endoscopes, as we have done previously using mechanical echo endoscopes. In a prospective study, images were obtained from eight patients with benign-appearing lymph nodes and 11 with malignant lymph nodes, as verified by fine-needle aspiration. Midband fit, slope, intercept, correlation coefficient, and root-mean-square (RMS) deviation from a linear regression of the calibrated power spectra were determined and compared between the groups. Significant differences were observable for mean midband fit, intercept, and RMS deviation (t test P < 0.05). For benign (n = 16) vs. malignant (n = 12) lymph nodes, midband fit and RMS deviation provided classification with 89 % accuracy and area under receiver operating characteristic (ROC) curve of 0.95 based on linear discriminant analysis. We concluded that the mean spectral parameters of the backscattered signals from electronic array echo endoscopy can provide a noninvasive method to quantitatively discriminate between benign and malignant lymph nodes.

High-frequency ultrasound M-mode monitoring of HIFU ablation in cardiac tissue

Effective real-time HIFU lesion detection is important for expanded use of HIFU in interventional electrophysiology (e.g., epicardial ablation of cardiac arrhythmia). The goal of this study was to investigate rapid, high-frequency M-mode ultrasound imaging for monitoring spatiotemporal changes in tissue during HIFU application. The HIFU application (4.33 MHz, 1000 Hz PRF, 50% duty cycle, 1 s exposure, 6100 W/cm2) was perpendicularly applied to porcine cardiac tissue with a high-frequency imaging system (Visualsonics Vevo 770, 55 MHz, 4.5 mm focal distance) confocally aligned. Radiofrequency (RF) M-mode data (1 kHz PRF, 4 s × 7 mm) was acquired before, during, and after HIFU treatment. Gross lesions were compared with M-mode data to correlate lesion and cavity formation. Integrated backscatter, echo-decorrelation parameters, and their cumulative extrema over time were analyzed for automatically identifying lesion width and bubble formation. Cumulative maximum integrated backscatter showed the best results ...