VTechWorks Repository :: Browsing by Author "Davalos, Rafael V."

Browsing by Author "Davalos, Rafael V."

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3D Coiling at the Protrusion Tip: New Perspectives on How Cancer Cells Sense Their Fibrous Surroundings
Mukherjee, Apratim (Virginia Tech, 2021-05-24)
Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer.
7.0-T Magnetic Resonance Imaging Characterization of Acute Blood-Brain-Barrier Disruption Achieved with Intracranial Irreversible Electroporation
Garcia, Paulo A.; Rossmeisl, John H. Jr.; Robertson, John L.; Olson, JohnD.; Johnson, Annette J.; Ellis, Thomas L.; Davalos, Rafael V. (PLOS, 2012-11-30)
The blood-brain-barrier (BBB) presents a significant obstacle to the delivery of systemically administered chemotherapeutics for the treatment of brain cancer. Irreversible electroporation (IRE) is an emerging technology that uses pulsed electric fields for the non-thermal ablation of tumors. We hypothesized that there is a minimal electric field at which BBB disruption occurs surrounding an IRE-induced zone of ablation and that this transient response can be measured using gadolinium (Gd) uptake as a surrogate marker for BBB disruption. The study was performed in a Good Laboratory Practices (GLP) compliant facility and had Institutional Animal Care and Use Committee (IACUC) approval. IRE ablations were performed in vivo in normal rat brain (n = 21) with 1-mm electrodes (0.45 mm diameter) separated by an edge-to-edge distance of 4 mm. We used an ECM830 pulse generator to deliver ninety 50-ms pulse treatments (0, 200, 400, 600, 800, and 1000 V/cm) at 1 Hz. The effects of applied electric fields and timing of Gd administration (25, +5, +15, and +30 min) was assessed by systematically characterizing IRE-induced regions of cell death and BBB disruption with 7.0-T magnetic resonance imaging (MRI) and histopathologic evaluations. Statistical analysis on the effect of applied electric field and Gd timing was conducted via Fit of Least Squares with a = 0.05 and linear regression analysis. The focal nature of IRE treatment was confirmed with 3D MRI reconstructions with linear correlations between volume of ablation and electric field. Our results also demonstrated that IRE is an ablation technique that kills brain tissue in a focal manner depicted by MRI (n = 16) and transiently disrupts the BBB adjacent to the ablated area in a voltage-dependent manner as seen with Evan’s Blue (n = 5) and Gd administration.
Ablation outcome of irreversible electroporation on potato monitored by impedance spectrum under multi-electrode system
Zhao, Yajun; Liu, Hongmei; Bhonsle, Suyashree P.; Wang, Yilin; Davalos, Rafael V.; Yao, Chenguo (2018-09-20)
Background Irreversible electroporation (IRE) therapy relies on pulsed electric fields to non-thermally ablate cancerous tissue. Methods for evaluating IRE ablation in situ are critical to assessing treatment outcome. Analyzing changes in tissue impedance caused by electroporation has been proposed as a method for quantifying IRE ablation. In this paper, we assess the hypothesis that irreversible electroporation ablation outcome can be monitored using the impedance change measured by the electrode pairs not in use, getting more information about the ablation size in different directions. Methods Using a square four-electrode configuration, the two diagonal electrodes were used to electroporate potato tissue. Next, the impedance changes, before and after treatment, were measured from different electrode pairs and the impedance information was extracted by fitting the data to an equivalent circuit model. Finally, we correlated the change of impedance from various electrode pairs to the ablation geometry through the use of fitted functions; then these functions were used to predict the ablation size and compared to the numerical simulation results. Results The change in impedance from the electrodes used to apply pulses is larger and has higher deviation than the other electrode pairs. The ablation size and the change in resistance in the circuit model correlate with various linear functions. The coefficients of determination for the three functions are 0.8121, 0.8188 and 0.8691, respectively, showing satisfactory agreement. The functions can well predict the ablation size under different pulse numbers, and in some directions it did even better than the numerical simulation method, which used different electric field thresholds for different pulse numbers. Conclusions The relative change in tissue impedance measured from the non-energized electrodes can be used to assess ablation size during treatment with IRE according to linear functions.
Advancements in Irreversible Electroporation for the Treatment of Cancer
Arena, Christopher Brian (Virginia Tech, 2013-05-03)
Irreversible electroporation has recently emerged as an effective focal ablation technique. When performed clinically, the procedure involves placing electrodes into, or around, a target tissue and applying a series of short, but intense, pulsed electric fields. Oftentimes, patient specific treatment plans are employed to guide procedures by merging medical imaging with algorithms for determining the electric field distribution in the tissue. The electric field dictates treatment outcomes by increasing a cell's transmembrane potential to levels where it becomes energetically favorable for the membrane to shift to a state of enhanced permeability. If the membrane remains permeabilized long enough to disrupt homeostasis, cells eventually die. By utilizing this phenomenon, irreversible electroporation has had success in killing cancer cells and treating localized tumors. Additionally, if the pulse parameters are chosen to limit Joule heating, irreversible electroporation can be performed safely on surgically inoperable tumors located next to major blood vessels and nerves. As with all technologies, there is room for improvement. One drawback associated with therapeutic irreversible electroporation is that patients must be temporarily paralyzed and maintained under general anesthesia to prevent intense muscle contractions occurring in response to pulsing. The muscle contractions may be painful and can dislodge the electrodes. To overcome this limitation, we have developed a system capable of achieving non-thermal irreversible electroporation without causing muscle contractions. This progress is the main focus of this dissertation. We describe the theoretical basis for how this new system utilizes alterations in pulse polarity and duration to induce electroporation with little associated excitation of muscle and nerves. Additionally, the system is shown to have the theoretical potential to improve lesion predictability, especially in regions containing multiple tissue types. We perform experiments on three-dimensional in vitro tumor constructs and in vivo on healthy rat brain tissue and implanted tumors in mice. The tumor constructs offer a new way to rapidly characterize the cellular response and optimize pulse parameters, and the tests conducted on live tissue confirm the ability of this new ablation system to be used without general anesthesia and a neuromuscular blockade. Situations can arise in which it is challenging to design an electroporation protocol that simultaneously covers the targeted tissue with a sufficient electric field and avoids unwanted thermal effects. For instance, thermal damage can occur unintentionally if the applied voltage or number of pulses are raised to ablate a large volume in a single treatment. Additionally, the new system for inducing ablation without muscle contractions actually requires an elevated electric field. To ensure that these procedures can continue to be performed safely next to major blood vessels and nerves, we have developed new electrode devices that absorb heat out of the tissue during treatment. These devices incorporate phase change materials that, in the past, have been reserved for industrial applications. We describe an experimentally validated numerical model of tissue electroporation with phase change electrodes that illustrates their ability to reduce the probability for thermal damage. Additionally, a parametric study is conducted on various electrode properties to narrow in on the ideal design.
Advancements in the Treatment of Malignant Gliomas and Other Intracranial Disorders With Electroporation-Based Therapies
Lorenzo, Melvin Florencio (Virginia Tech, 2021-04-19)
The most common and aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of large molecular agents to permeate through the blood-brain barrier (BBB); migration of highly invasive GBM cells beyond the solid tumor margin; and gross, macroscopic intratumor heterogeneity. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. Notably, irreversible electroporation (IRE) has been used for BBB disruption and nonthermal ablation of intracranial tumor tissues. Despite promising results, IRE can cause unintended muscle contractions and is susceptible to electrical heterogeneities. Second generation High-frequency IRE (H-FIRE) utilizes bursts of bipolar pulsed electric fields on the order of the cell charging time constant (~1 μs) to ablate tissue while reducing nerve excitation, muscle contraction, and is far less prone to differences in electrical heterogeneities. Throughout my dissertation, I discuss investigations of H-FIRE for the treatment of malignant gliomas and other intracranial disorders. To advance the versatility, usability, and understanding of H-FIRE for intracranial applications, my PhD thesis focuses on: (1) characterizing H-FIRE-mediated BBB disruption effects in an in vivo healthy rodent model; (2) the creation of a novel, real-time impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as an in situ technique to monitor ablation growth and to determine patient-specific ablation endpoints; (4) conducting a preliminary efficacy study of H-FIRE ablation in an orthotopic F98 rodent glioma model; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a novel, intraoperative impedance spectroscopy technique towards determining patient-specific ablation endpoints for intracranial H-FIRE procedures.
Advancing Maternal Health through Projection-based and Machine Learning Strategies for Reduced Order Modeling
Snyder, William David (Virginia Tech, 2024-06-12)
High-fidelity computer simulations of childbirth are time consuming, making them impractical for guiding decision-making during obstetric emergencies. The complex geometry, micro-structure, and large finite deformations undergone by the vagina during childbirth result in material and geometric nonlinearities, complicated boundary conditions, and nonhomogeneities within finite element (FE) simulations. Such nonlinearities pose a significant challenge for numerical solvers, increasing the computational time. Simplifying assumptions can reduce the computational time significantly, but this usually comes at the expense of simulation accuracy. The work herein proposed the use of reduced order modeling (ROM) techniques to create surrogate models that capture experimentally-measured displacement fields of rat vaginal tissue during inflation testing in order to attain both the accuracy of higher-fidelity models and the speed of lower-fidelity simulations. The proper orthogonal decomposition (POD) method was used to extract the significant information from FE simulations generated by varying the luminal pressure and the parameters that introduce the anisotropy in the selected constitutive model. In our first study, a new data-driven (DD) variational multiscale (VMS) ROM framework was extended to obtain the displacement fields of rat vaginal tissue subjected to ramping luminal pressure. For comparison purposes, we also investigated the classical Galerkin ROM (G-ROM). In our numerical study, both the G-ROM and the DD-VMS-ROM decreased the FE computational cost by orders of magnitude without a significant decrease in numerical accuracy. Furthermore, the DD-VMS-ROM improved the G-ROM accuracy at a modest computational overhead. Our numerical investigation showed that ROM had the potential to provide efficient and accurate computational tools to describe vaginal deformations, with the ultimate goal of improving maternal health. Our second study compared two common computational strategies for surrogate modeling, physics-based G-ROM and data-driven machine learning (ML), for decreasing the cost of FE simulations of the ex vivo deformations of rat vaginal tissue subjected to inflation testing to study the effect of a pre-imposed tear. Since there are many methods associated with each modeling approach, to provide a fair and natural comparison, we selected a basic model from each category. From the ROM strategies, we considered a simplified G-ROM that is based on the linearization of the underlying nonlinear FE equations. From the ML strategies, we selected a feed-forward dense neural network (DNN) to create mappings from constitutive model parameters and luminal pressure values to either the FE displacement history (in which case we denote the resulting model ML) or the POD coefficients of the displacement history (in which case we denote the resulting model POD-ML). The numerical comparisons of G-ROM, ML, and POD-ML took place in the reconstructive regime. The numerical results showed that the G-ROM outperformed the ML model in terms of offline central processing unit (CPU) time for model training, online CPU time required to generate approximations, and relative error with respect to the FE models. The POD-ML model improved on the speed performance of the ML, having online CPU times comparable to those of the G-ROM given the same size of POD bases. However, the POD-ML model did not improve on the error performance of the ML. In our last study, we expanded our investigation of ML methods for surrogate modeling by comparing the performance of a DNN similar to what was used previously to that of a convolutional neural network (CNN) using 1-D convolution on the input parameters from FE simulations of active vaginal tearing. The new FE simulations utilized a custom continuum damage model that provided material damage and failure properties to an existing anisotropic hyperelastic constitutive model to replicate experimentally-observed tear propagation behaviors. We employed our DNN and CNN models to create mappings from constitutive model parameters, geometric properties of the propagating tear, and luminal pressure values to either the full FE displacement history or the POD coefficients of the displacement history. The root-mean-square error (RMSE) with respect to the FE displacement history achieved by full order output ML predictions was reproducible with POD-ML using a basis of only dimension l=10. Additionally, an order of magnitude reduction in offline time was observed using POD-ML over full-order ML with minimal difference between DNN and CNN architectures. Differences in online computational costs between ML and POD-ML were found to be negligible, but the DNNs produced predictions slightly faster than the CNNs, though both online times were on the same order of magnitude. While convolution did not significantly aid the regression task at hand, POD-ML was demonstrated to be an efficient and effective approach for surrogate modeling of the FE tear propagation model, approximating the displacement history with RMSE less than 0.1 mm and generating results 7 orders of magnitude faster than the FE model. This set of baseline numerical investigations serves as a starting point for future computer simulations that consider state-of-the-art G-ROM and ML strategies, and the in vivo geometry, boundary conditions, material properties, and tissue damage mechanics of the human vagina, as well as their changes during labor.
Advancing Transcranial Focused Ultrasound for Noninvasive Neuromodulation of Human Cortex
Mueller, Jerel Keith (Virginia Tech, 2015-09-09)
Ultrasound waves are mechanical undulations above the threshold for human hearing, and have been used widely in both the human body and brain for diagnostic and therapeutic purposes. Ultrasound can be controlled using specially designed transducers into a focus of a few millimeters in diameter. Low intensity ultrasound, such as used in imaging applications, appears to be safe in adults. It is also known that ultrasound waves can penetrate through the skull and be focused within the brain for ablation purposes, employing the heat generation properties of high intensity focused ultrasound. High intensity focused ultrasound is thus used to irreversibly ablate brain tissue in localized areas without observable damage to intermediate tissue and vasculature. Ablation with high intensity focused ultrasound guided by magnetic resonance imaging is used for abolishing brain tumors, and experimentally for pain. Low intensity ultrasound can be utilized beyond imaging in neuroscience and neurology by focusing the ultrasound beam to investigate the structure and function of discrete brain circuits. In contrast to high intensity focused ultrasound, the effects of low intensity focused ultrasound on neurons are reversible. Considering the volume of work on high intensity focused ultrasound, low intensity focused ultrasound remains decidedly underdeveloped. Given the great potential for impact of low intensity focused ultrasound in both clinical and scientific neuromodulation applications, we sought to advance the use of low intensity focused ultrasound for noninvasive, transcranial neuromodulation of human cortex. This dissertation contains novel research on the use of low intensity transcranial focused ultrasound for noninvasive neuromodulation of human cortex. The importance of mechanical forces in the nervous system is highlighted throughout to expand beyond the stigma that nervous function is governed chiefly by electrical and chemical means. Methods of transcranial focused ultrasound are applied to significantly modulate human cortical function, shown using electroencephalographic recordings and behavioral investigations of sensory discrimination performance. This dissertation also describes computational models used to investigate the insertion behavior of ultrasound across various tissues in the context of transcranial neuromodulation, as ultrasound's application for neuromodulation is relatively new and crudely understood. These investigations are critical for the refinement of device design and the overall advancement of ultrasound methods for noninvasive neuromodulation.
Analysis of Interfacial Processes on Non-Wetting Surfaces
Hatte, Sandeep Shankarrao (Virginia Tech, 2022-10-04)
Non-wetting surfaces mainly categorized into superhydrophobic (SHS), lubricant-infused (LIS) and solid-infused surfaces (SIS), by virtue of their superior water repellant properties have wide applications in several energy and environmental systems. In this dissertation, the role of non-wetting surfaces toward the enhancement of condensation effectiveness is analyzed by taking into consideration the tube side and shell side individual interfacial energy transport processes namely, drag reduction, convection heat transfer enhancement, fouling mitigation and dropwise condensation heat transfer. First, an analytical solution is developed for effective slip length and, in turn, drag reduction and friction factor on structured non-wetting surfaces. Secondly, by combining the solution for effective slip length on structured non-wetting surfaces and the fractal characterization of generic multiscale rough surfaces, a theoretical analysis of drag reduction, friction factor, and convection heat transfer enhancement is conducted for scalable non-wetting surfaces. Next, fractal representation of rough surfaces is used to theoretical derive the dropwise condensation heat transfer performance on SHS and novel SIS surfaces. The aspect of dynamic fouling mitigation properties of non-wetting surfaces is explored by conducting systematic experiments. Using Taguchi design of experiments, this work for the first time presents a closed formed relationship of fouling mitigation quantified in terms of asymptotic fouling resistance with Reynolds number, foulant concentration and viscosity of the infusion material that represents the different surface types in a unified manner. Furthermore, it was observed that LIS and SIS offer excellent fouling mitigation compared to SHS and conventional smooth surfaces, however only SIS owing to the presence of solid-like infusion materials is observed to be robust for practical applications.
Analytical and Experimental Investigation of Insect Respiratory System Inspired Microfluidics
Chatterjee, Krishnashis (Virginia Tech, 2018-11-06)
Microfluidics has been the focal point of research in various disciplines due to its advantages of portability and cost effectiveness, and the ability to perform complex tasks with precision. In the past two decades microfluidic technology has been used to cool integrated circuits, for exoplanetary chemical analysis, for mimicking cellular environments, and in the design of specialized organ-on-a-chip devices. While there have been considerable advances in the complexity and miniaturization of microfluidic devices, particularly with the advent of microfluidic large-scale integration (mLSI) and microfluidic very-large-scale-integration (mVLSI), in which there are hundreds of thousands of flow channels per square centimeter on a microfluidic chip, there remains an actuation overhead problem: these small, complex microfluidic devices are tethered to extensive off-chip actuation machinery that limit their portability and efficiency. Insects, in contrast, actively and efficiently handle their respiratory air flows in complex networks consisting of thousands of microscale tracheal pathways. This work analytically and experimentally investigates the viability of incorporating some of the essential kinematics and actuation strategies of insect respiratory systems in microfluidic devices. Mathematical models of simplified individual tracheal pathways were derived and analyzed, and insect-mimetic PDMS-based valveless microfluidic devices were fabricated and tested. It was found that not only are these devices are capable of pumping fluids very efficiently using insect-mimetic actuation techniques, but also that the fluid flow direction and magnitude could be controlled via the actuation frequency alone, a feature never before realized in microfluidic devices. These results suggest that insect-mimicry may be a promising direction for designing more efficient microfluidic devices.
The Application of BioHeat Perfusion Sensors to Analyze Preservation Temperature and Quantify Pressure Ischemia of Explanted Organs
O'Brien, Timothy J. (Virginia Tech, 2015-03-09)
The development of an organ preservation system (primarily kidneys and livers, but could be adapted to fit hearts, lungs, and even limbs in the future) that can provide surgeons and doctors with real-time quantitative feedback on the health of the organ would be a significant improvement on current transplant practices. This organ transport system will provide surgeons and doctors the opportunity to make more educated decisions towards whether or not to proceed with organ transplantation. Here, we discuss the use Smart Perfusion's organ preservation system as a platform for determining the optimal perfusion temperature of an organ. Porcine kidneys were procured and perfused with a modified PBS solution on the Vasowave™. While on this organ preservation system, a heart emulating pressure waveform (90/50 mmHg) was generated and sent to the specimen. The pressure response, flow rate, temperature, pH, dissolved oxygen content, and conductivity of the fluid stream were all monitored throughout the duration of experimentation. In addition to inline sensors, IR imaging captured the surface temperature of the organ while on the system. Lastly, the use of a combined heat flux-temperature (CHFT) sensor, previously developed at Virginia Tech, was applied for the first time to monitor and measure local tissue perfusion of an explanted organ. A total of 12 experiments were performed (6 at a set fluid temperature of 15°C, and 6 at 20°C). All system data was collected, statistically evaluated and finally compared against blind histological readings (taken at the termination of each experiment at the hilum and pole) to investigate the effects of temperature on organ vasculature. The results of this experiment indicated that the effects of temperature on explanted kidneys can be affectively measured using a non-invasive bioheat perfusion sensor. Specifically, the lower temperature group of kidneys was measured to have lower perfusion. Furthermore, an enhancement to the CHFT sensor technology (CHFT+) was developed and tested for compliance. A controllable thin filmed heat resistor was added to the CHFT assembly to replace the current convective thermal event. This enhancement improved the measured heat flux and temperature signals and enables autonomy. Also, the thin and semi-flexible nature of the new CHFT+ sensor allows for perfusion measurements to be taken from the underside of the organ, permitting a quantitative measure of pressure ischemia. Results from a live tissue test illustrated, for the first time, the effects of pressure ischemia on an explanted porcine kidney.
Biaxial Mechanical Evaluation of Uterosacral and Cardinal Ligaments
Baah-Dwomoh, Adwoa Sarpong (Virginia Tech, 2018-03-06)
The uterosacral ligament (USL) and the cardinal ligament (CL) are two major suspensory tissues that provide structural support to the vagina/cervix/uterus complex. These ligaments have been studied mainly due for their role in the surgical repair for pelvic organ prolapse (POP). POP, which is the descent of a pelvic organ from its normal place towards the vaginal walls and into the vaginal cavity, affects an estimated 3.3 million women in the United States annually. Despite their important mechanical function, little is known about the elastic and viscoelastic properties of the USL and CL due to ethical concerns with in vivo testing of human tissues and the lack of accepted animal models. The goal of this first study is to help establish an appropriate animal model for studying the mechanics of these pelvic supportive ligaments. To achieve this, the first rigorous comparison of histological and planar equi-biaxial mechanical properties of the swine and human USLs was completed. Relative collagen, smooth muscle, and elastin contents were quantified from histological sections and the USL was found to have similar components in both species, with a comparable relative collagen content. Using the digital image correlation (DIC) method to calculate the in-plane Lagrangian strain, no differences in the peak strain during precon- ditioning/cyclic loading tests, secant modulus of the pre-creep/elastic response, and strain at the end of creep tests were detected in the USLs from the two species along both axial loading directions (the main in vivo loading direction and the direction that is perpendicular to it). Because these ligaments are subjected to repeated constant loads in vivo, the effect of re- peated biaxial loads at three different load levels (1 N, 2 N, or 3 N) on elastic and creep properties of the swine CL was investigated. The results showed that CL was elastically anisotropic, as statistical differences were found between the mean strains along the two axial loading directions for specimens at all three different load levels. The increase in strain over time by the end of the 3rd creep test was comparable along the axial loading direc- tions. The greatest mean normalized strain (or, equivalently, the largest increase in strain over time) was measured at the end of the 1st creep test, regardless of the equi-biaxial load magnitude or loading direction. Overall, these experimental findings validate the use of swine as an appropriate animal model and offer new knowledge of the mechanical properties of the USL and CL that can guide the development of better treatment methods such as surgical reconstruction for POP.
The Biological and Immunological Effects of Irreversible Electroporation and Combination Therapy Options for the Improving the Treatment of Pancreatic Cancer
Brock, Rebecca Michaela (Virginia Tech, 2020-06-05)
While cancer treatments have advanced for multiple cancers, pancreatic cancer remains a lethal cancer with few therapy options available. This is due to the inaccessibility of the tumor by surgical and thermal ablative means, high potential for chemoresistance and metastasis, and strongly immunosuppressive tumor microenvironment that makes new treatment measures ineffective in clinic. Irreversible electroporation (IRE) utilizes short, high voltage electrical pulses to form microlesions in cell membranes and induce cell death. While IRE has had significant impact in pancreatic cancer treatment in clinical trials, little is known on the biological and immunological effects of IRE on pancreatic cancer. By studying the effects of IRE on pancreatic tumor biology and the host immune system, I hypothesize I can identify potential combination therapy targets for IRE. I utilized in vitro, ex vivo, and in vivo animal models of both human and murine cancer to study the effects of IRE on pancreatic cancer progression and its potential for immunomodulation. My findings have shown that IRE can significantly delay cancer progression by inducing necroptosis in the tumor and altering the tumor microenvironment by increasing inflammatory signaling. IRE can also produce viable antigens for presentation to induce local and systemic immunosurveillance. However, these effects are limited by countering expression of programmed-cell death ligand 1 (PD-L1), a checkpoint protein that inhibits cytotoxic lymphocyte activity and allows the tumors to recur. The effects of IRE can therefore be expanded by multiple combination therapy approaches, such as chemotherapeutic application (potentially with nanoparticle packaging), PD-1/PD-L1 antibody immunotherapies, and small molecule inhibitors directed at tumor growth signaling that previously showed limited efficacy in clinic.
The Biomechanics of Tracheal Compression in the Darkling Beetle, Zophobas morio
Adjerid, Khaled (Virginia Tech, 2019-11-05)
In this dissertation, we examine mechanics of rhythmic tracheal compression (RTC) in the darkling beetle, Zophobas morio. In Chapter 2, we studied the relationship between hemolymph pressure and tracheal collapse to test the hypothesis that pressure is a driving mechanism for RTC. We found that tracheae collapse as pressure increases, but other physiological factors in the body may be affecting tracheal compression in live beetles. Additionally, as the tracheae compress, they do so in varying spatial patterns across the insect body. In chapter 3, we examined spatial variations in the taenidial spacing, stiffness, and tracheal thickness along the length of the tracheae. We related variations in Young's modulus and taenidial spacing with measurements of collapse dimples and found that spatial patterns of Young's modulus correlate with dimensions of collapse dimples. This correlation suggests an intuitive link between tracheal stiffness variations and the unique patterns observed in compressing tracheae. Lastly, in chapter 4, we studied the non-uniform collapse patterns in 3-D. By manually pressurizing the hemocoel and imaging using synchrotron microcomputed tomography (SR-µCT), we reconstructed the tracheal system in its compressed state. While previous studies used 2-D x-ray images to examine collapse morphology, ours is the first to quantify collapse patterns in 3-D and compare with previous 2-D quantification methods. Our method is also the first to make a direct measure of tracheal volume as the tracheal system compresses, similar to the phenomenon that occurs during rhythmic tracheal compression.
Buckling at the Fluid - Soft Solid Interface; A Means for Advanced Functionality within Soft Materials
Tavakol, Behrouz (Virginia Tech, 2015-09-02)
Soft materials and compliant structures often undergo significant deformation without failure, a unique feature making them distinct from classical rigid materials. These substantial deformations provide a means for faster or more energy efficient deformations, which can be achieved by taking advantage of elastic instabilities. We intend to utilize structural instabilities to generate advanced functionality within soft materials. In particular, we use the buckling of thin, flexible plates to control or enhance the flow of fluid in a micro channel. The buckling deformation is created or altered via two different stimuli, first a mechanical strain and then an electrical signal. We investigate the behavior of each system under different conditions experimentally, numerically, or theoretically. We also show that the coupled interaction between fluid and the soft film plays a critical role in the shape of deformation and consequently in the functionality of the mechanism. We first embed a buckled thin film in a fluid channel within a soft device. By applying a mechanical strain to the device, we show both experimentally and numerically that the height of the buckled film changes accordingly as does the flow rate. We then offer an analytical solution by extending the classical lubrication theory to higher-order terms as a means to more accurately describe the flow in a channel with a buckled thin film, and in general, the flow in channels with any constrictions provided the Reynolds number is low. Next, we use an electrical signal to make a confined dielectric film undergo out-of-plane buckling deformation. The thin film is sandwiched between two flexible electrodes and the mechanism is implemented in a microfluidic device to pump the fluid into a micro channel. We show that the critical buckling voltage at which the thin film buckles out of the plane is mainly a function of voltage while the shape of deformation and so the functionality of this mechanism depend considerably on the applied boundary conditions. Finally, we enhance the fluid-soft structure response of the actuating mechanism by substituting flexible electrodes with fluid electrodes, resulting in a significant increase in the actuation frequency as well as a reduction in the critical buckling voltage.
Burst sine wave electroporation (B-SWE) for expansive blood–brain barrier disruption and controlled non-thermal tissue ablation for neurological disease
Campelo, Sabrina N.; Salameh, Zaid S.; Arroyo, Julio P.; May, James L.; Altreuter, Sara O.; Hinckley, Jonathan; Davalos, Rafael V.; Rossmeisl, John H. Jr. (AIP Publishing, 2024-05-30)
The blood–brain barrier (BBB) limits the efficacy of treatments for malignant brain tumors, necessitating innovative approaches to breach the barrier. This study introduces burst sine wave electroporation (B-SWE) as a strategic modality for controlled BBB disruption without extensive tissue ablation and compares it against conventional pulsed square wave electroporation-based technologies such as high-frequency irreversible electroporation (H-FIRE). Using an in vivo rodent model, B-SWE and H-FIRE effects on BBB disruption, tissue ablation, and neuromuscular contractions are compared. Equivalent waveforms were designed for direct comparison between the two pulsing schemes, revealing that B-SWE induces larger BBB disruption volumes while minimizing tissue ablation. While B-SWE exhibited heightened neuromuscular contractions when compared to equivalent H-FIRE waveforms, an additional low-dose B-SWE group demonstrated that a reduced potential can achieve similar levels of BBB disruption while minimizing neuromuscular contractions. Repair kinetics indicated faster closure post B-SWE-induced BBB disruption when compared to equivalent H-FIRE protocols, emphasizing B-SWE’s transient and controllable nature. Additionally, finite element modeling illustrated the potential for extensive BBB disruption while reducing ablation using B-SWE. B-SWE presents a promising avenue for tailored BBB disruption with minimal tissue ablation, offering a nuanced approach for glioblastoma treatment and beyond.
Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth
Sano, Michael B.; Arena, Christopher B.; Bittleman, Katelyn Rose; DeWitt, Matthew R.; Cho, Hyung J.; Szot, Cchristopher S.; Saur, Dieter; Cissell, James M.; Robertson, John L.; Lee, Yong Woo; Davalos, Rafael V. (Nature Publishing Group, 2015-10-13)
Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 μs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.
Cell Death Characterization In Tumor Constructs Using Irreversible Electroporation
Prokop, Katherine Jane (Virginia Tech, 2013-10-04)
Pancreatic and prostate cancer are both prevalent cancers in the United States with pancreatic being one of the most aggressive of all cancers and prostate cancer being one of the most common, ranking as the number one cancer in men. Treatment of both cancers can be quite challenging as the anatomy of the pancreas and prostate, as well as the development and diagnosis of the disease can greatly limit treatment options. Therefore, it is necessary to develop new cancer treatments to help manage and prevent these cancers. Irreversible electroporation is a new non-thermal focal ablation therapy utilizing short, pulsed electric fields to damage cell membranes leading to cell death. The therapy is minimally invasive, involving the insertion of needle electrodes into the region of interest and lasts less than two minutes. Heat sink effects that thermal therapies experience near large blood vessels do not affect irreversible electroporation. This allows the treatment to be used on tumors near vasculature as well as critical structures without harming these vital regions. While irreversible electroporation is a promising new cancer therapy, further developments are necessary to improve treatment planning models. This work aims to further understand the electric field thresholds necessary to kill different types of cancer cells with a focus on pancreatic and prostate cancer. The work is done using an in vitro tumor (hydrogel) model as this model is better than traditional cell suspension studies, with added benefits over the immediate use of tissue and animal models.
Cell Migration on Opposing Rigidity Protein Gradients: Single Cell and Co-culture Studies
Jain, Gaurav (Virginia Tech, 2014-10-31)
Cell migration is a complex physiological process that is important from embryogenesis to senescence. In vivo, the migration of cells is guided by a complex combination of signals and cues. Directed migration is typically observed when one of these cues is presented to cells as a gradient. Several studies have been conducted into directed migration on gradients that are purely mechanical or chemical. Our goal was to investigate cellular migratory behavior when cells are presented with a choice and have to choose between increasing substrate rigidity or higher protein concentration. We chose to focus on this unique environment since it recapitulates several interfacial regions in vivo. We have designed novel hydrogels that exhibit dual and opposing chemical and mechanical profiles using photo-polymerization. Our studies demonstrate that durotaxis, a well-known phenomenon, can be reversed when cells sense a steep protein profile in the opposite direction. Fibroblasts were co-cultured with macrophages to obtain an understanding on how migration occurs when two different cell types are present in the same microenvironment. First, we investigated the migratory behavior of macrophages. These cell types exhibited a statistically significant preference to move towards the rigid/low collagen region of the interface. Interestingly, fibroblasts when co-cultured with macrophages, exhibited a preference for the low modulus-high collagen region of the interface. However, with the current sample size, these trends are statistically insignificant. On the contrary, the presence of fibroblasts in the cellular microenvironment did not result in the reversal of durotaxis exhibited by macrophages. Macrophages secreted significantly higher levels of secreted tumor necrosis factor (TNF-alpha) in mono-cultures in contrast to fibroblast-macrophage co-cultures. This trend could be an indication of macrophage plasticity between mono- and co-cultures. In summary, we have designed dual and opposing rigidity-protein gradients on a hydrogel substrate that can provide new insights into cellular locomotion. These results can be used to design biomimetic interfaces, biomaterial implants and for tissue engineering applications.
Cell-Fiber Interactions: A New Route to Mechano-Biological Investigations in Developmental and Disease Biology
Sheets, Kevin Tyler (Virginia Tech, 2014-11-03)
Cells in the body interact with a predominantly fibrous microenvironment and constantly adapt to changes in their neighboring physiochemical environment, which has implications in developmental and disease biology. A myriad of in vitro platforms including 2D flat and 3D gel substrates with and without anisotropy have demonstrated cellular alterations to subtle changes in topography. Recently, our work using suspended fibers as a new in vitro biological assay has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to cytoskeleton arrangement, including focal adhesion cluster lengths and nucleus shape indices, leading to altered migration speeds. It is hypothesized that these behaviors occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Thus, in this study, we investigate the role of fiber curvature and structural stiffness in force modulation of single cells attached to suspended fibers. Using our previously reported non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we present our findings on single cell inside-out and outside-in forces using fibers of three diameters (250 nm, 400 nm and 800 nm) representing a wide range of structural stiffness (3-45 nN/μm). To investigate cellular adaptability to external perturbation, we present the development of a first-of-its-kind force measurement 'nanonet' platform capable of investigating cell adhesion forces in response to symmetric and non-symmetric (injury model) loading. Our combined findings are multi-fold: (i) Cells on suspended fibers are able to form focal adhesion clusters approximately four times longer than those on flat substrates, which gives them potential to double their migration speeds, (ii) Nanonets as force probes show that the contractility-based inside-out forces are nearly equally distributed on both sides of the cell body, and that overall force magnitudes are dependent on fiber structural stiffness, and (iii) External perturbation can evenly (symmetric) or unevenly (non-symmetric) distribute forces within the cell, and the resulting bias causes diameter-dependent outside-in adhesion force response. Finally, we demonstrate the power of the developed force measurement platform by extending our studies to cell-cell junctional forces as well as single-cell disease models including cancer and aortic aneurysm.
Characterization of Ablation Thresholds for 3D-Cultured Patient-Derived Glioma Stem Cells in Response to High-Frequency Irreversible Electroporation
Ivey, J. W.; Wasson, E. M.; Alinezhadbalalami, N.; Kanitkar, A.; Debinski, Waldemar; Sheng, Z.; Davalos, Rafael V.; Verbridge, Scott S. (American Association for the Advancement of Science, 2019-04-28)
High-frequency irreversible electroporation (H-FIRE) is a technique that uses pulsed electric fields that have been shown to ablate malignant cells. In order to evaluate the clinical potential of H-FIRE to treat glioblastoma (GBM), a primary brain tumor, we have studied the effects of high-frequency waveforms on therapy-resistant glioma stem-like cell (GSC) populations. We demonstrate that patient-derived GSCs are more susceptible to H-FIRE damage than primary normal astrocytes. This selectivity presents an opportunity for a degree of malignant cell targeting as bulk tumor cells and tumor stem cells are seen to exhibit similar lethal electric field thresholds, significantly lower than that of healthy astrocytes. However, neural stem cell (NSC) populations also exhibit a similar sensitivity to these pulses. This observation may suggest that different considerations be taken when applying these therapies in younger versus older patients, where the importance of preserving NSC populations may impose different restrictions on use.We also demonstrate variability in threshold among the three patient-derived GSC lines studied, suggesting the need for personalized cell-specific characterization in the development of potential clinical procedures. Future work may provide further useful insights regarding this patient-dependent variability observed that could inform targeted and personalized treatment.

Browsing by Author "Davalos, Rafael V."

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