The objective of this thesis is to observe and characterize the early mechanical and biochemical events in osteocyte mechanotransduction. Physical forces have been increasingly implicated in normal physiological and pathological cellular activities of osteocytes. The mechanotransduction process in osteocytes involves spatiotemporally complex changes in cytoskeletal organization, signal activation, and whole cell mechanical properties. Most in vitro biophysical techniques currently available sacrifice either spatial or temporal resolution and are unable to visualize 3D cellular behavior on the millisecond time scale. Here, we develop a novel multi-channel quasi-3D microscopy technique to simultaneously visualize and measure whole-cell mechanics, intracellular cytoskeletal deformation, and biochemical signal activation under fluid shear flow.The technique was applied to visualize cell dilatation and cytoskeletal deformation in osteocytes under steady fluid shear flow. Analysis of the plasma membrane and either the intracellular actin or microtubule cytoskeletal networks provided characterization of their deformations over time. No volumetric dilatation of the whole cell was observed under flow, and both cytoskeletal networks experienced primarily tensile viscoelastic creep and recovery in all measured strain components. Intra- and inter- cellular mechanical heterogeneity was observed in both cytoskeletal networks. Cytoskeletal disruption pointed towards a unidirectional mechanical interaction where microtubule networks affected actin network strains, but not vice versa.The second study in this thesis investigated the effects of steady and oscillatory flow on the actin and microtubule networks within the same cell. Shear strain was the predominant strain in both steady and oscillatory flows, in the form of viscoelastic creep and elastic oscillations, respectively. Under oscillatory fluid shear flow, the actin networks displayed an oscillatory strain profile more often than the MT networks in all the strains tested and had a higher peak-to-trough magnitude. Taken together with the first study, the actin networks were determined to be the more responsive cytoskeletal networks in osteocytes to fluid flow and may play a bigger role in mechanotransduction.The final culminating study tracked [Ca+2]i and F-actin network strains simultaneously in a single osteocyte. We demonstrated novel osteocyte mechano- and transduction behavior where [Ca+2]i oscillations activate phasic actomyosin contractions using a smooth muscle-like mechanism. Fluid shear, ATP, and ionomycin induced [Ca+2]i signaling with a subsequent compression and recovery in actin strains of the cell, being most apparent in the height direction strain. This contraction was reversible over the period of hundreds of seconds. ML-7, a myosin light chain kinase inhibitor, significantly slowed down the kinetics of contraction initiation, but blebbistatin, a potent skeletal and non-muscle inhibitor, had no effect on the actin contraction. Furthermore, smooth muscle contraction-related proteins were detected by Western blot. The observation of muscle-like contractility in osteocytes demonstrates a possible positive feedback mechanism of osteocytes to activate mechanotransduction pathways.

The objective of this thesis is to observe and characterize the early mechanical and biochemical events in osteocyte mechanotransduction. Physical forces have been increasingly implicated in normal physiological and pathological cellular activities of osteocytes. The mechanotransduction process in osteocytes involves spatiotemporally complex changes in cytoskeletal organization, signal activation, and whole cell mechanical properties. Most in vitro biophysical techniques currently available sacrifice either spatial or temporal resolution and are unable to visualize 3D cellular behavior on the millisecond time scale. Here, we develop a novel multi-channel quasi-3D microscopy technique to simultaneously visualize and measure whole-cell mechanics, intracellular cytoskeletal deformation, and biochemical signal activation under fluid shear flow. The technique was applied to visualize cell dilatation and cytoskeletal deformation in osteocytes under steady fluid shear flow. Analysis of the plasma membrane and either the intracellular actin or microtubule cytoskeletal networks provided characterization of their deformations over time. No volumetric dilatation of the whole cell was observed under flow, and both cytoskeletal networks experienced primarily tensile viscoelastic creep and recovery in all measured strain components. Intra- and inter- cellular mechanical heterogeneity was observed in both cytoskeletal networks. Cytoskeletal disruption pointed towards a unidirectional mechanical interaction where microtubule networks affected actin network strains, but not vice versa. The second study in this thesis investigated the effects of steady and oscillatory flow on the actin and microtubule networks within the same cell. Shear strain was the predominant strain in both steady and oscillatory flows, in the form of viscoelastic creep and elastic oscillations, respectively. Under oscillatory fluid shear flow, the actin networks displayed an oscillatory strain profile more often than the MT networks in all the strains tested and had a higher peak-to-trough magnitude. Taken together with the first study, the actin networks were determined to be the more responsive cytoskeletal networks in osteocytes to fluid flow and may play a bigger role in mechanotransduction. The final culminating study tracked [Ca+2]i and F-actin network strains simultaneously in a single osteocyte. We demonstrated novel osteocyte mechano- and transduction behavior where [Ca+2]i oscillations activate phasic actomyosin contractions using a smooth muscle-like mechanism. Fluid shear, ATP, and ionomycin induced [Ca+2]i signaling with a subsequent compression and recovery in actin strains of the cell, being most apparent in the height direction strain. This contraction was reversible over the period of hundreds of seconds. ML-7, a myosin light chain kinase inhibitor, significantly slowed down the kinetics of contraction initiation, but blebbistatin, a potent skeletal and non-muscle inhibitor, had no effect on the actin contraction. Furthermore, smooth muscle contraction-related proteins were detected by Western blot. The observation of muscle-like contractility in osteocytes demonstrates a possible positive feedback mechanism of osteocytes to activate mechanotransduction pathways.

Cancer remains, one of the major public health problems in the United States as well as many other countries worldwide. According to According to the World Health Organization, cancer is currently the leading cause of death worldwide, accounting for 7.6 million deaths annually, and 25% of the annual death was due to Cancer during the year of 2011. In the long history of the cancer treatment field, many treatment options have been established up to date. Traditional procedures include surgical procedures as well as systemic therapies such as biologic therapy, chemotherapy, hormone therapy, and radiation therapy. Nevertheless, side-effects are often associated with such procedures due to the systemic delivery across the entire body. Recently technologies have been focused on localized therapy under minimally or noninvasive procedure with imaging-guidance, such as cryoablation, laser ablation, radio‐frequency (RF) ablation, and High Intensity F-ocused Ultrasound (HIFU). HIFU is a non-invasive procedure aims to coagulate tissue thermally at a localized focal zone created with noninvasively emitting a set of focused ultrasound beams while the surrounding healthy tissues remain relatively untreated. Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a dynamic, radiation-force-based imaging technique, which utilizes a single HIFU transducer by emitting an Amplitude-modulated (AM) beam to both thermally ablate the tumor while inducing a stable oscillatory tissue displacement at its focal zone. The oscillatory response is then estimated by a cross-correlation based motion tracking technique on the signal collected by a confocally-aligned diagnostic transducer. HMIFU addresses the most critical aspect and one of the major unmet needs of HIFU treatment, which is the ability to perform real-time monitoring and mapping of tissue property change during the HIFU treatment. In this dissertation, both the assessment and monitoring aspects of HMIFU have been investigated fundamentally and experimentally through development of both a 1-D and 2-D based system. The performance assessment of HMIFU technique in depicting the lesion size increase as well as the lesion-to-background displacement contrast was first demonstrated using a 3D, FE-based interdisciplinary simulation framework. Through the development of 1-D HMIFU system, a multi-parametric monitoring approach was presented where presented where the focal HMI displacement, phase shift (Δφ), and correlation coefficients were monitored along with thermocouple and PCD under the HIFU treatment sequence with boiling and slow denaturation. For HIFU treatments with slow denaturation, consistent displacement increase-then-decrease trend was observed, indicating tissue softening-then-stiffening and phase shift increased with treatment time in agreement with mechanical testing outcomes. The correlation coefficient remained high throughout the entire treatment time under a minimized broadband energy and boiling mechanism. Contrarily, both displacement and phase shift changes lacked consistency under HIFU treatment sequences with boiling due to the presence of strong boiling mechanism confirmed by both PCD and thermocouple monitoring. In order to facilitate its clinical translation, a fully-integrated, clinically 2D real-time HMIFU system was also developed, which is capable of providing 2D real-time streaming during HIFU treatment up to 15 Hz without interruption. Reproducibility studies of the system showed consistent displacement estimation on tissue-mimicking phantoms as well as monitoring of tissue-softening-then-stiffening phase change across 16 out of 19 liver specimens (Increasing rate in phase shift (Δφ): 0.73±0.69 %/s, Decreasing rate in phase shift (Δφ): 0.60±0.19 %/s) along with thermocouple monitoring (Increasing: 0.84±1.15 %/ °C, Decreasing: 2.03± 0.93%/ °C) and validation of tissue stiffening using mechanical testing. In addition, the 2-D HMIFU system feasibility on preclinical pancreatic tumor mice model was also demonstrated in vivo, where HMI displacement decreases were observed across three of five treatment locations on the kP(f)c model at 20.8±6.84, 18.6±1.46, and 24.0±5.43%, as well as across four of the seven treatment locations on the KPC model at 39.5±2.98%, 34.5±21.5%, 16.0±3.05%, and 35.0±3.12% along with H and E histological confirmation. In order to improve the quantitative monitoring aspect of HMIFU, a novel, model-independent method for the estimating Young's modulus based on strain profile was also implemented, where 1-D HMIFU system showed feasibilities on polyacrylamide phantom (EHMI/E ≈ 2.3) and liver specimen (EHMI/E ≈ 8.1), and 2-D HMIFU system showed feasibilities on copolymer phantom(EHMI/E ≈ 30.4), liver specimen(EHMI/E ≈ 211.3), as well as HIFU treated liver specimen(EHMI,end/EHMI,beginning ≈ 5.96). In conclusion, the outcomes from the aforementioned studies successfully showed the feasibility of both HMIFU systems in multi-parametric monitoring of HIFU treatment with slow denaturation and boiling, which prepares its stage towards clinical translation.

The work of my thesis is the cumulative result of 6 years of research in Prof. Michael P. Sheetz laboratory at the Biological Sciences Department of Columbia University, within the collaborative framework of the Nanotechnology Center for Mechanobiology, an interdisciplinary and multi-institutional center for the study of cell mechanics, involving, among other institutions, the Applied Physics department at Columbia University, and the Schools of Medicine of University of Pennsylvania, New York University, and Mt Sinai. In Chapter 1, I provide an overview of the field of mechanobiology, with an emphasis on the implications of cell-extracellular matrix and cell-cell attachment on cell function. In Chapter 2, I present the aims of the thesis, with a focus on the two cell systems used in the projects described: human mesenchymal stem cells, and T cells. Then, Chapters 3-5 represent the main body of my thesis, where I present detailed descriptions of the projects that I worked on and that successfully made it into scientific publications or that are in preparation for publication. In Chapter 3, I analyze how matrix chemistry and substrate rigidity affect human mesenchymal stem cell morphology in the context of lineage differentiation, and speculate on potential mechanisms that cells use to sense local rigidity. In Chapter 4, I present a new substrate design that facilitates live visualization of the interface formed between a T cell and an antigen presenting cell, i.e. the immunological synapse, and discuss the impact of intercellular forces on T cell activation. In Chapter 5, I explore the molecular mechanism of Cas-L mechanical activation at the immunological synapse of T cells, and demonstrate how Cas-L regulates T cell activation in the context of an immune response. Finally, in Chapter 6, I lay down the main conclusions of the thesis, and discuss ongoing projects that directly follow up on the results of this thesis.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by a high incidence of lethal ventricular arrhythmias, fibrofatty replacement of myocardium, and can account for up to 20% of sudden cardiac death (SCD) cases in the young. Typically involving autosomal dominant transmission, germline mutations in genes encoding desmosomal proteins have been identified as a cause of ARVC, although the pathogenesis of the disease is still unclear. While early detection and treatment can provide a normal life expectancy for the majority of patients, with less than 10% progressing to overt right ventricular failure, low genetic penetrance and epigenetic modifiers (such as endurance exercise) can make the condition difficult to diagnose. Addressing this clinical challenge requires a better understanding of the defective molecular mechanisms that underlie the disease. To that end, the goal of this dissertation is to provide insight into the effects of ARVC-causing mutant proteins on the mechanical and signaling properties of cardiac myocytes. Using elastography and histological techniques, we begin by characterizing the structural and mechanical properties of the native right ventricular myocardium, particularly the right ventricular apex (RVA). Because the RVA is a key site for development of arrhythmias and a potential pacing target, a careful characterization of its structure and mechanical properties are essential for understanding its role in cardiac physiology. In the first section of this dissertation, we perform a systematic analysis of the structural features and mechanical strains in the heart, focusing on the RVA region. More than half of ARVC patients exhibit one or more mutations in genes encoding desmosomal proteins. This has led many investigators to suggest that ARVC is a "disease of the desmosome" in which defective cell-cell adhesion plays a critical pathogenic role, although direct evidence for this hypothesis is lacking. To gain greater insights into potential mechanisms by which desmosomal mutations cause ARVC, we next characterize biomechanical properties and responses to shear stress (motivated by our results in the previous section) in neonatal rat ventricular myocytes expressing two distinct mutant forms of the desmosomal protein plakoglobin which have been linked to ARVC in patients. We show that ARVC-causing mutations in plakoglobin lead to altered cellular distribution of plakoglobin, without alterations in cell mechanical properties or certain early signaling pathways. The identification of defective molecular mechanisms that are common across ARVC-patients remains a strategic area of research. Specifically, recent studies have investigated the mechanistic basis for different ARVC-causing mutations in hopes of identifying common defects in a signaling pathway - information that could be used to develop diagnostic tests or identify therapeutic targets. In the last section of this dissertation, we investigate the effects of mutant plakophilin-2 expression, and repeat key experiments performed in the previous section to identify common defects in mechanical and signaling properties. We identify a common, underlying defect in ARVC pathogenesis. Specifically, we show that disease-causing mutations across different desmosomal proteins can cause the cell to respond abnormally to mechanical shear stress with respect to plakoglobin trafficking.

Walking is a state of continuous imbalance that requires a complex control strategy and cyclic activation of leg muscles to achieve successful inter‐limb coordination. Neuro‐musculoskeletal impairments, such as stroke, cerebral palsy, and spinal cord injury, affect one's ability to voluntarily contract muscles to normal amplitudes. This change in muscle activation pattern reduces the joint level torque generation and as a result impairs the ability to walk normally. Technological advances over the last two decades have resulted in the development of rigid link robotic exoskeletons that aim to improve gait deficits. These devices reduce repetitive and manual labor of therapists while providing objective measurement of the therapy during the gait rehabilitation. Despite the development of these robotic devices, no consensus has emerged about the superiority of robot-aided gait rehabilitation over the traditional methods. This may be because of the inherent complexity of the human musculoskeletal system and the constraints that rigid linked systems impose on the human movement. In this work, we present a cable-driven Active Tethered Pelvic Assist Device (A-TPAD) for gait rehabilitation that can apply a controlled external wrench to the human pelvis in any direction and at any point of the gait cycle for a specified duration. The A-TPAD does not add undesirable inertia on the user and does not constrain the user's motion during training. The A-TPAD provides a technological platform to scientifically study human adaptation in gait due to externally applied forces and moments on the pelvis. Human studies with the A-TPAD can motivate new gait rehabilitation paradigms which can potentially be used to correct gait deficits in human walking. The human nervous system is capable of modifying the motor commands in response to alterations in the movement conditions. Several studies have demonstrated the flexibility of human locomotion despite motor impairments and have shown the potential of using such paradigms for gait rehabilitation. In this work, we present a number of human experiments using the cable-driven A-TPAD to propose novel force interventions that induce adaptation in human gait kinematics and kinetics. In particular, stance phase gait interventions have been developed for gait rehabilitation of hemiparetic patients. In these interventions, the external force vector was applied to the pelvis to target weight bearing during walking and to promote longer stance durations. A single-session force training experiment with hemiparetic stroke patients was also conducted as a part of this work. It is shown that hemiparetic stroke patients improved the ground reaction force symmetry, forward propulsion effort, and stance phase symmetry during walking. In this work, the A-TPAD is also used to develop an intervention to apply external gait synchronized forces on the pelvis to reduce the user's effort during walking. The external forces were directed in the sagittal plane to assist the trailing leg during the forward propulsion and vertical deceleration of the pelvis during the gait cycle. A pilot experiment with five healthy subjects was conducted. This study provides a novel approach to study the role of external forces in altering the walking effort, such understanding is important while designing assistive devices for individuals who spend higher than normal effort during walking.

<p> The esophagus is critical for passage of oral bolus into the gastrointestinal tract. Diseases of the esophagus, such as malignancy, can necessitate resection of esophageal tissue. To maintain esophageal continuity with the remainder of the gastrointestinal tract, reconstruction is mandatory. Current reconstructive options are morbid and involve autologous conduits such as stomach, small bowel, or colon. An alternative tissue engineered conduit that facilitates esophageal regrowth to reduce the need for these morbid reconstructions would have significant clinical utility. Several critical challenges must be addressed in order to make these conduits a clinical reality. First, scaffolds should be designed to ideally mimic mechanical behavior of the native esophagus. To accomplish this, a non-destructive method to mechanically assess these constructs benchmarked to native esophagi is necessary before and after implantation. Second, scaffolds should be both biocompatible and mechanically stable <i> in vitro</i>; this would allow selection of desirable candidates for subsequent <i>in vivo</i> testing. Finally, <i>in vivo</i> testing of the esophageal conduit requires development of an analogous large animal model to human disease. <i>In vivo</i> large animal model testing is required as proof of concept for esophageal regeneration as a critical step toward future human use.3</p><p>

acase@tulane.edu / Progress towards understanding the underlying mechanisms of pelvic organ prolapse (POP) is limited, in part, due to a lack of information on the biomechanical properties and microstructural composition of the vaginal wall. Compromised vaginal wall integrity is thought to contribute to pelvic floor disorders. In particular, disruption of the elastin metabolism within the vaginal wall extracellular matrix has been highly implicated in POP pathogenesis; however, the role of elastin within the vaginal wall is not fully understood. In addition to the information produced from uniaxial testing, biaxial extension-inflation tests performed over a range of physiological values could provide additional insights into vaginal wall mechanical behavior (i.e. axial coupling and anisotropy) while preserving in vivo tissue geometry. Thus, the objective of this study is to identify the role of elastin in vaginal wall mechanics using physiologically relevant experimental and mathematical approaches. Our specific aims are thus: 1. Develop biaxial mechanical testing methods for assessing the mechanical properties of the murine vaginal wall in a physiological manner. 2. Establish a microstructurally-motivated constitutive model capable of describing the biaxial extension-inflation response of the nonpregnant murine vaginal wall. 3. Quantify the role of elastin in murine vaginal mechanical properties through enzymatic digestion of elastin with elastase.Vaginal tissue from female C57BL/6 mice underwent pressure-diameter and force-length preconditioning and testing within a pressure myograph device before and after elastase digestion. In order to mathematically interpret biaxial data, vaginal tissue was modeled using a 2D membrane approach. Several constitutive models were evaluated on their ability to describe vaginal wall mechanical behavior. Elastase digestion induced marked changes in biaxial mechanical properties, suggesting that elastin may play an important role in vaginal wall mechanical function. Constitutive model evaluation resulted in the selection of a diagonal two-fiber family strain energy function and suggests that collagen fibers within the vaginal wall extracellular matrix (ECM) may be primarily oriented diagonally with a slight preference towards the circumferential direction. Further, our results suggest that elastin-collagen interactions may be important for vaginal wall homeostasis. The present findings may help to understand the underlying mechanisms of POP and aid in the development of growth and remodeling models for improved assessment and prediction of changes in structure-function relationships with prolapse development. / 0 / Katy Robison

Nucleus replacement has recently come into favor as a possible treatment for Degenerative Disc Disease. Replacing degenerative nucleus tissue with a synthetic material that mimics healthy nucleus tissue may restore normal function and biomechanics to the disc and delay or obviate the need for more invasive procedures such as total disc replacement and fusion. This thesis evaluated a novel protein polymer hydrogel composed of silk and elastin as a nucleus replacement material. There are three experimental components; one in vivo and two in vitro portions. In the first experimental portion, a large animal model was developed to evaluate the biocompatibility of the material as well as the effect on surrounding boney and soft tissues. Three discs were evaluated in each animal; sham, discectomy and discectomy treated with hydrogel. Discs were evaluated at 4, 26 and 52 weeks. The hydrogel group showed a quiet cellular response, as well as decreased boney remodeling and fewer degenerative changes when compared to the discectomy group. The second experimental portion evaluated the biomechanics of 9 cadaveric motion segments loaded in axial rotation, lateral bending, flexion/extension (FE) and compression. Specimens were tested sequentially in the intact state, following annulotomy, discectomy and after hydrogel treatment. Range of Motion (ROM) in FE was shown to increase from the intact state (8.50+/-1.44˚) to the discectomy state (9.86+/-1.77˚) and decrease following hydrogel treatment (8.66+/-0.76˚) to be similar to the intact ROM. The third experimental portion investigates the effect of three commonly applied testing conditions on the mechanical properties of spinal segments. 27 motion segments were tested at 18˚C wrapped with Phosphate Buffered Saline (PBS), at 37˚C in a PBS bath, and at 37˚C and 100% humidity. Specimens were tested hourly for 6 hours. The heated conditions were shown to have lower stiffness and increased range of motion when compared 18 ˚C tests. Repeated testing with time increased neutral zone and ROM for all modes of bending. As tests are repeated over time, tissue properties change and may mask the ability of a nucleus replacement to restore biomechanics.

Juvenile red snapper are attracted to structure and settle onto low profile reefs, which serve as nursery grounds. Little is known about their life history during this time. However, recent studies from a shell bank in the NW Gulf of Mexico have shown higher growth rates for juveniles located on mud habitats adjacent to low profile reefs, perhaps due to varied prey availability and abundance. To further investigate the habitat needs of juvenile red snapper, individuals were collected from a low profile shell ridge (on-ridge) and adjacent mud areas (off-ridge) on Freeport Rocks, TX, and divided into three size classes (≤3.9 cm SL, 4.0-5.9 cm SL, ≥6 cm SL). Feeding morphology and kinematics were characterized and compared among size classes and between the two habitats. A dynamic jaw lever model was used to make predictions about feeding mechanics, and kinematic profiles obtained from high-speed videos of prey capture events validated the model’s predictive ability. Model output suggested an ontogenetic shift in feeding morphology from a juvenile feeding mode (more suction) to an adult feeding mode (more biting). Stomach contents revealed a concomitant shift in prey composition that coincided with the ontogenetic shift in feeding mode. The model also predicted that on-ridge juveniles would have faster jaw closing velocities compared to off-ridge juveniles, which had slower, stronger jaws. Analysis of prey capture events indicated that on-ridge juveniles demonstrated greater velocities and larger displacements of the jaws than off-ridge juveniles. Shape analysis was used to further investigate habitat effects on morphology. Off-ridge juveniles differed from on-ridge in possessing a deeper head and body. Results from model simulations, kinematic profiles, personal observations, and shape analysis all complement the conclusion that on-ridge juveniles exhibited more suction feeding behavior, whereas off-ridge juveniles used more biting behavior. Stomach contents demonstrated an early switch to piscivory in off-ridge juveniles compared to on-ridge juveniles, which may account for higher off-ridge growth rates. Habitat disparity, perhaps available prey composition, generated variations in juvenile feeding mechanics and consequently feeding behavior. This disparity may ultimately affect the growth rates and recruitment success of juvenile red snapper from different habitats.