The knee is the most common site of osteoarthritis (OA) and is one of the leading causes of disability in older adults affecting over 53 million people in the United States and more than 302 million people worldwide. These numbers are only expected to grow because of the rise of diseases such as obesity, demographic shifts to an older population, and a more sedentary lifestyle. The rise of obesity and a more sedentary lifestyle comes with increases in joint loading which along with the aging population creates worse outcomes in proprioception. All of which can contribute to worsening OA. Despite the great costs to quality of life and society, there is no cure for OA. Only treatments exist to treat the symptoms of OA; and since knee pain is one of the most common symptoms of OA, it is a powerful driver for treatment because of the disruptive nature it can have on quality of life. Therefore, many treatments focus on pain relief and exercise to reduce the pain and worsening of OA. Radio frequency nerve ablation (RFA) is a procedure that is increasingly being performed for those who want an alternative before resorting to or are not a good match for total knee arthroplasty (TKA). Because RFA is minimally invasive, it can be performed on an outpatient basis and has been shown to be effective in reducing pain for at least 24 months for most patients. Despite the benefits in pain reduction, little is known about the biomechanical effects of RFA and its consequences on proprioception. However, based on prior studies into the pain relieving effects of interventions such as celecoxib or HA injections, we can hypothesize that with a decrease in pain, knee loading increases. Therefore, the pain relieving effects of RFA may increase the incidence of OA. Furthermore, because the RFA procedure involves ablating nerves that carry sensory information, changes to proprioception are expected. However, currently there is no information regarding its effect on proprioception. Again, using prior research that studies the consequences of reduced proprioception on those with OA, we can hypothesize that with RFA, proprioception would be further reduced compared to the reductions experienced by people with OA already, and it may also lead to worsening OA outcomes. Despite the possible issue of worsening OA outcomes with RFA, the pain relieving effects cannot be discounted as it is one of the most disruptive symptoms of OA. Therefore, effects of RFA on knee biomechanics and proprioception should be studied to understand the long-term impacts of this procedure.

Irreversible electroporation (IRE) is a local ablation technique that has been shown to be both safe and effective in the treatment of solid tumors. The treatment typically consists of inserting needle electrodes directly into the treatment zone and applying high-voltage pulses with widths on the order of hundreds of microseconds. These pulses permeabilize tissue leading to loss of homeostasis among the cells in the treatment zone. Predicting these treatments is challenging as the electric field (EF) induced through the electrode configuration is heterogeneous and is affected by several adjustable parameters. Computational treatment planning models aim to provide a visualization of the treatment zone, and they rely on two critical pieces of information: the electric field distribution (EFD) within the tissue, and the lethal EF threshold for the target tissue type. This work primarily aims to quantify tissue properties necessary for computing the EFD for any electrode configuration, for both traditional IRE as well as next-generation high-frequency IRE treatments. Also included is the determination of pancreatic tumor lethal EF threshold using collagen tissue mimics. Additionally, this work builds on previous reports of an optimal resistance reached during IRE by examining the changes in patients' immune cell populations following treatment, and proposing a method of optimizing these populations by monitoring real-time current achieved during IRE. / Doctor of Philosophy / We are in dire need of new options in cancer therapy, especially in the treatment of tumors that are unresectable, particularly aggressive, or resistant to drugs. Irreversible electroporation (IRE) is a local tumor treatment that has been shown to safely and effectively destroy tumor tissue while leaving behind important structures like blood vessels. As IRE treatments depend on the electric field (EF) generated within the target tissue, it is difficult for clinicians to predict the amount of tissue that will be treated ahead of time. This work aims to collect and examine the information about tumors and the surrounding healthy tissue that is critical to models that can help visualize the treatment and ensure the tumor is exposed to enough lethal energy. Additionally, a new and improved, high-frequency version of IRE (H-FIRE) is explored in terms of its impact on how tissue behaves during the delivery of these types of pulses. In addition to informing models of these therapies, we also explore strategies that clinicians can employ during treatment in order to know when to stop in order to avoid over-treating the area.

Histotripsy is a noninvasive mechanical ablation method that uses focused ultrasound to disintegrate target tissues into acellular homogenate through the generation of acoustic cavitation and is currently being developed for numerous clinical applications. Histotripsy uses high-pressure (>10 MPa), short-duration (<15 cycles) pulses to cause the rapid expansion and collapse of nuclei at the focus resulting in large applied stress and strain in the adjacent tissue. At a sufficiently high pressure above the target medium's intrinsic cavitation threshold and an adequate number of applied pulses, cavitation "bubble clouds" create precise lesions with high fidelity to the region of the focus. Despite advances in histotripsy, additional research is still needed to better understand the acoustic cavitation nucleation process and its effects on therapies using focused ultrasound. This understanding is critical to better predict and control pulse dose for more rapid and efficient ablation procedures, to reduce off-target cavitation events for safer focused ultrasound therapies, and to localize ablation for high-precision procedures near critical structures or treatments without active imaging guidance. In this dissertation, I investigate the nucleation and dynamics of ultrasonically generated acoustic cavitation for novel applications of focused ultrasound. My Ph.D. thesis focuses on (1) investigating the effect of histotripsy pulsing parameters on bubble cloud cavitation nucleation, bubble dynamics, and ablation efficiency, (2) investigating the effect of nuclei characteristics on the threshold for cavitation nucleation and resulting bubble dynamics for therapeutic applications, and (3) developing methods alter select characteristics and dynamics of acoustic cavitation by adjusting pulsing parameters to optimize ablation efficiency in conventional and nanoparticle-mediated histotripsy. The culmination of this thesis will advance our understanding of the nucleation and behavior of acoustic cavitation from pulsed focused ultrasound and develop innovative systems to improve the efficacy, efficiency, and safety of clinical focused ultrasound therapies. / Doctor of Philosophy / Histotripsy is a noninvasive focused ultrasound method that precisely destroys target tissues such as tumors through the acoustic generation of cavitation and is currently being developed for numerous clinical applications. Histotripsy uses high-pressure, short-duration pulsed soundwaves to cause the bubbles to rapidly expand and collapse within a precise region called the focus. This rapid cavitation results in large mechanical strain in the targeted tissue. With increasingly higher pressure, numerous bubbles form in the shape of cavitation "bubble clouds" that create lesions, closely matching their shape, in the target tissue after a sufficient number of pulses have been applied. Despite advances in histotripsy, additional research is still needed to better understand the initiation of the acoustic cavitation process in histotripsy and its effects on focused ultrasound therapies. This understanding is critical to better predict and control ablation procedures, improve procedure efficiency, reduce off-target cavitation events for safer focused ultrasound therapies, and further increase ablation precision for procedures near critical structures or treatments without active image guidance. In this dissertation, I investigate the initiation, growth, and collapse of ultrasonically generated acoustic cavitation for novel applications of focused ultrasound. My Ph.D. thesis focuses on (1) investigating the effect of histotripsy pulsing parameters on bubble cloud cavitation initiation, bubble growth and collapse, and treatment efficiency, (2) investigating the effect of particle characteristics on the threshold for cavitation initiation and resulting bubble behavior for therapeutic applications, and (3) adjusting pulsing parameters to optimize ablation efficiency in conventional and particle mediated histotripsy. The culmination of this thesis will advance our understanding of the initiation and behavior of acoustic cavitation from pulsed focused ultrasound and develop innovative systems to improve the efficacy, efficiency, and safety of clinically focused ultrasound therapies.

Phenylethanolamine-N-methyltransferase (Pnmt), is the enzyme that catalyzes the conversion of noradrenaline to adrenaline. It has been found in the embryonic heart and in certain adult heart cells, including intrinsic cardiac adrenergic cells, intracardiac neurons, and cardiomyocytes, but their physiological role in the heart is not well understood. To determine the function of Pnmt-expressing cells in the developing heart, a novel genetically-targeted mouse model that causes selective cellular suicide of Pnmt-expressing cells was created by mating Pnmt-Cre Recombinase knock-in mice (PnmtCre/Cre) with ROSA26-eGFP-DTA (R26R+/DTA). The “cellular suicide” allele is the Diptheria Toxin A (DTA) gene fragment. Activation of the DTA suicide allele is dependent upon Cre expression, which is under the control of the endogenous Pnmt gene locus (i.e., expression is restricted to adrenaline-producing “adrenergic” cells). Ongoing studies in Dr. Ebert’s laboratory have shown that Pnmt-Cre/DTA mice have a loss of adrenergic cells in the adrenal gland and begin developing serious cardiac and neurological deficits within one month after birth. The purpose of my project is to examine the potential cardiac consequences of selective adrenergic cell ablation in this model. Aim 1 of this study is to analyze echocardiography data from mice with genetic ablation of adrenergic cells compared to age-matched (littermate) controls over the first 6-months after birth. Preliminary evidence indicates that there is substantial loss of function that progressively worsens with age in the ablation group compared to controls. Aim 2 of this study seeks to uncover evidence of adrenergic cell ablation in the heart using histological and immunofluorescence staining techniques. We predict that these experiments will provide physiological and anatomical evidence showing that Pnmt-expressing cells in the heart make significant contributions to cardiac development and function. This knowledge is expected to increase our basic understanding about the specific roles adrenergic cells play during heart, and could lead to the development of novel treatment strategies for certain types of cardiac defects in the future.

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Histotripsy is an emerging non-invasive, non-thermal, image-guided cancer ablation modality that has recently been approved for its first clinical trial in the United States (NCT04573881). Histotripsy utilizes focused ultrasound to generate acoustic cavitation within a tumor to mechanically fractionate targeted tissues. While pre-clinical work has demonstrated the feasibility of applying histotripsy to solid tumors including primary liver and renal tumors, there is still a need to investigate the potential of histotripsy to treat additional malignancies. In investigating the potential for treating other malignancies there are two avenues that need to be considered: 1) the feasibility for treating tissues with more complex stromal structures and 2) the ability of histotripsy to modulate the tumor microenvironment. To determine the safety and feasibility of additional applications of histotripsy, we conducted dose studies ex vivo on human tumors and human liver to establish dosimetry metrics for applying histotripsy to more fibrotic tumors such as cholangiocarcinoma while sparing nearby critical structures, such as bile ducts and blood vessels. Learning the safety dose-margins from the excised tissues, we performed an in vivo study using mice bearing patient-derived xenograft cholangiocarcinoma tumors. With this model, we were able to demonstrate our ability ablate the stiff cholangiocarcinoma tumors without causing any debilitating off- target damage. To gain a more robust understanding of the effects of histotripsy ablation on potentially difficult to treat tumors, we developed a porcine xenograft tumor model and utilized veterinary cancer patients. These studies have helped established protocols for utilizing histotripsy with ultrasound guidance to treat tumors that are more difficult to treat and can withstand mechanical ablation, including pancreatic adenocarcinoma, osteosarcomas, and soft tissue sarcomas. Pigs share many similarities with human anatomy and physiology, making them an ideal model organism for testing new medical devices and regimes for treating new targets. Using pigs, we were able to establish a procedure to utilize histotripsy to target the pancreas in vivo without causing any lasting or major side effects, such as off-target damage or pancreatitis. One limitation to the porcine model and veterinary patients, is the limitation of gaining rapid insight into the immunological effects of histotripsy. Established cancer mouse models offer the opportunity to rapidly test many organisms with an intact immune system. We used these mice to study pancreatic adenocarcinoma to determine the immune response after histotripsy ablation. For these tumors the general response was an increase in immune cell infiltration post-treatment and a shift in the tumor microenvironment to a more anti-tumor environment. The results of this dissertation provide insight into establishing protocols for treating new types of tumors with histotripsy and immunological effects that lay groundwork for improving future co-therapeutic treatment planning. Future work will aim to translate histotripsy into clinical applications and determining co-therapies that can help control metastasis. / Doctor of Philosophy / Histotripsy is a new medical therapy that can remove tumors without the need for surgery, with the first clinical trial in the United States starting this year, 2021. This therapy uses focused ultrasound waves to generate powerful microscopic bubbles that can rapidly destroy targeted tissues with a high-degree of precision. Early studies on histotripsy have demonstrated the ability of histotripsy to ablate tumors of the liver and kidneys. In order to be able to fully use this therapy on more difficult to target and treat cancers more studies are needed. Given that histotripsy uses physical forces to destroy targets, stronger, more fibrotic tumors and cancers that have begun to spread throughout the body will be more difficult to treat will need more than simple tumor removal to better treat these patients. Therefore, when investigating new cancer applications of histotripsy, it is important to consider the physical features of the tumors as well as the ability of histotripsy to initiate an immune response against the cancer. To determine the safety and feasibility of additional applications of histotripsy, we conducted dose studies on excised human tumors and human liver to see what doses of histotripsy are required to ablate stronger tumors, such as bile duct tumors. Learning the potential safety margins of doses from the excised tissues, we conducted a study using a mouse model to grow stiff, human tumors. With this model, we were able to show that it is possible to ablate the stiffer tumors without causing any major off-target damage. While it is useful to prove in excised tissues and mice that we can treat certain tumors, there is an additional need to study the therapy in a model that is more similar in size and anatomy to humans. Therefore, to gain a better understanding of the effects of histotripsy on potentially difficult to target and ablate tumors, we developed a novel porcine tumor model that can support the growth of human tumors and utilized veterinary cancer patients. These studies have helped established protocols for utilizing histotripsy to treat difficult to physically ablate tumors and difficult to ultrasound target tumors, including pancreatic and bone cancers. Established cancer mouse models offer the opportunity to rapidly test many organisms with an intact immune system. We used these mice to study pancreatic cancer to determine the immune response after histotripsy ablation. For this tumor type, while there were slight differences, the general response was an increase in immune cell infiltration of the tumors post-treatment and a shift to a stronger immune response against the tumor. The results of this dissertation provide insight into establishing protocols for treating new types of tumors with histotripsy and immune effects that lay groundwork for improving future co-therapeutic planning. Future work will aim to translate histotripsy into clinical applications and determining co-therapies that can help control body-wide disease.

Environmental response as determined by the cell-polymer interaction stands as the greatest restriction to the implementation of new polymeric materials. Cell-polymer interactions are most influenced by substrate surface free energy, surface chemistry, topography, and rigidity[1]. Alteration of these properties through surface treatment has become a common approach to attain the desired cellular interaction. This study investigates Kr-F excimer laser(248 nm) surface modification of poly(methyl methacrylate), glycol-modified poly(ethylene terephthalate), and polytetrafluoroethylene and its effect on the adhesion of Escherichia Coli K-12 bacteria. These three polymers were chosen for their very different mechanisms of ablation as well as their range of surface free energies and bacterial responses[2-4]. Polymers were ablated using a pulsed Kr-F excimer laser with a dose of 3.3x 10-9 W/cm2 per pulse. This high level of UV radiation was sufficient to cause significant surface damage on both PMMA and PTFE. PETG showed some signs of wavering in the surface and material removal was confirmed through optical microscopy. Due to the extensive damage associated with ablation, a much lower-powered, continuous beam Kr-F laser was used for contact angle samples. It delivered a dose of 1.27 W/cm2. Contact angle measurements were then taken which showed dose-dependent surface free energy in all three polymers. Following ablation, bacterial adhesion to PETG was improved two-fold, while it decreased in both PTFE and PMMA. Surface chemistry analysis supported the idea that the ablation occurred through chain scission, since there were no new surface groups created. There were significan texture modifications observed in PTFE and PMMA whicle PETG demonstrated the rolling structure characteristic of polyesters following laser ablation described in Wefers et al [4] and Hopp et al [5]. Contact angle measurements showed a correlation between radiation dose and surface free energy of all three polymers. / Master of Science

Osteosarcoma (OS) is the most common primary bone tumor with an annual incidence rate of 3-4 individuals per million particularly affecting children and young adults. The 5-year survival rate stands at 60-80% with the current standard of care for human OS patients who do not have metastatic disease at presentation, but this drops to 20% for patients with metastatic disease which frequently occurs in the lungs. OS is much more common in canines, with metastasis being the major contributor to mortality, the same as in humans. Metastatic OS warrants novel treatment strategies to improve prognosis and survival. High-frequency irreversible electroporation (H-FIRE) is a promising, non-thermal, minimally invasive technique that induces cell death by applying pulsed electric fields in targeted regions, potentially triggering an anti-tumor immune response that could also target and prevent metastases. Such a dual functionality of H-FIRE is uniquely suited to treat pulmonary metastatic OS. The goal of this thesis was to study the ablative and immunomodulatory effects of H-FIRE on OS in-vitro with the overall hypothesis that H-FIRE completely ablates OS cells, induces the release of damage-associated molecular patterns (DAMPs), and promotes pro-inflammatory immune activating signatures in macrophages and T cells. Using an in-vitro model, my master's thesis focused on 1) Determining the electric field strength that completely ablates OS cells 2) Evaluating the immunomodulatory effects of H-FIRE by co-culturing H-FIRE treated OS cells with macrophages and T cells separately. Our study has utilized murine, canine, and human OS and immune cells, thus demonstrating a unique cross-species approach, 3) Evaluating DAMPs (ATP, calreticulin, and HMGB1) post-H-FIRE ablation of human OS cells. Overall, our study showed that H-FIRE successfully ablated OS cells in-vitro, induced the release of DAMPs from treated cells, and promoted activation signatures in immune cells. This thesis provides foundational data for future investigations developing H-FIRE as an immunomodulatory strategy for treating metastatic OS. / Master of Science / Osteosarcoma (OS) is the most common primary bone tumor that majorly affects young adults and children with an incidence rate of 3-4 individuals per million per year. When metastasis occurs (i.e. OS spreads from its site of origin to other organs in the body), most frequently to the lungs, patients experience poor chances of recovery and survival. Currently, the treatment protocol followed for patients with metastatic OS largely includes complete surgical removal and chemotherapy both of which can be very grueling for patients. No significant improvement in the overall 5-year survival rate with current mainstay treatment has led to the urgent need of novel treatment modalities for treating patients with pulmonary metastatic OS. High-Frequency Irreversible Electroporation (H-FIRE) is a novel non-thermal tumor ablation strategy that utilizes electrical pulses to create pores on the cell membrane, thus leading to irreversible damage and cell death. These dying tumor cells release certain molecules and proteins that send danger signals to activate the body's own immune system against the tumor. H-FIRE with its dual function of destroying the targeted tumor region via electroporation and distant metastases via activating immune system is uniquely suited to treat pulmonary metastatic OS. This thesis is the first to investigate H-FIRE ablation and immunomodulation for OS. We hypothesized that H-FIRE can completely destructs OS cells, promotes the release of danger signals, and causes immune activation. Using an in-vitro model, this thesis focused on 1) Determining the electric field strength needed for complete OS cell destruction by H-FIRE 2) Evaluating the immune activation potential of H-FIRE by exposing these H-FIRE treated cells to immune cells like macrophages and T cells separately. We utilized human, mouse, and dog-derived OS cells to increase the biological and clinical relevance of our study. 3) Evaluating certain proteins that act as danger signals post-H-FIRE treatment of human OS cells. Overall, our results indicated that H-FIRE can successfully destruct OS cells in-vitro, promotes the release of danger signals, and induces immune activation. This thesis contributes to providing crucial preliminary data in the development of H-FIRE as a novel ablation and immunomodulation treatment strategy for pulmonary metastatic OS.

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