Master's Theses

Date of Award

2015

Document Type

Thesis

Department

Biomedical Engineering

First Advisor

Sihong Wang

Second Advisor

Mitchell Schaffler

Keywords

Ultrasound, Stem cells, Thermal sensors

Abstract

Osteoarthritis (OA) is a disease that affects millions of people annually, often the elderly, and limits their physical abilities. Current methods of treating OA are often very invasive, and expensive, costing the Medicare system billions of dollars annually. A minimally invasive therapy that can be localized to the affected area that provides both relief and a short recovery time is optimal for treating the disease. For targeted therapy and clinical use, therapeutic ultrasound in the 0.7 MHz to 3 MHz frequency range can provide relief and is convenient, portable and localized. Low-intensity pulsed ultrasound (LIPUS) has been proven in clinical studies to increase the rate of healing of non-union fractures in rat models, primarily through the mechanical component, and referring to LIPUS itself as a way of producing mechanical stress. LIPUS itself is mechanical energy that when transmitted to living tissue, become acoustic intensity waves that can induce thermal, and non-thermal effects that alter cell function when applied. In our prior work, the effects of thermal dosing on hMSC cultures induced to osteogenesis was explored, and it was determined that thermal dosing via heat shocking in an incubator for one hour upregulates alkaline phosphatase (ALP) activity and calcium deposition, indicators of bone formation. In this project, the thermal and mechanical effects of pulsed ultrasound on living tissue are combined to investigate the coupling of the two effects of ultrasound on hMSCs in a preliminary investigation based on our prior work that focused on thermal effects alone. The aim of this thesis is to create and calibrate an ultrasound stimulation device compatible with standard cuboidal ultrasonic transducers for in vitro direct therapeutic ultrasound stimulation. To emulate the thermal environment of the human body, the 2 device, and transducers are placed inside of an incubator at standard culture conditions (37°C, 5% CO2). To observe the thermal effect of therapeutic ultrasound, a high voltage load is placed onto two transducers. The transducers, a focused transducer with spherical focal length of 2 inches, and a plane wave transducer were selected. The focused transducer can target a point at the height of its focal length and deliver energy to the target at maximal intensity, whereas a plane wave transducer emits the wave in a shape similar to cylinder with a diameter equivalent to the lens diameter of the transducer. The resultant power load on the transducer can be increased which in turn deposits energy onto the target that is used to increase the temperature in the well by 2°C from standard culture conditions to 38°C - 39°C based on calibration results for the focused transducer. This temperature was selected based on prior experiments performed determined that heat shocking hMSCs induced to osteogenesis at that temperature upregulated ALP activity normalized for DNA content and calcium deposition. Using an array of high-precision thermistors, the increase in heat can be monitored and quantified to allow proper setting of the device’s transducers as well determine the optimal pattern of well heating. To validate the device’s effectiveness in vitro, the thermal effects of ultrasonic waves in the therapeutic frequency range on hMSCs that have been induced to differentiate down the osteogenesis pathway will be observed. Thermal dosing is provided by increasing the voltage load on the transducers to increase power output according prior calibrations, as the source of direct heat stimulation. It is therefore hypothesized that hMSCs directly exposed to pulsed ultrasound waves at power levels high enough to induce heating will experience an upregulation of ALP activity, higher than that of cells heat shocked with indirect heat. 3 This hypothesis is based on prior in vitro studies that monitored cell proliferation of ultrasound pulsed cultures followed by a proteomic analysis of those cultures, and our prior data where hMSCs were shown to have upregulation of ALP activity when heat shocked. Our data showed that hMSCs undergoing differentiation to osteogenesis displayed upregulation of ALP activity when exposed to targeted ultrasound when compared to osteogenic samples without ultrasound stimulation. hMSCs not exposed to targeted ultrasound, but on the same plate as the ultrasound stimulated samples also saw comparable upregulation of the enzyme, in addition to higher rates of calcium deposition. This demonstrated that despite targeting, propagation of the US wave affected another well horizontally, and upregulated two properties used to determine maturation of osteocytes and should spur further research into the effects of propagating waves in vitro and in vivo.

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