Engineering Microbubbles, Phase Shift Droplets and 3D Printed Scaffolds for Biomedical Acoustic Studies Open Access
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Ultrasound has a wide array of applications in diagnostics, therapeutics and characterization. In the first section of this work, the goal is to broaden the applications of ultrasound to extravascular interrogations such as tumor imaging and drug delivery. As an ideal imaging modality, contrast of ultrasound imaging is quite poor. To enhance the image quality, ultrasound contrast agents- micorbubbles (MB) - have been introduced. Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing microbubbles as contrast agents in situ as well as higher stability and the possibility of achieving smaller sizes. Introduction of phase shift nanodroplets as novel ultrasound contrast agents has opened new realms of applications in biomedical acoustics. Here, we acoustically measured the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with various liquid cores including perfluoropentane (PFP), perfluorohexane (PFH) and perfluorooctyle Bromide (PFOB). To optimize the ADV threshold, we varied acoustic parameters (excitation frequency and pulse length), physical parameters (droplet size) as well as host fluid properties (temperature and dissolved gas concentration). We found that ADV threshold increases as the boiling point of the liquid core increases. ADV threshold decreased with increasing temperature and increasing droplet size. Ultrasound pulse duration and dissolved gas concentration did not affect the ADV threshold value. The effect of frequency of excitation on the ADV threshold depended on the size and the liquid core which will be discussed in detail. In addition, the scattered response from vaporized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble (MB) suspension in magnitude as well as trends above the determined ADV threshold value. In the second chapter the acoustic and mechanical properties of 3D-printed porous poly-(ethylene glycol)-diacrylate (PEGDA) hydrogel scaffolds were investigated using an ultrasound pulse echo technique varying scaffold microstructures (solid, hexagonal and square pores). PEGDA is one of the most commonly used biomaterials and knowing its properties is important. Acoustic parameters such as speed of sound, acoustic impedance and attenuation coefficient as well as physical parameters such as pore structure, effective density and elastic moduli were determined. The results show that microstructure (porosity and pore geometry) plays a crucial role in defining properties of 3D-printed scaffolds, achieving the highest attenuation for the scaffold with hexagonal pores and showing a decrease in sound speed and elastic moduli with increasing porosity. The properties were also found to be similar to those of soft tissues, making PEGDA scaffolds a suitable candidate for tissue engineering applications. To evaluate their cellular performance, adhesion and proliferation of human mesenchymal stem cells (hMSCs) in these scaffolds were investigated. The porous scaffolds performed better than the solid one, recording the highest cell attachment and growth for the scaffold with the square pores. In the following chapter, we combine the use of these 3D-printed scaffolds made of PEGDA with low intensity pulsed ultrasound as the mechanical stimulation tool to enhance human mesenchymal stem cells (hMSCs) growth and differentiation. We also investigate the effect of pore geometry on cell growth.In the last chapter, we apply lipid-coated microbubbles along with low intensity pulsed ultrasound (LIPUS) for the first time to enhance proliferation and chondrogenic differentiation of hMSCs in a 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) hydrogel scaffold. The hMSC proliferation increased up to 40% after 5 days of culture in the presence of 0.5 % (v/v) microbubbles and LIPUS in contrast to 18% with LIPUS alone. We systematically varied the acoustic excitation parameters⎯excitation intensity, frequency and duty cycle⎯to find 30mW/cm2, 1.5 MHz and 20% duty cycle to be optimal for hMSC proliferation. A 3-week chondrogenic differentiation results demonstrated that combining LIPUS with microbubbles enhanced glycosaminoglycan (GAG) production by 17% (5% with LIPUS alone), and type II collagen production by 78% (44% by LIPUS alone). Therefore, integrating LIPUS and microbubbles appears to be a promising strategy for enhanced hMSC growth and chondrogenic differentiation, which are critical components for cartilage regeneration. The results offer possibilities of novel applications of microbubbles, already clinically approved for contrast enhanced ultrasound imaging, in tissue engineering.