Breast cancer shows a high affinity towards the bone, causing bone-related complications leading to poor clinical prognosis. Approximately 80% of breast cancer patients die within five years after primary cancer has metastasized to the bones. The tumor stage strongly influences the survival rates of patients with breast cancer that has spread to bone at the time of diagnosis. There are currently no effective therapeutics available for bone metastases due to the failure of animal models and the scarcity of human bone metastasized samples, as most patients with advance stages of cancer are already in palliative care. Therefore, it is imperative to develop translational models to elucidate disease mechanisms at the cellular and molecular level. Here, we report the development of tissue-engineered nanoclay-based bone-mimetic three-dimensional (3D) in vitro model for studying later stages of cancer pathogenesis at the metastatic bone site using osteogenically-differentiated human mesenchymal stem cells (MSCs) and human breast cancer cells (MDA-MB-231 and MCF-7). This 3D model provides an ideal microenvironment suitable for cell-cell and cell-matrix interactions while retaining the behavior of breast cancer cells with different metastatic potential along with mimicking mesenchymal to epithelial transition (MET) of breast cancer cells. Sequential cultures of MSCs with MCF-7 gave rise to tumoroids, while sequential cultures of MSCs with MDA-MB-231 formed disorganized clusters of cells with poor cell-cell adhesion. We further evaluated how cancer-derived factors and cytokines affect bone leading to up to metastasis and conferring drug resistance, respectively. Results showed that Wnt/β-catenin and interleukin-6 (IL-6) mediated IL-6/STAT3 pathways are responsible for bone-related complications and conferring drug resistance, respectively. Furthermore, we have utilized the 3D in vitro model to develop methods for non-invasive and rapid prediction of cancer progression using various biophysical techniques such as spectroscopy and nanoindentation. Spectroscopy methods showed significant contributions of proteins, lipids, and nucleic acids, while the nanoindentation method showed F-actin mediated softening of cancer cells during cancer progression at the metastatic bone site, respectively. Collectively, 3D in vitro model provides an ideal platform for studying the molecular mechanism of breast cancer progression at the metastatic bone site, drug development, and discovery of biomarkers for cancer progression.

For healthy individuals, wound healing mainly occurs without medical intervention, yet for the growing elderly, diabetic, or obese populations, as well as for those recovering from surgery, disregulated wound healing poses a serious health risk. Therefore, understanding the cellular processes regulating wound healing and correcting them when they go awry is essential for meeting these population’s healthcare needs. Wound healing is a complex process consisting of a suite of injury repair programs executed by cells in the injured tissue. While several of these programs have been previously described, there are many possible cellular signalling pathways that can mediate a given repair program, and its unclear which pathway mediates a specific process. In this work, we aimed to identify the key cellular signaling pathway that regulates the injury contraction process in a dermal microtissue on a chip model. We found that a balance of tissue forces generated via RhoA activation is critical for injury contraction, and that spatially localized RhoA activation can recruit new cells to participate in injury contraction. During our experiments, we also discovered and characterized a novel actin cytoskeleton-plasma membrane topology present in human dermal fibroblasts at the extreme end of cellular contractility. We also developed several technical advances: the real-time imaging and manipulation of calcium in 3D microtissues, the development of a reporter for smooth muscle actin and a labeled cellular fibronectin fusion protein, and the optimization of Forster Resonance Energy Transfer sensors. Taken together, our experimental results demonstrate the importance of RhoA-mediated force balance during injury contraction, which also has implications for scarring wound pathologies, while the tools we developed provide support for future investigations into the cellular signaling mediating injury repair programs.

The commercial field of radical polymerized polymers, such as polyvinyl alcohol, is very broad partly because they are easy to polymerize and cheap. One aspect that could improve their commercial range is to enhance their degradation rate. As the environmental aspect of polymers grows bigger an enhancement of biological degradation is a great improvement. This thesis deals with the prospect of polymerizing polyvinyl alcohol with degradable linkages in the main chain. In order to achieve the aim the monomer 2-methylene-1,3-dioxepane is successfully synthesized and characterized. The synthesis is followed by copolymerization of 2-methylene-1,3-dioxepane with vinylacetate at the feed compositions; 30/70, 50/50, 70/30 mol% respectively. The copolymerization was successful and reached over 90% conversion at the reaction time 3-4 hours with the conditions 60°C and 5mol% 2,2-Azobis(2-methylpropionitrile) as initiator. The copolymerization is followed by hydrolysis with potassium hydroxide or Candida Rugosa Lipase. The results show that chain scission occurs when the polymer is hydrolyzed by potassium hydroxide but not by lipase. There is also a tendency toward hydrolysis of the chain with lipase.

The use of 3D printing in biological applications is a new field of study given that 3D printing technology has become more available and user friendly. Possible uses include using existing 3D printing polymers to use in extracorporeal or in vitro devices, like Lab-on-a-Chip, and the development of new biologically derived materials to print cell-containing constructs. The latter concept is what is more commonly known as bioprinting. Our research had the goal of developing a bioprinting system including the printer, a bioink, and a feedback system for printing parameter optimization which could be done cheaply and within the reach of nearly any research lab. To make the bioprinter, we were able to take a popular plastic 3D printer and convert it to a bioprinter with 3D printed parts and the addition of a new motherboard. This came with great contribution from Carnegie Melon University. We were also able to improve upon the original design and, along with the new bioprinting capabilities, maintain the original capabilities of the plastic 3D printer. A new bioink was developed to work in coordination with this bioprinting system. Our lab has the luxury of having access to decellularized tissue, which provided a unique material to create a bioink which is derived from the extra-cellular matrix of porcine hearts. The final bioink protocol allows the users to make their own bioink, from easily obtainable tissue and determine their own concentration of the extra-cellular matrix/collagen within a range. Lastly, a feedback system was developed using a Raspberry Pi and camera module to provide real-time visual feedback of the bioprinting process which is otherwise very difficult to see and optimize parameters from. A protocol was developed to sequentially optimize the parameters for an open-source slicing software which governs the resolution of the bioprinter itself. In related research, the cytotoxicity and cell adherence properties of a printing resin for a microfluidic 3D printer were evaluated for use in Lab-on-a-Chip applications. The existing resin was tested and determined to be cytotoxic to cells and therefore not suitable for biological applications. We showed that a simple ethanol washing step and plasma treatment pulled the cytotoxic elements out of the polymer and modified the surface such that cells could attach and proliferate on the printed resin. Another printed resin was also tested which was determined to have no natural cytotoxicity, but the same plasma treatment was needed to allow for cell adherence.

This dissertation presents the fabrication of a silicate-based nanocomposite hydrogel with outstanding shear-thinning properties, viscoelastic behaviour, and water retention capacity. Due to their adaptable mechanical properties, bioavailability, and water retention capacity, these nanocomposite hydrogels have been extensively used for biomedical applications. Laponite nanoparticles are among the most utilized silicate-based minerals. These clay nanoparticles are composed of platelets that are positively charged on the edges and negatively charged on the surface. The high aspect ratio of the polyanionic surface of the Laponite nanoparticles can absorb and trap ionic functional groups with non-covalent interactions. These silicate-based nanocomposite hydrogels are produced by dispersing Laponite nanoparticles in deionized water, forming a homogenous colloid. The uniform dispersion of these nanoparticles in aqueous solutions forms a “house of cards” structure, which eliminates particle aggregation and improves their surface interaction with ionic compounds. The fabrication process is followed by the addition of the stable colloid to various organic and inorganic mixtures including, chitosan, alginate, graphene oxide, and gelatin. The chemical, physical, and mechanical properties of these nanocomposites are experimentally evaluated. Silicate-based nanocomposite hydrogels offer unique rheological characteristics, which facilitate the injection process while preserving the mechanical integrity of the construct following extrusion. The injectability of these nanocomposites was assessed by evaluating their shear-thinning properties through multiple rheological analyses. As per the definition of shear-thinning, the viscosity of nanocomposites is directly affected by the applied shear stress; the viscosity of these compositions decreases under shear stress and reverts to the original viscosity after removal of the force. Accordingly, nanocomposite hydrogels with shear-thinning properties can be utilized for extrusion-based 3D printing and for depositing drugs in localized tissue without the jeopardy of being washed away by circulating blood. In addition, the large number of surface interactions and cationic exchange capacity of Laponite nanoparticles improve electrostatic interactions between the nanocomposite components and a wide range of ionic compounds. Accordingly, these chemical properties facilitate the incorporation of stimuli-responsive materials into the polymeric structure of the nanocomposite, allowing for the utilization of these hydrogels in on-demand drug delivery applications. These properties of the silicate-based nanocomposite hydrogels are investigated through swelling and release studies, Fourier transforms infrared spectroscopy (FTIR), and zeta potential measurements. The results of these experiments indicate that the non-covalent electrostatic interactions and chemical properties of these hydrogels improve the solubility and loading efficiency of therapeutic agents. Silicate-based nanocomposite hydrogels may also be utilized for developing electrical conductive bioinks for extrusion-based three-dimensional (3D) printing. Adjusting the viscosity and shear-thinning properties of the hydrogel plays a significant role in the printability of a bioink. For instance, a highly viscous bioink disrupts extrusion, while a bioink with a low viscosity results in the formation of droplets instead of the desired cylindrical filaments. Optimized formulations of the nanocomposite hydrogels are investigated by conducting various mechanical property measurements. Consequently, the unique chemical and rheological properties of the proposed hydrogels make them superior candidates for drug delivery and tissue engineering applications. / Graduate / 2022-03-30

Cardiovascular disease is currently the leading cause of death in the U.S. that frequently requires bypass surgery using vascular grafts for treatment. Current limitations with fully synthetic grafts have led researchers to bioengineered alternatives that consist of a combination of vascular scaffolds and cells. A major challenge in creating a functional bioengineered vascular graft is development of a confluent endothelium on the lumen that is able to resist detachment under physiologic fluid flow. In addition, methodologies used to assess the growth and maturation of the endothelium in a noninvasive and dynamic manner are severely lacking. Therefore, the overall goal of this research is to advance the field of vascular tissue engineering by 1) creating methodologies to enhance EC adherence to a vascular graft and 2) development of a noninvasive and real-time imaging system capable of assessing the graft endothelium.  To achieve these objectives, three separate studies were performed. In the first study, electrospun scaffold fiber diameter and alignment were systematically varied to determine their effect on endothelial cell (EC) morphology and adherence under fluid flow. ECs on uniaxially aligned nanofibers displayed elongated and aligned morphologies leading to higher adherence to the scaffolds under physiologic levels of fluid flow as compared to those on randomly oriented scaffolds. In the second study, a fiber optic based (FOB) imaging system was developed to image fluorescent ECs through a thick electrospun scaffold.  Results demonstrated that the FOB imaging system was able to accurately visualize fluorescent ECs in a noninvasive manner through the thick and highly opaque scaffold. In the final study, the FOB imaging system was used to noninvasively quantify vascular graft endothelialization, EC detachment, and apoptosis through the vessel wall with greater imaging penetration depth than two-photon microscopy. Additionally, the FOB method was capable of continuously tracking EC migration and endothelialization of a bioengineered graft in a bioreactor. Overall, these results demonstrate that aligned scaffold topographies enhance EC adherence under fluid flow and the FOB imaging system is a promising tool to monitor endothelium development and response to fluid flow in a manner that has not previously been afforded using conventional imaging methods. / Ph. D.

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The drug development process, especially for anti-cancer therapies, continues to be highly inefficient. The current preclinical drug evaluation paradigm of human monolayer in vitro culture followed by small animal in vivo models results in a roughly 90% failure rate in clinical trials involving actual cancer patients. Our hypothesis then, is that there is a clear need for engineered, 3D, healthy and tumor tissue models capable of recapitulating patient physiology and disease, allowing for more accurate preclinical evaluation of anti-cancer drugs. Our hope, is that tissue engineering can provide us with valuable new insights into drug responses, over what we can currently achieve with existing models. Here, we present a new multi-tissue organs-on-a-chip microfluidic platform, Inter-Organ, designed to allow more comprehensive recapitulation of the disease seen in patients. We developed bioengineered tissues of primary and metastatic tumors across three cancer types, and integrated them into the Inter-Organ platform alongside healthy tissues like cardiac muscle known to cause failures in clinical trials for off-target drug toxicities. Overall, the development of these new cancer models and their culture in the Inter-Organ platform allowed us to more accurately predict the success of various drugs in clinical trials than existing models could. Finally, this tissue engineering approach allowed us to explore the relationships between specific constituents of the tumor microenvironment, recapitulate complex cancer processes like metastasis previously only done in small animal models, and identify new potential diagnostic and therapeutic targets.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18643号 / 医博第3942号 / 新制||医||1006(附属図書館) / 31557 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 三森 経世, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM