Preclinical screening during the development of new drugs is poorly predictive and costly, creating a significant interest from pharmaceutical companies, government agencies, and the public in the development of better preclinical tests. To create more predictive organ models, human derived stem cells can be coupled with biomimetic tissue engineering approaches to create physiologically relevant functional subunits of each tissue/organ within the body. However, existing methods of generating cardiomyocytes (CMs) and cardiac tissues from human induced pluripotent stem cells (hiPSC) derived CMs (hiPS-CMs) are relatively immature and produce tissues that resemble that of a fetal heart at best. This limits their use in therapeutic development and thus, methods to overcome their immature phenotype are of high importance. In pursuit of this goal, this dissertation focuses on the role of biophysical stimuli in driving the functional maturation of hiPSC-CMs to engineer cardiac muscle of high biological fidelity. In an effort to recapitulate the hierarchical structure and functionality of native heart tissue, methods to pattern cells at the nano- and microscale levels were developed and optimized towards the functional assembly of cardiac tissues at the macroscale. To address the challenges currently associated with hiPS-CM immaturity, the decoupled effects of electrical and electromechanical stimulation in driving cardiac maturation were investigated. Subsequently, optimal electromechanical stimulation regimens were established. Daily intervals of high intensity electromechanical training were shown to upregulate cardiac functionality and energetics, and thus, enhance maturation. Combining these methods enabled the development of a custom bioreactor capable of generating larger, more functionally mature hiPS-CM tissues. Mimicking the developmental increases in cardiac beating frequency, exposure of the resulting tissues to a dynamic electromechanical intensity training regimen matured hiPS-CMs beyond levels currently demonstrated within the field. Specifically, the engineered tissues recapitulated many of the molecular, structural, and functional properties of adult human heart muscle, including well developed registers of sarcomeres, networks of T-tubules, calcium homeostasis, and a positive force-frequency relationship. The enhanced functionality of the resulting bio-engineered adult-like myocardium enabled its utility in predicting drug cardiotoxicity and modeling human cardiac disease.

Cartilage is a tissue that is critical for skeletal function, yet its study has been limited by a lack of quantitative, non-destructive, three-dimensional imaging techniques that enable simultaneous interrogation of both bone and cartilage. Recently, methods of contrast-enhanced micro-computed tomography (CECT) have been developed that exploit the electrostatic interactions between ionic contrast agents and negatively charged glycosaminoglycans (GAGs) in cartilage, thus providing information about the composition and morphology of cartilage that was previously only available via destructive methods. The goal of this dissertation project was to apply CECT, a non-destructive, three-dimensional imaging method, to understand the how the morphology and composition of cartilage changes in response to injury and disease. First, CECT was applied to a model of growth plate injury to quantify changes to the cartilaginous tissue of the growth plate and formation of bone bridges within this tissue in response to injury. Using CECT, it was possible to identify increased thickness and CECT attenuation at the injury site. This result, paired with histological evidence of localized dysregulation of cellular activity, suggests that treatment designed to reduce bone bridge formation at the injury site should also consider the effects of the treatment on the adjacent cartilage. Second, CECT was applied to a collagen antibody-induced arthritis (CAIA) model to determine the role of the A2B adenosine receptor (A2BAR) in the arthritic deterioration of bone and cartilage. CECT scans demonstrated that loss of GAG in the cartilage preceded degeneration, but that ablation of the A2BAR in mice had little effect on the degenerative changes in bone and cartilage associated with CAIA. These results suggest that the A2BAR does not independently mediate these changes and that it may be necessary to target multiple adenosine receptors. Third, the ability of CECT to monitor the fracture healing and predict the stiffness of the cartilaginous fracture callus was assessed both at the level of the whole callus and at the level of the cartilage tissue. Callus stiffness was negatively correlated with the size of the callus and the amount of cartilage, while neither stiffness nor indentation modulus were correlated with CECT attenuation, suggesting that the stiffness of the cartilaginous fracture callus depends on the amount, rather than GAG content of cartilage. The work presented in this dissertation provides outlines changes in both bone and cartilage that occur in pathological conditions and provides new insights for both the treatment and assessment of these conditions.

Nanoparticle (NP) drug delivery is a major focus in the research community because of its potential to use existing drugs in safer and more effective ways. Chemotherapy encapsulation in NPs shields the drug from the rest of the body while it is within the NP, with less systemic exposure leading to fewer off-target effects of the drug. However, passive loading of drugs into NPs is a suboptimal method, often leading to burst release upon administration. This work explores the impact of incorporating the drug-polymer conjugate doxorubicin-poly (lactic-co-glycolic) acid (Dox-PLGA) into a lipid-polymer hybrid nanoparticle (LPN). The primary difference in using a drug-polymer conjugate for NP drug delivery is the drug’s release kinetics. Dox-PLGA LPNs showed a more sustained and prolonged release profile over 28 days compared to LPNs with passively loaded, unconjugated doxorubicin. This sustained release translates to cytotoxicity; when systemic circulation was simulated using dialysis, Dox-PLGA LPNs retained their cytotoxicity at a higher level than the passively loaded LPNs. The in vivo implication of preserving cytotoxic potency through a slower release profile is that the majority of Dox delivered via Dox-PLGA LPNs will be kept within the LPN until it reaches the tumor. This will result in fewer systemic side effects and more effective treatments given the higher drug concentration at the tumor site. An intriguing clinical application of this drug delivery approach lies in using Dox- PLGA LPNs to cross the blood-brain barrier (BBB). The incorporation of Dox-PLGA is hypothesized to have a protective effect on the BBB as its slow release profile will prevent drug from harming the BBB. Using induced pluripotent stem cells differentiated to human brain microvascular endothelial cells that comprise the BBB, the Dox-PLGA LPNs were shown to be less destructive to the BBB than their passively loaded counterparts. Dox-PLGA LPNs showed superior cytotoxicity against plated tumor cells than the passively loaded Dox LPNs after passing through an in vitro transwell BBB model. Dox-PLGA LPNs and drug-polymer conjugates are exciting alternatives to passively loaded NPs and show strong clinical promise of a treatment that is more potent with fewer side effects and less frequent administration.

Computational methods for predicting macromolecular complexes are useful tools for studying biological systems. They are used in areas such as drug design and for studying protein-protein interactions. While considerable progress has been made in this field over the decades, enhancing the speed and accuracy of these computational methods remains an important challenge. This work describes two different enhancements to the accuracy of ClusPro, a method for performing protein-protein docking, as well as an enhancement to the efficiency of global rigid body docking. SAXS is a high throughput technique collected for molecules in solution, and the data provides information about the shape and size of molecules. ClusPro was enhanced with the ability to SAXS data collected for protein complexes to guide docking by selecting conformations by how well they match the experimental data, which improved docking accuracy when such data is available. Various other experimental techniques, such as NMR, FRET, or chemical cross linking can provide information about protein-protein interfaces, and such information can be used to generate distance-based restraints between pairs of residues across the interface. A second enhancement to ClusPro enables the use of such distance restraints to improve docking accuracy. Finally, an enhancement to the efficiency of FFT based global docking programs was developed. This enhancement allows for the efficient search of multiple sidechain conformations, and this improved program was applied to the flexible computational solvent mapping program FTFlex. / 2018-07-09T00:00:00Z

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