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No description available.

The use of cell transplantation to treat enzyme deficiency disorders is limited by the immune response targeted against foreign tissue or the use of life-long immunosuppressants. Hiding cells from the immune system in an encapsulation device is promising. Cells encapsulated within an anionic calcium alginate hydrogel bead are protected through a semi-permeable membrane formed by polycation, poly-L-lysine (PLL). A final layer of alginate is added to hide the cationic PLL surface but this has proved to be difficult creating capsules which are prone to fibrotic overgrowth, blocking exchange of nutrients, waste and therapeutic enzymes through the capsule. For long term applications these capsules need to be both biocompatible and mechanically robust. This thesis aims to address the biocompatibility issue of high cationic surface charge by synthesizing polycations of reduced charge using N-(3- aminopropyl)methacrylamide hydrochloride (APM) and N-(2- hydroxypropyl)methacrylamide (HPM) and study the associated mechanical properties of the capsules using micropipette aspiration. Micropipette aspiration was applied and validated for alginate based capsules (gel and liquid core) to quantify stiffness. Varying ratios of APM were used to control the overall charge of the polycations formed while HPM was incorporated as a neutral, hydrophilic, nonfouling comonomer. The molecular weight (MW) was controlled by using reversible addition-fragmentation chain transfer (RAFT) polymerization. The biocompatibility of these polymers was tested by cell adhesion and proliferation of 3T3 fibroblasts onto APM/HPM copolymer functionalized surfaces and by solution toxicity against C2C12 myoblasts. The ability for the APM/HPM copolymers to bind to alginate and form capsules was also assessed, along with the integrity and stiffness of the capsule membrane with or without additional covalent cross-linking by reactive polyanion, poly(methacrylic acid-co-2-vinyl-4,4- dimethylazlactone) (PMV60). Thermo-responsive block copolymers of N-isopropylacrylamide (NIPAM) and 2- hydroxyethylacrylamide (HEA) were also synthesized as potential drug delivery nanoparticles, showing control over micelle morphology with varying NIPAM to HEA ratios. / Thesis / Doctor of Science (PhD) / The treatment of enzyme deficiency disorders by cell transplantation is limited by the immune attack of foreign tissue in absence of immunosuppressants. Cells protected in an encapsulation device has shown promise. Poly-L-lysine, a widely used membrane material in these protective capsules, binds to the anionic gel entrapping living cells because it is highly cationic. The high cationic charge is difficult to hide causing the immune system to build tissue around the capsule, preventing the encapsulated cells from exchanging nutrients and therapeutic enzymes. This thesis aims to replace poly-L-lysine by synthesizing a series of more biocompatible materials of decreasing cationic charge. These materials were studied for the ability to support tissue growth and form stable capsules. The membrane strength was measured using an aspiration method validated for these types of capsules. Reducing the cationic charge of the materials increased the biocompatibility of the capsule membrane but also made for weaker membranes.

Calcific aortic valve disease (CAVD) is one of the most common causes of cardiovascular disease in North America. Mechanical factors have been closely linked to the pathogenesis of CAVD and may contribute to the disease by actively regulating the mechanobiology of valve interstitial cells (VICs). Mechanical forces affect VIC function through interactions between the VIC and the extracellular matrix (ECM). Studies have shown that the transfer of mechanical stimulus during cell-ECM interaction depends on the local material properties at hierarchical length scales encompassing tissue, cell and cytoskeleton. In this thesis, biomechanical tools were developed and applied to investigate hierarchical cell-ECM interactions, using VICs and valve tissue as a model system. Four topics of critical importance to understanding VIC-ECM interactions were studied: focal biomechanical material properties of aortic valve tissue; viscoelastic properties of VICs; transduction of mechanical deformation from the ECM to the cytoskeletal network; and the impact of altered cell-ECM interactions on VIC survival. To measure focal valve tissue properties, a micropipette aspiration (MA) method was implemented and validated. It was found that nonlinear elastic properties of the top layer of a multilayered biomaterial can be estimated by MA by using a pipette with a diameter smaller than the top layer thickness. Using this approach, it was shown that the effective stiffness of the fibrosa layer is greater than that of the ventricularis layer in intact aortic valve leaflets (p<0.01). To characterize the viscoelastic properties of VICs, an inverse FE method of single cell MA was developed and compared with the analytical half-space model. It was found that inherent differences in the half-space and FE models of single cell MA yield different cell viscoelastic material parameters. However, under particular experimental conditions, the parameters estimated by the half-space model are statistically indistinguishable from those predicted by the FE model. To study strain transduction from the ECM to cytoskeleton, an improved texture correlation algorithm and a uniaxial tension release device were developed. It was found that substrate strain fully transfers to the cytoskeletal network via focal adhesions in live VICs under large strain tension release. To study the effects of cell-ECM interactions on VIC survival, two mechanical stimulus systems that can simulate the separate effects of cell contraction and cell monolayer detachment were developed. It was found that cell sheet detachment and disrupted cell-ECM signaling is likely responsible for the apoptosis of VICs grown in culture on thin collagen matrices, leading to calcification. The studies presented in this thesis refine existing biomechanical tools and provide new experimental and analytical tools with which to study cell-ECM interactions. Their application resulted in an improved understanding of hierarchical valve biomechanics, mechanotransduction, and mechanobiology.

Calcific aortic valve disease (CAVD) is one of the most common causes of cardiovascular disease in North America. Mechanical factors have been closely linked to the pathogenesis of CAVD and may contribute to the disease by actively regulating the mechanobiology of valve interstitial cells (VICs). Mechanical forces affect VIC function through interactions between the VIC and the extracellular matrix (ECM). Studies have shown that the transfer of mechanical stimulus during cell-ECM interaction depends on the local material properties at hierarchical length scales encompassing tissue, cell and cytoskeleton. In this thesis, biomechanical tools were developed and applied to investigate hierarchical cell-ECM interactions, using VICs and valve tissue as a model system. Four topics of critical importance to understanding VIC-ECM interactions were studied: focal biomechanical material properties of aortic valve tissue; viscoelastic properties of VICs; transduction of mechanical deformation from the ECM to the cytoskeletal network; and the impact of altered cell-ECM interactions on VIC survival. To measure focal valve tissue properties, a micropipette aspiration (MA) method was implemented and validated. It was found that nonlinear elastic properties of the top layer of a multilayered biomaterial can be estimated by MA by using a pipette with a diameter smaller than the top layer thickness. Using this approach, it was shown that the effective stiffness of the fibrosa layer is greater than that of the ventricularis layer in intact aortic valve leaflets (p<0.01). To characterize the viscoelastic properties of VICs, an inverse FE method of single cell MA was developed and compared with the analytical half-space model. It was found that inherent differences in the half-space and FE models of single cell MA yield different cell viscoelastic material parameters. However, under particular experimental conditions, the parameters estimated by the half-space model are statistically indistinguishable from those predicted by the FE model. To study strain transduction from the ECM to cytoskeleton, an improved texture correlation algorithm and a uniaxial tension release device were developed. It was found that substrate strain fully transfers to the cytoskeletal network via focal adhesions in live VICs under large strain tension release. To study the effects of cell-ECM interactions on VIC survival, two mechanical stimulus systems that can simulate the separate effects of cell contraction and cell monolayer detachment were developed. It was found that cell sheet detachment and disrupted cell-ECM signaling is likely responsible for the apoptosis of VICs grown in culture on thin collagen matrices, leading to calcification. The studies presented in this thesis refine existing biomechanical tools and provide new experimental and analytical tools with which to study cell-ECM interactions. Their application resulted in an improved understanding of hierarchical valve biomechanics, mechanotransduction, and mechanobiology.