Cartilage tissue engineering represents an exciting potential therapy for providing permanent and functional regeneration of healthy cartilage tissues, but these treatment options have yet to be successfully implemented in a clinical setting. One of the primary obstacles for cartilage engineering is obtaining a sufficient supply of cells capable of regenerating a functional cartilage matrix. Mesenchymal progenitors can easily be isolated from multiple tissues, expanded in vitro, and possess a chondrogenic potential, but it remains unclear what types or combinations of signals are required for lineage-specific differentiation and tissue maturation. The overall goal of this dissertation was to investigate how the coordination of biochemical stimuli with cues from mechanical forces and the extracellular matrix regulate the chondrogenesis of bone marrow stromal cells (BMSCs). These studies explored the potential for cyclic tensile loading and chondrogenic factors, TGF-1 and dexamethsone, to promote fibrochondrocyte-specific differentiation of BMSCs. The application of cyclic tensile displacements to cell-seeded fibrin constructs promoted fibrochondrocyte patterns of gene expression and the development of a fibrocartilage-like matrix. These responses were influenced by the specific loading conditions examined and the differentiation state of the BMSCs. Additionally, the roles of integrin adhesion and cytoskeletal organization in BMSC differentiation were examined within engineered hydrogels presenting controlled densities of biomimetic ligands. Adhesion to the arginine-glycine-aspartic acid (RGD) motif inhibited chondrogenesis in a density-dependent manner and was influenced by interactions with the f-actin cytoskeleton. Together, this research provided fundamental insights into the regulatory mechanisms involved in the chondrogenesis of mesenchymal progenitor cells.

Vocal fold scarring, caused by injury and inflammation, presents significant treatment challenges. Tissue engineering might be a promising treatment for vocal fold restoration or regeneration. It is important to investigate how scaffold properties alter cell behavior instead of screening thousand of materials, which is fundamental knowledge for rational scaffold design. This work studies how tuning only one parameter, mechanical strength of the hydrogel scaffold, influences the extracellular matrix production of encapsulated porcine vocal fold fibroblast (PVFF). PVFF cells were encapsulated by photopolymerization in 10 wt%, 20 wt%, and 30 wt% poly(ethylene glycol) diacrylate (PEGDA) hydrogels (MW 10,000), with the similar biochemical environment and network structure but different mechanical properties. Cell adhesive peptide, RGDS, was grafted into each hydrogel network to mimic a cell adhesive environment. The glycosaminoglycans (GAGs) production per cell increased from 10 wt% to 20 wt%, 30 wt% gels, with an increase in hydrogel stiffness. The collagen production per cell increased from 10 wt% to 20 wt% gels but no further increase occurred with the increasing modulus from 20 wt% to 30 wt% gels. Interestingly, in hydrogels of intermediate modulus (20% PEGDA hydrogels), the highest elastin per cell was observed compared with gels with higher and lower storage modulus after day 30. Histological analysis showed GAGs, collagen and elastin were distributed pericellularly. However, the organization of collagen type I appeared to be influenced by gel mechanical properties, which was confirmed by immunohistological analysis. Furthermore, the immunohistological analysis showed that the phenotype of PVFF is regulated by the stiffness of the PEG hydrogel. This study demonstrates that different levels of VFF ECM formation may be achieved by varying the mechanical properties of PEG hydrogels and validates a systematic and controlled platform for further research of cell-biomaterials interaction.

In the US alone, more than 500,000 coronary artery bypass procedures are performed annually. Tissue engineering shows the potential to construct functional grafts to overcome the limited availability of autologous saphenous veins, relatively poor elasticity and low compliance of synthetic materials (mainly Dacron and polytetrafluoroethylene). In order to meet the low modulus associate with myocyte differentiation, the high suture retention and an ultimate tensile strength (UTS) sufficient to withstand implantation and peak physiological stresses, we designed and characterized a multi-component scaffold comprised of polyurethane electrospun mesh layers bonded together by a fibrin hydrogel matrix. We have demonstrated this composite construct retains the high tensile strength and suture retention strength but displays a "J-shaped" mechanical response similar to that of native coronary artery. To improve our design, poly(ethylene glycol) diacrylate based hydrogel system was utilized as a blank slate to study the phenotypic regulation by cell material interactions. Fibrinogen, fibronectin, laminin and collagen type IV were incorporated into the hydrogel to mimic the stimuli from extracellular matrix (ECM) proteins. Surprisingly, no significant effect was detected on induction of smooth muscle cell (SMC) differentiation marker expression, activation of mitogen-activated protein (MAP) kinases pathway, or alteration of surface integrin expression profile. However, fibronectin showed repression of undesired phenotypes in SMC differentiation. In contrast to ECM proteins, glycosaminoglycans (GAGs) showed more influence on regulating SMC phenotype. By using a scaffold environment intended to be mimetic of early atherosclerosis, the impact of GAG identity on SMC foam cell formation was explored. We focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HA_IMW, ~400 kDa), the levels and/or distribution of which are significantly altered in atherosclerosis. CSC and DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HA_IMW gels. However, only SMCs in DS constructs maintained increased expression of adipocyte marker A-FABP relative to HA_IMW gels over 35 days of culture. Combined, our results suggested interesting roles for fibronectin and HA_IMW in repression of undesired phenotypes in SMC differentiation, which could give insights into rational design of novel biomaterials for vascular tissue engineering applications.

This work develops microfluidic technologies to advance the state-of-the-art in living cell-based assays. Current cell-based assay platforms are limited in their capabilities, particularly with respect to spatial and temporal control of external signaling factors, sample usage, and throughput. The emergence of highly quantitative, data-driven systems approaches to studying biology have added further challenges to develop assay technologies with greater throughput, content, and physiological relevance. The primary objectives of this research are to (i) develop a method to reliably fabricate 3-D flow networks and (ii) apply 3-D flow networks to the development and testing of microfluidic chamber arrays to query cellular response to soluble-matrix signal combinations and gradient signaling fields. An equally important objective is for the chamber arrays to be scaled efficiently for higher-throughput applications, which is another reason for 3-D flow networks. Two prototype chamber arrays are designed, modeled, fabricated, and characterized. Furthermore, tests are performed wherein cells are introduced into the chambers and microenvironments are presented to elicit complex responses. Specifically, soluble-matrix signaling combinations and soluble signal gradients are presented. The study of complex biological processes necessitates improved assay techniques to control the microenvironment and increase throughput. Quantitative morphological, migrational, and fluorescence readouts, along with qualitative observations, suggest that the chamber arrays elicit responses; however further experiments are required to confirm specific phenotypes. The experiments provide initial proof-of-concept that the developed arrays can one day serve as effective and versatile screening platforms. Understanding the integration of extracellular signals on complex cellular behaviors has significance in the study of embryonic development, tissue repair and regeneration, and pathological conditions such as cancer. The microfluidic chamber arrays developed in this work could form the basis for enhanced assay platforms to perform massively parallel interrogation of complex signaling events upon living cells. This could lead to the rapid identification of synergistic and antagonistic signaling mechanisms that regulate complex behaviors. In addition, the same technology could be used to rapidly screen potential therapeutic compounds and identify suitable candidates to regulate pathological processes, such as cancer and fibrosis.

Cell adhesion to extracellular matrices (ECM) is essential to numerous physiological and pathological processes. Cell adhesion is initiated by binding of the transmembrane integrin family of receptors to an ECM ligand such as fibronectin (FN). Once bound, integrins cluster together and form focal adhesions (FA). FAs serve as structural links and signal transduction elements between the cell and its extracellular environment. While a great deal of progress has been made in identifying the biochemical components that comprise focal adhesions and the roles they play in migration, cell spreading, and signaling, the contributions of these proteins to mechanical interactions between the cell and its environment remain poorly understood. A FA adhesion protein of particular importance is vinculin. When localized to focal adhesions, vinculin forms a ternary complex with talin and 1-integrin. This 1-integrin-talin-vinculin complex plays a central role in the regulation of FA assembly and cell spreading and migration. Nevertheless, the specific contribution to adhesive force generation of the 1-integrin-talin-vinculin complex remains poorly understood. The objective of this project was to analyze the role of vinculin in the cell adhesion strengthening process. Our central hypothesis is that vinculin modulates adhesion strength via regulating the size and/or composition of the integrin-talin-vinculin complex. We used a novel combination of biochemical reagents and engineering techniques along with quantitative and sensitive adhesion strength measurements to provide new insights into how the structure of vinculin contributes to cell adhesion strength.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2001. / Bibliography: leaves 184-201.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 86-103).

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 87-95). Also available in print.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 71-79).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.