The fundamental principle of tissue engineering is to use appropriate cell source, combined with scaffolds and bioactive factors to develop tissue constructs which restore, maintain or improve tissue function. There is increasing data emphasizing the importance of mechanical signals and extracellular matrix (ECM) proteins presented by the scaffold in determining stem cell fate/functions which are critical to tissue construct maturation and success of stem cell-based therapies. Cell-matrix adhesions are one of the major mechanosensing machineries cells use to convert information provided by ECM ligands and mechanical signals presented by scaffolds into intracellular biochemical signaling cascades which lead to particular functional responses. Therefore, understanding how ECM ligands and mechanical signals regulate cell-matrix adhesion formation and activation of associated intracellular signaling proteins is fundamental to rational design of biomaterial and loading protocol for optimal cell functional responses in tissue constructs. In this study, we attempted to understand the regulatory effects of external mechanical signal and exogenous ECM protein on cell-matrix adhesion formation and associated intracellular signaling proteins of human mesenhymal stem cells, and in particular, to test the hypothesis that mechanical stimulation or exogenous ECM protein can lead to adhesion maturation into 3D-matrix adhesions in 3D collagen environment. We used microencapsulation technique to embed cells in 3D collagen environment, forming disc-shaped hMSC-collagen constructs. By immunofluorescent staining and confocal microscopy, we visualized changes in size, morphologies and molecular composition of the adhesions. First of all, 2D adhesions of hMSCs were characterized. We showed that hMSCs form well-organized αv integrin-based focal adhesions and fibrillar adhesions in 2D culture. To investigate the regulatory effects of mechanical signals on adhesion signaling and maturation, we used micromanipulator-based loading device to impose dynamic compression to hMSC-collagen constructs. We found that dynamic compression lead to enlargement of integrin αv adhesions which recruit focal adhesion kinase (FAK), vinculin and extracellular signal-regulated kinase (ERK). In addition, FAK was activated at enlarged integrin αv adhesions and translocated to peri-nuclear region after compression, suggesting that loading induces activation of FAK signaling pathways through increased integrin αv clustering. Moreover, we demonstrated that dynamic compression can induce 3D-matrix adhesion formation, indicating the role of external force in integrin α5-based adhesion maturation in 3D collagen environment. We explored the effect of exogenous ECM proteins on adhesion maturation of hMSCs by adding fibronectin into cell-collagen mixture during fabrication of collagen constructs. Our results demonstrated that the exogenous fibronectin can induce α5 integrin-based adhesion maturation into 3D-matrix adhesions in our collagen constructs in a dose-dependent manner. This study demonstrated that the effect of external mechanical signals and exogenous ECM ligands on adhesion signaling and maturation of hMSCs in 3D collagen environment. Our findings contribute towards mechanobiology of hMSCs in 3D context. In particular, our results showed that exogenous proteins or external loading can lead to 3D-matrix adhesion formation, which may serve as a potential way to enhance biological functions of hMSCs in collagen constructs, facilitating stem cell-based therapies. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Physical factors in the environment of a cell affect its function and behavior in a variety of ways. There is increasing evidence that, among these factors, the geometric arrangement of receptor ligands plays an important role in setting the conditions for critical cellular processes. The goal of this thesis is to develop new techniques for probing the role of extracellular ligand geometry, with a focus on T cell activation. In this work, top-down molecular-scale nanofabrication and bottom-up selective self-assembly were combined in order to present functional nanomaterials (primarily biomolecules) on a surface with precise spatial control and single-molecule resolution. Such biomolecule nanoarrays are becoming an increasingly important tool in surface-based in vitro assays for biosensing, molecular and cellular studies. The nanoarrays consist of metallic nanodots patterned on glass coverslips using electron beam and nanoimprint lithography, combined with self-aligned pattern transfer. The nanodots were then used as anchors for the immobilization of biological ligands, and backfilled with a protein-repellent passivation layer of polyethylene glycol. The passivation efficiency was improved to minimize nonspecific adsorption. In order to ensure true single-molecule control, we developed an on-chip protocol to measure the molecular occupancy of nanodot arrays based on fluorescence photobleaching, while accounting for quenching effects by plasmonic absorption. We found that the molecular occupancy can be interpreted as a packing problem, with the solution depending on the nanodot size and the concentration of self-assembly reagents, where the latter can be easily adjusted to control the molecular occupancy according to the dot size. The optimized nanoarrays were used as biomimetic architectures for the study of T cell activation with single-molecule control. T cell activation involves an elaborate arrangement of signaling, adhesion, and costimulatory molecules organized into a stereotypic geometric structure, known as the immunological synapse, between T cell and antigen-presenting cell. Novel bifunctionalization schemes were developed to better mimic the antigen-presenting surfaces. Nanoarrays were functionalized by single molecules of UCHT1 Fab', and served as individual T cell receptor binding sites. The adhesion molecule ICAM-1 was bound to either static PEG background, or a mobile supported lipid bilayer. The minimum geometric requirements (receptor clustering, spacing and stoichiometry) for T cell activation was probed by systematic variation of the nanoarray spacing and cluster size. Out-of-plane spatial control of the two key molecules by way of nanopillar arrays was used to adjust the membrane bending and steric effects, which were essential for the investigation of molecular segregation in T cell activation. The results provide insights into the complicated T cell activation mechanism, with translational implications toward adoptive immunotherapies for cancer and other diseases. This single-molecule platform serves as a novel and powerful tool for molecular and cellular biology, e.g., receptor-mediated signaling/adhesion, especially when multiple ligands or membrane deformation are involved.

This thesis describes a comprehensive study of understanding the mechanism of rigidity sensing by quantitative analysis using submicron pillar array substrates. From mechanobiology perspective, we explore and study molecular pathways involved in rigidity and force sensing at cell-matrix adhesions with regard to cancer, regeneration, and development by quantification methods. In Chapter 2 and 3, we developed fabrication and imaging techniques to enhance the performance of a submicron pillar device in terms of spatial and temporal measurement ability, and we discovered a correlation of rigidity sensing forces and corresponding proteins involved in the early rigidity sensing events. In Chapter 2, we introduced optical effect arising from submicron structure imaging, and we described a technique to identify the correct focal plane of pillar tip by fabricating a substrate with designed-offset pillars. From calibration result, we identified the correct focal plane that was previously overlooked, and verified our findings by other imaging techniques. In Chapter 3, we described several techniques to selectively functionalize elastomeric pillars top and compared these techniques in terms of purposes and fabrication complexity. Techniques introduced in this chapter included direct labeling, such as stamping of fluorescent substances (organic dye, nano-diamond, q-dot) to pillars top, as well as indirect labeling that selectively modify the surface of molds with either metal or fluorescent substances. In Chapter 4, we examined the characteristics of local contractility forces and identified the components formed a sarcomere like contractile unit (CU) that cells use to sense rigidity. CUs were found to be assembled at cell edge, contain myosin II, α-actinin, tropomodulin and tropomyosin (Tm), and resemble sarcomeres in size (~2 μm) and function. Then we performed quantitative analysis of CUs to evaluate rigidity sensing activity over ~8 hours time course and found that density of CUs decrease with time after spreading on stiff substrate. However addition of EGF dramatically increased local contraction activity such that about 30% of the total contractility was in the contraction units. This stimulatory effect was only observed on stiff substrate not on soft. Moreover, we find that in the early interactions of cells with rigid substrates that EGFR activity is needed for normal spreading and the assembly of local contraction units in media lacking serum and any soluble EGF. In Chapter 5, we performed high temporal- and spatial-resolution tracking of contractile forces exerted by cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like CUs simultaneously moved opposing pillars in net steps of ~2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing.