Cell Culture Substrates Patterned With Anisotropic Topography And Stiffness And Their Applications In Tissue Engineering

The physical cues of cellular microenvironment such as topographies, stiffness, stress and strain play vital roles in regulation of processes such as cell growth, proliferation, differentiation, and death. Physical cues are more stable and permanent compared to biochemical cues in some cell behaviors regulation. Topography and stiff are more prominent than others in cell orientation, migration and differentiation regulation.Tissue engineering is the method for regenerating functional tissue equivalents based on biological principals and engineering technologies for research and transplant applications. The only tissues available for clinical practices are skin and cartilage. Muscles and blood vessels are two kind of tissues which are closest to clinical applications.Skeletal muscle tissues account for 40% to 45% of human weight. They are the drive forces for human motion and movement. A lot of reasons are responsible for skeletal injury, including muscle contusion in sports, muscle tears because of accident and muscle damages as a result of surgery. Additionally, muscle atrophy caused by diseases and muscle aging also make skeletal muscle regeneration an urgent issue.Blood vessel is the most important tissue of human body. Vascular diseases such as atherosclerosis are fetal. Tissues and organs lacking blood vessels will suffer from lacking of nutrition and oxygen and failure. The significant roles blood vessels play in tissues and organs make the fabrication of scaffold capable of regenerating functional blood vessels an vital issue. However, the complex structure and mechanical properties of blood vessel also make the fabrication of vascular scaffold full of challenges.The thesis presented managed to fabricate substrates and scaffold with anisotropic topography and stiffness using biocompatible materials and apply these substrates and scaffold into ring-shaped and striated skeletal muscle and vascular tissue engineering. The thesis provided new methods that will underpin the design of substrates of scaffolds for skeletal and vascular tissue engineering in vitro. The main results are listed as follows:1. Ring-shaped myofibers are important component of external urethral sphincter. The thesis revealed a simple way to engineer circular and elliptical ring-shaped myoblast patterns and maximize corresponding cell alignment simultaneously with supracellular constraints. To underlying the mechanism, finite element models of collective cell orientation under the influence of supracellular ring-shaped constraints were established. The experimental results showed that the dimensions and parallelism of the inner and outer boundaries of supracellular ring-shaped constraints can significantly affect cell orientation while curvature of curved constraints cannot. The theoretical results showed that forces can be transmitted through cell-cell junctions. Cells will orient along the directions of intercellular maximum principle stresses, which are parallel to the directions of ring-shaped constraints boundaries. When cells are highly confluent, information of constraints boundaries can be passed from cells located near the boundaries to cells located in the middle regions between the boundaries, making the later cells orient along the directions of the boundaries under the drive of intercellular forces. Highly oriented cells will facilitate myotubes generation. The conclusions of the thesis provide ring-shaped myotube regeneration with sound supports. Additionally, the substrate presented hold the potential to server as a compliant bottom layer of glass-attached myotubes with tissue like stiffness for cell-on-cell sphincter-like tissue engineering, which will benefit the drug screen process for urethral sphincter related diseases treatment and thransplant.2. Large-area and cost-efficiency skeletal muscle tissue engineering holds promising future. Anisotropic topography and stiffness play an important role in inducing myoblasts orientation towards myotubes regeneration. Under the consideration of cost and dimension, the thesis provide a mold-free method to fabricate topographic features on the surface of light-sensitive hydrogel through in site twice exposure. The method also brings spatially distributed stiffness corresponding to topography on hydrogel surface. The experimental results showed that the combination effects of anisotropic topography and stiffness in cell orientation regulation accelerated myotubes generation process. The method can fabricate large-scale topographies in a cost-efficiency manner. More importantly, it can be applied to other light-sensitive hydrogels, which makes it high potential to be used in tissue engineering.3. Blood vessel of arteries and veins are composite of axially aligned endothelial cells and circumferentially aligned smooth muscle cells. The thesis managed to fabricate diameter-controllable tubular structures with perpendicularly aligned constraints on the inner and outer surface of the structure. The inner constraints are axially patterned grooves fabricated through soft lithography and the outer constraints are circumferentially patterned topographies and stiffness fabricated through in site twice exposure. Both constraints are able to induce cells to align along constraints directions. In summary, the thesis successfully fabricated tubular structure with perpendicularly aligned constraints on both surfaces with hydrogels rather rigid materials that was used in electrospinning. The scaffolds fabricated and capability of the method to be applied in other materials makes them have potential applications in vascular tissue regeneration in vitro and in vivo.