LIGA-based Micro-shaping Processes for Unidirectional Micro/Mesoscale Structures Guiding Cell alignment and Myogenic Differentiation

Abstract

The present thesis is about the fabrication processes of unidirectional micro/mesoscale structures for cell-aligning platforms. Previous studies for topographical control of cells have focused on nano- or microscale structures below 10 μm. However, recently, micro- and mesoscale structures similar to or larger than the size of a single cell have received attention because the structures provide cells 3D microenvironments and induce intrinsic cell responses. Among the various structures, unidirectional structures have been widely used to induce cell alignment and related higher-order functions such as differentiation and tissue maturation. Previous fabrication processes for micro/mesoscale unidirectional structures, such as UV photolithography-based processes for a grooved-surface, only create the restricted structures having stepwise rectangular cross-section with shape edges. It is not relevant to in vivo structures. Tissues exhibiting the unidirectional characteristics, such as blood vessel, muscle, nerve, have smooth micro/mesoscale topography. In this regard, it basically is valuable to develop fabrication process that can modulate a cross-section shape of unidirectional structures for physiologically-relevant cell-aligning platforms. In order to modulate the cross-sectional shape of unidirectional structures, deep X-ray LIGA that can fabricate thick micro/mesoscale structures was used in this study. We established two types of LIGA-based shaping processes, LIGA-based micro-molding and micro-extrusion processes, for production of grooved-surfaces and fibers with a modulated cross-section. Grooved-surfaces with a modulated cross-sectional shape were produced by LIGA-based micro-molding process. Through deep X-ray lithography and nickel electroforming, metal mold inserts including microscale grooves with the cross-sectional shape of sinusoidal, triangular and rectangular waves at their sidewall were fabricated. . The vertically fabricated mold inserts were reorientated horizontally, and assembled to utilize the grooves at the sidewall as a mold surface. The assembled mold inserts were used as the mold for hot embossing and the mold core for injection molding. Using the assembled mold, polystyrene (PS) grooved surfaces with the modulated cross-sectional shape were successfully produced by hot embossing. PS dish-type platforms that can contain cell culture media were also produced by injection molding with the assembled mold core. Fibers with a modulated cross-sectional shape, i.e., shaped fibers, were produced by LIGA-based micro-extrusion process. To modulate a cross-sectional shape of fibers, nozzles with a cruciform aperture and a triangular aperture were fabricated by deep X-ray lithography and subsequent nickel electroforming. A pneumatic-type micro-extrusion system that can melt and pressurize a polymer was developed to use the fabricated nozzles. Polycaprolactone (PCL) shaped fibers were produced by the developed micro-extrusion system with the nozzles. The cross-section of the extruding fibers was distorted from the nozzle aperture shape to a circle due to the extrudate swell and surface tension of molten PCL. An evaluation factor, shape-ability, was introduced to quantify such distortion. The micro-extrusion process for shaped fiber was optimized with respect to the shape-ability. Utilizing the above established LIGA-based micro-molding and micro-extrusion processes, we developed two cell-aligning platforms in types of the grooved-surface and shaped fiber specialized in skeletal muscle. Skeletal muscle has unidirectional, smooth, and hierarchical structures. In this regard, we designed a basic cross-sectional shape of the unidirectional structures with considering sizes and hierarchy of muscle fibers and a fascicle for consistency of the two platforms. Furthermore, the both grooved-surface and shape-fiber were produced with same material, PCL. The basic cross-sectional shape is composed of mesoscale circle reflecting the fascicle and microscale sinusoidal waves reflecting the muscle fibers, and the sinusoidal waves are located at circumference of the mesoscale circle. The grooved-surface reflecting muscle structure was designed to have a cross-section which is the continuously-arranged halves of the basic cross-sectional shape, and we called it the hierarchical grooved-surface. To produce the hierarchical grooved-surface, a soft PDMS mold was employed in the LIGA-based micro-molding process, instead of nickel electroformed mold inserts. Template for the soft mold was achieved through reorientating and assembling of polymeric parts fabricated by X-ray lithography. And then, the template was casted with PDMS, resulting in the soft mold. The soft mold enabled us to replicate the hierarchical grooved-surface with inverse taper angle. In addition to the surface, a fiber having a cross-section analogous to the basic cross-sectional shape, called muscle-shaped fiber, was produced by the advanced LIGA-based micro-extrusion process. A nozzle aperture was designed to compensate for the distortion of fiber cross-section during micro-extrusion. Moreover, a screw-type micro-extrusion system was developed to overcome the disadvantage of low extrusion pressure in the previous pneumatic-type micro-extrusion system. The developed screw-type micro-extrusion system exhibited higher shape-ability compared with the previous one. In addition to improvement of the system, the nozzle fabrication process was simplified with help of thick resist photoresist film. The fibers produced by the screw-type extrusion system with the nozzles were evaluated to choose the proper nozzle for muscle-shaped fiber. Behavior of fibroblasts, myoblasts and adipose-derived stem cells (ASC) on the developed cell-aligning platforms was evaluated to investigate performances of the platforms. Alignment of fibroblasts on the sinusoidal wavy-surfaces produced by hot-embossing and injection molding was evaluated. As the height of the sinusoidal wave increased and the width decreased, the alignment of fibroblasts was improved. Myoblasts and ASC were cultured on the hierarchical grooved-surface reflecting the in vivo microenvironment of skeletal muscle. To investigate the effect of the hierarchical structure, corresponding microscale and mesoscale grooved-surfaces and a conventional stepwise grooved-surface were used as control groups. The hierarchical grooved-surface exhibited similar cell-aligning performance with respect to myoblasts compared with control groups. The formation and alignment of myotubes differentiated from myoblasts were highly improved and some myogenic genes were over-expressed on the hierarchical grooved-surface compared with that on other grooved-surfaces. The hierarchical grooved-surface showed the similar differentiating performance in ASC culture compared to the control group. To evaluate the effect of muscle-shaped fiber on myoblasts, a bundle-type scaffolds composed of the muscle-shaped fibers was fabricated. The morphological characteristics of myotubes, such as myotube area, length, diameter, and aspect ratio on the muscle-shaped fiber were similar or reduced compared to the conventional circular fiber, but myogenic index, the most important characteristic of myotube formation, was somewhat increased in the muscle-shaped fiber scaffold. The LIGA-based micro-shaping processes that can modulate a cross-sectional shape of unidirectional micro/mesoscale structures proposed in this study may drive the diversification and advancement of cell-aligning platforms. In addition to that, the developed platforms could be useful tools to understand the cell behavior on complex micro/mesoscale environments. Moreover, this study may become a good application example in the field of LIGA