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Thesis(masters) --์์ธ๋ํ๊ต ๋ํ์ :๊ธฐ๊ณํญ๊ณต๊ณตํ๋ถ,2010.2.Maste
๋ฉํฐ์ค์ผ์ผ ํ๋ฉด ํจํด์ ์ด์ฉํ ์ธํฌ์ ๊ฑฐ๋ ์กฐ์
ํ์๋
ผ๋ฌธ (๋ฐ์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ๊ธฐ๊ณํญ๊ณต๊ณตํ๋ถ, 2014. 2. ์ ๋๋ฆฌ.In this thesis, we study the effect of multiscale topographies with various density, size, spring constant, and slanted angle on the cellular behaviors. For this purpose, the multiscale polymeric patterns were fabricated by UV-assisted capillary force lithography (CFL) technique. Through the coating with extracellular matrix (ECM) proteins such as fibronectin and collagen, the multiscale topography can present physically bio-mimetic microenvironment to the cells.
First, we report on the effect of synthetic extracellular matrix (ECM) scaffold in the form of uniformly-spaced nanogrooved surfaces in dermal wound healing. The rate of wound coverage was measured on various nanotopographical densities with vertical or parallel orientation using nanogrooves of 550-nm width with three different gaps of 550, 1100, and 2750 nm (spacing ratio: 1:1, 1:2 and 1:5). Guided by the nanotopographical cues in the absence of growth factors in wound healing process, the cultured NIH-3T3 cells demonstrated distinctly different migration speed, cell division, and ECM production as dictated by the topographical density and orientation, whereas the proliferation rate turned out to be nearly the same. Based on our experimental results, the nanopattern of 1:2 spacing ratio yielded the best would healing performance in terms of migration speed, which seems similar to the natural organization of collagen fibers.
Next, we report the effect of feature size and orientation of multiscale topography on the migration of cancer cells. It is well known that tumor migration occurs in vivo following the basement membrane, microtracks, and lymphatic vasculature, showing predominant guidance by physical cues. Inspired by the nanoscale and microscale topographic guidance, we prepared flat, nano groove, and micro groove patterns. Furthermore, to emulate the reorganization of ECM by cancer cells and subsequent guided migration through reorganized ECM, the topographical orientation was also considered, by preparing groove, concentric, and radial patterns. When comparing the spreading of cell island, both collectively and individually migrating cells showed guided spreading in response to topographical orientation. However, the sensitivity to topography was more sensitive in the case of individually migrating cells. Microscopically, the topography not only induced polarization of intracellular elements such as f-actin and vinculin, but also modulated protein levels such as E-cadherin, ROCK2, and vinculin in response to the topographical size and orientation.
Finally, we study how sensitively cells can recognize underlying surface topography in the case of varying spring constants and varying slanted angles. To this end, nanopost arrays (diameter of 400 nm) having various stiffness (spring constant: 9.33, 345.58, and 5585.05) were fabricated with various height (2000 and 600 nm) and mechanical properties (19.8 and 320 MPa). On the vertical nanopillars with various spring constants, NIH-3T3 cells showed bi-axial alignment following the array, but the degree of alignment was decreased as the spring constant increases, demonstrating correlation with the bending of nanopillars. Furthermore, to understand underlying mechanism of mechanosensing in the case of nanopillars, slanted nanopillars with various angles (90, 75, 60, 45 and 30ห) with same diameter (400 nm) were prepared. On the relatively vertical nanopillars (such as 90 and 75ห), cells showed bi-axial alignment, but as the leaning angle increases (such as 30 and 45ห) cells showed uni-directional alignment along to the slanted orientation. According to the signaling inhibition, the alignment on the relatively vertical pillars was affected by Rac signaling pathways. However, the effect of Rac signaling inhibition decreases as the leaning angle increases.Abstract โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโi
List of tables โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโvii
List of figures โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโviii
Nomenclature โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโxiv
Chapter 1. Introduction โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ 1
Chapter 2. Effect of orientation and density of nanotopography in dermal wound healing 9
2-1. Introduction โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ9
2-2. Materials and methods โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ12
2-3. Results โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ17
2-3-1. Ex-vivo study of neonatal rat dermis โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ17
2-3-2. Experimental design โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ19
2-3-3. Time-dependent coverage of cell-free area โโโโโโโโโโโโโโโโโโโโโโโโโโโโโ22
2-3-4. Migration assay โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ24
2-3-5. Analysis of focal adhesion โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ27
2-3-6. Proliferation rate โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ34
2-3-7. Angle of cell division โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ36
2-3-8. Organization of produced ECM โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ40
2-4. Discussion โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ44
2-5. Summary โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ48
Chapter 3. Effect of topographical size and orientation in collective and individual cancer cell migration 49
3-1. Introduction โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ49
3-2. Materials and methods โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ51
3-3. Results โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ56
3-3-1. Design of multiscale topography โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ56
3-3-2. Topography-dependent collective and individual migration โโ58
3-3-3. Intracellular organization โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ64
3-3-4. Westernblot assay โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ67
3-4. Discussion โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ72
3-5. Summary โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ74
Chapter 4. The effect of spring constant and slanted angle on the alignment of cells 75
4-1. Introduction โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ75
4-2. Materials and methods โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ77
4-3. Results โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ83
4-3-1. The effect of spring constant โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ83
4-3-2. The effect of slanted angle โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ96
4-4. Discussion โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ110
4-5. Summary โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ113
Chapter 5. Summary โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ114
References โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ118
๊ตญ๋ฌธ์ด๋ก โโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโโ128Docto