나노 광조형 시스템으로 제작된 3 차원 인공지지체 패턴이 세포의 거동에 미치는 영향

Abstract

This thesis presents the development of nano-stereolithography system(NSTL) and its application to investigate the effect of scaffold pattern on the cell behavior in tissue engineering. Scaffolds, one of the three essential elements of tissue regeneration, have to degrade and disappear eventually to be replaced by the newly generated tissue when it was implanted in the human body. Scaffold provides the right kind of environment for cells to grow in vitro and in vivo by playing a supporting role. Recently, many research groups have fabricated scaffolds using various SFF(solid freefrom fabrication) technologies. SFF is a new technology that permits the control of internal/external geometry such as pore size, porosity, and line pattern of scaffold with reproducibility. In this study, a NSTL for manufacture of the scaffold with a precision of nanolevel is proposed by novel integration of the 3-axis nano positioning system and femto-second laser. Irradiated by femto-second laser, valence electrons obtain the energy equivalent to band gap energy by the absorption of two photons and come to the excited states. This phenomenon is called by 'Two photon absorption (TPA)' and permits the fabrication of 3D structure with the resolution as high as possible. The beam source of NSTL is Ti:sapphire laser that is operated in mode-lock at 800nm with a 100 femto second pulse width. When the CAD data of 3D structures were transferred into computer controller, NSTL controls the scanner, stage, shutter and CCD camera. Among them, CCD camera can be used to monitor the fabrication process in real time. Moreover, this system controls the coordinates of x-y-z axis by linear motors and linear encoders precisely. Then the laser beam, is activated by ON/OFF function of the shutter, is focused on the target point of resin and polymerizes the resin through pre-defined path. Most metazoan cell types require a surface on which to flatten out and divide. This cell is also called by "anchorage dependent cell" because it is possible to maintain their life only by the interaction with environmental surface. Therefore, a surface of scaffolds is very important factor for cell behavior. To investigate the effect of scaffold pattern on cell behavior, experiments were planned by the design of experiments (DOE)technique. Line width and line pitchwere selected as controllable factor for cell adhesion and cell proliferation. There are 3experimental levels of line width (1μm, 5μm, 10μm) and 4 experimental levels of line pitch (15μm, 30μm, 50μm, 80μm), respectively. Various kinds of patterned scaffolds with diverse line widths and pitches were fabricated by NSTL system. These scaffolds were seeded with MC3T3-E1, pre-osteoblast cell line, and were cultured in vitro for 7 days. To examine the effect of cell adhesion and proliferation on the scaffold patterns, CCK-8 assay was performed to quantify the living cell into each scaffold. 4 hours after seeding, the results of CCK-8 assay showed that cell adhesion characteristics of scaffolds with a pattern was excellent compared with that of nopattern substrate. However, there has no significant difference of cell adhesion according to scaffold patterns. The results of cell proliferation for 7 days indicated that scaffolds with 50μm line pitch showed the best proliferation among the experimental condition, whereas cells was hardly proliferated on scaffolds with 15μm line pitch. Additionally, we fabricated and evaluated 3D bi-pore scaffold with an external geometry of 4 x 4 x 1.5 mm3 by NSTL system to investigate the effect of bi-pore structure in cell behavior. To realize this goal, we also fabricated the scaffold with only global pore and compared the effectiveness of two types scaffold in cell adhesion and proliferation. Using CCK-8 assay, we demonstrated that bi-pore scaffold had better cell adhesion and proliferation than scaffold with only global pore. In this thesis, NSTL was successfully developed for the manufacture of 3D scaffolds with complex shape and precision of less than a micron. We fabricated various scaffolds with diverse line pitches and line widths using NSTL system and examined the effect of cell adhesion and proliferation on the scaffolds. With these results, we confirmed that the scaffold patterns, such as line width and line pitch, can affect cell behavior.