学位授与大学：京都大学 ; 取得学位: 博士(工学) ; 学位授与年月日: 2007-09-25 ; 学位の種類: 新制・課程博士 ; 学位記番号: 工博第2865号 ; 請求記号: 新制/工/1421 ; 整理番号: 25550 / Many conventional methods have been used to modify the wettability of the polymeric surfaces for the biomedical applications of the artificial bionic organ. Those methods are the chemical treatment, the ultraviolet (UV) irradiation, the plasma process and the ion implantation. Many artificial bionic organs, for example, an artificial heart, an artificial blood vessel, a device for prevention of thrombosis stent and an artificial endocranium have been developed for the physical or mental disability. For development of the high function of an artificial bionic organ, the data transmission between the brain neuron cells and the external electrical circuit, and the high biocompatible materials for the interface between brain and electrode are required. It is related to the technology of brain-computer interface (BCI), sometimes called a direct neural interface or a brain-machine interface. In case of the brain-controlled devices, the study of the brain memory is necessary. Then, the artificial pattern network of the brain cells cultured on the surface in vitro for simulation of the brain function is the concerned issue. The arrangement of a lot of neuron on a detection electrode is required. So, a formation method of the artificial neural network that arranged a neuron as technology for this purpose is demanded As for the neuron arrangement, there were the reports about the immobilization of neuron by fabrication of the three-dimension structure, and they could be divided into two methods from their manipulation. One is the arrangement with one-by-one manipulation and the other is the arrangement with self-assembly. The former method is the fabrication of many micro-structures and then arranged a neuron in a desired position with one-by-one manipulation to for a neuron network. For the brain memory stimulation, however, the neuron network from more than 10 millions of neurons is required. So, this method is not suitable. The latter is the fabrication of the carbon nanotube pillar to immobilize the neurosphere with self-assembly adhesion. Although this method could be formed the large neuron network, the neurosphere consists of several 1, 000 cells. So, it is very difficult to analyze the mechanism of data transformation. In contrast, by surface modification even if on the same surface to modify a geometric pattern, the cells can adhere along the modified pattern by using single culture on such surface. The neuron will migrate itself to adhere on the pattern. The self-assembly adhesion occur. This method is very useful for the neuron arrangement method. The surface modification of the polymeric materials to pattern the cell adhesion area as a network has been taken place by using many techniques such as the plasma process, the irradiations of UV and X-ray and the ion implantation. The ion implantation technique into the polymeric-material surface has more advantage than the other techniques since its abilities to control the micro-area, and to break down the tight bonding of polymer material. The ion implantation with positive ion without charge neutralization results in a charge-up problem due to the insulating properties of most polymers. This charge-up problem exerts a bad influence on the implantation control of ion dose and ion energy. The negative-ion implantation occurs almost “charge-up free” even if no external charge compensation. Then, the negative-ion implantation into polymeric surface has a very precise control to obtain very fine pattern. So, it is expected to control the adhesion size of about single cells (about several 10 μm). Since this study will be used for the application in the biomedical fields, the ion element should be considered to be harmless for the living body. Then, carbon is selected since it is main component of polymer materials and more familiar to cells. As above described, in this thesis, I use the carbon negative-ion implantation to modify the polymeric surface to obtain the pattern of the neuron with self-assembly-adhesion. As for the polymeric material in the biomedical fields, I selected polystyrene (PS) and silicone rubber (SR). In this research, the fundamental parameters for cell adhesion on the modified surface by carbon negative-ion implantation were described (Chapters 3, 4 and 5). As for the fundamental issue, the wettability relating to the atomic bonding state of the new functional group and the surface morphology (Chapter 3), the protein adsorption (Chapter 4), and also the adhesion of nerve-like cells on the pattern (Chapter 5) were examined. In these chapters, I clarified the relationship among them and the negative-ion implantation. Then, based on these phenomena, I have developed the new application techniques by negative-ion implantation for the adhesion patterning of neuron (Chapters 6 and 7). In the development of these techniques, I have proposed two methods since the neuronal cells required the special base surface to adhere. One is degradation method of the special base surface by which I tried to make an artificial neuron network (Chapter 6). The other is the patterning of the stem cell adhesion and differentiation into neuron with maintaining the adhesion position. So, the neuron patterns were formed on the pattern (Chapter 7). The obtained results are summarized as the following. In Chapter 3, the surfaces of the PS and SR were implanted by carbon negative ions at the energies of 5 – 20 keV and the doses of 1×1013 – 3×1016 ions/cm2. After the implantation, the change in the physical surface properties, relating to the adsorption properties of adhesive proteins, was described. The new atomic bonding, the surface morphology and the wettability were studied by XPS analysis, AFM and contact angle measurement, respectively. XPS analysis showed the formation of new oxygen function groups of hydroxyl and carbonyl on the implanted surfaces from the adsorption of the oxygen in the residual gas and in the moisture in the air on the ion-induced defects. These new bonds refer to the hydrophilicity for the wettability. The ion implantation sputtered and changed the surface morphology of surface roughness in order of several nm that dose not interfere to the protein adsorption and to cell culture. The wettability properties of the C¯-implanted surfaces of SCPS and SR were evaluated by measuring the change in contact angle. At first, the angles were measured by the water drop method. The contact angles of PS measured by water drop method decreased from 91° to 86° for the non-implantation to the implantation, respectively. Those of SR also decreased from 100° to 86°for the non-implantation to the implantation, respectively, even if the main chain bonds in SR are stronger than that in PS. The hydrophilic surfaces of PS and SR were obtained by carbon negative-ion implantation. Then, the contact angles were measured by the air bubble method. The sample was dipped in the water and the bubbles were injected on the surface. Then, the angle was evaluated from the arc circular of the bubble. After dipping in the water for 24 h, the average value of the angles decreased to 64° and to 52° for PS and SR, respectively. The more clearly hydrophilic properties were observed. In Chapter 4, I checked the adsorption properties of the adhesive protein and the poly-D-lysine (PDL) on the implanted surface. Generally, in the cell adhesion, the adhesive proteins exist between the cell surface and the surface. On the cell membrane, cells have specific receptors that anchor to the specific protein. So, the adsorptions of the adhesive proteins are necessary for the cell adhesion. In nature, protein has both hydrophobic and hydrophilic groups. Thus, the ultra hydrophobic and ultra hydrophilic surfaces are not suitable for protein adsorption. The adhesive proteins for the cell adhesion generally prefer to be adsorbed on the hydrophilic surface, which the contact angle is in the range of 40° – 80°. I evaluated the adsorption properties of adhesive protein such as type-I collagen, fibronectin and laminin and that of PDL on the modified surfaces of PS and SR by detecting the nitrogen atom with using XPS analysis. As a result, the adsorptions of the adhesive protein were almost improved with 1.2 – 3.3 times by carbon negative-ion implantation. In Chapter 5, the nerve-like cells of PC12h (rat adrenal pheochromocytoma) were cultured on the C¯-implanted surfaces of PS and SR to find out the fundamental condition for the neuron network formation. As a results, PC12h cells and their neurite outgrowth showed the self-assembly adhesion along the implanted pattern on both of PS and SR. The suitable condition of the ion implantation for the adhesion patterning of PC12h cells was about 1×1015 – 3×1015 ions/cm2. Almost no effect of energy in the range of 5 – 20 keV on the cell adhesion was observed. The effective minimum line width of the implanted region for the adhesion of single cell-body and single neurite outgrowth were about 5 and 2 μm, respectively. In Chapter 6, the brain neuronal cells require the specific surface culture, such as PDL. So, in this chapter, I used PDL coating on the PS and degraded it by the carbon negative-ion implantation. Two kinds of brain neuronal cells were used. One is newborn mouse brain neuronal cells (1 day) and the other is rat embryo brain cortex neuronal cells (16 – 18 days). As a result, obtained the effective ion dose for degradation of the adhesion at 1×1014 ions/cm2. The adhesion patterning of brain neuronal cells on the unmodified pattern of PDL could be achieved by carbon negative-ion implantation. In Chapter 7, I cultured the adult stem cells of rat mesenchymal stem cells (MSC), which has the multipotential to differentiation into many kinds of cell lines, especially into neuron, on the pattern region of the C¯-implanted surfaces of PS and SR. As a results, MSCs showed the self-assembly adhesion along the implanted pattern of PS and SR. Comparing to the adhesion patterning of PC12h cells, the adhesion patterning of MSCs required a lower ion dose to implant on the polymeric surfaces. By culturing with the culture medium supplementing withβ-Mercaptoethanol (BME) at concentration of 1 mM, the MSCs were induced to differentiate into neuronal cells. The adhesion patterning of the neuron-differentiated cells maintained on the implanted region was observed. By staining with anti-neuron-specific enolase, these differentiated cells were neurons. From all investigation, I clarified the change in the physical surface properties after the carbon negative-ion implantation into the polymeric surface and the mechanisms mentioned above. I showed the surface modification to obtain the hydrophilic surface by the ion-induced effect. This hydrophilic surface improved the protein adsorption properties. By using nerve-like cells, the ion implantation affecting to the cell adhesion were clarified. By the implantation through the micro-pattern mask, the cells adhered along the implanted pattern. The cells could adhere on the implanted area that was smaller than the cell size and their neurite also could adhere on the narrowed implanted area. So, I can obtain the self-assembly separation pattern of cell body adhesion and neurite outgrowth. For the application of patterning of real neuron, I coated the special surface with PDL and degraded it from patterning the negative-charge site on it by using carbon negative-ion implantation through a micro-pattern mask. I could pattern and form the neuron network of the brain neuron on the unmodified PDL. On the other hand, for the MSC, I also achieved the adhesion patterning by using carbon negative-ion implantation through a micro-pattern mask, and I succeeded the patterning of the neuron-differentiated cells from the adhered MSC with maintaining their adhesion pattern. As a conclusion, from all these researches, I achieved the cell-self-assembly adhesion and the patterning of the neuron network formation on the polymeric surfaces by using carbon negative-ion implantation. / Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13394号 / 工博第2865号 / 新制||工||1421(附属図書館) / 25550 / UT51-2007-Q795 / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 石川 順三, 教授 髙岡 義寛, 教授 小林 哲生 / 学位規則第4条第1項該当
| Identifer | oai:union.ndltd.org:kyoto-u.ac.jp/oai:repository.kulib.kyoto-u.ac.jp:2433/49145 |
| Date | 25 September 2007 |
| Creators | SOMMANI, Piyanuch |
| Contributors | 石川, 順三, 髙岡, 義寛, 小林, 哲生, ソムマニ, ピヤヌット |
| Publisher | 京都大学 (Kyoto University), 京都大学 |
| Source Sets | Kyoto University |
| Language | English |
| Detected Language | English |
| Type | DFAM, Thesis or Dissertation |
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