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I. 서론 8
1.1. 연구 배경 10
1.1.1. 기계적 자극 10
1.1.2. 골수기질간세포 15
1.1.3. 양극산화 16
1.2. 연구 목적 18
II. 재료 및 방법 19
2.1. 티타늄 시편 19
2.2. 양극산화처리된 티타늄 표면 분석 22
2.3. In vitro 실험 25
2.3.1. MG-63을 이용한 예비 실험 25
2.3.2. Human BMSCs를 이용한 본 실험 30
2.4. 통계 분석 35
III. 결과 36
3.1. 시편 특성 분석 결과 36
3.2. MG-63을 이용한 예비 실험 결과 42
3.3. Human BMSCs를 이용한 본 실험 결과 49
IV. 고찰 56
V. 결론 60
VI. 참고문헌 62
Abstract 67
감사의 글 70
TABLE 1. Processing conditions for anodic oxidation 21
TABLE 2. Classification of experimental groups of first preliminary test based on the patterns of IHP 26
TABLE 3. Classification of experimental groups of second preliminary and main test based on the patterns of IHP 26
TABLE 4. Surface roughness of each group(n=6) 37
TABLE 5. Compositions on the polished and anodic oxidized surfaces 40
Fig. 1. Schematic drawing of pressurizing system. 11
Fig. 2. Schematic drawings of axial and bi-axial extension system. 12
Fig. 3. Schematic drawings of in and out-of-plane circular substrate distention. 12
Fig. 4. Schematic drawing of four point bending system. 13
Fig. 5. Schematic drawings of cone and plate system and parallel plate flow chamber. 14
Fig. 6. Schematic structure of the electrical double layer at the titanium 17
Fig. 7. Schematic drawing of the anodizing apparatus. 21
Fig. 8. Ra roughness parameters. 23
Fig. 9. Contact angle(θ) of a liquid drop on the flat surface of a solid. 24
Fig. 10. Bioreactor: (a) frontal view of bioreactor, (b) control box, and (c) pressure chamber. 29
Fig. 11. Schematic diagram of mechanical stimulation(IHP) schedule: (a) first Preliminary test and (b) second preliminary test. 29
Fig. 12. Schematic diagram of mechanical stimulation(IHP) schedule in the main experiment using BMSCs. 31
Fig. 13. SEM morphology on the surfaces: (a) machined view(x1800), (b) anodic oxidized view(x1800), and (c) magnified view(x4500) of cracks at picture(b). 36
Fig. 14. XRD patterns of machined(M.) and anodic oxidized(A.) surfaces. 38
Fig. 15. EDX spectrums on the surfaces: (a) polishing and (b) anodic oxidation. 40
Fig. 16. Snap shot of wettability on the surfaces: (a) polishing and (b) anodic oxidation. 41
Fig. 17. DNA contents of MG-63 cells cultured on the cell culture plates(control, n=5) and the anodized titanium surfaces(Ano_Ti, n=5). 42
Fig. 18. DNA contents, results of the first preliminary test. The magnitude of pressure was 0.2 MPa(gauge pressure). Experimental groups were divided by patterns of IHP(pressurizing time(min)/resting... 45
Fig. 19. DNA contents, results of the second preliminary test, presented by (a) each experimental group and (b) the cultural time. The number of specimens of each experimental group was 5.... 46
Fig. 20. ALP activities of each experimental group(n=5) using MG-63. Experimental groups were divided by patterns of IHP(pressurizing time(min)/resting time(min)): no pressure (control), 2/5(Group 1), and... 47
Fig. 21. Calcium contents of each experimental group(n=5) using MG-63. Experimental groups were divided by patterns of IHP(pressurizing time(min)/resting time(min)): no pressure (control),... 48
Fig. 22. DNA contents of each experimental group(n=5) using BMSCs. Experimental groups were divided by patterns of IHP(pressurizing time(min)/resting time(min)). 49
Fig. 23. ALP activities of each experimental group(n=5) using BMSCs. Experimental groups were divided by patterns of IHP(pressurizing time(min)/resting time(min))(*: p〈0.05). 50
Fig. 24. Calcium contents of each experimental group (n=5) using BMSCs. Experimental groups were divided by patterns of IHP(pressurizing time(min)/resting time(min)): no pressure(control),... 51
Fig. 25. Fluoroscopic observations(×200) of osteocalcin stained with monoclonal bovine osteocalcin antibody and anti-mouse IgG(whole molecule)-FITC. Experimental groups were divided by patterns of IHP... 53
Fig. 26. SEM micrographs(×1,800) of BMSCs on the anodic oxidized surfaces. Experimental groups were divided by patterns of IHP(pressurizing time(min)/resting time(min)): no pressure(control),... 55
초록보기 더보기
The purpose of this study was set to investigate the celluar responses of BMSCs(Bone Marrow Stromal Cells) seeded onto anodic oxidized titanium surface under various simulated biophysical stimuli.
Before seeding the cells, the surface of disk-type specimens were analyzed by SEM, XRD, EDX. In addition to these, the contact angles for the hydrophile property roughness were measured.
As the preliminary tests, MG-63, osteoblast-like cells, were seeded onto the specimens and their proliferation and differentiation were quantified under various patterns of mechanical stimuli. Based on the patterns of stimuli, 3 test groups and 1 control group were set depending on resting period. They were stimulated for 3 hours/day for 2 days after 24 hours incubation for stabilization. The patterns of the stimulation was 2 min/5 min, 2 min/15 min, and 2 min/ 25min for stimulation/resting period, respectively. Three test groups were divided into 2 sub-groups again, which experienced extra 2-hour stimulation at day 5 of culture with same stimulation pattern. The cells were harvested after 7 days of culture and analyzed by quantifying DNA, ALP(alkaline phophotase) and calcium contents. The results from preliminary tests showed that additional stimulation did not affect cellular responses. However, groups of 2/5 and 2/15 without extra stimulation showed increased DNA contents with no significant difference. Therefore, the tests with BMSCs were carried out with 2/5, 2/15-stimulated groups and control group. The group stimulated with 2/15 showed significant increase in DNA contents after 14 days of culture. However, no significant increase was observed in other markers during the culture, even the trend showed that the mechanical stimuli somehow contributed in proliferation and differentiation.
Through the study, I came to the conclusions as follows: 1) The surface characteristics such as morphology and compositions provided more preferable environments to the cells to be proliferated and differentiation. 2) For the investigation of differentiation, cell-line such as MG-63 may not be useful. 3) The mesenchymal stem cell such as BMSC was found so sensitive to biomechanical and biophysical environments that cautions should be noted. 4) The mechanical stimuli obviously contributes to cellular responses such as proliferation and differentiation.
For the future studies related to tissue engineering combined with biomechanical stimuli, the optimal conditions such as patterns and types of stimulation should be investigated along with long-term animal studies.c techniques have been used more frequently to directly inspect the nasopharyngeal orifice of the E-tube. However, it is still required to develop a method to exactly estimate the function of E-tube. In this study, I have evaluated the morphologic and dynamic motion of nasopharyngeal orifice of the E-tube with 4mm 30˚ videoendoscope by comparing with the other existing tests of E-tubal function.
Subjects and methods : One hundred one E-tubes with chronic otitis media were selected. In order to study E-tube dilatory movements, I had performed the transnasal 4mm 30° videoendoscopic examination of the nasopharyngeal opening of the E-tube when patients were swallowing, and classified morphologic and dynamic findings of nasopharyngeal orifice of the E-tube into three categories as followings, Type A ; E-tube is opened widely on swallowing with normal mucosa(normal), Type B ; E-tube is not opened with normal mucosa(functional blockage), Type C ; E-tube is not opened with pathologic mucosa(mechanical blockage). I have investigated relationships between these categories and the results of other E-tube function tests, such as inflation deflation test, classification of otitis media and the amount of mastoid pneumatization of the diseased ear. Throughout the study, control group was 60 E-tubes that were free of ear pathologies.
Results : There were more type As in control group than in otitis media group via videoendoscopic findingsof nasopharyngeal orifice of E-tube. Videoendoscopic analysis of nasopharyngeal orifice of the E-tube had a close correlations with the results of inflation deflation test and classification of otitis media, but had no significant relationship with the degree of mastoid pneumatization. Simple otitis media had a relatively better tubalfunction than cholesteatomas and adhesive otitis medias.
Conclusion : Morphologic and dynamic examination of the nasopharyngeal orifice of the E-tube with videoendoscopic techniques may be an important and useful method to examine tubal function and its dysfunction. This technique also offers new direction to Eustachian tuboplasty, and according to development of technology, it may be possible to treat the patients with middle ear disease without damaging tympanic membrane through Eustachian tube.
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