臺灣博碩士論文加值系統

摘 要
本研究是以人工植體金屬材料-鈦(Titanium)金屬做為研究對象，針對材料特性、材料與細胞培養、材料與器官培養等三大主題的評估。首先就材料特性方面，可發現純鈦金屬表面TM經過酸蝕之後TA，其表面形成微米大小的蜂窩狀粗糙孔洞，其平均粗糙度(Ra)可達2.28μm；比一般酸蝕表面要大。根據表面成分分析發現TA較TM有較高比例的氧化表層。在細胞貼附與生長方面，發現不具毒性且有良好的生物相容性，由SEM觀察可發現細胞在TA的貼附形態較TM平坦且有較多的細小薄偽足(filopodia)攀附，預期在移植時應可增進植體表面和骨組織之間的機械咬合(mechanical interlocking)強度。在器官培養方面；將TM、TA與新生鼠頭蓋骨共培養，觀察在缺陷骨組織中的材料孔洞邊緣生長情形與鈣化程度。結果由倒立式光學顯微鏡發現，TA與TM皆具有骨引導作用，骨母細胞從缺陷骨組織孔洞邊緣往材料邊緣貼附；另外由SEM觀察與定量分析，發現器官培養五週後，TA的底部、側面、上表面的新生類骨細胞較TM多且較厚，TA鈣化程度也較TM明顯。因此TA材料為一理想人工植體。
關鍵字：鈦金屬、牙科植體，骨先驅細胞、骨整合、生物相容性。

Abstract
The object of this research is titanium, a metal material of synthetic implant, and this research evaluates respectively the three categories of (1) the nature of the material, (2) the material and cell culture, and (3) the material and organ culture. Having been etched, the surface layer of titanium has mm-sized beehive-like coarse holes, the roughness of which can be as big as 2.28μm, which is bigger than the holes commonly seen on the etching surface. We can find in the analysis on the component parts of the surface layer that the layer is more oxidized in TA than in TM. In cell adhesion and growth, we find that it is non-poisonous and bio-compatible. Through the SEM observation we find that the cell morphology is smoother in TA than in TM and there are more filopodia attached; therefore, the mechanical interlocking between the implant surface and osseous tissues can be expected to be enhanced. In organ culture, the frontal head bone of the newborn mouse is cultured with TM and TA, and we observed the growth and calcification of the defected bone tissue on the rims of the holes. Through the contra-photomicroscope, we observed that TA and TM are osteoconductive and that the bone cells attached themselves to the rim of the material from the rims of the holes. Furthermore, through the SEM observation and quantitative analysis, we found that, the organ having been cultured for five weeks, there were more and thicker cells on the bottom, side and surface of the TA than on those of TM, and the calcification of TA is more obvious than that of TM. To sum up, TA is an ideal implant material.
Key words：titanium, dental implant, osteoprogenitor cells, osseointegration, biocompatibility

目 錄
第一章 緒論…………………………………………………………......1
1-1 研究動機及目的………………………………………………….…...1
1-2 研究設計………………………………………………………………2
第二章 文獻回顧………………………………………….............……3
2-1人工植牙………………………………………………………….…....3
2-2鈦金屬材料特性…………………………………………………….....3
2-3牙科植體改質種類…………………………………………………….4
2-3.1 純鈦植體(Pure titanium)………………………………….…...4
2-3.2鈦電漿噴覆(Titanium plasma spray簡稱T.P.S.)植體……......5
2-3.3氫氧磷灰石覆蓋質體(Hydroxyapatite)……………………......5
2-3.4圓珠狀燒結覆蓋植體 (Ball-beads sintered implant)……...…..6
2-4人工牙科植體的演進………………………………………………....7
2-5植體與表面粗糙度的探討……………………………………..….....9
2-6表面粗糙度與細胞貼附的關係……………………………………....11
2-7利用生物機械測試(Removal torque value；RTV)
來評估骨和植體交介面的強度 ……………………………………...13
第三章 實驗材料與方法…………………………………………….16
3-1實驗流程………………………………………………………….......16
3-2實驗材料製備…………………………………………………….......18
3-3材料特性分析 …………………………………………………..…...19
3-3-1成分分析(ESCA) …………………………………………….….…19
3-3-2表面形態分析…………………………………………………........20
3-3-2.1掃描式電子顯微鏡（SEM）觀察材料表面特徵……..........20
3-3-2.2以探針式-3D表面粗糙儀測表面粗糙度…………….…….21
3-3-2.3原子力顯微鏡（Atomic force microscope，AFM）
觀察表面形態…………………………………………..…...23
3-4細胞培養評估………………………………………………….……..26
3-4-1 材料萃取液細胞培養……………………………………………..27
3-4-2材料與細胞培養……………………………………………............29
3-4-2.1新生鼠頭蓋骨骨母細胞萃取…………………………….......29
3-4-2.2細胞生長評估(DNA分析) …………………………………..30
3-4-2.3電子顯微鏡 (SEM)細胞形態觀察…………………….…….32
3-5器官培養評估…………………………………………………….......33
3-5-1新生幼鼠頭蓋骨器官培養--體外測試 ……………………….......34
3-5-2 器官培養實驗評估……………………………………………......36
3-5-2.1 定性分析………………………………………………..…...36
3-5-2.2 定量分析…………………………………………………….37
第四章 結果與討論…………………………………………………..38
4-1成分分析(ESCA)……………………………………………….…….38
4-2表面特性分析……………………………………………….………..39
4-2-1掃描式電子顯微鏡（SEM）材料表面觀察………..……….39
4-2-2微米粗糙度測量………………………………………...........39
4-2-3原子力顯微鏡（Atomic force microscope，AFM）
觀察表面形………………………………………………….....40
4-3細胞培養評估………………………………………………….……..41
4-3-1材料浸泡抽出液細胞培養…………………………….……….41
4-3-2 細胞貼附生長評估(DNA分析)……………………….……...42
4-4電子顯微鏡 (SEM)細胞形態觀察…………………………………..42
4-5器官培養實驗評估…………………………………………………...44
4-5-1定性分析…………………………………………………….……..44
4-5-2 定量分析…………………………………………………….…….47
第五章 結論與展望………………………………………………..…49
第六章 參考文獻……………………………………………....51
表目錄索引
表1.TM與TA表面元素成分分析(XPS)……………………….……….56
表2. TM與TA表面元素的鍵結能(binding energy)分析(XPS)…..........56
表3.TA與TM的微米粗糙度……………………………………………57
表4. 新生類骨組織生成面積佔材料孔洞總面積的比率……………...57
圖目錄索引
圖4-1(a)對照組TM (machined surface titanium)(b)實驗組TA
(acid etching titanium)………………………………………...…58
圖4-2(a)(b)TM之ESCA光譜與鍵結分析……………………………...59
圖4-2(a)(b)TA之ESCA光譜與鍵結分析……………………………....60
圖4-4(a)(b)(c)對照組TM表面的SEM照片： (a) 100×(b) 1000×
(c) 5000×……………………………………………………………….61
圖4-5(a)(b)(c)對照組TA表面的SEM照片：(a) 100×(b) 1000×
(c) 5000×……………………………………………………….…........62
圖4-6 (a) TM材料之表面形貌圖與截面圖……………………………...63
圖4-6 (b) TA材料之表面形貌圖與截面圖…………………….…........64
圖4-6 (C)TA與TM材料之表面相分佈圖……………………………..65
圖4-7 L929與TA、TM浸泡液(120h)做細胞毒性測試，培養24h、
48h的生長曲線直條…………………………………………………66
圖4-8(a)MG-63與TA、TM鈦金屬材料共培養24h、48h的生長曲
線直條圖……………………………………………………………….67
圖4-8(b)Osteoblast與TA、TM鈦金屬材料共培養24h、48h的生長
曲線直條圖。………………………………………………………….68
圖4-8(c)L929與TA、TM鈦金屬材料共培養24h、48h的生長曲線
直條圖。……………………………………………………………….69
圖4-9對照組(TM)、實驗組(TA)與L929細胞培養24h，貼附形態
觀察。 …………………………………………………………………70
圖4-10對照組(TM)、實驗組(TA)與MG-63細胞培養24h，貼附形
態觀察。 ………………………………………………………………71
圖4-11對照組(TM)、實驗組(TA)與Osteoblast細胞培養24h，貼附
形態觀察。 ……………………………………………………………72
圖4-12新生鼠頭蓋骨(a)正中央釘鑿一6 mm見方的洞(b)實驗組(TA)材料植入情形(c)對照組(TM)材料植入情形………………….……..….73
圖4-13 (a)(b)(c) 光學顯微鏡觀察對照組(TM)之鈦金屬材料於新生
鼠頭蓋骨中，經過一、三、五週培養後的情形。……………………….74
圖4-14 (a)(b)(c) 光學顯微鏡觀察實驗組(TA)之鈦金屬材料於新生
鼠頭蓋骨中，經過一、三、五週培養後的情形。………………………75
圖4-15以 SEM觀察不同材料於新生鼠頭蓋骨中經過五週培養，
材料底部新生骨細胞生長情形。(a)TM、(b)TA…………………….…76
圖4-16以 SEM觀察不同材料於新生鼠頭蓋骨中經過五週培養，
材料側面新生骨細胞生長情形。(a)TM、(b)TA……………….……..77
圖4-17以 SEM觀察TM材料於新生鼠頭蓋骨中經過五週培養，
材料表面新生骨細胞生長情形。(a)100× (b)400×………………..….78
圖4-18以 SEM觀察TA材料於新生鼠頭蓋骨中經過五週培養，
材料表面新生骨細胞生長情形。(a)100× (b)400×…………………...79
圖4-19 由光學顯微鏡觀察不同材料於新生鼠頭蓋骨中經過五
週培養，von Kossa’s染色，25 x。(a)TM、(b)TA…………………..........80

第六章文獻參考
1.Branemark PI, Breine U, Adell R, Hansson BO. Intraosseous anchorage of dental prostheses. I Experimental studies. Scand J Plast Reconstr Surg 1969；3：81.
2. Cho SA, Jung SK. A removal torque of the laser-treated titanium implants in rabbit tibia. Biomaterials 2003；24：4859-4863.
3. Gaggl A, Schultes G, Muller WD, Karcher H. Scanning electron
microscopical analysis of laser-treated titanium implants surfaces
a comparative study. Biomaterials 2000；21：1067-73.
4. Cho SA, Park KT. The removal torque of titanium screw inserted in rabbit tibia treated by dual acid etching.Biomaterials 2003；24： 3611—3617.
5. Rams TE, Roberts WE. Clinical and microbiologicals findings of newly inserted hydroxylapatite-coated and pure-titanium human dental implants. Clin. Oral Implants. Res 1991;2:121.
6. Gaggl A, Schultes G, MuKller WD, Karcher H. Scanning electron microscopical analysis of laser-treated titanium implant surfaces- comparative study. Biomaterials 2000；21：1067-1073.
7. 牙科植體論文集， 台北市牙科植體學學會編著，1999，21-34
8. 金屬鈦（理論與應用）， 賴耿陽編著，復漢出版社，1990，10-30
9. Schulte J.Manufacturing tolerances of the exrternal hex of six implant systems.Implant Dent.istry.Spring 1994:51-53.
10.Piattelli A,Scarano A,Piattelli M,Calabrese L.Direct bone formation on sand-blasted titanium implant：an experimental study. Biomaterials 1996；17：1015-1018.
11.Wahlbeck PG,Gilles PW, Ceram JA. Soc 1966；49：180.
12. 臨床口腔植體學，台北市牙科植體學會編著1999：24-30.
13. Schroeder A, Pohler O, Sutter F, Gewebsreaktion auf ein. Titan-Hohlzylinderim-plantat mit Titan-Spritz schichtoberflache Schweiz Monatsschr Zahnheilk 1976；86：713.
14. Cool SD et al. Interface mechanics and histology of titanium and hydroxylapatite-coated titanium for dental implants applications. International J of Oral&Macillofacial Implants 1987；2：15-22.
15. Delegard K, Gottlander M. Nobelpharma News Vol. 4, No. 3. 1990: 3.
16. Buser D, Schenk RK, Steinemann S, Fiorellini JP, Fox CH, Stich H. Influence of Surface Characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs. J. of Biomed Mater Res 1991；25：889-902,.
17. Baier RE, Meyer AE. Implant surface preparation. International J. of Oral& Maxillofacial Implants 1988；3：9-20.
18. Sul YT. On the bone response to oxidized titanium implants.Ph.D. thesis, Department of Biomaterials/Handicap Research,University of
Gothenburg, Sweden 2002.
19. Sung-Am C,Sang-Kyoo J. A removal torque of the laser-treated titanium implants in rabbit tibia.Biomaterials 2003; 24：4859—4863.
20. Bower KT,Keller JC,Randolph BA,Wick DC,Michasls CM. Optimization of surfaces micromorphology for enhanced osteoblast responses in vitro. Int J Oral Maxillofac Implants 1992；7：302-320.
21. Mustafa K, Wennerberg A, Wroblewski J, Hultenby K, Lope BS,
Arvidson K. Determining optimal surface roughness of TiO (2) blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone. Clin Oral Implant Res 2001；12：515-525.
22. Itala AI, Ylanen HO, Ekholm C, Karlsson KH, Aro HT. Pore diameter
of more than 100 mm is not requisite for bone ingrowth in rabbits. J
Biomed Mater Res 2000；58：679-683.
23. Wie H, Hero H, Solheim T. Hot isostatic pressing-processed
hydroxyapatite-coated titanium implants: light microscopic and
scanning electron microscopic investigation. Int J Oral Maxillofac
Implants 1998；13：837-844.
24. Kim H-M, Miyaji F, Kokubo T, Nakamura T. Preparation of bioactive Ti and its alloys via simple chemical surface treatment.J Biomed Mater Res 1996；32：409-417.
25. Overgaard S, Soballe K, Lind M, Bunger C. Resorption of hydroxyapatite and fluorapatite coatings in man. An experimental
study in trabecular bone. J Bone Jt Surg (Br) 1997；79：654-659.
26. Muller WD, Seliger K, Meyer J. The improvement of adhesion of
polymer of titanium surface after treatment with TEA-CO2 laser
irradiation. J Mater Sci Lett 1994；5：692-694.
27. Shigematsu I, Nakamura M, Saitou N, Shimojima K. Surface
hardening treatment of pure titanium by carbon dioxide laser.
J Mater Sci Lett 2000；19：967-970.
28. Buser D, Nydegger T, Oxiand T, et al. Influence of surface
characteristics on the interface shear strength between titanium implants and bone: a biomechanical study in the maxilla of miniature pigs. J Biomed Mater Res 1999 ; 45 : 75-83.
29. Martin JY, Schwartz Z. Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG-63). J Biomed Mater Res 1995；29：389-401.
30. Thomas KA, Cook JKJF, Cook SD, Jarcho M. The effect of surface macrotexture and hydroxylapatite coating on the mechanical strengths and histologic proles of titanium implant materials. J Biomed Mater Res 1987；21：1395—1414.
31. Wennerberg A, Ektessabi A, Albrektsson T, Johansson C,Andersson B. A 1-year follow-up of implants of differing surface roughness placed in rabbit bone. Int J Oral Maxillofac Implants 1997；12：486-494.
32. Batzer R, Liu Y, Cochran DL, Szmuckler-Moncler S, Dean DD,Boyan BD, Schwartz Z. Prostaglandine mediate the effects of titanium surface roughness on MG63 osteoblast-like cells and alter cell responsiveness to 1α,25-(OH)2D3. J Biomed Mater Res 1998；41：489-496.
33. Davies JE. Mechanisms of endosseous integration. Int J prosthodont. 1998；11：391-401.
34. Ahariz M, Mouhyi J, Louette P, Van Reck J, Malevez C, Courtois P. J Biomed Mater Res 2000；552：567.
35. Cacciafesta P, Hallam KR, Watkinson AC, Allen GC, Miles MJ, Jandt KD.Visualisation of human plasma fibrinogen adsorbed on titanium implant surfaces with different roughness. Sur.Sci 2001；491： 405-420.
36. Gemmell CH, Park JY. Initial blood interaction with endossteal implant Materials. Bone engineering, Davies JE. em squared inc. Toronto Canada 2000：p. 108-117.
37. Park JY, Davies JE. Red blood cell and platelet interactions with titanium implant surface. Clin Oral Irnpl Res 2000；11：530-539.
38. Dziedzic DM, Beaty KB, Brown GN, Heylmunt T, Davies JE. Bone growth in metallic bone healing chambers In: smith DC et al, eds. Proceedings of the 5th Biomaterials Congress. Volume 2. Toronto Ontario : University of Toronto Press 1996: 124.
39. Han CH, Johannson CB, Wennerberg A, Albrektsson T. Quantitative
and qualitative investigations of surface enlarged titanium and titanium alloy implants. Clin Oral Implant Res 1998；9：1-10.
40. D''Lima DD, Lemperle SM, Chen PC, Holmes RE, Colwell CW.Bone response to implant surface morphology. J Arthroplasty 1998；13：928-934.
41. Ronold HJ, Ellingsen JE. The use of a coin shaped implant for direct in situ measurement of attachment strength for osseointegrating
biomaterial surfaces. Biomaterials 2002；23：2201-2209.
42. Ronold HJ, Ellingsen JE. Effect of micro-roughness produced by TiO2 blasting-tensile testing of bone attachment by using coin-shaped implants. Biomaterials 2002；23：4211-4219.
43. Buser D, Nydegger T, Hirt HP, Cochran DL, Nolte LP. Removal torque values of titanium implants in the maxilla of miniature pigs. Int J Oral Maxillofac Implants 1998；13：611-619.
44. Lazzara RL, Porter SS, Testori T, Galante J, Zetterqvist L. A
prospective multi-center study evaluating loading of Osseotite
implants two months after placement: one-year results. J Esthet Dent 1998；10：280-289.
45. Lazzara RJ, Testori T, Trisi P, Porter SS, Weinstein PL. A human
histological analysis of osseotite and machined surfaces using mplants with two opposing surfaces. Jnt J Periodont Restor Dent 1999；19： 117-129.
46.Jandt KD.Atomic force microscopy of biomaterials surfaces and interfaces.Sur. Sci 2001；491：303-332.
47. Merritt K, Brown SA.Effect of protein and pH on pretting corrosion and ion release, J Biomed Mater Res 1988：22
48. Williams RL, Brown SA, Merrit K.Electrochemical studies on the
influence of proteins on the corrosion of implant alloys. J Biomed Mater Res 1998；9：181-186.
49. Huang LL,Sung HW,Tsai CC,Huang DM.Biocompatibilty study of a biological tissue fixed with a naturally occurring crosslinking reagent.J.Biomed Mater Res 1998；42：568-576.
50. James R,Hawker Jr.Chemiluminescence-based BrdU ELISA to measure DNA synthesis. Journal of Immunological Methods 2003；274：77-82.
51. Holy CE, Shoichet MS, Davies JE, J Biomed Mater Res 2000；51： p.376.
52. Fenga B, Wenga J, Yangb BC, Qua SX, Zhangb XD. Characterization of surface oxide lms on titanium and adhesion of osteoblast. Biomaterials 2003；24：4663—4670.
53. Oliveira1 PT, Nanci A. Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells. Biomaterials 2004；25：403—413.
54. Dalbya MJ, Giannarasa D, Riehlea MO, Gadegaarda N, Affrossmanb S, Curtisa ASG. Rapid broblast adhesion to 27nm high polymer demixed nano-topography. Biomaterials 2004；25：77—83.
55. Schwartz Z, Boyan BD. Underlying mechanisms at the bone-biomaterial interface. J Cell Biochem 1994；56：340-347.
56. Pillar RM,Deporter DA,Watson PA,Valiquettl N.Dental implamt design —Effect on bone remodeling. J Biomed Mater Res 1991；25：467-483.
57. Boyan BD, Batzer R, Kieswetter K, Liu Y, Cochran DL, Dean DD, Schwartz Z. Titanium surface roughness alters responsiveness of MG-63 osteoblast-like cells to 1α,25-(OH)2D3. J Biomed Mater Res 1998；39：77-85.
58. Prigodich RV,Vesely MR.Characterization of the complex between bovine osteocalcin and type I collagen.Archives of Biochemistry and Biophysics 1997；2：339-341.