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研究生:陳畊仲
研究生(外文):Ken-chung Chen
論文名稱:年齡及表面處理對於骨母細胞貼附鈦植入物之影響
論文名稱(外文):The effect of age and surface treatment on osteoblast reponse to titanium implant in vitro
指導教授:李澤民李澤民引用關係王東堯
指導教授(外文):Tzer-Min LeeTung-yiu Wong
學位類別:碩士
校院名稱:國立成功大學
系所名稱:口腔醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:69
中文關鍵詞:骨整合表面粗糙處理顎骨骨母細胞年齡
外文關鍵詞:agesurface roughness treatmentjaw bone osteoblastosseointegration
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人工植牙植入顎骨後,需有良好之骨整合才能進行後續之假牙補綴,骨整合即為宿主細胞與生物相容性材料之間產生一緊密之結合;因此,目前研究如何提高骨整合成功率多著重於研究材料本身特性及如何促進細胞與材料之間之交互作用;較少見有關宿主相關因素如年齡或性別對於骨整合是否有影響。僅有少數以股骨進行之相關實驗,報告年齡確實會影響骨整合,但缺乏顎骨相關之研究及結合材料表面處理之研究。故本研究目的是希望以顎骨分離出之骨母細胞於不同表面處理之鈦試片上進行研究,探討不同年齡來源之顎骨骨母細胞是否對骨整合過程造成影響及有何影響,與表面處理有何相關性。本實驗之材料與方法如下:組織來源為自不同年齡層之人體取出之上下顎骨;於體外成功培養出骨母細胞後,再將細胞種植於不同表面處理之鈦試片;各為噴砂處理、噴砂加酸蝕以及微弧陽極氧化。試片於表面處理後先行測試其表面粗糙度及以電子顯微鏡觀察表面型態,再將細胞培養於試片;於種植後第3,6,12,24小時以電子顯微鏡觀察細胞貼附性,及於第3、7、14天以鹼性磷酸脢活性(ALP activity),Alarmar blue assay監測細胞分化及增殖。實驗結果顯示,本次實驗所使用之方法可成功培養出顎骨骨母細胞,而細胞種植於試片後,於試片上生長之細胞型態並無太大差異;而於細胞增生方面,陽極氧化表現最好,而不同年齡層則無明顯差異;細胞分化方面於不同年齡層,不同表面之間並無差異。
故本實驗結論為年齡對於顎骨造骨母細胞於鈦試片上之型態、增生及分化無顯著影響,與部分臨床研究結果相符,但仍須更多之樣本,以得到更正確,更有證據之實驗結果。
Osseointegration define as “the direct structural and functional connection between living bone and the surface of a load-bearing artificial implant”,excellent osseointegration can reach a function bearing anchorage of an endoosseous implant in the surrounding bone. The host response and the properties of implant surface are considered as key factors for the process of its integration, but current data emphasize at material itself and cell-material interaction to increase success rate;however, little is known about the effects of aging on the molecular responses of bone cells, especially jaw bone osteoblasts , to implant materials and its surface teatment. The purpose of our study is to investigate the influence of different implant surface topographies and different age on the expression of differentiation/proliferation markers on jaw bone osteoblasts of human.Titanium disc with different surface treatments of sandblasting、sandblasting and acid etching、microarc oxidation surfaces were produced and surface roughness was detected by profilemeter and topography were observed by SEM. Tissue were harvested from human jaw bone and culture to osteoblast in vitro, then seed onto titanium disc.The behavior of different aged osteoblast from human grown on all surfaces were compared through determination of alkaline phosphatase activity and Alarmar Blue assay to determine cell differentiation and proliferation. And the result show that first, no difference at cell morphology between different surface treatment; second, MAO surface is better to proliferation but no difference between different age about cell proliferation; third, no difference of cell differentiation between different surface and age
So we conclude that no significant influence of host age to the interaction of jaw bone osteoblast and surface treatment, means that age maybe no significant influence to osseointegration; and it is compatible with some clinical studies. But the result need more samples to get more powerful evidence.
Chapter 1. Introduction ......................................................... 1

1.1背景………………………………………………………………..1
1.2 骨整合(osseointegration)及其過程……………………………...2
1.2.1骨整合過程之材料反應(material response)…………………....2
1.2.2骨整合過程之宿主反應(host response)………………………..2
1.3文獻回顧...........................................................................................4
1.3.1 材料特性………………………………………………………4
1.3.2 細胞與材料交互作用…………………………………………6
1.3.3 陽極氧化表面(micro-arc oxidation,MAO)…………………....7
1.3.4 宿主本身因素…………………………………………………9
1.4 研究背景........................................................................................10
1.5 研究目的…………………………………………………………12
1.6 研究重要性………………………………………………………12

Chapter 2. Materials and Methods ......................................13

2.1 實驗儀器與實驗用藥品………………………………………..13
2.1.1實驗儀器……………………………………………………..13
2.1.2實驗用藥品…………………………………………………..17
2.2實驗步驟與方法...........................................................................19
2.2.1 鈦試片及其表面處理.............................................................19
2.2.2表面處理……………………………………………………..19
2.2.2.1 噴砂(Sandblasting)……………………………………….19
2.2.2.2噴砂酸蝕(SLA)………………………………………..19
2.2.2.3陽極氧化(MAO)………………………………………….19
2.2.3 表面處理性質分析………………………………………….20
2.2.3.1 表面型態觀察……………………………………………20
2.2.3.2 表面粗糙度測量…………………………………………20
2.2.3.3表面粗糙度測量參數……………………………………..20
2.2.4 顎骨造骨母細胞之初級培養……………………………….20
2.2.4.1組織樣本來源…………………………………………….20
2.2.4.2 細胞培養流程……………………………………………21
2.2.5 造骨母細胞鑑定(characterization)………………………….22
2.2.6 細胞種植於試片…………………………………………….23
2.2.7 細胞型態觀察……………………………………………….24
2.2.8 細胞增生測定(Proliferation assay)…………………………24
2.2.9 細胞分化測定(Differentiation assay)……………………….25
2.3 實驗流程………………………………………………………..28
2.4 統計方法………………………………………………………..29

Chapter 3. Results.................................................................. 30

3.1骨組織樣本收集………………………….……………………...30
3.2顎骨細胞初級培養……………………………………………....31
3.2.1細胞型態……………………………………………………....31
3.2.2造骨母細胞鑑定……………………………………………...32
3.3鈦試片表面特性分析…………………………………………....33
3.3.1 表面型態觀察………………………………………………..33
3.3.2 表面粗糙度……………………..…………………………....35
3.4細胞型態觀察…………………………………….……………...36
3.5細胞增生測定(Proliferation assay)…………………………….46
3.5.1 表面處理差異………………………………………………..46
3.5.2 表面處理與年齡之影響……………………………………..47
3.6細胞分化測定(Differentiation assay)…………………………..51
3.6.1 表面處理差異………………………………………………..51
3.6.2 表面處理與年齡之影響……………………………………..52
3.6.3 實驗結果總結…………………………………………………56
3.6.3.1 樣本收集…………………………………………….……..56
3.6.3.2 細胞初級培養……………………………………………...56
3.6.3.3表面粗糙度………………………………………………....56
3.6.3.4 細胞型態…………………………………………………...56
3.6.3.5 細胞增生測定……………………………………………...56
3.6.3.6 細胞分化測定…………………………………………..….57

Chapter 4 Discussions……………………………………….58

4.1 細胞型態與表面處理……………………………………………58
4.2 細胞增生及分化與表面粗糙處理…………………………........58
4.3 年齡對骨細胞增生及分化之影響……………………………....59
Chapter 5 Conclusions………………………………………60
參考文獻……………………………………………………...61
附件一………………………………………………………...64
參考文獻
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