- 學位論文
鈦表面成骨細胞之遷移行為及貼附機制
Migration Behavior and Adhesion Mechanism of Osteoblasts on Titanium Surfaces
摘要
研究目的: 牙科純鈦植體手術植入骨內後，會與周圍骨骼緊密結合，稱為骨整合。鈦金屬表面擁有正確的化學組成與表面形貌能夠促進細胞的貼附及增加某些成骨細胞相關基因的表現，對於骨整合極為關鍵。在本論文中，將純鈦蒸鍍於蓋玻片表面，並適當控制其蒸鍍厚度成為半透明，使其可以用於觀察細胞在鈦表面的行為。此種鈦表面命名為“TiGlass”，首先以儀器進行了多項表面的鑑定，其後以此鈦表面進行了一系列的實驗，期能深入探討成骨細胞貼附在鈦表面的詳細機制。 材料與方法: 本論文中使用的“TiGlass”為半透明之鈦表面，由於其光學性質優良，可以應用於高倍數穿透式光學顯微鏡，使得原本無法以傳統方式觀察的不透明鈦金屬表面變為一可培養細胞及觀察的基質，能夠觀察正常細胞在鈦表面的生理及爬行，為雷射掃描共軛焦顯微鏡所無法達到之功能。“TiGlass”首先經過掃描式電子顯微鏡(SEM)，表面元素分析(EDX)，及原子力顯微鏡(AFM)等方式鑑定表面性質以便了解其物理特性。之後，“TiGlass”應用在成骨細胞的隨機遷移分析，量測成骨細胞附著後相關基因的表現，以及用於觀察全細胞免疫螢光染色。免疫螢光染色使用兩種抗體，進行雙重染色觀察不同抗原之間的相對分佈關係及分析關連性。首先，先觀察vinculin及F-actin之間的關係，然後分別於含有胎牛血清及不含胎牛血清的環境下分析vinculin及pFAK之間的關係，藉以了解focal adhesion之處的FAK是否有磷酸化及進行訊息傳遞。其後，以不同組合蛋白次元體(integrin subunit)針對vinculin進行雙重免疫螢光染色，縮小可能參與細胞貼附的integrin subunit種類。最後，挑出integrin subunit α5, αν, β3出來進一步分析，以integrin subunit α5, αν分別針對β3進行雙重染色，及以integrin subunit α5, αν, β3, ανβ3 針對 F-actin 雙重染色，藉以了解integrin subunit α5, αν,β3在成骨細胞貼附至鈦表面過程中所參與的角色。 結果: 由初級培養所得的大鼠顱骨成骨細胞在“TiGlass”表面的隨機遷移分析顯示蒸鍍在玻璃表面的鈦會增加成骨細胞的遷移速度，而且大幅縮短從附著在表面到開始遷移的時間差。使用MG-63 成骨細胞作相同的隨機遷移分析也得到極為類似的結果，可見鈦造成的此種效果與細胞種類無關。以平滑的“TiGlass”表面用於成骨細胞相關基因表現則發現與對照組之間沒有明顯差異。“TiGlass”用於穿透式螢光顯微鏡的細胞表面螢光觀察則可清楚看到actin filament 與vinculin 在空間上的互相重疊關係。利用雙重免疫螢光染色的方式，可以發現在focal adhesions之處的FAK均有磷酸化的現象，因而可以推測成骨細胞以integrin 附著在鈦及玻璃的表面。進一步研究成骨細胞在玻璃表面所形成的focal adhesions組成發現主要由αν 與β3 integrin subunits所構成。然而，除了上述兩者，成骨細胞在“TiGlass”表面的focal adhesions組成可能尚有integrin subunits α5參與其中。以integrin subunits α5，αν，β3，及 ανβ3與F-actin做雙重免疫螢光染色的結果則也顯示出integrin subunits α5對於成骨細胞貼附在鈦表面扮演輔助性角色。 結論: 將表面蒸鍍純鈦蓋玻片應用於細胞生物學可發現此層鈦鍍膜會改變成骨細胞的遷移行為模式，增強初期貼附能力及增加成骨細胞的遷移速度。利用顯微鏡作細胞表面螢光觀察則有助於分析成骨細胞在鈦表面貼附所產生的focal adhesion之組成。綜合一系列研究之結果，成骨細胞貼附在鈦表面所產生的focal adhesion主要由 integrin ανβ3所構成。但是除了對照組的玻璃表面也有的integrin ανβ3以外，鈦表面上的組成尚多了α5 subunit。此組成上的不同有可能是成骨細胞能夠在鈦表面上較早開始遷移及以較快的速度遷移的主要原因。
並列摘要
Purpose: Titanium with proper surface composition and topography is crucial for dental implant osseointegration, and has been known to enhance cell adhesion and promote expression of specific osteoblastic genes. In this study, a translucent titanium coating on glass coverslip, “TiGlass”, was introduced as a new tool for direct observation of cell behavior on titanium surface. The surface was characterized, and a series of studies were conducted to clarify the mechanism of osteoblastic adhesion on titanium surface. Material and Methods: The translucent titanium surface offers excellent optical characteristics that facilitate transmitted light observations under the optical microscopes, thereby transforming the opaque metal into an observable titanium matrix. With laser scanning confocal microscopy, only cells expressing fluorescent proteins could be observed on titanium surfaces. With TiGlass, migration of normal cells can now be observed under the optical microscopes. SEM, EDX, and AFM analyses were performed on TiGlass to understand its physical properties. Random migration analysis, osteoblastic gene expression, and immunofluorescence cell staining on TiGlass were then studied. In the immunofluorescent double staining, vinculin was first tested with F-actin, and then pFAK was tested with vinculin to see whether the focal adhesions were activated. Various integrin subunits were then tested with vinculin to study the composition of activated focal adhesions. Integrin subunit α5, αν were tested against β3; Integrin subunit α5, αν, β3, ανβ3 were tested with F-actin, respectively. Results: Random migration analysis of the primary rat calvarial osteoblasts on TiGlass revealed that the titanium coating enhanced the migration speed of the MG-63 osteoblasts, and significantly shortened the time lag for the initial migration behavior. The same phenomenon was found in random migration analysis of the rat calvarial osteoblasts. Further study of osteoblastic gene expression on this smooth titanium surface revealed no significant change. Co-localization of actin filament and vinculin was found on TiGlass under epifluorescent microscopy. Immunofluorescent double staining revealed all focal adhesions contained activated FAK on both surfaces. The osteoblast was inferred to made adhesion to titanium and glass through integrins. The focal adhesions on glass were found to be composed of integrin subunits αν and β3. However, on “TiGlass”, integrin subunits α5 might have supplemented the adhesion to titanium. Results from double staining of integrin subunits α5, αν, β3, and ανβ3 with F-actin also supported integrin subunits α5 might have involved in adhesion of titanium. Concluison: The application of translucent titanium-coated coverslip in cell-biology studies revealed that titanium coating altered the migration pattern of osteoblasts. The results suggested that titanium promotes initial adhesion and accelerates osteoblast migration. Epifluorescence microscopic studies were conducted to find out the composition of focal adhesion on titanium surface. Collectively, we may conclude the osteoblasts adhere to titanium coating through focal adhesions composed of most integrin ανβ3 and some integrin combination of α5 subunit, resulting in faster and earlier migration on titanium.