植體表面以原子層沉積技術奈米鍍膜氧化錫後之表現

Abstract

影響骨整合的因素很多，其中最重要的一個因素即是植體的表面處理。過去的研究顯示，植體表面經過微米等級處理後可以提高生物相容性及骨整合能力；而奈米數值範圍的表面處理可以增加細胞分化速度以及功能性表現。原子層沉積技術能在物體表面形成原子等級、厚度均勻且覆蓋率佳的鍍膜層，因此我們選擇使用原子層沉積技術作為本實驗的鍍膜方法。首先我們選用粗糙度與市面植體相仿，介於1-1.5 μm的商業級純鈦和鈦六鋁四釩之金屬試片，分別鍍上20 nm、50 nm、100 nm的氧化錫，接著進行材料分析及生物相容性的實驗：材料分析包含以電子顯微鏡觀察表面形態、原子力顯微鏡量測奈米等級粗糙度及3D形態、細微形狀測定儀測量微米等級粗糙度；細胞實驗部分包含第1、4、7、10、14天細胞存活率、第4、7、10、14天鹼性磷酸酶試驗、第4、7、10、14天骨鈣素試驗以及第1、4、7、10、14天免疫螢光染色。 掃描式電子顯微鏡可以觀測到未鍍膜試片表面具有各種大小，各種尺寸的不規則孔洞，鍍膜後的氧化錫顆粒，散佈在各種孔洞之中，隨著鍍膜厚度增加，顆粒堆積也相對明顯。在原子力顯微鏡下量測奈米等級粗糙度，發現鍍膜會增加表面粗糙度。細微形狀測定儀量測微米等級粗糙度，鍍膜後表面粗糙度反而下降。細胞存活率結果顯示，不論有無鍍膜皆有良好的生物相容性。在鹼性磷酸酶試驗中，所有組別均較控制組為佳，未鍍膜試片表現最好，氧化錫鍍膜組隨著厚度增加而遞減。骨鈣素試驗中，第14天未鍍膜組表現低於鍍膜組。免疫螢光染色結果顯示，控制組的細胞貼附完整且紡錘狀明顯，金屬試片組別的細胞骨架和偽足較為模糊，可能因為試片表面孔洞導致細胞往垂直方向生長，紡錘狀不明顯較偏向細梭狀。 綜合以上結果，因為20 nm鍍膜組在骨鈣素試驗表現較未鍍膜組佳，在其餘實驗中表性又較其餘鍍膜厚度佳，因此實際應用上可考慮20 nm氧化錫鍍膜組。

The surface treatment of dental implant plays an important role in osseointegration. According to the previous researches, the surface treatment of micron-scale can increase the biocompatibility and the osseointegration, while nano-level can increase performance of the cell differentiation. The atomic layer deposition technique provides uniform layer-by-layer film growth in atomic-level with excellent step coverage and conformal deposition on high aspect ratio structures. The uncoating and coating thickness in 20 nm, 50 nm, and 100 nm Tin oxide on Titanium and Ti-6Al-4V with roughness between 1-1.5 μm. The surface analysis was includes the observation of the surface morphology using scanning electron microscope, the roughness in nano-scale and the 3D configuration with atomic force microscope, and the roughness in micron-scale with surfcorder. The biocompatibility tests were including alamar Blue assay to measure of cell survival and growth, alkaline phosphatase assay and osteocalcin assay to valuate the mineralized markers in early and late stage respectively, and immunofluorescence assay to observe the cell morphology. The observation from scanning electron microscope indicated that the accumulation of particles increased with the rising coating thickness. When the thickness of coating was increased, the roughness in nanoscale was increased under the atomic force microscope. However, the surface roughness in micron-scale decreased under the sufcorder test. The results of alamar Blue assay, there were good biocompatibility in all groups. The results of ALP assay revealed that all groups a better outcome compared with TCPS, while the group without coating showed the best performance. The ALP level was decreased when the thickness of coating was increased. From the results of OCN assay, the uncoating groups showed least osteocalcin on 14th day. We can find out the cell adhesion and spindle-like shape are complete and significant in the control group with the observation from immunofluorescence assay. In the others group, the cytoskeleton and pseudopodium were not clear; it might be because the pores where HEMP grew were located in vertical wall of metal samples, and the cell body or pseudopodium was situated in the lateral surface of samples. Therefore, when the staining observed from the top view, the cytoskeleton and pseudopodium were defocused. To conclude, because the group of 20 nm in thickness performed better results of OCN than uncoated group, and better results of alarm Blue and ALP assays than other coating groups, we suggest 20 nm-SnO2 coating is best in practice.