探討不同植體表面處理對於骨分化及骨整合細胞之影響

The effects of implant surfaces treated in different methods on differentiation of bone marrow mesenchymal stem cells and osseointegration

鄧富元(Fu-Yuan Teng)

指導教授 ： 洪純正

高雄醫學大學/口腔醫學院/牙醫學系碩士班/博士(2016年)

摘要

鈦金屬因其優越高強度的機械性質、化學性質穩定度高及良好的生物相容性，因此運用在人工植牙的療程，突破了傳統牙科補綴治療的限制。半世紀以來純鈦金屬製成的人工牙根，其成功率都高達九成以上，但也隨著相關研究進行，對於人工植體的要求及改善，不再只是成功的骨整合，更希望能縮短骨整合時間，達到骨細胞、軟組織及人工植體三者間的相互配合達成美觀及健康的目的。
許多文獻都指出，植體上的表面粗糙度(roughness)、表面型態(topography)及氧化層(oxide films)為主要影響骨整合的因素。現今有許多表面處理方式，如噴砂(sandblasting)或陽極氧化(anodization)等，用來改質(modification)其表面的性質。因此，本論文第一部分【Study I】，我們運用現今常用的噴砂後酸蝕方法來改質鈦金屬表面之外，再使用tetracalcium phosphate(TTCP)來做第二次噴砂，並偵測其表面殘留物。結果顯示在運用TTCP噴砂之後，表面殘留氧化鋁明顯被TTCP顆粒取代。在細胞實驗中顯示TTCP能增強骨細胞的貼附與增生，此外對於上皮細胞及纖維母細胞則無不良影響。在鈦金屬植體表面測試貼附程度及細胞數目的交互相關性實驗中，發現上皮細胞相較纖維母細胞，對照組與改質後的植體表面具有較大改變，換言之上皮細胞較纖維母細胞較容易受植體表面特性所影響。更進一步發現植體表面改質後，其所產生的離子效應(ions effect)佔較重要的影響。根據本論文的第一部分研究，我們推測改質表面特性相較添加包覆離子特性物質，更能影響細胞在植體上的表現。
本論文第二部分【Study II】，我們運用另一常見於工業上改質金屬的方式"微電弧電漿氧化(micro-arc oxidation)"來改質植體表面。微電弧電漿氧化，是運用離子溶液環境中施加直流電壓，並產生微小電漿反應，在表面形成高温氧化局部環境，電漿高温氣化金屬表面與其接觸電解溶液氧化的相互作用影響下，將會形成多孔結構與氧化層結合的特殊表面，於鈦金屬表面生成銳鈦礦相二氧化鈦(Anatase-TiO2, A-TiO2)、金紅石相二氧化鈦(Rutile-TiO2, R-TiO2) ，等同於一般酸蝕噴砂的方式改良表面粗造度之外也改變二氧化鈦的結構，而二氧化鈦又是骨髓間質幹細胞形成骨母細胞及骨細胞的重要關鍵。不同於其他研究的是，本實驗我們運用H2SO4/H3PO4電解液當離子溶液以降低工作電壓，減少高壓電壓製作難度，實現結果工作電壓超過100V便開始有氧化反應，隨者電壓增加其平均粗糙度(average roughness)、孔洞大小與氧化層厚度都會增加，但在工作電壓超過200V下因孔洞變過大而使的多孔性質變差；而在工作電壓超過140V便開始有anatase的型態；在氧化層抗腐蝕能力上，隨著電壓提高而越佳，以工作電壓200V 具最強氧化層抗腐蝕能力。我們使用小鼠骨髓間質幹細胞在不同植體表面培養並進行骨化測試，發現骨髓間質幹細胞在工作電壓200V的改質植體表面表現較高的alkaline phosphatase(ALP)，且有明顯礦化的現象。統整以上，本論文研究顯示改善傳統噴砂後酸蝕改質植體表面的方式，不只是有表面粗糙度改變也進一步用新式微電弧電漿氧化方式。本論文最後部分【Study III】設計具有主動性質的仿生植體表面，以減少癒合時間並達到早期骨整合功能。骨形成蛋白2(Bone morphogenetic protein-2)是一個在成骨過程中，扮演重要的腳色。在本實驗中，運用合成具功能性矽烷薄膜，將BMP-2修飾在植體表面上。組別為: mechanical grinding (M)、電化學陽極處理修飾(electrochemical modification through potentiostatic anodization (ECH))、噴砂酸鹼處理(alkali heating, and etching (SMART))，並在這三組中有或無修飾植體表面的BMP-2進行活體外以及活體內實驗。在細胞測試中，ECH及SMART兩組修飾BMP-2，小鼠骨髓間質幹細胞的活性及分化能力有明顯提高。其中修飾過BMP-2的SMART，比其他組別更可以明顯的促進小鼠骨髓間質幹細胞在早期鹼性磷酸脢(ALP)的表現。在動物實驗中，修飾BMP-2的SMART組別表現出較高的骨整合以及骨生成的量。因此，在細胞及動物實驗，認為此修飾過骨形成蛋白2的SMART明顯促進細胞骨化及縮短骨整合的時間。

關鍵字

植體表面改質 ； 噴砂酸蝕 ； 離子效應 ； 微電弧電漿氧化 ； 骨形成蛋白2 ； 骨整合

並列摘要

Titanium is widely used in clinical because it’s excellent mechanical strength, highly stable chemical properties and biological compatibility. The breakthrough came in modern dental treatments with the application of titanium implants. The successful rate of dental implants is above 90 % during half century. With research of osseointegration in implant surfaces, we are progressive deepening in not only reducing healing time but also in achieving the harmony of osteoblasts, soft tissue and dental implants.
Many articles indicate that roughness, topography and oxide films of implant surface are significant factors affecting osseointegration. Many methods, such as sandblasting and anodization, can modify the characteristics of implant surface. The first part of this thesis 【Study I】, we used tetracalcium (TTCP) for secondly sandblasting after the commercial sandblasting and acid etching method. We detected the residual particles after secondly sandblasting and found majorly aluminum oxide replaced by TTCP. The residual particles, TTCP , can improve osteoblast the abilities of attachment and proliferation on the surface without affecting other cells, fibroblast and epithelium. We used titanium surfaces modified by this method to measure the relation of attachment and cell number among three differentiated cells; osteoblast, fibroblast and epithelium. We can find epithelium can be affected by the titanium surface than fibroblast. Moreover, we found the ions effect can play more a significantly role than coating on the surface.
Second part of this thesis【Study II】, implant surfaces were modified by the another industrial method, micro-arch oxidation (MAO). MAO is based on the conventional anodic oxidation of processing metals and alloys in aqueous electrolyte solutions accompanied by sparking micro-discharge due to dielectric breakdown, which is also regarded as the intrinsic feature differing from conventional anodizing. It synthesizes a film of titanium dioxide including anatase-TiO2 (A-TiO2), rutile-TiO2 (R-TiO2). Titanium dioxide has been proved playing a curial factor for bone marrow mesenchymal stem cells in osseointegration. In this study, we used H2SO4/H3PO4 as an electrolyte in order to reduce working voltage, and begun oxidation reaction after 100V. The porosity, surface roughness of the MAO coatings and thickness of oxide film increased with increasing working voltage. However, the property of porosity decreases as the working voltage was over 200 V. And anatase phase began to be detected as the working voltage was over 140 V.We cultured mouse bone marrow mesenchymal stem cells in different surfaces and detected mineralization. The highest expression of alkaline phosphatase (ALP) and alizarin red S were found and dominated mineralization in the titanium surface modified in the working voltage 200V. We can modify the characteristic of oxide film by micro-arch oxidation to improve differentiation of mouse bone marrow mesenchymal stem cell.
In the final part of this thesis【Study III】, we designed a biomimetic implant for reducing healing time and achieving early osseointegration to create an active surface. Bone morphogenetic protein-2 (BMP-2) is a strong regulator protein in osteogenic pathways. Due to hardly maintain BMP-2 biological function and specificity, BMP-2 efficient delivery on implant surfaces is the main challenge for the clinic application. In this study, a novel method for synthesizing functionalized silane film for superior modification with BMP-2 on titanium surfaces is proposed. Three groups were compared with and without BMP-2 on modified titanium surfaces in vitro and in vivo: mechanical grinding; electrochemical modification through potentiostatic anodization (ECH); and sandblasting, alkali heating, and etching (SMART). Cell tests indicated that the ECH and SMART groups with BMP-2 markedly promoted D1 cell activity and differentiation compared with the groups without BMP-2. Moreover, the SMART group with a BMP-2 surface markedly promoted early alkaline phosphatase expression in the D1 cells compared with the other surface groups. Compared with these groups in vivo, SMART silaning with BMP-2 showed superior bone quality and created contact areas between implant and surrounding bones. The SMART group with BMP-2 could promote cell mineralization in vitro and osseointegration in vivo, indicating potential clinical use.

並列關鍵字

modification implant surface ； sandblasting and acid etching ； ions effect ； micro-arch oxidation ； Bone morphogenetic protein-2 (BMP-2) ； osseointergation

參考文獻

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