氧化鋯植體經電漿表面處理以促進骨整合及抗菌效應

Abstract

氧化鋯陶瓷因具有良好的機械性質與化學穩定性，且擁有比鈦金屬更接近人體牙齒顏色，其骨整合能力又與鈦金屬相近，因此被認為是理想的植體材料，但因氧化鋯屬於生物惰性之材料，植體植入後與骨組織無法形成化學鍵結，且不易利用機械或化學等方式進行表面改質，限制其發展與臨床的應用。本研究將利用低溫電漿表面改質來優化，其有利於氧化鋯表面形成化學鍵結後，來提升其表面生物活性。電漿表面改質其優點除了具經濟效益外，可針對表面特性進行有選擇性的強化，而使材料本體特性保持不變，可與任何基材產生良好之附著性，且無孔洞等優點。本研究主要以低溫電漿表面改質技術進行，以電漿聚合方式將無機的氧化鋯表面上，形成有機矽烷氧類的界面層，再以化學或天然交聯劑將生長因子其抗菌分子固定於氧化鋯表面，製備出具有功能性且不具細胞毒性的材料。 本研究的第一部分中，以低溫電漿聚合沉積法，在氧化鋯基材上分別沉積六甲基二矽氧烷薄膜、二乙氧基二甲基矽烷薄膜與乙氧基三甲基矽烷薄膜，使氧化鋯表面形成有機界面層。實驗結果顯示，三種不同薄膜經電漿表面改質後對氧化鋯的性質影響，於接觸角測試中得知以六甲基二矽氧烷薄膜與乙氧基三甲基矽烷薄膜沉積於氧化鋯表面上最為穩定且未有老化的情形出現。在細胞毒性評估結果顯示，經三種不同薄膜沉積之氧化鋯表面並無細胞毒性產生，而在皮下植入試驗中觀察臨床生理狀況與組織病理切片分析顯示，與未處理的控制組相比乙氧基三甲基矽烷薄膜之發炎反應較為緩慢。本研究的第二部分中，以乙氧基三甲基矽烷薄膜沉基於氧化鋯表面後，再分別浸泡化學及兩種天然交聯劑以提供固定生長因子(Bone Morphogenetic Proteins 2 ,BMP2)和抗菌分子(Chlorhexidine ,CHX)於氧化鋯基材表面。其化學交聯劑為1-乙基-(3-二甲基氨基丙基)碳醯二亞胺/N-羥基琥珀醯亞胺，天然交聯劑分別是京尼平、原花青素。從實驗結果顯示，經電漿處理過後分別浸泡三種交聯劑，確實有效提高氧化鋯試片之親水性，且機械性質方面並無太大改變可保有原先氧化鋯良好的機械性質。經表面性質分析，證實交聯劑成功固定在氧化鋯基材表面上。在附著度實驗分析，其附著度均達到4B以上。經處理過後的表面也並無產生細胞毒性，而在皮下植入試驗中觀察臨床生理狀況與組織病理切片分析顯示，與未處理的控制組相比以1-乙基-(3-二甲基氨基丙基)碳醯二亞胺/N-羥基琥珀醯亞胺與京尼平組別，發炎反應相較之下較為和緩，且可較快進展到癒合時期的血管新生與纖維化等狀況。本研究的第三部份中，將生長因子和抗菌分子固定在改質後的氧化鋯表面上，將其改質後氧化鋯試片與MG-63類骨母細胞進行共培養，進行生物相容性與體外成骨分化以及氧化鋯植體在動物模式下之表現，另外透過與細菌共培養來評估其抗菌能力。實驗結果顯示，經MG63類骨母細胞測試後觀察其經改質過後，當表面有BMP2可以有助於細胞增殖的趨勢，三種交聯劑都明顯可以提升細胞生長，但其中以天然交聯劑京尼平最為顯著，且明顯地可促進MG63類骨母細胞在早期鹼性磷酸脢的表現，並且能夠誘導鈣化組織生成。在骨新生螢光標定觀察與未處理的氧化鋯植體相比，經表面改質後固定BMP2於氧化鋯植體，表現出較高的骨整合以及骨生成的量。抗菌結果顯示，經表面改質後固定CHX於氧化鋯表面上，對革蘭氏陰性菌有較佳的抗菌效果。 本研究結果顯示，將功能性生醫有機矽氧烷膜界面層沉積於生物惰性的氧化鋯材料，再利用化學或天然的交聯劑，提高生長因子及抗菌劑之化學鍵結強度，藉以提高與抗菌劑及骨分化誘導分子之化學鍵結，可有效優化氧化鋯，此氧化鋯具備(1)增加骨生長速率及骨癒合的能力以及(2)對抗細菌堆積的優勢，並有益於生醫材料發展以提高在臨床上的應用性。

Zirconia is a preferred material used in implant due to its excellent mechanical properties, chemical stability, and natural look color. In addition, zirconia implants can offer a similar success rate in osseointegration as titanium implants. However, zirconia is a bioinert material, and there is no chemical bonding between the bone tissue and the zirconia implant. Zirconia implant is hard to do surface modification by mechanical and chemical methods. This restricts its application in the dental implant field. To overcome this obstacle, plasma surface modification, which is a low-temperature technique, is used to modify chemical groups on zirconia. The plasma surface modification technique has an effective and economical surface treatment for many materials. The unique advantage of plasma surface modification is that surface characteristics can conduct selected enhancements without altering the material’s bulk properties. The thin film by plasma polymerization deposition can adhere to almost any substrate with the advantage of producing no pores. The purpose of this study was to evaluate the plasma surface modification to produce an active organosiloxane film and improve the biological activity of zirconia. It caused a condensation reaction with an amine of growth factors and antibacterial agents by a crosslinking solution. The growth factors and antibacterial agent-immobilized on the zirconia surface were prepared. The first part of this thesis was to investigate the characterization of the organosiloxane functional groups deposited on zirconia substrates using a plasma polymerization process with three different monomers hexamethyldisiloxane (HMDSO), diethoxydimethylsilane (DEODMS) and ethoxytrimethylsilane (EOTMS) coated on the zirconia substrate/implant. The results of the contact angle showed that the EOTMS film might provide a more proper wettability due to a less hydrophobic surface property than the other films. The hydrophobic of the zirconia substrate after plasma treatment decreased with the increasing aging time. The in-vitro cytocompatibility study showed that there was no cytotoxicity for the zirconia surface deposited with three different films. The in-vivo test on the safety of the implantation showed that the inflammatory reaction of the zirconia substrate deposited with EOTMS was slower than the other groups, and the content of polymorphonuclear cells and lymphocytes was also lower. In the second part, three different crosslinking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/ N-hydroxysuccinimide (NHS), genipin (GP), and proanthocyanidin (PC) were performed after plasma polymerization to immobilize the growth factor and antibacterial molecules on the surface. The results showed that all the crosslinking treatments increased the surface wettability. These surface modification treatments did not alter the mechanical properties of zirconia. The adhesion strength of surface modification treatments reaches 4B and above. The in vitro cytocompatibility study showed that there was no cytotoxicity on the zirconia surface of these three crosslinking agents. The in-vivo test on the safety of the implantation showed that the EDC/NHS and GP groups had the best appearance surface without any bleeding. In the third part, the EOTMS plasma surface treatment was processed on zirconia substrates. The samples immersed in an EDC/NHS, GP, and PC by an optimized crosslinking time. The treated zirconia substrates were then dipped into bone morphogenetic protein 2 (BMP2) (10 μg/ml) and Chlorhexidine (CHX) (0.2wt%) solution. The in-vitro biocompatibility, osteogenic differentiation, in-vivo animal studies (implant stability, periotest value), and antibacterial effect of surface modification treatments were measured. EDC/NHS, GP, and PC are chemicals to covalently immobilize BMP2 on the surface to promote osteogenesis and wound healing. The labeling fluorochrome observation results showed that the untreated zirconia dental implant did not have bone growth in the fourth week, while some red fluorescent outlines in both PD-EOTMS/EDC NHS/BMP2 and PD-EOTMS/GP/BMP2 groups, were indicated representing the formation of new bone. This result demonstrated that there is a strong beneficial effect of the different crosslinking agents in immobilizing CHX on the zirconia surface, and these materials are applicable in dental implants. The research confirmed that the zirconia implant surface with BMP2 immobilized could significantly promote cell ossification and shorten the time of osseointegration. We have therefore demonstrated that the surface modified with BMP2 through plasma polymerization enhanced the in vitro and in vivo biocompatibility of zirconia substrate/implant. The outcome of this study could be a useful reference for the clinical application in the future.