【作者】 李雪；

【导师】 周延民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2020， 博士

【摘要】 种植修复已成为牙齿缺失的常用修复方式之一。然而,种植体周围炎是种植修复常见并发症,如不及时干预,将导致种植修复的失败。种植体周围炎是以菌斑感染为始动因素的炎症性病变,其特征是周围软组织的炎症以及支撑骨组织的进行性吸收。当微生物,特别是致病菌黏附定植于种植体表面形成生物膜后,其毒力因子导致的内源性免疫失调成为引起种植体周围炎的潜在风险。因此提出一种抑制种植体表面细菌附着、同时减轻种植体周围炎症反应的新型表面改性策略是目前研究的趋势和热点。近年来,二氧化铈纳米粒子（CeO₂ NPs）由于其具有Ce³⁺、Ce⁴⁺共存发生氧化还原反应的特性,因此能模拟超氧化物歧化酶（SOD）和过氧化氢酶（CAT）对活性氧自由基（ROS）的清除能力。大量的研究表明,CeO₂ NPs能够缓解或减轻炎症,加速伤口愈合和抑制细菌生长。因此,CeO₂ NPs在种植体表面的抗菌抗炎改性中可以发挥重要的作用。此外,CeO₂ NPs具有多种形貌,通过合成反应条件的改变可以控制其晶体形态,提高CeO₂ NPs的催化活性。CeO₂ NPs的形貌同时能够影响各晶面上Ce³⁺离子与氧空穴的数量和迁移率。但是到目前为止,关于不同形貌CeO₂ NPs改性钛表面的抗菌和抗炎性能的研究尚未见报道。综上所述,我们拟通过控制水热反应条件合成棒状、立方体、八面体三种不同形貌的CeO₂ NPs,并利用其改性纯钛种植体表面,研究其对体内外抗菌、抗炎功能的影响,深入揭示同一物质不同形貌与其抗菌、抗炎作用的关系,阐明CeO₂ NPs形貌影响其抗菌、抗炎的机理。本研究具体内容分四部分实验展开:实验1不同形貌CeO₂纳米粒子的合成、表面处理及表征采用水热合成法,通过改变碱的浓度和反应温度、时间合成三种（棒状、立方体、八面体）不同形貌的CeO₂ NPs,并通过旋涂法将不同形貌的CeO₂ NPs旋涂于纯钛表面。X射线衍射（XRD）、透射电镜（TEM）、扫描电镜（SEM）、X射线光电子能谱分析（XPS）、接触角实验及傅立叶变换红外光谱（FTIR）评价其各项物理表征。实验2不同形貌CeO₂纳米酶活性及生物安全性评价通过总抗氧化能力检测试剂盒、总SOD活性检测试剂盒及过氧化氢酶检测试剂盒分别检测三种不同形貌CeO₂ NPs的酶活性及其各自的总抗氧化能力。采用细胞毒性实验以及溶血实验评价三种不同形貌CeO₂ NPs及其涂覆钛片后的生物安全性及其生物相容性。采用qPCR实验评价三种不同形貌CeO₂ NPs改性钛片表面的炎症反应。实验3不同形貌CeO₂纳米粒子及其改性纯钛表面的抗菌性能研究培养牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）、具核酸杆菌（Fusobacterium nucleatum,F.nucleatum）、血链球菌（Streptococcus sanguinis,S.sanguinis）三种代表性种植体周围炎相关细菌,通过早期黏附实验研究革兰氏阳性细菌S.sanguinis以及革兰氏阴性细菌F.nucleatum在三种不同形貌CeO₂NPs改性钛片表面的细菌早期定植能力。通过活死染色、扫描电镜（SEM）、细菌代谢活性检测、菌落形成单位（CFU）检测三种不同形貌CeO₂ NPs改性钛表面的抗菌性能。实验4不同形貌CeO₂纳米粒子改性纯钛表面的抗炎性能的研究由牙龈卟啉单胞菌脂多糖（P.gingivalis-LPS）刺激,进而构建体外小鼠巨噬细胞炎症模型,通过qPCR分别检测肿瘤坏死因子α（TNF-α）、白介素1β（IL-1β）及白介素6（IL-6）炎症因子的表达情况,并检测三种不同形貌CeO₂ NPs改性后的钛表面能否抑制NF-κB信号通路的活化从而起到抗炎的作用。构建大鼠背部钛片植入炎症反应模型,将负载三种不同形貌CeO₂ NPs的钛片植于大鼠背部,分别于3周、6周后切取植入钛片周围组织,通过qPCR、H&E染色、免疫组化（IHC）、以及免疫荧光染色（IF）的方法评价其抗炎性能。通过上述四部分实验,得出以下结果:1、三种不同形貌的CeO₂ NPs改性的纯钛表面均具有一定的抗菌功能,对于关键致病菌生物膜（P.gingivalis）的抑制可达到约2个数量级。2、八面体CeO₂ NPs具有最强的总抗氧化能力。3、在体外,立方体和八面体CeO₂ NPs改性的纯钛表面比棒状CeO₂ NPs改性的纯钛表面具有更好的抗炎作用。4、不同形貌的CeO₂ NPs改性纯钛表面均能抑制促炎型NF-κB信号通路的活化,其中八面体CeO₂ NPs改性纯钛表面的抑制效果最为明显。5、体内研究表明,不同形貌CeO₂ NPs改性纯钛表面均能够显著降低促炎因子的表达,其中八面体CeO₂ NPs改性纯钛表面显示出最强的抗炎作用。更多还原

【Abstract】 Titanium dental implants have been widely used in dentistry for prosthetic rehabilitations of lost teeth.However,peri-implantitis is a common complication of implant repair,if not timely intervention,will lead to failure.Clinically,peri-implantitis is an inflammatory lesion with bacterial plaque infection as the driving factor.It is characterized by the inflammation of surrounding soft tissues and the absorption of supporting bones.When microorganisms,especially pathogenic bacteria,adhere to the surface of the implant to form a biofilm,the endogenous immune disorders caused by their virulence factors become a potential risk of peri-implantitis.Therefore,preparation of an implant surface that could inhibit bacterial attachment and reduce inflammation is the current research trend and hot spot.In recent years,cerium dioxide（CeO₂）nanoparticles（NPs）have the ability to mimic the enzyme activities of superoxide dismutase（SOD）and catalase（CAT）due to the coexistence of Ce³⁺and Ce⁴⁺.NPs have a strong ability to scavenge active oxygen free radicals（ROS）,while giving them effective antibacterial and anti-inflammatory properties.In the past decade,a large number of studies have shown that CeO₂ NPs could alleviate or reduce inflammation,accelerate wound healing and inhibit bacterial growth.Therefore,CeO₂ NPs could play an important role in the antibacterial and anti-inflammatory modification of the implant surface.In addition,related studies have shown that controlling the morphology of CeO₂ NPs could improve the catalytic activity of CeO₂ NPs.The different morphologies of CeO₂NPs could affect the number and mobility of Ce³⁺ions and oxygen vacancies on each crystal plane.But so far,there have been no reports on the antibacterial and anti-inflammatory capabilities of CeO₂ NPs modified titanium surfaces with different morphologies.In summary,we intend to synthesize three different morphologies of CeO₂ NPs in rod,cube,and octahedron by controlling the hydrothermal reaction conditions,and study its antibacterial and anti-inflammatory functions of the modified titanium implant surfaces in vivo and in vitro.Deeply reveal the relationship between different morphologies of the same substance and its antibacterial and anti-inflammatory effects.Further clarify the mechanism of CeO₂ NPs morphologie affecting its antibacterial and anti-inflammatory effects.The specific content of this research is divided into four parts:Experiment 1:Synthesis,Surface Treatment and Characterization of CeO₂NPs with Different MorphologiesCeO₂ NPs with different morphologies（rod,cube,octahedron）were synthesized by changing the concentration of alkali,the reaction temperature/time using hydrothermal synthesis.Coating CeO₂ NPs with different morphologies on the surface of pure titanium by spin coating method.X-ray diffraction（XRD）,transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,X-ray photoelectron spectroscopy（XPS）,contact angle experiments,and Fourier transform infrared spectroscopy（FTIR）were used to evaluate the physical characteristics.Experiment 2:Evaluate the Nanozyme Activity and Biosafety of CeO₂ NPs with Different MorphologiesEnzyme activities of CeO₂ NPs with three different morphologies and their respective total antioxidant capacity were detected by total antioxidant capacity test kit,total SOD activity test kit,and catalase test kit.Cytotoxicity and hemolysis test were used to evaluate the biological safety and biocompatibility of three different morphologies of CeO₂ NPs and their coated titanium disks.Using qPCR experiments to evaluate the inflammatory response on the surface of three different morphologies CeO₂ NPs modified titanium disks.Experiment 3:Research on the Antibacterial Properties of CeO₂ NPs with Different Morphologies and their Modified Pure Titanium SurfaceCultivation of three representative peri-implantitis-associated pathogenic bacteria:Porphyromonas gingivalis（P.gingivalis）,Fusobacterium nucleatum（F.nucleatum）,Streptococcus sanguinis（S.sanguinis）.The early adhesion experiment was tested by Gram-positive bacteria S.sanguinis and Gram-negative bacteria F.nucleatum on the surface of titanium disks modified by three different morphologies of CeO₂ NPs.The antibacterial performance of three different morphologies of CeO₂ NPs modified titanium surface was detected by live/dead staining,scanning electron microscope（SEM）,bacterial metabolic activity detection,and colony forming unit（CFU）.Experiment 4:Research on the Anti-inflammatory Properties of CeO₂ NPs with Different Morphologies and their Modified Pure Titanium SurfaceIn vitro mouse macrophage inflammation model was established by P.gingivalis lipopolysaccharide（P.gingivalis-LPS）stimulation,and tumor necrosis factorα（TNF-α）,interleukin 1β（IL-1β）,interleukin 6（IL-6）were detected by qPCR.Explore whether the titanium surface modified by three different morphologies of CeO₂ NPs could inhibit the activation of NF-κB signaling pathway to play an anti-inflammatory role.Titanium disks loaded with three different morphologies of CeO₂ NPs were implanted on the back of rats to build a rat back inflammation model.Tissues surrounding the implanted titanium disks were excised 3 and 6 weeks later,respectively.Hematoxylin-eosin staining（H&E staining）,immunohistochemistry（IHC）,and immunofluorescence staining（IF）were used to evaluate its anti-inflammatory properties.Through the above 4 part experiments,the following results were obtained:1.All the three different morphologies of CeO₂ NPs modified pure titanium surfaces have certain antibacterial functions,and could inhibit about 2 logs for key pathogenic biofilms（P.gingivalis）.2.The total antioxidant capacity of octahedral CeO₂ NPs was the strongest.3.Cube and octahedral CeO₂ NPs modified titanium has better anti-inflammatory effects in vitro than rod CeO₂ NPs.4.All the three morphologies of CeO₂ NPs modified titanium could inhibit the activation of the pro-inflammatory NF-κB signaling pathway,but the inhibition effect of octahedron CeO₂ NPs was the most obvious.5.In vivo,the expression of pro-inflammatory factors was significantly reduced in all three groups.The octahedral CeO₂ NPs modification showed the strongest anti-inflammatory effect in all groups.更多还原