【作者】 刘燕；

【导师】 毛靖；

【作者基本信息】 华中科技大学 ， 口腔临床医学， 2011， 博士

【摘要】 1.研究背景与目的随着新型牙科材料和粘接技术的广泛应用,传统龋病治疗中“预防性扩展”的观念已逐渐被“微创治疗”这一新概念所替代。当代微创牙科学(minimally invasive dentistry, MID)的特点是对龋齿进行保守治疗,以保存其自身的再矿化潜能。因此,临床上的粘接基底往往是由正常牙本质和龋坏内层的脱矿牙本质混合而成。在龋病的发展过程中,龋坏内层的脱矿牙本质发生结构改变,如牙本质小管堵塞、牙本质硬度下降等,严重影响了临床上的有效粘接。有研究表明,与正常牙本质相比,粘接剂与脱矿牙本质界面间的微拉伸强度更低,混合层更厚且疏松。近年来,有关水吸收导致的混合层内树脂水解和胶原降解现象的报道增加,而混合层的降解将直接影响到牙色复合树脂的使用寿命。然而,到目前为止,有关如何提高树脂-脱矿牙本质界面间粘接强度的研究非常有限。研究这一问题对于进一步探索能有效保持复合树脂-脱矿牙本质粘接体完整性和延长复合树脂-脱矿牙本质粘接体持久性的方法至关重要。引导组织再矿化(guided tissue remineralization, GTR)是一种粒子介导的、非经典的新型仿生再矿化模式。与传统矿化方式不同,GTR通过使用牙本质基质蛋白(dentin matrix proteins, DMPs)类似物引导无定形磷酸钙(amorphous calcium phosphate, ACP)纳米前体在胶原纤维内部水间隔进行有序沉积,形成与自然牙本质结构类似的矿物质,且GTR机制并不依赖于胶原纤维内籽晶的外延生长。本实验组前期研究表明,GTR能够成功地再矿化5～8μm深的树脂渗透差的混合层,并恢复了自然矿化牙本质的硬度及蛋白水解酶的排除机制,从而保持了树脂-牙本质粘接界面的稳定性。龋坏内层脱矿牙本质的深度可达几百微米,但其胶原纤维仍然具备分子间的交联和明显的横纹样结构。因此,从生理学上看,脱矿牙本质具备再矿化的潜能。本研究从矿物质含量和超微结构两个方面检测GTR对脱矿牙本质及脱矿牙本质-树脂混合层的影响,拟通过GTR方式增强脱矿牙本质的硬度且延长脱矿牙本质-树脂界面间的粘接强度。研究分为三个部分：多聚磷酸盐在引导组织再矿化中的作用机制；传统再矿化法与引导组织再矿化法的再矿化效果比较；三偏磷酸钠底剂在引导组织再矿化脱矿牙本质-树脂混合层中的应用研究。2.研究材料与方法(1)重组单层的Ⅰ型胶原,并用1-乙基-3(3-二甲丙氨基)碳化亚胺(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC)/N-羟基丁二酰亚胺(N-hydroxysuccinimide, NHS)混合物交联重组的Ⅰ型胶原。将一部分重组的Ⅰ型胶原用不同浓度的三偏磷酸钠(sodium trimetaphosphate, STMP)或三聚磷酸钠(sodium tripolyphosphate, TPP)(0.313～2.5 wt%)分别磷酸化处理5 min,然后浸泡于含聚丙烯酸(polyacrylic acid, PAA)的模拟体液(simulated body fluid, SBF)/硅酸盐水门汀矿化体系(实验组)或SBF/硅酸盐水门汀矿化体系(模板对照组)；另一部分重组的胶原直接浸泡于含PAA的SBF/硅酸盐水门汀矿化体系(PAA对照组)或SBF/硅酸盐水门汀矿化体系(空白组)。采用傅立叶变换红外光谱术(fourier transform-infrared spectroscopy, FT-IR)评估磷酸化修饰后重组胶原的构象变化。矿化4-72 h后采用透射电子显微镜(transmission electron microscopy, TEM)观察晶体的大小、形态以及晶体在胶原内沉积的情况；同时采用选区电子衍射法(selected area electron diffraction, SAED)观察晶相和晶体的排列情况。(2)收集30颗新鲜拔除无龋坏的第三磨牙,用慢机垂直牙齿长轴于牙冠中部制备1mm厚的牙本质片。采用pH循环的方法制备60片脱矿深度为250-500μm,宽度为3mm部分脱矿的牙本质,以模拟临床上龋齿去腐后剩余的脱矿牙本质。选取24片脱矿深度为300±30μm样本并随机分为三组(N=8)：①传统再矿化组(top-down),矿化介质为含钙磷离子的矿化液；②引导组织再矿化组(bottom-up)矿化介质为含牙本质基质蛋白类似物的SBF/硅酸盐水门汀；③阴性对照组,矿化介质为SBF/硅酸盐水门汀。矿化4个月后分别采用显微计算机断层扫描仪(micro-computed tomography,μCT)和TEM检测矿化前后矿物质含量变化以及晶体在矿化胶原基质内的排列情况。(3)选取32片脱矿深度为300±30μm的人工脱矿牙本质片,一半样本用2.5 wt%STMP处理5 min作为实验组,另一半样本作为对照组(无STMP处理)。然后分别将实验组和对照组中样本随机分为两个亚组：一组样本表面涂布全酸蚀粘接剂One-Step (N=8),另一组样本表面不涂布任何粘接剂(N=8)。实验组的矿化介质为含PAA的SBF/硅酸盐水门汀,对照组为SBF/硅酸盐水门汀。矿化4个月后,分别用μCT和TEM检测矿化前后矿物质含量变化以及晶体在矿化胶原基质内的排列情况。3.研究结果(1)在无多聚磷酸盐和PAA存在时,胶原本身并不能引导再矿化。矿化24 h后,TEM镍网内可见较大的无定形磷酸钙(amorphous calcium phosphate, ACP)球(ca.1-3μm)形成,72 h后转化成晶体沉积在胶原纤维外,而胶原纤维内无矿化。模板对照组与空白组结果类似,但ACP相的尺寸稍小(2)当仅有PAA稳定ACP而无多聚磷酸盐磷酸化胶原时,矿化4h后,TEM镍网内可见大小约10-100nm ACP相形成,24 h后胶原出现膨胀样变,72 h后胶原恢复原有尺寸,胶原纤维内产生非分级化再矿化,即晶体在胶原内呈连续绳索状,而不是离散的相互重叠的板状排列；当多聚磷酸盐与PAA同时存在时,胶原呈分级化的纤维内矿化,纳米板状晶体在纤维内呈迭序排列,形成横纹样特征。(3)从矿物质含量上看,top-down的方法仅仅矿化基底部脱矿的牙本质,脱矿牙本质表面的矿物质含量在矿化前后无明显变化,矿化效果依赖于胶原基质中剩余矿物质的含量；而bottom-up的方法可以矿化整个脱矿的牙本质,从表面到基底部,矿化效果不依赖于矿物质的含量。(4)从超微结构上看,top-down方法主要是纤维外矿化；而bottom-up方法则同时存在纤维内和纤维外矿化,矿化后胶原组织中晶体的大小、方向及结构层次和天然矿化胶原类似。(5) STMP未处理的脱矿牙本质,无论有无粘接剂的渗透,4个月后均无再矿化发生。(6)μCT结果显示,在脱矿深度与矿物质含量的改变上,脱矿牙本质的矿化效果明显优于脱矿牙本质-树脂混合层,脱矿牙本质表层的粘接剂抑制了矿化成分向牙本质基底的渗透；TEM结果显示,矿化4个月后,矿物质稀疏的脱矿牙本质表面存在胶原变性。4.研究结论多聚磷酸盐在引导Ⅰ型胶原分级化纤维内再矿化过程中起着重要的作用,重现了与天然矿化胶原结构类似的横纹特征。top-down矿化机制依赖于胶原基质内剩余籽晶的外延生长,属于经典的离子介导的矿化模式；而bottom-up矿化机制却涉及到无定形磷酸钙纳米颗粒向晶体的转化,属于非经典的粒子介导的矿化模式。从矿物质含量和超微结构上看,STMP作为牙本质基质蛋白类似物能够特异性地与脱矿牙本质胶原结合,并诱导PAA稳定的无定形磷酸钙前体在纤维内的成核,形成与矿化牙本质结构类似的磷灰石纳米微晶有序地沉积在胶原纤维内间隙,促进了脱矿牙本质的再矿化。GTR通过bottom-up矿化机制恢复了矿化牙本质的硬度及蛋白水解酶的排除机制,从而保持了树脂-脱矿牙本质粘接界面的稳定性,是一种潜在而有效的保持复合树脂-脱矿牙本质粘接体完整性和延长复合树脂-脱矿牙本质粘接体持久性的方法。更多还原

【Abstract】 Objectives:Over the past few decades, scientific developments in dental materials and bond technology have changed dentistry’s approach to manage carious teeth. The hallmark of minimally invasive dentistry (MID) is conservative treatment of caries to preserve their potential for remineralization. Therefore, the clinical bonding substrate is likely to be a combination of normal dentin and caries-affected dentin after caries excavation. It has been shown that bond strengths to caries-affected dentin are significantly lower than those to normal dentin and the hybrid layer created in caries-affected dentin is thicker and more porous compared with that in normal dentin. This may be the result of the changed structures of caries-affected dentin, such as occluded dentin tubules and decreased hardness. However, durability studies on bond strengths to caries-affected dentin are still limited. Our previous studies have shown that the poor-infiltrated hybrid layer (5-8μm) has been successfully remineralized via guided tissue remineralization (GTR), which restores the enzyme exclusion and fossilization properties of naturally mineralized dentin. This proof-of-concept strategy is capable of preserving the longevity of resin-dentin bonds. It has been proved that the collagen matrix of caries-affected dentin demonstrates intermolecular crosslinks and normal cross-banding. And therefore, caries-affected dentin is physiologically remineralizable. As caries-affected dentin can extend several hundred microns below the excavated surface, our objectives in this study is to test the effect of GTR on remineralizing caries-affected dentin and the hybrid layer created in caries-affected dentin. Materials and Methods:(1) A single-layer of type I collagen fibrils was reconstituted over formvar-andcarbon-coated 400-mesh Ni TEM grids and cross-linked with 0.3 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/0.06M N-hydroxysuccinimide (NHS). Thereafter, half of TEM grids containing cross-linked collagen fibrils were phosphorylated with 0.313-2.5 wt% hydrolyzed sodium trimetaphosphate (STMP) or sodium tripolyphosphate (TPP) for 5 min. Remineralization was performed initially for 4-24 h to identify the optimal STMP/TPP concentration for further remineralization for 48-72 h. They were floated upside-down over 30μL GTR remineralizing medium containing simulated body fluid (SBF)/Portland Cement with polyacrylic acid (PAA) as a sequestration analog. The templating analog control consisted of STMP/TPP-treated collagen and SBF (no sequestration analog). The sequestration analog control consisted of reconstituted collagen and PAA-containing SBF (no templating analog). The collagen negative control (no sequestration and templating analogs) consisted of reconstituted collagen and SBF. Grids were examined unstained using transmission electron microscopy (TEM) and selected area electron diffraction (SAED) at 110 kV.(2) Thirty extracted non-carious human third molars were obtained with patient-informed consent under a protocol approved by the Human Assurance Committee of the Medical College of Georgia. A 1 mm thick dentin disk was prepared by making two parallel cuts perpendicular to the longitudinal axis of each tooth using a low-speed Isomet saw under water cooling. A 250-300μm thick layer of partially demineralized dentin was created by pH-cycling procedure to mimic caries- affected dentin below the excavated surface. The samples were then randomly divided into two groups:half of them were remineralized using a classical top-down approach and the rest using non-classical bottom-up approach. Micro-computed tomography (μCT) and TEM were employed to examine mineral uptake and apatite arrangement within the mineralized collagen matrix.(3) Artificial carious lesions with lesion depths of 300±30μm were created by pH-cycling.2.5% hydrolyzed STMP was applied to the artificial carious lesions to phosphorylate the partially-demineralized collagen matrix. Half of the STMP-treated specimens were bonded with One-Step. The adhesive and non-adhesive infiltrated specimens were remineralized in a Portland cement-SBF system containing PAA to stabilize amorphous calcium phosphate (ACP) as nanoprecursors.μCT and TEM were used to evaluate the results of remineralization after a 4-month period.Results:(1) In the collagen negative control, there was no remineralization. And ACP phases were bigger than those in the sequestration analog control. In the sequestration analog control,1000μg/mL PAA was included in the remineralization medium but did not treat the reconstituted collagen with STMP or TPP.(2) For the templating analog control, no sequestration analog was included in the remineralization medium and needle-shaped apatite was randomly precipitated over the surface of collagen fibrils after 72 h. Cross-banding patterns produced by discrete apatite crystallites could be identified only from partially-mineralized collagen fibrils in presence of both templating and sequestration analog.(3) In the view of mineral uptake, the top-down approach which relied on the mineral density could remineralize only the base of the partially demineralized dentin. Conversely, the entire partially demineralized dentin, including apatite-depleted collagen fibrils, can be completely remineralized by the bottom-up approach.(4) In absence of PAA and STMP as biomimetic analogs (control groups), there was no remineralization irrespective of whether the lesions were infiltrated with adhesive. From ultrastructure, extrafibrillar remineralization was predominantly observed in the top-down approach, while the bottom-up approach showed evidence of both intrafibrillar and extrafibrillar remineralization.(5) For the STMP-treated experimental groups immersed in PAA-containing SBF, specimens without adhesive infiltration were more heavily remineralized than those infiltrated with adhesive. Statistical analysis of the 4-monthμCT data revealed significant differences in the lesion depth, relative mineral content along the lesion surface and changes in the intergrated mineral loss (AZ) between the non-adhesive and adhesive experimental groups for all the three parameters.(6) TEM examination indicated that collagen degradation occurred in both the non-adhesive and adhesive control and experimental groups after 4 months of remineralization. And even the denatured collagen fibrils could be remineralized via GTR.Conclusions:There is no difference between STMP and TPP in templating hierarchical intrafibrillar apatite assembly in reconstituted collagen via GTR. The ability of using simple non-protein molecules to reproduce different levels of structural hierarchy in mineralized collagen fibrils indicates the ultimate simplicity in Nature’s biomineralization design principles. And this challenges the need for using more complex recombinant matrix proteins in bioengineering applications. The mechanisms involved in the classical top-down and non-classical bottom-up remineralization approaches appear to be different. The former probably relies on epitaxial growth of seed crystallites within the collagen fibrils. The latter involves transformation of ACP nanoparticles into apatite crystallites in the presence of biomimetic analogs. GTR using STMP as a biomimetic ananlog is a promising method to remineralize artificial carious lesions particularly in areas devoid of seed crystallites. Future studies should consider remineralizing the real caries-affected and caries-infected dentin via GTR. During remineralization process, MMP-inhibitors should be incorporated within the partially-demineralized collagen matrix to prevent collagen degradation.更多还原