Abstract
目的
根管治疗术是治疗牙髓和根尖周炎症最有效且常见的方法，在根管预备的过程中，化学冲洗起到关键作用。次氯酸钠(sodium hypochlorite，NaOCl)是目前临床广泛应用的根管冲洗液，然而它可能会在发挥杀菌、溶解功能的同时，影响根管治疗后牙本质与修复材料间的粘接强度，导致治疗效果不佳。因此，为保证良好的根管治疗效果，探究NaOCl对牙本质与修复材料间粘接的影响至关重要。本研究旨在探讨不同浓度的NaOCl处理对复合树脂与牙本质间粘接的影响，以及异抗坏血酸钠(sodium erythorbate，ERY)的作用，为根管治疗后牙本质的粘接提供临床指导。
方法
选取新鲜拔除的18~33岁青年的第三磨牙70颗，在水冷却下用慢速锯切割获得牙本质试件。实验分为10组，每组14个牙本质试件。其中5组分别为去离子水(deionized water，DW)组、0.5% NaOCl组、1% NaOCl组、2.5% NaOCl组和5.25% NaOCl组，分别用对应液体浸泡20 min，每5 min更换1次液体；剩余5组按照上述相同方法处理后，用DW冲洗1 min，10% ERY溶液浸泡5 min。每组从14个牙本质试件中取4个，在扫描电镜(scanning electron microscope，SEM)下观察，计算牙本质小管的开口数目并测量直径；每组剩余的10个牙本质试件，经自酸蚀粘接系统粘接及复合树脂固化后制备成粘接试件，用慢速锯切割后，每组获得横截面积约1 mm×1 mm大小的20个条状试件，其中10个条状试件于SEM下观察，分别从树脂突形成、树脂突长度、混合层形成3个方面评估粘接层形态；剩余10个进行微拉伸实验和断裂破坏类型分析。
结果
0.5% NaOCl、1% NaOCl、2.5% NaOCl、5.25% NaOCl组随着NaOCl浓度的升高，牙本质小管开口数目和直径均逐渐增加(均P<0.05)。DW、0.5% NaOCl、1% NaOCl、2.5% NaOCl、5.25% NaOCl组使用10% ERY后，牙本质小管开口数目和直径没有显著增加 (均P>0.05)。在SEM下，DW、0.5% NaOCl、1% NaOCl及2.5% NaOCl组随着NaOCl浓度的增高，树脂突形成评分逐渐上升；但5.25% NaOCl组的评分较2.5% NaOCl组显著降低(均P<0.05)；使用10% ERY与未使用组相比，树脂突形成评分差异均无统计学意义(均P>0.05)。在SEM下，5.25% NaOCl组的树脂突长度评分显著高于DW、0.5% NaOCl及1% NaOCl组(均P<0.05)，与2.5% NaOCl组比较差异无统计学意义(P>0.05)；使用10% ERY与未使用组相比，树脂突长度评分差异均无统计学意义(均P>0.05)。在SEM下，与DW组相比，2.5% NaOCl和5.25% NaOCl组混合层形成评分均显著降低(均 P<0.05)；2.5% NaOCl+10% ERY、5.25% NaOCl+10% ERY组的混合层形成评分分别显著高于2.5% NaOCl、5.25% NaOCl组(均P<0.05)。与DW组相比，0.5% NaOCl、1% NaOCl及2.5% NaOCl组微拉伸强度均增大，而5.25% NaOCl组则降低(均P<0.05)；2.5% NaOCl+10% ERY、5.25% NaOCl+10% ERY组与2.5% NaOCl、5.25% NaOCl组相比，微拉伸强度均增大(均P<0.05)。在断裂模式中，5.25% NaOCl组粘接面断裂的类型显著多于其他组(均P<0.05)，而5.25% NaOCl+10% ERY组较5.25% NaOCl组粘接面断裂的类型减少(P<0.05)。
结论
当NaOCl浓度低于2.5%时，其可增强复合树脂与牙本质间的粘接强度，并且粘接强度随着NaOCl浓度的增加而增强；但当NaOCl浓度高于5.25%时，则显著降低复合树脂与牙本质间的粘接强度；使用10% ERY能够恢复5.25% NaOCl处理对复合树脂与牙本质粘接强度的不良影响。
Keywords: 牙本质粘接, 次氯酸钠, 异抗坏血酸钠, 扫描电镜, 微拉伸实验
Abstract
Objective
Root canal therapy is the most effective and common method for pulpitis and periapical periodontitis. During the root canal preparation, chemical irrigation plays a key role. However, sodium hypochlorite (NaOCl), the widely used irrigation fluid, may impact the bonding strength between dentin and restorative material meanwhile sterilization and dissolving. Therefore, it’s important to explore the influence of NaOCl on the adhesion between dentin and restoration materials to ensure clinical efficacy. This study aims to explore the effect of NaOCl on dentine adhesion and evaluate the effect of dentine adhesion induced by sodium erythorbate (ERY), and to provide clinical guidance on dentin bonding after root canal therapy.
Methods
Seventy freshly complete extracted human third molars aged 18-33 years old, without caries and restorations were selected. A diamond saw was used under running water to achieve dentine fragments which were divided into 10 groups with 14 fragments in each group: 2 control [deionized water (DW)±10% ERY] and 8 experimental groups (0.5%, 1%, 2.5%, and 5.25% NaOCl±10% ERY). The dentine specimens in the control group (treated with DW) and the experimental groups (treated with 0.5% NaOCl, 1% NaOCl, 2.5% NaOCl, and 5.25% NaOCl) were immersed for 20 min using corresponding solutions which were renewed every 5 min. The other 5 groups were immersed in 10% ERY for 5 min after an initial washing with DW for 1 min. Then, we selected 4 dentine fragments from all 14 fragments in each group and the numbers and diameters of opening dentinal tubules were observed under scanning electron microscope (SEM). The other 10 dentine fragments from each group were used to make adhesive samples by using self-etch adhesive wand composite resin. All the above adhesive samples were sectioned perpendicular to the bonded interface into 20 slabs with a cross-sectional area of 1 mm×1 mm using a diamond saw under the cooling water, and then the morphology of 10 slabs in each group’s bonding interface was observed from aspects of formation of resin tags, depth of tags in dentin, and formation of hybrid layer under SEM. The other 10 slabs of each group’s microtensile bond strength and failure modes were also analyzed.
Results
Among the 0.5% NaOCl, 1% NaOCl, 2.5% NaOCl, and 5.25% NaOCl groups, the number and diameter of patent dentinal tubules gradually increased with the rise of concentration of NaOCl solution (all P<0.05). Among the DW, 0.5% NaOCl, 1% NaOCl, 2.5% NaOCl, and 5.25% NaOCl groups, the number and diameter of patent dentinal tubules increased after using ERY, but without significant difference (all P>0.05). Among the DW, 0.5% NaOCl, 1% NaOCl, and 2.5% NaOCl groups, the scores of formation of resin tags under SEM gradually increased with the increase of concentration of NaOCl solution, while the score in the 5.25% NaOCl group decreased significantly compared with the score of the 2.5% NaOCl group (P<0.05). There was no significant difference between using 10% ERY groups and without using 10% ERY groups (all P>0.05). The scores of length of the tags under SEM in the 5.25% NaOCl group was significantly higher than the scores of DW, 0.5% NaOCl, and 1% NaOCl groups (all P<0.05), and it was also higher than the score of the 2.5% NaOCl group, but without significant difference (P>0.05). There was no significant difference between using 10% ERY groups and without using 10% ERY groups (P>0.05). The scores of formation of hybrid layer under SEM in the 2.5% NaOCl and 5.25% NaOCl groups significantly decreased compared with the score of the DW group (all P<0.05). There were significant differences between the 2.5% NaOCl±10% ERY groups and between the 5.25% NaOCl±10% ERY groups (all P<0.05). Microtensile bond strength was greater in the 0.5% NaOCl, 1% NaOCl, and 2.5% NaOCl groups, but lower in the 5.25% NaOCl group than that in the DW group (all P<0.05). There were significant differences between the 2.5% NaOCl±10% ERY groups and between the 5.25% NaOCl±10% ERY groups (all P<0.05). The incidence of type “Adhesive” of failure modes in the 5.25% NaOCl group was significantly higher than that in other groups (all P<0.05), while the incidence of type “Adhesive” in the 5.25% NaOCl+10% ERY group was lower than that in the 5.25% NaOCl group (P<0.05).
Conclusion
The bonding strength to dentine increases with the increase of NaOCl concentration when the concentration lower than 2.5%; whereas it is decreased at a higher concentration (such as 5.25%). 10% ERY has a definite recovery effect on attenuated bonding strength to 5.25% NaOCl-treated dentine.
Keywords: dentine adhesive, sodium hypochlorite, sodium erythorbate, scanning electron microscope, microtensile bond strength
次氯酸钠(NaOCl)是目前临床使用最广泛的根管冲洗液[1]，可杀灭根管系统内及根管壁上定植的微生物，中和细菌产生的毒素，还可以溶解坏死的牙髓组织，且能在根管预备过程中起到润滑作用。
牙本质所含有机物中胶原蛋白约占18%，为所有有机物的85%~90%，主要为I型胶原。NaOCl溶液在杀菌、溶解组织的同时，会发挥其非特异性蛋白水解作用，溶解牙本质中的有机物质，进而改变牙本质的理化性能[2]。这可能影响根管治疗后牙本质与修复材料间的即刻粘接效果，而有效的即刻粘接可以防止修复体与牙体组织间边缘微渗漏的产生，防止修复体脱落[3]。
目前，NaOCl溶液处理对牙本质粘接的影响存在争议。多数学者认为NaOCl降低牙本质与修复材料间的粘接强度[4-6]，可能是牙本质胶原蛋白分解及NaOCl氯化反应生成的氧化自由基影响树脂聚合造成的[7]；也有部分学者[8-9]认为经NaOCl溶液处理后，深层及次级牙本质小管开放数目增多，进而导致牙本质与修复材料之间粘接强度增加。但上述研究所选用的NaOCl溶液浓度、使用时间及粘接系统等存在差异，得出的结论也不尽相同。
研究[10-11]表明：在粘接之前应用抗氧化剂可在一定程度上恢复NaOCl溶液处理后牙本质的粘接强度并且稳定树脂-牙本质粘接界面。现有研究[12]多采用抗坏血酸及其钠盐作为抗氧化剂，认为其除能恢复NaOCl溶液处理后牙本质的粘接强度外，亦可促进粘接树脂的聚合，但迄今尚无一致结论，有待进一步深入研究。异抗坏血酸钠(sodium erythorbate，ERY)作为一种抗坏血酸异构体所形成的盐，是目前国际上食品加工行业广泛应用的食品抗氧化剂，价格低廉且无毒副作用，具有与抗坏血酸相似的生理性能，但抗氧化能力强于抗坏血酸及抗坏血酸钠，10% ERY溶液的pH值为8~9，略高于10%抗坏血酸钠，对其在恢复NaOCl溶液处理后牙本质粘接强度中作用的研究较少。
本研究根据临床根管治疗的实际情况选择最常用的几种浓度的NaOCl溶液及合理的处理时间，尽可能地模拟髓室及冠方牙本质在根管治疗过程中受到NaOCl溶液冲洗的情况，旨在于更贴合临床的条件下探究NaOCl溶液处理或用NaOCl处理后再用ERY处理对牙本质粘接的影响并分析其潜在的机制。
1. 材料与方法
1.1. 牙本质试件的制备
收集2019年1至4月于中南大学湘雅医院口腔医学中心(口腔颌面外科)就诊拔除的新鲜第三磨牙70颗，患者年龄为18~33岁。牙冠完整无龋坏，未经牙体或牙髓治疗。将牙浸泡于去离子水(deionized water，DW)中，用手用刮治器去净表面残留软组织及牙结石后，储存于4 ℃ DW中，每周更换1次液体，3个月内使用。总实验流程见图1。
图1.
流程图
Figure 1 Flow chart
A: Collecting human third molars; B: Obtaining dentine fragments with a diamond saw; C: Selecting dentine fragments; D: Polishing dentine fragments with silicon carbides; E: Control and experimental groups; F: Analyzing dentine tubules’ morphology under the scanning electron microscope; G: Making adhesive samples; H: Cutting adhesive samples into sticks with a diamond saw; I: Analyzing bonding interfaces’ morphology under the scanning electron microscope; J: Testing microtensile bond strength.
在水冷却下用慢速锯(SYJ-150，沈阳科晶自动化设备有限公司)切割，每颗牙齿以髓室顶和底为标记作2条平行的切割线，去掉冠方及根方牙体组织，保留髓室部分，沿牙体长轴作纵向切割并去掉2侧牙釉质获得2个牙本质试件。随后，将靠近髓腔一侧的牙本质试件表面在DW冲洗下依次用180、240、320及600目碳化硅砂纸打磨以获得一个标准玷污层。
实验分为10组，每组14个牙本质试件，DW组、0.5% NaOCl组、1% NaOCl组、2.5% NaOCl组及5.25% NaOCl组分别用对应液体浸泡20 min，每 5 min更换1次液体以模拟临床根管治疗过程中其冲洗到髓室牙本质的情况。剩余5组分别先按照上述方法处理后，用DW冲洗1 min，最后浸泡于10% ERY溶液(江西省德兴市百勤异VC钠有限公司)中5 min。上述溶液均以DW配制。
1.2. 扫描电镜下观察牙本质小管形态
每组取4个经上述处理的牙本质试件，面对髓腔一侧的牙本质表面为观察面，行干燥、抽真空、喷金处理后置于扫描电镜(scanning electron microscope，SEM)(S-4800，日本日立公司)下，于1 000、5 000及20 000倍下观察牙本质小管开口形态。
在每组4个样本中心处放大1 000倍进行拍照采图，并参照Scelza等[13]计算牙本质小管开口数的方法，用Adobe Photoshop CC软件，按照原始图片像素制作由64个相同正方形组成的格子图案，随机抠出其中的16个正方形格子，将所得的图案与上述SEM所采照片重叠，计算每个正方形格子中牙本质小管开口数及并统计总数，代表此样本牙本质小管开口数目。将上述采集的图片以图像格式导入Image J图像分析软件后，首先根据图片上的比例尺矫正软件标尺，然后再定义灰度值以分辨牙本质小管，并标记小管边界(排除边缘不完整的小管)，最后选取小管开放区域计算小管的平均直径。
1.3. 粘接试件及条状试样的制备
每组取剩余10个经上述处理的牙本质试件，用DW冲洗1 min，将面对髓腔一侧的牙本质表面按照说明书要求，使用毛刷轻力双向涂擦，将一步法自酸蚀粘接系统(AdperTM Easy One，美国3M公司)涂布于待粘接面，操作步骤为：使用中等气流轻吹，干燥牙面，涂胶黏剂，轻吹使胶黏剂分布均匀，气枪与粘接面呈45°角，约保持1 cm距离，气流大小一致，光固化10 s。使用胶黏剂后，将复合树脂Z350(美国3M公司)分层堆砌于粘接面上形成约5 mm高的树脂块，每层厚度<2 mm，用LED光固化灯(SmartLite® FOCUS，美国登士柏西诺德公司)固化20 s，光强度1 000 MW/cm2。将制备好的粘接试件即刻储存于DW中，在37 ℃恒温水浴中保存24 h后进行微拉伸实验。
将上述恒温水浴后的粘接试件在水冷却下用慢速锯垂直于粘接界面切割成1 mm厚的片状，将试件旋转90°，再次纵向切割，得到横截面积约1 mm× 1 mm大小的条状试样，在体视显微镜20倍下观察，排除出现缺损、裂纹及气泡的试样，最后每个粘接试件可得到2个条状试样。
1.4. SEM下观察粘接层形态
每组取前述切割好的条状试样10个，用DW冲洗1 min，行干燥、抽真空、喷金处理后置于SEM下，分别于500、1 000倍下观察粘接层形态。参照Moda等[14]和Bitter等[15]的评分标准，列出相关观察指标的评价表格，并记录每个样本的评分(表1)。采用双盲法，如2名参与本研究的检查者意见不一致，则由2名检查者重新评估图像，直到达成共识。
表1.
扫描电镜下粘接层形态的评价标准
Table 1 Evaluation criteria for the morphology of adhesive layers under the scanning electron microscope
| 特征 | 评分 | 评价标准 |
|---|---|---|
| 树脂突的形成 | 0 | 无树脂突 |
| 1 | 少数树脂突 | |
| 2 | 均匀树脂突形成但少有侧枝 | |
| 3 | 长树脂突形成并有大量侧枝 | |
| 树脂突的长度 | 0 | 无树脂突 |
| 1 | 平均长度≤3 μm | |
| 2 | 平均长度3~8.9 μm | |
| 3 | 平均长度9~15 μm | |
| 4 | 平均长度≥15 μm | |
| 混合层的形成 | 0 | 无混合层 |
| 1 | 粘接面50%的面积有混合层形成 | |
| 2 | 粘接面有>50%但<100%的面积有 混合层形成 | |
| 3 | 整个粘接面均有混合层形成 |
1.5. 微拉伸实验及断裂破坏类型
通过测定试件在承受负荷发生断裂时的微拉伸强度来评价材料的粘接强度。每组取前述切割好的剩余条状试件10个，将试件两端固定在微拉伸测试仪(MicroTensileTester，美国Bisco公司)试样台上，使长轴与试样台平行且粘接面位于试样台正中。启动测试仪，速度1 mm/min，直至样本断裂，记录断裂时载荷峰值，微拉伸粘接强度=载荷峰值/粘接面积。在体视显微镜20倍下观察并记录试件断裂破坏类型，分为粘接界面破坏(Adhesive)、内聚破坏(Cohesive)及混合破坏(Mixed)3种。内聚破坏又可以分为牙本质内聚破坏和复合树脂内聚破坏。
1.6. 统计学处理
采用SPSS 22.0统计学软件进行数据分析，计量资料采用均数±标准差( ±s)表示，两组间比较采用t检验，多组间比较采用方差分析，两两比较采用LSD-t检验；计数资料采用构成比或率表示，组间比较采用χ2检验；等级资料多组间比较采用Kruskal-wallis检验，P<0.05为差异具有统计学意义。
2. 结 果
2.1. SEM下牙本质小管的形态
随着NaOCl浓度的升高，牙本质小管开口数目和直径逐渐增加(均P<0.05)，使用了10% ERY的各组牙本质小管开口数目和直径亦逐渐增加(均P<0.05)。使用10% ERY组与未使用组相比，牙本质小管开口数目和直径有所增加，但差异无统计学意义(均P>0.05，表2)。
表2.
次氯酸钠及异抗坏血酸钠处理后牙本质小管的开口 数目和直径(n=4， ±s)
Table 2 Numbers and diameters of opening dentinal tubules after NaOCl and sodium erythorbate (ERY) treatment (n=4, ±s)
| 组别 |
牙本质小管 开口数目/个 |
牙本质小管 开口直径/μm |
|---|---|---|
| DW | 20.15±6.31 | 1.20±0.12 |
| 0.5% NaOCl | 50.99±3.85* | 1.77±0.22* |
| 1% NaOCl | 63.31±6.81*† | 2.33±0.33*† |
| 2.5% NaOCl | 80.77±6.43*†‡ | 2.81±0.22*†‡ |
| 5.25% NaOCl | 94.09±6.03*†‡§ | 3.17±0.18*†‡§ |
| DW+10% ERY | 25.03±5.64 | 1.42±0.16 |
| 0.5% NaOCl+10% ERY | 55.81±1.82¶ | 1.99±0.26¶ |
| 1% NaOCl+10% ERY | 68.3±4.67¶ǁ | 2.53±0.38¶ǁ |
| 2.5% NaOCl+10% ERY | 85.87±5.54¶ǁ# | 3.01±0.21¶ǁ# |
| 5.25% NaOCl+10% ERY | 99.37±6.36¶ǁ#^ | 3.41±0.21¶ǁ#^ |
DW：去离子水；NaOCl：次氯酸钠；ERY：异抗坏血酸钠。与DW组比较，*P<0.05；与0.5% NaOCl组比较，†P<0.05；与1% NaOCl组比较，‡P<0.05；与2.5% NaOCl组比较，§P<0.05；与DW+10% ERY组比较，¶P<0.05；与0.5% NaOCl+10% ERY组比较，ǁP<0.05；与1% NaOCl+10% ERY组比较，#P<0.05；与2.5% NaOCl+10% ERY组比较，^P<0.05。
DW组、0.5% NaOCl组牙本质表面仍有大量玷污层覆盖，牙本质小管开口数目少且直径较小，使用10% ERY处理后暴露的牙本质小管开口稍有增加，但直径变化并不明显；1% NaOCl组有相对多的牙本质小管已暴露，但局部仍可见少许玷污层物质遮挡(图2)；2.5% NaOCl与5.25% NaOCl组的牙本质表面牙本质小管开口较大，数目较多，且与NaOCl浓度的增加成正比。经2.5% NaOCl与5.25% NaOCl处理后的管间牙本质表面呈现多孔隙结构，即脱蛋白牙本质，且随着浓度的增加，小孔隙分布更加密集，牙本质脱蛋白情况亦更加明显。在10% ERY处理之后，孔隙较未处理组明显减少，牙本质脱蛋白情况似有所改善，但牙本质小管开口数目及直径无明显改变(图3)。
图2.
扫描电镜下低浓度次氯酸钠及异抗坏血酸钠处理后的牙本质小管形态
Figure 2 Morphologies of dentinal tubules after low concentration of NaOCl and sodium erythorbate (ERY) treatment under the scanning electron microscope
A: DW group; B: DW+10% ERY group; C: 0.5% NaOCl group; D: 0.5% NaOCl+10% ERY group; E: 1% NaOCl group; F: 1% NaOCl+10% ERY group. In the DW and 0.5% NaOCl groups, a great deal of smear layer overlays on the surface of dentine, and diameter and number of dentinal tubules are comparatively small, while obviously increase in the 1% NaOCl group. In addition, number and diameter of dentinal tubules significantly increase in the ERY-treated groups compared with the non-ERY-treated groups.
图3.
扫描电镜下高浓度次氯酸钠及异抗坏血酸钠处理后的牙本质小管形态
Figure 3 Morphologies of dentinal tubules after high concentration of NaOCl and sodium erythorbate (ERY) treatment under the scanning electron microscope
A: 2.5% NaOCl (×1 000); B: 2.5% NaOCl (×5 000); b: 2.5% NaOCl (×20 000), where the black arrow point zooms in, *pores densely distributed; C: 2.5% NaOCl+10% ERY (×1 000); D: 2.5% NaOCl+10% ERY (×5 000); d: 2.5% NaOCl+10% ERY (×20 000), where the black arrow points zooms in, 〇 pores not that densely distributed; E: 5.25% NaOCl (×1 000); F: 5.25% NaOCl (×5 000); f: 5.25% NaOCl (×20 000), where the black arrow points zooms in, *pores densely distributed; G: 5.25% NaOCl+10% ERY (×1 000); H: 5.25% NaOCl+10% ERY (×5 000); h: 5.25% NaOCl+10% ERY (×20 000), where the black arrow pointing zooms in, 〇 pores not that densely distributed. Diameter and number of dentinal tubules are comparatively large in the 2.5% NaOCl (A and B) and 5.25% NaOCl (E and F) groups. Pores are significantly reduced in the 2.5% NaOCl+10% ERY (B, C and c) and 5.25% NaOCl+10% ERY (G, H and h) groups compared with the 2.5% NaOCl (A, B, and b) and 5.25% NaOCl (E, F, and f) groups, however, the diameter and number of dentinal tubules are rarely changed.
2.2. SEM下粘接层的形态
根据SEM下的观察结果及树脂突形成评分可知：除5.25% NaOCl组的评分较2.5% NaOCl组降低外，随着NaOCl浓度的增高，评分逐渐增加，树脂突形成逐渐变多、变粗且长度增加(P<0.05)。2.5% NaOCl组树脂突形成范围广、粗大且彼此相互交联，深部有侧枝形成；而5.25% NaOCl组虽然树脂突形成长度增加，却较细且彼此无明显交联。使用10% ERY组与未使用组相比，评分差异无统计学意义(P>0.05；图4，表3)。
图4.
扫描电镜下次氯酸钠及异抗坏血酸钠处理后的粘接层形态
Figure 4 Bonding interface morphology after NaOCl and sodium erythorbate (ERY) treatment under the scanning electron microscope
HL: Hybrid layer; RT: Resin tags; AD: Adhesive resin. A: Deionized water (DW); B: DW+10% ERY; C: 0.5% NaOCl; D: 0.5% NaOCl+10% ERY; E: 1% NaOCl; F: 1% NaOCl+10% ERY; G: 2.5% NaOCl; H: 2.5% NaOCl+10% ERY; I: 5.25% NaOCl; J: 5.25% NaOCl+10% ERY. In the DW (A), 0.5% NaOCl (C), 1% NaOCl (E), and 2.5% NaOCl (G) groups, with the increase of NaOCl solution concentration, the length of resin tag penetration gradually augmented, while the thickness and range of hybrid layer are gradually reduced. In the 5.25% NaOCl group, resin tags are relatively slimmer without significant cross links, though its length increased, and the hybrid layer formation is scarce or its thickness is minimal. After treated with ERY, the resin tag formation, depth of tag penetration and hybrid layer formationare rarely changed in the DW (A and B), 0.5% NaOCl (C and D), and 1% NaOCl (E and F) groups. After treated with ERY, the range and thickness of hybrid layer formation increased in the 2.5% NaOCl (G and H) and 5.25% NaOCl (I and J) groups.
表3.
扫描电镜下次氯酸钠及异抗坏血酸钠处理后粘接层形态的评价分数(n=10)
Table 3 Evaluated scores of bonding interface morphology after NaOCl and sodium erythorbate (ERY) treatment under scanning electron microscope (n=10)
| 组别 | 树脂突的形成评分/个 | 树脂突的长度评分/个 | 混合层的形成评分/个 | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 0 | 1 | 2 | 3 | 4 | 0 | 1 | 2 | 3 | |||
| DW | 8 | 2 | 0 | 0 | 9 | 1 | 0 | 0 | 0 | 0 | 0 | 6 | 4 | ||
| 0.5% NaOCl | 0 | 4 | 6 | 0 | 0 | 4 | 5 | 1 | 0 | 0 | 5 | 5 | 0 | ||
| 1% NaOCl | 0 | 3 | 7 | 0 | 0 | 3 | 6 | 1 | 0 | 1 | 4 | 5 | 0 | ||
| 2.5% NaOCl | 0 | 0 | 1 | 9 | 0 | 0 | 7 | 2 | 1 | 3 | 5 | 2 | 0 | ||
| 5.25% NaOCl | 0 | 3 | 7 | 0 | 0 | 0 | 0 | 1 | 9 | 8 | 2 | 0 | 0 | ||
| DW+10% ERY | 7 | 3 | 0 | 0 | 7 | 3 | 0 | 0 | 0 | 0 | 0 | 4 | 6 | ||
| 0.5% NaOCl+10% ERY | 0 | 5 | 5 | 0 | 0 | 3 | 6 | 1 | 0 | 0 | 4 | 6 | 0 | ||
| 1% NaOCl+10% ERY | 0 | 4 | 6 | 0 | 0 | 2 | 6 | 2 | 0 | 0 | 2 | 7 | 1 | ||
| 2.5% NaOCl+10% ERY | 0 | 0 | 0 | 10 | 0 | 0 | 6 | 2 | 2 | 0 | 0 | 1 | 9 | ||
| 5.25% NaOCl+10% ERY | 0 | 4 | 6 | 0 | 0 | 0 | 0 | 0 | 10 | 0 | 9 | 1 | 0 | ||
DW：去离子水；NaOCl：次氯酸钠；ERY：异抗坏血酸钠。每组10个条状试件，此表为每组每种评分下对应的试件数量，具体评分标准见表1。
根据SEM下的观察结果及树脂突长度评分可知：随着NaOCl溶液浓度的增高，树脂突长度逐渐增加，表现为树脂突加长。5.25% NaOCl组的评分高于0.5% NaOCl和1% NaOCl组(均P<0.05)，亦高于2.5% NaOCl组，但二者差异无统计学意义(P>0.05)。使用10% ERY组与未使用组相比，树脂突长度评分差异无统计学意义(P>0.05)，树脂突长度无明显改变(图4，表3)。
根据SEM下的观察结果及混合层的形成评分可知：随着NaOCl浓度的增高，评分逐渐下降，混合层的厚度及形成范围逐渐减小。与DW组相比，2.5% NaOCl、5.25% NaOCl组混合层形成评分显著降低(均P<0.05)，混合层形成极少或厚度极薄， 5.25% NaOCl组的混合层形成评分亦低于2.5% NaOCl组，但差异无统计学意义(P>0.05)。2.5% NaOCl+10% ERY、5.25% NaOCl+10% ERY组的混合层形成评分分别显著高于2.5% NaOCl、5.25% NaOCl组(均P<0.05)，混合层形成范围有所扩大且厚度增加；2.5% NaOCl+10% ERY组混合层形成的范围最大，厚度最厚(图4，表3)。
2.3. 微拉伸实验及断裂破坏类型
与DW组相比，0.5% NaOCl、1% NaOCl、2.5% NaOCl组微拉伸强度均增大(均P<0.05)，而5.25% NaOCl组则降低(P<0.05)。使用10% ERY处理的组别微拉伸强度高于未处理组，其中2.5% NaOCl+10% ERY、5.25% NaOCl+10% ERY组分别与2.5% NaOCl、5.25% NaOCl组相比，微拉伸强度均增大(均P<0.05，表4)。
表4.
次氯酸钠及异抗坏血酸钠处理后牙本质与树脂间的 微拉伸强度(n=10， ±s)
Table 4 Microtensile bond strength between dentine and resin after NaOCl and sodium erythorbate (ERY) treatment (n=10, ±s)
| 组别 | 微拉伸强度/MPa |
|---|---|
| DW | 45.79±3.68 |
| 0.5% NaOCl | 49.60±3.10* |
| 1% NaOCl | 50.75±5.12*† |
| 2.5% NaOCl | 55.73±3.64*†‡ |
| 5.25% NaOCl | 42.01±3.01*†‡§ |
| DW+10% ERY | 46.23±3.79 |
| 0.5% NaOCl+10% ERY | 50.51±3.29¶ |
| 1% NaOCl+10% ERY | 51.73±3.48¶ |
| 2.5% NaOCl+10% ERY | 59.11±2.41¶ǁ# |
| 5.25% NaOCl+10% ERY | 44.60±1.51ǁ#^ |
DW：去离子水；NaOCl：次氯酸钠；ERY：异抗坏血酸钠。与DW组比较，*P<0.05；与0.5% NaOCl组比较，†P<0.05；与1% NaOCl组比较，‡P<0.05；与2.5% NaOCl组比较，§P<0.05；与DW+10% ERY组比较，¶P<0.05；与0.5% NaOCl+10% ERY组比较，ǁP<0.05；与1% NaOCl+10% ERY组比较，#P<0.05；与2.5% NaOCl+10% ERY组比较，^P<0.05。
对微拉伸实验后条状试件的断裂类型进行统计，通过观察粘接界面发生断裂的比例，进一步判断NaOCl及10% ERY处理后牙本质与复合树脂间粘接面的情况，得到的各组断裂类型分布百分比见图5。其中DW和DW+10% ERY组的试件断裂类型以混合破坏居多，即断裂同时发生在粘接界面及树脂和牙本质内部；在5.25% NaOCl组的断裂模式分布中“Adhesive”类型显著高于DW、0.5% NaOCl、1% NaOCl及2.5% NaOCl组(均P<0.05)；同时，5.25% NaOCl+10% ERY组较5.25% NaOCl组“Adhesive”类型显著减少，混合破坏类型增加(均P<0.05)。
图5.
断裂模型分布
Figure 5 Incidence of failure modes among groups DW: Deionized water; NaOCl: Sodium hypochlorite; ERY: Sodium erythorbate. *P<0.05.
3. 讨 论
本实验观察到：在NaOCl溶液浓度较低时，随着其浓度升高，牙本质小管开口数目逐渐增多，直径逐渐增大，牙本质粘接强度亦有所增加；当用2.5% NaOCl溶液处理时，粘接强度最大。这可能是因为NaOCl溶液具有非特异性蛋白水解作用，在一定程度上溶解了玷污层中的有机物质及牙本质表面的胶原纤维，使得牙本质小管开放数目增多，次级牙本质小管开放，相应地，其树脂突长度亦逐渐增加，继而提高了机械固位力，增大了粘接强度。其中2.5% NaOCl组所形成的树脂突最粗大且深部侧枝形成，互相交联，成为一体。因此，在NaOCl溶液浓度较低时，虽然在SEM下观察与分析结果表明NaOCl溶液作用后，粘接面混合层的形成范围及厚度减小，但其对牙本质小管的打开及树脂突的渗透起促进作用，导致整体粘接强度有所增大。然而在NaOCl溶液浓度为5.25%的情况下，牙本质深层胶原纤维溶解明显，其对混合层形成的影响远大于其开扩牙本质小管口致树脂突长度增加的作用，导致整体粘接强度下降；在SEM下可以发现其混合层几乎不可见，且形成的树脂突虽然较长，但却较细、分布不均匀，同时无明显侧枝交联。
管间牙本质位于管周牙本质之间，胶原纤维含量较多，矿化程度较管周牙本质低，故前述结果中所观察到的2.5% NaOCl及5.25% NaOCl组牙本质表面密集分布的小孔隙主要位于管间，这可能是NaOCl溶液溶解牙本质胶原纤维形成的。在SEM下，5.25% NaOCl组小孔隙数量更多也更密集，意味着这种溶解作用更加严重，影响混合层的形成，使胶黏剂不能有效地渗入胶原纤维网和牙本质小管内。同样，本实验在SEM下观察到随着NaOCl溶液浓度的升高，混合层形成范围及厚度逐渐减小。而在使用ERY处理后，2.5%及5.25% NaOCl组牙本质表面的小孔隙分布明显没有之前密集，牙本质粘接强度亦较未使用组增加，考虑其一定程度上恢复了NaOCl溶液对牙本质粘接带来的不利影响。
ERY增加牙本质的粘接强度可能与以下因素有关：第一，在水中，NaOCl电离为Na离子与ClO离子，在pH值为4~7时，ClO离子主要以HClO的形式存在，在pH值为9左右时，ClO离子单独存在的比例更大[1]，而HClO溶解牙本质胶原蛋白的作用更强[16]。ERY的pH值为8~9，其在一定程度上可以降低HClO的比例，进而减小HClO对牙本质胶原蛋白的溶解作用。第二，ERY作为性能优良的抗氧化剂，可直接与NaOCl在水中电离出的主要成分强氧化剂HClO发生氧化还原反应，从而抑制其对氨基酸和胶原纤维的分解作用。第三，ERY通过与牙本质胶原纤维中的脯氨酸或赖氨酸进行羟基化反应，稳定维持并一定程度上恢复了胶原蛋白分子的结构，进而改善了混合层的状态[17-19]。最后，NaOCl在水中电离生成的HClO可以分解生成氧气及氧自由基，可能与胶黏剂中的单体等成分发生反应，使聚合反应链提前终止，导致牙本质的粘接强度下降[12,20]；而ERY作为抗氧化剂，可与HClO及氧化自由基等副产物发生氧化还原反应，继而降低其对聚合反应链的影响，促进粘接树脂聚合，恢复牙本质的粘接强度。
在条状试件断裂模型中，5.25% NaOCl组以发生在粘接层的断裂类型为主，且断裂数远远多于其他组，进一步说明了5.25% NaOCl溶液处理后出现了相对粘接薄弱区，这同时印证了牙本质微拉伸强度的降低，而在使用ERY后，该组发生在粘接层的断裂类型数目显著减少，微拉伸强度亦有所增加，再次说明了使用ERY能够在一定程度上恢复较高浓度NaOCl处理对牙本质粘接的不利影响。本实验中DW和DW+10% ERY对照组的试件断裂类型以混合破坏类型居多，断裂同时发生在粘接界面及树脂或牙本质内部，说明粘接力与内聚力大致相等，体现了修复材料、胶黏剂与牙本质之间的一体化[21]。
以往研究所选用的NaOCl溶液浓度多单一，本研究设置的浓度较多元，考虑到了临床常用浓度，且样本的选择及处理、作用时间及方法尽可能模拟了根管治疗过程中NaOCl溶液对髓室牙本质的冲洗作用，使得本实验得到的结果更具有真实性及可信度。本实验采用微拉伸实验评定牙本质的粘接强度，是目前较为合适的也是应用最广泛的评估牙本质与材料之间的粘接强度的研究方法，得到的结果准确而可靠；牙本质小管开口数目与直径的评估测量方法参照Scelza等[13]的方法，且本研究在测量过程中，严格遵循随机与盲法原则，以确保实验的准确性；而粘接层的评估方法类比Moda等[14]于激光共聚焦显微镜下定量评估粘接层的相关指标，较具有权威性。
现有研究多仅以微拉伸实验的宏观角度评估NaOCl溶液处理对牙本质粘接的影响，本研究同时采用SEM下观察和定量指标评估牙本质小管及粘接层的情况，宏观与微观相结合，结果更加清楚明晰且有说服力。但由于本实验的主要目的在于探索临床常用不同浓度的NaOCl对牙本质粘接的影响及ERY对其的作用，故控制了影响实验结果的其他变量，这可能导致研究不够全面，是仍需改进之处。另外，本实验采用的是收集离体牙标本进行体外研究的方法，且微拉伸实验法更侧重反映的是即刻粘接强度，因此，相关的动物和临床实验将是我们未来继续进行探索的方向。
综上所述，本研究结果表明：当冲洗用的NaOCl浓度低于2.5%时，复合树脂与牙本质间的粘接强度随着NaOCl的浓度增加而增大；但当浓度为5.25%时，复合树脂与牙本质间的粘接强度显著降低。ERY能够增大2.5% NaOCl对复合树脂与牙本质间粘接强度的增加作用；亦能够恢复高浓度5.25% NaOCl对复合树脂与牙本质粘接强度的不良影响。
利益冲突声明
作者声称无任何利益冲突
作者贡献
张碧晗 实验操作，论文撰写与修订；杨东辉 实验器材等准备和提供；朱喜蕾 协助实验，数据采集；周亚琴 数据采集，统计分析；朱沁怡 统计分析，图片处理；方厂云 实验设计，提供实验指导及论文修改。全体作者都阅读并同意最终的文本。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202202226.pdf
参考文献
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