兽研引言
保留牙齿的操作（如根管治疗）已不再仅与人医牙科相关，近几十年来在兽医领域的重要性日益增加（1–3）。材料、器械和影像学模式的进步扩大了伴侣动物（尤其是犬）牙髓治疗的适应证。
根管治疗被认为是牙科中技术要求最高的操作之一，需要多种器械、化学冲洗液和精心排序的临床步骤协同使用。这些步骤通常包括制备开髓洞形、去除病变牙髓组织、对根管系统进行机械和化学成形、使用合适的填充材料进行充填以消除细菌定植的潜在龛隙和通道，以及最终的冠部修复以防止微渗漏（4, 5）。
犬和猫的牙髓治疗与人医牙髓治疗存在显著差异，主要由于物种间在牙齿大小、根长、根管直径和髓腔形态方面的差异（1, 2）。这些解剖差异可使充分的清创和充填变得复杂，尤其是在大型犬齿中。
根管治疗可在单根牙和多根牙上进行。在兽医牙科中，具有重要战略和功能意义的牙齿，如上颌和下颌犬齿以及裂齿（上颌第四前臼齿和下颌第一臼齿），因其在抓握、咀嚼和工作性能中的重要作用而经常接受牙髓治疗（1, 6）。
保留这些牙齿对工作犬和服务犬尤为重要，包括警犬、军犬、猎犬和治疗犬，因为牙齿缺失可能显著损害其功能和生活质量（2）。
准确的术中及术后影像学检查对根管治疗的成功和随访评估均至关重要。传统牙科X线摄影仍是兽医实践中最常用的影像学模式，因为它易于获得、成本效益高，且可在不重新摆放患者体位的情况下术中应用。然而，其二维性质限制了对复杂根管形态的评估。
锥形束计算机断层扫描（CBCT）已成为牙科中成熟的影像学工具，可提供牙齿和根管系统的高分辨率三维可视化，并在临床和研究环境中显示出显著的诊断优势（7, 8）。在人医牙髓治疗中，CBCT已被证明对评估根管解剖、治疗结果和操作并发症特别有价值（7）。
本研究的主要目的是在犬的犬齿中比较两种机械驱动的往复式镍钛器械技术与手动镍钛器械技术，并使用高分辨率微型CT定量评估其对根管几何形态和充填质量的影响。次要目的是将术后CBCT上观察到的牙本质-填充材料界面透亮表现与相应的高分辨率微型CT表现进行定性比较。
我们假设三种器械技术对根管几何形态和充填质量的影响会有所不同。此外，我们假设术后CBCT上观察到的透亮界面区在使用高分辨率微型CT评估时不一定对应于真实的结构缺陷，如空隙或残留碎屑。
材料与方法
共分析了30颗因与本研究无关的原因拔除的下颌和上颌犬齿。牙齿固定于中性缓冲福尔马林中，随后保存于70%乙醇中。治疗前，每颗牙齿的微型CT扫描已作为前期研究的一部分获取，作为解剖参考。因此，本研究的样本量受限于具有符合预定义纳入标准的相应治疗前微型CT数据集的犬齿的可获得性。因此，未进行先验效能分析。
样本按照改良Triadan编号系统（104、204、304、404）根据体重、年龄和颌位（上颌/下颌）进行分类，并随机分配到三组（9）。30颗犬齿分为三组，每组10颗，分别分配到以下预备技术之一。每组内，五颗牙齿由博士前专科医师（操作者1号）进行器械操作，五颗由DAVDC/DEVDC认证的兽医牙科专科医师（操作者2号）进行器械操作。A组：iM3a的Control Cut Rotary Flex镍钛锉（使用iM3 C Flex马达机用操作）。
B组：Dentsplyc的Vet Flex和Preserve镍钛锉（使用iM3 C Flex马达机用操作）。
C组：手用锉：iM3d的镍钛锉（K-Reamer；图1）。
[图：三个器械组使用的牙髓器械（从左至右）：(A) A组：Control Cut旋转锉]
为在反映常规临床实践的同时标准化器械磨损，每根器械在更换为新器械套装前最多用于预备两颗牙齿。治疗前，每颗牙齿分别包埋于聚碳酸酯块中（2, 6），并接受标准化操作流程（预备、成像、化学预备、充填和修复；图2）。根管治疗方案遵循本诊所常规应用的标准临床程序。所用全部材料和器械的完整清单见补充表S1。
[图：代表性犬牙标本在根管预备前单独包埋于聚碳酸酯块中。]
开髓
每颗牙齿分别以釉牙骨质界（CEJ）高度固定于聚碳酸酯中，并干燥至少48小时。使用浸有96%乙醇的棉签进行釉质清洁和消毒。为模拟牙折，测量牙冠长度的25%，并在持续水冷却下用旋转金刚石锯片水平横断。随后使用直径1 mm的2号圆形金刚砂车针f，在A组和B组中向“折裂部位”方向制备深度至少6 mm的冠部洞腔。
在C组中，使用镍钛（Ni-Ti）手用锉，按多项研究所述在CEJ上方约2 mm处创建辅助开髓孔（1, 10）。随后使用Gates-Glidden（GG）车针（1-6号）g联合润滑剂Glydeh扩大冠部洞腔。车针被动引入根管，仅在退出时进行主动牙本质去除。
预备
使用不同规格的一次性标准神经针i进行牙髓摘除。确定第一个到达根尖的ISO编码根管锉（FF = 第一根锉）以确定以毫米计的工作长度，并通过X线片及在根管标尺上测量的硅胶止动片进行验证。后续根管锉按其递增的ISO规格选择，并始终将硅胶止动片置于预定工作长度处。
在A组中，Control Cut Rotary Flex镍钛锉以300 rpm和2.0 Ncm的标准化往复运动操作，旋转角度为顺时针150°和逆时针30°。选择较大的正向角度以对应器械的右手切割设计，其中主动切割发生在顺时针旋转期间。该设置为iM3 C Flex endomotor的推荐预设（11）。
在B组中，Vet Flex & Preserve镍钛锉c在标准化往复条件下以300 rpm和2.0 Ncm操作，采用逆时针150°和顺时针60°的旋转运动。由于器械设计对应左手切割配置，较大的角位移（150°）分配给逆时针阶段。这些参数根据制造商建议选择（12, 13）。
C组标本使用镍钛K扩孔锉d以顺时针¼转-提拉技术手动预备，同样为右手切割设计。
每使用一根根管锉后，均执行标准化的化学预备冲洗方案，该方案在三种预备技术中保持一致（14, 15）。使用专用侧孔冲洗针27Gj执行的冲洗程序包括以下步骤：首先，使用5.25%次氯酸钠k（NaOCl）冲洗和灌洗根管，随后用ISO 15d（K扩孔锉，镍钛）锉回锉以搅动牙本质碎屑。溶液根据制造商建议储存于4 °C，使用前恢复至室温（16）。随后使用空注射器连同冲洗针进行吸引。接着，使用0.9%无菌氯化钠l（NaCl）进行最终冲洗，再次进行吸引。
每使用一根根管锉均以Glydeh作为根管调理剂，以增强根管润滑并降低器械折断风险（17）。最终锉（LF = 最后一根锉）定义为在不改变既定工作长度的情况下刚好到达根尖的最大规格锉。三种技术均采用统一的化学冲洗方案。最终冲洗时，将17% EDTAm引入根管并留置1分钟后通过吸引去除。随后保留NaOCl一分钟，然后吸引去除。最后，用NaCl冲洗根管并用ISO标准化纸尖n干燥。
操作并发症
记录每颗牙齿在根管器械操作期间发生的操作并发症。对于每次事件，记录涉及的器械、碎片取出成功或失败以及执行操作的术者。同一牙齿发生的多次并发症记录为独立事件。如果折断器械取出失败，则受影响牙齿被排除进一步治疗，并由另一颗牙齿替换，以维持预定义的每组10个标本的样本量。操作并发症以绝对数和百分比进行描述性总结，包括按器械类型和术者经验分布的汇总。
除对操作并发症进行系统记录外，所有器械在使用前及使用过程中均进行肉眼检查，以观察宏观变形和表面附着碎屑。这些观察为定性观察，未进行标准化评分或组间统计学比较。
根管充填
根管充填过程的第一步是选择与最终锉的ISO尺寸相对应的合适牙胶尖。然后使用专用输送头涂布GuttaFlow bioseal。使用Lentulo螺旋输送器将GuttaFlow bioseal均匀分布于整个根管系统（18）。
单尖法操作中，将预先在5.25% NaOCl中消毒1 min（5）并在96%乙醇中冲洗以去除可能形成的表面沉积物的已预备牙胶尖插入根管。随后使用加热的挖匙切断牙胶尖。使用垂直加压器进行材料压实。用不同的挖匙清理冠部窝洞的牙本质壁，并按常规临床方案及术者判断获取标准化口内X线片（图3A–E）。
[图：代表性放射影像序列，展示根管治疗的不同阶段。(A) 治疗前放射影像]
修复
最终开髓孔修复采用所谓的“三明治技术”，涉及两种不同修复材料的粘接。在充填物上方涂布一层厚度不超过2 mm的玻璃离子水门汀，随后使用蓝光进行光聚合，光强500–1,000 mW/cm²，波长约470 nm，距离牙面1–1.5 mm，照射20 s。
然后按制造商说明涂布Adhese Universal Viva Pent（粘接剂），再次使用蓝光在距离牙面1–1.5 mm处光照20 s。最终修复使用Tetric EvoCeramu（可压实复合树脂）完成，同样光照20 s。
使用SHOFUv抛光器抛光复合树脂，以获得与周围牙釉质相匹配的光滑表面，最后在光照下使用G-Coat Nanosealw对表面进行封闭。
CBCT
所有治疗后的牙齿术后均使用CBCT（Planmed Verity®，Planmed Oy，Helsinki，Finland）进行扫描。CBCT扫描参数如下：扫描时间18 s，重建时间约30 s，体素大小0.2 mm，管电压84 kV，管电流4 mA，曝光时间10.0 s。图像采集参数根据制造商规格选择，并结合实验设置进行调整。容积数据集以DICOM格式导出以供后续分析（19）。
微型CT
所有扫描均使用微型CT μCT35系统（SCANCO Medical AG，Brüttisellen，Switzerland）采集。
未处理牙齿 mounted 于含70%乙醇的Parafilm密封样品架中。未处理牙齿的扫描参数为70 kVp / 114 μA，使用0.5 mm铝（Al）滤片。积分时间800 ms（探测器合并=2），角度增量0.36°。根据牙齿大小，未处理牙齿数据集中的各向同性体素大小分别为20 μm（大牙，20 mm样品架，20.5 mm视野）或12 μm（小牙，12 mm样品架，12.3 mm视野）。
处理后牙齿由修剪过的聚碳酸酯块包围， mounted 于20 mm样品架中（空气介质）。处理后牙齿的扫描参数为70 kVp / 114 μA，使用0.1 mm铜（Cu）滤片。积分时间800 ms（探测器合并=2），角度增量0.36°。处理后牙齿重建数据集中的各向同性体素大小为20 μm。
重建图像体积以DICOM（*.dcm）文件格式导出。
CBCT与微型CT比较
对治疗后CBCT数据集进行肉眼评估，观察牙本质-充填材料界面处的透射区。在随后的微型CT图像分析中，由一名研究操作者在体素水平对所有标本进行肉眼检查，特别关注CBCT上识别的透射性发现是否对应于相应微型CT数据集中的实际空隙、空气夹杂、碎屑堆积或结构不连续。CBCT与微型CT发现之间的比较为定性比较，未进行统计学分析。
图像处理与分析
图像配准与治疗前分割
将DICOM图像体积导入3D分析与可视化软件AMIRA（FEI Visualization Sciences Group，Thermo Fisher Scientific旗下，Mérignac Cédex，France，版本2023.2），并转换为Amira网格（*.am）文件格式。将治疗前和治疗后图像体积导入项目模板，并使用Register Images工具基于归一化互信息进行空间配准（刚性变换）。
为降噪，预处理微CT数据集使用双边滤波器进行滤波（解释：3D，核大小3*3*3，相似度=10,000）。使用Amira分割编辑器以固定阈值（2600）从牙髓中分割出矿化牙体。随后使用闭运算（3像素膨胀后接3像素腐蚀）对牙髓的初始分割进行进一步处理，然后使用Smooth Labels命令（大小=3，模式=3D体积）进行平滑。使用Material Statistics工具测量牙体和牙髓的体积。
中心线生成与横截面分析
接下来，基于断层图像（Ortho Slice模块）在牙髓中心手动放置标记点，为牙髓全长设置B样条（阶数：3，采样点201）。将B样条转换为空间图（Line Set to Spatial Graph模块），并使用Smooth Line Set模块进行平滑（平滑度：0.5，迭代次数：100）。最后，使用Tcl命令模块执行一个Tcl脚本——该脚本最初作为Johnson Chacko等(20)的补充材料发表，此处使用时对像素大小做了轻微修改。该脚本使用Trajectory沿牙髓空间图的每个点移动牙髓分割（二值标签场）的切片。切片的方向始终垂直于曲线相应位置的曲率。提取的牙髓二值分割掩膜横截面图像（Extract Image）导出为*.tif图像，并使用自定义Fiji宏进行分析，该宏测量了多个参数，如牙髓面积、周长、椭圆拟合的长轴和短轴、Feret直径、Feret角、圆度、圆整度和纵横比。自定义Fiji宏自动处理每个标本的全部201张横截面图像。尽管记录了多个几何参数，但本研究报告的锉-根管直径分析中使用Feret直径作为根管直径的度量。
由于每条空间图曲线恰好包含201个点（如B样条最初定义），从一张牙髓横截面图像到下一张的每一步恰好代表牙髓长度的0.5%。这使得尽管所测量牙齿大小不同，仍可比较不同牙齿间牙髓面积和形状的相对变化(20)（图4A–E）。
[图：用于治疗前后根管系统评估的Micro-CT图像处理和分析工作流程。(A) 治疗前]
治疗后分割与材料分类
治疗后微CT数据集使用非局部均值算法进行滤波（解释：3D；空间SD=5；强度SD=0.2；搜索窗口=10像素；局部邻域=3像素）。使用Amira分割编辑器再次分割牙体和填充后的牙髓。使用固定阈值分割牙体（下限阈值：2000，上限阈值11,000）以及牙髓填充材料和牙胶尖（下限阈值：11001），后者与牙体相比显示出高得多的X射线衰减。使用手动分割工具将处理后牙髓中的空气腔添加至牙髓标签，因此牙髓的分割代表了整个处理后的根管。使用Smooth Labels命令（大小：6，模式=3D体积）对分割的牙体和牙髓进行平滑。随后，复制牙髓腔室以便对修复材料进行分割。为牙胶尖、GuttaFlow bioseal水门汀和空气定义了单独的材料类别。每种材料使用固定掩膜阈值（空气：0–10,999，水门汀：11000–16,000，牙胶：28000–32,767）和区域增长工具进行分割，随后进行3D平滑（Smooth Labels，大小=3，模式=3D体积）。最后，通过仔细的视觉检查验证修复材料的分割。所得标签场保存用于定量分析。
体积与表面分析
为将定量分析限制在牙根部分，使用Volume edit工具手动勾画牙冠（感兴趣区域（ROI）），并在分割编辑器中将其从所有标签场（治疗前牙髓、治疗后牙髓、牙髓修复材料）中排除。然后使用Material Statistics工具分析治疗前后牙髓的牙根体积。使用Arithmetic工具（治疗前牙髓体积减去处理后牙髓体积）计算未处理牙髓的比例。体积测量值（单位mm3）从Amira导出至Microsoft Excel。
牙髓修复材料的体积也使用Material Statistics工具测量。此外，使用Generate Surface工具（算法模式：可重复，平滑：现有权重）从修复材料分割生成多边形表面模型。随后，使用Surface Area Volume工具（模式：面片）计算填充材料（GuttaFlow bioseal）/牙体、牙胶/牙体以及空气/牙体之间的界面面积（单位mm2）。
治疗后横截面分析
为对处理后的牙髓进行横截面评估，执行了来自未处理牙髓的空间图以及先前用于未处理牙髓的 Amira Tcl 脚本，以自动提取每个样本的 201 张横截面图像。这些图像再次使用自定义 FIJI（ImageJ）脚本进行处理，以量化牙髓和充填界面的几何参数。所得数据在 Microsoft Excel 中汇总并分析，以进行比较治疗前和治疗后状态的体积和表面比较。
锉至根管直径分析
为了将器械预备根管的尺寸与预期具有 2% 锥度的最终锉的尺寸进行比较，根据由 201 个点组成的预定义样条对弯曲牙髓长度进行标准化。对于锉至根管直径分析，使用从 Fiji 分析获得的 Feret 直径作为治疗后实际根管直径的度量，并根据相应的像素大小从像素转换为毫米。在 201 个标准化位置中的每一个位置，将治疗后根管的 Feret 直径与最终 2% 锥度器械在相应位置的计算直径进行比较，并确定两次测量之间的差异。对于每颗牙齿，随后计算所有 201 个位置的平均和中位差异，并用于器械组之间的统计比较。
统计分析
统计分析使用 IBM SPSS Statistics（版本 29；IBM Corp.，Armonk，NY，USA）进行。连续变量在适当情况下以平均值 ± 标准差（SD）和 95% 置信区间（95% CI）进行描述性总结。
为了评估器械预备技术对根管几何形态、充填质量和界面特征的影响，根据结局变量应用了不同的统计模型。根管直径与器械尺寸之间的差异以及内部空气体积使用线性模型进行分析，包括组别（A、B、C）、颌骨（上颌 vs. 下颌犬齿）及其交互作用（组别 × 颌骨）的固定效应。相比之下，治疗后/治疗前根管体积比和基于表面的界面测量使用单因素方差分析（ANOVA）进行分析，因为这些模型未包括颌骨。拟合的线性模型中未包括随机效应结构。表面分析包括对充填材料与牙齿结构、牙胶与牙齿结构以及空气与牙齿结构之间界面的定量测量。对于表现出非正态分布模式或异方差性的变量，在分析前额外应用了对数转换。从拟合模型得出的估计边际均值用于成对组间比较。每当检测到显著的组别主效应时，使用 Sidak's alpha 校正程序进行事后比较。在指示的情况下，根据牙齿位置（Triadan 104/204 vs. 304/404）进行了额外的亚组分析。
为了评估潜在的术者依赖性效应，进行了独立样本 t 检验，比较两位术者之间的体积和基于表面的结局变量。
通过目视检查残差图和 Q–Q 图来评估模型假设，以评估近似正态性和残差方差的同质性。所有统计检验均为双侧检验，统计显著性定义为 p < 0.05。
结果
锉至根管直径差异
治疗后根管直径与相应最终器械直径之间的差异使用每颗牙齿 201 个标准化位置计算的平均和中位差异进行评估。因此，较大的值代表预备根管直径与最终器械直径之间的较大差异。
对平均差异的分析显示组别具有显著主效应（F(2, 24) = 5.94，p = 0.008），而颌骨效应（p = 0.129）和组别 × 颌骨交互作用（p = 0.766）均未达到统计学显著性。
描述性分析显示，A 组的平均差异为 0.162 mm（SD = 0.144；95% CI：0.034–0.233），B 组为 0.098 mm（SD = 0.117；95% CI：0.000–0.186），C 组为 0.299 mm（SD = 0.155；95% CI：0.208–0.390），其中 C 组观察到最高的平均差异。
Sidak 校正的成对比较显示 C 组与 B 组之间存在显著差异（p = 0.010）。相比之下，C 组与 A 组之间（p = 0.055）以及 A 组与 B 组之间（p = 0.906）的比较未达到统计学显著性。
对中位差异的分析同样显示组别具有显著效应（F(2, 24) = 5.85，p = 0.009）和颌骨具有显著效应（F(1, 24) = 5.09，p = 0.033）。组别与颌骨之间的交互作用无统计学显著性（p = 0.729）。
中位差异的估计边际均值在 A 组为 0.129 mm（95% CI：0.043–0.214），B 组为 0.064 mm（95% CI：−0.015–0.144），C 组为 0.247 mm（95% CI：0.169–0.325），同样在 C 组观察到最大值。
Sidak 校正的事后比较显示 C 组与 B 组之间存在显著差异（p = 0.008），而 C 组与 A 组之间（p = 0.130）以及 A 组与 B 组之间（p = 0.609）的比较无统计学显著性。
按颌骨分组的亚组分析显示，组别对上颌犬齿（104/204）有显著影响（F(2, 24) = 3.59，p = 0.043），而下颌犬齿（304/404）未检测到显著的组别效应（p = 0.085）。
总体而言，均值和中位数分析均一致显示，C组治疗后实际根管直径与相应最终器械直径之间的差异最大。仅在C组与B组之间发现显著的成对差异，而涉及A组的比较在Sidak校正后无统计学意义。
操作并发症的发生率
30颗牙齿中有8颗（26.7%）发生操作并发症，共计10起独立事件。在若干病例中，同一颗牙齿记录了不止一种类型的并发症。
最常见的并发症与使用Gates-Glidden钻相关（4例，40%），其次为ISO 15号锉（3例，30%）、Path Finder器械（2例，20%）和ISO 70号锉（1例，10%）。
在受影响的8颗牙齿中，有5颗（62.5%）成功取出断片，而3例（37.5%）取出失败，不得不使用替代牙齿进行进一步分析。
按操作者经验分层后，10起并发症中有7起（70%）发生在博士前专科医师（1号操作者）实施的操作中，而3起（30%）发生在委员会认证的兽医牙科医师DAVDC/DEVDC（2号操作者）进行器械操作期间。
体积分析
治疗后与治疗前根管体积比（即治疗后根管体积与治疗前根管体积之比）在三种器械操作技术之间无显著差异（F(2,27) = 0.79，p = 0.464）。A组、B组和C组的平均治疗后与治疗前根管体积比分别为11.47 ± 4.91、11.33 ± 8.29和8.40 ± 4.60。使用Sidak校正事后检验进行成对比较，未发现任何组间显著差异（A vs. B：p = 1.000；A vs. C：p = 0.620；B vs. C：p = 0.652；图5）。
[图：按器械组划分的根管治疗后与治疗前体积比。A组：Control Cut旋转柔性镍钛锉。B组：]
空气体积分析
观察到组别对总空气体积有显著影响（F(2, 24) = 4.83，p = 0.017）。
经Sidak校正的成对比较显示，C组与A组之间无显著差异（p = 0.060），而C组的空气体积显著高于B组（p = 0.028）。A组与B组之间未观察到显著差异（p = 0.994）。
颌骨（p = 0.966）及组别×颌骨交互作用（p = 0.140）均不显著。估计边际均值A组为0.61 mm³、B组为0.46 mm³、C组为2.15 mm³。因此，C组表现出最高体积的内部空隙或未填充根管空间，表明与A组和B组相比其充填模式均匀性较差（图6）。
[图：按器械组划分的充填根管系统内空气体积（mm³）。A组：Control]
CBCT与微型CT比较
CBCT成像在多个标本的牙本质-充填材料界面显示透射区，可解释为潜在空隙或界面缺陷。然而，使用高分辨率微型CT进行的比较分析显示，这些位置无相应空隙、碎屑堆积或空气夹杂。相反，微型CT数据集显示充填材料与根管壁连续适配，表明这些透射区并不代表真正的结构缺陷。
定量微型CT分析进一步证实，内部空气体积有限，且主要局限于根管系统内，而非牙本质-充填界面处。在对应于CBCT透射区的位置缺乏结构不连续，提示这些发现归因于成像相关的对比效应，而非充填不完全或残留碎屑。
表面分析
表面分析显示，各组之间在任何评估参数上均无显著差异：充填材料表面积/牙齿（F(2, 27) = 0.31，p = 0.734）、牙胶表面积/牙齿（F(2, 27) = 0.20，p = 0.818）和空气表面积/牙齿（F(2, 27) = 0.16，p = 0.855）。
这些发现表明，尽管内部空气体积存在差异，但所有组间充填材料及界面的表面特征相当。
操作者比较
比较操作者表现的独立样本t检验显示，体积参数无显著差异（所有p > 0.25）。然而，每颗牙齿充填材料的表面积在操作者之间差异显著（t(28) = 2.18，p = 0.038），1号操作者（JW）产生的充填表面略大于2号操作者（MES）。这一发现提示充填或分割存在轻微的操作者依赖性变异，而总体体积学结果保持一致。
综上所述，体积分析显示治疗后总根管体积无显著组间差异，但C组表现出充填根管内空气体积增加的一致趋势。基于表面的指标在各组之间无显著变化，操作者间变异性极小。
讨论
本研究利用高分辨率微型CT评估了犬齿中不同的根管器械操作技术，并评估了其对根管几何形态、充填质量及操作安全性的影响。研究结果不仅有助于理解根管成形特征，还为术后影像的解读提供了见解。
关于CBCT评估，结果支持以下假设：在牙本质-充填材料界面观察到的透射区主要代表影像相关伪影，而非真正的治疗缺陷。使用高分辨率微型CT进行的对比分析表明，这些透射区对应于不同射线密度材料之间的对比度过渡，并不反映根管系统内残留的坏死牙本质、持续存在的碎屑或滞留空气(21)。微型CT数据集中不存在相应的空隙、碎屑堆积或结构不连续，这强烈提示CBCT所见应归因于束硬化效应或对比度相关成像现象，而非充填不充分或清创不彻底。这些观察结果强调在兽医牙髓病随访中谨慎解读CBCT所见的重要性，并突显了高分辨率成像在区分真实结构缺陷与成像方式相关伪影方面的附加诊断价值(22)（图7A,B）。
[图：代表性术后CBCT图像，显示牙本质-充填材料界面处相似的透射区]
与传统口内X线摄影相比，CBCT本身可提供根管解剖结构的三维可视化，并避免了二维成像固有的重叠和投影相关局限性。既往研究已证实CBCT在评估根管形态和牙髓治疗 outcomes 方面的诊断价值(23, 24)。然而，本研究并未系统比较CBCT与传统口内X线摄影。相反，我们的研究结果表明，CBCT上牙本质-充填材料界面观察到的透射区需要谨慎解读，因为相应的高分辨率微型CT评估表明，并非所有表观透射区都代表真实的结构不连续。因此，在解读术后CBCT检查时，认识此类成像相关现象非常重要。
在本机构，CBCT检查常规在根管治疗前后进行，并辅以术中口内X线片以实现连续的操作记录。随访检查包括两种成像方式，以促进对治疗成功、失败或无影像学疾病证据的评估。这种联合成像方法可提高诊断信心，尤其是在仅凭二维成像可能不足的复杂病例中(23, 25)。
除诊断评估外，本研究结果还为不同根管预备器械操作策略的生物力学后果提供了见解。高分辨率三维成像能够详细评估根管扩大和牙本质去除模式，从而更全面地评价成形质量和根牙本质的保留情况(26)。在有效根管扩大以实现充分清创与保留牙本质之间维持适当平衡，被认为对牙髓治疗牙齿的长期结构完整性至关重要(27)。
机用镍钛系统在锉尺寸与实际根管直径之间产生的差异往往小于手动器械操作。然而，经Sidak校正后，仅在B组和C组之间观察到统计学显著的配对差异(28)。尽管三种器械操作技术在术后与术前根管体积比方面未观察到显著差异，但手动器械操作往往导致更高的内部空气体积。配对比较显示C组空气体积显著大于B组，而C组与A组之间的差异在Sidak调整后未保持统计学显著性。多重比较校正后A组与C组之间缺乏统计学显著性，提示尽管观察到相似趋势，但差异幅度不足以在更保守的Sidak程序下证明稳健效应。
这些发现提示，机械器械操作的优势主要并非归因于更大的整体根管扩大，而是归因于更受控且可重复的成形。
改善的根管居中性以及器械尺寸与实际根管尺寸之间更紧密的对应关系，可能有助于更有效的清创，同时最大限度地减少不必要的根牙本质去除(4, 26)。因此，预备质量似乎较少取决于根管扩大程度，而更多取决于对原始根管解剖结构的精确尊重(23)。总体而言，这些发现表明，成形精确性而非根管扩大程度，可能代表兽医牙髓病学中预备质量的关键决定因素。
尽管本研究主要关注器械操作后的体积和几何变化，但体积增大并不一定反映器械与根管壁机械接触的比例。既往人类牙髓病学中的微型CT研究表明，即使根管显著扩大，根管壁的部分区域仍可能未被触及(26)。因此，体积变化和器械操作的根管壁表面应被视为成形效果评价的互补指标。
另一个需要考虑的因素是不同器械操作方案之间进入路径设计的差异。C组使用了辅助性近中进入路径，而A组和B组未使用。尽管镍钛器械具有柔韧性和形状记忆特性，但对于C组所用器械，建议采用辅助进入路径以提供更直接的路径到达弯曲根管根尖部分。适当的进入路径设计是犬齿牙髓治疗的重要组成部分，朝向根尖的直线进入路径有助于器械插入，同时最大限度减少不必要的牙体组织去除(29)。近中进入路径也被描述为犬牙髓治疗的标准方法(10)。因此，从理论上讲，C组的额外进入路径预期应有助于而非妨碍器械操作。然而，由于各组的进入路径设计未标准化，其对着形结果的潜在影响不能排除。因此，观察到的差异应被解释为反映各自的整体器械操作方案，而非仅反映器械系统本身。
在人类牙髓病学中，机用镍钛器械在成形能力、操作安全性和预备效率方面已显示出优于手动器械的优势(4, 28)。然而，必须区分许多人类牙髓病学研究中所评估的连续旋转运动与本研究中A组和B组机用系统所使用的往复运动。与连续旋转相比，往复运动学可能提高镍钛器械的抗循环疲劳能力，这在长而弯曲的根管器械操作中可能尤为重要(13)。因此，评估连续旋转镍钛系统的研究结果不应直接外推至本研究所探讨的往复系统。
在兽医牙髓病学中，犬齿长且常弯曲的根管对机械预备提出了特殊挑战。在此解剖背景下，镍钛器械相较于不锈钢器械的更高柔韧性可能具有优势，因为它有助于维持原始根管轨迹并减少弯曲根管中根管偏移的趋势(30)。在解释犬齿不同器械操作技术之间的差异时，这些解剖学和力学因素尤为重要(2, 6)。
然而，镍钛手用器械的力学特性也带来了固有的局限性。其超弹性和形状记忆行为虽然有助于适应根管弯曲，但可能限制故意将器械导向特定根管壁的能力，因此可能导致与部分根管表面的机械接触不完整。这一特性可能导致了C组中观察到的成形标准化程度较低和内部空气体积较大，尽管本研究中未直接量化根管壁接触情况。因此，C组的结果应仅在手动镍钛器械操作的背景下解释，不应推广至使用预弯不锈钢锉的手动器械操作。
器械组与颌骨之间接近显著的交互作用也可能反映了根形态的潜在解剖变异性。既往对犬齿的三维微型CT分析已证明根管弯曲度存在相当大的个体间变异，弯曲模式受体型和个体根形态的影响(30)。即使上颌和下颌犬齿之间不存在一致的体积差异，根管轨迹和弯曲度的细微变化也可能改变器械在预备过程中的力学行为。弯曲度增加或根管路径复杂可使成形对技术更为敏感，尤其是在使用更易发生偏移和根管偏移的手动器械时(31)。这些解剖特征可能增加根管预备的技术难度并易导致操作失误，尤其是在手动器械操作过程中(30)。这种解剖变异性可能部分解释了观察到的体积反应随颌骨变化的趋势。
C组观察到的内部空气体积增加最可能反映充填不够均匀，这通常与根管几何形态标准化程度较低相关(32–34)。尽管所有组均接受了相同的充填程序，但机用器械操作所实现的更可重复的根管几何形态可能有助于改善充填材料的适应性，从而减少内部空隙的发生(18)。
目前，比较犬齿不同牙髓器械预备技术的高分辨率微型CT定量证据仍然有限。既往研究主要集中于犬根管系统的解剖变异性，并已证实根管形态存在显著差异，这可能使彻底清创和充填变得复杂（1, 2）。本研究在这些观察基础上进一步提供了定量高分辨率微型CT证据，表明机用器械预备可实现更标准化的根管预备，且内部空隙少于手用器械预备。
技术与临床意义
观察到的空气体积和根管-器械偏差差异具有实际临床意义。空气空隙和未充填的根管空间可能破坏封闭性并增加细菌渗漏风险，而细菌渗漏仍是决定牙髓治疗长期成功的关键因素（23, 35）。因此，结果支持在兽医牙髓病学中临床使用机用镍钛系统，尤其是对于犬齿和裂齿等战略性牙齿。
此外，研究表明操作者经验可能在操作失误的发生中起重要作用，因为大多数并发症是在经验较少的操作者进行治疗时观察到的。尽管样本量有限，但器械相关并发症发生于约四分之一的标本中，部分牙齿出现多次操作失败。这些发现强调了犬齿牙髓预备相关的技术挑战，并提示操作者技能水平可能显著影响器械分离及相关并发症的发生率。
除定量分析的结果外，在器械操作过程中还注意到若干定性观察，此处作为偶然的、产生假设的发现进行报告，而非系统性的组间比较。除完全器械分离外，偶尔还观察到一种明显的塑性变形模式，其特征为螺旋刃可见解旋以及工作段在断裂前伸长。该模式与扭转应力积累一致，当器械尖端在根管内卡住而冠部继续旋转时即会发生，并可能作为器械失效前的宏观预警信号。此外，对新开封器械的目视检查偶尔发现使用前切割刃上附着细小金属碎屑；若未去除，此类颗粒可能作为异物被带入根管。由于这些观察未标准化或量化，应谨慎解读；然而，早期识别刃解旋和仔细检查新器械可能是简单的预防措施，其系统评估将是未来研究的有价值目标（4, 36）（图8A–C）。
[图：目视检查期间牙髓器械的代表性定性观察结果。(A) 塑性变形特征]
总体而言，这些观察强调了操作者熟练程度和预备质量对于实现可预测牙髓治疗结果的重要性（37）。这一解读与人类研究一致，后者表明操作者培训、技术表现与牙髓治疗成功之间存在强相关性（10, 38, 39）。
然而，C组内部空隙体积显著更大可能表明充填材料适应性不完全，这可能是由于手用器械预备所达到的根管几何形态标准化程度较低所致，尽管手用和机用器械均使用相同的镍钛合金。
局限性及未来方向
本研究存在若干局限性，在解读研究结果时应予以考虑。研究使用离体犬齿，这允许标准化预备和成像，但未考虑体内条件，如牙周膜支持、牙髓活力和生理性水分含量。
另一个需要考虑的问题是成像过程中样本的水合状态。治疗前扫描时牙齿浸没于70%乙醇中，而在包埋、开髓、器械预备、充填和修复后，治疗后扫描未重新浸没。因此，两次扫描之间牙体硬组织的一定程度脱水不能完全排除，且已有研究表明牙本质干燥可诱导牙本质微裂纹，原则上可能影响根管测量（40）。然而，在高分辨率治疗后micro-CT数据集中未观察到牙本质微裂纹，提示脱水不太可能在本研究所用空间分辨率下造成可检测的实质性结构损伤。此外，所有样本均接受了相同的处理和成像流程；因此，任何残留效应预期在各组间类似地存在，不太可能系统性地偏倚组间比较。充填及冠部修复的根管也可能限制了根管内部表面的干燥。尽管如此，绝对根管尺寸应谨慎解读，未来研究若能在治疗前后成像期间保持一致的水合状态，将有助于进一步减少这一潜在偏倚来源。
此外，相对较小的样本量（每个器械组10颗牙）是一个重要局限。由于样本量受限于相应治疗前micro-CT数据集的可用性，而非由先验效能分析确定，本研究可能效能不足，无法检测器械技术之间的较小差异。因此，无统计学显著性的结果不应必然解读为技术之间等效的证据。个体间解剖变异性可能进一步增加了相对较小各组内的变异性。未来研究应纳入更大样本量并进行前瞻性样本量计算，以及使用CBCT进行临床体内研究，以进一步评估本研究结果的临床可转化性。此外，评估不同器械预备方法后封闭能力和抗折性的纵向研究将补充本形态学分析。
基于固定灰度阈值的材料分割可能在空气体积定量中引入了轻微不准确性，尽管研究操作者在体素水平上进行了仔细验证。
尽管如此，应用于所有样本的标准化成像和分析方案很可能已将这一潜在误差来源降至最低。最后，尽管操作者因素对体积数据的影响有限，但观察到的经验较少操作者并发症更多的趋势强调了兽医牙科中专科技能培训的重要性。
结论
在本体外研究的局限性范围内，机器驱动的镍钛系统在犬牙中提供了比手动镍钛手用锉更具可重复性的根管预备和更均匀的充填。第一，尽管治疗后与治疗前根管体积比在各器械技术间无显著差异，但手动器械预备与器械尺寸和根管几何形态之间更大的差异相关，表明根管预备标准化程度较低。第二，手动器械预备观察到的内部空气体积增加提示与机器驱动技术相比充填均匀性较差。第三，使用高分辨率micro-CT评估时，CBCT上观察到的射线可透界面区主要代表成像相关现象而非真实结构缺陷，强调了对术后CBCT发现需谨慎解读的必要性。总体而言，这些发现支持使用机器驱动器械进行更可预测的根管预备，同时突出了CBCT在评估牙本质-充填材料界面方面的诊断局限性。需要进一步研究在临床条件下探讨不同器械技术的长期生物学和力学结局。
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Introduction
Tooth-preserving procedures such as root canal treatment are no longer exclusively relevant to human dentistry but have gained increasing importance in veterinary medicine over recent decades (1–3). Advances in materials, instrumentation, and imaging modalities have expanded the indications for endodontic treatment in companion animals, particularly in dogs.
Root canal treatment is considered one of the most technically demanding procedures in dentistry, requiring the coordinated use of multiple instruments, chemical irrigants, and carefully sequenced clinical steps. These steps typically include the preparation of an access cavity, removal of diseased pulp tissue, mechanical and chemical shaping of the root canal system, obturation with appropriate filling materials to eliminate potential niches and pathways for bacterial colonization, and definitive coronal restoration to prevent microleakage (4, 5).
Endodontic treatment in dogs and cats differs substantially from human endodontics, primarily due to species-specific variations in tooth size, root length, canal diameter, and pulp chamber morphology (1, 2). These anatomical differences can complicate adequate debridement and obturation, particularly in large canine teeth.
Root canal treatment may be performed in both single- and multi-rooted teeth. In veterinary dentistry, strategically and functionally important teeth, such as the maxillary and mandibular canine teeth as well as the carnassial teeth (maxillary fourth premolar and mandibular first molar) are frequently treated endodontically due to their essential role in prehension, mastication, and working performance (1, 6).
Preservation of these teeth is especially important in working and service dogs, including police, military, hunting, and therapy dogs, where tooth loss may significantly impair function and quality of life (2).
Accurate intraoperative and postoperative imaging is essential for both the success and follow-up evaluation of root canal treatment. Conventional dental radiography remains the most commonly used imaging modality in veterinary practice, as it is readily available, cost-effective, and allows intraoperative use without repositioning the patient. However, its two-dimensional nature limits the assessment of complex root canal morphology.
Cone-beam computed tomography (CBCT) has become an established imaging tool in dentistry, offering high-resolution three-dimensional visualization of teeth and root canal systems and has demonstrated significant diagnostic advantages in both clinical and research settings (7, 8). In human endodontics, CBCT has proven particularly valuable for evaluating canal anatomy, treatment outcomes, and procedural complications (7).
The primary aim of the present study was to compare two mechanically driven reciprocating Ni-Ti instrumentation techniques with manual Ni-Ti instrumentation in canine teeth of dogs and to quantitatively evaluate their effects on root canal geometry and obturation quality using high-resolution micro-CT. A secondary aim was to qualitatively compare radiolucent findings observed at the dentin–filling material interface on postoperative CBCT with the corresponding high-resolution micro-CT findings.
We hypothesized that the three instrumentation techniques would differ in their effects on root canal geometry and obturation quality. In addition, we hypothesized that radiolucent interface zones observed on postoperative CBCT would not necessarily correspond to true structural defects, such as voids or residual debris, when evaluated using high-resolution micro-CT.
Materials and methods
A total of 30 lower and upper dog canines, which were extracted for reasons unrelated to this study, were analyzed. Teeth were fixed in neutral buffered formalin and subsequently stored in 70% ethanol. Prior to treatment, a micro-CT scan of each tooth had been acquired as an anatomical reference as part of a preceding study. The sample size of the present investigation was therefore constrained by the availability of canine teeth with corresponding pre-treatment micro-CT datasets that met the predefined inclusion criteria. Consequently, an a priori power analysis was not performed.
The samples were categorized according to weight, age, and jaw position (upper/lower) following the modified Triadan numbering system (104, 204, 304, 404) and randomly assigned to three groups (9). The 30 canine teeth were divided into three groups of 10, each assigned to one of the following preparation techniques. Within each group, five teeth were instrumented by a pre doctoral specialist (operator no. 1) and five by a board-certified veterinary dentist DAVDC/DEVDC (operator no. 2).
Group A: Control Cut Rotary Flex Ni-Ti files by iM3a (machine-operated with the iM3 C Flex endomotorb).
Group B: Vet Flex and Preserve Ni-Ti files by Dentsplyc (machine-operated with the iM3 C Flex endomotorb).
Group C: Hand files: Ni-Ti files by iM3d (K-Reamer; Figure 1).
[图：Endodontic instruments used in the three instrumentation groups (From left to right): (A) Group A: Control Cut Rotary Fl]
To standardize instrument wear while reflecting routine clinical practice, each instrument was used for the preparation of a maximum of two teeth before being replaced with a new instrument set. Prior to treatment, each tooth was individually embedded in a polycarbonate blocke (2, 6) and subjected to standardized procedures (preparation, imaging, chemical preparation, filling, and restoration; Figure 2). The root canal treatment protocol followed the standard clinical procedure routinely applied at our clinic. A complete list of all materials and instruments used is provided in Supplementary Table S1.
[图：Representative canine tooth specimen individually embedded in a polycarbonate block prior to root canal preparation.]
Access
Each tooth was individually positioned in polycarbonate at the height of the cementoenamel junction (CEJ) and allowed to dry for at least 48 h. Enamel cleaning and disinfection were performed using cotton swabs soaked in 96% ethanol. To simulate a tooth fracture, 25% of the crown length was measured and horizontally sectioned using a rotating diamond disk under permanent water cooling. A round diamond bur #2f one mm in diameter was subsequently used to prepare a coronal cavity for at least 6 mm in depth toward the “fracture site” in groups A and B.
For group C, in which nickel–titanium (Ni-Ti) hand files were used, an auxiliary access hole was created as described in many studies approximately 2 mm above the CEJ (1, 10). The coronal cavity was subsequently enlarged using Gates-Glidden (GG) burs (sizes 1-6)g in combination with the lubricant Glydeh. The burs were introduced passively into the canal, and active dentin removal was performed exclusively during withdrawal.
Preparation
Pulpal extirpation was performed with single-use standard nerve needlesi of different sizes. The first ISO coded endodontic file to reach the apex (FF = first file) was identified to determine the working length in millimeters, which was verified radiographically and with a silicone stopper measured on an endodontic ruler. The following endodontic files were selected according to their increasing ISO size and a silicone stopper was always positioned at the predetermined working length.
In group A, Control Cut Rotary Flex Ni–Ti files were operated using a standardized reciprocating motion at 300 rpm and 2.0 Ncm, with rotational angles of 150° clockwise and 30° counterclockwise. The larger forward angle was selected to correspond to the instrument’s right-handed cutting design, in which active cutting occurs during clockwise rotation. The settings were used as the recommended presetting from the iM3 C Flex endomotor (11).
In group B, Vet Flex & Preserve Ni-Ti filesc were operated under standardized reciprocating conditions at 300 rpm and 2.0 Ncm, using a 150° counterclockwise and 60° clockwise rotational movement. As the instrument design corresponds to a left-handed cutting configuration, the larger angular displacement (150°) was assigned to the counterclockwise phase. These parameters were selected according to the manufacturer’s recommendations (12, 13).
The specimens in group C were instrumented manually using Ni-Ti K-reamersd with a clockwise ¼-turn–pull technique, also with a right-handed cutting design.
After each endodontic file, a standardized irrigation protocol for chemical preparation was applied which remained consistent across all three preparation techniques (14, 15). The irrigation procedure performed using a special side vented irrigation needle 27Gj consisted of the following steps: First, sodium hypochloritek 5.25% (NaOCl) was used to flush and irrigate the root canal, followed by recapitulation with an ISO 15d (K-Reamer, Ni-Ti) file to agitate dentinal debris. The solutions were stored at 4 °C according to the manufacturer’s recommendations and brought to room temperature prior to use (16). Suction was then performed using an empty syringe together with an irrigation needle. Subsequently, sterile sodium chloride 0.9%l (NaCl) was used for final irrigation, followed again by suction.
Glydeh was used as a root canal conditioner with each endodontic file to enhance canal lubrication and reduce the risk of instrument fracture (17). The final file (LF = last file) was defined as the largest file that just reached the apex without altering the established working length. A uniform chemical irrigation protocol was applied across all three techniques. For the final irrigation, 17% EDTAm was introduced into the root canal and left in place for 1 min before being removed with suction. This was followed by a one-minute retention of NaOCl, which was subsequently suctioned. Finally, the canal was irrigated with NaCl and dried using ISO-standardized paper pointsn.
Procedural complications
Procedural complications occurring during root canal instrumentation were recorded for each tooth. For each incident, the instrument involved, the success or failure of fragment retrieval, and the operator performing the procedure were documented. Multiple complications occurring in the same tooth were recorded as separate incidents. If retrieval of a fractured instrument was unsuccessful, the affected tooth was excluded from further treatment and replaced by another tooth to maintain the predefined group size of 10 specimens. Procedural complications were summarized descriptively as absolute numbers and percentages, including their distribution according to instrument type and operator experience.
In addition to the systematic documentation of procedural complications, all instruments were visually inspected before and during use for macroscopic deformation and adherent surface debris. These observations were qualitative and were not subjected to standardized scoring or statistical comparison between groups.
Obturation
The first step of the obturation procedure was the selection of an appropriate gutta-percha pointo corresponding to the ISO size of the final file. GuttaFlow biosealp was then applied using the designated application tip. A Lentulo spiral was used to distribute the GuttaFlow bioseal evenly throughout the root canal system (18).
For the single-cone technique, the prepared gutta-percha pointo, previously disinfected for 1 min in 5.25% NaOCl (5) and flushed in 96% ethanol to remove the surface deposits that may form, was inserted into the canal. The gutta-percha point was then severed using a heated excavator. Material compaction was performed using pluggersq for vertical compaction. The dentin wall of the coronal cavity was cleaned with different excavatorsr and standardized intraoral radiographs were obtained as part of the routine clinical protocol and at the operator’s discretion (Figures 3A–E).
[图：Representative radiographic sequence illustrating the different stages of root canal treatment. (A) Pre-treatment radiog]
Restoration
A so called “sandwich technique” was used for the final access restoration, involving the bonding of two different restorative materials. Glass ionomer cements was applied in a layer on top of the obturation not exceeding 2 mm in thickness, followed by light polymerization with blue light using 500–1,000 mW/cm2 and approximately 470 nm wavelength for 20 s at a distance of 1–1.5 mm from the tooth surface.
Adhese Universal Viva Pent (bonding agent) was then applied according to the manufacturer’s instructions and again light-cured with blue light for 20 s at a distance of 1–1.5 mm from the tooth surface. The final restoration was completed using Tetric EvoCeramu (packable composite), which was also light-cured for 20 s.
The composite was polished using SHOFUv polish to obtain a smooth surface matching the surrounding enamel and finally the surface was sealed with G-Coat Nanosealw under light-curing.
CBCT
All treated teeth were scanned postoperatively using a CBCT (Planmed Verity®, Planmed Oy, Helsinki, Finland). The CBCT scans were acquired using the following parameters: scan time 18 s, reconstruction time approximately 30 s, voxel size 0.2 mm, tube voltage 84 kV, tube current 4 mA and exposure time 10.0 s. Image acquisition parameters were selected according to the manufacturer’s specifications and adapted to the experimental setup. Volumetric datasets were exported in DICOM format for subsequent analysis (19).
Micro-CT
All scans were acquired using a micro-CT μCT35 system (SCANCO Medical AG, Brüttisellen, Switzerland).
Untreated teeth were mounted in Parafilm-sealed sample holders containing 70% ethanol. Scans of untreated teeth were acquired at 70 kVp / 114 μA and using a 0.5 mm Aluminum (Al) filter. Integration time was 800 ms (detector binning = 2) and the angular increment was 0.36°. Depending on the size of the tooth, isotropic voxel size in the datasets of untreated teeth was either 20 μm (large teeth, 20 mm sample holder, 20.5 mm field of view) or 12 μm (small teeth, 12 mm sample holder, 12.3 mm field of view).
Treated teeth surrounded by trimmed polycarbonate blocks were mounted in 20 mm sample holders in air. Scans of treated teeth were acquired at 70 kVp / 114 μA and using a 0.1 mm copper (Cu) filter. Integration time was 800 ms (detector binning = 2) and the angular increment was 0.36°. The isotropic voxel size in reconstructed datasets of treated teeth was 20 μm.
Reconstructed image volumes were exported in DICOM (*.dcm) file format.
CBCT and micro-CT comparison
Post-treatment CBCT datasets were visually assessed for radiolucent areas at the dentin–filling material interface. During subsequent micro-CT image analysis, all specimens were visually inspected at the voxel level by a study operator with particular attention to whether radiolucent findings identified on CBCT corresponded to actual voids, air inclusions, debris accumulations, or structural discontinuities in the corresponding micro-CT datasets. The comparison between CBCT and micro-CT findings was performed qualitatively and was not subjected to statistical analysis.
Image processing and analysis
Image registration and pre-treatment segmentation
DICOM image volumes were imported into the 3D analysis and visualization software AMIRA (FEI Visualization Sciences Group, part of Thermo Fisher Scientific, Mérignac Cédex, France, version 2023.2) and converted to Amira mesh (*.am) file format. Pre- and post-treatment image volumes were imported into a project template and spatially co-registered (rigid transformation) based on normalized mutual information using the Register Images tool.
For noise reduction, pre-treatment micro-CT datasets were filtered using a Bilateral filter (interpretation: 3D, kernel size 3*3*3, similarity = 10,000). The Amira Segmentation Editor was used to segment the mineralized tooth from the pulp using a fixed threshold (2600). The initial segmentation of the pulp was then further processed using a closing operation (3 pixel dilation followed by 3 pixel erosion) followed by smoothing using the command Smooth Labels (size = 3, mode = 3D volume). The volume of the tooth and pulp was measured using the Material Statistics tool.
Centerline generation and cross-sectional analysis
Next, a B-Spline (degree: 3, samples 201) was set for the entire length of the pulp by manually placing markers in the center of the pulp based on the tomograms (Ortho Slice module). The B-Spline was converted to a spatial graph (module Line Set to Spatial Graph) and smoothed using the module Smooth Line Set (smoothing: 0.5, iterations: 100). Finally, a Tcl script - originally published as supplement to Johnson Chacko et al. (20) and used here with slight modifications regarding the pixel size—was executed using the Tcl Command Module. This script used a Trajectory to move a Slice of the pulp segmentation (binary label field) along each point of the spatial graph of the pulp. The orientation of the slice was always perpendicular to the curvature at the respective position of the curve. The extracted cross-sectional images of the binary segmentation mask of the pulp (Extract Image) were exported as *.tif images and analyzed using a custom Fiji macro, which measured multiple parameters such as pulp area, perimeter, major and minor axis of ellipse fit, Feret’s diameter, Feret Angle, circularity, roundness, and aspect ratio. The custom Fiji macro automatically processed all 201 cross-sectional images of each specimen. Although multiple geometric parameters were recorded, Feret’s diameter was used as the measure of canal diameter for the file-to-canal diameter analysis reported in the present study.
As each spatial graph curve included exactly 201 points (as initially defined in the B-Spline), each step from one cross-sectional image of the pulp to the next represented exactly 0.5% of the pulp length. This allowed to compare relative changes in pulp area and shape across different teeth, despite the different size of measured teeth (20) (Figures 4A–E).
[图：Micro-CT image-processing and analysis workflow for pre- and post-treatment assessment of the root canal system. (A) Pre]
Post-treatment segmentation and material classification
Post-treatment micro-CT datasets were filtered using a non-local means algorithm (interpretation: 3D; spatial SD = 5; intensity SD = 0.2; search window = 10 px; local neighborhood = 3 px). The tooth and the filled pulp were segmented again using the Amira Segmentation Editor. Fixed thresholds were used for the segmentation of the tooth (lower threshold: 2000, upper threshold 11,000) and the pulp filling material and gutta percha point (lower threshold: 11001), which showed much higher X-ray attenuation compared to the tooth. Air cavities in the treated pulp were added to the pulp label using manual segmentation tools, thus the segmentation of the pulp represented the entire treated pulp canal. The segmented tooth and pulp were smoothed using the Smooth Labels command (size: 6, mode = 3D volume). Subsequently, the pulp compartment was duplicated to enable segmentation of restorative materials. Separate material classes were defined for gutta-percha point, GuttaFlow bioseal cement and air. Each material was segmented using fixed masking thresholds (air: 0–10,999, cement: 11000–16,000, gutta-percha: 28000–32,767) and region-growing tools, followed by 3D smoothing (Smooth Labels, size = 3, mode = 3D volume). Finally, the segmentation of restorative materials was verified by careful visual inspection. The resulting label field was stored for quantitative analysis.
Volumetric and surface analysis
To restrict the quantitative analysis to the root portion, the crown of the tooth (region of interest (ROI)) was manually outlined using the tool Volume edit and excluded from all label fields (pre-treatment pulp, post-treatment pulp, pulp restoration materials) in the Segmentation Editor. Root volumes of the pre- and post-treatment pulp were then analyzed using the Material Statistics tool. The Arithmetic tool (pre-treatment pulp volume minus treated pulp volume) was used to compute the proportion of pulp that remained untreated. Volume measurements (in mm3) were exported from Amira to Microsoft Excel.
The volume of pulp restoration materials was also measured using the Material Statistics tool. In addition, a polygon surface model was generated from the restoration material segmentation using the Generate Surface tool (algorithm mode: repeatable, smoothing: existing weights). Subsequently, the tool Surface Area Volume (mode: patches) was used to calculate interfaces (in mm2) between fill material (GuttaFlow bioseal)/tooth, gutta-percha/tooth, and air/tooth.
Post-treatment cross-sectional analysis
For cross-sectional evaluation of the treated pulp, the spatial graph from the untreated pulp and the Amira Tcl script used before for the untreated pulp was executed to automatically extract 201 cross-sectional images per sample. These images were again processed using a custom FIJI (ImageJ) scripts to quantify geometric parameters of the pulp and filling interfaces. The resulting data were compiled and analyzed in Microsoft Excel for volumetric and surface comparisons between pre- and post-treatment conditions.
File-to-canal diameter analysis
To compare the dimensions of the instrumented root canal with those expected from the final files featuring a 2% taper, the curved pulp length was standardized according to a predefined spline consisting of 201 points. For the file-to-canal diameter analysis, Feret’s diameter obtained from the Fiji analysis was used as the measure of the actual post-treatment canal diameter and was converted from pixels to millimeters according to the respective pixel size. At each of the 201 standardized positions, the Feret’s diameter of the post-treatment canal was compared with the calculated diameter of the final 2%-tapered instrument at the corresponding position, and the difference between both measurements was determined. For each tooth, the mean and median discrepancies across all 201 positions were subsequently calculated and used for statistical comparison between instrumentation groups.
Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics (Version 29; IBM Corp., Armonk, NY, USA). Continuous variables were summarized descriptively as mean ± standard deviation (SD) and 95% confidence intervals (95% CI), where appropriate.
To evaluate the effects of instrumentation technique on canal geometry, obturation quality and interface characteristics, different statistical models were applied depending on the outcome variable. Differences between canal diameter and instrument size as well as internal air volume were analyzed using linear models including the fixed effects of group (A, B, C), jaw (maxillary vs. mandibular canine teeth), and their interaction (group × jaw). In contrast, the post-treatment/pre-treatment canal volume ratio and surface-based interface measurements were analyzed using one-way analysis of variance (ANOVA), as jaw was not included in these models. No random-effects structure was included in the fitted linear models. Surface analyses comprised quantitative measurements of the interfaces between filling material and tooth structure, gutta-percha, and tooth structure, as well as air and tooth structure. For variables demonstrating non-normal distribution patterns or heteroscedasticity, logarithmic transformation was additionally applied prior to analysis. Estimated marginal means derived from the fitted models were used for pairwise group comparisons. Whenever significant main effects of group were detected, post-hoc comparisons were performed using Sidak’s alpha correction procedure. Additional subgroup analyses according to tooth position (Triadan 104/204 vs. 304/404) were conducted where indicated.
To assess potential operator-dependent effects, independent-samples t-tests were performed comparing volumetric and surface-based outcome variables between the two operators.
Model assumptions were evaluated by visual inspection of residual and Q–Q plots to assess approximate normality and homogeneity of residual variance. All statistical tests were two-sided and statistical significance was defined as p < 0.05.
Results
File-to-canal diameter discrepancy
The discrepancy between the post-treatment canal diameter and the corresponding final instrument diameter was evaluated using the mean and median differences calculated across the 201 standardized positions for each tooth. Larger values therefore represent a greater discrepancy between the prepared canal diameter and the diameter of the final instrument.
Analysis of the mean discrepancy revealed a significant main effect of group (F(2, 24) = 5.94, p = 0.008), whereas neither the effect of jaw (p = 0.129) nor the group × jaw interaction (p = 0.766) reached statistical significance.
Descriptive analysis showed mean discrepancies of 0.162 mm (SD = 0.144; 95% CI: 0.034–0.233) for group A, 0.098 mm (SD = 0.117; 95% CI: 0.000–0.186) for group B, and 0.299 mm (SD = 0.155; 95% CI: 0.208–0.390) for group C, with the highest mean discrepancy observed in group C.
Sidak-adjusted pairwise comparisons demonstrated a significant difference between groups C and B (p = 0.010). In contrast, the comparisons between groups C and A (p = 0.055) and between groups A and B (p = 0.906) did not reach statistical significance.
Analysis of the median discrepancy likewise demonstrated a significant effect of group (F(2, 24) = 5.85, p = 0.009) and a significant effect of jaw (F(1, 24) = 5.09, p = 0.033). The interaction between group and jaw was not statistically significant (p = 0.729).
Estimated marginal means for the median discrepancy were 0.129 mm (95% CI: 0.043–0.214) for group A, 0.064 mm (95% CI: −0.015–0.144) for group B, and 0.247 mm (95% CI: 0.169–0.325) for group C, again with the largest values observed in group C.
Sidak-adjusted post-hoc comparisons demonstrated a significant difference between groups C and B (p = 0.008), whereas the comparisons between groups C and A (p = 0.130) and between groups A and B (p = 0.609) were not statistically significant.
Subgroup analysis according to jaw revealed a significant effect of group for maxillary canine teeth (104/204; F(2, 24) = 3.59, p = 0.043), whereas no significant group effect was detected for mandibular canine teeth (304/404; p = 0.085).
Overall, both the mean and median analyses consistently showed the greatest discrepancy between the actual post-treatment canal diameter and the corresponding final instrument diameter in group C. Significant pairwise differences were identified only between groups C and B, whereas comparisons involving group A were not statistically significant after Sidak correction.
Incidence of procedural complications
Procedural complications occurred in 8 of 30 teeth (26.7%), accounting for a total of 10 individual incidents. In several cases, more than one type of complication was recorded for the same tooth.
The most frequent complications were associated with the use of Gates-Glidden burs (4 cases, 40%), followed by ISO size 15 files (3 cases, 30%), Path Finder instruments (2 cases, 20%) and ISO size 70 files (1 case, 10%).
Fragment removal was successful in 5 of the affected 8 teeth (62.5%), whereas in 3 cases (37.5%), retrieval was unsuccessful and a replacement tooth had to be used for further analysis.
When stratified by operator experience, 7 of the 10 complications (70%) occurred in procedures performed by a pre doctoral specialist (operator no.1), while 3 incidents (30%) occurred during instrumentation by a board-certified veterinary dentist DAVDC/DEVDC (operator no.2).
Volumetric analysis
The post-to-pre canal volume ratio, calculated as the ratio of the post-treatment canal volume to the pre-treatment canal volume, did not differ significantly among the three instrumentation techniques (F(2,27) = 0.79, p = 0.464). Mean post-to-pre canal volume ratios were 11.47 ± 4.91 in group A, 11.33 ± 8.29 in group B, and 8.40 ± 4.60 in group C. Pairwise comparisons using Sidak-adjusted post-hoc tests revealed no significant differences between any of the groups (A vs. B: p = 1.000; A vs. C: p = 0.620; B vs. C: p = 0.652; Figure 5).
[图：Post-to-pre root canal volume ratio according to instrumentation group. Group A: Control cut rotary flex Ni-Ti. Group B:]
Air volume analysis
A significant effect of group was observed for total air volume (F(2, 24) = 4.83, p = 0.017).
Pairwise Sidak-adjusted comparisons revealed no significant difference between groups C and A (p = 0.060), whereas group C exhibited significantly higher air volumes than group B (p = 0.028). No significant difference was observed between groups A and B (p = 0.994).
Neither jaw (p = 0.966) nor the group × jaw interaction (p = 0.140) was significant. Estimated marginal means were 0.61 mm3 for group A, 0.46 mm3 for group B and 2.15 mm3 for group C. Thus, group C demonstrated the highest volume of internal voids or unfilled canal spaces, indicating a less homogeneous obturation pattern compared with groups A and B (Figure 6).
[图：Internal air volume within the obturated root canal system according to instrumentation group (mm 3 ). Group A: Control ]
CBCT and micro-CT comparison
CBCT imaging revealed radiolucent areas at the dentin-filling material interface in multiple specimens, which could be interpreted as potential voids or interfacial defects. However, comparative analysis using high-resolution micro-CT demonstrated no corresponding voids, debris accumulations, or air inclusions at these locations. Instead, the micro-CT datasets showed continuous adaptation of the obturation material to the canal walls, indicating that these radiolucent zones did not represent true structural deficiencies.
Quantitative micro-CT analysis further confirmed that internal air volumes were limited and predominantly localized within the canal system rather than at the dentin-filling interface. The absence of structural discontinuities at sites corresponding to CBCT radiolucencies suggests that these findings are attributable to imaging-related contrast effects rather than incomplete obturation or residual debris.
Surface analysis
Surface analyses showed no significant differences among groups for any of the evaluated parameters: filling material surface/tooth (F(2, 27) = 0.31, p = 0.734), gutta-percha surface/tooth (F(2, 27) = 0.20, p = 0.818) and air surface/tooth (F(2, 27) = 0.16, p = 0.855).
These findings indicate that surface characteristics of the obturation materials and interfaces were comparable across all groups, despite volumetric differences in internal air volume.
Operator comparison
Independent-samples t-tests comparing operator performance showed no significant differences in volumetric parameters (p > 0.25 for all). However, the surface area of the filling material per tooth differed significantly between operators (t(28) = 2.18, p = 0.038), with operator no.1 (JW) producing slightly larger filling surfaces than operator no. 2 (MES). This finding suggests minor operator-dependent variability in obturation or segmentation, while overall volumetric outcomes remained consistent.
Taken together, the volumetric analyses revealed no significant group differences in total canal volume after treatment but group C showed a consistent tendency toward increased air volume within the obturated canals. Surface-based metrics did not vary significantly among groups and inter-operator variability was minimal.
Discussion
The present study evaluated different endodontic instrumentation techniques in canine teeth using high-resolution micro-CT and assessed their effects on canal geometry, obturation quality and procedural safety. The findings provide insight not only into shaping characteristics but also into the interpretation of postoperative imaging.
With regard to the CBCT assessment, the results support the assumption that radiolucent areas observed at the dentin-filling material interface predominantly represent imaging-related artifacts rather than true treatment deficiencies. Comparative analysis using high-resolution micro-CT demonstrated that these radiolucent zones corresponded to contrast transitions between materials of differing radiodensity and did not reflect residual necrotic dentin, persistent debris or entrapped air within the canal system (21). The absence of corresponding voids, debris accumulations or structural discontinuities in the micro-CT datasets strongly suggests that the CBCT findings were attributable to beam-hardening effects or contrast-related imaging phenomena rather than inadequate obturation or insufficient debridement. These observations emphasize the need for cautious interpretation of CBCT findings in veterinary endodontic follow-up and highlight the added diagnostic value of high-resolution imaging for distinguishing true structural defects from modality-related artifacts (22) (Figures 7A,B).
[图：Representative postoperative CBCT images demonstrating similar radiolucent zones at the dentin–filling material interfac]
Compared with conventional intraoral radiography, CBCT inherently provides three-dimensional visualization of root canal anatomy and avoids limitations related to superimposition and projection inherent to two-dimensional imaging. Previous studies have demonstrated the diagnostic value of CBCT for the assessment of root canal morphology and endodontic treatment outcomes (23, 24). However, the present study did not systematically compare CBCT with conventional intraoral radiography. Rather, our findings demonstrate that radiolucent areas observed at the dentin-filling material interface on CBCT require careful interpretation, as corresponding high-resolution micro-CT evaluation indicated that not all apparent radiolucencies represented true structural discontinuities. Awareness of such imaging-related phenomena is therefore important when interpreting postoperative CBCT examinations.
At our institution, CBCT examinations are routinely performed before and after root canal treatment and are complemented by intraoperative intraoral radiographs for continuous procedural documentation. Follow-up examinations include both imaging modalities to facilitate assessment of treatment success, failure, or the absence of radiographic evidence of disease. This combined imaging approach may enhance diagnostic confidence, particularly in complex cases where two-dimensional imaging alone may be insufficient (23, 25).
Beyond diagnostic assessment, the present findings provide insight into the biomechanical consequences of different instrumentation strategies for root canal preparation. High-resolution three-dimensional imaging enabled a detailed assessment of canal enlargement and dentin removal patterns, allowing a more comprehensive evaluation of shaping quality and preservation of radicular dentin (26). Maintaining an appropriate balance between effective canal enlargement for adequate debridement and preservation of dentin is considered essential for the long-term structural integrity of endodontically treated teeth (27).
Machine-driven Ni-Ti systems tended to produce a smaller discrepancy between file size and actual canal diameter than manual instrumentation. Following Sidak correction, however, a statistically significant pairwise difference was observed only between groups B and C (28). Although no significant differences in the post-to-pre canal volume ratio were observed among the three instrumentation techniques, manual instrumentation tended to result in higher internal air volumes. Pairwise comparisons demonstrated significantly greater air volumes in group C than in group B, whereas the difference between groups C and A did not remain statistically significant after Sidak adjustment. The absence of statistical significance between groups A and C after correction for multiple comparisons suggests that, although a similar trend was observed, the magnitude of the difference was insufficient to demonstrate a robust effect under the more conservative Sidak procedure.
These findings suggest that the advantages of mechanical instrumentation are not primarily attributable to greater overall canal enlargement but rather to more controlled and reproducible shaping.
Improved canal centering and a closer correspondence between instrument size and actual canal dimensions may facilitate more effective debridement while minimizing unnecessary removal of radicular dentin (4, 26). Consequently, preparation quality appears to depend less on the extent of canal enlargement than on the precision with which the original canal anatomy is respected (23). Collectively, these findings indicate that shaping precision, rather than the extent of canal enlargement, may represent a key determinant of preparation quality in veterinary endodontics.
While the present study primarily focused on volumetric and geometric changes following instrumentation, volumetric enlargement does not necessarily reflect the proportion of the canal wall mechanically contacted by the instruments. Previous micro-CT studies in human endodontics have demonstrated that areas of the root canal wall may remain untouched despite substantial canal enlargement (26). Therefore, volumetric changes and instrumented canal wall surface should be considered complementary measures of shaping efficacy.
An additional consideration is the difference in access design between the instrumentation protocols. An auxiliary mesial access was used in group C but not in groups A and B. Despite the flexibility and shape-memory properties of Ni-Ti instruments, an auxiliary access is recommended for the instruments used in group C to provide a more direct approach to the apical portion of the curved canal. Appropriate access design is an important component of endodontic treatment in canine teeth, with straight-line access toward the apex facilitating instrument insertion while minimizing unnecessary removal of tooth structure (29). Mesial access has also been described as a standard approach for endodontic treatment of canine teeth in dogs (10). Therefore, the additional access in group C would theoretically be expected to facilitate rather than impair instrumentation. Nevertheless, because access design was not standardized across all groups, its potential influence on shaping outcomes cannot be excluded. The observed differences should therefore be interpreted as reflecting the respective instrumentation protocols as a whole rather than the instrument systems alone.
In human endodontics, machine-driven Ni-Ti instrumentation has demonstrated advantages in shaping ability, procedural safety, and preparation efficiency compared with manual instrumentation (4, 28). However, an important distinction must be made between the continuous rotary motion evaluated in many human endodontic studies and the reciprocating motion used for the machine-driven systems in groups A and B of the present study. Reciprocating kinematics may increase the cyclic fatigue resistance of Ni-Ti instruments compared with continuous rotation, which may be particularly relevant during instrumentation of long and curved root canals (13). Accordingly, findings from studies evaluating continuously rotating Ni-Ti systems should not be directly extrapolated to the reciprocating systems investigated in the present study.
In veterinary endodontics, the long and frequently curved root canals of canine teeth present particular challenges for mechanical preparation. In this anatomical setting, the greater flexibility of Ni-Ti compared with stainless-steel instruments may be advantageous, as it facilitates maintenance of the original canal trajectory and reduces the tendency for canal transportation in curved canals (30). These anatomical and mechanical considerations are particularly relevant when interpreting differences between instrumentation techniques in canine teeth (2, 6).
Nevertheless, the mechanical properties of Ni-Ti hand instruments also impose inherent limitations. Their superelasticity and shape-memory behavior, while facilitating adaptation to canal curvature, may limit the ability to deliberately direct the instrument against specific canal walls and may therefore result in incomplete mechanical contact with portions of the canal surface. This characteristic may have contributed to the less standardized shaping and greater internal air volumes observed in group C, although canal-wall contact was not directly quantified in the present study. Accordingly, the findings for group C should be interpreted specifically in the context of manual Ni-Ti instrumentation and should not be generalized to manual instrumentation using pre-curved stainless-steel files.
The near-significant interaction between instrumentation group and jaw may also reflect underlying anatomical variability in root morphology. Previous three-dimensional micro-CT analyses of canine teeth have demonstrated considerable interindividual variation in root canal curvature, with curvature patterns influenced by body size and individual root morphology (30). Even in the absence of consistent volumetric differences between maxillary and mandibular canines, subtle variations in canal trajectory and curvature may alter the mechanical behavior of instruments during preparation. Increased curvature or complex canal pathways can render shaping more technique-sensitive, particularly when using manual instrumentation that is more prone to deviation and transportation (31). These anatomical characteristics may increase the technical difficulty of canal preparation and predispose to procedural errors, particularly during manual instrumentation (30). Such anatomical variability may partly explain the observed trend toward jaw-dependent differences in volumetric response.
The increased internal air volume observed in group C most likely reflects less homogeneous obturation, which is commonly associated with less standardized canal geometry (32–34). Although all groups underwent identical obturation procedures, the more reproducible canal geometry achieved with machine-driven instrumentation may have facilitated improved adaptation of the filling material and consequently reduced the occurrence of internal voids (18).
Only limited quantitative evidence is currently available comparing different endodontic instrumentation techniques in canine teeth using high-resolution micro-CT. Previous studies have primarily focused on the anatomical variability of canine root canal systems and have demonstrated substantial variation in canal morphology that may complicate complete debridement and obturation (1, 2). The present study expands upon these observations by providing quantitative high-resolution micro-CT evidence that machine-driven instrumentation achieves more standardized canal preparation and fewer internal voids than manual instrumentation.
Technical and clinical implications
The observed differences in air volume and canal-file discrepancy have practical clinical relevance. Air voids and unfilled canal spaces may compromise the hermetic seal and increase the risk of bacterial leakage, which remains a key determinant of long-term endodontic success (23, 35). The results therefore support the clinical use for machine-driven Ni-Ti systems in veterinary endodontics, particularly for strategic teeth such as canines and carnassials.
Moreover, the study indicates that operator experience may play an important role in the occurrence of procedural errors, as most complications were observed during treatments performed by a less experienced operator. Although the sample size was limited, instrumentation-related complications occurred in approximately one quarter of the specimens, with some teeth exhibiting multiple procedural failures. These findings emphasize the technical challenges associated with endodontic preparation of canine teeth and suggest that operator skill level may substantially influence the incidence of instrument separation and related complications.
Beyond the quantitatively analyzed outcomes, several qualitative observations were noted during handling of the instruments and are reported here as incidental, hypothesis-generating findings rather than as a systematic between-group comparison. In addition to complete instrument separation, a distinct pattern of plastic deformation was occasionally observed, characterized by visible unwinding of the helical flutes and elongation of the active working segment prior to fracture. This pattern is consistent with torsional stress accumulation, which occurs when the file tip binds within the canal while the coronal portion continues to rotate, and may act as a macroscopic warning sign preceding instrument failure. Furthermore, visual inspection of newly unpacked instruments occasionally revealed fine metallic debris adherent to the cutting flutes prior to use; if not removed, such particles could conceivably be introduced into the canal as foreign material. As these observations were not standardized or quantified, they should be interpreted with caution; nevertheless, early recognition of flute unwinding and careful inspection of new instruments may represent simple preventive measures, and their systematic evaluation would be a worthwhile objective for future studies (4, 36) (Figures 8A–C).
[图：Representative qualitative observations of endodontic instruments during visual inspection. (A) Plastic deformation char]
Collectively, these observations highlight the importance of both operator proficiency and preparation quality for achieving predictable endodontic outcomes (37). This interpretation is consistent with human studies demonstrating a strong correlation between operator training, technical performance and endodontic success (10, 38, 39).
However, the presence of significantly greater internal void volume in group C may indicate incomplete adaptation of the obturation material, potentially resulting from the less standardized canal geometry achieved by manual instrumentation, even though the same Ni-Ti alloy was used for both hand and motor-driven files.
Limitations and future directions
The present study has several limitations that should be considered when interpreting the findings. The study used extracted canine teeth, which allowed standardized preparation and imaging but did not account for in vivo conditions such as periodontal ligament support, pulp vitality and physiological moisture content.
An additional consideration concerns the hydration state of the specimens during imaging. Pre-treatment scans were acquired with the teeth immersed in 70% ethanol, whereas after embedding, access preparation, instrumentation, obturation, and restoration, the post-treatment scans were performed without re-immersion. A degree of dehydration of the dental hard tissues between the two scans can therefore not be entirely excluded, and drying of root dentin has been shown to induce dentinal microcracks that may, in principle, affect canal measurements (40). However, no dentinal microcracks were observed in the high-resolution post-treatment micro-CT datasets, suggesting that dehydration was unlikely to have caused substantial structural damage detectable at the spatial resolution used in the present study. In addition, all specimens underwent an identical handling and imaging sequence; therefore, any residual effect would be expected to apply similarly across groups and is unlikely to have systematically biased between-group comparisons. The obturated and coronally restored canals may also have limited drying of the internal canal surface. Nevertheless, absolute canal dimensions should be interpreted with appropriate caution, and future studies maintaining a consistent hydration state during pre- and post-treatment imaging would help to further minimize this potential source of bias.
Furthermore, the relatively small sample size, with 10 teeth per instrumentation group, represents an important limitation. As the sample size was constrained by the availability of corresponding pre-treatment micro-CT datasets rather than determined by an a priori power analysis, the study may have been underpowered to detect smaller differences between instrumentation techniques. Accordingly, non-significant findings should not necessarily be interpreted as evidence of equivalence between techniques. Interindividual anatomical variability may have further contributed to variability within the relatively small groups. Future studies should include larger sample sizes with prospective sample-size calculations and clinical in vivo investigations using CBCT to further evaluate the clinical translatability of the present findings. Additionally, longitudinal studies evaluating sealing ability and fracture resistance after different instrumentation methods would complement the present morphological analysis.
Material segmentation based on fixed gray-level thresholds may have introduced minor inaccuracies in air volume quantification, despite careful validation, at the voxel level by the study operator.
Nevertheless, the standardized imaging and analysis protocol applied to all specimens is likely to have minimized this potential source of error. Finally, although the operator factor showed limited influence on volumetric data, the observed trend toward more procedural complications by less experienced operators emphasizes the importance of specialized endodontic training in veterinary dentistry.
Conclusion
Within the limitations of this in vitro study, machine-driven Ni-Ti systems provided more reproducible root canal preparation and more homogeneous obturation than manual Ni-Ti hand files in canine teeth. First, although the post-to-pre canal volume ratio did not differ significantly among instrumentation techniques, manual instrumentation was associated with greater discrepancies between instrument size and canal geometry, indicating less standardized canal preparation. Second, the increased internal air volume observed with manual instrumentation suggests less homogeneous obturation compared with the machine-driven techniques. Third, radiolucent interface zones observed on CBCT predominantly represented imaging-related phenomena rather than true structural deficiencies when assessed using high-resolution micro-CT, emphasizing the need for cautious interpretation of postoperative CBCT findings. Collectively, these findings support the use of machine-driven instrumentation for more predictable root canal preparation while highlighting the diagnostic limitations of CBCT in assessing the dentin–filling material interface. Further studies are warranted to investigate the long-term biological and mechanical outcomes of different instrumentation techniques under clinical conditions.