Sample size calculation
The sample size was calculated using unpublished pilot data (n = 5) and the two-sided Welch's t-test for unequal variance at a significance level of alpha = 0.05 and a power of 0.9 (nQuery Advisor version 7, Statistical Solutions, Cork, Ireland). The sample size was evaluated as n = 15 for each group. Considering possible dropouts and a deviation of normality assumptions a sample size of n = 18 was used in the study.
Sample Preparation
Extracted human maxillary central incisors with similar dimensions, straight root canal, and mature apical foramen were collected and disinfected in accordance with the university’s policy. The teeth were cleaned with scalers and stored in 1% chloramine trihydrate solution. Crowns were removed using a diamond saw at slow speed (WOCO 50/Med, Conrad, Clausthal-Zellerfeld, Germany) to obtain a standardized 13 mm root length. A stereomicroscope (Stemi SV8, Zeiss, Oberkochen, Germany) at 12 x magnification was used to exclude teeth with carious lesions or pre-existing dentin defects. After numbering the teeth, the cross sections of the roots were measured at the level of the cutting surface in the mesio-distal and bucco-palatal direction with a digital caliper (Garant, Hoffmann, Munich, Germany). The area of the ellipsed root cross section (A) was calculated according to: A = π/4 x a x b (where a and b were the mesio-distal and bucco-palatal dimension in mm). Roots of extreme size were excluded. The remaining samples were randomized into six groups (two control and four experimental groups) of 18 roots each using the randomization software at the university’s institute of epidemiology and medical biometry. No significant differences were found between the groups regarding cross-sectional area (35.7 ± 3.6 mm2). To simulate the periodontal ligament with relatively uniform stress distribution, the roots were wrapped in one layer of latex rubber milk (Suter Kunststoffe, Jegenstorf, Switzerland) with a thickness of approximately 250 µm and embedded in acrylic resin (Technovit 4071, Heraeus Kulzer, Hanau, Germany) with the cervical root third being exposed.
Root Canal Treatment
In the negative control, the root canals were left untreated. Root canal treatments were performed by a single operator with (+) or without (–) MIE preparation. Canal patency was controlled with hand files ISO size #10 (K-file, Kerr, Orange, California, USA). The working length was set to 12 mm and K-files up to ISO size #20 were used to create a glide path. Canals in the + MIE group were prepared with nickel-titanium (NiTi) rotary files (Twisted File, Kerr) using the single-length technique in the file sequence of size #25, size #30, and size #35 in combination with 0.06 tapers up to size #40 and 0.04 taper. The files were rotated with a 4:1 reduction handpiece (WD-77 M, W & H, Buermoos, Austria) powered by a torque-control motor (Endo IT professional, VDW, Munich, Germany). During instrumentation, the canals were irrigated with 5 ml of 3% sodium hypochlorite (NaOCl) and 15% ethylenediaminetetraacetic acid (EDTA) solutions (Glyde File Prep, Dentsply Sirona, Ballaigues, Switzerland). After a flush with 5 ml distilled water, the canals were dried with paper points and filled according to the single-cone technique and respective manufacturer’s instructions. For conventional gutta-percha/sealer obturation (C), a calcium hydroxide sealer (Sealapex, Kerr) was mixed in equal amounts of base and catalyst paste for 15–20 s and applied to the canal walls using a lentulo spiral (VDW). The matched Twisted File gutta-percha cone (Kerr) was inserted in the canal to working length and condensed with a plugger 1 mm below the canal opening. For adhesive obturation (A), a self-adhesive methacrylate resin sealer (RealSeal SE, Kerr) was mixed using the automix syringe provided by the manufacturer and applied to the canal walls with the lentulo spiral. A matched polymer-based cone (Resilon, Kerr) was seated in the canal to working length and condensed 1 mm below the canal opening. The adhesive obturation was light cured for 40 s with an LED curing light (Bluephase, Ivoclar Vivadent, Schaan, Liechtenstein) at 1200 mW/cm2 light intensity. Canals of the –MIE group were prepared as those of the + MIE group, but then enlarged with Twisted File size #50 and 0.04 taper, followed by manual widening with K-files from ISO size #60 and ISO size #70 up to ISO size #80. During instrumentation, the canals were irrigated with 5 ml of 3% NaOCL and 15% EDTA. After a flush with 5 ml distilled water and drying with paper points, the canals were either left unfilled (positive control) or filled according to C and A, respectively. The canal orifices were filled with a temporary filling material (Cavit, 3M Espe, Seefeld, Germany).
Chewing Simulation And Vrf Testing
After storage in water for 24 h at 37°C, the samples were subjected to 1500 thermocycles in distilled water at 5–55°C with a dwelling time of 20 s in each bath and a transfer time of 5 s (Haake W15, Willytec, Gräfelfing, Germany). Mechanical loading was performed according to the staircase method starting at a load of 25 N at an angle of 10° to the axial direction of the roots in a chewing simulator (Standard 2002, Willytec). Every 20,000 cycles at a frequency of 2 Hz, the load was increased in increments of 25 N until 120,000 cycles were reached. The 1 mm unfilled canal space ensured that the force applied by the coneshaped metal antagonist with an angle of 120° was transmitted to the root dentin rather than the root canal filling. The diameter of the truncated cone was dimensioned in such a way that the metal tip fitted exactly into the canal space.
VRF resistance and crack formation was determined from the samples that survived chewing simulation. Pre-testing failures (PTFs) were recorded. The external root surfaces were examined under the microscope using a cold light source (Stemi SV8, Zeiss). Because of the latex milk, the roots could easily be removed from the acrylic blocks. Pictures were taken with a digital camera at 12–100 x magnification (3CCD Color Video Camera, Sony, Tokyo, Japan). Crack formation was analyzed per root third (cervical, middle, apical) as follows: (a) no defect, (b) craze line, (c) vertical crack, and (d) horizontal crack. Representative images of the defect patterns are shown in Fig. 1. Different defect patterns in the same root third were recorded, resulting in a maximum of nine defects per root. After microscopic analysis, the roots were returned to the acrylic blocks and subjected to VRF testing. The same antagonist as used for the chewing simulator was attached to the load cell of a universal testing machine (Zwicki 1120, Zwick, Ulm, Germany). The samples were loaded until fracture with a crosshead speed of 1 mm/min. The fracture load (N) was recorded when the force in the load-strain curve decreased by 30%.
Statistics
Statistical analysis was performed with IBM SPSS version 19 for Windows (Chicago, IL, USA). The significance level was set in advance at α = 0.05. As the Shapiro-Wilk test indicated that the data of the VRF resistance (P = 0.002) and crack formation (P = 0.0001) were not normally distributed, differences between the groups were compared with the non-parametric Kruskal-Wallis test. Post hoc multiple comparisons were performed using the Mann-Whitney-U-test with Bonferroni correction for 15 two-group comparisons.