Effect of apexification on occlusal resistance of immature teeth

The sample size was calculated with G*Power 3.1.9 (Universität Kiel, Kiel, Germany) to detect significant differences (α = 0.05, 85% power). The estimated sample size was 10 teeth in each group. A total of 50 single-rooted extracted human lower premolars with similar crown and root dimensions were used in this study. Teeth with an intact crown and root that had not undergone any treatments were included. Teeth with cervical abrasions were excluded to minimize possible micro-flexion under loading. An in vitro apexification model was prepared according to a previous study [10]. The mean crown dimensions of the collected teeth were 8.2 ± 0.7 mm in the maximum mesio-distal dimension and 9.5 ± 0.8 mm in crown height measured from the cusp tip to the cementoenamel junction (CEJ) at the buccal aspect. The crown dimensions were standardized to a maximum mesio-distal dimension of 7.0 mm and a crown height of 8.0 mm from the CEJ. To standardize root length, we cut approximately 3 mm of the apical root tip, resulting in an 8.0-mm root length below the CEJ measured at the buccal surface.

The teeth were accessed with a round carbide bur #4 (Komet, Rock Hill, SC), and their root canals were enlarged with a Gates Glidden bur #5 (Mani, Utsunomiya, Japan) to the working length. A simulated divergent open apex was generated by retrograde preparation with a tapered diamond bur (CR-11F, Mani, Utsunomiya, Japan). The root canals were irrigated with a 5.25% sodium hypochlorite (NaOCl) solution between instrumentation, and immersed in a 17% ethylenediaminetetraacetic acid solution (pH 7.2) for 1 min before a final flush with 5.25% NaOCl solution. All irrigants were activated using intracanal ultrasonic devices (P5 Newtron XS; Satelec Acteon group, Mérignac, France). Finally, the canals were copiously rinsed with sterile distilled saline and dried with sterile paper points.

The teeth were randomly assigned to the following experimental groups and obturated accordingly: group 1, full-length orthograde obturation of MTA (ProRoot MTA; Dentsply Tulsa Dental, Tulsa, OK); group 2, a 5-mm MTA apical plug with a composite core; and group 3, a 5-mm MTA apical plug and back-filling with warm gutta-percha (GP). MTA obturation was performed using the obturation technique suggested by Bogen and Kuttler [11]. ProRoot MTA was mixed with distilled water according to the manufacturer’s instructions and placed incrementally with a carrier gun. An SS K-file with a #100 size was used to compact the apical 3–4 mm, then progressively larger K-files were used for further compaction. The final coronal portion was tamped by using stainless steel hand pluggers to complete the root canal obturation. Proper placement and thickness were verified by radiographic examinations. Following obturation, the teeth were stored at 37 °C under 100% humidity for a week to allow complete setting of the filling materials. The access cavities were sealed using resin composite (Z250; 3 M ESPE, St Paul, MN) and bonding agent (Scotchbond multipurpose; 3 M ESPE). Teeth with CH-medicated canals (CleaniCal, Maruchi, Wonju, Korea) and untreated teeth with normal apices were tested as controls (n = 10 each). Teeth were stored at 37 °C with 100% humidity for 3 weeks until further analysis.

The instrument for measuring strain development under occlusal forces consisted of a 3-unit teeth assembly constructed with extracted human teeth, strain gauges, and a data acquisition board (Fig. 1). Two intact teeth with normal apices (adjacent teeth) and the treated tooth (experimental tooth) were arranged in a straight line in a single occlusal plane and embedded in a mold. The roots were double-covered with 0.13-mm paraffin film (Parafilm M; Sigma-Aldrich, St. Louis, MO) to simulate a periodontal ligament (PDL) space of approximately 0.25-mm thickness. Strain gauges (KFG-2-350-C1-11, Kyowa Electronic Instruments, Tokyo, Japan) were attached to the buccal surfaces of the teeth at 4 mm below the buccal cusp tip. Simulated food fabricated with a polyvinylsiloxane impression material (Exafine putty type, GC Corp., Tokyo, Japan) was placed between the occlusal plane of the teeth and the test zig of the universal testing machine (Instron 8871, Instron Co., Norwood, MA) [12].

The 3-unit teeth assembly model was calibrated to ensure that it was free of residual strain under default conditions (0 N). Data from the untreated teeth with normal apices were used to confirm the even distribution of occlusal stress under tested forces. Compressive occlusal forces of 50, 100, 200, and 300 N were applied with a universal testing machine, and strain values were simultaneously measured and recorded from each tooth. Strain measurements were repeated 20 times for each condition. The simultaneously recorded electronic measurements from each strain gauge were converted into occlusal force (N) measurements for each tooth. The occlusal resistance of each tooth was calculated as the percentage (%) of the occlusal force loaded in the treated tooth to the average occlusal force in the 3-teeth assembly, calculated from the summed values for all three strain gauges.

All analyses were performed using SPSS version 19 (IBM Corp., Armonk, NY, USA). Occlusal resistance data were pooled according to the apexification method and tested occlusal forces. All data were analyzed using two-way analysis of variance (ANOVA) with the type of apexification method and occlusal force as the main factors, followed by Tukey’s post-hoc comparisons. For each tested occlusal force, data were separately analyzed with one-way ANOVA and Tukey’s post-hoc comparisons. The significance level was set at α = 0.05.