**1. Introduction**

Root perforation is characterized as a communication between the root canal system and the surrounding periodontal tissues [1]. Perforations occurring due to dental caries or resorption are commonly defined as pathologic in nature [2], while iatrogenic perforations are usually related to inappropriate prosthodontic or endodontic treatment [3]. Up to 20% of endodontically treated teeth are diagnosed with root perforations, of which the majority are caused by various iatrogenic errors [4]. The most severe complication of root perforations is a persistent inflammation, breakdown of periodontal tissues and subsequent loss of bone attachment, ultimately leading to a tooth extraction [5]. Therefore, early diagnosis and appropriate perforation repair have a major influence on the long-term prognosis and survival of the affected tooth [4]. It is generally assumed that apical root perforation, which usually occurs because of endodontic instrumentation during the root canal preparation, has a good prognosis [6]. However, the managemen<sup>t</sup> of apical root perforations frequently poses a challenge even for experienced endodontists, as visualization and direct access to the perforation site, especially in moderately or severely curved root canals, can be

**Citation:** Drukteinis, S.; Bilvinaite, G.; Shemesh, H.; Tusas, P.; Peciuliene, V. The Effect of Ultrasonic Agitation on the Porosity Distribution in Apically Perforated Root Canals Filled with Different Bioceramic Materials and Techniques: A Micro-CT Assessment. *J. Clin. Med.* **2021**, *10*, 4977. https://doi.org/ 10.3390/jcm10214977

Academic Editors: Massimo Amato, Giuseppe Pantaleo and Alfredo Iandolo

Received: 4 October 2021 Accepted: 25 October 2021 Published: 27 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

remarkably complicated, with a significant risk of collateral treatment mishaps, errors and complications [2].

The main goal of apical root perforation repair is to obtain a persistent bacteria-tight apical seal to prevent the percolation of fluids, microorganisms and their byproducts in the periapical tissues, allowing the healing and reorganization of damaged tissues [7]. Before the introduction of mineral trioxide aggregate (MTA), various dental materials, such as amalgam or glass ionomer cement, were used to repair root perforations. However, MTA instantly gained popularity due to its favorable biological, physical and chemical properties, which ensured an overall success rate of perforation repair of more than 80% [3,7]. Nevertheless, modifications of the original MTA formulation have been recently made to overcome its poor handling characteristics and long setting time [8]. MTA Flow (MF) (Ultradent Products Inc., South Jordan, UT, USA) is a relatively new MTA-based repair material, consisting of a di- and tri-calcium silicate grey powder and a water-soluble silicone-based gel [9]. MF was developed to give the clinician a variety of mixing options and consistencies, facilitating the manipulation and delivery of the material into the root canal [10]. Due to the extremely small particle size of less than 10μm, MF can be prepared in a thin consistency and delivered to the perforation site using a 29-G needle [11].

Although MTA-based materials have been widely used for root perforation repair since their first introduction [12], various investigations of hydraulic calcium silicate-based cements (HCSC) have shown that BioRoot RCS (Septodont, Saint-Maur-des-Fosses, France) could be effectively used as a filler and seal the apical root perforation as well [13]. BioRoot RCS possesses all the necessary antibacterial, biocompatible and bioactive properties, which promote the regeneration of periapical tissues and contribute to the recruitment of osteoodontogenic stem cells within the apical environment [14]. Moreover, this material has the desirable dimensional stability and low solubility and provides high clinical success rates when used in conjunction with a single gutta-percha cone (SC) obturation technique [15–17]. In contrast to cold lateral compaction or various thermoplastic methods, the BioRoot RCS/single gutta-percha cone (BR/SC) obturation technique is clinically appealing due to its simplicity, as no superior clinical skills or any additional armamentarium and devices are needed [18]. However, the available data on the performance of the BR/SC technique used for an apical plug in apically perforated roots are still limited. There is only one study demonstrating the sealability of apical perforations using the BR/SC technique and porosity distribution in these fillings [19].

Ultrasonic devices have been successfully used in endodontics over the years for a wide range of clinical procedures, including root canal obturation [20,21]. It has been reported that ultrasonication of the sealers during the root canal filling procedure may increase their penetrability into the dentinal tubules and improve the interfacial adaptation between the filling material and the root canal wall [22,23]. Additionally, ultrasonic energy is capable of rearranging the material particles and removing the entrapped air and thus reducing the porosity [24,25]. Therefore, ultrasonic agitation has been recommended in order to improve the quality and homogeneity of root canal fillings [25,26]. However, most of the previous research has investigated the effect of ultrasonic agitation, applied to the sealers indirectly, and there are still no data available on the porosity distribution within the BR/SC and MF root canal fillings after the use of direct ultrasonication.

Micro-computed tomography (micro-CT) is a widely accepted non-destructive method to perform two-dimensional (2D) and three-dimensional (3D) assessments of root canal fillings using high-resolution images [27]. Micro-CT analysis, due to its high accuracy, can be used to determine the overall porosity of the fillings as well as to identify and quantify open and closed pores separately [28]. Therefore, the present study aimed to evaluate, by means of micro-CT analysis, the effect of direct ultrasonic agitation on the porosity distribution in BR/SC and MF root canal fillings used as apical plugs in artificially perforated and moderately curved roots of mandibular molars. The null hypothesis tested was that direct ultrasonic agitation significantly impacts the quality and homogeneity of BR/SC and MF apical plugs, decreasing their porosity.

#### **2. Materials and Methods**

#### *2.1. Specimen Selection and Preparation*

A total of 40 human mandibular first molars were selected for this study, under the approval of the local ethics committee (protocol no. EK-2). The minimum sample size was calculated using G\*Power 3.1.9.7 software (Heinrich Heine, Iniversität Düsseldorf, Düsseldorf, Germany) followed by t-test family, α error probability of 0.05 and 1-β error probability of 0.95. Therefore, the requirement of 16 root canals per group was determined. Teeth were extracted for medical reasons unrelated to the present study and were stored in a saline solution at room temperature until use. Only molars with two separate mesial root canals, fully developed root apices and moderately curved roots (10◦–20◦) were selected. The root curvature was determined on preoperative radiographs using Shilder's method [29].

The orifices of root canals were accessed conventionally by preparing endodontic cavities with high-speed Endo Access burs (Dentsply Sirona, Ballaigues, Switzerland) under copious water-cooling. The presence of two separate mesial root canals was confirmed radiographically using the size 10 K-file (Dentsply Sirona, Ballaigues, Switzerland) inserted to the full working length (WL). The WL of both mesial canals was determined by inserting a size 10 K-file into the root canal until the tip approached the apical foramen and was visible under 10× magnification (OPMI Pico, Carl Zeiss, Oberkochen, Germany). Afterwards, the WL was increased by 2 mm to over-instrument the root canal and simulate apical perforation. All mesial canals were enlarged beyond the apical foramen. The glide path was created using size 15 and 20 K-Flexofiles (Dentsply Sirona, Ballaigues, Switzerland), and the root canal shaping was performed with ProTaper NEXT (Dentsply Sirona, Ballaigues, Switzerland) nickel-titanium rotary instruments at the established WL in the following sequence: X1 (17/0.04), X2 (25/0.06), X3 (30/0.07), X4 (40/0.06) and X5 (50/0.06). Instruments were driven using an X-Smart (Dentsply Sirona, Ballaigues, Switzerland) endodontic motor at the rotation speed of 300 rpm and the torque of 1 Ncm.

After the use of each instrument, root canals were repeatedly irrigated with 5 mL 3% sodium hypochlorite (Ultradent Products Inc., South Jordan, UT, USA), while 5 mL of 18% ethylenediaminetetraacetic acid (Ultradent Products Inc., South Jordan, UT, USA) followed by 5 mL of distilled water was used for the final flush at the end of instrumentation. The irrigants were delivered using 29-G NaviTip needles (Ultradent Products Inc., South Jordan, UT, USA) attached to disposable syringes. Afterwards, the root canals were dried with paper points.

The imitation of surrounding periodontal tissues and the alveolar bone was achieved using prefabricated A-silicone (3M ESPE, Seefeld, Germany) blocks. Specimens were fixed in these blocks up to the cement-enamel junction after the coverage of apices with a polytetrafluoroethylene tape (Tesa SE, Norderstedt, Germany).

#### *2.2. Root Canal Obturation*

A true randomness generator (www.random.org, accessed on 25 October 2021) was used for random allocation of the samples into four equal experimental groups (10 teeth/20 canals per group), according to the material and technique selected and used for root canal obturation:

• BR/SC group—the root canals were filled with BioRoot RCS sealer and single Pro-Taper NEXT size X5 gutta-percha point (Dentsply Sirona, Ballaigues, Switzerland). The apical 4 mm of the gutta-percha point was cut with a sterile scalpel to fit the gutta-percha with a tug-back effect at a length 2 mm shorter than the perforated apical foramen. The sealer was mixed according to the manufacturer's instructions, inserted into the Skini syringe (Ultradent Products Inc., South Jordan, UT, USA) and subsequently delivered into the root canal via attached plastic Capillary Tip cannula (Ultradent Products Inc., South Jordan, UT, USA). The tip was inserted approximately 2 mm shorter than the perforation site, and the plunger of the syringe was gently pressed while withdrawing the plastic cannula until reaching the orifice level. After the injection of BioRoot RCS, the pre-fitted gutta-percha point was coated with a

thin amount of the sealer and gently inserted into the root canal 2 mm short of the perforated apex.


Postoperative radiographs were made immediately after the obturation of the root canals to evaluate the filling quality. The obturation procedure was repeated when a lack of homogeneity or inadequate filling length was observed. New radiographs were taken to confirm the quality of the root canal fillings afterwards. The heat carrier was used to cut the gutta-percha point at the orifice level in the BR/SC and BR/SC-UA groups. The endodontic access cavities of all specimens were sealed with temporary filling material Cavit ™-W (3M ESPE, Seefeld, Germany), and the teeth were stored at 37 ◦C and 100% humidity for 7 days to allow the filling materials to set completely.

All specimens were prepared and obturated by a single operator: an experienced endodontist.

#### *2.3. Micro-CT Analysis*

Teeth were scanned before and after root canal obturation with a high-resolution micro-CT scanner SkyScan 1272 (Bruker, Kontich, Belgium). The scanning parameters were set at 100 kV source voltage, 100 μA beam current, 9.9 μm isotropic resolution, 0.11 mm copper filter, 1073 ms exposure time, 0.4◦ rotation step and 360◦ rotation angle. The obtained images were reconstructed using NRecon v.1.6.9.18 software (Bruker, Kontich, Belgium) under a beam hardening correction of 20% and a ring artefact reduction factor of 6.

The CTAn v.1.14.4.1 software (Bruker, Kontich, Belgium) was used to analyze the quality of root canal fillings in the apical 5 mm. All grayscale images from the selected region of interest were converted to binary images using a global threshold method in a density histogram. The original and segmented scans were thoroughly compared to confirm the segmentation accuracy before further analysis with a custom-processing tool. Images obtained from pre-obturation scans were used for quantification of the root canal volume (CVol), while post-obturation images were used to determine volumes of filling

material (FVol) and closed pores (CPVol). The total volume of pores (VVol) and volume of open pores (OPVol) were calculated using the following formulas, respectively:

$$\begin{aligned} \mathbf{V}\_{\text{Vol}} &= \mathbf{C}\_{\text{Vol}} - \mathbf{F}\_{\text{Vol}}, \\ \mathbf{OP}\_{\text{Vol}} &= \mathbf{V}\_{\text{Vol}} - \mathbf{CP}\_{\text{Vol}}. \end{aligned}$$

Afterwards, the percentage volume of open (%OPVol) and closed (%CPVol) pores was determined as follows:

$$\% \text{OP} \text{V}\_{\text{Vol}} = \text{OP}\_{\text{Vol}} / \text{C}\_{\text{Vol}} \times 100,$$

%CPVol = CPVol/CVol × 100

The evaluation of micro-CT images was performed by a single person who was blinded to data regarding the root canal filling material and technique.

#### *2.4. Statistical Analysis*

The porosity distribution between experimental groups was compared using a nonparametric Kruskal-Wallis test followed by the Mann-Whitney test due to a non-normal distribution of the data and validated with the Shapiro-Wilk test. All comparisons were performed using SPSS 25.0 software (SPSS Inc., Chicago, USA), with the significance level set at *p* < 0.05.
