**4. Discussion**

Root perforations are one of the most common complications observed in modern endodontology [4]. Regardless of recent advances in the field of endodontic instruments and devices, the mechanical preparation of curved root canals still remains a significant challenge, even for experienced clinicians [30]. It has been reported that the risk of root perforation occurring strongly correlates with the degree of root canal curvature, and the prevalence of apical root perforations is significantly higher in molars as compared to other teeth [2,31]. Therefore, mandibular first molars with a moderate curvature of mesial roots were selected in the present study to maximize its clinical relevance.

The managemen<sup>t</sup> of root perforation is a time-dependent procedure, where hermetic physical seal is crucial to improve the prognosis and survival of the affected tooth [32]. It has been reported that up to 52–79% of the root canal may remain unprepared, regardless of the instruments or instrumentation technique used [33], and no currently available irrigation protocol is capable of completely disinfecting the entire root canal system [34]. Therefore, the obturation phase of the endodontic treatment has undeniable importance

in order to create an unfavorable environment for the microorganisms left inside the root canal system after the preparation and to prevent their penetration into periapical tissues [4,35]. The homogeneity of root canal obturation highly depends on the porosity of the fillings [25], as open pores communicating with dentinal walls may create an excellent pathway for microleakage and eventually decrease the success rate and outcome of endodontic treatment [28,36]. Closed pores are considered to be less clinically relevant, as they represent empty spaces completely surrounded by filling material [37]. Nevertheless, it has been shown that this type of porosity may negatively affect the physical properties of the material, such as hardness and strength [36,38]. Therefore, the quantification of both open and closed pores is necessary to evaluate the quality of root canal fillings properly. Previously, various porosity and leakage measuring approaches, such as dye staining, glucose or radioactive isotope penetration, protein loss, scanning electron microscopy, mercury and capillary flow porometry, were applied to assess the sealing feature of the material used [39]. However, the significant limitations of these methods, e.g., the need to section the samples and hence the creation of artifacts, led to micro-CT being the technique of choice for accurate 3D evaluation of root canal fillings [20]. Therefore, micro-CT analysis was used in the present study to quantify and qualify the pores within the apical plugs. The isotropic resolution was set at 9.9 μm, as it has been shown that a voxel size of 11.2 μm or less is a reliable cutoff value to assess the filling porosity [36,40], even though there is always a risk of tiny pores left undetected due to a high radiopacity of the material used [18].

Techniques and materials applied for root perforation repair have not been standardized. However, MTA is generally assumed to be a benchmark for sealing various types of root perforations [32]. MF is one of the newest MTA-based repair materials, which surpasses traditional MTA in terms of clinical applicability due to its superior handling and delivery characteristics as well as faster setting time and thus increased washout resistance [10,19]. Moreover, MF retains all desirable biological properties of the original MTA, such as biocompatibility and bioactivity, which are the crucial requirements for perforation repair material exposed to periodontal tissues [41]. The biocompatibility and bioactivity are attributed mainly to the continuous calcium ion release and the formation of calcium phosphate apatite crystals, which induce the regeneration and remineralization of adjacent hard tissues while also reducing the porosity of filling material [28,41]. Nevertheless, a previous study has shown that, despite all the improvements and advantageous characteristics, MF results in highly porous apical plugs [19]. These results are in accordance with the present study, in which both MF groups (with/without ultrasonic agitation) exhibited a high porosity. The incidence of pores within MF fillings can be attributed to the increased water-to-cement ratio used during the mixing procedure to achieve a highly flowable consistency of the cement. It has been reported that excess water in the mixture eventually dries off and leaves pores that are not filled by hydration products [42]. Additionally, bismuth oxide, which is added to the MF composition as a radiopacifier, can negatively affect the sealing features by interfering with the hydration reaction and leaving more unreacted water within the filling [43]. Instead of bismuth oxide, some HCSC formulations, e.g., BioRoot RCS, contain zirconium oxide, which appears to have no impact on the material porosity [44]. These findings may correlate with the results of the present study, where significantly more homogeneous apical plugs were observed in both BR/SC groups than the MF groups.

Sealing apical root perforations with BioRoot RCS, together with a modified SC obturation technique, was proposed mainly due to the simplicity and effectiveness previously reported in in vitro and in vivo studies [16,17,19]. The concept of the SC obturation technique refers to the desirable physico-chemical properties of BioRoot RCS [14,15], which was designed as a biological filler [45], and to the tapered gutta-percha cone, acting as a piston on the flowable sealer [46]. As reported previously, the insertion of the tapered gutta-percha cone creates hydraulic pressure, which improves the material distribution throughout the root canal [47]. Therefore, the gutta-percha cone may be considered as the

main factor leading to the significant differences between the BR/SC and MF groups. No porosity associated with gutta-percha cones was observed in the present study through micro-CT analysis. Therefore, the superior overall homogeneity of BR/SC apical plugs can be attributed to the use of solid gutta-percha cones.

Attempts to minimize the occurrence of pores within BR/SC and MF fillings by using ultrasonic agitation were made in our previous study, which demonstrated that neither of these techniques was able to produce pore-free apical plugs [19]. The effect of ultrasonic application mainly refers to the acoustic energy transmission and the formation of cavitation bubbles, which eventually implode, increasing the temperature and the pressure inside the root canal [21]. According to previous investigations, which have reported significantly better results in terms of porosity after the use of indirect ultrasonication, the increased pressure may remove the entrapped air, disperse agglomerated particles, reduce their surface friction and provide a more efficient incorporation of filler particles into the organic matrix, with no changes in particle size or material composition [23,25,48]. Additionally, the pressure generated during ultrasonic agitation may lead to superior interfacial adaptation between the filling material and the root canal wall, with better tubular penetration as well [21,22]. However, these advantageous effects of ultrasonic application did not provide more homogeneous apical plugs in the present study; the increased percentages of open and closed pores were observed in both BR/SC-UA and MF-UA groups. Therefore, the null hypothesis was rejected.

The lower overall homogeneity of ultrasonicated apical plugs could be attributed to the direct ultrasonic agitation, resulting in excessive vibratory forces. It has been reported that excessive ultrasonic energy potentially can lead to air incorporation into the filling material and thus contribute to the higher porosity [26,49]. However, the use of direct ultrasonic agitation should not be directly associated with less homogenous root canal fillings, since it has been reported that indirect ultrasonication may also increase the porosity [20]. Instead of ultrasonication type, more attention should be paid to the agitation time, which is potentially directly related to both the rearrangemen<sup>t</sup> of cement particles and the heat generation [20,50]. The ultrasonic agitation of 10 s was selected in the present study in accordance with Sisli et al. [8], who agitated 5 mm apical plugs for 10 s and afterwards reported a lower incidence of pores. It has been reported that a short agitation time may create a shock-like effect, and the duration of 5 to 10 s is necessary to rearrange the cement particles and decrease the porosity [20]. On the other hand, the prolonged agitation time may be responsible for the increase in temperature, ultimately leading to water loss from HCSC [22,49]. Even though the number of published studies evaluating the temperature changes in filling materials is still limited, there are few reports in the literature indicating that ultrasonic agitation is capable of raising the temperature inside the root canal by 2 ◦C [7], which can be sufficient to increase the water desorption occurring at temperatures as low as 20 ◦C [51]. The water loss may alter the rheological properties of the material and increase the porosity [7,51], which is considered the result of spaces between unhydrated cement particles [42]. Nevertheless, it can be speculated that indirect ultrasonic application is not prone to these adverse effects of temperature changes, as ultrasonic energy is transmitted to the material through the gutta-percha cone, plugger or another instrument. This would explain the contradictory findings in terms of porosity obtained between the present study and previous investigations [8,24], which also performed ultrasonic agitation for 10 s. However, it is difficult to directly compare the present study results with the available literature, as they differ in too many aspects, including the type and properties of filling material, the application technique, ultrasonication type and duration, assessment method, etc.

The present study suggests that all apical plugs, regardless of the obturation technique used, may potentially lead to microleakage, as none of the fillings was pore-free, and the percentages of open pores surpassed the closed porosity in all experimental groups. Nevertheless, MF apical plugs (with/without ultrasonic agitation) demonstrated significantly higher percentages of open and closed pores as compared to the BR/SC obturation

technique. Therefore, reinforcing the findings of Benavides-García et al. [52], it can be concluded that MF prepared in a thin consistency should not be the material of choice for apical root perforation repair. Even though there is still no clear evidence what porosity level is critical, the significantly higher amount of pores observed in both MF groups may theoretically contribute to a worse outcome of endodontic treatment [27,28]. On the other hand, it has been shown that HCSC reduces their porosity with time in the presence of tissue fluids [42]. Therefore, the results of the present study should be evaluated with caution, as it is impossible to fully reproduce the clinical conditions using in vitro models. Further studies are needed to determine the clinical efficacy of BR/SC and MF obturation techniques in apically perforated and moderately curved roots and to confirm the adverse impact of direct ultrasonic agitation on the quality and homogeneity of root canal fillings.
