## 4 Discussion  
Implantation of biomaterials into the tibia of rats has been widely used in dental research and is a valid experimental model for secondary screening to determine biocompatibility [28; 32; 33; 34]. Although the results cannot be directly correlated to what occurs in human periapical tissues, the rat model is highly standardized and allows a comparison between materials. ESHF and AHPS were implanted in diaphysseal bone in which the natural mechanisms of endochondral ossification are absent. Therefore, only reparative new bone formation is to be expected after implantation of the materials. Medical grade silicone tubes were used as carriers for the test materials because silicone tubes by themselves have proven biocompatibility, while offering conservation of the integrity of the samples during preparation of the histologic sections [28; 32]. The implantation method used in this study brings the freshly prepared sealers into direct contact with bone tissues, which simulates what may accidentally occur during root canal obturation. It should be noted that ESHF is a recently introduced sealer that has been recommended for use in conjunction with the WVCT because the material is resistant to heat [22; 24]. Although previous reports showed that the sealer demonstrated biocompatibility when implanted into the subcutaneous connective tissue of rats [27], the evaluation of its biocompatibility and bioactivity in bone after it was subjected to high temperatures is essential to provide relevant data on its safety and efficacy for clinical use. In this study, AHPS was also tested because previous in vitro and in vivo experiments [1; 5; 6; 7; 35; 36] demonstrated that the material showed favorable physicochemical and biological properties. These are the two reasons why AHPS is frequently used by clinicians. Furthermore, AHPS has been widely used as the gold standard for comparison with other materials. In a study by [PERSON] et al. [37], AHPS specimens were subjected to 100 \({}^{\circ}\)C for 1 min and the results showed that the physicochemical properties of the sealer underwent modifications. However, the effect of these modifications on bone tissue has not been investigated. According to a study by [PERSON] et al. [38], the highest temperature developed inside root canals via heat carriers during the WVCT ranges from 40 \({}^{\circ}\)C to 70 \({}^{\circ}\)C. However, these temperatures may vary according to the experimental model used. Thus, as per protocol, both ESHF and  
Figure 4: Photomicrographs of tissue reaction to the silicone tubes. (**A**) Representative specimen of a 10-day specimen showing a mild inflammatory reaction (white arrow). Note the presence of a blood vessel and the bone marrow (black arrow). BC: cortical bone (Hematoxylin and Eosin; original magnification \(\times\)400). (**B**) After 30 days the thickness of the fibrous connective capsule decreased without the presence of inflammatory cells (white arrow). The bone marrow is indicated by the black arrow. BC: cortical bone (Hematoxylin and Eosin; original magnification \(\times\)400). (**C**) After 90 days, there was a thin fibrous capsule contained mature fibroblasts and absence of inflammatory cells (white arrow). The bone marrow is indicated by the black arrow. BC: cortical bone (Hematoxylin and Eosin; original magnification \(\times\)400). AHPS were heated to 100 \({}^{\circ}\)C with the purpose of mimicking the high temperature that may occur in a clinical situation. The results of the current study showed that after 30 days ESHF caused a lower inflammatory reaction compared to AHPS, due to the different chemical composition of the materials. Independent of the inflammatory reaction caused by the surgical trauma during implantation, the initial inflammatory response to freshly prepared AHPS may be caused by the release of formaldehyde and polyamines [39]. The amines behaved as setting initiators and are necessary for the polymerization of the sealer.