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Article

Tissue Response to a Heat Resistant Silicate-Based and an Epoxy Resin-Based Endodontic Sealer Implanted in Rat Tibias

by
Osvaldo Zmener
1,2,
Cornelis H. Pameijer
3,*,
Roberto Della Porta
1,2 and
Romina de Lucca
4
1
Department of Specialized Endodontics, Faculty of Medical Sciences, University of El Salvador, Buenos Aires 1425, Argentina
2
Argentine Dental Association, Buenos Aires 1425, Argentina
3
Department of Reconstructive Sciences, School of Dental Medicine, University of Connecticut, Farmington, CT 06030, USA
4
Department of Histology and Embryology, Faculty of Odontology, University of Buenos Aires, Buenos Aires 1425, Argentina
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(18), 10075; https://doi.org/10.3390/app131810075
Submission received: 19 August 2023 / Revised: 30 August 2023 / Accepted: 5 September 2023 / Published: 7 September 2023
(This article belongs to the Special Issue Bioactive Dental Materials: A Paradigm Shift in Dentistry?)
Browse Figures
Versions Notes

Abstract

:
Introduction: The effect of high temperatures on the properties of endodontic sealers during warm compaction of gutta-percha may be a matter of concern. This study aimed to evaluate the effect of heat on the biocompatibility and bioactivity of EndoSequence BC Sealer HiFlow (ESHF; Brasseler, Savannah, GA, USA) and AH Plus (AHPS; Dentsply, De Trey, Konstanz, Germany) when implanted into the bone tissue of rat tibias. Methods: Medical-grade silicone tubes containing freshly prepared ESHF or AHPS were heated to 100 °C and then cooled down to 65 °C before being implanted in the tibias of 24 Wistar rats. The outer walls of the tubes served as controls. After 10, 30 and 90 days, the animals were euthanized and the implants and their surrounding tissues were dissected, fixed in formalin, and processed for microscopic evaluation. Results: After 10 days postoperatively, a severe inflammatory reaction without reactionary bone formation was observed in contact with ESHF and AHPS. The severity of the reaction had decreased at the 30-day observation period for both sealers but only ESHF samples showed new bone formation adjacent to the sealer. After 90 days, no inflammatory cells were found in contact with ESHF, while a thin fibrous tissue capsule and complete bone reparation of the surrounding areas were observed in contact with the material. For AH Plus, a fibrous connective tissue containing scarce remaining inflammatory cells could be observed in most of the samples, however, in the absence of new bone formation. No significant differences (p > 0.05) between ESHF and AHPS were found at the 10-day observation period. At the 30 and 90-day, significant differences (p < 0.05) between both materials were observed. The reaction to the controls showed significant differences with ESHF and AHPS for all experimental periods. Significant differences (p < 0.05) for the total effect of time were also found between both sealers (p < 0.05). Conclusions: At the end of the experiment, heated ESHF reacted as a biocompatible/bioactive material and stimulated continued development of new healthy bone. Although AHPS was tolerated well by the surrounding tissues, the sealer did not promote new reparative bone formation.

1. Clinical Relevance

The results of the study suggest that heated EndoSequence BC Hi Flow sealer demonstrated biocompatibility and retained its bioactive properties and was able to form new bone when implanted in the tibias of rats. Although AH Plus exhibited biocompatibility, it did not show the formation of new bone at any of the time periods of evaluation and cannot therefore be considered as a bioactive root canal sealer.

Introduction

The current concept among clinicians is that after debridement, disinfection and preparation, complete obturation of the root canal space with a biocompatible/bioactive material constitutes the key for successful endodontic therapy [1]. For this purpose, different materials and techniques have been advocated for filling root canals, while gutta-percha and a sealer cement are still the most widely used [2,3]. In this respect, the warm vertical compaction of gutta-percha technique (WVCT) has been used to obtain a three dimensional root canal obturation [4]. Among a large variety of endodontic sealers, the epoxy resin-based sealer AH Plus (AHPS; Dentsply Sirona, Konstanz, Germany) is widely used in conjunction with gutta-percha and is considered the gold standard. AH Plus is a paste/paste epoxy resin-based material showing excellent physicochemical properties [5,6] and is well tolerated by living tissues [1,7]. More recently, calcium silicate-based sealers have gained popularity among clinicians and are being used for root canal obturation [8,9,10,11,12,13]. Previous reports have shown that these sealers showed promise for in vivo human clinical trials [14]. Among a variety of calcium silicate-based sealers, EndoSequence BC sealer (ESBC; Brasseler, Savannah, GA, USA), which is also known as iRoot SP (Innovative Bioceramix Inc., Vancouver, BC, Canada), has been an attractive alternative for clinicians due to its favorable properties [15]. ESBC is an injectable pre-mixed and ready-to-use sealer containing calcium silicate, calcium phosphate, zirconium oxide, calcium hydroxide, additional fillers and thickening agents [16]. The sealer is dimensionally stable and possesses high flowability and excellent sealing properties [17]. Previous studies demonstrated that ESBC possesses biocompatibility and has bioactive osteo-inducing properties [18,19,20]. For root canal obturation ESBC is indicated for use with a single cone (SCT) [21] or the warm vertical compaction technique (WVCT) [22]. Fernández et al. [22] reported that the WVCT with ESBC is more effective than the SCT for in vitro obturation in an artificial root canal system. However, Qu et al. [23] cautioned that the increase in temperature during the WVCT may alter the physical properties of ESBC and could negatively affect the quality of the obturation.
To overcome this problem, a new hydraulic high-temperature-resistant silicate-based sealer named EndoSequence BC Hi Flow (ESHF; Brasseler) has recently been introduced. According to the manufacturer, the chemistry of the sealer is like ESBC, but it flows better due to its lower viscosity. Zhang et al. [24] reported that this sealer exhibited excellent results in oval-shaped premolars when used in combination with the WVCT. Previous in vitro [25] and recent in vivo implantation studies in subcutaneous connective tissue in rats [26] showed that ESHF demonstrated biocompatibility and bioactive properties through forming apatite crystals while promoting macrophage polarization. To the best of our knowledge, little is known about the above-mentioned properties of ESHF or AHPS in bone after having been exposed to high temperatures. It is conceivable that due to exposure to high temperatures the chemical composition of the sealers is negatively affected causing them to be less clinically effective. The aim of the present study was to evaluate the effect of high temperatures on the biocompatibility and osteogenic capacity of ESHF and AHPS when implanted in the tibias of rats. The null hypothesis was that there are no statistically significant differences in biocompatibility between ESHF and AHPS after exposure to high temperatures and that the bioactivity of ESHF is not affected, regardless of the period of observation.

2. Material and Methods

2.1. Preparation of Samples

The protocol for this study was approved by the Research Ethics Committee of the Argentine Dental Association, Buenos Aires, Argentina (Protocol #0123). The sample size was calculated using the F test family, ANOVA, of G* Power 3.1 software [27]. A minimum of twenty-four (n = 24) animals was indicated as the appropriate sample size. Forty-eight (n = 48) medical grade silicone tubes (Raholin SRL, CABA, Buenos Aires, Argentina) closed at one end, measuring 1.5 mm long with an outer diameter of 1.0 mm and internal lumen of 0.5 mm were autoclaved and then divided into two groups of twenty-four (n = 24). The use of silicone tubes has previously been reported as a reliable technique [28]. In Group 1, the tubes were filled flush with ESHF, while group 2 received AHPS. Care was taken to prevent smearing of the sealers on the outside of the tubes. The pre-mixed sealer and AHPS were used according to the manufacturers’ instructions.
After filling the tubes of both groups, they were placed in separate glass plates and inserted in an oven at a temperature of 100 °C and 100% humidity for 1 min to simulate the heat caused by the WVCT. They were then cooled to 37 °C for 1 min and immediately implanted in the tibias of 24 white male Wistar rats weighing approximately 250 g each. The outer wall of the silicone tube served as negative controls (NEC). The husbandry and management of the animals met the requirements of the ISO 10993-1 (2018) [29] and ISO 10993-2, (2018) standards [30] as well as the International Regulatory Requirements for the care and use of laboratory animals [31].

2.2. Sample Implantation

The animals were anesthetized via administration of an intraperitoneal injection of ketamine chloride (14 mg/kg body weight) and acepromazine (10 mg/kg body weight). The tibias were shaved, and the skin disinfected with 5% iodine in alcohol. The implantation technique was performed under sterile conditions according to the procedures described in a previous report [28]. Briefly, with a scalpel, a 1.5 cm longitudinal incision was made at the lateral aspect of the anterior border of the tibias. After the incision, muscles, ligaments, and the periosteum were displaced by blunt dissection, thus exposing the underlying bone. To avoid overheating, a cylindrical opening 1.5 mm deep was prepared by manually rotating a sterilized 1.2 mm diameter end-cutting bur in the diaphyseal bone of each tibia at approximately 8 mm from the lateral external side. Bleeding was controlled by rinsing with saline and the openings were lightly dried with sterile gauze. Then, two silicone tubes were placed in each rat. One tube with ESHF was placed in the right tibia while the other side received AHPS. The tubes were placed so that the open ends contacted the marrow space while the closed ends were level with the cortical bone. After the wounds were closed with silk sutures, the animals were placed in cages maintained at a temperature of 22–24 °C with 12 h light/dark cycles and access to food and water ad libitum. After 10, 30 and 90 days the animals were euthanized in groups of 8 (n = 8) by means of an overdose of the anesthetic solution. The tibias were dissected and fixed in 10% neutral buffered formalin. The solution was replenished after 48 h, and the samples fixed for 8 days. Following decalcification in ethylenediaminetetraacetic acid (EDTA), the specimens were processed for routine paraffin embedding. Longitudinal sections approximately 6 μm thick were obtained from the center of the implants and stained with hematoxylin and eosin (H&E). From these sections, three of them were randomly selected and used for histological evaluation.

2.3. Evaluation of Tissue Reaction

To determine the tissue reaction to ESHF and AHPS, the areas in direct contact with the test materials were analyzed using a light stereomicroscope (LECO Corp., St. Joseph, MI, USA) coupled with a digital Sony Cyber-Shot DSC-W180 camera (Sony Corp, Tokyo, Japan). Photographs were taken at different magnifications from three arbitrarily selected sections representing the center of each specimen. The images were transferred to a computer and analyzed with the Image J 1.38Ximage-analyzer software (National Institutes of Health, Bethesda, MD, USA). The tissue reaction was viewed at different magnifications on the three randomly selected sections limited to a predetermined area measuring 1000 × 850 µm located at the open end of the silicone tubes. The inflammatory reactions were scored according to the following parameters: none, presence of a fibrous capsule with new healthy bone trabeculae formation (NHBF) and absence of inflammatory cells; mild, fibrous capsule, NHBF and presence of few remaining inflammatory cells; moderate, presence of some concentration or clusters of polymorphonuclear leukocytes, lymphocytes, plasmocytes and macrophages without NHBF; severe, presence of large accumulations of polymorphonuclear leukocytes, lymphocytes, plasmocytes, macrophages, some foreign-body giant cells and congested capillaries. Mean scores for ESHF and AHPS were calculated from the sum of the scores for all the parameters analyzed. All measurements were performed by two calibrated examiners who were blinded to the treatments. The sealers were considered biologically acceptable if the tissue reaction was scored as none or mild.

2.4. Statistical Analysis

Statistical analysis was performed with the SPSS Version 21 software (IBM Corp., Chicago, IL, USA). Data were analyzed with the Wilcoxon Signed Rank test to determine if there was a statistically significant difference between ESHF and AHPS at each observation period. The total effect of time and material upon the tissue reaction was determined using the Kruskal–Wallis and the Dunn’s multiple comparison test. The significance level was set at p < 0.05.

3. Results

At the end of each observation period, the animals showed satisfactory wound healing while all implants remained in situ. The inter-examiner agreement showed a Cohen’s Kappa value of 0.89. The intra-examiner reproducibility was determined on 30-day post-operative samples via analyzing 20 randomly selected histologic slides. In this case, the Cohen’s Kappa value ranged from 0.85 to 0.90. Mean values of the number of specimens for each tissue reaction category at different experimental periods are shown in Table 1.

3.1. ESHF and AHPS

After 10 days, the tissue response to ESHF and AHPS was scored as Severe. A granulomatous tissue composed of lymphocytes, polymorphonuclear leucocytes, macrophages, blood vessels and some areas of necrosis were observed in contact with either material (Figure 1A–D).
After 30 days, the reaction to both materials had diminished. ESHF scored a tissue reaction of Mild. A juvenile NHBF covered by a thin fibrous capsule had progressed with a tendency to cover the open end of the tubes. Below the capsule, a few inflammatory cells were still present. This picture was common for all specimens of the ESHF group (Figure 2A,B). Tissue reaction to AHPS was scored as Mild (one specimen) and Moderate (seven specimens). Under a discrete initial fibrous tissue formation, a fibro-granulomatous tissue containing clusters of lymphocytes, macrophages, plasmocytes, blood vessels and islets of necrosis were observed (Figure 2C,D).
At the end of the experiment (90 days), all samples with ESHF received scores of None. Complete bone deposition of healthy new bone trabeculae lined by active osteoblast-like cells was observed (Figure 3A,B). The NHBF was covered by a thin fibrous capsule free of inflammatory cells. In contact with AHPS (Figure 3C,D), the tissue reaction was scored as Mild. There was a dense fibrous connective tissue formation containing a few persistent lymphocytes, but no NHBF was observed.

3.2. Controls

After 10 days, a mild inflammatory reaction was observed in contact with the controls. An immature fibrous capsule containing a few inflammatory cells and some congested blood vessels was present. The thickness of the fibrous connective capsule decreased progressively over 30 and 90 days, showing the presence of mature fibroblasts, however, without inflammatory cells (Figure 4A–C).
In all observation periods, the tissue reaction to the controls significantly differed from ESHF and AHPS (p < 0.05). No significant differences (p > 0.05) between ESHF and AH Plus were observed after 10 days of implantation. However, significant differences (p < 0.05) between both materials were found at the 30- and 90-day observation periods. Significant differences for the total effect of time were also found between both ESHF and AH Plus (p < 0.05). Therefore, the null hypothesis was partially accepted.

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 diaphyseal 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 Camilleri et al. [37], AHPS specimens were subjected to 100 °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 Donnermeyer et al. [38], the highest temperature developed inside root canals via heat carriers during the WVCT ranges from 40 °C to 70 °C. However, these temperatures may vary according to the experimental model used. Thus, as per protocol, both ESHF and AHPS were heated to 100 °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. Since the setting time of AHPS is reduced by heat, it has been shown that heating may disintegrate the amine group, thus leading to incomplete polymerization of the sealer [38]. Karapinar-Kazandag et al. [39] suggested that most of the unpolymerized components of the sealer may be released and act as irritants to the surrounding tissues. The severity of the inflammatory reaction decreased over time, and after 90 days a dense fibrous connective tissue containing only a few remaining inflammatory cells were found in contact with the sealer. Although at the end of the experiment AHPS appeared to be well tolerated by the surrounding bone tissues, reparative NHBF was not observed in any of the observation periods. Our findings agree with Giacomino et al. [20] who demonstrated that AHPS does not possess osteogenic potential.
The initial inflammatory reaction caused by freshly implanted ESHF may be due to the strong alkaline pH and the greater calcium and hydroxyl ion release from calcium silicate-based sealers [40]. After 30 days, this early reaction was significantly reduced, while an incipient NHBF was observed. At the end of the study, no inflammatory cells were seen in the areas in contact with ESHF. The contact area was completely replaced by healthy reparative bone formation, which was the result of prolonged release of bioactive molecules from the sealer [25]. However, our results differ from Camilleri et al. [37] who reported that heating of calcium silicate-based sealers may affect their biomineralization property. In our study, heating of ESHF to 100 °C did not impede the reparative NHBF induction property of the sealer. This may be explained by the tricalcium and dicalcium of ESHF remaining unaltered during heating, even to 100 °C [41]. According to previous reports [41,42,43], this desirable mineralization action derived from the persistence of alkalinity and from the release of bone morphogenetic protein 2, together with alkaline phosphatase, are the two key factors that contribute to the mineralization activity of the sealer. Moreover, as is the case with other calcium silicate-based sealers, ESHF has a Ca/P ratio almost like human bone, which is considered a benefit to its biocompatibility/bioactivity [44]. It should be emphasized, however, that the findings reported here should not be interpreted as being equivalent to clinical outcomes. Only in vivo experiments in humans can confirm or reject our findings.

5. Conclusions

Within the limitations of this study, it can be concluded that after heating ESHF to 100 °C, its biocompatibility and bioactivity properties remained intact as evidenced when implanted in bone tissue of the rat tibia. After heating, AHPS appeared to be well tolerated by the surrounding tissues, but only ESHF promoted new bone formation when observed after 30 and 90 days. Confirmation of the findings reported here can only be obtained through clinical use in humans. Its use cannot be considered experimental as both sealers have demonstrated to be biocompatible.

Author Contributions

Investigation: O.Z., C.H.P. and R.D.P.; conceptualization: O.Z., R.d.L. and C.H.P.; validation: R.D.P. and R.d.L.; writing—original draft preparation: O.Z.; writing—review and editing: C.H.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Approval of the Research Ethics Committee of the Argentine Dental Association, Buenos Aires, Argentina (Protocol #0123).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. At 10 days. (A) Photomicrograph of a representative specimen of ESHF showing a dense inflammatory reaction (white arrow) in contact with the sealer (empty space). Black arrow: bone marrow; BC: cortical bone (Hematoxylin and Eosin; original magnification ×100). (B) Higher magnification from the square area in (A) showing a granulomatous tissue in which polymorphonuclear leucocytes (PN), lymphocytes (L) and plasmocytes (PL) were observed. White arrow indicates the presence of an islet of necrotic area (Hematoxylin and Eosin; original magnification ×400). (C) Photomicrograph of a representative specimen of AHPS showing an inflammatory reaction and a necrotic area (white arrow) in contact with the sealer (empty space). Below, black arrows indicate the presence of reduced marrow space (Hematoxylin and Eosin; original Magnification ×100). (D) Higher magnification from the square area in (C). This shows a granulomatous tissue containing polymorphonuclear leucocytes, lymphocytes, macrophages, and blood vessels (BV). Note the presence of a free bone particle (white arrow) and a reduced bone marrow area (black arrow) (Hematoxylin and Eosin; original magnification ×400).
Figure 1. At 10 days. (A) Photomicrograph of a representative specimen of ESHF showing a dense inflammatory reaction (white arrow) in contact with the sealer (empty space). Black arrow: bone marrow; BC: cortical bone (Hematoxylin and Eosin; original magnification ×100). (B) Higher magnification from the square area in (A) showing a granulomatous tissue in which polymorphonuclear leucocytes (PN), lymphocytes (L) and plasmocytes (PL) were observed. White arrow indicates the presence of an islet of necrotic area (Hematoxylin and Eosin; original magnification ×400). (C) Photomicrograph of a representative specimen of AHPS showing an inflammatory reaction and a necrotic area (white arrow) in contact with the sealer (empty space). Below, black arrows indicate the presence of reduced marrow space (Hematoxylin and Eosin; original Magnification ×100). (D) Higher magnification from the square area in (C). This shows a granulomatous tissue containing polymorphonuclear leucocytes, lymphocytes, macrophages, and blood vessels (BV). Note the presence of a free bone particle (white arrow) and a reduced bone marrow area (black arrow) (Hematoxylin and Eosin; original magnification ×400).
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Figure 2. At 30 days. (A) Photomicrograph of a representative specimen of ESHF. In contact with the material (empty space), new healthy bone has formed (white arrow) which advances to the open end of the tube. Below, the area of the bone marrow is still in disarray. BC: cortical bone (Hematoxylin and Eosin; original magnification ×40). (B) Higher magnification of the square area in (A), showing a thin fibrous tissue capsule covering the NHBF (white arrow). Below, some persistent inflammatory cells can be observed (thin black arrow). Note the presence of a congested blood vessel (BV) and the bone marrow (heavy black arrow) (Hematoxylin and Eosin; original magnification ×400). (C) Photomicrograph of a representative specimen of AHPS. Dense fibro-granulomatous tissue is in contact with the material (empty space) (white arrow). BC: cortical bone (Hematoxylin and Eosin; original magnification ×40). (D) Higher magnification of the square area in (C). In contact with the material (empty space), the presence of initial fibrous tissue formation can be seen (white arrow). Below it is a fibro-granulomatous tissue (FGT) containing islets of necrotic tissue (black arrow) and blood vessels (BV). BC: cortical bone (Hematoxylin and Eosin; original magnification ×400).
Figure 2. At 30 days. (A) Photomicrograph of a representative specimen of ESHF. In contact with the material (empty space), new healthy bone has formed (white arrow) which advances to the open end of the tube. Below, the area of the bone marrow is still in disarray. BC: cortical bone (Hematoxylin and Eosin; original magnification ×40). (B) Higher magnification of the square area in (A), showing a thin fibrous tissue capsule covering the NHBF (white arrow). Below, some persistent inflammatory cells can be observed (thin black arrow). Note the presence of a congested blood vessel (BV) and the bone marrow (heavy black arrow) (Hematoxylin and Eosin; original magnification ×400). (C) Photomicrograph of a representative specimen of AHPS. Dense fibro-granulomatous tissue is in contact with the material (empty space) (white arrow). BC: cortical bone (Hematoxylin and Eosin; original magnification ×40). (D) Higher magnification of the square area in (C). In contact with the material (empty space), the presence of initial fibrous tissue formation can be seen (white arrow). Below it is a fibro-granulomatous tissue (FGT) containing islets of necrotic tissue (black arrow) and blood vessels (BV). BC: cortical bone (Hematoxylin and Eosin; original magnification ×400).
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Figure 3. At 90 days. (A) Photomicrograph of a representative specimen of ESHF showing a fibrous connective tissue capsule (FC) in contact with the material (empty space) and healthy new bone formation (NHBF). Note that the size of the bone marrow space (white arrow) has been reduced and is randomly distributed within the new bone trabeculae. Note also that the bone trabeculae are lined with active osteoblast-like cells (black arrow) (Hematoxylin and Eosin; original magnification ×40). (B) Higher magnification of the square area in (A). Note the fibrous connective tissue capsule (FC) which covers the new reparative bone trabeculae (NHBF). Also note the presence of normally distributed bone marrow cells (white arrow) (Hematoxylin and Eosin; original magnification ×400). (C) Photomicrograph of a representative specimen of AHPS. In contact with the material (empty space) showing a thick fibrous connective tissue (white arrow). BC: cortical bone (Hematoxylin and Eosin; original magnification ×40). (D) Higher magnification of the square area in (C). (A) Thick fibrous tissue (FT) containing some remaining inflammatory cells, a wide blood vessel (BV), and a reduced area of bone marrow cells (white arrow) can be seen. BC: cortical bone (Hematoxylin and Eosin; original magnification ×400).
Figure 3. At 90 days. (A) Photomicrograph of a representative specimen of ESHF showing a fibrous connective tissue capsule (FC) in contact with the material (empty space) and healthy new bone formation (NHBF). Note that the size of the bone marrow space (white arrow) has been reduced and is randomly distributed within the new bone trabeculae. Note also that the bone trabeculae are lined with active osteoblast-like cells (black arrow) (Hematoxylin and Eosin; original magnification ×40). (B) Higher magnification of the square area in (A). Note the fibrous connective tissue capsule (FC) which covers the new reparative bone trabeculae (NHBF). Also note the presence of normally distributed bone marrow cells (white arrow) (Hematoxylin and Eosin; original magnification ×400). (C) Photomicrograph of a representative specimen of AHPS. In contact with the material (empty space) showing a thick fibrous connective tissue (white arrow). BC: cortical bone (Hematoxylin and Eosin; original magnification ×40). (D) Higher magnification of the square area in (C). (A) Thick fibrous tissue (FT) containing some remaining inflammatory cells, a wide blood vessel (BV), and a reduced area of bone marrow cells (white arrow) can be seen. BC: cortical bone (Hematoxylin and Eosin; original magnification ×400).
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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 ×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 ×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 ×400).
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 ×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 ×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 ×400).
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Table 1. Number (%) of samples in each group categorized according to the inflammatory score and the observation period.
Table 1. Number (%) of samples in each group categorized according to the inflammatory score and the observation period.
Inflammatory Response10 Days30 Days90 Days
ESHFAHPSESHFAHPSESHFAHPS
n (%)n (%)n (%)n (%)n (%)n (%)
None0 (0)0 (0)0 (0)0 (0)8 (100)0 (0)
Mild0 (0)0 (0)8 (100)1 (12.50)0 (0)8 (100)
Moderate0 (0)0 (0)0 (0)7 (87.50)0 (0)0 (0)
Severe8 (100)8 (100)0 (0)0 (0)0 (0)0 (0)
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MDPI and ACS Style

Zmener, O.; Pameijer, C.H.; Della Porta, R.; de Lucca, R. Tissue Response to a Heat Resistant Silicate-Based and an Epoxy Resin-Based Endodontic Sealer Implanted in Rat Tibias. Appl. Sci. 2023, 13, 10075. https://doi.org/10.3390/app131810075

AMA Style

Zmener O, Pameijer CH, Della Porta R, de Lucca R. Tissue Response to a Heat Resistant Silicate-Based and an Epoxy Resin-Based Endodontic Sealer Implanted in Rat Tibias. Applied Sciences. 2023; 13(18):10075. https://doi.org/10.3390/app131810075

Chicago/Turabian Style

Zmener, Osvaldo, Cornelis H. Pameijer, Roberto Della Porta, and Romina de Lucca. 2023. "Tissue Response to a Heat Resistant Silicate-Based and an Epoxy Resin-Based Endodontic Sealer Implanted in Rat Tibias" Applied Sciences 13, no. 18: 10075. https://doi.org/10.3390/app131810075

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