Journal Pre-proof

Biocompatibility and bioactive potential of new calcium silicate-based endodontic

sealers: Bio-C Sealer and Sealer Plus BC

Evelin Carine Alves Silva, DDS, MSc, Mario Tanomaru-Filho, DDS, PhD, Guilherme,
Ferreira da Silva, DDS, PhD, Mateus Machado Delfino, DDS, MSc, Paulo Sérgio
Cerri, DDS, PhD, Juliane Maria Guerreiro-Tanomaru, DDS, PhD
PII: S0099-2399(20)30498-2
DOI: https://doi.org/10.1016/j.joen.2020.07.011
Reference: JOEN 4623

To appear in: Journal of Endodontics

Received Date: 3 May 2020

Revised Date: 4 July 2020
Accepted Date: 7 July 2020

Please cite this article as: Alves Silva EC, Tanomaru-Filho M, da Silva G,F, Delfino MM, Cerri PS,
Guerreiro-Tanomaru JM, Biocompatibility and bioactive potential of new calcium silicate-based
endodontic sealers: Bio-C Sealer and Sealer Plus BC Journal of Endodontics (2020), doi: https://
doi.org/10.1016/j.joen.2020.07.011.

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Copyright © 2020 Published by Elsevier Inc. on behalf of American Association of Endodontists.

Biocompatibility and bioactive potential of new calcium silicate-based endodontic
sealers: Bio-C Sealer and Sealer Plus BC
Evelin Carine Alves Silva, DDS, MSc1, Mario Tanomaru-Filho, DDS, PhD1, Guilherme
Ferreira da Silva, DDS, PhD2, Mateus Machado Delfino, DDS, MSc1, Paulo Sérgio
Cerri, DDS, PhD3, and Juliane Maria Guerreiro-Tanomaru, DDS, PhD1
1 Department of Restorative Dentistry, School of Dentistry, São Paulo State University (Unesp),
Araraquara, São Paulo, Brazil.
2 Department of Dentistry, Unisagrado, Bauru, SP, Brazil
3 Department of Morphology, School of Dentistry, São Paulo Stat

University (Unesp),
Araraquara, São Paulo, Brazil.
Acknowledgments
This study was financed by Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - (CAPES) - Finance Code 001; and was supported by São Paulo Research
Foundation - FAPESP (2017/14305-9, 2017/19049-0).
The authors deny any conflicts of interest related to this study
Address for correspondence and reprint requests:
Prof. Dr. Mário Tanomaru Filho
Department of Restorative Dentistry, Araraquara Dental School, São Paulo State
University-UNESP, Rua Humaitá, 1680, PO 331, CEP 14.801-903, Araraquara, SP,
Brazil. Tel: +55-16-3301-6390. Fax: +55-16-3301-6392. E-mail address:
tanomaru@uol.com.br
 1

1 Biocompatibility and bioactive potential of new calcium silicate-based endodontic sealers:

2 Bio-C Sealer and Sealer Plus BC
3
4 Abstract
5
6 Introduction: Bio-C Sealer (BC) and Sealer Plus BC (SPBC) are new ready-to-use bioceramic
7 endodontic sealers. The aim of the study was to evaluate biocompatibility and bioactive potential
8 of BC and SPBC sealers in comparison to AH Plus (AHP) in subcutaneous tissue of rats.
9 Methods: Polyethylene tubes filled with materials and empty tubes, as control group, were
10 implanted in the subcutaneous tissues of rats. After 7, 15, 30 and 60 days, the tubes with
11 connective tissue were removed and inflammatory cells / mm2 (IC) and immunolabeled cells for
12 interleukin-6 (IL-6) were evaluated. Osteocalcin (OC) and von Kossa analysis were also
13 performed. Data were submitted to ANOVA and Tukey tests, with a significance of 5%. Results:
14 At 7 days, SPBC showed lower IC than BC (p <0.05). AHP exhibited greater immunolabeled
15 cells for IL-6 (p <0.05). After 15 days, BC showed lower IC and IL-6 when compared to other
16 materials. At 30 days, SPBC and AHP showed higher values for IC (p <0.05). After 60 days,
17 calcium silicate sealers did not show statistical difference (p>0.05) for IC and IL-6, with values
18 lower than AHP (p <0.05). The materials showed positive structures to von Kossa. BC exhibited
19 osteocalcin labeling in all periods. SPBC showed osteocalcin labeling from 15 to 60 days. AH
20 Plus and the control group did not exhibit osteocalcin labeling. Conclusions: Bio-C Sealer and
21 Sealer Plus BC sealers are biocompatible and present bioactive potential.
22
23 Key words: Calcium silicate, biocompatibility, root canal filling material, endodontics.
24
25
26
27
28
29
30
31
 2

1 INTRODUCTION
2 Premixed calcium silicate-based sealers are developed presenting proper
1
3 physicochemical and biological properties . EndoSequence BC and TotalFill BC Sealer (FKG
4 Dentaire AS, Switzerland) are endodontic sealers composed of calcium silicates, zirconium
5 oxide, monobasic calcium phosphate, and ready-to-use. These materials have biocompatibility,
6 release calcium ions, present high pH, dimensional stability and radiopacity2,3,4. Zirconium oxide
7 used as radiopacifier in a calcium silicate-based material stimulate fibroblast proliferation and
8 collagen formation in subcutaneous tissue of rats5.
9 New calcium silicate-based ready-to-use endodontic sealers have been developed. Bio-C
10 Sealer (Angelus, Brazil) is a new sealer composed of tricalcium silicate, dicalcium silicate,
11 tricalcium aluminate, calcium oxide, zirconia oxide, silicon oxide, polyethylene glycol, iron
12 oxide. The physico-chemical properties of Bio-C Sealer are different from TotalFill BC and AH
13 Plus sealers, presenting the shortest setting time and highest solubility, but with low dimensional
14 change6. When compared to Bio-C Repair, a repair calcium silicate ready-to-use material, Bio-C
15 Sealer presented less cell viability, however, it was better than AH Plus7. The Sealer Plus BC
16 (SPBC; MK Life, Brazil) is composed of calcium disilicate, nanoparticulate calcium trisilicate
17 and zirconium oxide. This sealer presents alkaline pH, releases calcium ions and has proper
18 setting time and radiopacity. When compared to AH Plus (Dentsply De Trey Gmbh, Konstanz,
19 Germany), Sealer Plus BC showed higher solubility, less radiopacity and higher pH8. When
20 compared to MTA Fillapex and AH Plus, Sealer Plus BC showed greater biocompatibility in the
21 subcutaneous of rats9.
22 Biocompatibility and bioactivity are important properties for endodontic sealers, since
23 they maintain contact with the periapical tissues and can influence repair 3,5,9. Both properties are
24 directly related to the composition of the material. It is well reported that calcium silicate sealers
25 promote low cytotoxicity and induce mild/moderate inflammatory reaction3-5,7,9. y
26 Therefore, the aim of this study was to evaluate the biocompatibility and bioactive
27 potential of two bioceramic sealers, in comparison with AH Plus. The null hypothesis is that the
28 difference between the compositions of the materials would not interfere in the tissue reaction
29 induced by bioceramics or epoxy resin sealers.
30
31 MATERIALS AND METHODS
 3

1 The materials used in this study, their manufacturers, compositions and proportions are
2 included in Table 1.
3
4 The research protocol was approved by the Ethical Committee for Animal Research of
5 São Paulo (CEUA, Process number 35/2018). Twenty-four male Holtzman rats (Rattus
6 norvegicus albinus) were used and distributed in four groups (n = 6): sealers and control group
7 (empty polyethylene tubes). The materials were inserted in polyethylene tubes implanted in
8 dorsal subcutaneous sites. Four tubes were inserted per animal, one from each group (ISO-
9 10993-6, 2007). The animals were anesthetized with ketamine (80 mg/kg body weight, Virbac do
10 Brasil Indústria e Comércio Ltda., São Paulo, SP, Brazil) and xylazine (4 mg/kg body weight,
11 União Química, São Paulo, SP, Brazil). After 7, 15, 30 and 60 days, the animals were euthanized
12 with anesthetic overdose, the implants with adjacent tissues were removed.

13 Histological procedures
14 The implants and surrounding tissues were removed and immersed for 72 hours in a 4%
15 formaldehyde solution and pH adjusted to 7.2. After fixation, the specimens were dehydrated,
16 diaphanized, immersed in liquid paraffin (60ºC) and embedded in paraffin. Longitudinal sections
17 with 6 μm thickness were obtained. Non-serial sections were stained with hematoxylin and eosin
18 (HE) to estimate the number of inflammatory cells in the capsules and thickness of the capsules
19 adjacent to the materials.

20 Numerical density of inflammatory cells

21 The numerical density of inflammatory cells (IC) was evaluated using a light microscope
22 (BX51, Olympus, Tokyo, Japan) and an image analysis system (Image Pro-Express 6.0,
23 Olympus). In each implant, three H&E-stained sections of the capsule were selected at intervals
24 of at least 100 mm. In each section, a standardized field 0.09 mm2 of the capsule adjacent to the
25 opening of the tube implanted was analyzed, totaling 0.27 mm2 per implant. In each area, the
26 total number of IC (neutrophils, lymphocytes, plasma cells, and macrophages) was counted using
27 the image analysis system at 695 magnification. Thus, the number of ICs/mm2 was obtained for
28 each implant10-12.
29
30 Thickness of capsules
 4

1 The thickness (in µm) of the capsules adjacent to the implanted tubes was measured.
2 Three images of non-serial sections stained with HE of each specimen were captured. The
3 thickness of the capsules was estimated in the middle portion from its surface adjacent to the
4 material to its limit with the adjacent tissues. After obtaining the values, the average was
5 calculated from the measurements obtained from the three sections for each specimen.
6
7 Immunohistochemical detection of IL-6
8 For the detection of IL-6, mouse anti-IL-6 antibody (Abcam, Cambridge Science, UK;
9 code Ab 9324,) was used. The sections were incubated overnight in a humidified chamber with
10 anti-IL-6 antibody. Subsequently, the sections were incubated in Labeled StreptAvidin-Biotin kit
11 (Universal Dako LSAB, Dako Inc., Carpinteria, CA, USA; K0675). After washing, peroxidase
12 activity was revealed by the 3,3'-diaminobenzidine chromogen (ImmPACTTM DAB Vector,
13 Burlingame, CA, United States). The sections were counterstained with Carazzi's hematoxylin.
14 So, the number of IL-6-immunolabeled cells per mm2 of the capsule was counted at 695X using
15 an image analysis system (Image Pro-Express 6.0, Olympus). For each tube implanted, the IL-6
16 positive cells were counted in a standardized area (0.09 mm2). In each group, the values were
17 divided by the total area, and then, the number of IL-6/mm2 was obtained10,12.
18

19 Immunohistochemical detection of osteocalcin

20 The sections were incubated with rabbit anti-osteocalcin antibody (1:200; Sigma-Aldrich
21 Co., Saint Louis, Missouri, USA; code SAB1306277). After 16 hours, in a humidified chamber,
22 the sections were incubated in Labeled StreptAvidin-Biotin kit (Universal Dako LSAB, Dako
23 Inc., Carpinteria, CA, USA; K0675). Subsequent to buffer washes, peroxidase activity was
24 revealed by the 3,3'-diaminobenzidine chromogen (ImmPACTTM DAB Vector, Burlingame,
25 CA, United States). The sections were counterstained with Carazzi's hematoxylin. The number of
26 osteocalcin-immunolabeled cells was performed similarly to IL-6.

27 von Kossa reaction and analysis under polarized light

28 The sections were immersed in a 5% silver nitrate solution. Posteriorly immersed in a 5%
29 sodium hyposulfite solution. The sections were then stained with picrosirius-red and mounted in
30 resinous medium (Permount®, Fisher Scientific, New Jersey, USA11,12). Sections close to those
 5

1 subjected to von Kossa were deparaffinized, dehydrated .The unstained sections were analyzed
2 under a light microscope equipped with polarization filters (Olympus, BX51).
3 Statistical analysis
4 The statistical analysis was obtained with the aid of the GraphPad Prism 5 software
5 (Jandel Scientific, Sausalito, CA, USA). The data were evaluated by the two-way ANOVA
6 followed by the Tukey test. The level of significance considered was p ≤0.05.

8 Results

9 Numerical density of inflammatory cells

10 At 7 days, SPBC showed lower values than BC (p <0.05) (Fig. 1A and 1B). After 15
11 days, BC showed lower IC than SPBC and AHP (Fig 1E, 1F and 1G). At 30 days, there was no
12 statistical difference between SPBC and AHP for IC (p >0.05) (1J and 1K). At 60-day period,
13 BC and SPBC showed no statistical difference between them (p >0.05). AHP presented the
14 highest IC values when compared to the others (p <0.0001) (1O) (Table 2).
15
16 Thickness of the capsules adjacent to the implants
17 The capsules exhibited a moderate inflammatory reaction. At 7 days, BC and SPBC
18 showed no statistical difference (p >0.05). At 15 and 30 days, there was no statistical difference
19 between BC and AHP (p >0.05). SPBC exhibited the highest values. After 60 days, all materials
20 showed reduction in the thickness of the capsules, with no statistical difference between them
21 (Table 2).
22
23 Immunohistochemical detection of IL-6
24 At 7 days, the number of immunostained inflammatory cells/mm2 was significantly
25 higher compared to the other periods (p <0.05). AHP exhibited the highest values (p <0.0001)
26 (Fig 2C). At 15 and 30 days, BC exhibited less marking when compared to SPBC (p <0.05). At
27 60 days, BC, SPBC and control showed the lowest values (Figure 2I, 2J and 2L), with no
28 difference between BC and SPBC (p >0.05). The AHP presented the highest values in all the
29 periods analyzed (p <0.0001) (Table 2).
 6

1 Immunohistochemical detection of osteocalcin

2 At 7 days, only BC group (Figure 2E) exhibited positive marking for osteocalcin. At 15
3 and 30 days, BC and SPBC had immunopositive cells. After 60-day period BC showed higher
4 number of labeled cells compared to SPBC (p <0.05) (Table 2) (Figure 2M and 2N). AH Plus
5 sealer and the control group did not show positive marking in any period (Figure 2G, 2H, 2O and
6 2P).
7
8
9 von Kossa reaction and analysis under polarized light
10 BC, SPBC and AHP presented positive structures to von Kossa method in all periods
11 analyzed (Fig 3A, 3B, 3C, 3G, 3H and 3I). The control group did not show positive structures.
12 BC and SPBC showed birefringent structures in all periods spread by the adjacent tissues, and
13 AH Plus presented structures located only on the surface of the capsules. The control group did
14 not exhibit birefringent structures.
15
16 Discussion
17 The Bio-C Sealer was compared with Sealer Plus BC and AH Plus for up to 60 days to
18 assess tissue repair and bioactive potential. The present study demonstrated that Bio-C Sealer
19 and Sealer Plus BC induced a less intense inflammatory process than AH Plus, and exhibited
20 positive marking for osteocalcin. Bio-C sealer presents tricalcium aluminate, calcium oxide,
21 silicon oxide, iron oxide and polyethylene glycol, as dispersant agent, which is different from
22 other bioceramic sealers. Sealer Plus BC presents calcium disilicate, nanoparticulate calcium
23 trisilicate, zirconium oxide, and the dispersant agent is not described. These differences may
24 interfere in the physicochemical properties and also in the biocompatibility and bioactive
25 potential. Therefore, the null hypothesis was rejected, as differences were observed in tissue
26 responses induced by bioceramics and epoxy-based sealers.
27 At 7 days, all materials showed moderate inflammatory reaction, with presence of plasma
28 cells, neutrophils, macrophages and giant cells close to the sealer particles. The control group
29 exhibited the lowest number of inflammatory cells and a thick capsule. Considering that
30 polyethylene tubes are inert, the inflammatory reaction observed in the control group may be
31 related to the surgical procedure11. Bio-C Sealer and Sealer Plus BC have been biocompatible
 7

1 over time. However, in the initial periods an inflammatory reaction was observed in the
2 subcutaneous tissues12. The setting reaction of these materials promotes the formation of calcium
3 and hydroxyl ions (OH-), and the alkaline pH stimulates the recruitment of inflammatory cells
4 and the production of cytokines13-15. The large number of inflammatory cells observed in the
5 initial periods may be related to the elevated pH observed for bioceramic materials9,13,14,16.
6 IL-6 is a proinflammatory cytokine that may activate and modulate specific cells, playing
7 an important role in inflammatory reaction and in bone resorption3,12. Our findings revealed that
8 sealers promoted significant increase in the number of IL-6 immunolabeled cells in the capsules,
9 especially, after 7 days. However, the increase was greater for AH Plus compared to Bio-C
10 Sealer and Sealer Plus BC in all experimental periods. The gradual and significant reduction in
11 inflammatory cells and IL-6 immunolabeled cells suggests that the capsules showed an intense
12 remodeling process in the initial periods. In addition, the presence of well-oriented collagen
13 fibers bundles in the capsules indicates that initial inflammatory reaction was replaced, after 60
14 days, by a dense connective tissue. This hypothesis is reinforced by the reduction in the capsule
15 thickness over the evaluated periods.
16 AH Plus presented the highest inflammatory reaction and IL-6 immunolabeled cells after
17 60 days. It is possible that the epoxy resin released from AH Plus delays the healing process9. It
18 was already demonstrated that bioceramic sealers are less cytotoxicity than AH Plus17,18 and that
19 they present biocompatibility after extended periods19. Furthermore, when analyzed in the
20 subcutaneous tissue of rats, AH Plus sealer presented greater quantity of IL-6 immunolabeled
21 cells when compared to MTA sealer11. A systematic review that investigated the gene expression
22 of various cells in response to different tricalcium silicate cements showed that these materials
23 were related to significantly increased gene expression related to periapical regeneration20.
24 Tricalcium silicate materials are usually related to biocompatibility and osteoblastic
25 differentiation20,21,22. Organic matrix of mineralized tissues can be evaluated by
26 immunohistochemistry to detect proteins such as osteocalcin, secreted by osteoblasts, identified
27 as marker of mature osteoblasts9, and by the von Kossa histochemical technique to evaluate
28 calcium precipitates. SPBC did not show positive marking for osteocalcin at 7 days. SPBC
29 showed immunopositive cells for osteocalcin from 15 to 60 days, and BC presented in all
30 periods. The ability to induce mineral deposition by using immunohistochemical analysis for
31 osteocalcin was demonstrated for the calcium silicate-based cement Bio-C Pulpo (Angelus), a
 8

1 ready-to-use repair material23. López-Gárcia et al. 20197 showed that the Bio-C Sealer is able to
2 promote cellular viability, cell survival and cell migration of periodontal ligament stem cells.
3 In order to identify amorphous calcite deposits, the von Kossa birefringence technique
4 was performed. Bio-C Sealer and Sealer Plus BC presented irregular structures that were positive
5 to von Kossa staining with calcium deposits in the adjacent capsules at 7 days. AH Plus
6 exhibited von Kossa positivity, but did not show positive marking for osteocalcin in any
7 evaluated period. Carvalho et al. 201724 showed that bioactivity is not expected for AH Plus.
8 Calcium silicate sealers release calcium favoring mineralization and differentiation of dental
9 pulp cells13. The reaction process of calcium ions and carbon dioxide leads to the formation of
10 calcite crystals, birefringent structures that may lead to the formation of calcified structures25,26.
11 Positivity for von Kossa and birefringent to polarized light are usually performed to evaluate the
12 mineralization potential of calcium silicate cements27,28. The calcium silicate-based sealers
13 showed birefringent structures in all analyzed periods.
14 Biocompatibility and bioactivity are important properties as endodontic sealers maintain
15 contact with periapical tissues and may influence tissue repair3,5,9. In the present study, Bio-C
16 Sealer and Sealer Plus BC presented biocompatibility since they allow the regression of
17 inflammatory reaction faster than AH Plus. Furthermore, both bioceramic sealers show bioactive
18 potential, exhibiting osteocalcin imunopositive cells and structures that were positive to von
19 Kossa staining in the capsules.
20
21 Conclusion
22 Bio-C Sealer and Sealer Plus BC are biocompatible to be used in close contact with
23 periapical tissue, inducing a mild inflammatory reaction and favoring repair. In addition, both
24 sealers may contribute to the periapical tissue mineralization process, as they demonstrate
25 bioactive potential.
26
27
28 The authors deny any conflicts of interest related to this study.
29
30
31
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1 References
2 1- Silva Almeida LH, Moraes RR, Morgental RD, Pappen FG. Are Premixed Calcium
3 Silicate-based Endodontic Sealers Comparable to Conventional Materials? A Systematic Review
4 of In Vitro Studies. J Endod. 2017 Apr;43(4):527-535.
5
6 2- Zhou HM, Shen Y, Zheng W, et al. Physical properties of 5 root canal sealers. J Endod
7 2013; 39: 1281-6.
8
9 3- Fonseca TS, Silva GF, Tanomaru-Filho M, et al. In vivo evaluation of the inflammatory
10 response and IL-6 immunoexpression promoted by Biodentine and MTA Angelus. Int End J
11 2016; 49: 145-153.
12
13 4- Zhou H, Du T, Shen Y, et al. In vitro cytotoxicity of calcium silicate–containing endodontic
14 sealers. J Endod 2015; 41: 56-61.
15
16 5- Silva GF, Guerreiro-Tanomaru JM, Fonseca TS, Bernardi MIB, Sasso-Cerri E,Tanomaru-
17 Filho M, Cerri PS. Zirconium oxide and niobium oxide used as radiopacifiers in a calcium
18 silicate-based material stimulate fibroblast proliferation and collagen formation. . Int End Journal
19 2017; 50: 95-108.
20
21 6- Zordan-Bronzel CL, Torres FF, Tanomaru-Filho M, et al. Evaluation of Physicochemical
22 properties of a new calcium silicate–based Sealer, Bio-C Sealer. J Endod 2019; 45: 1248–1252.
23
24 7- López-García S , Lozano A , García-Bernal D, et al. Biological Effects of New
25 Hydraulic Materials on Human Periodontal Ligament Stem Cells. J Clin Med;.2019; 14: 1216.
26
27 8- Mendes AT, da Silva PB, Só BB, et al. Evaluation of Physicochemical Properties of New
28 Calcium Silicate-Based Sealer. Brazil Dental J 2018; 29: 536-540.
29
30 9- Benetti F, De Azevedo Queiroz I, Conti LC, et al. Cytotoxicity and biocompatibility of a new
31 bioceramic endodontic sealer containing calcium hydroxide. Brazil Oral Research 2019; 33: 1-9.
32
33 10- Andrade A, Silva GF, Camilleri J, et al. Tissue response and immunoexpression of
34 interleukin 6 promoted by tricalcium silicate–based Repair Materials after Subcutaneous
35 Implantation in Rats. J Endod 2018; 44: 458–4639.
36
37 11- Viola NV, Guerreiro-Tanomaru JM, da Silva GF, et al. Biocompatibility of an experimental
38 MTA sealer implanted in the rat subcutaneous: quantitative and immunohistochemical
39 evaluation. J Bio Material Research Part B 2012; 100: 1773-81.
40
41 12- Silva GF, Tanomaru-Filho M, Bernardi MI, et al. Niobium pentoxide as radiopacifying agent
42 of calcium silicate-based material: evaluation of physicochemical and biological properties. Clin
43 Oral Investig 2015; 19: 2015-253.
44
 10

1 13- Koutroulis A, Kuehne SA, Cooper PR, Camilleri J.The role of calcium ion release on
2 biocompatibility and antimicrobial properties of hydraulic cements. Scientific Reports 2019; 9:
3 1-10.
4
5 14- Fonseca TS, Silva GF, Guerreiro-Tanomaru JM, Sasso-Cerri E, et al. Mast cells and
6 immunoexpression of FGF-1 and Ki-67 in rat subcutaneous tissue following the implantation of
7 Biodentine and MTA Angelus. Int Endod Journal 2019; 2: 54–67.
8
9 15- Vosoughhosseini S, Lotfi M, Shahi S et al. Influence of white versus gray mineral trioxide
10 aggregate on inflammatory cells. J Endod 2008; 34: 715-717.
11
12 16- Zordan-Bronzel CL, Tanomaru-Filho M, Rodrigues EM,et al. Cytocompatibility,bioactive
13 potential and antimicrobial activity of an experimental calcium silicate‐based endodontic sealer.
14 Int Endod Journal 2019; 52: 979-986.
15
16 17- López-García S, Pecci-Lloret MR ,Guerrero-Gironés J, et al. Comparative Cytocompatibility
17 and Mineralization Potential of Bio-C Sealer and TotalFill BC Sealer. Materials 2019; 22: 3087.
18
19 18- Rodriguez-Lozano FJ, GarciIa-Bernal D, Onate-Sanchez RE, et al. Calcium silicate sealers
20 and dental stem cells. Int Endod J 2017; 50: 67-76.
21
22 19-Taha N, Safadi R, Alwedai M. Biocompatibility Evaluation of EndoSequence Root Repair
23 Paste in the Connective Tissue of Rats. J Endod 2016; 42: 1523-1528.
24
25 20- Rathinam E, Rajasekharan S, Chitturi RT, Declercq H, Martens L, De Coster P. Gene
26 Expression Profiling and Molecular Signaling of Various Cells in Response to Tricalcium
27 Silicate Cements: A Systematic Review. J Endod. 2016 Dec;42(12):1713-1725.
28
29 21- Giacomino CM, Wealleans JA, Kuhn N, Diogenes A. Comparative Biocompatibility and
30 Osteogenic Potential of Two Bioceramic Sealers. J Endod. 2019 Jan;45(1):51-56.
31
32 22- Saraiva JA, Fonseca T, Silva G et al. Reduced interleukin-6 immunoexpression and
33 birefringent collagen formation indicate that MTA Plus and MTA Fillapex are biocompatible.
34 Biom. Mater 2018; 13: 1-14.
35
36 23- Cosme-Silva L, Gomes-Filho JE, Benetti F, Dal-Fabbro R, Sakai VT, Cintra LTA, Ervolino
37 E, Viola NV. Biocompatibility and immunohistochemical evaluation of a new calcium silicate-
38 based cement, Bio-C Pulpo. Int Endod J. 2019 May;52(5):689-700.
39
40 24- Carvalho CN, Grazziotin-Soares R, Candeiro GT et al. Micro Push-out Bond Strength and
41 Bioactivity Analysis of a Bioceramic Root Canal Sealer. Iranian Endodontic Journal 2017; 12:
42 343-348.
43
44 25- Peng W, Liu W, Zhai W. Effect of Tricalcium Silicate on the Proliferation and Odontogenic
45 Differentiation of Human Dental Pulp Cells. J Endod 2011; 37: 1240-1246.
46
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1 26- Khalil I, Naaman A, Camilleri J. Properties of Tricalcium Silicate Sealers. J Endod 2016;
2 42: 1529-1535.
3
4 27- Bueno CR, Valentim M, Marque VA et al. Biocompatibility and biomineralization
5 assessment of bioceramic-, epoxy-, and calcium hydroxide-based sealers. Braz. Oral Res 2016;
6 30: 1-9.
7
8 28- da Fonseca TS, Silva GF, Guerreiro-Tanomaru JM, Delfino MM, Sasso-Cerri E, Tanomaru-
9 Filho M, Cerri PS. Biodentine and MTA modulate immunoinflammatory response favoring bone
10 formation in sealing of furcation perforations in rat molars. Clin Oral Investig. 2019
11 Mar;23(3):1237-1252.
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18
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20
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1
2
3 Figure legends

4
5 Figure 1 - Photomicrographs of the sections showing portions of the capsules adjacent to the
6 opening of the implanted tubes (T) after 7 (A-D), 15 (E-H), 30 (I-L) and 60 (M-P) days of
7 implantation. The capsules show numerous inflammatory cells, mainly lymphocytes, plasma
8 cells and macrophages. In the capsules of the control group (Figure 1D), the inflammatory cells
9 (arrows) have a lower amount, with an evident presence of fibroblasts (Fb). The capsules of the
10 AH Plus and BC group show a greater number of inflammatory cells (Figure 1A and 1C).
11 Figures 1E to 1H present capsules after 15 days with a reduction in the total number of cells.
12 The AH Plus group exhibited a greater number of cells (Figure 1G). At 30 days, the capsules of
13 AH Plus (Figure 1K) and SPBC (1J) exhibit a greater number of inflammatory cells, compared
14 to the other groups, while BC (Figure 1I) already have lower amount of inflammatory cells. The
15 control group (Figure 1L) has fibroblasts arranged between the collagen fibers. Figures 1M-1P -
16 show capsules after 60 days. The capsules of BC (Figure 1M) and SPBC (Figure 1N) exhibit
17 mainly fibroblasts (Fb) located between bundles of collagen fibers. The AH Plus sealer capsule
18 (Figure 1O) has more inflammatory cells compared to the other groups. Fb, fibroblasts; BV,
19 blood vessels. Bars: 18 µm.
20
21 Figure 2 - Photomicrographs showing portions of the capsules adjacent to the opening of the
22 subcutaneous implanted tubes for 7 (A-D) and 60 (E-H) IL-6 imunopositive cells and 7 days (I-
23 L) 60 (M-P) OC imunopositive cells. The capsules contain several IL-6 immunopositive cells,
24 inflammatory cells (arrows), in the period of 7 days, at 60 days reduced marking is observed in
25 all groups. AH Plus (Figure 2K) with greater quantity. At 7 days, only Bio-C showed marking
26 for OC, at 60 days Bio-C and Sealer Plus BC showed marking for OC. Bars: 18 µm.
27
28 Figure 3 - Photomicrographs of sections showing portions of capsule adjacent to the opening of
29 the tubes implanted in the subcutaneous tissue submitted to the von Kossa reaction after 7 (A, B,
30 E, F, I, J) and 60 (C, D, G, H, K, L) days. The BC (Figures 3A and 3G), SPBC (Figures 3B and
31 3H), AH Plus (Figures 3C and 3I) capsules exhibit positive von Kossa structures (black / brown)
32 and 3D, 3E, 3F, 3J, 3K and 3L compatible birefringent structures. Positive structures for calcium
33 precipitation. Von Kossa and Picrosirius-red. Bars: 18 µm.
34
35
Tables

Table 1 - Endodontic sealers used, manufactures and proportions.

Sealers Composition Manufacturers Proportion

Tricalcium Silicate, Dicalcium Silicate,

Bio C- Tricalcium Aluminate, Calcium Oxide, Angelus, Londrina,
Ready to use
Sealer (BC) Zirconia Oxide, Silicon Oxide, Brazil
Polyethylene Glycol, Iron Oxide.

Sealer Plus Calcium disilicate, nanoparticulate MK Life, Porto

Ready to use
BC (SPBC) calcium trisilicate, zirconium oxide. Alegre, Brazil

Paste A: epoxy bisphenol-A resin and

epoxy bisphenol-F, calcium tungstate Dentsply DeTrey
AH Plus 1g:1g (paste/
(CaWO4), zirconium oxide (ZrO2), GmbH, Konstanz,
(AHP) paste)
silica, iron oxide. Paste B: dibenzyl- Germany
diamine, aminoadamantane, CaWO4,
ZrO2, silica, silicone.
Table 2 - Capsule thickness (µm), number of inflammatory cells (IC) per mm2 and
number of immunopositive cells for IL-6 and osteocalcin per mm2.Bio-C (BC), Sealer
Plus BC (SPBC), AH Plus (AHP) and Control (CG) after 7, 15, 30 and 60 days.

BC SPBC AHP CG

thickness 285±79b;1 270±97b;1 343±62a;1 152±72c;1

7 days

IC 775±26a;1 690±40b;1 865±63a;1 239±32c;1

`

IL-6 549±7b;1 451±5c;1 782±8a;1 210±6d;1

OC 18±5a;1 0±0b;1 - -

thickness 252±59b;1 309±142a;1 242±18b;1 184±9b;1

15 days

IC 346±49c;2 480±48b;2 681±64a;2 232±13d;1

IL-6 253±4b;2 357±4c;2 531±6a;2 169±3d;2
OC 31±6a;2 11±4b;2 - -

thickness 232±42b;1 333±160a;2 221±22b;2 109±6c,1

30 days

IC 362±27b;3 440±17a;2 468±41a;3 173±22c,2

IL-6 201±2c;2 292±4b,3 454±4a;2 132±4d,2
OC 20±5a;1 11±7a;2 - -

thickness 172±60a;2 178±36a;3 194±41a;2 111±35a,1

60days

IC 233±40b;3 129±32b;3 352±84a;4 66±12c,3

IL-6 146±3b;3 193±3b,3 279±6a;3 79±2c,3
OC 46±9a;3 14±3b;2 - -

Mean (standard deviation).

The comparison between groups in the same period is indicated by superscript letters on
the line. Same letters = no statistically significant difference.
The comparison between intervals in the same group is indicated by numbers
superscript in the columns; same numbers = no statistically significant difference.
Tukey's test (p ≤ 0.05)