Cytotoxicity, inflammation, biomineralization, and immunoexpression of IL-1β and TNF-α promoted by a new bioceramic cement

Abstract Aim To evaluate the cytotoxicity, biocompatibility and mineralization capacity of BIO-C PULPO, and MTA. Methodology L929 fibroblasts were cultured and MTT assay was used to determine the material cytotoxicity on 6, 24, and 48 h. A total of 30 male rats (Wistar) aged between 4 and 6 months, weighing between 250 and 300 g were used. Polyethylene tubes containing BIO-C PULPO, MTA, and empty tubes were implanted into dorsal connective tissue. After the experimental periods (7, 15, 30, 60, and 90 days) the tubes were histologically analyzed using hematoxylin-eosin (H&E), immunolabeling of IL-1β and TNF-α, and von Kossa staining, or without staining for polarized light analysis. The average number of inflammatory cells was quantified; the mineralization assessment was determined by the area marked in μm2 and semiquantitative immunolabeling analyses of IL-1β and TNF-α were performed. Then, data underwent statistical analysis with a 5% significance level. Results It was observed that BIO-C PULPO and MTA presented cytocompatibility at 6, 24, and 48 similar or higher than control for all evaluated period. On periods 7 and 15 days, BIO-C PULPO was the material with the highest number of inflammatory cells (p<0.05). On periods 30, 60, and 90 days, BIO-C PULPO and MTA presented similar inflammatory reactions (p>0.05). No statistical differences were found between Control, BIO-C PULPO, and MTA for immunolabeling of IL-1β and TNF-α in the different periods of analysis (p<0.05). Positive von Kossa staining and birefringent structures under polarized light were observed in all analyzed periods in contact with both materials, but larger mineralization area was found with BIO-C PULPO on day 90 (p<0.05). Conclusion BIO-C PULPO was biocompatible and induced mineralization similar to MTA.


Introduction
Pulpotomy is the treatment for reversible pulpal lesions and it can be performed on permanent or deciduous teeth. After removal of the coronary pulp, a material must be chosen for insertion in the pulp region and this is a relevant factor that influences successful treatment. [1][2][3] This material should be biocompatible, bactericidal, harmless to tissue, and promote healing of the pulp tissue. 4,5 Furthermore, conservative treatment of the dental pulp has the advantage of preserving the vital function of the pulp, including the defensive mechanism and proprioceptive function of the teeth. 2 Some materials have already been used in pulpotomy, such as Formocresol (FC), 3 Calcium Hydroxide (CH), 4 Ferric Sulfate (FS), 3 Mineral Trioxide Aggregate (MTA), [6][7][8] and Biodentine (Biodentine; Septodont Inc., Saint-Maur-des-Fosses, France). [6][7] MTA is considered the gold standard for pulpotomies and other clinical applications for its physical-chemical and biological properties. 1,2,[5][6][7][8][9] MTA is a biocompatible material, that presents hydrophilic properties enabling its use even in the presence of moisture. 7   Preparing of the extract The preparing of material extracts was performed according to previous study and also according to ISO 10993-5:2009. 15,16 Disks containing these materials were prepared under aseptic conditions by using a sterile cylindrical polyethylene tube (diameter, 5 mm; height, 3 mm). Disks were kept in a 5% CO 2 incubator at 37º C for 6 h for final setting. 14,16 Then, disks with materials were removed from the mold and sterilized with ultraviolet light for 1 h. 14 Each disk was immersed into 1 mL DMEM with 10% FBS and incubated in a humidified atmosphere containing 5% CO 2 for 24 hours, according to ISO 10993-5:2009. 15 Then, disks were discarded, and the supernatants (eluate extract) were collected and filtered through a sterile 0.22-mm filter (Sigma-Aldrich, St Louis, MO), to remove any suspended particles from the materials in the extracts. 14,17 L929 fibroblasts were seeded into the 96-well plates (10 4 cells/ well) and incubated for 24 h in a humidified air atmosphere of 5% CO 2 at 37º C to enable cell attachment. The undiluted extract (1:1) 16 was used for 6, 24, and 48 h. The L929 fibroblasts cell cultured in medium without extract served as the control. The MTT (3-(4,5-dimethylthiazol-2-yl The rats were maintained in cages with three animals each, with free access to food and water, under 12 hour light/dark cycle and temperature of 22°C ± 2°C.
Polyethylene tubes were implanted according to previous studies. 5,14,16,17,[19][20][21] The animals received intravenous dypirone (0.03 mg per 100 g of body weight) during the first three days. Also, rats' feeding and drinking pattern and possible changes in behavioral profile were assessed.   Table 1). There was no significant difference for von Kossa and for birefringent structures to polarized light (Table 1)   Comparison between the different groups not even time of analysis

Inflammatory response
The average number of inflammatory cells in the tube was determined on days 7, 15, 30, 60, and 90 and data were compared at each timepoint (Table 1)

Immunohistochemistry
The immunoreactivity patterns for IL-1β and TNF-α are showed in Table 1 5,20,29,30 This study showed In another study, 21 BIO-C PULPO and white MTA-Ang induced a similar inflammatory response in all periods. However, in this other study, inflammation analysis was performed by scores, and paraffin was used as the method of including the specimens, which differs from our study, that used quantitative analysis of the inflammatory cells as well as using the resin as a material for inclusion. Resin (glycol methacrylate) embedding for the histological process presents some good aspects: preservation of cell morphology, enabling the definition of inflammatory process degree. 24 Probably, the difference in the evaluation of inflammatory process between these studies may be due to both method of evaluation and inclusion method. However, considering the difficulty to perform immunohistochemical in resin slices, paraffin was also used to get around this negative point. The BIO-C PULPO has components that promote adequate tissue response. The zirconium oxide was used as a radiopacifier in BIO-C PULPO once bismuth oxide has drawbacks related to the discoloration, which can be attributed to its interaction with the collagen present in tooth tissue. It has been established that zirconia oxide used as radiopacifier did not interact with collagen and may prevent tooth discoloration, which reinforce the hypothesis that bismuth oxide is associated with MTA darkening even in the white formulation. 13 Besides, zirconium oxide causes a lower inflammatory reaction in tissues compared to bismuth oxide. 36 Moreover, the manufacturer states that silicon dioxide was added to bring viscosity to the material, thus improving its insertion into the cavity. Furthermore, distilled water was replaced with a liquid containing distilled water, plasticizing material, calcium chloride, and methylparaben. The plasticizer provides higher plasticity, improving material handling and insertion into cavity. However, the manufacturer did not disclose the plasticizer composition. Calcium chloride was added to provide shorter setting time, 37 iron oxide was added to act as pigmentation agent, similar to that found in MTA 7 and methylparaben was inserted as a preservative. 38 Calcium fluoride was inserted in BIO-C PULPO. The endodontic materials that contain fluoride compounds have an improved penetrability into dentin and they also present increased mineralization. 39 Specifically, the addition of calcium fluoride improves the ability of forming apatite. 40 Once BIO-C PULPO is a bioceramic, J Appl Oral Sci. 2020;28:e20200033 11/13 it was expected that positive von Kossa and also birefringent structures in polarized light were similar or superior to the white MTA-Ang. It was confirmed by a larger area in the BIO-C PULPO group for von Kossa on day 90, which was significantly different compared to the white MTA-Ang, thus suggesting a longer time for calcium carbonate formation, possibly due to its formulation.
Calcium silicate together with calcium fluoride, calcium aluminate, and calcium hydroxide promote deposition of mineralized structures in BIO-C PULPO.
In calcium silicate-based material bioactivity occurs after hydration. 25,41 The contact of materials with tissue triggers the reaction of Ca 2+ ions with OH − ions derived from water, forming the calcium hydroxide, that creates a highly alkaline environment. 37 The calcium ions react with carbon dioxide present in the tissues, originating calcite crystals and a reduced inflammatory process. 25 It has been suggested that these crystals originated from the precipitation of calcium carbonate could stimulate the deposition of mineralized tissue detected by von Kossa and also birefringent structures in polarized light. 41 Also, these calcified structures in subcutaneous investigations are a sign of osteoinductivity of the tested material. 5,20,27,41 This type of study is the first stage of research on the use of new endodontic materials and, therefore, it should be interpreted with caution. However, its execution is greatly relevant since the use of cells and animals enable work in a highly controlled environment, focusing only on variables necessary for a preliminary understanding of the material response during future clinical use.
Conclusion BIO-C PULPO presents similar results to the control in the metabolic activity of cell and it induces tissue reaction similar to white MTA-Ang including stimulation of mineralized tissue formation. Therefore, BIO-C PULPO was considered a biocompatible material.

Conflict of Interest
The authors declare no conflict of interest.