Evaluation of dentinogenesis inducer biomaterials: an in vivo study

Abstract When exposure of the pulp to external environment occurs, reparative dentinogenesis can be induced by direct pulp capping to maintain pulp tissue vitality and function. These clinical situations require the use of materials that induce dentin repair and, subsequently, formation of a mineralized tissue. Objective: This work aims to assess the effect of tricalcium silicate cements and mineral trioxide aggregate cements, including repairing dentin formation and inflammatory reactions over time after pulp exposure in Wistar rats. Methodology: These two biomaterials were compared with positive control groups (open cavity with pulp tissue exposure) and negative control groups (no intervention). The evaluations were performed in three stages; three, seven and twenty-one days, and consisted of an imaging (nuclear medicine) and histological evaluation (H&E staining, immunohistochemistry and Alizarin Red S). Results: The therapeutic effect of these biomaterials was confirmed. Nuclear medicine evaluation demonstrated that the uptake of 99mTc-Hydroxymethylene diphosphonate (HMDP) showed no significant differences between the different experimental groups and the control, revealing the non-occurrence of differences in the phosphocalcium metabolism. The histological study demonstrated that in mineral trioxide aggregate therapies, the presence of moderate inflammatory infiltration was found after three days, decreasing during follow-ups. The formation of mineralized tissue was only verified at 21 days of follow-up. The tricalcium silicate therapies demonstrated the presence of a slight inflammatory infiltration on the third day, increasing throughout the follow-up. The formation of mineralized tissue was observed in the seventh follow-up day, increasing over time. Conclusions: The mineral trioxide aggregate (WhiteProRoot®MTA) and tricalcium silicate (Biodentine™) present slight and reversible inflammatory signs in the pulp tissue, with the formation of mineralized tissue. However, the exacerbated induction of mineralized tissue formation with the tricalcium silicate biomaterial may lead to the formation of pulp calcifications


Introduction
Some clinical situations such as deep cavities, severe crown trauma, and iatrogenic situations may lead to pulp tissue exposure of the external oral environment.
Dental pulp has a natural potential for tissue repair, which leads to the formation of reparative dentin. It has been well documented that dental pulp can form a hard tissue barrier (dentin bridge) after directly capping the pulp or pulpotomy. During reparative dentinogenesis, the original odontoblasts at the site of exposure are destroyed and replaced by newly differentiated odontoblast-like cells. This process involves progenitor cells migration to the lesion site and subsequent proliferation and differentiation of these cells into odontoblasts. Thus, when the dental pulp tissue is exposed to the external environment, reparative dentinogenesis can be induced by pulp capping, to maintain pulp tissue vitality and function. [1][2][3][4] Direct pulp capping consists of biocompatible materials and bio-conductors application in the exposure zone of the tissue in order to seal the communication, acting as a barrier, and at the same time protecting the pulp complex and consequently preserving its vitality. The fundamental characteristics of these materials are their biocompatibility, which includes antibacterial capacity and properties that induce tissue healing; cytocompatibility, and ability to seal the lesion. 5 Several authors report that mineralized tissue induction formation by pulp cells is the main function of the biomaterial used for this type of therapy. 5,6 The direct pulp capping regenerative treatment objective is the induction of odontoblast-like cells differentiation and consequently the formation of tertiary dentin in the exposure area with tissue structure reorganization. Some characteristics are common to most biomaterials indicated for therapeutics, protecting the pulp tissue vitality and which induce reparative dentin. These characteristics include high pH, antimicrobial activity and release of calcium ions. 4,[7][8][9][10] Calcium hydroxide is the most popular agent for direct and indirect pulpal capping and for maintaining pulp vitality due to its ability to release hydroxyl and calcium ions after dissolution. This biomaterial is the most studied and documented in several cellular, animal, and clinical studies and presents satisfactory results with success rates up to 80%, which is considered gold standard. 6,11,12 However, there are some disadvantages such as poor adhesion to dentin, high solubility, and mechanical instability and consequent dissolution of the dentin bridged material with multiple tunnel defects. 11, 13,14 Developed in the 1990s, mineral trioxide-based cements received great attention, initially as a retrograde filling material, and its indications were expanded as a material for direct pulp capping.
Although there are clinical evidence of the calcium hydroxide-based cements and mineral trioxide aggregates use in direct pulp capping therapies with satisfactory results, the best clinical performance in relation to each other is inconclusive. 6 During the experiment all animals were submitted to normal maintenance and nutrition, with an ambient temperature of 22°C and 12-h light-dark cycle.
The 45 Wistar Han rats used were randomly divided into four groups in a split-mouth study design: two control groups, and two test groups as shown in Figure 1. All animals handled were weighed and anesthetized with 77% ketamine (25 mg/kg) and 33% chlorpromazine (25 to 40 mg/kg) intraperitoneally.
In the right first mandibular molars no intervention was performed in the negative control (Group 1). After disinfection of the teeth dental surface with 0.12% chlorhexidine, the pulp exposures were performed in the mandibular left first molars with a spherical diamond drill bit 008 in multiplier contra-angle and finished with the aid of a K 10 file. The cavity was irrigated with 2% sodium hypochlorite (DentaFlux; Paul, Minnesota, USA) glass ionomer cement. In both test groups, the WhiteProRoot ® MTA (Group 3) and Biodentine ™ (Group 4) were used to cap the exposed In total 15 animals, corresponding to five animals from each intervention group, were killed at three, seven and twenty-one days after the surgical procedure, by anesthetic overdose.

Acquisition of molecular imaging by Nuclear Medicine
In this study, the animals were anesthetized at three, seven and twenty-one days after treatment with the biomaterials with ketamine 50 mg/ml  The objective of H&E staining was to assess

Grade 0
No inflammatory signs and no presence or with the appearance of a few inflammatory cells in the pulp area corresponding to the exposure zone.

Grade 1
Light inflammatory infiltrate with the presence of cells, such as polymorphonuclear leukocytes (PMNLs) and mononuclear leukocytes (MNLs).

Grade 2
Moderate cellular inflammatory infiltrate involving the coronary pulpal tissue.

Grade 3
Severe cellular inflammatory infiltrate involving the coronary pulp tissue or with abscess characteristics.

Pulp tissue disorganization Characterization
Grade 0 Normal tissue.

Grade 1
Disorganization of the odontoblast layer, but normal coronary pulp tissue.

Grade 2
Total disorganization of pulp tissue morphology.

Results
During the research period, the animals showed a healthy appearance with motor activity and normal breathing.
Nuclear medicine assessment using 99m Tc-HMDP All samples collected from the studied animals were included in the functional study. The results obtained after 99m Tc-HMDP administration revealed no statistically significant differences (p>0.05), when comparing each group in the periods of three, seven and twenty-one days, as can be seen in Figure 5. When the differences between those groups with different materials were assessed at each stage, it was found that there are no statistically significant differences (p>0.05).

Biomaterials therapy analysis
All samples collected from the studied animals were included in the histological analysis. The choice of images was random.
Normal pulp morphology as the baseline can be observed in Group 1 within all stages and all stain techniques. In Figures 6 and 11, the histological results can be observed after biomaterials therapies at three, seven and twenty-one days of follow-up.
On day three (Figures 6 and 7), a substantial amount of inflammatory cell infiltration could be

Grade 1
Slight deposition of hard tissue immediately below the exposure zone.

Grade 2
Moderate hard tissue deposition immediately below exposure zone.

Grade 3
Intense deposition of hard tissue immediately below the exposure zone, characteristic of a complete dentin bridge.  In Group 2, the pulp polyp is visible with a disorganization of the tissue inside the pulp chamber (red asterisk). In Group 3, shows a decrease in inflammatory infiltrate without mineralized tissue formation (yellow arrows). Group 4 shows an intense inflammatory infiltrate especially in the area near the exposure (black arrows) and the formation of small focuses of mineralized tissue in the exposure location (blue arrows) On days three and seven, the inflammatory reaction decreased gradually in Group 3, but increased in Group 4.
On day seven (Figures 8 and 9), the matrix calcification could be observed, and it was more visible in the Biodentine ™ group (Figure 8). In the treatment with WhiteProRoot ® MTA ( Figure 8A), the presence of mild inflammatory infiltrate with neutrophil polymorphonuclear cells, tissue morphology maintenance, and presence of mineralized deposits near the exposure zone were observed, without being characteristic of a complete dentin bridge. Figure   8B shows an increase in calcium deposition in areas already identified by H&E staining. In treatment with Biodentine™ with H&E staining (Figure 8C), intense inflammatory infiltrate could be observed in the exposure zone with mineralized tissue presence. Figure   8D shows the moderate deposition of hard tissue immediately below the exposure zone and a slight deposition in other areas of the pulp tissue.

T h e s i t e s w h e r e W h i t e P r o Ro o t ® M TA wa s administered (Group 3), intense inflammatory
infiltration was observed after three days of follow-up ( Figures 6, 7A and 7B), with pulp tissue morphology disorganization. However, these inflammatory characteristics were present only at an early stage, which a gradual decrease in its intensity were observed as well as pulp tissue reorganization after seven days of follow-up (Figures 8, 9A and 9B). In addition, we also  (Figures 6, 7C and 7D). These inflammatory signs increased in intensity over follow-up stages, accompanied by increased disorganization of cell morphology, which was observed after seven and twenty-one days (Figures 8, 9C, 9D and 10). The formation of mineralized tissue was observed after seven days of follow-up ( Figure 9C and 9D), with the appearance of some mineralized zones near the exposure zone. At twenty-one days, several mineralized zones were observed near the exposure area and throughout the pulp tissue, called generalized pulp calcifications (Figure 10).

The evolution of the inflammatory response and
mineralized tissue formation throughout the study stages corroborate the histological findings ( Figure   12). Figure 11-Images of histological sections of groups 2, 3 and 4 at 21 days of follow -up stained with H&E and by immunohistochemistry for DSP. The A image corresponding to group 2 (positive control) was stained with H&E and obtained with 100x magnification. Red arrow -intense inflammatory infiltrate with total disorganization of the pulp tissue. Image B corresponding to Group 3 (WhiteProRoot ® MTA therapy) was stained with H&E and obtained with 100x magnification. Black arrows -deposition of mineralized tissue near the exposure zone, immediately adjacent to the dentin tissue, revealing an incomplete dentin bridge. The image C corresponding to Group 4 (Biodentine™ therapy) was stained by immunohistochemistry for the DSP and obtained with 100x magnification. Yellow arrows -intense DSP marking next to the pulp exposure zone As previously mentioned, the anatomical and histological similarity of rat mandibular molars to human molars was also observed in this study. 43,44 The dentin structure that surrounds all the pulp tissue was observed, with the visualization of the dentinal tubules such as humans' one. Several cell populations can be observed in the pulp tissue, namely fibroblasts, blood cells, nerve cells, undifferentiated cells, and odontoblasts, arranged at the periphery of the entire pulp tissue. In anatomical terms, rat's first molar is similar to human's lower molars, both in regard to the coronary structure, with three cusps, and in the root structure, with two mesial and distal roots. Considering the results obtained were observed an increase in calcification. Calcium accumulation will not represent an increase in the radiopharmaceutical uptake between the groups, since its retention into the cell, require the presence of phosphate.
Some authors report that MTA-based cement induces pulp tissue recovery as well as reparative dentin formation superior to calcium hydroxidebased cement in the dog, monkey, and rat models. 45 In another study, carried out with miniature pigs, the mineralized tissue formation in a dentin bridge shape was observed similarly with WhiteProRoot ® MTA and Biodentine™. 46 Other authors conclude that mineralized tissue formation with Biodentine™ therapy translates into thicker and more morphologically organized dentin bridges than those using MTA-based cements. 47,48 In other studies, reparative dentin formation has demonstrated in wells restored with Biodentine™ in miniature pigs, with a significant deposition increase when compared with calcium hydroxide-based cement. 49 In this study, the presence of dentin sialoprotein was assessed by immunohistochemistry, showing an increase in its expression after twenty-one days of follow-up with both biomaterials. These histological findings corroborate with data obtained in the Alizarin Red S staining, proving its direct relation to mineralized tissue formation. Although the intensity of DSP marking did not reveal differences between the studied biomaterials, the observation of mineralized tissue formation showed significant differences, with a higher Other materials, such as the demineralized bone matrix, have also been tested in direct pulp capping therapies in the rat model, with promising results and superior to calcium hydroxide-based cement, showing their ability to promote repair through dentin bridges. 20 The biocompatibility of dental materials is essential to prevent inflammatory reactions appearance as well as enable tissue regeneration. The subcutaneous implantation method is a valid methodology to determine the biocompatibility of the materials. Some authors have adopted this methodology, showing that Biodentine™ exhibits an initial inflammatory response, but this response is rapidly followed by connective tissue formation, indicating the absence of tissue irritation. 51 In other subcutaneous compatibility studies in rats, the authors demonstrated that the number of inflammatory cells and IL-6 was significantly higher with Biodentine™ when compared to MTA after seven and fifteen days of follow-up. However, after 60 days, a significant regression of the inflammatory reaction was observed, and both materials showed capsules with numerous fibroblasts and collagen fibers. 52 Although in methodological terms there is a great disparity between the studies as already mentioned, there is generally a consensus regarding the induction of mineralized tissue formation with both biomaterials, ProRoot ® MTA and Biodentine ™ . 53-55 The superior performance of calcium hydroxide-based cements is also consensual. Concerning the presence of an intense inflammatory reaction throughout the followup stages of the studies, some controversy persists.
The inflammatory reaction of the pulp tissue after treatments with ProRoot ® MTA is mild to moderate, as previously discussed in most studies. In treatments with Biodentine™, some authors report a more intense initial inflammatory reaction with a decrease over time while others report the opposite, as found in our study.