Topography of Primary Molar Pulp Chamber Floor: A Scanning Electron Microscopy and Micro-Computed Tomography Analysis

Topography of primary molar


Introduction
The pulp chamber floor of deciduous molars presents peculiarities such as reduced dentin thickness, resorption areas, and the presence of accessory canals [1].These conditions favor communications of the coronal pulp region with the periodontal region, which is where the successor's tooth is under development [2,3].
Pulpo-periodontal communications enable diffusion of bacterial metabolism products [4] and substances used in pulp therapies to the periodontal region [5].In addition, the communication routes can explain the presence of radiolucent areas in the root regions of deciduous molars with infected pulp [6].
Accessory canals are the result of failures in the formation of the Hertwig epithelial sheath during the root formation [7].These failures are caused by modifications in the odontoblast differentiation phase associated with discontinuation of dentin formation, giving rise to small spaces [8].In vital teeth, the spaces are filled by connective tissue and blood vessels and allow physiological pulpoperiodontal communications [9,10].In teeth with pulp necrosis, these tissues are replaced by the bacteria and endotoxins of the root canal system microbiome, and these are virulence factors involved in inflammatory reactions in the periapical and periradicular tissues [11][12][13].
An associated clinical complication of pulpal infection in the floor region of the pulp of deciduous molars is damage to the ameloblasts of premolars in development, increasing the chances of developing defects in the tooth enamel [5,11,14,15].Such defects are frequent causes of aesthetic complaints, dentin sensitivity and retention factors of dental biofilm [16].
There is a gap in the literature that addresses the topography of the pulp chamber floor of primary molars, and it is believed that in the region, there is permeability related to several factors, among them the presence of accessory channels [17,18].From this perspective, the aim of this in vitro study was to determine the frequency, shapes, diameters and communication with the periodontal tissues of accessory canals in the pulp chamber floor of deciduous molars.

Sample Selection and Preparation
The study was approved by the Research Ethics Committee (Opinion number 2,100,116) and followed the ethical principles of the Declaration of Helsinki.
Primary molars used in the study were extracted when the permanent successor had a root formation of 2/3 or more and the children were aged nine to 11 years.The sample was consecutive and consisted of healthy human primary molars from children of both sexes who were patients of the children's dental clinic.
The samples were taken between August 2017 and November 2017, regardless to ethnicity or socioeconomic status.Molars, which presented integrity in furcation region, were included in the study.Sixteen primary molars were used in this study: four upper first molars, four lower first molars, four upper second molars and four lower second molars.After extraction, the teeth were kept in saline solution (NaCl 0.9%) until analysis.
The characterization of the accessory canals in the molar pulp chamber floor was determined according to the classification of Paras et al. [19] and Sharma et al. [20].The evaluation of the frequency, shape (round, oval, or irregular) and diameter of the canals was performed by scanning electron microscopy (SEM).To determine the type (blind, true, hidden) and communication with the periodontal tissues of the accessory canals, micro-computed tomography (micro-CT) was used.Both evaluations were carried out for each tooth.

Analysis by Scanning Electron Microscopy (SEM)
After the analysis using micro-CT, the molars were sectioned at a thickness of two mm above the amelocemental junction and two mm apical to the floor of the pulp chamber.20 The cuts were made with a high-concentration diamond wafering blade (4" x 0.12" x 0.5" Lapmaster) coupled to a precision cutter Isomet 1000 (Isomet, Buehler Ltd., Lake Bluff, IL, USA).
After sectioning, the specimens were immersed in a 3% sodium hypochlorite solution for 10 minutes to remove organic debris [20].They were then put into individual pots with distilled water in ultrasonic tanks for five minutes to complement the removal of pulp remnants [1,2].Next, they were dehydrated by increasing 70%, 95%, and 100% ethyl alcohol concentrations, with four exchanges every 15 minutes.The specimens were then arranged in a desiccator (Marconi MA 410/CF), at room temperature, for 24 h [20].
The specimens were mounted on stubs using double-sided carbon tape and placed in the vacuum chamber of a gold coating unit (Bal-tec SCD 050 Sputter Coater).After, they were submitted to SEM analysis (JEOL -JSM 5600 PV scanning microscope, Tokyo, Japan), with magnifications from 18X to 400X.The frequency and diameter of the accessory canals were recorded.

Analysis by Micro-CT
The teeth were dried with gauze compresses and scanned in the Skyscan 1174 apparatus (Bruker, Kontich, Belgium) using 50 kV, 800 mA, 0.8 step rotation, 360° rotation around the vertical axis, voxel size of 11.9 µm and matrix of 1304 x 1024 pixels.The image of each specimen was reconstructed, containing the area of the furcation region pulp chamber floor of molars, using the software NRecon (Bruker, Kontich, Belgium).
For viewing of the data on coronal, transaxial and sagittal sections of the region of furcation, the software DataViewer (Bruker, Kontich, Belgium) was used.
To determine the type and communication with the periodontal tissues of the accessory canals at the floor of the pulp, designs in three dimensions (3D) were constructed by binarization of the dentin and root canal system, using the software CTAn and CTVol (Bruker, Kontich, Belgium).The images were evaluated by two experienced and calibrated operators with experience in the analysis of the root and root canal morphologies obtained using micro-CT technology.A double-blind evaluation was performed and a third evaluator in cases of disagreement.

Data Analysis
The data were recorded in an elaborate form then tabulated and analyzed using the software Statistical Package for the Social Sciences (SPSS ® Statistics for Windows, version 22.0, IBM Corp., Armonk, NY, USA).Descriptive analyses of the quantity, shape and diameter of the accessory canals were performed.
To analyze the diameter difference between the upper and lower molar canals, the Mann-Whitney test was used since the data did not have a normal distribution according to the Shapiro-Wilk test.A p-value <0.05 was considered statistically significant.

Results
The SEM micrographs of the deciduous molars revealed accessory canals on half of selected teeth.
Most of these were located in the upper molars (Table 1).The shape of the founded accessory canals is presented in Figure 1.The irregular form was the most frequent accessory canal shape, with 63.6% in the upper molars and 50% in the lower molars (Table 2).There was no difference in the means of accessory canal diameters between the upper and lower molars (p=0.083)(Table 3).
The micro-CT analysis revealed permeability in the furcation region without the involvement of the pulp chamber floor in two specimens corresponding to lower first and second molars.Communication with the periodontal tissues was observed in three specimens.A first molar had an accessory canal connecting the floor of the pulp chamber to the palatine canal and accessory canals true type, connecting the floor of the pulp with the furcation region on two maxillary molars.Blind accessory canals were detected on the floor of the pulp chamber of two upper molars.In the four upper second molars, intercanal communications were found (Figure 2).
In the present study, half of the molars had accessory channels.However, only 18.7% had pulpoperiodontal communications; that is, they had patency.Through the three-dimensional analysis, it was also possible to observe blind-type canals.Blind-type canals are those that begin and end within the dentin [19,26].These canals may acquire communication with the periodontal tissues when resorption progression occurs in the external furcation regions of the deciduous primary molars with necrotic pulps [11].
Concerning the shape of the accessory canals, irregular canal shape was the most frequent, and this result was discordant with the results of Sharma et al. [20], in which an oval shape was predominant.Further studies should be performed to assess whether there is a relationship between accessory canals and communication with the periodontal tissues of the pulp floor of deciduous molars.
In canals with large diameter, there may be an increased risk of dentin permeability with consequent diffusion of chemicals, biologicals, endotoxins or substances used in endodontic therapies for the periodontal spaces [5,12,22,27].The clinical importance of this finding may be related to the presence of radiolucent images in the interradicular regions of the deciduous molars with pulp infections [6].
The presence of accessory canals was more frequent in maxillary molars; however, there was no statistically significant difference but suggesting that these teeth have a higher communication with the periodontal tissues in the furcation region when compared to the lower molars [2].However, the presence of accessory canals is not the only factor influencing the communication with the periodontal tissues of the molar furcation region; dentin thickness and root resorption areas should also be considered [1].Therefore, further studies should be performed to evaluate the thickness of dentin and the different degrees of resorption in the furcation region of molars with necrotic pulp.
Failures in endodontic treatments are related to the presence of residual microorganisms in the root canal system in the furcation and periapical regions [27,28].Accessory canals of molars with pulp necrosis become a favorable environment for bacterial housing and proliferation [29], which may spread to the periodontal region, where the premolars are in development [5,7].
The study has a sample size limitation that can be justified because it is a consecutive sample that should meet the eligibility criteria; however, other studies should be done using a more significant sample size and that can be inferred for the population.Thus, the degree of applicability or generalization of the results needs to be analyzed with caution.In addition, it has also been pointed out that age, sex and ethnicity influence tooth morphology [18].Another limitation is related to the MicroCT method that some anatomical features should not be visible because they are smaller than the pixel size in micrometers.

Figure 2 .
Figure 2. Axial micro-CT sections and 3D reconstructions of deciduous molars: (a) first molar with types blind and true accessory canals; (b) lower first molar without the presence of an accessory canal; (c) upper second molar with blind-type accessory canal and chamber canal.

Table 1 . Descriptive analysis of deciduous molar accessory canals. Accessory Canals Frequency of Accessory Canals Primary Molar Number of Teeth
SD: Standard-Deviation; N: Number Of Accessory Canals; Min: Minimum; Max: Maximum.

Table 3 . Mean and standard deviation (SD) of diameter (µm) of accessory canals and association between arches.
SD: Standard Deviation; * Mann-Whitney test.