Morphology Analysis of Human Fourth Molars Using Computed Microtomography – Pilot Study

Analyze the morphological characteristics of fourth molars from non-syndromic patients using computed microtomography (microCT). Five human fourth molars (case group) and other three third molars (control group) were obtain from Biobank of UFPR Dentistry School. The teeth were submitted to macroscopic and microCT analysis. Sample characteristics: 60% of the teeth from the lower arch, mainly from the right lower arch (40%). The results showed that eighty percent of the teeth were impacted and all of them presented normal morphology. The microCT revealed that one of the fourth molars had hypercementosis. Concerning the root canal anatomy, all of the fourth molars studied had just one principal root canal. Four teeth had one collateral canal that ended at the lateral face of the root. The volume average of root canal, dentine and enamel were respectively 9.27 mm, 122.64 mm and 74.71 mm. The averages between these variables in third molars were 46.98 mm, 473.25 mm and 198.34 mm, respectively. Proportionally, the volume of the fourth molars was five times less than the third molars, but the dentine and enamel volume were, respectively, 3.8 and 2.7 times less than third molars. The fourth molar teeth usually showed only one principal root canal and it can be associated with other alterations, such as hypercementosis. Although the fourth molars were smaller, the proportion of enamel volume is bigger than third molar in comparison.


Introduction
Forensic age and race estimation is an important element of anthropological research, as it produces one of the primary sources of data that researchers use to establish the identity of a person living or the identity of unknown bodily remains 1 . The presence and characteristics of a supernumerary tooth may be a useful tool in human identification 2 .
Once an ST is detected, its presence is documented in the dental records.
ST are a unique entity such that its position, in relation to surrounding structures, is unique to a particular individual and becomes an important identification point in forensic odontology as it is always documented and can be compared with previous radiographs. There are numerous case reports which state the importance of identification of an individual based on the dental records such as radiographs, case sheets, study models, and supernumerary teeth 3 .
The permanent dentition is composed of 32 teeth among maxilla and mandibula jaws. Occasionally some individuals may have teeth in excess number than usual and these teeth are called supernumerary, which is a dental anomaly known as hyperdontia 4,5 . According to Refoua et al 6 , it may result from horizontal proliferation or hyperactivity of the permanent or primary dental lamina. There are many theories about supernumerary teeth, but the etiology is still unknown.
Hyperdontia is more frequent at permanent dentition 5 and its incidence may vary between permanent and primary dentition in 0.1 a 2.1% e 0.3 a 0.8% respectively 7-10 .
The supernumerary teeth may have normal form (supplementary teeth) or abnormal form (rudimentary teeth) such as conical shape or may be a dental tissue mass 9. Studies have revealed that supernumerary teeth are more frequent on upper teeth and among male individuals [11][12][13][14][15][16] . Hyperdontia can be classified according to its location. Central incisors supernumerary teeth are called mesiodens; when they appear on molar region they are classified as paramolars; and those distal to the third molar as distomolars 8,17 . Supernumerary teeth can cause eruption disorders, dental crowding, tooth reabsorption and odontogenic cists and tumors 18 .
Hyperdontia diagnosis is generally based on x-ray exams by counting the number of teeth. Panoramic x-ray is the ideal technique to find fourth molars since they are often impacted due to lack of space [19][20][21] .  MicroCT images are useful tools in assessment of age of an individual 21 and a non-destructive analytic procedure that is able to characterize samples by determining their morphology and analyzing their structural properties 22 .
The objective of this pilot study was to evaluate the morphological characteristics of human fourth molars through computer microtomography (microCT).

Materials and methods
This study was submitted and accepted by the Ethical Committee of the Federal University of Paraná (UFPR) through the approval number 1.618.336 (Attachment1).
The sample was composed of 10 human teeth divided into two groups: 5 fourth molars (case group) and 5 third molars (control group). Third molars were chosen as control group due to their similar anatomy. As inclusion criteria, preserved human teeth diagnosed as third and fourth molars were included in the study. As exclusion criteria teeth with any damage from surgical removal drill or enamel defects were excluded.
One fourth molar from three different individuals and two fourth molars from another individual were used. All participants agreed with the donation to the biobank and signed the legal terms.
All the sample teeth extractions were performed at the Surgical Clinic of UFPR Dentistry School. After the surgical procedure the specimen were immersed in a saline solution and then cleaned to be steam sterilized by autoclave. University's Biobank collected data as gender, age and systemic health from the patients' files.
The specimens were taken to Rocks and Minerals Laboratory (LAMIR) located on UFPR campus to be microCT analyzed. Initially the root volume was analyzed since its x-ray absorption rate is different from the rest of the tooth, followed by the root tissue analysis that wouldn't contain enamel, only dentin. When the coronal portion was examined, where enamel and dentin coexist, it was necessary to make a binarization to distinguish the two structures that have very different x-ray absorption rates. Finally, the enamel volume was isolated to allow the sum of the total volume of dentin.
The statistical treatment was composed by descriptive analysis under mean and standard deviation form. Table 1 presents the clinical characteristics of the teeth donators and shows a gender balance. The age average of the individuals who donated the fourth molars when they were removed was 21.6 years old. Although 5 fourth molars have been collected, two came from the same individual. The majority of fourth molars were from upper jaw (60%) and 40% from the right side of mandibula. Eighty percent were impacted and 100% had normal morphology. Following the microCT analysis it was observed that one of the fourth molars had hypercementosis (Figure 1). Regarding the root, canal system anatomy the microCT revealed that all fourth molars showed only one main canal and four of them had a collateral canal discharging into the lateral wall of the root (Figure 2). In concern to the third molars, one of the specimens had three root canals. Contrariwise, the other third molars had only two root canals and one of them showed a deformation at the apical portion and accessory canals.  Correspondingly, the volume of the fourth molars was 5 times smaller comparing to third molars. As for dentin and enamel volume, the values were 3.8 and 2.7 times smaller than the third molars' volume.  It can be identified that the third molars percentage of volume of the three different structures (root canal, dentin and enamel) was higher than what was found on the fourth molars. Taking the fourth molars into account, the percentage of enamel volume in relation to the total volume was higher than in the third molars.

Discussion
Age and race estimation are common tasks in Forensic Dentistry. Dental structures are the hardest and well protected structures in the body. These structures resist decomposition and high temperatures and are among the last ones to disintegrate after death. Dental features such as tooth morphology, number of teeth, variations in shape and size, restorations, pathologies, missing tooth, wear patterns, crowding of the teeth, color and position of the tooth, rotations and other peculiar dental anomalies give every individual a unique identity. In absence of ante-mortem dental records for comparison, the teeth can help in the determination of age, sex and race, which can give further clues regarding human identification 1,2,20 . The presence of a fourth molar can be a key factor for individual recognition and understanding its morphology may help to identify age and sex.
This study investigated the morphological characteristics of fourth molars trough microCT images. Although a small sample was analyzed, the pilot study results showed that the fourth molars may have further alterations such as hypercimentosis. Besides, the microCT revealed that in regard to the internal anatomy, the fourth molars showed only one main root canal but presented collateral canals.
Odontogenesis is the complex process of tooth development and it has been characterized with a series of reciprocal interactions between epithelial and mesenchymal cells leading to tissue differentiation [19][20][21][22] . Teeth are formed initially as appendices of epithelia and its morphogenesis is regulated by the integration of epithelial and subjacent neural crest from mesenchyme. Mammal's dentition consists on a group of very specialized teeth known as incisors, canines and molars, which come from different areas of the oral epithelia 19 .
Hyperdontia is the term used to describe extra or supernumerary teeth that exceed the 20 deciduous teeth or 32 permanent teeth 23 . Atavism, by definition, is the reappearance in an individual of characteristics of some remote ancestor 24 and it supports some theories for fourth molars development. Hyperdontia may occur due to a division of tooth germ and also as a consequence of genetic and circumstantial factors [25][26][27][28] . However, according to some studies 29 the final development post permanent dentition arises on account of late dental lamina conception, occurring after the permanent dentition is concluded.
In addition, the morphological analysis of the fourth molars the microCT also enables the evaluation of root canal, dentin and enamel. This technique was previously used by Núñez et al 30 to measure the volume and thickness of mineral density of rats' incisors enamel. Until the present day it couldn't be found similar studies to be compared to this one. Meanwhile, it was possible to calculate the percentage of volume occupancy of root canal, dentin and enamel and when its values were compared to the control group it was observed that third molars had a bigger value. Moreover, although one of the fourth molars had microdontia, the percentage of enamel in relation to the total was higher than the third molars.
The prevalence of supernumerary deciduous and permanent teeth ranges between 0.1 a 0.8% [8][9][10][11] . In this study, however, after six months of sample searching, it was possible to gather only 5 teeth with this anomaly. Although some studies 10,11,[13][14][15] suggest that supernumerary teeth are more common in male upper jaw there was no difference among male and female gender in this experiment. Also, in this sample the fourth molar were more frequent in the lower jaw (60%), in contrast to the literature.
Despite the limitations of the present study, it raises contributions in relation to humans fourth molars morphology and it is going be followed up by a second part study where enamel crystallinity will be analyzed using different techniques such as x-ray diffraction an infrared light absorption.

Conclusions
Understanding supernumerary teeth morphology provides useful epidemiological and forensics information. Based on this pilot study discoveries it can be concluded that forth molars typically present one main root canal and that they may be associated to other alterations like hypercementosis. Moreover, it can be verified that the fourth molar has a proportionally bigger enamel volume when compared to third molars.