Advanced Dental Applications for Scanning and 3D Printing TechnologyView this Special Issue
Dental Radiographic/Digital Radiography Technology along with Biological Agents in Human Identification
The heavy casualties associated with mass disasters necessitate substantial resources to be managed. The unexpectedly violent nature of such occurrences usually remains a problematic amount of victims that urgently require to be identified by a reliable and economical method. Conventional identification methods are inefficient in many cases such as plane crashes and fire accidents that have damaged the macrobiometric features such as fingerprints or faces. An appropriate recognition method for such cases should use features more resistant to destruction. Forensic dentistry provides the most appropriate available method for the successful identification of victims using careful techniques and precise data interpretation. Since bones and teeth are the most persistent parts of the demolished bodies in sudden mass disasters, scanning and radiographs are unrepeatable parts of forensic dentistry. Forensic dentistry as a scientific method of human remain identification has been considerably referred to be efficient in disasters. Forensic dentistry can be used for either “sex and age estimation,” “Medical biotechnology techniques,” or “identification with dental records,” etc. The present review is aimed at discussing the development and implementation of forensic dentistry methods for human identification. For this object, the literature from the last decade has been searched for the innovations in forensic dentistry for human identification based on the PubMed database.
In the past decade, an alarming rise in criminal and casualty incidences has been observed . The contemporary considerable prevalence of violent and criminal actions has necessitated applying modern methods for criminal investigations . Additionally, the present incidence of casualties associated with mass disasters (MD), such as travel and transport accidents, terrorism, and unusual climatic conditions, needs novel efficient methods for MD victim identification . The legal discipline of using medical facts for victim identifications is called “forensic medicine” . Forensic has a Latin root in the term of forense or forensis which means a forum, public, or marketplace where the legal issues are discussed . Forensic dentistry or forensic odontology is defined as the knowledge of dentistry as related to the law and is one of three primary identifiers recognized by Interpol for victim identification in multicasualty incidents or MDs [2, 5]. The scanning techniques including different types of digital radiographs and photographs have an approval role in forensic dentistry . The diversity of dental patterns among human individuals has facilitated the accurate identification of them . It has been said that one of the most common AM evidences applied for human identification is their dental radiographs which have a crucial role in forensic sciences . Moreover, some macrobiometric features are recognizable when the entire skeleton is available which helps for example identify the victim gender with 100% accuracy. However, in most MD cases, the victim bodies are more damaged than be visually identifiable. The modern identification processes are very helpful in such cases. These methods use microscopy and molecular examinations of the remained sources which often include the skull and teeth . Using forensic dentistry as a proven reliable scientific method in legal identification cases dates back to the 1950s and 1960s in the developed countries of Europe and the United States . Since then, using forensic dentistry has been extensively helpful for human identification especially in MDs, and progressing the new methods has made its application increasingly more applicable . Not only do dental identification mainly benefits from detection of damages inflicted to the jaws, oral tissues, and teeth, it can help to suspect elimination or potential identity . Forensic dentists are required to process, review, and evaluate the collected evidence from dental remains in the form of scientific and objective data and present them to legal authorities . Forensic dentistry has significantly changed in the past decades from being occasionally used to playing a key role in identification procedures . Since it is simple, user-friendly, and not expensive in comparison with other methods, forensic odontological comparison is considered one of the three principal identifiers designated by INTERPOL for use in identifying the victims of a multicasualty incident. Its positive outcome is considered sufficient to permit personal identification without further support from other methods . The present review study is aimed at having a systematic survey on the role of forensic scanning technologies in the identification of individuals contributed to a crime or disaster. Recent human identification researches in the field of dental records are shown in Table 1.
2. Dental Parameters Used in Sex and Age Estimation
Age, sex, and race are the very basic and fundamental characteristics generally used for defining and identifying every human individual . The age of children up to puberty can be estimated from the developmental stages of their teeth in dental radiographs and scanning since they are minimally influenced by malnutrition and hormonal and pathological disorders [17, 18]. Among different techniques used in forensic science, scanning ways are the hallmark . Parameters that show “sexual dimorphism” are very helpful for determining the sexuality of the victims. Sexual dimorphism is the considerable differences that a parameter shows in size, stature, and/or appearance depending on sexuality. The second important variable required to be determined within the biological profile of a victim is their age at the time of being missed or dead. The recovered skeletal remains represent classic features applied for this purpose . Except for bone morphology, age can be estimated in a range of decades using dental findings too . In the following, dental parameters that help to identify the sex, age, and race of an unknown person are listed.
2.1. Radiographic Estimation
For this aim, three methods are used. First is the age determination using a developmental process to wit clinically visualization of formation, eruption, and calcification of teeth [18, 22]. The second method is radiographically using intraoral periapical radiographs, bitewing radiographs, and OPG. The radiographic images must include developing teeth of interest to be evaluated based on the selected development standards . The third and last method is the radiographic staging technique of the mandibular third molar tooth development using the DI combined with the skeletal maturity using radiographs from hand wrist and CVMI [22, 24]. Since accurate age determination becomes increasingly difficult after developing the third molar, the aging procedure and regressive conversions of teeth are the only helpful assessing methods at adult age . A variety of structures can help to reach radiographic estimation. From different types of techniques for achieving this goal, the evaluation of third molar, maxillary and frontal sinus and cervical vertebral maturation are the most practical one which can raise the accuracy of estimation.
2.1.1. Development of the Third Molar
Third molars and their radiographic images are used extensively for age assessment in the range of 9-23 years because of their long periods of the crown and root developments . Third molar mineralization is frequently completed before 21; however, it has been observed to be completed before 18 in some populations . Assessing the maturity of the third molar is a noninvasive method recognized as the best age estimation way because its radiographic images can be easily obtained. This is while no radiographic images from many other skeletal aging indicators are available . Third molar development can be also explored by CT scans and X-ray free imaging MRI methods. These methods are noninvasive and practical and present 3D images of the third molar. Also, geometric distortion does not make any magnification errors in them. MRI is mostly used in countries where radiation is not recommended or allowed .
2.1.2. Maxillary Sinus
MS is the first among the paranasal sinuses (frontal, ethmoid, maxillary, and sphenoid sinuses) that develop (at 10 weeks of intrauterine life) . The dimensions of the maxillary sinuses are considered reliable indicators for age determination since they are interestingly shown to remain intact in accidents and casualties such as explosions, warfare, and aircraft crashes, where the skull and other bones are mostly disfigured terribly . MS volume can be under the influence of several environmental and physiological factors such as breathing patterns, dental problems, the anatomy of the body, gender, age, ethnicity, and climate . In cases of incomplete skeletons, the ratio of maxillary sinus width and height to other bones can be used for sex identification. Figure 1(a) shows the method of measuring the width and height of the maxillary sinuses . Traditional X-ray technique has been mostly substituted by CT and MRI for imaging the paranasal sinuses . CT is a popular method for MS imaging  and brings out a 3D shaded image of the MS surface (Figure 1(b)) that illustrates MS involved inside the reconstructed head (Figure 1(b)) . For viewing the MS, radiographs are mostly taken from the occipitomental view (Waters’ projection) of the skull .
2.1.3. Frontal Sinus
The frontal sinus  is a highly unique tissue that rarely changes throughout life that facilitates identity assessment using linear and area measurements in all genders . The frontal sinus also barely endures aplasia providing a reliable assessment through comparing antemortem and postmortem radiographs of FS . Different FS measurements are shown in the Caldwell view (Figure 1(c)) . Posteroanterior (PA) radiograph provides the best view of the frontal sinus by the Caldwell technique. Lateral cephalograms, CT, and CBCT are other techniques occasionally used for studying the frontal sinus .
2.1.4. Cervical Vertebrae
Another indicator for assessing the bone development extent is the maturation degree of CVM . The modification in the size and shape of cervical vertebrae (mostly second, third, and fourth vertebrae of the neck) in both genders ideally represents the pubertal growth period of craniofacial bones during adolescence and young adulthood . The morphological analysis of these vertebrae (C2, C3, and C4) is used for assessing for example the lower border concavity, and the CV body shape changes during skeletal maturation . Cephalometar HF V1 is another reliable method for assessing the CVM stages. The GUI of the application is shown in Figure 1(d) .
The mandible or lower jaw is the largest and toughest bone in the face that forms the lower part of the skull. The mandible is a horizontally curved body having two rami and a convex ascending from the posterior to the anterior of the face . Mandibular morphological parameters such as the gonial and antegonial angles of mandible, mental and mandibular foramen, as well as the mandibular canal, change during life and between genders . According to Albalawi et al., the lines from the left and right gonion to menton (Gn-M0) form an angle that provides helpful anthropological data for dental and medicolegal practices . Gn-M0 angle and minimum ramus breadth and height show statistically significant dimorphism between the two genders making them reliable indicators for sex demarcation .
2.2.1. Mental Foramen
MF appears in different shapes (round, oblong, slit-like, or irregular) in radiographs. It is a partially or completely corticated radiolucent area  that shows significant changes in height according to age . OPGs facilitate surveying the complete mandible leading in a more accurately determining the vertical and horizontal measurements of the mental foramen . The CBCT technology is increasingly used for the 3D locating of MF because of its magnification-free high-resolution imaging potential and precision .
The calcified tissue around the teeth which contains the periodontal ligament fibers is an acellular part of the dentine called cementum. Cementum forms the attachment site of teeth to the part of the jaw that holds them which is known as the alveolar bone . The continual construction process of cementum and its conserved model during life has made cement-chronology possible. Cementochronology is a potent method for directly evaluating the chronological age and determining the season at death . The apical areas of cementum are thicker than parts at the CEJ, and its shape and texture are stable within an individual’s life . The underlying mechanism is the racemization reaction of aspartic acid in a constant manner  that produces a connective tissue in the form of incremental layers surrounding tooth roots and creates an appearance of concentric lines . These circular lines are called salter lines, and each pair of them represents one year of life (Figure 2(a)). The number of salter lines provides a biological record that represents the estimated age of the victim .
The secondary dentin starts forming after completing the tooth root and primary dentine. Since secondary dentin formation is a continuous process throughout life, its amount is applicable for age estimation. Some physical or chemical insults or dental caries can affect the regularity of the secondary dentin (Figure 2(b)) . One of the applicable related biomarkers is the quantity of root dentine translucency that is determined using vernier calipers or digital aids . Environmental factors and pathological processes minimally affect this parameter, and it can be macroscopically examined in both thorough teeth and sectioned teeth. Root dentine translucency appears symmetrically distributed on both sides of the jaws [52, 53]. An example of a scanned image of a tooth section is shown in Figure 2(c) .
2.5. Dental Pulp Chamber
The innermost soft core of pulp is protected by cementum, dentin, and enamel which is the outermost covering of the tooth crown. These tissues have a hard structure that is resistant to decomposition .
2.5.1. Radiographic Aspect
A parameter in correlation with the chronological age of victims is the size of their pulp chamber that can be obtained from the radiographic examination of their teeth . Since the volume of the pulp chamber changes during life , the pulp chamber volume is measured using the pulp/tooth area ratio method in both panoramic and periapical radiographs. Also, the PV/TV ratio and its relation with age are now extensively applied in clinical dental practice using 3D images . According to Ravindra et al., the apical pulp area reduces as individuals get older in an increasing manner through age. The changes in the apical area are more obvious than in the middle area or the pulpal floor probably because of the age-related modifications of the cementum and dentine. In this regard, the related literature reports that despite the apical pulp area, the mean size of the middle and coronal pulp does not change .
2.5.2. Histologic Aspect
The sex of a subject is histologically determined through exploring the presence of a sex chromatin body (Barr body) consisting of a condensed, inactive X chromosome mainly in the somatic cell nucleus. The Barr body can be observed in various cells using most of the nuclear stains and is regarded as representative of genetic femaleness (Figure 2(d)). The commonly used staining methods of Barr body include H/E, thionine, Papanicolaou, Feulgen, cresyl violet, Giemsa, aceto-orcein, and under fluorescence such as AO. Barr body analysis for sex determination is considered the most reliable method when the tooth is the only evidence that remained at the crime scene [57–60].
2.5.3. Advanced Techniques
In addition to the above-mentioned examinations, the dental pulp tissue is also used for PCR analysis and AMEL identification. Both of these advanced techniques help determine the sex of victims. Dental pulp-containing fibroblasts are an excellent source of DNA . From the authors’ standpoints, as digital scans especially OPGs are more reachable and economical way than histologic analysis, they may be a more practical method in sex and age estimation. The above-mentioned parts illustrate the importance of scanning methods and even a single tooth in forensic science. Not only have the radiographs an important role in sex and age estimation, but also the presence of a single tooth can raise the accuracy of estimation dramatically.
3. Dental Records
The extreme hardness of teeth preserves them intact in a dead body for a long time . Then, the AM dental data and material such as dental records, X-rays, CT scans, dental model, and full-face photographs can be obtained from the missing person’s dental practitioner by the local police and get interpreted by a forensic odontologist . Therefore, every dentist is required to accurately record all dental data and maintain them for any probable legal circumstances . The unicity of dentition in every particular individual has made it a resistant analog to fingerprints  which is greatly useful in cases of highly demolished bodies for example by fire . The reason is the hard tissue of teeth and dental restorations that is extremely resistant to destruction even in cases of charred and decomposed bodies . Among the different techniques used in forensic dentistry, scanning ways are the most practical ones . To use dentition records for identification, an official office document referred to as the patient’s chart or dental profiles is developed that contains all patient dental treatments . In the cases of mass casualties, a forensic odontologist is generally responsible for comparing the antemortem (AM) and postmortem (PM) data in the patient’s chart and extracting the matches which support identification . AM data are usually obtained from the private clinics where the victim has records of dental treatments such as radiography, prescription, dental casts, and photographs while the PM data is obtained during cadaveric examinations . The most common AM evidence applied for human identification is their dental radiographs . Recently, forensic dental evidence has helped to primarily solve many incident cases of rapes, trafficking, terrorist attacks, homicides, and natural disasters [1, 68].
3.1. Digital Radiography
The diversity of human dentition comes from the number of teeth  in 6 different types (incisor, lateral incisor, canine, premolar, and molar) and the unique dental treatment which is not the same between every two persons . Figure 3(a) illustrates the comparison points in the dental radiographs for identification which include the number and arrangement of teeth, dental anatomy, caries, coronal or hidden restorations, periodontal bone loss, bone pathology, trabecular and crestal bone topography, nutrient canals, anatomical landmarks of the teeth and jaws, and the size and morphology of both maxillary/frontal sinuses and nasal aperture . Teeth that are missed, rotated, spaced, extra, and/or impacted can affect the dentition number and arrangement. Bases under fillings, pins, root canal fillings, posts, and implants are the hidden features that are only seen by radiography .
3.1.1. Orthopantomography (Panoramic Radiography)
Orthopantomography or panoramic radiographs are the panoramic X-ray scanning of the upper and lower jaws that provide a broad radiologic graph from the human dentition. Because of the low radiation dose and short time required for imaging, dental panoramic radiography  has become a broadly applied standard method in dentistry as complementary information and initial examinations for oncological treatments [71–73]. Accordingly, orthopantomography is now practically used for victim identification in several MDs including war casualties . During recording PM data, the corpse fixation system allows acquiring a reliable panoramic graph (Figure 3(b)) . In Figure 3(c), the most commonly observed dental pattern is illustrated . Even though OPGs are one of the routine methods in human identification, since they have a lot of effective information, the only limitation in using the panoramic technique is its sensitivity to misalignments of the body which may lead to image distortion .
3.1.2. Lateral Cephalometry
A lateral cephalogram is an X-ray of the craniofacial area from the lateral angle that displays a variety of anatomical points and distinctions of architectural and morphological structure and intra-cranial details, simultaneously. Therefore, it has an exemplary application in skull analysis and evaluation . According to Sassouni, some niceties such as bigonial width, cranial height from the mastoid to vertex, bimaxillary breadth, height from bi-gonial width to temporal crest, maximum cranial breadth, frontal sinus breadth, incisor height, and facial are referred to be evaluated in lateral skull radiographs and compared in the ante and postmortem records . As it is said in the previous part, like OPG, lateral cephalometry is sensitive to misplacement of the head in the head position part of a machine. Also, magnification of AM and PM images may result in distortion and it should not be neglected in human identification.
3.1.3. Frontal Radiography (Posteroanterior or PA Cephalometric Analysis)
Frontal sinuses or PA cephalometric analysis are other individual traits known for their high variations among people that make them suitable evidence for forensic purposes . Pattern matching in PA cephalometric analysis is performed using both Caldwell-oriented film projections and occipitomental or Water’s view. The frontal sinus is better displayed in Caldwell’s view while Water’s view provides a slightly foreshortened image. According to Nikam et al., the variables of frontal sinus can be measured for forensic identification by drawing a certain tangent to the baseline and dividing the sinus area into four parts. The general sinus variables include the number of complete sinus cavities, number of partial sinus lines, maximum overall height above baseline, maximum overall width, and number of complete sinus cavities left of the septum, as well as some partial sinus lines in the main cavity, number of scalloped arcades on the main cavity, the maximum height of quadrant above baseline, maximum height of the main cavity above baseline, maximum width of the main cavity from the tangent line, and maximum width of the main cavity on both left and right sides of the sinus (Figure 3(d)) .
3.1.4. Cone Beam Computed Tomography (CT)
Another frequently used method in forensic investigations for providing AM data is computed tomography known as CT . MSCT or cone beam images can produce a panoramic or 3D image of teeth, peripheral soft tissues, nerve pathways, and connected bones in a single scan by some posttreatment processing . The AM 3D scan data can be compared with the PM data from CT or CBCT scans or a 3D scanner (Figure 3(e)) . Some newly developed software such as Dentascan (GE Health care, UK) has helped more precise identification with fewer artifacts by reformatting the panoramic images . However, several factors such as cone beam or dental restorations can produce different types of CT artifacts which can impede comparison between AM and PM radiographs . Dental reconstructions cause artifacts because of their increased radio-opacity .
3.2. Forensic Digital Photography
Especial devices such as digital cameras are developed for recording PM dental information such as oral photographs and mouth gags . Generally, forensic photography using cameras needs to follow certain rules, including securing the crime scene for providing proper evidence, evaluating the conditions (e.g., light, weather, and camera settings), shooting the entire scene using both wide-angle and close up shots for showing the relationship among pieces of evidence, recording the location, injuries, and condition of victims, using the right angles and eliminating probable distance distortions, and locating evidence markers using the first shot of entire crime scene. Photographers need to use alternate light sources such as lasers, blue/green lights, and colored filters to detect fingerprints, bite marks, and footprints .
3.2.1. Facial and Intraoral Photographs
In forensic cases of human abuse and bite mark analysis, facial and intraoral photography is the most common and easiest diagnostic method of obtaining and maintaining evidence . In cases that the face of the deceased is recognizable, the oral photographs can be applied for direct identification, while in cases of completely disfigured faces, the intraoral photographs are more applicable, because the intraoral photographs can display certain useful data about the hard tissue such as fluorosis, abrasion, tooth attrition, enamel decalcification, enamel cracks and fractures, and lower canine anatomy . Also, smile photographs are significant evidence of the victim’s teeth through life .
3.3. Dental Appliance and Restoration
Dental appliances and accessories such as full or partial dentures, decorative accessories or orthodontic appliances, bleaching trays, occlusal splints, and mouth guards are also used to identify the dentition or mouth of a specific victim or suspect .
3.3.1. Denture Marking
The denture marking of prosthetic and orthodontics appliances is now considered very important evidence for forensic identification in different medicolegal issues . Denture marking is also applicable in nonforensic cases such as identifying a lost denture wearer suffering from amnesia or senility, loss of memory, and other psychiatric cases. Also, a denture with marking can be conveniently returned to the owner if it is lost and found. Regarding forensic issues, denture marking has an important role in identifying the unknown cases of homicide and suicide, as well as victims of fire, explosion, floods, earthquake, plane crash, or war . Therefore, denture labeling provides a rapid and reliable method other than fingerprinting for identifying unknown individuals in the laboratory. For this aim, the denture marking is preferred to contain the name of the owner with or without other identifiers such as social security number, driver’s license number, and city code . Also, a coding method is invented by Queiroz et al. referred to as the DPid system which randomly generates individual 2D data matrix codes along with a 5-digit alpha/numeric token which is individually unique. Forensic investigators can scan this code by either a smartphone or a tablet equipped with a 2D Code Reader App. There is also a DPid website that authorities can log into and enter the patient’s unique code and identify the denture device owner. The DPid system has been proved to be an efficient tool for solving forensic cases involving dental prosthesis (Figures 4(c) and 4(d)) .
3.3.2. Dental Restoration
The country or region where the dental restoration has been performed can be recognized from the quality and type of treatment and materials. For example, in Central and South America, the anterior teeth are prevalently restored using silver or gold color metal crowns. While in Eastern Europe, these teeth are frequently restored using full cast metal crowns with acrylic facings . Also, according to many dental anthropological studies, the morphological characteristics of teeth can be used for determining the race of the victim. For example, the Carabelli cusp, having few dental cusps, and simplified fissure patterns can be indicators of a Caucasoid race, while Asians are characterized by shoveled incisors and complex fissure patterns with the normal count of dental cusps .
3.4. Dental Cast
The dental cast provides a 3D model of both maxillary and mandibular arches that facilitate evaluating the malocclusions, morphology, and anatomy of the victim’s teeth. These models are certainly proper for assessing the abrasions, attrition, and fractures in the enamel . Also, using dental casts, certain odontometric parameters such as mandibular and maxillary canine indices, the size of mandibular canine, maxillary canine, and maxillary first molar, as well as the cumulative size of all teeth are used for determining the gender of the deceased .
3.4.1. Palatoscopy (Palatal Rugoscopy)
Palatoscopy or palatal rugoscopy is the knowledge of palatal rugae that can be much helpful in cases of fingerprint unavailability such as decomposed and burned bodies [89, 90]. Different tissues such as the lips, cheek, tongue, and the buccal pad of fat, teeth, and jawbones preserve the internally located palatal rugae from trauma and high temperatures . As shown in Figures 4(a) and 4(b) respectively, two main methods of classification for identification purposes have been suggested for the rugae patterns: Martin dos Santos classification (1983) and Thomas classification (1983) . On the other hand, a significant association had been reported between the rugae form and ethnicity which is considered a potential method for victim identification by forensic odontologists .
3.4.2. Intercanine Width
In forensic odontology, the mandibular canines exhibit the greatest dimorphism between females and males . These maxillary canines have several characteristics that make them a proper candidate for identifying the sexuality of victims. These teeth are less engaged with periodontal diseases, plaque, and calculus and abrasion by brushing. Above all, they are the least exposed to extraction due to aging .
Despite the importance of every method of identification, because of the nature of mass disasters, the methods based on hard tissues may be a more reliable way than the techniques based on soft tissues. For this reason, the scans and radiographs are used more in forensic dentistry, and the presence of PM photographs may be less useful than radiographs. It is clear that special restorations and the presence of AM dental casts are so valuable and can increase the accuracy of identification.
4. Medical Biotechnology Techniques
An important identifier that is increasingly extensively used today is DNA fingerprinting, DNA profiling, or gene typing which includes extracting sets of codes encrypting the DNA configuration of an individual . DNA fingerprint of every person is as unique as their fingerprint and much more precise. The DNA samples are obtained from the remnants in the crime scenes, victims, suspects, and/or inanimate objects around them (Figure 5(a)) . In cases of lacking antemortem records and the poor state of corpses’ preservation, DNA testing is applied . In criminal and missing person cases or MS tragedies, DNA can also be obtained from the human dental remains for being used in DNA typing . The sequence of tooth-extracted DNA from the unidentified subject can be then matched with known antemortem DNA samples such as stored blood, toothbrush, hairbrush, clothing, cervical smear, biopsy, or isolated samples from a parent or sibling to identify the unknown person . Dental DNA samples can be better preserved and undergo more successful typing compared to other bone-extracted DNA samples (e.g., from long bones with thick cortical tissue like femurs) because they are highly protected in the enamel .
4.1. Techniques of Identification
The individual-specific sequence used for gene typing includes the polymorphic repetitive DNA that constitutes 20-30% of the noncoding DNA or junk DNA which contains >95% of the whole genome, while the protein-coding segments of DNA (genes) contain only 2-5% of entire cellular DNA and are highly preserved among species. Several functions have been hypothesized for the noncoding DNA including spacer in a single copy. On the other hand, the repetitive sequence appears as LTR or midi satellites, STR or mini satellites, and interspersed repetitive sequences (Figure 5(b)) . There are three main methods of DNA fingerprints; (1) RFLP, (2) VNTR, and (3) STR or simple sequence repeat (SSR). Autosomal STR genotyping is the most popular one but, RFLP and VNTR do not require much amount of DNA. This is a valuable feature since DNA fragments found from the forensic scene are usually extremely scarce for being analyzed and too long for being amplified by PCR. Then, STR analysis is applicable with short sequences of DNA, much easier to be handled by PCR, and less time-consuming because it does not require the probe-hybridization .
Dental DNA can also be used for evaluating the age based on DNA methylation and estimating the biogeographical ancestry based on the sequence of the mtDNA and Y-Haplotype. These assessments provide essential information in forensic investigations. DNA methylation is an age-associated modification providing a promising biomarker with relatively acceptable accuracy for forensic chronological age estimation showing a mean absolute deviation of only 3-5 years . In comparison with nuclear STRs, mtDNA is comparable to more distant relatives; then, it is better applicable in cases of missing persons. The analysis of mtDNA is also valuable and helpful in cases of obtaining little or no nuclear DNA from the crime scene . In cases of analyzing relationships among multiple male contributors, the Y chromosome is especially useful because it is only inherited by men directly from their fathers . However, a problem associated with using tooth-extracted DNA is the coextraction of calcium and collagen especially from the enamel tissue . Another problem can occur due to environmental contaminants such as humic acid, fulvic acid, and metals. Also, microorganisms can negatively affect DNA purity during extraction and amplification processes . DNA extraction methods include tooth- and saliva-related approaches. The most important recent studies in the field of sex and age estimation, as well as DNA dental fingerprinting, are summarized in Table 2. The release of endogenous intracellular nucleases with the beginning of the postmortem phase causes decomposing DNA. But DNA contained in teeth is highly preserved against the enzymatic degradation with the naturally hard mineral and low porous tissue of the tooth. Generally, the tooth is constructed from three parts of cementum, dentin, and enamel (Figure 5(c)) . However, the exact anatomy of each tooth is different which is important to know to achieve the maximum DNA yield in studies. For example, it will help to know that the palatine upper and the distal lower molars or the most subsequent have the widest root canal, and the canine tooth has the longest canal within the same arch. Then, these teeth are the best sources for DNA extraction . DNA molecules are obtained from almost all parts of the teeth except the enamel .
The dental pulp in the radicular and coronal portion of the teeth is the oldest source for DNA odontological forensic investigations [107–109]. Containing a great number of odontoblasts, fibroblasts, endothelial cells, undifferentiated mesenchymal cells, and nucleated cells of the blood, pulpal tissue provides a favorable amount of DNA to be isolated . Therefore, in an intact fresh tooth, the dental pulp is considered the best source of DNA . Another preferred source for DNA extraction is dentine which is a hard dense bony tissue protected by cementum and enamel . Dentine is generated from odontoblasts present in the peripheral layer in a columnar form that is extended toward the thickness of the dentin . AAR is also an accredited method for age estimation using dentine based on the nonenzymatic covalent modification of proteins . Raman microspectrometry is another modern, highly selective, and noninvasive technology for age estimation that provides the chemical structure of a molecular fingerprint . Considering that the DNA content of pulp can be negatively impacted by dental diseases, aging, and postmortem cellular degradation, some studies have sought other sources of nuclear DNA, especially in moist environments. Related publications explain that extracted DNA from different dental tissues is variable in quality and prioritize cementum for its less vulnerability to dental diseases or raising chronological age. Also, these reports hypothesize an important role for cellular cementum in the adaptation to occlusion and tooth movement after the eruption . Also, the mitochondrial and nuclear DNA obtained from the tooth may reduce in quality and quantity due to the chronological age and dental disease and show variable efficiencies for STR typing. Also, while dentine-recovered DNA highly depends on the presence/absence of pulp, both nuclear and mitochondrial DNA can be finely extracted from the cementum especially when teeth remnants are degraded or ancient . Large DNA strands with high molecular weight are commonly collected from the oral fluid at crime scenes from bite marks, cigarette butts, postage stamps, envelopes, etc. . Compared to the blood as a source of DNA, saliva benefits from technical advantages such as easier and less invasive collection as well as no religious conflict, especially in infant subjects, children, and elderly subjects, and does not have the challenges . Using modern technologies, only 0.1 ml of saliva samples are sufficient for obtaining enough applicable DNA . The chronological age is detected from the level of salivary DNA methylation, and gender identification is exerted via measuring the salivary content of testosterone, whole saliva flow rates, etc. . The individual characteristics evoked from the left salivary traces that can present age, gender, personal data, and health status are used for creating one’s salivary signature (Figure 5(d)) . In a comparative analysis by Watanabe et al., the sensitivity and stability of RNA-based and amylase-based markers were examined under different storage conditions and the RNA method was suggested as a supplementary method for the conventional amylase-based identification method . Age, gender, and race are also determined by protein profiling and evaluating the total salivary protein concentration using the standard baseline of the protein variations . Screening the α-amylase activity is a sensitive, simple, and cost-effective method for indicating the saliva presence; however, it is low specific . Then, preserving the α-amylase stability is a must for catalytic and immunological forensic saliva investigations . Different eating patterns, oral hygiene measures, humidity, climate, temperature, or even disease outbreaks can be used for recognizing various geographical locations since any locales result in a different salivary microbial community in composition and function . Methods based on various oral resident bacteria such as Streptococcus salivarius, Streptococcus mutans, and Veillonella atypica are more specific than protein-based methods . One of the most important species in forensic microbiology is S. mutans which is substituted in the mouth during birth and remain there throughout life. The most frequent genotyping analysis for oral microbial species is PCR-restriction fragment length polymorphism . Saliva is routinely used as roadside testing for detecting the level of ethanol or psychotropic drug (e.g., cannabis) abuse. Corresponding THC is detectable in saliva due to smoking cocaine, while its secretion from serum to the oral cavity through saliva takes approximately 10 h .
DNA extraction could be one of the most valuable techniques among all methods of human identification, but the sensitivity of techniques, high prices, and time-consuming methods are its undeniable weakness.
5. Lip Print
The soft tissues of lips with their wrinkles and grooves on the labial mucosa provide applicable evidence for personal identification and criminal investigation called lip print [123, 124]. The study of the individually unique characteristic pattern of the sulci labiorum, similar to the fingerprints, is called cheiloscopy [124, 125]. Some information about the contributors in a crime scene such as the number of the people, their genders, cosmetics used, and the pathological transformations of lips can be figured out from the lip prints . The advantage of lip prints is that they can be recognized conveniently since they recover after trauma, inflammation, and diseases such as herpes ; however, its pattern may change due to pathology, postsurgical alteration, or loss of support due to loss of anterior teeth . The grooves of the lip also change based on the open/close status of the mouth since they can be well-defined or ill-defined in close and open states, respectively. Legibly, they are difficult to interpret in the open position . Although some studies implicate certain lip groove patterns to be related to each of the sexes or geographically distinct populations, this specificity is not completely proved . Recent identification researches using lip prints are summarized in Table 3.
5.1. History and Classification
Initially, cheiloscopy (Cheilo means lips in Greek) was noted by anthropologists, and Fischer was the one who described this epithet in 1902 . Three decades later, criminologists such as French Edmond Locard started to use the trace of lip furrows for human identification [131, 132]. Afterward, lip prints are classified based on their consistent groove patterns which resist many afflictions [132, 133]. Several lip print classifications have been established among which the most popular is the one made by Suzuki and Tsuchihashi [10, 126, 134, 135]. According to their classification, lips are categorized in four groups: (1) thin lips that are frequent in European Caucasian, (2) medium lips that are the most frequent type, (3) thick or very thick lips that are commonly seen in African Americans and usually associated with a lip cord inversion, and (4) mix lips which are often seen in Orientals .
5.2. Methods of Identification
The variety of lip print analysis methods has brought up an inconsistency in the related literature all over the world . Among these various methods are photographing, direct prints onto paper, and generating lips 3D casts which finely display the lips groves using dental impression materials . Comparison methods for lip prints (e.g., picture/lens magnification) have been facilitated using a computer . For utilizing digital methods, clear lip print photographs in a standardized position are required (Figure 6(a)) . Different substances including aluminum powder, silver metallic powder, silver nitrate powder, plumb carbonate powder, fat black aniline dyer, cobalt oxide, lysochrome dye, or fluorescent dyes can be used for visualization of the latent lip prints . Aluminum and magnetic powder are the most common substances usually used for identifying and tracing lip prints left in crime scenes . The lipsticks or cellophane tapes are usually used for manual generating lip print traces in scientific studies [125, 127, 141–143]. Lipsticks contain several compounds, oils, or waxes in their complex constitution . In cases of similar colors of lip print and its background or the trace being old, fluorescent agents are used for visualizing the lip’s traces . Furthermore, a study by Ramakrishnan et al. has suggested using persistent lipstick, cellophane sheets, and lysochrome dyes for sex determination (Figure 6(b)). They also maintained the latent lip prints in a digital database . Another adjuvant tool employing lip prints in forensic investigations is lip outline patterns. Maloth et al. have shown that lip outline patterns have the potential to be used for human identification since they are individually unique .
6. Bite Mark
An arguable area of forensic odontology which is mostly applicable in homicide, rape, sexual assault, robbery, and child abuse criminal cases is bite mark analysis [35, 147]. Although the bite marks are unique to individuals even in identical twins, accepting bite mark evidence in courts needs fundamental validation and high scrutiny investigations to ensure its reliability [148, 149]. Various scientific principles and factors are required to be considered to make the bite mark applicable for personal identification. The injury site, size, and age, as well as the skin mobility, the degree of trauma, and the state of structures underlying the skin in the injured area are some factors that need to be considered . Many studies impact the more accuracy and reproducibility of bite marks obtained on food items than those on the skin .
Human bite marks are usually left by the incisors, canines, and premolars that create two opposing U-shaped arches. The open spaces between U shapes may contain hematoma due to soft tissue compression. The most prominent marks are created by maxillary canines with a normal distance of 25–40 mm . Recent human identification researches of bite marks are summarized in Table 3.
6.1. Bite Mark Types
From a forensic odontology viewpoint, teeth marks comprise three main types: (1) bite marks on comestibles, (2) bite marks on the assailant body due to victim’s self-defense, and (3) bite marks left on an assault or murder victim’s body usually in cases of sexual harassments which are mostly found on the breast, neck, or cheek . The injuries caused by the human bite are divided into two categories based on the force they have applied on the skin to lose its integrity (closed fist injury or fight bite) or to breach and probably avulse the tissue (occlusive bite injury) .
6.2. Methods of Identification
A comprehensive description of reliable methods of assessing the bite marks is available on the latest Manual published by the ASFO, in the section titled “Bite Mark Pattern Recognition and Collection from Humans and Inanimate Objects: Non-Invasive Analysis” . According to this manual, several techniques can be used to analyze the bite mark patterns. One of the most accurate models for identifying human bite marks is dental casts. Also, registering the bite marks of volunteers on the clay, wax sheet, styrofoam sheet, and human skin by overlay is recommended by the American Board of Forensic Odontology . Overlays are obtained by hand tracing, xerographic images, or through X-ray films. Then, the impressions generated by these life-sized overlays are surveyed by comparing with the bite mark evidence from the crime scene or the suspect’s teeth . Cone Beam Computed Tomography is another suggested forensic technique especially for analyzing the bite marks in foodstuffs . The position of the body can cause differences in the bite mark appearance (Figure 6(c)) . An indirect technique has been applied by Daniel and Pazhani for victim identification using computer-assisted overlay generation. In this method, life-size photographs of dental casts are used for generating overlays from anterior dentition. For this purpose, the “magic wand” wizard tool in Adobe Photoshop CS4 software is applied (Figure 6(d)). An adequate matching between these overlays and the real bite mark pattern obtained from the physical evidence (such as foodstuff) can be met by superimposing one over the other in various angles . Computer-generated overlays are the most popular and reliable method of overlay generation .
Lip print and bite marks are practical in forensic science. However, as they are soft tissue-related methods, in disasters with serious facial damage or fires, they are useless and human identification needs more precise methods which are markedly in relation with hard tissues like bone and teeth along with approaches based on scanning radiographs.
7. Blood Group
Since decades ago, the ABO blood categorization methodology has been considered a reliable medicolegal identification system based on the antigen-antibody reactions of every individual’s RBCs membrane and remains unchanged throughout life . Based on the ABO system, people are categorized into groups of A, B, AB, or O blood groups . There are enough ABO antigens in dental tissues to be used for identifying even highly decomposed bodies . Another combined antigen used in ABO system identification is the Rh . There are also other antigens and blood group systems, but they are not as applicable as the ABO and Rh groups in practice, since they are weak or the corresponding antibodies are not conveniently available . Recent blood group applications in forensic researches and human identification studies are summarized in Table 3.
7.1. Teeth-Related Diagnosis
Although pulp soft tissue is all surrounded by dental hard tissues, the ABO antigens diffused from both blood and saliva can be isolated from the tooth pulp since it contains a lot of blood vessels [2, 163, 165]. However, the distribution of these factors gradually decreases from the pulp cavity wall toward the enamel and dentin edge . ABO factors are also found in dentinal tubules . Siracusa (1923) developed the AE technique firstly, and Kind (1960) modified his method. However, later on, more modifications have been applied to improve AE sensitivity, specificity, and resistance to external interfering agents. AE is extensively used for detecting the blood group from various forensic evidence such as dried stains, tissues, secretions, and teeth. An important pro of this method is the reusability of its prepared and processed antigenic material . More recently, Kumar et al. have suggested a validated method for obtaining red cell agglutination from the dental samples (Figure 6(e)) . Furthermore, there are other methods such as absorption elusion, hemagglutination, PCR, and histochemical techniques for determining the ABO/Rh group. Among all, PCR shows high sensitivity and specificity putting it at the highest priority .
7.2. Saliva-Related Diagnosis
As mentioned before, water-soluble antigens are hypothesized to be infused from the saliva to the tooth tissue (infusion sedimentation theory) . Therefore, several studies have been allocated to developing new techniques for detecting ABO factors from saliva with 100% accuracy. Two main methods currently established for this aim include the AI method and the absorption-elution method or AE which is easier and simpler . SPR imaging is another ABO detecting method based on the interactions between immobilized biomolecules and DNA-protein or cells in the solution phase. SPR imaging has been used to indicate another blood categorizing type called ABH system with 100% accuracy .
The ABO and Rh groups can raise the accuracy of forensic human identification. Since pulp tissue is protected by tooth outer layers, detection of these groups may be done more precisely in comparison with saliva or a drop of blood which are vulnerable to environmental contamination. So, it is another reason why the presence of only a single tooth can help dramatically during various identification methods.
Forensic dentistry or odontology is attracting an increasing concern for its importance and efficiency in identifying victims of different tragedies and mass disasters. This article is aimed at having a comprehensive survey on the most significant aspects of forensic dentistry including dental radiographs and scanning, sex and age estimation, medical-biological techniques, blood grouping, lip print, and bite mark identification. This review attempted to highlight the conventional and new approaches of forensic odontology in each aforementioned part. From our standpoints, because of the ease of use, the velocity of techniques, and being cost-benefit, the role of dental radiographs and scans are more effective in comparison with others in oral-and-maxillofacial related approaches of human identification. The reason for that is they illustrate resistant-to-disasters hard tissues in a facial complex like bone and teeth and they act faster than histologic approaches along with being more economical. Two key factors are required for efficiently applying dental records in human identification. These factors include regular oral health follow-ups and preserving high-quality dental records in the form of dental charts, radiographs, photographs, impressions, casts, etc. Different patterns and anatomy of teeth, jawbones, and sinuses (including the missing, filled, and decayed teeth) also increase the specificity of dental identification methods. Usage of other characteristics such as lip print patterns, bite marks, oral microbiome, and salivary biomarker databanks, and ABO blood groups of individuals can help radiographs and scanning methods to reach more precise detection. This is clear that each method has its limitation. The scanning approaches are highly based on the quality of imagination, magnification, the accuracy of measurements, and the correct interpretation of results. On the other hand, soft tissue-related methods are under the influence of the severity of the disaster, the situation of the PM environment, and the accuracy of the histopathologic analysis. We hope that our attempt in extracting maximum information about forensic dentistry will be beneficial to society in human identification.
9. Future Direction
Ten years ago when the new technology of CT had been accepted to be routinely used for human identification purposes, the 3D surface comparison had just emerged. Today, the 3D surface comparison is the main technique for gender determination. We expect further development and rapidly advancing forensic odontology technologies related to imagery such as CT and CBCT. Also, 3D datasets including CT and AM 3D surface scan data are expected to play an important role in applying forensic odontology in DVI. From our standpoints, 3D approaches like CBCTs are the future of DVI because they provide valuable information. The reason why they are so important is the fact that not only would be the presence of teeth variations clear in advance techniques but also various types of significant radiographs like OPGs, lateral and PA cephalometric can be extracted in one CBCT and precise measurements can be accomplished easier in these practical modes of digital scans. 3D AM dental profile and PM virtual models can be prepared using 3D intraoral scanners becoming an indispensable toolset for forensic investigations. Like any other field, statistical and computational analyses, as well as modeling techniques, are extensively used in designing and implementing researches about identification methods. In the same regard, digital forensics as an integral part of forensic researches has considerably reduced the costs of technology and increased the accuracy of forensic investigations. Also, advances in molecular biology technologies have helped more efficient DNA extraction from less available material even under adverse conditions. Forensic biorobots for DNA extraction, laser microetching for labeling of metallic prostheses, Raman spectroscopic analysis of dentin for age estimation, forensic thanatology for investigation of every phenomenon related to death, virtual autopsy, intraoral scanners for improving the accuracy of bite mark impressions, and retouched images for fraudulent purposes are some advanced tools and methods used in forensic investigations. However, as always, conventional methods hands in hands with advanced technologies exert more convenient and accurate results. It is wise to mention that using an interdisciplinary approach using both forensic medicine, forensic dentistry with the emphasis on advanced scanning technology will enhance the accuracy of clarifying a questionable identity in a legal jurisdiction.
|AAR:||Aspartic acid racemization|
|AGEs:||Advanced glycation end products|
|CBCT:||Cone Beam Computed Tomography|
|CPCH:||Coronal pulp cavity height|
|CSI officers:||Crime scene investigation officers|
|CVM:||Cervical vertebral maturation|
|CVMI:||Cervical vertebrae maturation indicators|
|DPid:||Dental prosthetics identification|
|DPR:||Dental panoramic radiography|
|DVI:||Disaster victim identification|
|Gn-M0:||Angle of the intersected lines from the left and right gonion to menton|
|GUI:||Graphical user interface|
|ICP:||Iterative closest point|
|LTR:||Long tandem repeats|
|MCI:||Mandibular canine index|
|MDCT:||Multislice Computed Tomography|
|MRI:||Magnetic resonance imaging|
|NGS:||Next generation sequencing|
|PCA:||Principal component analysis|
|PCR:||Polymerase chain reaction|
|RBCs:||Red blood cells|
|RFLP:||Restriction fragment length polymorphism|
|SPR:||Surface plasmon resonance imaging|
|STR:||Short tandem repeat|
|VNTR:||Variable number of tandem repeats|
|ASFO:||American Society of Forensic Odontology.|
This article is a review and does not contain any studies with human or animal performed by any of the authors.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
The authors would like to acknowledge the useful comments given by colleagues.
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