The Hidden Secrets of the Dental Calculus: Calibration of a Mass Spectrometry Protocol for Dental Calculus Protein Analysis.

Dental calculus is a solid deposit that forms and accumulates on the tooth surface, entrapping oral microorganisms, biomolecules, and other micro-debris found in the oral cavity. A mass spectrometry analysis of its protein content opens a vista into the subject's diet, oral flora, and even some aspects of health, thus providing new insight and expanding our knowledge of archaic cultures. Multiple experimental protocols have been proposed for the optimal extraction of proteins from dental calculus. Herein, we compared various experimental conditions in order to calibrate and validate a protocol for protein extraction. Our results show that a high concentration of acetic acid followed by mechanical crushing and sonication provided the highest protein yield, while acetone precipitation enabled the identification of more distinct proteins. We validated this protocol using archeological samples, identifying human and microbial proteins in specimens from the eighth and seventeenth centuries (approximately 250-1300 years ago). These findings demonstrate that the developed protocol is useful for studying excavated archaeological samples and that it might be utilized to explore the biohistory, dietary habits, and microbiome of archaic populations.

The dental remains from the Early Upper Paleolithic of Manot Cave, Israel.

This study presents the dental remains discovered at Manot Cave (MC), Western Galilee, Israel. The cave contains evidence for human occupation during the Early Upper Paleolithic period (46-33 ka) mainly of Early Ahmarian (∼46-42 ka) and Levantine Aurignacian (∼38-34 ka) cultural levels. Six teeth (three deciduous and three permanent) were found at the site, of which four could be thoroughly analyzed. The morphology of the teeth was qualitatively described and analyzed using traditional and geometric morphometric methods. A large comparative sample was used in order to assess the morphological affiliation of the Manot specimens with other Homo groups. The results provided equivocal signals: the upper first premolar (MC-9 P3) is probably modern human; the upper deciduous second molar (MC-10 dm2) and the upper second permanent molar (MC-8 M2) might be modern humans; the lower second deciduous molar (MC-7 dm2) might be Neanderthal. Owing to the small sample size and the almost total lack of distinctive characteristics, our outcome could not supply conclusive evidence to address the question of whether Manot Aurignacian population came from Europe or descended from the local Ahmarian population.

Are chin and symphysis morphology facial type-dependent? A computed tomography-based study.

INTRODUCTION: The chin is a major determinant of the facial profile; hence, it plays a major role in orthodontics and orthognathic surgery. It is thus essential to follow and better understand its expression in different facial types. The major objectives of the current study were to characterize morphometrically the chin and symphysis and reveal their association with different facial types. METHODS: Computed tomography scans of the head and neck of 311 adults (163 males, 148 females; age range, 18-95 years) were classified into 3 facial types: short, average, and long. Height, width, projection, inclination, thickness, and area were measured on the chin and symphysis. RESULTS: The majority of the population (70%) manifested an average facial type; the other 30% were almost equally distributed between short and long facial types. The long facial type was more common among females and the short facial type among males. Chin projection, area, and size were significantly greater in short-faced patients. Chin width in males was similar for all facial types, whereas, in females, chin width was the widest in the short facial type and the narrowest in the long facial type. Symphysis height was significantly greater in long-faced patients in both sexes. The mandibular incisors' inclination relative to the mandibular plane was not significantly associated with the chin or symphysis morphology. CONCLUSIONS: Chin and symphysis morphology is facial type-dependent. Orthodontists and maxillofacial surgeons should be aware of the complex relationship between facial types and chin/symphysis size and shape when planning treatment.

Age estimation of fragmented human dental remains by secondary dentin virtual analysis.

Unlike bones, teeth are remarkably resilient and can withstand severe trauma, making age assessment based on the dentition essential for forensic analysis. Modern techniques for age estimation focus on pulp-chamber volume measurements using radiographs and computerized tomography (CT); however, these are applicable only for complete teeth (i.e., with intact crown and root). In the current study, we developed a new approach using high-resolution micro-computerized tomography (µCT) to visualize the secondary dentin (SD), an inner layer surrounding the pulp which accumulates with age, thus facilitating age estimation of fragmented and broken teeth.The growth pattern of the SD with age was analyzed for 77 lower premolars from two anthropological collections. A comparison of SD virtual segmentation and histological measurement was highly correlative (ICC = 0.95). SD was measured per volume (mm3) of a 1 mm thick slice directly below the cemento-enamel junction. Regression analysis using SD measurements increased the success rates of age estimation (82%) compared with the "gold-standard" pulp/dentin method (54%) in the range of ± 10 years. The accuracy of age estimation based on SD analysis was improved to a range of 7-8 years.The SD method thus allows age estimation with greater prediction rates and better accuracy based on only a small fragment of a tooth in a non-invasive manner. This novel methodology is easy to use, accessible, and bears implications in various fields such as forensic sciences and anthropological research.

Sex estimation using computed tomography of the mandible.

Sex estimation of skeletal parts is of great value even in the DNA era. When computed tomography (CT) facilities were introduced to forensic institutes, new possibilities for sex estimation emerged. The aim of this study was to develop a CT-based method for sex estimation using the mandible. Twenty-five CT-based measurements of the mandible were developed and carried out on 3D reconstructions (volume rendering) and cross sections of the lower jaw of 438 adult individuals (214 males and 224 females). Intraobserver and interobserver variances of the measurements were examined using intraclass correlation coefficient (ICC) analysis. Five discriminant functions were developed using different states of completeness of the mandible. The success rates of these equations were cross validated twice. The measurements were found to be highly reliable (for intraobserver 0.838 < ICC < 0.995 and for interobserver 0.71 < ICC < 0.996). For a complete mandible, the correct classification rate was 90.8%. For incomplete mandibles, the correct classification rates varied from 72.9 to 85.6%. Cross-validation tests yielded similar success rates, for the complete mandible 89% and for the incomplete mandible 67.5 to 89%. We concluded that CT techniques are appropriate for estimating sex based on the mandible size and shape characteristics. Suggested discriminant functions for sex estimation are given with data on the correct classification rates.

Rate and pattern of interproximal dental attrition.

Although occlusal and interproximal attrition occur because of diverse etiology and present dissimilar features, both progress with age. The objectives of this study were to reveal the rate and pattern of development of interproximal attrition facets (PAF) with age and to compare those with occlusal attrition (OA) changes. Five-hundred and ninety-four teeth were collected from 198 skulls (of adults, 20-71 yr of age). Three mandibular teeth [first premolar (P1), second premolar (P2), and first molar (M1)] were examined for PAF size and OA rate. Interproximal attrition and OA followed similar patterns of development until subjects reached 40-45 yr of age, after which they took different paths: PAF did not increase in size and were not as large as in younger groups, regardless of facet location, whereas OA continued to progress. The PAF changes with age differed between premolars and molars, unlike OA, which presented a similar rate for all teeth studied. Although OA scores presented significantly moderate correlations with age, PAF area size demonstrated low correlations with age. Low, but significant, correlations were found between the rate of OA and that of PAF. However, PAF and OA exhibited different patterns of development with age. Premolars and molars presented dissimilar development of PAF, which is probably caused by a unique attrition pattern in the molar teeth, different morphology, and force vectors.

Proximal attrition facets: morphometric, demographic, and aging characteristics.

Although interproximal attrition is considered to be limited in modern populations, it has important clinical implications. However, in contrast to occlusal attrition, proximal attrition receives limited scientific attention. The main purpose of the current study was to fill this void. Seven-hundred and sixty-five teeth were collected from 255 skulls of subjects 18-75 yr of age. For each individual, three mandibular teeth (the first and second premolars and the first molar) were examined for proximal attrition facets (PAFs). The results provide detailed information on the size, shape, and location of the facets according to age cohort, gender, and ethnicity. The validity of the method used to measure the facets was also examined. The major findings were as follows: PAFs are usually located on the upper half of the crown proximal aspect; in each tooth, the mesial facet is more lingually positioned and the distal facet is more buccally positioned; the majority of the facets are subrectangular in shape; the size of the facets tends to increase in an anteroposterior direction (from premolars to molars); and facet size and location are age- and sex-dependent and ethnicity-independent. It is our recommendation that dentists bear in mind that interproximal attrition is a dynamic, long-term process and needs to be considered in many clinical scenarios.

Three-Dimensional Surface Texture Analysis of Fluoride's Effect on Enamel Erosion.

Enamel erosion has become a common clinical finding that often impairs dental esthetics and function. In the current study, we aimed to implement the three-dimensional surface texture analysis (3DST) method to explore the protective effect of fluoride on surface texture prior to erosive conditions. For each of the 50 teeth used in this study, the polished buccal enamel surface was divided into three separate areas: the first area was untouched polished enamel, the two other surfaces were immersed in 0.3% citric acid for 30 s. One was treated with high-concentration (19,000 ppm) sodium fluoride (NaF) solution prior to acid attack, and the other had no treatment prior to acid exposure. Enamel surface texture and step height measurements were obtained using a high-resolution disk scanning confocal microscope, and SEM images were also acquired. Surfaces treated with fluoride showed fewer variations in 3-D surface texture parameters than the eroded surface compared to the control group (p = 0.001). This was in accordance with the SEM descriptive images. The findings indicate that pre-fluoridated enamel areas were less affected by the acid and showed similar features to the untouched enamel. Moreover, a protective effect of the fluoride treatment against irreversible enamel damage was noted as the surface loss (step-height) was significantly reduced (p = 0.03). The study showed that 3DST analysis is a valuable methodology for detecting and quantifying subtle differences between the surfaces. When exploring the combination of all surface texture parameters, it was revealed that the pre-fluoridated eroded enamel surfaces showed considerable similarity to the untouched enamel.

Response to Comment on "A Middle Pleistocene Homo from Nesher Ramla, Israel".

Marom and Rak claim, on the basis of a few mandibular features, that the Nesher Ramla (NR) Homo is a Neanderthal. Their comments lack substance and contribute little to the debate surrounding the evolution of Middle Pleistocene Homo. Limitations and preconceptions in their study prevented them from achieving resolution beyond a dichotomous interpretation of the NR as either a Neanderthal or a modern human.

Variation in Chin and Mandibular Symphysis Size and Shape in Males and Females: A CT-Based Study.

The chin is a unique anatomical landmark of modern humans. Its size and shape play an important role from the esthetic perspective. However, disagreement exists in the dental and anthropological literature regarding the sex differences in chin and symphysis morphometrics. The "sexual selection" theory is presented as a possible reason for chin formation in our species; however, many other contradictory theories also exist. This study's aims were therefore to determine how chin and symphysis size and shape vary with sex, and to discuss "sexual selection" theory as a reason for its formation. Head and neck computed tomography (CT) scans of 419 adults were utilized to measure chin and symphysis sizes and shapes. The chin and symphysis measures were compared between the sexes using an independent-samples t-test, a Mann-Whitney test, and the F-statistic. The chin width was significantly greater in males than in females (p < 0.001), whereas the chin height, area, and size index were significantly greater in females (p < 0.001). Symphysis measures did not differ significantly between the sexes. Size accounted for 2-14% of the chin variance and between 24-33% of the symphysis variance. Overall, the chin was found to be a more heterogeneous anatomical structure than the symphysis, as well as more sexually dimorphic.

Changes in human mandibular shape during the Terminal Pleistocene-Holocene Levant.

The transition to food production, exploitation of 'secondary' products (e.g., milk), and advances in cookware technology have affected all aspects of human life. The aim of the present study was to follow changes in mandibular form and shape throughout the terminal Pleistocene-Holocene Levant. The hemimandibles of four populations were included in this study: Natufian hunter-gatherers (n = 10), Pre-pottery Neolithic early farmers (n = 6), Chalcolithic farmers (n = 9), Roman-Byzantine (n = 16), and modern (n = 63) populations. A surface mesh of each mandible was reconstructed from CT or surface scans. Changes in mandibular form and shape were studied using the Procrustes-based geometric morphometrics method. Univariate and multivariate analyses were carried out to examine differences in size and shape between the studied populations. Our results reveal considerable temporal changes in mandibular shape throughout the Holocene Levant, mainly between the pre-agricultural population (the Natufian) and the succeeding ones, and between the post-industrial (the Modern) and the pre-industrial populations. A tendency for a reduction in mandibular size was identified between the pre-agricultural population and the farmers. Most regions of the mandible underwent shape changes. In conclusion, substantial changes in mandibular shape occurred throughout the Holocene Levant, especially following the agricultural revolution. These changes can be explained by the "masticatory-functional hypothesis".

Human mandibular shape is associated with masticatory muscle force.

Understanding how and to what extent forces applied to the mandible by the masticatory muscles influence its form, is of considerable importance from clinical, anthropological and evolutionary perspectives. This study investigates these questions. Head CT scans of 382 adults were utilized to measure masseter and temporalis muscle cross-sectional areas (CSA) as a surrogate for muscle force, and 17 mandibular anthropometric measurements. Sixty-two mandibles of young individuals (20-40 years) whose scans were without artefacts (e.g., due to tooth filling) were segmented and landmarked for geometric morphometric analysis. The association between shape and muscle CSA (controlled for size) was assessed using two-block partial least squares analysis. Correlations were computed between mandibular variables and muscle CSAs (all controlled for size). A significant association was found between mandibular shape and muscle CSAs, i.e. larger CSAs are associated with a wider more trapezoidal ramus, more massive coronoid, more rectangular body and a more curved basal arch. Linear measurements yielded low correlations with muscle CSAs. In conclusion, this study demonstrates an association between mandibular muscle force and mandibular shape, which is not as readily identified from linear measurements. Retrodiction of masticatory muscle force and so of mandibular loading is therefore best based on overall mandibular shape.

The earliest modern humans outside Africa.

To date, the earliest modern human fossils found outside of Africa are dated to around 90,000 to 120,000 years ago at the Levantine sites of Skhul and Qafzeh. A maxilla and associated dentition recently discovered at Misliya Cave, Israel, was dated to 177,000 to 194,000 years ago, suggesting that members of the Homo sapiens clade left Africa earlier than previously thought. This finding changes our view on modern human dispersal and is consistent with recent genetic studies, which have posited the possibility of an earlier dispersal of Homo sapiens around 220,000 years ago. The Misliya maxilla is associated with full-fledged Levallois technology in the Levant, suggesting that the emergence of this technology is linked to the appearance of Homo sapiens in the region, as has been documented in Africa.

A possible case of cherubism in a 17th-century Korean mummy.

Cherubism is a benign fibro-osseous disease of childhood limited specifically to the maxilla and mandible. The progressive replacement of the jaw bones with expansile multilocular cystic lesions causes eventual prominence of the lower face, and hence the classic "cherubic" phenotype reflecting variable extents of jaw hypertrophy. Histologically, this condition has been characterized as replacement of the normal bone matrix with multicystic pockets of fibrous stroma and osteoclastic giant cells. Because of radiographic features common to both, primarily the presence of multiloculated lucencies with heterogeneous "ground-glass" sclerosis on CT imaging, cherubism was long mistaken for a craniofacial subtype of fibrous dysplasia. In 1999, however, the distinct genetic basis for cherubism was mapped to chromosome 4p16.3 and the SH-3 binding protein SH3BP2. But while there are already three suspected cases of fibrous dysplasia amongst archaeological populations, no definitive cases of cherubism have yet been reported in historical populations. In the current study we describe micro- and macro-structural changes in the face of a 17th century Joseon Dynasty Korean mummy which may coincide with the clinic-pathologic and radiologic features of cherubism.

Malocclusion in early anatomically modern human: a reflection on the etiology of modern dental misalignment.

Malocclusions are common in modern populations. Yet, as the study of occlusion requires an almost intact dentition in both the maxilla and mandible, searching for the ultimate cause of malocclusion is a challenge: relatively little ancient material is available for research on occlusal states. The Qafzeh 9 skull is unique, as its preserved dentition allowed us to investigate the presence and manifestations of malocclusion. The aim of this study was thus to examine the occlusal condition in the Qafzeh 9 specimen in light of modern knowledge regarding the etiology of malocclusion. We revealed a pathologic occlusion in the Qafzeh 9 skull that probably originated in the early developmental stage of the dentition, and was aggravated by forces applied by mastication. When arch continuity is interrupted due to misalignment of teeth as in this case, force transmission is not equal on both sides, causing intra-arch outcomes such as mesialization of the teeth, midline deviation, rotations and the aggravation of crowding. All are evident in the Qafzeh 9 skull: the midline deviates to the left; the incisors rotate mesio-buccally; the left segment is constricted; the left first molar is buccally positioned and the left premolars palatally tilted. The inter-arch evaluation revealed anterior cross bite with functional shift that might affect force transmission and bite force. In conclusion, the findings of the current study suggest that malocclusion of developmental origin was already present in early anatomically modern humans (AMH) (the present case being the oldest known case, dated to ca. 100,000 years); that there is no basis to the notion that early AMH had a better adjustment between teeth and jaw size; and that jaw-teeth size discrepancy could be found in prehistoric populations and is not a recent phenomenon.