Tooth Cementum Research Articles

Motherhood at Early Bronze Age Unterhautzenthal, Lower Austria

This article utilises skeletal evidence (n = 57) from settlement features and graves at Unterhautzenthal, Lower Austria, to outline our methodological approach to researching motherhood in prehistory. Unterhautzenthal includes the grave of a pregnant teenager, a triple burial of a woman with two children and a family grave of a man, woman and baby; additional women’s graves include remains of neonates and young children. Comparing archaeological context information with osteobiographical data allows us to draw inferences about the social status of women and the ways Bronze Age motherhood was conceptualised. The archaeological approach includes a gender and age analysis of material culture and Social Index calculations. The osteological analyses include age at death, sex, body height, health indicators, and pathologies, with an emphasis on pelvic changes. Physical traces that may relate to strain through pregnancy and childbirth were explored in detail. In addition to morphological assessment of the entire skeletal collection, we applied tooth cementum annulation analysis, 14C dating, and δ13C/δ15N isotope analysis to selected individuals. These data, in conjunction with demographic modelling, enable us to draw conclusions about women’s age at first pregnancy and the average number of children per woman, as well as the cultural and social context of motherhood.

Hypercementosis Associated with ENPP1 Mutations and GACI

Mineralization of bones and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. This study for the first time analyzed the effect of decreased PPi on dental development in individuals with generalized arterial calcification of infancy (GACI) due to loss-of-function mutations in the ENPP1 gene. Four of the 5 subjects reported a history of infraocclusion, overretained primary teeth, ankylosis, and/or slow orthodontic tooth movement, suggesting altered mineral metabolism contributing to disrupted tooth movement and exfoliation. All subjects had radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. High-resolution micro–computed tomography (micro-CT) analyses of extracted primary teeth from 3 GACI subjects revealed 4-fold increased cervical cementum thickness (P = 0.00007) and a 23% increase in cementum density (P = 0.009) compared to age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 mutant mouse molars revealed 4-fold increased acellular cementum thickness (P = 0.002) and 5-fold increased cementum volume (P = 0.002), with no changes in enamel or dentin. Immunohistochemistry identified elevated ENPP1 expression in cementoblasts of human and mouse control teeth. Collectively, these findings reveal a novel dental phenotype in GACI and identify ENPP1 genetic mutations associated with hypercementosis. The sensitivity of cementum to reduced PPi levels in both human and mouse teeth establishes this as a well-conserved and fundamental biological process directing cementogenesis across species (ClinicalTrials.gov NCT00369421).

Determining Age and Season of Death by Use of Incremental Lines in Norwegian Reindeer Tooth Cementum

ABSTRACTUsing incremental patterns in tooth cementum is a powerful tool for age assessment. Recent developments have shown that the method has a large potential as an indicator of the season of death of the individual in question. In this paper, the results of a study comparing thin sections of teeth of the modern Hardangervidda reindeer population to those of Iron Age, eleventh, and thirteenth century reindeer hunting stations from the same area are presented. The tooth wear stages were compared to the actual age of the individual (based on the number of incremental lines and the age at eruption). It was established that in the studied reindeer populations wear stages sometimes result in too low an age estimate. Having identified the start of the period of deposition for rest lines in the modern reindeer population as early autumn and winter, analysis of the outermost layer in the tooth cementum at the archaeological sites points to a prolonged hunting season in the thirteenth century, while in the earlier phases hunters were probably present on fewer and shorter visits.

Mineral features of connective dental hard tissues in hypoplastic amelogenesis imperfecta.

To explore the mineral features of dentin and cementum in hypoplastic Amelogenesis imperfecta AI teeth. Forty-four (44) teeth cleaned and free of caries were used: 20 control and 24 affected by hypoplastic amelogenesis imperfecta. Thirty-two teeth were studied by pQCT, cut in sections, and analyzed under microradiography, polarized light microscopy, and confocal Raman spectroscopy. Eight teeth were observed under scanning electron microscope. Four teeth were used for an X-ray diffraction. The mineral density data were analyzed statistically with the Mann-Whitney U test, using GraphPad InStat software. Both coronal dentin and radicular dentin were less mineralized in AI teeth when compared to control (respectively 6.2% and 6.8%; p<.001). Root dentinal walls were thin and irregular, while the cellular cementum layers were thick, reaching sometimes the cervical region of the tooth. Regular dentinal tubules and sclerotic dentin areas were noticed. Partially tubular or cellular dysplastic dentin and hyper-, normo-, or hypomineralized areas were noticed in the inter-radicular areas of hypoplastic AI teeth. The main mineral component was carbonate hydroxyapatite as explored by Raman spectroscopy and X-ray diffraction. Dentin and cementum in hypoplastic AI teeth are (i) hypomineralized, (ii) constituted of carbonate hydroxyapatite, and (iii) of non-homogenous structure.

Rural populations of the red fox Vulpes vulpes show little evidence of reproductive senescence

The ageing theory predicts fast and early senescence for fast-living species. We investigated whether the pattern of senescence of a medium-sized, fast-living and heavily-culled mammal, the red fox (Vulpes vulpes), fits this theoretical prediction. We used cross-sectional data from a large-scale culling experiment of red fox conducted over six years in five study sites located in two regions of France to explore the age-related variation in reproductive output. We used both placental scars and embryos counts from 755 vixens’ carcasses aged by the tooth cementum method (age range: 1–10), as proxies for litter size. Mean litter size per vixen was 4.7±1.4. Results from Generalized Additive Mixed Models revealed a significant variation of litter size with age. Litter size peaked at age 4 with 5.0±0.2 placental scars and decreased thereafter by 0.5 cubs per year. Interestingly, we found a different age-specific variation when counting embryos which reached a plateau at age 5–6 (5.5±0.2) and decreased slower than placental scars across older ages, pointing out embryo resorption as a potential physiological mechanism of reproductive senescence in the red fox. Contrary to our expectation, reproductive senescence is weak, occurs late in life and takes place at an age reached by less than 11.7% of the population such that very few females exhibit senescence in these heavily culled populations.

Uneven distribution of enamel, dentine and cementum in cheek teeth of domestic horses (Equus caballus): A micro computed tomography study.

BackgroundHypsodont equine cheek teeth possess large dental crowns, resting partly in the bony alveolus. Over a horse’s life cheek teeth erupt continuously to compensate for occlusal wear of 3–4 mm per year. Parts of the crown initially resting in the bony alveolus become progressively exposed at the occlusal surface with time. Hitherto, it is unclear whether the typical structure of the equine occlusal surface, composed of a complex arrangement of enamel, dentin and cementum, remains constant or undergoes structural changes with age. Therefore, we tested the hypothesis that the occlusal surface composition does not remain constant by a quantitative analysis of the dental substances at multiple levels along the dental crown of equine cheek teeth.MethodsMicro-computed tomography scans of 20 upper cheek teeth and 16 lower cheek teeth from 19 domestic horses were morphologically analysed using imaging and measurement software. Area for individual dental substances was measured at different levels from the apex to the occlusal surface. The data was statistically analysed to detect changes in the area of individual substance along the dental crown. The area of peripheral cementum was measured separately for levels inside and outside the bony alveolus.ResultsIn both, upper and lower cheek teeth, enamel area decreased in an apical direction, while dentine area increased. Peripheral Cementum increased dramatically in the occlusal/coronal extra-alveolar position.ConclusionWith increasing age the occlusal surface content of dentine increases while the content of enamel decreases. These changes are considered relevant for the detailed explanation of forage disruption in horses as well as for the recommendation of concepts in equine dentistry.

Tissue-specific mineralization defects in the periodontium of the Hyp mouse model of X-linked hypophosphatemia

X-linked hypophosphatemia (XLH) is a dento-osseous disorder caused by inactivating mutations in the PHEX gene, leading to renal phosphate wasting and hypophosphatemia, and impaired mineralization of bones and teeth. In the oral cavity, recent reports suggest a higher susceptibility of XLH patients to periodontitis, where patients present with impaired tooth cementum – a bone-like tissue involved in tooth attachment to the jaw bones and post-eruption tooth positioning – and a higher frequency of intrabony defects. In the present study, the pathobiology of alveolar bone and tooth cementum was investigated in the Hyp mouse, the murine analog of XLH. PHEX deficiency in XLH/Hyp dramatically alters the periodontal phenotype, with hypoplasia of tooth root cementum associated with a lack of periodontal ligament attachment and the presence of an immature apatitic mineral phase of all periodontal mineralized tissues. Challenging the Hyp periodontium in two surgical experimental models – ligature-induced periodontal breakdown and repair, and a model of tooth movement adaptation inducing cementum formation – we show that bone and cementum formation, and their healing, are altered. Bone and cementum mineralization appear similarly disturbed, where hypomineralized pericellular matrix surrounds cells, and where the protein osteopontin (OPN, a mineralization inhibitor) accumulates in a tissue-specific manner, most notably in the perilacunar matrix surrounding osteocytes. Although the pathobiology is different between XLH/Hyp bone and cementum, our results show a major XLH phenotype in oral mineralized tissues consistent with variations in patient susceptibility to periodontal disorders.

Distinct post-translational features of type I collagen are conserved in mouse and human periodontal ligament.

Specifics of the biochemical pathways that modulate collagen cross-links in the periodontal ligament (PDL) are not fully defined. Better knowledge of the collagen post-translational modifications that give PDL its distinct tissue properties is needed to understand the pathogenic mechanisms of human PDL destruction in periodontal disease. In this study, the post-translational phenotypes of human and mouse PDL type I collagen were surveyed using mass spectrometry. PDL is a highly specialized connective tissue that joins tooth cementum to alveolar bone. The main function of the PDL is to support the tooth within the alveolar bone while under occlusal load after tooth eruption. Almost half of the adult population in the USA has periodontal disease resulting from inflammatory destruction of the PDL, leading to tooth loss. Interestingly, PDL is unique from other ligamentous connective tissues as it has a high rate of turnover. Rapid turnover is believed to be an important characteristic for this specialized ligament to function within the oral-microbial environment. Like other ligaments, PDL is composed predominantly of type I collagen. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including hydroxylation, glycosylation and cross-linking. The chemistry, placement and quantity of intermolecular cross-links are believed to be important regulators of tissue-specific structural and mechanical properties of collagens. Type I collagen was isolated from several mouse and human tissues, including PDL, and analyzed by mass spectrometry for post-translational variances. The collagen telopeptide cross-linking lysines of PDL were found to be partially hydroxylated in human and mouse, as well as in other types of ligament. However, the degree of hydroxylation and glycosylation at the helical Lys87 cross-linking residue varied across species and between ligaments. These data suggest that different types of ligament collagen, notably PDL, appear to have evolved distinctive lysine/hydroxylysine cross-linking variations. Another distinguishing feature of PDL collagen is that, unlike other ligaments, it lacks any of the known prolyl 3-hydroxylase 2-catalyzed 3-hydroxyproline site modifications that characterize tendon and ligament collagens. This gives PDL a novel modification profile, with hybrid features of both ligament and skin collagens. This distinctive post-translational phenotype may be relevant for understanding why some individuals are at risk of rapid PDL destruction in periodontal disease and warrants further investigation. In addition, developing a murine model for studying PDL collagen may be useful for exploring potential clinical strategies for promoting PDL regeneration.

A preliminary application of dental cementum incremental analysis to determine the season-of-death of equids from the Xujiayao site, China

Seasonality study of Paleolithic archaeological faunal assemblages is important for reconstructing modes of animal death and ancient hominin strategies for prey acquisition. The dental cementum of animal teeth records the season of an individual animal in which it died, providing the necessary evidence for determining any patterns in season of death. This article presents the procedure and preliminary results of the application of cementum incremental analysis to equid teeth, which comprise the dominant component of the fauna from the Xujiayao Site—an early Late Pleistocene archaeological site in China. Results show multi-seasonal use in both the upper and lower layers of this site. Furthermore, attention is given to the method’s future application in seasonality studies at Paleolithic archaeological sites in China.

The role of fluoride and chlorhexidine in preserving hardness and mineralization of enamel and cementum after gamma irradiation.

The purpose of this study was to evaluate the effect of 0.05% sodium fluoride and 0.12% chlorhexidine mouthwashes on the micro-hardness of tooth enamel and cementum that was exposed to therapeutic doses of gamma radiation. Sixty extracted human teeth were divided into two groups, one was irradiated, the other was not irradiated. The two groups were further subdivided into three subgroups, which were each treated either with 0.05% sodium fluoride or with 0.12% chlorhexidine; the third subgroup served as a control. After demineralization-remineralization cycling, teeth from the irradiated groups showed a significantly lower micro-hardness when compared to those from the non-irradiated groups. Both in the irradiated and non-irradiated groups, teeth from the control subgroups showed a significantly lower micro-hardness, as compared to teeth treated with sodium fluoride and chlorhexidine. For non-irradiated enamel samples, those treated with chlorhexidine showed a significantly less micro-hardness compared to those treated with sodium fluoride. In contrast, irradiated enamel showed no significant difference in micro-hardness, whatever treatment (chlorhexidine or sodium fluoride) was applied. For cementum, treatment with chlorhexidine resulted in a significantly lower micro-hardness compared to sodium fluoride, both for the irradiated and non-irradiated groups. It is concluded that gamma irradiation with therapeutic doses typically used for head and neck carcinoma treatment has a direct effect in reducing micro-hardness of tooth enamel and cementum. Mouthwash protocols including, for example, application of 0.05% sodium fluoride or 0.12% chlorhexidine three times per day for 6weeks, can protect enamel and cementum against the reduction in hardness and demineralization caused by gamma irradiation. Sodium fluoride offers more protection compared to chlorhexidine.

SPARC and the N-propeptide of collagen I influence fibroblast proliferation and collagen assembly in the periodontal ligament.

The periodontal ligament (PDL) is a fibrous connective tissue that anchors tooth cementum into alveolar bone. Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein known to influence collagen fiber assembly in the PDL. In contrast, functional properties of the N-propeptide of collagen I, encoded in exon 2 of the COL1A1 gene, are poorly understood. In this study, the PDL of collagen I exon 2-deleted (wt/ko), SPARC-null (ko/wt), and double transgenic (ko/ko) mice were evaluated in terms of cellularity, collagen area, fiber morphology, and extraction force and compared to WT (wt/wt) mice. Picro sirius red staining indicated a decrease in total PDL collagen content in each of the transgenic mice compared to WT at 1 and 3 month age points. At 12 months, only SPARC-null (ko/wt) and double-null PDL demonstrated less total collagen versus WT. Likewise, an increase in thin PDL collagen fibers was observed at 1 and 3 months in each transgenic, with increases only in SPARC-null and double-null mice at 12 months. The force required for tooth extraction was significantly reduced in SPARC-null versus exon 2-deleted and WT mice, whereas double-null mice demonstrated further decreases in force required for tooth extraction. The number of proliferating fibroblasts and number and size of epithelial rests of Malassez were increased in each transgenic versus WT with double-null PDL exhibiting highest levels of proliferation and rests of Malassez at 1 month of age. Consistent with increases in PDL collagen in exon-2 deleted mice, with age, numbers of rests decreased at 12 months in this genotype. These results demonstrate for the first time a functional role of the N-propeptide in regulating collagen fiber assembly and cell behavior and suggest that SPARC and the N-propeptide of collagen I have distinct activities in regulating collagen fiber assembly and fibroblast function.

From the forests to teeth: Visual crossdating to refine age estimates in marine mammals

Estimation of the age of individuals is a fundamental parameter for understanding the biology of individuals and ecology of populations. Incremental growth deposits in tooth dentine or cementum, known as growth layer groups (GLGs), have been used to estimate the age of toothed marine mammals (odontocetes, pinnipeds) since the 1950s (Scheffer and Myrick 1980). In most species, it is assumed that GLGs are deposited annually (Hohn 2009). Validation of this assumption has only been possible in a small number of marine mammal species (e.g., Bowen et al. 1983, Myrick et al. 1984, Hohn et al. 1989, Lockyer 1993, Oosthuizen 1997), due to impracticalities in obtaining validated ages (Evans et al. 2002, Hohn 2009). For species in which validation of age is not possible, absolute age or the accuracy of age estimates cannot be confirmed (Campana 2001). Further, the precision of estimates (i.e., the closeness of repeat counts from an individual tooth) may not necessarily reflect the best estimate of age and, at present, an objective method to assist with identification of GLGs for age estimation purposes has not been established (Evans et al. 2002, Campana and Stewart 2014).

Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum.

Large-scale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. We present a comparative analysis of DNA preservation in these two substrates obtained from the same human skulls, across a range of different ages and preservation environments. Both substrates display significantly higher endogenous DNA content (average of 16.4% and 40.0% for teeth and petrous bones, respectively) than parietal skull bone (average of 2.2%). Despite sample-to-sample variation, petrous bone overall performs better than tooth cementum (p = 0.001). This difference, however, is driven largely by a cluster of viking skeletons from one particular locality, showing relatively poor molecular tooth preservation (<10% endogenous DNA). In the remaining skeletons there is no systematic difference between the two substrates. A crude preservation (good/bad) applied to each sample prior to DNA-extraction predicted the above/below 10% endogenous DNA threshold in 80% of the cases. Interestingly, we observe signficantly higher levels of cytosine to thymine deamination damage and lower proportions of mitochondrial/nuclear DNA in petrous bone compared to tooth cementum. Lastly, we show that petrous bones from ancient cremated individuals contain no measurable levels of authentic human DNA. Based on these findings we discuss the pros and cons of sampling the different elements.

Do cranial suture age and growth layer groups correlate in South American pinnipeds?

Age is one of the most important life history parameters required to understand the dynamics of mammalian populations. Growth Layers Groups (GLGs) are incremental units of calcified tissue in the teeth (dentine and cementum), which represent a pattern of cyclical deposition that can be counted. However, the estimation of absolute age in GLGs demands a skull with teeth, the permission to destroy part of a tooth, equipment to cut the teeth, and experienced GLGs readers. In 1954 Sivertsen proposed an alternative method using cranial suture age (CSA) to establish age categories. However, there are no studies validating the CSA in relation to GLGs. Thus, this study examined whether there is a correlation between age categories proposed by the CSA and chronological age in years from GLGs of South American fur seals (Arctocephalus australis) (N = 52) and of South American sea lions (Otaria flavescens) (N = 37). 93% of the skulls ofA. australisand 83.8% ofO. flavescenscorresponded accurately to the age in years estimated by each cranial suture age range. These results indicated the existence of high correspondence between the CSA and the GLGs age (r: 0.491 forA. australisandr: 0.675 forO. flavescens). However, an adaptation to Sivertsen's method is recommended: using only eight sutures (excluding the premaxillary-maxillary suture for CSA analysis, due to its late fusion), and updating the intervals for cranial sutures, that correspond to 16–32 = adults, 11–15 = young and 8–10 = pups.

Immunohistochemical Localization of Osteopontin - A Comparative Study on Periodontally Healthy and Diseased Tooth Root Surface

A Great deal of effort has been focused on trying to determine the presence and localization of factors responsible for cementogenesis, as well as in repair and regeneration of periodontal tissues. Bone sialoprotein and osteopontin are the two major non-collagenous proteins in cementum that appeared to have important roles in cementogenesis and regeneration. Aim: To investigate the expression and distribution of osteopontin on diseased root surface in comparison with healthy non-diseased root surface in by immunohistochemical technique using osteopontin mouse monoclonal antibody [NCL-O-PONTIN] (Novocastra) Materials and Methods: Fifty-Five teeth which included 25 permanent healthy human teeth and 30 permanent teeth affected by advanced periodontitis were collected. All teeth were initially fixed in 10% neutral buffered formalin. Following fixation and demineralization, specimens were embedded in paraffin, sectioned and exposed to antibodies against osteopontin. Stained sections were assessed using digital photomicroscope. Results: In healthy root surface staining intensity was found to be mild to moderate in the cemental surface and matrix. Moderate staining was found in relation to periodontal ligament. In diseased root surface there was absence of immunostaining for osteopontin (OPN) in exposed cementum. In, the apical part of the diseased tooth, a strong staining for OPN was seen along the cementum surfaces adjacent to the periodontal ligament. Conclusion: The absence of staining for OPN in all areas of exposed cementum (absence of overlying periodontal ligament) in diseased teeth can affect the ability for regeneration and new connective tissue attachment onto the previously denuded root surface.

Hertwig's epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by TGF-β.

In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwig's epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-β regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-β was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-β1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-β1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-βsignaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.

Microscopic Differences in Cementum Structure and Mineral Composition of Teeth Extracted from Patients with Gingivitis, Chronic Periodontitis and Aggressive Periodontitis. A Preliminary Comparative Study

Background: During the progression of periodontal diseases, cementum undergoes alterations in its structure and mineral composition. Aim: To detect different alterations in structure and mineral composition of cementum according to different periodontal statuses. Subjects and Methods: A true experimental study carried out among six extracted human teeth (One gingivitis of badly decayed tooth (used as control), two chronic periodontitis and three aggressive periodontitis (teeth were extracted due to severe bone loss and mobility). Specimens were taken from different parts of the cementum of the extracted teeth. Each specimen was put in one millilitre of sodium hypochlorite 10 % concentration for 24 hours, then dehydrated by 100% alcohol for half an hour and cementum structures was examined under Scanning Electron Microscope. Student t-test was used to determine the difference between the groups with the level of significance set at P value ≤0.05. Results: There is difference in structure and minerals components of cementum between the specimens of gingivitis, chronic and aggressive periodontitis. Chronic periodontitis has higher amount of minerals with cracks on cementum. Aggressive periodontitis has hypoplasia in cementum, cracks and there is unequal distribution of phosphate and calcium on cemental surface. Conclusion: Different diseases in the periodontium can cause different changes in cementum structure and minerals composition.

Raporturile tisulare la nivelul joncţiunii smalţ/cement

Tooth enamel/cementum junction represents an important area in the tooth regarding the incorporation of mineral tissues that bear high pressure forces. The level of the junction is important in measuring the depth of the periodontal sac and the level of attachment. On the tooth enamel/cementum junction, the tooth enamel, cementum and dentin connect in an unregulated curve. We will present, in this article, the morphology of the toot enamel/ cementum junction, the identification methods and it´s importance in clinical practice.

The circles of life: age at death estimation in burnt teeth through tooth cementum annulations.

Age at death estimation in burnt human remains is problematic due to the severe heat-induced modifications that may affect the skeleton after a burning event. The objective of this paper was to assess if cementochronology, which focuses on the cementum incremental lines, is a reliable method of age estimation in burnt remains. Besides the classical approach based on the counting of incremental lines, another approach based on the extrapolation of incremental lines taking into account the cement layer thickness and the incremental line thickness was investigated. A comparison of the performance of the two techniques was carried out on a sample of 60 identified monoradicular teeth that were recently extracted at dentist offices and then experimentally burnt at two maximum temperatures (400 and 900°C). Micrographs of cross-sections of the roots were taken via an optical microscope with magnification of ×100, ×200 and ×400. Incremental line counting and measurements were carried out with the ImageJ software. Age estimation based on incremental line counting in burnt teeth had no significant correlation with chronological age (p = 0.244 to 0.914) and led to large absolute mean errors (19 to 21years). In contrast, age estimation based on the extrapolation approach showed a significant correlation with known age (p = 0.449 to 0.484). In addition, the mean absolute error of the latter was much smaller (10 to 14years). The reason behind this discrepancy is the heat-induced dimensional changes of incremental lines that affect their visibility and individualization thus complicating line counting. Our results indicated that incremental lines extrapolation is successful at solving this problem and that the resulting age estimation is much more reliable.

Six1 is required for mouse dental follicle cell and human periodontal ligament‐derived cell proliferation

The periodontal ligament (PDL) is a connective tissue that attaches the tooth cementum to the alveolar bone and is derived from dental follicle cells (DFCs). The DFCs form fibroblasts, osteoblasts, cementoblasts, and PDL stem cells (PDLSCs). We previously reported homeobox transcription factor Six1 expression in mouse DFCs. However, the role of Six1 in periodontal tissue development is largely unknown. In this study, we analyzed SIX1 expression in mouse periodontal tissue cells during postnatal development and adulthood. We also addressed the role of SIX1 in mouse periodontium development and in human cultured PDL-derived cells (PDLCs). In mouse development, SIX1 production was abundant in DFCs and PDL cells by 2weeks, but it was greatly diminished in the PDL at 4weeks and in adults. Although the SIX1-positive cell distribution was sparse in the adult PDL, SIX1-positive cells were observed with low expression levels. We used 5-ethynyl-2'-deoxyuridine (EdU) for cell labeling to reveal numerous EdU/SIX1-double positive cells at 2weeks; however, a few EdU-positive cells remained at 4weeks. The proportion of DFCs that incorporated EdU was significantly lower in Six1-deficient mice compared with wild-type mice at E18.5. In human PDLCs, SIX1 was intensely expressed, and SIX1-knockdown using siRNA reduced proliferating PDLCs. Our results suggest that SIX1 is a key proliferation regulator in mouse DFCs and human PDLCs, which provides novel insight into Six family gene function in mammals.