Cementum annulation and age determination in Homo sapiens. II. Estimates and accuracy

The cementum annulation aging technique was evaluated in a sample of 80 clinically extracted premolars (age range 11–70 years). Demineralized thin sections (7μm) stained with hematoxylin were used. The correlation (r) between age and adjusted count (number of annulations added to age of tooth eruption) was 0.78 for the entire sample (N = 73) and 0.86 for a subsample in which teeth with periodontal disease were excluded (N = 55). Standard error of the estimates ranged from 4.7 to 9.7 years depending on sex and health status of the tooth. The technique provided significantly better estimates for females than for males. The overall inaccuracy (mean absolute error) of the technique was 6.0 years, with a bias (mean error) of 0.26 years. Reduced major axis regression of adjusted count on age produced a slope of 0.797 for the entire sample and 0.889 for the nonperiodontal disease subsample. These slopes are consistent with a hypothesis of annual deposition of cementum rings given a decrease in cementogenesis with increasing age.     

COUNTS OF GROWTH LAYER GROUPS IN CEMENTUM AND DENTINE IN RINGED SEALS (PHOCA HISPIDA)

We compared counts of growth layer groups (GLGs) in the dentine of un-decalcified, unstained cross-sections and in the cementum of decalcified, stained longitudinal sections of canine teeth from 144 ringed seals ( Phoca hispida ). Although there was a statistically significant correlation until approximately 10 GLGs, about 75% of paired readings at ≤ 10 cementum GLGs disagreed. After 10 GLGs, the number of GLGs in the cementum usually was greater. The maximum GLG count in cementum was 33, compared to a maximum in dentine of only 19. Interobserver differences in median counts were not statistically significant using cementum or dentine counts. Regression analysis revealed that for cementum in female seals, readers differed at higher counts (P < 0.05), and for dentine, there was a constant difference of about 0.6 GLGs ( P < 0.05) for male seals and 1.1 GLGs ( P < 0.05) for female seals. Counting GLGs in the cementum of decalcified and stained longitudinal sections provided higher counts and more agreement between readers, and it was the better of the methods examined for ageing ringed seals.     

Incremental lines in root cementum of human teeth —A reliable age marker?

In root cementum of teeth, alternating dark and light lines become visible in cross-sections under the light microscope. These lines bear an apparent resemblance to the annual rings of trees. Numerous studies have been done to correlate the number of cementum lines with the dental age by examining a great number of teeth of known age. Our study used a different approach. If lines in root cementum develop in an annual rhythm and are thus comparable to annual rings of trees, the same or at least a very similar number of these structures should be found in all areas of the root cementum of the same tooth. We counted cementum lines in the buccal, lingual, distal and mesial region of different sections, all from the middle third of the same root. This was repeated in eight teeth. To our surprise, we had immense difficulty in counting reproducible line numbers in the same cementum area at repeated counts. Nevertheless, the same tooth was found to differ markedly in the number of lines in different sections as well as in different regions of the same sections. These differences cannot be ascribed to variations caused by difficulties with reproducible line counting. Therefore, we are more than skeptical about the reliability of counting lines in root cementum as a method for determining the age of human teeth.     

Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues

Affinity-purified antibodies have been used in combination with the peroxidase-antiperoxidase technique to study the distribution of osteonectin and collagen types I and III in porcine dental tissues. Tissue sections (2 mm thick), including unerupted (fetal) or erupted (adult) teeth, were fixed in periodate-lysine-paraformaldehyde, demineralized in 12% w/v ethylenediaminetetraacetic acid, and after embedding, 6 micron sections were prepared for immunolocalization. Strong staining for osteonectin was observed in dentine of unerupted teeth and in the associated alveolar bone. Light to moderate staining was observed in the dental pulp, stratum intermedium, stellate reticulum, and the reticular elements in the endosteal spaces. In erupted teeth, osteonectin staining in dentine was concentrated around dentinal tubules and the associated alveolar bone stained with variable intensity. Cementum was poorly stained. However, the periodontal ligament and reticular material in the endosteal spaces showed moderate to strong staining. Weaker staining was apparent in the pulp and lamina propria of the gingiva. In comparison, type I collagen showed a similar distribution to osteonectin in both fetal and adult tissues, whereas type III collagen was generally restricted to the periodontal ligament, reticular elements of the endosteal spaces, and Sharpey's fibers in bone and cementum. Both odontoblast and ameloblast layers in fetal tissues stained for osteonectin and type III collagen.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components

The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.     

Age estimation of the White rhinoceros (Ceratotherium simum)

Age estimation criteria for the southern White rhinoceros ( Ceratotherium simum simum ) are presented both for free-ranging live animals and for cranial material. These are based on: (i) size appearance and horn development of live animals; (ii) stages of tooth eruption; (iii) tooth wear classes; (iv) attrition in height of the first molar tooth; (v) counts of cementum lines visible in tooth sections. Selected measurements are presented for live animals, skulls and horns.
For live animals, eight size classes are distinguished, seven of these covering immature animals up to ten years of age. Sixteen tooth wear classes are established, based on eruption and surface wear of maxillary dentition. Chronological ages were assigned from individually known animals followed in the field, and from skulls from animals for which exact records of age were available, or which could be assigned to an age category from appearance at death. Cementum line counts corresponded approximately with age in years, despite difficulties in interpreting lines. Some variability was observed, possibly related to nutritional conditions.
The maximum cementum line count obtained indicates a longevity of at least 40 years. Full body weight and socio-sexual maturity are attained by males between 10 and 15 years of age, while females first give birth between six and eight years of age. Sequences and times of tooth eruption are similar to those reported for the Black rhinoceros ( Diceros bicornis ).
Comparative cranial and body measurements are presented for the northern subspecies ( Ceratotherium simum cottoni ).     

Cementum annulus counts provide a means for age determination in Macaca mulatta (primates, anthropoidea)

14 teeth of 8 rhesus macaques (Macaca mulatta) of known age were analyzed to assess the usefulness of cementum annuli counts as a means of estimating chronological age. Methods used were histological examination of stained thin sections by light microscopy, and examination of polished and etched epoxy-embedded sections by scanning electron microscopy. In 11 of 14 cases, the known chronological ages of the individuals fell within the predicted age ranges based on cementum annuli counts; in 2 other cases, it fell within half a year of the ranges. Cementum annulus counts can provide valuable information about the age of primates living in semitropical environments. This is the most accurate method for aging skeletally adult primates that has yet been tested on animals of known age.     

Comparison of two histological techniques for age determination in small cetaceans

Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as "good quality" for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.     

Sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars

Methods of measuring tissue area from images of longitudinal thin tooth sections have been used to assess sexual dimorphism in the permanent dentition. The aim of this study was to demonstrate the extent of sexual dimorphism within the coronal tissue proportions of permanent mandibular canines and premolars, using area measurements of the enamel and dentine-pulp core. The sample consisted of embedded "half-tooth" sections from 45 individuals, all of known age-at-death and sex, collected from the St. Thomas' Anglican Church historic (1821-1874) cemetery site in Belleville, ON, Canada. The relative dentine-pulp area of the third premolars and canines displayed high levels of sexual dimorphism, as well as statistically significant mean differences between the sexes. The male canines and premolars have significantly more dentine than their female counterparts, as well as relatively more dentine with respect to overall crown size. The female canines and premolars have significantly more enamel relative to overall crown area than those of the males. These results suggest that relative area measures of crown tissues are more predictable measures of sexual dimorphism than absolute measures, and tissue proportions may remain constant despite intrasex variation in overall tooth crown size.     

A new method for the automated age-at-death evaluation by tooth-cementum annulation (TCA)

A valid age at death estimation is required in historical and also forensic anthropology. Tooth cementum annulation (TCA) is a method for age at death estimation of adult individuals. The method is based on light microscope images taken from tooth-root cross sections. The age is then estimated by manually counting the cementum incremental lines and adding this to the chronological age at the assumed point of tooth eruption. Manual line counting, however, is time consuming, potentially subjective and the number of individual counts is insufficient for statistical evaluations. Software developed for the automated evaluation of TCA images, that uses Fourier analysis and algorithms for image analysis and pattern recognition is presented here. It involves "line-by-line" scanning and the counting of gray scale peaks within a selected region-of-interest (ROI). Each scanning process of a particular ROI yields up to 400 counts that are subsequently statistically evaluated. This simple and time saving program seeks to substitute manual counting and supply consistent and reproducible results as well as reduce the demand of human error by eliminating unavoidable factors such as subjectivity and fatigue.     

Histometric features for the grading of prostatic carcinoma

Histometric features for the objective grading of prostatic adenocarcinoma in histologic specimens were analyzed in five cases each of well, moderately and poorly differentiated lesions. Tissue sections from the selected cases were stained by the Feulgen method and digitized by a video-based microphotometer. Twenty total fields were recorded for each grade: ten at high resolution (an image sampling of 0.5 micron per pixel) and ten at low resolution (0.8 micron per pixel), with two fields per case recorded at each resolution. The images were segmented by an automated expert system-guided scene segmentation procedure. The performance of that procedure was measured by comparing the automated counts of nuclei in the segmented fields to the visual counts made by a pathologist in the same fields. For well, moderately and poorly differentiated cases, respectively, the nuclear counts made by the expert system at high resolution were 2.7%, 4.2% and 4.7% higher than the visual counts (as estimated from a total of 6,628 nuclei), but 1.2%, 2.5% and 1.1% lower at low resolution (10,329 nuclei). High-resolution features and tissue textural features were computed for each case. The high-resolution features showed good separation between the three groups of cases. The tissue textural features showed consistent separation between well and moderately differentiated cases. The relaxation of the spatial resolution (to 0.8 micron/pixel spacing) did not affect the selection of features, but led to less separation between the data from different grades. In conclusion, the automated system performed satisfactorily in distinguishing sections of prostatic tumors of varying degrees of differentiation.     

Incremental lines of dental cementum in biological age estimation

The aim of this study is to evaluate the use of counting incremental lines of dental root cementum for biological age determination, and to compare it with alternative methods. Two samples were taken: 51 teeth from 49 individuals of known age obtained at the Stomatological Clinic, Vilnius University, as well as the canine teeth from the remains of 48 individuals from the mass graves of Tuskulenai in Vilnius (inhumed 1944-47). In the latter sample, the chronological age of 43 individuals was known through personal identification. Undecalcified teeth were sectioned with the Leica SP 1600 microtome diamond saw, and incremental line count as a blind test was made on sections of 35 to 100 microns thickness. Incremental line count was possible in 82-86 percent of cases. The results of three independent counts showed that intra-observer bias has no significant impact. Biological age was estimated by adding incremental line number to the average age of tooth eruption. It was found that mean absolute error was 6.46 years for the 1st sample, 6.27 years for the 2nd sample, and in some cases exceeded 10 years. For the 2nd sample, the results were compared to those of other methods such as endocranial suture ossification, pubic symphysis morphology and the "combined" method of Nemeskéri. All four methods yield a similar correlation in regard to an individual's chronological age. The highest correlation was found for the combined method, and the lowest one for public symphysis morphology. All correlations had a similar standard error. Thus our assessment is less enthusiastic than in some past studies; it is suggested that the incremental lines rather have a similar use as other methods.     

BIASES IN DOLPHIN AGE STRUCTURE DUE TO AGE ESTIMATION TECHNIQUE1

The use of different tooth-preparation techniques resulted in widely different estimates of age in a sample of bottlenose dolphins, Tursiops truncatus. Teeth from 30 animals were prepared using the two most prevalent techniques reported in the literature for this species, unstained sections and decalcified and stained thin sections, and the resulting paired counts of growth layers were compared. Estimates from the two methods were identical or at least placed the specimen in the same age class in only five cases, ranging in age from 2 to 22 yr. Otherwise, the results fell into one of two categories: when the estimates were close (± 3-yr difference, n= 15), counts from unstained sections generally were higher (13 cases, age from unstained sections 2-20 yr); when the counts were more disparate, estimates from stained sections always were higher (6-31 yr difference, n= 10, age from unstained sections 12-27 yr and corresponding ages from stained sections of 27-47). Previous studies of age estimation in known-age bottlenose dolphins indicate that stained sections allow accurate estimates of age and demonstrate that maximum lifespan approaches or exceeds 50 yr. In contrast, the results herein suggest that using unstained sections for age estimation may result in imprecise or biased age-structure data.     

Improved Method for Determination of Respiring Individual Microorganisms in Natural Waters

A method is reported that combines the microscopic determinations of specific, individual, respiring microorganisms by the detection of electron transport system activity and the total number of organisms of an estuarine population by epifluorescence microscopy. An active cellular electron transport system specifically reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, which is recognized as opaque intracellular deposits in microorganisms stained with acridine orange. In a comparison of previously described sample preparation techniques, a loss of >70% of the counts of INT-reducing microorganisms was shown to be due to the dissolution of INT-formazan deposits by immersion oil (used in microscopy). In addition, significantly fewer fluorescing microorganisms and INT-formazan deposits, both ≤0.2 μm in size, were found for sample preparations that included a Nuclepore filter. Visual clarity was enhanced, and significantly greater direct counts and counts of INT-reducing microorganisms were recognized by transferring microorganisms from a filter to a gelatin film on a cover glass, followed by coating the sample with additional gelatin to produce a transparent matrix. With this method, the number of INT-reducing microorganisms determined for a Chesapeake Bay water sample was 2-to 10-fold greater than the number of respiring organisms reported previously for marine or freshwater samples. INT-reducing microorganisms constituted 61% of the total direct counts determined for a Chesapeake Bay water sample. This is the highest percentage of metabolically active microorganisms of any aquatic population reported using a method which determines both total counts and specific activity.     

Age estimation and growth layer patterns in teeth of crabeater seals: A comparison of techniques

Age estimation of marine mammals provides important information about ecological and life history parameters. Counting growth layer groups (GLGs) in the dentine and cementum of teeth is the most common technique for age estimation in pinnipeds. In this study, we used acid-etched canines (n = 38) and decalcified stained postcanine sections (n = 40) to calibrate readings in mummified crabeater seals (Lobodon carcinophaga). A subsample of this group received a prior cleaning treatment of boiling the teeth with hydrogen peroxide (H₂O₂). Ages ranged from 0 to 31 years. Dentine from canines and cementum from postcanines showed higher estimated ages (maximum 31 years) than dentine from postcanines (maximum 24 years), mainly in those teeth that did not receive prior treatment. Boiling with H₂O₂ has shown to affect cementum structure and, thus, results in underestimated ages, but dentine did not seem to be affected. The results presented here showed that ages can be estimated for the crabeater seal regardless of which tooth/tissue or method is used, at least for seals <13 years old. However, nontreated, stained thin sections of postcanine teeth are recommended because more reliable age estimations in older crabeater seals are obtained.     

Probit and survival analysis of tooth emergence ages in a mixed-longitudinal sample of chimpanzees (Pan troglodytes).

Tooth emergence data from a mixed-longitudinal sample of 58 chimpanzees of known age were analyzed using probit and survival techniques to produce median emergence ages, ranges of variability, and emergence sequences for primary and permanent teeth. Between-group comparisons were made to test for statistically significant differences in emergence ages. No such differences were found between right and left sides, or between maxilla and mandible, for any primary or permanent teeth. Male-female comparisons did demonstrate significant emergence-age differences for some teeth, although they were not always bilaterally symmetrical. More complete data are required to further clarify the nature of sex differences in tooth emergence in chimpanzees. Regression models for age prediction from the number of emerged teeth were generated and indicate that males achieve a given number of emerged teeth at a significantly later age than females. However, when fewer than five teeth have emerged, males are predicted to be younger than females. The sizable root mean square error values for these models suggest that this method of age prediction has limited usefulness owing to the amount of variability in timing of tooth emergence in chimpanzees. The implications of these data for studies on tooth emergence in early hominids are addressed.     

Permanent tooth emergence in Gujjars of Punjab, India.

Patterns of permanent tooth emergence in Gujjars were studied in a cross-sectional sample of 483 children ranging in age from 6 to 13 years. Females were markedly advanced in permanent tooth emergence times over males, but no such sex differences were observed in sequence of emergence. Differences between median emergence times of right and left side antimers were significant for only 4 of 28 instances (14.29%), namely central incisors, mandibular first molars in males and lateral maxillary incisors in females. In general mandibular teeth except premolars tended to emerge earlier than their maxillary counterparts. The quiescent period between first and second tooth emergence stages was longer in males than in females. Mandibular depth and morphological facial length were very significantly correlated (p < 0.01) with the number of permanent teeth present in the oral cavity.     

The actin filament content of hair cells of the bird cochlea is nearly constant even though the length, width, and number of stereocilia vary depending on the hair cell location

By direct counts off scanning electron micrographs, we determined the number of stereocilia per hair cell of the chicken cochlea as a function of the position of the hair cell on the cochlea. Micrographs of thin cross sections of stereociliary bundles located at known positions on the cochlea were enlarged and the total number of actin filaments per stereocilium was counted and recorded. By comparing the counts of filament number with measurements of actin filament bundle width of the same stereocilium, we were able to relate actin filament bundle width to filament number with an error margin (r2) of 16%. Combining this data with data already published or in the process of publication from our laboratory on the length and width of stereocilia, we were able to calculate the total length of actin filaments present in stereociliary bundles of hair cells located at a variety of positions on the cochlea. We found that stereociliary bundles of hair cells contain 80,000-98,000 micron of actin filament, i.e., the concentration of actin is constant in all hair cells with a range of values that is less than our error in measurement and/or biological variation, the greatest variation being in relating the diameters of the stereocilia to filament number. We also calculated the membrane surface needed to cover the stereocilia of hair cells located throughout the cochlea. The values (172-192 micron 2) are also constant. The implications of our observation that the total amount of actin is constant even though the length, width, and number of stereocilia per hair cell vary are discussed.     

Recommendations for the forensic diagnosis of sex and age from skeletons.

In both diagnostic fields a two-stage strategy is recommended: to first use "field" methods that are quick and easy but more imprecise and then "laboratory" methods that are time consuming but more precise. In preparing skeletal work, individuality of a skeleton should be checked, traces of diseases sought and time since death assessed. For sexing non-adults, the field methods are tooth mineralisation, long bone length and a few morphological skull and pelvis characteristics, for adults it is the morphology of pelvis and skull, and for both age groups the advanced laboratory method is molecular biology. For ageing non-adults the methods are mineralisation of teeth, long bone length and epiphysis development. For ageing adults the advanced laboratory method is aspartic acid racemisation. Less accurate laboratory methods are cement ring counts and histology of bones and teeth. Quick morphological methods using the pubic symphysis and other traits in combinations follow. Finally, cranial sutures and tooth number give a quick and rough impression. For the selection of a method and the assessment of its value the stochastic error produced for the reference sample is the decisive criterion; it should also be used to assess the reliability of a single diagnosis. Prerequisites for all work with skeletons are not only a complete knowledge of the relevant biology as well as specific techniques but also initial detailed instructions and with forensic applications, personal experience.