Dietary health does affect histological age assessment: an evaluation of the Stout and Paine (1992) age estimation equation using secondary osteons from the rib

An age at death estimation equation that uses rib histological variables presented by Stout and Paine was used to evaluate a skeletal population of individuals with a known age at death and cause of death from either malnutrition or the niacin deficiency disease pellagra. The sample was comprised of 26 autopsied black South Africans. Histological analysis of mounted thin sections involved the microscopic measurement of cortical area and a count of the number of intact and fragmentary secondary osteons for the entire cross-section of the rib. Rib osteon population density values were then calculated for each case. It was found that this equation under-aged individuals on average by 29.2 years. Overall, secondary osteon size and Haversian canals tended to be larger than expected, while cortical bone area was less when compared with a control population. The implications of these findings are critical given that many of the skeletal remains examined by forensic anthropologists come from marginalized backgrounds, including malnutrition. This research suggests that measurements based on healthy cases may not be useful in an analysis of individuals with poor diet and health. It is argued that new standards for histological age assessment methods need to be created that account for variation in the health status of individuals examined by forensic anthropologists.     

Quantification of Osteon Morphology Using Geometric Histomorphometrics

Many histological methods in forensic anthropology utilize combinations of traditional histomorphometric parameters which may not accurately describe the morphology of microstructural features. Here, we report the novel application of a geometric morphometric method suitable when considering structures without anatomically homologous landmarks for the quantification of complete secondary osteon size and morphology. The method is tested for its suitability in the measurement of intact secondary osteons using osteons digitized from transverse femoral diaphyseal sections prepared from two human individuals. The results of methodological testing demonstrate the efficacy of the technique when applied to intact secondary osteons. In providing accurate characterization of micromorphology within the robust mathematical framework of geometric morphometrics, this method may surpass traditional histomorphometric variables currently employed in forensic research and practice. A preliminary study of the intersectional histomorphometric variation within the femoral diaphysis is made using this geometric histomorphometric method to demonstrate its potential.     

儿童骨骼特征的组织学研究

目的研究儿童骨骼的组织学特征,为儿童骨骼残片的法医学鉴定提供科学依据。方法提取儿童四肢长骨骨片,制作骨骼的组织切片,在显微镜下观察内、外环骨板,骨单位,骨细胞的组织学结构,比较儿童骨骼与动物及成人骨骼的组织学区别。结果儿童骨骼的骨单位形态与成人相似,骨单位间存在大量的内层骨板,与动物的骨组织特征相似。结论根据骨组织学特征,可以确定儿童的骨骼残片。     

Analysis of histomorphometric variables: Proposal and validation of osteon definitions

Histological analysis of bone tissue has been used to explore a variety of questions relating to age-at-death, habitual behaviors, health, and nutritional stress. Identification of intact and fragmentary osteons is of key interest to many researchers in these studies, yet the definitions of these features vary between researchers making cross-study comparisons problematic. Furthermore, histological variable definitions are often ambiguous or require subjective classifications by the observer. As a result, and as indicated by previous studies, observer error and misclassification of certain variables, namely intact and fragmentary osteons, can be significant. This study proposes new definitions for intact and fragmentary osteons that are designed to limit observer subjectivity and also explore efficacy of combining osteon types into one variable. A sample of 30 6th rib cross-sections from a modern forensic population was used to test the validity of the proposed definitions. Observations of intact osteon population density (OPD(I)) and fragmentary osteon population density (OPD(F)) were made by three observers for each cross-section. These observations were used to explore the interobserver error associated with the proposed definitions and determine if combining variables into one variable (OPD) mitigates persisting classification difficulties. Results indicate that the proposed definitions significantly reduce interobserver error and misclassification of intact and fragmentary osteons. However, the interobserver error associated with fragmentary osteons is still high. Evaluation of the variables independently indicates that combining variables has potential to reduce the predictive strength of an age estimation model and the ability to interpret age-related bone remodeling.     

Differentiating fragmented human and nonhuman long bone using osteon circularity

Distinguishing between human and nonhuman bone is important in forensic anthropology and archeology when remains are fragmentary and DNA cannot be obtained. Histological examination of bone is affordable and practical in such situations. This study suggests using osteon circularity to distinguish human bone fragments and hypothesizes that osteons will more closely resemble a perfect circle in nonhumans than in humans. Standard histological methods were used, and circularity was determined using an image analysis program, where circularity was controlled for by Haversian canal measurements. Homogeneity was first tested for multiple variables within human and nonhuman samples. No significant differences were found between human sexes (p = 0.657) or among nonhuman species (p = 0.553). Significant differences were found among intraskeletal elements of both humans (p = 0.016) and nonhumans (p = 0.013) and between pooled samples of humans and nonhumans (p < 0.001). Results of this study indicate that osteon circularity can be used to distinguish between fragmented human and nonhuman long bone.     

丛状骨和骨单位带的种属特征及法医学应用价值

目的 探讨丛状骨和骨单位带的种属特征及其法医学应用价值。方法 人及猪、牛、羊、狗长骨中段横切磨片35张，在光学显微镜下观察骨组织中丛状骨和骨单位带的形态学特征。结果 丛状骨和骨单位带在猪、牛、羊、狗的密质骨中广泛存在，人骨组织中几乎不见；同种动物不同个体之间骨组织学结构也存在差异。结论 观察丛状骨和骨单位带是根据人骨鉴别种属的重要排除指标。     

Differences in osteon banding between human and nonhuman bone

The objective of this paper is to compare patterns of osteon organization in human and nonhuman bone. A linear organization of Haversian systems in nonhuman bone, where osteons line up in rows, has been reported but has not been quantified. The present research provides a quantitative examination of this observation through a comparative analysis of the femoral midshaft from human and nonhuman bone. Femoral midshaft thin sections from 60 humans were compared to femoral midshaft sections from nine sheep and six miniature swine. The presence or absence of osteon banding was recorded and, if present, described. Results indicate that 2 out of 60 human sections and 5 out of 15 nonhuman sections exhibit osteon banding (chi2 = 9.46; p < 0.01). Further, the type of banding present in the human and nonhuman samples is easily distinguished, indicating that human and nonhuman bone can be distinguished where handing is present in this study.     

Population-specific histological age-estimating method: a model for known African-American and European-American skeletal remains

Previously developed histological age-estimating methods have been based on samples lacking interpopulation variability. A comparison of age-associated rib histomorphometrics between an European-American sample and an African-American sample indicates that ethnicity can have a significant effect on osteon population density (OPD), osteon cross-sectional area (On.Ar), and relative cortical area (Ct.Ar/Tt.Ar). Based upon these findings, new histological age-predicting formulae are presented that are recommended when estimating age for African-American or European-American skeletal remains. A general formula that is applicable to remains of unknown ethnicity is also provided.     

Assessment of age at death by microscopy: unbiased quantification of secondary osteons in femoral cross sections

The microscopic method of age at death determination was introduced by Kerley in 1965 [E.R. Kerley, The microscopic determination of age in human bone, Am. J. Phys. Anthropol, 23 (1965) 149-163.]. However, even though the method has been revised several times, there remain some fundamental issues concerning the reliability of the methods. This is because several basic histological features seen in a cross section of a bone have to be quantified according to the method, but the definition, and hence quantification, of these features leaves room for subjectivity. In a previous study we found that some of these features (osteon fragments and Haversian canals) could not be identified reliably. Only secondary osteons could be identified with a low inter and intra observer error. Furthermore, since the histological features are quantified in only parts of an entire bone cross section, the selection of these parts or areas is a potential source of bias. Finally, unless an unbiased method is used for addressing features on the borders of the selected areas, this will also introduce error. These issues have not been addressed specifically in previous studies. In this study, we used the methods of stereology to choose the regions of interest, as well as for dealing with border phenomena, and we only counted secondary osteons. Our results show a statistically significant increase in the median number of osteons per area unit with increasing age at death. However, this was after exclusion of one outlier. This result is probably due to the limited sample size (N = 24). As such, this study is preliminary, but does warrant applying the described techniques to a larger sample.     

Estimation of age at death by tibial osteon remodeling in an autopsy series

Estimation of age at death by histological methods in tibiae has been found to be inaccurate in individuals less than 55 years of age. This study describes reasons for the inaccurate age estimates and provides a new regression equation for age estimation. Bone cores were removed from tibiae in 53 individuals at autopsy ranging in age from 17 years to 53 years of age; 48 males and 5 females. The sample contained 41 whites and 12 blacks. Variables ascertained from each core were: cortical thickness, cortical bone density, secondary osteon number, area, and perimeter, and Haversian canal area and perimeter. The results showed significant differences in secondary osteon number, area, and perimeter between groups aged less than and greater than 35 years of age. Multiple linear regression analysis indicated osteon number to be the best predictor of age in this sample aged less than 55 years of age. Age at death was accurately estimated for 11 additional forensic cases using osteon number in tibiae. No significant histological differences were observed between males and females and between blacks and caucasians.     

Chronic Alcohol Abuse Leads to Low Bone Mass with No General Loss of Bone Structure or Bone Mechanical Strength,

Chronic alcohol abuse (CAA) has deleterious effects on skeletal health. This study examined the impact of CAA on bone with regard to bone density, structure, and strength. Bone specimens from 42 individuals with CAA and 42 individuals without alcohol abuse were obtained at autopsy. Dual‐energy X‐ray absorptiometry (DEXA), compression testing, ashing, and bone histomorphometry were performed. Individuals with CAA had significantly lower bone mineral density (BMD) in the femoral neck and significantly lower bone volume demonstrated by thinner trabeculae, decreased extent of osteoid surfaces, and lower mean wall thickness of trabecular osteons compared to individuals without alcohol abuse. No significant difference was found for bone strength and structure. Conclusion: CAA leads to low bone mass due to a decrease in bone formation but with no destruction of bone architecture nor a decrease in bone strength. It is questionable whether this per se increases fracture risk.     

Intra- and inter-observer variation in histological criteria used in age at death determination based on femoral cortical bone

The microscopic method of age at death determination was introduced by Kerley in 1965. The method, which relies on the quantification of selected elements in cortical bone tissue, has been widely used, and several other researchers have modified or added to the method. Yet, very few studies have been carried out dealing with the intra- and inter-observer error. Furthermore, when such studies have been completed, the statistical tools for assessing variability have not been adequate. This study presents the results of applying simple quantitative statistics on several counts of microscopic elements as observed on photographic images of cortical bone, in order to assess intra- and inter-observer error. Overall, substantial error was present at the level of identifying and counting secondary osteons, osteon fragments and Haversian canals. Only secondary osteons can be reliably identified, precluding the use of osteon fragments and Haversian canals. The observers in this study included experienced and inexperienced users of the microscopic method, yet the variability was uniformly large for all observers, suggesting fundamental problems in definition and identification of the structural elements. Until more rigorous definitions of such elements have been agreed upon, the use of microscopical methods must be discouraged as a sole or uncontrolled method of evaluating age at death.     

人与动物骨组织特征的比较研究

目的研究区别骨骼残片是否为人类骨骼的方法。方法提取人体骨骼及鱼类、两栖类、爬行类、鸟类、哺乳类、灵长类等动物的骨骼标本制做骨骼组织学片,在显微镜下观察,将显微镜下的图像录入电脑进行分析。结果内、外环骨板,骨单位,骨细胞的显微镜下的结构,人类与动物存在明显差别。结论根据骨骼的组织学特征可以区别人类与动物的骨骼。     

New Formulas to Estimate Age at Death in Maya Populations Using Histomorphological Changes in the Fourth Human Rib*

Abstract: This study develops new histomorphological algorithms for Maya populations’ human ribs and tests the applicability of published algorithms. Thin sections from the fourth rib of 36 individuals of known age were analyzed under polarized light microscopy. Osteon population density (OPD, the concentration of intact and fragmented osteons per mm²), cortical area (CA), and osteon size (OS) were recorded. Seven algorithms were calculated, using all combinations of variables, and compared to the performance of published formulas. The OPD‐based formulas deviate from the known age 8.7 years on average, while those from OS and CA deviate between 10.7 and 12.8 years. In comparison, our OPD‐based algorithms perform better than the one by Stout and Paine and much better than Cho et al. In conclusion, algorithms should be developed using OPD for different ethnic groups; although Stout and Paine’s can be used for Maya and maybe Mesoamerican individuals.     

Assessment of histomorphological features of the sternal end of the fourth rib for age estimation in Koreans

The aim of this study was to assess the histomorphological features of the fourth rib and to develop age-predicting equations for Koreans. Sixty-four rib samples (36 males and 28 females) obtained from forensic cases were used for developing equations. Two thin sections (<100-microm thick) per sample were prepared by manual grinding. Multivariate analysis of covariance revealed statistically significant differences in age-adjusted histomorphological variables between sexes. Using stepwise regression analysis, osteon population density and average osteon area were correlated with unknown sex (r2 = 0.826), and sex plus two histomorphological variables provided the best results for an age-predicting equation given the assumption of knowing the sex of a specimen (r2 = 0.839). Average Haversian canal area had little influence on age estimation for male or female samples, and relative cortical area was not significantly related to age for any specimen.     

A Four‐Stage Method of Age at Death Estimation for Use in the Subadult Rib Cortex*,†

Abstract: Age estimation in the subadult skeleton can be rather precise when the epiphyses and dentition are present, but incomplete or commingled remains still present a challenge. Histomorphometric age‐at‐death estimation methods developed for use on adults are based on the age‐associated accumulation of osteons. In the growing skeleton, there is a poor correlation between osteon numbers and age until the latter half of the second decade. As a result, there has been no histological aging method for use in subadults. The analysis of the rib cortex of 72 subadults ranging in age from 2 to 21 years has identified a series of developmental changes in the bone microstructure that can be used to estimate age. This qualitative method utilizes the systematic changes in rib cortical morphology to classify ribs into one of four age phases. This method can be applied to immature skeletons in forensic, archaeological, and paleontological contexts.     

Effects of obesity on talar micro- and macro-morphology

The addition of information regarding obesity status to the forensic anthropological biological profile could significantly contribute to the identification of human skeletal remains since over 40% of the U.S. adult population is currently obese. This study examines the differences in talar shape and trabecular bone structure between obese and non-obese individuals. A sample of 20 obese and 20 non-obese divided evenly by sex was selected from the Texas State University Donated Skeletal Collection. Tali were imaged using x-ray computed tomography (voxel size: 28–38.7 μm). Image stacks were processed to produce binary images as well as trabecular thickness and spacing maps. Landmark-based geometric morphometric analyses were conducted to quantify shape variation. Shape coordinates were used to locate 100 geometrically homologous volumes of interest within each talus. Bone volume fraction, trabecular thickness, and trabecular spacing were extracted at each volume of interest. Within each sex, a one-way ANCOVA was used to determine if significant differences exist between obese and non-obese individuals in trabecular bone after controlling for age. The size of the talus as well as subtle aspects of shape were found to distinguish the sexes. The results further indicate that bone volume fraction significantly differs between obese and non-obese males. In females, bone volume fraction is correlated with age but does not differ between obese and non-obese. The study demonstrates that bone microstructure is a promising approach to estimating body mass or body mass index category but age effects diminish the potential for the talus to be used alone.     

Estimation of age from the femur of Japanese cadavers

The purpose of this study is to estimate the age of cadavers by histomorphometry of the femur. Seventy-two Japanese males ranged from 43 days to 92 years old and 26 females ranged from 2 to 88 years old were used. The thickness of sections was adjusted at 50 to 70 microns by grinding with sand paper. The sections were not decalcificated. They were stained with Villanueva's bone staining powder and with thionin dye. Microradiographs of the sections were obtained by the soft X-ray apparatus. The area, maximum and minimum diameter, and perimeter of the perfect osteon and Haversian canal were measured. In addition, the type II osteon number, osteon fragment number, and area of triangle were also determined. All these parameters were examined by an image analyzer. The parameters of the osteon showed high correlation coefficient with age (magnitude of r > 0.77), while those of the Haversian canal were low (magnitude of r < 0.11). All parameters were subjected to multiple regression analysis for producing a multiple regression equation of age estimation. For the stepwise selecting method, the perimeter of osteon, maximum length of the Haversian canal and osteon fragment number were selected for the equation. Their multiple r2 and standard error of estimation were 0.8874 and 6.39, respectively. For the forward selection method, in addition to the above items, three parameters, the maximum length of Haversian canal, triangle area, osteon fragment number were selected. Their multiple r2 and standard error of estimation were 0.9484 and 4.884, respectively. Bone staining was useful to clarify the demarcation between osteon and fragment, leading to an increase in the accuracy of age estimation. However, the entire range from birth to 90 years was difficult to cover for precise age estimation.     

The Frontal Sinus Cavity Exhibits Sexual Dimorphism in 3D Cone‐beam CT Images and can be Used for Sex Determination

Few studies have assessed the use of the frontal sinus cavities for sex differentiation. In this study, a new methodology was used to assess the reliability of CBCT images of the frontal sinus cavity for determining sex based on the sexual dimorphism found in this anatomical structure. The survey sample consisted of 130 scans that were reconstructed three‐dimensionally. 3D images of the cavity volume in the frontal, lateral, and basal views were exported in TIFF. The following variables were measured in a second program: area, perimeter, bounding rectangle, ellipse fit, circularity, aspect ratio, roundness, solidity, and Feret's diameter. The methodology demonstrated the existence of sexual dimorphism with an accuracy of 80.0% in the logistic regression model. The basal view had the greatest explanatory power in the final model. This methodology may be used as an alternative way of determining a reliable biological profile during the analysis of skeletal remains.     

Rib Variation at Multiple Locations and Implications for Histological Age Estimation*

Existing histological age estimation methods using the rib were developed mainly from the midshaft; however, in forensic practice, uncertainty of sampling location often arises due to fragmented or previously sampled ribs. The potential for error increases when sampling location is uncertain and utilizing a section beyond the midshaft (either anterior or posterior) may result in erroneous age estimates. Additionally, there is debate within the field regarding the minimum number of sections needed for accurate age estimation. The aim of this research is to determine the importance of the midshaft distinction for age-at-death assessment and the necessity of analyzing serial sections by evaluating histological variables at sampling locations along the length of the rib. Three seriated histological sections at three sampling locations (anterior, midshaft, and posterior) were obtained from sixth ribs of ten postmortem human subjects. Cortical area (Ct.Ar) and osteon population density (OPD) were collected from each section (n = 90). Significant differences were determined in Ct.Ar between sampling locations, demonstrating the variation present along the length of the rib. A comparison of OPD at sampling locations revealed significant differences, suggesting that sampling site is critical to accurate age estimates. When sampling location is uncertain, a more anterior section should be taken. Analysis of serial sections within locations revealed no significant differences in OPD or Ct.Ar, supporting the practice of collecting data from one section for age estimation. While an age estimate can be achieved through the analysis of one section, best practice suggests reading two sections to capture intraindividual variation.