Age estimation in the combined long bones and ribs by histomorphometry: Past, present, and future.

Numerous age estimation methods in unidentified bone have been a long time developing for application in forensic anthropology. The histomorphometric technique is one of the alternative methods that relied upon the evaluation of the cortical bone microstructure over the lifespan as a result of the remodeling process in bone. Remodeling is a sophisticated event occurring from the coupled function of bone formation and resorption cells for maintaining mineral homeostasis and repairment of microdamage in bone tissue. Products derived from remodeling are primary changes in the osteon or haversian system in various regions in the cortical bone, including periosteum, endosteum, and trabecular bone. Throughout life, bone remodeling rate with osteon alteration can be predictable. In the forensic field, histological methods are getting more attention due to the unavailability of macroscopic methods. Histomorphometry approach can be accomplished in fragmentary or incomplete bone remains indicating the limited use of gross morphological methods. In addition, the microscopic methods can aid to increase the more accuracy of analyses and diminish the biased subjective assessment for determining age. Most histomorphometry method utilizes a cross-section of the midshaft of the long bones including the mandible, rib, and clavicle. This review provides the basic knowledge of bone biology and anatomy, several age-estimating methods of histology, and crucial factors for age methods. Studies regarding overall age determination methods from the past until now contribute to obtaining more benefits for developing methods of histomorphometry using human bone in forensic identification.

Bone histomorphometry of the clavicle in a forensic sample from Albania.

Forensic assessment of skeletal material includes age estimation of unknown individuals. When dealing with extremely fragmented human remains that lack macro-features used in age estimation, histological assessment of the skeletal elements can be employed. Historically, microscopic methods for age assessment used by forensic anthropologists have been available since 1965. Several skeletal elements have been used for this purpose. Among them, the clavicle has garnered very little attention. The purpose of this study is to explore the validity of clavicular histomorphometry as an age marker in a modern Balkan sample. This study examined a modern clavicular autopsy sample from Albania. The sample consisted of 33 individuals of known age and cause of death. Data were collected for micro-anatomical features including osteon population density (OPD) and cortical area. Intra- and inter-observer errors were assessed through technical error of measurement (TEM) and R coefficient. A validation study was performed in order to test the accuracy of existing histological formulae. Regression analysis was run to developed age prediction models with the best models tested through cross-validation and the comparison between OPD for the Albanian sample and a European-American sample examined. Intra- and inter-observer error TEM results demonstrated values falling within the limits of acceptance. The existing histological methods did not perform accurately on the sample under study. Regression equations for Albanians produced age estimations deviating 8 and 11 years from known age. Cross-validation on the most accurate regression formula which includes OPD as a single variable demonstrated similar mean errors. Statistically significant differences were observed between the Albanian and the European-American population when the two samples were compared. The research presented is the fifth article published and the fifth population explored on clavicular microstructure. The potential of histology to estimate age on the Albanian population is shown here; however, population effect, diet and health status might be considered. Further inclusion of individuals will corroborate our preliminary findings.

Using histomorphometry for human and nonhuman distinction: A test of four methods on fresh and archaeological fragmented bones.

Positive identification of human remains is the very first step in anthropological analysis, and the task may be particularly difficult in the case of fragmented bones. Histomorphometry methods have been developed to discriminate human from nonhuman bones, based on differences in the size and shape of Haversian systems between the two groups. Those methods all focus on a very specific type of bone, section, and zone. Therefore, the objective of this study was to test the efficiency of four histomorphometric methods on a sample of fragmented bones. The sample is composed of 37 archaeological and fresh specimens, 25 nonhumans (Bos taurus, Equus caballus, Sus scrofa, Capreolus, Canis familiaris, Cervus elaphus, Ovis, and Capra) and 12 humans (Homo sapiens). Eight histomorphometric criteria were collected from all intact osteons visible on each fragment and then inserted into the corresponding discriminate function of each method. The results were compared with the real origin to establish rates of correct classification for each method. The methods of Martiniaková et al. (2006) and Crescimanno and Stout (2012) obtained very low percentages of good classification (32 % and 67 %). Those of Cattaneo et al. (1999) obtained 94 % correct classification, but only after a correction of the units of measurement for Haversian canal area in their formula. The methods of Dominguez and Crowder (2012) obtained an 86 % rate for well-classified specimens. Some of the methods tested here contain errors in the original publication that make them unusable in their current state. Plus, it seems that histomorphometric methods developed from specific areas are more difficult to apply to fragments. A reduced number of intact osteons analyzed may partially affect the reliability of the method by being unrepresentative of the entire microstructure. Therefore, this study demonstrates that one should be cautious with the use of histomorphometric methods to distinguish human and nonhuman fragmented bone until further research can refine these methods to achieve greater reliability.

The impact of burial period on compact bone microstructure: Histological analysis of matrix loss and cell integrity in human bones exhumed from tropical soil.

Human bone histological analysis is a useful tool to assess post mortem diagenesis and to predict successful nuclear DNA typing of forensic material. This study is part of a series of studies developed by the authors intended to improve the understanding of post mortem diagenesis and to develop applications for DNA analysis of skeletal species from tropical soils, in order to optimize genetic and anthropological protocols. The aim of this study was to analyze the impact of burial period on the integrity of exhumed compact bone microstructure from tropical climate. In fragments of exhumed human femora from 39 individuals from the same cemetery (exhumed group) and 5 fresh femora from routine autopsies (control group), sections stained by hematoxylin-eosin were analyzed in order to measure bone microstructural integrity. We found that bone integrity index in exhumed group was negatively influenced by the period of burial (r = -0.37, p < 0.05) and highly significantly decreased (p < 0.0001) in comparison to control group. The period of burial and nitric acid decalcification time was positively correlated (r = 0.51; p < 0.01), leading to imply a bone petrification process during inhumation. Exhumed group showed higher level of matrix bone loss (p < 0.001), as expected, and 87% of cases analyzed were "tunneled" as described by Hackett. Bone integrity index and bone matrix tend to decrease in bones buried in tropical soil between 8-14 years of inhumation. This period is short if we consider cases in which there are preserved bones interred for longer periods in other environments. These data must be considered in cases where genetic identification of exhumed skeletons from tropical environment is required. The diagenesis in these bones and the variations of results found are discussed, clarifying some challenges for forensic laboratories, especially in DNA analysis.

Histomorphometric analysis of osteocyte lacunae in human and pig: exploring its potential for species discrimination.

In recent years, several studies have focused on species discrimination of bone fragments by histological analysis. According to literature, the most consistent distinguishing features are Haversian canal and Haversian system areas. Nonetheless, there is a consistent overlap between human and non-human secondary osteon dimensions. One of the features that have never been analyzed for the purpose of species discrimination is the osteocyte lacuna, a small oblong cavity in which the osteocyte is locked in. The aim of this study is to verify whether there are significant quantitative differences between human and pig lacunae within secondary osteons with similar areas. Study sample comprises the midshaft of long bones (humerus, radius, ulna, femur, tibia, and fibula) of a medieval human adult and a juvenile pig. Sixty-eight secondary osteons with similar areas have been selected for each species and a total of 1224 osteocyte lacunae have been measured. For each osteon, the total number of lacunae was counted, and the following measurements were taken: minimum and maximum diameter, area, perimeter, and circularity of nine lacunae divided between inner, intermediate, and outer lacunae. Statistical analysis showed minimal differences between human and pig in the number of lacunae per osteons and in the minimum diameter (P > 0.05). On the contrary, a significant difference (P < 0.001) has been observed in the maximum diameter, perimeter, area, and circularity. Although there is the need for further research on different species and larger sample, these results highlighted the potential for the use of osteocyte lacunae as an additional parameter for species discrimination. Concerning the difference between the dimensions of osteocyte lacunae based on their position within the osteon (inner, intermediate, and outer lacunae), results showed that their size decreases from the cement line towards the Haversian canal both in human and pig.

Testing the accuracy of a new histomorphometric method for age-at-death estimation.

Histomorphometric methods for age-at-death estimation constitute alternatives to macroscopic ones in the forensic context. However, all new methods must be tested on independent samples to justify their use and assure their accuracy. The main goal of this study was to test a new age-at-death method presented by Goliath et al. (2016) on a sample of 29 decalcified femur sections from individuals autopsied in the Institute of Legal Medicine of Montpellier, France. The formula proposed by the authors was not efficient to estimate the age-at-death in our sample, with only four out of 29 individuals well estimated. Despite this result, the same relationship between age and histomorphometric criteria was found, though at a lower intensity than what Goliath et al. previously found in their study. The use of a different bone preparation technique from Goliath et al. does not allow us to propose a definitive conclusion on the accuracy of this method.

Immunohistochemical analysis of bone metabolism in osteonecrosis of the femoral head.

Osteonecrosis of the femoral head occurs because of the suppression of blood circulation. At the level of the area affected by the ischemic phenomenon, there are two types of events, in the first stage there is necrosis of the cellular elements, followed by a reparatory phase of the spongy bone tissue. The objective of the study was the histological and immunohistochemical (IHC) analysis of bone metabolism in the repair phase. We observed the action of the main cells involved in the remodeling, the osteoblasts and the osteoclasts, following the reaction of the markers of their activity: osteoprotegerin, osteonectin, osteopontin. We included 23 patients diagnosed with femoral head osteonecrosis, stage II, Ficat and Arlet classification, biological material required for histological and IHC analysis being obtained during hip arthroplasty. Regardless of the age or presence of risk factors, the reaction to osteoprotegerin was mildly positive, being only highlighted at the level of the reactive dividing line, being absent in the other areas, indicating a reduced activity of inhibiting differentiation and activation of osteoclasts, also highlighted with classical histology methods, the affected area being well-defined and we could observe the necrotic tissue resorption by osteoclasts. The intense positive reaction of osteopontin and osteonectin, especially at the line of demarcation, is due to the increase in the number of osteoblasts required for the synthesis of neoformation bone tissue. We believe that the aspects revealed by our study can be a track in finding new-targeted therapies useful in stopping the development of the disease.

Histomorphological analysis of the variability of the human skeleton: forensic implications.

One of the fundamental questions in forensic medicine and anthropology is whether or not a bone or bone fragment is human. Surprisingly at times for the extreme degradation of the bone (charred, old), DNA cannot be successfully performed and one must turn to other methods. Histological analysis at times can be proposed. However, the variability of a single human skeleton has never been tested. Forty-nine thin sections of long, flat, irregular and short bones were obtained from a well-preserved medieval adult human skeleton. A qualitative histomorphological analysis was performed in order to assess the presence of primary and secondary bone and the presence, absence and orientation of vascular canals. No histological sections exhibited woven or fibro-lamellar bone. Long bones showed a higher variability with an alternation within the same section of areas characterized by tightly packed secondary osteons and areas with scattered secondary osteons immersed in a lamellar matrix. Flat and irregular bones appeared to be characterized by a greater uniformity with scattered osteons in abundant interstitial lamellae. Some cases of "osteon banding" and "drifting osteons" were observed. Although Haversian bone represent the most frequent pattern, a histomorphological variability between different bones of the same individual, in different portions of the same bone, and in different parts of the same section has been observed. Therefore, the present study has highlighted the importance of extending research to whole skeletons without focusing only on single bones, in order to have a better understanding of the histological variability of both human and non-human bone.

Histological estimation of age at death in amputated lower limbs: Issues of disuse, advanced age, and disease in the analysis of pathological bone.

Histological studies of healed bone tissue following amputation are relatively rare in the literature. This study describes the histomorphological features of femoral thin sections from six uni- and bi-lateral amputees of documented age and sex. Thin sections were cut from the midshaft of both the right and left femora from each amputee and analyzed following standard forensic methods for histological estimation of age at death from the human femur. The histological age at death estimations for the thin sections from amputated bone were consistently lower than the actual chronological age of each individual, suggesting that the effects of amputation prohibit the effective use of age at death estimation methods. The nature of each amputation is unknown, which suggests that alternative factors could be responsible for the slowed bone turnover seen in the thin sections from the amputated bone. First, it is reasonable to assume that the amputations in this sample could have resulted from complications of diabetes mellitus rather than trauma so the possible effects on bone remodeling due to disease are explored. Second, the mobility of the decedents following their amputations is unknown so the histomorphological results could be due to disuse osteoporosis.

Characterization of bone diagenesis by histology in forensic contexts: a human taphonomic study.

The diagenesis of a bone in the postmortem period causes an identifiable deterioration in histology. This degradation is characterized by a collagenous alteration, which can be observed very early. In order to develop a method for determining a postmortem interval for medico-legal use, two ribs collected from six human bodies were studied prospectively over 2 years. Each bone was studied after staining with Sirius red to demonstrate the degradation of collagen as a function of time. This study demonstrated a time-based bone alteration characterized by the architectural degradation of the lamellar bone, without any microbial influence in this postmortem period. The staining was carried out by using Sirius red and correlated this alteration with a collagenic degradation by chemical hydrolysis owing to the affinity of this dye to the amino acids lysine, hydroxylysine, and arginine. Our work asserts that human bone samples that were studied in a controlled environment and analyzed for 24 months underwent a diagenetic trajectory whose main element was collagen hydrolysis.

Cortical Histomorphometry of the Human Humerus During Ontogeny.

Modeling and remodeling are two key determinants of human skeletal growth though little is known about the histomorphometry of cortical bone during ontogeny. In this study, we examined the density and geometric properties of primary and secondary osteons (osteon area and diameter, vascular canal area and diameter) in subperiosteal cortical bone from the human humerus (n = 84) between birth and age 18 years. Sections were removed from the anterior midshaft aspect of humeri from skeletons. Age-at-death was reconstructed using standard osteological techniques. Analyses revealed significant correlation between the histomorphometric variables and age. Higher densities of primary osteons occurred between infancy and 7 years of age but were almost completely replaced by secondary osteons after 14 years of age. The geometry of primary osteons was less clearly related to age. Secondary osteons were visible after 2 years of age and reached their greatest densities in the oldest individuals. Osteon size was positively but weakly influenced by age. Our data imply that modeling and remodeling are age-dependent processes that vary markedly from birth to adulthood in the human humerus.

The Micro-Taphonomy of Cold: Differential Microcracking in Response to Experimental Cold-Stresses.

Cold is a central feature of environments at higher latitudes and elevations. Thus, cold-induced taphonomic changes are relevant in many forensic contexts. Fifty-two lamb bone segments were used to assess the impact of cold, freeze-thaw cycles, freeze-drying, and water immersion on microstructural cracking of bone in a series of controlled exposure experiments. For each bone segment, three thin sections were examined under a light microscope. Cold exposure caused taphonomic changes in the form of microscopic cracking. Transverse cracks occurred in all treatments, whereas osteonal cracks were restricted to rapid freezing treatments. Type of cold exposure had a statistically significant effect on both the total number of cracks and each type of crack observed. Skeletal microcracking could potentially be used as a taphonomic indicator of postmortem bone exposure to sub-zero temperatures. The type and prevalence of this damage could also be used to distinguish between different types of cold exposure.

Bone natural autofluorescence and confocal laser scanning microscopy: Preliminary results of a novel useful tool to distinguish between forensic and ancient human skeletal remains.

The fast, high-throughput distinction between palaeoanthropological/archaeological remains and recent forensic/clinical bone samples is of vital importance in the field of medico-legal science. In this paper, a novel dating method was developed using the autofluorescence of human bones and the confocal laser scanning microscope as the means to distinguish between archaeological and forensic anthropological skeletal findings. Human bones exhibit fluorescence, typically induced by natural antibiotics that are absorbed by collagen, and provide secondary, exogenous fluorophores. However, primary natural fluorescence (or autofluorescence) caused by enigmatic endogenous fluorophores is also present as a micro-phenomenon, whose nature is still obscure. Here, we show that the endogenous fluorophores are mucopolysaccharides of the Rouget-Neumann sheath and, more relevant, that the intensity of the natural fluorescence in human bone decreases in a relationship to the antiquity of the samples. These results suggest that the autofluorescence of bone is a promising technique for the assessment of skeletal remains that may be potentially of medico-legal interest. A larger study is proposed to confirm these findings and to create a predictive model between the autofluorescence intensity and the time since death.

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.

Postmenopausal osteoporosis - clinical, biological and histopathological aspects.

Osteoporosis is one of the most common disorders in postmenopausal women, affecting the quality of life and increasing the risk for fractures in minor traumas. Changes in the bone microarchitecture causes static changes in the body and affects motility. In this study, we analyzed two groups of women, one with physiological menopause and one with surgically induced menopause. The diagnosis of osteoporosis was suspected based on the clinical symptoms and confirmed by assessing bone mineral density by the dual-energy X-ray absorptiometry (DEXA). Comparing some clinical and biological aspects there was noted that a much higher percentage of women with surgically induced menopause exhibited increases in body mass index, changes in serum lipids, cholesterol, triglycerides, blood glucose, serum calcium, magnesemia and osteocalcin. In contrast, no significant differences were observed in the histopathological aspects of bone tissue examined from these two groups. In all patients, there was identified a significant reduction in the number of osteocytes and osteoblasts, the expansion of haversian channels, reducing the number of trabecular bone in the cancellous bone with wide areola cavities often full of adipose tissue, non-homogenous demineralization of both the compact bone and the cancellous bone, atrophy and even absence of the endosteal, and the presence of multiple microfractures. Our study showed that early surgically induced menopause more intensely alters the lipid, carbohydrate and mineral metabolism, thus favoring the onset of osteoporosis.

Variation in osteon histomorphometrics and their impact on age-at-death estimation in older individuals.

Histomorphometric studies have reported relations between osteon size and age; however, data focused on the shape of osteons is sparse. The purpose of this study was to determine how osteon circularity (On.Cr) varies with age in different skeletal elements. Regression analysis was used to evaluate the relationship between age and osteon shape and size. We hypothesized that age would be negatively related to osteon size (area, On.Ar) and positively related to osteon shape (On.Cr). On.Cr and On.Ar were determined for the ribs and femora of 27 cadaveric specimens with known age-at-death. As predicted, age was significantly related to osteon size and shape for both the femur and rib. With age, there was a decrease in size and an increase in circularity. No relationship between sex and On.Cr was detected. An age predicting model, including On.Cr, On.Ar and OPD, is proposed to improve our ability to estimate age-at-death, especially for older individuals.

Palatal Osseous Choristoma.

Osseous choristoma of soft tissue in the oral cavity is rare. The choristoma is a tumor-like mass of normal cells in an abnormal location. In this report, a case of osseous choristoma of the palate in a 37-year-old male patient was reported. Approximately 6 × 5 mm pedunculated healthy colored and hard mass was observed in the median palate. The mass was surgically removed. Microscopic examination of the hematoxylin and eosin-stained sections of the mass revealed that a dense lamellar bone nodule was surrounded by fibrous connective tissue under the squamous epithelium. Immunohistochemical analysis revealed that the mass was negative for S-100 protein, cytokeratin, and epithelial membrane antigens. According to these findings, the final pathologic diagnosis confirmed that the mass was an osseous choristoma.

Histological determination of the human origin from dry bone: a cautionary note for subadults.

Anthropologists are frequently required to confirm or exclude the human origin of skeletal remains; DNA and protein radioimmunoassays are useful in confirming the human origin of bone fragments but are not always successful. Histology may be the solution, but the young subadult structure could create misinterpretation. Histological tests were conducted on femur and skull of 31 human subjects. Each sample was observed focusing on presence or absence of fibrous bone, lamellar bone, radial lamellar bone, plexiform bone, reticular pattern, osteon banding, Haversian bone, primary osteons, secondary osteon and osteon fragments. Samples were divided into five age classes; 1 (<1 year), 2 (1-5 years), 3 (6-10 years), 4 (11-15 years) and 5 (16-20 years). Regarding femurs, class 1 presented the following: 87.5% fibrous bone, 37.5% plexiform bone, 12.5% reticular pattern and 12.5% lamellar bone radially oriented. Class 2 showed 37.5% of fibrous bone, 12.5% of reticular pattern and 37.5% of osteon banding. In the higher age classes, the classical human structures, lamellar bone and osteons were frequently visible, except for one case of reticular pattern, generally considered a distinctive non-human structure. The situation appeared different for the skull, where there was a lack of similar information, both in human and non-human. An analysis of the percentage of lamellar bone and osteons was conducted on femur and skull fragments. A trend of increase of primary osteon number and a decrease of the lamellar bone area has been detected in the femur. The present study has therefore shed some light on further pitfalls in species determination of subadult bone.