Histomorphometric age assessment of the Boxgrove 1 tibial diaphysis.

Histomorphometric analysis of a medial midshaft chip from the Middle Pleistocene (ca. 500 ka BP) hominid tibia from Boxgrove, U.K. provides a modal age-at-death estimate at the end of the fourth decade of life. This makes Boxgrove 1 one of the older known and systematically aged Middle Pleistocene hominid specimens, and it reinforces the pattern of an underrepresentation of older adults observed in Middle and Late Pleistocene archaic Homo samples.

Computer-assisted 3D reconstruction of serial sections of cortical bone to determine the 3D structure of osteons.

The objective of this study was to create three-dimensional (3D) images for the histomorphological study of osteons. Medical imaging technology was used to register digitized 2D images of serial decalcified histological sections of bone, to segment the tissues of interest from the surrounding tissues, and to create 3D reconstructions from the segmented structures. Examination of the 3D reconstructions did not support suggestions in the literature that osteons have a spiraling organization. In contrast, the 3D reconstructions indicated that osteons have a complex pattern of organization that is dominated by branching. Examination of the reconstructions also suggested that osteons described in the literature as being dumbbell shaped are actually artifacts of the plane of sectioning. This study demonstrated the applicability of imaging and visualization technology developed for the 3D reconstruction of medical images to the reconstruction of digitized 2D images of serial sections of bone and additionally demonstrated the feasibility of using 3D reconstructions for the histomorphological study of osteons.

Morphology of the drifting osteon.

Drifting osteons were followed longitudinally through the cortex of human and baboon long bones using serial sections. Direction of transverse drift was recorded at different cross-sectional levels of the same systems, and maximum angular change in drift direction was measured for each system. Most drifting osteons exhibit: (1) substantial ( approximately 90 degrees ) variation in the direction of transverse drift along their longitudinal axes, (2) intermittent regions of concentric (type I) morphology, and (3) change in drift direction over time, evident at single cross-sectional levels. Additionally, 3-dimensional reconstruction reveals that the basic multicellular units (BMUs) responsible for creating drifting osteons are morphologically distinct from the cutting-cone-closing-cone model BMUs that produce other types of osteons. The stimulus involved in the activation and guidance of drifting BMUs is unclear, but it is likely that the complex strain environment experienced by long bone cortices exerts a significant influence on their morphology.

Methods for improving the efficiency of estimating total osteon density in the human anterior mid-diaphyseal femur.

In order to preserve whole bone integrity and minimize destruction, paleohistologists often rely on histomorphometric data obtained from small areas (1.5-50 mm2) sampled within the anterior mid-diaphyseal femur. Because bone exhibits significant histological variation, the validity of results based on such sampling is questionable. The accuracy of various subareas (columns, rows, squares approximating dimensions and locations assessed by paleohistologists) in predicting total osteon density in the anterior mid-diaphyseal femur is assessed in the present study. Thirty-five specimens (12.7 mm wide, 100 microm thick, average area 56.7 mm2) were chosen at random from a skeletal population of 94 Inuits and Pueblo agriculturists. The specimens were photographed and enlarged; an acetate grid (12 columns, 10 rows, 120 squares, square = 1 mm2 of bone surface) was superimposed over the photograph; and secondary osteons and fragments were identified. Alternate columns (50% total area, T.Ar) predicted over 98% of entire section total osteon density. Two column combinations (15% T.Ar), separated by at least one column, predicted 91 to 95% of total osteon density. Individual column (8% T.Ar) predictability ranged from 48 to 86%. Two row combination (32 to 40% T.Ar) predictability values ranged from 86 to 95%. Individual rows (<1 to 20% T.Ar) predicted from 45 to 92% of total variation. Combinations of squares approximating areas and locations assessed by other paleohistologists ranged in predictability values from 80 to 94%. The results demonstrate that subareas of as little as 15% predict 95% of variation in total osteon density in the entire anterior mid-diaphyseal femoral section. A minimization of histological area evaluated without the loss of accuracy allows for a minimization of time invested in data collection and the utilization of partially damaged specimens.

Brief communication: a test and correction of the clavicle method of Stout and Paine for histological age estimation of skeletal remains.

The histological method developed by Stout and Paine ([1992] Aln. J. Phys. Antropol. 87:111-115) for estimating age at death using the clavicle is tested on a known age independent sample from a nineteenth century cemetery near Spitalfriedhof St. Johann in Basel, Switzerland. The mean absolute difference between reported ages and histologically predicted ages is 5.5 years. Mean predicted age for the sample is different from mean reported age. This difference is accounted for by differences in the age distributions between the original autopsy sample used to derive the histological age-predicting formula and the cemetery sample, and an inherent loss of reliability of histological age predictions for the skeletal remains of older individuals. A new formula based upon the combined original autopsy sample of Stout and Paine (1992) and the Swiss cemetery sample is presented. It is recommended that this formula be used when estimating ages for older individuals or archaeological skeletal samples.

Bone remodeling rates and skeletal maturation in three archaeological skeletal populations.

Cortical bone remodeling rates for rib samples from three archaeological populations and a modern autopsy sample were determined using an algorithm developed by Frost (Frost [1987a] Calcif. Tissue Res. 3:211-237). When plotted against the relative antiquities for population samples, histomorphometric variables; i.e., activation frequency (mu rc), net bone formation (netVf,r,t), and mean annual bone formation rate (Vf,r,t), exhibit a concordant trend of increased cortical bone remodeling activity levels over time. Two intensive foraging populations, Windover and Gibson, are similar for all bone remodeling parameters and have the lowest remodeling activity levels among the samples. The more recent Ledders sample, which is reported to practice agricultural subsistence, is consistently intermediate between these and a modern autopsy sample. Although there appear to be differences in bone formation rates among the populations it is concluded that these differences cannot be attributed to differences in bone remodeling rates among the populations, but rather are reflecting different effective ages of adult compacta for their ribs. These findings suggest that the earlier populations, particularly Windsor and Gibson, appear to have reached skeletal maturity at an older age than observed for modern.

Estimation of age at death using cortical histomorphometry of the sternal end of the fourth rib.

Given the often fragmentary nature of unidentified human remains, and the importance of using multiple criteria to estimate age at death, it is essential to have a variety of methods that use different anatomical sampling sites. In this study, osteon population densities (OPDs) were determined from transverse sections removed from an area immediately adjacent to the sternal ends of 60 autopsy rib samples. Regression analysis was performed using age at death as the dependent variable and OPD as the independent variable. The results of a "training set/test set" strategy to evaluate the performance of the histological age predicting model indicates that it provides reasonably reliable and accurate age estimates. A multiple regression model using both OPD and the mean age for a rib's morphological age according to the phase method of Iscan et al. [7,8] is also presented. This later age predicting model is recommended when both methods are applicable.

Brief communication: bone remodeling rates: a test of an algorithm for estimating missing osteons.

Frost (1987a) proposed an algorithm for estimating the number of missing osteons that correspond to observed osteon population densities (OPD). Such an algorithm should allow more accurate estimates of bone remodeling rates for skeletal remains for which in vivo labeling is not possible. In order to validate the algorithm, it was tested on an autopsy sample of 44 ribs. Estimates of activation frequency (mu RC) and bone remodeling rate (Vf,r,t) using the new algorithm are in reasonable agreement with age-matched tetracycline-based values. Although mean values for activation frequencies (mu RC) and bone formation rate (Vf,r,t) generated by the algorithm were generally lower, they fell below 1 standard error for only an age category that included all ages above the 5th decade. It is now appropriate to apply the algorithm to archaeological skeletal remains.

Brief communication: histological age estimation using rib and clavicle.

Histological methods for estimating age at death using osteon population densities for the rib, clavicle, and rib and clavicle combined are presented. Predicting formulas were generated from a sample of 40 individuals of known age, sex, and race. Independent samples of 12 ribs and 7 clavicles were used to test the formulas. Mean differences between known and predicted ages were 1.1 years, 2.6 years, and 3.4 years for the clavicle, rib and clavicle combined, and rib formulas respectively. An analysis of variance found no significant differences among the means for predicted and known ages. Since the formula based upon rib and clavicle combined has the higher standard error and r2, and includes data from different bones, it should provide better overall accuracy and reliability, and is recommended whenever both bones are available.

Percent osteonal bone versus osteon counts: the variable of choice for estimating age at death.

Ahlqvist and Damsten's (1969) modification of the Kerley (1965) method for histological age estimation uses percent osteonal bone, rather than actual osteon counts, in order to eliminate the difficulty of distinguishing between intact and fragmentary osteons. Since their method has been developed for the femur only, and several more recent methods have been proposed that utilize percent osteonal bone, a study was undertaken to ascertain the relative value of percent osteonal bone compared with osteon counts to estimate age at death for the radius, tibia, and fibula. First the question of how much of the cross-section of a bone should be sampled was addressed by comparing the results of regression against age for percent osteonal bone derived from sampling only four fields with those derived from the entire cross-section of the radius. A significant age association was found only when the entire cross-section was sampled. In order to evaluate the relative merit of using either percent osteonal bone, or osteon counts to estimate age, each variable was regressed against age. Significant correlation coefficients were found for all three bones when the independent variable was osteon counts. When percent osteonal bone was employed, a significant correlation was found only for the radius. Stepwise linear regression found osteon counts for the fibula alone to be the best age predictor. Finally, a repeated measures analysis of variance revealed that percent osteonal bone and osteon counts both differ among the three bones within an individual. Based upon these results, osteon counts, rather than percent osteonal bone, should be the variable of choice when developing histological age predicting methods.

Bones, blood, pellets, glass, and no body.

A man was found guilty of killing his wife, although her body was never found. The case centered on her car, which contained fragments of bone, glass, shotgun pellets, and dried blood. Deoxyribonucleic acid (DNA) fingerprinting techniques were used to establish the decedent's identity. Examination of the bone fragments revealed that they were from the skull. These two pieces of information, added to other evidence, proved that the defendant's wife had received a fatal injury in her car, and a guilty verdict was rendered.

Bone fragments a body can make.

Data obtained from various analytical techniques applied to a number of small bone fragments recovered from a crime scene were used to provide evidence for the occurrence of a fatality. Microscopic and histomorphometric analyses confirmed that the fragments were from a human skull. X-ray microanalysis of darkened areas on the bone fragments revealed a chemical signature that matched the chemical signature of a shotgun pellet recovered at the scene of the crime. The above findings supported the deoxyribonucleic acid (DNA) fingerprint evidence which, along with other evidence, was used to convict a man for the murder of his wife, even though her body was never recovered.

The use of histomorphology to estimate age.

The purpose of this paper is to discuss some of the major factors that can affect age-at-death predictions when using histomorphological methods. Although evidence suggests that some of the currently available methods are more reliable and accurate, and there are a number of factors other than chronological age that can affect bone remodeling, histomorphological methods, when properly applied, are valuable tools for anthropology and forensic medicine. It is suggested that both accuracy and reliability are maximized when the histomorphometrics of as many anatomical sampling sites as possible are sampled and the resultant ages are averaged.

The use of bone histomorphometry in skeletal identification: the case of Francisco Pizarro.

Two lines of approach are utilized in a histomorphometric analysis of a rib sample from the postcranial remains believed to be those of Francisco Pizarro. Using a newly developed age predicting formula, age at death is estimated to be 62 years. This age agrees with several ages reported in historical documents. The histomorphometry of cortical area/total area (C/T) ratio, mean osteonal cross-sectional area, and mean annual Haversian bone formation rate conform to those of a healthy individual in his early sixties.

The effects of long-term immobilization on the histomorphology of human cortical bone.

The cortical bone histomorphometrics, total visible osteon density, and mean osteonal cross-sectional area were determined for the major long bones and sixth ribs of two individuals with neurological deficit. One was a multiple sclerosis patient who had been in a wheelchair for 15 years. The other was a quadriplegic as a result of poliomyelitis. Statistically significant differences in osteon densities occurred only in the case of the quadriplegic. Nevertheless, in that subject, the total visible osteon densities for bones of the right arm were not statistically different from these of their age-matched (control) radii. Medical history records revealed that there had been partial use of this limb. These results support the belief that mechanical stress is an important factor in the maintenance of normal cortical bone remodeling. In addition, since there were subnormal osteon densities and normal mean osteonal cross-sectional areas, immobilization appears to be characterized by reduced activation frequency with a normal amount of bone turnover per BMU.

Effects of field size when using Kerley's histological method for determination of age at death.

The field size at which a bone is read affects the results obtained when using Kerley's histological method for age estimation, even after applying the recommended correction factor. Whereas there is no tendency for any one of three field sizes tested to consistently underestimate or overestimate age, a field size closest to that used by Kerley in his original study had significantly lower variances for its age estimates, and thus provides greater reliability. This particular field size yields more precise estimates because it is sampling a pattern and number of structures more similar to that of Kerley. Correction factors cannot equalize the counts of osteons and osteon fragments because of spatial variations in the distributions of these histological structures. A field size similar to that used by Kerley in gathering the data from which he developed his regression equations must be used to assure that the same pattern and number of structures is being sampled. For this reason, we suggest a field size as close to 2.06 mm2 as possible be used when employing Kerley's method.

The relative accuracy and reliability of histological aging methods.

Histological aging methods of Kerley and of Ahlqvist and Damsten were applied to bone samples from thirteen individuals of known age at death. Relative accuracy and reliability were determined for six of Kerley's predicting formulas for the femur, fibula and tibia, and his profile method, Ahlqvist and Damsten's femoral predicting formulas, and age determined by averaging ages predicted by Kerley's six formulas. Averaging age estimates by Kerley's six formulas (mean regression) was found to produce the overall greatest accuracy and reliability. Dividing the sample into two age groups (13 - 51 and 60 - 102 years) altered the results only slightly. Kerley's femoral intact osteon formula produced the greatest accuracy for individuals in the younger age category, while his fibular osteon fragment formula was most accurate for older ages. Mean regression produced the greatest reliability for all age classes. Based upon both accuracy and reliability, averaging age predictions by Kerley's regression formulas appears to be the method of choice for broad application of histological aging.

Histomorphometric determination of formation rates of archaeological bone.

Archaeological rib samples were subjected to quantitative histologic analysis to determine rates of cortical bone formation. Histologic features are usually well enough preserved to permit the determination of mean annual Haversian bone formation rate averaged over the life span of the individual. Moreover, gross estimates of aging archaeological bone correlate well with histologic parameters expected for particular ages. Age-associated changes in bone histomorphology in extinct populations have remained essentially unchanged for at least 1,600 years. Bone formation rates determined for these populations agree with age-matched values determined for extant Homo sapiens. A relatively high frequency of pathologic conditions reported by others for the Ledders population may be reflected by the wide range of histomorphometric parameters present in the ribs of these individuals. On the basis of morphophysiologic relationships in extant populations, it can be assumed that mean annual osteonal creation frequency, and mean annual Haversian bone formation rate can be reliably determined in extinct populations. To our knowledge, this is the first time a dynamic physiologic parameter has been measured in an extinct population of H. sapiens.

Histological analysis of undecalcified thin sections of archeological bone.

Archeological bone often lends itself to histological analysis. Sections from bone samples approximately one thousand years old may show as much structural preservation as those only a few hundred years old; hence, it appears that the degree of preservation is not necessarily affected by time. Enough structure may be preserved to permit the diagnosis of metabolic disorders of bone which might go undetected by other methods. This type of analysis can be utilized to accept or reject individual remains suspected of being pathological on the basis of other less precise diagnostic techniques.