C.D.L. Thomas

Age-related changes in cortical porosity of the midshaft of the human femur

Complete cross‐sections from the femoral midshaft of 180 individuals of known height and weight, aged 21–97 y, from a modern Australian population were examined using automatic video image analysis to quantify total subperiosteal porosity (TSPP). More specifically, the aim was to investigate whether age changes were similar in both sexes in (1) total subperiosteal area (TSPA), cortical area (CA) and medullary area (MA), (2) intracortical porosity (ICP), and (3) the respective contributions to TSPP made by MA and intracortical void area (ICVA). Our findings indicated that both sexes showed a significantly greater height normalised TSPA in the 70s as compared with the 20s. Males had consistently larger bones with a greater height normalised CA. In both sexes CA showed a tendency to increase till the 7th decade and then to decline, more so in females. MA approximately trebled in females and doubled in males over the age range studied. Although ICP also increased, from 4–6% in young adults to over 9% in the elderly, it showed a significant difference between the sexes only in the 3rd decade, being greater in males at this stage. By contrast, TSPP became significantly greater in females (from that recorded in the 3rd decade) by the time they reached the 50s, while in males this did not occur till the 80s. It increased from ∼25% in young adults of both sexes to ∼50% in females and ∼37% in males in their 80s. However, in the elderly there was great variability in both sexes in the appearance of bones from individuals of similar chronological age. Some bones differed little from those in younger subjects, others showed greatly increased ICP, still others displayed reduced cortical widths with low ICP. The femoral midshaft resembles other skeletal sites in that age changes in TSPP are more marked in females than males.

Regional variations in cortical modeling in the femoral mid-shaft: sex and age differences.

Modern lifestyle changes may result in site-specific alterations in the skeleton. Our aim was to determine sex and age differences in regional geometry at the mid-femur. Complete cross sections from 113 individuals aged 20-97 years from a modern Australian population were obtained. A further subsample of 24, in whom the precise orientation of specimens was known, was subsequently collected. Microradiographs were made of 100-microm sections and the bone was analyzed using image processing software (Optimas, Media Cybernetics). The periosteal boundary was extracted automatically and the centroid of the periosteal outline was calculated. Fourier shape analysis was used to delineate the endocortical surface. Radial and cortical widths in each quadrant were determined. The posterior was identified by the linea aspera, and the medial and lateral were indistinguishable and therefore grouped together. For analysis, the entire sample was divided into three groups: young (20-40 years), middle (41-60 years), and old (61+ years). Raw and height-normalized values were analyzed with SPSS using t-tests, analysis of variance, and Tukeys honestly significant difference (HSD) tests. The results show that with age the femoral mid-shaft in both sexes becomes larger and more circular, with a slight shift towards the anterior. Apposition is least on the posterior and resorption greatest on the anterior, the latter being particularly evident in postmenopausal females. The greatest sex differences are seen in the middle years, lessening again in the old. We conclude that differential circumferential modeling in response to functional and postural changes occurs in both sexes with age.

An automated analysis of intracortical porosity in human femoral bone across age

The matrix of human cortical bone is arranged around a network of vascular spaces (hereafter referred to as “pores”). Our aim was to investigate age‐related differences in human cortical porosity (total pore area divided by cortical bone area), pore size and number, and surface to volume ratios, while adjusting for sex, height, and weight. Ninety‐six specimens of entire transverse sections of human femoral diaphysis, from subjects aged 21–92 years, were examined. We used our established automated image acquisition and analysis system which measures pores from entire sections of multiple specimens of bone. Over 400,000 pores were recorded. Results showed a greater porosity in older bone (p < 0.01) but marked variation in porosity for any given age. The cohort median, of the specimen medians, of pore cross‐sectional area was 2050 μm2. Older specimens did not have more pores than younger specimens but had a greater proportion of larger pores (p < 0.05) and greater intraspecimen variation in pore size (p < 0.001). The pore surface to bone matrix volume ratio was a median 2.3 mm2/mm3. This varied more than 4‐fold between individuals but did not relate to age. No simple relationships were found between any of the measured parameters and either sex, height, or weight, even after adjustment for age. We conclude that the greater porosity in older specimens is due to greater pore size rather than a larger number of pores. Age, however, explains little of the inter‐individual variation in the parameters studied.

Ontogenetic Patterning of Cortical Bone Microstructure and Geometry at the Human Mid‐Shaft Femur

The bone growth process has long‐lasting effects on adult bone structure and mechanical adaptation, yet the tissue level dynamics of growth are poorly studied. The specific aims of this study were to (1) quantify changes in bone size and shape through ontogeny, (2) describe the distribution of tissue types and patterns of cortical drift and expansion through ontogeny, and (3) explore relationships between cortical drift and ontogenetic variation geometric size and shape. The study utilized 14 juvenile (ages 2–19) mid‐shaft femur blocks removed at autopsy from individuals who died suddenly. Eighty‐μm‐thick sections were imaged using polarized and brightfield microscopy. For descriptive purposes the sample was divided into five age groups. Features of collagen fiber matrix orientation, vascularity (e.g., pore orientation and density), and osteocyte lacunar density and shape were used to classify primary and secondary tissue types in LM images. This information, combined with evaluation of resorptive versus depositional bone surfaces, was used to identify cortical drift direction. A pattern of posterior and medial drift was identified at the mid‐shaft femur in the toddler years. The drift pattern shifts antero‐laterally in late childhood, predating the appearance of a more adult‐like geometry. On the basis of the presence of transitional fibrolamellar bone complex, growth is more rapid during the toddler years and peri‐puberty, and slower in early to late childhood and in later adolescence. Extensive variability in histological and geometric organization typifies the sample, particularly beginning in late childhood. The potential implications of this variability for adult fracture risk warrant further study. Anat Rec, 2009.

Visualization of 3D osteon morphology by synchrotron radiation micro-CT

Cortical bone histology has been the subject of scientific inquiry since the advent of the earliest microscopes. Histology – literally the study of tissue – is a field nearly synonymous with 2D thin sections. That said, progressive developments in high‐resolution X‐ray imaging are enabling 3D visualization to reach ever smaller structures. Micro‐computed tomography (micro‐CT), employing conventional X‐ray sources, has become the gold standard for 3D analysis of trabecular bone and is capable of detecting the structure of vascular (osteonal) porosity in cortical bone. To date, however, direct 3D visualization of secondary osteons has eluded micro‐CT based upon absorption‐derived contrast. Synchrotron radiation micro‐CT, through greater image quality, resolution and alternative contrast mechanisms (e.g. phase contrast), holds great potential for non‐destructive 3D visualization of secondary osteons. Our objective was to demonstrate this potential and to discuss areas of bone research that can be advanced through the application of this approach. We imaged human mid‐femoral cortical bone specimens derived from a 20‐year‐old male (Melbourne Femur Collection) at the Advanced Photon Source synchrotron (Chicago, IL, USA) using the 2BM beam line. A 60‐mm distance between the target and the detector was employed to enhance visualization of internal structures through propagation phase contrast. Scan times were 1 h and images were acquired with 1.4‐μm nominal isotropic resolution. Computer‐aided manual segmentation and volumetric 3D rendering were employed to visualize secondary osteons and porous structures, respectively. Osteonal borders were evident via two contrast mechanisms. First, relatively new (hypomineralized) osteons were evident due to differences in X‐ray attenuation relative to the surrounding bone. Second, osteon boundaries (cement lines) were delineated by phase contrast. Phase contrast also enabled the detection of soft tissue remnants within the vascular pores. The ability to discern osteon boundaries in conjunction with vascular and cellular porosity revealed a number of secondary osteon morphologies and provided a unique 3D perspective of the superimposition of secondary osteons on existing structures. Improvements in resolution and optimization of the propagation phase contrast promise to provide further improvements in structural detail in the future.

Bimodal distribution of osteocyte lacunar size in the human femoral cortex as revealed by micro-CT

Tomographic reconstructions of sections of human femoral bone were created from x-ray data sets taken using synchrotron radiation of 26.4 keV and with isotropic voxels 1.47 μm on a side. We demonstrate that it is possible to segment the data to isolate both the osteocyte lacunae and the Haversian canals in the bone as well as identifying osteon boundaries. From this information a wealth of data relating to bone structure becomes available. The data were used to map the spatial positions of the osteocyte lacunae, relative to the Haversian canals and of the osteon boundaries. The dimensions and volume of the imaged osteocyte lacunae were measured for close to 10,000 lacunae. When averaged over the 11 osteons measured, osteocyte densities varied from 4×10(4)per mm(3) close to the Haversian canals to about 9×10(4)per mm(3) at 80% of osteon radius. The nearest-neighbour distances varied from 10 μm to 40 μm with a peak at 23 μm and an approximately normal distribution. The distribution of lacunar long-axis length was also approximately normal with a small positive skew and the peak value was 8 μm with a range from 3 μm to 20 μm. The most significant finding from this study was that the distribution of the measured volumes of osteocyte lacunae had two distinct peaks, one at 200 μm(3) and a second at 330 μm(3).

Intrapopulation variability in mineralization density at the human femoral mid-shaft

One of several microstructural variables known to affect the mechanical properties of bone is the degree of mineralization of bone matrix. The aim of this study was to examine mineralization density, and its variability with age and sex, from a biomechanical perspective. Histological sections, prepared from mid‐shaft femora obtained at autopsy from 40 individuals, were imaged using quantitative backscattered electron microscopy. Each cross‐section montage was divided into 48 segments according to anatomical position. Mean grey‐level values were quantified for each segment. One‐way anova with Tukey HSD post hoc tests were used to test for differences in mineralization between segments, age groups and sexes. Results showed a decrease in overall degree of mineralization density with adult age, but an increase in its coefficient of variation. Degree of mineralization was significantly lower in the periosteal third of the cortex, particularly in the antero‐lateral aspect. This pattern was most prevalent amongst the youngest individuals in the sample. Whereas males between ages 45–64 years had a higher average degree of mineralization than females, the opposite was true of the older age group. Mineralization significantly decreased between middle and older age groups in males, but not in females. Despite limited consistencies in the location of high and low average mineralization bone through the cortex, the degree of interindividual variation, even within a single age and sex group, overwhelmed population level trends. The patterns of variability identified in this study are consistent with results of an analysis of collagen fibre orientation using the same sample material.

Relationships among microstructural properties of bone at the human midshaft femur.

Mineralization density and collagen fibre orientation are two aspects of a bones microstructural organization that influence its mechanical properties. Previous studies by our group have demonstrated a distinctly non‐random, though highly variable, spatial distribution of these two variables in the human femoral cortex. In this study of 37 specimens, these variables are examined relative to one another in order to determine whether regions of bone demonstrating higher or lower mineralization density also demonstrate a prevalence of either transversely or longitudinally oriented collagen fibres. An analysis of rank‐transformed collagen fibre orientation (as determined by circularly polarized light) and mineralization density (as determined by backscattered electron microscopy) data sets demonstrated that areas of low mineralization density (predominantly in the anterior‐lateral cortex) tended to correspond to regions of higher proportions of longitudinally oriented collagen fibres. Conversely, areas of higher mineralization density (postero‐medially) tended to correspond to regions of higher proportions of transversely oriented collagen fibres. High variability in the sample led to generally low correlations between the two data sets, however. A second analysis focused only on the orientation of collagen fibres within poorly mineralized bone (representing bone that was newly formed). This analysis demonstrated a lower proportion of transverse collagen fibres in newly formed bone with age, along with some significant regional differences in the prevalence of collagen fibres of either orientation. Again high variability characterized the sample. These results are discussed relative to the hypothesized forces experienced at the midshaft femur.

Determination of age at death using combined morphology and histology of the femur.

Bone is characterised by age‐related morphological and histological changes. We have previously established an automated method of recording bone morphometry and histology from entire transverse sections of cortical bone. Our aim was to determine whether data acquired using this automated system were useful in the prediction of age. Ninety‐six specimens of human femoral middiaphysis were studied from subjects aged 21–92 y. Equations predicting specimen age were constructed using macroscopic data (total subperiosteal area (TSPA), periosteal perimeter (PP), endosteal perimeter (EP), cortical bone area (CA) and moments of area) and microscopic data (the number, size and diversity of pores and intracortical porosity) together with sex, height and weight. Both TSPA and PP were independent predictors of age but the number of pores was not a significant predictor of age in any equation. The age predicted by these equations was inaccurate by more than 8 y in over half the subjects. We conclude that we could not predict age at a clinically acceptable level using data from our automated system. This most likely reflects an insensitivity to regional age‐related changes in bone histology because we recorded data from each entire cortex. Automated bone measurement according to cortical region might be more useful in the prediction of age. The inclusion of TSPA together with PP as independent predictors of age raises the possibility that a future measure of periosteal shape at the femoral diaphysis could also be helpful in the prediction of age. The accuracy reached with the relatively simple methods described here is sufficient to encourage the development of image‐analysis systems for the automatic detection of more complex features.

A morphometric study of the cortex of the human femur from early childhood to advanced old age

One hundred and ninety-five mid-shaft femoral bone samples were collected at the Victorian Institute of Forensic Pathology, Melbourne, Australia. These samples came from individuals aged 1-97 years, who had dies unexpectedly and were considered healthy immediately before death. From the samples, transverse sections 100-200 micronM thick were cut on a sawing microtome. Three groups of sections [from persons aged 19-21 (n = 9), 39-41 (n = 9), and 59-61 years (n = 9) at death] were adjusted to a thickness of 100 +/- 5 microns by hand-lapping and microradiographs were taken of these sections. Automatic image analysis methods were used to measure the area and perimeter of the femoral cortex for all 195 sections. Additionally, the number and areas of all holes within the cortex were measured for those sections that were microradiographed. The results show that, even though the number of Haversian canals measured from the entire bone cortex changes with age, the spread of values at any given age makes these measurements of limited value for age determination. From this it may be inferred that all methods that rely, to any extent, upon measurements of this parameter are likely to be flawed.