Ultrastructure of Bone: Hierarchical Features from Nanometer to Micrometer Scale Revealed in Focused Ion Beam Sections in the TEM.

The ultrastructure of bone has been widely debated, in part due to limitations in visualizing nanostructural features over relevant micrometer length scales. Here, we employ the high resolving power and compositional contrast of high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) to investigate new features in human bone with nanometer resolution over microscale areas. Using focused ion beam (FIB)-milled sections that span an area of 50 µm2, we have shown how most of the mineral of cortical human osteonal bone occurs in the form of long, thin polycrystalline plates (mineral lamellae, MLs) which are either flat or curved to wrap closely around collagen fibrils. Close to the collagen fibril (< 20 nm), the radius of curvature matches that of the fibril diameter, while at greater distances, MLs form arcs with much larger radii of curvature. In addition, stacks of closely packed planar (uncurved) MLs occur between fibrils. The curving of mineral lamellae both around and between the fibrils would contribute to the strength of bone. At a larger scale, rosette-like clusters of fibrils are noted for the first time, arranged in quasi-circular arrays that define tube-like structures in alternating osteonal lamellae. At the boundary between adjacent osteonal lamellae, the orientation of fibrils and surrounding mineral lamellae changes abruptly, resembling the "orthogonal" patterns identified by others (Reznikov et al. in Acta Biomater 10:3815-3826, 2014). These features spanning nanometer to micrometer scale have implications for our understanding of bone structure and mechanical integrity.

Three-dimensional reconstruction of Haversian systems in human cortical bone using synchrotron radiation-based micro-CT: morphology and quantification of branching and transverse connections across age.

This study uses synchrotron radiation-based micro-computed tomography (CT) scans to reconstruct three-dimensional networks of Haversian systems in human cortical bone in order to observe and analyse interconnectivity of Haversian systems and the development of total Haversian networks across different ages. A better knowledge of how Haversian systems interact with each other is essential to improve understanding of remodeling mechanisms and bone maintenance; however, previous methodological approaches (e.g. serial sections) did not reveal enough detail to follow the specific morphology of Haversian branching, for example. Accordingly, the aim of the present study was to identify the morphological diversity of branching patterns and transverse connections, and to understand how they change with age. Two types of branching morphologies were identified: lateral branching, resulting in small osteon branches bifurcating off of larger Haversian canals; and dichotomous branching, the formation of two new osteonal branches from one. The reconstructions in this study also suggest that Haversian systems frequently target previously existing systems as a path for their course, resulting in a cross-sectional morphology frequently referred to as 'type II osteons'. Transverse connections were diverse in their course from linear to oblique to curvy. Quantitative assessment of age-related trends indicates that while in younger human individuals transverse connections were most common, in older individuals more evidence of connections resulting from Haversian systems growing inside previously existing systems was found. Despite these changes in morphological characteristics, a relatively constant degree of overall interconnectivity is maintained throughout life. Altogether, the present study reveals important details about Haversian systems and their relation to each other that can be used towards a better understanding of cortical bone remodeling as well as a more accurate interpretation of morphological variants of osteons in cross-sectional microscopy. Permitting visibility of reversal lines, synchrotron radiation-based micro-CT is a valuable tool for the reconstruction of Haversian systems, and future analyses have the potential to further improve understanding of various important aspects of bone growth, maintenance and health.

[Species Identification Based on Morphological Variability of Femur].

OBJECTIVE: To study the morphological characteristics of femurs of adult human and 11 kinds of adult animals from cattle, horses, pigs, goats, sheep, dogs, cats, rabbits, geese, ducks, chickens, and to establish an effective species identification method among various species. METHODS: The 4 cm mid-diaphyseal segment of the femur from adult human (older than 20 years old) at autopsy was obtained. Addi-tionally, the 4 cm ones from 11 kinds of adult animals were obtained. After decalcification, all femurs were made into slices, and then were observed by optical microscope. The 25 indexes were selected and analyzed by step discriminant analysis according to differences between human and mammal, human and poultry, and human and 11 kinds of animals. RESULTS: The histological structure of bone mineral density of middle part of femur had obvious characteristics among the species. And the morphology and number of osteon showed the trend of obvious biological evolution. There were 11 indexes with significant differences between human and 11 kinds of animals to establish some mathematical models to discriminate all species. The correct discrimination rate was 96.3% between human and mammal. The correct discrimination rate was up to 100% between human and poultry, and was 89.4% among human, mammal and poultry. CONCLUSION: The mathematical models have good correct discrimination rate among human and the other animals, which could be applied in the practical species identification cases.

The long bone deformity of osteogenesis imperfecta III: analysis of structural changes carried out with scanning electron microscopic morphometry.

The wedges of the mid-diaphyseal osteotomies carried out to correct the femoral and/or tibial native deformity in type III osteogenesis imperfecta (OI III) were used to study the remodeling patterns and lamellar organization at the level of the major deformity. Histology and scanning electron microscopy (SEM) morphology showed abnormal cortical remodeling characterized by the failure to form a cylinder of compact bone with a regular marrow canal. Atypical, flattened, and large resorption lacunae with a wide resorption front on one side and systems of parallel lamellae on the opposite side were observed, resembling those formerly reported as drifting osteons. SEM morphometry documented a higher percentage of nonossified vascular/resorption area (44.3 %) in OI than in controls (13.6 %), a lower density of secondary osteons, and lower values for the parameters expressing the individual osteon size. The mean osteon total area, the mean central canal area, and the mean osteon bone area of two selected, randomized populations of secondary osteons were significantly higher (p < 0.001, p = 0.028, and p < 0.001, respectively) in control bones than in OI. The mean ossified matrix area was not significantly different, but the mean secondary osteon number and mean density were higher in controls (both p < 0.001). Osteon wedges were carried out to correct the native deformity of OI III and morphologic analysis suggested that the abnormal remodeling pattern (with "drifting osteons") may result from the altered load and tensile stresses on the deformed tubular bones.

A quantitative collagen fibers orientation assessment using birefringence measurements: calibration and application to human osteons.

Even though mechanical properties depend strongly on the arrangement of collagen fibers in mineralized tissues, it is not yet well resolved. Only a few semi-quantitative evaluations of the fiber arrangement in bone, like spectroscopic techniques or circularly polarized light microscopy methods are available. In this study the out-of-plane collagen arrangement angle was calibrated to the linear birefringence of a longitudinally fibered mineralized turkey leg tendon cut at variety of angles to the main axis. The calibration curve was applied to human cortical bone osteons to quantify the out-of-plane collagen fibers arrangement. The proposed calibration curve is normalized to sample thickness and wavelength of the probing light to enable a universally applicable quantitative assessment. This approach may improve our understanding of the fibrillar structure of bone and its implications on mechanical properties.

Morphoscopic analysis of experimentally produced bony wounds from low-velocity ballistic impact.

Understanding how bone behaves when subjected to ballistic impact is of critical importance for forensic questions, such as the reconstruction of shooting events. Yet the literature addressing microscopic anatomical features of gunshot wounds to different types of bone is sparse. Moreover, a biomechanical framework for describing how the complex architecture of bone affects its failure during such impact is lacking. The aim of this study was to examine the morphological features associated with experimental gunshot wounds in slaughtered pig ribs. We shot the 4th rib of 12 adult pigs with .22 mm subsonic bullets at close range (5 cm) and examined resultant wounds under the light microscope, scanning electron microscope SEM and micro tomograph µCT. In all cases there was a narrow shot channel followed by spall region, with evidence of plastic deformation with burnishing of the surface bone in the former, and brittle fracture around and through individual Haversian systems in the latter. In all but one case, the entrance wounds were characterized by superficially fractured cortical bone in the form of a well-defined collar, while the exit wounds showed delamination of the periosteum. Inorganic residue was evident in all cases, with electron energy dispersive spectroscopy EDS confirming the presence of carbon, phosphate, lead and calcium. This material appeared to be especially concentrated within the fractured bony collar at the entrance. We conclude that gunshot wounds in flat bones may be morphologically divided into a thin burnished zone at the entry site, and a fracture zone at the exit.

Numerical modeling of oxygen distributions in cortical and cancellous bone: oxygen availability governs osteonal and trabecular dimensions.

Whereas recent work has demonstrated the role of oxygen tension in the regulation of skeletal cell function and viability, the microenvironmental oxemic status of bone cells remains unknown. In this study, we have employed the Krogh cylinder model of oxygen diffusion to predict the oxygen distribution profiles in cortical and cancellous bone. Under the assumption of saturation-type Michaelis-Menten kinetics, our numerical modeling has indicated that, under steady-state conditions, there would be oxygen gradients across mature osteons and trabeculae. In Haversian bone, the calculated oxygen tension decrement ranges from 15 to 60%. For trabecular bone, a much shallower gradient is predicted. We note that, in Haversian bone, the gradient is largely dependent on osteocyte oxygen utilization and tissue oxygen diffusivity; in trabecular bone, the gradient is dependent on oxygen utilization by cells lining the bone surface. The Krogh model also predicts dramatic differences in oxygen availability during bone development. Thus, during osteon formation, the modeling equations predict a steep oxygen gradient at the initial stage of development, with the gradient becoming lesser as osteonal layers are added. In contrast, during trabeculum formation, the oxygen gradient is steepest when the diameter of the trabeculum is maximal. Based on these results, it is concluded that significant oxygen gradients exist within cortical and cancellous bone and that the oxygen tension may regulate the physical dimensions of both osteons and bone trabeculae.

The shape modulation of osteoblast-osteocyte transformation and its correlation with the fibrillar organization in secondary osteons: a SEM study employing the graded osmic maceration technique.

Cortex fractured surface and graded osmic maceration techniques were used to study the secretory activity of osteoblasts, the transformation of osteoblast to osteocytes, and the structural organization of the matrix around the cells with scanning electron microscopy (SEM). A specialized membrane differentiation at the base of the cell was observed with finger-like, flattened processes which formed a diffuse meshwork. These findings suggested that this membrane differentiation below the cells had not only functioned in transporting collagen through the membrane but also in orienting the fibrils once assembled. Thin ramifications arose from the large and flat membrane foldings oriented perpendicular to the plane of the osteoblasts. This meshwork of fine filaments could not be visualized with SEM because they were obscured within the matrix substance. Their 3-D structure, however, should be similar to the canalicular system. The meshwork of large, flattened processes was no more evident in the cells which had completed their transformation into osteocytes.

A Method to Interpolate Osteon Volume Designed for Histological Age Estimation Research.

Aging adult skeletal material is a crucial component of building the biological profile of unknown skeletal remains, but many macro- and microscopic methods have challenges regarding accuracy, precision, and replicability. This study developed a volumetric method to visualize and quantify histological remodeling events in three dimensions, using a two-dimensional serialized approach that applied circular polarizing microscopy and geographic information systems protocols. This approach was designed as a tool to extend current histological aging methodologies. Three serial transverse sections were obtained from a human femoral midshaft. A total sample size of 6847 complete osteons from the three sections was identified; 1229 osteons connected between all sections. The volume of all connected osteons was interpolated using ArcGIS area calculations and truncated cone geometric functions. Each section was divided into octants, and two random samples of 100 and of 30 connected osteons from each octant were generated. Osteon volume was compared between the octants for each random sample using ANOVA. Results indicated that the medial aspect had relative uniformity in osteon volume, whereas the lateral aspect showed high variability. The anterolateral-lateral octant had significantly smaller osteon volume, whereas the posterior-posterolateral octant had significantly larger osteon volume. Results also indicated that a minimum of 100 osteons is statistically more robust and more representative of normal osteon distribution and volume; the use of 30 osteons is insufficient. This research has demonstrated that osteon volume can be interpolated using spatial geometry and GIS applications and may be a tool to incorporate into adult age-at-death estimation techniques.

Bone microstructure in juvenile chimpanzees.

The growth, development, and maintenance of bone are influenced by genetic and environmental variables. Understanding variability in bone microstructure among primates may help illuminate the factors influencing the number and size of secondary osteons. The purpose of this study is to assess the bone microstructure in 8 humeral and 12 femoral sections of 12 juvenile chimpanzees, aged 2-15.3 years, and one adult chimp. Secondary osteons were counted and measured for 16 fields per section. Results indicate that the femur exhibits a mean osteon population density (OPD) of 4.46 +/- 2.34/mm(2), mean Haversian canal area of 0.0016 +/- 0.0007 mm(2), and mean osteon area of 0.033 +/- 0.006 mm(2). The humerus has a mean OPD of 4.72 +/- 1.57/mm(2), mean Haversian canal area of 0.0013 +/- 0.0003 mm(2), and mean osteon area of 0.033 +/- 0.005 mm(2). Differences are not significant between the humerus and femur, possibly indicating similar mechanical demands during locomotion. Osteon population density exhibits a moderate correlation with age (r = 0.498) in the femur of the juvenile chimps, but the adult chimp has an OPD of 10.28/mm(2), suggesting that osteons likely accumulate with age. Females exhibit higher osteon densities in the periosteal envelope compared to males in the humerus, indicating more remodeling during periosteal expansion. Overall similarities between chimpanzees and humans as well as previously published data on Late Pleistocene hominids (Abbott et al.: Am J Phys Anthropol 99 1996 585-601) suggest that bone microstructure has been stable throughout human evolution.

Anatomy of the intracortical canal system: scanning electron microscopy study in rabbit femur.

The current model of compact bone is that of a system of longitudinal (Haversian) canals connected by transverse (Volkmann's) canals. Models based on histology or microcomputed tomography lack the morphologic detail and sense of temporal development provided by direct observation. Using direct scanning electron microscopy observation, we studied the bone surface and structure of the intracortical canal system in paired fractured surfaces in rabbit femurs, examining density of canal openings on periosteal and endosteal surfaces, internal network nodes and canal sizes, and collagen lining of the inner canal system. The blood supply of the diaphyseal compact bone entered the cortex through the canal openings on the endosteal and periosteal surfaces, with different morphologic features in the midshaft and distal shaft; their density was higher on endosteal than on periosteal surfaces in the midshaft but with no major differences among subregions. The circumference measurements along Haversian canals documented a steady reduction behind the head of the cutting cone but rather random variations as the distance from the head increased. These observations suggested discontinuous development and variable lamellar apposition rate of osteons in different segments of their trajectory. The frequent branching and types of network nodes suggested substantial osteonal plasticity and supported the model of a network organization. The collagen fibers of the canal wall were organized in intertwined, longitudinally oriented bundles with 0.1- to 0.5-mum holes connecting the canal lumen with the osteocyte canalicular system.

Compositional and mechanical properties of growing cortical bone tissue: a study of the human fibula.

Human cortical bone contains two types of tissue: osteonal and interstitial tissue. Growing bone is not well-known in terms of its intrinsic material properties. To date, distinctions between the mechanical properties of osteonal and interstitial regions have not been investigated in juvenile bone and compared to adult bone in a combined dataset. In this work, cortical bone samples obtained from fibulae of 13 juveniles patients (4 to 18 years old) during corrective surgery and from 17 adult donors (50 to 95 years old) were analyzed. Microindentation was used to assess the mechanical properties of the extracellular matrix, quantitative microradiography was used to measure the degree of bone mineralization (DMB), and Fourier transform infrared microspectroscopy was used to evaluate the physicochemical modifications of bone composition (organic versus mineral matrix). Juvenile and adult osteonal and interstitial regions were analyzed for DMB, crystallinity, mineral to organic matrix ratio, mineral maturity, collagen maturity, carbonation, indentation modulus, indicators of yield strain and tissue ductility using a mixed model. We found that the intrinsic properties of the juvenile bone were not all inferior to those of the adult bone. Mechanical properties were also differently explained in juvenile and adult groups. The study shows that different intrinsic properties should be used in case of juvenile bone investigation.

In vivo linear microcracks of human femoral cortical bone remain parallel to osteons during aging.

Previous studies have examined the density of microdamage within the cortex of long bones mostly from the viewpoint that is perpendicular to the long axis of the bone. The goal of the present work is to conduct a systematic characterization of the microcracks from a viewpoint that is parallel to the long axis of a load-bearing bone, the femur, so as to gain a better understanding of the size, shape and orientation of the microdamage. Longitudinal cross sections were taken at the mid-diaphysis of femurs from 13 male donors (23-85 years old) after being stained with basic fuchsin. The number of cracks, their lengths and orientation with respect to osteons were characterized using brightfield and UV-epifluorescent imaging. The mean crack density was 0.1118+/-0.0417 mm(-2) in the longitudinal plane and it significantly increased with age. The median crack length along the longitudinal plane did not change with age. The crack length in the posterior quadrant was significantly lower than anterior, medial and lateral quadrants. Less than 3% of the cracks were longer than 1 mm, indicating the presence of 'in vivo macroscopic' cracks in bone tissue. It was observed that the 99% of the cracks had angles that were less than 25 degrees with the osteons (median angle of 4.2 degrees with an interquartile range of 5.8 degrees ), indicating that the majority in vivo linear microcracks are parallel to osteons. This parallelism did not differ between quadrants nor changed with age. The remarkably stagnant crack length and crack orientation across decades of aging suggest that either physiological loading profile leading to these in vivo microcracks are not changing notably with age, or, microcrack and osteonal orientations may be relatively insensitive to age-related changes in locomotion. In conclusion, in vivo linear microcracks of the femoral mid-shaft grow in planes parallel to osteons and their lengths do not increase with age.

The effects of increased intracortical remodeling on microcrack behaviour in compact bone.

The behaviour of microdamage in bone is related to its microstructural features and thus has an important role in tissue structural properties. However, it is not known how cracks behave in areas of increased intracortical remodeling. More remodeling creates wider variation in the properties of the primary microstructural features of cortical bone, namely osteons. This situation may occur after treatment involving parathyroid hormone or events such as menopause/ovariectomy. High turnover was modeled in this study by using ovariectomy (OVX) to induce surgical menopause in sheep. We hypothesized that osteon age would influence microcrack behaviour during propagation. Five fluorochrome dyes were administered intravenously at different time-points over 12 months post-OVX to label remodeling sites and all animals were then euthanized. Compact bone specimens (2x2x36 mm) were harvested from the right metatarsal. Samples were cyclically loaded to failure and then histological analyses were carried out. Cracks were categorized by length into three groups; short (<100 mum), intermediate (100-300 mum) and long (>300 mum). Numerical crack density (Cr.Dn) of long cracks was greater in controls compared with OVX. Controls also displayed a higher crack surface density (Cr.S.Dn) compared with OVX (p<0.05). The behaviour of short cracks did not differ between old and new osteons, but intermediate and long cracks preferentially stopped at newer osteons compared with older ones (p<0.05). This mechanism may have an important role in terms of prolonging fatigue life. We conclude that recently formed secondary osteons have a unique influence on propagating microcracks compared with older osteons. Therefore localized remodeling levels should be considered when studying microcrack behaviour in bone.

Orientation of collagen at the osteocyte lacunae in human secondary osteons.

This work characterizes an aspect of human bone micro-structure, pertinent to fracture initiation and arrest. It addresses how the orientation of elementary components proximate to osteocyte lacunae influences secondary osteon micro-biomechanics. New data at the perilacunar region concerning orientation of collagen-apatite, and prior data on collagen orientation outside the perilacunar region, are incorporated in a novel simulation of osteons to investigate how orientation relates to strains and stresses during mechanical testing. The perilacunar region was observed by confocal microscopy within single lamellar specimens, isolated from osteons. The specimens were separated by extinct or bright appearance in transverse section under circularly polarizing light. This is because synchrotron diffraction and confocal microscopy had established that each type, away from the perilacunar region, corresponds to specific dominant collagen orientation (extinct lamellae's dominant collagen forming small angles with the original osteon axis, while the bright lamellae's forms larger angles). Morphometry of serial confocal images of each perilacunar region showed collagen orientation generally following the orientation of canaliculi, circumambiently-perpendicular to the lacuna. The lacunae tilted relative to the lamellar walls were more numerous in extinct than in bright lamella. Their apices were less likely in extinct than bright lamella to show collagen following the canalicular orientation. The simulation of osteocyte lacunae in osteons, under tension or compression loading, supports the hypothesis that collagen orientation affects strains and stresses at the equatorial perilacunar region in conjunction with the presence of the lacuna. We further conjecture that collagen orientation diverts propagation of micro-cracks initiating from apices.

Alveolar process anabolic activity in C3H/HeJ and C57BL/6J inbred mice.

BACKGROUND: The purpose of this study was to characterize bone activity of the alveolar process in C3H/HeJ (C3H) and C57BL/6J (B6) inbred mice. Based on observations in other animal species, we hypothesized that the bone-formation rate/bone surface (BFR/BS) is greater in the alveolar process compared to the body of the mandible and that the bone anabolic activity is greater in the alveolar process of the mandible than in the maxilla. We also examined the alveolar process of C3H and B6 mice for the presence of secondary osteons. METHODS: Jaws from 17-week-old C3H and B6 female mice (N = 15/group) were harvested. Histomorphometric parameters were evaluated in sections from the alveolar process, each of which included at least one molar root. RESULTS: In C3H and B6 mice, BFR/BS was not significantly different (P >0.05) between the alveolar process and the body of the mandible. In C3H mice, BFR/BS was significantly greater (P = 0.05) in the mandible compared to the maxilla. BFR/bone volume (BV) was not significantly different (P >0.05) between C3H mandible and maxilla. In the B6 inbred mouse, BFR/BS and BFR/bone volume (BV) were not significantly different (P >0.05) between jaws. After analyzing 165 bone sections, we identified 25 secondary osteons. CONCLUSIONS: The surface anabolic activity was not different between the body and the alveolar process of the mandible. The surface activity was greater in the C3H mandible than in the maxilla. Although secondary osteonal bone remodeling existed in the C3H and B6 alveolar bone, this process was not a consistent finding.

Raman imaging of two orthogonal planes within cortical bone.

The lamellar bone's strength is mainly affected by the organization of its mineralized collagen fibers and material composition. In the present study, Raman microspectroscopic and imaging analyses were employed to study a normal human femoral midshaft bone cube-like specimen with a spatial resolution of approximately 1-2 microm. Identical bone lamellae in both longitudinal and transverse directions were analyzed, which allowed us to separate out orientation and composition dependent Raman lines, depending on the polarization directions. This approach gives information about lamellar bone orientation and variation in bone composition. It is shown that the nu1 PO4 to amide I ratio mainly displays lamellar bone orientation; and nu2 PO4 to amide III and CO3 to nu2 PO4 ratios display variation in bone composition. The nu2 PO4 to amide III ratio is higher in the interstitial bone region, whereas the CO3 to nu2 PO4 ratio has lower values in the same region. The present study provides fresh insights into the organization of a lamellar bone tissue from two orthogonal orientations.

Are distributions of secondary osteon variants useful for interpreting load history in mammalian bones?

BACKGROUND/AIMS: In cortical bone, basic multicellular units (BMUs) produce secondary osteons that mediate adaptations, including variations in their population densities and cross-sectional areas. Additional important BMU-related adaptations might include atypical secondary osteon morphologies (zoned, connected, drifting, elongated, multiple canal). These variants often reflect osteonal branching that enhances toughness by increasing interfacial (cement line) complexity. If these characteristics correlate with strain mode/magnitude-related parameters of habitual loading, then BMUs might produce adaptive differences in unexpected ways. METHODS: We carried out examinations in bones loaded in habitual torsion (horse metacarpals) or bending: sheep, deer, elk, and horse calcanei, and horse radii. Atypical osteons were quantified in backscattered images from anterior, posterior, medial, and lateral cortices. Correlations were determined between atypical osteon densities, densities of all secondary osteons, and associations with habitual strain mode/magnitude or transcortical location. RESULTS: Osteon variants were not consistently associated with 'tension', 'compression', or neutral axis ('shear') regions, even when considering densities or all secondary osteons, or only osteon variants associated with relatively increased interfacial complexity. Similarly, marrow- and strain-magnitude-related associations were not consistent. CONCLUSION: These data do not support the hypothesis that spatial variations in these osteon variants are useful for inferring a habitual bending or torsional load strain history.

A method for measuring the three-dimensional orientation of cortical canals with implications for comparative analysis of bone microstructure in vertebrates.

The orientation of vascular canals in cortical bone can reveal information about the growth rate and loading environment of a bone. For example, in birds it has been proposed that a high proportion of circumferential canals (a laminar cortex) is related to fast growth or torsional loading related to active flight. In this paper we present a method to measure the three dimensional (3D) orientation of vascular canals. Image data are obtained from micro-CT and two angles are measured: phi, determining how longitudinal a canal is; and theta, determining whether a canal is radial or circumferential. This method can measure the orientation of each canal contained in the scanned images. Here we demonstrate the approach on two samples - a rat tibia and a hawk humerus. This method offers a direct (3D) method for quantifying features of canal orientation, such as the degree of laminarity, and can be applied easily and non-destructively to multiple species and bones. The growth and development of the cortical canal network and its impact on factors such as bone strength and bone quality remains relatively unexplored. Our method provides a new tool to examine the impact of the orientation of cortical bone canals on bone and explore the origins of cortical canals formed during modelling and remodeling. This method has applications in comparative bone biology, small animal models, and human bone studies.

Teriparatide increases bone formation in modeling and remodeling osteons and enhances IGF-II immunoreactivity in postmenopausal women with osteoporosis.

UNLABELLED: Transiliac bone biopsies were obtained from 55 women treated with teriparatide or placebo for 12-24 months. We report direct evidence that modeling bone formation at quiescent surfaces was present only in teriparatide-treated patients and bone formation at remodeling sites was higher with teriparatide than placebo. INTRODUCTION: Recombinant teriparatide [human PTH(1-34)], a bone formation agent for the treatment of osteoporosis when given once daily subcutaneously, increases biochemical markers of bone turnover and activation frequency in histomorphometry studies. MATERIALS AND METHODS: We studied the mechanisms underlying this bone-forming action of teriparatide at the basic multicellular unit by the appearance of cement lines, a method used to directly classify surfaces as modeling or remodeling osteons, and by the immunolocalization of IGF-I and IGF-II. Transiliac bone biopsies were obtained from 55 postmenopausal women treated with teriparatide 20 or 40 microg or placebo for 12-24 months (median, 19.8 months) in the Fracture Prevention Trial. RESULTS: A dose-dependent relationship was observed in modeling and mixed remodeling/modeling trabecular hemiosteons. Trabecular and endosteal hemiosteon mean wall thicknesses were significantly higher in both teriparatide groups than in placebo. There was a dose-dependent relationship in IGF-II immunoreactive staining at all bone envelopes studied. The greater local IGF-II presence after treatment with teriparatide may play a key role in stimulating bone formation. CONCLUSIONS: Direct evidence is presented that 12-24 months of teriparatide treatment induced modeling bone formation at quiescent surfaces and resulted in greater bone formation at remodeling sites, relative to placebo.