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The growth plate is a cartilaginous layer present from the gestation period until the end of puberty where it ossifies joining diaphysis and epiphysis. During this period several endocrine, autocrine, and paracrine processes within the growth plate are carried out by chondrocytes; therefore, a disruption in cellular functions may lead to pathologies affecting bone development. It is known that electric fields impact the growth plate; however, parameters such as stimulation time and electric field intensity are not well documented. Accordingly, this study presents a histomorphometrical framework to assess the effect of electric fields on chondroepiphysis explants. Bones were stimulated with 3.5 and 7 mV/cm, and for each electric field two exposure times were tested for 30 days (30 min and 1 h). Results evidenced that electric fields increased the hypertrophic zones compared with controls. In addition, a stimulation of 3.5 mV/cm applied for 1 h preserved the columnar cell density and its orientation. Moreover, a pre-hypertrophy differentiation in the center of the chondroepiphysis was observed when explants were stimulated during 1 h with both electric fields. These findings allow the understanding of the effect of electrical stimulation over growth plate organization and how the stimulation modifies chondrocytes morphophysiology., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Biomechanical testing using image-based deformation detection techniques such as digital image correlation (DIC) offer optical contactless methods for strain and displacement measurements of biological tissues. However, given the need of most samples to be speckled for image correlation using sprays, chemical alterations with impact on tissue mechanicals may result. The aim of this study was to assess the impact of such surface coating on the mechanical properties of rat bones, under routine laboratory conditions including multiple freeze-thaw cycles., Methods: Two groups of rat bones, highly-uniform and mixed-effects, were assigned to six subgroups consisting of three types of surface coating (uncoated, commercially-available water- and solvent-based sprays) and two types of bone conditions (periosteum attached and removed). The mixed-effects group had undergone an additional freeze-thaw cycle at - 20 degrees. All bones underwent a three-point bending test ranging until material failure., Results: Coating resulted in similar and non-significantly different mechanical properties of rat bones, indicated by elastic moduli, maximum force and bending stress. Scanning electron microscopy showed more pronounced mechanical alterations related to the additional freeze-thaw cycle, with fewer cracks being present in a bone from the highly-uniform group., Conclusions: This study has concluded that surface coating with water- or solvent-based sprays for enhancing image correlation for DIC and having an additional freeze-thaw cycle do not significantly alter mechanical properties of rat bones. Therefore, this method may be recommended as an effective way of obtaining a speckled pattern.

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., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: One of the hardest tasks in developing or selecting grafts for bone substitution surgery or tissue engineering is to match the structural and mechanical properties of tissue at the recipient site, because of the large variability of tissue properties with anatomical site, sex, age and health conditions of the patient undergoing implantation. We investigated the feasibility of defining a quantitative bone structural similarity score based on differences in the structural properties of synthetic grafts and bone tissue., Methods: Two biocompatible hydroxyapatite porous scaffolds with different nominal pore sizes were compared with trabecular bone tissues from equine humerus and femur. Images of samples' structures were acquired by high-resolution micro-computed tomography and analyzed to estimate porosity, pore size distribution and interconnectivity, specific surface area, connectivity density and degree of anisotropy. Young's modulus and stress at break were measured by compression tests. Structural similarity distances between sample pairs were defined based on scaled and weighted differences of the measured properties. Their feasibility was investigated for scoring structural similarity between considered scaffolds or bone tissues., Results: Manhattan distances and Quadrance generally showed sound and consistent similarities between sample pairs, more clearly than simple statistical comparison and with discriminating capacity similar to image-based scores to assess progression of pathologies affecting bone structure., Conclusions: The results suggest that a quantitative and objective bone structural similarity score may be defined to help biomaterials scientists fabricate, and surgeons select, the graft or scaffold best mimicking the structure of a given bone tissue.

Reattachment and healing of tendon to bone poses a persistent clinical challenge and often results in poor outcomes, in part because the mechanisms that imbue the uninjured tendon-to-bone attachment with toughness are not known. One feature of typical tendon-to-bone surgical repairs is direct attachment of tendon to smooth bone. The native tendon-to-bone attachment, however, presents a rough mineralized interface that might serve an important role in stress transfer between tendon and bone. In this study, we examined the effects of interfacial roughness and interdigital stochasticity on the strength and toughness of a bimaterial interface. Closed form linear approximations of the amplification of stresses at the rough interface were derived and applied in a two-dimensional unit-cell model. Results demonstrated that roughness may serve to increase the toughness of the tendon-to-bone insertion site at the expense of its strength. Results further suggested that the natural tendon-to-bone attachment presents roughness for which the gain in toughness outweighs the loss in strength. More generally, our results suggest a pathway for stochasticity to improve surgical reattachment strategies and structural engineering attachments., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: The cn/cn dwarf mouse is caused by a loss-of-function mutation in the natriuretic peptide receptor 2 (NPR-2) gene which helps positively regulate endochondral longitudinal bone growth. The gene mutation corresponds to that in the human skeletal dysplasia Acromesomelic Dysplasia Maroteaux type (AMDM). This study assesses histomorphometric, ultrastructural and radiographic correlates of the growth abnormality., Methods: Ten litters of cn/cn and cn/+littermates at ages ranging from 2.5 to 6.5 weeks were studied by skeletal radiographs, histomorphometry and physeal ultrastructure. Skeletal radiographs were done on 2 cn/cn and 2 cn/+littermates at 5 weeks of age. Humeral, femoral, and tibial lengths were measured from 34 intact bones (17 cn/cn, 17 cn/+) at 2.5 to 6.5 weeks. Growth plate histomorphometry in 50 bones (26 cn/cn and 24 cn/+) determined the hypertrophic zone/entire physeal cartilage ratios in 204 sections (87 cn/+, 117 cn/cn) at 3 time periods (2.5-3, 4-4.5, and 6-6.5 weeks). Electron microscopy assessed 6 cn/cn and 6 cn/+age and site-matched physeal cartilage., Results: Cn/cn mice were two thirds the size of the cn/+. Cn/cn bones were normal in shape or only minimally deformed except for the radius with mid-diaphyseal bowing. Length ratios of cn/cn humeri, femurs, and tibias were a mean of 0.65 (± 0.03, n = 34, 17 ratios) compared to cn/+bones. The main physeal abnormality was a markedly shortened hypertrophic zone with the ratio of hypertrophic zone to entire physis 0.17 (± 0.063) in the cn/cn and 0.30 (± 0.052) in the cn/+mice. Ratio assessments were similar comparing humeral, femoral, and tibial growth plates as were ratios from each of the 3 time periods. Ultrastructural assessments from the resting zone to the lower hypertrophic zone-metaphyseal junction showed no specific individual cell abnormalities in cn/cn compared to cn/+physes., Conclusions: The disorder causes a shortened physeal hypertrophic zone but normal ultrastructure of cn/cn chondrocytes points to abnormality primarily affecting the hypertrophic zone rather than a structural cell or matrix synthesis problem.

Background and Objective: A precise means to cut bone without significant thermal or mechanical injury has thus far remained elusive. A novel non-ionizing ultrafast pulsed picosecond infrared laser (PIRL) may provide the solution. Tissue ablation with the PIRL occurs via a photothermal process with thermal and stress confinement, resulting in efficient material ejection greatly enhanced through front surface spallation photomechanical effects. By comparison, the Er:YAG laser (EYL) ablates via photothermal and cavitation-induced photomechanical effects without thermal or acoustic confinement, leading to significant collateral tissue injury. This study compared PIRL and EYL bone ablation by infrared thermography (IRT), environmental scanning electron microscopy (ESEM), and histology., Study Design: Prospective, comparative, ex vivo animal model., Setting: Optics laboratory., Subjects and Methods: Ten circular area defects were ablated in ex vivo chicken humeral cortex using PIRL and EYL at similar average power (~70 mW) under IRT. Following fixation, ESEM and undecalcified light microscopy images were obtained and examined for signs of cellular injury., Results: Peak rise in surface temperature was negligible and lower for PIRL (1.56 °C; 95% CI, 0.762-2.366) compared to EYL ablation (12.99 °C; 95% CI, 12.189-13.792) (P < .001). ESEM and light microscopy demonstrated preserved cortical microstructure following PIRL ablation in contrast to diffuse thermal injury seen with EYL ablation. Microfractures were not observed., Conclusion: Ablation of cortical bone using the PIRL generates negligible and significantly less heat than EYL ablation while preserving cortical microstructure. This novel laser has great potential in advancing surgical techniques where precision osseous manipulation is required.

Background: Noncemented revision arthroplasty is often complicated by the presence of bone implant gaps that reduce initial stability and biologic fixation. Demineralized bone matrix has osteoinductive properties and therefore the potential to enhance gap healing and porous implant fixation., Questions/purposes: We determined at what times and to what extent demineralized bone matrix promotes gap healing and bone ingrowth around a porous implant., Methods: We inserted porous titanium implants into the proximal metaphyses of canine femora and humeri, with an initial 3-mm gap between host cancellous bone and implants. We left the gaps empty (control; n = 12) or filled them with either demineralized bone matrix (n = 6) or devitalized demineralized bone matrix (negative control; n = 6) and left them in situ for 4 or 12 weeks. We quantified volume healing of the gap with new bone using three-dimensional micro-CT scanning and quantified apposition and ingrowth using backscattered scanning electron microscopy., Results: The density of bone inside gaps filled with demineralized bone matrix reached 64% and 93% of surrounding bone density by 4 and 12 weeks, respectively. Compared with empty controls and negative controls at 4 and 12 weeks, gap healing using demineralized bone matrix was two to three times greater and bone ingrowth and apposition were up to 15 times greater., Conclusions: Demineralized bone matrix promotes rapid bone ingrowth and gap healing around porous implants., Clinical Relevance: Demineralized bone matrix has potential for enhancing implant fixation in revision arthroplasty.

It is known that low bone quality, caused for instance by osteoporosis, not only increases the risk of fractures, but also decreases the performance of fracture implants; yet the specific mechanisms behind this phenomenon are still largely unknown. We hypothesized that especially peri-implant bone microstructure affects implant stability in trabecular bone, to a greater degree than more distant bone. To test this hypothesis we performed a computational study on implant stability in trabecular bone. Twelve humeral heads were measured using micro-computed tomography. Screws were inserted digitally into these heads at 25 positions. In addition, at each screw location, a virtual biopsy was taken. Bone structural quality was quantified by morphometric parameters. The stiffness of the 300 screw-bone constructs was quantified as a measure of implant stability. Global bone density correlated moderately with screw-bone stiffness (r2=0.52), whereas local bone density was a very good predictor (r2=0.91). The best correlation with screw-bone stiffness was found for local bone apparent Young's modulus (r2=0.97), revealing that not only bone mass but also its arrangement in the trabecular microarchitecture are important for implant stability. In conclusion, we confirmed our hypothesis that implant stability is affected by the microstructural bone quality of the trabecular bone in the direct vicinity of the implant. Local bone density was the best single morphometric predictor of implant stability. The best predictability was provided by the mechanical competence of the peri-implant bone. A clinical implication of this work is that apparently good bone stock, such as assessed by DXA, does not guarantee good local bone quality, and hence does not guarantee good implant stability. New tools that could quantify the structural or mechanical quality of the peri-implant bone may help improve the surgical intervention in reaching better clinical outcomes for screw fixation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Kaposiform hemangioendothelioma (KHE) is a rare vascular neoplasm of low malignant potential that mainly affects infants and adolescents. The tumor almost exclusively occurs in somatic soft tissue or the retroperitoneum. We report herein two cases of primary KHE occurring in a long bone without cutaneous changes with long-term follow up in young patients. The patients were a 9-year-old girl and 5-year-old boy presenting with lytic lesions of the femur and humerus, respectively, without cutaneous lesions. Histologically, the neoplasms were comprised of nodules of spindle- to oval-shaped cells growing in an infiltrative fashion. The neoplastic cells formed poorly canalized or slit-like blood vessels alternating with solid spindle areas. Immunohistochemical studies showed that the tumor cells expressed CD31, CD34 and Fli1, but not HHV8, LNA-1 or GLUT1. D2-40 stained the neoplastic spindle cells and lymphatic channels adjacent to vascular lobules. The girl remains well with 15 years and 6 months follow up after a second complete excision. The boy has no signs of recurrence or metastasis nearly 5 years after local complete excision. To our best knowledge, this is the first report in the English literature of primary long bone occurrences of KHE without cutaneous changes with long-term follow up., (© 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

For testing of fixation devices such as suture anchors used in rotator cuff repair often animal bones are used. They are easily obtained, inexpensive and some have been found to be similar to human bone. But can we rely on the results drawn from these studies in our daily surgical practice? The purpose of this study was to compare the trabecular bone mineral density, the trabecular bone volume fraction and the cortical layer thickness in the greater tubercle in different species to evaluate their influence on primary stability of suture anchors under a cyclic loading protocol representing the physiologic forces placed on rotator cuff repairs in vivo. Bovine and ovine humeri are not suitable for suture anchor testing. The statistical significances for pullout forces between the anchors varied from species to species. Therefore, no very applicable information can be obtained from testing suture anchors in ovine or bovine humeri with regard to ultimate failure loads in human humeri. The ultimate failure load seems to depend mainly on the cortical thickness and on the subcortical trabecular bone quality., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The functional significance of three-dimensional trabecular bone architecture in the primate postcranial skeleton has received significant interest over the last decade. Some previous work has produced promising results, finding significant relationships between femoral head trabecular bone structure and hypothesized locomotor loading in leaping and nonleaping strepsirrhines. Conversely, most studies of anthropoid femoral head bone structure have found broad similarity across taxonomic and locomotor groups. The goal of this study is to expand on past analyses of anthropoid trabecular bone structure by assessing the effects of differential limb usage on the trabecular bone architecture of the forelimb and hindlimb across taxa characterized by diverse locomotor behaviors, including brachiation, quadrupedalism, and climbing. High-resolution x-ray computed tomography scans were collected from the proximal humerus and proximal femur of 55 individuals from five anthropoid primate species, including Symphalangus syndactylus, Papio sp., Presbytis rubicunda, Alouatta caraya, and Pan troglodytes. Trabecular bone structural features including bone volume fraction, anisotropy, trabecular thickness, and trabecular number were quantified in large volumes positioned in the center of the humeral or femoral head. Femoral head trabecular bone volume is consistently and significantly higher than trabecular bone volume in the humerus in all taxa independent of locomotor behavior. Humeral trabecular bone is more isotropic than femoral trabecular bone in all species sampled, possibly reflecting the emphasis on a mobile shoulder joint and manipulative forelimb. The results indicate broad similarity in trabecular bone structure in these bones across anthropoids., ((c) 2010 Wiley-Liss, Inc.)

Many studies have described the effects of exercise restriction on the mammalian skeleton. In particular, human and animal models have shown that reduction in weight bearing leads to generalised bone loss and deterioration of its mechanical properties. The aim of this study was to assess the effect of prolonged exercise restriction coupled with heavy calcium demands on the micro-structural, compositional and mechanical properties of the avian skeleton. The tibiae and humeri of 2-year-old laying hens housed in conventional caging (CC) and free-range (FR) housing systems were compared by mechanical testing and micro-computed tomography (microCT) scanning. Analyses of cortical, cancellous and medullary bone were performed. Mechanical testing revealed that the tibiae and humeri of birds from the FR group had superior mechanical properties relative to those of the CC group, and microCT scanning indicated larger cortical and lower medullary regions in FR group bones. Cancellous bone analysis revealed higher trabecular thickness and a higher bone volume fraction in the FR group, but no difference in mineral density. The biomechanical superiority of bones from the FR group was primarily due to structural rather than compositional differences, and this was reflected in both the cortical and cancellous components of the bones. The study demonstrated that prolonged exercise restriction in laying hens resulted in major structural and mechanical effects on the bird skeleton., (Copyright (c) 2008 Elsevier Ltd. All rights reserved.)

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.

Bone microstructure of domestic herbivores is still not completely understood. Indeed, works focused on the bone histology of numerous Mammalian species frequently led to misunderstandings because of the high number of variations such as the kind of bone, section orientation, species, breed and age. Moreover, attempts to identify the species in archaeozoological studies by a mere qualitative approach have not been encouraging and in recent years quantitative methods, based on image processing and statistical analysis, have appeared. The present study was undertaken to determine whether morphometrical and morphological differences exist in the compact bone structure of the femur and humerus between horses and cows. Measurements such as area, perimeter, minimum and maximum diameter of osteons and Haversian canals as well as the osteonal density were carried out on cross sections of eight humeri and eight femurs of the two herbivores investigated. In agreement with other authors, the qualitative investigation confirmed that the compact bone of horses and cows can be classified as dense Haversian tissue. Osteons of the horse were more numerous and composed of a higher number of well-defined lamellae when compared with the cow. Diameter, perimeter and area of osteons and Haversian canals were always higher in horses than in cows and this pattern could be related to the different locomotor behaviour of these animals.

The perichondral ossification of the limb long bones in the quail embryo is investigated, in this study, by means of light and electron microscopy. Longitudinal sections of the humerus, radius, ulna, femur, tibia and fibula stained with haematoxylin-eosin were examined by the light microscope. Ultrathin cross sections were selected for the electron microscope as well. Light microscopic analysis showed that the ossification began at the same time in the long bones of the wing and leg. At the embryonic day 6, all the cartilaginous rudiments consisted of three zones. The central zone composed of hypertrophic chondrocytes, a second zone on either side of the central zone, which consisted of flattened cells and a third zone, which represented the epiphyseal region. A thin sheath of osteoid and a bi-layered perichondrium-periosteum surrounded the central zone of the cartilaginous rudiments of the long bones. The perichondrium consisted of a layer of osteoblasts, in contact with the cartilage, and a layer of fibroblasts. At the embryonic day 7, the thickness of the calcified osteoid ring increased and a vasculature appeared between the layer of osteoblasts and the layer of fibroblasts. At the embryonic day 8, a second sheath of periosteal bone began to be formed. Concurrently, vascular and perivascular elements began to invade the cartilage. The ossification spread towards the distal ends of both the diaphysis. At the electron microscopic level, the osteoblasts of the perichondium showed cytoplasmatic characteristics of cells involved in protein synthesis. The perichondral ossification is the first hallmark of the osteogenesis in the long bones. The observations reported above, are in accordance with previous studies in the chick embryo.

Recently published data for fracture in human humeral cortical bone are analyzed using cohesive-zone models to deal with the nonlinear processes of material failure. Such models represent the nonlinear deformation processes involved in fracture by cohesive tractions exerted by the failing material along a fracture process zone, rather than attributing all damage to a process occurring at a single point, as in conventional linear-elastic fracture mechanics (LEFM). The relationship between the tractions and the net displacement discontinuity across the process zone is hypothesized to be a material property for bone. To test this hypothesis, the cohesive law was evaluated by analyzing published load vs. load-point displacement data from one laboratory; the calibrated law was then used to predict similar data taken for a different source of bone using a different specimen geometry in a different laboratory. Further model calculations are presented to illustrate more general characteristics of the nonlinear fracture of bone and to demonstrate in particular that LEFM is not internally consistent for all cases of interest. For example, the fracture toughness of bone deduced via LEFM from test data is not necessarily a material constant, but will take different values for different crack lengths and test configurations. LEFM is valid when the crack is much longer than a certain length scale, representative of the length of the process zone in the cohesive model, which for human cortical bone ranges from 3 to 10mm. Since naturally occurring bones and the specimens used to test them are not much larger than this dimension for most relevant orientations, it is apparent that only nonlinear fracture models can give an internally consistent account of their fracture. The cohesive law is thus a more complete representation of the mechanics of material failure than the single-parameter fracture toughness and may therefore provide a superior measure of bone quality. The analysis of fracture data also requires proper representation of the approximately orthotropic elasticity of the bone specimen; if the specimen is incorrectly assumed to be isotropic, the initial measured compliance cannot be reproduced to within a factor of four and the fracture toughness deduced from the measured work of fracture will be overestimated by approximately 30%.

Several previous studies of bone repair have shown 2- to 4-fold increases in bone formation following local delivery of exogenous transforming growth factor-beta (TGF-beta). Here, we use quantitative backscatter electron microscopy to test the effect of TGF-beta1 on mineralization of regenerated bone by examining tissue samples from a previously published canine study in which we found increased bone formation. In the experiment, the proximal humeri of 10 male canines were implanted bilaterally for 28 days with porous-coated implants in the presence of a 3 mm gap between the surface of the implant and the host bone. Implants placed in the left humeri were treated with TGF-beta1 at a dose of either 120 microg (n = 5) or 335 microg (n = 5), and the implants placed in the contralateral humeri served as untreated controls. Quantitative backscatter scanning electron microscopy was used to assess the volume fraction of bone and its degree of mineralization in the 3 mm gaps. The calibrated grayscale mean and median values were depressed compared to the controls in the high dose group (p = 0.048 and p = 0.041, respectively), suggesting that high dose TGF-beta delayed or inhibited mineralization of newly formed osteoid.

Although fatigue damage in bone induced by cyclic loading has been recognized as a problem of clinical significance, few fracture mechanics based studies have investigated how incipient cracks grow by fatigue in this material. In the present study, in vitro cyclic fatigue experiments were performed in order to quantify fatigue-crack growth behavior in human cortical bone. Crack-growth rates spanning five orders of magnitude were obtained for the extension of macroscopic cracks in the proximal-distal direction; growth-rate data could be well characterized by the linear-elastic stress-intensity range, using a simple (Paris) power law with exponents ranging from 4.4 to 9.5. Mechanistically, to discern whether such behavior results from "true" cyclic fatigue damage or is simply associated with a succession of quasi-static fracture events, cyclic crack-growth rates were compared to those measured under sustained (non-cyclic) loading. Measured fatigue-crack growth rates were found to exceed those "predicted" from the sustained load data at low growth rates ( approximately 3 x 10(-10) to 5 x 10(-7) m/cycle), suggesting that a "true" cyclic fatigue mechanism, such as alternating blunting and re-sharpening of the crack tip, is active in bone. Conversely, at higher growth rates ( approximately 5 x 10(-7) to 3 x 10(-5) m/cycle), the crack-growth data under sustained loads integrated over the loading cycle reasonably predicts the cyclic fatigue data, indicating that quasi-static fracture mechanisms predominate. The results are discussed in light of the occurrence of fatigue-related stress fractures in cortical bone.

Objective: To observe the microstructure of human long bone with scanning electron and atomic force microscopes to understand the ultrastructural organization and the composition and the morphology of the bone collagen and minerals., Method: Fresh human cadaveric humeral bone were fixed and dehydrated to prepare the ground sections and thin slices, respectively, which were observed for bone microstructure using optical, scanning electron and atomic force microscopy, respectively., Results: Under optical microscope, the bone lacunae were in circular permutation around the Haversian canal, and the canaliculi communicated the lacunae and Haversian canal or between the lacunae. Atomic force microscopy represented distinct morphology of the large clusters of collagen, and the collagen fibers thickened in the mineralized zone and appeared in the shape of overlapping discs, whereas the calcium-phosphorus crystal displayed ellipse cylinders. The size, shape and relation of the canaliculi and lacunae were observed clearly. The bone matrix was observed in circular arrangement along the Haversian canal and the calcium-phosphorus crystal was in regular alignment under scanning electron microscope., Conclusion: Atomic force microscopy can help analyze the three-dimensional configuration of calcium-phosphorus crystal and bone collagen as well as the canaliculi and lacuna. The canaliculi serve as the channels mediating alimentation and molecular signals into the lacuna.

Osteocytes are believed to be the mechanical sensor cells in bone. One potential physical mechanism for the mechanosensing process is that osteocytes directly sense the deformation of the substrate to which they are attached. However, there is a fundamental paradox in this theory: tissue-level strains in whole bone are typically <0.2%, yet an extensive range of in vitro experiments show that dynamic substrate strains must be at least an order of magnitude larger in order for intracellular biochemical responses to occur. Recently, a theoretical model was developed (You et al. J. Biomech., 2001; 34:1375-1386) that provides a possible mechanism by which mechanical loading-induced fluid flow in the lacuno-canalicular system, under routine physical activity, can produce cellular-level strains on the osteocyte processes that are at least one order of magnitude larger than bone tissue deformations. This would resolve the fundamental paradox mentioned above. In this work we experimentally confirm and quantify the essential ultrastructural elements in this model: 1) the presence of the transverse elements that bridge the pericellular space surrounding the osteocyte process, which interact with the fluid flow and lead to an outward hoop tension on the process; and 2) the presence of bundled F-actin in the osteocyte processes, which resists the outward hoop tension and limits the cell process membrane deformation. Morphological data to support these assumptions are scant. Special staining techniques employing ruthenium III hexamine trichloride (RHT) were developed to elucidate these structures in the humeri of adult mice., (Copyright 2004 Wiley-Liss, Inc.)

This work investigated the origin and development of microcirculation in the rat humeral head and the expression of vascular endothelial growth factor (VEGF) as a factor supporting the vascular growth and the development of the secondary ossification centers. Sixty rats aging 1, 3-4, 6-8, 11, and 21 days, 5 weeks, and 4 and 8 months were used. Samples of humeral head were collected for histology and immunohistochemistry for VEGF. Some animals were perfused with Mercox resin in order to obtain vascular corrosion casts (vcc) observed by scanning electron microscopy (SEM). No cartilage canals were present at birth. At 6 days postnatal, blood vessels coming from the perichondrium and the region near the capsule attachment invaded the cartilage; at 11 days postnatal, signs of calcification were present and within the third week some bone trabeculae were formed. Just before the vascular invasion of the epiphysis, a positive reaction for VEGF was localized in chondrocytes of the epiphyseal cartilage close to the capsule insertion. During the development and expansion of the secondary ossification center, VEGF expression was higher in chondrocytes but decreased when epiphysis was diffusely ossified. VEGF was expressed also by mesenchymal cells present in and around the fibrous tissue where the secondary ossification center will develop. SEM vcc confirmed that vessels penetrating into the epiphysis arose merely from the periosteal and the capsular networks, and vascular connections with the diaphyseal circulation were not evident. These observations demonstrated that VEGF production by chondrocytes begun some days after birth, supported the rapid vascular growth from the surrounding soft tissues, and was chronologically related to the development of the secondary ossification center in rat proximal humerus. Finally, the possible role of VEGF as mediator of angiogenesis and, at least indirectly, as a trigger factor also in the ossification and the bone remodeling of the secondary ossification centers has been discussed., (Copyright 2004 Wiley-Liss, Inc.)

We examined the effects of long-term bisphosphonate (BP, pamidronate) administration at a therapeutic dose (1.5 mg/kg/day) on the distribution, structure, and vacuolar-type H(+)-ATPase expression of osteoclasts, and the resulting trabecular bone volume and structure in ovariectomized (OVX) mature rats. Six-month-old female rats were allocated to sham-operated control, untreated-OVX, and BP-administered OVX groups. Postoperatively, BP was administered intraperitoneally once a day to OVX rats for up to 30 days. On postoperative days 14, 30, and 60, all of the rats were killed and the distal metaphyseal area of the dissected humeri was examined. Quantitative backscattered-electron image analysis revealed that the trabecular bone volume/unit medullary area in untreated OVX rats was significantly (P < 0.05) lower than that in sham-operated controls at 30 and 60 days postoperation. BP administration significantly (P < 0.05) increased trabecular bone volume at 14, 30, and 60 days postoperation in BP-administered OVX rats compared to both sham-operated and untreated OVX rats. Compared to untreated OVX rats, the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts along the bone trabeculae in BP-administered OVX rats was not significantly decreased on days 14 and 30, but was significantly decreased on day 60. Ultrastructurally, BP administration caused the disappearance of both the ruffled border (RB) and the clear zone (CZ) structures, and decreased the expression of vacuolar-type H(+)-ATPase in most osteoclasts, but did not significantly induce apoptosis of osteoclasts detected by the terminal dUTP nick end-labeling (TUNEL) method. Our results suggest that long-term BP administration significantly reduces bone and calcified cartilage resorption through impairment of the structure and bone-resorbing function of osteoclasts, and thereby effectively maintains trabecular bone volume and structure in ovariectomy-induced acute estrogen deficiency in mature rats., (Copyright 2003 Wiley-Liss, Inc.)

Osteoprotegerin (OPG) is an osteoblast-derived secreted member of the tumour necrosis factor receptor superfamily that inhibits osteoclastogenesis. Mice that are OPG-deficient have severe bone loss, including growth plate cartilage destruction. Using OPG-deficient mice as a useful animal model, we attempted to clarify differentiation and ultrastructural features of osteoclasts located on destructed growth plate cartilage and trabecular bone matrix. In the humerus and femur of OPG homozygous (-/-) mice, adjacent to the growth plate cartilage, bone trabeculae without a calcified cartilage core were characteristically formed at the metaphyseal side of the medullary cavity, which resulted in an irregular chondrocyte distribution and arrangement in growth plate cartilage. During growth plate cartilage destruction, osteoclasts positive for tartrate-resistant acid phosphatase showed unusual localization on both type-II collagen-positive cartilage and type-I collagen-positive trabecular bone matrix at the ossification centre of the epiphyseal/metaphyseal border. Although multinucleated osteoclasts were distributed within open lacunar canals in the growth plate, those on uncalcified cartilage matrix lacked a ruffled border. Facing the calcified cartilage matrix within lacunar canals, osteoclasts showed irregularly formed ruffled borders. After growth plate destruction, a thin bone layer was deposited on the remaining cartilage surfaces by invading osteoblasts. Osteoclasts formed prominent ruffled border structures on bone matrix, deposited on the remaining growth plate cartilage. These results suggest that, in OPG (-/-) mice, terminal osteoclast differentiation requires the presence of newly produced bone matrix, as the coupled phenomenon of bone formation and resorption, as well as osteoblast-derived cytokines.

Different lines of evidence suggest that trabecular bone architecture contains a functional signal related to an organism's locomotor behavior. An understanding of the interspecific and intraspecific variation in extant nonhuman primate trabecular structure is needed to evaluate its usefulness as a tool to reconstruct the locomotor habits of extinct primates. High-resolution X-ray computed tomography (HRXCT) is a new imaging approach with a resolution in the tens of microns that allows nondestructive access to the internal structure of bony elements. Previous studies indicate that such resolution is necessary to accurately quantify structural parameters of trabecular bone. The primary goal of this study was to test the accuracy of HRXCT by comparing stereological measurements from HRXCT images and histological thin sections of cancellous bone taken from the proximal femur and humerus of baboons. To this end, 11 bone samples were scanned on an HRXCT scanner and then thin-sectioned to reveal the scanned plane. HRXCT images were thresholded using a modified half-maximum height protocol. The stereological measurements included bone volume fraction (BV/TV), trabecular number (Tb.N), bone surface to volume ratio (BS/BV), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). The measurement errors on the HRXCT images were 10.90% for BV/TV, 6.06% for Tb.N, 14.19% for BS/BV, 14.33% for Tb.Th, and 7.09% for Tb.Sp, but none of these measurements were significantly different from the histological standards (alpha = 0.05). A second goal of this study was to examine the influence of thresholding, a necessary step in any morphometric study using computed tomography, on the accuracy of the quantitative morphometry. Threshold values derived from a modified half-maximum height protocol showed that parameters derived from the region of interest (area in which stereological measurements were later taken) produced better reconstructions of the actual bone structure than threshold values derived from more inclusive areas of bone. We conclude that HRXCT can accurately reconstruct the complex architecture of trabecular bone, and that thresholding is a nontrivial step in trabecular bone studies, with even slight changes in the protocol greatly affecting the morphometric data. HRXCT represents a valuable analytical tool that should be of interest to a great many researchers in physical anthropology because it allows nondestructive access to internal morphology, thereby preserving valuable and limited skeletal collections., (Copyright 2002 Wiley-Liss, Inc.)

Scanning electron microscopy (SEM) has occasionally been used by anthropologists and forensic scientists to look at morphological characteristics that certain implements leave on bone. However, few studies have addressed techniques or protocols for assessing quantitative differences between tool marks on bone made by different bladed implements. In this study, the statistical variation in cut mark width was examined between control and test samples on bone using a scalpel blade, paring knife, and kitchen utility knife. Statistically significant differences (p < .0005) were found between cut marks made by the same knife under control and test conditions for all three knife types used in the study. When the control sample and test samples were examined individually for differences in mean variation between knife types, significant differences were also found (p < .0005). While significant differences in cut mark width were found, caution should be used in trying to classify individual cut marks as being inflicted by a particular implement, due to the overlap in cut mark width that exists between different knife types. When combined, both quantitative and qualitative analyses of cut marks should prove to be more useful in trying to identify a suspect weapon. Furthermore, the application of SEM can be particularly useful for assessing many of these features.

To clarify the early process of recovery at the bone-tendon interface, we used light microscopy and SEM to examine the process of anchoring of collagen fibers to bone in a canine model. At two weeks, tendon, scar tissue, woven bone and lamellar bone were present at the insertion site. SEM revealed anchoring of collagen fibril bundles of the scar to the woven bone. By 4 weeks, the number of anchoring fibers had increased and a parallel arrangement of fibers was observed. SEM demonstrated deep penetration of fibers into the woven bone layer. In addition, the fibers were observed to project into and intermingle with the scar tissue. By 6 weeks, the anchoring fibers had developed fully and were distributed densely over the interface. SEM also revealed that the collagen fibril bundles in the scar tissue had connected with the collagen fibrils of the woven bone by way of the anchoring bundles. The woven bone was identifiable throughout the early stages of recovery as the interface between soft tissue and hard tissue. Throughout all experimental periods, no staining was observed at the interface of the tendon and bone by Saffranin-O. The formation of woven bone was important during early recovery of the tendon-bone interface prior to the completion of fibrocartilage-mediated insertion.

Until recently, detailed analyses of the architecture of nonhuman primate cancellous bone have not been possible due to a combination of methodological constraints, including poor resolution imaging or destructive protocols. The development of micro-computed tomography (microCT) and morphometric methods associated with this imaging modality offers anthropologists a new means to study the comparative architecture of cancellous bone. Specifically, microCT will allow anthropologists to investigate the relationship between locomotor behavior and trabecular structure. We conducted a preliminary study on the trabecular patterns in the proximal humerus and femur of Hylobates lar, Ateles paniscus, Macaca mulatta, and Papio anubis to investigate the quantitative differences in their trabecular architecture and evaluate the potential of microCT in anthropological inquiry. MicroCT allows the researcher to evaluate variables beyond simple two-dimensional orientations and radiographic densities. For example, this methodology facilitates the study of trabecular thickness and bone volume fraction using three-dimensional data. Results suggest that density-related parameters do not reliably differentiate suspensory-climbing species from quadrupedal species. However, preliminary results indicate that measurements of the degree of anisotropy, a measure of trabecular orientation uniformity, do distinguish suspensory-climbing taxa from more quadrupedal species. The microCT method is an advance over conventional radiography and medical CT because it can accurately resolve micron-sized struts that make up cancellous bone, and from these images a wide array of parameters that have been demonstrated to be related to cancellous bone mechanical properties can be measured. Methodological problems pertinent to any comparative microCT study of primate trabecular architecture are discussed., (Copyright 2001 Wiley-Liss, Inc.)

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the alpha1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of alpha1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.

The processes of bone wound healing after maxillary molar extraction in ovariectomized aged rats were examined by means of quantitative backscattered electron image analysis and energy-dispersive X-ray microanalysis. Six-month-old female rats were either sham-operated or underwent bilateral ovariectomy (OVX), and 60 days postoperatively, the maxillary first molars were extracted. On post-extraction days 7, 30, and 60, the dissected and resin-embedded maxillae were micromilled in the transverse direction through the extracted alveolar sockets, and new bone formation on the buccal maxillary bone surface and within the extracted alveolar sockets was examined. In both sham-operated control and OVX rats, new bone formation was recognized on the buccal bone surface, as well as within the extracted sockets, and increased daily through to day 60. In comparison to sham-operated controls, new bone formation in OVX rats was significantly decreased both on the buccal bone surface and within the extracted sockets. Our results suggest that bone wound healing by new bone formation after maxillary molar extraction is significantly decreased in OVX-induced osteoporosis., (Copyright 2000 Wiley-Liss, Inc.)

Chondrocytes at the lower zone of the growth plate must be eliminated to facilitate longitudinal growth; this is generally assumed to involve apoptosis. We attempted to provide definitive electron-microscopic evidence of apoptosis in chondrocytes of physes and chondroepiphyses in the rabbit. We were, however, unable to find a single chondrocyte with the ultrastructure of 'classical' apoptosis in vivo, although such a cell was found in vitro. Instead, condensed chondrocytes had a convoluted nucleus with patchy chromatin condensations while the cytoplasm was dark with excessive amounts of endoplasmic reticulum. These cells were termed 'dark chondrocytes'. A detailed study of their ultrastructure combined with localisation methods in situ suggested a different mechanism of programmed cell death. In addition, another type of death was identified among the immature chondrocytes of the chondroepiphysis. These cells had the same nucleus as dark chondrocytes, but the lumen of the endoplasmic reticulum had expanded to fill the entire non-nuclear space, and all cytoplasm and organelles had been reduced to dark, worm-like inclusions. Since these cells appeared to be 'in limbo', they were termed 'paralysed' cells. It is proposed that 'dark chondrocytes' and 'paralysed cells' are examples of physiological cell death which does not involve apoptosis. It is possible that the confinement of chondrocytes within their lacunae, which would prevent phagocytosis of apoptotic bodies, necessitates different mechanisms of elimination.

In the present study, we tested the hypothesis that tension and bending, rather than compression alone, determine the functional adaptation of subchondral bone in incongruous joints. We investigated whether tensile stresses in the subchondral bone of the humero-ulnar articulation are affected by the direction of muscle and joint forces, and whether the tensile stresses are large enough to cause microstructural adaptation, specifically a preferential alignment of the trabeculae and the subchondral collagen fibres. Using a previously validated finite element model of the human humero-ulnar joint, we calculated the contact pressure, the principal compressive and tensile stresses, and the strain energy density in the subchondral bone for various flexion angles. A bicentric (ventro-dorsal) pressure distribution was found in the joint at 30 degrees to 120 degrees of flexion, with contact pressures of up to between 2.5 and 3 MPa in the ventral and dorsal aspects of the ulnar joint surface, but less than 0.5 MPa in the centre. The principal tensile stress in the subchondral bone of the trochlear notch quantitatively exceeded the principal compressive stress at low flexion angles (maximum 8.2 MPa), and the distribution of subchondral strain energy density differed substantially from that of the contact stress (r=-0.72 at 30 degrees and r=+0.58 at 90 degrees of flexion). No important tensile stress was computed in the trochlea humeri. On contact radiography, we found sagittally orientated subarticular trabeculae in the notch, running tangential to the surface. Furthermore, we observed sagittally orientated split lines in the subchondral bone of the notch of 20 cadaver joints, suggesting a ventro-dorsal orientation of the collagen fibres. The trochlea humeri, on the other hand, did not show a preferential direction of the subchondral split lines, these findings confirming the predictions of tensile stresses in the model. We conclude that, due to the important contribution of tension to subchondral bone stress, the distribution of subchondral density cannot be directly employed for assessing the long term distribution of joint pressure at the cartilage surface. The magnitude of the tensional stress varies considerably with the direction of the muscle and joint forces, and it appears large enough to cause functional adaptation of the subchondral bone on a microstructural level.

Normal and pathological (osteochondrotic and osteoarthrotic) pig articular cartilages from medial humeral and femoral condyles were studied by scanning electron microscopy. The pathological cartilages showed primitive osteochondrotic lesions with progressive aspects according to the severity of the pathology (flaking, fibrillation and cracks) and other superficial changes (micro-undulation, micro-fissurations, clefts) that appeared to be a consequence of the action of intense mechanical stresses such as shearing forces, compressive deformation, friction of the articular surfaces. The observation of deep lesions such as cracks and fractures at or near the interface between cartilage and calcified zone, frequently observed both at medial condyle and at intercondylar crista of the humerus, often evolving in cartilagineous flaps, were related to excessive tangential and shear forces induced by an abnormal articular topography resulting in joint instability. This pathological joint dynamic could be also worsened by an anomalous leg conformation (cross-legs) and/or by an increased occurrence of environmental micro- and macrotrauma (impact loading). Also in this case, the frequency and severity of the lesions can be increased if the deep layer is affected by osteo-chondrotic lesions. The results stress the pathogenetic importance of mechanical load in initiating and worsening the articular lesions in pigs; they also suggest that the resulting alterations can be influenced by a pre-existing different maturity or pathological condition of the cartilage.

Continuous treatment with calcitonin (CT) to inhibit osteoclastic bone resorption results in acquired resistance. The mechanisms of this "escape" phenomenon are not yet established. The aim of this study was to examine the effects of continuous treatment with CT on the generation of osteoclasts and calcitonin receptor (CTR) expression in mouse bone marrow cultures. This was done by daily CT treatment of mouse bone marrow cultures from day 0, when only undifferentiated mononuclear precursors of osteoclast-like cells were present, or commencing from day 6, when differentiated osteoclast-like cells were abundant. The response to CT treatment was determined by quantitation of cells positive for tartrate-resistant acid phosphatase (TRAP) and binding of 125I-salmon CT. Calcitonin receptor and TRAP mRNA levels were determined using semi-quantitative reverse transcription/polymerase chain reaction. When cultures were treated with CT from day 0, TRAP-positive multinucleated cells appeared. These cells expressed only very low levels of CTR or CTR mRNA and were morphologically indistinguishable from osteoclast-like cells formed in control cultures. They also displayed the ability to resorb bone. Continuous CT treatment of cultures from day 6 rapidly reduced the CTR mRNA levels, with a t1/2 of 6 to 12 h, and these levels remained low thereafter. 125I-salmon CT binding capacity, as determined by autoradiography, was lost in parallel. These effects were specific for the CTR since there was no consistent effect on TRAP mRNA levels. Based on these data, we suggest that the "escape" phenomenon may result from a prolonged CT-induced loss of CT responsiveness due, at least in part, both to reduced synthesis of CTR, and to the appearance in bone of CTR-deficient osteoclasts.

Enhancement of bone ingrowth with transforming growth factor-beta was evaluated in a canine model. Ten dogs had bilateral implantation of a titanium-fiber-metal-coated rod in the proximal part of the humerus. A three-millimeter gap between the outer surface of the porous coating and the surrounding cancellous bone was created to impair bone ingrowth. All of the implants were plasma-flame-sprayed with hydroxyapatite and tricalcium phosphate. In each animal, one implant was also treated with recombinant transforming growth factor-beta 1 while the other implant, which was not so treated, served as a paired control. Two doses of transforming growth factor-beta 1 were used: 335 micrograms in five animals and 120 micrograms in the other five. At four weeks, the amount of bone ingrowth in the implants that had been treated with 120 micrograms of transforming growth factor-beta 1 was threefold higher than that in the paired controls (p = 0.009), but with the numbers available there was no significant increase in bone ingrowth with the higher dose. The amount of new-bone formation in the three-millimeter gaps adjacent to the treated implants was twice that in the gaps of the paired controls, regardless of the dose. The differences between the treated and control implants with regard to the architecture of the new bone in the gap indicate that the mechanism of action of transforming growth factor-beta 1 may include both proliferation of osteoprogenitor cells and production of matrix by committed osteoblasts. Compared with the findings in a previous study in which this canine model was used, the data from the present investigation indicate that enhancement of bone ingrowth in implants that have been treated with a combination of a hydroxyapatite-tricalcium phosphate coating and transforming growth factor-beta 1 may exceed that obtainable with grafting of the gap with autogenous cancellous bone.

The effect of adrenaline on glycogen breakdown in different skeletal muscle fibre types was investigated in the epitrochlearis muscle in vitro. Histochemical studies showed that adrenaline stimulated glycogen breakdown in all three major fibre types, with a higher absolute glycogen breakdown in type IIB fibres compared to type IIA and type I fibres. In biochemical studies we found that the glycogenolytic rate decreased during prolonged incubation with adrenaline, although the percentage phosphorylase in the a form and the concentration of glucose-6-phosphate (G-6-P) remained high. In the dose response studies we found an EC50 of 2.2 x 10(-8) M adrenaline for adrenaline stimulated glycogen breakdown, with an EC50 of 2.0 x 10(-7) M for adrenaline stimulated accumulation of G-6-P, excluding G-6-P as the key inhibitor of phosphorylase a activity.

Cancellous bone mass decreases following ovariectomy in rodents, providing a useful model for post-menopausal bone loss in humans. This study describes and quantifies the longer-term changes in cancellous bone structure in the ovariectomized (OVX) rat. Rats were OVX or sham-OVX at 100 days of age and bones were collected 540 days later. Lumbar vertebral bodies were prepared for microradiography and structural analyses (nodal analyses and star volume analyses) of cancellous bone. Proximal humerii were prepared for scanning electron microscopy (SEM). Microradiography confirmed the loss of cancellous bone from the central spongiosa regions of the vertebral bodies and the humerii in the OVX rats. Changes in trabecular structural elements included relative increases in the number of free to free, cortical to free, cortical to node struts and decreases in the node to node struts in the OVX animals compared with controls. There were increases in average lengths of the node to free, node to node, and free to free trabecular struts in the OVX animals. The marrow star volume was increased in the OVX animals indicating a greater trabecular separation in these animals compared with controls. Viewed by SEM, metaphyseal trabeculae in the controls consisted of rods and plates but in the OVX animals the remaining trabeculae were mostly longitudinal rods with smaller transverse connecting rods. The remaining bone in the OVX animals was found in the lateral metaphyseal areas and is consistent with maintenance of the structural capacity of the bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Six clinically and patho-histologically proven cases of chondroblastoma were studied with electron microscopic and immunohistochemical methods. The tumor tissues of chondroblastoma exhibited biphasic pattern i.e., chondroid and cellular area. In the chondroid area, small and round tumor cells contained many filaments in the cytoplasm and small processes in the cell wall. Many glycogen granules were present in the tumor cells in some cases. Intercellular matrix was immunohistochemically positive for S-100 protein and showed fine collagen fibers were very similar to those of articular and epiphysial cartilage. Many portions of mitochondria, cell wall and matrix of chondroblastoma tissue clearly exhibited calcification but were not definitely ossification. In the cellular area, tumor cells were composed of small immature mesenchymal and clear large degenerated tumor cells, histiocytes with lysosomes and osteoclast-like multinuclear giant cells. Immunohistochemical studies in the cellular area revealed that there were many tumor cells positive for lysozyme, alpha 1-antitrypsin and alpha 1-antichymotrypsin. In the transitional zone between chondroid and cellular area, degenerated tumor cells were found. The chondroblastoma was composed of chondroid and histiocytic areas which were very similar to those of chondromyxoid fibroma. The present study appears to demonstrate that chondroblastoma originates from a mixture of chondrocytic and histiocytic tumor cells but not from articular and epiphysial cartilage.

The ultrastructural features of quinolone-induced arthropathy were studied in the humeral and femoral heads of nine skeletally immature Beagle dogs (3 months old) that were dosed orally with difloxacin at 300 mg/kg body weight and euthanatized 24, 36, or 48 hours later in groups of three. Three age-matched dogs were given a placebo and euthanatized after 48 hours. Mitochondria in chondrocytes had significantly greater cross-sectional areas (P less than 0.05) in electron micrographs from dogs euthanatized after 48 hours of treatment than did those in other groups. There was also a significantly greater percentage of chondrocytes with swollen mitochondria in treated dogs than in the controls (P less than 0.05). These changes preceded the necrosis observed in some chondrocytes in the dogs of the 48-hour group. Disruption of extracellular matrix was first observed in the pericellular matrix of necrotic chondrocytes, indicating that this change was secondary to the changes in chondrocytes. Fissures within cartilages apparently resulted from the loss of the normal association of proteoglycans with collagen fibrils.

The elbows of 13 puppy cadavers were dissected, samples were taken for light and electron microscopy, and the thickness of the articular cartilage of the distal humerus and proximal ulna was measured. Throughout post natal development differences were found in the arrangement of the growth plate and articular chondrocytes. At birth, the articular surface had remnants of a fibrous limiting membrane that was continuous with the perichondrium, a finding not previously recorded in dogs. Orientation of the collagen fibrils within the matrix of the articular cartilage was initially lacking but became established by three weeks. In the humerus cartilage canals were present up to 12 weeks old. The articular cartilage of the humeral condyle varied in thickness across the joint surface, being thicker on the medial than on the lateral side; it was also thicker at the apex of the medial coronoid process. These regions of thick cartilage correspond with the sites where cartilage defects arise in elbow osteochondrosis. No histological evidence was found that the medial cornoid process of the ulna is a separate centre of ossification.

Osteopenia is a recognized complication of diabetes mellitus in humans and experimental animals. We recently found that tetracyclines prevent osteopenia in the streptozotocin-induced diabetic rat and that this effect was associated with a restoration of defective osteoblast morphology (Golub et al., 1990). The present study extends these initial ultrastructural observations by assessing osteoblast function in the untreated and tetracycline-treated diabetic rats. After a 3-week protocol, non-diabetic control and diabetic rats, including those orally administered a tetracycline, minocycline (MC), or a non-antimicrobial tetracycline analog (CMT), were perfusion-fixed with an aldehyde mixture; the humeri were dissected and processed for ultracytochemical localization of alkaline phosphatase (ALPase) and Ca-ATPase activities. Some rats from each experimental group received an intravenous injection of 3H-proline as a radioprecursor of procollagen, and the humeri were processed for light microscopic autoradiography. In addition, the osteoid volume in each experimental group was quantitatively examined by morphometric analysis of electron micrographs. During the diabetic state, active cuboidal osteoblasts in the endosteum of control rats were replaced by flattened bone-lining cells that contained few cytoplasmic organelles for protein synthesis (Golgi-RER system), and active transport (mitochondria). Treating diabetic rats with MC, and even more so with CMT, appeared to "restore" osteoblast structure. During diabetes, bone-lining cells incorporated little 3H-proline or secreted little labeled protein and produced only a very thin osteoid layer. Tetracycline administration to the diabetics increased both the incorporation of 3H-proline by osteoblasts and their secretion of labeled protein toward the osteoid matrix, in a pattern similar to that seen in the non-diabetic controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The occasional humeral growth retardation or premature closure of the growth plate medially, documented among more than 120 solitary bone cysts reviewed, was reason to undertake a thorough study of the growth plate in the area of the cyst. The findings in the hyaline zone disclose a considerable number of vessels and slits of the cartilage that are broadened toward the proliferative and hypertrophic zones. In some places they communicate with the vessels or are lined with endothelium. Ultrastructurally, changes of a destructive nature are disclosed, involving the growth plate zone and corresponding to the most active metabolic processes. In the altered chondrocytes substantial increases in glycogen granules and lipid droplets are observed, as well as reduction of the cellular organelles. In rather severely affected cells changes in the nuclei, lysosomal structures, and myelin-like lamellar bodies are established. The disappearance of cellular organelles and the fragmentation of cytoplasm are interpreted as a final phase of the above-listed changes. In conclusion, the cyst is considered a probable primary cause of the changes described.

The ultrastructural examination of four osteosarcomas (osteogenic, undifferentiated, and pleomorphic) is described. There are three types of tumor cells. Most of the cells are held in contact by desmosome-like tight junctions; they are atypical osteoblasts with cytoplasmic processes, dilated rough endoplasmic reticulum, mitochondria carrying calcific inclusions, lipid droplets surrounded by glycogen, and intracellular fine filamentous fibers. Other cells exhibiting varying degrees of osteoblastic maturity are also seen with multilobed nuclei, a clear cytoplasm, and straight bordered membranes. The last type is chondroid with abundant deposits of glycogen, lipid droplets, and undilated rough endoplasmic reticulum. The matrix is composed of fibrils without periodicity, scattered and deteriorated collagen fibers, and focal calcium deposits of hydroxyapatite crystals as in embryonal bone, dentine, or callus bone.

Malignant change is to be expected in about 1% of patients with Paget's disease. Usually these are osteosarcomas. Fibrosarcomas, benign and malignant giant cell tumours and chondrosarcomas are less common. The present case concerns the development of an immunoblastic sarcoma in the humerus, which was affected by Paget's disease. Only one other similar case is recorded in the literature. Other myelogenous tumours, such as plasmocytomas, are very rare in association with Paget's disease. Consequently there is no evidence for a causal connection between Paget's disease to these tumours, whereas sarcomas arising from bine tissue may be regarded as a form of malignant degeneration of Paget's disease.

A unique osseous tumor, which arose in the humerus of a 44-year-old white man, is reported. The lesion was designated atypical fibrous histiocytoma because of the absence of atypical mitoses despite prominent nuclear pleomorphism of tumor cells. The tumor was composed mainly of spindle cells, tightly packed in a storiform pattern. Clear histiocytes and tumor giant cells were occasionally seen. Toward the proximal and distal ends, the tumor showed increasing fibrogenesis, and the ends were composed of areas of packed clear histiocytes and acellular myxoid matrix with focal calcification. Ultrastructurally, five cell types comprised the tumor, but fibroblast-like cells predominated. Histiocyte-like, xanthomatous, giant and undifferentiated cells were observed only occasionally. The patient has been followed for five years after disarticulation without developing evidence of distant metastasis.

Two patients are reported with a chondroid form of osteosarcoma, whose structure was also analyzed submicroscopically. The clinical and radiologic picture and the rapid lethal progress of the disease indicated an osteosarcoma, while both histologic and electron-microscopic investigation suggested the prevalence of malignant chondroid. Progress in the treatment of osteosarcoma makes it essential for diagnostic, therapeutic and prognostic reasons to reintroduce subclassification of these tumors in an attempt to correlate their morphological picture with their biologic properties. According to our experience to date, it seems that the cartilaginous component in no way ameliorates the rapid malignant penetration of the osteosarcoma. It is essential to distinguish these tumors from the usually much less malignant chondrosarcomas and to place the chondroblastic osteogenic sarcoma among in spite of the clear predominance of the malignant chondroid, and thus also introduce highly radical and combined therapy.