Cortical bone age estimates from historically known adults.

Adult skeletal remains from 19th century historic cemeteries were used to test the accuracy and precision of age estimations based on the cortical bone histological technique proposed by Thompson [6, 7]. Comparison of estimates made from anterior microscopic fields versus peripheral fields (N = 29 femora) indicates general age agreement, with 6/29 demonstrating marked differences between sampling locations. Three cases of unilateral trauma show no consistent effect on bone age estimates. Among sixteen adults of known age, the ages of eight were accurately estimated; five were estimated to be significantly older and three were estimated to be younger than they were. It is argued that cortical erosion/diagenetic destruction may contribute to overestimation by obscuring the periosteal regions required for application of the technique. While not without its shortcomings, it is concluded that the Thompson technique is relatively robust and merits further development.     

Long bone histology of Ophiacodon reveals the geologically earliest occurrence of fibrolamellar bone in the mammalian stem lineage

Shared histological characteristics have been observed in the bone matrix and vascularity between Ophiacodontidae and the later therapsids (Synapsida). Historically, this coincidence has been explained as simply a reflection of the presumed aquatic lifestyle of Ophiacodon or even a sign of immaturity. Here we show, by histologically sampling an ontogenetic series of Ophiacodon humeri, as well as additional material, the existence of fibrolamellar bone (FLB) in the postcranial bones of a pelycosaur. Our findings have reaffirmed what previous studies first described as fast growing tissue, and by proxy, have disproven that the highly vascularized cortex is simply a reflection of young age. This tissue demonstrates the classic histological characteristics of true FLB. The cortex consists of primary osteons in a woven bone matrix and remains highly vascularized throughout ontogeny, providing evidence for fast skeletal growth. Overall, the FLB tissue we have described in Ophiacodon is more advanced or “mammal-like” in terms of the osteonal development, bone matrix, and skeletal growth than what has been described thus far for any other pelycosaur taxon. With regards to the histological record, our results remain inconclusive as to the preferred ecology of Ophiacodon due to a similar cortical vascularity pattern exhibited by other carnivorous pelycosaurs. Our findings have set the evolutionary origins of FLB and high skeletal growth rates back approximately 20 million years to the Early Permian, and by phylogenetic extension perhaps the Late Carboniferous.     

新石器时代人骨遗骸中古代DNA的提取及X—Y染色体同源基因片段的PCR扩增

从中国新石器时代人骨遗骸中提取出古代ＤＮＡ。通过聚合酶链式反应技术扩增得到Ｘ－Ｙ染色体上的单考贝同源基因片段。由于扩增的基因片段长度具有性别多态性,从而为古代人骨和牙齿提供了分子生物学的性别鉴定。     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

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

The histological method developed by Stout and Paine ([1992] Am. J. Phys. Antropol. 87:111–115) for estimating age at death using the clavicle is tested on a known age independent sample from a nineteenth century cemetery near Spitalfriedhof St. Johann in Basel, Switzerland. The mean absolute difference between reported ages and histologically predicted ages is 5.5 years. Mean predicted age for the sample is different from mean reported age. This difference is accounted for by differences in the age distributions between the original autopsy sample used to derive the histological age-predicting formula and the cemetery sample, and an inherent loss of reliability of histological age predictions for the skeletal remains of older individuals. A new formula based upon the combined original autopsy sample of Stout and Paine (1992) and the Swiss cemetery sample is presented. It is recommended that this formula be used when estimating ages for older individuals or archaeological skeletal samples. © 1996 Wiley-Liss, Inc.     

The assaying of haemoglobin using luminol chemiluminescence and its application to the dating of human skeletal remains

The luminol chemiluminescence reaction has, for some time, been used as a tool for the detection of haemoglobin at crime scenes. More recently, the luminol test has been suggested as a possible tool for estimating the post‐mortem interval (PMI) of skeletal remains. The preliminary results from the following study indicate that the chemiluminescent luminol test is a relatively easy and economical method for distinguishing between remains of medico‐legal (≤100 years) and historical (>100 years) interest. The femur was the preferred bone for PMI measurements using the luminol test, due to its robustness and relative resistance to diagenesis. Initial results suggest that bone that was historical in nature, produced a demonstrably weaker reaction than that of medico‐legal interest. These results suggest that the luminol test is a promising technique, albeit with some limitations, for the assessment of skeletal material that may be potentially of medico‐legal interest. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid Decalcification Using Microwaves for in Situ Hybridization in Skeletal Tissues

In situ hybridization histochemistry is the sole tool available for detecting the localization and expression of specific RNA on histological sections under various in vivo conditions. For this paper, we examined the effect of microwave exposure on the time needed for decalcification of skeletal tissues and on the preservation of sensitivity for hybridization signals. Our data show that the use of microwave decalcification reduces the decalcification period while preserving intense hybridization signals for mouse al chain of procollagen type I mRNA in osteogenic cells in bone. The use of microwave treatment to decalcify skeletal tissues may prevent delay in obtaining experimental results or the loss of signals during in situ hybridization.     

Growth requires bone resorption at particular skeletal elements in a teleost fish with acellular bone (Oreochromis niloticus, Teleostei: Cichlidae)

To date, little is known about bone resorption during skeletal development in teleostean fish with acellular bone. We report here about bone resorption with regard to growth in the tilapia Oreochromis niloticus. Nine skeletal elements obtained from growing juveniles were examined using histological and histochemical methods, and transmission electron microscopy (TEM). Tartrate-resistant acid phosphatase (TRAP) served as a marker for bone resorbing cells (osteoclasts), alkaline phosphatase (ALP) was used to identify osteoblasts, and alizarin staining indicated sites of bone formation. TRAP-activity was located at those skeletal elements where growth requires bone resorption, and at sites of cartilage degeneration. No TRAP-activity was found at those skeletal elements where resorption was not required for growth. The examination of the praeopercular shaft leads to a model of bone enlargement, including bone resorption by TRAP-positive cells located at the endosteal bone surface and bone formation by ALP-positive cells located at the periosteal bone surface. TRAP-positive cells were mononucleated and lacked a ruffled border. They appeared either as cell aggregates (resembling the shape of multinucleated giant cells) or as flat cells (resembling bone lining cells). Problems of osteoclast identification in bony fish are discussed.     

Compressive axial mechanical properties of rat bone as functions of bone volume fraction,apparent density and micro-ct based mineral density

Mechanical testing has been regarded as the gold standard to investigate the effects of pathologies on the structure–function properties of the skeleton. With recent advances in computing power of personal computers, virtual alternatives to mechanical testing are gaining acceptance and use. We have previously introduced such a technique called structural rigidity analysis to assess mechanical strength of skeletal tissue with defects. The application of this technique is predicated upon the use of relationships defining the strength of bone as a function of its density for a given loading mode. We are to apply this technique in rat models to assess their compressive skeletal response subjected to a host of biological and pharmaceutical stimulations. Therefore, the aim of this study is to derive a relationship expressing axial compressive mechanical properties of rat cortical and cancellous bone as a function of equivalent bone mineral density, bone volume fraction or apparent density over a range of normal and pathologic bones.We used bones from normal, ovariectomized and partially nephrectomized animals. All specimens underwent micro-computed tomographic imaging to assess bone morphometric and densitometric indices and uniaxial compression to failure.We obtained univariate relationships describing 71–78% of the mechanical properties of rat cortical and cancellous bone based on equivalent mineral density, bone volume fraction or apparent density over a wide range of density and common skeletal pathologies. The relationships reported in this study can be used in the structural rigidity analysis introduced by the authors to provide a non-invasive method to assess the compressive strength of bones affected by pathology and/or treatment options.     

Adolescent health in medieval Serbia: signs of infectious diseases and risk of trauma

Although pattern of health in adults has been frequently assessed in past human populations, health status of adolescents as a distinct life stage has usually been overlooked. Inconsistency in number and meaning of recognised age categories in anthropological literature, as well as chronological age ranges used to define them, further complicate the interpretation of adolescent health. In this study, we analysed signs of pathological conditions on skeletal remains of 81 adolescents from a medieval site of Stara Torina (northern Serbia). Diagnostic palaeopathological procedures comprised gross examination, digital radiography, and histological analysis.Skeletal signs of anaemia such as cribra orbitalia and other porotic phenomena as well as signs of non-specific bone infection were observed frequently, while evidence of bone trauma was recorded in a very low percentage of individuals. In addition, we recorded two conditions relatively rarely observed in palaeopathological contexts: a case of skull and vertebral asymmetry indicative of congenital muscular torticollis, and a case of a fibrous cortical defect on distal femur. Comparison with available information from other medieval adolescent samples from Serbia demonstrated that while mortality was relatively constant throughout the sample, Stara Torina showed a much higher occurrence of bone disease. Characteristics of observed skeletal conditions, supported by available historical reports, suggest that the health of medieval adolescents in the examined population was most significantly affected by infectious processes.     

Embedding of bone samples in methylmethacrylate: a suitable method for tracking LacZ mesenchymal stem cells in skeletal tissues.

Considerable research has been focused on the use of bone marrow-derived mesenchymal stem cells (MSCs) for the repair of non-unions and bone defects. To date, the question of whether transplanted MSCs survive and engraft within newly formed tissue remains unresolved. The development of an easy and reliable method that would allow cell fate monitoring in transplant recipients is a pressing concern for the field of tissue engineering. To demonstrate the presence of transplanted cells in newly formed bone, we established a xenograft nude rat model allowing the detection of murine LacZ MSCs in vivo. MSCs were isolated from transgenic lacZ mice, seeded onto bioabsorbable collagen sponges, and transplanted to repair a calvarial defect in nude rats. As a preliminary step, the histological procedure was adapted to optimize the detection of LacZ cells in bone tissue embedded in methylmethacrylate (MMA). Four fixatives and four fixation times were evaluated. Among all the fixatives tested, 2% formaldehyde/0.2% glutaraldehyde at 4C for 4 days gave the best results for X-gal staining at pH 7.4 on both cell cultures and bone explants. All fixatives were effective for immunodetection of beta-gal. In the chimeric LacZ/nude rat animal model, MSCs were detected in vivo for up to 4 weeks after implantation and contributed to the repair and the neovascularization of the bone defect. LacZ is a suitable phenotypic marker to track MSCs in skeletal tissues embedded in MMA.     

Gap junctions in skeletal development and function

Gap junctions play a critical role in the coordinated function and activity of nearly all of the skeletal cells. This is not surprising, given the elaborate orchestration of skeletal patterning, bone modeling and subsequent remodeling, as well as the mechanical stresses, strains and adaptive responses that the skeleton must accommodate. Much remains to be learned regarding the role of gap junctions and hemichannels in these processes. A common theme is that without connexins none of the cells of bone function properly. Thus, connexins play an important role in skeletal form and function.     

The Ice Man's diet as reflected by the stable nitrogen and carbon isotopic composition of his hair.

Establishing the diets of ancient human populations is an integral component of most archaeological studies. Stable isotope analysis of well-preserved bone collagen is the most direct approach for a general assessment of paleodiet. However, this method has been limited by the scarcity of well-preserved skeletal materials for this type of destructive analysis. Hair is preserved in many burials, but is often overlooked as an alternative material for isotopic analysis. Here we report that the stable carbon and nitrogen isotope values for the hair of the 5200 year-old Ice Man indicates a primarily vegetarian diet, in agreement with his dental wear pattern. Whereas previous investigations have focused on bone collagen, the stable isotope composition of hair may prove to be a more reliable proxy for paleodiet reconstruction, particularly when skeletal remains are not well preserved and additional archaeological artifacts are unavailable.     

Age-dependent fatigue behaviour of human cortical bone

Despite a general understanding that bone quality contributes to skeletal fragility, very little information exits on the age-dependent fatigue behavior of human bone. In this study four-point bending fatigue tests were conducted on aging bone in conjunction with the analysis of stiffness loss and preliminary investigation of nanoindentation based measurements of local tissue stiffness and histological evaluation of resultant tensile and compressive damage to identify the damage mechanism responsible for the increase in age-related bone fragility. The results obtained show that there is an exponential decrease in fatigue life with age, and old bone exhibits different modulus degradation profiles than young bone. In addition, this study provides preliminary evidence indicating that during fatigue loading, younger bone formed diffuse damage, lost local tissue stiffness on the tensile side. Older bone, in contrast, formed linear microcracks lost local tissue stiffness on the compressive side. Thus, the propensity of aging human bone to form more linear microcracks than diffuse damage may be a significant contributor to bone quality, and age related fragility in bone.     

Noggin suppression enhances in vitro osteogenesis and accelerates in vivo bone formation

Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.     

Growth and textural ageing in long bones of the American alligator Alligator mississippiensis (Crocodylia: Alligatoridae)

Growth series of femora, tibiae, and humeri of the American alligator Alligator mississippiensis were examined to assess the relationship between bone surface textures and relative skeletal maturity. Element texture types were compared with both size-based and size-independent maturity estimates. Selected elements were thin sectioned to observe the histological structures underlying various surface textures. Results suggest little to no relationship between bone textures and skeletal maturity in Alligator . Controlling for additional factors suspected to affect textural variation – sexual dimorphism, seasonally interrupted growth, wild vs. captive habitat, and geographical range – provides little resolution. Indeterminate growth is almost certainly a factor; however, this alone cannot explain all observed variability. Histological analyses reveal that highly porous surface textures are often associated with zones composed of fibrolamellar bone; smoother textures are generally underlain by lamellar zones or annuli. Textures of intermediate porosity may be associated with more than one histological pattern. Until the factors affecting bone texture changes in modern crocodylians are better understood, it is recommended that the textural ageing method be applied with caution to studies of fossil archosaurs with crocodylian-like or unknown growth regimes.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 150 , 1–39.     

From mouse to man: redefining the role of insulin-like growth factor-I in the acquisition of bone mass

The insulin-like growth factor system (IGF) has been linked to the process of bone acquisition through epidemiologic analyses of large cohorts and in vitro studies of bone cells. But the exact relationship between expression of IGF-I in bone and skeletal homeostasis or pathologic conditions, such as osteoporosis, remains poorly defined. Recent advances in genomic engineering have resulted in the development of better in vivo models to test the role of IGF-I during development and maintenance of the adult skeleton. It is now established that skeletal expression of IGF-I is critical for differentiative bone cell function. It may also be essential for the full anabolic effects of parathyroid hormone on trabecular bone and for some component of biomineralization. Evidence from conditional mutagenesis studies suggests that serum IGF-I may represent more than a storage depot or permissive factor during the final phase of skeletal acquisition. This work re-examines the original tenets of the "somatomedin hypothesis" in light of these newer mouse models and their remarkable skeletal phenotypes. The implications are far reaching and suggest that newer approaches for manipulating the IGF regulatory system may one day be useful as therapeutic adjuncts for the treatment of osteoporosis.     

Hierarchical grouping analysis and skeletal materials

The technique of hierarchical grouping analysis is examined with respect to its use as a tool in grouping skeletal remains, utilizing both continuous and certain types of discontinuous anthropometric variables. The method is found to be useful when tested with Amerind skeletal materials at the micro-evolutionary level, and as a technique for sexual differentiation within human and non-human primate populations. The application of this form of cluster analysis to dental data is examined.     

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization,cellular and structural detail

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.