Bone typology and growth rate: testing and quantifying 'Amprino's rule' in the mallard (Anas platyrhynchos)

Periosteal bone histology expresses its rate of deposition. This fundamental relationship between bone structure and growth dynamics, first assumed by Amprino many decades ago, was quantified in preliminary studies, but never statistically tested. Moreover, the precise typological characters of bone tissue linked to growth rate remained poorly known. Here, we present the first statistical analysis of 'Amprino's rule', measured on comprehensive growth series of the mallard, Anas platyrhynchos. Growth rates were assessed by fluorescent labelling. Bone typology was described according to Ricqlès' typological classification. Results show that the presence and proportion of primary osteons, two consequences of bone initial porosity at the time of its deposit, are strongly related to bone growth rate. However, no significant relationship between primary osteons orientation and bone growth rate could be detected, at least for osteonal orientations (longitudinal, laminar and reticular) and growth rates values observed in mallard long bones. These results suggest that Amprino's rule holds for some major typological characters of primary compact bone tissues (i.e. primary osteons presence and proportion). However, it is irrelevant to some other characters (i.e. osteonal orientation), the meaning of which remains to be discovered.     

Evolutionary Patterns of Bone Histology and Bone Compactness in Xenarthran Mammal Long Bones

Bone microstructure reflects physiological characteristics and has been shown to contain phylogenetic and ecological signals. Although mammalian long bone histology is receiving increasing attention, systematic examination of the main clades has not yet been performed. Here we describe the long bone microstructure of Xenarthra based on thin sections representing twenty-two species. Additionally, patterns in bone compactness of humeri and femora are investigated. The primary bone tissue of xenarthran long bones is composed of a mixture of woven, parallel-fibered and lamellar bone. The vascular canals have a longitudinal, reticular or radial orientation and are mostly arranged in an irregular manner. Concentric rows of vascular canals and laminar organization of the tissue are only found in anteater bones. The long bones of adult specimens are marked by dense Haversian bone, a feature that has been noted for most groups of mammals. In the long bones of armadillos, secondary osteons have an oblique orientation within the three-dimensional bone tissue, thus resulting in their irregular shape when the bones are sectioned transversely. Secondary remodeling is generally more extensive in large taxa than in small taxa, and this could be caused by increased loading. Lines of arrested growth are assumed to be present in all specimens, but they are restricted to the outermost layer in bones of armadillos and are often masked by secondary remodeling in large taxa. Parameters of bone compactness show a pattern in the femur that separates Cingulata and Pilosa (Folivora and Vermilingua), with cingulates having a lower compactness than pilosans. In addition, cingulates show an allometric relationship between humeral and femoral bone compactness.     

The effect of T-2 toxin on bone microstructure in rabbits

T-2 toxin is the most toxic of the trichothecene mycotoxins. Its effect on bone microstructure is still unknown. This study focuses on acute effects of the T-2 toxin on compact and trabecular bone tissues structure of rabbits after a single intramuscular administration. Experimental E group (n?=?4) consisted of animals which were intramuscularly injected with T-2 toxin at dose 0.08 mg.kg^?1 body weight 72 h before slaughter. Group C (n?=?4) without T-2 toxin application served as a control. An absence of primary vascular longitudinal bone tissue near endosteal surfaces, its deposition on periosteal surfaces and a lower density of secondary osteons in the middle part of the substantia compacta were observed in both females and males injected with T-2 toxin. On the contrary, morphometrical analysis of the compact bone showed no demonstrable alternations in the sizes of primary osteons’ vascular canals, Haversian canals or secondary osteons between rabbits from E and C groups. Also, no significant effects of the T-2 toxin on trabecular bone morphometry and cortical bone thickness were observed between rabbits of either sex. The single intramuscular application of T-2 toxin at the dose used in our study affects only qualitative histological characteristics of the compact bone in rabbits.     

Bone regeneration within a coralline hydroxyapatite implant.

The hypothesis that incomplete resorption of osteons in an autogenous cortical bone graft may limit its replacement by new bone regeneration was explored by implanting a hydroxyapatite replica of a coral skeletal structure into bone gaps. This implant contained channels and interconnections similar to those in osteon-evacuated bone grafts. In 6 implanted mandibular defects in dogs, two of which were examined at two, 4, and 6 months, 11 percent, 46 percent, and 88 percent of the implant areas were filled with regenerated bone. The regenerated bone was a woven type at two months, but changed to a lamellar type by 6 months. In two implanted defects examined at 12 months, biodegradation of 29 percent of the implant had occurred. The bone regeneration was physiological, the implant was biocompatible, and the biodegradation began after the bone had regenerated.     

Histological structure of the cancellous bone layer in Bothriolepis canadensis (Antiarchi, Placodermi)

The Placodermi are extinct basal gnathostomes which had extensive dermal and perichondral bone, but which lacked the endochondral bone which characterizes the more derived bony fishes. Thin sections of bone from a specimen of the antiarch placoderm Bothriolepis canadensis, from the Escuminac Formation (Frasnian, Upper Devonian), Québec, Canada, reveal that part of the cancellous layer in its dermal and endoskeletal bone formed from perichondral bone trabeculae growing around cartilage spheres. The resultant structure mimics that of osteichthyan endochondral bone. The layout and dimensions of this polygonal mosaic patterning of the bone trabeculae and flattened cartilage spheres resemble those of the prismatic layers of calcified cartilage in chondrichthyans. If the lack of endoskeletal bone in chondrichthyans is a derived character, then the structure identified in B. canadensis could represent a 'template' for the formation of prismatic calcified cartilage in the absence of bone.     

Heparanase mRNA expression during fracture repair in mice

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.     

Interfacial Strength of Cement Lines in Human Cortical Bone

In human cortical bone, cement lines (or reversal lines) separate osteons from the interstitial bone tissue, which consists of remnants of primary lamellar bone or fragments of remodeled osteons. There have been experimental evidences of the cement line involvement in the failure process of bone such as fatigue and damage. However, there are almost no experimental data on interfacial properties of cement lines in human cortical bone. The objective of this study is to design and assemble a precision and computer controlled osteon pushout microtesting system, and to experimentally determine the interfacial strength of cement lines in human cortical bone by performing osteon pushout tests. Thirty specimens were prepared from humeral diaphyses of four human subjects. Twenty specimens were tested under the condition of a small hole in the supporting plate, in which the cement line debonding occurred. The cement line interfacial strength ranged from 5.38 MPa to 10.85 MPa with an average of 7.31±1.73 MPa. On the other hand, ten specimens were tested under the condition of a large hole in the supporting plate, in which the shear failure inside osteons was observed. The specimens tested under the condition of the large hole resulted in an average shear strength of 73.71±15.06 MPa, ranging from 45.97 MPa to 93.74 MPa. Therefore, our results suggest that the cement line interface between osteon and interstitial bone tissue is weaker than that between bone tissue lamellae.     

Two orinetational features of compact bone as predictors of split-line patterns

Ways of determining orientation patterns in compact bone are needed for specimens which are not available for split-line analysis. Weathering cracks have been shown to be essentially identical in orientation to split-lines, but they are more often than not absent or very few in number. Two other features of compact bone which promise to extend the range of orientational analysis are illustrated and described. These are (1) red or brown lines near the surface, and (2) numerous light-colored filaments, at or near the surface, laid parallel to each other. The latter manifestation has been observed more frequently and usually gives a more complete picture of orientational patterns. Specimens showing the two indicators were decalcified and processed for split-line patterns, which were essentially identical to the indicators in orientation. The first type of indicator is shown microscopically to be a manifestation of resorption spaces and immature osteons containing coagulated blood. The second represents oriented mature osteons in monkey pelves, but the microscopic basis in a cat pelvic bone is not clear.     

The healing of facial bone fractures by the process of secondary union

The mechanism of healing of facial bone fractures was investigated in a rabbit model. Twelve New Zealand white rabbits underwent surgically induced fractures of the right infraorbital rim and fracture ostectomies (4 to 5 mm) of the left infraorbital rim. Animals were sacrificed 2, 4, and 8 weeks postfracture. Bone, including periosteum, obtained from each fracture or fracture osteoctomy site was divided longitudinally for hematoxylin and eosin staining, fluorescent microscopy, microangiography, and microradiography. Sequential fluorochrome labels of oxytetracycline (30 mg/kg), alizarin complexone (30 mg/kg), DCAF (20 mg/kg), and xylenol orange (90 mg/kg) were administered 24 hours preoperatively and at 1, 2, 4, and 8 weeks postfracture. All fracture and fracture ostectomy sites demonstrated vascular ingrowth, mineralization, and woven bone formation by 2 to 4 weeks postoperatively, beginning with a cartilage precursor. Subsequently, the woven bone was replaced with remodeled lamellar bone, resulting in complete bony healing by 8 weeks postoperatively. These steps were substantiated by microscopic, microradiographic, and radiologic examination of the specimens. This study demonstrates that fractures of the facial bones in a rabbit model heal by a process of new bone formation that resembles secondary union in endochondral bones.     

Changes in the Microvasculature and Interstitium of Aged Human Mandibles and Maxillae1

The present study describes the age changes to the microvasculature and connective tissue interstitium of the osteons and periosteums of aged human mandibles and maxillae. The mandibles and maxillae obtained from 14 and 19 year old males, respectively, were also studied. In the nutrient canals of the aged osteons, the walls of the arterioles and venules stained intensely PAS positive, and alcian blue negative. The walls of the blood capillaries were thick and strongly PAS positive. There was a deposition of PAS positive material in the connective tissue stroma of the nutrient canals which progressed to the obliteration of the canal space. Many of the nutrient canals exhibited diffuse calcification within the connective tissue interstitium localized around the blood vessels. The lacunae and canaliculi of those osteons in which the nutrient canals were partially or completely obliterated were filled with PAS material. None of these histochemical changes were seen in the osteons of young individuals. The microvasculature of the aged periosteum showed similar changes. The periosteal tissue consisted of thick collagenous bundles and few osteogenic cells. There was a thin darkly stained amorphous calcified layer forming the bone surface.     

Developmental basis of limb length in rodents: evidence for multiple divisions of labor in mechanisms of endochondral bone growth

SUMMARY Mammals are remarkably diverse in limb lengths and proportions, but the number and kind of developmental mechanisms that contribute to length differences between limb bones remain largely unknown. Intra- and interspecific differences in bone length could result from variations in the cellular processes of endochondral bone growth, creating differences in rates of chondrocyte proliferation or hypertrophy, variation in the shape and size of chondrocytes, differences in the number of chondrocytes in precursor populations and throughout growth, or a combination of these mechanisms. To address these questions, this study compared cellular mechanisms of endochondral bone growth in cross-sectional ontogenetic series of the appendicular skeleton of two rodent species: the mouse ( Mus musculus ) and Mongolian gerbil ( Meriones unguiculatus ). Results indicate that multiple cellular processes of endochondral bone growth contribute to phenotypic differences in limb bone length. The data also suggest that separate developmental processes contribute to intraspecific length differences in proximal versus distal limb bones, and that these proximo-distal mechanisms are distinct from mechanisms that contribute to interspecific differences in limb bone length related to body size. These developmental "divisions of labor" are hypothesized to be important features of vertebrate limb development that allow (1) morphology in the autopods to evolve independently of the proximal limb skeleton, and (2) adaptive changes in limb proportions related to locomotion to evolve independently of evolutionary changes in body size.     

Molecular ontogeny of the skeleton

From a traditional viewpoint, skeletal elements form by two distinct processes: endochondral ossification, during which a cartilage template is replaced by bone, and intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts. There are inherent difficulties with this historical classification scheme, not the least of which is that bones typically described as endochondral actually form bone through an intramembranous process, and that some membranous bones may have a transient chondrogenic phase. These innate contradictions can be circumvented if molecular and cellular, rather than histogenic, criteria are used to describe the process of skeletal tissue formation. Within the past decade, clinical examinations of human skeletal syndromes have led to the identification and subsequent characterization of regulatory molecules that direct chondrogenesis and osteogenesis in every skeletal element of the body. In this review, we survey these molecules and the tissue interactions that may regulate their expression. What emerges is a new paradigm, by which we can explain and understand the process of normal- and abnormal-skeletal development.     

Evidence for Phosphoprotein Microspheres in Bone

Bone sialoprotein and osteopontin are bone-specific phosphoproteins, but their function is uncertain and their ultrastructural associations remain unclear. Insight into their role was sought by special attention to their general distribution and specific morphology under the high-power optical microscope. Their extracellular staining characteristics were examined in cryosections of adult rat skeletal tissues using two immunohistochemical methods. The two proteins were clearly evident in immature woven bone of endochondral and intramembranous origin (although cartilage was negative, even when calcified). In mature lamellar bone, bone sialoprotein remained ubiquitous, while osteopontin was confined to cement lines and other relatively discrete sites of past and present resorption activity, particularly near blood vessels. In neither case was the distribution of the stain structureless and diffuse. Invariably (except when non-specific), it was sharply defined and had the form of microspheres measuring approximately 1 m in diameter. In both immature and mature regions, these objects appeared in sheets, chains or groups in a pattern that was evidently coincident with a similar structural arrangement found within the inorganic phase of bone. It was concluded that phosphoproteins are not randomly located throughout the collagenous matrix but are apparently integral to calcified microsphere populations, and it is suggested that these structures are well placed to control the chemical State of the mineral over their surfaces and influence remodelling.     

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n=45) received daily injections of alendronate (n=27) or sterile saline solution as control (n=18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.     

Influence of interstitial bone microcracks on strain-induced fluid flow

It is well known that microcracks act as a stimulus for bone remodelling, initiating resorption by osteoclasts and new bone formation by osteoblasts. Moreover, microcracks are likely to alter the fluid flow and convective transport through the bone tissue. This paper proposes a quantitative evaluation of the strain-induced interstitial fluid velocities developing in osteons in presence of a microcrack in the interstitial bone tissue. Based on Biot theory in the low-frequency range, a poroelastic model is carried out to study the hydro-mechanical behaviour of cracked osteonal tissue. The finite element results show that the presence of a microcrack in the interstitial osteonal tissue may drastically reduce the fluid velocity inside the neighbouring osteons. This fluid inactive zone inside osteons can cover up to 10% of their surface. Consequently, the fluid environment of bone mechano-sensitive cells is locally modified.     

Animal welfare in modern dog breeding

Background

While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits.

Methods

Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed.

Results

Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter.

Conclusion

The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.     

Rethinking the nature of fibrolamellar bone: an integrative biological revision of sauropod plexiform bone formation

We present novel findings on sauropod bone histology that cast doubt on general palaeohistological concepts concerning the true nature of woven bone in primary cortical bone and its role in the rapid growth and giant body sizes of sauropod dinosaurs. By preparing and investigating longitudinal thin sections of sauropod long bones, of which transverse thin sections were published previously, we found that the amount of woven bone in the primary complex has been largely overestimated. Using comparative cellular and light‐extinction characteristics in the two section planes, we revealed that the majority of the bony lamina consists of longitudinally organized primary bone, whereas woven bone is usually represented only by a layer a few cells thin in the laminae. Previous arguments on sauropod biology, which have been based on the overestimated amount, misinterpreted formation process and misjudged role of woven bone in the plexiform bone formation of sauropod dinosaurs, are thereby rejected. To explain the observed pattern in fossil bones, we review the most recent advances in bone biology concerning bone formation processes at the cellular and tissue levels. Differentiation between static and dynamic osteogenesis (SO and DO) and the revealed characteristics of SO‐ versus DO‐derived bone tissues shed light on several questions raised by our palaeohistological results and permit identification of these bone tissues in fossils with high confidence. By presenting the methods generally used for investigating fossil bones, we show that the major cause of overestimation of the amount of woven bone in previous palaeohistological studies is the almost exclusive usage of transverse sections. In these sections, cells and crystallites of the longitudinally organized primary bone are cut transversely, thus cells appear rounded and crystallites remain dark under crossed plane polarizers, thereby giving the false impression of woven bone. In order to avoid further confusion in palaeohistological studies, we introduce new osteohistological terms as well as revise widely used but incorrect terminology. To infer the role of woven bone in the bone formation of fast‐growing tetrapods, we review some aspects of the interrelationships between the vascularity of bone tissues, basal metabolic rate, body size and growth rate. By putting our findings into the context of osteogenesis, we provide a new model for the diametrical limb bone growth of sauropods and present new implications for the evolution of fast growth in vertebrates. Since biomechanical studies of bone tissues suggest that predominant collagen fibre orientation (CFO) is controlled by endogenous, functional and perhaps phylogenetic factors, the relationship between CFO and bone growth rate as defined by Amprino's rule, which has been the basis for the biological interpretation of several osteohistological features, must be revised. Our findings draw attention to the urgent need for revising widely accepted basic concepts of palaeohistological studies, and for a more integrative approach to bone formation, biomechanics and bone microstructural features of extant and extinct vertebrates to infer life history traits of long extinct, iconic animals like dinosaurs.     

Metaplasia of chondrocytes into osteoblasts

Hypertrophic chondrocytes, commonly considered as terminal cells responsible for apoptotic elimination in endochondral osteogenesis, have the potential to switch their metabolic role and enter osteoblastic differentiation, based on histochemical, immunohistochemical, biochemical and cytological analysis. During endochondral osteogenesis, some osteocytes are derived from hypertrophic chondrocytes. Also non-hypertrophic chondrocytes are able to transform into osteogenic cells, and the bone thus formed is termed "transchondroid bone". In this review a summary and discussion of reports on chondrocyte transdifferentiation is given.     

Composition of the cement line and its possible mechanical role as a local interface in human compact bone

Human compact bone may be viewed as a fiber reinforced composite material in which the secondary osteons act as the fiber reinforcements. The cement line, which is the interface between the 'fibers' (osteons) and extraosteonal bone matrix, may impart important mechanical properties to compact bone. The nature of these properties is not known partly because the composition of the cement line is unknown. This analysis examines the constituents of the osteon cement line using scanning electron microscopy and X-ray microprobe analysis to address its biomechanical functions as a local interface. The analysis suggests that the cement line is a region of reduced mineralization which may contain sulfated mucosubstances. This composition is consistent with the hypothesis that the cement line provides a relatively ductile interface with surrounding bone matrix, and that it provides the point specific stiffness differences, poor 'fiber'-matrix bonding and energy transfer qualities required to promote crack initiation but slow crack growth in compact bone.