Mechanical hysteresis loops from single osteons: technical devices and preliminary results

The purpose of this paper is to describe an original apparatus for recording hysteresis loops from single osteons and a special microgrinding lathe for preparing osteonic samples for testing. The results obtained by testing isolated osteons, and composite bone samples ground to the same size as an osteon sample justify one to draw the following main conclusions: at an equal degree of calcification, longitudinal osteons show larger hysteresis loops under compression and alternate osteons show larger hysteresis loops under tension; there seems to be little chance of acquiring detailed information on the mechanical effects of osteon calcification recording hysteresis loops; collagen orientation in lamellae is the main factor determining the kind of hysteresis loops displayed by a composite bone sample.     

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.     

Scaling of Haversian canal surface area to secondary osteon bone volume in ribs and limb bones

Studies of secondary osteons in ribs have provided a great deal of what is known about remodeling dynamics. Compared with limb bones, ribs are metabolically more active and sensitive to hormonal changes, and receive frequent low‐strain loading. Optimization for calcium exchange in rib osteons might be achieved without incurring a significant reduction in safety factor by disproportionally increasing central canal size with increased osteon size (positive allometry). By contrast, greater mechanical loads on limb bones might favor reducing deleterious consequences of intracortical porosity by decreasing osteon canal size with increased osteon size (negative allometry). Evidence of this metabolic/mechanical dichotomy between ribs and limb bones was sought by examining relationships between Haversian canal surface area (BS, osteon Haversian canal perimeter, HC.Pm) and bone volume (BV, osteonal wall area, B.Ar) in a broad size range of mature (quiescent) osteons from adult human limb bones and ribs (modern and medieval) and various adult and subadult non‐human limb bones and ribs. Reduced major axis (RMA) and least‐squares (LS) regressions of HC.Pm/B.Ar data show that rib and limb osteons cannot be distinguished by dimensional allometry of these parameters. Although four of the five rib groups showed positive allometry in terms of the RMA slopes, nearly 50% of the adult limb bone groups also showed positive allometry when negative allometry was expected. Consequently, our results fail to provide clear evidence that BS/BV scaling reflects a rib versus limb bone dichotomy whereby calcium exchange might be preferentially enhanced in rib osteons. Am J Phys Anthropol 151:230–244, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Peripheral vascularization of the dermal laminae of the equine hoof

The vascular architecture of the dermal laminae was studied by scanning electron microscopy of vascular corrosion casts. Ultrastructurally, the laminar vasculature consisted of arterioles, capillaries, venules and veins, arranged in a sheet-like network. Through the laminae, arterioles ran parallel to the solar surface and branched at two levels to form a continuous arteriolar arcade, parallel to the hoof wall. Capillaries originating from these arcades formed hairpin loops joining the marginal vein prior to forming an axially situated venous network. Additional capillaries were also given off by the arterioles, forming an abaxially arranged capillary plexus.     

Distribution of osteonic and interstitial components in the human femoral shaft with reference to structure, calcification and mechanical properties

This paper analyzes the distribution of osteons and interstitial bone in the femoral compacta according to their structure, degree of calcification and mechanical properties. Three cross sections, 100 microns thick, each located 1 cm from the next, were prepared by grinding from the middle third of a human femoral shaft. Starting from the premise that, in lamellar bone, lamellae whose fiber bundles and crystallites have a longitudinal course withstand loading by tension, whereas those whose fiber bundles and crystallites have a transversal course withstand loading by compression, each osteon and fragment of interstitial bone has been given a number recording the percentage of its surface consisting of lamellae with transversally oriented fiber bundles and crystallites (bright under the polarizing microscope). The degree of calcification of the same structures was determined micro-radiographically. The distribution of both osteons and interstitial bone was assessed using a tungsten grid for reference. The total surface of each bone microstructure, and the percentage of that surface consisting of bright lamellae, were all calculated using a Zeiss Video-plan. Our results confirm the view that the distribution of both osteons and interstitial bone is mainly related to their structure--and hence to their mechanical properties. In addition, bone remodeling seems to be most active in areas capable of supporting tensile stress.     

Mechanical properties of porcine femoral cortical bone measured by nanoindentation

This study uses a nanoindentation technique to examine variations in the local mechanical properties of porcine femoral cortical bone under hydrated conditions. Bone specimens from three age groups (6, 12 and 42 months), representing developing bone, ranging from young to mature animals, were tested on the longitudinal and transverse cross-sectional surfaces. Elastic modulus and hardness of individual lamellae within bone's microstructure: laminar bone, interstitial bone, and osteons, were measured. Both the elastic modulus and hardness increased with age. However, the magnitudes of these increases were different for each microstructural component. The longitudinal moduli were higher than the transverse moduli. Dehydrated samples were also tested to allow a comparison with hydrated samples and these resulted in higher moduli and hardness than the hydrated samples. Again, the degree of variation was different for each microstructural component. These results indicate that the developmental changes in bone have different rates of mechanical change within each microstructural component.     

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.     

Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice

Abstract

We investigated the presence and alteration of lymphatic vessels in joints of arthritic mice using a whole-slide imaging system. Joints and long bone sections were cut from paraffin blocks of two mouse models of arthritis: meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) and TNF transgene (TNF-Tg)-induced rheumatoid arthritis (RA). MLI-OA mice were fed a high fat diet to accelerate OA development. TNF-Tg mice were treated with lymphatic growth factor VEGF-C virus to stimulate lymphangiogenesis. Sections were double immunofluorescence stained with anti-podoplanin and alpha-smooth muscle actin antibodies. The area and number of lymphatic capillaries and mature lymphatic vessels were determined using a whole-slide imaging system and its associated software. Lymphatic vessels in joints were distributed in soft tissues mainly around the joint capsule, ligaments, fat pads and muscles. In long bones, enriched lymphatic vessels were present in the periosteal areas adjacent to the blood vessels. Occasionally, lymphatic vessels were observed in the cortical bone. Increased lymphatic capillaries, but decreased mature lymphatic vessels, were detected in both OA and RA joints. VEGF-C treatment increased lymphatic capillary and mature vessel formation in RA joints. Our findings suggest that the lymphatic system may play an important role in arthritis pathogenesis and treatment.     

Specifications for machining the bovine cortical bone in relation to its microstructure

Until date, many devices have been developed for cutting human bones during orthopedic surgeries. However, bones are anisotropic material, and their machining characteristics depend on the tool feed direction. In this study, microcutting of the bovine cortical bone is performed and its structure observed under a microscope. Furthermore, the formation of cutting chips and measurement of the cutting force during bone machining are dynamically observed while considering the anisotropy of bone tissue. In particular, the fracture of secondary osteons and crack propagation in bones are observed and analyzed. The results indicate that when the cut depth exceeds 20 μm and is greater than the interval of concentric lamellae, cracks are formed together with chips. A new method for bone machining is proposed. This method is based on the characteristics of crack propagation in bones and is expected to produce low mechanical stress and realize highly efficient and precise machining of living tissues such as bones.     

Morphometrics of compact bone: an example from Sudanese Nubia.

Morphometric analysis of compact femoral tissue was applied to a prehistoric population from Sudanese Nubia. Microradiographs of thin sections from below the lesser trochanter were examined. A total of 74 adults (40 females, 34 males) from the X-Group population (A.D. 350–550) were used to determine the underlying processes of bone remodeling in skeletal growth and maintenance. The relationship of bone turnover to the age of onset, patterning and frequency of cortical bone loss (osteoporosis) as a function of age and sex was examined. The cortical thickness, cortical area and formation/resorption frequencies were determined. Males exhibit a 4.9% net loss in cortical area, while females show a 10.7% loss. A substantial amount of female loss occurs in the third decade, with a slight gain in the fourth and a steady loss up to the sixth decade. These trends were further explored and refined histologically. The periosteal and endosteal frequencies for males show a variable but definite decrease in mineralization (i.e., increased numbers of osteons in the forming stage) and a slight increase in resorption. Third decade females show a marked difference with high frequencies of resorption spaces and forming osteons. It is suggested that in this population, stress related to childbearing and childrearing may be effecting the frequencies of formation foci and resorption spaces.     

Micro-biomechanics vs macro-biomechanics in cortical bone. A micromechanical investigation of femurs deformed by bending

Previous studies have shown that longitudinal and transverse lamellae in compact bone signal the presence of tensile and compressive forces, respectively. On this basis an investigation has been carried out to ascertain the distribution of lamellae in femoral shafts that have been deformed by bending. To do this, a series of undecalcified plane-parallel sections 100 microns thick were prepared, and the proportion of transversely oriented collagen and crystallites in the lamellae was measured, using circularly polarized light as an illuminating source and a Quantimet 720 image-analyzing computer. It has been concluded that the distribution of the two types of lamellae is in accordance with the need to compensate for the deformity produced by the bending of the bones, and that an obvious relationship exists between the macroscopic features of the femurs and their modified microscopic structures.     

Development and growth of long bones in European water frogs (Amphibia: Anura: Ranidae), with remarks on age determination

Differentiation and development of long bones were studied in European water frogs: Rana lessonae, R. ridibunda, and R. esculenta. The study included premetamorphic larvae (Gosner Stage 40) to frogs that were 5 years old. Femora, metatarsal bones, and proximal phalanges of the hindlimb exhibit the same pattern of periosteal bone differentiation and the same pattern of growth. Longitudinal and radial growth of these bones was studied by examination of the diaphyses and epiphyses, particularly where the edge of periosteal bone is inserted into the epiphysis. The periosteum seems to be responsible for both longitudinal and radial growth. Investigation of the formation, length, and arrangement of lines of arrested growth reveals that the first line is present only in the middle 25-35% of the length of the diaphysis of an adult bone; therefore, only the central portion of the diaphysis should be used for age estimation in skeletochronological studies. Comparison of the shapes and histological structures of epiphyses in the femur, metatarsal bones, and phalanges revealed that epiphyseal cartilages are composed of an inner and outer part. The inner metaphyseal cartilage has distinct zones and plugs the end of the periosteal bone cylinder; its role in longitudinal growth is questioned. The outer epiphyseal cartilage is composed of articular cartilages proper, in addition to lateral articular cartilages. Differences in the symmetry of the lateral articular cartilages of distal epiphyses of the femur and toes may reflect adaptations to different kinds of movements at the knee and in the foot.     

Establishment of the capillary-receptor connections of laminar bodies in the hand muscles of fetuses and newborn infants

The first findings of capillary-receptor connections of the laminar bodies in the hand short muscles are related with 5--6 months of embryogenesis. These connections become gradually more complex by the time of birth and in early postnatal life. Arterial portions of the capillaries penetration to the laminar bodies, arrange along the border of the internal cone and the external capsule and within the internal cone itself, along the course of the axonal cylinder. Venous capillary loops form a dense network on the surface of the laminar bodies. In the group accumulations of the laminar bodies (reflexogenic zones) the capillary network is denser than in identical areceptor zones of the hand muscles.     

Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RS™): In vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo

We have previously demonstrated that multilayered periosteal sheets prepared from the explant culture of alveolar periosteum serve as a promising osteogenic grafting material in periodontal tissue regeneration. For the preparation of more potent periosteal sheets, we examined the applicability of stem-cell culture media. Compared to the control medium (Medium 199 + 10% FBS), periosteal sheets expanded with MesenPRO-RS? medium exhibited these features: Cells grew three-dimensionally and deposited collagen in the extracellular spaces to form thicker multilayers of cells. Chondrocytic markers were not significantly upregulated. Contractile force was generated in proportion with the increased thickness of the periosteal sheets and the formation of cytoplasmic α-smooth muscle actin fibers. However, myofibroblastic markers were not significantly upregulated. The surface marker CD146 was substantially upregulated, while both CD73 and CD105 were downregulated. Alkaline phosphatase, a representative osteoblastic marker, was not upregulated by osteogenic induction. However, these expanded periosteal sheets exhibited substantially stronger osteogenic differentiation when implanted in nude mice. Therefore, despite our reservations, MesenPRO medium effectively expanded the cells contained in periosteal sheets to promote the formation of thicker multilayers of cells in vitro, and these enhanced periosteal sheets expressed increased osteogenic potential at implantation sites in vivo. In conjunction with data indicating that CD146-positive cells were notably expanded and the recently proposed concept that CD146 is a marker for osteogenic progenitor cells found in the bone marrow stroma, our findings suggest that MesenPRO medium improves the preparation of highly osteogenic periosteal sheets suitable for clinical application largely through the induction of CD146-positive cells.     

Setup of a bone aging experimental model in the rabbit comparing changes in cortical and trabecular bone: Morphological and morphometric study in the femur

Bone aging was studied in an experimental model (rabbit femur) in three populations aged 0.5, 1.5, and 7.5 years. Cortical bone histology was compared with a data set from a 1.5‐month‐old population of an earlier published paper. From 0.5‐year‐old onward, the mean femur length did not increase further. Thereafter, the mean marrow area increased and the cortical area decreased significantly with aging. This was associated with a structural pattern transformation from plexiform to laminar and then Haversian‐like type. The distal meta‐epiphysis bone trabecular density of the oldest populations also was significantly lower in specific regions of interest (ROI). Percentage sealed primary vascular canals in laminar bone significantly increased with aging without variation of percentage sealed secondary osteons. Remodeling rate reflected by the density of cutting cones did not significantly change among the age populations. These data suggest that laminar bone vascular pattern is more functional in the fast diaphyseal expansion but not much streamlined with the renewal of blood flow during secondary remodeling. Bone aging was characterized by: 1) secondary remodeling subendosteally; 2) increment of sealed primary vascular canals number; 3) increased calcium content of the cortex; 4) cortical and trabecular bone mass loss in specific ROIs. Taken together, the present data may give a morphological and morphometric basis to perform comparative studies on experimental models of osteoporosis in the rabbit. J. Morphol. 276:733–747, 2015. © 2015 Wiley Periodicals, Inc.     

New insights from bone microanatomy of the Late Triassic Hyperodapedon (Archosauromorpha,Rhynchosauria): implications for archosauromorph growth strategy

Bone microanatomy of multiple postcranial skeletal elements of several individuals of Hyperodapedon collected from India is reported. This reveals that fibrolamellar bone tissue is predominant in the mid‐ and inner cortices, whereas the peripheral region of the cortex is composed of either parallel‐fibred and/or lamellar bone. The pattern of primary osteons mostly ranges between laminar and subplexiform. Such predominance of fibrolamellar bone tissue in the cortex suggests an overall fast growth, which slowed down considerably later in ontogeny. Four distinct ontogenetic stages are identified based on the bone microstructure. During the juvenile stage, growth was fast and continuous, but it became punctuated during the early and late sub‐adult stages. In adult individuals, growth was slow and showed periodic interruption but did not stop completely, suggesting that Hyperodapedon had an indeterminate growth strategy. Interelemental histovariations affecting cortical thickness, organization of the vascular network, incidence of growth rings and extent of secondary reconstruction are noted. Throughout ontogeny, the femora show higher cortical thickness than humeri and tibiae, suggesting differential appositional growth rate between the skeletal elements. Differences in cortical thickness are noted in the ribs, which suggest differential functional constraints based on anatomical site‐specific occurrences. Although fibrolamellar bone tissue became progressively more dominant towards the archosaurs, there are considerable variations in the growth patterns of the archosauromorphs. This is exemplified by the bone microstructure of Hyperodapedon, which deviates from the generalized slow‐growth pattern proposed for all basal archosauromorphs, suggesting that rapid growth was already present in the archosauromorphs. The cortical thickness of various long bones of Hyperodapedon bears similarity with that of several extant terrestrial quadrupeds, suggesting that Hyperodapedon was essentially a terrestrial quadruped.     

LYSTROSAURUS MURRAYI (THERAPSIDA, DICYNODONTIA): BONE HISTOLOGY, GROWTH AND LIFESTYLE ADAPTATIONS

Abstract:  Examination of the bone microstructure of Lystrosaurus murrayi from India and South Africa reveals a predominance of fibrolamellar bone tissue, which suggests rapid periosteal osteogenesis and an overall fast growth. Four distinct ontogenetic stages have been identified based on tissue type, organization of the primary osteons, incidence of growth rings, secondary reconstruction and endosteal bone deposition. An indeterminate growth strategy is proposed for Lystrosaurus . Inter-elemental histovariability suggests differential growth rate of the skeletal elements within the same individual, and among different individuals. The high cortical thickness of the dorsal ribs, an extensive secondary reconstruction in the cortical region of different skeletal elements that resulted in erosionally enlarged channels from the perimedullary to the midcortical region, and trabecular infilling of the medullary region even in the diaphyseal sections of the limb bones suggest at least a semi-aquatic lifestyle for L. murrayi .     

The bending properties of single osteons

The bending properties of two fully calcified osteon types (longitudinal and alternate) have been investigated in 62 cylindrical samples by applying the technique of three-point bending loading. The bending of each sample was recorded using a microwave micrometer based on the cavity and pulse technique. It has been shown that alternate osteons are better able to withstand bending stress than longitudinal ones. This result offers a definitive explanation for the high concentration of transverse lamellae in pathologically bowed bone shaft.