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.     

Structure of the erosive lacunae on the periosteal and endosteal surfaces of bone

The periostal and endostal surfaces of the femur, ribs and lumbar vertebrae have been investigated in people at the age of 20-90 years. The erosive lacunae on the surface of the bone are revealed as recesses of various form and size. They are usually encountered as groups forming a resorptive focus, or fuse into large zones of resorption. Comparing the data of the light optic investigation and the results of SAM studies of the bone surface, it is possible to distinguish the erosive lacunae with an active process of resorption from the lacunae, where the process proceeds slowly or is not yet fully completed. On the periostal surface the resorptive foci are revealed more often, while on the endostal surface--large zones of resorption are seen. The erosive lacunae are often situated in the ostial parts of the vascular canals. No age dependence is noted in morphological manifestation of the resorption process on the investigated osseous surfaces of a mature person.     

Comparative Anatomy of Resin Canals of the Needles of Picea Grown in China

The comparative anatomy of the leaves of 21 species and 5 varieties of the genus Picea has been investigated with special reference to their resin canals. Resin canals are found in the needles of all Picea species under study. They are always arranged in longitudinal series, mostly being interrupted by transverse partitions of mesophyll except in a few species where they exhibit two disinterrupted canals. The number of the canal segments in a needle is quite variable in different species, and even in different needles of the same species. Their arrangement, according to the frequency of the occurrence of two-canals, one-canal and no canal in the middle transection of the needles and from the observation of the clearing specimens, can be classified into four groups (fig. 1). It would seem preferable that the characteristic difference of the resin canals, which might be of texonomic importance, lie in their discrepancy in the relative distribution of the resin canals rather than in the number or the relative length of the canals in each needle. This study also suggests that those with two canals lying along the lateral angles of the needles might be considered as the primitive types, from which other forms have been derived. Long canals may be broken into short cystic segments or subsequently degenerated into residue canals located only on one side of the needles.     

Detection of DNA in Ancient Bones Using Histochemical Methods

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian “Casti Amanti” house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4′ -′ 6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.     

Metrical analysis of the basicranium of extant hominoids and Australopithecus

The size and shape of the basicranium (seen in norma basilaris) in Homo, Gorilla, Pan, Pongo, and Australopithecus have been studied by recording the relative disposition of midline and bilateral bony landmarks. Fifteen linear measurements and two angles were used to relate the landmarks. The relatively longer and narrower cranial base of Gorilla, Pan, and Pongo is clearly contrasted with the wider, shorter cranial base in Homo sapiens. When the same observations were made on two “robust” and two “gracile” australopithecine crania, marked differences were found between the taxa. In the two “robust” specimens, the foramen magnum is located relatively further forward, and the axis of the petrous temporal bone is aligned more nearly with the coronal plane than in the two “gracile” crania. The implications of this apparent parallelism in basicranial morphology between Homo sapiens and the “robust” australopithecines are discussed.     

Observations of microdamage around osteocyte lacunae in bone

Tensile microdamage was examined using laser scanning confocal microscopy in beam specimens of bovine, equine and human long bones loaded in vitro and whole specimens of rat ulnae loaded in vivo. Microcracks were observed to initiate frequently at osteocyte lacunae. The implication is that osteocyte lacunae act as stress concentrating features in bone. This association provides a potential mechanism for the detection of strain and/ or damage by osteocytes in bone.     

The ultrafine structure of the body wall of sexually mature thorny-headed worms Echinorhynchus gadi (Acanthocephala)]

Studies of the fine structure of the adult acanthocephalan Echinorhynchus gadi have given a new information on the structure and organization of the body wall of these parasitic helminths. Their body surface is covered by glycocalyx of mucopolysaccharide nature. Just under it there is the surface membrane which has numerous invaginations forming a network of branching canals from which membrane vesicles are isolating. In their turn these canals pass through "the cytoplasmic canals" of the cortical matrix. Between the surface membrane and cortical matrix there is the base plate. These three structures form the striped layer underlain by the felt layer. It is formed by three layers of fibrous strands (one circular and two longitudinal), which are parallel to the body surface. These strands consist of loosely laid fibrils. The lowest layer is a radial one which occupies 2/3 of the body wall. It consists of the radial strands beginning from the cortical matrix and ending at the basement membrane. Numerous lipid droplets and glycogen granules are formed here. Two types of fibrils with 0.26 and 0.05 diameter have been detected for the first time. The radial layer in the cytoplasm was found to have crystalline structures and polymembrane bodies, numerous nuclei with light karyoplasm and distinct nucleoli. The location of the nuclei is of two types: either in the cytoplasm or in the "lacunae". We have shown that the "lacunae" are specialized sites of the cytoplasm whose boundaries are marked by the fibres of two types. Besides, this type of the acanthocephalan was found to have two "giant lacunae" extending along the body.(ABSTRACT TRUNCATED AT 250 WORDS)     

气候变化下千金榆在我国潜在分布区预测

气候变化严重影响植物的地理分布格局,植物分布对气候变化响应的区域差异性也将为引种和保存提供重要的指导.本研究基于ArcGIS与MaxEnt模型,利用176个千金榆地理信息和13个气候变量,对当前和未来的生态位进行重建.结果表明:模型模拟当代潜在分布区可信度极高,测试集与训练集AUC值分别为0.973和0.957.主要核心适宜区集中在秦岭、长白山及其各自的邻近区域,其他为零星的“岛屿”式分布.贵州、江西、云南、福建等地未发现千金榆的分布,但存在一定的适宜分布区.随着未来气候变暖,千金榆生态适宜区明显增加,主要表现为“向高海拔地区收缩”、“北扩”和“东扩”.但核心适宜区略有减少,具体表现为“南缩”、“中稳”和“北扩”.千金榆的分布对气候变暖的响应存在明显的区域性,在东部江苏、安徽等地,因其独特的地理位置与气候环境,该地区开始成为千金榆生态适宜区;在较低纬度的南方,原有的低海拔地区可能不再适宜千金榆生存;中部秦岭地区气候为南北的渐变区,有较强的缓冲能力,气候变暖对其分布区影响不大;较高纬度的长白山地区及其邻近区域愈发适宜千金榆生存.     

Modeling microdamage behavior of cortical bone

Bone is a complex material which exhibits several hierarchical levels of structural organization. At the submicron-scale, the local tissue porosity gives rise to discontinuities in the bone matrix which have been shown to influence damage behavior. Computational tools to model the damage behavior of bone at different length scales are mostly based on finite element (FE) analysis, with a range of algorithms developed for this purpose. Although the local mechanical behavior of bone tissue is influenced by microstructural features such as bone canals and osteocyte lacunae, they are often not considered in FE damage models due to the high computational cost required to simulate across several length scales, i.e., from the loads applied at the organ level down to the stresses and strains around bone canals and osteocyte lacunae. Hence, the aim of the current study was twofold: First, a multilevel FE framework was developed to compute, starting from the loads applied at the whole bone scale, the local mechanical forces acting at the micrometer and submicrometer level. Second, three simple microdamage simulation procedures based on element removal were developed and applied to bone samples at the submicrometer-scale, where cortical microporosity is included. The present microdamage algorithm produced a qualitatively analogous behavior to previous experimental tests based on stepwise mechanical compression combined with in situ synchrotron radiation computed tomography. Our results demonstrate the feasibility of simulating microdamage at a physiologically relevant scale using an image-based meshing technique and multilevel FE analysis; this allows relating microdamage behavior to intracortical bone microstructure.     

Visualization of acidic compartments in cultured osteoclasts by use of an acidotrophic amine as a marker for low pH

Using the acidotrophic amine 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP) as a marker for low pH and immunofluorescence cytochemistry, we examined acidic compartments of osteoclasts cultured on cover glasses or bone slices, where they could resorb the bone surface, forming resorptive lacunae. DAMP-positive structures were seen as vesicular and tubular forms in the cytoplasm, indicating lysosomes and endosomes. Not only the osteoclastic cytoplasm but also the extracellular area around the ruffled border and resorptive lacunae were stained with DAMP, suggesting acidic regions. Immunofluorescence was localized predominantly on the substratum side of actively resorbing osteoclasts, whereas an evenly distributed staining pattern was seen in the nonactive cell. The most intensive reaction was seen at the advancing front of resorptive lacunae within the actively resorbing osteoclasts. The distribution pattern of DAMP seemed to be correlated with the osteoclastic activity, since osteoclasts exhibit alternating resorption and migration phases during the bone-remodeling cycle. In this culture system, the resorptive lacunae were left behind after the osteoclasts had completed resorption and migrated along the bone surface. These exposed resorptive lacunae were also stained with DAMP, which were presumably kept at an acidic pH. The effect of treatment with monensin, chloroquine, ammonium chloride, or nigericin was varied in terms of the immunoreactivity for DAMP, but not complete abolition of the staining was obtained. Weak bases such as chloroquine or ammonium chloride inhibited both intra- and extracellular immunoreactivity. Immunoreactivity for the vacuolar type of proton ATPase (V-ATPase) was demonstrable in the cytoplasm of the osteoclasts but was weakened by the addition of bafilomycin. Immunofluorescence of the resorptive lacunae was still retained even after the treatment with bafilomycin and acetazolamide. Besides, both bafilomycin and acetazolamide reversibly inhibited cellular acidity as judged by DAMP immunocytochemistry, which agrees with the fact that ostoeclastic acidification results from the action of vacuolar proton-pump ATPase coupled with carbonic anhydrase.     

Spatial organization of the lacunar-canalicular system in the structures of bone lamellae

By means of light microscopical techniques and scanning electron microscopy spatial organization of the lacunar-canalicular system (LCS) has been studied in structures of a mature lamellar bone. A method for making corrosive casts of osseous lacunae and canaliculi is suggested, owing to which their spatial organization can be analysed. Certain data on interconnections of the osseous lacunae with each other and with vascular canals and natural surfaces of the bone are presented. The role of LCS as a component of the microcirculatory bed of the lamellar bone is discussed.     

The relative effects of collagen fiber orientation, porosity, density, and mineralization on bone strength

This investigation determined the relative importance of collagen fiber orientation, porosity, density, and mineralization in determining the tensile strength of bovine cortical bone. Thirty-nine specimens were tested for failure stress and the values of eight histologic and compositional parameters: collagen fiber orientation, wet and dry apparent density, percent mineralization of the bone matrix, and several components of porosity (Haversian canals, Volkmann's canals, and plexiform vascular spaces). Linear regression analysis showed that collagen fiber orientation was consistently the single best predictor of strength. Mineralization of the bone matrix was generally a poor predictor of strength. Density and porosity ranked between these variables in importance. Multiple regression equations containing all significantly correlated variables achieved correlation coefficients of 0.607 for plexiform bone and 0.881 for osteonal bone. Also, separate analysis of plexiform and osteonal specimens showed that the latter group was weaker even though it was less porous, apparently because it had collagen fibers which were less longitudinally oriented. This study suggests it is feasible to develop better empirical formulae for the prediction of cortical bone strength than are currently available if a variety of variables is introduced. Additional data are needed to confirm these results.     

Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar occlusion reveals failure and delay of remodeling in aged human bone

Aging decreases the human femur’s fatigue resistance, impact energy absorption, and the ability to withstand load. Changes in the osteocyte distribution and in their elemental composition might be involved in age‐related bone impairment. To address this question, we carried out a histomorphometric assessment of the osteocyte lacunar distribution in the periosteal and endosteal human femoral cortexes of 16 female and 16 male donors with regard to age‐ and sex‐related bone remodeling. Measurements of the bone mineral density distribution by quantitative backscattered electron imaging and energy dispersive X‐ray analysis were taken to evaluate the osteocyte lacunar mineral composition and characteristics. Age‐dependent decreases in the total osteocyte lacunar number were measured in all of the cases. This change signifies a risk for the bone’s safety. Cortical subdivision into periosteal and endosteal regions of interest emphasized that, in both sexes, primarily the endosteal cortex is affected by age‐dependent reduction in number of osteocyte lacunae, whereas the periosteal compartment showed a less pronounced osteocyte lacunar deficiency. In aged bone, osteocyte lacunae showed an increased amount of hypermineralized calcium phosphate occlusions in comparison with younger cases. With respect to Frost’s early delineation of micropetrosis, our microanalyses revealed that the osteocyte lacunae are subject to hypermineralization. Intralacunar hypermineralization accompanied by a decrease in total osteocyte lacunar density may contribute to failure or delayed bone repair in aging bone. A decreased osteocyte lacunar density may cause deteriorations in the canalicular fluid flow and reduce the detection of microdamage, which counteracts the bone’s structural integrity, while hypermineralized osteocyte lacunae may increase bone brittleness and render the bone fragile.     

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.     

Quantitative electron microscope observations of the collagen fibrils in rat-tail tendon

An electron microscope study of collagen fibrils from fixed tail tendons of rats has revealed that from some time shortly after birth until maturity, the fibril diameters have a bimodal distribution. The “two” types of fibril are indistinguishable in both transverse and longitudinal section. Unfixed specimens of eight-week-old-tail tendon showed a similar bimodal distribution of diameters though the positions of the peak values compared to fixed specimens of an eight-week-old-tail tendon were shifted upwards by about 30%. It has also been shown quantitatively that the polar collagen fibrils are directed randomly “up” and “down” with respect to their neighbors. Whilst it has been suggested by others that anastomosis is a feature of collagen structure, the results presented here do not support this hypothesis. Fibrillar units ～ 140 Å in diameter have been observed and the possibilities that these are elastic fibers or the breakdown products of collagen fibrils have been considered.     

Brief communication: Paleohistopathological analysis of pathology museum specimens: Can periosteal reaction microstructure explain lesion etiology?

The assertion that the microstructure of periosteal new bone formation can be used to differentiate between disease etiologies (Schultz: Yrbk Phys Anthropol 44 2001 106–147; Schultz: Identification of pathological conditions in human skeletal remains, 2nd ed. London: Academic Press 2003 73–109) was tested in a pilot‐study, using diagnosed bone specimens from St George's Hospital Pathology Museum, London, UK. Embedded bone specimens exhibiting pathological periosteal new bone formation were examined using scanning electron microscopy in back‐scattered electron imaging mode (SEM‐BSE). The results suggest that several histological features (i.e. Grenzstreifen, Polsters, and sinuous lacunae) deemed to be diagnostic of specific pathological conditions are of no specific diagnostic value, as they are encountered in pathological conditions of differing disease etiology. These results tie in with a previous investigation demonstrating a lack of diagnostic qualitative or quantitative characteristics seen in the macroscopic and radiographic appearance of periosteal reactions (Weston: Am J Phys Anthropol 137 2008 48–59). Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Ossification of the antler in the Lapland reindeer (Rangifer tarandus tarandus)

A histological and histochemical study of biopsy specimens from the Lapland reindeer antler indicated that the intercellular matrix of the cartilage that forms the partitions of longitudinal channels becomes calcified at virtually the initial stage of formation. The lacunae at the peripheries of the cartilaginous partitions are invaded by osteoblasts in a process comparable to endochondral ossification. The very centres of the partitions evidently become directly converted into bone without the presence of e.g. osteoclasts. The osseous partitions are remodelled by osteoclasts. The antler increases in diameter by periosteal apposition.     

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.     

Stress-concentrating effect of resorption lacunae in trabecular bone

Analyses of the distributions of stress and strain within individual bone trabeculae have not yet been reported. In this study, four trabeculae were imaged and finite elements models were generated in an attempt to quantify the variability of stress/strain in real trabeculae. In three of these trabeculae, cavities were identified with depths comparable to values reported for resorption lacunae ( approximately 50 microm)-although we cannot be certain, it is most probable that they are indeed resorption lacunae. A tensile load was applied to each trabeculum to simulate physiological loading and to ensure that bending was minimized. The force carried by each trabecula was calculated from this value using the average cross sectional area of each trabecula. The analyses predict that very high stresses (>100 MPa) existed within bone trabecular tissue. Stress and strain distributions were highly heterogeneous in all cases, more so in trabeculae with the presumptive resorption lacunae where at least 30% of the tissue had a strain greater than 4000 micoepsilon in all cases. Stresses were elevated at the pit of the lacunae, and peak stress concentrations were located in the longitudinal direction ahead of the lacunae. Given these high strains, we suggest that microdamage is inevitable around resorption lacunae in trabecular bone, and may cause the bone multicellular unit to proceed to resorb a packet of bone in the trabeculum rather than just resorb whatever localized area was initially targeted.