A Method to Interpolate Osteon Volume Designed for Histological Age Estimation Research.

Aging adult skeletal material is a crucial component of building the biological profile of unknown skeletal remains, but many macro- and microscopic methods have challenges regarding accuracy, precision, and replicability. This study developed a volumetric method to visualize and quantify histological remodeling events in three dimensions, using a two-dimensional serialized approach that applied circular polarizing microscopy and geographic information systems protocols. This approach was designed as a tool to extend current histological aging methodologies. Three serial transverse sections were obtained from a human femoral midshaft. A total sample size of 6847 complete osteons from the three sections was identified; 1229 osteons connected between all sections. The volume of all connected osteons was interpolated using ArcGIS area calculations and truncated cone geometric functions. Each section was divided into octants, and two random samples of 100 and of 30 connected osteons from each octant were generated. Osteon volume was compared between the octants for each random sample using ANOVA. Results indicated that the medial aspect had relative uniformity in osteon volume, whereas the lateral aspect showed high variability. The anterolateral-lateral octant had significantly smaller osteon volume, whereas the posterior-posterolateral octant had significantly larger osteon volume. Results also indicated that a minimum of 100 osteons is statistically more robust and more representative of normal osteon distribution and volume; the use of 30 osteons is insufficient. This research has demonstrated that osteon volume can be interpolated using spatial geometry and GIS applications and may be a tool to incorporate into adult age-at-death estimation techniques.

Compositional and mechanical properties of growing cortical bone tissue: a study of the human fibula.

Human cortical bone contains two types of tissue: osteonal and interstitial tissue. Growing bone is not well-known in terms of its intrinsic material properties. To date, distinctions between the mechanical properties of osteonal and interstitial regions have not been investigated in juvenile bone and compared to adult bone in a combined dataset. In this work, cortical bone samples obtained from fibulae of 13 juveniles patients (4 to 18 years old) during corrective surgery and from 17 adult donors (50 to 95 years old) were analyzed. Microindentation was used to assess the mechanical properties of the extracellular matrix, quantitative microradiography was used to measure the degree of bone mineralization (DMB), and Fourier transform infrared microspectroscopy was used to evaluate the physicochemical modifications of bone composition (organic versus mineral matrix). Juvenile and adult osteonal and interstitial regions were analyzed for DMB, crystallinity, mineral to organic matrix ratio, mineral maturity, collagen maturity, carbonation, indentation modulus, indicators of yield strain and tissue ductility using a mixed model. We found that the intrinsic properties of the juvenile bone were not all inferior to those of the adult bone. Mechanical properties were also differently explained in juvenile and adult groups. The study shows that different intrinsic properties should be used in case of juvenile bone investigation.

Ultrastructure of Bone: Hierarchical Features from Nanometer to Micrometer Scale Revealed in Focused Ion Beam Sections in the TEM.

The ultrastructure of bone has been widely debated, in part due to limitations in visualizing nanostructural features over relevant micrometer length scales. Here, we employ the high resolving power and compositional contrast of high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) to investigate new features in human bone with nanometer resolution over microscale areas. Using focused ion beam (FIB)-milled sections that span an area of 50 µm2, we have shown how most of the mineral of cortical human osteonal bone occurs in the form of long, thin polycrystalline plates (mineral lamellae, MLs) which are either flat or curved to wrap closely around collagen fibrils. Close to the collagen fibril (< 20 nm), the radius of curvature matches that of the fibril diameter, while at greater distances, MLs form arcs with much larger radii of curvature. In addition, stacks of closely packed planar (uncurved) MLs occur between fibrils. The curving of mineral lamellae both around and between the fibrils would contribute to the strength of bone. At a larger scale, rosette-like clusters of fibrils are noted for the first time, arranged in quasi-circular arrays that define tube-like structures in alternating osteonal lamellae. At the boundary between adjacent osteonal lamellae, the orientation of fibrils and surrounding mineral lamellae changes abruptly, resembling the "orthogonal" patterns identified by others (Reznikov et al. in Acta Biomater 10:3815-3826, 2014). These features spanning nanometer to micrometer scale have implications for our understanding of bone structure and mechanical integrity.

A method for measuring the three-dimensional orientation of cortical canals with implications for comparative analysis of bone microstructure in vertebrates.

The orientation of vascular canals in cortical bone can reveal information about the growth rate and loading environment of a bone. For example, in birds it has been proposed that a high proportion of circumferential canals (a laminar cortex) is related to fast growth or torsional loading related to active flight. In this paper we present a method to measure the three dimensional (3D) orientation of vascular canals. Image data are obtained from micro-CT and two angles are measured: phi, determining how longitudinal a canal is; and theta, determining whether a canal is radial or circumferential. This method can measure the orientation of each canal contained in the scanned images. Here we demonstrate the approach on two samples - a rat tibia and a hawk humerus. This method offers a direct (3D) method for quantifying features of canal orientation, such as the degree of laminarity, and can be applied easily and non-destructively to multiple species and bones. The growth and development of the cortical canal network and its impact on factors such as bone strength and bone quality remains relatively unexplored. Our method provides a new tool to examine the impact of the orientation of cortical bone canals on bone and explore the origins of cortical canals formed during modelling and remodeling. This method has applications in comparative bone biology, small animal models, and human bone studies.

The application of heat-deproteinization to the morphological study of cortical bone: A contribution to the knowledge of the osteonal structure.

Observation of heat-deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin-embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat-deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat-deproteinized samples, as compared with dehydrated controls. In both samples, the X-ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat-deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech. 79:691-699, 2016. © 2016 Wiley Periodicals, Inc.

Three-dimensional reconstruction of Haversian systems in human cortical bone using synchrotron radiation-based micro-CT: morphology and quantification of branching and transverse connections across age.

This study uses synchrotron radiation-based micro-computed tomography (CT) scans to reconstruct three-dimensional networks of Haversian systems in human cortical bone in order to observe and analyse interconnectivity of Haversian systems and the development of total Haversian networks across different ages. A better knowledge of how Haversian systems interact with each other is essential to improve understanding of remodeling mechanisms and bone maintenance; however, previous methodological approaches (e.g. serial sections) did not reveal enough detail to follow the specific morphology of Haversian branching, for example. Accordingly, the aim of the present study was to identify the morphological diversity of branching patterns and transverse connections, and to understand how they change with age. Two types of branching morphologies were identified: lateral branching, resulting in small osteon branches bifurcating off of larger Haversian canals; and dichotomous branching, the formation of two new osteonal branches from one. The reconstructions in this study also suggest that Haversian systems frequently target previously existing systems as a path for their course, resulting in a cross-sectional morphology frequently referred to as 'type II osteons'. Transverse connections were diverse in their course from linear to oblique to curvy. Quantitative assessment of age-related trends indicates that while in younger human individuals transverse connections were most common, in older individuals more evidence of connections resulting from Haversian systems growing inside previously existing systems was found. Despite these changes in morphological characteristics, a relatively constant degree of overall interconnectivity is maintained throughout life. Altogether, the present study reveals important details about Haversian systems and their relation to each other that can be used towards a better understanding of cortical bone remodeling as well as a more accurate interpretation of morphological variants of osteons in cross-sectional microscopy. Permitting visibility of reversal lines, synchrotron radiation-based micro-CT is a valuable tool for the reconstruction of Haversian systems, and future analyses have the potential to further improve understanding of various important aspects of bone growth, maintenance and health.

Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon.

Modular tissue engineering holds great potential in regenerating natural complex tissues by engineering three-dimensional modular scaffolds with predefined geometry and biological characters. In modular tissue-like construction, a scaffold with an appropriate mechanical rigidity for assembling fabrication and high biocompatibility for cell survival is the key to the successful bioconstruction. In this work, a series of composite hydrogels (GH0, GH1, GH2, and GH3) based on a combination of methacrylated gelatin (GelMA) and hydroxyapatite (HA) was exploited to enhance hydrogel mechanical rigidity and promote cell functional expression for osteon biofabrication. These composite hydrogels presented a lower swelling ratio, higher mechanical moduli, and better biocompatibility when compared to the pure GelMA hydrogel. Furthermore, on the basis of the composite hydrogel and photolithograph technology, we successfully constructed an osteon-like concentric double-ring structure in which the inner ring encapsulating human umbilical vascular endothelial cells (HUVECs) was designed to imitate blood vessel tubule while the outer ring encapsulating human osteoblast-like cells (MG63s) acts as part of bone. During the coculture period, MG63s and HUVECs exhibited not only satisfying growth status but also the enhanced genic expression of osteogenesis-related and angiogenesis-related differentiations. These results demonstrate this GelMA-HA composite hydrogel system is promising for modular tissue engineering.

Shear deformation and fracture of human cortical bone.

Bone can be viewed as a nano-fibrous composite with complex hierarchical structures. Its deformation and fracture behaviors depend on both the local structure and the type of stress applied. In contrast to the extensive studies on bone fracture under compression and tension, there is a lack of knowledge on the fracture process under shear, a stress state often exists in hip fracture. This study investigated the mechanical behavior of human cortical bone under shear, with the focus on the relation between the fracture pattern and the microstructure. Iosipescu shear tests were performed on notched rectangular bar specimens made from human cortical bone. They were prepared at different angles (i.e. 0°, 30°, 60° and 90°) with respect to the long axis of the femoral shaft. The results showed that human cortical bone behaved as an anisotropic material under shear with the highest shear strength (~50MPa) obtained when shearing perpendicular to the Haversian systems or secondary osteons. Digital image correlation (DIC) analysis found that shear strain concentration bands had a close association with long bone axis with an average deviation of 11.8° to 18.5°. The fracture pattern was also greatly affected by the structure with the crack path generally following the direction of the long axes of osteons. More importantly, we observed unique peripheral arc-shaped microcracks within osteons, using laser scanning confocal microscopy (LSCM). They were generally long cracks that developed within a lamella without crossing the boundaries. This microcracking pattern clearly differed from that created under either compressive or tensile stress: these arc-shaped microcracks tended to be located away from the Haversian canals in early-stage damaged osteons, with ~70% developing in the outer third osteonal wall. Further study by second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) microscopy revealed a strong influence of the organization of collagen fibrils on shear microcracking. This study concluded that shear-induced microcracking of human cortical bone follows a unique pattern that is governed by the lamellar structure of the osteons.

Nonvascular transport distraction osteogenesis in bone formation and regeneration. Is it an accidental phenomenon?

PURPOSE: To explore the osteogenic mechanism of nonvascular transport distraction osteogenesis (NTDO) by constructing mandibular defects in dogs. METHODS: Sixty adult dogs were randomly divided into three groups with 20 dogs in each group. Canine mandibular defect models of NTDO were constructed. Animals were euthanized 1, 4 and 12 weeks after distraction, and the transport disc and surrounding tissue were collected and fixed. Histochemical staining using hematoxylin and eosin (H&E) and electron microscopic observations were used to examine bone regeneration. RESULTS: Distraction bone regeneration was observed in the distraction gap and around the transport disc, and osseous connections had formed between new bone and the transport disc after one week. Osteoclasts gathered around the transport disc, and bone absorption pit formation could be seen. After 4 weeks of distraction, the new bone around the transport disc was close to maturity with thick sclerostin on the middle of the transport disc. After 12 weeks the new bone and the transport disc were fully integrated, and were difficult to distinguish by H&E staining and electron microscopy. CONCLUSIONS: Canine mandibular defects were successfully repaired by NTDO resulting in ideal new bone formation and fully recovered mandibular physiological function. The surrounding tissues, including musculoskeletal tissues, the periosteum and other soft tissues and the nonvascular transport disc, together contribute to bone regeneration and neovascularization in NTDO.

[Species Identification Based on Morphological Variability of Femur].

OBJECTIVE: To study the morphological characteristics of femurs of adult human and 11 kinds of adult animals from cattle, horses, pigs, goats, sheep, dogs, cats, rabbits, geese, ducks, chickens, and to establish an effective species identification method among various species. METHODS: The 4 cm mid-diaphyseal segment of the femur from adult human (older than 20 years old) at autopsy was obtained. Addi-tionally, the 4 cm ones from 11 kinds of adult animals were obtained. After decalcification, all femurs were made into slices, and then were observed by optical microscope. The 25 indexes were selected and analyzed by step discriminant analysis according to differences between human and mammal, human and poultry, and human and 11 kinds of animals. RESULTS: The histological structure of bone mineral density of middle part of femur had obvious characteristics among the species. And the morphology and number of osteon showed the trend of obvious biological evolution. There were 11 indexes with significant differences between human and 11 kinds of animals to establish some mathematical models to discriminate all species. The correct discrimination rate was 96.3% between human and mammal. The correct discrimination rate was up to 100% between human and poultry, and was 89.4% among human, mammal and poultry. CONCLUSION: The mathematical models have good correct discrimination rate among human and the other animals, which could be applied in the practical species identification cases.

Species Identification Based on Morphological Variability of Femur / 法医学杂志

OBJECTIVE@#To study the morphological characteristics of femurs of adult human and 11 kinds of adult animals from cattle, horses, pigs, goats, sheep, dogs, cats, rabbits, geese, ducks, chickens, and to establish an effective species identification method among various species.@*METHODS@#The 4 cm mid-diaphyseal segment of the femur from adult human (older than 20 years old) at autopsy was obtained. Addi-tionally, the 4 cm ones from 11 kinds of adult animals were obtained. After decalcification, all femurs were made into slices, and then were observed by optical microscope. The 25 indexes were selected and analyzed by step discriminant analysis according to differences between human and mammal, human and poultry, and human and 11 kinds of animals.@*RESULTS@#The histological structure of bone mineral density of middle part of femur had obvious characteristics among the species. And the morphology and number of osteon showed the trend of obvious biological evolution. There were 11 indexes with significant differences between human and 11 kinds of animals to establish some mathematical models to discriminate all species. The correct discrimination rate was 96.3% between human and mammal. The correct discrimination rate was up to 100% between human and poultry, and was 89.4% among human, mammal and poultry.@*CONCLUSION@#The mathematical models have good correct discrimination rate among human and the other animals, which could be applied in the practical species identification cases.

Chemical etching in processing cortical bone specimens for scanning electron microscopy.

Transverse and longitudinal sectioning of undecalcified cortical bone is a commonly employed technique for investigating the lamellar structure of the osteons. Since a flat surface is required, the specimen has to be grinded and then polished. Whereas the smear of debris and inorganic/organic deposits left by these treatments cannot be removed by ultrasonication alone, a chemical treatment of the specimen surface with either a basic or an acid etching solution is currently employed. A further effect of the latter can be the enhancement of the lamellar bone pattern. The kind of etching solution, its pH, the concentration of etchants, and the contact time significantly affect the sectioned surface when it is observed with scanning electron microscopy (SEM). The etching procedures can severely influence the obtained images. Homogeneous cortical bone specimens were sampled from the first metatarsal of two fresh human subjects. One or two cut surfaces were exposed to different acid and basic solutions in bonded conditions. Considering the type of chemical agents, the solution pH, and the exposure time of the specimens, the effects of several etching media have been investigated and compared. Strong etching, either acid or basic produced surface decalcification and severe damage of the collagen matrix, compromising any morphological or morphometric analysis. Weak acid etching (for example citric and acetic acid), even though causing distinctive alteration of the sample, enhanced the visibility of the lamellar pattern, while the polyphosphate treatment of the surface decalcified a thin layer matrix, ensuring a good visibility of fibrils and avoiding rough distortions.

Local microarchitecture affects mechanical properties of deposited extracellular matrix for osteonal regeneration.

Multiple biomimetic approaches have been attempted to accelerate the regeneration of functional bone tissue. While most synthetic scaffolds are designed to mimic the architecture of trabecular bone, in the current study, cortical bone-like extracellular matrix was regenerated in vitro within organized structures. Biphasic calcium phosphate (BCaP) and hydroxyapatite (HAp) scaffolds were developed with longitudinal microchannels (250 µm diameter) that resembled native osteons in cortical bone. BCaP and HAp scaffolds had a compressive strength of 7.61±1.42 and 9.98±0.61 MPa respectively. The constructs were investigated in vitro to evaluate the organization and stiffness of the extracellular matrix (ECM) formed by human fetal osteoblasts (HFObs) cultured inside the microchannels. The ECM deposited on the BCaP scaffolds was found to have a higher micro-hardness (h) (1.93±0.40 GPa) than the ECM formed within the HAp microchannels (h=0.80±0.20 GPa) (p<0.05) or native bone (h=0.47-0.74 GPa). ECM deposition within the microchannels resembled osteoid organization and showed a significant increase in both osteoid area and thickness after 24 days (p<0.001). These observations indicate that controlled microarchitecture, specifically cylindrical microchannels, plays a fundamental role in stimulating the appropriate cellular response aimed at recreating organized, cortical bone-like matrix. These findings open the door for researchers to develop a new generation of cortical bone scaffolds that can restore strong, organized bone.

Bone repair of critical size defects treated with mussel powder associated or not with bovine bone graft: histologic and histomorphometric study in rat calvaria.

The objective of this study was to evaluate the bone repair of critical size defects treated with mussel powder with or without additional bovine bone. Critical size defects of 5 mm were realized in the calvaria of 70 rats, which were randomly divided in 5 groups - Control (C), Autogenous Bone (AB), Mussel Powder (MP), Mussel Powder and Bovine Bone (MP-BB) and Bovine Bone (BB). Histological and histomorphometric analysis were performed 30 and 90 days after the surgical procedures (ANOVA e Tukey p < 0.05). After 30 days, the measures of remaining particles were: 28.36% (MP-BB), 26.63% (BB) and 8.64% (MP) with a statistically significant difference between BB and MP. The percentage of osseous matrix after 30 days was, AB (55.17%), 23.31% (BB), 11.66% (MP) and 10.71% (MP-BB) with statistically significant differences among all groups. After 90 days the figures were 25.05% (BB), 21.53% (MP-BB) and 1.97% (MP) with statistically significant differences between MP-BB and MP. Percentages of new bone formation after 90 days were 89.47% (AB), 35.70% (BB), 26.48% (MP-BB) and 7.37% (MP) with statistically significant differences between AB and the other groups. Within the limits of this study, we conclude that mussel powder, with or without additional bovine bone, did not induce new bone formation and did not repair critical size defects in rat calvaria.

The effects of freezing, boiling and degreasing on the microstructure of bone.

The histology of bone has been a useful tool in research. It is commonly used to estimate the age of an individual at death, to assess if the bone is of human or non-human origin and in trauma analysis. Factors that affect the histology of bone include age, sex, population affinity and burning to name but a few. Other factors expected to affect bone histology are freezing, boiling and degreasing but very little information is available for freezing and the effect thereof, and it is unknown if boiling and degreasing affects bone histology. The aim of this study was to assess the effects of freezing, freezing and boiling, and freezing, boiling and degreasing on the histological structure of compact bone. Five cadaver tibiae were frozen at -20°C for 21 days followed by segments being boiled in water for three days and degreased in trichloroethylene at 82°C for three days. Anterior midshaft sections were prepared as ground sections and for Scanning Electron Microscopy (SEM). Quantitatively, there were no significant differences between freezing, boiling and degreasing; however, qualitative differences were observed using SEM. After being frozen the bone displayed cracks and after boiling the bones displayed erosion pits on the surface. It is suggested that further research, using different durations and temperatures for boiling and freezing be undertaken on bone samples representing different ages and various skeletal elements.

The long bone deformity of osteogenesis imperfecta III: analysis of structural changes carried out with scanning electron microscopic morphometry.

The wedges of the mid-diaphyseal osteotomies carried out to correct the femoral and/or tibial native deformity in type III osteogenesis imperfecta (OI III) were used to study the remodeling patterns and lamellar organization at the level of the major deformity. Histology and scanning electron microscopy (SEM) morphology showed abnormal cortical remodeling characterized by the failure to form a cylinder of compact bone with a regular marrow canal. Atypical, flattened, and large resorption lacunae with a wide resorption front on one side and systems of parallel lamellae on the opposite side were observed, resembling those formerly reported as drifting osteons. SEM morphometry documented a higher percentage of nonossified vascular/resorption area (44.3 %) in OI than in controls (13.6 %), a lower density of secondary osteons, and lower values for the parameters expressing the individual osteon size. The mean osteon total area, the mean central canal area, and the mean osteon bone area of two selected, randomized populations of secondary osteons were significantly higher (p < 0.001, p = 0.028, and p < 0.001, respectively) in control bones than in OI. The mean ossified matrix area was not significantly different, but the mean secondary osteon number and mean density were higher in controls (both p < 0.001). Osteon wedges were carried out to correct the native deformity of OI III and morphologic analysis suggested that the abnormal remodeling pattern (with "drifting osteons") may result from the altered load and tensile stresses on the deformed tubular bones.

Orientation and size-dependent mechanical modulation within individual secondary osteons in cortical bone tissue.

Anisotropy is one of the most peculiar aspects of cortical bone mechanics; however, its anisotropic mechanical behaviour should be treated only with strict relationship to the length scale of investigation. In this study, we focus on quantifying the orientation and size dependence of the spatial mechanical modulation in individual secondary osteons of bovine cortical bone using nanoindentation. Tests were performed on the same osteonal structure in the axial (along the long bone axis) and transverse (normal to the long bone axis) directions along arrays going radially out from the Haversian canal at four different maximum depths on three secondary osteons. Results clearly show a periodic pattern of stiffness with spatial distance across the osteon. The effect of length scale on lamellar bone anisotropy and the critical length at which homogenization of the mechanical properties occurs were determined. Further, a laminate-composite-based analytical model was applied to the stiffness trends obtained at the highest spatial resolution to evaluate the elastic constants for a sub-layer of mineralized collagen fibrils within an osteonal lamella on the basis of the spatial arrangement of the fibrils. The hierarchical arrangement of lamellar bone is found to be a major determinant for modulation of mechanical properties and anisotropic mechanical behaviour of the tissue.

Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures.

Lamellar bone is a major component of most mammalian skeletons. A prominent component of individual lamellae are parallel arrays of mineralized type I collagen fibrils, organized in a plywood like motif. Here we use a dual beam microscope and the serial surface view (SSV) method to investigate the three dimensional collagen organization of circumferential lamellar bone from rat tibiae after demineralization and osmium staining. Fast Fourier transform analysis is used to quantitatively identify the mean collagen array orientations and local collagen fibril dispersion. Based on collagen fibril array orientations and variations in fibril dispersion, we identify 3 distinct sub-lamellar structural motifs: a plywood-like fanning sub-lamella, a unidirectional sub-lamella and a disordered sub-lamella. We also show that the disordered sub-lamella is less mineralized than the other sub-lamellae. The hubs and junctions of the canalicular network, which connect radially oriented canaliculi, are intimately associated with the disordered sub-lamella. We also note considerable variations in the proportions of these 3 sub-lamellar structural elements among different lamellae. This new application of Serial Surface View opens the way to quantitatively compare lamellar bone from different sources, and to clarify the 3-dimensional structures of other bone types, as well as other biological structural materials.

Morphometry and patterns of lamellar bone in human Haversian systems.

The lamellar architecture of secondary osteons (Haversian systems) has been studied with scanning electron microscopy (SEM) in transverse sections of human cortical bone. Na(3) PO(4) etching was used to improve the resolution of the interface between neighboring lamellae and the precision of measurements. These technical improvements permitted testing of earlier morphometry assumptions concerning lamellar thickness while revealing the existence of different lamellar patterns. The mean lamellar thickness was 9.0 ± 2.13 µm, thicker and with a wider range of variation with respect to earlier measurements. The number of lamellae showed a direct correlation with the lamellar bone area, and their thickness had a random distribution for osteonal size classes. The circular, concentrical pattern was the more frequently observed, but spiral and crescent-moon-shaped lamellae were also documented. Selected osteons were examined by either SEM or SEM combined with polarized light microscopy allowing comparisons of corresponding sectors of the osteon. The bright bands observed with polarized light corresponded to the grooves observed in etched sections by SEM. The dark bands corresponded to the lamellar surface with the cut fibrils oriented approximately longitudinally along the central canal axis. However, lamellae with large and blurred bright bands could be observed, which did not correspond to a groove observed by SEM. These findings are in contrast with the assumption that all the fibril layers within a lamella are oriented along a constant and unchangeable angle. The different lamellar patterns may be explained by the synchronous or staggered recruitment and activation of osteoblasts committed to the osteon's completion.

Alterations in the osteocyte lacunar-canalicular microenvironment due to estrogen deficiency.

While reduced estrogen levels have been shown to increase bone turnover and induce bone loss, there has been little analysis of the effects of diminished estrogen levels on the lacunar-canalicular porosity that houses the osteocytes. Alterations in the osteocyte lacunar-canalicular microenvironment may affect the osteocyte's ability to sense and translate mechanical signals, possibly contributing to bone degradation during osteoporosis. To investigate whether reduced estrogen levels affect the osteocyte microenvironment, this study used high-resolution microscopy techniques to assess the lacunar-canalicular microstructure in the rat ovariectomy (OVX) model of postmenopausal osteoporosis. Confocal microscopy analyses indicated that OVX rats had a larger effective lacunar-canalicular porosity surrounding osteocytes in both cortical and cancellous bone from the proximal tibial metaphysis, with little change in cortical bone from the diaphysis or cancellous bone from the epiphysis. The increase in the effective lacunar-canalicular porosity in the tibial metaphysis was not due to changes in osteocyte lacunar density, lacunar size, or the number of canaliculi per lacuna. Instead, the effective canalicular size measured using a small molecular weight tracer was larger in OVX rats compared to controls. Further analysis using scanning and transmission electron microscopy demonstrated that the larger effective canalicular size in the estrogen-deficient state was due to nanostructural matrix-mineral level differences like loose collagen surrounding osteocyte canaliculi. These matrix-mineral differences were also found in osteocyte lacunae in OVX, but the small surface changes did not significantly increase the effective lacunar size. The alterations in the lacunar-canalicular surface mineral or matrix environment appear to make OVX bone tissue more permeable to small molecules, potentially altering interstitial fluid flow around osteocytes during mechanical loading.