Lacunar Density Research Articles

Ontogenetic changes in the histological features of zonal bone tissue of ruminants: A quantitative approach

Bone histology is a powerful tool to explore the growth patterns of vertebrates. There is a broad consensus that a deeper understanding of bone development in living taxa is still lacking. Here, we aim to explore the ontogeny of the fibrolamellar (FLC) zonal bone of mammals by studying histological sections of the femoral growth series of 84 wild ruminants. Our results indicate that large ruminants do not preserve a complete ontogenetic record of primary bone growth, so it is necessary to use methods of age retrocalculation. Our study also stresses the ontogenetic variation in histological features of the FLC-zonal bone (vascular orientation, vascular and osteocyte lacunae density) that may reflect the slowdown in growth associated with the onset of physiological maturity. We conclude that the transition from FLC bone to lamellar bone (EFS) in ruminants records the fundamental life history trade-off between growth and reproduction (reproductive maturity).

Investigating histomorphometric relationships at the human femoral midshaft in a biomechanical context.

Cortical bone histomorphometry utilised in human and animal bone biology studies has demonstrated that osteon densities and their geometric properties may be in a relationship with biomechanical load application. Further research is required to investigate mutual links between bone histological variables to elucidate their usefulness in future biomechanical studies. Here, a series of correlations exploring bone biology relationships at the human midshaft femur were performed using a large sample. Mean intact, fragmentary and total osteon population densities, Haversian canal diameter and area, osteon area, as well as osteocyte lacunae density were measured along the sub-periosteal cortex in sections removed from the posterior midshaft aspect of modern human male (n=233) and female (n=217) femora (total n=450). Parametric and non-parametric correlations between the histology variables were sought in the entire sample, as well as within age and sex sub-groups. Several significant positive and negative correlations explaining a large proportion of data variation were found. Haversian canal area, diameter, and osteon area were positively correlated. As the density of osteocyte lacunae increased, Haversian canals and osteons became smaller. As osteons increased in density, so did osteocyte lacunae, but Haversian canal and osteon area became smaller. Results were consistent across age and sex groups. Findings suggest that an increased rate of bone remodelling is associated with a decrease in geometrical properties of osteons. An increased density of osteocyte lacunae and osteons indicates the involvement of bone maintenance cells in remodelling potentially induced by mechanical stimuli. Future histomorphometry studies will benefit from examining multiple bone histology variables due to many mutual bone biology relationships that exist at the human midshaft femur.

Microstructure and compressive mechanical properties of cortical bone in children with osteogenesis imperfecta treated with bisphosphonates compared with healthy children

Osteogenesis imperfecta (OI) is a genetic disorder characterized by a change in bone tissue quality, but little data are available to describe the factors involved at the macroscopic scale. To better understand the effect of microstructure alterations on the mechanical properties at the sample scale, we studied the structural and mechanical properties of six cortical bone samples from children with OI treated with bisphosphonates and compared them to the properties of three controls. Scanning electron microscopy, high resolution computed tomography and compression testing were used to assess these properties. More resorption cavities and a higher osteocyte lacunar density were observed in OI bone compared with controls. Moreover, a higher porosity was measured for OI bones along with lower macroscopic Young’s modulus, yield stress and ultimate stress. The microstructure was impaired in OI bones; the higher porosity and osteocyte lacunar density negatively impacted the mechanical properties and made the bone more prone to fracture.

Osteohistological variation in growth marks and osteocyte lacunar density in a theropod dinosaur (Coelurosauria: Ornithomimidae).

BackgroundOsteohistological examinations of fossil vertebrates have utilized a number of proxies, such as counts and spacing of lines of arrested growth (LAGs) and osteocyte lacunar densities (OLD), in order to make inferences related to skeletochronology and mass-specific growth rates. However, many of these studies rely on samplings of isolated bones from single individuals. These analyses do not take individual variation into account, and as a result may lead to misleading inferences of the physiology of extinct organisms. This study uses a multi-element, multi-individual sampling of ornithomimid dinosaurs to test the amount of individual variation in the aforementioned osteohistological indicators. Based on these results we also assess the conclusions of previous studies that tested paleohistological hypotheses using isolated elements.ResultsLAG number was found to be consistent within the hind limb bones of each individual, with the exception of the fibula, which preserves one additional LAG. Considerable differences in LAG spacing were found between elements of the sampled individuals, with larger variation found in elements of the foot compared with the femur, fibula, and tibia. Osteocyte lacunar density ranged between 29000 and 42000 osteocyte lacunae per mm3, and was found to vary more between hind limb bones of an individual and within bones, than between the average values of individuals.ConclusionsThe variation between hind limb elements in LAG number and LAG spacing suggests that direct comparisons of these elements may be misleading, and that LAG spacing is not a reliable proxy for mass-specific growth rates of an individual. Sampling of multiple bones should be performed as an internal check of model-based LAG retro-calculation and growth equations. The observation that osteocyte lacunar density varies more between individual bone elements than between average individual values suggests that the choice of sampled element can greatly influence the result, and care should be taken to not bias interpretations of the physiology of fossil tetrapods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0231-y) contains supplementary material, which is available to authorized users.

Accessing osteocyte lacunar geometrical properties in human jaw bone on the submicron length scale using synchrotron radiation μCT.

The architectural properties of the osteocyte cell network provide a valuable basis for understanding the mechanisms of bone remodelling, mineral homeostasis, ageing and pathologies. Recent advances in synchrotron microtomography enable unprecedented three-dimensional imaging of both the bone lacunar network and the extracellular matrix. Here, we investigate the three-dimensional morphological properties of osteocyte lacunae in human healthy and bisphosphonate-related osteonecrotic jaw bone based on synchrotron X-ray computed tomography images, with a spatial isotropic voxel size of 300 nm. Bisphosphonate-related osteonecrosis of the jaw is a relatively new disease with increasing incidence, which remains poorly understood. A step forward in elucidating this malady is to assess whether, and how, the morphology of the osteocyte lacunar network is modified in the affected jaw tissue. We evaluate thousands of cell lacunae from five specimens of which three originate from patients diagnosed with bisphosphonate-associated osteonecrosis. In this exploratory study, we report three-dimensional quantitative results on lacunar volumes (296-502 μm(3)), shape (approximated by an ellipsoidal shape with principal axes a > b > c, such that a = 2.2b and a = 4c) and spatial distribution (i.e., 50% of the mineralized matrix volume is located within 12 μm to the closest lacunar boundary) at submicron resolution on such specimens. We observe that the average lacunar volumes of the bisphosphonate-related osteonecrotic jaw specimens were within the range of volumes found in the two specimens originating from healthy donors and conclude that lacunar volumes are not the key element in the course of bisphosphonate-related osteonecrotic jaw. In three out of five specimens we observe lacunar volume sizes in segmented osteons to be significantly different compared to lacunar volumes in the adjacent tissue regions. Furthermore, we quantify the number of lacunae containing small dense objects (on average 9%). In contrast to lacunar morphology we report the lacunar density (16,000-50,000 per mm(3)) to be different in jaw bone tissue compared to what has been reported in femoral sites.

Normal variation in cortical osteocyte lacunar parameters in healthy young males.

The most abundant cell in bone, osteocytes form an interconnected system upon which the regulation of healthy bone relies. Although the complete nature of the role of osteocytes has yet to be defined, they are generally accepted to play a part in the sensing of load and the initiation of damage repair. A previous study conducted by our group identified variation of up to 30% in osteocyte lacunar density and morphological parameters between regions of a single cross-section of human femoral shaft; that study, however, was limited to a single individual. The aim of the current study was to determine whether this pattern consistently occurs in healthy young male femora. Anterior, posterior, medial and lateral blocks were prepared from the proximal femoral shaft of seven males and synchrotron radiation micro-CT imaged. Average lacunar densities (± SD) from the anterior, posterior, medial and lateral regions were 23 394 ± 1705, 30 180 ± 4860, 35 946 ± 5990 and 29 678 ± 6081 lacunae per mm(3) of bone tissue, respectively. These values were significantly different between the anterior and both the medial and posterior regions (P < 0.05). The density of the combined anterior and posterior regions was also significantly lower (P = 0.006) than the density of the combined medial and lateral regions. Although no difference was found in predominant orientation, shape differences were found; with the combined anterior-posterior regions having lacunae that were significantly more elongated and less flat than the combined medial-lateral values (P < 0.001). As expected, in this larger study, there was a dramatic difference in lacunar density between the medial and anterior region (up to ~ 54%). The study clearly demonstrates that the high variation seen in osteocyte lacunar density as well as other lacunar parameters, noted in a number of biomechanical, age and pathology studies, are well within the range of normal variation; however, the reasons for and consequences of this variation remain unclear. Lacunar parameters including abundance and shape are being increasingly incorporated into computational modeling of bone biology and this paper represents a more comprehensive description of normal healthy lacunae.

Reduced diaphyseal strength associated with high intracortical vascular porosity within long bones of children with osteogenesis imperfecta

Osteogenesis imperfecta is a genetic disorder resulting in bone fragility. The mechanisms behind this fragility are not well understood. In addition to characteristic bone mass deficiencies, research suggests that bone material properties are compromised in individuals with this disorder. However, little data exists regarding bone properties beyond the microstructural scale in individuals with this disorder.Specimens were obtained from long bone diaphyses of nine children with osteogenesis imperfecta during routine osteotomy procedures. Small rectangular beams, oriented longitudinally and transversely to the diaphyseal axis, were machined from these specimens and elastic modulus, yield strength, and maximum strength were measured in three-point bending. Intracortical vascular porosity, bone volume fraction, osteocyte lacuna density, and volumetric tissue mineral density were determined by synchrotron micro-computed tomography, and relationships among these mechanical properties and structural parameters were explored.Modulus and strength were on average 64–68% lower in the transverse vs. longitudinal beams (P<0.001, linear mixed model). Vascular porosity ranged between 3 and 42% of total bone volume. Longitudinal properties were associated negatively with porosity (P≤0.006, linear regressions). Mechanical properties, however, were not associated with osteocyte lacuna density or volumetric tissue mineral density (P≥0.167). Bone properties and structural parameters were not associated significantly with donor age (P≥0.225, linear mixed models).This study presents novel data regarding bone material strength in children with osteogenesis imperfecta. Results confirm that these properties are anisotropic. Elevated vascular porosity was observed in most specimens, and this parameter was associated with reduced bone material strength. These results offer insight toward understanding bone fragility and the role of intracortical porosity on the strength of bone tissue in children with osteogenesis imperfecta.

The resistance of cortical bone tissue to failure under cyclic loading is reduced with alendronate

Bisphosphonates are the most prescribed preventative treatment for osteoporosis. However, their long-term use has recently been associated with atypical fractures of cortical bone in patients who present with low-energy induced breaks of unclear pathophysiology. The effects of bisphosphonates on the mechanical properties of cortical bone have been exclusively studied under simple, monotonic, quasi-static loading. This study examined the cyclic fatigue properties of bisphosphonate-treated cortical bone at a level in which tissue damage initiates and is accumulated prior to frank fracture in low-energy situations. Physiologically relevant, dynamic, 4-point bending applied to beams (1.5mm×0.5mm×10mm) machined from dog rib (n=12/group) demonstrated mechanical failure and micro-architectural features that were dependent on drug dose (3 groups: 0, 0.2, 1.0mg/kg/day; alendronate [ALN] for 3years) with cortical bone tissue elastic modulus (initial cycles of loading) reduced by 21% (p<0.001) and fatigue life (number of cycles to failure) reduced in a stress-life approach by greater than 3-fold with ALN1.0 (p<0.05). While not affecting the number of osteons, ALN treatment reduced other features associated with bone remodeling, such as the size of osteons (−14%; ALN1.0: 10.5±1.8, VEH: 12.2±1.6, ×103μm2; p<0.01) and the density of osteocyte lacunae (−20%; ALN1.0: 11.4±3.3, VEH: 14.3±3.6, ×102 #/mm2; p<0.05). Furthermore, the osteocyte lacunar density was directly proportional to initial elastic modulus when the groups were pooled (R=0.54, p<0.01). These findings suggest that the structural components normally contributing to healthy cortical bone tissue are altered by high-dose ALN treatment and contribute to reduced mechanical properties under cyclic loading conditions.

Nano-structural, compositional and micro-architectural signs of cortical bone fragility at the superolateral femoral neck in elderly hip fracture patients vs. healthy aged controls

To unravel the origins of decreased bone strength in the superolateral femoral neck, we assessed bone structural features across multiple length scales at this cortical fracture initiating region in postmenopausal women with hip fracture and in aged-matched controls. Our combined methodological approach encompassed atomic force microscopy (AFM) characterization of cortical bone nano-structure, assessment of mineral content/distribution via quantitative backscattered electron imaging (qBEI), measurement of bone material properties by reference point indentation, as well as evaluation of cortical micro-architecture and osteocyte lacunar density. Our findings revealed a wide range of differences between the fracture group and the controls, suggesting a number of detrimental changes at various levels of cortical bone hierarchical organization that may render bone fragile. Namely, mineral crystals at external cortical bone surfaces of the fracture group were larger (65.22nm±41.21nm vs. 36.75nm±18.49nm, p<0.001), and a shift to a higher mineral content and more homogenous mineralization profile as revealed via qBEI were found in the bone matrix of the fracture group. Fracture cases showed nearly 35% higher cortical porosity and showed significantly reduced osteocyte lacunar density compared to controls (226±27 vs. 247±32#/mm2, p=0.05). Along with increased crystal size, a shift towards higher mineralization and a tendency to increased cortical porosity and reduced osteocyte lacunar number delineate that cortical bone of the superolateral femoral neck bears distinct signs of fragility at various levels of its structural organization. These results contribute to the understanding of hierarchical bone structure changes in age-related fragility.

Osteohistological variation in growth marks and osteocyte lacunar density in a theropod dinosaur (Coelurosauria: ornithomimidae)

Osteohistological examinations of fossil vertebrates have utilized a number of proxies, such as counts and spacing of lines of arrested growth (LAGs) and osteocyte lacunar densities (OLD), in order to make inferences related to skeletochronology and mass-specific growth rates. However, many of these studies rely on samplings of isolated bones from single individuals. These analyses do not take individual variation into account, and as a result may lead to misleading inferences of the physiology of extinct organisms. This study uses a multi-element, multi-individual sampling of ornithomimid dinosaurs to test the amount of individual variation in the aforementioned osteohistological indicators. Based on these results we also assess the conclusions of previous studies that tested paleohistological hypotheses using isolated elements. LAG number was found to be consistent within the hind limb bones of each individual, with the exception of the fibula, which preserves one additional LAG. Considerable differences in LAG spacing were found between elements of the sampled individuals, with larger variation found in elements of the foot compared with the femur, fibula, and tibia. Osteocyte lacunar density ranged between 29000 and 42000 osteocyte lacunae per mm3, and was found to vary more between hind limb bones of an individual and within bones, than between the average values of individuals. The variation between hind limb elements in LAG number and LAG spacing suggests that direct comparisons of these elements may be misleading, and that LAG spacing is not a reliable proxy for mass-specific growth rates of an individual. Sampling of multiple bones should be performed as an internal check of model-based LAG retro-calculation and growth equations. The observation that osteocyte lacunar density varies more between individual bone elements than between average individual values suggests that the choice of sampled element can greatly influence the result, and care should be taken to not bias interpretations of the physiology of fossil tetrapods.

3D osteocyte lacunar morphometric properties and distributions in human femoral cortical bone using synchrotron radiation micro-CT images

Osteocytes, the most numerous bone cells, are thought to be actively involved in the bone modeling and remodeling processes. The morphology of osteocyte is hypothesized to adapt according to the physiological mechanical loading. Three-dimensional micro-CT has recently been used to study osteocyte lacunae. In this work, we proposed a computationally efficient and validated automated image analysis method to quantify the 3D shape descriptors of osteocyte lacunae and their distribution in human femurs. Thirteen samples were imaged using Synchrotron Radiation (SR) micro-CT at ID19 of the ESRF with 1.4μm isotropic voxel resolution. With a field of view of about 2.9×2.9×1.4mm3, the 3D images include several tens of thousands of osteocyte lacunae. We designed an automated quantification method to segment and extract 3D cell descriptors from osteocyte lacunae. An image moment-based approach was used to calculate the volume, length, width, height and anisotropy of each osteocyte lacuna. We employed a fast algorithm to further efficiently calculate the surface area, the Euler number and the structure model index (SMI) of each lacuna. We also introduced the 3D lacunar density map to directly visualize the lacunar density variation over a large field of view. We reported the lacunar morphometric properties and distributions as well as cortical bone histomorphometric indices on the 13 bone samples.The mean volume and surface were found to be 409.5±149.7μm3 and 336.2±94.5μm2. The average dimensions were of 18.9±4.9μm in length, 9.2±2.1μm in width and 4.8±1.1μm in depth. We found lacunar number density and six osteocyte lacunar descriptors, three axis lengths, two anisotropy ratios and SMI, that are significantly correlated to bone porosity at a same local region.The proposed method allowed an automatic and efficient direct 3D analysis of a large population of bone cells and is expected to provide reliable biological information for better understanding the bone quality and diseases at cellular level.

Osteoblast-osteocyte transformation. A SEM densitometric analysis of endosteal apposition in rabbit femur.

Transformation of osteoblasts into osteocytes is marked by changes in volume and cell shape. The reduction of volume and the entrapment process are correlated with the synthesis activity of the cell which decreases consequently. This transformation process has been extensively investigated by transmission electron microscopy (TEM) but no data have yet been published regarding osteoblast-osteocyte dynamic histomorphometry. Scanning electron microscope (SEM) densitometric analysis was carried out to determine the osteoblast and open osteocyte lacunae density in corresponding areas of a rabbit femur endosteal surface. The lining cell density was 4900.1 ± 30.03 n mm(-2), the one of open osteocyte lacunae 72.89 ± 22.55 n mm(-2). This corresponds to an index of entrapment of one cell every 67.23 osteoblasts (approximated by defect). The entrapment sequence begins with flattening of the osteoblast and spreading of equatorial processes. At first these are covered by the new apposed matrix and then also the whole cellular body of the osteocyte undergoing entrapment. The dorsal aspect of the cell membrane suggests that closure of the osteocyte lacuna may be partially carried out by the same osteoblast-osteocyte which developed a dorsal secretory territory. A significant proportion of the endosteal surface was analysed by SEM, without observing any evidence of osteoblast mitotic figures. This indicates that recruitment of the pool of osteogenic cells in cortical bone lamellar systems occurs prior to the entrapment process. No further additions occurred once osteoblasts were positioned on the bone surface and began lamellar apposition. The number of active osteoblasts on the endosteal surface exceeded that of the cells which become incorporated as osteocytes (whose number was indicated by the number of osteocyte lacunae). Therefore such a balance must be equilibrated by the osteoblasts' transformation in resting lining cells or by apoptosis. The current work characterised osteoblast shape changes throughout the entrapment process, allowing approximate calculation of an osteoblast entrapment index in the rabbit endosteal cortex.

Preliminary analysis of osteocyte lacunar density in long bones of tetrapods: all measures are bigger in sauropod dinosaurs.

Osteocytes harbour much potential for paleobiological studies. Synchrotron radiation and spectroscopic analyses are providing fascinating data on osteocyte density, size and orientation in fossil taxa. However, such studies may be costly and time consuming. Here we describe an uncomplicated and inexpensive method to measure osteocyte lacunar densities in bone thin sections. We report on cell lacunar densities in the long bones of various extant and extinct tetrapods, with a focus on sauropodomorph dinosaurs, and how lacunar densities can help us understand bone formation rates in the iconic sauropod dinosaurs. Ordinary least square and phylogenetic generalized least square regressions suggest that sauropodomorphs have lacunar densities higher than scaled up or comparably sized mammals. We also found normal mammalian-like osteocyte densities for the extinct bovid Myotragus, questioning its crocodilian-like physiology. When accounting for body mass effects and phylogeny, growth rates are a main factor determining the density of the lacunocanalicular network. However, functional aspects most likely play an important role as well. Observed differences in cell strategies between mammals and dinosaurs likely illustrate the convergent nature of fast growing bone tissues in these groups.

Bone histology as an approach to providing data on certain key life history traits in mammals: Implications for conservation biology

The knowledge of the life histories of wild mammals is of crucial importance in the field of conservation management. The endangered status of many species calls for faster data collection that can be used in risk assessment and, ultimately, for designing conservation policies. This study is pioneering the potential of bone histology to provide data on life history traits crucial for conservation biology in long-lived mammals. Long bone cross-sections show pronounced annual cycles of growth arrest allowing application of skeletochronology (counts of lines of arrested growth ‘LAGs’). Consequently, the number of LAGs within the primary fast-growing bone tissue up to the outer cortical slow-growing bone tissue corresponds to the age at first reproduction; whereas the age at death can be estimated by the total number of rest lines throughout the whole of bone cross-section. Furthermore, the diameters of successive growth rings as well as the osteocyte lacuna density may shed light on growth rates. We use the endangered desert dwelling antelope Addax nasomaculatus as a case study. By analyzing different ontogenetic stages in five Addax individuals (three captive and two wild specimens) from a museum collection, we show that bone histology may be a reliable tool for determining certain key life history traits. In our sample, the wild Addax female attained reproductive maturity at three years, whereas the male specimens, both the captive and the wild ones, reached maturity at four years. This is congruent with data from other large antelopes with male-biased size dimorphism, but differs slightly from data on sexual maturity previously published for wild Addax. Moreover, quantification of osteocyte lacunae in both adult males provides a higher cell density in the captive one than in the wild one suggesting the strong effect of constant resources supply in individuals from zoos on growth rates. While age at first reproduction and longevity are essentials parameters to carry out demographic models, growth rates may allow evaluation of the health status of wild populations. This approach may provide useful data on life history traits when applied to bones collected in the wild.

Femoral osteocyte lacunar density, volume and morphology in women across the lifespan

Osteocytes are believed to be the primary agents of mechanosensing in bone. Due to this important role in the structure-function relationship of bone, osteocytes and the spaces they occupy (lacunae) are of increasing interest. Changes in lacunae with age are of particular interest in women since they are more susceptible to bone loss and fragility associated with senescent diseases including osteoporosis. This study's purpose was to test whether differences exist in lacunar density (lacunae/mm(3) of bone), orientation and morphology in the cortex of adult women spanning the human lifespan. Anterior blocks from the femoral shaft from 30 women aged 20-86years were imaged by synchrotron-radiation micro-CT. No significant relation between lacunar density and age was detected. A significant reduction in lacunar volume with age (p<0.001) was observed, alongside changes in lacunar morphology. When divided into two groups (<50 and >50years) the younger group's lacunae were ∼30% larger and were flatter (p<0.001) and less equant (spherical) (p<0.001). To our knowledge the observation that lacunar volume and morphology change over the human lifespan is novel, potentially resulting from preferential surface infilling within the extracellular space. The functional impact of this infilling is unclear but such a change in scale likely impacts the mechanosensing function of the osteocyte network. Limitations in resolution prevented us from assessing if this infilling is associated with disruption of the canaliculi. This hypothesis warrants further investigation as, if confirmed, it would represent a profound negative impact on the osteocyte network and may provide new insights into age-related bone loss.

Morphometric analysis of osteonal architecture in bones from healthy young human male subjects using scanning electron microscopy

The shape and structure of bones is a topic that has been studied for a long time by morphologists and biologists with the goal of explaining the laws governing their development, aging and pathology. The osteonal architecture of tibial and femoral mid-diaphyses was examined morphometrically with scanning electron microscopy in four healthy young male subjects. In transverse sections of the mid-diaphysis, the total area of the anterior, posterior, lateral and medial cortex sectors was measured and analysed for osteonal parameters including osteon number and density, osteon total and bone area and vascular space area. Osteons were grouped into four classes including cutting heads (A), transversely cut osteons (B), longitudinally cut osteons (C) and sealed osteons (D). The morphometric parameters were compared between the inner (endosteal) and outer (periosteal) half of the cortex. Of 5927 examined osteons, 24.4% cutting heads, 71.1% transversely cut osteons, 2.3% longitudinally cut osteons and 2.2% sealed osteons were found. The interosteonic bone (measured as the area in a lamellar system that has lost contact with its own central canal) corresponded to 51.2% of the endosteal and 52.4% of the periosteal half-cortex. The mean number of class A cutting heads and class B osteons was significantly higher in the periosteal than in the endosteal half-cortex (P<0.001 and P<0.05, respectively), whereas there was no significant difference in density. The mean osteon total area, osteon bone area and vascular space area of both classes A and B were significantly higher (P<0.001 for all three parameters) in the endosteal than in the periosteal half-cortex. The significant differences between the two layers of the cortex suggest that the osteoclast activity is distributed throughout the whole cortical thickness, with more numerous excavations in the external layer, but larger resorption lacunae closer to the marrow canal. A randomly selected population of 109 intact class B osteons was examined at higher magnification (350×) to count osteocyte lacuna and to analyse their relationship with osteon size parameters. The distribution frequency of the mean number of osteocyte lacunae increased with the increment in the sub-classes of osteon bone area, whereas the density did not show significant differences. The number of osteocyte lacunae had a direct correlation with the osteon bone area and the mean osteon wall thickness, as well as the mean number of lamellae. The osteocyte lacunae density showed an inverse relationship. These data suggest a biological regulation of osteoblast activity with a limit to the volume of matrix produced by each cell and proportionality with the number of available cells in the space of the cutting cone (total osteon area). The collected data can be useful as a set of control parameters in healthy human bone for studies on bone aging and metabolic bone diseases.

Osteogenic Efficacy Enhancement of Kaempferol Through An Engineered Layer-By-Layer Matrix: A Study in Ovariectomized Rats

A layer-by-layer matrix (LBL) comprising kaempferol (LBL-KEM) was prepared for improved osteogenic action. The LBL-KEM consisted of alternate layers of sodium alginate and protamine sulfate, which were sequentially deposited on the preformed kaempferol (KEM)-loaded CaCO3 core (CaCO3-KEM) by LBL self-assembly. The LBL matrix developed was evaluated for layer growth by ζ-potential and size alterations after self-assembly of each layer. Its physicochemical properties and intestinal absorption pattern were characterized and its pharmacokinetic behavior, mineralization of bone marrow cells, bone mineral density, bone strength, microcrack formation and estrogenicity were evaluated after oral administration. The entrapment efficiency of KEM was 94 ± 2% and the cumulative %KEM released from LBL-KEM was 19.2 and 63.5% at pH 1.4 and 7.4, respectively, after 24 h. Stepwise polyelectrolyte assembly onto initially positively charged particles (+21.2 mV) resulted in alterations between -28.5 and +10.9 mV. A final ζ-potential of -8.9 mV was obtained after terminal surface modification with sodium deoxycholate. Fluorescein isothiocyanate-labeled LBL matrix was diffused into the basolateral lacteal region upon oral administration to rats. The area under the KEM serum concentration curve following oral administration of LBL-KEM to rats was 2479 ± 682 ng·h/ml, nearly twofold higher than free KEM. The concentration-time profile in bone marrow indicated improved penetration and retention of KEM on administration of LBL-KEM. Treatment with LBL-KEM restored bone mineralization, bone mineral density, microcrack formation and empty osteocyte lacunae density in ovariectomized (OVx) rats, which was significantly (p < 0.05) improved in femoral diaphysea, tibial head and vertebrae compared with free KEM treatment. Administration of LBL-KEM to growing female rats for 4 weeks resulted in no estrogenicity when compared with OVx rats. The data suggests that LBL matrix enhanced drug delivery, improved pharmacokinetics and maintained better bone quality under OVx conditions.

Intra‐element variation in osteocyte lacunar density and morphology in healthy young male cortical bone

We previously reported considerable intra‐element variation in osteocyte lacunar properties, with density differences up to 30% across a cross‐section of the male (20 yrs) femoral cortex related to the biomechanical axes. The purpose of this research was to determine whether this pattern of variation occurs in a larger sample. Anterior (A), posterior (P), medial (M) and lateral (L) blocks (2×2 mm) were prepared from the proximal femoral shaft of 7 healthy males aged 20–34 and synchrotron micro‐CT imaged. Average lacunar density (± sd) from the APML regions were 22,961 ± 1991, 28,264 ± 4186, 32,082 ± 7731 and 30,525 ± 1977 per mm3 of bone, respectively. Consistent with the pattern observed in the previous study, density was significantly different between the A and the L region (p&lt;0.05). The density of the combined A and P regions was also lower (p=0.006) than that of the combined M and L regions. The differences in density were mirrored by shape differences; with the combined AP region having more elongate (p=0.004) and flattened (p=0.001) lacunae, than those of the ML region. The consistent appearance of this pattern of variation in a larger sample suggests considerable normal variation occurs in the lacunar properties of healthy young males. The underlying functional significance of the observed differences likely relates to localized variations in loading conditions as the pattern corresponds well with mechanical axes.Grant Funding Source: Saskatchewan Health Research Fund

ANÁLISE HISTOMORFOLÓGICA E HISTOMORFOMÉTRICA DO TECIDO ÓSSEO MADURO DE Glossophaga soricina (PHYLLOSTOMIDAE:CHIROPTERA)

Bone tissue has different models of vascularization, cellular distribution, mineralization and remodeling among mammals species. A variety of dietary habits associated with the mode of locomotion and habitat required from bats the establishment of different flying styles and some skeletal adaptations. This study aimed at examining the microscopic characteristics of mature bone tissue of Glossophaga soricina (Phyllostomidae, Chiroptera). Twelve animals of both genders were used, in which the right humerus were dissected, weighed, decalcified and submitted to routine histological processing. Semi-serial cuts of 5 micrometers were stained with hematoxylin and eosin (H.E.), picrosirius red and silver nitrate 50%. The histological preparations were subjected to histological and histomorphometric analysis. Lacunae density was significantly higher in humerus of females when compared to males (33.96 x 27.80, p = 0.02). Microscopic analysis indicated the presence of parallel collagen fibers distributed in the bone matrix. Lacunes presented various shapes and canaliculi are well distributed and individualized. Few Havers systems and canals were observed. The mature bone tissue of the humerus Glossophaga soricina share microscopic features with other mammals, however, differences in the structural organization are peculiar to this species. KEYWORDS: bone; Glossophaga soricina; histomorphometry.

Changes to the cell, tissue and architecture levels in cranial suture synostosis reveal a problem of timing in bone development

Premature fusion of cranial sutures is a common problem with an incidence of 3-5 per 10,000 live births. Despite progress in understanding molecular/genetic factors affecting suture function, the complex process of premature fusion is still poorly understood. In the present study, corresponding excised segments of nine patent and nine prematurely fused sagittal sutures from infants (age range 3-7 months) with a special emphasis on their hierarchical structural configuration were compared. Cell, tissue and architecture characteristics were analysed by transmitted and polarised light microscopy, 2D-histomorphometry, backscattered electron microscopy and energy-dispersive-x-ray analyses. Apart from wider sutural gaps, patent sutures showed histologically increased new bone formation compared to reduced new bone formation and osseous edges with a more mature structure in the fused portions of the sutures. This pattern was accompanied by a lower osteocyte lacunar density and a higher number of evenly mineralised osteons, reflecting pronounced lamellar bone characteristics along the prematurely fused sutures. In contrast, increases in osteocyte lacunar number and size accompanied by mineralisation heterogeneity and randomly oriented collagen fibres predominantly signified woven bone characteristics in patent, still growing suture segments. The already established woven-to-lamellar bone transition provides evidence of advanced bone development in synostotic sutures. Since structural and compositional features of prematurely fused sutures did not show signs of pathological/defective ossification processes, this supports the theory of a normal ossification process in suture synostosis - just locally commencing too early. These histomorphological findings may provide the basis for a better understanding of the pathomechanism of craniosynostosis, and for future strategies to predict suture fusion and to determine surgical intervention.