7T MRI of distal radius trabecular bone microarchitecture: How trabecular bone quality varies depending on distance from end-of-bone.

PURPOSE: To use 7T magnetic resonance imaging (MRI) to determine how trabecular bone microarchitecture varies at the epiphysis, metaphysis, and diaphysis of the distal radius. MATERIALS AND METHODS: The distal radius of 24 females (mean age = 56 years, range = 24-78 years) was scanned on a 7T MRI using a 3D fast low-angle shot sequence (0.169 × 0.169 × 1 mm). Digital topological analysis was applied at the epiphysis, metaphysis, and diaphysis to compute: total trabecular bone volume; trabecular thickness, number, connectivity, and erosion index (a measure of network resorption). Differences and correlations were assessed using standard statistical methods. RESULTS: The metaphysis and epiphysis had 83-123% greater total bone volume and 14-16% greater trabecular number than the diaphysis (both P < 0.0001). The erosion index was significantly higher at the diaphysis than the metaphysis and epiphysis (both P < 0.01). The most elderly volunteers had lower trabecular number (<66 years mean 0.29 ± 0.01; ≥66 years, 0.27 ± 0.02, P < 0.05) and higher erosion index (<66 years mean 1.18 ± 0.17; age ≥66 years, mean 1.42 ± 0.46, P < 0.05) at the epiphysis; differences not detected by total trabecular bone volume. CONCLUSION: 7T MRI reveals trabecular bone microarchitecture varies depending on scan location at the end-of-bone, being of overall higher quality distally (epiphysis) than proximally (diaphysis). Age-related differences in trabecular microarchitecture can be detected by 7T MRI. The results highlight the potential sensitivity of 7T MRI to microarchitectural differences and the potential importance of standardizing scan location for future clinical studies of fracture risk or treatment response. LEVEL OF EVIDENCE: 3 J. Magn. Reson. Imaging 2017;45:872-878.

Morphological features of healing of experimental defect of long bones diaphysis under the conditions of implantation of biphasic osteoplastic material / Características morfológicas del proceso de reparación de un defecto experimental en diáfisis de huesos largos postimplantación de material bifásico osteoplástico

Purpose: to study the healing process of a defect of the compact bone tissue after the implantation of osteoplastic material «easygraft CRYSTAL¼. Methods: the experiment was conducted on 24 Wistar rats. In the middle third of the diaphysis of the femur we created a perforated defect of 2.5 mm diameter in the medullary canal, which was filled with osteoplastic material «easy-graft CRYSTAL¼. Fragments of the injured bones were studied on the 60th and 120th days by light microscopy with morphometry and scanning electron microscopy. Results: it was found that in the area of the defect of the compact bone tissue «easy-graft CRYSTAL¼ shows high biocompatibility, osteoconductive properties and provides stability to the volume of the defect due to good integration with the bone tissue of regenerate and absence of signs of resorption of osteoplastic material throughout the period of experiment. (AU)

Objetivo: estudiar el proceso de reparación de un defecto del tejido óseo compacto después de la implantación de material osteoplástico (easy-graft CRYSTAL). Métodos: el experimento se realizó en 24 ratas Wistar. Se realizó en el tercio medio de la diáfisis del fémur un defecto de 2,5 mm de diámetro en el canal medular que se rellenó de material de osteoplástico (easy-graft CRYSTAL). Los huesos lesionados fueron estudiados en los días 60ª y 120ª mediante microscopía óptica y microscopía electrónica de barrido. Resultados: se encontró que en la zona del defecto del tejido óseo compacto el injerto mostró alta biocompatibilidad y propiedades osteoconductivas y proporcionó estabilidad al volumen del defecto debido a la buena integración con el tejido óseo y ausencia de signos de resorción de material osteoplástico durante todo el período experimental. (AU)

Regionally-derived cell populations and skeletal stem cells from human foetal femora exhibit specific osteochondral and multi-lineage differentiation capacity in vitro and ex vivo.

BACKGROUND: Adult skeletal stem cells (SSCs) often exhibit limited in vitro expansion with undesirable phenotypic changes and loss of differentiation capacity. Foetal tissues offer an alternative cell source, providing SSCs which exhibit desirable differentiation capacity over prolonged periods, ideal for extensive in vitro and ex vivo investigation of fundamental bone biology and skeletal development. METHODS: We have examined the derivation of distinct cell populations from human foetal femora. Regionally isolated populations including epiphyseal and diaphyseal cells were carefully dissected. Expression of the SSC marker Stro-1 was also found in human foetal femora over a range of developmental stages and subsequently utilised for immuno-selection. RESULTS: Regional populations exhibited chondrogenic (epiphyseal) and osteogenic (diaphyseal) phenotypes following in vitro and ex vivo characterisation and molecular analysis, indicative of native SSC maturation during skeletal development. However, each population exhibited potential for induced multi-lineage differentiation towards bone (bone nodule formation), cartilage (proteoglycan and mucopolysaccharide deposition) and fat (lipid deposition), suggesting the presence of a shared stem cell sub-population. This shared sub-population may be comprised of Stro-1+ cells, which were later identified and immuno-selected from whole foetal femora exhibiting multi-lineage differentiation capacity in vitro and ex vivo. CONCLUSIONS: Distinct populations were isolated from human foetal femora expressing osteochondral differentiation capacity. Stro-1 immuno-selected SSCs were isolated from whole femora expressing desirable multi-lineage differentiation capacity over prolonged in vitro expansion, superior to their adult-derived counterparts, providing a valuable cell source with which to study bone biology and skeletal development.

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal.

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial. HSCs are rare and few can be found in thin tissue sections or upon live imaging, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α-catulin(GFP)), we discover that α-catulin(GFP) is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α-catulin-GFP(+)c-kit(+) cells give long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP(+)c-kit(+) cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α-catulin-GFP(+)c-kit(+) cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP(+)c-kit(+) cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr(+)) and Cxcl12(high) niche cells, and approximately 85% of HSCs were within 10 µm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67(+)) and non-dividing (Ki-67(-)), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr(+)Cxcl12(high) cells throughout the bone marrow.

MicroRNA-146a regulates human foetal femur derived skeletal stem cell differentiation by down-regulating SMAD2 and SMAD3.

MicroRNAs (miRs) play a pivotal role in a variety of biological processes including stem cell differentiation and function. Human foetal femur derived skeletal stem cells (SSCs) display enhanced proliferation and multipotential capacity indicating excellent potential as candidates for tissue engineering applications. This study has examined the expression and role of miRs in human foetal femur derived SSC differentiation along chondrogenic and osteogenic lineages. Cells isolated from the epiphyseal region of the foetal femur expressed higher levels of genes associated with chondrogenesis while cells from the foetal femur diaphyseal region expressed higher levels of genes associated with osteogenic differentiation. In addition to the difference in osteogenic and chondrogenic gene expression, epiphyseal and diaphyseal cells displayed distinct miRs expression profiles. miR-146a was found to be expressed by human foetal femur diaphyseal cells at a significantly enhanced level compared to epiphyseal populations and was predicted to target various components of the TGF-ß pathway. Examination of miR-146a function in foetal femur cells confirmed regulation of protein translation of SMAD2 and SMAD3, important TGF-ß and activin ligands signal transducers following transient overexpression in epiphyseal cells. The down-regulation of SMAD2 and SMAD3 following overexpression of miR-146a resulted in an up-regulation of the osteogenesis related gene RUNX2 and down-regulation of the chondrogenesis related gene SOX9. The current findings indicate miR-146a plays an important role in skeletogenesis through attenuation of SMAD2 and SMAD3 function and provide further insight into the role of miRs in human skeletal stem cell differentiation modulation with implications therein for bone reparation.

Differential effects of exercise on tibial shaft marrow density in young female athletes.

CONTEXT: Increased mechanical loading can promote the preferential differentiation of bone marrow mesenchymal stem cells to osteoblastogenesis, but it is not known whether long-term bone strength-enhancing exercise in humans can reduce marrow adiposity. OBJECTIVE: Our objective was to examine whether bone marrow density (MaD), as an estimate of marrow adiposity 1) differs between young female athletes with contrasting loading histories and bone strengths and 2) is an independent predictor of bone strength at the weight-bearing tibia. DESIGN: Mid-tibial MaD, cortical area (CoA), total area, medullary area, strength strain index (SSI), and cortical volumetric bone mineral density (vBMD) (total, endocortical, midcortical, and pericortical) was assessed using peripheral quantitative computed tomography in 179 female athletes involved in both impact and nonimpact loading sports and 41 controls aged 17-40 years. RESULTS: As we have previously reported CoA, total area, and SSI were 16% to 24% greater in the impact group compared with the controls (all P < .001) and 12% to 18% greater than in the nonimpact group (all P < .001). The impact group also had 0.5% higher MaD than the nonimpact and control groups (both P < .05). Regression analysis further showed that midtibial MaD was significantly associated with SSI, CoA, endocortical vBMD, and pericortical vBMD (P < .05) in all women combined, after adjusting for age, bone length, loading groups, medullary area, muscle cross-sectional area, and percent fat. CONCLUSION: In young female athletes, tibial bone MaD was associated with loading history and was an independent predictor of tibial bone strength. These findings suggest that an exercise-induced increase in bone strength may be mediated via reduced bone marrow adiposity and consequently increased osteoblastogenesis.

Increasing participation of sclerostin in postnatal bone development, revealed by three-dimensional immunofluorescence morphometry.

Confocal immunofluorescence tiling imaging revealed the spatio-temporal distributions of osterix and sclerostin in femurs from 3-day-old, 2-week-old and 4-week-old rats to be reciprocally exclusive at the tissue level. Further quantitative three-dimensional immuno fluorescence morphometry demonstrated the increasing distribution of sclerostin in the osteocytic lacuno-canalicular system specifically in diaphysis, which paralleled the cooperative participation and depletion of osterix and ß-catenin in adjacent periosteum cells. Treating MC3T3-E1 cells with BIO (a GSK3 inhibitor) induced the stabilization of ß-catenin and nuclear translocation of osterix, and negatively regulated osteocalcin/BGLAP and Dmp1. These results collectively demonstrate that the increasing distribution of sclerostin in diaphyseal cortical bone appears to be involved in the attenuation of osterix and ß-catenin in adjacent periosteum cells, thus possibly contributing to osteoblast maturation and reducing the osteoblast formation at this bone site. Our confocal microscopy-based imaging analyses provide a comprehensive and detailed view of the spatio-temporal distribution of sclerostin, ß-catenin and osterix at the tissue to subcellular level in a coherent manner, and uncovered their spatio-temporal cooperation in postnatal bone development, thus providing evidence that they link skeletogenic growth and functional bone development.

Separation of newly formed bone from older compact bone reveals clear compositional differences in bone matrix.

In long bone diaphyses, woven bone forms first and then transitions into a more mineralized compact bone tissue. Prior evidence suggests that the non-collagenous protein composition of woven bone may be distinct from that of more mature bone tissue, particularly with respect to a diverse group of phosphorylated, extracellular matrix proteins. To critically test this hypothesis, we developed an in situ approach to isolate newly formed bone from more mature bone within the same long bone, and combine this anatomical approach with Western blotting to make relative comparisons of 7 phosphorylated matrix proteins important for bone physiology and biomineralization. Interestingly, 75 kDa bone sialoprotein (BSP), 63 kDa osteopontin, and the 75 kDa form of bone acidic glycoprotein-75 (BAG-75) were enriched in primary bone as opposed to more mature cortical bone, while osteonectin, fetuin A, matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein-1 (DMP-1) appeared to be equally distributed between these two bone tissue compartments. Analyses also revealed the presence of larger sized forms of osteopontin (and to a lesser degree BSP) mostly in newly formed bone, while larger forms of BAG-75 were mostly detected in more mature cortical bone. Smaller sized forms of DMP-1 and BAG-75 were detected in both newly formed and more mature bone tissue extracts, and they are likely the result of proteolytic processing in vivo. Intact DMP-1 (97 kDa) was only detected in unmineralized matrix extracts. These findings indicate that newly formed bone exhibits a non-collagenous matrix protein composition distinct from that of more mature compact bone even within the same long bone, and suggest that the temporal fate of individual non-collagenous proteins is variable in growing bone.

A weighted osteon morphotype score outperforms regional osteon percent prevalence calculations for interpreting cortical bone adaptation.

Using circularly polarized light microscopy,we described a weighted-scoring method for quantifying regional distributions of six secondary osteon morphotypes(Skedros et al.: Bone 44 (2009) 392-403). This osteon morphotype score (MTS) strongly correlated with "tension" and "compression" cortices produced by habitual bending. In the present study, we hypothesized that the osteon MTS is superior to a relatively simpler method based on the percent prevalence (PP) of these osteon morphotypes. This was tested in proximal femoral diaphyses of adult chimpanzees and habitually bent bones: calcanei from sheep, deer, and horses, radii from sheep and horses, and third metacarpals (MC3s) from horses. Sheep tibiae were examined because their comparatively greater torsion/shear would not require regional variations in osteon morphotypes. Predominant collagen fiber orientation (CFO), a predictor of regionally prevalent/predominant strain mode, was quantified as image gray levels (birefringence). Ten PP calculations were conducted. Although PP calculations were similar to the osteon MTS in corroborating CFO differences between "tension" and "compression" cortices of the chimpanzee femora and most of the habitually bent bones, PP calculations failed to show a compression/tension difference in equine MC3s and sheep radii. With the exception of the prevalence of the "distributed" osteon morphotype, correlations of PP calculations with CFO were weak and/or negative. By contrast, the osteon MTS consistently showed positive correlations with predominant CFO. Compared with the osteon MTS and predominant CFO, regional variations in PP of osteon morpho types are not stronger predictors of nonuniform strain distributions produced by bending.

Experimental determination of the permeability in the lacunar-canalicular porosity of bone.

Permeability of the mineralized bone tissue is a critical element in understanding fluid flow occurring in the lacunar-canalicular porosity (PLC) compartment of bone and its role in bone nutrition and mechanotransduction. However, the estimation of bone permeability at the tissue level is affected by the influence of the vascular porosity in macroscopic samples containing several osteons. In this communication, both analytical and experimental approaches are proposed to estimate the lacunar-canalicular permeability in a single osteon. Data from an experimental stress-relaxation test in a single osteon are used to derive the PLC permeability by curve fitting to theoretical results from a compressible transverse isotropic poroelastic model of a porous annular disk under a ramp loading history (2007, "Compressible and Incompressible Constituents in Anisotropic Poroelasticity: The Problem of Unconfined Compression of a Disk," J. Mech. Phys. Solids, 55, pp. 161-193; 2008, "The Unconfined Compression of a Poroelastic Annular Cylindrical Disk," Mech. Mater., 40(6), pp. 507-523). The PLC tissue intrinsic permeability in the radial direction of the osteon was found to be dependent on the strain rate used and within the range of O(10(-24))-O(10(-25)). The reported values of PLC permeability are in reasonable agreement with previously reported values derived using finite element analysis (FEA) and nanoindentation approaches.

Effect of the vitamin D receptor on bone geometry and strength during gestation and lactation in mice.

The vitamin D receptor (VDR) plays an important role in maintaining calcium homeostasis, acting as a mediator of transcellular calcium absorption and bone remodeling. Mice lacking a functional VDR have an abnormal skeletal phenotype, which is rescued by feeding a high-calcium diet. In this study, the role of the VDR in maintaining bone geometry and strength during gestation and lactation, when increased demands are placed on the calcium regulatory channels, was examined using a knockout mouse model. A rescue diet was used to counteract the decrease in calcium absorption in the gut that results from the absence of the VDR. Structural and compositional characteristics of the femur were compared between VDR knockout and wild-type mice following 9 and 16 days of gestation and 5 and 10 days of lactation using generalized linear models. Overall, the knockout mice had 6.5% lower cortical area, 23% lower trabecular volume fraction, and 9% lower bending stiffness than wild-type mice. However, the maximum moment of inertia of the femoral diaphyses, ultimate bending load, ash fraction, and trabecular thickness were not significantly different between knockout and wild-type mice. Only the mineral content exhibited interdependence between genotype and time point. Taken together, the results show that the VDR affects the quantity of mineralized bone tissue in the femoral diaphysis and metaphysis independently of reproductive status. However, the moments of inertia were similar between genotypes, resulting in similar bone stiffness and strength despite lower mineral content and cross-sectional area.

Chondrocyte apoptosis enhanced at the growth plate: a physeal response to a diaphyseal fracture.

Post-traumatic overgrowth of growing long bones is a common clinical phenomenon in paediatric traumatology and is the result of an enhanced stimulation of the nearby growth plate after fracture. To date, the exact post-fractural reactions of the growth plate are poorly understood. The aim of this study has been to determine the impact of fracture on the frequency of chondrocyte apoptosis of the growth plate. Rats sustained a mid-diaphyseal closed fracture of the left tibia or were left untreated. All animals were killed 3, 10, 14 or 29 days after trauma. The left and right tibiae were harvested and apoptotic chondrocytes of the proximal tibial growth plate were detected by TUNEL staining. The apoptosis percentage of physeal chondrocytes was statistically compared among fractured bones, intact contra-lateral bones and control bones. The physeal apoptosis rate of the fractured bone was significantly higher than that of the contra-lateral intact bone (valid for all evaluated days) and the control bone (valid from day 10 onwards). Contra-lateral intact tibiae never showed significantly higher apoptosis rates compared with control tibiae. Thus, mid-diaphyseal fracture influences the nearby growth plate by stimulating chondrocyte programmed cell death, which is associated with cartilage resorption and bone replacement. The lack of a significant difference between the intact contra-lateral and the intact control bone suggests that fracture only has a local effect that contributes to the greater apoptosis rate of the adjacent physis.

Sex-related variation in compact bone microstructure of the femoral diaphysis in juvenile rabbits.

BACKGROUND: While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits. METHODS: Seventeen clinically healthy 2-3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70-80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed. RESULTS: Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter. CONCLUSION: The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.

Transplantation of a vascularized rabbit femoral diaphyseal segment: mechanical and histologic properties of a new living bone transplantation model.

A new vascularized bone transplantation model is described, including the anatomy and surgical technique of isolating a rabbit femoral diaphyseal segment on its nutrient vascular pedicle. The histologic and biomechanical parameters of pedicled vascularized femoral autotransplants were studied following orthotopic reimplantation in the resulting mid-diaphyseal defect. Vascularized femur segments were isolated in 10 rabbits on their nutrient pedicle, and then replaced orthotopically with appropriate internal fixation. Postoperative weightbearing and mobility were unrestricted, and the contralateral femora served as no-treatment controls. After 16 weeks, the bone flaps were evaluated by x-ray (bone healing), mechanical testing (material properties), microangiography (quantification of intraosseous vasculature), histology (bone viability), and histomorphometry (bone remodeling). Bone healing occurred by 2 weeks, with further callus remodeling throughout the survival period. Eight transplants healed completely, while two had a distal pseudarthrosis. Microangiography demonstrated patent pedicles in all transplants. Intraosseous vessel densities were comparable to nonoperated (control) femora. We found ultimate strength and elastic modulus to be significantly reduced when compared to normal controls. Viable bone, increased mineral apposition rate, and bone turnover were demonstrated in all transplants. The method described, and the data provided will be of value for the further study of isolated segments of living bone, and in particular, for investigations of reconstruction of segmental bone loss in weight-bearing animal models. This study also provides important normative data on living autologous bone flap material properties, vascularity, and bone remodeling. We intend to use this method and data for comparison in subsequent studies of large bone vascularized allotransplantation.

[Changes of the femoral bone microstructure in the reindeer during the fetal period of ontogenesis].

The diaphysis of the femoral bone was studied morphometrically in 30 reindeer fetuses aged 2-7.5 months. The formation of the diaphyseal cavity of the femoral bone took place from 3 till 7.5 months, rapidly progressing during all the fetal period. The thickness of the periosteum in the epiphysis and the diaphysis of the bone increased from 2 to 6 months inclusive, whereupon it decreased by the time of birth. The cartilaginous tissue in the epiphyses was present from 2 till 7.5 months; its growth was registered up to 3 months in a proximal epiphysis, while it continued till 4 months in a distal epiphysis. Later on, the thinning of the cartilage was noted till the birth. The thickness of spongy substance of bone epiphyses increased with the fetal age. The osteons in the diaphysis of the femoral bone were formed in 2-month-old fetus, their numbers were found to increase with age. The compact substance of the diaphysis of the femoral bone increased in thickness till 5 months, whereupon the process of thinning of tissue till the time of birth, was noted.

Small oscillatory accelerations, independent of matrix deformations, increase osteoblast activity and enhance bone morphology.

A range of tissues have the capacity to adapt to mechanical challenges, an attribute presumed to be regulated through deformation of the cell and/or surrounding matrix. In contrast, it is shown here that extremely small oscillatory accelerations, applied as unconstrained motion and inducing negligible deformation, serve as an anabolic stimulus to osteoblasts in vivo. Habitual background loading was removed from the tibiae of 18 female adult mice by hindlimb-unloading. For 20 min/d, 5 d/wk, the left tibia of each mouse was subjected to oscillatory 0.6 g accelerations at 45 Hz while the right tibia served as control. Sham-loaded (n = 9) and normal age-matched control (n = 18) mice provided additional comparisons. Oscillatory accelerations, applied in the absence of weight bearing, resulted in 70% greater bone formation rates in the trabeculae of the metaphysis, but similar levels of bone resorption, when compared to contralateral controls. Quantity and quality of trabecular bone also improved as a result of the acceleration stimulus, as evidenced by a significantly greater bone volume fraction (17%) and connectivity density (33%), and significantly smaller trabecular spacing (-6%) and structural model index (-11%). These in vivo data indicate that mechanosensory elements of resident bone cell populations can perceive and respond to acceleratory signals, and point to an efficient means of introducing intense physical signals into a biologic system without putting the matrix at risk of overloading. In retrospect, acceleration, as opposed to direct mechanical distortion, represents a more generic and safe, and perhaps more fundamental means of transducing physical challenges to the cells and tissues of an organism.

Combination therapy of Nigella sativa and human parathyroid hormone on bone mass, biomechanical behavior and structure in streptozotocin-induced diabetic rats.

Extracts of the seeds of Nigella sativa (NS), an annual herbaceous plant of the Ranunculaceae family, have been used for many years for therapeutic purposes, including their potential anti-diabetic properties. The aim of the present study was to test the hypothesis that combined treatment with NS and human parathyroid hormone (hPTH) is more effective than treatment with NS or hPTH alone in improving bone mass, connectivity, biomechanical behaviour and strength in insulin-dependent diabetic rats. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) at a single dose of 50mg/kg. The diabetic rats received NS (2ml/kg/day, i.p.), hPTH (6microg/kg/day, i.p.) or NS and hPTH combined for 4 weeks, starting 8 weeks after STZ injection. The beta-cells of the pancreatic islets of Langerhans were examined by immunohistochemical methods. In addition, bone sections of femora were processed for histomorphometry and biomechanical analysis. In diabetic rats, the beta-cells were essentially negative for insulin-immunoreactivity. NS treatment (alone or in combination with hPTH) significantly increased the area of insulin immunoreactive beta-cells in diabetic rats; however, hPTH treatment alone only led to a slightly increase in the insulin-immunoreactivity. These results suggest that NS might be used in a similar manner to insulin as a safe and effective therapy for diabetes and might be useful in the treatment of diabetic osteopenia.

Spatial distribution of osteocyte lacunae in equine radii and third metacarpals: considerations for cellular communication, microdamage detection and metabolism.

Osteocytes, which are embedded in bone matrix, are the most abundant cells in bone. Despite the ideal location of osteocytes to sense the local environment and influence bone remodeling, their functions, and the relative importance of these functions, remain controversial. In this study, we tested several hypotheses that address the possibilities that population densities of osteocyte lacunae (Ot.Lc.N/B.Ar) correlate with strain-, remodeling- or metabolism-related aspects of the local biomechanical environments of mid-third diaphyseal equine radii and third metacarpals from skeletally mature animals. Ot.Lc.N/B.Ar data, quantified in multiple cortical locations, were analyzed for possible correlations with (1) structural and material characteristics (e.g., cortical thickness, percent ash, secondary osteon population density, mean osteon cross-sectional area, and predominant collagen fiber orientation), (2) strain characteristics, including prevalent/predominant strain magnitude and mode (tension, compression, shear), (3) hypothesized strain-mode-related microdamage characteristics, which might be perceived by osteocyte 'operational' networks, and (4) variations in remodeling dynamics and/or metabolism (i.e. presumably higher in endocortical regions than in other transcortical locations). Results showed relatively uniform Ot.Lc.N/B.Ar between regions with highly non-uniform strain and strain-related environments and markedly heterogeneous structural and material organization. These results suggest that population densities of these cells are poorly correlated with mechanobiological characteristics, including local variations in metabolic rate and strain magnitude/mode. Although osteocytes hypothetically evolved both as strain sensors and fatigue damage sensors able to direct the removal of damage as needed, the mechanisms that govern the distribution of these cells remain unclear. The results of this study provide little or no evidence that the number of osteocyte lacunae has a functional role in mechanotransduction pathways that are typically considered in bone adaptation.

Human femoral neck has less cellular periosteum, and more mineralized periosteum, than femoral diaphyseal bone.

Periosteal expansion enhances bone strength and is controlled by osteogenic cells of the periosteum. The extent of cellular periosteum at the human femoral neck, a clinically relevant site, is unclear. This study was designed to histologically evaluate the human femoral neck periosteal surface. Femoral neck samples from 11 male and female cadavers (ages 34-88) were histologically assessed and four periosteal surface classifications (cellular periosteum, mineralizing periosteum, cartilage, and mineralizing cartilage) were quantified. Femoral mid-diaphysis samples from the same cadavers were used as within-specimen controls. The femoral neck surface had significantly less (P<0.05) cellular periosteum (18.4+/-9.7%) compared to the femoral diaphysis (59.2+/-13.8%). A significant amount of the femoral neck surface was covered by mineralizing periosteal tissue (20-70%). These data may provide an alternate explanation for the apparent femoral neck periosteal expansion with age and suggest the efficiency of interventions that stimulate periosteal expansion may be reduced, albeit still possible, at the femoral neck of humans.

Studies on action of menaquinone-7 in regulation of bone metabolism and its preventive role of osteoporosis.

The effect of menaquinone-7 (MK-7) on bone components and bone resorbing factors induced-bone resorption using the femoral-diaphyseal and - metaphyseal tissues obtained from elderly female rats in vitro were examined. Calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) in the diaphyseal and metaphyseal tissues in elderly females rats were significantly decreased as compared with that of young rats, indicating that aging causes a deterioration of bone formation. The presence of MK-7 (10(-6)-10(-5) M) caused a significant prevention of reduction of biochemical components. On the other hand, the bone-resorbing factor, parathyroid hormone (1-34) (PTH; 10(-7) M) and prostaglandin E(2) (PGE(2); 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. This decreases was completely inhibited in the presence of MK-7 (10(-7)-10(-5) M). In addition, MK-7 (10(-7)-10(-5) M) completely prevented the PTH (10(-7) M) or PGE(2) (10(-5) M) induced increases in medium glucose consumption and lactic acid production by bone tissues, Furthermore, the effect of the prolonged intake of dietary MK-7 on bone loss in ovariectomized rats was investigated. As a result, it was found that the intake of experimental diets containing the fermented soybean (natto) with supplemental MK-7 caused significant elevations of MK-7 and gamma-carboxylated osteocalcin concentration, a bio marker of bone formation, in the serum of both ovariectomized rats and normal subjects, suggesting that MK-7 may play an important role in the prevention of age-related bone loss.