Sclerostin does not play a major role in the pathogenesis of skeletal complications in type 2 diabetes mellitus.

In contrast to previously reported elevations in serum sclerostin levels in diabetic patients, the present study shows that the impaired bone microarchitecture and cellular turnover associated with type 2 diabetes mellitus (T2DM)-like conditions in ZDF rats are not correlated with changes in serum and bone sclerostin expression. INTRODUCTION: T2DM is associated with impaired skeletal structure and a higher prevalence of bone fractures. Sclerostin, a negative regulator of bone formation, is elevated in serum of diabetic patients. We aimed to relate changes in bone architecture and cellular activities to sclerostin production in the Zucker diabetic fatty (ZDF) rat. METHODS: Bone density and architecture were measured by micro-CT and bone remodelling by histomorphometry in tibiae and femurs of 14-week-old male ZDF rats and lean Zucker controls (n = 6/group). RESULTS: ZDF rats showed lower trabecular bone mineral density and bone mass compared to controls, due to decreases in bone volume and thickness, along with impaired bone connectivity and cortical bone geometry. Bone remodelling was impaired in diabetic rats, demonstrated by decreased bone formation rate and increased percentage of tartrate-resistant acid phosphatase-positive osteoclastic surfaces. Serum sclerostin levels (ELISA) were higher in ZDF compared to lean rats at 9 weeks (+40 %, p < 0.01), but this difference disappeared as their glucose control deteriorated and by week 14, ZDF rats had lower sclerostin levels than control rats (-44 %, p < 0.0001). Bone sclerostin mRNA (qPCR) and protein (immunohistochemistry) were similar in ZDF, and lean rats at 14 weeks and genotype did not affect the number of empty osteocytic lacunae in cortical and trabecular bone. CONCLUSION: T2DM results in impaired skeletal architecture through altered remodelling pathways, but despite altered serum levels, it does not appear that sclerostin contributes to the deleterious effect of T2DM in rat bone.

Bone mineralization: from tissue to crystal in normal and pathological contexts.

Bone is a complex and structured material; its mechanical behavior results from an interaction between the properties of each level of its structural hierarchy. The degree of mineralization of bone (bone density measured at tissue level) and the characteristics of the mineral deposited (apatite crystals) are major determinants of bone strength. Bone remodeling activity acts as a regulator of the degree of mineralization and of the distribution of mineral at the tissue level, directly impacting bone mechanical properties. Recent findings have highlighted the need to understand the underlying process occurring at the nanostructure level that may be independent of bone remodeling itself. A more global comprehension of bone qualities will need further works designed to characterize what are the consequences on whole bone strength of changes at nano- or microstructure levels relative to each other.

Effects of strontium on the quality of bone apatite crystals: a paired biopsy study in postmenopausal osteoporotic women.

UNLABELLED: In paired biopsies of osteoporotic women treated with either strontium ranelate or a placebo for 36 months, characteristics of bone apatite crystals were not influenced by the presence of strontium. The mean rate of substitutions of calcium by strontium ions was 4.5 %. INTRODUCTION: The potential effect of strontium (Sr) on bone apatite crystals was investigated in paired biopsies of osteoporotic women treated with either strontium ranelate (SrRan) or a placebo for 36 months. METHODS: In ten paired biopsies, crystallinity, apparent length and width/thickness of crystals, interplanar distances, and lattice parameters of unit cells were assessed by X-ray diffraction and selected area electron diffraction. RESULTS: All these parameters, reflecting crystal and unit cell characteristics, were not influenced by the presence of Sr and were similar in SrRan and placebo groups after 36 months of treatment. The mean rate of substitutions of calcium by Sr ions was 4.5 %. CONCLUSION: Overall, the quality of bone apatite crystals was maintained after 36 months of treatment with SrRan.

Intrinsic properties of osteomalacia bone evaluated by nanoindentation and FTIRM analysis.

Osteomalacia is a pathological bone condition consisting in a deficient primary mineralization of the matrix, leading to an accumulation of osteoid tissue and reduced bone mechanical strength. The amounts, properties and organization of bone constituents at tissue level, are known to influence its mechanical properties. It is then important to investigate the relationship between mechanical behavior and tissue composition at this scale in order to provide a better understanding of bone fragility mechanisms associates with this pathology. Our purpose was to analyze the links between ultra-structural properties and the mechanical behavior of this pathological bone tissue (osteomalacia) at tissue level (mineral and osteoid separately, or global). Four bone biopsies were taken from patients with osteomalacia, and subsequently embedded, sectioned, and polished. Then nanoindentation tests were performed to determine local elastic modulus E, contact hardness Hc and true hardness H for both mineralized and organic bone phases and for the global bone. The creep of the bone was also studied using a special indentation procedure in order to assess visco-elasto-plastic (creep) bone behavior. This allowed a detailed study of the rheological models adapted to the bone and to calculate the parameters associated to a Burgers model. Ultra-structural parameters were measured by Fourier Transform InfraRed Microspectroscopy (FTIRM) on the same position as the indents. The use of rheological models confirmed a significant contribution from the organic phase on the viscous character of bone tissue. The elastic E and the elasto-plastic Hc deformation were correlated to both collagen maturity and Mineral/Matrix. The pure plastic deformation H was only correlated to the mineral phase. Our data show that mineral phase greatly affects mechanical variables (moduli and viscosities) and that organic phase (as illustrated in osteoid tissue) may play an important role in the creep behavior of bone. In conclusion, this study brings mechanical and physicochemical values for osteoid and mineral phases.

In osteoporotic women treated with strontium ranelate, strontium is located in bone formed during treatment with a maintained degree of mineralization.

UNLABELLED: In postmenopausal osteoporotic women and up to 3 years of treatment with strontium ranelate, strontium was present only in recently deposited bone tissue resulting from formation activity during the period of treatment. Strontium was shown to be dose-dependently deposited into this newly formed bone with preservation of the mineralization. INTRODUCTION: Interactions between strontium (Sr) and bone mineral and its effects on mineralization were investigated in women treated with strontium ranelate. METHODS: Bone biopsies from osteoporotic women were obtained over 5-year strontium ranelate treatment from phases II and III studies. Bone samples obtained over 3-year treatment were investigated by X-ray microanalysis for bone Sr uptake and focal distribution, and by quantitative microradiography for degree of mineralization. On some samples, Sr distribution (X-ray cartography) was analyzed on whole sample surfaces and the percentage of bone surface containing Sr was calculated. Bone Sr content was chemically measured on whole samples. RESULTS: In treated women, Sr was exclusively present in bone formed during treatment; Sr deposition depended on the dose with higher focal content in new bone structural units than in old ones constantly devoid of Sr, even after 3-year treatment. A plateau in global bone Sr content was reached after 3 years of treatment. Cartography illustrated the extent of surfaces containing Sr, and formation activity during strontium ranelate treatment was higher in cancellous than in cortical bone. Mineralization was maintained during treatment. CONCLUSION: The quality of bone mineral was preserved after treatment with strontium ranelate, supporting the safety of this agent at the bone tissue level.

Bone remodeling and bone matrix quality before and after menopause in healthy women.

Acceleration of remodeling activity after menopause leads to bone loss and fragility, however, whether this is associated with modifications of bone matrix quality has been less studied. The impact of variation in bone remodeling rate on bone matrix has been studied mainly in pathologies or anti-osteoporotic treatments. However, in healthy women this has been less studied. We analyzed, at the global level, bone matrix quality in bone biopsies from 3 groups of healthy women (20 per group): 1) before menopause (PreM), 2) 1 year after menopause (PostM, paired biopsies with preM), and 3) 14 (±9) years after menopause (LT-PostM). The mean degree of mineralization (DMB) and heterogeneity index (HI) of mineralization were assessed by X-ray microradiography on whole bone matrix; intrinsic properties (mineral/organic ratio, mineral maturity, mineral crystallinity, collagen maturity) were assessed by Fourier Transform Infrared microspectroscopy, microhardness by microindentation, both at a global level and calculated by mean of several measurements over the whole tissue area. In PostM compared to PreM (bone remodeling rate had doubled), mean DMB measured on the entire bone plane (whole bone matrix) of the sample was not different. HI was increased in trabecular bone indicating a higher heterogeneity of mineralization. However, in PostM, mineral/organic ratio (trabecular) and microhardness (cortical and trabecular) were decreased, whereas mineral/collagen maturation or crystal size/perfection were unchanged. Thus, in PostM, the local mineral content and microhardness were first affected. In LT-PostM (bone remodeling rate was 3 times higher), the mean DMB was still not different. However, the mineral/organic ratio, microhardness, mineral maturity, crystallinity all were lower compared to PreM and PostM, in both cortical and trabecular bone. Bone remodeling rate was negatively correlated with microhardness, DMB, mineral/organic and crystallinity. This suggests that increases in bone remodeling rates after menopause have a direct impact on bone quality by inducing the formation of more extensive "immature" bone areas, but the amount of immature bone does not cause modification of the global DMB.

Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

The role of mineralization and organic matrix in the microhardness of bone tissue from controls and osteoporotic patients.

Degree of mineralization of bone (DMB) is a major intrinsic determinant of bone strength at the tissue level but its contribution to the microhardness (Vickers indentation) at the intermediary level of organization of bone tissue, i.e., Bone Structural Units (BSUs), has never been assessed. The purpose of this study was to analyze the relationship between the microhardness, the DMB and the organic matrix, measured in BSUs from human iliac bone biopsies. Iliac bone samples from controls and osteoporotic patients (men and women), embedded in methyl methacrylate, were used. Using a Vickers indenter, microhardness (kg/mm2) was measured, either globally on surfaced blocks or focally on 100 microm-thick sections from bone samples (load of 25 g applied during 10 sec; CV=5%). The Vickers indenter was more suited than the Knoop indenter for a tissue like bone in which components are diversely oriented. Quantitative microradiography performed on 100 microm-thick sections, allowed measurement of parameters reflecting the DMB (g/cm3). Assessed on the whole bone sample, both microhardness and DMB were significantly lower (-10% and -7%, respectively) in osteoporotic patients versus controls (p<0.001). When measured separately at the BSU level, there were significant positive correlations between microhardness and DMB in controls (r2=0.36, p<0.0001) and osteoporotic patients (r2=0.43, p<0.0001). Mineralization is an important determinant of the microhardness, but did not explain all of its variance. To highlight the role of the organic matrix in bone quality, microhardness of both osteoid and adjacent calcified matrix were measured in iliac samples from subjects with osteomalacia. Microhardness of organic matrix is 3-fold lower than the microhardness of calcified tissue. In human calcanei, microhardness was significantly correlated with DMB (r2=0.33, p=0.02) and apparent Young's modulus (r2=0.26, p=0.03). In conclusion, bone microhardness measured by Vickers indentation is an interesting methodology for the evaluation of bone strength and its determinants at the BSU level. Bone microhardness is linked to Young's modulus of bone and is strongly correlated to mineralization, but the organic matrix accounts for about one third of its variance.

An in vitro model to test the contribution of advanced glycation end products to bone biomechanical properties.

We developed an in vitro model which provides the ability to test the effects of advanced glycation end products (AGEs), specifically pentosidine (PEN) and one of its inhibitors, the aminoguanidine (AMG), on cortical bone. This model allows modification of the extent of collagen cross-linking, while controlling other factors known to influence bone strength. In this in vitro model, young bovine cortical bone specimens were incubated in phosphate-buffered saline (PBS)+/-ribose (RIB, an inducer of AGEs formation)+/-AMG for 15 days at 37 degrees C. The mineral and organic matrix as well as biomechanical properties were examined. We found that (i) incubation+/-treatments did not induce collagen denaturation compared to specimens that were not incubated; (ii) neither treatment or incubation time effected the concentration of trivalent enzymatic cross-links pyridinoline and deoxypyridinoline. The non-enzymatic cross-link PEN was undetectable in specimens that were not incubated or that were incubated in PBS or AMG alone. However, PEN concentration increased significantly in specimens incubated with RIB, whereas ribose-induced PEN formation was markedly inhibited by AMG. (iii) Incubation+/-treatments did not change the mineral maturity, crystallinity or microhardness assessed by X-ray diffraction, X-ray microscopy analyses, FTIRM and micro-indentation tests. (iv) PEN concentration was not associated with biomechanical properties assessed by 3-point bending. In conclusion, this in vitro incubation model of young bovine cortical bone induced physiologic concentrations of PEN in the RIB+AMG group and is the first to show that AMG inhibits ribose-induced formation of PEN cross-links in bone while not affecting the organic and mineral phases. AGE concentration did not influence bending mechanical properties; however, the simple 3-point bending test we used was likely inadequate to demonstrate effects of AGEs on mechanical properties.

Evidence for a dense and intimate innervation of the bone tissue, including glutamate-containing fibers.

The recent demonstration in bone cells of receptors for glutamate (Glu), a major neuromediator, suggests that Glu may also act as a signaling molecule in bone and regulate bone cell metabolism. Although bone is known to be innervated, the distribution and characteristics of nerve fibers in this tissue have not been well documented. We have studied the anatomical distribution of nerve fibers and the presence of glutamate-immunoreactive ones in sections of long bones from neonatal, 15-, and 25-day-old rats, using immunocytochemistry with antibodies directed against several neuronal markers and Glu. We showed by electron microscopy that bone is rich in nerve-like processes running along vessels adjacent to bone trabeculae, in the vicinity of hematopoietic cells and bone cells. Immunocytochemical studies at the tissue and cellular level confirmed the presence of a dense network of thin nerve processes immunolabeled for neurofilament 200, tyrosine hydroxylase, and microtubule associated protein-2, three markers of nerve fibers. Some of these nerve processes showed local dilatations in contact with medullary cells and bone cells that were immunolabeled for synaptophysin, a nerve terminal marker. Glu was largely expressed in these thin nerve processes in proximity to bone cells. These findings show evidence for a dense and intimate network of nerve processes in bone, some of which were containing Glu, suggesting glutamatergic innervation in bone.

A comparative analysis of strategies for isolation of matrix vesicles.

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations. The second isolation method does not use proteases but rather starts with differential centrifugation, followed by a fractionation on a sucrose gradient. The first method results in a homogeneous population of MVs with higher cholesterol/lipid content, alkaline phosphatase activity, and mineral formation rate as compared with MVs isolated by the second method. The second method leads to higher protein diversity as compared with MVs isolated according to the first method. Due to their distinct protein composition, lipid-to-protein and cholesterol-to-phospholipid ratios, and differences in rates of mineral formation, both types of isolated MVs are crucial for proteomic analysis and for understanding the regulation of mineralization process at the molecular level.