OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

Dietary supplementation with nacre reduces cortical bone loss in aged female mice.

Aging is associated with detrimental bone loss leading to fragility fractures in both men and women. Notably, a majority of bone loss with aging is cortical, as well as a large number of fractures are non-vertebral and at the non-hip sites. Nacre is a product of mollusks composed of calcium carbonate embedded in organic components. As our previous study demonstrated the protective effect of nacre supplementation on trabecular bone loss in ovariectomized rats, we sought to evaluate the effect of dietary nacre on bone loss related to aging in female mice which do not suffer true menopause as observed in women. The current study compared the effect of a 90-day long nacre-supplemented diet to that of Standard or CaCO3 diets on both bone mass and strength in 16-month-old C57BL/6 female mice. Multiple approaches were performed to assess the microarchitecture and mechanical properties of long bones, analyze trabecular histomorphometry, and measure bone cell-related gene expressions, and bone turnover markers. In the cortex, dietary nacre improved cortical bone strength in line with lower expression levels of genes reflecting osteoclasts activity compared to Standard or CaCO3 diets (p < 0.05). In the trabeculae, nacre-fed mice were characterized by a bone remodeling process more active than the other groups as shown by greater histomorphometric parameters and osteoblast-related gene expressions (p < 0.05). But these differences were not exhibited at the level of the trabecular microarchitecture at this age. Collectively, these data suggest that dietary nacre should be a potential candidate for reducing aging-associated cortical bone loss in the elderly.

Material and nanomechanical properties of bone structural units of cortical and trabecular iliac bone tissues from untreated postmenopausal osteoporotic women.

The differences in bone nanomechanical properties between cortical (Ct) and trabecular (Tb) bone remain uncertain, whereas knowing the respective contribution of each compartment is critical to understand the origin of bone strength. Our purpose was to compare bone mechanical and intrinsic properties of Ct and Tb compartments, at the bone structural unit (BSU) level, in iliac bone taken from a homogeneous untreated human population. Among 60 PMMA-embedded transiliac bone biopsies from untreated postmenopausal osteoporotic women (64 ± 7 year-old), >2000 BSUs were analysed by nanoindentation in physiological wet conditions [indentation modulus (elasticity), hardness, dissipated energy], by Fourier transform infrared (FTIRM) and Raman microspectroscopy (mineral and organic characteristics), and by X-ray microradiography (degree of mineralization of bone, DMB). BSUs were categorized based on tissue age, osteonal (Ost) and interstitial (Int) tissues location and bone compartments (Ct and Tb). Indentation modulus was higher in Ct than in Tb BSUs, both in Ost and Int. dissipated energy was higher in Ct than Tb, in Int BSUs. Hardness was not different between Ct and Tb BSUs. In Ost or Int BSUs, mineral maturity (conversion of non-apatitic into apatitic phosphates) was higher in Ct than in Tb, as well as for collagen maturity (Ost). Mineral content assessed as mineral/matrix (FTIRM and Raman) or as DMB, was lower in Ct than in Tb. Crystallinity (FTIRM) was similar in BSUs from Ct and Tb, and slightly lower in Ct than in Tb when measured by Raman, indicating that the crystal size/perfection was quite similar between Ct and Tb BSUs. The differences found between Ost and Int tissues were much higher than the difference found between Ct and Tb for all those bone material properties. Multiple regression analysis showed that Indentation modulus and dissipated energy were mainly explained by mineral maturity in Ct and by collagen maturity in Tb, and hardness by mineral content in both Ct and Tb. In conclusion, in untreated human iliac bone, Ct and Tb BSUs exhibit different characteristics. Ct BSUs have higher indentation modulus, dissipated energy (Int), mineral and organic maturities than Tb BSUs, without difference in hardness. Although those differences are relatively small compared to those found between Ost and Int BSUs, they may influence bone strength at macroscale.

Bone Mineral and Organic Properties in Postmenopausal Women Treated With Denosumab for Up to 10 years.

In postmenopausal women with osteoporosis, denosumab (DMAb) therapy through 10 years resulted in significantly higher degree of mineralization of bone, with a subsequent increase from years 2-3 to year 5 and no further difference between years 5 and 10. Our aim was to assess the variables reflecting the quality of bone mineral and organic matrix (Fourier transform infrared microspectroscopy), and the microhardness of bone (Vickers microindentation). Cross-sectional assessments were performed in blinded fashion on iliac bone biopsies from osteoporotic women (72 from FREEDOM trial, 49 from FREEDOM Extension trial), separately in cortical and cancellous compartments. After 2-3 years of DMAb, mineral/matrix ratio and microhardness of cortical bone were significantly higher compared with placebo, whereas mineral maturity, mineral crystallinity, mineral carbonation, and collagen maturity were not different in both bone compartments. Through 5 years of DMAb, mineral carbonation was significantly lower and mineral/matrix ratio, mineral maturity, and crystallinity were significantly higher versus 2-3 years and were not different between 5 and 10 years, with the exception of mineral maturity in cancellous bone. These data support a transition of mineral to more mature crystals (within physiological range) and the completeness of secondary mineralization within 5 years of DMAb treatment. Microhardness in cortical and cancellous compartments was significantly lower at 5 years of DMAb versus 2-3 years and was not different from years 5 to 10. The lower microhardness at years 5 and 10 is likely the result of maturation of the organic matrix in a persistently low state of bone remodeling over 5 and 10 years. © 2022 American Society for Bone and Mineral Research (ASBMR).

Bone matrix quality in paired iliac bone biopsies from postmenopausal women treated for 12 months with strontium ranelate or alendronate.

Bone quality is altered mainly by osteoporosis, which is treated with modulators of bone quality. Knowledge of their mechanisms of action is crucial to understand their effects on bone quality. The goal of our study was to compare the action of alendronate (ALN) and strontium ranelate (SrRan) on the determinants of bone quality. The investigation was performed on over 60 paired human iliac biopsies. Paired samples correspond to biopsies obtained from the same patient, one before treatment (baseline) and one after 12 months of treatment, in postmenopausal women with osteoporosis. Vibrational spectroscopy (Raman and FTIRM) and nanoindentation were used to evaluate the effect of both drugs on bone quality at the ultrastructural level. Outcomes measured by vibrational spectroscopy and nanoindentation are sensitive to bone age. New bone packets are distinguished from old bone packets. Thus, the effect of bone age is distinguished from the treatment effect. Both drugs modify the mineral and organic composition in new and old bone in different fashions after 12 months of administration. The new bone formed during ALN administration is characterized by an increased mineral content, carbonation and apatite crystal size/perfection compared to baseline. Post-translational modifications of collagen are observed through an increase in the hydroxyproline/proline ratio in new bone. The proteoglycan content is also increased in new bone. SrRan directly modulates bone quality through its physicochemical actions, independent of an effect on bone remodeling. Strontium cations are captured by the hydrated layer of the mineral matrix. The mineral matrix formed during SrRan administration has a lower carbonate content and crystallinity after 12 months than at baseline. Strontium might create bonds (crosslinks) with collagen and noncollagenous proteins in new and old bone. The nanomechanical properties of bone were not modified with either ALN or SrRan, probably due to the short duration of administration. Our results show that ALN and SrRan have differential effects on bone quality in relation to their mechanism of action.

Low doses of uranium and osteoclastic bone resorption: key reciprocal effects evidenced using new in vitro biomimetic models of bone matrix.

Uranium is widely spread in the environment due to its natural and anthropogenic occurrences, hence the importance of understanding its impact on human health. The skeleton is the main site of long-term accumulation of this actinide. However, interactions of this metal with biological processes involving the mineralized extracellular matrix and bone cells are still poorly understood. To get a better insight into these interactions, we developed new biomimetic bone matrices containing low doses of natural uranium (up to 0.85 µg of uranium per cm2). These models were characterized by spectroscopic and microscopic approaches before being used as a support for the culture and differentiation of pre-osteoclastic cells. In doing so, we demonstrate that uranium can exert opposite effects on osteoclast resorption depending on its concentration in the bone microenvironment. Our results also provide evidence for the first time that resorption contributes to the remobilization of bone matrix-bound uranium. In agreement with this, we identified, by HRTEM, uranium phosphate internalized in vesicles of resorbing osteoclasts. Thanks to the biomimetic matrices we developed, this study highlights the complex mutual effects between osteoclasts and uranium. This demonstrates the relevance of these 3D models to further study the cellular mechanisms at play in response to uranium storage in bone tissue, and thus better understand the impact of environmental exposure to uranium on human bone health.

Duration-Dependent Increase of Human Bone Matrix Mineralization in Long-Term Bisphosphonate Users with Atypical Femur Fracture.

Bisphosphonates (BPs) are the most widely used drugs for the treatment of osteoporosis but prolonged use of BPs might increase the risk of atypical femur fracture (AFF). There are only a few studies that address the bone material quality in patients on long-term BP treatment with or without AFFs. We analyzed 52 trans-iliac bone biopsies from patients on long-term BP therapy with (n = 26) and without (n = 26) AFF. At the microscopic level, the degree of mineralization of bone (DMB) was assessed on whole bone by X-ray digitized microradiography while microhardness by Vickers microindentation, and bone matrix characteristics by Fourier transform infrared microspectroscopy (FTIRM) (mineral/organic ratio, mineral maturity and crystallinity, and collagen maturity) were measured at random focal areas. The AFF patients were treated longer than non-AFF patients (9.7 ± 3.3 years versus 7.9 ± 2.7 years). As expected, bone remodeling was low in both groups, without difference between them. The AFF group had significantly higher DMB in cortical bone (+2.9%, p = .001), which remained so after adjusting for treatment duration (p = .007), and showed a trend in cancellous bone (+1.6%, p = .05). Consistent with higher DMB, heterogeneity index (HI) was lower in the AFF than in the non-AFF group, illustrating lower heterogeneity of mineralization in the AFF group. A significant positive correlation between the duration of treatment and DMB in cortical bone was found in AFF, and not in the non-AFF group. Microhardness and bone matrix characteristics were similar between groups. We conclude that the AFF group had a duration-dependent increase in DMB leading to a significantly higher DMB than the non-AFF. Because BPs have high affinity to bone mineral and lining the walls of the osteocyte lacunae, the accumulation of matrix-bound BPs in AFF could lead to inhibition of the osteocyte cytoskeleton blunting their response to mechanical strains, a hypothesis to be further investigated. © 2021 American Society for Bone and Mineral Research (ASBMR).

Expression of the type 1 lysophosphatidic acid receptor in osteoblastic cell lineage controls both bone mineralization and osteocyte specification.

Lysphosphatidic acid (LPA) is a major natural bioactive lipid mediator whose biological functions affect multiple organs. These include bone as demonstrated by global Lpar1-knockout mice (Lpar1-/-) which present a bone growth defect. LPA acts on all bone cells including osteoblasts, that are responsible for bone formation, and osteoclasts, which are specialized cells that resorb bone. LPA appears as a potential new coupling molecule during bone remodeling. LPA1 is the most ubiquitous LPA receptor among the six LPA receptor family members (LPA1-6). To better understand the specific role of LPA via its receptor LPA1 in osteoblastic cell lineage we generated osteoblast-specific Lpar1 knockout mice (Lpar1-∆Ob) by crossing Lpar1flox/flox and Osx:Cre+ mouse lines. Lpar1-∆Ob mice do not recapitulate the bone defects of Lpar1-/- mice but revealed reduced bone mineralization and decreased cortical thickness, as well as increased bone porosity associated with an augmentation in the lacunae areas of osteocyte and their apoptotic yield. In vitro, primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts revealed a remarkable premature expression of alkaline phosphatase, reduced cell proliferation associated with decreased YAP-P nuclear accumulation, and reduced mineralization activity. Osteocyte specification is markedly impaired as demonstrated by reduced expression of early (E11) and late (DMP1, DKK1, SOST) osteocyte markers ex vivo in enriched osteocytic fractions of Lpar1-∆Ob mouse bone explants. In addition, E11 expression and dendrite formation induced by FGF2 are markedly impaired in both primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts. Taken together these results suggest a new role for LPA in bone mass control via bone mineralization and osteocyte function.

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

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

Uranium Effect on Osteocytic Cells In Vitro.

Once absorbed in the body, natural uranium [U(VI)], a radionucleotide naturally present in the environment, is targeted to the skeleton which is the long-term storage organ. We and others have reported the U(VI) negative effects on osteoblasts (OB) and osteoclasts (OC), the main two cell types involved in bone remodeling. In the present work, we addressed the U(VI) effect on osteocytes (OST), the longest living bone cell type and the more numerous (> 90%). These cells, which are embedded in bone matrix and thus are the more prone to U(VI) long-term exposure, are now considered as the chief orchestrators of the bone remodeling process. Our results show that the cytotoxicity index of OST is close to 730 µM, which is about twice the one reported for OB and OC. However, despite this resistance potential, we observed that chronic U(VI) exposure as low as 5 µM led to a drastic decrease of the OST mineralization function. Gene expression analysis showed that this impairment could potentially be linked to an altered differentiation process of these cells. We also observed that U(VI) was able to trigger autophagy, a highly conserved survival mechanism. Extended X-ray absorption fine structure analysis at the U LIII edge of OST cells exposed to U(VI) unambiguously shows the formation of an uranyl phosphate phase in which the uranyl local structure is similar to the one present in Autunite. Thus, our results demonstrate for the first time that OST mineralization function can be affected by U(VI) exposure as low as 5 µM, suggesting that prolonged exposure could alter the central role of these cells in the bone environment.

Anisotropic elastic properties of human femoral cortical bone and relationships with composition and microstructure in elderly.

The strong dependence between cortical bone elasticity at the millimetre-scale (mesoscale) and cortical porosity has been evidenced by previous studies. However, bone is an anisotropic composite material made by mineral, proteins and water assembled in a hierarchical structure. Whether the variations of structural and compositional properties of bone affect the different elastic coefficients at the mesoscale is not clear. Aiming to understand the relationships between bone elastic properties and compositions and microstructure, we applied state-of-the-art experimental modalities to assess these aspects of bone characteristics. All elastic coefficients (stiffness tensor of the transverse isotropic bone material), structure of the vascular pore network, collagen and mineral properties were measured in 52 specimens from the femoral diaphysis of 26 elderly donors. Statistical analyses and micromechanical modeling showed that vascular pore volume fraction and the degree of mineralization of bone are the most important determinants of cortical bone anisotropic mesoscopic elasticity. Though significant correlations were observed between collagen properties and elasticity, their effects in bone mesoscopic elasticity were minor in our data. This work also provides a unique set of data exhibiting a range of variations of compositional and microstructural cortical bone properties in the elderly and gives strong experimental evidence and basis for further development of biomechanical models for human cortical bone. STATEMENT OF SIGNIFICANCE: This study reports the relationships between microstructure, composition and the mesoscale anisotropic elastic properties of human femoral cortical bone in elderly. For the first time, we provide data covering the complete anisotropic elastic tensor, the microstructure of cortical vascular porosity, mineral and collagen characteristics obtained from the same or adjacent samples in each donor. The results revealed that cortical vascular porosity and degree of mineralization of bone are the most important determinants of bone anisotropic stiffness at the mesoscale. The presented data gives strong experimental evidence and basis for further development of biomechanical models for human cortical bone.

Third harmonic generation imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone.

The lacuno-canalicular network (LCN) hosting the osteocytes in bone tissue represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. Using third-harmonic generation (THG) microscopy with sub-micrometer resolution, we investigate the impact of microgravity on the 3D LCN structure in mice following space flight. A specific analytical procedure to extract the LCN characteristics from THG images is described for ex vivo studies of bone sections. The analysis conducted in different anatomical quadrants of femoral cortical bone didn't reveal any statistical differences between the control, habitat control and flight groups, suggesting that the LCN connectivity is not affected by one month space flight. However, significant variations are systematically observed within each sample. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.

Variables Reflecting the Mineralization of Bone Tissue From Fracturing Versus Nonfracturing Postmenopausal Nonosteoporotic Women.

Women with equivalent areal bone mineral densities may show a different fracture incidence due to differences in bone intrinsic quality. Previously, Fourier transform infrared spectroscopic imaging (FTIRI) on the same iliac bone biopsies reported here, showed that the only significantly different variable was the carbonate/phosphate ratio, which was decreased in the fracturing group. Nanoindentation showed that fracturing bone was less mechanically heterogeneous than nonfracturing bone and could propagate damage (microcracks) more easily. The hypothesis is that fracturing women have reduced mineralization of bone tissue compared to nonfracturing women. Transiliac bone biopsies were collected from fracturing (n = 60, 62.5 ± 7.4 years old) and nonfracturing (n = 60, 62.3 ± 7.3 years old) postmenopausal women, to assess the mineralization of bone tissue using digitized microradiography. The degree of mineralization of bone (DMB, g/cm3) and the heterogeneity index (HI, g/cm3) of the DMB were calculated for cancellous (canc), cortical (cort) and total bone. Results were compared to variables from nanoindentation, FTIRI, and histomorphometry. DMB and HI were not significantly different between fracturing and nonfracturing groups. In the nonfracturing group, cort and canc HI were weakly negatively associated with cort and canc DMB (r' = -0.388, p < 0.003; r' = -0.532, p < 0.0001, respectively). In the fracturing group, DMB and HI were negatively correlated only in canc (r' = -0.295, p = 0.024). DMB and HI were not associated with nanoindentation variables. Cort and canc DMB were positively associated with mineral-to-matrix ratio measured by FTIRI (ratio between mineral and organic matrix representing the relative mineralization of the collagen matrix), and negatively associated with carbonate/phosphate ratio. None of the DMB variables were strongly associated with any of the histomorphometric variables. In conclusion, bone mineralization was not significantly different between fracturing and nonfracturing postmenopausal women, suggesting that bone fragility could be partly due to other variables, such as changes in hydration of bone matrix or an increase of non-enzymatic crosslinks in bone collagen. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Skeletal impairment in Pierson syndrome: Is there a role for lamininß2 in bone physiology?

INTRODUCTION: Pierson syndrome is caused by a mutation of LAMB2, encoding for laminin ß2. Clinical phenotype is variable but usually associates congenital nephrotic syndrome (CNS) and ocular abnormalities. Neuromuscular impairment has also been described. METHODS: We report on a 15-year old girl, suffering from Pierson Syndrome, who developed severe bone deformations during puberty. This patient initially displayed CNS and microcoria, leading to the clinical diagnosis of Pierson syndrome. Genetic analysis revealed a truncating mutation and a splice site mutation of LAMB2. The patient received a renal transplantation (R-Tx) at the age of 3. After R-Tx, renal evolution was simple, the patient receiving low-dose corticosteroids, tacrolimus and mycophenolate mofetil. At the age of 12, bone deformations progressively appeared. At the time of bone impairment, renal function was subnormal (glomerular filtration rate using iohexol clearance 50mL/min per 1.73m2), and parameters of calcium/phosphate metabolism were normal (calcium 2.45mmol/L, phosphorus 1.30mmol/L, PTH 81ng/L, ALP 334U/L, 25OH-D 73nmol/L). Radiographs showed major deformations such as scoliosis, genu varum and diffuse epiphyseal abnormalities. A high resolution scanner (HR-pQCT) was performed, demonstrating a bone of "normal low" quantity and quality; major radial and cubital deformations were observed. Stainings of laminin ß2 were performed on bone and renal samples from the patient and healthy controls: as expected, laminin ß2 was expressed in the control kidney but not in the patient's renal tissue, and a similar pattern was observed in bone. CONCLUSION: This is the first case of skeletal impairment ever described in Pierson syndrome. Integrin α3ß1, receptor for laminin ß2, are found in podocytes and osteoblasts, and the observation of both the presence of laminin ß2 staining in healthy bone and its absence in the patient's bone raises the question of a potential role of laminin ß2 in bone physiology.

Erratum: One-month spaceflight compromises the bone microstructure, tissue-level mechanical properties, osteocyte survival and lacunae volume in mature mice skeletons.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy.

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.

One-month spaceflight compromises the bone microstructure, tissue-level mechanical properties, osteocyte survival and lacunae volume in mature mice skeletons.

The weightless environment during spaceflight induces site-specific bone loss. The 30-day Bion-M1 mission offered a unique opportunity to characterize the skeletal changes after spaceflight and an 8-day recovery period in mature male C57/BL6 mice. In the femur metaphysis, spaceflight decreased the trabecular bone volume (-64% vs. Habitat Control), dramatically increased the bone resorption (+140% vs. Habitat Control) and induced marrow adiposity invasion. At the diaphysis, cortical thinning associated with periosteal resorption was observed. In the Flight animal group, the osteocyte lacunae displayed a reduced volume and a more spherical shape (synchrotron radiation analyses), and empty lacunae were highly increased (+344% vs. Habitat Control). Tissue-level mechanical cortical properties (i.e., hardness and modulus) were locally decreased by spaceflight, whereas the mineral characteristics and collagen maturity were unaffected. In the vertebrae, spaceflight decreased the overall bone volume and altered the modulus in the periphery of the trabecular struts. Despite normalized osteoclastic activity and an increased osteoblast number, bone recovery was not observed 8 days after landing. In conclusion, spaceflight induces osteocyte death, which may trigger bone resorption and result in bone mass and microstructural deterioration. Moreover, osteocyte cell death, lacunae mineralization and fatty marrow, which are hallmarks of ageing, may impede tissue maintenance and repair.

Nanoscale modifications in the early heating stages of bone are heterogeneous at the microstructural scale.

Nanoscale studies of bone provide key indicators to evidence subtle structural changes that may occur in the biomedical, forensic and archaeological contexts. One specific problem encountered in all those disciplines, for which the identification of nanostructural cues could prove useful, is to properly monitor the effect of heating on bone tissue. In particular, the mechanisms at work at the onset of heating are still relatively unclear. Using a multiscale approach combining Raman microspectroscopy, transmission electron microscopy (TEM), synchrotron quantitative scanning small-angle X-ray scattering imaging (qsSAXSI) and polarized light (PL) microscopy, we investigate the ultrastructure of cortical bovine bone heated at temperatures < 300°C, from the molecular to the macroscopic scale. We show that, despite limited changes in crystal structure, the mineral nanoparticles increase in thickness and become strongly disorganized upon heating. Furthermore, while the nanostructure in distinct anatomical quadrants appears to be statistically different, our results demonstrate this stems from the tissue histology, i.e. from the high degree of heterogeneity of the microstructure induced by the complex cellular processes involved in bone tissue formation. From this study, we conclude that the analysis of bone samples based on the structure and organization of the mineral nanocrystals requires performing measurements at the histological level, which is an advantageous feature of qsSAXSI. This is a critical aspect that extends to a much broader range of questions relating to nanoscale investigations of bone, which could also be extended to other classes of nanostructured heterogeneous materials.

Impaired rib bone mass and quality in end-stage cystic fibrosis patients.

BACKGROUND: Advancements in research and clinical care have considerably extended the life expectancy of cystic fibrosis (CF) patients. However, with this extended survival come comorbidities. One of the leading co-morbidities is CF-related bone disease (CFBD), which progresses with disease severity and places patients at high risk for fractures, particularly of the ribs and vertebrae. Evidence that CF patients with vertebral fractures had higher bone mineral density (BMD) than the nonfracture group led us to postulate that bone quality is impaired in these patients. We therefore examined rib specimens resected at the time of lung transplant in CF patients to measure parameters of bone quantity and quality. METHODS: In this exploratory study, we analysed 19 end-stage CF and 13 control rib specimens resected from otherwise healthy lung donors. BMD, bone microarchitecture, static parameters of bone formation and resorption and microcrack density of rib specimens were quantified by imaging, histomorphometric and histological methods. Variables reflecting the mineralization of ribs were assessed by digitized microradiography. The degree of bone mineralization (g/cm3) and the heterogeneity index of the mineralization (g/cm3) were calculated for trabecular and cortical bone. RESULTS: Compared to controls, CF ribs exhibited lower areal and trabecular volumetric BMD, decreased trabecular thickness and osteoid parameters, and increased microcrack density, that was particularly pronounced in specimens from patients with CF-related diabetes. Static parameters of bone resorption were similar in both groups. Degree of mineralization of total bone, but not heterogeneity index, was increased in CF specimens. CONCLUSION: The combination of reduced bone mass, altered microarchitecture, imbalanced bone remodeling (maintained bone resorption but decreased formation), increased microdamage and a small increase of the degree of mineralization, may lead to decreased bone strength, which, when coupled with chronic coughing and chest physical therapy, may provide an explanation for the increased incidence of rib fractures previously reported in this population.

Nonenzymatic Glycation and Degree of Mineralization Are Higher in Bone From Fractured Patients With Type 1 Diabetes Mellitus.

Low-energy fractures are frequent complications in type 1 diabetes mellitus patients (T1DM). Modifications of bone intrinsic composition might be a potential cause of fragility observed in diabetic subjects. Advanced glycation end products (AGEs) were found in numerous connective tissues from T1DM patients. However, whether AGEs are present at high levels in bone matrix from diabetic subjects is unknown. Moreover, whether elevated AGEs in the bone matrix impair mineralization has not been addressed in humans. The purposes of this study were 1) to determine whether bone matrix from fracturing and nonfracturing T1DM contained more AGEs than bone from healthy patients (CTL), and 2) to compare the degree of mineralization of bone and hardness between fracturing and nonfracturing T1DM versus CTL. We analyzed iliac crest bone biopsies from 5 fracturing T1DM patients, 5 nonfracturing T1DM patients, and 5 healthy subjects, all age- and sex-matched. AGEs (pentosidine) in bone matrix was measured by high-performance liquid chromatography separately in trabecular and cortical bone. The degree of mineralization of bone (DMB) was assessed by digitized microradiography, and mechanical properties by micro- and nanohardness tests. Trabecular bone from fracturing T1DM exhibited significantly higher levels of pentosidine than CTL (p = 0.04) and was more mineralized than nonfracturing T1DM (p = 0.04) and CTL (p = 0.04). Trabecular bone was not significantly different in pentosidine between nonfracturing T1DM and CTL. Cortical bone from nonfracturing T1DM was not significantly different from CTL. Positive correlations were found between HbA1c and pentosidine (r' = 0.79, p < 0.003) and between HbA1c and DMB (r' = 0.64, p < 0.02). Both modifications could lead to less flexible bone (reduced modulus of elasticity) and a tendency toward low-energy fractures in T1DM patients.