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.

Key topographic parameters driving surface adhesion of Porphyromonas gingivalis.

Dental implant failure is primarily due to peri-implantitis, a consequence of bacterial biofilm formation. Bacterial adhesion is strongly linked to micro-/nano-topographies of a surface; thus an assessment of surface texture parameters is essential to understand bacterial adhesion. In this study, mirror polished titanium samples (Ti6Al4V) were irradiated with a femtosecond laser (fs-L) at a wavelength of 1030 nm (infrared) with variable laser parameters (laser beam polarization, number, spacing and organization of the impacts). Images of 3-D topographies were obtained by focal variation microscopy and analyzed with MountainsMap software to measure surface parameters. From bacteria associated with peri-implantitis, we selected Porphyromonas gingivalis to evaluate its adhesion on Ti6Al4V surfaces in an in vitro study. Correlations between various surface parameters and P. gingivalis adhesion were investigated. We discovered that Sa value, a common measure of surface roughness, was not sufficient in describing the complexity of these fs-L treated surfaces and their bacterial interaction. We found that Sku, density and mean depths of the furrows, were the most accurate parameters for this purpose. These results provide important information that could help anticipate the bacterial adhesive properties of a surface based on its topographic parameters, thus the development of promising laser designed biofunctional implants.

COVID-19 Clusters in Belgian Nursing Homes: Impact of Facility Characteristics and Vaccination on Cluster Occurrence, Duration and Severity.

COVID-19 severely affected nursing home residents from March 2020 onwards in Belgium. This study aimed to model the impact of vaccination and facility characteristics on cluster occurrence, duration and severity in this setting. Possible clusters were identified between June 2020 and January 2022, based on the Belgian COVID-19 surveillance in nursing homes. Median attack rates (AR) among residents and staff, case hospitalization rates (CHR) and case fatality rates (CFR) were calculated. A negative binomial model was used to identify the association between nursing home characteristics and the number of cases, hospital admissions and deaths and the duration of the cluster. A total of 2239 clusters were detected in more than 80% of nursing homes. Most of these (62%) occurred before the start of COVID-19 vaccination (end of December 2020). After vaccination, the number of clusters, the AR among residents and staff, the CHR and the CFR dropped. Previous cluster(s) and vaccination decreased the number of cases, hospital admissions and deaths among residents. Previous cluster experience and having started vaccination were protective factors. We recommend continued implementation of targeted interventions such as vaccination, large-scale screening and immediate implementation of additional infection prevention and control measures.

Hindlimb unloading in C57BL/6J mice induces bone loss at thermoneutrality without change in osteocyte and lacuno-canalicular network.

Impaired mechanical stimuli during hindlimb unloading (HLU) are believed to exacerbate osteocyte paracrine regulation of osteoclasts. We hypothesized that bone loss and deterioration of the osteocyte lacuno-canalicular network are attenuated in HLU mice housed at thermoneutrality (28 °C) compared with those housed at ambient temperature (22 °C). Following acclimatization, 20-week-old male C57BL/6J mice were submitted to HLU or kept in pair-fed control cages (CONT), for 5 days (5d) or 14d, at 22 °C or 28 °C. In the femur distal metaphysis, thermoneutral CONT mice had higher bone volume (p = 0.0007, BV/TV, in vivo µCT, vs. 14dCONT22) whilst osteoclastic surfaces of CONT and HLU were greater at 22 °C (5dCONT22 + 53 %, 5dHLU22 + 50 %, 14dCONT22 + 186 %, 14dHLU22 + 104 %, vs matching 28 °C group). In the femur diaphysis and at both temperatures, 14dHLU exhibited thinner cortices distally or proximally compared to controls; the mid-diaphysis being thicker at 28 °C than at 22 °C in all groups. Expression of cortical genes for proteolytic enzyme (Mmp13), markers for osteoclastogenic differentiation (MCSF, RANKL), and activity (TRAP, Ctsk) were increased following 22 °C HLU, whereas only Ctsk expression was increased following 28 °C HLU. Expression of cortical genes for apoptosis, senescence, and autophagy were not elevated following HLU at any temperature. Osteocyte density at the posterior mid-diaphysis was similar between groups, as was the proportion of empty lacunae (<0.5 %). However, analysis of the lacuno-canalicular network (LCN, fluorescein staining) revealed unstained areas in the 14dHLU22 group only, suggesting disrupted LCN flow in this group alone. In conclusion, 28 °C housing influences the HLU bone response but does not prevent bone loss. Furthermore, our results do not show osteocyte senescence or death, and at thermoneutrality, HLU-induced bone resorption is not triggered by osteoclastic activators RANKL and MCSF.

YAP promotes cell-autonomous immune responses to tackle intracellular Staphylococcus aureus in vitro.

Transcriptional cofactors YAP/TAZ have recently been found to support autophagy and inflammation, which are part of cell-autonomous immunity and are critical in antibacterial defense. Here, we studied the role of YAP against Staphylococcus aureus using CRISPR/Cas9-mutated HEK293 cells and a primary cell-based organoid model. We found that S. aureus infection increases YAP transcriptional activity, which is required to reduce intracellular S. aureus replication. A 770-gene targeted transcriptomic analysis revealed that YAP upregulates genes involved in autophagy/lysosome and inflammation pathways in both infected and uninfected conditions. The YAP-TEAD transcriptional activity promotes autophagic flux and lysosomal acidification, which are then important for defense against intracellular S. aureus. Furthermore, the staphylococcal toxin C3 exoenzyme EDIN-B was found effective in preventing YAP-mediated cell-autonomous immune response. This study provides key insights on the anti-S. aureus activity of YAP, which could be conserved for defense against other intracellular bacteria.

Protective Effect on Bone of Nacre Supplementation in Ovariectomized Rats.

Nacre has emerged as a beneficial natural product for bone cells and tissues, but its effect was only studied by gavage in the ovariectomized mouse model. We sought to assess the antiosteoporotic effect of nacre through a nutritional supplementation in the ovariectomized rat model. Sixteen-week-old female Wistar rats were either Sham-operated or bilateral ovariectomized (OVX) and then fed with standard diet (Sham and OVX groups) or standard diet supplemented with either 0.25% CaCO3 or nacre (OVX CaCO3 and OVX Nacre group, respectively) for 28 days (n = 10/group). The bone microarchitecture was assessed at appendicular and axial bones by micro-computed tomography (µCT). Histomorphometric analysis was performed to determine cellular and dynamic bone parameters. Bone metabolism was also evaluated by biochemical markers and gene expression levels. Nacre-based diet prevented the OVX-induced bone loss better than that of the CaCO3 supplement, given the significant changes in trabecular bone volume fraction (BV/TV) both at the femoral distal metaphysis (difference, 35%; p = 0.004) and at the second lumbar spine (difference, 11%; p = 0.01). Trabecular osteoclast surfaces (Oc.S/BS) were also 1.5-fold lower at the tibial proximal metaphysis in OVX Nacre group compared with OVX CaCO3 group (p = 0.02). By principal component analysis (PCA), OVX Nacre group formed a cluster away from OVX group and with a trend closest to Sham group. These data were consistent with biological measurements demonstrating a positive profile related to nacre supplementation, which blunted an increase in serum CTX level and enhanced serum P1NP secretion 14 days post-OVX compared with CaCO3 supplementation. Bmp2 mRNA expression in OVX Nacre group was +1.76-fold (p = 0.004) and +1.30-fold (p = 0.20) compared with OVX and OVX CaCO3 groups, respectively. We conclude that supplementation with nacre could effectively limit bone loss induced by estrogen deficiency just after OVX in rats by modulating the negative imbalance of bone turnover. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

YAP Transcriptional Activity Dictates Cell Response to TNF In Vitro.

YAP/TAZ are transcription co-factors recently described responsive to pro-inflammatory cytokines and involved in inflammatory-related disorders. However, the role of tumor necrosis factor (TNF), a major pro-inflammatory cytokine, on YAP signaling is not well understood and controversial. Here, we observe in vitro, using wild type and YAP knockout HEK293 cells, that TNF triggers YAP nuclear translocation and transcriptional activity, thus being dependent on Rho family of GTPases. In response to TNF, YAP transcriptional activity orientates cell fate toward survival. Transcriptional analysis with Nanostring technology reveals that YAP modulates TNF-induced increase in fibro-inflammatory pathways such as NF-κB, inflammasomes, cytokines or chemokines signaling and pro-fibrotic pathways involving TGF-ß and extracellular matrix remodeling. Therefore, in response to TNF, YAP acts as a sustainer of the inflammatory response and as a molecular link between inflammation and fibrotic processes. This work identifies that YAP is critical to drive several biological effects of TNF which are involved in cancer and inflammatory disorders.

YAP/TAZ: Key Players for Rheumatoid Arthritis Severity by Driving Fibroblast Like Synoviocytes Phenotype and Fibro-Inflammatory Response.

Objective: The role of YAP/TAZ, two transcriptional co-activators involved in several cancers, was investigated in rheumatoid arthritis (RA). Methods: Fibroblast like synoviocytes (FLS) from patients with RA or osteoarthritis were cultured in 2D or into 3D synovial organoids. Arthritis rat model (n=28) and colitis mouse model (n=21) were used. YAP/TAZ transcriptional activity was inhibited by verteporfin (VP). Multiple techniques were used to assess gene and/or protein expression and/or localization, cell phenotype (invasion, proliferation, apoptosis), bone erosion, and synovial stiffness. Results: YAP/TAZ were transcriptionally active in arthritis (19-fold increase for CTGF expression, a YAP target gene, in RA vs. OA organoids; p<0.05). Stiff support of culture or pro-inflammatory cytokines further enhanced YAP/TAZ transcriptional activity in RA FLS. Inhibiting YAP/TAZ transcriptional activity with VP restored a common phenotype in RA FLS with a decrease in apoptosis resistance, proliferation, invasion, and inflammatory response. Consequently, VP blunted hyperplasic lining layer formation in RA synovial organoids. In vivo, VP treatment strongly reduced arthritis severity (mean arthritic index at 3.1 in arthritic group vs. 2.0 in VP treated group; p<0.01) by restoring synovial homeostasis and decreasing systemic inflammation. YAP/TAZ transcriptional activity also enhanced synovial membrane stiffening in vivo, thus creating a vicious loop with the maintenance of YAP/TAZ activation over time in FLS. YAP/TAZ inhibition was also effective in another inflammatory model of mouse colitis. Conclusion: Our work reveals that YAP/TAZ were critical factors during arthritis. Thus, their transcriptional inhibition could be relevant to treat inflammatory related diseases.

Therapeutic Potential of Anti-Interferon α Vaccination on SjS-Related Features in the MRL/lpr Autoimmune Mouse Model.

Sjögren's syndrome (SjS) is a frequent systemic autoimmune disease responsible for a major decrease in patients' quality of life, potentially leading to life-threatening conditions while facing an unmet therapeutic need. Hence, we assessed the immunogenicity, efficacy, and tolerance of IFN-Kinoid (IFN-K), an anti-IFNα vaccination strategy, in a well-known mouse model of systemic autoimmunity with SjS-like features: MRL/MpJ-Faslpr/lpr (MRL/lpr) mice. Two cohorts (with ISA51 or SWE01 as adjuvants) of 26 female MRL/lpr were divided in parallel groups, "controls" (not treated, PBS and Keyhole Limpet Hemocyanin [KLH] groups) or "IFN-K" and followed up for 122 days. Eight-week-old mice received intra-muscular injections (days 0, 7, 28, 56 and 84) of PBS, KLH or IFN-K, emulsified in the appropriate adjuvant, and blood samples were serially collected. At sacrifice, surviving mice were euthanized and their organs were harvested for histopathological analysis (focus score in salivary/lacrimal glands) and IFN signature evaluation. SjS-like features were monitored. IFN-K induced a disease-modifying polyclonal anti-IFNα antibody response in all treated mice with high IFNα neutralization capacities, type 1 IFN signature's reduction and disease features' (ocular and oral sicca syndrome, neuropathy, focus score, glandular production of BAFF) improvement, as reflected by the decrease in Murine Sjögren's Syndrome Disease Activity Index (MuSSDAI) modelled on EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI). No adverse effects were observed. We herein report on the strong efficacy of an innovative anti-IFNα vaccination strategy in a mouse model of SjS, paving the way for further clinical development (a phase IIb trial has just been completed in systemic lupus erythematosus with promising results).

Parathyroid Hormone Remodels Bone Transitional Vessels and the Leptin Receptor-Positive Pericyte Network in Mice.

Intermittent parathyroid hormone (iPTH) is anti-osteoporotic and affects bone vessels. Transitional capillaries close to the bone surface, which express both endomucin (Edm) and CD31, bear leptin receptor-expressing (LepR) perivascular cells that may differentiate into osteoblasts. Increased numbers of type H endothelial cells (THEC; ie, Edmhi /CD31hi cells assessed by flow cytometry, FACS) are associated with higher bone formation in young mice. We hypothesized that iPTH administration impacts transitional vessels by expanding THECs. Four-month-old C57/Bl6J female mice were injected with PTH 1-84 (100 µg/kg/d) or saline (CT) for 7 or 14 days. We quantified LepR+ , CD31+ , Edm+ cells and THECs by FACS in hindlimb bone marrow, and Edm/LepR double immunolabelings on tibia cryosections. Additionally, we analyzed bone mRNA expression of 87 angiogenesis-related genes in mice treated with either intermittent or continuous PTH (iPTH/cPTH) or saline (CT) for 7, 14, and 28 days. iPTH dramatically decreased the percentage of THECs by 78% and 90% at days 7 and 14, respectively, and of LepR+ cells at day 14 (-46%) versus CT. Immunolabeling quantification showed that the intracortical Edm+ -vessel density increased at day 14 under iPTH. In the bone marrow, perivascular LepR+ cells, connected to each other via a dendrite network, were sparser under iPTH at day 14 (-58%) versus CT. iPTH decreased LepR+ cell coverage of transitional vessels only (-51%), whereas the number of LepR+ cells not attached to vessels increased in the endocortical area only (+ 49%). Transcriptomic analyses showed that iPTH consistently upregulated PEDF, Collagen-18α1, and TIMP-1 mRNA expression compared with CT and cPTH. Finally, iPTH increased immunolabeling of endostatin, a Collagen-18 domain that can be cleaved and become antiangiogenic, in both endocortical (79%) and peritrabecular transitional microvessels at day 14. Our results show that iPTH specifically remodels transitional vessels and suggest that it promotes LepR+ cell mobilization from these vessels close to the bone surface. © 2019 American Society for Bone and Mineral Research.

Early sclerostin expression explains bone formation inhibition before arthritis onset in the rat adjuvant-induced arthritis model.

Periarticular bone loss in rheumatoid arthritis (RA) is considered to be mainly related to synovial inflammation. However, strong bone loss has also described at the time of arthritis onset. Recently, a paradoxical exacerbation of joint damage was described when blocking sclerostin in various arthritis models. Thus, we aimed to determine kinetics of bone loss and its mechanisms in the adjuvant induced arthritis (AIA) rat model of RA. AIA was induced (n = 35) or not (n = 35) at day 0. In addition to well-known arthritis at day 12, we showed with 3D-imaging and histomorphometry that bone microstructural alterations occurred early from day 8 post-induction, characterized by cortical porosity and trabecular bone loss. Active osteoclastic surfaces were increased from day 8 with RANKL upregulation. More surprisingly SOST and DKK1 were overexpressed from day 6 and followed by a dramatic decrease in bone formation from day 8. At the time of arthritis onset, SOST and DKK1 returned to control values, but frizzled related protein 1 (SFRP1), proinflammatory cytokines, and MMPs started to increase. Bone alterations before arthritis onset reinforce the hypothesis of an early bone involvement in arthritis. Kinetics of osteocyte markers expression should be considered to refine Wnt inhibitor treatment strategies.

Effects of short-term dry immersion on bone remodeling markers, insulin and adipokines.

BACKGROUND: Dry immersion (DI), a ground-based model of microgravity previously used in Russia, has been recently implemented in France. The aim of this study was to analyze early events in a short-term DI model in which all conditions are met to investigate who is first challenged from osteo- or adipo-kines and to what extent they are associated to insulin-regulating hormones. METHODS: Twelve healthy men were submitted to a 3-day DI. Fasting blood was collected during pre-immersion phase for the determination of the baseline data collection (BDC), daily during DI (DI24h, DI48H and DI72h), then after recovery (R+3h and R+24h). Markers of bone turnover, phosphocalcic metabolism, adipokines and associated factors were measured. RESULTS: Bone resorption as assessed by tartrate-resistant acid phosphatase isoform 5b and N-terminal crosslinked telopeptide of type I collagen levels increased as early as DI24h. At the same time, total procollagen type I N- and C-terminal propeptides and osteoprotegerin, representing bone formation markers, decreased. Total osteocalcin [OC] was unaffected, but its undercarboxylated form [Glu-OC] increased from DI24h to R+3h. The early and progressive increase in bone alkaline phosphatase activities suggested an increased mineralization. Dickkopf-1 and sclerostin, as negative regulators of the Wnt-ß catenin pathway, were unaltered. No change was observed either in phosphocalcic homeostasis (calcium and phosphate serum levels, 25-hydroxyvitamin D, fibroblast growth factor 23 [FGF23]) or in inflammatory response. Adiponectemia was unchanged, whereas circulating leptin concentrations increased. Neutrophil gelatinase-associated lipocalin [lipocalin-2], a potential regulator of bone homeostasis, was found elevated by 16% at R+3h compared to DI24h. The secretory form of nicotinamide phosphoribosyl-transferase [visfatin] concentrations almost doubled after one day of DI and remained elevated. Serum insulin-like growth factor 1 levels progressively increased. Fasting insulin concentrations increased during the entire DI, whereas fasting glucose levels tended to be higher only at DI24h and then returned to BDC values. Changes in bone resorption parameters negatively correlated with changes in bone formation parameters. Percent changes of ultra-sensitive C-reactive protein positively correlated with changes in osteopontin, lipocalin-2 and fasting glucose. Furthermore, a positive correlation was found between changes in FGF23 and Glu-OC, the two main osteoblast-/osteocyte-derived hormones. CONCLUSION: Our results demonstrated that DI induced an unbalanced remodeling activity and the onset of insulin resistance. This metabolic adaptation was concomitant with higher levels of Glu-OC. This finding confirms the role of bone as an endocrine organ in humans. Furthermore, visfatin for which a great responsiveness was observed could represent an early and sensitive marker of unloading in humans.

Macrotopographic closure promotes tissue growth and osteogenesis in vitro.

While the impact of substrate topographies at nano- and microscale on bone cell behavior has been particularly well documented, very few studies have analyzed the role of substrate closure at a tissular level. Moreover, these have focused on matrix deposition rather than on osteoblastic differentiation. In the present work, mouse calvaria cells were grown for 15days on hydroxyapatite (HA) ceramics textured with three different macrogrooves shapes (**100µm): 1 sine and 2 triangle waveforms. We found that macrotopography favors cell attachment, and that bone-like tissue growth and organization are promoted by a tight "closure angle" of the substrate geometry. Interestingly, while Flat HA controls showed little marker expression at the end of the culture, cells grown on macrogrooves, and in particular the most closed (triangle waveform with a 517µm spatial period) showed a fast time-course of osteoblast differentiation, reaching high levels of gene and protein expression of osteocalcin and sclerostin, a marker of osteocytes. STATEMENT OF SIGNIFICANCE: Many in vitro studies have been conducted on topography at nano and microscale, fewer have focused on the influence of macrotopography on osteoblasts. Ceramics with a controlled architecture were obtained throught a 3D printing process and used to assess osteoblast behavior. Biocompatible, they allowed the long-terme survival of osteoblast cells and the laying of an important bone matrix. V-shaped grooves were found to accelerates osteoblast differentiation and promote bone-like tissue deposition and maturation (osteocyte formation), proportionately to angle closure. Such macrostructures are attractive for the design of innovative implants for bone tissue engineering and in vitro models of osteogenesis.

Bone Shaft Revascularization After Marrow Ablation Is Dramatically Accelerated in BSP-/- Mice, Along With Faster Hematopoietic Recolonization.

The bone organ integrates the activity of bone tissue, bone marrow, and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP+/+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (×4) and vascular volume (×7) than BSP+/+. Vessel numbers in the shaft of ablated BSP+/+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin- (×3) as well as LSK (Lin- IL-7Rα- Sca-1hi c-Kithi , ×2) and hematopoietic stem cells (HSC: Flt3- LSK, ×2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin- was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants. In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. J. Cell. Physiol. 232: 2528-2537, 2017. © 2016 Wiley Periodicals, Inc.

Fat and Sucrose Intake Induces Obesity-Related Bone Metabolism Disturbances: Kinetic and Reversibility Studies in Growing and Adult Rats.

Metabolic and bone effects were investigated in growing (G, n = 45) and mature (M, n = 45) rats fed a high-fat/high-sucrose diet (HFS) isocaloric to the chow diet of controls (C, n = 30 per group). At week 19, a subset of 15 rats in each group (HFS or C, at both ages) was analyzed. Then one-half of the remaining 30 HFS rats in each groups continued HFS and one-half were shifted to C until week 27. Although no serum or bone marrow inflammation was seen, HFS increased visceral fat, serum leptin and insulin at week 19 and induced further alterations in lipid profile, serum adiponectin, and TGFß1, TIMP1, MMP2, and MMP9, suggesting a prediabetic phenotype and cardiovascular dysfunction at week 27 more pronounced in M than G. These events were associated with dramatic reduction of osteoclastic and osteoid surfaces with accelerated mineralizing surfaces in both HFS age groups. Mineral metabolism and its major regulators were disturbed, leading to hyperphosphatemia and hypocalcemia. These changes were associated with bone alterations in the weight-bearing tibia, not in the non-weight-bearing vertebra. Indeed in fat rats, tibia trabecular bone accrual increased in G whereas loss of trabecular bone in M was alleviated. At diaphysis cortical porosity increased in G and even more in M at week 27. After the diet switch, metabolic and bone cellular disturbances fully reversed in G, but not in M. Trabecular benefit of the obese was preserved in both age groups and in M the age-related bone loss was even lighter after the diet switch than in prolonged HFS. At the diaphysis, cortical porosity normalized in G but not in M. Hypocalcemia in G and M was irreversible. Thus, the mild metabolic syndrome induced by isocaloric HFS is able to alter bone cellular activities and mineral metabolism, reinforce trabecular bone, and affect cortical bone porosity in an irreversible manner in older rats.

The impairment of osteogenesis in bone sialoprotein (BSP) knockout calvaria cell cultures is cell density dependent.

Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors.

Blocking the expression of both bone sialoprotein (BSP) and osteopontin (OPN) impairs the anabolic action of PTH in mouse calvaria bone.

Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1-84 (iPTH) over the calvaria of BSP knockout (-/-) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and -/- mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from -/- mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5× higher in -/- versus +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in -/- cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP +/+ calvaria, while it blunted iPTH effects in -/- mice, reduced to a modest increase in periosteum thickness. In -/- (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins.

Absence of bone sialoprotein (BSP) alters profoundly hematopoiesis and upregulates osteopontin.

Matrix proteins of the SIBLING family interact with bone cells, extracellular matrix and mineral and are thus in a key position to regulate the microenvironment of the bone tissue, including its hematopoietic component. In this respect, osteopontin (OPN) has been implicated in the hematopoietic stem cell (HSC) niche as negative regulator of the HSC function. We investigated the impact on hematopoietic regulation of the absence of the cognate bone sialoprotein (BSP). BSP knockout (-/-) mice display increased bone marrow cellularity, and an altered commitment of hematopoietic precursors to myeloid lineages, leading in particular to an increased frequency of monocyte/macrophage cells. The B cell pool is increased in -/- bone marrow, and its composition is shifted toward more mature lymphocyte stages. BSP-null mice display a decreased HSC fraction among LSK cells and a higher percentage of more committed progenitors as compared to +/+. The fraction of proliferating LSK progenitors is higher in -/- mice, and after PTH treatment the mutant HSC pool is lower than in +/+. Strikingly, circulating levels of OPN as well as its expression in the bone tissue are much higher in the -/-. Thus, a BSP-null bone microenvironment affects the hematopoietic system both quantitatively and qualitatively, in a manner in part opposite to the OPN knockout, suggesting that the effects might in part reflect the higher OPN expression in the absence of BSP.

Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and progressive establishment of high trabecular bone mass.

Adult Ibsp-knockout mice (BSP-/-) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice, while impairing primary mineralization.

Intermittent PTH(1-84) is osteoanabolic but not osteoangiogenic and relocates bone marrow blood vessels closer to bone-forming sites.

Intermittent parathyroid hormone (PTH) is anabolic for bone. Our aims were to determine (1) whether PTH stimulates bone angiogenesis and (2) whether vascular endothelial growth factor (VEGF A) mediates PTH-induced bone accrual. Male Wistar rats were given PTH(1-84) daily, and trabecular bone mass increased 150% and 92% after 30 and 15 days, respectively. The vascular system was contrasted to image and quantify bone vessels with synchrotron radiation microtomography and histology. Surprisingly, bone vessel number was reduced by approximately 25% and approximately 40% on days 30 and 15, respectively. PTH redistributed the smaller vessels closer to bone-formation sites. VEGF A mRNA expression in bone was increased 2 and 6 hours after a single dose of PTH and returned to baseline by 24 hours. Moreover, anti-VEGF antibody administration (1) blunted the PTH-induced increase in bone mass and remodeling parameters, (2) prevented the relocation of bone vessels closer to bone-forming sites, and (3) inhibited the PTH-induced increase in mRNA of neuropilin 1 and 2, two VEGF coreceptors associated with vascular development and function. In conclusion, PTH(1-84) is osteoanabolic through VEGF-related mechanism(s). Further, PTH spatially relocates blood vessels closer to sites of new bone formation, which may provide a microenvironment favorable for growth.