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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.

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.

Synovitis is usually observed before loss of articular function in rheumatoid arthritis (RA). In addition to the synovium and according to the "Inside-Outside" theory, bone compartment is also involved in RA pathogenesis. Then, we investigated time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant-induced arthritis (AIA) model. Lewis female rats were longitudinally monitored from arthritis induction (day 0), with early (day 10) and late (day 17) steps. Trabecular and cortical microarchitecture parameters of four ankle bones were assessed by microcomputed tomography. Gene expression was determined at sacrifice. Arthritis occurred at day 10 in AIA rats. At this time, bone erosions were detected on four ankle bones, with cortical porosity increase (+67%) and trabecular alterations including bone volume fraction (BV/TV: -13%), and trabecular thickness decrease. Navicular bone assessment was the most reproducible and sensitive. Furthermore, strong correlations were observed between bone alterations at day 10 and arthritis severity or bone loss at day 17, including predictability of day 10 BV/TV to day 17 articular index (R2 = 0.76). Finally, gene expression at day 17 confirmed massive osteoclast activation and interestingly provided insights on strong activation of bone formation inhibitor markers at the joint level. In rat AIA, bone loss was already observed at synovitis onset and was predicted late arthritis severity. Our results reinforced the key role of subchondral bone in arthritis pathogenesis, in favour to the "Inside-Outside" theory. Mechanisms of bone loss in rat AIA involved resorption activation and formation inhibition changes. J. Cell. Physiol. 232: 1318-1325, 2017. © 2016 Wiley Periodicals, Inc.

Microgravity-related cytoskeletal disorganization is associated with an altered balance between osteoblastogenesis and adipogenesis of multipotent cells. Strontium chloride is known to increase osteoblastogenesis and repress adipogenesis, but its effects in microgravity-related conditions have not been established. Our goal was to investigate early events in this process, focusing on RhoGTPases as controllers of cytoskeletal organization leading to stem cell commitment. We cultivated C3H10T1/2 on microspheres using a rotating wall vessel bioreactor (NASA) in order to simulate microgravity-related conditions in adipogenesis and osteoblastogenesis conditions independently. We observed that rotating wall vessel cultures presented increased adipogenesis, while osteoblastogenesis was reduced. Strontium-treated multipotent cells presented a significant repression in adipogenesis (−90 %, p, Countering Bone Loss in Space A chemical element naturally found for instance in seafood or grains, could counter bone loss from long-term spaceflight. Alain Guignandon and colleagues from the Université de Lyon à St-Etienne in France exposed multipotent embryonic fibroblasts to microgravity conditions similar to those found in space. They found the balance shifted in these stem cells from differentiating to bone-forming cells (osteoblasts) to differentiating to fatty-tissue forming cells (adipocytes). When the cells were treated with strontium, the shift toward osteoblastogenesis was regained. Strontium achieves this by sustaining the activity of two proteins that play a role in bone development but are suppressed in space. Strontium’s effect on the proteins could happen via release of vascular endothelial growth factor, which, under normal gravity conditions, plays a role in committing the cell to differentiation into osteoblasts rather than adipoyctes.

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-β 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.

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. J. Cell. Physiol. 230: 568–577, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

The effects of 4 weeks of endurance training in conditions of normoxia or hypoxia on muscle characteristics and blood lactate responses after a 5-min constant-load exercise (CLE) at 90% of the power corresponding to the maximal oxygen uptake were examined at sea-level in 13 sedentary subjects. Five subjects trained in normobaric hypoxia (HT group, fraction of oxygen in inspired gas = 13.2%), and eight subjects trained in normoxia at the same relative work rates (NT group). The blood lactate recovery curves from the CLE were fitted to a biexponential time function: La(t) = La(0) + A1(1 - e- gamma 1.t) + A2(1 - e- gamma 2.t), where the velocity constants gamma 1 and gamma 2 denote the lactate exchange and removal abilities, respectively, A1 and A2 are concentration parameters that describe the amplitudes of concentration variations in the space represented by the arterial blood, La(t) is the lactate concentration at time t, and La(0) is the lactate concentration at the beginning of recovery from CLE. Before training, the two groups displayed the same muscle characteristics, blood lactate kinetics after CLE, and gamma 1 and gamma 2 values. Training modified their muscle characteristics, blood lactate kinetics and the parameters of the fits in the same direction, and proportions among the HT and the NT subjects. Endurance training increased significantly the capillary density (by 31%), citrate synthase activity (by 48%) and H isozyme proportion of lactate dehydrogenase (by 24%), and gamma 1 (by 68%) and gamma 2 (by 47%) values. It was concluded that (1) endurance training improves the lactate exchange and removal abilities estimated during recovery from exercises performed at the same relative work rate, and (2) training in normobaric hypoxia results in similar effects on lactate exchange and removal abilities to training in normoxia performed at the same relative work rates. These results, which were obtained non-invasively in vivo in humans during recovery from CLE, are comparable to those obtained in vitro or by invasive methods during exercise and subsequent recovery.

Bone matrix, mainly composed of type I collagen and apatite, is constantly modified during the bone remodeling process, which exposes bone cells to various proportions of mineralized collagen within bone structural units. Collagen-mineralized substrates have been shown to increase osteoblast activities. We hypothesized that such effects may be explained by a rapid secretion of specific growth factors and/or deposition of specific matrix proteins. Using MC3T3-E1 seeded for 32 h on collagen substrates complexed with various apatite contents, we found that pre-osteoblasts in contact with mineralized collagen gave rise to a dose-dependent deposit of Vascular Endothelial Growth Factor-A (VEGF-A) and RGD-containing proteins such as osteopontin (OPN) and fibronectin (FN). This RGD-matrix deposition reinforced the cell adhesion to collagen-mineralized substrates. It was also observed that, on these substrates, this matrix was elaborated concomitantly to an increased cell migration, allowing a homogeneous coverage of the sample. This particular surface activation was probably done firstly to reinforce cell survival (VEGF-A) and adhesion (OPN, FN) and secondly to recruit and prepare surfaces for subsequent bone cell activity.

Since VEGF-A is involved in mechanically induced bone gain and because vegf exists under 6 isoforms exerting various biological effects, we studied vegf isoform expression and VEGF protein production in osteoblastic cells (rat Ros17/2.8 and human osteoblasts) submitted to 4 mechanical regimens. Mechanical regimens (1% stretch deformation) were designed with a fixed number of cycles (450) delivered at various frequencies (0.05 to 5 Hz). We found a negative correlation (R(2)=0.76, p