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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.

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-1 hi c-Kit hi , ×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., (© 2016 Wiley Periodicals, Inc.)

Little is known about middle and inner ear development during the second and third parts of human fetal life. Using ultra-high resolution Microcomputed Tomography coupled with bone histology, we performed the first quantitative middle and inner ear ossification/mineralization evaluation of fetuses between 17 and 39 weeks of gestational age. We show distinct ossification paces between ossicles, with a belated development of the stapes. A complete cochlear bony covering is observed within the time-frame of the onset of hearing, whereas distinct time courses of ossification for semicircular canal envelopes are observed in relation to the start of vestibular functions. The study evidences a spatio-temporal relationship between middle and inner ear structure development and the onset of hearing and balance, critical senses for the fetal adaptation to birth.

Syndesmophyte occurrence and axial bone loss were investigated in the heterozygous Tg187 tumor necrosis factor (TNF) transgenic mouse model (Tg-huTNF) of arthritis. Female and male Tg-huTNF mice were compared to wild-type mice (WT) at 2, 4, 6, 8, and 10 weeks. Syndesmophytes, intervertebral disc space, osteoclasts, osteoid surface, and vertebra microarchitecture were assessed by histomorphometry and microcomputed tomography. No spontaneous syndesmophyte formation was detected in Tg-huTNF compared to WT mice. However, increased porosity was observed mainly in peridiscal lumbar vertebra. Accordingly, bone microarchitecture parameters were altered in Tg-huTNF mice, with decrease in bone volume fraction, and trabecular number and thickness after 6 weeks compared to WT (p < 0.05). Osteoclast count and surface were increased (p < 0.01). Moreover, the non-mineralized (osteoid) surface was also increased in Tg-huTNF after 6 weeks (p < 0.01). Despite increased osteoclast and osteoid surfaces, an imbalance between both was observed in favour of osteoid surface at the early phase and then to osteoclast surface. These results demonstrated an axial bone loss in the Tg-huTNF model, additional to the common limb arthritis, related to overexpression of TNF. However, the absence of syndesmophyte and the increase of osteoid surface suggested that chronic inflammation might block bone mineralisation. Finally, the relative increased osteoid surface was not enough to compensate the high osteoclast activity.

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., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Surface improvement of implants is essential for achieving a fast osseo-integration. Technically, the creation of a precise pattern on a titanium alloy surface is challenging. Here, the femtosecond laser was chosen as an innovative technology for texturing with accuracy a nano-micro topography. By adjusting the laser parameters, three biomimetic textures were fabricated on the titanium surface: micropits with nano-ripples in the pits, micropits with nano-ripples around the pits, and a texture with only nano-ripples. Mesenchymal stem cells (MSCs, C3H10T1/2) grown on these surfaces displayed altered morphometric parameters, and modified their focal adhesions in term of number, size, and distribution depending on surface type. These results indicate that the MSCs perceived subtle differences in topography. Dynamic analyses of early cellular events showed a higher speed of spreading on all the textured surfaces as opposed to the polished titanium. Concerning commitment, all the laser-treated surfaces strongly inhibited the expression of adipogenic-related genes (PPARϒ2, C/EBPα) and up-regulated the expression of osteoblastic-related genes (RUNX2, osteocalcin). Interestingly, the combination of micropits to nano-ripples enhanced their osteogenic potential as seen by a twofold increase in osteocalcin mRNA. Alkaline phosphatase activity was increased on all the textured surfaces, and lipid production was down-regulated. The functionalization of metallic surfaces by this high-resolution process will help us understand the MSCs' interactions with substrates for the development of textured implants with predictable tissue integrative properties.

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., (© 2014 Wiley Periodicals, Inc., A Wiley Company.)

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.

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.

Bone sialoprotein (BSP) is highly expressed in early bone deposition and may play a part in primary bone mineralization. We previously showed that while BSP−/− mice have a mild secondary bone phenotype and are responsive to mechanical (unloading) and hormonal (ovariectomy, parathyroid hormone (PTH)) challenges, repair of a cortical bone defect, which involves primary bone deposition is significantly delayed in these mice. In the present study, we investigated the role of BSP in a pure model of primary bone modeling. Bone marrow was ablated by trans-epiphysis aspiration in the femora of BSP+/+ and BSP−/− mice, and 7 days post surgery μCT analysis showed vigorous new bone formation in the shaft of BSP+/+ animals but much less in BSP−/− mice. After 14 days, the volume of medullary bone was significantly decreased as expected in BSP+/+ mice, while it remained stable in the BSP−/−. Osteoid thickness and surface were higher in BSP−/− at day 7, suggesting delayed mineralization, while osteoclast surface and number were significantly lower at day 14, a stage of high medullary bone resorption. At day 7, mRNA expression of early osteoblast marker genes (RUNX2, osterix, alkaline phosphatase, osteopontin) did not differ between the two genotypes, while markers of terminal differentiation (MEPE, DMP1, osteocalcin) as well as receptor activator of NF-kappaB ligand (RANKL) and tartrate-resistant acid phosphatase (TRAP) were significantly lower in BSP−/− than in BSP+/+ mice. PTH treatment maintained the volume of medullary bone up to 12 days after ablation in BSP+/+ mice, but failed to do so in BSP−/− mice. PTH significantly increased bone formation rate in both genotype, while it reduced osteoclast number and surface in BSP+/+, but not in BSP−/− medullary bone. In summary, medullary bone formation after marrow ablation is blunted in BSP−/− mice, with delayed resorption and impaired response to PTH. These findings confirm the hypothesis of a crucial role for BSP in primary ossification, which has long been suspected for mineralization, but here extends to bone deposition and turnover.

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., (Copyright © 2011 American Society for Bone and Mineral Research.)

Lysophosphatidic acid (LPA) is a lipid mediator that acts in paracrine systems via interaction with a subset of G protein-coupled receptors (GPCRs). LPA promotes cell growth and differentiation, and has been shown to be implicated in a variety of developmental and pathophysiological processes. At least 6 LPA GPCRs have been identified to date: LPA1-LPA6. Several studies have suggested that local production of LPA by tissues and cells contributes to paracrine regulation, and a complex interplay between LPA and its receptors, LPA1 and LPA4, is believed to be involved in the regulation of bone cell activity. In particular, LPA1 may activate both osteoblasts and osteoclasts. However, its role has not as yet been examined with regard to the overall status of bone in vivo. We attempted to clarify this role by defining the bone phenotype of LPA1((-/-)) mice. These mice demonstrated significant bone defects and low bone mass, indicating that LPA1 plays an important role in osteogenesis. The LPA1((-/-)) mice also presented growth and sternal and costal abnormalities, which highlights the specific roles of LPA1 during bone development. Microcomputed tomography and histological analysis demonstrated osteoporosis in the trabecular and cortical bone of LPA1((-/-)) mice. Finally, bone marrow mesenchymal progenitors from these mice displayed decreased osteoblastic differentiation. These results suggest that LPA1 strongly influences bone development both qualitatively and quantitatively and that, in vivo, its absence results in decreased osteogenesis with no clear modification of osteoclasis. They open perspectives for a better understanding of the role of the LPA/LPA1 paracrine pathway in bone pathophysiology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bone sialoprotein (BSP) and osteopontin (OPN) belong to the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members interact with bone cells and bone mineral. Previously, we showed that BSP knockout (BSP(-/-) ) mice have a higher bone mass than wild type (BSP(+/+) ) littermates, with very low bone-formation activity and reduced osteoclast surfaces and numbers. Here we report that approximately twofold fewer tartrate-resistant acid phosphatase (TRACP)-positive cells and approximately fourfold fewer osteoclasts form in BSP(-/-) compared with BSP(+/+) spleen cell cultures. BSP(-/-) preosteoclast cultures display impaired proliferation and enhanced apoptosis. Addition of RGD-containing proteins restores osteoclast number in BSP(-/-) cultures to BSP(+/+) levels. The expression of osteoclast-associated genes is markedly altered in BSP(-/-) osteoclasts, with reduced expression of cell adhesion and migration genes (αV integrin chain and OPN) and increased expression of resorptive enzymes (TRACP and cathepsin K). The migration of preosteoclasts and mature osteoclasts is impaired in the absence of BSP, but resorption pit assays on dentine slices show no significant difference in pit numbers between BSP(+/+) and BSP(-/-) osteoclasts. However, resorption of mineral-coated slides by BSP(-/-) osteoclasts is markedly impaired but is fully restored by coating the mineral substrate with hrBSP and partly restored by hrOPN coating. In conclusion, lack of BSP affects both osteoclast formation and activity, which is in accordance with in vivo findings. Our results also suggest at least some functional redundancy between BSP and OPN that remains to be clarified., (Copyright © 2010 American Society for Bone and Mineral Research.)

Bone sialoprotein (BSP) and osteopontin (OPN) are both highly expressed in bone, but their functional specificities are unknown. OPN knockout (-/-) mice do not lose bone in a model of hindlimb disuse (tail suspension), showing the importance of OPN in bone remodeling. We report that BSP(-/-) mice are viable and breed normally, but their weight and size are lower than wild-type (WT) mice. Bone is undermineralized in fetuses and young adults, but not in older (> or =12 mo) BSP(-/-) mice. At 4 mo, BSP(-/-) mice display thinner cortical bones than WT, but greater trabecular bone volume with very low bone formation rate, which indicates reduced resorption, as confirmed by lower osteoclast surfaces. Although the frequency of total colonies and committed osteoblast colonies is the same, fewer mineralized colonies expressing decreased levels of osteoblast markers form in BSP(-/-) versus WT bone marrow stromal cultures. BSP(-/-) hematopoietic progenitors form fewer osteoclasts, but their resorptive activity on dentin is normal. Tail-suspended BSP(-/-) mice lose bone in hindlimbs, as expected. In conclusion, BSP deficiency impairs bone growth and mineralization, concomitant with dramatically reduced bone formation. It does not, however, prevent the bone loss resulting from loss of mechanical stimulation, a phenotype that is clearly different from OPN(-/-) mice.

Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Young's modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.

Aims: Beta2 adrenergic agonists are widely used in therapeutics and as doping agents by athletes. However, their effects on bone tissue, especially bone microarchitecture, remain poorly understood. Using three-dimensional (3D) microtomography, dual-energy X-ray absorptiometry, biomechanical testing and enzyme-linked immunosorbent assay, we evaluated the effects of two beta2 agonists, clenbuterol and salbutamol, on bone in growing rats., Methods: Twelve-week-old Wistar female rats (N = 39), divided in 3 groups, received during 6 weeks either salbutamol (4 mg/kg/day), clenbuterol (2 mg/kg/day) or normal saline (0.5 ml/kg/day) by subcutaneous injections., Results: After 6 weeks, the salbutamol and clenbuterol groups displayed lower bone mineral content (BMC), femoral length and cortical width than controls. Clenbuterol treatment further reduced bone mineral density. Bone microarchitecture was clearly altered by clenbuterol, as evidenced by lower trabecular number (-40.40%; P < 0.001), connectivity and trabecular bone volume (-42.85%; P < 0.001), leading to lower ultimate force. Clenbuterol significantly increased muscle mass (P < 0.01) and reduced fat mass when compared to controls. Salbutamol did not seem to have any effect on bone microarchitecture or body composition. Both beta2 agonists increased the bone resorption marker (C-terminal collagen crosslinks) without any change of a bone formation marker. At the end of the treatment, a drop in leptin was seen in the clenbuterol group only. Leptin levels were correlated with BMC (r = 0.69, P = 0.003)., Conclusion: These results confirm the deleterious effect of beta2 agonists on bone mass and show the negative effects of clenbuterol on trabecular bone microarchitecture. Bone loss occurred independently from muscle mass but was related to fat mass. A leptin-mediated effect on bone tissue seems likely. These pathophysiological effects may have important consequences in human therapeutics and doping.

Unlabelled: Physiological angiogenesis during bone remodeling is undefined. Treadmill-running rats displayed bone marrow angiogenesis concomitant with bone formation increase and resorption decrease and upregulation of VEGF and its R1 receptor mRNA in proximal tibia. VEGF blockade over 5 weeks of training fully prevented the exercise-induced bone mass gain., Introduction: We investigated the role of vascular endothelial growth factor (VEGF) and angiogenesis in the osteogenic response to exercise., Materials and Methods: Nine-week-old male Wistar rats were treadmill-trained at 60% Vo(2max) for various periods. Bone and vascular histomorphometry was performed after 2- and 5-week experiments. On-line RT PCR for VEGF and its receptors R1 and R2 was done after a 10-day experiment. In the 5-week experiment, running rats received either a VEGF inhibitory antibody or a placebo., Results: After 2 weeks, tibial BMD did not change; however, vessel number in the proximal metaphysis increased by 20% in running versus sedentary rats. In running rats, vessel number correlated positively (r = 0.88) with bone formation rate and negatively (r = -0.85) with active resorption surfaces. After 10 days of training, upregulation of VEGF and VEGF receptor R1 mRNA was detected in periosteum and metaphyseal bone. VEGF blockade in 5-week trained rats fully prevented the exercise-induced increase in metaphyseal BMD (9%) and cancellous bone volume (BV/TV; 25%), as well as the increased vessel number (25%). In 5-week placebo-treated running rats, bone formation rate returned to initial values, whereas osteoclastic surfaces continued to decline compared with both sedentary and anti-VEGF-treated running rats., Conclusion: VEGF signaling-mediated bone angiogenesis is tightly related to exercise-induced bone cellular uncoupling and is indispensable for bone gain induced by exercise.

Immobilization affects bone formation. However, the mechanisms regulating the decrease in osteoblast recruitment remain unclear. The aim of our study was to determine in vivo osteoblastic proliferation after short-term immobilization among the different bone compartments. Twelve Wistar 5-wk-old rats were assigned to two groups: six tail-suspended animals for 6 days and their six age-related controls. Osmotic minipumps, each containing 40 mg of bromodeoxyuridine (BrdU), were implanted intraperitoneally at day 4 until euthanasia. Histomorphometric measurements found a significantly lower bone volume in primary (ISP, -22%) and secondary spongiosa (IISP, -37%) in unloaded rats compared with their age-related controls. BrdU immunohistochemistry showed that the proliferation capacity of osteogenic precursors in ISP (-29%) and preosteoblasts in IISP (-80%) and in periosteum as well as bone marrow cells (-40%) was lowered by unloading. We demonstrated in vivo for the first time that 6-day tail suspension induced a significant decrease in proliferation of periosteal and trabecular preosteoblasts in ISP and IISP as well as in bone marrow cells.