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.

Hypergravity Increases Blood-Brain Barrier Permeability to Fluorescent Dextran and Antisense Oligonucleotide in Mice.

The earliest effect of spaceflight is an alteration in vestibular function due to microgravity. Hypergravity exposure induced by centrifugation is also able to provoke motion sickness. The blood-brain barrier (BBB) is the crucial interface between the vascular system and the brain to ensure efficient neuronal activity. We developed experimental protocols of hypergravity on C57Bl/6JRJ mice to induce motion sickness and reveal its effects on the BBB. Mice were centrifuged at 2× g for 24 h. Fluorescent dextrans with different sizes (40, 70 and 150 kDa) and fluorescent antisense oligonucleotides (AS) were injected into mice retro-orbitally. The presence of fluorescent molecules was revealed by epifluorescence and confocal microscopies in brain slices. Gene expression was evaluated by RT-qPCR from brain extracts. Only the 70 kDa dextran and AS were detected in the parenchyma of several brain regions, suggesting an alteration in the BBB. Moreover, Ctnnd1, Gja4 and Actn1 were upregulated, whereas Jup, Tjp2, Gja1, Actn2, Actn4, Cdh2 and Ocln genes were downregulated, specifically suggesting a dysregulation in the tight junctions of endothelial cells forming the BBB. Our results confirm the alteration in the BBB after a short period of hypergravity exposure.

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.

Similar effect of co-administration of methotrexate and folic acid for the treatment of arthritis compared to separate administration.

OBJECTIVES: MTX is the recommended first-line treatment for RA associated with folic acid (FA) to reduce side effects related to MTX. Here, we proposed to test a co-administration of MTX with FA in the rat adjuvant-induced arthritis (AIA) on efficacy. MATERIAL AND METHODS: AIA was induced in female Lewis rats and treated with MTX in three groups. The first group of rats received only MTX (n = 13), whereas the second received MTX and FA on the same day (n = 14). The third group received FA one day after MTX (n = 14). Arthritic index (AI), ankle circumference (AC), ankle microcomputed tomography, and blood tests assessed arthritis severity and MTX tolerance. RESULTS: AI and AC were similar in MTX groups at various time points. Bone erosion and bone loss parameters were similar in all groups. MTX-PG1 was found at similar levels in various MTX groups and correlated negatively with arthritis severity. Finally, haematology and metabolic parameters were found at a similar level in MTX groups. CONCLUSION: Co-administration of MTX with FA on the same day did not reduce efficacy compared with FA application one day after MTX. Thus, co-administration of MTX and FA could be more convenient and improve compliance in patients.

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.

Improvement of Pain Management by Nefopam in a Rat Adjuvant-Induced Arthritis Model.

Introduction: The adjuvant-induced arthritis (AIA) model is widely used in research to investigate arthritis pathogenesis. Hind paw inflammation is the main outcome in this model with high loss of mobility function partly related to pain. However, analgesics such as non-steroidal anti-inflammatory drugs or opioid drugs interfere with the inflammation process related to arthritis, thus reducing their beneficial use in this model. Therefore, we investigated the effect of nefopam on arthritis development in order to improve pain management in the AIA model. Methods: Female Lewis rats were randomly divided into two groups, and each group received an injection of Mycobacterium butyricum on defining day (D) 0. At D6, rats (n = 10) received nefopam (intraperitoneally or orally) or NaCl 0.9% IP or 1% sucrose in water (n = 5 for each). Rats were monitored with the arthritic index (AI) and ankle circumference. Pain was assessed by scoring based on behavioral indicators. Histology, RT-qPCR, and microcomputed tomography were performed. Results: The clinical parameter AI and ankle circumference were not different in both groups at various time points. However, pain score was significantly lower in the nefopam group at the early stage of the disease. At a later stage of the disease, inflammation was mildly lower whereas bone erosion and bone loss parameters increased in the nefopam group. Conclusion: Nefopam provided a slight reduction in the level of pain at the arthritis onset without reducing arthritis severity and bone loss in the rat AIA model. However, it should be administrated orally for a shorter period to avoid inflammation reduction in the long run.

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.

Hypergravity as a gravitational therapy mitigates the effects of knee osteoarthritis on the musculoskeletal system in a murine model.

Insights into the effects of osteoarthritis (OA) and physical interventions on the musculoskeletal system are limited. Our goal was to analyze musculoskeletal changes in OA mice and test the efficacy of 8-week exposure to hypergravity, as a replacement of physical activity. 16-week-old male (C57BL/6J) mice allocated to sham control and OA groups not centrifuged (Ctrl 1g and OA 1g, respectively) or centrifuged at 2g acceleration (Ctrl 2g and OA 2g). OA 1g displayed decreased trabecular bone in the proximal tibia metaphysis and increased osteoclastic activity and local TNFα gene expression, all entirely prevented by 2g gravitational therapy. However, while cortical bone of tibia midshaft was preserved in OA 1g (vs. ctrl), it is thinner in OA 2g (vs. OA 1g). In the hind limb, OA at 1g increased fibers with lipid droplets by 48% in the tibialis anterior, a fact fully prevented by 2g. In Ctrl, 2g increased soleus, tibialis anterior and gastrocnemius masses. In the soleus of both Ctrl and OA, 2g induced larger fibers and a switch from type-II to type-I fiber. Catabolic (myostatin and its receptor activin RIIb and visfatine) and anabolic (FNDC5) genes dramatically increased in Ctrl 2g and OA 2g (p<0.01 vs 1g). Nevertheless, the overexpression of FNDC5 (and follistatine) was smaller in OA 2g than in Ctrl 2g. Thus, hypergravity in OA mice produced positive effects for trabecular bone and muscle typology, similar to resistance exercises, but negative effects for cortical bone.

Unloading-Induced Cortical Bone Loss is Exacerbated by Low-Dose Irradiation During a Simulated Deep Space Exploration Mission.

Spaceflight-induced bone losses have been reliably reproduced in Hind-Limb-Unloading (HLU) rodent models. However, a considerable knowledge gap exists regarding the effects of low-dose radiation and microgravity together. Ten-week-old male C57BL/6J mice, randomly allocated to Control (CONT), Hind-Limb Unloading (HLU), and Hind-Limb Unloading plus Irradiation (HLUIR), were acclimatized at 28 °C, close to thermoneutral temperature, for 28 days prior to the 14-day HLU protocol. HLUIR mice received a 25 mGy dose of X-ray irradiation, simulating 14 days of exposure to the deep space radiation environment, on day 7 of the HLU protocol. Trabecular bone mass was similarly reduced in HLU and HLUIR mice when compared to CONT, with losses driven by osteoclastic bone resorption rather than changes to osteoblastic bone formation. Femoral cortical thickness was reduced only in the HLUIR mice (102 µm, 97.5-107) as compared to CONT (108.5 µm, 102.5-120.5). Bone surface area was also reduced only in the HLUIR group, with no difference between HLU and CONT. Cortical losses were driven by osteoclastic resorption on the posterior endosteal surface of the distal femoral diaphysis, with no increase in the numbers of dead osteocytes. In conclusion, we show that low-dose radiation exposure negatively influences bone physiology beyond that induced by microgravity alone.

Effects of centrifugation and whole-body vibrations on blood-brain barrier permeability in mice.

Modifications of gravity levels induce generalized adaptation of mammalian physiology, including vascular, brain, muscle, bone and immunity functions. As a crucial interface between the vascular system and the brain, the blood-brain barrier (BBB) acts as a filter to protect neurons from pathogens and inflammation. Here we compare the effects of several protocols of hypergravity induced by centrifugation and whole-body vibrations (WBV) on BBB integrity. The immunohistochemistry revealed immunoglobulin G (IgG) extravasation from blood to hippocampal parenchyma of mice centrifuged at 2 × g during 1 or 50 days, whereas short exposures to higher hypergravity mimicking the profiles of spaceflight landing and take-off (short exposures to 5 × g) had no effects. These results suggest prolonged centrifugation (>1 days) at 2 × g induced a BBB leakage. Moreover, WBV were similarly tested. The short exposure to +2 × g vibrations (900 s/day at 90 Hz) repeated for 63 days induced IgG extravasation in hippocampal parenchyma, whereas the progressive increase of vibrations from +0.5 to +2 × g for 63 days was not able to affect the IgG crossing through the BBB. Overall, these results suggest that the BBB permeability is sensitive to prolonged external accelerations. In conclusion, we advise that the protocols of WBV and centrifugation, proposed as countermeasure to spaceflight, should be designed with progressively increasing exposure to reduce potential side effects on the BBB.

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.

High-acceleration whole body vibration stimulates cortical bone accrual and increases bone mineral content in growing mice.

Whole body vibration (WBV) is a promising tool for counteracting bone loss. Most WBV studies on animals have been performed at acceleration <1g and frequency between 30 and 90Hz. Such WBV conditions trigger bone growth in osteopenia models, but not in healthy animals. In order to test the ability of WBV to promote osteogenesis in young animals, we exposed seven-week-old male mice to vibration at 90Hz and 2g peak acceleration for 15min/day, 5 days/week. We examined the effects on skeletal tissues with micro-computed tomography and histology. We also quantified bone vascularization and mechanosensitive osteocyte proteins, sclerostin and DMP1. Three weeks of WBV resulted in an increase of femur cortical thickness (+5%) and area (+6%), associated with a 25% decrease of sclerostin expression, and 35% increase of DMP1 expression in cortical osteocytes. Mass-structural parameters of trabecular bone were unaltered in femur or vertebra, while osteoclastic parameters and bone formation rate were increased at both sites. Three weeks of WBV resulted in higher blood vessel numbers (+23%) in the distal femoral metaphysis. After 9-week WBV, we have not observed the difference in structural cortical or trabecular parameters. However, the tissue mineral density of cortical bone was increased by 2.5%. Three or nine weeks of 2g/90Hz WBV treatment did not affect longitudinal growth rate or body weight increase under our experimental conditions, indicating that these are safe to use. These results validate a potential of 2g/90Hz WBV to stimulate trabecular bone cellular activity, accelerate cortical bone growth, and increase bone mineral density.

The role of the SIBLING, Bone Sialoprotein in skeletal biology - Contribution of mouse experimental genetics.

Bone Sialoprotein (BSP) is a member of the "Small Integrin-Binding Ligand N-linked Glycoproteins" (SIBLING) extracellular matrix protein family of mineralized tissues. BSP has been less studied than other SIBLING proteins such as Osteopontin (OPN), which is coexpressed with it in several skeletal cell types. Here we review the contribution of genetically engineered mice (BSP gene knockout and overexpression) to the understanding of the role of BSP in the bone organ. The studies made so far highlight the role of BSP in skeletal mineralization, as well as its importance for proper osteoblast and osteoclast differentiation and activity, most prominently in primary/repair bone. The absence of BSP also affects the local environment of the bone tissue, in particular hematopoiesis and vascularization. Interestingly, lack of BSP induces an overexpression of OPN, and the cognate protein could be responsible for some aspects of the BSP gene knockout skeletal phenotype, while replacing BSP for some of its functions. Such interplay between the partly overlapping functions of SIBLING proteins, as well as the network of cross-regulations in which they are involved should now be the focus of further work.

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.

Effects of chronic hypergravity: from adaptive to deleterious responses in growing mouse skeleton.

One of the most important but least studied environmental factors playing a major role in bone physiology is gravity. While the knowledge of deleterious effects of microgravity on the skeleton is expanding, little is known about hypergravity and its osteogenic potential. Centrifugation was used to assess effects of 21-day continuous 2- or 3-g acceleration on femur and L2-vertebra of 7-wk-old male C57BL/6 mice. Under 3 g, body mass growth slowed down, and deleterious skeletal effects were found (P < 0.05 compared with control): cortical thinning, osteoclasts surface increase (+41% in femur, +20% in vertebra), and bone formation rate decrease (-34% in femur, -38% in vertebra). A 2-g centrifugation did not reduce body mass and improved trabecular volume (+18% in femur, +13% in vertebra) and microarchitecture (+32% connectivity density in femur, +9% trabecular thickness in vertebra, P < 0.05 compared with control). Centrifugation at 2 g also decreased osteoclast surfaces (-36% in femur, -16% in vertebra) and increased the extent of mineralized surfaces (+31% in femur, +48% in vertebra, P < 0.05 compare to control). Quantitative immunohistochemistry revealed an increase of dentin matrix acidic phosphoprotein 1 (DMP1) and decrease of sclerostin (+60% and -35% respectively, P < 0.001 compared with control) in the femur cortex of 2-g mice. In the distal femur metaphysis, the number and volume of blood vessels increased by 22 and 44%, respectively (P < 0.05 compared with control). In conclusion, the effects of continuous hypergravity were bone compartment-specific and depended on the gravity level, with a threshold between beneficial 2-g and deleterious 3-g effects.

Assessment of bone vascularization and its role in bone remodeling.

Bone is a composite organ that fulfils several interconnected functions, which may conflict with each other in pathological conditions. Bone vascularization is at the interface between these functions. The roles of bone vascularization are better documented in bone development, growth and modeling than in bone remodeling. However, every bone remodeling unit is associated with a capillary in both cortical and trabecular envelopes. Here we summarize the most recent data on vessel involvement in bone remodeling, and we present the characteristics of bone vascularization. Finally, we describe the various techniques used for bone vessel imaging and quantitative assessment, including histology, immunohistochemistry, microtomography and intravital microscopy. Studying the role of vascularization in adult bone should provide benefits for the understanding and treatment of metabolic bone diseases.

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.