Impact of an obesogenic diet program on bone densitometry, micro architecture and metabolism in male rat.

International audienceABSTRACT: Background The relationships between fat mass and bone tissue are complex and not fully elucidated. A high-fat/high-sucrose diet has been shown to induce harmful effects on bone micro architecture and bone biomechanics of rat. When such diet leads to obesity, it may induce an improvement of biomechanical bone parameters in rodent. Here, we examined the impact of a high-fat/high-sucrose diet on the body composition and its resulting effects on bone density and structure in male rats. Forty three Wistar rats aged 7 months were split into 3 groups: 1 sacrificed before diet (BD, n=14); 1 subjected to 16 weeks of high-fat/high-sucrose diet (HF/HS, n=14); 1 subjected to standard diet (Control, n=15). Abdominal circumference and insulin sensitivity were measured and visceral fat mass was weighed. The bone mineral density (BMD) was analyzed at the whole body and tibia by densitometry. Microcomputed tomography and histomorphometric analysis were performed at L2 vertebrae and tibiae to study the trabecular and cortical bone structures and the bone cell activities. Osteocalcin and CTX levels were performed to assess the relative balance of the bone formation and resorption. Differences between groups have been tested with an ANOVA with subsequent Scheffe post-hoc test. An ANCOVA with global mass and global fat as covariates was used to determine the potential implication of the resulting mechanical loading on bone. RESULTS: The HF/HS group had higher body mass, fat masses and abdominal circumference and developed an impaired glucose tolerance compared to Control group (p<0.001). Whole body bone mass (p<0.001) and BMD (p<0.05) were higher in HF/HS group vs. Control group. The trabecular thickness at vertebrae and the cortical porosity of tibia were improved (p<0.05) in HF/HS group. Bone formation was predominant in HF/HS group while an unbalance bone favoring bone resorption was observed in the controls. The HF/HS and Control groups had higher total and abdominal fat masses and altered bone parameters vs. BD group. Conclusions The HF/HS diet had induced obesity and impaired glucose tolerance. These changes resulted in an improvement of quantitative, qualitative and metabolic bone parameters. The fat mass increase partly explained these observations

Mechanical signals modulated vascular endothelial growth factor-A (VEGF-A) alternative splicing in osteoblastic cells through actin polymerisation.

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<0.0001) between production of soluble VEGF and mechanical stretch frequency and a positive correlation (R(2)=0.99, p<0.0001) between production of matrix-bound VEGF and mechanical stretch frequency. mRNA expressions of soluble VEGF isoforms (121, 165) were specifically expressed under low frequency while matrix-bound VEGF isoforms (206, 189, 165, 145) were specifically expressed under high frequency in human osteoblasts. As f-actin stress fiber formation was significantly increased selectively in high frequency conditions, we disrupted actin fibers in Ros17/2.8 and found that immobilisation of VEGF was abolished. Conversely, Jasplakinolide treatment which increases stress fiber formation was able to mimic high frequency stretch-induced immobilisation of VEGF. Thus, we speculate that the stretch-induced increase in cell tension is responsible for matrix-bound vegf isoform production. Mechanically induced selection of soluble or matrix-bound VEGF production may modify osteoblast and endothelial cell crosstalk crucial during osteogenesis and fracture healing

The impairement of bone formation and mineralization in BSP−/− mouse calvaria cell cultures is partly rescued by increasing cell density

International audienceBone sialoprotein regulates osteoblast activity and bone formation. In knockout (BSP-/-) mouse bone marrow (BM) stromal cell cultures, the pool of osteoprogenitor (OP) cells (CFU-F number) is not different from wild type (+/+), nor is their early differentiation (same numbers of alkaline phosphatase positive colonies =CFU-ALP, although these are smaller), while the number of osteoblast, mineralized colonies (CFU-OB) is dramatically reduced. Because ossification of newborn BSP-/- mouse calvaria is delayed, we analysed the impact of the mutation on in vitro osteogenesis in cultures of mouse calvaria cells (MCC), isolated from 6 days old mice by collagenase digestion. In contrast to BM, CFU-F, CFU-ALP and CFU-OB numbers were lower in BSP-/- MCC cultures. Consistent with less OP, BSP-/- cultures displayed lower proliferation and delayed growth. In MCC cultures seeded at 5000cell/cm2 osteoblast marker expression did not differ between genotypes until D6. By D14 (=first CFU-OBs) ALP, Coll1, OSX, Runx2 as well as terminal differentiation markers, OCN, PHEX, DMP1 and MEPE increased strongly in BSP+/+ cultures but was low/absent in BSP-/-, with no mineralization. In contrast, osteopontin (OPN) was over-expressed in BSP-/- dishes. At high density (≥25000cell/cm2), marker levels were similar for both genotypes, and BSP-/- cultures mineralized. OPN is a potent inhibitor of mineralization, and was reported to be a substrate for PHEX. Very low PHEX expression in low density BSP-/- cultures suggests that OPN is less degraded and might inhibit mineralization. Increased PHEX expression at higher density would permit OPN degradation and mineralization. Lack of BSP thus reduces MCC culture clonogenicity, differentiation and activity, consistent with lower bone formation in vivo. A BSP-/- bone microenvironment may alter proliferation/cell fate in early OP, explaining the smaller size of CFU-PAL observed in BM cultures

The impairement of bone formation and mineralization in BSP−/− mouse calvaria cell cultures is partly rescued by increasing cell density

International audienceBone sialoprotein regulates osteoblast activity and bone formation. In knockout (BSP-/-) mouse bone marrow (BM) stromal cell cultures, the pool of osteoprogenitor (OP) cells (CFU-F number) is not different from wild type (+/+), nor is their early differentiation (same numbers of alkaline phosphatase positive colonies =CFU-ALP, although these are smaller), while the number of osteoblast, mineralized colonies (CFU-OB) is dramatically reduced. Because ossification of newborn BSP-/- mouse calvaria is delayed, we analysed the impact of the mutation on in vitro osteogenesis in cultures of mouse calvaria cells (MCC), isolated from 6 days old mice by collagenase digestion. In contrast to BM, CFU-F, CFU-ALP and CFU-OB numbers were lower in BSP-/- MCC cultures. Consistent with less OP, BSP-/- cultures displayed lower proliferation and delayed growth. In MCC cultures seeded at 5000cell/cm2 osteoblast marker expression did not differ between genotypes until D6. By D14 (=first CFU-OBs) ALP, Coll1, OSX, Runx2 as well as terminal differentiation markers, OCN, PHEX, DMP1 and MEPE increased strongly in BSP+/+ cultures but was low/absent in BSP-/-, with no mineralization. In contrast, osteopontin (OPN) was over-expressed in BSP-/- dishes. At high density (≥25000cell/cm2), marker levels were similar for both genotypes, and BSP-/- cultures mineralized. OPN is a potent inhibitor of mineralization, and was reported to be a substrate for PHEX. Very low PHEX expression in low density BSP-/- cultures suggests that OPN is less degraded and might inhibit mineralization. Increased PHEX expression at higher density would permit OPN degradation and mineralization. Lack of BSP thus reduces MCC culture clonogenicity, differentiation and activity, consistent with lower bone formation in vivo. A BSP-/- bone microenvironment may alter proliferation/cell fate in early OP, explaining the smaller size of CFU-PAL observed in BM cultures

Functionalization of matrices by cyclically stretched osteoblasts through matrix targeting of VEGF.

International audienceVascular endothelial growth factor A (VEGF) plays a central role in load-induced bone gain. We previously showed that increasing cyclic stretch frequency from 0.05 to 5 Hz induce parallel increased in entrapment of VEGF (mVEGF) into osteoblast secreted extracellular matrix. We ask in this study if mVEGF could be protective against apoptotic signals and biologically active in vitro on endothelial cell migration as well as in vivo on angiogenesis. We established that mechanically-induced VEGF entrapment using stretched silicone membrane was saturable after 3 exposures at high frequency stretches (5Hz). We found that mVEGF stimulates microvascular cells migration and enhanced angiogenesis more importantly than VEGF 165 controls suggesting the absence of potent anti-angiogenic factors in our functionalized matrices. Indeed we found that the anti-angiogenic factors, tissue inhibitor of metalloproteinase (TIMP2) and pigment epithelium-derived factor (PEDF) were specifically downregulated for 5 Hz stretch and that the release of these potent factors was increased for low frequency of stretch (0.05 Hz). This study qualifies high frequency cyclic stretch as an interesting approach for surfaces activation of deformable biomaterials

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

International audienceInsights 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 TNF alpha 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

Impact of an obesogenic diet program on bone densitometry, micro architecture and metabolism in male rat

Abstract Background The relationships between fat mass and bone tissue are complex and not fully elucidated. A high-fat/high-sucrose diet has been shown to induce harmful effects on bone micro architecture and bone biomechanics of rat. When such diet leads to obesity, it may induce an improvement of biomechanical bone parameters in rodent. Here, we examined the impact of a high-fat/high-sucrose diet on the body composition and its resulting effects on bone density and structure in male rats. Forty three Wistar rats aged 7 months were split into 3 groups: 1 sacrificed before diet (BD, n = 14); 1 subjected to 16 weeks of high-fat/high-sucrose diet (HF/HS, n = 14); 1 subjected to standard diet (Control, n = 15). Abdominal circumference and insulin sensitivity were measured and visceral fat mass was weighed. The bone mineral density (BMD) was analyzed at the whole body and tibia by densitometry. Microcomputed tomography and histomorphometric analysis were performed at L2 vertebrae and tibia to study the trabecular and cortical bone structures and the bone cell activities. Osteocalcin and CTX levels were performed to assess the relative balance of the bone formation and resorption. Differences between groups have been tested with an ANOVA with subsequent Scheffe post-hoc test. An ANCOVA with global mass and global fat as covariates was used to determine the potential implication of the resulting mechanical loading on bone. Results The HF/HS group had higher body mass, fat masses and abdominal circumference and developed an impaired glucose tolerance (p  Conclusions The HF/HS diet had induced obesity and impaired glucose tolerance. These changes resulted in an improvement of quantitative, qualitative and metabolic bone parameters. The fat mass increase partly explained these observations.</p

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

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, n¼45) and mature (M, n¼45) rats fed a high-fat/high-sucrose diet(HFS)isocaloric to the chow diet of controls (C, n¼30 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 TGFb1, 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 inMwas alleviated. At diaphysis cortical porosity increased in G and even more inMat 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 inMthe 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. © 2015 American Society for Bone and Mineral Research