Marie-Hélène Lafage-Proust

Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts.

BACKGROUND Microgravity has been thought to induce osteoporosis because of reduced weight-bearing. However, up to now, few data have been available about its precise nature and timecourse. METHODS We measured bone mineral density (BMD) at the distal radius and tibia in 15 cosmonauts of the Russian MIR space station who sojourned in space either 1 (n=two), 2 (two), or 6 months (11). After recovery periods of similar duration to the space missions, BMD was measured for the 2-month and 6-month crews. FINDINGS Neither cancellous nor cortical bone of the radius was significantly changed at any of the timepoints. On the contrary, in the weight-bearing tibial site, cancellous BMD loss was already present after the first month and deteriorated with mission duration. In tibial cortices, bone loss was noted after a 2-month flight. In the 6-month group, cortical bone loss was less pronounced than that for cancellous bone. In some individuals, tibial deterioration was great. Actual BMD did not depend on preceding cumulative periods spent in space. During recovery, tibial bone loss persisted, suggesting that the time needed to recover is longer than the mission duration. INTERPRETATION In space, despite physical training, bone loss is an adaptive process that can become pathological after recovery on Earth. Striking interindividual variations in bone responses seem to suggest a need for adequate crew preselection. Targeted treatment or prevention strategies would be useful, not only for space purposes, but also for the increasing number of osteoporotic patients on Earth.

Effects of whole body vibration on the skeleton and other organ systems in man and animal models : What we know and what we need to know

Previous investigations reported enhanced osseous parameters subsequent to administration of whole body vibration (WBV). While the efficacy of WBV continues to be explored, scientific inquiries should consider several key factors. Bone remodeling patterns differ according to age and hormonal status. Therefore, WBV protocols should be designed specifically for the subject population investigated. Further, administration of WBV to individuals at greatest risk for osteoporosis may elicit secondary physiological benefits (e.g., improved balance and mobility). Secondly, there is a paucity of data in the literature regarding the physiological modulation of WBV on other organ systems and tissues. Vibration-induced modulation of systemic hormones may provide a mechanism by which skeletal tissue is enhanced. Lastly, the most appropriate frequencies, durations, and amplitudes of vibration necessary for a beneficial response are unknown, and the type of vibratory signal (e.g., sinusoidal) is often not reported. This review summarizes the physiological responses of several organ systems in an attempt to link the global influence of WBV. Further, we report findings focused on subject populations that may benefit most from such a therapy (i.e., the elderly, postmenopausal women, etc.) in hopes of eliciting multidisciplinary scientific inquiries into this potentially therapeutic aid which presumably has global ramifications.

Tail Suspension Induces Bone Loss in Skeletally Mature Mice in the C57BL/6J Strain but Not in the C3H/HeJ Strain

We assessed the effects of tail‐suspension in two skeletal genetic backgrounds, the high C3H/HeJ (C3H) and low C57BL/6J (B6) bone masses inbred mice (male, 4‐months old). Cancellous bone mass and structural parameters were evaluated in distal femoral metaphysis by three dimensional microcomputed tomography. Bone cellular activities were evaluated by histomorphometry and measurements of alkaline phosphatase activity (ALP) and osteocalcin in blood and deoxypyridinoline (D‐pyr) in urine. In C3H mice, 2‐ and 3‐week unloading experiments were performed. After an early and transient decrease in body weight, a 2‐week suspension period resulted in stimulation of both bone formation rate by 45% and active osteoclastic surfaces by 19%. D‐pyr did not change, but ALP and osteocalcin levels increased by 18% and 72%, respectively, in 2‐week suspended mice, and osteocalcin remained elevated by 30% in the 3‐week suspended mice. Such cellular modifications allowed the C3H mice to maintain their initial bone mass and trabecular structural parameters even after a 3‐week suspension period. In B6 mice, 1‐ and 2‐week unloading experiments were performed. Tail suspension resulted in decreased body weight during the first days followed by an incomplete recovery during the second week of unloading. The resorption activity was unaffected by any suspension time period, whereas a decrease of 42.5% in bone formation rate and of 21.5% in ALP were seen by the end of the first week of suspension, both values being restored after a 2‐week suspension period. At this latter time, trabeculae were thinner, leading to a 24.5% cancellous bone loss. Trabecular number and connectivity, rod‐plate index, and degree of anisotropy were not modified. We concluded that C3H mice constituted a unique model in which genetic background overwhelmed the usual effects of reduced biomechanical usage in bone, whereas B6 mice, compared with the standardized rat model, offered an alternative model of bone loss in a mature skeleton.

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

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

Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three‐dimensional contractile collagen gels

Studies performed at tissular (three‐dimensional, 3‐D) or cellular (two‐dimensional, 2‐D) levels showed that the loading pattern plays a crucial role in the osteoblastic physiology. In this study, we attempted to investigate the response of a 3‐D osteoblastic culture submitted to either no external stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (33%). The proliferation rate was greater in TG than in FRG (+52%) but remained low in both DGs. Gel dimensions were related to the collagen concentration and on a minor extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well‐developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: a first increase at day 2, followed by a decrease from day 4 to 14, and a second increase above initial values at day 18. Microanalysis‐x indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3‐D collagen matrices, played a significant role on the phenotypic modulation of osteoblast‐like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3‐D cultured osteoblast‐like cells. J. Cell. Biochem. 76:217–230, 1999.

Weight gain reverses bone turnover and restores circadian variation of bone resorption in anorexic patients

The present study was conducted in order to describe the variations and circadian rhythm of biochemical markers of bone remodelling at baseline and after weight gain in patients with aneroxia nervosa (AN).

MAP and src kinases control the induction of AP-1 members in response to changes in mechanical environment in osteoblastic cells

The activating protein-1 (AP-1) complex plays a critical role in bone physiology, including its response to strain. We studied gene expression and nuclear translocation kinetics of the seven AP-1 members, after substrate deformation (Flexcell) or simulated microgravity (Clinostat), in osteoblastic ROS17/2.8 cells. Gene expression and nuclear translocation of all the AP-1 members were induced, under both conditions, with differences in their kinetics, except fosB mRNA in the Clinostat. Downregulation of protein kinase C (PKC) and COX1/2 or inhibition of ERK1/2, p38(MAPK) or src kinases had no major effect on AP-1 mRNA expression in the Flexcell. In contrast, ERK1/2, p38(MAPK) and src kinases treatment blocked nuclear translocation of almost all the AP-1 members in both models, except Fra-1, JunD after deformation and Fra-1, JunB after clinorotation. Thus, changes in the osteoblastic mechanical environment induced a dramatic induction of most of the AP-1 members with specific kinetics and involved MAPK and src kinase pathways, which differed whether the cells were stretched or clinorotated.

Transmigration: A New Property of Mature Multinucleated Osteoclasts†

Even though it is assumed that multinucleated osteoclasts are migrating cells on the bone surface to be resorbed, we show that they can also selectively transmigrate through layers of cells usually found in the bone microenvironment. This activity is associated with c‐src and MMPs and can be stimulated by bone metastatic breast cancer cells, a process blocked by bisphosphonate treatment.

Chronic kidney disease bone and mineral disorder (CKD–MBD) in apolipoprotein E-deficient mice with chronic renal failure

BACKGROUND Chronic kidney disease (CKD) is associated with disorders of mineral and bone metabolism (MBD) which include renal osteodystrophy and vascular calcifications. This is of clinical concern because the high risk of cardiovascular (CVD) complications observed in uremic patients may be linked with bone disease. In this context, our aim was to characterize the bone lesions in CKD-apolipoprotein E-deficient mice (apoE(-/-)) and analyze their relationships with the vascular calcifications which these animals develop rapidly in this model. With ApoE being also involved in bone metabolism, we compared the effects of CRF on the bone of apoE(-/-) mice to those observed in wild type mice (WT) of the same genetic background, C57/BL6. METHODS After CRF creation or sham surgery, 10 week-old female apoE(-/-) and WT mice were randomized to 4 groups (n=10-14/group) and fed with standard diet. Eight weeks later, animals were euthanized. Serum, aorta and femur were sampled. Femurs were imaged with 3-dimensional microtomography (microCT) and processed for bone histomorphometry (BHM). Additional quantitative histology was performed on atherosclerotic and calcified lesions in the aortas of apoE(-/-) mice. RESULTS First, apoE(-/-) mice exhibited higher cortical (10%) and trabecular (31%) bone mass than WT. CRF led to a further increase in trabecular BV/TV in WT and in apoE(-/-) mice (10.2% and 77.2%, respectively). We observed a similar increase in osteoid surface and osteoblastic parameters in CRF mice of both genotypes while resorption parameters were less augmented by CRF in apoE(-/-) mice. Finally, based on either BHM or microCT we found positive correlations between the extent of atherosclerotic lesions and bone volume parameters, and between the size of plaque calcification and osteoclast parameters in apoE(-/-) mice. CONCLUSION ApoE deficiency is associated with an increase in bone mass and volumetric mineral density in 20 week-old female mice. Bone mass is further increased, whereas bone mineral density is decreased, in response to CRF in association with histological features of osteitis fibrosa. Finally, our findings of correlations between changes in bone and aortic lesions in apoE(-/-) mice, are compatible with the hypothesis of a link between bone and vascular disease and require further study.

Absence of bone sialoprotein (BSP) impairs cortical defect repair in mouse long bone

Matrix proteins of the SIBLING family interact with bone cells and with bone mineral and are thus in a key position to regulate bone development, remodeling and repair. Within this family, bone sialoprotein (BSP) is highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. We recently reported that mice lacking BSP (BSP-/-) have very low trabecular bone turnover. In the present study, we set up an experimental model of bone repair by drilling a 1 mm diameter hole in the cortical bone of femurs in both BSP-/- and +/+ mice. A non-invasive MRI imaging and bone quantification procedure was designed to follow bone regeneration, and these data were extended by microCT imaging and histomorphometry on undecalcified sections for analysis at cellular level. These combined approaches revealed that the repair process as reflected in defect-refilling in the cortical area was significantly delayed in BSP-/- mice compared to +/+ mice. Concomitantly, histomorphometry showed that formation, mineralization and remodeling of repair (primary) bone in the medulla were delayed in BSP-/- mice, with lower osteoid and osteoclast surfaces at day 15. In conclusion, the absence of BSP delays bone repair at least in part by impairing both new bone formation and osteoclast activity.