How to obtain an integrated picture of the molecular networks involved in adaptation to microgravity in different biological systems?

Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.

How are cell and tissue structure and function influenced by gravity and what are the gravity perception mechanisms?

Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap "Biology in Space and Analogue Environments" focusing on "How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?" The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.

How do gravity alterations affect animal and human systems at a cellular/tissue level?

The present white paper concerns the indications and recommendations of the SciSpacE Science Community to make progress in filling the gaps of knowledge that prevent us from answering the question: "How Do Gravity Alterations Affect Animal and Human Systems at a Cellular/Tissue Level?" This is one of the five major scientific issues of the ESA roadmap "Biology in Space and Analogue Environments". Despite the many studies conducted so far on spaceflight adaptation mechanisms and related pathophysiological alterations observed in astronauts, we are not yet able to elaborate a synthetic integrated model of the many changes occurring at different system and functional levels. Consequently, it is difficult to develop credible models for predicting long-term consequences of human adaptation to the space environment, as well as to implement medical support plans for long-term missions and a strategy for preventing the possible health risks due to prolonged exposure to spaceflight beyond the low Earth orbit (LEO). The research activities suggested by the scientific community have the aim to overcome these problems by striving to connect biological and physiological aspects in a more holistic view of space adaptation effects.

Key topographic parameters driving surface adhesion of Porphyromonas gingivalis.

Dental implant failure is primarily due to peri-implantitis, a consequence of bacterial biofilm formation. Bacterial adhesion is strongly linked to micro-/nano-topographies of a surface; thus an assessment of surface texture parameters is essential to understand bacterial adhesion. In this study, mirror polished titanium samples (Ti6Al4V) were irradiated with a femtosecond laser (fs-L) at a wavelength of 1030 nm (infrared) with variable laser parameters (laser beam polarization, number, spacing and organization of the impacts). Images of 3-D topographies were obtained by focal variation microscopy and analyzed with MountainsMap software to measure surface parameters. From bacteria associated with peri-implantitis, we selected Porphyromonas gingivalis to evaluate its adhesion on Ti6Al4V surfaces in an in vitro study. Correlations between various surface parameters and P. gingivalis adhesion were investigated. We discovered that Sa value, a common measure of surface roughness, was not sufficient in describing the complexity of these fs-L treated surfaces and their bacterial interaction. We found that Sku, density and mean depths of the furrows, were the most accurate parameters for this purpose. These results provide important information that could help anticipate the bacterial adhesive properties of a surface based on its topographic parameters, thus the development of promising laser designed biofunctional implants.

Polarization of Femtosecond Laser for Titanium Alloy Nanopatterning Influences Osteoblastic Differentiation.

Ultrashort pulse lasers have significant advantages over conventional continuous wave and long pulse lasers for the texturing of metallic surfaces, especially for nanoscale surface structure patterning. Furthermore, ultrafast laser beam polarization allows for the precise control of the spatial alignment of nanotextures imprinted on titanium-based implant surfaces. In this article, we report the biological effect of beam polarization on human mesenchymal stem cell differentiation. We created, on polished titanium-6aluminum-4vanadium (Ti-6Al-4V) plates, a laser-induced periodic surface structure (LIPSS) using linear or azimuthal polarization of infrared beams to generate linear or radial LIPSS, respectively. The main difference between the two surfaces was the microstructural anisotropy of the linear LIPSS and the isotropy of the radial LIPSS. At 7 d post seeding, cells on the radial LIPSS surface showed the highest extracellular fibronectin production. At 14 days, qRT-PCR showed on the same surface an increase in osteogenesis-related genes, such as alkaline phosphatase and osterix. At 21 d, mineralization clusters indicative of final osteoinduction were more abundant on the radial LIPSS. Taken together, we identified that creating more isotropic than linear surfaces enhances cell differentiation, resulting in an improved osseointegration. Thus, the fine tuning of ultrashort pulse lasers may be a promising new route for the functionalization of medical implants.

YAP Transcriptional Activity Dictates Cell Response to TNF In Vitro.

YAP/TAZ are transcription co-factors recently described responsive to pro-inflammatory cytokines and involved in inflammatory-related disorders. However, the role of tumor necrosis factor (TNF), a major pro-inflammatory cytokine, on YAP signaling is not well understood and controversial. Here, we observe in vitro, using wild type and YAP knockout HEK293 cells, that TNF triggers YAP nuclear translocation and transcriptional activity, thus being dependent on Rho family of GTPases. In response to TNF, YAP transcriptional activity orientates cell fate toward survival. Transcriptional analysis with Nanostring technology reveals that YAP modulates TNF-induced increase in fibro-inflammatory pathways such as NF-κB, inflammasomes, cytokines or chemokines signaling and pro-fibrotic pathways involving TGF-ß and extracellular matrix remodeling. Therefore, in response to TNF, YAP acts as a sustainer of the inflammatory response and as a molecular link between inflammation and fibrotic processes. This work identifies that YAP is critical to drive several biological effects of TNF which are involved in cancer and inflammatory disorders.

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.

Osteocytes and Weightlessness.

PURPOSE OF REVIEW: Osteocytes are considered to be the cells responsible for mastering the remodeling process that follows the exposure to unloading conditions. Given the invasiveness of bone biopsies in humans, both rodents and in vitro culture systems are largely adopted as models for studies in space missions or in simulated microgravity conditions models on Earth. RECENT FINDINGS: After a brief recall of the main changes in bone mass and osteoclastic and osteoblastic activities in space-related models, this review focuses on the potential role of osteocytes in directing these changes. The role of the best-known signalling molecules is questioned, in particular in relation to osteocyte apoptosis. The mechanotransduction actors identified in spatial conditions and the problems related to fluid flow and shear stress changes, probably enhanced by the alteration in fluid flow and lack of convection during spaceflight, are recalled and discussed.

Regulation of SMC traction forces in human aortic thoracic aneurysms.

Smooth muscle cells (SMCs) usually express a contractile phenotype in the healthy aorta. However, aortic SMCs have the ability to undergo profound changes in phenotype in response to changes in their extracellular environment, as occurs in ascending thoracic aortic aneurysms (ATAA). Accordingly, there is a pressing need to quantify the mechanobiological effects of these changes at single cell level. To address this need, we applied Traction Force Microscopy (TFM) on 759 cells coming from three primary healthy (AoPrim) human SMC lineages and three primary aneurysmal (AnevPrim) human SMC lineages, from age and gender matched donors. We measured the basal traction forces applied by each of these cells onto compliant hydrogels of different stiffness (4, 8, 12, 25 kPa). Although the range of force generation by SMCs suggested some heterogeneity, we observed that: 1. the traction forces were significantly larger on substrates of larger stiffness; 2. traction forces in AnevPrim were significantly higher than in AoPrim cells. We modelled computationally the dynamic force generation process in SMCs using the motor-clutch model and found that it accounts well for the stiffness-dependent traction forces. The existence of larger traction forces in the AnevPrim SMCs were related to the larger size of cells in these lineages. We conclude that phenotype changes occurring in ATAA, which were previously known to reduce the expression of elongated and contractile SMCs (rendering SMCs less responsive to vasoactive agents), tend also to induce stronger SMCs. Future work aims at understanding the causes of this alteration process in aortic aneurysms.

Multiparametric investigation of non functionalized-AGuIX nanoparticles in 3D human airway epithelium models demonstrates preferential targeting of tumor cells.

Liquid deposit mimicking surface aerosolization in the airway is a promising strategy for targeting bronchopulmonary tumors with reduced doses of nanoparticle (NPs). In mimicking and studying such delivery approaches, the use of human in vitro 3D culture models can bridge the gap between 2D cell culture and small animal investigations. Here, we exposed airway epithelia to liquid-apical gadolinium-based AGuIX® NPs in order to determine their safety profile. We used a multiparametric methodology to investigate the NP's distribution over time in both healthy and tumor-bearing 3D models. AGuIX® NPs were able to target tumor cells in the absence of specific surface functionalization, without evidence of toxicity. Finally, we validated the therapeutic potential of this hybrid theranostic AGuIX® NPs upon radiation exposure in this model. In conclusion, 3D cell cultures can efficiently mimic the normal and tumor-bearing airway epitheliums, providing an ethical and accessible model for the investigation of nebulized NPs.

Ultrafast Laser Processing of Nanostructured Patterns for the Control of Cell Adhesion and Migration on Titanium Alloy.

Femtosecond laser texturing is a promising surface functionalization technology to improve the integration and durability of dental and orthopedic implants. Four different surface topographies were obtained on titanium-6aluminum-4vanadium plates by varying laser processing parameters and strategies: surfaces presenting nanostructures such as laser-induced periodic surface structures (LIPSS) and 'spikes', associated or not with more complex multiscale geometries combining micro-pits, nanostructures and stretches of polished areas. After sterilization by heat treatment, LIPSS and spikes were characterized to be highly hydrophobic, whereas the original polished surfaces remained hydrophilic. Human mesenchymal stem cells (hMSCs) grown on simple nanostructured surfaces were found to spread less with an increased motility (velocity, acceleration, tortuosity), while on the complex surfaces, hMSCs decreased their migration when approaching the micro-pits and preferentially positioned their nucleus inside them. Moreover, focal adhesions of hMSCs were notably located on polished zones rather than on neighboring nanostructured areas where the protein adsorption was lower. All these observations indicated that hMSCs were spatially controlled and mechanically strained by the laser-induced topographies. The nanoscale structures influence surface wettability and protein adsorption and thus influence focal adhesions formation and finally induce shape-based mechanical constraints on cells, known to promote osteogenic differentiation.

Laser-Based Hybrid Manufacturing of Endosseous Implants: Optimized Titanium Surfaces for Enhancing Osteogenic Differentiation of Human Mesenchymal Stem Cells.

Additive manufacturing (AM) is becoming increasingly important in the orthopedic and dental sectors thanks to two major advantages: the possibility of custom manufacturing and the integration of complex structures. However, at smaller scales, surface conditions of AM products are not mastered. Numerous non-fused powder particles give rise to roughness values (Sa) greater than 10 µm, thus limiting biomedical applications since the surface roughness of, e.g., metal implants plays a major role in the quality and rate of osseointegration. In this study, an innovative hybrid machine combining AM and a femtosecond laser (FS) was used to obtain Ti6Al4V parts with biofunctional surfaces. During the manufacturing process, the FS laser beam "neatly" ablates the surface, leaving in its path nanostructures created by the laser/matter interaction. This step decreases the Sa from 11 to 4 µm and increases the surface wettability. The behavior of human mesenchymal stem cells was evaluated on these new AM+FS surfaces and compared with that on AM surfaces and also on polished surfaces. The number of cells attached 24 h after plating is equivalent on all surfaces, but cell spreading is higher on AM+FS surfaces compared with their AM counterparts. In the longer term (days 7 and 14), fibronectin and collagen synthesis increase on AM+FS surfaces as opposed to AM alone. Alkaline phosphatase activity, osteocalcin production, and mineralization, markers of osteogenic differentiation, are significantly lower on raw AM surfaces, whereas on the AM+FS specimens they display a level equivalent to that on the polished surface. Overall, these results indicate that using an FS laser beam during the fabrication of AM parts optimizes surface morphology to favor osteoblastic differentiation. This new hybrid machine could make it possible to produce AM implants with functional surfaces directly at the end of AM, thereby limiting their post-treatments.

Curvotaxis directs cell migration through cell-scale curvature landscapes.

Cells have evolved multiple mechanisms to apprehend and adapt finely to their environment. Here we report a new cellular ability, which we term "curvotaxis" that enables the cells to respond to cell-scale curvature variations, a ubiquitous trait of cellular biotopes. We develop ultra-smooth sinusoidal surfaces presenting modulations of curvature in all directions, and monitor cell behavior on these topographic landscapes. We show that adherent cells avoid convex regions during their migration and position themselves in concave valleys. Live imaging combined with functional analysis shows that curvotaxis relies on a dynamic interplay between the nucleus and the cytoskeleton-the nucleus acting as a mechanical sensor that leads the migrating cell toward concave curvatures. Further analyses show that substratum curvature affects focal adhesions organization and dynamics, nuclear shape, and gene expression. Altogether, this work identifies curvotaxis as a new cellular guiding mechanism and promotes cell-scale curvature as an essential physical cue.

RhoGTPase stimulation is associated with strontium chloride treatment to counter simulated microgravity-induced changes in multipotent cell commitment.

Microgravity-related cytoskeletal disorganization is associated with an altered balance between osteoblastogenesis and adipogenesis of multipotent cells. Strontium chloride is known to increase osteoblastogenesis and repress adipogenesis, but its effects in microgravity-related conditions have not been established. Our goal was to investigate early events in this process, focusing on RhoGTPases as controllers of cytoskeletal organization leading to stem cell commitment. We cultivated C3H10T1/2 on microspheres using a rotating wall vessel bioreactor (NASA) in order to simulate microgravity-related conditions in adipogenesis and osteoblastogenesis conditions independently. We observed that rotating wall vessel cultures presented increased adipogenesis, while osteoblastogenesis was reduced. Strontium-treated multipotent cells presented a significant repression in adipogenesis (-90 %, p < 0.001 PPARyD8) and an activation of osteoblastogenesis (+95 %, p < 0.001 bone sialoprotein and osteopontin D8), even in gravity altered conditions. We established that concomitant RhoA/Rac1 activations were associated with osteoblastogenesis enhancement and adipogenesis limitation in uncommitted cells. As vascular endothelial growth factor splicing is mechanosensitive and its signaling is central to stem cell commitment, we investigated vascular endothelial growth factor production, isoforms and receptors expressions in our conditions. We observed that vascular endothelial growth factor and receptors expressions were not significantly affected, but we found that presence of soluble vascular endothelial growth factor was associated with RhoA/Rac1 activations, whereas sequestration of vascular endothelial growth factor by cells was associated with RhoA/Rac1 inhibitions. We propose that strontium triggers secretion of vascular endothelial growth factor and the subsequent Rac1 and RhoA activations leading to repression of adipogenesis and osteogenesis stimulation validating strontium as a counter measure for microgravity-induced alteration of cell commitment.

TNAP stimulates vascular smooth muscle cell trans-differentiation into chondrocytes through calcium deposition and BMP-2 activation: Possible implication in atherosclerotic plaque stability.

Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PPi), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PPi, and PPi provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications.

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.

Stimulation of Bone Repair with Ultrasound.

This chapter reviews the different options available for the use of ultrasound in the enhancement of fracture healing or in the reactivation of a failed healing process: LIPUS, shock waves and ultrasound-mediated delivery of bioactive molecules, such as growth factors or plasmids. The main emphasis is on LIPUS, or Low Intensity Pulsed Ultrasound, the most widespread and studied technique. LIPUS has pronounced bioeffects on tissue regeneration, while employing intensities within a diagnostic range. The biological response to LIPUS is complex as the response of numerous cell types to this stimulus involves several pathways. Known to-date mechanotransduction pathways involved in cell responses include MAPK and other kinases signaling pathways, gap-junctional intercellular communication, up-regulation and clustering of integrins, involvement of the COX-2/PGE2 and iNOS/NO pathways, and activation of the ATI mechanoreceptor. Mechanisms at the origin of LIPUS biological effects remain intriguing, and analysis is hampered by the diversity of experimental systems used in-vitro. Data point to clear evidence that bioeffects can be modulated by direct and indirect mechanical effects, like acoustic radiation force, acoustic streaming, propagation of surface waves, heat, fluid-flow induced circulation and redistribution of nutrients, oxygen and signaling molecules. One of the future engineering challenge is therefore the design of dedicated experimental set-ups allowing control of these different mechanical phenomena, and to relate them to biological responses. Then, the derivation of an 'acoustic dose' and the cross-calibration of the different experimental systems will be possible. Despite this imperfect knowledge of LIPUS biophysics, the clinical evidence, although most often of low quality, speaks in favor of the clinical use of LIPUS, when the economics of nonunion and the absence of toxicity of this ultrasound technology are taken into account.

Femtosecond laser nano/micro patterning of titanium influences mesenchymal stem cell adhesion and commitment.

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.

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.