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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. Supplementary information The online version of this article (10.1007/s10237-020-01412-6) contains supplementary material, which is available to authorized users.

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. Summary 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.

International audience; Femtosecond laser texturing allows one to produce various surface relief ranging from nano- and micro ones to much more complex multi-scale pattern. In this context, the main advantages of using ultra-short laser pulses are known to be in a reduction of thermal effects, of debris, and of the surface contamination. Additionally, combinations of laser-induced micro- and nano- reliefs have strong capacities to alternate cell-surface interactions and even to guide cell behavior. Despite previously demonstrated link of cell behavior with wetting properties, the definition of the best laser treatment condition remains challenging. In particular, the difficulty arises from the change in surface wettability with time after laser treatment, but also due to annealing, sterilization, ultra-sound or cold plasma treatment. The reasons for these changes are not yet well understood. For this, femtosecond laser irradiation of titanium-based surfaces is used. As a result, multi-scale textures are produced with high precision. A series of simulations were also performed to determine the properties of these surfaces. Their wetting properties were tested and the capacities to guide stem cells were evaluated. Several templates with different sizes and motifs have been tested. In addition, human stem cell cultures (HSC) have been cultivated on several structured surfaces. The results reveal that HSCs preferentially place their nuclei in the pits with a diameter about the cell size, while the cell were looking for the hydrophilic areas for the attachment of their periphery. As a result, wetting maps can serve to predict cellular behavior. To better understand the involved mechanisms, wetting tests were performed for several surface reliefs and the results were compared to the results of our modeling. The obtained results are particular helpful in bioengineering, for cellular tests, for the treatment of various prosthesis, as well as of dental implants.

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.

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 &lsquo, spikes&rsquo, 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.

Introduction et objectifs Le cancer broncho-pulmonaire demeure un probleme de sante publique. Le gold standard du traitement est la chirurgie pouvant etre remplacee et/ou associee a de la radiotherapie. La contrainte principale etant les effets deleteres de la radiotherapie sur le tissu sain environnant. L’utilisation de nouveaux agents radio-sensibilisants de haut numero atomique tels que le gadolinium (Z = 64) offre un nouveau potentiel therapeutique, afin d’augmenter l’efficacite de l’irradiation, tout en limitant la dose locale. Les nanoparticules AGuIX® a base de gadolinium sont de nouveaux composes theranostiques (ciblage tumoral versus action radio-sensibilisante) dont il est crucial d’evaluer leur toxicite, ainsi que leur comportement cellulaire in vitro et in vivo [1] , [2] . Materiels et methodes Nous avons utilise des modeles innovants de culture 3D normaux (MucilAirTM, Epithelix) et tumoraux (OncoCilAirTM, OncoTheis), representatifs de l’epithelium respiratoire humain [3] , [4] . Ces modeles cellulaires ont ete exposes aux nanoparticules AGuIX® par des depots liquides en surface apicale, afin d’imiter une aerosolisation dans les voies respiratoires, ce qui represente une strategie prometteuse pour cibler les tumeurs broncho-pulmonaires [5] . Nous avons utilise une methodologie multiparametrique (microscopie versus cytometrie et ICP/Inductively Coupled Plasma Spectrometry) pour etudier leur bio-toxicite et internalisation. Resultats Les AGuIX® sont depourvues de toxicite et capables de cibler preferentiellement les cellules tumorales en l’absence d’un effet EPR (Enhanced Permeability Retention effect) decrit in vivo, et ceci sans une fonctionnalisation specifique de surface. Enfin, nous avons valide leur capacite radio-sensibilisante, lors d’une irradiation a 4 Gy dans le modele tumoral OncoCilAirTM. Conclusion Notre etude a permis de conforter le potentiel theranostique de ces nanoparticules AGuIX®. De plus, ces cultures humaines 3D MucilAirTM et OncoCilAirTM constituent des modeles in vitro de pointe permettant de s’affranchir eventuellement d’etudes in vivo ; et qui sont particulierement bien adaptes pour des investigations nano-toxicologiques et pre-cliniques en therapie ciblee.

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.

International audience; The contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 µN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 µN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue.

19-23 novembre 2018; In vivo les cellules sont en contact avec diverses caractéristiques topographiques qui couvrent plusieurs ordres de taille et d'organisation. L'intégration cellulaire de ces caractéristiques topographiques affecte de multiples aspects de la physiologie de la cellule. Un grand nombre d'études ont mis en évidence que les cellules sont sensibles aux nanotopographies ou aux structures géométriques à l'échelle des cellules. Les biotopes naturels présentent également des caractéristiques topographiques beaucoup plus grandes qui sont souvent courbées et lisses, tels que les parois des vaisseaux sanguins, les cavités des cellules osseuses, les acini ou d'autres corps cellulaires. On sait très peu de choses sur la façon dont les cellules lisent et intègrent les courbures à leur propre échelle et sur les mécanismes menant à l'intégration de tels signaux physiques. Nous rapportons ici un nouveau sens cellulaire que nous appelons «curvotaxie» qui permet aux cellules de réagir aux variations de courbure à l'échelle cellulaire, un trait omniprésent des biotopes cellulaires. Nous développons des surfaces 3D sinusoïdales ultra-lisses présentant des modulations de courbure dans toutes les directions, et contrôlons le comportement des cellules sur ces paysages topographiques simplifiés. Nous montrons que les cellules évitent les régions convexes pendant leur migration et se positionnent dans des vallées concaves. La modélisation computationnelle combinée à l'analyse fonctionnelle et à l'imagerie en direct montre que la curvotaxie repose sur une interaction dynamique entre le noyau et le cytosquelette de la cellule. D'autres analyses montrent que la courbure du substrat affecte la forme du noyau cellulaire, les tensions intracellulaires, l'organisation et la dynamique des adhésions focales et l'expression des gènes. Au total, ce travail identifie la curvotaxie comme un nouveau mécanisme de guidage et qui favorise la courbure à l'échelle de la cellule comme un signal physique essentiel entièrement intégré par les cellules. Mots clés : biomatériau, cellules, topographie

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, Countering Bone Loss in Space A chemical element naturally found for instance in seafood or grains, could counter bone loss from long-term spaceflight. Alain Guignandon and colleagues from the Université de Lyon à St-Etienne in France exposed multipotent embryonic fibroblasts to microgravity conditions similar to those found in space. They found the balance shifted in these stem cells from differentiating to bone-forming cells (osteoblasts) to differentiating to fatty-tissue forming cells (adipocytes). When the cells were treated with strontium, the shift toward osteoblastogenesis was regained. Strontium achieves this by sustaining the activity of two proteins that play a role in bone development but are suppressed in space. Strontium’s effect on the proteins could happen via release of vascular endothelial growth factor, which, under normal gravity conditions, plays a role in committing the cell to differentiation into osteoblasts rather than adipoyctes.

Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [−50% in silenced, scrambled (SiScr) controls vs. −15% for SiRac1], the number of F-actin fibers (−60% in SiScr controls vs. −10% for SiRac1), and the depletion of matrix-bound VEGF (−40% in SiScr controls vs. −8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects.—Guignandon, A., Faure, C., Neutelings, T., Rattner, A., Mineur, P., Linossier, M.-T., Laroche, N., Lambert, C., Deroanne, C., Nusgens, B., Demets, R., Colige, A., Vico, L. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells.

HSVEC behavior under physiological shear stress in vitro is investigated on PET surfaces micropatterned with both RGDS and WQPPRARI peptides. This technique allows (i) creating geometries on surface to guide cell orientation under shear stress and (ii) controlling surface chemical composition in order to modulate cell behavior. Under shear stress, endothelial cells adhere on patterned PET surfaces and present a more rapid orientation in flow direction in comparison to cells cultured on homogeneous surfaces. Micropatterned surfaces presenting a large surface area ratio of RGDS/WQPPRARI peptides induce fibrillar adhesion, while surfaces presenting an equal RGDS/WQPPRARI peptides surface area ratio preferentially induce focal adhesion.

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.

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. © 2011 American Society for Bone and Mineral Research

The effects of low-magnitude, high-frequency (LMHF) mechanical stimulation on osteoblastic cells are poorly understood. We have developed a system that generates very small (15-40 μe), high-frequency (400 Hz, sine) deformations on osteoblast cultures (MC3T3-E1). We investigated the effects of these LMHF stimulations mainly on extracellular matrix (ECM) synthesis. The functional properties of this ECM after decellularization were evaluated on C3H10T1/2 mesenchymal stem cells (MSCs). LMHF stimulations were applied 20 min once daily for 1, 3, or 7 days in MC3T3-E1 culture (1, 3, or 7 dLMHF). Cell number and viability were not affected after 3 or 7 dLMHF. Osteoblast response to LMHF was assessed by an increase in nitric oxide secretion, alteration of the cytoskeleton, and focal contacts. mRNA expression for fibronectin, osteopontin, bone sialoprotein, and type I collagen in LMHF cultures were 1.8-, 1.6-, 1.5-, and 1.7-fold higher than controls, respectively (P < 0.05). In terms of protein, osteopontin levels were increased after 3 dLMHF and ECM organization was altered as shown by fibronectin topology after 7 dLMHF. After decellularization, 7 dLMHF-ECM or control ECM was reseeded with MSCs. Seven dLMHF-ECM improved early events such as cell attachment (2 h) and focal contact adhesion (6 h) and, later (16 h), modified MSC morphological parameters. After 5 days in multipotential medium, gene-expression changes indicated that 7 dLMHF-ECM promoted the expression of osteoblast markers at the expense of adipogenic marker. LMHF stimulations of osteoblasts are therefore efficient and sufficient to generate osteogenic matrix.

Plasticity is the ability to give rise to cell types whose phenotype is different from that of the source tissue. Osteoblasts originate in progenitors located in the bone marrow or around blood vessels. Marrow stromal cells can differentiate into adipocytes, in part at the expense of osteoblasts. The osteoblast–adipocyte balance is influenced by systemic factors, chiefly hormones, and local factors in the microenvironment, as well as by mechanical loads, which induce or suppress the activity of transcription factors crucial to the differentiation of each cell type. New insights into the molecular mechanisms involved in controlling the osteoblast–adipocyte balance are unlocking doors to a vast array of innovative treatment strategies.

Resume La plasticite cellulaire est la capacite de certaines cellules a se differencier en types cellulaires dont le phenotype est different, voire sans rapport avec celui de leur tissu d'origine. Les osteoblastes proviennent de la differenciation de progeniteurs qui sont situes dans la moelle osseuse ou a la peripherie des vaisseaux. Les cellules stromales de moelle peuvent se differencier, entre autres, en adipocytes en partie aux depens des osteoblastes. Des facteurs systemiques le plus souvent hormonaux, ou locaux, apportes par le microenvironnement et les contraintes mecaniques sont capables de moduler la balance osteoblaste/ adipocytes en induisant ou reprimant l'activite de facteurs de transcription necessaires a leur differenciation. Une meilleure connaissance des bases moleculaires de ce controle ouvre de nombreuses perspectives therapeutiques.

Because a lack of mechanical information favors the development of adipocytes at the expense of osteoblasts, we hypothesized that the peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent balance between osteoblasts and adipocytes is affected by mechanical stimuli. We tested the robustness of this hypothesis in in vivo rodent osteogenic exercise, in vitro cyclic loading of cancellous haversian bone samples, and cyclic stretching of primary stromal and C3H10T1/2 cells. We found that running rats exhibit a decreased marrow fat volume associated with an increased bone formation, presumably through recruitment of osteoprogenitors. In the tissue culture model and primary stromal cells, cyclic loading induced higher Runx2 and lower PPARgamma2 protein levels. Given the proadipocytic and antiosteoblastic activities of PPARgamma, we studied the effects of cyclic stretching in C3H10T1/2 cells, treated either with the PPARgamma activator, Rosiglitazone, or with GW9662, a potent antagonist of PPARgamma. We found, through both cytochemistry and analysis of lineage marker expression, that under Roziglitazone cyclic stretch partially overcomes the induction of adipogenesis and is still able to favor osteoblast differentiation. Conversely, cyclic stretch has additive effects with GW9662 in inducing osteoblastogenesis. In conclusion, we provide evidence that mechanical stimuli are potential PPARgamma modulators counteracting adipocyte differentiation and inhibition of osteoblastogenesis.

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.

Whole body vibration (WBV) is a promising tool for counteracting bone loss. Most WBV studies on animals have been performed at acceleration

A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions.

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.

We showed that cyclic strain (CS) of osteoblastic cells induced tyrosine phosphorylation of two homologous tyrosine kinases FAK and PYK2, and of two homologous adaptor proteins paxillin and Hic5, with similar kinetics. Immunostaining showed that all four proteins were localized to focal contacts in controls. In contrast, the dynamics of their subcellular localization observed after CS differed. While FAK and paxillin remained at the focal contact, Hic-5 and PYK2 translocated outside ventral focal contacts as early as 30 min after CS and were sequestered by the cytoskeleton. Co-immunoprecipitation showed that the association of PYK2/Hic-5 and PYK2/FAK increased with time after strain while that of paxillin and Hic-5 decreased. Altogether these results suggested that CS regulates focal contact activity in osteoblasts by modulating PYK2-containing complexes in particular by shuttling out of the focal contact the adaptor Hic-5 and favoring the anchorage of FAK within contacts.

The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.

We compared quantitatively vinculin-related adhesion parameters in osteoblastic cells submitted to opposite mechanical stresses, i.e., low deformation and frequency strain regimens (stretch condition) and microgravity exposure (relaxed condition). Cyclic deformation induced a biphasic response comprising new focal contacts formation followed by their clustering in ROS cells. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. We previously demonstrated that 1% cyclic deformations at 0.05 Hz during a daily 10 min episode over 7 days stimulated ROS 17/2.8 growth as compared to static culture whereas relaxed ROS proliferated similarly to static culture (BC). To evaluate whether the proliferation (stretch) or the survival (relaxed) status of ROS cells influences focal contact organization, we inhibited ERKs proliferative-dependent pathway. Inhibition of proliferation by PD98059 was overcome although not fully restored by stretch. Furthermore stretch-induced clustering of vinculin-positive contacts still occurs in the presence of ERKs inhibitor, whereas the increase in focal contact number is abolished. In conclusion, we showed that focal contacts are mechanoeffectors and that hyper-mechanical stimulation could up regulate focal contacts size as compared to hypo-mechanical that down regulate clusterization.

Summary 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.

HSVEC behavior under physiological shear stress in vitro is investigated on PET surfaces micropatterned with both RGDS and WQPPRARI peptides. This technique allows (i) creating geometries on surface to guide cell orientation under shear stress and (ii) controlling surface chemical composition in order to modulate cell behavior. Under shear stress, endothelial cells adhere on patterned PET surfaces and present a more rapid orientation in flow direction in comparison to cells cultured on homogeneous surfaces. Micropatterned surfaces presenting a large surface area ratio of RGDS/WQPPRARI peptides induce fibrillar adhesion, while surfaces presenting an equal RGDS/WQPPRARI peptides surface area ratio preferentially induce focal adhesion.

International audience; Analysis of bone microvascularization has generally been performed from 2D histology. The method proposed in this study enables for the first time to simultaneously analyze, in 3D, the microvascularization and bone microstructure in a rat model. The method is based on the use of quantitative synchrotron micro-computed tomography (SR-μCT) coupled to an automatic image analysis procedure. It was applied to investigate the effect of intermittent parathyroid hormone (PTH) administration on angiogenesis and osteogenesis in rats. Rats were posthumously injected with a contrast agent and subsequently imaged. The algorithm allowed the reconstruction and the segmentation of both bone microstructure and microvascularization in cortical and trabecular regions. A large set of 3D quantitative parameters were then extracted from the bone and vascular networks. In particular, we propose a new parameter, utilizing the availability of both microstructures to relate the two, which we dub the vascular-trabecular interdistance (VTI). Due to the short acquisition times of SR-μCT and the efficiency of the image analysis algorithm, a large data set was analyzed, which permitted statistical analysis of the measured parameters. Statistical analysis confirmed that treatment with PTH significantly modulated several bone and vessel parameters, including the VTI.