E Potier

J Noailly

K Ito

Directing bone marrow-derived stromal cell function with mechanics

Keywords: Mechanoregulation, Bone marrow-derived stromal cells, Mesenchymal stem cells, Mechanical stimulation

Because bone marrow-derived stromal cells (BMSCs) are able to generate many cell types, they are envisioned as source of regenerative cells to repair numerous tissues, including bone, cartilage, and ligaments. Success of BMSC-based therapies, however, relies on a number of methodological improvements, among which better understanding and control of the BMSC differentiation pathways. Since many years, the biochemical environment is known to govern BMSC differentiation, but more recent evidences show that the biomechanical environment is also directing cell functions. Using in vitro systems that aim to reproduce selected components of the in vivo mechanical environment, it was demonstrated that mechanical loadings can affect BMSC proliferation and improve the osteogenic, chondrogenic, or myogenic phenotype of BMSCs. These effects, however, seem to be modulated by parameters other than mechanics, such as substrate nature or soluble biochemical environment. This paper reviews and discusses recent experimental data showing that despite some knowledge limitation, mechanical stimulation already constitutes an additional and efficient tool to drive BMSC differentiation.

Introduction

Over the last decades, tissue engineering has been proposed as a method to repair or regenerate a growing number of tissues and organs (skin, bone, heart, pancreas, etc.). This therapeutic strategy consists of transplantation of ex-vivo expanded regenerative cells, sometimes associated with a biomaterial scaffold and/or molecular agents for tissue reconstruction. Many cell types are envisioned as source for those regenerative cells: from organ/tissue specific cells (chondrocytes, skin fibroblast, etc.) to totipotent embryonic stem cells [START_REF] Muschler | Engineering principles of clinical cell-based tissue engineering[END_REF]. One of the most promising sources of regenerative cells, however, might be the bone marrow. Evidence that bone marrow contains cells having an important proliferation capacity and being able to form bone and cartilage was first showed by [START_REF] Friedenstein | The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells[END_REF] in the late 60s. Since then, many studies have established that these cells can differentiate in vitro into chondrocytes, osteoblasts, adipocytes, and myoblasts [START_REF] Chamberlain | Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing[END_REF][START_REF] Krampera | Mesenchymal stem cells for bone, cartilage, tendon and skeletal muscle repair[END_REF] (Fig. 1A). They are currently referred either as bone marrow-derived mesenchymal stem cells (as they can give rise to different mesenchymal tissues) or bone marrow-derived stromal cells (as they are isolated from the supportive structure (stroma) of the bone marrow, BMSCs). Multipotent BMSCs constitute one of the most promising sources of regenerative cells, because, as adult cells, they (i) can be harvested from the patient, minimizing risks of rejection and pathogen transmission; (ii) possess high proliferate rates, allowing the implantation of a large number of regenerative cells; and (iii) are able to give rise to several differentiated cell types, expanding the number of potential applications.

As a consequence, they have been used in numerous animal models to repair bone defects [START_REF] Kadiyala | Cultureexpanded, bone marrow-derived mesenchymal stem cells can regenerate a critical sized segmental bone defect[END_REF][START_REF] Kon | Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones[END_REF]Petite et al., 2000), cartilage lesions [START_REF] Guo | Repair of large articular cartilage defects with implants of autologous mesenchymal stem cells seeded into beta-tricalcium phosphate in a sheep model[END_REF][START_REF] Uematsu | Cartilage regeneration using mesenchymal stem cells and a threedimensional poly-lactic-glycolic acid (PLGA) scaffold[END_REF], and infarcted hearts [START_REF] Dai | Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short-and long-term effects[END_REF][START_REF] Fazel | Cell transplantation preserves cardiac function after infarction by infarct stabilization: augmentation by stem cell factor[END_REF][START_REF] Tomita | Autologous transplantation of bone marrow cells improves damaged heart function[END_REF]. Few clinical studies, however, have been conducted to date: BMSC transplantation has been shown to be capable of increasing bone mass in children with osteogenesis imperfecta [START_REF] Horwitz | Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta[END_REF], repairing large bone defect [START_REF] Quarto | Repair of large bone defects with the use of autologous bone marrow stromal cells[END_REF], reconstructing a distal phalanx [START_REF] Vacanti | Replacement of an avulsed phalanx with tissue-engineered bone[END_REF], improving cardiac function after acute myocardial infarction [START_REF]Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction[END_REF], and repairing cartilage defects in osteoarthritis patients [START_REF] Wakitani | Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees[END_REF]. Although these results are promising, they were not able to establish that tissue engineering approaches were superior to standard treatments. But, it is the general belief that with further advancement, tissue engineering protocols will eventually be helpful in clinics.

To improve such protocols, it is important to understand and control the pathways guiding uncommitted BMSCs to become differentiated and functional cells. Since many years the differentiation of BMSCs has been induced by various cocktails of biochemical agents and growth factors, such as dexamethasone, βglycerophophate and ascorbic acid for osteogenesis [START_REF] Jaiswal | Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro[END_REF][START_REF] Pittenger | Multilineage potential of adult human mesenchymal stem cells[END_REF] or serum free medium and transforming growth factor β (TGFβ) for chondrogenesis [START_REF] Johnstone | In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells[END_REF][START_REF] Pittenger | Multilineage potential of adult human mesenchymal stem cells[END_REF]. More recently several studies have established that the cell mechanical environment can also play a role in cell function and that it is possible to control BMSC proliferation and differentiation with mechanical loadings. This paper reviews recent published works addressing the mechanical regulation and stimulation of BMSC proliferation and differentiation. Parameters eventually affecting cell response to mechanical loadings are also discussed.

Stimulating cells

In vivo Mechanobiology

Starting from studies of the late 19th century on bone internal structure [START_REF] Meyer | Die Architectur der Spongiosa[END_REF], the idea that body, organ, or tissue structure is associated with its mechanical environment or function has become widely accepted. More recently, investigations have also explored in vivo mechanical adaptation of soft connective tissues, such as articular cartilage [START_REF] King | Cyclical articular joint leads to cartilage thinning and osteopontin production in a novel in vivo rabbit model of repetitive finger flexion[END_REF][START_REF] Saadat | Long-term cyclical in vivo loading increases cartilage proteoglycan content in a spatially specific manner: an infrared microspectroscopic imaging and polarized light microscopy study[END_REF][START_REF] Wong | Articular cartilage functional histomorphology and mechanobiology: a research perspective[END_REF], intervertebral disc [START_REF] Brickley-Parson | Is the chemistry of collagen in the intervertebral discs an expression of Wolff's law? A study of the human lumbar spine[END_REF][START_REF] Maclean | In vivo intervertebral disc remodeling: kinetics of mRNA expression in response to a single loading event[END_REF][START_REF] Stokes | Intervertebral disc adaptation to wedging deformation[END_REF], circulatory system tissues [START_REF] Ben Driss | Arterial expansive remodeling induced by high flow rates[END_REF][START_REF] Opitz | Phenotypical plasticity of vascular smooth muscle cells-effect of in vitro and in vivo shear stress for tissue engineering of blood vessels[END_REF][START_REF] Van Gieson | Enhanced smooth muscle cell coverage of microvessels exposed to increased hemodynamic stress in vivo[END_REF]Lehoux, 2006), and tendons and ligaments [START_REF] Hayashi | Biomechanical studies of the remodeling of knee joint tendons and ligaments[END_REF]. Many of these investigations have suggested an association of cell shape, tissue composition and micro-structure to different types of mechanical environments. Although in vivo mechanical environments result from geometryand muscle activity-dependent external loads that are not definitively known, some general patterns have been identified. Much experimental evidences suggest that, among other extracellular matrix (ECM) components, production of proteoglycans by chondrocytes is mostly stimulated by cyclic hydrostatic pressures, collagen type I produced by fibroblasts, chondrocytes, or smooth muscle cells usually appears in direct, shear-induced, or pressure-induced (vessels) tensile environments, and collagen type II production by chondrocytes or fibroblasts is rather affected by compressive loads.

In vivo remodeling of tendons and ligaments has been extensively investigated and illustrates perfectly the natural changes occurring in soft tissues and associated cells in response to different loadings. Under in vivo altered mechanical loadings, complete or partial phenotype changes from ligament to bone have been observed [START_REF] Tsukamoto | Repetitive tensile stress to rat caudal vertebrae inducing cartilage formation in the spinal ligaments: a possible role of mechanical stress in the development of ossification of the spinal ligaments[END_REF]. Under normal physiological loadings, entheses or wrap-around tendons are a good illustration of how gradients in cell and tissue phenotypes, i.e. from fibrous to fibro-cartilaginous, can be closely related to gradients in local mechanical environments, i.e. from tensile to compressive [START_REF] Matyas | Stress governs tissue phenotype at the femoral insertion of the rabbit MCL[END_REF]Benjamin and Ralphs, 1998). Entheses link purely fibrous to calcified tissues and are believed to act as dissipaters to progressively reduce the strain energy density from the strong but deformable tendons/ligaments to the stiff but less deformable adjacent bone [START_REF] Shaw | Structurefunction relashionships of entheses in relation to mechanical load and exercise[END_REF]. At given points of this transition, cell phenotype and ECM were shown to be partly controlled by the local mechanical environment [START_REF] Kotani | The effect of spinal fixation and destabilization on the biomechanical and histologic properties of spinal ligaments[END_REF][START_REF] Benjamin | Where tendons and ligaments meet bone: attachment sites ('entheses') in relation to exercise and/or mechanical load[END_REF]. Although the origin of enthesis cells is still a matter for discussion, progenitor cells contained in the endotenon (connective tissue between the strands in a tendon) and/or in the adjacent bone marrow have been proposed as precursors [START_REF] Benjamin | Where tendons and ligaments meet bone: attachment sites ('entheses') in relation to exercise and/or mechanical load[END_REF]. Note that such assumption has been partly raised by the improved healing of chondral entheses after implantation of tendon grafts covered with BMSCs [START_REF] Lim | Enhancement of tendon grafts osteointegration using mesenchymal stem cells in a rabbit model of anterior cruciate ligament reconstruction[END_REF].

Healing of cartilage defects in knee joints, including subchondral bone disruption, is also believed to involve progenitor cells [START_REF] Appleton | Forced mobilization accelerates pathogenesis: characterization of a preclinical surgical model of osteoarthritis[END_REF][START_REF] Metsäranta | Evidence for insufficient chondrocytic differentiation during repair of full-thickness defects of articular cartilage[END_REF][START_REF] Buckwalter | Joint injury, repair, and remodeling. Roles in post-traumatic osteoarthritis[END_REF]. In such cases, tissue mechanical environment was shown to significantly influence the type of produced ECM. An in vivo study on skeletally mature monkeys showed that production of collagen type II and hyaline tissue repair were higher with imposed intermittent passive flexion of the knee than with cast immobilization [START_REF] Buckwalter | Osteochondral repair of Primate knee femoral and patellar articular surfaces: implications for preventing post-traumatic osteoarthritis[END_REF]. However, repaired cartilage obtained under motion had lower aggregate modulus and higher permeability than undamaged articular cartilage. Actually, with cast immobilization, muscles are still tonic and maintain the tissue under compression, while passive flexion motion increases shear deformations in the damaged tissue. Thus, at short term, cast immobilization most likely preserved a higher relative amount of hydrostatic pressure, known to favor glycosaminoglycan (GAG) synthesis, largely responsible for the properties of normal articular cartilage [START_REF] Katta | The effect of glycosaminoglycan depletion on the friction and deformation of articular cartilage[END_REF][START_REF] Wong | Articular cartilage functional histomorphology and mechanobiology: a research perspective[END_REF].

In bone healing or distraction osteogenesis, progenitor cells are also suspected to play a key role, through successive formation of fibrous, cartilaginous, and osseous tissues, controlled by the local mechanical environment. Such hypothesis has been successfully used in different theoretical models that have been able to predict, more or less phenomenologically, in vivo bone fracture healing [START_REF] Isaksson | A mechano-regulatory bone-healing model incorporating cellphenotype specific activity[END_REF][START_REF] Lacroix | A mechano-regulation model for tissue differentiation during fracture healing: analysis of gap size and loading[END_REF], loaddependent tissue growth and remodeling [START_REF] García-Aznar | Computational simulation of fracture healing: influence of interfragmentary movement on the callus growth[END_REF][START_REF] Isaksson | Remodeling of fracture callus in mice is consistent with mechanical loading and bone remodeling theory[END_REF], and cartilage repair [START_REF] Kelly | Mechanoregulation of stem cell differentiation and tissue regeneration in osteochondral defects[END_REF]. In all these models, predicted phenotypes were mainly controlled by deviatoric strains and parameters associated with volumetric strains, e.g. fluid flow.

In vitro systems

Most of the studies looking at BMSC phenotype modulation by mechanical means have aimed to reproduce in vitro one component of the in vivo mechanical environments under highly controlled conditions. To improve chondrogenic differentiation, for instance, BMSCs have been exposed to hydraulic pressure, trying to reproduce the hydrostatic stress experienced by cartilage. To this end, BMSCs have been put under hydrostatic pressure [START_REF]Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro[END_REF][START_REF] Finger | Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure[END_REF][START_REF] Miyanishi | Dose-and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro[END_REF][START_REF] Scherer | The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro[END_REF]. They have also been seeded into hydrogels (agarose [START_REF] Campbell | Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells[END_REF][START_REF] Huang | Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[END_REF][START_REF] Mauck | Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading[END_REF][START_REF] Mouw | Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells[END_REF], fibrin [START_REF] Pelaez | Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds. Stem cells and Development[END_REF], hyaluronan/gelatin [START_REF] Ito | Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds[END_REF], etc.) and exposed to carrier compression [START_REF] Ito | Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds[END_REF][START_REF] Campbell | Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells[END_REF][START_REF] Huang | Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[END_REF][START_REF] Mauck | Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading[END_REF][START_REF] Mouw | Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells[END_REF][START_REF] Pelaez | Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds. Stem cells and Development[END_REF][START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF] (Fig. 2B). It is difficult to compare these two types of mechanical stimulation. Hydrostatic pressure is a single component of the compressive environment in cartilage. In contrast, carrier compression involves a mixture of codependent pressure, strain, fluid flow, and gradients, and is more similar to the in vivo mechanical environment of loaded articular cartilage. They both, however, led to equivalent improvement of chondrogenic differentiation of BMSCs, with increased expression of early markers [START_REF] Finger | Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure[END_REF][START_REF] Huang | Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading[END_REF][START_REF] Miyanishi | Dose-and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro[END_REF][START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF] and enhanced deposition of cartilaginous ECM [START_REF]Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro[END_REF][START_REF] Ito | Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds[END_REF][START_REF] Mauck | Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading[END_REF][START_REF] Miyanishi | Dose-and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro[END_REF][START_REF] Mouw | Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells[END_REF][START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF].

To drive osteogenesis, BMSCs have been exposed to substrate strain, mimicking bone matrix deformation during loading (gait, motions, etc.) by scaffold stretching resulting in uniaxial (Fig. 2C) [START_REF] Byrne | Gene expression by marrow stromal cells in a porous collagenglycosaminoglycan scaffold is affected by pore size and mechanical stimulation[END_REF][START_REF] Diederichs | Effects of repetitive and short time strain in human bone marrow stromal cells[END_REF][START_REF] Haasper | Cyclic strain induces FosB and initiates osteogenic differentiation of mesenchymal cells[END_REF][START_REF] Jagodzinski | Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells[END_REF] or bi-or equi-axial strain (Fig. 2D) [START_REF] Friedl | Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro[END_REF][START_REF] Mauney | Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro[END_REF][START_REF] Qi | Mechanical strain induces osteogenic differentiation: Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells[END_REF][START_REF] Simmons | Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway[END_REF][START_REF] Sumanasinghe | Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression[END_REF][START_REF] Thomas | Bone marrow stromal cells are load responsive in vitro[END_REF][START_REF] Ward | Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[END_REF][START_REF] Yoshikawa | Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation[END_REF]. Scaffold strains resulted in up-regulation of early markers of osteogenic differentiation [START_REF] Friedl | Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro[END_REF][START_REF] Haasper | Cyclic strain induces FosB and initiates osteogenic differentiation of mesenchymal cells[END_REF][START_REF] Qi | Mechanical strain induces osteogenic differentiation: Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells[END_REF][START_REF] Ward | Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[END_REF], as well in improved deposition of bone proteins and calcification of the ECM [START_REF] Diederichs | Effects of repetitive and short time strain in human bone marrow stromal cells[END_REF][START_REF] Mauney | Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro[END_REF][START_REF] Simmons | Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway[END_REF][START_REF] Ward | Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[END_REF][START_REF] Yoshikawa | Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation[END_REF].

Some studies have also explored the effects of shear stress on osteoblastic differentiation of BMSCs (Kreke and Goldstein, 2004;[START_REF] Kreke | Effect of intermittent shear stress on mechanotransductive signaling and osteoblastic differentiation of bone marrow stromal cells[END_REF][START_REF] Li | Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation[END_REF]) (Fig. 2A). Current hypotheses propose that bone matrix deformation during gait results in the oscillatory movement of interstitial fluid inside the canalicular network, resulting in application of shear stress to bone cell processes. Finally, flow perfusion of 3D porous scaffolds has also been used to induce osteogenesis. Though the primary reason to use such systems was to improve nutrient supply and waste removal in thick scaffolds, flow perfusion results in BMSC exposure to (uncontrolled and heterogenous) shear stress [START_REF] Rodan | Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner[END_REF][START_REF] Goldstein | Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds[END_REF]Holtorf et al., 2005a;[START_REF] Meinel | Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow[END_REF][START_REF] Sikavitsas | Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor[END_REF][START_REF] Sikavitsas | Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces[END_REF][START_REF] Stiehler | Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells[END_REF][START_REF] Zhao | Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: experiments and hydrodynamic modeling[END_REF] (Fig. 2A). This shear stress has been showed to have an effect by itself. Increased viscosity of the perfused medium, resulting in larger shear stress at the same solute flow rates, enhanced calcium deposition by rat BMSCs [START_REF] Sikavitsas | Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces[END_REF]. Applying shear stress to BMSCs, either controlled (fluid flow) or uncontrolled (flow perfusion), led to increased expression of early markers [START_REF] Jagodzinski | Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells[END_REF], Jagodzinski et al., 2008;[START_REF] Scaglione | Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system[END_REF][START_REF] Stiehler | Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells[END_REF] and improved osteogenic maturation of the ECM [START_REF] Rodan | Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner[END_REF]Holtorf et al., 2005a;[START_REF] Jagodzinski | Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture[END_REF][START_REF] Kreke | Effect of intermittent shear stress on mechanotransductive signaling and osteoblastic differentiation of bone marrow stromal cells[END_REF][START_REF] Kreke | Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner[END_REF]Kreke and Goldstein, 2004;[START_REF] Meinel | Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow[END_REF][START_REF] Sikavitsas | Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces[END_REF][START_REF] Zhao | Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: experiments and hydrodynamic modeling[END_REF].

Finally, substrate strain (uniaxial [START_REF] Kurpinski | Anisotropic mechanosensing by mesenchymal stem cells[END_REF][START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF][START_REF] Engelmayr | Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: implications for engineered heart valve tissues[END_REF][START_REF] Gong | Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs)[END_REF][START_REF] Hamilton | Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications[END_REF][START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF][START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF]; Figs. 2C andD) has also been used to improve BMSC differentiation into vascular smooth muscle cells (VSMCs) for vascular tissue repair [START_REF] Engelmayr | Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: implications for engineered heart valve tissues[END_REF][START_REF] Hamilton | Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications[END_REF][START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF][START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF], as these tissues mainly experience circumferential traction.

Affecting orientation

One of the first cell functions affected by mechanical loading is cell spreading. Substrate strain, indeed, has been shown to induce elongation and alignment of BMSCs [START_REF] Hamilton | Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications[END_REF][START_REF] Kurpinski | Anisotropic mechanosensing by mesenchymal stem cells[END_REF][START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF][START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF]. The orientation of such alignment, however, was reported to be perpendicular [START_REF]Effects of cyclic mechanical stretching on the mRNA expression of tendon/ligament-related and osteoblast-specific genes in human mesenchymal stem cells[END_REF][START_REF] Hamilton | Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications[END_REF][START_REF] Kurpinski | Anisotropic mechanosensing by mesenchymal stem cells[END_REF][START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF] as well as parallel [START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF] to the direction of the applied strain. These differences may be explained by the different substrates used: 2D collagen type I coated substrates [START_REF] Hamilton | Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications[END_REF][START_REF] Kurpinski | Anisotropic mechanosensing by mesenchymal stem cells[END_REF][START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF] versus 3D fibrin gels [START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF]. In 2D systems, BMSCs are hypothesized to re-orientate to minimize stretch forces applied to the cell bodies [START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF]. In stretched 3D gels, elongation and orientation of the pores in the strain direction may impede BMSC re-orientation perpendicular to this strain.

Controlling proliferation

Mechanical loadings can also affect cell proliferation, although their exact effects are still unclear. Many types of mechanical stimulations have been, indeed, shown to improve (Fig. 3) [START_REF] Rodan | Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner[END_REF]Holtorf et al., 2005a;[START_REF] Jagodzinski | Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture[END_REF][START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF][START_REF] Pelaez | Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds. Stem cells and Development[END_REF][START_REF] Qi | Mechanical strain induces osteogenic differentiation: Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells[END_REF][START_REF] Riddle | Chemotransport contributes to the effect of oscillatory fluid flow on human bone marrow stromal cell proliferation[END_REF][START_REF] Sikavitsas | Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces[END_REF][START_REF] Song | Mechanical stretch promotes proliferation of rat bone marrow mesenchymal stem cells. Colloids and Surfaces[END_REF][START_REF] Yoshikawa | Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation[END_REF] as well as to decrease [START_REF] Diederichs | Effects of repetitive and short time strain in human bone marrow stromal cells[END_REF][START_REF] Gong | Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs)[END_REF][START_REF] Hamilton | Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications[END_REF][START_REF] Simmons | Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway[END_REF][START_REF] Zhao | Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: experiments and hydrodynamic modeling[END_REF] BMSC growth. Mechanical loadings have also been reported to have no effects on cell proliferation [START_REF]Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro[END_REF][START_REF] Goldstein | Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds[END_REF][START_REF] Huang | Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[END_REF]Kreke and Goldstein, 2004;[START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF][START_REF] Thomas | Bone marrow stromal cells are load responsive in vitro[END_REF]. These findings seem independent from the type of stimulation (shear stress, compressive load, or substrate stretch), the cell species (human, rat, or goat), or the cell culture medium (containing FBS, dexamethasone, osteogenic supplement, or TGFβ) used. Nevertheless, the diversity of the in vitro systems (2D, 3D, substrate nature, cell species, medium, etc.) and of the loading protocols (duration, frequency, amplitude, time in static conditions before loading, etc.) may explain this wide variety in responses.

Driving differentiation

Beyond the question of proliferation, one of the main goals of mechanobiology applied to BMSCs is to drive and to improve their differentiation in mature cells in order to ultimately enhance the mechanical and the regenerative properties of engineered tissues.

Osteogenic pathway (Fig. 1B)

The application of flow perfusion [START_REF] Jagodzinski | Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture[END_REF][START_REF] Stiehler | Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells[END_REF], fluid flow [START_REF] Scaglione | Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system[END_REF], and scaffold strechting [START_REF] Ward | Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[END_REF][START_REF] Haasper | Cyclic strain induces FosB and initiates osteogenic differentiation of mesenchymal cells[END_REF][START_REF] Jagodzinski | Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells[END_REF][START_REF] Qi | Mechanical strain induces osteogenic differentiation: Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells[END_REF] stimulates the gene expression levels of two early osteogenic markers: osterix (Osx) and runt-related transcription factor 2 (Runx2). Both are transcription factors required for bone formation [START_REF] Ducy | A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development[END_REF][START_REF] Nakashima | The novel zinc fingercontaining transcription factor osterix is required for osteoblast differentiation and bone formation[END_REF] and regulating commitment of undifferentiated BMSCs to the osteogenic lineage. Runx2 expression seems to be highly sensitive to loading amplitude with inhibited expression for low flow perfusion rates (0.1 vs 10 ml/min) [START_REF] Bjerre | Flow perfusion culture of human mesenchymal stem cells on silicatesubstituted tricalcium phosphate scaffolds[END_REF][START_REF] Jagodzinski | Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture[END_REF] and increased expression for high substrate strain (2% vs 8%) [START_REF] Haasper | Cyclic strain induces FosB and initiates osteogenic differentiation of mesenchymal cells[END_REF][START_REF] Jagodzinski | Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells[END_REF]. This inductive effect, however, can be abolished by application of high shear stress (0.001 vs 1 Pa) [START_REF] Li | Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation[END_REF][START_REF] Scaglione | Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system[END_REF]. Fluid flow, flow perfusion, hydrostatic pressure, and substrate stretching enhance the formation of an ECM through the regulation of gene [START_REF] Friedl | Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro[END_REF][START_REF] Haasper | Cyclic strain induces FosB and initiates osteogenic differentiation of mesenchymal cells[END_REF][START_REF] Jagodzinski | Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells[END_REF][START_REF] Kim | ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure[END_REF][START_REF] Kreke | Effect of intermittent shear stress on mechanotransductive signaling and osteoblastic differentiation of bone marrow stromal cells[END_REF][START_REF] Scaglione | Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system[END_REF][START_REF] Stiehler | Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells[END_REF] and protein [START_REF] Diederichs | Effects of repetitive and short time strain in human bone marrow stromal cells[END_REF] expression of collagen type I. Such loadings also improved the differentiation of BMSCs into osteoprogenitors by increasing their alkaline phosphatase (ALP) activity [START_REF] Rodan | Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner[END_REF][START_REF] Bjerre | Flow perfusion culture of human mesenchymal stem cells on silicatesubstituted tricalcium phosphate scaffolds[END_REF][START_REF] Friedl | Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro[END_REF][START_REF] Goldstein | Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds[END_REF]Holtorf et al., 2005b;Holtorf et al., 2005a;[START_REF] Jagodzinski | Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells[END_REF][START_REF] Kim | ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure[END_REF][START_REF] Mauney | Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro[END_REF][START_REF] Meinel | Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow[END_REF][START_REF] Sikavitsas | Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces[END_REF][START_REF] Stiehler | Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells[END_REF][START_REF] Zhao | Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats[END_REF]. This differentiation process can be observed as early as after 3 days but may require up to 20 days, with the fastest maturation observed under daily stretching conditions. Fluid flow, flow perfusion, scaffold stretching, and hydrostatic pressure also contribute to further maturation of BMSCs and ECM, by inducing gene [START_REF] Bjerre | Flow perfusion culture of human mesenchymal stem cells on silicatesubstituted tricalcium phosphate scaffolds[END_REF][START_REF] Jagodzinski | Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells[END_REF][START_REF] Kim | ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure[END_REF]Kreke and Goldstein, 2004;[START_REF] Li | Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation[END_REF][START_REF] Scaglione | Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system[END_REF] and protein [START_REF] Rodan | Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner[END_REF]Holtorf et al., 2005a;Holtorf et al., 2005b;[START_REF] Jagodzinski | Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture[END_REF][START_REF] Kim | ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure[END_REF][START_REF] Kreke | Effect of intermittent shear stress on mechanotransductive signaling and osteoblastic differentiation of bone marrow stromal cells[END_REF][START_REF] Yoshikawa | Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation[END_REF] expression of osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP) (non collagenous bone proteins, playing roles in matrix mineralization and osteoblast adhesion processes). Finally, flow perfusion (Fig. 4) [START_REF] Rodan | Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner[END_REF]Holtorf et al., 2005a;Holtorf et al., 2005b;[START_REF] Meinel | Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow[END_REF][START_REF] Sikavitsas | Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces[END_REF][START_REF] Stiehler | Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells[END_REF][START_REF] Zhao | Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats[END_REF] and scaffold stretching (Fig. 5) [START_REF] Mauney | Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro[END_REF][START_REF] Simmons | Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway[END_REF][START_REF] Ward | Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[END_REF] improve the late stage of osteogenic differentiation increasing calcium deposition in the ECM. This process seems sensitive to loading duration as it is mainly observed for continuous stimulations.

Chondrogenic pathway (Fig. 1B)

The application of hydrostatic pressure [START_REF] Finger | Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure[END_REF][START_REF] Miyanishi | Dose-and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro[END_REF], scaffold compression [START_REF] Huang | Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading[END_REF][START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF], and stretching [START_REF] Friedl | Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro[END_REF] significantly stimulates the expression of the early chondrogenic marker sox9, a transcription factor involved all along the chondrogenic pathway [START_REF] Lefebvre | Transcriptional control of chondrocyte fate and differentiation[END_REF]. Compressive loads (hydrostatic pressure and scaffold compression) improved the formation of unspecific ECM with increased collagen type I gene expression [START_REF] Ito | Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds[END_REF][START_REF] Finger | Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure[END_REF][START_REF] Mouw | Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells[END_REF][START_REF] Wagner | Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium[END_REF], but also enhance the cartilaginous differentiation of BMSCs through gene expression of collagen type II and aggrecan [START_REF] Ito | Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds[END_REF][START_REF] Huang | Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[END_REF][START_REF] Huang | Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading[END_REF][START_REF] Mauck | Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading[END_REF][START_REF] Miyanishi | Dose-and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro[END_REF][START_REF] Mouw | Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells[END_REF][START_REF] Pelaez | Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds. Stem cells and Development[END_REF][START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF][START_REF] Wagner | Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium[END_REF]. Scaffold compression (Fig. 6), hydrostatic pressure, and scaffold stretching enhance, not only short term transcriptional activity of chondrogenic markers but also long term GAG deposition [START_REF]Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro[END_REF][START_REF] Ito | Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds[END_REF][START_REF] Mauck | Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading[END_REF][START_REF] Mcmahon | Involvement of stretch-activated ion channels in strainregulated glycosaminoglycan synthesis in mesenchymal stem cell-seeded 3D scaffolds[END_REF][START_REF] Miyanishi | Dose-and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro[END_REF][START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF]. This ECM maturation (and cell differentiation), however, requires prolonged exposure to mechanical loads (with 5-14 days of stimulation).

Finally, mechanical stimulations can affect chondrogenic terminal differentiation by inducing chondrocyte hypertrophy. In vivo, this stage of differentiation precedes vascular invasion of cartilage, chondrocyte apoptosis, and subsequent replacement by osteoblasts. This terminal differentiation, however, should be avoided for cartilage tissue engineering, as it may lead to BMSC death. Several studies reported no effects of mechanical loadings on collagen type X (characterizing this stage of differentiation) [START_REF] Ito | Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds[END_REF][START_REF] Friedl | Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro[END_REF][START_REF] Huang | Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[END_REF], though scaffold compression can enhance its gene expression [START_REF] Campbell | Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells[END_REF].

Myogenic pathway (Fig. 1B)

So far few studies have concentrated on mechanical stimulations driving vascular smooth muscle cell (VSMC) differentiation of BMSCs when compared to the extensive work done on bone and cartilage phenotype, though myogenic differentiation is also influenced by mechanical loading. The application of scaffold stretching, indeed, induces increased gene and protein expression of calponin [START_REF] Kurpinski | Anisotropic mechanosensing by mesenchymal stem cells[END_REF][START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF], SM22α [START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF], α smooth muscle actin (αSMA) [START_REF] Engelmayr | Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: implications for engineered heart valve tissues[END_REF][START_REF] Hamilton | Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications[END_REF][START_REF] Kobayashi | Mechanical stress promotes the expression of smooth muscle-like properties in marrow stromal cells[END_REF][START_REF] Nieponice | Mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix[END_REF], and smooth muscle-myosin heavy chain (SM-MHC) [START_REF] Kobayashi | Mechanical stress promotes the expression of smooth muscle-like properties in marrow stromal cells[END_REF], all contractile apparatus proteins involved in VSMC cytoskeleton. SM22α regulation, however, seems to be dependent on the type of stimulation as equiaxial strain is reported to decrease its expression when uniaxial strain increases it [START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF].

All these studies provide evidence that BMSCs can adapt their phenotype in response to their mechanical environment. It is, however, difficult to determine which type of mechanical loading specifically enhanced osteogenesis, chondrogenesis, or myogenesis, as similar mechanical stimulations (e.g. 10% strain by scaffold stretching) can improve all three phenotypes. Moreover, mechanical loading has been reported to have no or negative effects on BMSC differentiation. Shear stresses (induced by fluid flow or flow perfusion) and strains (caused by scaffold stretching), for example, have been shown to decrease Runx2, OCN, and BSP expression [START_REF] Bjerre | Flow perfusion culture of human mesenchymal stem cells on silicatesubstituted tricalcium phosphate scaffolds[END_REF][START_REF] Byrne | Gene expression by marrow stromal cells in a porous collagenglycosaminoglycan scaffold is affected by pore size and mechanical stimulation[END_REF][START_REF] Scaglione | Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system[END_REF][START_REF] Stiehler | Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells[END_REF] as well as calcium deposition [START_REF] Sikavitsas | Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor[END_REF]. Compressive (by scaffold compression or hydrostatic pressure) loads have also been reported to have no effects on collagen type II and/or aggrecan gene expression [START_REF] Campbell | Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells[END_REF][START_REF] Finger | Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure[END_REF][START_REF] Scherer | The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro[END_REF]. These chondrogenic markers can also be inhibited by hydrostatic pressure [START_REF] Scherer | The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro[END_REF] or scaffold bending [START_REF] Ward | Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[END_REF]. As for myogenic phenotype (αSMA, calponin, and SM22α protein expression), it can be inhibited by substrate strain [START_REF] Park | Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells[END_REF]. These discrepancies might be explained by the broad range of loading patterns, substrates, and biochemical environments, rather selected from empirical knowledge, than from a thorough understanding of the mechanotransduction phenomena. Furthermore, the local mechanical conditions at the cell level have not been well controlled. In many prior investigations, the loading has been applied to the boundary of the construct. This is especially true for cells in 3D or scaffold environments. Often the mechanical conditions have been calculated considering homogenous linear continuum mechanics principals. And, although even more sophisticated constitutive models have been employed in some cases, they have not been rigorously validated. Hence, the actual mechanical stimulation of the cell may not be that which we can assume from the applied macroscopic loading conditions.

Modulating the effects

The effects of mechanical loading can be, indeed, modulated by the loading pattern applied: stimulation amplitude, duration, and frequency can all alter cell response. For example, osteogenic differentiation (calcium deposition) is shear stress-dependent in fluid flow systems (from 0.3 to 3 ml/min [START_REF] Rodan | Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner[END_REF]) and is improved by increased viscosity of cell culture media [START_REF] Sikavitsas | Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces[END_REF]. Chondrogenic differentiation (GAG and collagen content, aggrecan gene expression) is also reported to depend on load magnitude, in hydrostatic pressure systems (from 0.1 to 10 MPa [START_REF] Miyanishi | Dose-and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro[END_REF]). Regarding duration of loading, several studies demonstrate improved differentiation (calcium deposition and runx2 expression for osteogenesis; GAG content and aggrecan and collagen type II expression for chondrogenesis) for longer duration of mechanical stimulations (from 1 to 8 h/day of stretching [START_REF] Diederichs | Effects of repetitive and short time strain in human bone marrow stromal cells[END_REF]Haasper et al., 2008), scaffold compression [START_REF] Huang | Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading[END_REF][START_REF] Mauck | Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading[END_REF][START_REF] Terraciano | Differential response of adult and embryonic mesenchymal progenitor cells to mechanical compression in hydrogels[END_REF], or hydrostatic pressure [START_REF]Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro[END_REF]). Even if most of the studies use frequencies of 0.5 or 1 Hz (considered as physiological loading frequencies during gait), frequency can also influence cell response as demonstrated by higher aggrecan and collagen type II gene expression for 1 Hz than for 0.1 Hz scaffold compression [START_REF] Pelaez | Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds. Stem cells and Development[END_REF].

Another factor influencing BMSC response to mechanical loading is the substrate on/in which cells are seeded. First, the mechanical properties (elasticity) of the matrix itself can direct BMSC proliferation, with higher proliferation for stiffer (7500 Pa) than for softer (250 Pa) matrices [START_REF] Winer | Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli[END_REF], and differentiation, with more rigid (34 kPa) matrices favoring osteogenesis and softer substrates (11 kPa) favoring myogenesis [START_REF] Engler | Matrix elasticity directs stem cell lineage specification[END_REF] (Fig. 7).

Second, the response of BMSC to mechanical loading is also dependent on the biochemical nature of the scaffold. For example, increased gene expression of OPN and BSP induced by fluid flow is abolished by calcium phosphate coating, due to higher expression in static control for this type of substrate [START_REF] Scaglione | Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system[END_REF]. Fibronectin coating, on the other hand, improves protein expression of calponin by BMSCs exposed to strain, when collagen type I coating or control substrate do not [START_REF] Gong | Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs)[END_REF]. These differences might be explained by the involvement of different integrins in the BMSC adhesion to these substrates.

Integrins are transmenbrane receptors thought to act as mechanosensors. They are composed of an α and a β sub-unit and bind to ECM proteins on one side of the membrane and to cytoskeletal proteins on the other side (Fig. 8). With 18α and 8β sub-units, 24 heterodimers are known, each of which can bind to a definite ligand. Integrins form focal adhesions, insuring cell adhesion as well as signal transduction informing the cell about the ECM. Applying external pulling forces on these focal adhesions can activate, through the cytoskeletal proteins, different signal transduction pathways, all leading to the phosphorylation of ERK1/2, which, in turn, can up/down regulate different transcription factors [START_REF] Iqbal | Molecular regulation of mechanotransduction[END_REF]. Different integrins, however, will bind to, and signal through, different transduction pathways, resulting in different tuning of ERK1/2 activation. It appears, then, that mechanosensing might be matrix dependent, as shown with a mouse myoblast cell line [START_REF] Grossi | Mechanical stimulation increases proliferation, differentiation and protein expression in culture: stimulation effects are substrate dependent[END_REF]. Adhesion to fibronectin and to collagen, for example, involve different sets of integrins, with α5β1 and α4β1 for fibronectin and α1β1 and α2β1 for collagen [START_REF] Docheva | Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system[END_REF]Hynes, 2002), potentially explaining the different cell responses observed with BMSCs seeded on these substrates in Gong and Niklason's study [START_REF] Gong | Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs)[END_REF].

BMSC sensitivity to mechanical stimulation seems also dependent on the soluble biochemical environment. In several studies, mechanical loading alone is enough to improve the expression of different osteogenic markers at the gene level (Runx2, osteocalcin, osteopontin, etc. (Chen et al., 2008;[START_REF] Friedl | Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro[END_REF][START_REF] Kreke | Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner[END_REF][START_REF] Li | Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation[END_REF][START_REF] Qi | Mechanical strain induces osteogenic differentiation: Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells[END_REF]), though very few report terminal differentiation of BMSCs (calcium deposition) with mechanical stimuli only [START_REF] Ward | Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[END_REF]. The addition of dexamethasone, a glucocorticoid often added into differentiation media for BMSCs, seems to enhance the sensitivity of BMSCs with higher calcium deposition and OPN expression by BMSCs exposed to strain or shear (Holtorf et al., 2005a;[START_REF] Mauney | Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro[END_REF]. As for the chondrogenic pathway, effects of mechanical loading in absence of TGFβ (inducer of BMSC chondrogenesis) are unclear. Some studies show this growth factor as not necessary for improvement of chondrogenic phenotype and cartilaginous matrix deposition by mechanical stimulation [START_REF] Campbell | Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells[END_REF][START_REF] Huang | Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells[END_REF][START_REF] Mauck | Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading[END_REF], when others demonstrate that TGFβ is required [START_REF] Finger | Differential effects on messenger ribonucleic acid expression by bone marrow-derived human mesenchymal stem cells seeded in agarose constructs due to ramped and steady applications of cyclic hydrostatic pressure[END_REF][START_REF] Mouw | Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells[END_REF][START_REF] Scherer | The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro[END_REF].

The influence of these biochemical substances on BMSC responses to mechanical loads might be explained by synergistic effects of both biochemical and mechanical stimulations reciprocally needed to stimulate specific signaling pathways. It is also possible that BMSCs need to reach a threshold state of differentiation (improved by dexamethasone and TGFβ) and/or to start to deposit matrix to efficiently relay the mechanical stimuli. Studies reporting enhanced induction of osteogenesis [START_REF] Thomas | Bone marrow stromal cells are load responsive in vitro[END_REF][START_REF] Zhao | Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: experiments and hydrodynamic modeling[END_REF], chondrogenesis [START_REF] Mouw | Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells[END_REF], and myogenesis [START_REF] Kobayashi | Mechanical stress promotes the expression of smooth muscle-like properties in marrow stromal cells[END_REF] by mechanical stimulations of BMSCs previously differentiated in static conditions support this hypothesis.

Conclusion

BMSCs represent one of the most promising sources of regenerative cells for the repair of numerous tissues. Though several animal and clinical studies have demonstrated their potential, a better understanding and control of their proliferation and differentiation is required to fulfill their potential. One of the critical point is to encourage and maintain the appropriate cell metabolism and phenotype. Most of the studies conducted so far have focused on biochemical stimulation and gene transfer to do so, but more recent data report that BMSC proliferation and differentiation are also affected by their mechanical environment. Mechanical stimulation, therefore, represents an additional and an efficient tool to improve and maintain BMSC differentiation. Providing correct mechanical environments both in vitro and in situ will lead to improved engineered tissues and to a greater eventual success in the clinics. (A) Under the appropriate induction BMSCs are able to give rise to adipocytes, osteoblats, chondroblasts and myocytes. (B) Differentiation pathways of BMSCs: (a) osteogenesis is characterized by the involvement of osterix (Osx, aka SP7) and runt-related transcription factor 2 (Runx2, aka cbfa-1), increased alkaline phosphatase (ALP) activity, collagen type I (COL1), osteopontin (OPN, aka SPP1), osteocalcin (OCN, aka BGLAP), and bone sialoprotein (BSP) gene and protein expression, and calcification of the extra-cellular matrix (ECM) [START_REF] Aubin | Mesenchymal stem cells and osteoblast differentiation[END_REF]; (b) chondrogenesis is characterized by the involvement of the SRY (sex determining region Y)-box 9 (Sox-9) transcription factor, collagen type II (COL2), tenascin (Tnc), aggrecan (Acan), and cartilage oligomeric matrix protein (COMP) isoform gene and protein expression, and glycosaminoglycan (GAG) deposition [START_REF] Lefebvre | Transcriptional control of chondrocyte fate and differentiation[END_REF]; (c) myogenesis (for vascular smooth muscle cells, VSMCs) is characterized by collagen type I (COL1), matrix metallo-proteinases (MMPs), α smooth muscle actin (aSMA), smooth muscle-myosin heavy chain (SM-MHC), calponin (Cnn1), and SM22α (aka Tagln) gene and protein expression [START_REF] Rensen | Regulation and characteristics of vascular smooth muscle cell phenotypic diversity[END_REF]. (Reprinted from Journal of Biomechanics, 41, Jagodzinski, M., Breitbart, A., Wehmeier, M., Hesse, E., Haasper, C., Krettek, C., Zeichen, J., Hankemeier, S., Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture, 1885-1891, copyright (2008) with permission from Elsevier.) Human BMSCs were seeded into bovine bone matrix and subjected, either to flow perfusion (10 ml/min; solid circles), or to flow perfusion and scaffold compression (10 ml/min, 10% strain, 0.5 Hz, hollow circles), or left in static culture (solid triangles). n=7; *: p<0.03. (Reprinted from Journal of Biomechanics, 36, Simmons, C. A., Matlis, S., Thornton, A. J., Chen, S., Wang, C. Y., Mooney, D. J., Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway, 1087-1096, copyright (2003) with permission from Elsevier.) Mechanical strain was applied to human BMSCs through bending of a collagen coated silicon membrane (3% strain; 0.25 Hz). Osteogenic differentiation (calcium deposition) was evaluated with von Kossa staining and biochemical assay. Values presented are means±SD; n=3; *: p<0.05 vs unstrained control. (Reprinted from Biomechanics and Modeling in Mechanobiology, 6, Mauck, R. L., Byers, B. A., Yuan, X., Tuan, R. S., Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading, 113-125, copyright (2007) with permission from Springer.) Bovine BMSCs were seeded into agarose scaffolds and exposed either to unconfined compression (10% deformation, 180 min/day, 1 Hz) or to free-swelling conditions for 5 days. Both groups were then maintained in free-swelling conditions for 23 more days. After 3 days of culture, GAG deposition was evaluated with Alcian Blue staining in free-swelling (A) and dynamically loaded (B) samples. Scale bar=50 μm. GAG content was determined by biochemical assay (C). n=3; *: p<0.05. (Reprinted from Cell, 126, Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E., Matrix elasticity directs stem cell lineage specification, 677-689, copyright (2006) with permission from Elsevier.) Human BMSC aggregates were seeded on variably compliant polyacrylamide gels and cultured in identical media conditions. On the softest substrate (0.1-1 kPa) BMSCs displayed neurogenic marker (β3 tubulin). On moderately stiff substrate (8-17 kPa), BMSCs upregulated myogenic transiption factor expression (MyoD1). On stiffer substrate (25-40 kPa) BMSCs expressed osteogenic transcription factor (cbfa1 aka Runx2). Scale bar=5 μm. They bind to extra-cellular matrix proteins (such as collagen, fibronectin, etc.) on one side of the cell membrane and to cytoskeletal proteins (such as actin, talin, etc.) on the other side.

Figure 1 .

 1 Figure 1. Differentiation potential of bone marrow-derived stromal cells.

Figure 2 .

 2 Figure 2. Common techniques for mechanical stimulation of cells (Brown, 2000). (A) Application of fluid-induced shear stresses: cells are seeded on a 2D substrate (such as coated glass slides) and exposed to laminar fluid flow (a), are seeded onto a 3D porous scaffold (such as titanium fiber mesh) and exposed to medium perfusion through the scaffold (b), or to turbulent flow by medium steering (c). (B) Application of compressive loads: cells are seeded into a hydrogel (such as agarose) and exposed to unconfined (eventually confined) compression between platens (a) or to hydrostatic pressure (b). (C) Scaffold stretching (uniaxial): cells are seeded onto 2D or into 3D scaffolds (such as coated silicon or collagen I gel) hold between a fixe and a moving grip (a), or exposed to four point bending (b). (D) Scaffold stretching (bi-or equi-axial): cells are seeded on a 2D substrate (such as collagen coated silicon membranes) deformed by applied vacuum or fluid pressurization (a) or by prong or platen displacement (b).

Figure 3 .

 3 Figure 3. Effects of flow perfusion (and compressive loading) on BMSC proliferation (Jagodzinski et al., 2008).

Figure 4 .

 4 Figure 4. Effects of strain on osteogenic differentiation of BMSCs (Simmons et al., 2003).

Figure 5 .

 5 Figure 5. Effects of flow perfusion on osteogenic differentiation of BMSCs (Simmons et al., 2003).(From Bancroft, G. N., Sikavitsas, V. I., van den, D. J., Sheffield, T. L., Ambrose, C. G., Jansen, J. A., Mikos, A. G. (2002) Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. Proc Natl Acad Sci USA 99: 12600-12605, copyright (2002) National Academy of Sciences, U.S.A.) Rat BMSCs were seeded into a titanium fiber mesh and exposed to different flow rates (0.3, 1, or 3 ml/min) for 16 days. Calcium content of cultured scaffold, with *: highest, **: second highest, ***: third highest calcium content. Each group statistically different (p<0.05) from all other groups.

Figure 6 .

 6 Figure 6. Effects of scaffold compression on chondrogenic differentiation of BMSCs (Mauck et al., 2007).

Figure 7 .

 7 Figure 7. Effects of matrix elasticity on BMSC differentiation (Engler et al., 2006).

Figure 8 .

 8 Figure 8. Integrins receptors. Integrins are transmenbrane receptors composed of an α and a β sub-unit.

Conflict of interest statement

None of the authors of the submitted review paper, E. Potier, J. Noailly, and K. Ito "Directing mesenchymal stromal cell function with mechanics", have any financial and/or personal relationships with other people or organisations that could inappropriately influence (bias) this work.