SKELETAL BIOLOGY (DB BURR, SECTION EDITOR)

Mechanical Regulation of Skeletal Development

Rebecca Rolfe & Karen Roddy & Paula Murphy

Published online: 7 March 2013# Springer Science+Business Media New York 2013

Abstract Development of the various components of anormal skeleton requires highly regulated signalling sys-tems that co-ordinate spatial and temporal patterns of celldivision, cell differentiation, and morphogenesis. Muchwork in recent decades has revealed cascades of molecularsignalling, acting through key transcription factors to regu-late, for example, organized chondrogenic and osteogenicdifferentiation. It is now clear that mechanical stimuli arealso required for aspects of skeletogenesis but very little isknown about how the mechanical signals are integrated withclassic biochemical signalling. Spatially organized differen-tiation is vital to the production of functionally appropriatetissues contributing to precise, region specific morphol-ogies, for example transient chondrogenesis of long boneskeletal rudiments, which prefigures osteogenic replacementof the cartilage template, compared with the production ofpermanent cartilage at the sites of articulation. Currently alack of understanding of how these tissues are differentiallyregulated hampers efforts to specifically regenerate stablebone and cartilage. Here, we review current research revealing

the influence of mechanical stimuli on specific aspects ofskeletal development and refer to other developing systemsto set the scene for current and future work to uncover themolecular mechanisms involved.We integrate this with a briefoverview of the effects of mechanical stimulation on stemcells in culture bringing together developmental and tissueengineering aspects of mechanoregulation of cell behavior. Abetter understanding of the molecular mechanisms that linkmechanical stimuli to transcriptional control guiding cell dif-ferentiation will lead to new ideas about how to effectivelyprime stem cells for tissue engineering and regenerativetherapies.

Keywords Skeletal development . Mechanical stimulation .

Bone . Ossification . Joint . Chondrogenesis . Articularcartilage . Regenerative therapies . Mechanotransduction

Introduction

To better understand the basis of degenerative skeletal dis-eases such as osteoporosis and osteoarthritis we not onlyneed to uncover mechanisms of bone and cartilage mainte-nance, but also the molecules and cues that drive skeletalformation in the embryo. A deep understanding of develop-mental processes is also required to harness the potential ofstem cells to regenerate bone and cartilage; we need to knowwhich stimuli are required to prime site specific, function-ally appropriate, differentiation pathways. Such therapieshold great promise however it is clear that we are still somedistance away from knowing how to guide the formation offunctionally appropriate bone and cartilage to replace in-jured and degenerating tissue.

The central role of mechanical stimuli on bone mainte-nance and adaptation has been a fundamental part of bonebiology since Wolff’s Law was formulated in the 19th

R. Rolfe :K. Roddy : P. MurphyDepartment of Zoology, School of NaturalSciences, Trinity College, Dublin, Ireland

R. Rolfe :K. Roddy : P. MurphyTrinity Centre for Bioengineering, Trinity College,Dublin, Ireland

P. Murphy (*)School of Natural Sciences, Trinity College,Dublin, Irelande-mail: paula.murphy@tcd.ie

Present Address:K. RoddyCardiff School of Biosciences, Life SciencesBuilding, Museum Avenue,Cardiff CF10 3AX, UK

Curr Osteoporos Rep (2013) 11:107–116DOI 10.1007/s11914-013-0137-4

century, and was incorporated by Harold Frost into hisMechanostat theory where local mechanical effects are in-tegrated with and adjusted by the biochemical system [1, 2].Although the effect of mechanical stimulation on the for-mation of the skeletal system in the embryo has been lesswidely considered in the past (reviewed in [3]), evidence hasexisted for some time that mechanical forces generated byembryonic muscle contractions are required for normalskeletogenesis. Such evidence comes from 2 sources: con-genital muscle disorders where foetal muscle contractionsare reduced in utero and from the results of experimentalimmobilization of model animals. Recent work is beginningto show how mechanical stimuli impact specific develop-mental processes. As suggested by Frost in the Mechanostattheory, these mechanical influences must be integrated withmolecular processes at a cellular level. Although more workis required to reveal the points of integration between bio-physical and biochemical signals guiding skeletal develop-ment, discoveries revealing the impact of biophysicalstimuli on other developing systems are providing valuableclues. The stage is now set for synergistic advances on thequestion of mechanoregulation of skeletal development.Here we review current research revealing the influence ofmechanical stimuli on skeletal development, specificallyoutlining the developmental processes that are impacted,the influence on progenitor cells in culture as well as prin-ciples emerging from other developing systems. This workwill prime future research to address key challenges inmaintaining healthy skeletal tissue and improving regenera-tive capacities.

Mechanical Influences on Developmental Processes

Our understanding of how tissue differentiation is controlledin the developing embryo and how shape and structure isestablished has progressed enormously in recent years. Apicture has emerged of proliferating progenitor cellsresponding to layers of positional information that guidecellular differentiation and tissue morphogenesis with anemphasis on the role played by locally produced molecularsignals and the response of cells through activation of spe-cific sets of transcription factors. In more recent years it hasalso become apparent that we must expand these ideas toincorporate evidence that the molecular pathways are tightlyintegrated with physical cues and mechanical signals expe-rienced by the developing cell [4, 5]. For example, elegantwork on mesenchymal condensations during tooth develop-ment has shown that cell compression is a necessary step forthe modulation of gene expression and cell differentiation inresponse to classic signaling from epithelium to mesen-chyme [6•]. Several recent studies on Drosophila develop-ment have shown the importance of mechanical signals in

cell polarization, in particular impacting the planar cellpolarity (PCP) pathway, which shares components of theWnt signaling pathway [7, 8].

Bone and Joint Development and the Influenceof Biophysical Stimuli from Muscle Contractions

Limb skeletogenesis begins with the condensation ofprechondrogenic mesenchymal cells at the core of the limbbud in a y shaped pattern, prefiguring the future rudiments;eg, humerus, radius, and ulna in the case of the forelimb,with more distal elements added progressively [9].Chondrogenic differentiation begins to define the individualrudiments, and the future elbow/knee joint region becomesvisibly distinct, composed of flatter more dense cells; theinterzone [10–12]. The cartilage that prefigures each futurebone is transient and begins a process of maturation at themid diaphysis in a well defined sequence of events involv-ing hypertrophy and eventual replacement by bone (coordi-nated periosteal and endochondral ossification). Thissequence of events progresses proximally and distally ineach rudiment with growth plates persisting at the epiphysesto allow continued elongation. The site of the future syno-vial joint at the interface of the rudiments is made up of 3layers of cells that are histologically and molecularly dis-tinct; the chondrogenous layers contouring the ends of therudiments and the intervening intermediate layer wherecavitation will later take place. Cell lineage marking ofinterzone cells in the mouse (expressing Gdf5) shows thatthey will give rise to all joint tissues including the articularcartilage and synovium with very little contribution to un-derlying epiphyseal bone [13], while marking of cells thatexpress Matrilin 1 (within the rudiments) shows that they donot contribute to articular cartilage [14]. Therefore the cellsthat will give rise to transient cartilage in the rudiments andarticular cartilage of the joint derive largely from distinctterritories early in development (although there may be anintervening population of cells that can still contribute toboth tissues). In contrast to transient cartilage, articularcartilage has a stable phenotype for the healthy lifetime ofthe joint with a unique striated ‘zonal’ architecture [15].This tissue is vulnerable to breakdown with age and indisease states such as osteoarthritis (OA).

The musculoskeletal system develops in a co-ordinatedfashion and this is seen clearly in the developing limb wheremuscle forms from cells that migrate into the limb bud,adjacent to the condensing mesenchyme cells that will formthe cartilage template of the future skeleton [16]. This is alsocoordinated with the appearance of tendon specific markersin cells at the sites of muscle attachment to the rudimentsand the gradual morphogenesis of functional tendons [12,16]. The forming muscle masses begin to contract precisely

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as the cartilage template is taking shape [12]. These closelyassociated developing tissues can therefore influence eachother in a number of ways; through paracrine signallingbetween cell populations and through mechanical influ-ences. While developing muscle is known to secrete para-crine signals [17] and signalling between developing tendonprecursors and skeletal cells at the point of tendon attach-ment has been shown to be important in bone ridge initiation[18], direct evidence has also implicated mechanical forcesgenerated by muscle contractions as necessary stimuli forthe normal development of skeletal rudiments. Indicationsof this came from the described effects of congenital neuro-muscular diseases, such as spinal muscular atrophy, whichcan cause partial or full intrauterine immobilization [19]leading to abnormal formation of long bones and suscepti-bility to fracture in the infant. Direct evidence came fromanimal models where the mechanical environment can bealtered in a number of ways including in vivo immobiliza-tion of the musculature or the use of mouse mutants wherethe skeletal rudiments develop with reduced, absent, or non-contractile muscle (reviewed in [3]).

Similar findings in different animal models (chick,mouse, zebrafish), using either immobilization or absence/reduction of muscle, definitively separate paracrine andmechanical influences. Integrating these findings allows usto draw general conclusions about the specific aspects ofskeletogenesis that require appropriate mechanical stimula-tion from adjacent twitching muscles for normal progres-sion, including joint formation, ossification, and rudimentshape (morphogenesis) (Fig. 1). This underlines the impor-tance of early movement for coordinated development of thewhole musculoskeletal system.

Joint Formation

The accessibility of the chick embryo, where drug inducedimmobilization can be carried out with relative ease,allowed early demonstration of the particular sensitivity ofjoint formation to paralysis [30–34]. Immobilization led tojoint fusions in extreme cases with loss of cavitation, butalso loss of associated structures such as articular surfaces,menisci, and patella. This led to the general conclusion thatpositioning of the joints is not dependent on mechanical inputfrom the muscle, but the relatively late event of cavitation issensitive (reviewed in [11]). More recently, the use of severaldifferent mouse mutants where embryos develop in the ab-sence ofmuscle (Myf5nlacZ/nlacZ/MyoD–/– [20••, 35••], Splotch[20••, 35••]), or reduced (Myf5nlacZ/+/MyoD–/– [20••, 36]), orimmobile muscle (Mdg–/– [35••], or Dock1–/– [37] (ourunpublished results)), have shown similar joint fusions withparticular sensitivity of the shoulder and elbow joints [20••].We recently carried out examination of the effect of immobili-zation on development of the knee joint in the chick including

3D imaging of the developing region and the expression ofgenes that are either markers of emerging joint tissues or areknown regulators of joint development [23••]. We also used3D imaging to inform a computational model of musclecontractions in the developing knee region and showedcorrespondence between the predicted patterns of biophys-ical stimuli and the observed effects[23••, 38•]. We foundthat cellular organization (3 distinct layers) and the defini-tion of tissue territories within the developing joint werelost, together with the expression of locally restricted reg-ulatory molecules (eg, BMP2, FGF2) as well as genesinvolved in hyaluronan function (HAS2 and CD44), impli-cated in the cavitation process. This shows that the cavita-tion process cannot be isolated as the sensitive event andreflects more an impact on a continuous process of localpatterning, defining multiple component tissues of the ma-ture joint including articular cartilage (Fig. 1). We proposethat the process of tissue definition requires appropriatemechanical stimuli, which act as a form of positional infor-mation guiding position-specific differentiation. This issupported by muscleless mouse studies where cellular or-ganization is also lost within the forming elbow and shoul-der joints [20••] and where canonical Wnt signaling,necessary for the progressive development of the joint oncedefined [39, 40], is lost [35••]. We therefore need to con-sider mechanical stimuli as contributing to the spatial orga-nization of location specific differentiation programs ratherthan the cavitation process in isolation.

A notable difference between the effects seen inimmobilized chick and mouse embryos is that forelimbsshow greater sensitivity in the mouse whereas similareffects are seen in knee and elbow joints in the chick.Simulations of the biophysical stimuli produced by musclecontractions in both species and by passive movement(displacement of the limb), only relevant in the mousewhere embryos develop in utero, offer a possible expla-nation for this [41••]. Due to the position, size, and shapeof the hindlimb, simulation of passive displacement pre-dicted much higher stimuli impacting the rudiment tissues;this could compensate to some extent for the lack of selfgenerated movement. However, this does not precludeother possible species specific differences in local regula-tory mechanisms in fore and hindlimbs.

In addition to the loss of tissue territories, immobilizationleads to changes in joint shape, in particular the shape of theemerging condyles in both the chick [23••] and mouse (seeMorphogenesis below).

Ossification

As described above, endochondral ossification begins withhypertrophy of chondrocytes at the mid- diaphysis of eachlong bone and the coordinated formation of an adjacent

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periosteal bone collar, progressing proximally and distally.Initial descriptions of Myf5/MyoD double mutant, musclelessmouse embryos did not examine the ossification pattern atearly stages of skeletogenesis but later stage rudiments(E18.5) showed alteration of size [24, 25], lack of fusion ofpalatal shelves [24] and effects on secondary ossification sites[42]. Sites of secondary cartilage in the avian jaw also dependon mechanical stimulation [43]. In the chick embryo we usedcomputer simulations of embryonic muscle contractions topredict that peak biophysical stimuli (eg, stress, strain, hydro-static pressure) are experienced by cells at the mid-diaphysisprior to the initiation of ossification, spreading proximally and

distally ahead of the wave of hypertrophy [21]. This led us tosuggest a functional link between peak stimuli and maturationof chondrocytes toward hypertrophy/ ossification, corroborat-ed by demonstrating reduced ossification at early stages of theprocess in immobilized embryos [22]. Therefore we felt itimportant to examine early stages of ossification in musclelessmouse embryos and showed that compared with stage-matched controls, ossification was reduced and abnormal inthe scapula, humerus and femur [20••]. So although ossifica-tion proceeds, the altered pattern of initiation and progressionshows that the process is not normal (Fig. 1). Gomez et al.revealed increased cortical thickness of the femur at later

Fig. 1 Lack of mechanical stimulation from muscle contractions im-pacts several aspects of skeletal development: the mouse forelimb andchick hindlimb are used here to summarize the effects on ( )ossification [eg, 20••, 21, 22], ( ) joint formation [eg, 23, 25••,26••, 29••] and ( ) rudiment shape (morphogenesis) [eg, 20••, 23••,24, 25, 26••, 27, 28••]. Mouse embryos show strongest (but notexclusive) effects on the rudiments and joints of the forelimb. The datashown in boxes a, b, and c serve as examples of the evidence of impacton each process; a mid diaphysis of the mouse humerus at TS23(E14.5) in control and Spd mutant (muscle-less), stage matchedembryos, showing expression of Spp1. The Spp1 gene is normallyexpressed in the hypertrophic zone [29]. Despite the appearance ofhypertrophic chondrocytes at the mid-diaphysis of immobile Spdembryos, no Spp1 gene expression is detected (our unpublished

results); b In the knee joint of immobilized chick embryos, lack ofdefinition of joint territories is shown by histology (note the absence oforganized cell layers within the presumptive joint) and gene expressionpatterns (as indicated), which normally define the territories of theforming joint; the periarticular cartilage of the rudiment (PthrP), theintermediate layer of the interzone (Bmp2). The schematic summarizeseffects of immobilization on the knee joint; c The effect on rudimentmorphogenesis is exemplified by the change in shape of the distal femur;the diagram represents outlines of control (blue) and immobilized (red)sections, overlaid (plane of section indicated). Note the change in heightof the condyles (black brackets), narrowing of the intercondylar fossa(colored brackets), and reduction in outgrowth of typical condylar spurs(arrow heads), particularly at the interface with the fibula (tf trocleafibularis groove). Images in b and c adapted fromRoddy et al. 2011 [23••]

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stages [25] and more recently, Sharir et al. (2011) usedmicroCTof late stage long bones from non-contractile musclemutants (Cacnasmdg/Cacnasmdg) to show that the normalasymmetric pattern of mineral deposition specific to eachbone is lost in the absence of contractions, compromisingthe mechanical performance of the bone [26••]. This is asso-ciated with a difference in osteoblast distribution. So musclegeneratedmechanical stimuli are required for normal initiationof ossification and normal local bone growth, which reflectsthe hypomineralized and fragile bones seen in human infantswith reduced uterine movement [19].

Morphogenesis (Rudiment Shape)

Each skeletal rudiment and joint of the limb can be identi-fied by its unique, species specific, size and shape, appro-priate to its contribution to a coordinated, refined system.Shape emerges through the local modulation of cellularprocesses, such as cell proliferation, differentiation, extra-cellular matrix synthesis, cell shape and size, but little isknown about mechanisms that coordinate such processes tosculpt shape in the embryo. In the mature system, muscleloads affect precise bone shape, suggestive of coordinatedevolution between muscle loads and the ability of the skel-eton to bear the loads, and now evidence is accumulatingfrom a number of different animal models and experimentalimmobilizations, that muscle loads also contribute to aspectsof rudiment shape during development. 3D imaging ofconstituent tissues across developmental stages showed thatthe intricate shape of the chick knee joint emerges followingthe initiation of muscle contractions (HH28-34), with dra-matic changes to typical protrusions (eg, condyles) on therudiment termini following the appearance of tendonmarkers at the points of muscle attachment (HH32-34), sothat the knee joint at HH34 includes all the typical shapecharacteristics of the adult knee [12]. Elevated cell prolifer-ation rates, corresponded to regions of higher dynamic stim-uli predicted by Finite Element analysis and to the regionsunder relative expansion during development [38•]. Wefurther showed that these typical characteristics are lost inimmobilized embryos, corresponding with specific changesin spatial patterns of cell proliferation and altered geneexpression patterns of specific tissue markers and regulatorygenes including Pthrp, which is known to control chondro-cyte proliferation [23••]. Reduced chondrocyte proliferationwas also recorded in growth plates of immobilized chickembryos [27].

In addition to affecting local patterns of cell proliferation,mechanical stimuli have also recently been implicated in theprocess of chondrocyte intercalation and column stackingimportant for rudiment elongation. In immobilized andmuscleless zebrafish, pharyngeal cartilage morphology isabnormal with shorter elements and smaller and rounder

chondrocytes lacking typical columnar organization [28••].In mouse mutants with immobile muscle (Spd), reducedrudiment length was not accompanied by lower cell num-bers in growth plates, but a difference in cell shape wasnoted. Unlike in immobile zebrafish, cell columns did formbut the columns were significantly shorter and it wassuggested that the normal process, which combines shortcolumns into elongated columns, does not take place [28••].Although the molecular mechanisms driving chondrocyteintercalation are not fully known, the Wnt/PCP pathway[44, 45], and Wnt5a in particular [44], has been implicatedin chondrocyte polarity and limb elongation.

Integration of Mechanical Stimuli and MolecularMechanisms: theMechanistic Basis of aMechanoresponse

The molecular mechanisms that control cartilage hypertro-phy and ossification are relatively well defined [46–48],involving complex interactions between major signalingpathways; in particular a well described feedback loop be-tween Ihh and Pthrp controls maturation to hypertrophy andthe balance between ossification and elongation. Althoughcontrol of joint development is comparatively less wellunderstood, many of the same signaling pathways and roleplayers have been implicated but with very distinct interac-tions and outcomes (reviewed in [15]). It is therefore inter-esting that both ossification and joint definition are alteredwhen muscle contractions are absent or reduced [20••]. It isclear that a balance of antichondrogenic and chondrogenicfactors is needed in the joint and is likely required tomaintain the stable phenotype of the articular cartilage,preventing maturation and hypertrophy; multiple alterationsthat tip the balance toward hypertrophy are associated withOA (reviewed in [15, 49]). Early specification of the cells ofthe future joint is clearly important and many regulatorygenes show restricted expression in these cells, howeverworking out specific functions has been challenging. BMPsignaling and its modulation through the antagonist Nogginis clearly important since Noggin inactivation leads to lossof joints [50]. Wnt signaling has also emerged as pivotalwith very elegant gain of function and loss of functionexperiments showing it is required in particular to maintainthe joint once specified [39, 40]; several assays showchondrogenic effects of BMP and antichondrogenic effectsof Wnt activity.

The similarity of the effects seen when muscle is eitherabsent or immobile and in both chick and mouse systemsshows that common aspects of the phenotypes, on bothossification and joint development, are caused by alteredmechanical stimuli. However we know nothing about howmechanical signals are integrated with other sources ofpositional cues and the cellular and molecular mechanisms

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that are implicated in differentiation and tissue patterning:This represents a major gap in our knowledge that is achallenge to address. Stretch Activated Ion Channels(SACs) have been implicated as 1 possible mechanism ofmechanotransduction in chondrocytes [51] however, analy-sis of secondary cartilage induction in avian embryos showsthat different mechanisms are at play in different sites andthat SACs are not involved in secondary cartilage formationat the enthesis [43]. The characterization of effects inmuscleless mouse embryos in particular, establishes an ex-perimental system that allows us to hone in on embryonicmuscle contractions as the source of mechanical stimulation,and its impact on cellular differentiation and morphogenesisin developing skeletal rudiments and informs the collectionof tissue samples (stimulated and unstimulated) for furtheranalysis. We have carried out transcriptome analysis ofrudiments under normal stimulation (control) and in theabsence of limb muscle (Pax3Spd/Spd) using both microarrayand whole transcriptome sequencing (Rolfe et al. in prepa-ration). Having defined that the earliest phenotypic impactwas detected at Theiler stage (TS)23 (usually at embryonicday 14.5) at the outset of ossification and early patterning oftissue territories within the joint [20••], and that the humerusand associated shoulder and elbow joints are the most se-verely affected, we chose this developmental time and tissuefor analysis. Analysis of the differentially expressed (DE)gene sets using Gene Ontology terms showed strong specif-ic enrichment of genes associated with development anddifferentiation, cytoskeletal organization, ExtraCellularMatrix (ECM) and cell adhesion, providing important cluesto the molecular mechanisms impacted by the altered stim-uli. The DE gene sets also showed enormous enrichment forcomponents of multiple cell signalling pathways withknown roles in skeletal development (eg, BMP, FGF,Notch, Hedgehog) and in particular Wnt signalling (Rolfeet al. in preparation). The molecular mechanisms impactedby mechanical stimuli in other developing systems(reviewed in [4, 5]) might provide some clues to commonmediators. Although much remains to be elucidated, it isclear that mechanical signals impact classic developmentalsignaling pathways including Wnt [7, 8, 52], can be sensedthrough cytoskeletal tension, possibly through integrins anddeformation of other ECM components [53–56], and isoften mediated by the Rho-Rock/ MyoII pathway [5].

In addition to providing insight into the mechanismsdisturbed, this work also provides target genes and pathwaysfor further analysis, to test hypotheses addressing molecularmechanisms involved in the mechanoresponse. Rot andKablar (2013) have recently reported a microarray analysisof maxilla/palate complex tissues in control vs muscleless(Myf5:MyoD double nulls) E18.5 embryos [57] arising fromtheir previous observation of cleft palate [24]. While thisscreen revealed genes associated with human congenital

cleft palate, and also included genes encoding developmen-tal regulators (signalling pathway components and transcrip-tion factors), there was very little overlap in the preciseindividual genes disregulated in these different developingsystems.

Response of Progenitor Cells in Culture to MechanicalStimuli: Application to Regenerative Therapies

A major challenge for the effective development of stem celltherapies for skeletal disorders is defining conditions re-quired for priming of stable differentiation to produce ap-propriate tissues for repair and regeneration. As outlinedabove, cells that will form transient cartilage in the limbskeletal rudiment (will progress through hypertrophy and bereplaced by bone) and cells at the rudiment termini that willform permanent articular cartilage, are already distinct earlyin development and experience different signalling environ-ments, both biophysical and biochemical. Current attemptsto differentiate adult derived stem cells in culture invariablyinduce transient chondrogenesis with progression to hyper-trophy (eg [58, 59]), a situation that is wholly unsatisfactoryfor articular repair and regeneration. A major outstandingdevelopmental question is how articular cartilage progenitorcells are distinguished and then maintained in the embryo? Itappears that even mature articular cartilage cells can beinduced to undergo chondrogenic maturation and hypertro-phy by treatment with 5-azacytidine [60]. Instability is alsonaturally seen with age and in OA. The specific mechanicalenvironment of the developing and mature joint contributeto the maintenance of the stable phenotype as altering me-chanical stimulation leads to loss of articular territories[23••]. Understanding the basis of this maintenance andthe contribution of mechanical stimuli would be a majorstep forward in development of new approaches to promotestable chondrogenesis of mesenchymal stem cells (MSCs),resistant to hypertrophy. Conversely, this knowledge couldalso be critical in enabling improved endochondral boneregeneration [61]. The concept of applying knowledgegained from the developing embryo to in vitro priming ofstem cells for regenerative therapies is fundamental and isgaining attention in the field of tissue engineering (reviewedin [62, 63]).

A wealth of evidence shows that various mechanicalstimuli influence the differentiation of progenitor cells iso-lated from adult sources (reviewed in [64, 65]). These stud-ies demonstrate the importance of mechanical stimuli buthave varied results depending on cell context [64] — forexample compression can enhance chondrogenesis in MSCsbut it depends on when the load is applied and in whatcellular environment [66–68]. From a developmental per-spective this is not surprising: context dependent responses

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are a common feature of development. Part of the variabilityis likely to be due to the issue of heterogeneity among MSCpopulations from different sources [69] and under differentpurification protocols; optimal cell sources, and purificationstrategies need to be defined for specific tissue regenerationapproaches, requiring extensive analysis to investigate cellresponses [63]. Again knowledge gained from the develop-ing embryo would be important in defining appropriatemarkers to assess cellular response. This will be requiredbefore any therapy can enter the clinic.

Despite variability in response, some generalities can bedrawn from the work on biophysical stimulation of stem cellsto date. The computational model generated by Prendergast etal. (1997) proposed that a balance of fluid flow and shearstrain determines the spatial and temporal pattern of MSC cellfate and this prediction has been corroborated by variousexperimental results including work in bone chambers or 3Dscaffolds [70, 71]. Hydrostatic Pressure (HP) has been largelyassociated with chondrogenic differentiation whereas tensilestrain and fluid induced shear stress are generally shown toinduce osteogenesis [72]. HP, which occurs during loading ofthe joint, is predicted by finite element modeling to promotecartilage formation and suppress endochondral ossification[73, 74]. Experimentally cyclic HP applied to MSCs in de-fined medium was found to enhance chondrogenesis, increas-ing expression of chondrogenic markers [75–77]. Severalparameters, including magnitude, frequency, onset, and dura-tion of HP stimuli are important in the response [78–80].Application of HP has recently been shown to increase thefunctional properties of cartilageous tissues [81] and tosynergize with TGFβ signalling in generating a more stablechondrogenic phenotype with reduced levels of hypertrophicmarkers [82].

Cyclic tensile strain is prominent in the superficial regionof cartilage tissue and at the cartilage-bone interface and ispredicted (with octahedral shear stress) to stimulate matura-tion and ossification [83] and is experimentally associatedwith enhanced osteogenic differentiation of stem cells.Tensile strain applied in a 2-dimensional culture caused aswitch in cell fate from adipogenic to osteogenic differenti-ation via up-regulation of canonical Wnt responsive genes[84]. Wnt signalling (through β-catenin) was also implicat-ed in the osteogenic differentiation of bone marrow derivedcells embedded in alginate-gels exposed to dynamic tension[85]. Similarily short-term cyclic tensile strain reduced therate of MSC proliferation and induced osteogenic differen-tiation [86]. Conversely, Connolly et al. (2010) found thatcyclic tensile loading of bone marrow stromal cells promot-ed fibrocartilage-like differentiation [87]. Fluid-inducedshear stress has also been shown to promote osteogenicdifferentiation [88–90] through RhoA/ROCK2 and en-hanced tension in the actin cytoskeleton [89]. In a similarstudy oscillatory fluid-flow induced β-catenin nuclear

translocation and up-regulation of Wnt associated proteins,Wnt5a, and Ror2, which are both involved in RhoA activa-tion [88] and ultimately Runx2 expression [89]. A signifi-cant decrease in β-catenin/N-cadherin association followingfluid-flow was also shown, resulting in an increase in cyto-plasmic β-catenin [88], suggesting a role for adherens junc-tions as mechanosensors. In 3-dimensional cultures acontinuous level of flow-induced shear stress showed anincrease in collagen synthesis, and an increase in the tensilemechanical properties of the tissue [91]. Application offluid-flow perfusion increased ECM deposition comparedwith static control, demonstrating a potential beneficial ef-fect also on chondrogenic differentiation [92].

Our work modeling biophysical stimuli generated bymuscle contractions in developing skeletal rudiments pre-dicted a number of interesting patterns corresponding todifferentiation events, corroborated by phenotypic effectsin abnormal mechanical environments: (1) a sustained peri-od of cyclic stress at the perichondrium promotes chondro-cyte hypertrophy at the core, stimulating subsequent bonecollar formation [21]; and (2) modelling of stimuli in thejoint region indicated that the articular cartilages and patelladevelop under the influence of very specific patterns ofbiophysical stimuli; the chondrogenous layers emerge fromlocations that experience distinct patterns of elevated fluidflow while the patella develops under much higher magni-tudes of stress, fluid velocity, and pore pressure [38•].

Conclusions

In order to effectively apply biophysical stimuli to cellulardifferentiation regimes for regenerative therapies, we needto know more about how cells in the developing embryorespond, in different contexts, across space and time as thetissues emerge. The accumulating evidence reviewed hereshows that several aspects of normal skeletogenesis requireappropriate mechanical stimulation generated by embryonicmovement; tissue patterning during joint formation, ossifi-cation, and rudiment morphogenesis (Fig. 1). We now needto discover the precise molecular events guided by mechan-ical stimuli and how mechanical and biochemical signals areintegrated in order to apply this knowledge in therapeuticapproaches. Clearly the effects of biophysical stimuli willdepend on the cell context and other aspects of the signal-ling milieu. Therefore, more testing is needed on definedpopulations of progenitor cells to reproduce the integratedbiochemical and mechanical signalling environment [82].Bioreactor systems could be developed to reproduce multi-ple aspects of in vivo developmental conditions aimed atcreating tissues with improved and more stable functionalproperties. It would be unrealistic to expect to reproduce allconditions that guide normal development of, for example,

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articular cartilage, however improved knowledge of theintegrated mechanisms and their outcome, combined withtailor made bioreactor systems would allow definition ofessential differentiation regimes toward establishing regen-erative therapies for diseased or aged joints.

Conflicts of Interest R Rolfe declares no conflicts of interest; KRoddy declares no conflicts of interest; and P Murphy declares noconflicts of interest.

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