Mechanical Influences on Morphogenesis of the Knee Joint Revealed through Morphological ...

14 Pages·2011·2.1 MB·English

Mechanical Influences on Morphogenesis of the Knee
Joint Revealed through Morphological, Molecular and
Computational Analysis of Immobilised Embryos
Karen A. Roddy1,2, Patrick J. Prendergast2, Paula Murphy1,2*
1DepartmentofZoology,SchoolofNaturalSciences,TrinityCollegeDublin,Dublin,Ireland,2TrinityCentreforBioengineering,SchoolofEngineering,TrinityCollege
Dublin,Dublin,Ireland
Abstract
Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle
contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that
biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis
influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint
interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent
Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of
computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital
representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes
knowntoregulatejointdevelopment.Thisworkrevealedtheprecisechangestoshape,particularlyinthedistalfemur,that
occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of
mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the
emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of
characteristicinterzonegeneexpressionandabnormalexpressionofcartilagemarkers.Thisworkshowsthatlocaldynamic
patternsofbiophysicalstimuligeneratedfrommusclecontractionsintheembryoactasasourceofpositionalinformation
guiding patterningand morphogenesis ofthedeveloping kneejoint.
Citation:RoddyKA,PrendergastPJ,MurphyP(2011)MechanicalInfluencesonMorphogenesisoftheKneeJointRevealedthroughMorphological,Molecularand
ComputationalAnalysisofImmobilisedEmbryos.PLoSONE6(2):e17526.doi:10.1371/journal.pone.0017526
Editor:SudhaAgarwal,OhioStateUniversity,UnitedStatesofAmerica
ReceivedDecember14,2010;AcceptedFebruary3,2011;PublishedFebruary28,2011
Copyright: (cid:2) 2011 Roddy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyaTCDOverheadInvestmentPlan(OIP)InterdiscipinaryAward,WellcomeTrustprojectgrant(083539/Z/07/Z)andScience
FoundationIreland(ProgrammeAward02/IN1/B267).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationof
themanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction rudiments (including the prominent condyles of the distal femur)
dictate the shape of thearticular surfaces of theknee while other
Eachskeletalrudimentandjointofthelimbcanbeidentifiedby joint structures such as articular cartilages, menisci and the
itsunique,speciesspecific,sizeandshape.Theseindividualshapes synovium derive from cells in the joint interzone [6,7]. The
emerge by the local modulation of cellular processes, such as cell emergenceofkneejointshapeandformmustthereforeinvolvethe
proliferation, differentiation, extracellular matrix synthesis, cell localregulationofgrowthinthecartilaginousrudimentsandtissue
shapeandsize[1],creatingcomplexshapesfromrelativelysimple differentiation withinthejointinterzone.
initial morphologies. Skeletal morphogenesis is regulated by a Several lines of evidence show that contraction of the
combination of inductive regulatory signals produced by the developing embryonic musculature is required for normal
constituent tissues [reviewed in 2,3,4]. While such networks of skeletogenesis.Humancongenitalmalformations[8,9]andanimal
molecular regulatory signals are clearly essential to the correct models where muscle contractions are removed or altered using
establishmentofspatialpatterning,thereisevidencethatfeatures neuromuscular blocking agents [10,11,12,13], surgery [11] or
ofthephysicalenvironment,suchasmechanicalforcesinducedby explant culture [14,15,16] and mouse mutants where no skeletal
muscle contraction, contribute to regulatory mechanisms govern- muscleforms[17,18,19,20],leadtounderdeveloped,brittleandin
ingmorphogenesis.Wefocusonthedevelopingchickkneejointas some cases misshapen skeletal elements [11,12,21,22]. Joints
aconvenientmodeltoinvestigatehowmechanicalforcesintegrate appear to be particularly sensitive with immobilisation leading to
with cellular and molecular events to impact the emerging loss of joint structures such as the cavity, articular surfaces and
properties oftheskeleton. patella [10,11,12,19,20,21,23]. It is unknown how mechanical
We have previously shown that the complex 3D shape of the stimulation derived from movement can influence rudiment and
kneejointemergesfollowingtheinitiationofmusclecontractions, joint morphogenesis but computational modelling has been used
between chick embryonic stages Hamburger and Hamilton topredictmechanicalloadsactingonthetissues.Previousstudies
(HH)28 and HH34 [5]. The interfacing ends of the cartilaginous usedFiniteElement(FE)modellingtopredictmechanicalforcesin
PLoSONE | www.plosone.org 1 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
simplified representations of the skeleton to investigate joint loadsinhibitcellmatrixsynthesisandgrowth[49,50,51,52]while
formation [24],endochondral ossification[25,26],theemergence low magnitude dynamic loading stimulates matrix synthesis
ofthefemoralbicondylarangle[27]anddevelopmentaldysplasia [49,50,51,53].Forexample,thedynamiccompressionofcartilage
of the hip [28]. We previously [29] created a FE model from explants by approximately 3% was shown to stimulate matrix
morphologically accurate 3D data captured from the developing synthesis while graded levels of static compression did not [49].
chick tibiotarsus to simulate the dynamic patterns of stimuli Mechanicalforcesareknowntoinfluencetheexpressionofcertain
generated by muscle contraction. A striking correspondence genes in mechanically stimulated cells when compared to non
between the patterns of stimuli and the dynamics of ossification stimulatedcells[52,54,55].Suchgeneshavebeencalledmechan-
was noted and we further showed that in an altered mechanical osensitiveormechanoresponsiveandevidenceexiststhatseveralof
environmentossificationwasreducedandtheinvivoexpressionof the molecules involved in the development of the joint and
a number of genes involved in bone formation was altered [13]. regulation of ossification are mechanosensitive, at least in a cell
More recently we used a similar approach to create a culture context. Such genes include the previously mentioned
morphologically accurate FE model of knee joint development BMP2, CD44, b1 integrin subunit, FGF2, FGFR2 and PTHLP
[30], indicating that the tempero-spatial pattern of mechanical (Table1).Alimitednumberofstudieshaveinvestigatedalterations
stimuli generated in the distal femur by muscle contraction intheexpressionpatternsofregulatorygenesindevelopingtissues
correspondswithaspectsofthepatternofshapechangesandwith in vivo in response to immobilisation, showing for example
differentialratesofcellproliferationinthefemoralcondyles.This alteration in FGF2 [37], IHH and COLX [22]. Such in vivo
ledtotheproposalthatmechanicalforcescouldactasaphysical studies have the advantage of demonstrating mechanosensitivity
form of positional information, generating local patterns that withina specific developmental contextandalso makeit possible
modulate cellular events such as cell proliferation and differenti- to relate the changes in gene expression to changes in tissue
ation,thereby guiding tissuemorphogenesis. differentiation andmorphogenesis.
A number of key molecules regulating cartilage growth Inthispaperwefurtherexplorethelinkbetweenlocalpatternsof
and differentiation [31,32,33,34,35,36] and joint formation biophysicalstimuligeneratedbyembryonicmusclecontractionsand
[32,37,38,39,40,41] have been identified. PTHLP(also known as thegenerationofshapeandstructureintheaviankneejoint.Inovo
PTHrP) has been shown to act in a regulatory loop with Ihh to immobilisationwasusedtoalterthemechanicalenvironmentduring
maintain a pool of proliferating chondrocytes at the epiphysis of developmentandchangesintheresultingstructureandshapeofthe
long bones [34,42]. BMP and FGF family members act in an knee joint region were revealed. Shape in particular was analysed
antagonistic relationship to co-ordinate differentiation and prolif- following 3D imaging ofcontrol andimmobilised specimensusing
eration processes during development of the skeletal rudiment Optical Projection Tomography (OPT). To explore cellular
[43]. A large number of genes, including BMP2, FGF2, FGFR2, processes impacted by mechanical stimulation, patterns of cell
PTHLH, b1 integrin (ITGB1), CD44 and HAS2, are expressed proliferation in the distal femur were compared between immobi-
specifically in the joint region. Several of these gene products lised and control specimens. A FEmodel of rigid muscle paralysis
regulatechondroctyegrowthanddifferentiationwhileotherssuch was used to determine how the local mechanical information
asCD44andHAS2regulatejointcavitationthroughtheactionof producedbymusclecontractionswouldbealteredbyparalysisand
hyaluronan[44,45,46].CD44encodesoneofthemajorreceptors inturnhowthiscompareswiththealterationsinjointshapeandcell
for hyaluronan while HAS2 encodes an enzyme involved in its proliferationobserved.Finallytheeffectofalteredmechanicalforces
synthesis. Inhibition of a5b1 integrin leads to ectopic joint ontheexpressionofregulatorygeneswasinvestigatedusinginsitu
formation while missexpression causes the inhibition of joint hybridisation. Genes were chosen for analysis based on previous
formation leading tofused long bones[47]. experimental evidence of a regulatory role in the process of joint
Verylittleisknownabouthowcellsrespondtomechanoregula- formation and an indication of mechanosensitivity in another
tion,especiallyinaninvivodevelopingsystem.Mechanoregulation cellular context (Table 1). The findings support and further the
ofchondrocyteproliferationandbiosynthesishasbeenextensively hypothesis that patterns of biophysical stimuli generated by the
studied in a wide range of culture systems, including explants, contracting musculature act as a type of positional information
monolayer and 3D scaffolds [reviewed in 48] where it has been during skeletal morphogenesis,impacting the molecular regulation
proposedthatcontinuousloadsorhighfrequency,highmagnitude ofcellproliferationandtissuepatterning.
Table1. Summaryof regulatorygenes selectedforanalysisbased onfunctional evidenceand mechanosensitivity.
Gene Evidenceofskeletalfunction Evidenceofmechanosensitivity
BMP2 chondrocytematurationandproliferation[31,35,85], Distractionosteogenesis(invivo)[86]
CD44 jointcavityformation[44] inculture[71,87]
HAS2 jointcavityformation[88] noevidence
b1integrin interzoneformation[47] inexplants[89]
WNT9a interzonespecification[90,91] noevidence
PTHLP maintainschondrocyteproliferation[31,34,42] inculture[92]
FGF2 jointcavityformation[93],chondroctyematuration[43,94] invivo[93]
FGFR2 proliferationofosteoprogenitorcells[95] inculture[96]
COL2A1 ECMmatrixcomponent[97],markerofproliferatingchondrocyes invivo[20]
TNC ArticularcartilageECMmatrixcomponent[98,99] invivo[98]
doi:10.1371/journal.pone.0017526.t001
PLoSONE | www.plosone.org 2 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
Results revealed absence of the inter-articular ligaments (Figure. 1F and
H),thechondrogenouslayersandmenisci(Figure1JandL).The
Abnormal development of the knee joint following expressionofbothmarkersalsoappearedtobereducedorabsent
muscle immobilisation inthejointcapsuleandpatellaregion(Figure1F,J)inimmobilised
Comparison of embryos immobilised with 0.5% DMB for 4–5 joints.
days, commencing on day 4.5 of incubation, and control
specimens,revealedanumberofconsistentabnormalities.Staging Shape changes in the knee joint
oftheembryos,usingtheHamburgerandHamiltoncriteria[56], To reveal shape changes in the knee joint following immobi-
insuredthatonlystagematchedspecimenswerecompared.Drug lisation, 3D analysis of Alcian blue stained, OPT scanned
treated embryos showed previously reported effects of immobili- specimens following 4 or 5 days of immobilisation was carried
sationincludingspinalcurvatureandjointcontracture(notshown) out (n=17 and 32 for immobilised specimens on days 4 and 5,
[10,21].Specificallyinthekneejoint,histologicalsectionsshowed n=16and18forcontrols).3Ddigitalrepresentationsofeachknee
a general reduction in the separation of the rudiments, altered jointspecimencouldbeorientedtoviewcomparablesections[57]
cellular organisation in the interzone with no clear definition of andtakemeasurementsthatcapturecharacteristicaspectsofshape
chondrogenous layers and no sign of cavitation in the altered including the width of the proximal tibiotarsus and fibula, the
mechanicalenvironmentofimmobilisedspecimens(Figure1A–D separationofthetibiotarsusandfemur(interzone)andtheheight
and K,L). Additional alterations to knee joint associated tissues and width of the condyles and intercondylar fossa of the distal
wererevealedthroughmarkergeneexpressionanalysis.Collagen femur (individual measurements detailed in Figure 2). Statistical
typeIIalpha1(COL2A1)geneexpressionmarksthejointcapsule, analysis of the measurements showed that immobilisation had a
developing ligaments and tendons and initial appearance of the significant effect on specific morphological features of the knee
patella, in addition to the cartilaginous rudiments in control (Table 2). Immobilisation caused a significant reduction in the
specimens at this stage (Figure 1E and G). Tenascin C (TNC) width of the proximal epiphysis of the tibiotarsus and fibula
expressionalsomarksthejointcapsuleandpatellaandrevealsthe (Table2).Thedistalendofthefemur,showedareductioninthe
chondrogenous layers, the perichondrium and the appearance of height of both condyles in the dorso-ventral orientation but no
the menisci in the joint interzone (Figure 1I and K). In significant reduction in width following 4 days of immobilisation
immobilisedkneejoints,expressionanalysisofthesetissuemarkers (Table2),eitheratthemidlineorventralaspect.Thesameoverall
Figure1.Anatomicalchangesinthekneejointsofimmobilisedembryos.Longitudinalsectionsthroughthechickkneejointofcontroland
immobilisedembryos(4.5daysofimmobilisation)atlow(A,B,E,F,I,J)andhighmagnification(C,D,G,H,K,L).Histologicalsections(E–D)were
stainedusingalcianblueandcounterstainedwithhaematoxlyinandeosin.OthersectionsshowtheexpressionofCOL2A1andTNCmRNAincontrol
andimmobilisedkneejoints.c;cavity,cl;chondrogenouslayers,jc;jointcapsule,lg;ligament,m;meniscus,p;patella,pc;perichondrium,t;tendon.
Scalebar0.5mm(A,B,E,F,I,J)and0.1mm(C,D,G,H,K,L).
doi:10.1371/journal.pone.0017526.g001
PLoSONE | www.plosone.org 3 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
Figure2.Overviewofmorphometricanalysisofthekneejoint.3Dvolumerepresentationofthehindlimb(A–B)atHH35withguidelinesin
redshowsthelocationofconsistentvirtualsectionstakenthroughthehindlimbsofallspecimens(C–G).Individualmeasurementsareindicatedby
linesi-xii.Scalebar1mm.
doi:10.1371/journal.pone.0017526.g002
effects on shape were seen following 4 and 5 days of following 4 and 5 days of treatments respectively (p,0.001,
immobilisation except that a reduction in the width of condyles Table 2).
in the ventral aspect became apparent with extended treatment Thestrongestandmostconsistentchangeinshapewasseenin
(Table 2,highlighted). thewidthoftheintercondylarfossa;reducedby41.6%(P,0.001)
The apparent reduction in the separation of rudiments in the at the midline and 44.7% (p,0.001) at the ventral side of the
knee joint noted from histological sections was confirmed here femurfollowing4daysofimmobilisation(Table2).Thisreduction
through a significant reduction in the size of the interzone in the separation of the femoral condyles is still obvious after 5
separating the femur and the tibiotarsus; 30.1% and 35.6% days ofimmobilisation (30.2and36%,p,0.001).
Table2.Comparisonofmeanmorphometricmeasurementsofcontrolandimmobilisedkneejointsfollowingimmobilisationfor4
or5days.
Day4 Day5
Control Immob %reduction significance Control Immob %reduction significance
TibiotarsusEpiphysealwidth(i) 1.18 0.94 19.65 F =60.43,,0.001 1.19 1.01 15.06 F =62.75,p,0.001
(1,29) (1,46)
FibulaEpiphysealwidth(ii) 0.55 0.45 18.44 F(1,29)=25.61,p,0.001 0.57 0.46 20.29 F(1,46)=90.92,p,0.001
Interzone(iii) 0.08 0.06 30.12 F(1,29)=35.55,p,0.001 0.10 0.07 35.59 F(1,46)=41.61,p,0.001
FemurHeight LateralCondyle(iv) 0.96 0.79 18.26 F =36.40,p,0.001 1.02 0.88 13.22 F(1,46)=58.62,p,0.001
(1,29)
Intercondylarfossa(iv) 0.30 0.27 10.33 F(1,29)=4.19,p=0.05 0.32 0.30 6.64 F(1,46)=7.93,p=0.007
MedialCondyle(v) 0.81 0.70 12.98 F =23.22,p,0.001 0.82 0.72 12.49 F =23.37,p,0.001
(1,29) (1,46)
FemurWidth LateralCondyle(vi) 0.40 0.39 NS 0.46 0.43 NS
midline
Intercondylarfossa(vii) 0.29 0.17 41.63 F =36.67,p,0.001 0.25 0.18 30.18 F =28.51,p,0.001
(1,29) (1,46)
MedialCondyle(iix) 0.30 0.30 NS 0.34 0.32 NS
FemurWidth LateralCondyle(ix) 0.42 0.39 NS 0.50 0.42 15.41 F(1,46)=27.46,p,0.001
Ventral
Intercondylarfossa(x) 0.31 0.17 44.69 F(1,29)=18.40,p,0.001 0.31 0.20 35.95 F(1,46)=98.37,p,0.001
MedialCondyle(xi) 0.29 0.27 NS 0.33 0.28 13.88 F =24.18,p,0.001
(1,46)
Aparticularlyinterestingpatternwithrespecttothewidthoftheintercondylarfossaishighlightedinbold.Percentagedifferencesbetweenthemeanlengthsofthe
measurementsareshownwiththeassociatedstatisticalsignificance.
doi:10.1371/journal.pone.0017526.t002
PLoSONE | www.plosone.org 4 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
This and other detailed aspects of local shape changes in the thelateralcondylealsoappearedtobeflattenedbyimmobilisation
distal femur were visualised by outlining cartilage (alcian blue (Figure 3C). Five days of immobilisation caused greater simplifi-
stained) in comparable sections of 3D reconstructions. Outlines cation.Inparticularnotethereductioninheightofthemedialand
were generated using the sections shown in Figure 2 C, D and F lateralcondylesinthedorsalaspect(Figure3D,brackets)andthe
andphysically overlaidso that themedial andlateralsidesof the reduced outgrowths on the ventral aspect of the condyles
femora in the sections were parallel and the midpoints of the (arrowheads Figure 3D) in the region of the trochlea fibularis
intercondylar fossa were overlapping. Overlaying outlines of this grovewherethefemurinterfaceswiththefibulaenablingsmooth
characteristic view of control and immobilised specimens high- movement in later life. A flattening of the articular surfaces was
lighted the effect of rigid paralysis on the emergence of shape in nowapparent inbothcondyles (Figure 3E,F).
the femoral condyles (Figure 3A–F). Without muscle contraction
thegeneralshapeofthekneejointismuchsimpler,jointsurfaces Alteration of cell proliferation patterns in immobilised
are flattened (e.g. flattening of the lateral condyle shown in specimens
Figure3CandF)andfunctionaloutgrowthsarelost.After4days A comparison of the proportion of proliferating cells in five
ofimmobilisationthereductioninwidthoftheintercondylarfossa selectedlocationsofthedistalfemurincontrolandexperimentally
wasveryobvious,particularlyventrally(Figure3A).Thesurfaceof immobilised embryos (4 days of immobilisation) was performed
Figure3.Comparisonofcartilageshapeandcellproliferationindistalfemoraofcontrol(blue)andimmobilised(red)embryos.
Outlinesofthecartilageanlaga(A–F)wereextractedfromvirtualsections(Fig.2,C,D,F).Aminimumoffouroutlinespertreatmentwereoverlaid.
Arrowheadsindicateregionofreducedoutgrowthintheimmobilisedanimals.*indicatesflatteningofrudimentsurfaces.Blackbracketsshowextent
of growth reductions in the dorsal aspect of the condyles. Coloured brackets compare width of the intercondylar fossa of control (red) and
immobilised(blue)embryos(A,D).Thedistributionofproliferatingchondroctyesinthefemuracross5regions,representedinG(boxes1–5),was
comparedincontrol(blue)andimmobilised(red)embryos(h).tf;trochleafibularis,IF;intercondylarfossa.
doi:10.1371/journal.pone.0017526.g003
PLoSONE | www.plosone.org 5 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
(Figure 3G,H). Looking across the locations in control animals, muscles), as induced by DMB, to compare predicted biophysical
similar proportions of proliferating cells were observed across the stimuli in immobilised as compared to normal developing joints.
femur head except for a slightly lower proportion in the region The obvious change to the mechanical environment in the rigid
adjacenttotheintercondylarfossa.Theeffectoftreatmentoncell model is the loss of dynamic stimulation associated with a
proliferation was investigated using a generalised linear mixed contraction cycle since muscle tension is constant. In addition,
effects model where multiple sections were nested within changes in the predicted patterns of the local mechanical
individuals in order to take account of the nested nature of the environmentareindicated,includingreductionstothemagnitude
data.Combiningdataacrossthelocations,treatmentsignificantly of loads and a general simplification of the spatial pattern of
reduced the proportion of proliferating cells by an average of stimuli (Figures4 and5).
11.8/1000 chondrocytes (s.e.=3.7, df=4, p=0.03) (not shown). Focusing on the distal femur (Figure 4), the most obvious
Examining each location separately, immobilisation caused a pattern of stimuliobservedinthenormalmodels wasthedistinct
significantreductionintheproportionofproliferatingcellsinfour peakcentredontheregionadjacenttotheintercondylarfossaand
out of the five locations with the ventral aspect of the medial ageneralelevationofstimuliinthedorsalregionsofbothcondyles
condyle being the only region unaffected (Figure 3H). These (Figure4E–G,Flex,Extend).Whilethispeakwasstillpredictedfor
changes correspond to the major alterations observed in joint patterns of Von Mises stress and Minimum Principal stress
shapefollowingimmobilisationwithlargereductionsinthedorsal (compression)(Figure4E,G)underrigidparalysis,itisreducedfor
side of both condyles and no apparent reduction in the ventral patterns ofMaximumPrincipalstress(tension) (Figure4F).Thus,
aspect of the medial condyle (Figure 3D). Narrowing of the while the normal joint develops under the influence of dynamic
intercondylar fossa was one of the strongest effects of immobili- patternsoftensionandcompression(compareFlexandExtendfor
sation revealed by the morphometric study (Table 2) and the each)therigidparalysismodelislargelyexperiencingcompression.
proportion of proliferating cells was reduced in the cartilage
rudiment adjacent tothedorsalaspect ofthefossa. Biophysical stimuli patterns in the interzone region
correspondwiththelocationofanatomicalfeaturesthat
Effect of immobilisation on patterns of biophysical are disrupted following rigid paralysis
stimuli in the developing knee The FE simulations of biophysical stimuli in the developing
We previously constructed a FE model to predict biophysical chick knee joint comparing normal muscle contraction and rigid
stimuliduringanextension/flexioncycleofmusclecontractionin paralysis, as described above, were also used to examine the
the developing knee joint region [30]. We adapted this model to mechanical environment in the interzone region between the
represent rigid limb paralysis (simultaneous contraction of all skeletalrudiments(Figure5).Themodelsweregeneratedusingthe
Figure4.PredictedpatternsofbiophysicalstimuliinFEmodelsofimmobilised(boxed,rightcolumnofeachpanel)comparedto
normaldevelopingfemora.IllustrationsshowtheFEmesh(A),theplacementoftheloadsforbothflexion(B)andextension(C)andtheplaneof
section(D)showninE,FandG.ThepatternsofVonMisesstress(E),Maximum(tension)(F)andMinimum(compression)(G)Principalstress,were
captured in equivalent sections through the distal femur (indicated in D) at HH30 (row i), HH32 (ii) and HH34 (iii). Patterns at mid flexion and
extensionareshownforthenormalmodelswhereastheconstantpatternduringrigidparalysisisshownforimmobilised.Stimulipatternsfornormal
contractionsarecapturedfromsimulationspreviouslydescribed[30].
doi:10.1371/journal.pone.0017526.g004
PLoSONE | www.plosone.org 6 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
Figure5.ComparisonoftheVonMisesStress(B),FluidVelocity(C)andPorePressure(D)withinsectionsofthedevelopingknee
interzoneregionatHH32underrigidparalysis(boxedonright)andnormalmusclecontractions.Locationofsections,isindicatedby
redlinesinA.*indicateslocationofthepatellaregion(B),redarrowindicatespatternofelevatedfluidvelocityinthepresumptivechondrogenous
layer(C),bluearrowsindicatepeakinporepressureattheintermediatelayer(D).Flex;midflexion,Extend;midextension,Immob;Immobilisation.
Theimagesfornormalcontractionsarecapturedfromsimulationspreviouslydescribed[30].
doi:10.1371/journal.pone.0017526.g005
first direct estimates of mechanical properties of cartilage and therighthindlimbforalcianbluestaining/OPTscanningandthe
interzone tissue in the developing chick knee using nanoindenta- lefthindlimbforgeneexpressionanalysis.Threecandidategenes,
tion, as previously described [30]. The general pattern of stimuli b1 integrin, FGFR2 and WNT9a showed no difference in
did not vary between stages, thus Figure 5 presents the results at expression pattern between control and immobilised specimens
HH32 only,the midpointinthestudy. (notshown).Figure6showsdifferencesintheexpressionobserved
The patterns of stimuli predicted under normal muscle in control and immobilised embryos on longitudinal sections
contractions indicated that several territories and tissues develop- through theknee joint(plane ofsection as inFigure 2D).
ingwithintheinterzoneexperiencespecificpatternsofstimulation InthekneejointatthisstagePTHLHexpressionwasdetected
[initially described in 30, Figure 5]. While the patella normally only at the very proximal and distal ends of the cartilaginous
develops under dynamic magnitudes of stress, fluid velocity and anlagen, restricted to the periarticular cartilage where the
pore pressure (Figure 5B–D, indicated by *; compare Flex and rudiments oppose (Figure 6A, A’). Immobilisation disrupts the
Extend), in rigid paralysis, the territory of elevated stimulation is characteristicpatternofPTHLHsothatitisnolongerrestrictedto
restrictedandthedynamicaspectislost.Thisisofparticularnote the periarticular regions but is detected across the interzone
sincethepatellafailstoappearinimmobilisedembryos(Figure1). (Figure 6B, B’) (n=5/5). The expression pattern in immobilised
Appearanceofthechondrogenouslayersisalsolostinimmobilised animals appears to have ‘‘fuzzy’’ boundaries compared to the
embryos. The chondrogenous layers emerge in a location that more clearly defined territories in control specimens. This
experiencesdynamicpatternsofelevatedfluidvelocity(Figure5C. surprising finding of expression across the joint region of a gene
indicated by red arrow) while the intermediate layer, separating normally restricted to cells within cartilage rudiments is compli-
the two chondrogenous layers, emerges under a pattern of mented by detection of COL2A1 transcripts in some cells
dynamically elevatedpore pressure(Figure5D,indicatedbyblue spanning the interzone in immobilised individuals (n=7/7),
arrow). Under rigid paralysis, elevated fluid velocity in the similar totheexpression ofPTHLP (Figure 6C,D,C’D’).
presumptive chondrogenous layer and elevated pore pressure in Expression of FGF2 in control knee joints was detected within
the presumptive intermediate layer is still predicted (Figure 5, the chondrogenous layers of the interzone, part of the forming
C,D), although in less extensive territories, but the pattern is no meniscus,theregionofthefuturepatella,prominentlywithinthe
longer dynamic. cranial cnemialcrest (Figure 6E)andsurrounding thedeveloping
tendons (not shown). Immobilisation resulted in a specific
The expression of genes that regulate joint alteration to the spatial expression of FGF2 with expression no
morphogenesis is altered in immobilised embryos longerdetectedinthechondrogenouslayersatthepointwherethe
Specific changes were observed in the shape of cartilage femurisclosesttothetibiotarsus(n=5/5)(Figure6F)i.e.normally
rudiments and theappearance of tissues associated with theknee continuous expression in the chondrogenous layers is disrupted.
joint in immobilised embryos (Figures 1 and 3). To explore the Expression in the presumptive patella region and meniscus was
molecular basis of these changes, a number of regulatory genes also absent in immobilised animals whereas expression in the
implicated in cartilage growth or joint cavity formation were cranial cnemial crestwas unchanged(Figure 6F).
selected for expression analysis, comparing immobilised and In control specimens BMP2 transcripts were detected in the
controlembryos.Thecandidategenes(Table1)werealsoselected perichondriumalongthelengthoftherudiments,theintermediate
on the basis of some evidence of mechanosensitivity in another layer of the interzone, the developing patella and joint capsule
context(e.g.cellculture).Geneexpressionanalysiswascarriedout (Figure6G).WhenBMP2expressionwasanalysedinimmobilised
onthesameembryosusedformorphometricanalysisabove;using embryos,clear,elevatedexpressionwasnolongerdetectedinthe
PLoSONE | www.plosone.org 7 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
Figure6.Expressionofcandidatemechanosensitivegenesincontrolandimmobilisedspecimensonlongitudinalsectionsthrough
the knee joint. Images A’–D’ (scale bar 0.1mm) show the knee region of A to D (scale bar 0.5mm) at a higher magnification. cc; capsular
condensation,cl;chondrogenouslayer,iz;intermediatelayer,jf;jointfusion,m;meniscus,p;patella,par;periarticularcartilage,t;tendon,tc;tibial
crest.
doi:10.1371/journal.pone.0017526.g006
intermediate layer (n=10/10) (Figure 6H). Expression in the inthedevelopingkneejointandweinvestigatedtheimpactofsuch
perichondrium and joint capsule remained unchanged and some immobilisation on the tissues of the developing joint at
expressionofBMP2withinthepatellacouldbedetectedalthough morphological and molecular levels. We showed that when
theexpressionlevelandsizeoftheexpressiondomainappearedto dynamic stimulation is removed, patterning of the interzone and
be reduced (not shown). joint morphogenesis are altered with very specific changes to the
Similar to BMP2 expression, CD44 and HAS2 have very shapeofthecartilagerudimentsandabnormaldefinitionoftissue
definedexpressionwithinthepresumptivejointlineregioninthe territories within the presumptive joint. The altered shape of the
intermediate layer of the interzone (Figure 6I, k). HAS2 is also cartilaginousrudimentsisaccompaniedbyregionspecificchanges
expressed in the most distal part of the inter-patella-femoral fat in cell proliferation. The altered definition of tissues within the
pad adjacent to the tibiotarsus (Figure 6I). CD44 also shows jointinterzoneisshownnotonlybytheexpressionoftissuemarker
additionalexpressionintheregionofthefuturepatella(Figure6K), genes but also by altered expression of regulatory genes that are
themuscleblocksandcellssurroundingtheligaments(notshown). involved insteering differentiation andmorphogenesis.
Expression of both genes was lost from the intermediate layer of The dependence of correct joint development on stimulation
immobilised joints (Figure 6J,L). This loss occurred in all from muscle contractions was previously shown by similar
specimens analysed (8 assayed for HAS2, 7 for CD44). Specific immobilisation studies in the chick [10,11,21,23,58] and using
CD44 expression in the region of the future patella was lost in genetically altered mice that have absent, reduced or non-
immobilised specimens (Figure 6L). In addition, both genes contractile muscle [19,20]. However in the altered mechanical
showed elevated expression throughout the inter-patella-femoral environment of mouse models, Nowlan et al [19] showed that
fat pad ofimmobilised embryos (Figure6J,L). forelimbs are more affected than hindlimbs and that while the
elbowjointisseverelyaffected,thekneejointappearsnormal.In
Discussion thecurrentworkwecomparedtheeffectsofimmobilisationonthe
kneeandelbowjointsinthechickandfoundthattheelbowjoint
Blocking muscle contractions in the chick embryo alters the showed similar but no more severe alterations (not shown)
biophysicalenvironmentofthedevelopingmusculoskeletalsystem. highlighting a clear and intriguing difference between avian and
Inthisworkweusedcomputationalmodellingtodemonstratehow mammalian models. Computational modelling of the mouse
rigidimmobilisationwouldaffectthemechanicalstimuligenerated model demonstrated thatpassivedisplacementofembryoniclimbs
PLoSONE | www.plosone.org 8 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
inuteroduetomovementofthemotherandnormallittermateswould forces the expression patterns of marker genes Tenascin C and
produce greater stimulation of the hindlimbs than the forelimbs CollagentypeIIalpha1(COL2A1)andregulatorygenesPTHLP,
providing a possible source of compensation for the reduced BMP2, FGF2, CD44 and HAS2 were altered (summarised in
stimulation, particularly in the hindlimbs (unpublished data). The Figure 7).Normal expression of FGF2, BMP2,CD44 andHAS2
in ovo situation of the chick embryo means less passive movement was disrupted or lost specifically in the interzone regions of
from external sources and therefore a greater reliance on muscle immobilised embryos which acquire cartilage like tissue charac-
contractionstogeneratemechanicalstimuliinthehindlimbs. teristics as indicated by the inappropriate activation of COL2A1
Previous studies demonstrated that knee joints in immobilised andPTHLPexpression.Inadditionboundariesofgeneexpression
chickembryosfailtocavitate[10,11,21,23,58,59,60]buttherehas between cartilage rudiments and the interzone were less distinct
beenverylittleemphasisonchangestotheshapeofthejoint.Here suggesting either cell movement and cell mixing between the
weusedmorphometricanalysisof3Ddigitalrepresentationsofthe territories or transdifferentiation of cells in the interzone to a
specimenstopinpointtheshapefeaturesofthedistalfemurthatare cartilaginous character; the latter interpretation is supported by
dependent on extrinsically produced stimuli from muscle contrac- thefindingsofKahnetal[20]ofaberrantexpressionofCOL2A1
tions,showingalinkbetweentheshapechanges,changesinlocalcell inlineagelabeledinterzonecells(descendedfromGdf5expressing
proliferation and predicted biophysical stimuli. Specific changes cells) in immobile mouse embryo limbs. Finite element models
included simplification of the shape of the medial and lateral predictedthatthepatternsofbiophysicalstimulicreatedbymuscle
condylesofthedistalfemurwithflatteningofthecondylesandthe contractions correspond with the emergence of specific tissues in
absenceofcharacteristicspurs.Thestrongesteffectwasseenonthe thejoint,suggestingthattheycouldcontributetothepatterningof
separation of the condyles with a consistent narrowing of the these tissues [30]. For example the chondrogenous layers which
intercondylarfossa.Celldivisioncontributestomorphogenesisofa ultimately form the articular cartilages are predicted to develop
tissue when proliferation rates differ in a location specific manner under dynamic elevation of fluid velocity and stress. We propose
andwepreviouslyshowedthatcellproliferationisgreaterinregions from these findings that correct differentiation of interzone cells
ofthedistalfemurthatgrowmostbetweenstagesHH30andHH34 andthemaintenanceof interzone celltype aredependent onthe
[30],forexamplethemedialcomparedtothelateralcondyle.Here mechanical environment to which they are exposed. It has been
weshowthatinimmobilisedembryoscellproliferationratesinthe shown that cultured interzone cells initially express the interzone
cartilaginous rudiments are reduced, specifically where dynamic marker Gdf5, however after several days in culture the cells
mechanicalstimulationispredictedtobestrongest(dorsalaspectof resemblechondrocytesandexpressmarkerssuchasCollagentype
both condyles and the region of intercondylar fossa) and where II [7]. This strongly supports the conclusion that following initial
greatest shape changes are observed in immobilised specimens specification of the interzone, maintenance of the territory and
(reductionofheightofthecondylesandwidthoftheintercondylar further differentiation toward cell types of particular articular
fossa).Wethereforesuggestthatlocalpatternsofbiophysicalstimuli structures isdependent onmechanical stimulation.
contribute to the regulatory mechanisms controlling local growth. Classical descriptions of joint development divide the process
Previousinvitrostudieshaveshownthatmechanicalstimulationcan into two separate phases: interzone specification and cavitation.
influencecelldivisionandmatrixbiosynthesis[49,50,51,53]butwe Earlier immobilisation studies suggested that only the cavitation
show a location specific effect in vivo, relevant to morphogenetic phase is sensitive to mechanical stimulation since the interzone
changes. The altered mechanical environment of rigid paralysis formsbutfailstocavitatewhencontractionsarealtered[reviewed
mightresemblethatofastaticallyloadedculture.Staticloadshave in 61,62]. Our findings show that under rigid paralysis the
been found to inhibit biosynthesis of articular cartilage while organisation of joint territories is altered as discussed above.
dynamic loading, such as in normal muscle contraction cycles, Therefore mechanical stimulation impacts cellular processes
increases synthesis and proliferation of chondrocytes [50,53]. involved in the definition of tissues and cellular differentiation
Comparing the effects of rigid and flaccid paralysis (i.e. static priortocavitation.Thisshowsthatmechanicalstimulationshould
comparedtozeroload),Osborneetal.[12]foundthatrigidparalysis notbeseenashavingamoldingeffectonintrinsicmorphogenetic
ledtoagreaterreductioninthewidthoftheepiphesesindicatingthat processes [62,63] but as influencing the processes fundamentally.
staticloadingmayhaveaninhibitoryeffectongrowth. It also shows the importance of viewing joint development as a
Currentlymuchofwhatisknownaboutcartilagegrowthinlong seriesofinterlinkedevents,asarguedbyLambeetal[61];events
bonesrelatestolongitudinalgrowthandtheassociatedregulationof thatareimpactedbymechanicalstimulationfromanearlystage.
theossificationprocess;verylittleisknownaboutthecontroloflocal Thedatapresentedhereshowthatmultipleaspectsofkneejoint
outgrowths and protrusions such as features of the condyles. patterning and morphogenesis are affected when mechanical
Changes seen in the expression of PTHLP, FGF2 and BMP2 in stimulation is altered (cell proliferation, cell differentiation and
immobilised animals have possible implications for the observed tissue boundaries with consequential alterations to the shape of
altered shape of the femoral condyles due to their roles in the rudiment epiphyses and the structure of the joint and associated
regulationofdiaphysealcartilagegrowth[31].PTHLPisknownto tissues).Thesecompoundeffectssupportourpreviouslyproposed
maintainapoolofproliferatingchondrocytesintherudimentswith hypothesis [30] that local patterns of biophysical stimuli create a
therateofchondrocyteproliferationwithinthispoolmodulatedby type of positional information that contributes to the correct
BMPsandFGFsaspartofaIHH/PTHLPfeedbackloop.Regional patterningofemergingtissuesinthejoint.FiniteElementanalysis
expression of these potential growth modulating molecules could waspreviouslyusedtopredictpatternsofbiophysicalstimuliinthe
influence local growth patterns within the femoral condyles. The normal developing joint and here we used the same approach to
altered expression of the genes encoding these molecules and our simulate rigid paralysis showing changes in the stimuli patterns
previousdemonstrationofmechanosensitivityofIHHexpressionin that correspond with the major changes observed in immobilised
thedevelopingtibiotarsus[29]indicatethatmechanismsregulating embryos. In rigid paralysis all muscles are in tetanus. Modelling
cartilagegrowthareaffectedbyimmobilisation. this situation showed a reduction of stimuli in femoral condyles
Immobilisation also impacted theprocess of cell differentiation corresponding to sites of reduced proliferation and shape change
in the interzone, as indicated by changes in characteristic gene andthereplacementofacomplexdynamicpatternofstimulibya
expressionpatternsandhistology.Intheabsenceofnormalmuscle simplified, static environment. The observed changes in growth
PLoSONE | www.plosone.org 9 February2011 | Volume 6 | Issue 2 | e17526
MechanicalInfluencesonKneeJointMorphogenesis
Figure 7. Representation of the altered patterns of regulatory gene expression due to immobilisation. Colour coded expression
patternsofmarkersandregulatorygenesofinterestinbothcontrolandimmobilisedsections.cc;capsularcondensation,cl;chondrogenouslayer,il;
intermediatelayer,IPFp;inter-patella-femoralfatpad,jf;lossofjointlinedefinition,m,meniscus,MFc;medialfemoralcondyle,p;patella,tc;tibial
crest,tib;tibiotarsus.Note:BMP2andFGF2areexpressedinthecapsule(notshowninlegend).
doi:10.1371/journal.pone.0017526.g007
and proliferation may arise because of either the reduced cilia are particularly interesting in the present context because of
stimulation or the lack of dynamic stimulation or a combination theassociationbetween ciliaand hedgehogsignaling [78].
of both. In the interzone the patterns of stimuli predicted under The regulatory genes analysed in this study were previously
normal and immobilised situations is similar but of course the definedasmechanosensitivebasedoninvitroassays(Table1).Here
forces are static rather than dynamic underlining the importance we show that spatial restriction of the gene expression patterns of
ofdynamicstimuli,alsoobservedintheresponseofcellsinculture PTHLP, BMP2, FGF2, HAS2, CD44, COL2A1 and TNC is
[49,50]. However, alterations to the stimuli patterns may be responsive to biophysical stimuli in an in vivo context (Figure 7)
greater than predicted here for a number of reasons. The rigid revealingpotentialkeymediatorsofmechanicalstimulationofjoint
model was based on normal morphology and assumed normally development that warrant closer analysis. A key question is if the
functioning tendons but tendon development is known to be genes respond directly to mechanical stimuli or if they lie
negativelyaffectedbyimmobilization[64]sotransferofloadsmay downstreamofothermoredirectmediators.Ifwecandemonstrate
be compromised. Also the altered shape of the rudiments in inanappropriateassaysystemthattheresponseisdirectweopenthe
immobilizedspecimensmayaltertheforcesbutthisisunlikelyto possibilityofrevealingthecellularmechanismsthatlinksmechanical
have a large effect since we previously found that the general stimulationwithgeneregulation.Thisisakeyfocusoffuturework.
pattern of the forces do not change dramatically with shape Understanding the input of mechanical signals in the stable
changes over time [30]. So our predictions may be conservative differentiation of skeletal tissues is of particular importance in
andactualchangestothebiophysicalenvironmentinimmobilized attempts to regenerate tissue for replacement therapies including
embryos maybe moreextreme than predicted. therapies for patients with articular cartilage defects such as
Despite numerous examples of mechanoresponsiveness of arthritis. A wide range of different stimuli and culture methods
tissues and cells and a long list of mechanosensitive genes have been used to recapitulate the process of articular cartilage
demonstrated in culture, with a growing number demonstrated development with varying success [69,79,80]. One particular
in vivo [reviewed in 65], we know very little about the biological problem is preventing chondrogenic cells from undergoing
mechanisms that integrate biophysical stimuli with gene regula- hypertrophy as they would in endochondral ossification [81]. A
tion. Several possible sensory mechanisms including integrins, better understanding of how the interzone develops and in
stretch activated ion channels and the primary cilium have been particular the mechanical requirements for articular cartilage
indicated in cellular mechanotransduction [66,67,68,69,70]. A development in embryos provides useful information on the type
number of proteins have been shown to be phosphorylated as a and magnitude of loads which could be applied to cultures to
result of mechanical stimulation including MAP kinases like produce cartilage of the appropriate type and quality for
ERK1/2 [71]. Also, mechanical stimulation of human adult regenerativetherapies.Thepresentworkindicatesthatconditions
articular chondrocytes in culture results in transient tyrosine that increase interstitial fluid flow might be beneficial in the
phosphorylation of the protein kinase pp125FAK, the focal regenerationofarticularcartilage.Torecapitulatestablecartilage
adhesion protein paxillin, and the multifunctional signaling differentiation, theprocess needs tobe betterunderstood.
molecule b-catenin [67]. Primary Cilia have also been proposed
as a cellular antenna capable of detecting mechanical strains Materials and Methods
[72,73] and have been identified on both adult and embryonic
chondrocytes [74,75,76]. A number of ECM receptors are In ovo immobilisation
expressed on cilia [77] leading to the proposal that cilia may Fertilised chick eggs were purchased from Enfield Broiler
transduce mechanical forces from the ECM to the cell. Primary Breedersandincubated(SolwayNatureform)at37.5uCand70%
PLoSONE | www.plosone.org 10 February2011 | Volume 6 | Issue 2 | e17526