Materials design principles of ancient fish armour

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1 Mterils design priniples of nient fish rmour ARTICLES BENJAMIN J. F. BRUET *, JUHA SONG *, MARY C. BOYCE AND CHRISTINE ORTIZ Deprtment of Mterils Siene nd Engineering, Msshusetts Institute of Tehnology, 77 Msshusetts Avenue, Cmridge, Msshusetts 9, USA Deprtment of Mehnil Engineering, Msshusetts Institute of Tehnology, 77 Msshusetts Avenue, Cmridge, Msshusetts 9, USA *These uthors ontriuted eqully to this work e-mil: Pulished online: 7 July 8; doi:.8/nmt Knowledge of the struture property funtion reltionships of derml sles of rmoured fish ould enle pthwys to improved ioinspired humn ody rmour, nd my provide lues to the evolutionry origins of minerlized tissues. Here, we present multisle experimentl nd omputtionl pproh tht revels the mterils design priniples present within individul gnoid sles from the living fossil Polypterus seneglus. This fish elongs to the nient fmily Polypteride, whih first ppered 96 million yers go during the Creteous period nd still retins mny of their hrteristis. The mehnisti origins of penetrtion resistne (pproximting iting ttk) were investigted nd found to inlude the juxtposition of multiple distint reinforing omposite lyers tht eh undergo their own unique deformtion mehnisms, unique sptil funtionl form of mehnil properties with regions of differing levels of grdtion within nd etween mteril lyers, nd lyers with n undetetle grdtion, lod-dependent effetive mteril properties, irumferentil surfe rking, orthogonl mirorking in lminted sulyers nd geometrilly orrugted juntions etween lyers. Derml rmour in fish first ppered t the eginning of the Pleozoi period during the Ordoviin with the rise of the Ostroderms 5 Myr go nd ws ommon mong these erliest fishes. In 857, the geologist Hugh Miller speulted on the mehnil design of gnoid sles y drwing nlogies with the priniple of the two tles nd diploe of the humn skull, the priniple of the vriously rrnged ots of the humn stomh, nd the priniple of Oliver Cromwell s fluted pot.. Romer in 9 hypothesized tht derml rmour served s protetion from predtors 4 nd susequently numer of dditionl mehnil nd non-mehnil funtions hve een proposed 5 7. As nient fish eme more predeous 8, their rmour evolved in terms of their multilyered mteril strutures nd overll geometries; for exmple, lrger pltes roke up into mny smller ones, the thikness of vrious lyers deresed, nd the numer of lyers deresed, ll of whih would hve deresed weight, improved flexiility nd mnoeuvrility nd inresed speed. Interestingly, mny prllels n e found etween the evolution of rmour in the niml world nd humn-designed engineered ody rmour, oth of whih seem to e ontrolled y lne etween protetion nd moility, to mximize survivility 9. The design strtegies used y minerlized iologil tissues, in generl, re n re of gret interest nd muh progress hs een mde in the understnding of onepts suh s rystlline nuletion, growth nd morphology, iomromoleulr interltion nd reinforement, modultion of rystl texture, stiliztion of morphous phses, smll length sle effets, heterogeneity nd so on 6. However, little is known out the mehnil properties of individul derml rmour pltes nd sles. For exmple, the mehnil roles of the mthemtil form of mteril property vritions (suh s grdients) oth within nd etween vrious lyers, the numer of lyers, the lyer nd juntion thiknesses, strutures nd geometries, the onstitutive lws of eh lyer nd so on, nd more importntly the reltionship of these prmeters to lrger sle iomehnil performne nd environmentl stresses (suh s predtory or territoril ttks). Here, we use multisle experimentl nd omputtionl pproh, developed from our previous works 5,7, to eluidte suh design priniples, in prtiulr with regrds to multilyering nd grding. The generl methodologies presented here re pplile to rod rnge of multilyered protetive exoskeletl mterils (for exmple rusten 8, inset 9 nd gstropod mollus ) nd le to yield mehnisti understnding of how different orgnisms were designed, nd possily evolved, to sustin the lods they experiene in their environment. Suh fundmentl knowledge, in prtiulr thret design reltionships, holds gret potentil for the development of improved iologilly inspired struturl mterils, for exmple soldier, first responder nd militry vehile rmour pplitions 9. In this report, we fous on fsinting model system, the interloking, qud-lyered, minerlized sles from Polypterus seneglus, whih elongs to the fish fmily Polypteride (ppering 96 Myr go in the Creteous period ), nd still lives tody, primrily t the ottoms of freshwter, muddy shllows nd esturies in Afri. P. seneglus sles hve retined mny hrteristis of the derml rmour of nient pleonisoids, nd re known to possess multilyered struture (Fig. ) of four different orgni inorgni nnoomposite mteril lyers (from outer to inner surfe): gnoine, type of enmel (thikness µm), dentine (thikness 5 µm), isopedine (thikness 4 µm) nd one sl plte (thikness µm) (ref. ). Instrumented nnoindenttion ws used to mesure the penetrtion resistne, s well s elsti nd plsti mehnil properties, sptilly through the four lyers tht ompose nture mterils ADVANCE ONLINE PUBLICATION

2 mm Soket Peg Bony plte Axil ridge Anterior proess 5 µm Anterior proess Peg 5 µm d e Bone µm µm f g 5 nm nm Figure Struture of Polypterus seneglus sle., P. seneglus skeleton 4., Snning eletron mirogrph (SEM) of outer sle surfe., SEM of the inner sle surfe. d, Optil mirogrph of the ross-setion of n individul sle showing four lyers; only frtion of the ony sl plte is visile. e, Bksttered eletron mirosopy imge of the ross-setion of the gnoine dentine juntion. f, SEM imge of gnoine nnorystls. g, SEM imge of gnoine ross-setion. nture mterils ADVANCE ONLINE PUBLICATION

3 E O P modulus (GP) Epoxy Modulus Hrdness Bone 6 4 H O P hrdness (GP) Distne (µm) E O P modulus (GP) Modulus Hrdness Bone 6 4 H O P hrdness (GP) Lod (µn) 5 4 Bone Lod Unlod FEA Experiment 5 5 Mteril lyer Depth (nm) d e nm 5 nm Figure Mehnil properties derived from nnoindenttion experiments ross the ross-setion of the different mteril lyers of n individul P. seneglus sle. All dt represent n verge of three prllel olumns of 5 indents eh., 5 µn mximum lod; verge indenttion modulus, E O P, nd hrdness, H O P, oth redued from Oliver Phrr nlysis 4 s funtion of distne ross the sle ross-setion. The vertil error rs represent one stndrd devition. Epoxy is epoxy used for emedding., Averge E O P nd H O P for eh of the different lyers derived from 5-µN-mximum-lod nnoindenttion urves. The vertil error rs represent one stndrd devition for tht prtiulr dtset., Averge nnoindenttion lod depth urves for ll four mteril lyers. Horizontl error rs represent one experimentl stndrd devition. d, TMAFM mplitude imges of the residul indents (5 µn mximum lod) on the ross-setion of eh of the four mteril lyers. Top left to ottom right: gnoine, dentine, isopedine nd one. e, TMAFM phse imge of the residul indenttion (5 µn mximum lod) on the isopedine ross-setion. the ross-setion of n individul sle from P. seneglus. A representtive dtset is presented in Fig. ; the Oliver Phrr 4 lulted indenttion modulus, E O P, nd hrdness, H O P, oth derese with distne from the outer to the inner surfes of the sle (from 6 GP to 7 GP nd from 4.5 GP to.54 GP, respetively; Fig. ). Eh mteril lyer ws found to hve signifintly different mehnil properties ompred with the others (p <.5), exept for one ompred with isopedine. The highest verge indenttion modulus ( 6 GP) nd hrdness ( 4.5 GP) of the outer gnoine lyer is onsistent with its nture mterils ADVANCE ONLINE PUBLICATION

4 known highly minerlized (<5% orgni 5 ) non-ollgenous struture omposed of rodlike, pseudoprismti rystllites of ptite (Fig. f,g) 6. The ollgenous dentine lyer hs redued minerl ontent ompred with gnoine, ut is more minerlized thn the osseous sl plte, onsistent with the mehnil property trends oserved in Fig., (E O P nd H O P 9 GP nd. GP, respetively). The third lyer, isopedine, onsists of uniform superimposition of orthogonl ollgenous lyers tht form plywood-like struture nd derese in minerliztion with distne towrds the inner surfe of the sle 7. The innermost thik sl osseous plte is omposed of suession of vsulrized one lmelle, with the mjor xis of the ollgen firils pproximtely prllel to the sle surfe. Besides the sptil orgniztion of their fires, isopedine nd one lso differ in the lrger dimeter of the isopedine ollgen firils ( nm versus nm, respetively) nd in the thikness of its sulyers ( µm versus < µm, respetively) 8. Averged E O P nd H O P for one were found to e slightly less thn isopedine (Fig. ), ut this differene ws not sttistilly signifint (p >.5). Multiple ross-setionl indents were tken on n individul sle, on different sles from the sme niml nd on sles from numer of different nimls, nd the trends desried ove were onsistently otined. The grdtion in mteril properties ws lulted s the slope of dt suh s tht shown in Fig.. An pproximtely negtive liner grdtion (from the outer to inner surfe) in oth E O P nd H O P exists within the outer gnoine (.6 GP µm nd. GP µm, respetively) nd underlying dentine (. GP µm nd.5 GP µm, respetively) lyers. nd one were found to show no detetle grdtion. The gnoine dentine nd dentine isopedine juntions (Fig., dotted lines) possessed steep grdtion reltive to their neighouring lyers. For the gnoine dentine juntion the grdients in E O P nd H O P were found to e 5.9 GP µm nd.44 GP µm, respetively. For the dentine isopedine juntion, the grdients in E O P nd H O P were found to e. GP µm nd. GP µm, respetively. The gnoine dentine juntion ws oserved to show n exeedingly shrp (<5 nm) nd orrugted juntion etween the lyers (Fig. e). The verged experimentl lod depth dt for eh lyer were fitted to n isotropi, elsti perfetly plsti finite element nlysis (FEA) omputtionl model (Fig. ), whih represents the threedimensionl Berkovih indenter geometry s two dimensionl, xisymmetri, onil-like nd rigid. A finite residul depth oserved experimentlly fter unloding (Fig. ) nd residul impressions diretly imged y tpping mode tomi fore mirosopy (TMAFM, Fig. d,e) onfirm the plsti nture of indenttion. In these simultions, the indenttion modulus, E, nd the yield stress, σ Y, were determined y est fit to the verged loding nd unloding experimentl dt (Fig. ), with the indenttion modulus lulted from the FEA-predited unloding slope 4 mthed to the experimentlly lulted verge E O P (Fig. ). Figure shows good greement etween the FEA preditions nd the experimentl dt, giving σ Y vlues of,, 4, 5 nd 8 MP for the gnoine, dentine, isopedine nd one, respetively. An extensive prmetri study showed tht the inlusion of post yield strin hrdening (liner isotropi hrdening, liner kinemti hrdening nd Rmerg Osgood isotropi hrdening) in the simultions hd miniml effet in providing n improved predition of the nnoindenttion lod depth dt nd the estimted yield stress. All mteril lyers were oserved to undergo mehnil hysteresis (Fig. ), nd energy dissiption (lulted s the re of the verge indenttion loding unloding yle) inresed with distne from the outer surfe of the sle inwrd from gnoine 5.98 pj to one.79 pj. The role of the lol multilyered nd grded miromehnil properties in the lrger-length-sle iomehnil miroindenttion of the entire P. seneglus sle ws explored y onstruting two xisymmetri, two-dimensionl FEA models (Fig. ). The first, disrete, model (Fig. ) ws omposed of the four mteril lyers with thiknesses orresponding to their experimentlly mesured vlues (Fig. d), where eh lyer ws ssumed to possess isotropi, elsti perfetly plsti onstitutive ehviour with E nd σ Y tken s those redued from FEA preditions of the verged lod depth dt in Fig.. The seond, grdient, model (Fig. ) ws lso omposed of the four mteril lyers with thiknesses orresponding to their experimentlly mesured vlues (Fig. d) nd ssumed isotropi elsti perfetly plsti mteril ehviour, ut inorported liner grdients in E (sled y the mesured E O P grdient vlues) nd σ Y (sled with the mesured H O P grdient vlues) within the gnoine nd dentine lyers, s well s the gnoine dentine nd dentine isopedine juntions. The preditions of these two multilyered models were ompred with three simultions of single homogeneous elsti perfetly plsti lyer of mteril with thikness equivlent to tht of the entire sle ( 4 µm): gnoine, dentine nd one. Virtul miroindenttion experiments were then rried out using n xisymmetri, onil-like, rigid indenter (emulting n xisymmetri equivlent Viker s mirohrdness tip, end rdius.7 µm, onil ngle 7. ) (Fig. ). The two multilyered model preditions oth trked the gnoine lod depth urve up to. N, nd then t inresing penetrtions fell in etween the ll gnoine nd ll dentine simultions (Fig. ). For the multilyered simultions, t mximum lod of.5 N, the indenter hd penetrted 45% of the wy into the gnoine lyer (Fig. ). There ws miniml differene oserved in the lod depth ehviour for the disrete nd grdient models, inditing tht the overll mirolyering (not the grdients) provides the effetive mrosopi effetive mehnil lod depth resistne (whih does not suggest tht grdients do not ply role in penetrtion resistne, disussed further elow). The effetive ehviour of the mirolyered strutures ws further exmined y reduing eh lod depth ehviour to n effetive O P modulus, effetive mirohrdness nd energy dissiption. Interestingly, lod-dependent effetive modulus nd mirohrdness re predited for the multilyered models, whih is not the se for the homogeneous systems (Fig.,d). The effetive modulus for these two models is seen to derese nonlinerly from lose to tht of ll gnoine t smll mximum lods to vlues slightly elow tht of ll dentine y mximum lod of N (orresponding to penetrtion depth of µm, Fig. ). Hene, the underlying miromehnis of the multilyered struture diretly govern the effetive mrosopi indenttion modulus, where the indenter inresingly senses the more omplint dentine lyer s the indenttion lod is inresed. For the multilyered simultions, the effetive mirohrdness is lso predited to derese sigmoidlly etween vlues orresponding to ll gnoine (t smll mximum lods <. N) nd ll dentine ( N mximum lod, orresponding to penetrtion depth of µm, Fig. d). The multilyer mirohrdness preditions were ompred with experimentlly mesured Vikers hrdness (mesured using TMAFM nd optil mirosopy) on n individul P. seneglus sle nd the mgnitude nd lod dependeny were urtely ptured (Fig. d). The effetive energy dissiption of the mirolyered models were found to inrese nonlinerly with mximum lod (Fig. e) nd fell in etween the ll-gnoine nd ll-dentine urves. Figure 4 shows the stress ontours nd plsti equivlent strin fter unloding (mximum lod of N) for the simulted miroindenttion multilyer models s ompred with tht of the 4 nture mterils ADVANCE ONLINE PUBLICATION

5 E (GP) 55 8 μm σ Y (MP), 5 5 μm 46 μm 5 μm 5 Juntion Distne (μm) 5 75 μm 45 μm μm 5 75 Distne (μm) Juntion Bone μm 8 4 μm 8 4 Bone Lod (N) Lod Unlod Effetive modulus (GP) Depth (μm) Mximum lod (N) d 6 e. Effetive mirohrdness (GP) 5 4 Experiment OM AFM FEA model Dissiption energy ( 6 J) Mximum lod (N) Mximum lod (N) Figure Preditions of effetive miroindenttion mehnil properties of P. seneglus sle vi multilyered FEA simultions., Multilyered strutures of disrete (left) nd grdient (right) models with their orresponding elsti modulus nd yield strength distriutions (entre)., Simulted miroindenttion lod-versus-depth urves., Simulted effetive indenttion modulus. d, Simulted effetive mirohrdness nd experimentlly mesured vlues (OM, optil mirosopy; AFM, tomi fore mirosopy). e, Simulted effetive energy dissiption. ll-gnoine mteril. The grdient juntion is oserved to provide trnsitionl region for stress redistriution etween lyers of differing mterils (Fig. 4), s opposed to the rupt hnges oserved in the disrete model (Fig. 4). Suh smoother stress grdients re expeted to mitigte interfe filure nd inrese interfil toughness, therey providing inresed penetrtion resistne. Although the disrete nd grdient multilyered systems hieve similr mrosopi effetive indenttion modulus nd mirohrdness, the grdient se miromehnilly hieves more grdul stress redistriution, providing roustness to interfil filures. The plsti equivlent strin ontours revel the greter depths nd greter re of plsti strining for the multilyer nture mterils ADVANCE ONLINE PUBLICATION 5

6 Von Mises. GP.49 S.5 GP.8 S.7 GP.8 S.4 GP.6. S.4 GP.6.8 Plsti equivlent strin.. Pressure.9 GP µm Juntion Juntion Figure 4 Simultion ontours of stress, plsti strin nd pressure fields of P. seneglus sle vi multilyered FEA simultions., FEA preditions of von Mises stress field, S (norml stress on the plne perpendiulr to the xis), S (norml stress on the plne perpendiulr to the xis) nd pressure t mximum depth when fully loded, nd S (sher stress on the plne perpendiulr to the xis ting in the diretion), S (norml stress on the plne perpendiulr to the xis) nd plsti equivlent strin fter fully unloded for three models: ll gnoine (), disrete () nd grdient () models for -N-mximum-lod indenttion. system ompred with the ll-gnoine model, due to the inresed plsti strin of the underlying dentine, whih possesses lower σ Y ompred with gnoine; this orreltes with the greter dissiption seen in the lod depth urves (Fig.,e). The simultions lso show tht the mgnitude of the plsti strin in the gnoine lyer of the multilyered system is lower thn tht experiened in the 6 nture mterils ADVANCE ONLINE PUBLICATION

7 µm µm 5 d 4 Depth (µm) FEA Experiment Experiment Distne (µm) 5 µm e f 6 µm 5 µm Figure 5 Topogrphi profiles, residul impressions of miroindenttion nd frture of n individul P. seneglus sle., TMAFM mplitude imge of -N-mximum-lod miroindenttion., Optil mirogrph of -N-mximum-lod miroindenttion., FEA predition of the residul depth topogrphy otined from the grdient multilyer simultion nd experimentl height profiles from TMAFM imges (Fig. 5) for -N-mximum-lod miroindenttion. d, Optil mirogrph of N miroindenttion showing irumferentil rks. e, SEM of ross-setion of isopedine lyers. f, Bksttered eletron mirogrph of ross-setion of gnoine nd dentine lyers. ll-gnoine system, even though the overll depth of penetrtion in the multilyer ( µm) is more thn twie tht of the ll-gnoine system (.6 µm), diret result of the dentine deformtion ommodting the imposed penetrtion depth. The N mximum lod is pproprite to estimte rittle-filure ondition for the gnoine euse N mirohrdness experiments were found to irumferentilly rk in some ses nd only plstilly strin in other ses (Fig. 5) inditing tht the N se is t the order of meeting rk initition onditions. Contours of the rdil stress, S (Fig. 4), revel elevted tensile surfe vlues (.66 GP) t the perimeter of the ontt re (in the sme region where the irumferentil rks re found), suggesting mximum-norml-rittle-stress filure ondition of.66 GP. The vlues re lower nd the regions of elevted stress re smller in the grdient se thn the disrete se, inditing n importnt smoothing role of the grdient in reduing the surfe stresses. A seond prominent surfe filure ondition is often rdil rks, whih were not oserved in these mirohrdness experiments; this is onsistent with the simultion results, whih show the surfe irumferentil stress vlues, S, tht would nture mterils ADVANCE ONLINE PUBLICATION 7

8 e needed to initite rdil rks to e <S. (Prmetri omputtionl studies on the effet of the gnoine thikness show tht the experimentlly oserved vlues (5 µm) yield S > S, whih promotes irumferentil rking (oserved experimentlly Fig. 5d), whih is highly dvntgeous euse it lolly onfines the deformtion t the indenttion site. For thiker or thinner gnoine lyer, S remins lrge, whih promotes undesirle rdil rking, whih n led to tstrophi filure of the lyer.) As seen in other multilyer systems 9, the interfil norml nd sher stresses were found to e severe on unloding (S, S, Fig. 4). The stresses re found to e similr for the disrete nd grdient systems with the expettion of pek sher stress present on the interfe of the disrete system, whih is sent from the grdient system. Exellent greement etween simultion nd experiment ws lso demonstrted in omprison of the experimentlly mesured residul topogrphy (Fig. 5). The.5 N ses deformed in n elsti plsti mnner with no rks in most ses ( 9 of indents; Fig. 5,). For N, 5% of the smples showed irumferentil rking. Cirumferentil rks were oserved in ll of the. N ses (Fig. 5d). Although minerlized derml fish sles serve mny funtionl roles simultneously 5 7, we limit our disussion here to protetive iomehnil spets 4. The primry predtors of P. seneglus re known to e its own speies or its rnivorous verterte reltives 4, nd iting tkes ple during territoril fighting nd feeding. In nient times, mny lrge inverterte predtors existed. For exmple, the rnivorous eurypterid (thought to e predtor of the rmoured ostroderms) ws gint rthropod tht possessed iting mouth prts, grsping jws, lws, spines nd spiked til 4. Additionlly, it hs een suggested tht the ploderm Dukleosteus terelli possessed ite fore lrge enough to punture through the minerlized exoskeleton of its prey, to onsume the flesh underneth 8. Mehnilly, suh predtory ttks would represent dynmi (for P. seneglus ) penetrting lod (pproximted y the multilyered indenttion simultions), whih involves sptilly lolized, multixil nd lrgely ompressive stress field. P. seneglus shows multilyered sle design over mirometresized length sle, suggesting tht the purpose of this design is for resistne to mehnil lods nd deformtions existing over these smll length sles s well. For exmple, the teeth of similr-sized P. seneglus were mesured to possess n end-rdius of 4.7±8.8 µm, rnging from. to 44. µm. A finite-element geometri representtion of the teeth of P. seneglus ws reted nd FEA indenttion simultions rried out using these meshes (whih re muh lrger thn the stndrd Vikers nnoindenter, end rdius.7 µm, reported in the urrent mnusript) using the multilyered sle model. All of the trends nd onlusions of the urrent pper remined the sme using this fish-toothrepresenttion indenttion. Hene, the mirometre length sle nd indenttion mode of deformtion within individul sles re physiologilly importnt with regrd to mitigting penetrtion nd filitting protetion, working in onjuntion with the lrger, multisle struture of the rmour, whih enles signifint mount of iomehnil flexiility nd mnoeuvrility of the entire fish ody. In onlusion, here we report on the fsinting, omplex nd multisle mterils design priniples of nient fish rmour in the ontext of its speifi primry environmentl thret (penetrting iting ttks) nd mehnilly protetive funtion. One overrhing mehnil design strtegy is the juxtposition of multiple distint reinforing lyers, eh of whih hs its own unique deformtion nd energy dissiption mehnisms. As the stiff gnoine trnsfers lod through the gnoine dentine juntion, the underlying softer, more omplint dentine lyer dissiptes energy vi plstiity (t high enough lods). The gnoine thikness ws seleted () to simultneously ess the dvntgeous mehnil properties of the gnoine (hrdness, stiffness) nd underlying dentine (energy dissiption), () to redue weight while mintining the required mehnil properties nd () to promote the dvntgeous irumferentil rking mehnism (S > S), rther thn disdvntgeous rdil rking. The mteril lyer sequene is lso ritil; for exmple, reversing the gnoine nd dentine lyers in virtul miroindenttion leds to mgnified interfil tensile norml nd sher stresses, promoting delmintion (dt not shown). The presene of the dditionl isopedine strtified lyer with its plywood-like struture n serve s seond line of defene for deeper penetrtions, preventing tstrophi rk propgtion nd inresing energy dissiption nd frture toughness. Mirorking ws oserved to e onfined within the sulyers of the isopedine (Fig. 5e), where eh lyer hs rks running in pproximtely orthogonl diretions (onsistent with the known struture) ut rrested t the edge of eh sulyer. The underlying one ts s the se skeletl physil support struture for norml physiologil loding onditions, where plsti deformtion (if fored eyond the elsti limit vi trumti event) tkes ple vi mehnisms suh s nnogrnulr frition, ollgen firil sher 4 nd denturtion 5, rk initition propgtion 6, introrgni extensiility nd srifiil ond rupture 4. The qudlyered design of P. seneglus sles enles weight redution of up to %, ompred with ilyer of gnoine nd dentine of the sme thikness, with miniml degrdtion of effetive mehnil properties (tht is, mirohrdness, modulus, energy dissiption). Additionlly, we hve oserved tht the gnoine dentine juntion in P. seneglus sles is le to rrest rks (Fig. 5f), s in mmmlin teeth 7. Lolized internl frtures nd dethment of piees of the gnoine lyer re seen, wheres the gnoine dentine juntion onsistently remins intt (Fig. 5f), whih my e srifiil mehnism. The juntions etween mteril lyers re lerly funtionlly grded, tht is, they possess grdul sptil hnge in properties motivted y the performne requirements nd re le to promote lod trnsfer nd stress redistriution, therey suppressing plstiity, rresting rks, improving dhesion nd preventing delmintion etween dissimilr mteril lyers 8. Lst, the orrugted juntion etween the lyers is expeted to led to sptilly heterogeneous stresses nd higher net interfil ompression, lso resisting delmintion 9. Suh multisle mterils priniples my e inorported into the design of improved engineered iomimeti struturl mterils. METHODS EXPERIMENTAL DETAILS Anesthesi of living P. seneglus ws indued y pproximtely 5 min of immersion in uffered.% (y weight) solution of triine methne sulphonte (MS). Surgery ws rried out nd row of 5 sles ws removed during whih nesthesi ws mintined y.5% solution of MS. After the surgery, the wter of the tnk ws treted with tetryline tlets for two weeks to prevent infetion nd within two months the removed sles were regenerted. All experiments were performed in ordne with federl guidelines nd regultions nd pproved y the MIT Committee on Animl Cre (Protool ). MIT hs n Animl Welfre Assurne on file with the Offie for Lortory Animl Welfre (Assurne numer A-5-). Freshly extrted sles from living dult P. seneglus were emedded in room-temperture uring epoxy (Lotite Fixmster Poxy-Pk 8) nd setioned using dimond-impregnted nnulr wfering sw (Buehler Isomet 5) t 8 9 r.p.m. The smples were polished on polishing wheel (South By Tehnology, Model 9) with 5 µm, 6 µm nd µm sili nnoprtiles on soft pd, nd gin with 5 nm sili nnoprtiles on loth pd in distilled wter. Smples were stored in phosphte-uffered sline solution. Smples 8 nture mterils ADVANCE ONLINE PUBLICATION

9 were imged in JEOL JSM-66 SEM or JEOL JSM-67F field emission SEM. Bksttered eletron mirosope imges were tken with JEOL JSM-67F. Optil mirosopy imges were tken with Nikon Elipse L5. Nnoindenttion experiments were onduted in mient onditions using Hysitron Trioindenter using Berkovih dimond proe tip, following our previously reported methods 5,7. Artifts due to surfe roughness, surfe slope, residul indent overlp nd so on were refully voided, s desried in detil previously 5,7. Unpired Student t-tests were rried out to evlute whether vrious dtsets were sttistilly different from one nother, using p <.5 s the minimum riterion. Vikers mirohrdness experiments were onduted in mient onditions. The proe-tip end rdius ws mesured using TMAFM (.7 µm). TMAFM imging ws rried out with Multimode SPM IV (Veeo) nd Si ntilevers (Veeo, V-shped, squre pyrmidl proe tip, k (nominl spring onstnt).4 N m ; R tip (nominl proe-tip end rdius of urvture) nm). FINITE ELEMENT ANALYSIS A nonliner FEA nnoindenttion model ws used, whih pproximted the three-dimensionl Berkovih nnoindenttion experiments using two-dimensionl, onil-like xisymmetri indenter. The ury of the two-dimensionl xisymmetri pproximtion ws ssessed y omprison with fully three-dimensionl simultion results; the modulus nd yield stress vlues otined in the two-dimensionl simultions differed from those from three dimensions y less thn % ( rnge within ny experimentl omprison signifine). The indenter geometry ws hosen to orrespond to the experimentlly determined indenter ross-setionl tip-re funtion. The indenter tip ws modelled s rigid. The indented smples were modelled s isotropi, elsti perfetly plsti using four-node iliner xisymmetri qudrilterl elements (CAX4R in ABAQUS element lirry). Lrge-deformtion theory nd fritionless ontt etween the indenter nd mteril were ssumed. Full three-dimensionl nnoindenttion simultions were lso onduted on smples with two lyers oriented norml to the surfe, simulting severl indenttions with eh indenttion progressively loser to the lyer lyer interfe to ssess the role of ny onstrint of neighouring lyer on the indenttion lod depth ehviour. FEA models of miroindenttion followed similr methodology to the xisymmetri nnoindenttion simultions. The indenter ws tken to e rigid, with onil tip geometry, emulting Vikers mirohrdness tip with rdius of.7 µm (mesured y AFM). Mesh onvergene studies were used to ssess the needed mesh for urte solutions to the lod depth nd stress ontours. Reeived 4 Jnury 8; epted June 8; pulished 7 July 8. Referenes. Hoedemn, J. J. Nturlists Guide to Fresh Wter Aqurium Fish (Sterling Pulishing Co., Ok Tree Press Co., New York, London, nd Sydney, 974).. Colert, E. H. Evolution of the Vertertes: A History of Bkoned Animls Through Time (Wiley, New York, 955).. Miller, H. The Cruise of the Betsy or Summer Holidy in the Herides with Rmles of Geologist or Ten Thousnd Miles of Fossiliferous Deposits of Sotlnd (Gould nd Linoln, Boston, 857). 4. Romer, A. S. Eurypterid influene on verterte history. Siene 78, 4 7 (9). 5. Ørvig, T. in Pro. 4th Noel Symp. (ed. Ørvig, T.) 7 97 (Almqvist nd Wiskell, Stokholm, 968). 6. Long, J. H. & Nipper, K. S. The importne of ody stiffness in undultory propulsion. Am. Zool. 6, (996). 7. Rshi, W. & Tit, C. Funtionl spets of ploid sles: A review nd updte. Aust. J. Mr. Freshw. Res. 4, 47 (99). 8. Anderson, P. S. L. & Westnet, M. W. Feeding mehnis nd ite fore modelling of the skull of Dunkleosteus terrelli, n nient pex predtor. Biol. Lett., (7). 9. Ariszewski, T. & Cornell, J. in Bio-Inspirtion: Lerning Cretive Design Prinipi (ed. Smith, I. F. C.) (Springer, Berlin, 6).. Weiner, S. & Adddi, L. Design strtegies in minerlized iologil mterils. J. Mter. Chem. 7, (997).. Go, H., Ji, B., Jger, I. L., Arzt, E. & Frtzl, P. Mterils eome insensitive to flws t nnosle: Lessons from nture. Pro. Ntl Ad. Si. USA, ().. Buehler, M. J. Moleulr nnomehnis of nsent one: Firillr toughening y minerliztion. Nnotehnology 8, 9 (7).. Currey, J. D. The design of minerlised hrd tissues for their mehnil funtions. J. Exp. Biol., (999). 4. Fntner, G. E. et l. Srifiil onds nd hidden length dissipte energy s minerlized firils seprte during one frture. Nture Mter. 4, 6 66 (5). 5. Ti, K., Do, M., Suresh, S., Plzoglu, A. & Ortiz, C. Nnosle heterogeneity promotes energy dissiption in one. Nture Mter. 6, (7). 6. Blooh, G. et l. Evlution of new modulus mpping tehnique to investigte mirostruturl fetures of humn teeth. J. Biomeh. 7, (4). 7. Bruet, B. J. F. et l. Nnosle morphology nd indenttion of individul nre tlets from the gstropod mollus Trohus nilotius. J. Mter. Res., 4 49 (5). 8. Re, D., Shs, C. & Romno, P. The rusten exoskeleton s n exmple of struturlly nd mehnilly grded iologil nnoomposite mteril. At Mter. 5, (5). 9. Brkdze, N., Enders, S., Gor, S. & Arzt, E. Lol mehnil properties of the hed rtiultion utile in the eetle Phnod mrgint (Coleopter, Sreide). J. Exp. Biol. 9, 7 7 (6).. Ortiz, C. & Boye, M. C. Bioinspired struturl mterils. Siene 9, 5 54 (8).. Dget, J., Gyet, M., Meunier, F. J. & Sire, J.-Y. Mjor disoveries on the derml skeleton of fossil nd reent polypteriforms: A review. Fish Fisheries, 4 ().. Koder, H. et l. Jurssi Fishes: Seletion, Cre, Behvior (T. F. H. Pulitions, New Jersey, 994).. Crroll, R. L. Verterte Pleontology nd Evolution (W. H. Freemn nd Compny, New York, 988). 4. Oliver, W. C. & Phrr, G. M. An improved tehnique for determining hrdness nd elsti modulus using lod nd displement sensing indenttion experiments. J. Mter. Res. 7, (99). 5. Ørvig, T. in Struturl nd Chemil Orgniztion of Teeth (ed. Miles, A. E.) Ch., 45 (Ademi, New York, 967). 6. Meunier, F. J. Reherhes histologiques sur le squelette dermique des Polypteride. Arhives de Zoologie Expérimentle et Générle, (98). 7. Meunier, F. J. Os ellulire, os ellulire et tissus dérivés hez les Ostéihthyens: Les phénomènes de l ellulristion et de l perte de minérlistion. L Année Biologique 6, (987). 8. Sire, J.-Y. From gnoid to elsmoid sles in the Atinopterygin fishes. Neth. J. Zool. 4, 75 9 (99). 9. Jyhndrn, R., Boye, M. C. & Argon, A. S. Design of multilyer polymeri otings for indenttion resistne. J. Comput.-Aided Mter. Design, 5 66 (995).. Mrkey, M. J., Min, R. P. & Mrshll, C. R. In vivo rnil suture funtion nd suture morphology in the extnt fish Polypterus: Implitions for inferring skull funtion in living nd fossil fish. J. Exp. Biol. 9, 85 (6).. Luder, G. V. Evolution of the feeding mehnism in primitive tinopterygin fishes; funtionl ntomil nlysis of Polypterus, Lepisosteus, nd Ami. J. Morphol. 6, 8 7 (98).. Gemll, S. & Brtsh, P. Arhiteture of the integument in lower Teleostomes: Funtionl morphology nd evolutionry implitions. J. Morphol. 5, 9 9 ().. Ti, K., Ulm, F.-J. & Ortiz, C. Nnogrnulr origins of the strength of one. Nno Lett. 6, 5 55 (6). 4. Gupt, H. S. et l. Nnosle deformtion mehnisms in one. Nno Lett. 5, 8 (5). 5. Ti, K., Qi, H.-J. & Ortiz, C. Effet of minerl ontent on the nnoindenttion properties nd nnosle deformtion mehnisms of ovine tiil ortil one. J. Mter. Si.: Mter. Med. 6, (5). 6. Nll, R. K., Kinney, J. H. & Rithie, R. O. Mehnisti frture riteri for the filure of humn ortil one. Nture Mter., (). 7. Imeni, V., Kruzi, J. J., Mrshll, G. W. & Rithie, R. O. The dentin enmel juntion nd the frture of humn teeth. Nture Mter. 4, 9 (5). 8. Suresh, S. Grded mterils for resistne to ontt deformtion nd dmge. Siene 9, (). 9. Shimizu, D. & Mho, G. A. Funtionl signifine of the mirostruturl detil of the primte dentino-enmel juntion: A possile exmple of exption. J. Humn Evolut. 5, (7). 4. Humphries, J. Polypterus seneglus, online digitl morphology. Aessed June, 7 t < seneglus/whole/> (Mihign Museum of Zoology) (ummz 958). (). Aknowledgements The uthors thnk the MIT Deprtment of Mterils Siene nd Engineering Nnomehnil Testing Fility, the Ntionl Siene Foundtion MIT Center for Mterils Siene nd Engineering, the Centre de Reherhe de l Mtière Condensée et des Nnosienes (CRMC-N) t Université de Mrseille-Luminy, Frne, the MIT Interntionl Siene nd Tehnology Inititives Frne Seed Fund nd the US Army through the MIT Institute for Soldier Nnotehnologies (ontrt numer DAAD-9--D), s well s R. Jensen nd T. Weersooriy from the US Army Reserh Lortory nd T. Imholt from Rytheon for disussions. The ontent does not neessrily reflet the position of the government nd no offiil endorsement should e inferred. The uthors would lso like to thnk E. Chen, J. H. Choi, J. Kim, J. Y. Mo, M. D. Msro nd E. R. Pfeiffer for ssisting with initil smple preprtion nd preliminry dt, A. Bronnet for ssistne with eletron mirosopy nd G. Luder (Hrvrd University) for rrying out the sle-removl surgery. Author informtion Reprints nd permission informtion is ville online t Correspondene nd requests for mterils should e ddressed to C.O. nture mterils ADVANCE ONLINE PUBLICATION 9