Materials Design Principles of Ancient Fish Benjamin Bruet, Juha Song, Mary C

Home , Fish scale, Ganoine

Materials Design Principles of Ancient Fish Benjamin Bruet, Juha Song, Mary C. Boyce Christine Ortiz,* Associate Professor *Department of Materials Science and Engineering, MIT

Materials of Interest Exoskeletal Tissues Musculoskeletal Tissues Seashell nacre (Bruet, et al. JMR 2005) Cartilage (Han, et al. Biophys. J. 2007)

Berkovich diamond probe tip 1 mm

71º PCM Cell c

8 μm 500 nm

0.8 μm

Armored Fish Bone (Tai, et al. Nature Materials 2007)

50 nm Motivation

Musculoskeletal Tissues (Medical) • The degradation of load-bearing musculoskeletal tissues during aging, injury, and / or disease affects millions of people worldwide. • The application of experimental and theoretical nanotechnologies to the field of musculoskeletal tissues and tissue engineering holds great promise for significant and rapid advancements towards tissue repair and/or replacement and even possibly a cure for people afflicted with ailments such as osteoarthritis. Exoskeletal Tissues (Engineering) (Ashby, 1999) • Inspiration and guidance for improved and increasingly advanced structural engineering materials; e.g. body armor and vehicle protection for defense and security applications. • Learning the basic principles of “mechanical property amplification" via complex, hierarchical and heterogeneous nanostructures that undergo a wide variety of deformation mechanisms at multiple length scales. Comparison of natural and historic engineered armor

Tomasz Arciszewski and Joanna Cornell I.F.C. Smith (Ed.): EG-ICE 2006, LNAI 4200 Natural armor design : Maximize survivability Maximum Protection Maximum Mobility Tradeoff 1. Maximize energy absorption 1. Maximize flexibility 2. Maximize energy dissipation 2. Decrease Weight 3. Minimize deformations 4. Minimize penetration 5. Maximize visual impact 6. Maximize noise/sound impact -Metabolic costs; - materials available in the environment -Hygiene : lack of flexibility may increase vulnerability to parasites -Thermoregulation

Tomasz Arciszewski and Joanna Cornell I.F.C. Smith (Ed.): EG-ICE 2006, LNAI 4200 Today’s human body armor

Maximum Maximum Mobility Protection/Survivability Tradeoff Modern armors exist which can protect a person against even the high-velocity rounds fired by assault, battle or sniper rifles→too bulky and heavy for practical use

1. Flat plate inserts to protect vital organs only -The heart, lungs, central nervous systems, etc. are usually well protected, Helmet to Not Mobile Enough & Not Effective Against Shrapnel protect head. Limbs rarely armored or Blast 2. Weight reduction desirable. 3. Minimal protection from blast (improvised explosive devices, IED’s) 4. Multi-hit capability desirable. Today’s human body armor ; Fort Polk, Louisiana Gareth McKinley Christine Ortiz Greg Rutledge Karen Gleason

John Joannopolous Ned Thomas Today’s human body armor – Fort Polk, Louisiana Polypterus senegalus : An ancient armored fish

23 Turning maneuver 1 (total time: 2.6 s)3

• Dates back to the Cretaceous period > 60 million yrs ago • Lives in freshwater, swampy, shallow floodplains and estuaries in Africa • Has a rudimentary lung (“swim bladder”) which allows them to leave the water for extended periods (1-2 days) • Territorial fighting; prone to in-fighting with its own species and conspecifics; jaw structure and skull are capable of supporting powerful bites1 • Interlocking, quad-layered mineralized armored scales while still maintaining body flexibility and swimming at high speeds2,3 1Kodera et al. (1994), 2Gembella et al. (2002), 3Tytell et al. (2005) Microscale geometry of P. senegalus armored scales and joints

SEM image of scale top outer surface SEM image of peg (p) and socket (so) joint

Gemballa, J. Morph. 2002 Cross-sectional multilayered structure of P. senegalus mineralized fish scales

1 2

1 mm Ganoine

100 μm Dentin • Quadlayered microstructure where each material is an organic-inorganic bionanocomposite1 Isopedine

-Ganoine : outer layer, highly mineralized Bone 10 μm (>95%), noncollagenous enamel2,3 -Dentin : collagen / nanocrystalline apatite, reduced mineral content compared to ganoine5,6 -Isopedine : microstratified collagenous layers4 -Bone : collagenous lamellae5 1Sire (1990), 2Ørvig (1967), 3, 4Meunier (1980, 1987), 5Daget et al. (2001), 6Nalla et al. (2005) Mineral content of cross-section of P. senegalus fish scales Back scattered electron (BSEM) microscopy image Curved ganoine geometry, differentiated dentin thickness : capable of better isopedine energy absorption, deformations of a shell structure are bone smaller than those of a flat surface under the same impact force. Ultrastructure of ganoine from P. senegalus scales ● Tapping mode AFM of the ganoine surface showed dense array of hemispherical nanoasperities (diam ~ 50-100nm, height ~20nm)

● Higher magnification SEM imaging of the ganoine showed nanocrystals, hydroxyapatite (diameter ~50 nm, length 100’s of nm), consistent with the nanoasperity size found at surface. SEM image of cryo-fractured ganoine Ultrastructure of ganoine from P. senegalus scales outer surface

tubercule

SEM image of a cryo-fractured cross-section of the ganoine Back-scattered electron microscopy of ganoine- dentin interface in P. senegalus scales

ganoine ● Unique corregated microstructure, spatially Heterogeneous stresses and a higher net interfacial compression which could serve to prevent delamination dentin Sub-microlayered structure of isopedine in P. senegalus scales

high mineral content Crack arresting at ganoine-dentin interface in P. senegalus scales Back scattered electron (BSEM) microscopy image Objectives : Materials design principles of P. senegalus mineralized fish scales •To quantify the local mechanical properties (e.g. modulus, yield stress) spatially through the four material layers in the cross-section of an individual scale and to establish structure-property relationships of each layer •To incorporate these results into a larger-scale computational model of the entire scale to determine the effect of local spatial variations, e.g. gradients, etc. on larger scale biomechanical properties; in this case penetration resistance Ganoine

3 Ganoine Dentin 2 1 Dentin Isopedine

Isopedine Bone Bone 10 μm x x xxxxxxx x x •To relate multiscale structure-property design relationships to biomechanical function Methods : Mechanical properties of the four material layers in individual P. senegalus scales • Estimation of indentation modulus & hardness1 using instrumented

nanoindentation Berkovich 500 3 400 E Maximum Load O-P 2 N) S N) H 1 μ (500 μN) μ 300 O-P

Dentin

Load ( Load 200 Indenter Load ( 100 200 nm 3 μm 0 (RTip ~ 275 nm) 0 20 40 60 80 100 120 140 Depth (nm) fish scale Depth (nm) • Determination of yield stress via finite element analysis (FEA) - Elastic perfectly-plastic using 4-node bilinear axisymmetric quadrilateral elements Indenter Tip - Tip area function (TAF) of 2D tip is as same Indented Material as one of 3D experimental Berkovich tip 3 2 1Oliver & Pharr (1992) 1 50 μm Multilayered and graded mechanical properties of four material layers of an individual P. senegalus scale

inner surface Dent. Isop. Gan. 1 3

N) 2 μ

80 Bone Modulus Bone IsopedineIsopedine Ganoine 5 HardnessO-P Modulus Bone Ganoine 4 Load ( 60 O-P Hardness DentinDentin 3 40 Epoxy ` 2 20 1 ModulusModulus (GPa) (GPa) Hardness (GPa) (GPa)Hardness Hardness

Depth (nm) 0 0 O-P Modulus (GPa) O-P Hardness (GPa)

0 50 100 150 200 250 300 350 400 • Layers ordered from the outer stiffest and DistanceDistanceμ ( m)(μm) hardest surface (ganoine) to the most Junction compliant and softest inner (bone) Ganoine • Negative Gradation within ganoine and Isopedine Dentin dentin layers; no measurable gradation in isopedine and bone

330 340 350 360 370 380 390 400 410 420 •FEA shows good agreement with exp. data 330 340 350 360 370 380 390 400 410 Distance (μm) (σY ~ 2000, 400, 215 and 180 MPa from outer to inner layers) AFM imaging of residual indents in each layer

Dent. Isop. Phase Image Ganoine

Isop. Bone

• Each layer exhibits residual plastic deformation at 500 μN • Nanoasperities appear flattened in the indented area Virtual microindentation of multilayered scale cross-section using finite element analysis

O-P Modulus (GPa) YieldYield strength Strength (MPa) (MPa) Discrete Modulus (GPa) 1000 1500 2000 Gradient 2000 2000 14.5 13.5 14.5 13.5 500 180 400 400 215 215 180 10 20 30 40 50 60 25 55 25 55 0 0 0 0 0 0 0 Gan Gan 8 μm

5 μm 25 55 51030400 380 100 75 50 25 55 51030400 380 100 75 50 25 25 5 μm 25 5 μm 5 μm 25 Junction 46 μm Distance (μ Dent Distance (μ Dent 50 50 50 50 Distance ( Distance (

3 μm 3 μm Junction

3 μm 75 75 75 3 μm Isop 75 45 μm

μ Isop μ m) m) m) m) 100 100 100 All Gan 100 Bone All Dent Bone All Bone 380 8 400 380 380 3 400 380 Discrete 300 μm 400 20 μm 1 2 Gradient 400 xxxxxxxxxxx xx xxxxxxxxxxxxx x

• The material of each layer is assumed to be elastic-perfectly plastic

• Experimentally determined (nanoindentation) material properties (E, σY) incorporated • 2D conical tip provides an axisymmetric equivalent of Vickers tip (used experimentally). • Discrete and gradient models are compared to All Ganoine, All Dentin and All Bone models. Virtual microindentation of multilayered scale cross-section using finite element analysis Microlayering provides the effective mechanical penetration resistance of the entire scale

Gradient Discrete P 0.5 All Dentin surface 0.4 All Ganoine 0.3 3 Fish scale All Bone 1 2 0.2 P, Load (N) 0.1

0.0 0 123456 7 δ, Depth (μm) •Multilayer P-δ exhibits ganoine behavior up to P = ~ 0.1 N (~1 μm) • Multilayer, at P > 0.1 N, is more compliant than ganoine •Multilayer P –δ substantially stiffer and harder than dentin • Discrete and gradient interfaces have nearly identical P – δ behavior Multilayer structure exhibits load-dependent effective indentation modulus and hardness

Experiment x AFM OM Effective microhardness (GPa)

Effective modulus (GPa) Maximum load (N) Maximum load (N)

FEA models Discrete All Gan All Bone Gradient All Dent

• Low-load properties coincident with ganoine properties • Decreasing modulus and hardness with increasing load reflects deformation of underlying dentin layers • A (validation) between simulation and experiment - Load-dependent behavior & residual topography Experimental validation of microindentation simulations of entire scale (Vickers) 1 N - AFM 1 N - Optical

• FEA and AFM- measured residual footprint show good agreement 2 N - Optical • Loads above 1N consistently induce cracks around the microindent Multilayered simulations of entire scale :

Stress: GPa Mechanisms of penetration resistance *S S22S22 S11S11 *PEEQ Stress contour and PEEQ at 1N max. load 2.32 1.66 1.60 0.5 * PEEQ: Plastic equivalent strain, S: Von Mises • Multilayer vs. ganoine 0 -3.70 -3.15 0 - Larger volume of material plastically deforms in multilayer (more dissipation in multilayer) - Tensile radial stress (S22) is larger than

3 circumferential stress (S11) : consistent with All Gan 2 circumferential cracking at P > 1N. 1 2 1 N 2 N 1 3 Junction Residual Discrete topography 10 μm 15 μm • Multilayer discrete vs. gradient - Gradients provide lower stress levels and Junction smoother stress gradients through junctions, Gradient providing increased robustness to interfacial failure and hence, penetration resistance Microcomputed tomography of a scale • uses X-rays to create cross-sections of a 3D-object that later can be used to recreate a virtual model without destroying the original model with a resolution of the order of a few micrometers

20 mm

x z Sectioned MicroCT images of a fish scale with a resolution of 12 μm A standard tool for the assessment of the 3D < Sample information from MicroCT analysis >architecture of various objects such as bone Bone Total Bone Thickness Separation Bone Mineral Volume Conn-Dens. Sample Name Volume Volume [mm] [mm] Density Density [1/mm3] [mm3] [mm3] ±SD ±SD [mg HA/ccm] (BV/TV) 0.2267 0.0558 Wet scale 2.8856 2.6089 0.9041 18.0208 1093.079 ±0.0907 ±0.0208 0.2188 0.0448 Dried scale 2.2613 1.9967 0.883 14.5935 1210.263 ±0.082 ±0.0189 3D model construction of P. senegalus scale Generation of 2D mesh in Mimics

Mesh optimization

Generation of 3D mesh in Abaqus

3D mesh conversion FEA model of a single scale

Simple bending test >> complicated Undeformed shape situations can be applied P

Von Mises Stress Deformed shape 67.4 GPa

33.7

x x 0.1

Schematic drawing of bending model μCT to create 3D model of rows of scales

Interlocking model of five scales

Incorporate connective fibers in the joints μCT to create 3D model of rows of scales

Interlocking model of three scales

Interlocking model of five scales Incorporated connective fibers in the joints Whole Fish Biomechanics

23 1 Multiscale Mechanics of Fish Armor: From High Deformation Collagenous Connective Fibers to Graded Fish Scale Structure Properties to Detailed Geometric Structure –Deformation Interplay: Highly Flexible Protective Armor FISH ARMOR SCALES: FISH ARMOR SCALES: DETAILED MICROMECHANICAL EFFECTIVE ANISOTROPIC MODELING FINITE DEFORMATION BEHAVIOR

FISH BODY: EFFECTIVE FINITE DEFORMATION VISCOELASTIC TISSUE BEHAVIOR Complex Scale Geometry Enables Fish Scale Micromechanical Detail: Intricate Scale-to-Scale Connectivity And Deformation (MicroCT for geometry)

Collagenous Connective Fibers Enable Large Graded Stiffness Stretching and Hardness Optimize Scale Properties Gemballa, J. Morph. 2002 Mechanical / materials design principles of P. senegalus armor 1) Multilayered provides effective Increasing penetration resistance Length Scale 2) Graded mechanical properties and unique geometry reinforces interfaces 3) Lower stiffness, lower yield strength inner layers absorb energy, increase fracture toughness 4) Brittle ganoine layer - sacrificial? 5) Geometry of shell (curved) reduces deformations, increases energy absorption 6) Peg and socket mechanism increases mobility 7) Peg and socket reinforced by complex fibrous macromolecular system Biomechanical Function : Mechanics-Threat Relationships • P. senegalus exhibits a multilayered scale design over a micrometer-sized length scale, suggesting that the purpose of this design is for mechanical loads and deformations which exist over these small length scales as well - different than the larger length scale of tooth enamel / dentin (layer thicknesses ~ mm) undergoing masticatory stresses. -structural adaptations for transferring and minimizing stress, maximizing fracture toughness

- Virtually studying effect of interfacial geometry, layer thicknesess, gradation, etc. Biomechanical Function : Mechanics-Threat Relationships

• Territorial fighting; prone to in-fighting with its own species and conspecifics; jaw structure and skull are Protection capable of supporting powerful bites (Kodera et al. from predator (1994), will cannibalize penetrating attacks • Has a rudimentary lung (“swim bladder”) which allows them to leave the water for extended periods (1-2 days) Friction/wear protection •Withstand periods of adverse weather on mud, (http://science.jrank.org/pages/985/Bony-Fish.html) rocks, etc. •“sand-blasting"; abrasive action of sand particles dispersed in seawater (Holten-Anderson, et al. MRS Symp. Proc. 844 2005)

•control of body stiffness and undulatory wave motion during steady swimming (Long, J. H. & Nipper, K. S. Am Zool 36, 678–694 (1999) Lower right jaw Magnification: x 200 From back to front 200 μm 200 μm

200 μm 200 μm 9) r = 9.0 μm 10) r = 12.7 μm

14.7 +/- 8.8 μm (Min 3.0, Max 44.0)

100 μm 100 μm 11) r = 12) r = 20.9 μm 23.1 μm

100 μm 100 μm Other multilayered and graded biological systems : Seashell nacre Scanning electron microscope images of the microstructure of the shell of T. niloticus outer shell surface nacre

side view ↑ c-axis 10 μm top view c-axis 10 μm ● μm/mm multilayered structure, inner nacreous layer has microscale “brick and mortar” structure ~95 wt.% of it is calcium carbonate ~5 wt.% biomacromolecules ● individual nacre tablets are complex organic-inorganic biocomposites in and of themselves composed of nanograins (~ 30 nm) with embedded biomacromolecules Iron-plated shell and dermal sclerites

0.5 mm

● gastropod mollusk (snail) living a sedentary life near deep sea hydrothermal vents ¾High pressure (2,415 to 2,460m under water where pressure is 250 times greater than that on land) ¾High temperature and temperature gradient (superheated (~350ºC) sulfide- rich vent effluents mixed with cold (2ºC) oxygen-rich sea water) ¾Absence of sunlight ¾Toxic levels of sulfides and heavy metals Iron-plated shell and dermal sclerites

Internal Chemical Explosion of the Bombardier Beetle

● The only known animal with the ability to create repeated internal explosions (i.e. reaction of hydroquinone and hydrogen peroxide catalysed by catalase and peroxidase) in a 1 mm combustion chamber gland causing the pulsed high pressure ejection of a defensive spray which is composed of scalding hot (100oC) water and steam, along with quinine which causes a stinging sensation and a loud startling popping noise.

Beheshti + Int. Jnl. of Design and Nature (2006) Internal Chemical Explosion of the Bombardier Beetle

Beheshti + Int. Jnl. of Design and Nature (2006)