microstructural characterization of the bony plated armor...

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Bovine femur bone 0 100 200 300 400 500 600 0 2 4 6 8 10 10 15 20 25 30 35 40 45 50 Vicker's hardness (MPa) Tail width (mm) Tail length (bone #) Tail width Ventral hardness Dorsal hardness Bovine femur bone 0 100 200 300 400 500 UT seahorse bony plate DP seahorse bony plate DM seahorse bony plate Vicker's hardness (MPa) Microstructural features and surface roughness of bony plates The bony plated armor in the prehensile tail of a seahorse is a complex inorganic/organic composite with unique microstructural features (Fig 4). Each plate is a thin, V-shaped bone with a narrow shaft (male joint) and rounded inset (female joint) positioned at the front and back of the V-junction, respectively. The overall shape of the bony plates resembles a 3-dimensional coordinate system axis, where the two plate wings (right/left and ventral/dorsal) are positioned along the x and y axes and the male shaft projects along the z axis. The outer surfaces of the plate wings have a central, vein-like strut and small tendrils running the length of the wings for added structural support. At the junction of the plate wings and the male joint, a distinct ridge of rounded nodules and pores makeup the four pointed corners of each tail segment. The inner surface of the entire bony plate is relatively smooth, with directionally oriented mineral and protein fibers. Hierarchical structure of bony plates Seahorses have a prehensile tail composed of approximately 140 bony plates arranged in rings of four overlapping corner plates per tail segment. Fig 1 shows the tail cross-section (skin removed) and the hierarchical structure of the bony plated armor. The bony plates are inorganic/organic composites composed of approximately 45 wt% inorganic, 38 wt% organic, and 17 wt% water. For clarity, the tail segments are numbered, as seen in Fig 1, according to Bruner et al. [5]. Microstructural characterization of the bony plated armor in the prehensile tail of seahorses Michael M Porter 1 , Ana Bertha Castro Ceseña 1 , Ekaterina Novitskaya 1 , Marc A Meyers 1,2 , Joanna McKittrick 1,2 1 Materials Science and Engineering Program; 2 Department of Mechanical and Aerospace Engineering; University of California, San Diego, USA. Background At the University of California San Diego, we look at natural biological materials for insight to design and fabricate new bio-inspired materials and devices. Our group has investigated many biological materials designed for structural support and protection, including: bovine bone, elk antler, sheep and ram horn, armadillo carapace, porcupine quill, bird beaks, turtle shell, crab exoskeleton, abalone shell, and fish scales [1-4]. To add to our collection of characterized biological materials, we studied the microstructure of the bony plated armor in the prehensile tail of the seahorse (Hippocampus kuda). Seahorses, commonly known for their equine profile and vertical swimming posture, are amazingly complex fish with a variety of unique characteristics. Seahorses have a head like a horse, eyes that move independently like a chameleon, a brood pouch like a kangaroo, camouflage skin like a gecko, and a flexible prehensile (i.e., grasping and holding) tail like a monkey [5-7]. From a mechanics perspective, the bony plated armor in the seahorse tail is a fantastic example of a multifunctional material that provides the seahorse structural support, protection, and even the ability to bend in a strict spiral and grasp objects. In this work, the bony plated armor in seahorses was characterized with 3D optical microscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and micro-hardness testing. Fig 1. Right side view of a seahorse (unspecified species) showing the dorsal and ventral sides, the bone number of each tail segment, the tail cross-section, and the hierarchical structure of a bony plate from the macro to nano scales. Untreated (UT), deproteinated (DP), and demineralized (DM) bony plates The bony plates in seahorses are composed of 45 wt% calcium phosphate, 38 wt% protein, and 17 wt% water. To analyze the mineral and protein constituents individually, untreated (UT) bony plates were compared to those treated with 12.5% NaClO for 24 hrs (deproteinated - DP) and 0.6 N HCl for 24 hrs (demineralized - DM), respectively. Elemental analysis of the bony plates with energy dispersive X-ray spectroscopy (EDX) confirmed the compositions (Fig 6). Fracture surfaces of the bony plates illustrate their hierarchical complexity, showing multiple inorganic/organic layers of directionally aligned fibers at the submicron scale (Fig 6a-c). The deproteinated bony plates exhibited a brittle fracture behavior, while the untreated and demineralized plates were much tougher and more elastic. The hardness of the untreated bony plates (270 MPa) is higher than would be expected from a simple mixture of its mineral (336 MPa) and protein (168 MPa) constituents: H mix = φ m H m + φ p H p where φ is the wt% and H is the hardness of the mineral (m) and protein (p) constituents, respectively. The higher than expected hardness of the untreated bony plates is likely due to the complex structural hierarchy. Fig 4. SEM micrographs of the untreated bony plates of a seahorse (Hippocampus kuda), illustrating the various surface features and roughness at several different locations, the 3-dimensional shape axes and the fiber orientation of the inner plate surface. Bony plate (front view) Female joint (inner surface) Female joint (bottom edge) Female joint (top edge) Plate wing (inner surface) Plate wing (outer surface) Male joint (outer surface) Ridge pore (outer surface) Ridge nodules (outer surface) 500μm 500μm 500μm 100μm 500μm 20μm 200μm 20μm 50μm 1μm 1μm 1μm 1μm 1μm 1μm 1μm 1μm Micro-hardness of bony plates at different locations For protection, the bony plates must be sufficiently hard, yet tough enough to withstand stresses induced by bending and grasping. The female joint is nearly 10% softer than the male, enabling it to absorb stresses caused by joint movement (see right). The top ridge is the hardest region of the bone, which may serve as a hard protective shield against high impact (see right). Compared to bovine femur bone (~67 wt% mineral, ~500 MPa), the bony plates in seahorses have a much lower mineral content (45 wt%) and hardness (270 MPa). The superior toughness and overlapping nature of the bony plates may enhance tail flexibility, resulting in greater resistance to crushing and brittle fracture. Fig 5. (Top image) Representative illustration of a bony plate and the measured micro-hardness (Vicker’s hardness) at various locations (data from dorsal plates 13-19 see Fig 1). (Plot) Tail width and micro-hardness of the bony plated armor (unspecified surface locations) versus seahorse tail length by bone # [5] (see Fig 1). Micro-hardness of bovine femur bone is shown for comparison. Demineralized bone (0.6 N HCl, 24 hr) Deproteinated bone (12.5% NaClO, 24 hr) Untreated bone (washed) (b) Fracture surface of DP bone 20μm Elemental analysis (EDX) C: 43.11 wt % O: 40.25 wt % P: 6.95 wt % Ca: 9.68 wt % Elemental analysis (EDX) O: 50.37 wt % P: 15.66 wt % Ca: 33.97 wt % Elemental analysis (EDX) C: 67.22 wt % O: 32.78 wt % N: trace 20μm (c) Fracture surface of DM bone 20μm (a) Fracture surface of UT bone Fig 6. (Top) EDX elemental analysis and (Bottom) representative SEM micrographs showing fracture surfaces and fiber orientations of the plate wing of untreated (left), deproteinated (middle), and demineralized (right) bony plated armor. 1μm 1μm 1μm Fig 7. Micro-hardness and SEM insets of untreated (UT), deproteinated (DP), and demineralized (DM) seahorse bony plates compared to bovine femur bone. (All data from dorsal plates 13-19 at unspecified locations see Fig 1). Fig 2. Time course of seahorse tail subjected to 12.5% NaClO in ambient conditions. Treatment used to determine the size, placement, overlap, and sequence of bony plates. 5mm (a) 0 min, NaClO (b) 5 min, NaClO (c) 30 min, NaClO (d) 60 min, NaClO 5mm 5mm 5mm Fig 3. Images of overlapping bony plates after bleaching: (a) longitudinal overlap, (b) lateral overlap, and (c) random overlap sequence of dorsal plates over the tail length. Overlap and sequence of bony plates The bony plates overlap longitudinally and laterally giving seahorses the necessary flexibility for axial and lateral bending, grasping and holding [6]. To visualize the succession of overlapping plates, several seahorse tails were partially deproteinated with 12.5% NaClO for 0-60 mins (Fig 2). The longitudinal overlap between two adjacent plates is much like a joint, where a male shaft fits neatly into its female counterpart (Fig 3a). On the right and left sides of the tail, the ventral plates always overlap the dorsal plates (Fig 3b). The right-left (left-right) overlap on the dorsal and ventral sides of the tail, however, are randomly sequenced from segment to segment, and may be distinct to each individual seahorse, similar to a human fingerprint (Fig 3c). Longitudinal overlapping allows seahorses to bend their tails ventrally in a strict spiral. Slight lateral bending may occur concurrently with ventral bending [6]; although, the ventral- dorsal overlap seems to prevent significant lateral movement. (c) Random overlap sequence of dorsal plates (b) Lateral overlap (a) Longitudinal overlap 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Male Female 500μm Ventral Dorsal 500μm 30 Conclusions The bony plated armor in the prehensile tail of the seahorse is a multifunctional material that provides structural support, protection, and mobility. The bony plates are arranged in rings of four overlapping plates per tail segment, with approximately 35 segments spanning the length of the tail. When compared to bovine femur bone, the bony plates in the seahorse tail have a much lower mineral content and average hardness. The distribution of hardness across a single bony plate, however, seems to be tailored to specific tasks - harder on the outer surface for protection, and softer at the overlapping joints for mobility. The structural hierarchy of the mineral and protein constituents, along with the distinct hardness distribution and overlapping nature of the bony plates, gives the prehensile tail its exceptional toughness and flexibility required to perform specific functions. 270 (MPa) 280 200 320 0 2 3 4 KeV 1 C N O 0 2 3 4 KeV 1 Ca P O Ca C Ca 0 2 3 4 KeV 1 Ca Ca O P Ca 1μm 1μm 1μm Acknowledgements We would like to thank Leslee Matsushige, Phil Hastings, H.J. Walker and Fernando Nosratpour of Scripps Institute of Oceanography, UCSD, for providing the seahorse specimens, and Ryan Anderson of CalIT2, UCSD, for help in SEM/EDX. This work was supported by the National Science Foundation, Ceramic Program Grant 1006931. References [1] Chen, P.Y., et al., Journal of the Mechanical Behavior of Biomedical Materials, 2008. 1(3): p. 208-226. [2] Chen, P.Y., et al., Acta Biomaterialia, 2009. 5(2): p. 693-706. [3] McKittrick, J., et al., Materials Science and Engineering C-Materials for Biological Applications, 2010. 30(3): p. 331-342. [4] Lee, S., et al., Materials Science and Engineering: C, 2011. 31(4): p. 730-739. [5] Bruner, E., et al., International Journal of Morphology., 2008. 26(2): 247-262. [6] Hale, M., Journal of Morphology., 1996. 227: 51-65 [7] Consi, T.R., et al., Journal of Morphology., 2001. 248:80-97. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Macro Vertebrae Muscle Rings of bony plated armor Vein-like strut Hollow channel Micro Nano Bony plate Fibrous layers Pores containing mineralized fibers for skin attachment Channels of fiber bundles Mineralized layers Organic fibers

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Page 1: Microstructural characterization of the bony plated armor ...meyersgroup.ucsd.edu/research_posters/2012/Hawaii Seahorse Poster.pdfUT seahorse bony plate DP seahorse bony plate DM seahorse

Bovine femur bone

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10 15 20 25 30 35 40 45 50

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MP

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Tail

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mm

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Tail length (bone #)

Tail width Ventral hardness Dorsal hardness

Bovine femur bone

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100

200

300

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500

UT seahorse bony plate DP seahorse bony plate DM seahorse bony plate

Vic

ker'

s h

ard

ne

ss

(MP

a)

Microstructural features and surface roughness of bony plates

The bony plated armor in the prehensile tail of a seahorse is a complex inorganic/organic composite with unique

microstructural features (Fig 4). Each plate is a thin, V-shaped bone with a narrow shaft (male joint) and rounded inset

(female joint) positioned at the front and back of the V-junction, respectively. The overall shape of the bony plates

resembles a 3-dimensional coordinate system axis, where the two plate wings (right/left and ventral/dorsal) are

positioned along the x and y axes and the male shaft projects along the z axis. The outer surfaces of the plate wings

have a central, vein-like strut and small tendrils running the length of the wings for added structural support. At the

junction of the plate wings and the male joint, a distinct ridge of rounded nodules and pores makeup the four pointed

corners of each tail segment. The inner surface of the entire bony plate is relatively smooth, with directionally

oriented mineral and protein fibers.

Hierarchical structure of bony plates

Seahorses have a prehensile tail composed of approximately 140 bony plates arranged in rings of four

overlapping corner plates per tail segment. Fig 1 shows the tail cross-section (skin removed) and the

hierarchical structure of the bony plated armor. The bony plates are inorganic/organic composites

composed of approximately 45 wt% inorganic, 38 wt% organic, and 17 wt% water. For clarity, the tail

segments are numbered, as seen in Fig 1, according to Bruner et al. [5].

Microstructural characterization of the bony plated armor in the prehensile tail of seahorses

Michael M Porter1, Ana Bertha Castro Ceseña1, Ekaterina Novitskaya1, Marc A Meyers1,2, Joanna McKittrick1,2

1Materials Science and Engineering Program; 2Department of Mechanical and Aerospace Engineering;

University of California, San Diego, USA. Background

At the University of California San Diego, we look at natural biological materials for insight to design and

fabricate new bio-inspired materials and devices. Our group has investigated many biological materials

designed for structural support and protection, including: bovine bone, elk antler, sheep and ram horn,

armadillo carapace, porcupine quill, bird beaks, turtle shell, crab exoskeleton, abalone shell, and fish scales

[1-4]. To add to our collection of characterized biological materials, we studied the microstructure of the

bony plated armor in the prehensile tail of the seahorse (Hippocampus kuda).

Seahorses, commonly known for their equine profile and vertical swimming posture, are amazingly

complex fish with a variety of unique characteristics. Seahorses have a head like a horse, eyes that move

independently like a chameleon, a brood pouch like a kangaroo, camouflage skin like a gecko, and a

flexible prehensile (i.e., grasping and holding) tail like a monkey [5-7]. From a mechanics perspective, the

bony plated armor in the seahorse tail is a fantastic example of a multifunctional material that provides the

seahorse structural support, protection, and even the ability to bend in a strict spiral and grasp objects. In

this work, the bony plated armor in seahorses was characterized with 3D optical microscopy, scanning

electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and micro-hardness testing.

Fig 1. Right side view of a seahorse (unspecified species) showing the dorsal and ventral sides, the bone number of each tail

segment, the tail cross-section, and the hierarchical structure of a bony plate from the macro to nano scales.

Untreated (UT), deproteinated (DP), and demineralized (DM) bony plates

The bony plates in seahorses are composed of 45 wt% calcium phosphate, 38 wt% protein, and 17 wt%

water. To analyze the mineral and protein constituents individually, untreated (UT) bony plates were

compared to those treated with 12.5% NaClO for 24 hrs (deproteinated - DP) and 0.6 N HCl for 24 hrs

(demineralized - DM), respectively. Elemental analysis of the bony plates with energy dispersive X-ray

spectroscopy (EDX) confirmed the compositions (Fig 6). Fracture surfaces of the bony plates illustrate

their hierarchical complexity, showing multiple inorganic/organic layers of directionally aligned fibers at

the submicron scale (Fig 6a-c). The deproteinated bony plates exhibited a brittle fracture behavior, while

the untreated and demineralized plates were much tougher and more elastic. The hardness of the untreated

bony plates (270 MPa) is higher than would be expected from a simple mixture of its mineral (336 MPa)

and protein (168 MPa) constituents: Hmix = φm Hm + φp Hp where φ is the wt% and H is the hardness of

the mineral (m) and protein (p) constituents, respectively. The higher than expected hardness of the

untreated bony plates is likely due to the complex structural hierarchy.

Fig 4. SEM micrographs of the untreated bony plates of a seahorse (Hippocampus kuda), illustrating the various surface features and

roughness at several different locations, the 3-dimensional shape axes and the fiber orientation of the inner plate surface.

Bony plate (front view)

Female joint (inner surface) Female joint (bottom edge) Female joint (top edge)

Plate wing (inner surface)

Plate wing (outer surface) Male joint (outer surface)

Ridge pore (outer surface)

Ridge nodules (outer surface)

500μm

500μm 500μm 100μm

500μm

20μm 200μm

20μm 50μm

1μm

1μm 1μm 1μm

1μm

1μm 1μm 1μm

Micro-hardness of bony plates at different locations

For protection, the bony plates must be sufficiently hard, yet tough enough to withstand

stresses induced by bending and grasping. The female joint is nearly 10% softer than the

male, enabling it to absorb stresses caused by joint movement (see right). The top ridge is

the hardest region of the bone, which may serve as a hard protective shield against high

impact (see right). Compared to bovine femur bone (~67 wt% mineral, ~500 MPa), the

bony plates in seahorses have a much lower mineral content (45 wt%) and hardness (270

MPa). The superior toughness and overlapping nature of the bony plates may enhance tail

flexibility, resulting in greater resistance to crushing and brittle fracture.

Fig 5. (Top image) Representative illustration of a bony plate and the measured micro-hardness (Vicker’s hardness) at various locations (data

from dorsal plates 13-19 – see Fig 1). (Plot) Tail width and micro-hardness of the bony plated armor (unspecified surface locations) versus

seahorse tail length by bone # [5] (see Fig 1). Micro-hardness of bovine femur bone is shown for comparison.

Demineralized bone

(0.6 N HCl, 24 hr)

Deproteinated bone

(12.5% NaClO, 24 hr)

Untreated bone

(washed)

(b) Fracture surface of DP bone

20μm

Elemental analysis (EDX) C: 43.11 wt %

O: 40.25 wt %

P: 6.95 wt %

Ca: 9.68 wt %

Elemental analysis (EDX) O: 50.37 wt %

P: 15.66 wt %

Ca: 33.97 wt %

Elemental analysis (EDX) C: 67.22 wt %

O: 32.78 wt %

N: trace

20μm

(c) Fracture surface of DM bone

20μm

(a) Fracture surface of UT bone

Fig 6. (Top) EDX elemental analysis and (Bottom) representative SEM micrographs showing fracture surfaces and fiber

orientations of the plate wing of untreated (left), deproteinated (middle), and demineralized (right) bony plated armor.

1μm 1μm 1μm

Fig 7. Micro-hardness and SEM insets of untreated (UT), deproteinated (DP), and demineralized (DM) seahorse bony plates

compared to bovine femur bone. (All data from dorsal plates 13-19 at unspecified locations – see Fig 1).

Fig 2. Time course of seahorse tail subjected to 12.5%

NaClO in ambient conditions. Treatment used to determine

the size, placement, overlap, and sequence of bony plates.

5mm

(a) 0 min, NaClO (b) 5 min, NaClO

(c) 30 min, NaClO (d) 60 min, NaClO

5mm 5mm

5mm

Fig 3. Images of overlapping bony plates after bleaching:

(a) longitudinal overlap, (b) lateral overlap, and (c) random

overlap sequence of dorsal plates over the tail length.

Overlap and sequence of bony plates

The bony plates overlap longitudinally and laterally giving seahorses the necessary flexibility for axial and

lateral bending, grasping and holding [6]. To visualize the succession of overlapping plates, several

seahorse tails were partially deproteinated with 12.5% NaClO for 0-60 mins (Fig 2). The longitudinal

overlap between two adjacent plates is much like a joint, where a male shaft fits neatly into its female

counterpart (Fig 3a). On the right and left sides of the tail, the ventral plates always overlap the dorsal

plates (Fig 3b). The right-left (left-right) overlap on the dorsal and ventral sides of the tail, however, are

randomly sequenced from segment to segment, and may be distinct to each individual seahorse, similar to

a human fingerprint (Fig 3c). Longitudinal overlapping allows seahorses to bend their tails ventrally in a

strict spiral. Slight lateral bending may occur concurrently with ventral bending [6]; although, the ventral-

dorsal overlap seems to prevent significant lateral movement.

(c) Random overlap sequence of dorsal plates

(b) Lateral overlap (a) Longitudinal overlap

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Male Female

500μm Ventral

Dorsal

500μm

30

Conclusions

The bony plated armor in the prehensile tail of the seahorse is a multifunctional material that provides

structural support, protection, and mobility. The bony plates are arranged in rings of four overlapping

plates per tail segment, with approximately 35 segments spanning the length of the tail. When compared to

bovine femur bone, the bony plates in the seahorse tail have a much lower mineral content and average

hardness. The distribution of hardness across a single bony plate, however, seems to be tailored to specific

tasks - harder on the outer surface for protection, and softer at the overlapping joints for mobility. The

structural hierarchy of the mineral and protein constituents, along with the distinct hardness distribution

and overlapping nature of the bony plates, gives the prehensile tail its exceptional toughness and flexibility

required to perform specific functions.

270 (MPa)

280

200

320

0 2 3 4 KeV 1

C

N O

0 2 3 4 KeV 1

Ca P

O Ca C

Ca

0 2 3 4 KeV 1

Ca

Ca

O

P

Ca

1μm 1μm

1μm

Acknowledgements

We would like to thank Leslee Matsushige, Phil Hastings, H.J. Walker and Fernando Nosratpour of Scripps Institute of

Oceanography, UCSD, for providing the seahorse specimens, and Ryan Anderson of CalIT2, UCSD, for help in SEM/EDX.

This work was supported by the National Science Foundation, Ceramic Program Grant 1006931.

References

[1] Chen, P.Y., et al., Journal of the Mechanical Behavior of Biomedical Materials, 2008. 1(3): p. 208-226.

[2] Chen, P.Y., et al., Acta Biomaterialia, 2009. 5(2): p. 693-706.

[3] McKittrick, J., et al., Materials Science and Engineering C-Materials for Biological Applications, 2010. 30(3): p. 331-342.

[4] Lee, S., et al., Materials Science and Engineering: C, 2011. 31(4): p. 730-739.

[5] Bruner, E., et al., International Journal of Morphology., 2008. 26(2): 247-262.

[6] Hale, M., Journal of Morphology., 1996. 227: 51-65

[7] Consi, T.R., et al., Journal of Morphology., 2001. 248:80-97.

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Macro

Vertebrae

Muscle

Rings of bony plated armor

Vein-like strut

Hollow channel

Micro

Nano

Bony plate

Fibrous layers

Pores containing mineralized fibers for skin attachment

Channels of fiber bundles

Mineralized layers

Organic fibers