Supporting Information for adma.201701607

I. Analysis of calcium contents of baleen plate in different regions

Referring to the elemental analysis of baleen by Aubin et al[1], baleen plate was divided and cut into two groups of solid shell and tubular layer, each having three portions of pieces. All solid shell and tubular layer samples were further cut into tiny pieces (less than 20 µm thick), charred at 250oC and ashed at 550oC for at least 2 h. All ashes were dissolved in concentrated nitric acid (65-68%) for 1 day, then all solutions were diluted by adding ultra-pure water and centrifuged. The solutions were analyzed for calcium (Ca) content by an inductive coupled plasma emission spectrometer (PerkinElmer Optima 7000 DV). The Ca content of each powder can be calculated and Ca contents of the solid shell and tubular layer samples were obtained in this fashion.

Results show that solid shell samples have a Ca content of 0.115% and the tubular layer shows an average Ca content of 0.311% (dry weight); meaning that the tubular layer has a Ca content 2.7 times that of the solid shell region. This indicates that the tubular layer is the mineral rich region, which agrees with the x-ray micro-computed tomography of baleen in Figure 1.

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II. SEM with Energy dispersive x-ray (EDX) of baleen plate

Elemental mapping through SEM with EDX on the transverse section of the baleen plate (Figure S1) shows that calcium (Ca) and phosphorus (P) are often distributed within intertubular regions.

Figure S1. Elemental mapping of baleen plate. (a) SEM of the transverse section of the baleen plate; distributions of (b) carbon, C, (c) Sulphur, S, (d) oxygen, (e) phosphorus, (f) calcium, Ca.

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III. Fiber orientation in the tubular lamellae and the solid shell, and nanoindentation on transverse and longitudinal sections.

Figure S2. Longitudinally fractured baleen plate (SEM image in the center) showing the microscale longitudinal fibers that compose the solid shell (left) and longitudinal tubules in the tubular layer (right).

For nanoindentation, the baleen plate was cut into segments with transverse and longitudinal sections exposed and polished (Figure S4-a). The tubular region and the solid shell regions on both sections were indented. A nanoindentation testing machine (Nano Hardness Tester, Nanovea, CA, USA) and a Berkovich diamond tip (Poisson’s ratio of 0.07 and elastic modulus of 1,140 GPa) was used. All specimens were indented with 20 mN of maximum force, at loading and unloading rates of 40 mN/min, and 20 seconds of creep. The hardness and reduced Young’s moduli were calculated according to ASTM E2546 by the Oliver Pharr method, which is installed in the Nanovea tester (detail in [2]).

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Figure S3. Nanoindentation results on longitudinal section of shell (Long-s, shell), longitudinal section of tubule (Long-s,tubule), transverse section of shell (Tran-s,shell), and transverse section of tubule (Tran-s,tubule). For uniaxial aligned fiber composites, indenting parallel to the fibers will generate higher hardness and modulus than indenting perpendicular to the fibers. [3] The results here demonstrate that the solid shell is composed of longitudinally aligned fibers and the fibers in the tubular lamellae are also longitudinal.

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IV. Experimental procedure and determination of the J-integral

Figure S4. Materials and experimental procedures. (a) Photograph of the two bowhead whale baleen plates used in this study (left) and schematic illustration showing longitudinal, thickness, and transverse orientations (right). (b) Only transverse (Tran-Ori) and longitudinal (Long-Ori) sections were made, and tested in three-point flexure. (c) Fracture toughness tests, loaded in the longitudinal orientation (Long-Ori) and the transverse orientation (Tran-Ori). (d) Example of a cyclic load-unload experiment for one fracture toughness specimen, from which the J-integral value of each cycle is calculated. The surface in blue corresponds to the plastic area of the sixth load-unload cycle Apl (6 ).(e) J-R curve of one specimen, constructed from (d). (f) Impact compression tests, showing loading cases parallel to (Para-Ori) and perpendicular to (Perp-Ori) the longitudinal direction, and the corresponding observation surfaces.

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J-R curves for each specimen were established from cyclic load-unload tests (Figure S4-d): the material was loaded until the onset of cracking, then partially unloaded to obtain the elastic and plastic portions of the response, a process that was repeated by incrementally increasing the crack length, until the end of the test (~ 12 cycles). The crack front of each specimen was recorded by light microscopy to obtain the crack length and crack propagation (∆a) for each cycle. The J-integral of a load-unload cycle is calculated conforming to the ASTM 1820 standard:

J i=J el(i)+J pl (i) (i=1~12)

The elastic portion Jel is based on linear-elastic fracture mechanics:

Jel (i)=K I (i)

2

E(1−ν2)

where E is the elastic modulus from three-point flexure (Figure S4-b), ν is the Poisson ratio (0.3), and K I(i) is the Mode I stress-intensity factor, which can be calculated from the force-displacement curve. The plastic component J pl is calculated through:

J pl(i)=[J pl(i−1)+( ηpl

W −a(i−1))( A pl( i)−A pl(i−1)

B )]⋅[1−0.9( a(i )−a (i−1)

W −a( i−1 ) )] where ηpl=1.9, and Apl (i) is the plastic area under the force-displacement curve of the load-unload curves of cycle i. An example of Apl(6) is shown in Figure S4-d, and the corresponding J6 is marked on Figure S4-e. After obtaining all J-integral values for all cycles, the J-R curve is established for each sample (Figure S4-e). At least three valid sets of load-unload cyclic tests were analyzed in this study.

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V. Three-point flexure of baleen plate and hydration effects on fracture toughness

Figure S5. Three-point flexure of baleen plates. (a) Loading in the longitudinal orientation (Lg-Ori), in which baleen plates fracture into two at failure. The fracture site shows a smooth profile in the solid shell when viewing from the external surface due to the presence of longitudinal fibers, but shows a tortuous profile in the tubular layer when looking onto the loading surface (transverse section), indicating that the crack propagates along the boundaries of the tubules or along the interface between concentric tubular lamellae. Mineral-rich regions can be found between concentric tubules, which are preferred sites for microcracking, and thus crack path. (b) Loading in the transverse orientation (Tr-Ori), in which breaking the plate was difficult; specimens would bend into a bow shape rather than fracture. (c) Three-point flexure results of

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baleen plates in different loading orientations and hydration conditions, from which modulus values were used as input for calculating the J-integral values. (d) Schematic showing the orientations of fibers and tubules in the baleen with relation to the loading directions, and corresponding stress profile. In longitudinal bending, the external load is mostly sustained by the soft matrix; this loading condition squeezes and/or separates the fibers and tubules. In transverse bending, the external load is sustained by the longitudinal fibers and tubules; this loading orientation compresses and/or stretches the fibers and tubules. Water molecules plasticize the soft matrix, which is between the ordered, stronger fibers and tubules. Therefore, the material is stiffer and stronger in the transverse orientation, and hydration plays a more significant role in the softening response of the material in the longitudinal orientation.

From the equations in SI IV, it can be noted that the elastic portion of the J-integral, and thus the J-integral, is inversely proportional to the elastic modulus, and scales with K I

2. The stress near the crack tip region scales linearly with KI, according to linear elastic fracture mechanics, and can be assimilated to the bending strength (σ2) in SI IV. As a result, the value of the J-integral is affected by changes in the σ 2/E ratio.

In transverse loading, from ambient dry to hydrated conditions, the elastic modulus decreases by ~3 times while the strength decreases by 3.7 times (yielding a scaling ratio of ~0.22 with hydration). Hence, the J-integral values decrease significantly with the effect of hydration due to a dominant decrease of the bending strength. In longitudinal loading, from ambient dry to hydrated conditions, the elastic modulus decreases by a factor of 12 while the strength decreases by 3 times (scaling ratio ~1.3), suggesting a slight increase of the J-integral value, as observed experimentally.

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VI. Quasi-static and dynamic compressive behavior of baleen plates in ambient dry (Dry) and hydrated (Hyd) conditions in parallel (Para-Ori) and perpendicular (Perp-Ori) loading configurations.

Figure S6. Quasi-static and dynamic compression of baleen plate. Compressive stress-strain curves of baleen plate in (a) ambient dry conditions loaded along the parallel orientation (Para-Ori), (b) ambient dry conditions loaded along the perpendicular orientation (Perp-Ori), (c) hydrated conditions loaded along Para-Ori (Hyd-Para-Ori), and (d) hydrated conditions loaded along Perp-Ori (Hyd- Perp-Ori). All samples were tested at strain rates of 10-3, 10-1 and 103 s-1. Inserted images in each graph show the failure mechanisms for each orientation at the strain rates of 10-3 s-1 (yellow frame) and 103 s-1 (pink frame).

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Table S1. Compressive results of baleen plates

Strain rate (S-

1)

Elastic modulus (GPa)

Stress at peak /before plateau (MPa)

Strain at peak/ plateau

Dry-Para-Ori

10-3 3.1±0.19 116.93±3.26 0.05±0.003

10-1 3.3±0.18 146.87±2.68 0.06±0.004

103 4.3±0.17 184.90±17.4 0.05±0.008

Dry-Perp-Ori

10-3 1.8±0.22 80.48±5.37 0.08±0.013

10-1 1.6±0.14 91.92±2.93 0.08±0.083

103 4.2±0.22 102.60±6.61 0.04±0.00

Hyd-Para-Ori

10-3 1.1±0.08 19.09±1.20 0.02±0.001

10-1 1.0±0.07 22.90±2.22 0.03±0.004

103 1.8±0.39 60.13±1.32 0.05±0.008

Hyd-Perp-Ori

10-3 0.1±0.009 7.73±0.30 0.08±0.005

10-1 0.2±0.04 12.02±0.52 0.11±0.005

103 1.0±0.17 36.21±2.27 0.08±0.009

Impact resistance is determined by the ability of the structure to withstand deformation without fracturing. A higher stiffness can either increase or decrease impact resistance; if the toughness is increased, the impact resistance increases; conversely, if it decreases, the opposite takes place. In the hydrated condition, whale baleen exhibits lower stiffness than in dry condition, and significantly higher deformability. Thus, hydration significantly enhances the ability of the material to absorb the energy from the impact.

However, a higher strain-rate sensitivity for wet samples is reported, competing with softening and added resilience provided by hydration (see Figure 3 and Figure S6). As discussed in the text and Supporting Information VI, the strain-rate sensitivity significantly increases (mdry~0.02-0.03 for dry samples vs. mhy~0.09-0.11 for hydrated samples) from ambient dry to hydrated conditions. This is due to an increased viscoelastic component in deformation for the hydrated specimens. If this increased strain-rate sensitivity is accompanied by an increased fracture toughness, an improved impact resistance is expected. Baleen exhibits clearly decreased damage in the hydrated condition (minor cracks in static loading and no fragmentation in dynamic loading), as shown in Figure S6. Water molecules interact with keratin proteins, resulting in a plasticizing effect and an increase in material deformability, without critical failure. Although we did not perform dynamic toughness tests, the dynamic compression tests and

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evaluation of the specimens suggest a higher toughness, and therefore impact resistance, under high strain-rate loading.

VII. Fracture behavior of baleen plates in different loading rates (0.01 and 0.1 s -1) in both ambient dry and hydrated conditions.

Figure S7. Fracture behavior of baleen plates for different loading rates: (a-b) transverse orientation J-R curves in ambient dry and hydrated conditions loaded at 0.01 and 0.1 s-1; (c-d) longitudinal orientation J-R curves in ambient dry and hydrated conditions loaded at 0.01 and 0.1 s-1.

The fracture behavior can be influenced by the hydration level (water molecules act as a swelling agent to increase intermolecular chain spacing and break down secondary bonds to increase chain mobility[4]) and loading rate (by viscoelasticity of the keratin molecules).

In the longitudinal orientation, the external load is mostly sustained by the matrix, which softens under the effect of hydration, slightly increasing the value of the J-integral (SI V), from

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ambient dry to hydrated conditions (at similar strain rates). For hydrated samples, an increase in strain rate increases the failure load, but J-integral values remain comparable.

In the transverse orientation, the longitudinal fibers and tubules sustain most of the external load. From ambient dry to hydrated conditions (at a similar strain rate), baleen plates show lower J-integral values for the latter, due to the notable decrease in strength (SI V). For hydrated samples, higher strain rates increase both the stiffness and the strength of the material (by frictional effects between the matrix and the fibers), therefore resulting in higher J-integral values.

Comparable results have been reported for the tubular structure of horn keratin, with strengths in the tubule direction almost twice as high as perpendicularly to tubules (77.3 and 44.9 MPa, respectively),[5] in which the latter mainly exhibits failure by delamination.

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VIII. Important structural features of whale baleen for biomimetic design, and different patterns of mineral distributions in the tubular layer of the baleen plate.

The structural features that are important for the fracture and impact resistant behavior of whale baleen are:

the nanoscale intermediate filaments and mineral crystals embedded in an amorphous matrix, which increase the stiffness and strength of the material,

the microscale tubular lamellae, which control the direction of the crack propagation in fracture, and buckle and shear under impact,

the sandwich-tubular structure, which boosts the flexural stiffness and the strength with reduced increase in weight.

These construct the hierarchical structure of baleen, and deformation at the different length scales leads to enhanced fracture resistance.

Baleen plates from different species of whales show similar sandwich-tubular structure but different mineral distribution patterns in the tubular layer, as shown in the schematic of the tubular region in Figure S8-a (Types I and II are summarized from [1,6,7], Type III is from the present work):

(I) Multiple thin rings of mineralized tubule lamellae (with an increasing degree of mineralization as one goes towards the inner tubule) separated by layers of unmineralized lamellae, embedded in an unmineralized intertubular region;

(II) Single or a couple of calcified lamellar rings in the tubules, with a mineralized intertubular region;

(III) Randomly mineralized sites, mostly in the intertubular region.

For a structural representative model of the whale baleen (Figure S8-b), type I was selected as the reference model for the tubules. The tubular layer is then sandwiched by a solid shell with a filament-matrix composite structure. CATIA (computer-aided three-dimensional interactive application) and SolidWorks were implemented to generate structural models suitable for 3D printing.

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Figure S8. (a) Mineral distribution patterns in tubular layer of baleen plates, observed in different species: (I) mineralized thin lamellae separated by intervening layers of unmineralized lamellae, embedded in an unmineralized intertubular region, seen in high calcium content baleen of rorqual whales; (II) single or a couple of calcified tubular lamellae, in a mineralized intertubular region, from low calcium content baleen of rorqual whales ([1,6,7]). (III) randomly mineralized regions mostly within intertubular material, from bowhead whale baleen. (b) A sandwich-tubular structural model of the baleen plate, designed via computer-aided software for 3D printing.

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IX. Details of materials used in 3D printed baleen prototype.

The materials used are provided by the manufacturer of the 3D printer (Stratasys). Standard tensile testing specimens (ASTM D638-14) were separately printed for each material (three specimens per material) and tested in tension for quantification of the mechanical properties. The results are reported in Table S2.

Table S2. Mechanical properties of the three materials used for 3D printing of a structural model of whale baleen.

Material used

Elastic modulus (MPa)

Tensile strength (MPa)

Tensile strain Volume fraction in prototype

Matrix TangoBlack

0.2 0.36 1.96 0.48

Filament RGD 8730 490 50.3 0.15 0.46Mineral RGD 525 567 58.9 0.14 0.06

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X. Design of the structural models I, II, III and IV, and compression test results.

Figure S9. Design of a series of structural models, from I, II, III to IV, with addition of a structural feature each time.

Figure S10. (a) Representative compressive behavior of printed baleen prototypes in parallel direction showing the strain-rate stiffening and the damage mechanisms similar to those of natural baleen plate. (b) Images of the crack tip region during fracture toughness tests on printed baleen prototypes, taken in the transverse orientation, showing identical toughening mechanisms.

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Figure S11. Baleen-inspired design and mechanical response of the structural models. (a) Structural model (Model IV) of baleen plate, showing the hierarchical structural features. (b) Photograph of the printed baleen plate model; green arrows indicate the loading direction for c-f. (c-f) Compressive behavior under different loading rates of the structural models I, II, III and IV.

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Table S3. Compression test results of the structural models I, II, III and IV under different strain rates, with loading parallel to the direction of the fibers and tubules.

Strain rate (s-1) Yield stress (MPa)Elastic modulus

(MPa)

Model I

10-4 0.44±0.01 2.29±0.0310-2 0.48±0.03 5.84±0.3780.28 0.65±0.004 15.19±0.68

Model II

10-4 0.95±0.01 5.95±0.30110-2 1.10±0.02 23.14±0.610.28 2.82±0.04 52.23±1.48

Model III

10-4 0.92±0.01 5.61±0.00110-2 1.43±0.21 23.73±0.400.28 2.66±0.01 49.03±0.09

Model IV

10-4 1.34±0.03 5.96±0.0710-2 1.35±0.22 21.63±3.370.28 3.44±0.22 47.56±5.14

Table S4. Curve fitting for each model group, plotted for the yield stress (σ ) as a function of strain rate (ε̇), σ=m∗log10 ε̇+b, where m indicates the strain rate sensitivity.

Model I Model II Model IIII Model IVm 0.058 0.51 0.49 0.57

Table S5. Summary of the mechanical properties of models I, II, III and IV, ranked from first to last.

Model I Model II Model III Model IVStrength and stiffness

4th 2nd 3rd 1st

Viscoelasticity 4th 2nd 3rd 1stWeight (from lightest to heaviest)

1st 4th 2nd 3rd

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References

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[2] B. Wang, M. A. Meyers, Acta Biomater. 2017, 48, 270.

[3] B. Wang, M. A. Meyers, Adv. Sci. 2017, 4, 1.

[4] B. Wang, W. Yang, J. McKittrick, M. A. Meyers, Prog. Mater. Sci. 2016, 76, 229.

[5] M. W. Trim, M. F. Horstemeyer, H. Rhee, H. El Kadiri, L. N. Williams, J. Liao, K. B. Walters, J. McKittrick, S. J. Park, Acta Biomater. 2011, 7, 1228.

[6] F. G. E. Pautard, Nature 1963, 199, 9531.

[7] L. J. Szewciw, D. G. de Kerckhove, G. W. Grime, D. S. Fudge, Proc. Biol. Sci. 2010, 277, 2597.

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