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Mechanical properties and structure of silkworm cocoons: A comparative study of Bombyx mori, Antheraea assamensis, Antheraea pernyi and Antheraea mylitta silkworm cocoons
Materials Science and Engineering C 33 (2013) 3206–3213
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Mechanical properties and structure of silkworm cocoons: A comparative study of Bombyx mori, Antheraea assamensis, Antheraea pernyi and Antheraea mylitta silkworm cocoons J. Zhang a, J. Kaur a, R. Rajkhowa a, J.L. Li a, X.Y. Liu b, X.G. Wang a, c,⁎ a b c
Australian Future Fibres Research and Innovation Centre, Institute for Frontier Materials, Deakin University, VIC 3217, Australia Biophysics and Micro/Nanostructures Lab, Department of Physics, Faculty of Science, National University of Singapore, 117542, Singapore School of Textile Science and Engineering, Wuhan Textile University, Wuhan 430073, China
a r t i c l e
i n f o
Article history: Received 28 September 2012 Received in revised form 17 January 2013 Accepted 29 March 2013 Available online 6 April 2013 Keywords: Silkworm cocoon Mechanical properties Fracture toughness Calcium oxalate crystal Biological materials
a b s t r a c t As a protective shell against environmental damage and attack by natural predators, the silkworm cocoon has outstanding mechanical properties. In particular, this multilayer non-woven composite structure can be exceptionally tough to enhance the chance of survival for silkworms while supporting their metabolic activity. Peel, out-of-plane compression and nano-indentation tests and micro-structure analysis were performed on four types of silkworm cocoon walls (domesticated Bombyx mori, semi-domesticated Antheraea assamensis and wild Antheraea pernyi and Antheraea mylitta silkworm cocoons) to understand the structure and mechanical property relationships. The wild silkworm cocoons were shown to be uniquely tough composite structures. The maximum work-of-fracture for the wild cocoons (A. pernyi and A. mylitta) was approximately 1000 J/m 2, which was almost 10 times the value for the domesticated cocoon (Bombyx mori) and 3 ~ 4 times the value for the semi-domesticated cocoon (A. assamensis). Calcium oxalate crystals were found to deposit on the outer surfaces of the semi-domesticated and wild cocoons. They did not show influence in enhancing the interlaminar adhesion between cocoon layers but exhibited much higher hardness than the cocoon pelades. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In nature, very thin and lightweight silkworm cocoons can protect silkworms from physical attacks from predators or the environment while supporting their metabolic activity. Evolved over thousands of years' natural selection, silkworm cocoons are hierarchical and multifunctional. Their biological functions such as defence against natural enemies, thermal regulation and anti-bacterial function are essential for the survival of silkworms residing inside. These functions are attracting increasing attention from researchers to understand the structure and property of this important biological material and to design and develop next-generation bio-mimic protective materials [1–4]. A cocoon is a multilayer composite material formed by continuous twin silk filaments (fibroin) bonded by silk gum (sericin). The silk spinning and cocoon building process has evolved over thousands of years through natural selection [5]. Silk is produced as an aqueous solution in the posterior section of the silkworm's gland [6]. This solution is extruded through two spinnerets and coated and fastened together with silk gum (sericin) into twin filaments (bave) during ⁎ Corresponding author. Tel.: +61 3 52272894; fax: +61 3 52272539. E-mail address: [email protected] (X.G. Wang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.03.051
coagulation. A compact cocoon is then formed when a silkworm, along with spinning, wraps the bave around its body through a gyrating motion of its head and cyclically bending and stretching of its body with different shapes in a programmed manner [7,8]. Depending upon silkworm species and rearing environment, cocoons vary in weight, thickness, colour and stiffness. Compared with domesticated silkworms such as Bombyx mori (B. mori), wild silkworms are reared in the open environment and conceivably need much greater protection from environmental, biotic and physical hazards. For example, silkworms are immobilised when it is cold and therefore cannot move in response to environmental threats; research has shown that wild silkworm cocoons produced for winter are more robust than their summer counterparts [9]; wild silkworm cocoons have also shown higher tensile strength than domesticated silkworm cocoons [10]. Silk is a natural macromolecular protein polymer composed of two proteins: a central protein called fibroin which is covered by another protein known as sericin. Silk fibroin has been increasingly used in biomedical applications due to its excellent biocompatibility, controllable degradation rates and remarkable mechanical properties when fabricated into different forms such as fibres, films and 3D scaffolds [11]. The sericin protein can be cross-linked, copolymerised, and blended with other macromolecular materials, especially artificial polymers, to produce materials with improved properties and they have been used for cosmetic and
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Table 1 Geometrical parameters and nominal density of cocoons. Cocoon type
B. mori A. assamensis A. pernyi A. mylitta
Cocoon thickness (μm)
Cocoon nominal density (kg/m3)
Fibre bonding length from outer surface (μm)
Fibre bonding length from inner surface (μm)
393 277 387 260
377 516 711 693
12.64 24.50 35.68 34.63
8.98 25.32 29.07 31.97
± ± ± ±
21 29 31 26
± ± ± ±
15 28 44 54
± ± ± ±
1.61 2.03 3.36 5.03
± ± ± ±
Twin silk fibres
0.72 2.5 4.19 4.90
Table 2 Average layer thickness of cocoons for peel tests. Cocoon type
Layer 1 (μm)
Layer 2 (μm)
Layer 3 (μm)
B. mori A. assamensis A. pernyi A. mylitta
135 90 190 105
75 80 160 165
80 ± 14 105 ± 35 – –
± ± ± ±
21 28 14 7
± ± ± ±
21 0 57 21
Fig. 2. The non-woven cocoon structure from the A. pernyi.
medical applications. In recent years, much attention has been given to non-mulberry cocoon types such as Antheraea pernyi and Antheraea mylitta species, owing to their superior properties over mulberry type. Despite the rapid growth of research interest in silk fibres [12–14], silk protein [15,16] and the silk extrusion process [17,18], limited studies have been conducted to understand the structure and functions of silkworm cocoons. For example, Zhao et al. tested the tensile properties of B. mori cocoons and found graded mechanical properties from the outer layers to inner layers [1,19]; Chen et al. recently examined a wide range of silkworm cocoons to correlate their tensile, compressive and gas diffusion properties with the cocoon structure [2,3]; Roy et al. observed interesting directional carbon dioxide transfer through the cocoon to impart a conducive environment for the survival of the pupa [4]. The challenging requirements placed on the cocoon in the natural environment substantially influence the performance of protection shells of silkworms. Calcium oxalate crystals are found on the outer surfaces of many cocoon types [2], which may have unique functionalities towards efficient protection of
the silkworm inside. Understanding their roles in the structural properties of cocoons may also open up new avenues for the development of advanced multi-scale energy absorbing materials. This study aims to understand the differences in structure and mechanical properties between domesticated and wild silkworm cocoons reared under different conditions, which will be of importance to related research in understanding and utilizing non-mulberry silk materials. For example, the temperate oak tasar (A. pernyi) silk fibre has intermediate mechanical properties between the B. mori silkworm silk and the Nephila clavipes spider silk. The amino acid sequence of its silk fibroin is surprisingly similar to that of the partial sequence of the major ampullate silk spidroin I of the N. clavipes silk [20]. The research in A. pernyi silk will have great implication to bioengineered super-tough “spider silks”. Amongst the natural biomaterials available, silkworm silk protein fibroin possesses unique mechanical strength, biocompatibility and relative ease in fabrication into diverse morphologies [21].
Fig. 1. Morphologies of silk cocoons. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta. The outer and inner surface morphologies of each type are shown in the second row and the third row, respectively. The insets show the crystals on the cocoon outer surfaces.
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Pmax Paverage
5000
e d
Peeling Load (mN)
781 cm-1
-1
1317 cm
1618 cm
-1
a 4000 3000 2000 1000
c 0
1
b
2 3 Cocoon Type
4
a
b
1500 1200
The superior mechanical property, non-cytotoxic property and low level of inflammatory response of the non-mulberry silk fibroin make it an excellent candidate material for tissue engineering [22]. Cocoon materials including the tropical tasar (A. mylitta) and muga (Antheraea assamensis) silk protein fibroin can be suitably fabricated into scaffolds that embrace much higher compressive strength over those made from other naturally derived materials such as collagen and chitosan. The cocoons studied were domestic B. mori, semi-domestic A. assamensis, and wild A. pernyi and A. mylitta silkworm cocoons. Abbreviated names of B. mori, A. assamensis, A. pernyi and A. mylitta will be used in research discussion. The mechanical performance of different cocoons was examined by peel tests, out-of-plane compression tests and nano-indentation test.
Work of Fracture (J/m2)
Fig. 3. FTIR spectra of cocoon outer layers and commercial calcium oxalate crystals. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta; (e) commercial calcium oxalate powder.
800
900
600 600 400 300
200
Peel Strength (N/m)
1200
1000
0
0 1
2
3
4
Cocoon Type Fig. 5. Comparison of peeling properties among four types of cocoons. (a) Average peeling load and maximum peeling load; (b) work of fracture and peel strength.
Fig. 4. Loading curves for peel tests on different cocoons.
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Fig. 6. Fracture surface of peeled cocoon layers. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta. The SEM images on the right are enlargements of particular regions in the lower magnification images shown on the left.
The structural morphologies and the fracture surfaces were observed by scanning electron microscopy (SEM).
oxalate crystals were purchased from Sigma Aldrich and used as received.
2. Materials and methods
2.2. Methods
2.1. Materials
The geometrical parameters of cocoons were measured and summarised in Table 1. The thickness of cocoon walls was measured with a vernier caliper. The 180 degree peel test, which is modified from the ASTM standard test D 1876-08 used for peel resistance of adhesives [23], was performed on a Lloyd LR30K tester with a 100 N load cell. A loading rate of 2 mm/min was applied to delaminate cocoon samples with the dimension of 20 mm × 5 mm. The cocoon samples
B. mori and A. assamensis silkworm cocoons were purchased from silk rearing houses in North East India; A. mylitta cocoons were collected from Central India and A. pernyi cocoons were collected from Northern China. They were received as stifled cocoons, as commonly used prior to reeling silk filament for textile applications. Calcium
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Compressive Stress (MPa)
a
indentation depth). The morphologies of the cocoons and the fracture surfaces from failed samples were investigated by a scanning electron microscope (FEG-SEM Zeiss Supra 55VP). The silk fibre bonding length (i.e. the width of the single fibre) was taken from measuring 50 fibres from the SEM images and determined from the peak value of the histogram graphs of size distribution. Fourier Transform Infrared (FTIR) Spectroscopy-Attenuated Total Reflectance (ATR) measurements were performed on the outer surface of cocoons with a Bruker Vertex 70 FTIR spectrometer. All spectra were recorded at room temperature and signal-averaged over 64 scans at a resolution of 2 cm−1.
14
4
12
2
3
10
1
8 6 4
3. Results and discussion
0.00
0.02
0.04
0.06
0.08
Compressive Extension (mm)
Compressive Modulus (MPa)
b
60 50 40 30 20 10 0
1
2
3
4
Cocoon Type Fig. 7. Out-of-plane compressive properties of cocoons. (a) Curves from mechanical testing; (b) modulus comparison.
were peeled artificially for a length of 5 mm before they were pulled apart by the tester. The layers were numbered according to the sequence from the outer to inner layers (e.g. layer 1 is the outermost layer). Three samples for each type of cocoons were peeled into multiple layers. For the B. mori and A. assamensis cocoons, 9 peel tests were performed for each type; for the A. pernyi and the A. mylitta cocoons, 6 peel tests were performed for each type. The average thickness of peeled layers for each cocoon type is shown in Table 2. The average peeling load for each loading curve was determined as the average of load readings at a 2 mm increment of crosshead motion and the peel strength was determined by the average peeling load divided by sample width. The work of fracture (WOF) was obtained by the work under the peeling load vs. propagation extension curves divided by the delamination area. The maximum values of peel properties obtained from different layers were used for comparison among the domesticated, semidomesticated and wild cocoon types. Compression tests were performed perpendicular to the plane of cocoon wall on an Instron 30 K tester. The outer surface of cocoon walls was the compression side while the other side was the supporting side. A loading rate of 20 N/min was applied to characterise the compressive behaviour of rectangular cocoon samples with the dimension of 10 mm × 10 mm. Three samples were tested for each type of cocoons. A crystal pellet was prepared from 0.04 g of the purchased calcium oxalate crystal powder through compression using the SpectroPress (Chemplex Industries) with a load of 9 tons. The hardness tests were conducted on the inner surface of cocoon walls (pelades) and the crystal pellet using an ultra-micro indentation system (UMIS II). The maximum indentation loads of 45 mN and 1 mN were applied to the cocoon inner surface and the crystal pellet, respectively, through using a spherical indentor (the contact area A, equals to 24.5 ht2, where ht is the
3.1. Cocoon morphology The cocoon structure can be considered as a porous matrix of sericin reinforced by randomly oriented continuous fibroin fibres [19]. The silkworm cocoons showed a non-woven composite structure with twin silk fibres coated with sericin (Fig. 1 and Fig. 2). The wild cocoons (A. pernyi and A. mylitta) have a more compact structure than the domesticated (B. mori) and semi-domesticated (A. assamensis) ones. As shown in Table 1, the nominal density of A. pernyi and A. mylitta cocoons is approximately twice that of the B. mori cocoon and is about 40% higher than the A. assamensis cocoon. For most of the cocoon types, the fibre bonding length is smaller for the cocoon inner surface than the outer surface, although the A. assamensis cocoon shows comparable values between the outer and inner surfaces. The fibre bonding length for the semi-domesticated cocoons and wild cocoons are 100% ~ 300% larger than the domesticated cocoon. As shown in the insets of Fig. 1b, 1c and 1d, cubic crystals can be visualised on the outer surfaces of A. assamensis, A. pernyi and A. mylitta cocoons, loosely deposited on the surface of silk fibres and stacked in the pores. These crystals have been identified as calcium oxalates [2], which show unique functionality such as preferential gating of CO2 from cocoon inside to outside and temperature regulation to maintain a physiological temperature inside the cocoon irrespective of the surrounding environment [4]. In Fig. 3, the FTIR-ATR spectra showed absorption peaks at 1618 cm−1, 1317 cm−1 and 781 cm−1 for the calcium oxalate commercial powder. Similarly, all three peaks are present in the cocoon types of A. assamensis, A. pernyi and A. mylitta. The absence of the peaks at 1317 cm−1 and at 781 cm−1 indicates that the B. mori silk cocoon outer layer does not contain calcium oxalate crystals. Since the peak at 1618 cm−1 is the Amide I peak caused by the silk protein itself [24], all silk cocoons had absorbance peaks at this wave length. 3.2. Interlaminar peel resistance of cocoon layers Fig. 4 shows the loading curves for peel tests on different cocoons. For the B. mori cocoon, the peeling loads were less than 1 N and the load values were comparable for three peeled layers. For the A. assamensis cocoon, the peeling loads were within the range of 0.5 N to 2 N during stable delamination propagation; the inner layer, i.e. layer 3, was easier to peel apart than the outer layers, indicated by the lower peeling loads. In the case for both A. pernyi and A. mylitta cocoons, the peeling curves became more fluctuated with much higher loads (the maximum load values reached 4 ~ 5 N); the outer layers (layer 1) were more difficult to separate. This further indicates that delamination resistance of different B. mori layers was similar from outer to inner cocoon surfaces; however, the delamination resistance of A. pernyi and A. mylitta cocoon layers was stronger in the outer layers. It indicates that the outer layers of these two types of wild cocoons are tougher than the inner layers, which results from the more stringent requirement from the wild to protect the pupa against physical attack from natural predators and harsh surroundings. In
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a Fibre breakage
b
Fibre patches
c Fibre splits
d Fibre rupture
Fig. 8. Fracture surface of cocoons after compression tests perpendicular to the plane. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta. The SEM images show the outer surface (left column) and the inner surface (right column) of cocoons.
addition, the wild cocoons showed higher peak loads and larger load drops during delamination, signifying much greater peel resistance and interlaminar fracture toughness over the domesticated cocoon type. The comparison of peel resistance among four cocoon types was made based on the layers where the highest peeling loads occurred (Fig. 5). The maximum peeling load (Pmax) was 0.73 N and the average peeling load (Paverage) was 0.34 N for the B. mori cocoon. By comparison, the A. assamensis cocoon showed Pmax of 1.4 N and Paverage of 1.2 N. Much higher peeling loads were found for the wild cocoons, i.e. a Pmax of 4.4 N and 3.7 N and a Paverage of 2.5 N and 2.4 N for the A. pernyi and A. mylitta cocoons, respectively. The semi-domesticated cocoon A. assamensis showed an approximate 100% increase in both Pmax and Paverage compared with the domestic B. mori cocoon and the wild
cocoons (A. pernyi and A. mylitta) showed further remarkable 200% and 150% enhancement over the semi-domesticated A. assamensis cocoon, respectively. The maximum work of fracture (WOF) of all tested cocoon layers was about 1000 J/m2 from the A. pernyi outer layer, suggesting the highest bonding energy. The WOF and peel strength calculated for the wild cocoons (A. pernyi and A. mylitta) were about 10 times of the domestic cocoon (B. mori) and were 3 ~ 4 times of the semi-domestic cocoon (A. assamensis). As a result, the cocoon structure of wild silkworms is substantially tougher than that of the domesticated silkworms, presumably caused by the need for higher degree of protection against attack from predators and the harsh physical environment. The peel fracture surfaces of different cocoon types are revealed in the SEM images (Fig. 6). Four cocoon types showed different levels of
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50
4
3
40
1
2 Indentation Load (mN)
Indentation Load (mN)
a
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30 20 10
1.0
5
0.8 0.6 0.4 0.2 0.0 0.0
0.1 0.2 0.3 Indentation depth (µ m)
0 0
10
20
30
40
50
60
Indentation Depth (µm)
3500
70
3000
60
2500
50
2000 1500
40
1000
30
200 150 100 50 0
20 10 0 1
2
3
4
Hardness (MPa)
Maximum Indentation Depth (µm)
b
5
Material Type Fig. 9. Indentation tests performed on the inner surface of cocoons and calcium oxalate crystals. (a) Loading curves; (b) comparison of maximum indentation depth and hardness.
silk fibre damage after the peel tests. As shown in Fig. 6a, slight fibre damage can be observed at the intersections where B. mori silk fibres meet. However, fibre splitting is clearly shown on the fracture surface of A. assamensis cocoon layers (Fig. 6b). The fracture surfaces of A. pernyi and A. mylitta showed more severe fibre damage. The silk fibres of these wild cocoons split into many fibrils indicating the stronger fibre/matrix adhesion and the higher bonding between different layers (Fig. 6c and d). For the A. pernyi cocoon, the calcium oxalate crystals existed at the outer surface only, evidenced by the fracture surface of the first peeled layer shown in Fig. 6c, where no crystals can be seen. Both A. assamensis and A. mylitta cocoons have cubic crystals scattered on the fracture surfaces, however, these crystals do not seem to affect the peel resistance of their host cocoons, since the major failure mode of all cocoon layers is silk fibre damage as noticed in Fig. 6. The stronger matrices, rougher fibre surface of wild cocoons and larger bonding area between layers all make an important contribution to the substantially higher peel resistance. 3.3. Out-of-plane compressive properties Compression tests perpendicular to the plane direction of cocoon walls were performed and the results are summarised in Fig. 7. The compressive deformation was not recorded until the porous cocoon samples were consolidated by compression while the stress reached about 4 MPa. The wild cocoons (A. pernyi and A. mylitta) and semidomesticated cocoon (A. assamensis) were stiffer than the domesticated cocoon (B. mori). It has been proposed that the compressive stress is linked to the density of the porous samples [3,25]. The density of different types of cocoons follows the increasing sequence of B. mori, A. assamensis, A. mylitta and A. pernyi as summarised in Table 1, which is also the sequence for the increase of the compressive moduli. The fracture surfaces on both sides of the cocoon walls were observed and shown in Fig. 8. Despite the severe damage revealed on the outer
surface (the left column in Fig. 8), the inner surface of the cocoon walls remained intact (the right column in Fig. 8). Under compression, the B. mori silk fibres broke or fractured at regions close to the intersections of non-woven network (Fig. 8a). Various changes occur during compression of non-woven fibrous structures, including bending of individual fibres, change in the free distance between two contact points, and fibre-to-fibre slippage [26]. As mentioned previously in Section 3.2, the bonding energy between filaments of B. mori cocoons is much lower than the A. pernyi, A. mylitta and A. assamensis cocoons, therefore the bending of filaments and filament-to-filament slippage play a much more important role in compression for the B. mori cocoon walls compared with other cocoon types. Fig. 8b shows the compression surface of the A. assamensis cocoons: it is interesting to find that the silk fibres from outermost layer were completely compressed into patches and the scattered calcium oxalate crystals were pressed into these patches and filled the cracks and gaps, forming a flattened surface after compression. Abundant crystals therefore shared the compressive loads with the silk filaments at the same plane and protected the inner layers from compressive failure. Fig. 8c and d demonstrates the compressive failure that occurred in the two types of wild cocoons (A. pernyi and A. mylitta) respectively. Fibre splits (A. pernyi) and fibre rupture (A. mylitta) were clearly observed at regions where large amounts of silk fibres interconnected and therefore had higher surface profiles (height). In contrast to the failed filaments, all the crystals observed on the fracture surfaces were intact, which indicates superior mechanical properties to the silk fibres and sericin. 3.4. Nano-indentation on inner surface of cocoons and calcium oxalate crystals It has been noted that the innermost layer of silkworm cocoon (pelade) has superior strength and modulus to the corresponding thickness-averaged values of a complete silkworm cocoon [19]; therefore the indentation measurement on pelades gives a good indication of comparative properties of the inner layers and the cocoon structure. Fig. 9 shows that the pelade of the A. assamensis cocoon has the lowest hardness due to its highest maximum indentation depth (ht) under the same applied maximum indentation load (45 mN). The A. pernyi pelade exhibited a hardness of 125 MPa, which is remarkably higher than the other three cocoon types with hardness lower than 25 MPa. More importantly, the hardness of the calcium oxalate crystals was around 2 GPa and this extremely high hardness explains why these crystals remained intact after compression tests. Despite the unique functions these crystals have in influencing gas diffusion through cocoon walls [3,4], they did not show clear effect on enhancing interlaminar adhesion (e.g. peel resistance) in the outer layers of cocoons. However, more importantly, these extremely hard crystals formed the first defence layer against penetration and other physical attacks from outside the cocoon structure. The cocoons with crystals on the outer layer (A. assamensis, A. pernyi and A. mylitta) showed higher compressive modulus than the one without crystals (B. mori). Similarly, hard coatings with micro- and nano-particles are also able to enhance the Young's modulus of their substrates [27,28]. In nature, the calcium oxalate crystals exist in plants as well, where their presence usually points towards a well-adapted protective function, including mechanical support of soft tissues [29,30]. 4. Conclusions Four types of silk cocoons including domesticated (B. mori), semidomesticated (A. assamensis) and two wild types (A. pernyi and A. mylitta) were investigated in this paper. The wild silkworm cocoons showed superior interlaminar peel resistance, out-of-plane compressive modulus and hardness to the domestic type. The semi-domestic A. assamensis cocoon exhibited medium mechanical
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performance under interlaminar peeling and out-of-plane compressive loads despite of the low mechanical property of the pelade. Calcium oxalate crystals deposited loosely on the outer surface/layers of the A. assamensis, A. pernyi and A. mylitta cocoons. They were not shown to improve interlaminar fracture toughness but exhibit extremely high hardness, forming the first defensive layer against physical attacks from predators and the environment.
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Ms Jasjeet Kaur is a PhD student working on silkworm cocoon structure–property correlation to study the protective roles of the various cocoon components at the Institute for Frontier Materials, Deakin University.
Acknowledgement The authors would like to acknowledge the funding support from the Australian Research Council (ARC) through a project DP 120100139.
References [1] H.P. Zhao, X.Q. Feng, S.W. Yu, W.Z. Cui, F.Z. Zou, Polymer 46 (2005) 9192–9201. [2] F. Chen, D. Porter, F. Vollrath, Mater. Sci. Eng. C 32 (2012) 772–778. [3] F. Chen, D. Porter, F. Vollrath, J. R. Soc. Interface (2012), http://dx.doi.org/ 10.1098/rsif.2011.0887. [4] M. Roy, S. Meena, T. Kusurkar, S. Singh, N. Sethy, K. Bhargava, S. Sarkar, M. Das, Biointerphases 7 (2012) 1–11. [5] R. Manohar Reddy, M.K. Sinha, B.C. Prasad, J. Entomol. 7 (2010) 129–140. [6] C. Craig, Annu. Rev. Entomol. 42 (1997) 231–267. [7] L.P. Lounibos, Anim. Behav. 23 (1975) 843–853. [8] T. Kaise, M. Miura, H. Morikawa, W. Iwasa, J. Insect Biotechnol. Sericology 72 (2003) 171–175. [9] H.V. Danks, Eur. J. Entomol. 101 (2004) 433–437. [10] F. Chen, D. Porter, F. Vollrath, Phys. Rev. E 82 (2010) 041911–041917. [11] N. Kasoju, U. Bora, Adv. Healthc. Mater. 1 (2012) 393–412. [12] Q. Yuan, J. Yao, X. Chen, L. Huang, Z. Shao, Polymer 51 (2010) 4843–4849. [13] N. Du, Z. Yang, X.Y. Liu, Y. Li, H.Y. Xu, Adv. Funct. Mater. 21 (2011) 772–778. [14] N.C. Tansil, Y. Li, C.P. Teng, S. Zhang, K.Y. Win, X. Chen, X.Y. Liu, M.Y. Han, Adv. Mater. 23 (2011) 1463–1466. [15] J.G. Hardy, T.R. Scheibel, Prog. Polym. Sci. 35 (2010) 1093–1115. [16] J. Shi, S. Lua, N. Du, X. Liu, J. Song, Biomaterials 29 (2008) 2820–2828. [17] G. Askarieh, M. Hedhammar, K. Nordling, A. Saenz, C. Casals, A. Rising, J. Johansson, S.D. Knight, Nature 465 (2010) 236–238. [18] C. Riekel, B. Madsen, D. Knight, F. Vollrath, Biomacromolecules 1 (2000) 622–626. [19] H.P. Zhao, X.Q. Feng, W.Z. Cui, F.Z. Zou, Eng. Fract. Mech. 74 (2007) 1953–1962. [20] C. Fu, D. Porter, X. Chen, F. Vollrath, Z. Shao, Adv. Funct. Mater. 21 (2011) 729–737. [21] C. Patra, S. Talukdar, T. Novoyatleva, S.R. Velagala, C. Mühlfeld, B. Kundu, S.C. Kundu, F.B. Engel, Biomaterials 33 (2012) 2673–2680. [22] N. Kasoju, R.R. Bhonde, U. Bora, Mater. Lett. 63 (2009) 2466–2469. [23] ASTM standard D 1876-08, Standard test method for peel resistance of adhesives (T-peel test), ASTM international, 1–3, 2008. [24] P. Taddei, P. Monti, G. Freddi, T. Arai, M. Tsukada, J. Mol. Struct. 651–653 (2003) 433–441. [25] C. Zhou, C. Dai, G.D. Smith, Holzforschung 62 (2008) 201–208. [26] V.K. Kothari, A. Das, J. Text. Inst. 84 (1993) 16–30. [27] J. Douce, J.P. Boilot, J. Biteau, L. Scodellaro, A. Jimenez, Thin Solid Films 466 (2004) 114–122. [28] R.H. Brzesowsky, G. De with, S. Van den Cruijsem, I.J.M. Snijkers- Hendrickx, W.A.M. Wolter, J.G. Van Lierop, J. Non-Cryst. Solids 241 (1998) 27–37. [29] A. Schneider, Bot. Gaz. 32 (1901) 142–144. [30] R.F. Vincent, T.H.J. Harry, Bot. Rev. 46 (1980) 361–427.
Dr Jin Zhang is a research fellow in biological composites at the Australian Future Fibres Research and Innovation Centre of Deakin University. Her research interests are focussed on biological composites, multifunctional materials, fibre-reinforced polymer matrix composites, nano-composites and composite manufacture. She obtained her PhD degree from Deakin University, Australia.
Dr Rangam Rajkhowa is a research fellow in green natural fibres at the Australian Future Fibres Research and Innovation Centre at Deakin University Australia. His main research area is processing, characterisation and application of natural fibres. In particular he focusses on structure–property relationships of protein fibres and protein fibre based products. He is also involved with fibre powder production and applications such as silk and wool powders. Rajkhowa obtained a Masters' degree in Fibre Science and Technology from the Indian Institute of Technology Delhi and PhD from Deakin University Australia.
Dr Jingliang Li is working at the Institute for Frontier Materials of Deakin University as a research fellow. His research interests include (1) theoretical understanding of the formation mechanisms of soft functional materials with 3D fiber networks. On the basis of this, to develop novel strategies towards the design of supramolecular architectures with controllable functionalities; (2) natural fibers and related composite materials for advanced applications such as optoelectronics and optical sensing; (3) novel approaches to wool yellowing; (4) novel functional nano-particles for bioapplications such as cancer treatment.
Prof. Xiangyang Liu is the leader of the Biophysics and Micro/Nanostructures Group at National University of Singapore. His background is in biophysics, nanosciences and technologies, crystallisation, bio-functional materials. He has published high profile papers in leading journals including Nature.
Prof. Xungai Wang is a leading researcher in fibre science and technology. He is the 2005 recipient of Fiber Society Distinguished Achievement Award. Professor Wang is a Fellow of the Textile Institute. In recent years, Professor Wang has been investigating new applications for fibres and textiles, and new ways of adding value to fibres and textiles. Professor Wang holds a PhD in fibre science and technology from the University of New South Wales, Sydney.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Mechanical properties and structure of silkworm cocoons: A comparative study of Bombyx mori, Antheraea assamensis, Antheraea pernyi and Antheraea mylitta silkworm cocoons J. Zhang a, J. Kaur a, R. Rajkhowa a, J.L. Li a, X.Y. Liu b, X.G. Wang a, c,⁎ a b c
Australian Future Fibres Research and Innovation Centre, Institute for Frontier Materials, Deakin University, VIC 3217, Australia Biophysics and Micro/Nanostructures Lab, Department of Physics, Faculty of Science, National University of Singapore, 117542, Singapore School of Textile Science and Engineering, Wuhan Textile University, Wuhan 430073, China
a r t i c l e
i n f o
Article history: Received 28 September 2012 Received in revised form 17 January 2013 Accepted 29 March 2013 Available online 6 April 2013 Keywords: Silkworm cocoon Mechanical properties Fracture toughness Calcium oxalate crystal Biological materials
a b s t r a c t As a protective shell against environmental damage and attack by natural predators, the silkworm cocoon has outstanding mechanical properties. In particular, this multilayer non-woven composite structure can be exceptionally tough to enhance the chance of survival for silkworms while supporting their metabolic activity. Peel, out-of-plane compression and nano-indentation tests and micro-structure analysis were performed on four types of silkworm cocoon walls (domesticated Bombyx mori, semi-domesticated Antheraea assamensis and wild Antheraea pernyi and Antheraea mylitta silkworm cocoons) to understand the structure and mechanical property relationships. The wild silkworm cocoons were shown to be uniquely tough composite structures. The maximum work-of-fracture for the wild cocoons (A. pernyi and A. mylitta) was approximately 1000 J/m 2, which was almost 10 times the value for the domesticated cocoon (Bombyx mori) and 3 ~ 4 times the value for the semi-domesticated cocoon (A. assamensis). Calcium oxalate crystals were found to deposit on the outer surfaces of the semi-domesticated and wild cocoons. They did not show influence in enhancing the interlaminar adhesion between cocoon layers but exhibited much higher hardness than the cocoon pelades. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In nature, very thin and lightweight silkworm cocoons can protect silkworms from physical attacks from predators or the environment while supporting their metabolic activity. Evolved over thousands of years' natural selection, silkworm cocoons are hierarchical and multifunctional. Their biological functions such as defence against natural enemies, thermal regulation and anti-bacterial function are essential for the survival of silkworms residing inside. These functions are attracting increasing attention from researchers to understand the structure and property of this important biological material and to design and develop next-generation bio-mimic protective materials [1–4]. A cocoon is a multilayer composite material formed by continuous twin silk filaments (fibroin) bonded by silk gum (sericin). The silk spinning and cocoon building process has evolved over thousands of years through natural selection [5]. Silk is produced as an aqueous solution in the posterior section of the silkworm's gland [6]. This solution is extruded through two spinnerets and coated and fastened together with silk gum (sericin) into twin filaments (bave) during ⁎ Corresponding author. Tel.: +61 3 52272894; fax: +61 3 52272539. E-mail address: [email protected] (X.G. Wang). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.03.051
coagulation. A compact cocoon is then formed when a silkworm, along with spinning, wraps the bave around its body through a gyrating motion of its head and cyclically bending and stretching of its body with different shapes in a programmed manner [7,8]. Depending upon silkworm species and rearing environment, cocoons vary in weight, thickness, colour and stiffness. Compared with domesticated silkworms such as Bombyx mori (B. mori), wild silkworms are reared in the open environment and conceivably need much greater protection from environmental, biotic and physical hazards. For example, silkworms are immobilised when it is cold and therefore cannot move in response to environmental threats; research has shown that wild silkworm cocoons produced for winter are more robust than their summer counterparts [9]; wild silkworm cocoons have also shown higher tensile strength than domesticated silkworm cocoons [10]. Silk is a natural macromolecular protein polymer composed of two proteins: a central protein called fibroin which is covered by another protein known as sericin. Silk fibroin has been increasingly used in biomedical applications due to its excellent biocompatibility, controllable degradation rates and remarkable mechanical properties when fabricated into different forms such as fibres, films and 3D scaffolds [11]. The sericin protein can be cross-linked, copolymerised, and blended with other macromolecular materials, especially artificial polymers, to produce materials with improved properties and they have been used for cosmetic and
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Table 1 Geometrical parameters and nominal density of cocoons. Cocoon type
B. mori A. assamensis A. pernyi A. mylitta
Cocoon thickness (μm)
Cocoon nominal density (kg/m3)
Fibre bonding length from outer surface (μm)
Fibre bonding length from inner surface (μm)
393 277 387 260
377 516 711 693
12.64 24.50 35.68 34.63
8.98 25.32 29.07 31.97
± ± ± ±
21 29 31 26
± ± ± ±
15 28 44 54
± ± ± ±
1.61 2.03 3.36 5.03
± ± ± ±
Twin silk fibres
0.72 2.5 4.19 4.90
Table 2 Average layer thickness of cocoons for peel tests. Cocoon type
Layer 1 (μm)
Layer 2 (μm)
Layer 3 (μm)
B. mori A. assamensis A. pernyi A. mylitta
135 90 190 105
75 80 160 165
80 ± 14 105 ± 35 – –
± ± ± ±
21 28 14 7
± ± ± ±
21 0 57 21
Fig. 2. The non-woven cocoon structure from the A. pernyi.
medical applications. In recent years, much attention has been given to non-mulberry cocoon types such as Antheraea pernyi and Antheraea mylitta species, owing to their superior properties over mulberry type. Despite the rapid growth of research interest in silk fibres [12–14], silk protein [15,16] and the silk extrusion process [17,18], limited studies have been conducted to understand the structure and functions of silkworm cocoons. For example, Zhao et al. tested the tensile properties of B. mori cocoons and found graded mechanical properties from the outer layers to inner layers [1,19]; Chen et al. recently examined a wide range of silkworm cocoons to correlate their tensile, compressive and gas diffusion properties with the cocoon structure [2,3]; Roy et al. observed interesting directional carbon dioxide transfer through the cocoon to impart a conducive environment for the survival of the pupa [4]. The challenging requirements placed on the cocoon in the natural environment substantially influence the performance of protection shells of silkworms. Calcium oxalate crystals are found on the outer surfaces of many cocoon types [2], which may have unique functionalities towards efficient protection of
the silkworm inside. Understanding their roles in the structural properties of cocoons may also open up new avenues for the development of advanced multi-scale energy absorbing materials. This study aims to understand the differences in structure and mechanical properties between domesticated and wild silkworm cocoons reared under different conditions, which will be of importance to related research in understanding and utilizing non-mulberry silk materials. For example, the temperate oak tasar (A. pernyi) silk fibre has intermediate mechanical properties between the B. mori silkworm silk and the Nephila clavipes spider silk. The amino acid sequence of its silk fibroin is surprisingly similar to that of the partial sequence of the major ampullate silk spidroin I of the N. clavipes silk [20]. The research in A. pernyi silk will have great implication to bioengineered super-tough “spider silks”. Amongst the natural biomaterials available, silkworm silk protein fibroin possesses unique mechanical strength, biocompatibility and relative ease in fabrication into diverse morphologies [21].
Fig. 1. Morphologies of silk cocoons. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta. The outer and inner surface morphologies of each type are shown in the second row and the third row, respectively. The insets show the crystals on the cocoon outer surfaces.
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Pmax Paverage
5000
e d
Peeling Load (mN)
781 cm-1
-1
1317 cm
1618 cm
-1
a 4000 3000 2000 1000
c 0
1
b
2 3 Cocoon Type
4
a
b
1500 1200
The superior mechanical property, non-cytotoxic property and low level of inflammatory response of the non-mulberry silk fibroin make it an excellent candidate material for tissue engineering [22]. Cocoon materials including the tropical tasar (A. mylitta) and muga (Antheraea assamensis) silk protein fibroin can be suitably fabricated into scaffolds that embrace much higher compressive strength over those made from other naturally derived materials such as collagen and chitosan. The cocoons studied were domestic B. mori, semi-domestic A. assamensis, and wild A. pernyi and A. mylitta silkworm cocoons. Abbreviated names of B. mori, A. assamensis, A. pernyi and A. mylitta will be used in research discussion. The mechanical performance of different cocoons was examined by peel tests, out-of-plane compression tests and nano-indentation test.
Work of Fracture (J/m2)
Fig. 3. FTIR spectra of cocoon outer layers and commercial calcium oxalate crystals. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta; (e) commercial calcium oxalate powder.
800
900
600 600 400 300
200
Peel Strength (N/m)
1200
1000
0
0 1
2
3
4
Cocoon Type Fig. 5. Comparison of peeling properties among four types of cocoons. (a) Average peeling load and maximum peeling load; (b) work of fracture and peel strength.
Fig. 4. Loading curves for peel tests on different cocoons.
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Fig. 6. Fracture surface of peeled cocoon layers. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta. The SEM images on the right are enlargements of particular regions in the lower magnification images shown on the left.
The structural morphologies and the fracture surfaces were observed by scanning electron microscopy (SEM).
oxalate crystals were purchased from Sigma Aldrich and used as received.
2. Materials and methods
2.2. Methods
2.1. Materials
The geometrical parameters of cocoons were measured and summarised in Table 1. The thickness of cocoon walls was measured with a vernier caliper. The 180 degree peel test, which is modified from the ASTM standard test D 1876-08 used for peel resistance of adhesives [23], was performed on a Lloyd LR30K tester with a 100 N load cell. A loading rate of 2 mm/min was applied to delaminate cocoon samples with the dimension of 20 mm × 5 mm. The cocoon samples
B. mori and A. assamensis silkworm cocoons were purchased from silk rearing houses in North East India; A. mylitta cocoons were collected from Central India and A. pernyi cocoons were collected from Northern China. They were received as stifled cocoons, as commonly used prior to reeling silk filament for textile applications. Calcium
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Compressive Stress (MPa)
a
indentation depth). The morphologies of the cocoons and the fracture surfaces from failed samples were investigated by a scanning electron microscope (FEG-SEM Zeiss Supra 55VP). The silk fibre bonding length (i.e. the width of the single fibre) was taken from measuring 50 fibres from the SEM images and determined from the peak value of the histogram graphs of size distribution. Fourier Transform Infrared (FTIR) Spectroscopy-Attenuated Total Reflectance (ATR) measurements were performed on the outer surface of cocoons with a Bruker Vertex 70 FTIR spectrometer. All spectra were recorded at room temperature and signal-averaged over 64 scans at a resolution of 2 cm−1.
14
4
12
2
3
10
1
8 6 4
3. Results and discussion
0.00
0.02
0.04
0.06
0.08
Compressive Extension (mm)
Compressive Modulus (MPa)
b
60 50 40 30 20 10 0
1
2
3
4
Cocoon Type Fig. 7. Out-of-plane compressive properties of cocoons. (a) Curves from mechanical testing; (b) modulus comparison.
were peeled artificially for a length of 5 mm before they were pulled apart by the tester. The layers were numbered according to the sequence from the outer to inner layers (e.g. layer 1 is the outermost layer). Three samples for each type of cocoons were peeled into multiple layers. For the B. mori and A. assamensis cocoons, 9 peel tests were performed for each type; for the A. pernyi and the A. mylitta cocoons, 6 peel tests were performed for each type. The average thickness of peeled layers for each cocoon type is shown in Table 2. The average peeling load for each loading curve was determined as the average of load readings at a 2 mm increment of crosshead motion and the peel strength was determined by the average peeling load divided by sample width. The work of fracture (WOF) was obtained by the work under the peeling load vs. propagation extension curves divided by the delamination area. The maximum values of peel properties obtained from different layers were used for comparison among the domesticated, semidomesticated and wild cocoon types. Compression tests were performed perpendicular to the plane of cocoon wall on an Instron 30 K tester. The outer surface of cocoon walls was the compression side while the other side was the supporting side. A loading rate of 20 N/min was applied to characterise the compressive behaviour of rectangular cocoon samples with the dimension of 10 mm × 10 mm. Three samples were tested for each type of cocoons. A crystal pellet was prepared from 0.04 g of the purchased calcium oxalate crystal powder through compression using the SpectroPress (Chemplex Industries) with a load of 9 tons. The hardness tests were conducted on the inner surface of cocoon walls (pelades) and the crystal pellet using an ultra-micro indentation system (UMIS II). The maximum indentation loads of 45 mN and 1 mN were applied to the cocoon inner surface and the crystal pellet, respectively, through using a spherical indentor (the contact area A, equals to 24.5 ht2, where ht is the
3.1. Cocoon morphology The cocoon structure can be considered as a porous matrix of sericin reinforced by randomly oriented continuous fibroin fibres [19]. The silkworm cocoons showed a non-woven composite structure with twin silk fibres coated with sericin (Fig. 1 and Fig. 2). The wild cocoons (A. pernyi and A. mylitta) have a more compact structure than the domesticated (B. mori) and semi-domesticated (A. assamensis) ones. As shown in Table 1, the nominal density of A. pernyi and A. mylitta cocoons is approximately twice that of the B. mori cocoon and is about 40% higher than the A. assamensis cocoon. For most of the cocoon types, the fibre bonding length is smaller for the cocoon inner surface than the outer surface, although the A. assamensis cocoon shows comparable values between the outer and inner surfaces. The fibre bonding length for the semi-domesticated cocoons and wild cocoons are 100% ~ 300% larger than the domesticated cocoon. As shown in the insets of Fig. 1b, 1c and 1d, cubic crystals can be visualised on the outer surfaces of A. assamensis, A. pernyi and A. mylitta cocoons, loosely deposited on the surface of silk fibres and stacked in the pores. These crystals have been identified as calcium oxalates [2], which show unique functionality such as preferential gating of CO2 from cocoon inside to outside and temperature regulation to maintain a physiological temperature inside the cocoon irrespective of the surrounding environment [4]. In Fig. 3, the FTIR-ATR spectra showed absorption peaks at 1618 cm−1, 1317 cm−1 and 781 cm−1 for the calcium oxalate commercial powder. Similarly, all three peaks are present in the cocoon types of A. assamensis, A. pernyi and A. mylitta. The absence of the peaks at 1317 cm−1 and at 781 cm−1 indicates that the B. mori silk cocoon outer layer does not contain calcium oxalate crystals. Since the peak at 1618 cm−1 is the Amide I peak caused by the silk protein itself [24], all silk cocoons had absorbance peaks at this wave length. 3.2. Interlaminar peel resistance of cocoon layers Fig. 4 shows the loading curves for peel tests on different cocoons. For the B. mori cocoon, the peeling loads were less than 1 N and the load values were comparable for three peeled layers. For the A. assamensis cocoon, the peeling loads were within the range of 0.5 N to 2 N during stable delamination propagation; the inner layer, i.e. layer 3, was easier to peel apart than the outer layers, indicated by the lower peeling loads. In the case for both A. pernyi and A. mylitta cocoons, the peeling curves became more fluctuated with much higher loads (the maximum load values reached 4 ~ 5 N); the outer layers (layer 1) were more difficult to separate. This further indicates that delamination resistance of different B. mori layers was similar from outer to inner cocoon surfaces; however, the delamination resistance of A. pernyi and A. mylitta cocoon layers was stronger in the outer layers. It indicates that the outer layers of these two types of wild cocoons are tougher than the inner layers, which results from the more stringent requirement from the wild to protect the pupa against physical attack from natural predators and harsh surroundings. In
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a Fibre breakage
b
Fibre patches
c Fibre splits
d Fibre rupture
Fig. 8. Fracture surface of cocoons after compression tests perpendicular to the plane. (a) B. mori; (b) A. assamensis; (c) A. pernyi; (d) A. mylitta. The SEM images show the outer surface (left column) and the inner surface (right column) of cocoons.
addition, the wild cocoons showed higher peak loads and larger load drops during delamination, signifying much greater peel resistance and interlaminar fracture toughness over the domesticated cocoon type. The comparison of peel resistance among four cocoon types was made based on the layers where the highest peeling loads occurred (Fig. 5). The maximum peeling load (Pmax) was 0.73 N and the average peeling load (Paverage) was 0.34 N for the B. mori cocoon. By comparison, the A. assamensis cocoon showed Pmax of 1.4 N and Paverage of 1.2 N. Much higher peeling loads were found for the wild cocoons, i.e. a Pmax of 4.4 N and 3.7 N and a Paverage of 2.5 N and 2.4 N for the A. pernyi and A. mylitta cocoons, respectively. The semi-domesticated cocoon A. assamensis showed an approximate 100% increase in both Pmax and Paverage compared with the domestic B. mori cocoon and the wild
cocoons (A. pernyi and A. mylitta) showed further remarkable 200% and 150% enhancement over the semi-domesticated A. assamensis cocoon, respectively. The maximum work of fracture (WOF) of all tested cocoon layers was about 1000 J/m2 from the A. pernyi outer layer, suggesting the highest bonding energy. The WOF and peel strength calculated for the wild cocoons (A. pernyi and A. mylitta) were about 10 times of the domestic cocoon (B. mori) and were 3 ~ 4 times of the semi-domestic cocoon (A. assamensis). As a result, the cocoon structure of wild silkworms is substantially tougher than that of the domesticated silkworms, presumably caused by the need for higher degree of protection against attack from predators and the harsh physical environment. The peel fracture surfaces of different cocoon types are revealed in the SEM images (Fig. 6). Four cocoon types showed different levels of
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4
3
40
1
2 Indentation Load (mN)
Indentation Load (mN)
a
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30 20 10
1.0
5
0.8 0.6 0.4 0.2 0.0 0.0
0.1 0.2 0.3 Indentation depth (µ m)
0 0
10
20
30
40
50
60
Indentation Depth (µm)
3500
70
3000
60
2500
50
2000 1500
40
1000
30
200 150 100 50 0
20 10 0 1
2
3
4
Hardness (MPa)
Maximum Indentation Depth (µm)
b
5
Material Type Fig. 9. Indentation tests performed on the inner surface of cocoons and calcium oxalate crystals. (a) Loading curves; (b) comparison of maximum indentation depth and hardness.
silk fibre damage after the peel tests. As shown in Fig. 6a, slight fibre damage can be observed at the intersections where B. mori silk fibres meet. However, fibre splitting is clearly shown on the fracture surface of A. assamensis cocoon layers (Fig. 6b). The fracture surfaces of A. pernyi and A. mylitta showed more severe fibre damage. The silk fibres of these wild cocoons split into many fibrils indicating the stronger fibre/matrix adhesion and the higher bonding between different layers (Fig. 6c and d). For the A. pernyi cocoon, the calcium oxalate crystals existed at the outer surface only, evidenced by the fracture surface of the first peeled layer shown in Fig. 6c, where no crystals can be seen. Both A. assamensis and A. mylitta cocoons have cubic crystals scattered on the fracture surfaces, however, these crystals do not seem to affect the peel resistance of their host cocoons, since the major failure mode of all cocoon layers is silk fibre damage as noticed in Fig. 6. The stronger matrices, rougher fibre surface of wild cocoons and larger bonding area between layers all make an important contribution to the substantially higher peel resistance. 3.3. Out-of-plane compressive properties Compression tests perpendicular to the plane direction of cocoon walls were performed and the results are summarised in Fig. 7. The compressive deformation was not recorded until the porous cocoon samples were consolidated by compression while the stress reached about 4 MPa. The wild cocoons (A. pernyi and A. mylitta) and semidomesticated cocoon (A. assamensis) were stiffer than the domesticated cocoon (B. mori). It has been proposed that the compressive stress is linked to the density of the porous samples [3,25]. The density of different types of cocoons follows the increasing sequence of B. mori, A. assamensis, A. mylitta and A. pernyi as summarised in Table 1, which is also the sequence for the increase of the compressive moduli. The fracture surfaces on both sides of the cocoon walls were observed and shown in Fig. 8. Despite the severe damage revealed on the outer
surface (the left column in Fig. 8), the inner surface of the cocoon walls remained intact (the right column in Fig. 8). Under compression, the B. mori silk fibres broke or fractured at regions close to the intersections of non-woven network (Fig. 8a). Various changes occur during compression of non-woven fibrous structures, including bending of individual fibres, change in the free distance between two contact points, and fibre-to-fibre slippage [26]. As mentioned previously in Section 3.2, the bonding energy between filaments of B. mori cocoons is much lower than the A. pernyi, A. mylitta and A. assamensis cocoons, therefore the bending of filaments and filament-to-filament slippage play a much more important role in compression for the B. mori cocoon walls compared with other cocoon types. Fig. 8b shows the compression surface of the A. assamensis cocoons: it is interesting to find that the silk fibres from outermost layer were completely compressed into patches and the scattered calcium oxalate crystals were pressed into these patches and filled the cracks and gaps, forming a flattened surface after compression. Abundant crystals therefore shared the compressive loads with the silk filaments at the same plane and protected the inner layers from compressive failure. Fig. 8c and d demonstrates the compressive failure that occurred in the two types of wild cocoons (A. pernyi and A. mylitta) respectively. Fibre splits (A. pernyi) and fibre rupture (A. mylitta) were clearly observed at regions where large amounts of silk fibres interconnected and therefore had higher surface profiles (height). In contrast to the failed filaments, all the crystals observed on the fracture surfaces were intact, which indicates superior mechanical properties to the silk fibres and sericin. 3.4. Nano-indentation on inner surface of cocoons and calcium oxalate crystals It has been noted that the innermost layer of silkworm cocoon (pelade) has superior strength and modulus to the corresponding thickness-averaged values of a complete silkworm cocoon [19]; therefore the indentation measurement on pelades gives a good indication of comparative properties of the inner layers and the cocoon structure. Fig. 9 shows that the pelade of the A. assamensis cocoon has the lowest hardness due to its highest maximum indentation depth (ht) under the same applied maximum indentation load (45 mN). The A. pernyi pelade exhibited a hardness of 125 MPa, which is remarkably higher than the other three cocoon types with hardness lower than 25 MPa. More importantly, the hardness of the calcium oxalate crystals was around 2 GPa and this extremely high hardness explains why these crystals remained intact after compression tests. Despite the unique functions these crystals have in influencing gas diffusion through cocoon walls [3,4], they did not show clear effect on enhancing interlaminar adhesion (e.g. peel resistance) in the outer layers of cocoons. However, more importantly, these extremely hard crystals formed the first defence layer against penetration and other physical attacks from outside the cocoon structure. The cocoons with crystals on the outer layer (A. assamensis, A. pernyi and A. mylitta) showed higher compressive modulus than the one without crystals (B. mori). Similarly, hard coatings with micro- and nano-particles are also able to enhance the Young's modulus of their substrates [27,28]. In nature, the calcium oxalate crystals exist in plants as well, where their presence usually points towards a well-adapted protective function, including mechanical support of soft tissues [29,30]. 4. Conclusions Four types of silk cocoons including domesticated (B. mori), semidomesticated (A. assamensis) and two wild types (A. pernyi and A. mylitta) were investigated in this paper. The wild silkworm cocoons showed superior interlaminar peel resistance, out-of-plane compressive modulus and hardness to the domestic type. The semi-domestic A. assamensis cocoon exhibited medium mechanical
J. Zhang et al. / Materials Science and Engineering C 33 (2013) 3206–3213
performance under interlaminar peeling and out-of-plane compressive loads despite of the low mechanical property of the pelade. Calcium oxalate crystals deposited loosely on the outer surface/layers of the A. assamensis, A. pernyi and A. mylitta cocoons. They were not shown to improve interlaminar fracture toughness but exhibit extremely high hardness, forming the first defensive layer against physical attacks from predators and the environment.
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Ms Jasjeet Kaur is a PhD student working on silkworm cocoon structure–property correlation to study the protective roles of the various cocoon components at the Institute for Frontier Materials, Deakin University.
Acknowledgement The authors would like to acknowledge the funding support from the Australian Research Council (ARC) through a project DP 120100139.
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Dr Jin Zhang is a research fellow in biological composites at the Australian Future Fibres Research and Innovation Centre of Deakin University. Her research interests are focussed on biological composites, multifunctional materials, fibre-reinforced polymer matrix composites, nano-composites and composite manufacture. She obtained her PhD degree from Deakin University, Australia.
Dr Rangam Rajkhowa is a research fellow in green natural fibres at the Australian Future Fibres Research and Innovation Centre at Deakin University Australia. His main research area is processing, characterisation and application of natural fibres. In particular he focusses on structure–property relationships of protein fibres and protein fibre based products. He is also involved with fibre powder production and applications such as silk and wool powders. Rajkhowa obtained a Masters' degree in Fibre Science and Technology from the Indian Institute of Technology Delhi and PhD from Deakin University Australia.
Dr Jingliang Li is working at the Institute for Frontier Materials of Deakin University as a research fellow. His research interests include (1) theoretical understanding of the formation mechanisms of soft functional materials with 3D fiber networks. On the basis of this, to develop novel strategies towards the design of supramolecular architectures with controllable functionalities; (2) natural fibers and related composite materials for advanced applications such as optoelectronics and optical sensing; (3) novel approaches to wool yellowing; (4) novel functional nano-particles for bioapplications such as cancer treatment.
Prof. Xiangyang Liu is the leader of the Biophysics and Micro/Nanostructures Group at National University of Singapore. His background is in biophysics, nanosciences and technologies, crystallisation, bio-functional materials. He has published high profile papers in leading journals including Nature.
Prof. Xungai Wang is a leading researcher in fibre science and technology. He is the 2005 recipient of Fiber Society Distinguished Achievement Award. Professor Wang is a Fellow of the Textile Institute. In recent years, Professor Wang has been investigating new applications for fibres and textiles, and new ways of adding value to fibres and textiles. Professor Wang holds a PhD in fibre science and technology from the University of New South Wales, Sydney.