Available online at www.sciencedirect.com Acta Biomaterialia 4 (2008) 587–596 www.elsevier.com/locate/actabiomat Structure and mechanical properties of crab exoskeletons Po-Yu Chen *, Albert Yu-Min Lin, Joanna McKittrick, Marc Andre´ Meyers Department of Mechanical and Aerospace Engineering and Materials Science and Engineering Program, University of California, San Diego, La Jolla, CA 92093-0411, USA Received 15 June 2007; received in revised form 27 November 2007; accepted 18 December 2007 Available online 17 January 2008 Abstract The structure and mechanical properties of the exoskeleton (cuticle) of the sheep crab (Loxorhynchus grandis) were investigated. The crab exoskeleton is a natural composite consisting of highly mineralized chitin–protein fibers arranged in a twisted plywood or Bouligand pattern. There is a high density of pore canal tubules in the direction normal to the surface. These tubules have a dual function: to trans- port ions and nutrition and to stitch the structure together. Tensile tests in the longitudinal and normal to the surface directions were carried out on wet and dry specimens. Samples tested in the longitudinal direction showed a convex shape and no evidence of permanent deformation prior to failure, whereas samples tested in the normal orientation exhibited a concave shape. The results show that the com- posite is anisotropic in mechanical properties. Microindentation was performed to measure the hardness through the thickness. It was found that the exocuticle (outer layer) is two times harder than the endocuticle (inner layer). Fracture surfaces after testing were observed using scanning electron microscopy; the fracture mechanism is discussed. Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Arthropod exoskeletons; Biomineralization; Bouligand structure; Biological composite; Chitin 1. Introduction is further divided into two parts, the exocuticle (outer) and the endocuticle (inner), which have similar composi- Arthropods are the largest animal phylum. They include tion and structure. The endocuticle makes up around 90 the trilobites, chelicerates, myriapods, hexapods, and crus- vol.% of the exoskeleton. The exocuticle is stacked more taceans. All arthropods are covered by an exoskeleton, densely than the endocuticle. The spacing between layers which is periodically shed as the animal grows. The exo- varies from species to species. Generally, the layer spacing skeleton of arthropods consists mainly of chitin. In the case in the endocuticle is about three times larger than that in of crustaceans, there is a high degree of mineralization, typ- the exocuticle [6]. Fig. 1a is a SEM micrograph showing ically calcium carbonate, which gives mechanical rigidity. the epicuticle, exocuticle, and endocuticle. The arthropod exoskeleton is multifunctional: it sup- A striking feature of arthropod exoskeletons is their ports the body, resists mechanical loads, and provides envi- well-defined hierarchical organization, which reveals differ- ronmental protection and resistance to desiccation [1–5]. ent structural levels, as shown in Fig. 1b. At the molecular The outermost region is the epicuticle, a thin, waxy layer level, there are long-chain polysaccharide chitins that form which is the main waterproofing barrier. Beneath the epicu- fibrils, 3 nm in diameter and 300 nm in length. The fibrils ticle is the procuticle, the main structural part, which is pri- are wrapped with proteins and assemble into fibers of marily designed to resist mechanical loads. The procuticle about 60 nm in diameter. These fibers further assemble into bundles. The bundles then arrange themselves parallel to each other and form horizontal planes. These planes are * Corresponding author. Tel.: +1 858 531 4571. stacked in a helicoid fashion, creating a twisted plywood E-mail address: [email protected] (P.-Y. Chen). structure. A stack of layers that have completed a 180° 1742-7061/$ - see front matter Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2007.12.010 588 P.-Y. Chen et al. / Acta Biomaterialia 4 (2008) 587–596 Fig. 1. (a) SEM micrograph of a cross-sectional fracture surface showing three different layers in the exoskeleton: epicuticle, exocuticle, and endocuticle. (b) Hierarchical structure of the exoskeleton of sheep crab, Loxorhynchus grandis. Chitin fibrils (3 nm in diameter) wrapped with proteins form a fiber of 60 nm in diameter. Fibers further assemble into bundles, which form horizontal planes (x–y plane) superposed in a helicoid stacking, creating a twisted plywood structure (180° rotation). In the z-direction there are ribbon-like tubules, 1 lm wide and 0.2 lm thick, running through the pore canals. rotation is referred to as a Bouligand structure. These et al. [19]. Melnick et al. [20] studied the hardness and structures repeat to form the exocuticle and endocuticle toughness of exoskeleton of the Florida stone crab, Meni- [7–11]. The same Bouligand structure is also characteristic ppe mercenaria, which exhibits a dark color (ranging from of collagen networks in compact bone, cellulose fibers in amber to black) on the chelae (tips of the claw) and walk- plant cell walls and other fibrous materials [12]. In crab ing legs. The dark material was much harder and tougher exoskeletons, the minerals are in the form of calcite or than the light-colored material from the same crab chela. amorphous calcium carbonate, deposited within the chi- The most comprehensive study is the one by Raabe and tin–protein matrix [11,13–16]. co-workers on the American lobster, Homarus americanus In the direction normal to the surface (the z-direction), [6,21–25]. as shown in Fig. 1b, there are well-developed, high-density However, the mechanical properties in the direction nor- pore canals containing long, flexible tubules penetrating mal to the surface have not yet been investigated. In this through the exoskeleton. These tubules play an important study, the mechanical properties of the sheep crab exoskel- role in the transport of ions and nutrition during the for- eton in the longitudinal direction (the y-direction) and the mation of the new exoskeleton after the animals molt [17]. z-direction were measured. The exoskeleton is highly aniso- The mechanical properties of crustacean exoskeletons tropic, both in structure and mechanical properties. The (mud crab, Scylla serrata and prawn, Penaeus mondon) motivation for this study is to understand the relationship were first investigated by Hepburn et al. [18] and Joffe between structure and mechanical properties in different P.-Y. Chen et al. / Acta Biomaterialia 4 (2008) 587–596 589 directions. The sheep crab (Loxorhynchun grandis), which machine (LECO M-400-H1, Leco Co., Michigan, USA) resides in California and Baja California, was used for this equipped with a Vickers indenter was used. A load of study. The sheep crab can reach a maximum span of 1 m 0.245 N was applied for 15 s, and a further 45 s was and a mass of about 4 kg [26,27]. The relatively large size allowed to elapse before the diagonals of the indentation and thick exoskeleton (the average thickness in the merus were measured. Twelve indentations were made through (top section) of the walking legs is about 2.5 mm) enable the thickness of exoskeleton at 100 lm intervals and 10 the mechanical tests in the z-direction. Another motivation parallel series of tests were performed on each sample (giv- for this investigation is the establishment of the mecha- ing a total of 120 indentations). Two adjacent indentations nisms by which nature develops strong and tough materials were kept at least 100 lm apart from each other to avoid using relatively weak (chitin, proteins and calcium carbon- the residual stress and strain hardening effect. ate) constituents. A range of hard natural materials has The specimens used for tensile and compressive testing been investigated, such as the intricate tiled structure of in the longitudinal direction were taken from the merus the nacreous component of abalone shells [28–30], the of the first and second pairs of walking legs (Fig. 2). Tensile sandwich structure of toucan beaks [31,32], silk [33] and testing was carried out in the longitudinal direction of the others [2], yielding results that have significant technologi- crab walking legs, whereas tensile testing in the transverse cal applications. direction was not conducted due to the limitation of sample size and curvature (the merus of walking legs is tubular in 2. Experimental methods shape, about 12 cm in length and 2 cm in diameter). After muscle removal, the specimens were dissected into rectan- Three living adult male sheep crab, 15.4 ± 0.8 cm in car- gles approximately 3 cm in length and 1 cm in width. The apace width, were obtained from a local seafood market. rectangles were ground to a thickness of about 1.8 mm The crabs were in intermolt stage, in which the exoskeleton and subsequently inserted into a laser cutting machine; is completely developed and fully mineralized [11,34]. They the dog-bone shape, which was programmed into the were then kept in a 150 gallon tank with constant circula- machine, had a length of 25.4 mm, width of 6.35 mm, gage tion of fresh sea water (mean temperature 16 °C, mean length of 6.35 mm and gage width of 2.29 mm. Thirty-four salinity 33.60 ppt) and fed with fish weekly at the Scripps pieces were cut from three different crabs to prepare two Institution of Oceanography until specimens were required. equal sets of samples, one in a dry condition and one The tank was curtained, limiting the exposure to exterior wet. Seventeen samples were air-dried, while others were lighting, creating a similar ambience to their natural habi- kept in seawater to prevent desiccation during preparation. tat. Sample preparation was done as stated in the NIH However, this could not be done for an indefinite period Guide for Care and Use of Laboratory Animals.