The Hierarchical Architecture of Chitin: The Insect and Crustacean Structural Building Material

The demand for new, sophisticated multifunctional materials has brought natural structural composites into the focus, since these materials have been optimized in their functions in a long selection and adaptation process during evolution. Biological structural materials differ fundamentally from most man-made structural materials in being inherently structurally heterogeneous.

This heterogeneity arises from a multitude of different constituents already at the molecular level including mainly various organic molecules like proteins or sugars but also inorganic matter, mostly in the form of calcium based minerals. The constituents are produced and/or provided by the organisms which also control the formation of subassemblies. These can have different compositions, shapes, sizes and spatial distribution, leading to the hierarchical organization found in most structural biological materials throughout the flora and fauna, like wood, vertebrate bones and teeth, mollusk shells, arthropod exoskeletons, and skeletal elements of other invertebrates.

The complexity of these materials is further increased by different composition in different positions of the same structure, like the degree of mineralization, fluid content and the resulting variation in the type and density of internal interfaces. This structural heterogeneity implies that the mechanical properties of such materials are also heterogeneous at different length scales. The highest level of hierarchy in biological structural materials can be either the whole organism like for example a tree respectively its smaller subunits like the stem or single branches or, like in the case of animals, a single bone of a vertebrate or an appendage segment of an arthropod. These functional units as a whole have ideal mechanical properties for their individual purposes which are optimized for the occurring loads. The overall mechanical properties of a functional unit rarely reflect the bulk properties of the material ingredients constituting them, but rather depend on their geometry and topological arrangement. In some biological materials like bone or teeth the mechanical properties of the bulk material are well investigated. Already at this macroscopic scale significant variations have been observed between different locations and correlations have been shown to specific loading requirements.

At the macroscopic level, the mechanical response to external loads represents the integral result of the mechanical behavior of the individual composite constituents and their hierarchical structural arrangement on different length scales in the bulk material. This structural heterogeneity and the decreasing size of the constituents at the lower hierarchicy levels is exactly the point where the study of biological structural materials becomes very challenging. It also implies, that the mechanical properties that can be measured in such materials depend strongly on the observed length scale and may vary from one hierarchical level to another. Depending on the structure, mechanical anisotropy also occurs on different hierarchical levels. 

Arthropoda are one of the most successful and diverse groups of organisms and their members like insects, crustaceans and chelicerates have adapted to virtually every habitat on earth.

One common feature of all arthropods is the presence of exoskeletons formed by their outer integument, which is referred to as cuticle, whose basic material is the polysaccharide chitin. In every arthropod species, this cuticle is a functional unit designed and differentiated to perform all the functions required for survival in its specific biological environment. Among other functions like acting as a selective chemical barrier between body and environment, the basic function of the exoskeleton is to provide stability to the body and enable movement through the formation of joints and attachment sites for muscles. In order to grow, arthropods have to shed their old exoskeleton and replace it with a new, larger one frequently in a process termed molt. In most crustaceans, body stability is achieved by the formation of a rigid cuticle in the load bearing parts which is often reinforced by the incorporation of nanoscopic biominerals. 
The American lobster H. americanus is a large sized crustacean. Its body can be divided into the head (cephalon), the thorax and the tail (abdomen). The first three pairs of walking legs which are attached to the thorax end in true pincers. Characteristically, the first pair is enlarged to massive and flattened claws which are referred to as crusher claw and pincher claw according to their biological function. In the rigid parts of the exoskeleton of H. americanus one can distinguish at least five levels of hierarchy ranging from the macroscopic to the nanoscopic scale. Macroscopically, the cuticle consists of three layers forming a functional unit.  A fourth thin membranous layer is present between the three main layers and the epidermal cells during intermolt, the period between two molts. The external epicuticle is thin and waxy and serves as a permeability barrier to the environment. It consists mainly of long chain hydrocarbons, esters of fatty acids, and alcohols and normally contains no chitin or minerals. The mechanically relevant procuticle comprises the exocuticle and the endocuticle. Both layers consist of planes formed by parallel arrays of mineralized chitin-protein fibers. These planes are stacked with the long axis of the fibers in every plane rotating gradually around the normal axis of the cuticle, thereby creating a twisted plywood structure. The stacking height of the twisted plywood is defined by the distance in which the superimposed fiber layers complete a 180° rotation. In the exocuticle the stacking height is considerably smaller than in the endocuticle, most probably caused by a larger rotation angle of the chitin protein fibers. The 50-250 nm thick fibers are composite structures themselves as they are composed of clustered nanofibrils with diameters between 2 and 5 nm and lengths of about 300 nm with various amounts of nanoscopic calcite and amorphous calcium carbonate (ACC) particles located between them. The nanofibrils consist of 18-25 chitin molecules wrapped by proteins. The sugar molecule chitin is the insoluble linear polymer of ß-1,4-linked N-acetylglucosamine residues. Chitin occurs in three different polymorphic forms that differ in the arrangement of the molecular chains in the crystal cell. In -chitin, which is the most abundant crystalline variant, the chains are arranged in an anti-parallel fashion. In β-chitin the chains are parallel. Alpha-chitin is a mixture of gamma- and β-chitin with two parallel chains in one direction and the third one in the opposite direction. The three forms can be found in parts of the same organism. Chitin is the second most abundant natural polymer on earth after cellulose and the basic constituent not only of the crustacean cuticle but of the arthropod exoskeleton in general, including insects, chelicerates and myriapods. It also occurs in mollusk shells, fungal cell walls and various other organisms.The crystalline alpha-chitin typically predominates in the exoskeleton of large crustaceans. Besides the twisted plywood structure a second design principle can be found in the lobster cuticle. Due to a well-developed pore canal system a honeycomb-like structure is generated as numerous canals penetrate the cuticle perpendicular to its surface. Each pore canal contains a long, soft and probably flexible tube which has an elliptical-like cross section with the long axis of the ellipse parallel to the fiber orientation in each plane. Additionally, the pore canals contain chitin-protein fibers oriented perpendicular to those forming the twisted plywood structure. Due to the rotation of the twisted plywood structure the outer shape of each tube resembles a twisted ribbon. Similar pore canals also occur in the cuticle of crabs like Carcinus maenas. The combination of a honeycomb-like structure and a twisted plywood structure with tightly connected lamellae should lead to remarkable mechanical properties and an anisotropic deformation behavior. The unmineralized parts of the lobster cuticle like joint membranes have a similar structural hierarchy, namely, a chitin-protein fiber twisted plywood as basic construction principle, but they lack all mineral components. Additionally the pore canal system is weakly developed and pore canals are rarely detected.