Br-rich tips of calcified crab claws are less hard but more fracture resistant: A comparison of mineralized and heavy-element biological materials

We find that the spoon-like tips of the chelipeds (large claws) of the crab Pachygrapsus crassipes differ from the rest of the claw in that they are not calcified, but instead contain about 1% bromine—thus they represent a new example of a class of structural biological materials that contain heavy elements such as Zn, Mn, Fe, Cu, and Br bound in an organic matrix. X-ray absorption spectroscopy data suggest that the bromine is bound to phenyl rings, possibly in tyrosine. We measure a broad array of mechanical properties of a heavy-element biological material for the first time (abrasion resistance, coefficient of kinetic friction, energy of fracture, hardness, modulus of elasticity and dynamic mechanical properties), and we make a direct comparison with a mineralized tissue. Our results suggest that the greatest advantage of bromine-rich cuticle over calcified cuticle is resistance to fracture (the energy of fracture is about an order of magnitude greater than for calcified cuticle). The greatest advantage relative to unenriched cuticle, represented by ant mandible cuticle, is a factor of about 1.5 greater hardness and modulus of elasticity. The spoon-like tips gain additional fracture resistance from the orientation of the constituent laminae and from the viscoelasticity of the material. We suggest that fracture resistance is of greater importance in smaller organisms, and we speculate that one function of heavy elements in structural biological materials is to reduce molecular resonant frequencies and thereby increase absorption of energy from impacts.     

Design and mechanical properties of insect cuticle

Since nearly all adult insects fly, the cuticle has to provide a very efficient and lightweight skeleton. Information is available about the mechanical properties of cuticle-Young's modulus of resilin is about 1 MPa, of soft cuticles about 1 kPa to 50 MPa, of sclerotised cuticles 1-20 GPa; Vicker's Hardness of sclerotised cuticle ranges between 25 and 80 kgf mm(-2); density is 1-1.3 kg m(-3)-and one of its components, chitin nanofibres, the Young's modulus of which is more than 150 GPa. Experiments based on fracture mechanics have not been performed although the layered structure probably provides some toughening. The structural performance of wings and legs has been measured, but our understanding of the importance of buckling is lacking: it can stiffen the structure (by elastic postbuckling in wings, for example) or be a failure mode. We know nothing of fatigue properties (yet, for instance, the insect wing must undergo millions of cycles, flexing or buckling on each cycle). The remarkable mechanical performance and efficiency of cuticle can be analysed and compared with those of other materials using material property charts and material indices. Presented in this paper are four: Young's modulus-density (stiffness per unit weight), specific Young's modulus-specific strength (elastic hinges, elastic energy storage per unit weight), toughness-Young's modulus (fracture resistance under various loading conditions), and hardness (wear resistance). In conjunction with a structural analysis of cuticle these charts help to understand the relevance of microstructure (fibre orientation effects in tendons, joints and sense organs, for example) and shape (including surface structure) of this fibrous composite for a given function. With modern techniques for analysis of structure and material, and emphasis on nanocomposites and self-assembly, insect cuticle should be the archetype for composites at all levels of scale.     

Worms under Pressure: Bulk Mechanical Properties of C. elegans Are Independent of the Cuticle

The mechanical properties of cells and tissues play a well-known role in physiology and disease. The model organism Caenorhabditis elegans exhibits mechanical properties that are still poorly understood, but are thought to be dominated by its collagen-rich outer cuticle. To our knowledge, we use a novel microfluidic technique to reveal that the worm responds linearly to low applied hydrostatic stress, exhibiting a volumetric compression with a bulk modulus, κ = 140 ± 20 kPa; applying negative pressures leads to volumetric expansion of the worm, with a similar bulk modulus. Surprisingly, however, we find that a variety of collagen mutants and pharmacological perturbations targeting the cuticle do not impact the bulk modulus. Moreover, the worm exhibits dramatic stiffening at higher stresses—behavior that is also independent of the cuticle. The stress-strain curves for all conditions can be scaled onto a master equation, suggesting that C. elegans exhibits a universal elastic response dominated by the mechanics of pressurized internal organs.     

Structure,composition and properties of naturally occurring non-calcified crustacean cuticle

Crustaceans are known for their hard, calcified exoskeleton; however some regions appear different in colour and opacity. These include leg and cheliped tips in the grapsid crab, Metopograpsus frontalis. The chelipeds and leg tips contain only trace levels of calcium but a significant mass of the halogens, chlorine (Cl) and bromine (Br). In contrast, the carapace is heavily calcified and contains only a trace mass of Cl and no Br. In transverse section across the non-calcified tip regions of cheliped and leg the mass percent of halogens varies with position. As such, the exoskeleton of M. frontalis provides a useful model to examine a possible correlation of halogen concentration with the physical properties of hardness (H) and reduced elastic modulus (E_r), within a chitin-based matrix. Previously published work suggests a correlation exists between Cl concentration and hardness in similar tissues that contain a metal (e.g. zinc). However, in M. frontalis H and E_r did not vary significantly across cheliped or leg tip despite differences in halogen concentration. The non-calcified regions of M. frontalis are less hard and less stiff than the carapace but equivalent to values found for insect cuticle lacking metals. Cheliped tips showed a complex morphological layering that differed from leg tips.     

Annotation and expression analysis of cuticular proteins from the tobacco hornworm,Manduca sexta

The insect cuticle is a unique material that covers the exterior of the animal as well as lining the foregut, hindgut, and tracheae. It offers protection from predators and desiccation, defines body shape, and serves as an attachment site for internal organs and muscle. It has demonstrated remarkable variations in hardness, flexibility and elasticity, all the while being light weight, which allows for ease of movement and flight. It is composed primarily of chitin, proteins, catecholamines, and lipids. Proteomic analyses of cuticle from different life stages and species of insects has allowed for a more detailed examination of the protein content and how it relates to cuticle mechanical properties. It is now recognized that several groups of cuticular proteins exist and that they can be classified according to conserved amino acid sequence motifs. We have annotated the genome of the tobacco hornworm, Manduca sexta, for genes that encode putative cuticular proteins that belong to seven different groups: proteins with a Rebers and Riddiford motif (CPR), proteins analogous to peritrophins (CPAP), proteins with a tweedle motif (CPT), proteins with a 44 amino acid motif (CPF), proteins that are CPF-like (CPFL), proteins with an 18 amino acid motif (18 aa), and proteins with two to three copies of a C-X₅-C motif (CPCFC). In total we annotated 248 genes, of which 207 belong to the CPR family, the most for any insect genome annotated to date. Additionally, we discovered new members of the CPAP family and determined that orthologous genes are present in other insects. We established orthology between the M. sexta and Bombyx mori genes and identified duplication events that occurred after separation of the two species. Finally, we utilized 52 RNAseq libraries to ascertain gene expression profiles that revealed commonalities and differences between different tissues and developmental stages.     

Quasi-Static and Dynamic Nanoindentation of Some Selected Biomaterials

This study details an investigation of the viscoelastic behavior of some biomaterials （nacre, cattle horn and beetle cuticle） at lamellar length scales using quasi-static and dynamic nanoindentation techniques in the materials＇ Transverse Direction （TD） and Longitudinal Direction （LD）. Our results show that nacre exhibits high fracture toughness moving towards a larger cam- paniForm as the stress frequency varies from 10 Hz to 200 Hz. Elytra cuticle exhibits the least fracture toughness presenting little energy dissipation in TD. It was initially speculated that the fracture toughness of the subject materials would be directly related to energy-dissipating mechanisms （mechanical hysteresis）, but not the maximum value of the loss tangent tan＆ However, it was found that the materials＇ elastic modulus and hardness are similar in both the TD and LD when assessed using the quasi-static nanoindentation method, but not dynamic nanoindentation. It is believed that the reported results can be useful in the design of new crack arrest and damping materials based on biological counterparts.     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

Cuticular microstructures turn specular black into matt black in a stick insect

The stick insect Peruphasma schultei stands out from other insects by its deep matt black cuticle. We tested whether the appearance of P. schultei is due to microstructures of the cuticle, a phenomenon that has recently been described for the velvet black scales of the Gaboon viper. The shiny black stick insect Anisomorpha paromalus served as a control. We found that the P. schultei cuticle is characterised by two different types of microstructures, tall elevations with a maximum size of 18 μm and small structures with a height of 4 μm. Other than in the snake, P. schultei microstructures do not bear nanostructures. The microstructures scatter light independently of the viewing angle. This causes the matt appearance of the cuticle, whereas pigments are responsible for the black colouration, resulting in a maximum reflectance of 2.8% percent. The microstructures also cause the hydrophobic properties of the cuticle with contact angles near 130°. Resin replicas and bleaching of the cuticle strongly support these results. Moreover, the matt black cuticle has a higher heat absorption compared to the control. We discuss the selective benefit of the matt black appearance of P. schultei in the context of behaviour, ecology and phylogeny.     

Enzymatic activities involved in incorporation of N-acetyldopamine into insect cuticle during sclerotization

During sclerotization of insect cuticle, N-acetyldopamine (NADA) is enzymatically oxidized before reaction with cuticular proteins. Not all oxidized NADA reacts with cuticular structural materials, a small fraction reacts with water or other available low molecular weight compounds to give soluble products. Various types of cuticle were incubated with excess NADA and the products studied by reversed phase high performance liquid chromatography (RP-HPLC) to obtain information on the enzymatic activities in the cuticle. The occurrence of at least two enzymes competing for NADA and present in different proportions in the various types of cuticle can explain the results. NADA may be incorporated into cuticle via α,β-dehydro-NADA (β-sclerotization) or via quinone methides and o-quinones, and the actual course of sclerotization will depend upon the relative activities of the enzymes involved. The various pathways may all be used simultaneously.     

Mechanical properties of calcified exoskeleton from the neotropical millipede, Nyssodesmus python

The calcified exoskeleton of millipedes plays a crucial role in resisting large forces developed during burrowing locomotion. I measured morphological and mechanical properties of cuticle from the neotropical forest floor millipede, Nyssodesmus python (Diplopoda: Polydesmidae), which ranges in body mass from 2 to 7 g. Scaling of thickness of the cuticle with respect to body mass followed predictions of geometric similarity. Both fracture strength and Young's modulus increased with body mass in females but not in males. In spite of their smaller size, male millipedes were still stronger, on average, than female millipedes. Mean fracture strength of millipede cuticle was 124 MPa, and Young's modulus was 17 GPa. Both of these values exceed measurements from typical insect cuticle, suggesting that calcium salts may play a role in stiffening and strengthening the millipede exoskeleton. Because of the high density of calcified millipede cuticle (1660 kg/m³), stiffness and strength relative to body weight remain comparable to values for other insect cuticles. These results corroborate a previous hypothesis that absolute not specific strength and stiffness have been selective factors in the evolution of millipede cuticle, and that bulkiness of the exoskeleton has been minimized through the deposition of calcium salts.     

Micromechanics of smooth adhesive organs in stick insects: pads are mechanically anisotropic and softer towards the adhesive surface

Animals have evolved adhesive structures on their legs to cling to the substrate during locomotion. Here we characterise the ultrastructure and mechanical properties of adhesive pads in Carausius morosus (Phasmatodea) using atomic force microscopy (AFM) as well as transmission and scanning electron microscopy (TEM, SEM). The smooth adhesive arolium has a soft cuticle consisting of principal rods, which branch into finer fibres near the surface. Indentation experiments showed that the pad material consists of distinct layers with different mechanical properties. The 100–300 nm thick outermost layer consisting of the cuticulin envelope and the epicuticle is extremely soft and resilient (mean effective Young’s modulus 12 kPa), while the subjacent procuticle is a much stiffer material (mean effective Young’s modulus 625 kPa). AFM contact mode imaging revealed that the cuticle is mechanically anisotropic, which can be explained by its fibrillar inner structure. We propose that the described layered structure of smooth adhesive pads, consisting of materials decreasing in stiffness towards the outer surface, represents a superior design to conform and adhere to substrates with roughnesses at different length scales. This design principle could be easily implemented in technical adhesives, and thus has a potential to inspire biomimetic applications.     

Novel elasticity measurements reveal C. elegans cuticle stiffens with age and in a long-lived mutant

Changes in biomechanical properties have profound impacts on human health. C. elegans might serve as a model for studying the molecular genetics of mammalian tissue decline. Previously, we found that collagens are required for insulin signaling mutants' long lifespan and that overexpression of specific collagens extends wild-type lifespan. However, whether these effects on lifespan are due to mechanical changes during aging has not yet been established. Here, we have developed two novel methods to study the cuticle: we measure mechanical properties of live animals using osmotic shock, and we directly perform the tensile test on isolated cuticles using microfluidic technology. Using these tools, we find that the cuticle, not the muscle, is responsible for changes in the “stretchiness” of C. elegans, and that cuticle stiffness is highly nonlinear and anisotropic. We also found that collagen mutations alter the integrity of the cuticle by significantly changing the elasticity. In addition, aging stiffens the cuticle under mechanical loads beyond the cuticle's healthy stretched state. Measurements of elasticity showed that long-lived daf-2 mutants were considerably better at preventing progressive mechanical changes with age. These tests of C. elegans biophysical properties suggest that the cuticle is responsible for their resilience.     

Nanomechanical properties of wing membrane layers in the house cricket (Acheta domesticus Linnaeus)

Many insect wings change shape dynamically during the wingbeat cycle, and these deformations have the potential to confer energetic and aerodynamic benefits during flight. Due to the lack of musculature within the wing itself, the changing form of the wing is determined primarily by its passive response to inertial and aerodynamic forces. This response is in part controlled by the wing’s mechanical properties, which vary across the membrane to produce regions of differing stiffness. Previous studies of wing mechanical properties have largely focused on surface or bulk measurements, but this ignores the layered nature of the wing. In our work, we investigated the mechanical properties of the wings of the house cricket (Acheta domesticus) with the aim of determining differences between layers within the wing. Nanoindentation was performed on both the surface and the interior layers of cross-sectioned samples of the wing to measure the Young’s modulus and hardness of the outer- and innermost layers. The results demonstrate that the interior of the wing is stiffer than the surface, and both properties vary across the wing.     

Damage,repair and regeneration in insect cuticle: The story so far,and possibilities for the future

The exoskeleton of an insect can contain countless specializations across an individual, across developmental stages, and across the class Insecta. Hence, the exoskeleton's building material cuticle must perform a vast variety of functions. Cuticle displays a wide range of material properties which are determined by several known factors: the amount and orientation of the chitin fibres, the constituents and degree of cross-linking and hydration of the protein matrix, the relative amounts of exo- and endocuticle, and the shape of the structures themselves. In comparison to other natural materials such as wood and mammal bone, relatively few investigations into the mechanical properties of insect cuticle have been carried out. Of these, very few have focussed on the need for repair and its effectiveness at restoring mechanical stability to the cuticle. Insect body parts are often subject to prolonged repeated cyclic loads when running and flying, as well as more extreme “emergency” behaviours necessary for survival such as jumping, wedging (squeezing through small holes) and righting (when overturned). What effects have these actions on the cuticle itself? How close to the limits of failure does an insect push its body parts? Can an insect recover from minor or major damage to its exoskeleton “bones”? No current research has answered these questions conclusively.     

Soft layered prostheses for arthritic hip joints: a study of materials degradation

A great deal of interest is being shown in compliant bearing surfaces for artificial joints. These produce very low friction because of the fluid-film lubrication that they exhibit, and therefore should produce lower wear than current prosthetic materials as the two surfaces of the joint are completely separated by a film of synovial fluid. However, one problem with soft elastic materials in vivo is that the elastomers may degrade with time. Specimens of four polyurethanes were kept in Ringer's solution at 37°C for about 4 months to investigate changes in mechanical properties and the mode of lubrication with time of immersion. The materials tested were a polyether-urethane (E57), a polyester-urethane (E58), an aromatic polyether-urethane (P1) and an aliphatic polyether-urethane (A1). Samples of E57 showed a decrease in hardness and elastic modulus of 21% and 28% respectively. This was much better than E58 which showed decreases of 32% and 42% respectively. Better results were achieved with A1 (hardness change 2%) and P1 (decrease in hardness of 14% and modulus 17%). When tested in a hip-function simulator, A1 showed virtually no change in its very low coefficient of friction (0.004), P1 showed an increase of 60%, while E57 and E58 both showed a doubling in friction over the course of the study. The modes of in vitro degradation were considered and surface effects determined to be most damaging. Currently the best candidate for a compliant material in hip replacement is the aliphatic polyether-urethane which maintains fluid-film lubrication.     

Variations in the mechanical properties of Alouatta palliata molar enamel

Teeth have provided insights into many topics including primate diet, paleobiology, and evolution, due to the fact that they are largely composed of inorganic materials and may remain intact long after an animal is deceased. Previous studies have reported that the mechanical properties, chemistry, and microstructure of human enamel vary with location. This study uses nanoindentation to map out the mechanical properties of Alouatta palliata molar enamel on an axial cross‐section of an unworn permanent third molar, a worn permanent first molar, and a worn deciduous first molar. Variations were then correlated with changes in microstructure and chemistry using scanning electron microscopy and electron microprobe techniques. The hardness and Young's modulus varied with location throughout the cross‐sections from the occlusal surface to the dentin‐enamel junction (DEJ), from the buccal to lingual sides, and also from one tooth to another. These changes in mechanical properties correlated with changes in the organic content of the tooth, which was shown to increase from ～6% near the occlusal surface to ～20% just before the DEJ. Compared to human enamel, the Alouatta enamel showed similar microstructures, chemical constituents, and magnitudes of mechanical properties, but showed less variation in hardness and Young's modulus, despite the very different diet of this species. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.     

Reliability and Validity of Quantifying Absolute Muscle Hardness Using Ultrasound Elastography

Muscle hardness is a mechanical property that represents transverse muscle stiffness. A quantitative method that uses ultrasound elastography for quantifying absolute human muscle hardness has been previously devised; however, its reliability and validity have not been completely verified. This study aimed to verify the reliability and validity of this quantitative method. The Young’s moduli of seven tissue-mimicking materials (in vitro; Young’s modulus range, 20–80 kPa; increments of 10 kPa) and the human medial gastrocnemius muscle (in vivo) were quantified using ultrasound elastography. On the basis of the strain/Young’s modulus ratio of two reference materials, one hard and one soft (Young’s moduli of 7 and 30 kPa, respectively), the Young’s moduli of the tissue-mimicking materials and medial gastrocnemius muscle were calculated. The intra- and inter-investigator reliability of the method was confirmed on the basis of acceptably low coefficient of variations (≤6.9%) and substantially high intraclass correlation coefficients (≥0.77) obtained from all measurements. The correlation coefficient between the Young’s moduli of the tissue-mimicking materials obtained using a mechanical method and ultrasound elastography was 0.996, which was equivalent to values previously obtained using magnetic resonance elastography. The Young’s moduli of the medial gastrocnemius muscle obtained using ultrasound elastography were within the range of values previously obtained using magnetic resonance elastography. The reliability and validity of the quantitative method for measuring absolute muscle hardness using ultrasound elastography were thus verified.     

A comparison of the cuticular properties of the female ticks <Emphasis Type="Italic">Ixodes pacificus</Emphasis> and <Emphasis Type="Italic">Amblyomma hebraeum</Emphasis> (Acari: Ixodidae) throughout the feeding period

The mechanical properties of the cuticle of Ixodes pacificus (Ip) are compared to those of Amblyomma hebraeum (Ah) from our earlier work. The 10-fold size difference between the species is expected to lead to significant differences in mechanical properties, because cuticular stretch depends on high internal hydrostatic pressure during the rapid phase of engorgement. We demonstrate here: (1) The cuticle of partially fed Ip is less stiff and viscous than that of Ah. (2) A stretch-recoil cycle in both ticks consists of recoverable deformation (ESv) and permanent deformation (ESp); ESp is higher in Ip, and increases sharply during the slow phase of engorgement, but not in Ah. (3) Injected dopamine (DA) increases ESp and reduces all measures of stiffness and viscosity, suggesting that a catecholaminergic neurotransmitter plays a fundamental role in modulating mechanical properties of the cuticle. However, unlike Ah, DA’s effect was not different from that of the control (1.2% NaCl). Mere insertion of the needle may have punctured the gut, causing the release of perhaps a catecholamine that increases ESp, an hypothesis supported by the fact that inserting a needle without any injection also caused an increase in ESp. (4) Stretch reduces ESp, but subjecting loops to pH 6.5 in vitro restores it. (5) Despite the smaller size of Ip, later onset of the rapid phase of engorgement, a thinner cuticle and different mechanical properties all reduce the internal pressure needed for stretch.     

Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle

Protein fractions of insect cuticles with different mechanical properties have related values of polarity and hydrophobicity. Hydrophobicity is important for the self-assembly of cuticle which is produced prior to the moult and in plasticization of cuticle. The cuticles of soft-bodied fly larvae are quite distinct from those of exopterygotes (e.g. locusts) and this can be related to the chemistry and mode of tanning. The properties of cuticular proteins are compared: the proteins of the pliant cuticles most closely resemble globulins, and the proteins in stiff cuticles are more like fibrous and hydrophobic structural proteins. Changes in the environment of the proteins may alter their shape and hence the mechanical properties of the cuticle.