We investigated the ultrastructure of the modern calcitic brachiopods Megerlia truncata (Linnaeus) and Terebratalia transversa (Sowerby) with SEM, electron backscattering diffraction and microhardness indentation. The outer, primary shell layer can be regarded as a nanocrystalline thin film that forms a hard protective coating around the inner, much softer secondary layer that can be expressed as an inorganic/organic fibre composite. The fibrous, curved growth of the secondary shell layer crystals occurs in arbitrary directions perpendicular to the <0 0 0 1> triad symmetry direction of calcite and is most likely obtained by simple confinement to a protein sheath rather than by biomolecular adsorbates blocking growth of any specific crystal face. The curvature of the fibres is caused by rearrangements of the secreting cell array during growth, whereby the existing crystal lattice is not distorted. It serves as a substrate for continued crystal growth. Thus biologically mediated calcite crystallization is a purposeful process and seems to be significantly different to the inorganic crystallization of calcite.

Implants, tissue engineering scaffold materials, drug delivery and bio-micro electromechanical systems (BioMEMs) all use polymer or hydrogel materials. These applications require both mechanical performance and successful integration of the material into a biological environment. Mechanical strength, storage and loss moduli, wear resistance and surface adhesion properties are all critical in biomedical device design and can be determined using nanoindentation. The difficulty of obtaining large samples of specialized materials, and the complexity of testing soft materials in traditional materials testing apparatus, make nanoindentation an attractive alternative. Our previous research using nanoindentation to measure the surface mechanical properties of non-hydrated polymers led to improvement in nanoindentation testing protocols. One of the major challenges in using this technique for hydrogels and tissues is maintaining and controlling hydration of the materials during the test. Here we describe the design of a microfluidic platform for nanoindentation that facilitates continuous hydration of hydrogel samples and high throughput nanomechanical testing. Data from creep experiments on synthetic, hydrated poly-2-hydroxyethyl methacrylate (poly-HEMA) are presented. In addition, we show data to validate the materials properties determined from nanomechanical testing by complementary testing. Finally, the data is fitted to a phenomenological model for viscoelastic materials, specifically the three-element standard linear solid model used by Cheng et al. [1].

Cortical bone is an anisotropic material, and its mechanical properties are determined by its composition as well as its microstructure. Mechanical properties of bone are a consequence of the proportions of, and the interactions between, mineral, collagen and water. Mid-shaft palmar cortical tissue from the equine third metacarpal bone is relatively dense and uniform with low porosity. The mainly primary osteons are aligned to within a few degrees of the long axis of the bone. Beams of compact cortical bone were prepared to examine effects of dehydration and embedding and to study contribution of collagen and mineral to nano-scale material properties. Five beams were tested: untreated (hydrated); 100% ethanol (dehydrated); or embedded in poly-methylmethacrylate (PMMA) for one normal, one decalcified, and one deproteinated bone sample. Elastic modulus was obtained by nanoindentation using spherical indenters, with the loading direction transverse [1] and longitudinal to the bone axis. By selectively removing water, mineral and organic components from the composite, insights into the ultrastructure of the tissue can be gained from the corresponding changes in the experimentally determined elastic moduli.

The mechanical properties of the mussel byssal thread have been investigated via nano-indentation, with the emphasis on the differences between the cuticle and the fibrous interior. The cuticle hardness was found to be 30–40% higher than that of the underlying fibrous interior. In contrast, the Young's moduli in the two regions were virtually identical to one another. Elemental analysis via energy dispersive spectroscopy indicated surprisingly high levels of Al and Br in the cuticle considering the low amounts found in seawater. A potential role of Al in byssal thread mechanics is discussed in light of the unique capability of the cuticle to accommodate strains of 70% by the underlying fibrils in the core without delamination.

We have previously devised a simple method for quantifying the creep resistance of silk fibres – i.e. the ability of silk to maintain dimensional stability while supporting a constant load. We demonstrated (C. Smith et al., J. Arachnol. 31:421–424, 2003) that creep of spider dragline is significant at stresses that are small compared to the conventional yield strength. In addition, the existence of a limiting creep stress was revealed: if samples are loaded smoothly and quickly to a constant stress lying above the limiting creep stress, they break within a few seconds of the stress being applied. The magnitude of the limiting creep stress is equal to approximately one fifth of the fracture stress recorded in conventional constant strain rate tests (in which the stress is applied more gradually).

Here we develop the method and broaden the study of creep to investigate the effects of immersion in water and in ethanol. Experiments are conducted by attaching a small weight (an appropriate number of office staples) to an approximately 20 cm length of dragline from Nephila clavipes spiders, and monitoring extension as a function of time while the samples are suspended vertically in the liquid. The silk is previously conditioned (immersed without imposing any geometrical constraint) in the liquid, to allow it to supercontract or relax if the liquid promotes such a response. Allowance for buoyancy is made when calculating the net force applied to samples by the weight of the staples. Water is found to have a plasticizing effect; it exacerbates the rate of creep in response to a given small load, and it decreases the limiting creep stress. Ethanol, a desiccant, delays the transition from creep dominated by changes in chain conformation to creep dominated by chain slip, and increases the limiting creep stress. These results draw attention to an important limitation that must be overcome if biomimetic silk is to be used in applications where dimensional stability is required while loads are being supported for long times, especially in a wet environment. The results also point to the molecular origins of the creep sensitivity, and thus to ways of making silk and silk analogues less susceptible to creep.

Based on ‘universal’ mechanical building blocks inherent to all different bones, this paper shows the integration of two classically separated fields of bone biomechanics, namely bone micromechanics[1] on the one hand, and bone poromechanics[2] on the other. Indeed, despite the complex hierarchical organization of bone, it was recently possible to identify such elementary components at the micro and nanolevel of the material for the explanation of the diversity of macroscopic (poro-)elastic properties of different bones. The mechanical properties (i.e. elasticity) of these elementary components are (within limits of experimental scatter) the same for all bones; they are ‘universal’, i.e. independent of tissue type, species, and anatomical location. The mechanical interaction between these elementary components (mechanical morphology) and the dosages of these components in different tissues determine the macroscopic material properties.

Continuum micromechanics has turned out as well-suited theoretical tool to derive tissue-dependent elastic properties at different length scales from upscaling the elasticity of ‘universal’ building blocks from the nanometer scale. Once respective micromechanics models are developed and validated, poro-micromechanics allows for the quantification of poroelastic properties such as the Biot and Skempton coefficients, as functions of the volume fractions of mineral, collagen, and the micropore space (Haversian and Volkmann canals, and the inter-trabecular space). Skempton coefficients, related to undrained conditions, allow for quantification of the marrow pressure rise due to impact loading, as can be shown by model predictions of non-destructive impact experiments. Moreover, the aforementioned poroelastic properties enter the governing equations for wave propagation in anisotropic porous media. They allow for the prediction of undrained, fast and slow waves, as is verified by comparison of model results with experimental findings.

Several mechanical models, in which a material is treated as a composite of crystalline and amorphous and/or interphase material, were used to predict the tensile modulus of spider dragline along the fiber direction. The models included the Voigt average (which assumes that the fibers/crystals are continuous, and that the strain is the same in all components of the composite); a modified Halpin-Tsai model (which is suitable for predicting the longitudinal elastic modulus for short aligned fiber composites, and is thus more appropriate for silk); and the shear-lag or Cox model (which is a modification of the Voigt average that takes into account a discontinuous nature of stiff fibers/crystals and the resulting shear stress in the amorphous matrix). The latter two models yielded close approximations of an experimentally measured elastic modulus of Latrodectus hesperus (black widow spider) dragline under conditions of controlled temperature and humidity, given realistic inputs for the moduli of the individual components and the percent crystallinity. A literature model for the stress-strain behavior of silk [1] was also considered, in the context of our experimental results from transmission electron microscopy (TEM) and X-ray diffraction (XRD) studies of L. hesperus dragline. TEM and XRD results indicated a bimodal size distribution of ordered regions; one population of crystals has a mean size of 2 nm, and another spans the size range 40–120 nm. The average elastic modulus measured from L. hesperus dragline is 23 GPa - close to the 25 GPa theoretical modulus for the case of large crystals in Termonia's model. The tensile strength of L. hesperus dragline is ca. 1.7 GPa, close to the case predicted for small crystals in Termonia's model. A combination of the small and large crystals could explain the forced elongation behavior of L. hesperus dragline.

This study uses various characterisation techniques on the razor shell (Ensis siliqua), to relate the shell's microstructure to its mechanical properties. Scanning electron microscopy (SEM) has shown that the outer and inner regions of the shell are composed of simple and complex crossed lamellar microstructures respectively. These layers are interspersed by prismatic layers of a completely different crystallographic orientation. Nanoindentation and microhardness measurements have shown that the structure is anisotropic, and Raman band shifts have been observed within these indented/deformed areas of shell, showing that the microstructure deforms rather than generating surface damage. The use of energy variable synchrotron X-ray diffraction has shown that the calcium carbonate crystals of the shell are preferentially orientated as a function of depth and that opposing residual stresses exist at the outer and inner regions of the shell. This study has analysed several microstructural features of the shell and provided an insight into how they prevent failure of the material.

The risk of dental caries increases with the use of orthodontic appliances, and it does not only depend on a patient's oral hygiene. Caries cause teeth enamel demineralization close to the orthodontic bracket. The hardness of the teeth consequently decreases, and they become brittle and loose. The type of adhesive used to fix brackets may or may not reduce enamel demineralization. Previous studies have shown that a resin-modified glass ionomer (RMGI) inhibits demineralization. The purpose of this work is to evaluate the effect of a fluoride-releasing bracket on mechanical properties of dentin and enamel on regions adjacent to orthodontic brackets bonded with RMGI and composite resin cements. The remineralization effect due to fluoride on teeth was assessed by measuring hardness and reduced elastic modulus on dentin, enamel and dentin-enamel junction (DEJ) around the bracket area by nanoindentation methods. Nanoindentation was performed using a TriboScope from Hysitron. For this purpose two sets of polished samples were prepared: one sample was a cut along a longitudinal section and the other one along a cross-section of a tooth with orthodontic treatment using RMGI. Anisotropy in the mechanical properties of the teeth is observed along each direction.

Bone is a composite material with a mineral hydroxyapatite (HA) phase and an organic collagen-based phase. Each phase represents about half the material by volume. The precise arrangement of these components at the ultrastructural level is unclear but of great interest in understanding the mechanical functionality of bone. Nanoindentation tests show that the elastic modulus of bone is primarily distributed between 10 GPa and 30 GPa. In this study we examine different ultrastructural arrangements of collagen and apatite phases, to test different proposed models for bone composite ultrastructure within the same finite element modeling framework. Different configurations of the composite are considered, including (a) a compliant phase with stiff reinforcing particles, (b) a stiff phase with compliant reinforcing particles, and (c) an interpenetrating two-phase (co-continuous) composite. An elastic modulus of 100 GPa is used for the mineral phase and 100 MPa for the organic phase, with volume fraction of each phase fixed at 0.5. Stiff phase continuity (as the only continuous phase in 2D and 3D or as one of two continuous phases in 3D) gives rise to effective composite elastic modulus values of 25–35 GPa, similar to the experimental results for bone modulus. Isotropic models with compliant phase continuity only give rise to moduli around 300 MPa, far below experimental results. Anisotropy was evaluated by calculating effective moduli in parallel and transverse directions relative to the primary axes of rectangular particles. High aspect ratio, stiff particles embedded in a compliant matrix do result in a substantially stiffened composite in the direction of the particles when compared to symmetric particles. However, this configuration results in a material with an effective elastic modulus of 2 GPa along the particle direction but a transverse modulus of only 250 MPa. Decreased interparticle spacing in the direction of loading was the mechanism for stiffening parallel to the particle long axis, demonstrating an indirect effect of particle aspect ratio. Although many bone models have considered the mineral as a particle reinforcement phase, the current results suggest this arrangement would not give rise to a material with bonelike properties, particularly when transverse modulus is considered, regardless of the particle geometry. Some degree of continuity of the mineral phase is required for bone-like elastic modulus values and thus a partially to fully co-continuous ultrastructural arrangement of phases is supported.

Salivary pellicle is an organic biofilm formed by the physisorption of proteins and carbohydrates onto the surface of dental enamel exposed to the oral environment. The pellicle has several key roles in oral physiology including lubrication and reduction of friction between teeth during mastication, as well as chemical protection of the enamel against acidic solutions. However, pellicle proteins are known to react with dietary compounds to cause extrinsic staining on the tooth surface.

In this study, nanoindentation and AFM have been used in vitro to examine the acquired salivary pellicle formed in vivo on dental enamel. The mechanical properties, growth, structure and morphology of pellicle grown in vivo on human enamel surfaces have been analysed. In addition, the effects of dietary agents such as polyphenols on the pellicle's morphology and properties have been studied.

It was found that initial adsorption of proteins on the enamel surface occurred within 30 seconds of exposure to the oral cavity, with full growth achieved within 2 hours. Differences in the properties of the pellicles such as surface adhesion, and time dependent effects due to polyphenol interaction were measured using nanoindentation. It was seen that the polyphenol interaction has a significant effect on these properties. These results suggest that the stained pellicle is mechanically stiffer, but also less viscous and more fluid like. This could explain why traditional tooth brushing techniques do not efficiently remove this layer.

Studies have been carried out on the structures and mechanical characteristics of an unusual family of sea sponges under the classification of Hexactinellida, genus Euplectella. The sponge spicules have been of interest to materials scientists because of their potentially important optical, coupled with mechanical, properties. The structures of the class Hexactinellida are characterized by a concentric ring appearance in the cross-section, which is a composite of hydrated silica, coupled with silicatein as a thin layer at the ring interfaces. The mechanical behavior and the toughness of the spicules have been examined with the aid of a special fiber testing method, coupled with scanning electrom microscopy (SEM) observations. It appears that there may be common mechanisms underlying toughness in rigid natural composites with high ratios of mineral/organic phase. In addition, novel pressurization tests of a portion of the sponge skeleton have provided information about the resilience of the skeleton, which resembles a selfsupporting glass winding of a cylindrical composite structure.

We recently developed an integrated imaging platform that combines single molecule evanescent wave fluorescence imaging (and spectroscopy) with in situ scanning probe microscopy. The advantages, challenges, and potential represented by this coupled tool will be described in the context of the structure-function characteristics of nanostructured biomaterials and thin lipid films.

A biomimetic process involving in situ mineralization of hydroxyapatite (HAP) is used to design new composite biomaterials for bone tissue engineering. Surface and bulk properties of HAP composites have been studied for hydroxyapatite mineralized in absence (ex situ) of polyacrylic acid (PAAc) and in presence (in situ) of PAAc. XRD studies show existence of structural disorder within in situ HAP. It has been observed that PAAc increases the rate of crystallization. FTIR studies indicate calcium deficiency in structure of both in situ and ex situ HAP. PAAc provides favorable sites for nucleation of HAP. During crystallization of HAP, PAAc dissociates to form carboxylate ion, which binds to HAP. Porous and solid composites of in situ and ex situ HAP with polycaprolactone (PCL) in 50:50 ratio have been made to evaluate their applicability as bone scaffold. Mechanical tests on solid samples indicate ex situ HAP/PCL composites have higher elastic modulus (1.16 GPa) than in situ HAP/PCL composites (0.82 GPa). However, in case of porous composites, in situ HAP/PCL composites are found to have higher elastic modulus (29.5 MPa) than ex situ HAP/PCL composites (10.4 MPa). Nanoindentation tests were also performed at different loads to evaluate mechanical properties of the composites. In situ HAP mineralized using non-degradable polymers has thus been shown to improve mechanical response in porous composites.

The Center for Commercial Applications of Combustion in Space (CCACS) at the Colorado School of Mines is currently using combustion synthesis to produce several advanced materials. These materials include ceramic, intermetallic, and metal-matrix composites in both porous and dense form. Currently, NiTi – TiC intermetallic ceramic composites are under investigation for use as a bone replacement material. The NiTi intermetallic has the potential to provide a surface that is capable of readily producing an oxide layer for corrosion resistance. The TiC ceramic has the potential to increase the hardness and wear resistance of the bulk material that can improve the performance lifetime of the implant. Processing parameters are critical to the production of the NiTi – TiC composite and will be discussed. These parameters can lead to the formation of substoichiometric TiC and nickel rich NiTi that changes the overall mechanical and material properties. In addition, the size of the TiC particles present within the bulk product varies with porosity. Both porous and dense samples have been mechanically analyzed employing micro-indentation techniques as well as compression tests in an attempt to characterize the mechanical response of these composites. The effects of the TiC particles, the formation of Ni3Ti intermetallic and the effects of porosity on the overall mechanical and material properties will be discussed.

The fundamental problem of micromechanics is the prediction of overall response of a composite material given the properties or response of the individual constituents and their internal geometric arrangement. The recently developed High-Fidelity Generalized Method of Cells is a promising micromechanics model whose predictive capability has been demonstrated for infinitesimal deformations in the presence of inelastic constituent behavior. The extension of this micromechanics model to the finite-deformation regime and incorporation of the quasi-linear viscoelasticity theory for the constituent response extends the range of this model's applicability to the bio-engineering area. Herein, an application involving the response of mitral valve chordae tendineae is presented that demonstrates the model's capability to mimic experimentally-observed response of this class of biological tissues rooted in their characteristic microstructures.

Functional tissue engineering establishes functional criteria for design and manufacture of the scaffold matrix for repair and replacement. It also provides useful and strategic information in mechanical stimulation of the cells seeded in the matrix before and after surgical placement to enhance the success of tissue engineering. Biomechanics plays an important role in accomplishing these requirements by assessing the in vivo environment and the material properties.

A nanocomposite of silkworm silk and single wall carbon nanotubes (SWNT) was produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun and the morphological, chemical and mechanical properties of the electrospun nanofibers were examined. The mechanical properties of the SWNT reinforced fiber show increases in Young's modulus up to 460 % in comparison with the un-reinforced aligned fiber.

Osteopontin (OPN), a phosphorylated glycoprotein, is among the most abundant non-collageneous bone matrix proteins produced by osteoblasts and osteoclasts. OPN has been implicated in bone formation, resorption and remodeling. However, previous studies have presented contradictory results regarding the effect of OPN on the mechanics and microstructure of bone. This study has used nanoindentation to identify local variations in elastic modulus and hardness of OPN deficient (OPN -/-) and wild-type control (OPN+/+) mouse bones. Specifically, the study has looked at changes in the mechanical properties of OPN-/- and OPN+/+ mouse bones with the mouse's age. Cortical sections of femurs from different age groups ranging from 3 weeks to 58 weeks were tested and compared. The results suggest that there are large, abrupt variations in mechanical properties across the femur's radial section for 3-week-old mouse bone. The hardness (H) drops significantly towards the inner and outer sections so the cortical bone has a mean H=3.66 GPa with a standard deviation of 2.44 GPa. In contrast, the hardness of the 58-week-old mouse bone had a standard deviation of 0.35 GPa and a mean H=1.45 GPa. The hardness across the radial axis of the 58-week-old bone was found to be quite uniform. The elastic modulus showed similar variations to the hardness with respect to age and position on the bone. We conclude that the mechanical properties of the mouse bones decrease substantially with maturity, and statistically the hardness and elastic modulus are more uniform in mature bones than young ones. Surprisingly we found a similar variation in both OPN-/- and OPN+/+ bones, with no statistically significant difference in the mechanical properties of the OPN -/- and OPN+/+ bones. The results for OPN-/- and OPN+/+ mouse bones are particularly important as control of OPN activity has been postulated as a potential treatment for bone pathologies that exhibit a change in the bone mineralization, such as osteoporosis, osteopetrosis and Paget's disease. Understanding the effects of OPN on bone mechanics is a vital step in the development of these new treatments.

Nacre, also known as mother-of-pearl, is the iridescent layer found inside some mollusk species such as oyster or abalone. It is made of relatively weak materials, but its hierarchical microstructure is so well optimized that its macroscopic mechanical properties are far superior to those of its constituents. For this reason there is a great interest in nacre as a source of inspiration for novel designs of composites. Despite many years of research on nacre, an accurate characterization of its constituents is lacking. In this work nacre was tested as a layered composite material using low depth indentation and uniaxial compression. The first test was modeled using finite element analysis and the second test was modeled as a Reuss composite in compression. A micromechanical model of the interface was also pursued to gain insight on the relevance of the interface features such as tablet roughness and biopolymer hydrated response. The results of the two experiments were combined to solve an inverse problem that yielded the needed properties for tablets and interfaces. These findings are expected to make possible computational models of nacre with a new degree of accuracy and therefore contribute to a better understanding of the mechanisms leading to its remarkable properties.