In-situ Ramanspectra were collected on the N. clavipes spider dragline silk under a tensile deformation rate of 15mm/min. The most prominent features on the spectra were due to those bands near 1100 cm-1, which present as a sensitive probe to structural changes associated with side-chains of silk peptide. A downshift of Raman bands at 1095 cm-1 and 1089 cm-1 was detected with increasing strain. Furthermore, an increase in the intensity of the Raman band at 1062 cm-1 due to the vibration of trans structure without lateral coupling was prominent at certain strain levels. This was interpreted in terms of a morphology transition from the random configuration to the trans conformation modulated by the reorganization of the hydrogen bonding among the side-chain.

During normal wound healing, a fibrin clot is formed within the first few minutes and is replaced, over several days, by collagen and other extracellular matrix components which are populated with fibroblasts [Clark, 1996]. This process leads to the rebuilding of dermal tissue on which the epidermal tissue is slowly rebuilt. In severe acute and chronic wounds dermal replacement materials that mimic these processes are needed. Current trends are to use scaffolds as structural substitutes as well as carriers for growth factors and cells for wound treatment. Fibrin-based sealants have been used over the last 30 years in hemostasis and tissue sealing applications and in the last 5 years as a scaffold. Fibrin sealant consists of two primary components, fibrinogen and thrombin which form a fibrin clot when mixed. However, a basic understanding of the microstructure/property relationships of fibrin/collagen constructs is not yet fully established. Furthermore, while previous studies showed that cells proliferate and differentiate within 3D fibrin clots, their effect on structural mechanics of the fibrin clot has not been examined.

Here, an indentation protocol was established to determine the effects of fibrin/collagen biochemistry and time-dependent cellular response on the elastic parameters of the constructs. Specifically, 4 ml fibrin/collagen constructs were prepared with varying compositions of human-derived fibrinogen (5 - 33 mg/ml), thrombin (1 or 2 U/ml) and bovine collagen (0, 25, 50, 75, 100 weight % of 2.4 mg/ml collagen). Constructs were prepared with/without the presence of human foreskin fibroblasts (ATCC NIH3T3) seeded at a density of 100K cells/ml. Using a 3-mm diameter punch indenter, the indentation load-displacement response was measured after 1, 5 and 10 days of incubation at 37°C in a humidified air/5% CO2 atmosphere. Four replicates per experimental condition were prepared, and three indentations per replicate were performed.

For the unpopulated fibrin, there was a linear (R=0.983) relationship between the indentation stiffness and fibrinogen concentration. Also, there was a nonlinear relationship between indentation stiffness and concentration of collagen in the fibrin/collagen constructs. Finally, although in most cases there was no measurable change in the stiffness of the cell-populated tissue constructs with incubation time, in one of the cell-populated formulations, however, the indentation stiffness decreased steadily with increasing incubation time. These results are analyzed within the context of existing analytical micromechanics models for the relationships between scaffold structure, e.g., porosity / fibril diameter, and stiffness.

Layered manufacturing (LM) using stereolithography (SL) of aqueous polymer solutions was accomplished so three-dimensional (3D) tissue engineered scaffolds with complex distributions of bioactive agents could be produced. Successful LM with embedded channel architectures required investigation of hydrogel thickness or cure depth as a function of photoinitiator type and concentration, energy dosage, and polymer concentration in solution. Poly(ethylene glycol) dimethacrylate (PEG-dma) with an average molecular weight of 1000 in quantities of 20% and 30% (w/v) was prepared in distilled water. Different concentrations of two photoinitiators (PIs), Sarcure1121 (2-hydroxy-2-methyl-1-phenyl-1-propanone) and Irgacure 2959 (2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone), were used to vary gel thickness at select energy dosages by controlling the scan speed of the SL machine's ultraviolet scanning system. Gel thickness was a strong function of PI type and concentration, energy dosage, and PEG-dma concentration, especially at the low PI concentrations required for implantation. The gel thickness curves were utilized to demonstrate LM for two construct geometries where different layer thicknesses were required to successfully fabricate the constructs. This work demonstrates the effective use of SL as a processing technique for complex 3D tissue scaffolds and addresses some practical considerations associated with the use of hydrogels in LM.

The material bone consists of a biopolymer matrix (collagen) reinforced with mineral nanoparticles (carbonated hydroxylapatite), forming a natural composite which builds up a dense shell on the exterior and a network of struts with a mean diameter of 200μm in the core of many bones. The architecture of the foamy inner part of bones (spongiosa) is determined by loading conditions. The architecture strongly influences the mechanical properties of cellular solids together with the apparent density and the material it consists of. In addition, the ingrowth of bone cells into porous implants depends on pore size, size distribution and interconnectivity. From this it is clear that the possibility to design the architecture of a bone replacement material is beneficial from a biological as well as a mechanical point of view. Our approach uses rapid prototyping methods, ceramic gelcasting and sintering to produce cellular structures with designed architecture from hydroxylapatite and other bioceramics.

The influence of sintering temperature and atmosphere on the physical properties of these scaffolds was investigated with x-ray diffraction and scanning electron microscopy. Furthermore, the cell ingrowth behaviour was determined in cell culture experiments, using the praeosteoblastic cell line MC3T3-E1, derived from mouse calvariae. The cell ingrowth behaviour was evaluated during a culture period of two and three weeks, by light microscopy and afterwards by histology after embedding and Giemsa-staining.

The phase composition of the material was found to change with increasing sintering temperature and its surface characteristics was influenced by the sintering atmosphere. These changes also affected the cell ingrowth behaviour. In some experiments, the osteoblasts-like cells were found to cover the whole external and internal surface of the scaffold. The cells produced extracellular matrix consisting of collagen, which eventually filled nearly all the pores. In particular, the cells had the tendency to fill any crack or opening in the scaffolds, and to generally smooth the surfaces.

In conclusion, rapid prototyping and ceramic gelcasting allows the freeform fabrication of porous bioceramics with controlled architecture. Such structures made of hydroxylapatit were found to support the growth of mouse osteoblasts.

The effective elastic modulus of composite materials results from a combination of elastic moduli of the component phases. Recent efforts to understand the mechanical behavior of calcified tissues in bones and teeth require estimates of the component phase properties, which are difficult to establish independently. A three-phase system, based on naturally occurring bone, is therefore examined by a combined nanoindentation and finite element modelling approach to better understand the proportions and properties of the component phases. Bone samples were prepared in four two- or three-phase composite configurations as follows: (1) as a dehydrated mineral-protein composite (with some void space); (2) similarly dehydrated mineral-protein composite but with polymethylmethacrylate (PMMA) resin filling the voids resulting in three solid phases; (3) as a PMMA-mineral composite following protein removal and replacement with PMMA, and (4) as a PMMA-protein composite following mineral removal and replacement with PMMA. Effective component volume fractions and elastic moduli for each phase in each system were computed based on the composite nanoindentation results. Finite element models of the two- and three-phase systems were constructed to explore the structural anisotropy of the composite systems, as demonstrated in the nanoindentation tests, and to examine the sensitivity of the composite results to changes in the assumed component properties.

Cancellous bone plays an important load-bearing role in the skeleton, yet relatively little is known about the microstructure-mechanical property relationships of the tissue at the sub-10 [.proportional]m level. Cancellous tissue is characterized by a layered microstructure with variable proportions of collagen and mineral. The lamellar material is substantially stiffer than the interlamellar material at the nanomechanical level. However, the microstructural origin of the observed differences in mechanical properties of these structures has not been investigated. In this study, second harmonic generation microscopy was used to examine collagen in human vertebral cancellous bone. At the same location in the tissue, nanoindentation was used to assess the indentation modulus of lamellar and interlamellar bone. The stiff lamellae corresponded to areas of highly ordered, collagen-rich material, while the compliant interlamellar regions corresponded to areas of unoriented or collagen-poor material. The lamellar bone was approximately 30% stiffer and contained approximately 50% more oriented collagen than the interlamellar bone. These observed differences in the mechanical properties and collagen content and organization of lamellar and interlamellar tissue are consistent with previous scanning electron microscopy studies showing greater mineral and collagen content and organization in lamellar bone. Given the well-known coupling between collagen and mineral in bone tissue, the mineral distribution may mirror that of the aligned collagen. However, similar measurements of local variations in mineral content are needed to confirm this hypothesis and may provide additional insights into the tissue nanomechanical behavior.

Bone is a composite material composed of collagen, carbonated apatite mineral, water, and other non-collagenous proteins. The bone structure inside human body is under constant remodelling. The mechanical properties of bone and their dynamic changes during remodelling are crucial to the health and quality of life.

In this study, the elastic and viscoelastic properties of a 73 year-old female cortical bone were investigated at the lamellar level. This was realized by a nanoindentation technique equipped with dynamic loading function. 325 indentations were made in individual Haversian systems and interstitial bone at both dry and wet condition, and under two different loading frequencies. The results showed no statistically significant differences in elastic modulus between Haversian systems and interstitial bone. There were no systematic differences in modulus across the cortex except for a slight drop at the periosteal site. The lamellar structure of both Haversian system and interstitial bone is viscoelastic with water playing a significant role to the properties. When dry bone is re-hydrated, elastic modulus decreases and loss tangent increases.

To understand the inelastic response of bone, a two-part investigation has been conducted. In the first, a flexural test protocol has been designed and implemented that monitors the axial and transverse strains on both the tensile and compressive surfaces of cortical bone. The results are used to assess the relative contributions of dilatation and shear to the inelastic deformation. Unload/reload tests have characterized the hysteresis and provided insight about the mechanisms causing the strain. These tests reveal strain healing attributed to sacrificial bonds. The second part devises a model for the stress/strain response, based on a recent assessment of the nano-scale organization of the collagen fibrils and mineral platelets. The model rationalizes the inelastic deformation in tension, as well as the permanent strain and hysteresis.

Mice are commonly considered the model mammal for many biomedical studies. In this work, mouse and human dentin were compared to specify structural and mechanical differences to establish a baseline for comparison of dental tissues between these species. Atomic force microscopy revealed tubules of about 1.0 to 1.6 μm in diameter as the main structural feature in dentin of both species. Nanoindentation yielded the elastic modulus about 15% lower in murine intertubular dentin while the hardness was almost equal. Dynamic stiffness mapping confirmed the lower elastic properties and also revealed that the peritubular region of increased mineralization around tubules is drastically reduced or maybe absent in murine dentin of this age. This study suggests that structural and mechanical differences need to be considered when murine dentin is used as a model system.

Many proteins have been experimentally observed to exhibit a force-extension behavior with a characteristic repeating pattern of a nonlinear rise in force with imposed displacement to a peak, followed by a significant force drop upon reaching the peak (a “saw-tooth” profile) due to successive unfolding of modules during extension. This behavior is speculated to play a governing role in biological and mechanical functions of natural materials and biological networks composed of assemblies of such protein molecules. In this paper, a constitutive model for the finite deformation stress-strain behavior of crosslinked networks of modular macromolecules is developed. The force-extension behavior of the individual modular macromolecule is represented using the Freely Jointed Chain (FJC) statistical mechanics model together with a two-state theory to capture unfolding. The single molecule behavior is then incorporated into a formal continuum mechanics framework to construct a constitutive model. Simulations illustrate a relatively smooth “yield”-like stress-strain behavior of these materials due to activate unfolding in these microstructures.

We present experiments on the mechanical and structural gradients through the cuticle of homarus americanus (lobster). The exocuticle (outer layer) is characterized by a very fine woven structure of the chitin-protein matrix (Bouligand structure) and by a high stiffness (8.5–9.5 GPa). The hardness increases within the exocuticle between the surface region (130 MPa) and the region close to the interface to the endocuticle (270 MPa). In the endocuticle which is characterized by a much coarser twisted plywood (Bouligand) structure both, the stiffness (3–4.5 GPa) and hardness (30–55 MPa) are much smaller than in the exocuticle. The transition in mechanical properties and structure between the exo- and endocuticle is abrupt.

Bone is a hierarchically structured mineral-organic composite material that has to bear static and dynamic mechanical loads applied by body weight and locomotion. Bone mechanical properties are influenced by a number of factors, depending on the particular hierarchical levels. The high stiffness of bone material is mainly achieved by reinforcement with calcium phosphate mineral platelets. A model for this elementary structure level consists of an arrangement of staggered mineral bricks, embedded in collagen matrix, which provides both stiffness and toughness. The mechanical properties depend on the amount, shape and arrangement of the mineral particles but also on the properties of the collagen-rich matrix.

One of the difficulties in assessing the properties of hierarchical structures is the inherent inhomogeneity of the tissue: Parameters such as stiffness or calcium content vary throughout a bone section. Such type of investigations becomes more meaningful by a combination of two complementary methods e.g. quantitative backscattered electron imaging (qBEI) and nanoindentation. The local Ca-content, representing the degree of the mineralization, is extracted from the qBEI measurements, whereas the local mechanical properties, elastic modulus and hardness, are measured by nanoindentation, a miniaturized hardness testing using a small diamond tip. The measured correlations between local Ca-content and mechanical strength help, for example, to verify biomechanical models based on the nanocomposite structure of bone. They may also shed new light on bone diseases such as osteoporosis or osteogenesis imperfecta.

Strain stiffening of protein networks is explored by means of a finite strain analysis of a two-dimensional network model of cross-linked semiflexible filaments. The results show that stiffening is caused by non-affine network rearrangements that govern a transition from a bending dominated response at small strains to a stretching dominated response at large strains. Thermally-induced filament undulations only have a minor effect; they merely postpone the transition.

In order to facilitate locomotion and limb movement many animals store energy elastically in their tendons. The formation of crosslinked collagen fibers in tendons results in the conversion of weak, liquid-like embryonic tissues into tough elastic solids that can store energy and perform work. Collagen fibers in the form of fascicles are the major structural units found in tendons. The purpose of this paper is to review the literature on collagen self-assembly and tendon development and to relate this information to the development of elastic energy storage in nonmineralizing and mineralizing tendons. Of particular interest is the mechanism by which energy is stored in tendons during locomotion. In the turkey, much of the force generated by the gastrocnemius muscle is stored as elastic energy during tendon deformation and not within the muscle. As limbs move, the tendons are strained, causing the collagen fibers in the extracellular matrices to stretch. Through the analysis of turkey tendons, collagen fibers, and a molecular model, it is hypothesized that elastic energy is stored in the flexible regions of the collagen molecule. Data from the molecular model, mineralized fibers, and turkey tendons show that the presence of calcium and phosphate ions causes an increase in elastic energy stored per unit strain. Based on the theoretical modeling studies, the increase in stress with strain is a result of the initiation of stretching of the rigid regions of collagen molecules.

High resolution atomic force microscopy (AFM) images of bovine trabecular bone fracture surfaces reveal individual fibrils coated with extrafibrillar mineral particles. The mineral particles are distinctly different in different regions. In some regions the particles have average dimensions of (70 ± 35) nm along the fibrils and about half that across the fibrils. In other regions they are smaller and rounder, of order (53 ± 14) nm both along and across the fibrils. In other regions they are smaller and rounder, of order (25 ± 15) nm both along and across the fibrils, with more rounded top surfaces.

Significantly, we rarely observed bare collagen fibrils. If the observed particles can be verified to be native extrafibrillar mineral, this could imply that the fractures which created the observed areas propagated within the mineralized extrafibrillar matrix.

The jaws of the marine worm Nereis sp. are made of protein fibers and are reinforced by zinc. Here we study a transverse section through the jaw using optical microscopy and nanoindentation. Optical microscopy images demonstrate a complex microstructure which includes channels that extend throughout the jaw. We suggest that these channels may be related to jaw remodeling. The mechanical results reveal spatial variations in both indentation hardness and reduced modulus. Specifically, the toothed side of the jaw (used for grasping food) is harder than the remainder of the jaw and the very exterior surface is hardest.

Most non-traumatic fractures occur in teeth that have been treated, for example restored or endodontically repaired. It is therefore essential to evaluate the structure and mechanical properties of altered forms of dentin. One such altered dentin is transparent (sclerotic) dentin, which forms gradually with aging. Accordingly, in the present study, we seek to study differences in the structure, i.e., dentinal mineral concentration, mineral crystallite size, and the mechanical properties, i.e., elastic moduli, fracture toughness and fatigue behavior, of normal and transparent root dentin. The mineral concentration, measured by x-ray computed tomography, was found to be significantly higher in transparent dentin, with the majority of the increase being due to the closure of the tubule lumens. Crystallite size, as measured by small angle x-ray scattering, appeared to be slightly reduced in transparent dentin, although the difference was not statistically significant. The elastic properties remained unchanged, although transparent dentin showed almost no yield/post-yield behavior. The fracture toughness was lowered by roughly 20%, while the fatigue resistance was deleteriously affected at high stress levels. These results are discussed in terms of the altered microstructure of transparent dentin.

Using a stochastic lattice model we have studied the architectural changes of trabecular bone occurring while the structure is remodeled. Our model considers the mechanical feedback loop, which control the remodeling process. A fast algorithm was employed to solve approximately the mechanical problem. A general feature of the model is that a networklike structure emerges, which further coarsens while the bone volume fraction remains unchanged. Decreasing the mechanical response of the system by either lowering the external load or the internal mechano-sensitivity leads not only to a reduction of the bone volume fraction, but results in topological changes of the trabecular bone architecture, where the loss of horizontal trabeculae is the most obvious effect.

Energy-variable synchrotron x-ray diffraction was applied to probe local structure in different kinds of aragonitic seashells with spatial resolution. All investigated specimens revealed lattice distortions of biogenic aragonite with respect to the geological mineral. The obtained data are analyzed in terms of residual strains induced by intercalating organic macromolecules.

The exoskeleton of the lobster Homarus americanus is a multiphase bio-composite which consists of a fibrous organic matrix (crystalline α-chitin and various types of non-crystalline proteins) and embedded biominerals (mainly calcite). In this study we present experimental data about the microscopic and mesoscopic structure of this material.