For more than two decades, the edible clam Ruditapes philippinarum has been affected by the Brown Ring Disease (BRD), a bacterial infection characterized by the formation of a brown organic deposit in the internal side of the valves. Although this infection is often lethal, in some cases specimens can overcome it by remineralizing over the organic deposit. The goal of the present study is to compare biochemically and immunologically the shell matrices of repaired and healthy zones. Our data suggest that the repair zones exhibit certain variability, which would be the direct consequence of a modification of the secretory regime of calcifying tissues responsible of the repair process.

Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein which plays an important role in mineralized tissue formation. Dmp1 null adult mice are ricketic and osteomalacic and are a model for hypophosphatemic rickets [1]. Mutation in humans results is Autosomal Recessive Hypophosphatic Rickets [1]. The degree of bone mineralization significantly contributes to bone tissue mechanical properties, but precise relationships and interactions between chemical and mechanical variables are unknown. The objective of this study was to relate the differences in chemical properties in the Dmp1 wildtype (WT) and null (KO) mouse femoral cortical bone to their mechanical properties by using FTIR imaging and Scanning Acoustic Microscopy (SAM). Interactive mechanical (elastic modulus) and chemical images (i.e., mineral/matrix ratios) were generated from the same region of bone at a lateral resolution of ˜10 um. Mechanical analysis showed that elastic modulus, 75 percentile around 7.1 GPa, was ˜60% less in Dmp1 KO than that in WT, in which elastic modulus was 75 percentile around 15.2 GPa. The mineral-to-matrix ratios in Dmp1KO (4.96±1.63) were ˜2 times lower than that in Dmp1 WT (8.65±1.14). The mineral crystallinity and collagen crosslink ratios were not significantly different between KO and WT. Conclusions: The results relate the bone elastic modulus changes in Dmp1WT and KO mice with chemical changes within a specific bone site. These measurements provide a new tool for describing the variability of bone chemical and mechanical properties.

The freshwater Zebra Mussel, Dreissena polymorpha, was accidently released into the Great Lakes approximately 20 years ago. Since then it has spread rapidly, thanks in part to its ability to adhere to hard substrates, resulting in serious environmental and economic consequences. Like the marine mussels, attachment of the Zebra Mussel is achieved by means of its byssus, a series of proteinaceous threads that connect the animal to surfaces via secreted adhesive plaques. While the byssus of the Zebra Mussel is superficially similar to those of its marine counterparts, significant structural and compositional differences suggest that further investigation of the adhesion mechanisms in this freshwater species is warranted. Here we examine for the first time the detailed distribution of DOPA (3,4-dihydroxyphenylalanine)-containing proteins in the Zebra Mussel plaque and threads, as well as the enzyme responsible for their cross-linking. We show that the plaque-substrate interface retains the greatest amount of DOPA after aging, consistent with an adhesive role, while in the threads and bulk of plaque DOPA is presumably cross-linked for cohesive strength. We report also on a remarkably uniform layer ˜10 nm thick on the underside of the plaque, which is most likely responsible for adhesion.

From an evolutionary viewpoint, the molluscan nacre constitutes a fascinating object. This microstructure appeared early, in the Lower Cambrian period, about 530 million years ago, and since then, has been kept unchanged until today. Nacre is restricted to the conchiferan mollusks, where it occurs in t least three main classes, bivalves, gastropods and cephalopods. The aim of the present study is to investigate whether all nacres are built from the same “macromolecular tools”, proteins of the nacre matrix. To this end, we studied three new nacre models, the freshwater bivalve Unio pictorum, the cephalopod Nautilus macromphalus, and the gastropod Haliotis asinina, to which we applied a combined biochemical and proteomic characterization of their respective nacre matrices. The results of our approach, that can be defined as “shellomics” (proteomics applied to shell proteins) shed a new light on the macroevolution of nacre matrix proteins and on the in vitro design of nacre-like biomaterials.

Mms6 is a small acidic protein which is tightly bound to magnetite in the bacterium Magnetospirillum magneticum AMB-1. Mms6 has been previously shown to promote iron-binding capacity as well as modulate the size and morphology of magnetic iron oxide crystals in vitro. In this study, we synthesized iron oxide crystals by using a monolayer-modified substrate. A self-assembled monolayer of octadecyltrimethoxysilane was modified on a silicon substrate. Recombinant Mms6 protein was attached to the substrate through the hydrophobic interactions between the protein molecules and the monolayer. The immobilization of protein molecules on the substrate surface was confirmed by fluorescent labeling of these molecules and subsequent fluorescence microscopy. This protein-modified substrate was then used as a template for iron oxide crystal formation in a ferrous solution. Scanning electron microscopy revealed site-specific formation of iron oxide crystals in substrate regions with immobilized proteins. This use of proteins might provide an alternative method for the bottom-up fabrication of nano-sized magnetic particles.

Dental remineralization may be achieved by mediating the interactions between tooth surfaces with free ions and biomimetic peptides. We recently developed octuplet repeats of aspartate-serine-serine (DSS-8) peptide, which occurs in high abundance in naturally occurring proteins that are critical for tooth remineralization. In this paper, we evaluated the possible role of DSS-8 in dentin remineralization. Human dentin specimens were demineralized, exposed briefly to DSS-8 solution, and then exposed to concentrated ionic solutions that favor remineralization. Dentin nano-mechanical behaviors, hardness and elastic modulus, at various stages of treatment were determined by nanoindentation. The phase, microstructure and morphology of the resultant surfaces were characterized using grazing incidence X-ray diffraction, variable pressure scanning electron microscopy, and atomic force microscopy, respectively. Nanoindentation results show that DSS-8 remineralization effectively improves the mechanical and elastic properties of native dentin. Moreover, the hardness and elastic modulus for the DSS-8 treated dentin were significantly higher than surfaces remineralized without DSS-8.

Like the lobsters, some terrestrial crabs and other crayfishes, the Australian red claw crayfish, Cherax quadricarinatus, elaborates in its stomach wall calcium storage structures called gastroliths. For understanding the cyclic elaboration and stabilization of these amorphous calcified structures, we studied the organic matrix (OM) of these paired biomineralizations. After decalcification with acetic acid, we analysed the proteinaceous components of an acetic acid-insoluble fraction by two-dimensional electrophoresis. Nine spots were digested by trpsin and the tryptic peptides were sequenced by nanoLC-nanoESI-MS/MS mass spectrometry. About 100 peptidic sequences were compared to sequences previously registered in the databases. Seven of the partially sequenced organic matrix polypeptides are probably new proteins. Another one corresponds to the previously sequenced protein, GAP65, from Cherax quadricarinatus and the last one, which migrates in electrophoresis at around 25 kDa, presents strong homology with the crustacyanin-A2 subunit of Homarus gammarus.

A technique combining both atomic force microscopy (AFM) and scanning electron microscopy (SEM) is used to evaluate the mechanical properties of individual collagen fibrils from the fractured surface of antler. SEM is used to locate individual mineralized collagen fibrils and allow visualization of the attachment of these fibrils to the end of an AFM probe. Tensile testing of individual collagen fibril to failure was performed using the AFM with resultant stress-strain curves obtained. Tensile strengths of up to 0.18GPa are found for some individual collagen fibrils, indicating the presence of mineral in improving mechanical performance. Consideration of the SEM operating parameters indicates that the amount of time the sample is within the SEM vacuum can affect the resultant mechanical behavior of individual fibrils.

The purpose of this study was to histologically and mechanically appraise the in vivo bone-bonding abilities of K2TinO2n+1 coated and uncoated Ti-15Mo-3Nb (TMN) implants. According to GB/T16886.6－1997 biological evaluation of medical devices Part 6:Tests for local effects after implantation, the two types of implants were implanted into the proximal metaphyses of Chinese white rabbits’ femurs for 12, 26 and 52 weeks and investigated by pushing out test, scanning electron microscopy (SEM) attached to an energy-dispersive X-ray micro-analyzer (EDX) and light microscopy. The bone-bonding abilities of the K2TinO2n+1 biocoating /Ti-15Mo-3Nb (KBT) gradient biomaterial implants were higher than those of T implants at different periods of implantation. The K2TinO2n+1 biocoating (KB) could stimulate new bone rapid formation at the early stages of implantation. And the implants with the biocoating eventually bonded to bone directly, with no intervening soft tissue layer, that was an osseocoalescence. However, the type of bone-bonding between TMN titanium alloy implants and bone was a simple osseocoaptation. The more excellent bone-bonding ability of the KBT implants should be attributed to the superficial characteristics, the bioactivity of low potassium titanate and biostability of high potassium titanate.

Alport Syndrome is a genetic disease characterized by the breakdown of the glomerular basement membrane (GBM) around blood vessels in the kidney, leading to kidney failure in most patients. It is the second most inherited kidney disease in the US, and many other symptoms are associated with the disease, including hearing loss and ocular lesions. Here we probe the molecular level mechanisms of this disease utilizing a bottom-up computational materiomics approach focused on the mutation associated with the most severe form of Alport Syndrome. Since the GBM is under constant mechanical loading due to blood flow, changes in mechanical properties due to amino acid mutations may be critical in the symptomatic GBM breakdown seen in Alport Syndrome patients. Through full-atomistic simulations in explicit solvent, the effects of a single-residue glycine substitution mutation are studied in a short segment of a collagen type IV tropocollagen molecule. Major changes are observed at the single molecule level of the mutated sequence, including a bent shape of the structures after equilibration with the kink located at the mutation site and a significant alteration of the molecule’s stress-strain response and stiffness.

To gain basic insight into the impact of non-biological features on cells’ behaviour, primary skin-cells, keratinocytes and fibroblasts, were cultured on amine-functionalized or carboxy-functionalized planar, nano- or microstructured surfaces. Sintered layers of silica nano- or microparticles were used to fabricate structures in the range of naturally occurring structure-sizes. Organo-chemical functionalization was achieved using organo-functional silanes. Primary human keratinocytes and fibroblasts were isolated from human foreskin and cultivated on the modified interfaces. Both cell-types displayed specific proliferation behaviour, depending on surface topography and chemical functionalization: Keratinocytes showed significantly better proliferation on amino-functionalized surfaces than on carboxy-functionalized surfaces. On amino-functional surfaces decree-topography. Fibroblasts, in contrast, tended to proliferate stronger on carboxylated surfaces. Immunohistological staining proofed that actin and vinculin, which is involved in the formation of focal adhesions, were expressed on all modified surfaces, thus revealing intact cytoskeleton and cell-substrate contacts.

To augment or replace defective, diseased, or impaired human digits, the design and development of tissue-engineered phalanges are important and include a middle phalanx model. This construct consists in part of two square-shaped biodegradable polyglycolic acid (PGA) scaffolds (1 x 1 x 0.2 cm in length, width and thickness, respectively) seeded with cartilage cells (chondrocytes) obtained from young calves. One such seeded scaffold is sutured to each end of a rectangular-shaped scaffold (˜2 x 0.7 x 0.5 cm in length, width and thickness) serving as the midshaft of the model. To examine the biological regenerative capacity of these biomimetic composites, midshafts were left uncovered or wrapped with periosteum, a tissue from calves giving rise to cartilage and bone. Midshafts were composed of poly(L-lactide-ε-caprolactone) [P(LA-CL)] or one of two ceramics, hydroxyapatite (HA) or β-tricalcium phosphate (β-TCP), admixed with P(LA-CL). When engineered middle phalanx models were implanted and grown for up to 20 weeks under dorsal skin flaps of athymic (nude) mice, resulting constructs varied in their midshaft bone and end plate cartilage composition and structure. Harvested from mice at 20 weeks, all constructs (n = 3 for each type) without periosteum developed viable end plate cartilage as determined by Safranin-O staining for chondrocyte-secreted proteoglycans but cells were not organized as in normal growth plate cartilage of human digits. Midshafts remained devoid of cells and mineral. Implanted for the same time 20 week period, constructs of P(LA-CL) (n = 3), HA-P(LA-CL) (n = 3), or β-TCP-P(LA-CL) (n = 3) and enclosed by periosteum each developed viable end plate cartilage whose chondrocytes were organized into columns resembling normal growth plate cartilage of digits. Midshafts mineralized through the normal process of endochondral ossification. While these features were common to all periosteum-wrapped composites, specific differences occurred between them, apparently depending on midshaft copolymer composition. In particular after 10 or 20 weeks of implantation, gene expression of end plate chondrocytes varied in their levels of type II collagen, aggrecan (proteoglycan), or bone sialoprotein, all markers for development of normal cartilage extracellular matrix and mineralization. These results indicate that the composition of midshaft scaffolds comprising middle phalanx models of human digits affects the composition and structure of both midshaft bone and end plate cartilage of constructs. Continuing studies are defining more completely the relationships between structure and composition of bone and cartilage tissues developed and properties of their underlying copolymer scaffolds in these biomineralized models.

Certain design strategies appear repeatedly in a variety of biological structures. One such motif consists of a soft and pliable interface joining much larger and stiffer elements. Examples include the craniofacial sutures between the bones of the skull, the sutures between the bony plates in shell of turtles and the periodontal ligament between teeth and their sockets. Yet the detailed mechanics of these systems are not fully understood.

Turtles are believed to have existed already in the early Triassic, about 200 million years ago. They are thus one of the oldest non-extinct vertebrates. Their shell is therefore a particularly attractive subject for investigation since it has developed and conserved through such an extremely long evolutionary process and has achieved a highly optimized structure.

The turtle shell has a ‘sandwich’ structure typical of flat bones like the skull of vertebrates. It consists of two external, relatively thin sheets of dense bone (internal endocortical and external exocortical bone plates) which contain very few voids, and between them a thick and very porous spongy bone layer. At the mid-distance between adjacent ribs the dermal bones are separated by soft sutures which have a unique and complex 3-D shape.

The primary function of the shell is to protect the turtle from external trauma, and therefore it has to be stiff. However excessive stiffness may result in microdamage accumulation as a result of everyday activities like minor impact, and decrease the efficiency of respiration and locomotion. We speculate that the structure and architecture of the sutures allow easy deformation of the shell at small loads but cause it to become considerably more rigid at larger loads, reminiscent of composite materials with interlocking elements. We hypothesize that this mechanical property is related to the putative function of the suture in the turtle shell.

In order to examine this hypothesis we studied samples obtained from shells of the red eared slider turtle (Chrysemys scripta elegans). We used several imaging techniques (micro-computed tomography, scanning electron microscopy and light microscopy), histology and mechanical testing. Based on these observations we present a concept of the structure-mechanics relationship of the shell, and present a simple mathematical model of the deformation pattern of the suture-containing samples in 3-point bending tests and compare its predictions to our experimental results.

Diatoms are unicellular eukaryotic algae found in fresh and marine water. Each cell is surrounded by an outer shell called a frustule that is composed of highly structured amorphous silica. Diatoms are able to transform silicic acid into these sturdy intricate structures at ambient temperatures and pressures, whereas the chemical synthesis of silica-based materials typically requires extremes of temperature and pH. Cationic polypeptides, termed silica affinity proteins (or silaffins) recently identified from dissolved frustules of specific species of diatoms are clearly involved and have been shown to initiate the formation of silica in solution. The relationship between the local environment of catalytic sites on these peptides, which can be influenced by the amino acid sequence and the extent of aggregation, and the observed structure of the silica is not understood. Moreover, the activity of these peptides in promoting silicification at lipid membranes has not yet been clarified. In this work we developed a model system to address some of these questions. We studied peptide adsorption to Langmuir monolayers and subsequent silicification using X-ray reflectivity and grazing incidence X-ray diffraction. The results demonstrate the lipid affinity of the parent sequences of several silaffin peptides. Further, the results show that the membrane-bound peptides promote the formation of interfacial nanoscale layers of amorphous silica at the lipid-water interface that vary in structure according to the peptide sequence.

Diatoms are unicellular, photosynthetic microalgae that live in marine and freshwater environments. The cell walls of diatoms are composed of biosilica and have exceedingly hierarchical ornate nanostructures. Consequently, these nanostructures have long been regarded as the paradigm for future silica nanotechnology. We have coated diatom Pinnularia sp. biosilica with a thin film of CdS using a chemical bath deposition technique. Possible uses for these CdS coated diatoms include the development of new nanodevice fabrication techniques and optoelectronic applications. Electron microscopy techniques were utilized to study their morphologies. Their electrical characteristics were investigated using an Agilent 4156C precision semiconductor parameter analyzer and a Cascade probe station. The CdS coating was found to be dense, adherent and nanostructured. The diatoms coated with CdS exhibited both metallic and semiconductor diode behavior.

Wood and paper are ubiquitous in societies around the world and are largely taken for granted as part of traditional industries with no new science to learn. Many of the technologies used in the forest products industry have been gained empirically through experience. The complexities of wood are now yielding to newer tools and we are beginning to see how the mechanical, optical and other physical properties of wood are related to hierarchical structures based on 2 to 10 nm diameter several hundred nm long fibers of nanocrystalline cellulose (NCC). The liberation of these NCC’s is allowing their re-assembly into remarkably strong structures. Examples will be given of the nature of these building blocks and structures assembled from them. Examples will include nanocomposites as well as very high strength “paper”. Paper is another example of a process whereby nanofibrils are released and then re-assembled with the use of “retention, drainage and formation aides” to make substrates we call paper with remarkable strength to weight performance. Other disciplines call this process “self-assembly” and the “aids” as necessary surfactants and additives to control structure and performance. Glossy magazine papers, for example, have approximately 10 micron thick coatings of white minerals and latex binders which are increasingly of nano dimensions. The coatings are assembled in structures to provide optical barrier performance (opacity) as well as controlled ink interaction with the necessary strength to survive printing and handling. These coatings are frequently similar in structure to seashells and, from this knowledge, progress has been made in understanding the mechanisms at play in achieving higher strength coatings. More recently kaolin clays have been introduced with mean crystal thicknesses in the range 20 to 40 nm instead of the usual 100 to 140 nm. These clays show useful strength performance and represent what may be called pragmatic nanoclays. Novel chemistries based on biomimetic learnings are emerging to displace the conventional starch or latex binders. Examples will be given of protocols for moving toward higher strength systems.

Despite much progress in recent years, the nature of microcracking in bone at the nano-meter scale is still not well understood. This is partly due to the complexity of bone's hierarchical structure, but also to the difficulty of detecting cracks at very fine scales. Bone microcracking is typically detected using fluorescent dye staining techniques followed by optical or laser microscopy examinations. However, fluorescence-based methods are limited to sub-micron resolution and do not fit three-dimensional imaging such as micro-CT or high resolution imaging such as electron microscopy. This pilot study explores the potential of a heavy metal staining technique to label nano-sized cracks in bone that could be detected by electron microscopy and, albeit at a larger scale, by micro-computed tomography. Upon further development, the method described here may lead to the nano-meter scale characterization of bone microcracking.

Bone grafts need to comply with some criteria of biocompatibility, including favoring neovascularization, new bone formation, and discourage inflammatory response and graft rejection. It is also expected that these materials should have mechanical properties similar to those of natural bone, that is, having enough pores to permit osteoprogenitor cells and vascular endothelium penetration but maintaining strength and flexibility.

Here, a new resistant and flexible tridimensional multilayered bioceramic composite was obtained by using chitosan and hydroxyapatite in combination with cells and their associated growth factors from the bone marrow tissue, allowing the development of a biocompatible bone graft.

This multilayered graft made out of chitosan functionalized with phosphate groups and mineralized with calcium phosphate (hydroxyapatite) was analyzed with scanning electron microscopy (SEM), X ray diffraction (XRD), energy dispersive X-ray analysis (EDX) and Fourier transform infrared spectroscopy (FTIR) to assess the degree of phosphorylation and the amount of hydroxyapatite present in the graft. The composite was further characterized by mechanical testing (Vicker's microhardness test), in vitro osteoblasts culture citotoxicity tests.

The results showed that this multilayered graft has mechanical properties comparable to that of trabecular bone and it was capable to keed osteogenic cells alive.

Biology has evolved several strategies for attachment of sedentary animals. In the bivalves, byssi abound and the best known example being the protein-based byssus of the blue mussel and other Mytilidae. In contrast the bivalve Anomia sp. has a single calcified thread. The byssus is hierarchical in design and contains several different types of structures as revealed by scanning electron microscopy images. The mechanical properties of the byssus are probed by nanoindentation. It is found that the mineralized part of the byssus is very stiff with a reduced modulus of about 67 GPa and a hardness of ˜3.7 GPa. This corresponds to a modulus roughly 20% smaller than that of pure calcite and a hardness that is about 20% larger than pure calcite. The results reveal the importance of microstructure on mechanical performance.

The suckers that line the arms and tentacles of squid are equipped with rigid toothed ring-like elements that increase the gripping power during prey capture and handling. The sucker rings of the Humboldt squid Dosidicus gigas, are fully proteinaceous and contain nanotubules with diameters ranging from 100 to 250 nm. It has been shown previously that the ensuing porosity is a prime determinant of the local elastic modulus [A. Miserez et al., Adv. Mater. <b>21</b>, 401 (2009)]. Here additional nanoindentation data are presented together with structural analyses. The nanomechanical data support our model that the measured modulus is determined by the local porosity. The dry moduli reach ca. 8 GPa and are reduced about two-fold in the hydrated state. This surprisingly small reduction is discussed in relation to possible chemistries responsible for assembly of these structures.