Collagen biomineralization is a complex process and the controlling factors, at the molecular level, are not well understood. The periodontium, the set of tissues involved in tooth anchorage, is an excellent model with which to investigate the controlling mechanisms of mineralization, as there are both mineralized and non-mineralized tissues in close proximity. More specifically, the periodontal ligament (PDL) becomes mineralized along a sharp front of about 200nm at the root of the tooth. We present here a model of collagen biomineralization based on demineralized sections mouse periodontium. The fixed tissue ensures both insoluble protein content and collagen structure of both mineralized and unmineralized tissues remain intact.. When exposed to mineralizing solutions, ultra-thin cryo-sections demonstrate selective remineralization, with preferential depostion of mineral in the previously mineralized tissues. Direct TEM characterization of the mineral formation within the tissues shows growth of mineral along collagen fibers, with banding patterns suggestive of intrafibrillar mineralization. Depending on the composition of the mineralizing solution, the mineral that forms is either amorphous calcium phosphate or oriented apatite. We additionally use a top down approach, which involves the enzymatic removal of non-collagenous proteins and/or proteoglycans, in order to evaluate the roles of these macromolecules in governing mineralization.

Type I collagen is the most abundant protein in the body and forms the scaffolding upon which many tissues are built. The building block of collagen-based tissues is the nanoscale collagen fibril. Based on the work of Hodge and Petruska, the primary morphological characteristic of Type I collagen fibrils, the D-periodic spacing, was shown to be 67 nm. Using atomic force microscopy (AFM), recent studies demonstrated that normal collagen exists with a distribution of D-spacing values in a variety of tissues. Disease states have been shown to significantly alter this distribution in bone, but these studies took place in air and mechanical properties were not measured. AFM imaging of collagen fibrils in fluid is not trivial. Since collagen absorbs water and swells, imaging in fluid often leads to degradation of image resolution and quality, making quantitative analysis difficult. Because fibrils exist in a hydrated environment in vivo, the effect of dehydration on collagen is an important factor to consider. The current study was undertaken to analyze the role of hydration on morphology and mechanics in normal Type I collagen. 6 month old male mice from a Sv129/CD-1/C57BL/6S background were used with prior approval (ASP 09-023). Individual tendon fibers were removed from the tail of each mouse and placed in phosphate buffered saline. Each fiber was rinsed in water, placed on a glass slide and flattened with curved forceps. Imaging took place in air or fluid. 2D Fast Fourier Transforms were performed on individual fibrils to determine the value of the D-spacing (n=60-70 per sample from at least 3 locations). The AFM cantilever was then used to indent the samples. Five locations on individual fibrils were indented and force curves were acquired. Indent modulus was calculated from the unloading curve using the Sneddon or the Derjaguin-Muller-Toporov model. In total, 61 dry fibrils were indented versus 53 wet fibrils. D-spacing and indentation modulus were compared in wet versus dry samples using Studentâ?Ts t-tests. To investigate differences in distributions of fibril morphology, a Kolmogorov-Smirnov test was performed in each group. For all tests, a value of p<0.05 was considered significant. The overall mean value for D-spacing was 67.8 ± 0.8 nm for dry samples and 67.7 ± 0.7 nm for wet samples, respectively (p=0.811). A distribution of spacings existed in wet and dry fibrils, but these distributions were indistinguishable (p=0.130). The modulus in dry versus wet tendons differed by 3 orders of magnitude (1.52 ± 0.86 GPa dry, 2.98 ± 0.61 MPa wet; p<0.001). In conclusion, the current study demonstrates that while the D-periodic spacing of individual collagen fibrils in tendon does not depend on hydration, mechanical properties do. Current studies are underway to thoroughly investigate this phenomenon and to determine how these properties change with disease in tendon, dentin and bone.

The often astonishing materials properties of crystalline biominerals are generally related to the hierarchical assembly of specifically interacting organic and inorganic components. A yet unfulfilled dream of many scientists is to synthesize new materials with similar advanced properties applying Natureâ?Ts biomimeralization strategies.[1] An absolute prerequisite for the design of such hybrid materials with predetermined structure and properties is to unravel the mechanisms of biologically and biomimetically controlled mineral formation. The in situ study of the development of mineral formation can make an important contribution to the understanding of the processes involved in biomineralization.[2] CryoTEM has been demonstrated as a method to investigate the early stages of mineral formation without removing the developing particles from their aqueous environment. [3, 4] These investigations revealed that the formation of both calcium carbonate and calcium phosphate is preceded by an amorphous phase, which itself is formed through the assembly and aggregation of nanometer building blocks termed â?opre-nucleation clustersâ? [5,6]. They also revealed the role of the templating surfaces and matrices in these processes. Using high resolution cryoTEM in combination with several other in-situ techniques we will demonstrate in detail the structure of these â?oclustersâ? and their role in the formation of the subsequent amorphous and crystalline phases. [1] N.A.J.M. Sommerdijk, H. Cölfen, MRS bulletin, 35, 116 (2010). [2] A. Dey, G. de With, N.A.J.M. Sommerdijk, Chem. Soc. Rev. 92, 381, (2010). [3] B.P. Pichon, P.H. H. Bomans, P.M. Frederik N. A. J. M. Sommerdijk, J. Am. Chem. Soc. 130, 4034 (2008). [4] E.M. Pouget, P.H.H. Bomans, J.A.C.M. Goos, P.M. Frederik, G.de With, N.A.J.M. Sommerdijk, Science, 323, 1455 (2009). [5] A. Dey, P.H.H. Bomans, F.A. Müller, J. Will, P.M. Frederik, G. de With and N.A.J.M. Sommerdijk, Nature Mater, 9 1010 (2010). [6] F. Nudelman, K. Pieterse, A. George, P.H.H. Bomans, H. Friedrich1, L. J. Brylka1, P.A.J. Hilbers, G. de With N.A.J.M. Sommerdijk, Nature Mater, 9 1004 (2010)

The use of bioactive glasses in the form of porous three-dimensional scaffolds for bone repair applications has been receiving considerable interest in recent years. However, bioactive glass scaffolds have been limited to the repair of low-load bone defects because of their low strength. We have used innovative processing techniques to create porous and strong bioactive glass scaffolds for potential application in the repair of loaded bone. Scaffolds of 13-93 glass (53SiO2, 6Na2O, 12K2O, 5MgO, 20CaO, 4P2O5, wt %) with an oriented microstructure of columnar pores (porosity = 50%; pore size = 50â?"150 µm), prepared by unidirectional freezing of camphene-based suspensions, showed a compressive strength of 47 ± 5 MPa. In comparison, scaffolds of 13-93 glass with a microstructure similar to dry human trabecular bone, prepared by a polymer foam replication technique, had a compressive strength of only 11 ± 1 MPa (porosity = 80%; pore size = 100â?"500 µm). In the present work, the ability of â?oorientedâ? 13-93 bioactive glass scaffolds to support bone regeneration and mineralization was evaluated in a non-healing rat calvarial defect model were implanted for 12 and 24 weeks. The controls consisted of â?otrabecularâ? 13-93 scaffolds and empty defects. The mechanical response of the scaffolds was evaluated in vivo (after implantation in rat calvarial defects for 12 and 24 weeks) using a diametral compression test. Evaluation of stained sections showed new bone formation within the trabecular and oriented bioactive glass implants by 12 weeks. Histomorphometry showed no significant difference in the amount of new bone infiltration between the trabecular scaffolds (19 ± 9%) and the oriented scaffolds (18 ± 3%). However, bone formation was found preferentially on the dural side of the oriented scaffolds, whereas bone ingrowth occurred both on the dural side and into the edges of the trabecular scaffolds. For both groups of scaffolds, new bone formation increased with an increase in the implantation time from 12 to 24 weeks. The amount of bone ingrowth was 36 ± 11% in the trabecular scaffolds and 24 ± 3% in the oriented scaffolds after 24 weeks. The scaffolds showed an â?oelasto-plasticâ? response after implantation for 12 and 24 weeks in rat calvarial defects. Both groups of scaffolds showed an increase in strength as the implantation time increased from 12 to 24 weeks. For the same implantation time, the oriented scaffolds had a higher strength than the trabecular scaffolds. Together, the results indicate that 13-93 bioactive glass scaffolds with the trabecular microstructure are promising for the regeneration of bone in low-load sites. Because of their higher strength and ability to support bone regeneration, oriented 13-93 bioactive glass scaffolds are promising implants for loaded bone repair.

While there have been advances in developing biomedical scaffolds for bone substitutes and tissue engineering, the rapid restoration of tissue biomechanical function remains an important challenge, emphasizing the need to replicate structural and mechanical properties of natural bone using engineered, novel, bioscaffold designs. The mechanical properties of bone scaffolds are important since bone regeneration must be done in such a way that the scaffoldâ?Ts properties closely mimic the surrounding tissue properties and can carry the required structural loads. Current bone scaffolds suffer from a lack of tunability, as well as variations in mechanical and viscoelastic properties. Furthermore, they need to be equipped with a highly interconnected deep porosity since it facilitates both the seeding and structural diffusion of cells and organic nutrients. Biodegradability/biocompatibility is also an essential characteristic so that scaffolds can be preferably entirely absorbed by the body without the need for additional surgical removal and the onset of potentially undesired biological effects. We show a novel, bioengineered, moldable platform for bone regeneration composed of porous bionanocomposite scaffolds made of components that are normally found in bone tissue (calcium, collagen, carbonate, sodium, and phosphorous). To accommodate high- or low-stress environments, the hardness and modulus (stiffness) of these scaffolds can be tuned in a wide range (MPa - GPa), while maintaining the required viscoelasticity. Our approach to control the mechanical properties is based on a new formulation of mineralized bioscaffolds by incorporation of calcium carbonate in which, calcium and phosphorous are in the form of both calcium polyphosphate (CPP) and hydroxyapatite (HAP). The variation in the calcium carbonate concentration allows tuning of CPP in the bioscaffold to tailor the degree of mineralization and mechanical and viscoelastic properties that closely match those of natural bone. Our results demonstrate an ideal framework for new bone scaffold designs for advanced bone substitute applications.

Tissue engineering scaffolds must provide a matrix with interconnecting porosity that promotes rapid bone formation while possessing sufficient strength to prevent fracture under physiological loads until full integration and healing are attained. However, the challenge to regenerate bone for practical clinical situations still persists. In this presentation we describe attempts to develop a range of bone- and nacre-like structural materials using freeze-casting technique, which utilizes the intricate structure of ice to create hybrid materials with complex lamellar and/or mortar and brick structures modeled across several length-scales. The challenge is to develop a new generation of implant materials that will combine the advantages of ceramics, in particular their inertness, with a mechanical response comparable to those of implant alloys. The unique properties of ceramic materials, including their outstanding corrosion resistance and excellent aesthetics, make them appealing candidates for many implant applications. However, their inferior mechanical properties, particularly fracture toughness, until now have hampered their commercial use, especially in load-bearing situations. This is a particular problem when designing porous implants or implants with rough and porous surfaces for better osseointegration. While the porosity will improve osseointegration, increased porosity can easily result in diminished strength and fracture at relatively low loads. Through new fabrication technologies, these limitations may be overcome. Our results show ceramic-polymer hybrid materials with toughness well in excess of those expected from a rule of mixtures construction. The architecture and properties of the synthetic materials are compared to their natural counterparts in order to identify the mechanisms that control mechanical behavior over multiple dimensions and propose new design concepts to guide the synthesis of hybrid/hierarchical structural materials with unique mechanical responses. The ultimate goal is to produce materials and therapies that will bring state-of-the-art technology to the bedside and improve quality of life and current standards of care. Support is provided by the National Institute of Health under grant number NIH/NIDCR 1 R01 DE015633

Natural composites like bone and nacre exhibit unique combinations of strength, toughness and self-healing abilities that are unmatched by engineering materials. This is achieved through the formation of complex hierarchical architectures and molecular scale interfacial engineering. Freeze casting is a powerful technique to produce bio-inspired hierarchical structured composites mimicking the natural design of nacre. Despite promising preliminary results, many issues need to be addressed in order to take full advantage of freeze casting and understand the role of bio-inspiration in the development of advanced synthetic structural composites. With regards to structural control, a main challenge is to increase the volume fraction of the ceramic phase in freeze casted composites to values close to nacre (~95 vol %). In addition, the proteinous nano-layer in nacre has a key function in the multi-scale toughening mechanisms. However, we do not know yet how to replicate the properties of this thin layer in synthetic materials to provide maximum energy dissipation and increased strength. In this work, nacre-inspired ceramic-based composites containing a very high ceramic content (~95%) and nano layers of different soft phases (e.g. polymers or carbon) are produced by a novel approach combining freeze casting and spark plasma sintering SPS. The relationship between the processing conditions and the material structure will be discussed. The role of soft phases will be assessed through the combination of mechanical testing and structural characterization in order to identify specific damage and toughening mechanisms down to sub-micron length scales.

Strontium (Sr), as a natural element found in bone and teeth, has the ability to both enhance the proliferation of osteoblasts and to effectively block osteoclastic bone resorption. As such, Sr plays an important role in the metabolism of bone, a nanocomposite material that consists mostly of calcium phosphate (Ca-P) mineral. There has been increasing evidence for the osteoinductive potential of Sr-substituted Ca-P ceramics. Therefore, the motivation for our research was to develop Sr substituted Ca-P coatings that mimic the natural function of bone for hard tissue repair and replacement. In this study, Sr-substituted Ca-P coatings containing different molar ratios of strontium and calcium were successfully formed on titanium substrates by electrochemical deposition. Self-assembled Sr-substituted Ca-P coatings were obtained with various morphologies (needle-like, flake-like or faceted micro crystals) and phase composition. These surface morphologies were observed through scanning electron microscopy (SEM). The coating composition and crystal phase were investigated by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). To evaluate the cytocompatibility and bioactivity of the coatings, in vitro experiments were performed using MC3T3-E1 osteoblast-like cells. The findings from our study suggest that the morphology and composition of the coatings may be impacted by strontium dose in the electrolyte during electrochemical deposition. As a result, the incorporation of strontium in certain phases may enhance the biocompatibility and osteoconductivity of the Ca-P Ceramics. The mechanism of the formation of different phases is currently being investigated.

Recent developments in biomineralization and biomaterials have demonstrated that nano-calcium phosphate particles play an important role in the formation of hard tissues in nature. It is suggested that the basic inorganic building blocks of biominerals such as bone and enamel are nano-sized calcium phosphate although their hierarchical structures differ. Tens to hundreds of nano-blocks, under the control of organic additives, can combine into self-assembled materials. It is confirmed experimentally that enamel- or bone-like structure can be biomimetically achieved by oriented aggregation by using nano-calcium phosphates as the raw materials. During this nano assembly, biologic molecules specifically determine the structural characteristics of the final architecture. Furthermore, the precursor role of amorphous phase in the assembly of biomaterials is also important, in which amorphous calcium phosphate functions by linking nanocrystals and then is incorporated by phase transformation. Based upon this biomimetic mechanism, we remodel bone-like and enamel-like structures and mechanics by using a cooperative effect of nano apatite particles (building block) and biomolecules (regulator) under physiological conditions. The resulted apatite layer has the similar crystallographic and tectonic properties as those of the natural materials. Especially, the apparent restorations of bone or enamel hardness using the mechanically stable materials are particularly significant, demonstrating a successful strategy for the in vitro repair. This study highlights the utilizations of biomineralization principles such as nano assembly, organic controls in tissue regenerations.

Bone tissue types consist of a nanoscale mesh of collagen fibrils impregnated with mineral (carbonated apatite) particles inside and on their surface, along with a small fraction of noncollageneous proteins. These constituent elements assemble into fibre bundles, lamellae and osteons forming a hierarchical structure and mechanical deformation at interfaces between elements at each level is expected to be critical to the high work to fracture of such systems. While these interfacial mechanisms are clearly demonstrated at the microscale (crack reorientation inside and between lamellae, and crack bridging) the corresponding mechanisms at the nanoscale are controversial especially in the realm of macroscopic inelasticity. We investigate the operative toughening mechanisms in antler, an exceptionally tough, low mineralized bone type. Using in situ tensile testing combined with synchrotron small angle X â?" ray scattering and diffraction, the fibrillar and mineral strain are measured in the zone of inelastic deformation during uniaxial and cyclic loading. Evidence of fibrillar plasticity is clearly seen, with minimal hysteresis, suggesting a permanent structural deformation at the nanoscale. By modifying a previously proposed two-level staggered model for tensile deformation of nanocomposites, we find that these results are very well explained by an interfacial sliding between the mineral and collagen components inside the fibril. The model can be generalized to macroscopically higher mineralization where it matches experimental measurements of elastic moduli well. We then demonstrate that these nanoscale mechanisms can be severely disrupted in metabolic bone diseases, using a murine model of hypophosphatemic rickets (HPR) generated by ENU-mutagenesis. The strain in the fibrils increased and effective fibril modulus decreased, both significantly, in rachitic bone compared to wild type. We propose a structural mechanism of partial (patchy) mineralization in HPR mice to explain these results. Our results show the ability of in situ synchrotron X-ray scattering techniques, when combined with micromechanics, to identify toughening mechanisms at the nanoscale, as well as the functional reasons for reduction of skeletal tissue mechanical competence in bone diseases at this length scale.

The structure of human cortical bone evolves over multiple length-scales from its basic constituents of collagen and hydroxyapatite at the nanoscale to osteonal structures at near-millimeter dimensions, which all provide the basis for its mechanical properties. To resist fracture, boneâ?Ts toughness is derived intrinsically through plasticity (e.g., fibrillar sliding) at structural-scales typically below a micron and extrinsically (i.e., during crack growth) through mechanisms (e.g., crack deflection/bridging) generated at larger structural-scales. Biological factors such as aging and irradiation lead to a markedly increased fracture risk, which is often associated with a loss in bone mass (bone quantity). However, we find that these factors can also significantly degrade the fracture resistance (bone quality) over multiple length-scales. Using in situ small-/wide-angle x-ray scattering/diffraction to characterize sub-micron structural changes and synchrotron x-ray computed tomography and in situ fracture-toughness measurements in the scanning electron microscope to characterize effects at micron-scales, we show how age-related structural changes at differing size-scales degrade both the intrinsic and extrinsic toughness of bone. Specifically, we attribute the loss in toughness to increased non-enzymatic collagen cross-linking which suppresses plasticity at nanoscale dimensions and to an increased osteonal density which limits the potency of crack-bridging mechanisms at micron-scales. The link between these processes is that the increased stiffness of the cross-linked collagen requires energy to be absorbed by â?oplasticâ? deformation at higher structural levels, which occurs by the process of microcracking. We propose analogous mechanisms for the embrittlement of bone due to x-irradiation.

Bone can be subject to a wide range of irradiation doses in a number of instances such as medical treatments for cancer and sterilization (~ 30-60 kGy) and space travel (~ 2Gy). Also, synchrotron x-rays are being increasingly used to determine the structure and mechanical properties of bone, where doses on the order of 0.2 kGy/s are imparted to the sample. These doses accumulate over long periods of time over multiple measurements, and result in significant amount of irradiation dose on the sample. It is thus important to determine how the mechanical properties of bones are affected by such treatments and experimental conditions. Whereas the mineral phase of bone is less susceptible to radiation damage, the properties of the collagen phase have been found to change due to mechanisms such as increased cross-linking or chain scission. The collagen-mineral interface, which is made up of a large number of ionic, hydrogen and Van der Waals â?"type bonds, has also been shown to degrade with irradiation. To investigate the effect of irradiation on the elastic properties of bone, bovine bone samples are subject to a combination of mechanical loading (0 and -60 MPa) and varying levels of x-ray (doses (70 keV, 5 -3800 kGy). The evolution of the apparent modulus (ratio of applied stress to phase elastic strain) and residual strains in the collagen fibrils and the mineral phase is studied via scattering experiments. The apparent moduli of the mineral (26±1.2 GPa) and fibrils (13±1.9 GPa) do not exhibit a systematic change with increasing radiation doses up to 3800 kGy, indicating that the effectiveness of load transfer to the mineral phase via the collagen phase remains unchanged at these radiation levels. This is possible since the elastic modulus of the material is mostly dependent on the mineral phase which is more resistant to damage. The residual strains in the mineral phase are found to relax (i.e., become less compressive) with increase in radiation dose as well as number of load-unload cycles. The decay in residual strains due to irradiation dose was found to be significantly greater than that due to mechanical loading-unloading, and hypothesized to occur due to damage at the HAP-collagen interface. Also, analysis of x-ray diffraction peak widths show an asymmetric sharpening of the peaks as a function of radiation dose. This suggests that the mineral platelets that are highly strained relax at a faster rate than those that are less strained. Moreover, an overall residual strain homogenization is found to occur with dose. These sensitive experiments provide important insights about the deformation mechanisms occurring at the nanoscale at the HAP-collagen interface, which is an important consideration in the overall mechanical behavior of bone. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

Current high-resolution techniques called upon to address structure-function relationships challenge us to further specify our descriptions of structure. As the hierarchical components of engineered materials approach the nanoscale, distinctions such as "amorphous" and "crystalline" defy simple application. Yet, these atomic arrangements, for both organic and inorganic material components, may still strongly dictate the nucleation and growth in biological and biomimetic systems. In this context, we present recent scanning transmission soft x-ray spectro-microscopy measurements of amorphous and crystalline calcium phosphate mineral. The synchrotron technique, known as STXM, enables mineral particles imbedded in organic matrix to be mapped with 20-nm spatial resolution, obtaining x-ray absorption spectra at each imaged pixel with 150 meV energy resolution. We will discuss analysis of these spectra in the contexts of experimental challenges and artifacts, and show how STXM can be used in future to delineate mineralized structures. In our calcium phosphate samples, the Ca L-edge spectra of crystalline and amorphous standards are nearly identical. This is very interesting in light of the "Posner cluster" interpretation of bone mineral from decades past, and the present discussions on non-classical nucleation pathways. We will show the extent to which these phases may be distinguished by careful quantitative analysis of the spectra, and discuss the chemical information so obtained.

Probing the early events that determine the nucleation pathway and final mineral structure is one of the challenges in understanding templated growth of biominerals. Much research has been conducted in biomimetic systems using organothiol self-assembled monolayers (SAMs) to template calcite nucleation. Despite the many advances in knowledge regarding templated nucleation, important aspects of the process remain poorly understood such as a quantitative measure of the energetics of directed nucleation and the structural evolution of incipient nuclei. We present in situ transmission electron microscopy (TEM) observations of crystal nucleation and growth in solution at nanometer scale and video rates. This capability is enabled by the combination of a custom designed TEM stage and fluid cell. Significantly, the design of the cell and holder ensures temperature and electrochemical control to initiate reactions of interest, such as the onset of crystal nucleation. We show that calcium carbonate nucleates on a bare electrode via nanoparticles of what appears to be a metastable precursor phase â?" most likely amorphous calcium carbonate â?" followed by consolidation and faceting of the crystalline material, and show how to extend the technique to observe oriented nucleation on organic templates.

To be successful orthopaedic or dental implants must fulfill a unique set of requirements to be used effectively in the oral environment. Here we show the basis of a novel silicate-based biomaterial based around the synthetic mineral clinopyroxenes systems. These are Q2 (metasilicate) chains with a variety of cations (e.g. Mg2+, Ca2+, Na+, Al3+ etc.) acting as counter ions. The various cations are incorporated in the chain of clinopyroxenes resulting in different minerals found in nature, such as diopside (CaMgSi2O6) and jadeite (NaAlSi2O6). These silicate based materials are preferred as dental implants and cements in hard tissue engineering due to their superior biocompatibility and bioactivity. These ceramics can be formed from the stoichiometric melt quenched glass and the extent of nanocrystalisation can, in part, be determined by the heat treatment of these systems. However, the degree of nanocrystalisation, is difficult to determine by conventional x-ray scattering due to Debye-Scherrer scattering. Therefore, in addition to powder X-ray diffraction (PXRD) we will use solid state NMR spectroscopy. The latter technique is an extremely powerful technique for probing both amorphous and poorly crystalline glass ceramics systems, as it is can determine the local structure irrespective of the degree of order present. A series of soda-lime-phospho-alumiosilicate glasses were synthesized and their bioactivity was determined by analysis of ion release profiles of the glass compositions was presented, and the presence of hydroxycarbonated apatite (HCA) formation was evaluated by adding to simulated body fluid (SBF) according to Kokuboâ?~s method.

Protein self-assembly is a commonplace phenomenon in living systems and is frequently responsible for development of one, two and three dimensional functional structures. In nature, matrix protein self-assembly plays a significant role in the growth and organization of the mineral in hard tissues (e.g. tooth and bone). Thus an understanding of the pathways and mechanisms of matrix protein assembly is important for successful hard tissue engineering. While many studies of matrix protein assembly have been performed on bulk solutions, in vivo these proteins are likely to be in contact with charged biological surfaces composed of lipids, proteins, or minerals. Here, we report the results of an in situ high-resolution AFM study of self-assembly by amelogenin - the principal protein of the extracellular matrix in developing enamel - in contact with two different charged substrates: hydrophilic negatively charged bare mica and positively charged 3-aminopropyl triethoxysilane (APS) silanized mica. First we demonstrate an AFM-based protocol for determining the size of both amelogenin monomers and oligomers. Using this protocol, we find that, although porcine amelogenin exists primarily as ~26 nm in diameter nanospheres in bulk solution at pH8.0, it behaves dramatically differently upon interacting with charged substrates and exhibits complex substrate-dependent assembly pathways and dynamics. On positively charged APS-treated mica surfaces, amelogenin forms a relatively uniform population of decameric oligomers which then transforms into two main populations: higher-order assemblies of oligomers and amelogenin monomers, while on negatively charged bare mica surfaces, it forms a film of monomers that exhibits tip-induced desorption and patterning. The present study represents a successful attempt to identify the size of amelogenin oligomers and to directly monitor assembly and disassembly dynamics on surfaces. The findings have implications for amelogenin-controlled calcium phosphate mineralization in vitro and may offer new insights into in vivo self-assembly of matrix proteins, as well as their control over hard tissue formation. The authors gratefully acknowledge funding from the NIH-NIDCR, ARRA (DE-013414S2)

One of the most fascinating aspects of calcite biominerals is their intricate and curved morphology, quite different from the normal rhombohedral crystal habit of geologic calcite. These morphologies are achieved via 2 amorphous calcium carbonate (ACC) precursor mineral phases (1). In this talk we will show that in sea urchin larval spicules two distinct phase transitions occur: ACCH2Oâ?'ACCâ?'calcite (2). Both transitions are significantly slower in biogenic than in synthetic phases (3), hence activation barriers must be introduced by inhibitory proteins. We present a novel assay to determine which proteins exhibit the inhibitory functions in vitro. 1.Y Politi, RA Metzler, M Abrecht, B Gilbert, FH Wilt, I Sagi, L Addadi, S Weiner, and PUPA Gilbert. Mechanism of transformation of amorphous calcium carbonate into calcite in the sea urchin larval spicule. Procs. Natl. Acad. Sci. USA 105, 17362-17366, 2008. 2.AV Radha, TZ Forbes, CE Killian, PUPA Gilbert, and A Navrotsky. Transformation and crystallization energetics of synthetic and biogenic amorphous calcium carbonate. Procs. Natl. Acad. Sci. USA 107, 16438â?"16443, 2010. 3.YUT Gong, CE Killian, IC Olson, NP Appathurai, RA Metzler, AL Amasino, FH Wilt, PUPA Gilbert. Phase Transitions in Sea Urchin Larval Spicules. Submitted.

Bone is a dynamic tissue continuously being adapted to its ambient requirements such as mechanical stimulation and the mineral balance of the body. During the formation of new bone, osteoblasts get embedded in the mineralizing collagen matrix which they synthesize, becoming osteocytes. Osteocytes form a dense network throughout the whole bone tissue â?" the osteocyte lacunar canalicular network â?" which is a direct image of the location of osteoblasts during bone formation. Osteocytes are well known to be the mechanosensor of bone tissue, orchestrating the process of bone formation. Moreover they are considered to be capable of directly interacting with their mineralized perilacunar matrix, hence facilitating an exchange of mineral ions. However, the precise role of osteocytes during mineral homeostasis is still unknown. Investigating the organization of the osteocyte network together with concomitant analysis of submicron mineral properties not only provides information on the dynamics of extracellular matrix and mineral deposition but also improves the understanding about the role of osteocytes during mineral homeostasis. In this regard, we examined several bone types using confocal laser scanning microscopy, polarized light microscopy as well as back-scattered electron imaging (BSE) and found a strong correspondence between the organization of the osteocyte network and the arrangement of the extracellular collagen matrix. From comparing the spatial arrangement of unorganized woven and highly organized lamellar bone, we propose that already initial coordination of osteoblasts is essential in order to form bone material featuring long range organization with highly arranged osteocyte networks. Hereby, modestly arranged woven bone â?" only showing rudimentary osteocyte networks â?" can act as a substrate on which osteoblasts can align and collectively deposit highly organized lamellar bone. Further examination of submicron mineral properties by scanning small angle x-ray scattering (sSAXS) shows that the occurrence of highly arranged osteocyte networks strongly correlates with mineral properties of mature bone material, which is substantially altered in the direct vicinity of the osteocytes. This supports the idea that osteocytes can control bone material properties in their vicinity. In this regard, formation of highly organized bone material accompanying the occurrence of highly organized osteocyte networks is not only important in terms of bone mechanics but furthermore might be crucial for the ability of osteocytes to directly participate in processes of bone mineral homeostasis.

Tyrannosaurid and hadrosaurid long bones were investigated with various electron microscopy and analytical x-ray techniques to further understand the degree of preservation of original cortical bone structural features following millions of years of diagenesis. Modern struthionid and rheid long bones were also investigated. All specimens were obtained from the University of California Museum of Paleontology. Analyzed structural features, ranging from the nano- to the microscale, were selected for their central importance in current structure/property studies of modern cortical bone tissues. Gaining insight into diagenetic consequences for these structural features further creates the possibility of discerning the mechanical properties of dinosaur or other extinct bone tissues by analogy. For analysis, a combination of wavelength-dispersive x-ray spectroscopy and x-ray diffraction was used to determine the bulk mineral phase and composition. Sub-micron compositional heterogeneity of bone mineral was characterized with energy-dispersive x-ray spectroscopy (EDS). Bioapatite nanoplate size ranges and morphologies were determined from a combination of bulk small-angle x-ray scattering curves and localized transmission electron microscope (TEM) images. Microfibril architecture models were constructed from TEM dark-field tomographic analyses. Microscale features, including osteon sizes, osteon densities, and osteocyte lacunae densities, were measured from back-scattered electron images of polished transverse bulk bone sections. All techniques employed in this study have been previously applied to modern bone within the materials community. Our analyses demonstrate that partial conversion of fossil bioapatite to fluorapatite, documented as enrichment in fluorine with simultaneous retention of the apatite phase, occurs in an architecturally conservative fashion. That is, nanoplate sizes and morphologies observed in the dinosaur bones were highly consistent with sizes and morphologies documented for modern bones. Further, despite substantial collagen loss in the fossil bones, the microfibrillar structures remained highly intact. Microscale features were almost universally preserved with diagenetic changes exhibiting clear markers, such as pressure-induced cracking and mineral infilling of porous regions. This work strongly suggests that even the most ancient fossil bones may contain substantial structural detail preserved from the nano- to the microscale levels of the structural hierarchy. This work further demonstrates the potential to infer structure/property relationships in ancient bone tissues that have suffered varying degrees of diagenetic alteration. Such potential has tremendous implications for all materials fields interested in bone quality or biomimetic materials.

Many biological fibers with mechanical function, for example from tendon collagen [1], silk [2], plant cell walls [3], mussel byssus [4] or whelk egg capsules [5], are built in a hierarchical way based on smaller subunits. The structural design of the subunits and â?" even more â?" of the interfaces between them [6] govern the mechanical behavior of these fibers, which simultaneously require reasonable stiffness to transmit loads and toughness to prevent fracture. In many cases, stiffness seems to be achieved by a multitude of weak and reversible bonds, such as hydrogen bonds [2-3] or metal coordination [4], rather than strong covalent binding. The advantage is that these weaker (sacrificial) bonds may break without completely disrupting the entire fiber and, thus, increase toughness either by providing considerable elongation (revealing hidden length by the unfolding of proteins) [4] or by structural transformations [5]. Similar mechanisms have also been proposed to operate in bone [7]. In this paper, we take a thermodynamic viewpoint and model the stress-strain curves of some of the fibers using concepts based on the theory of displacive phase transformations [8]. This highlights the importance of cooperativity in the mechanical action of weak bonds. [1] R. Puxkandl et al, Phil. Trans. Roy. Soc. London B 357, 191-197 (2002); A. Masic et al. Biomacromolecules dx.doi.org/10.1021/bm201008b (2011). [2] S. Keten et al., Nature Mater 9, 359â?"367 (2010). [3] J. Keckes et al., Nature Mater. 2, 810-814 (2003). [4] M.J. Harrington, J.H. Waite, Adv. Mater. 21, 440-444 (2009); M.J. Harrington et al., J. Struct. Biol. 167, 47-54 (2009) [5] A. Miserez et al., Nature Mater. 8, 910 - 916 (2009) [6] P. Fratzl, I. Burgert, H.S. Gupta, Phys. Chem. Chem. Phys. 6, 5575 - 5579 (2004); J.W. C. Dunlop, R. Weinkamer, P. Fratzl, Materials Today 3, 70 - 78 (2011) [7] G.E. Fantner et al., Nature Mater 4, 612-616 (2005); H.S. Gupta et al., Proc. Natl. Acad. Sci. USA 103, 17741-46 (2006).

Hydroxyapatite (HA) [Ca10(PO4)6(OH)2] is the main mineral constituent of human bone. Understanding its interaction with water and amino acids is crucial when thinking of applications in the medical field. Using density functional theory, we have previously studied the energetic, vibrational and electronic properties of HA surfaces. Here we focus on a (100)-oriented surface, terminated with two PO4, three Ca ions and two OH molecules per surface unit cell. This surface has the lowest energy under the experimentally relevant OH-rich conditions. We find a red-shift in the vibrational stretch modes of the OH pairs at the bare surface from 3660 cm-1 in bulk HA to 3570 cm-1. The degenerate OH libration mode at 700 cm-1 in bulk HA splits to 835 cm-1 and 560 cm-1. The structural relaxation due to surface formation decays within the first few Angstroms beneath the surface. We study the interaction between the H2O molecules and this surface for sub-monolayer coverage up to one full monolayer of water and for a 10 Ã. thick water layer. We deduce the vibrational spectrum of the water molecules and further analyze the surface reconstruction due to the water deposition.

Type I collagen is the major organic component of most mineralized vertebrate tissues including Bone, Dentin, and Cementum. In order to evaluate the role of collagen structure in mineralization, we use cryo Transmission Electron Microscopy (cryo-TEM) to evaluate the structure of hydrated collagen fibrils from closely associated non-mineralized and mineralized mouse tissues: Periodontal Ligament (PDL), Cementum, and Dentin. The PDL, a non-mineralized connective tissue, inserts into the bone of the tooth socket and into the mineralized Cementum on the tooth root to anchor root to bone. Collagen fibrils are continuous between PDL and Cementum, yet there is a sharp transition between mineralized and unmineralized tissue. This juxtaposition of mineralized and non-mineralized tissues presents an opportunity to observe differences between collagen fibrils which span a mineralized interface. Using cryo-TEM and image averaging techniques, we show that there are surprisingly few differences in periodicity and banding structure in collagen fibrils from Periodontal Ligament, Cementum, and Dentin, pointing to the role of non-collagenous macromolecules in control of mineralization at this interface.

Well controlled apatite nanocrystlas in bone composites provide the favorable mechanical properties for bone. The organic component of bone acts as an important biomineralization regulator. However, the biological mechanisms of apatite crystal growth and crystal size control have still not been entirely elucidated. The role of small organic molecules such as citrate, which are quite abundant in bone (5 wt% of the organic components), have been somewhat ignored in the search for a simple model to investigate the fundamental mechanisms of the biomineralization. In this work, the influence of supersaturation and citrate concentrations on the crystal growth rates and morphology development have been studied using a combination of macroscopic and conductimetrc constant composition (CC) to provide a new insight into the mechanisms of crystal growth and stability. The apatite crystal growth rate increased from 2.3E-8 mol HAP/(m2.min) to 3.2E-7 mol HAP/(m2.min) with an increase in supersaturation (Ïf) from 8 to 14. At lower supersaturation (Ïfâ?¤9), the crystal growth rate decreased with time but underwent an increase at Ïfâ?¥10. Kinetic analysis of the CC data showed that the effective reaction order changed from 2.0 to 4.4 at a relative supersaturationÏfâ?^9.5. SEM results showed that the crystal morphology changed from nanorods to aggregated ribbons after growth at higher supersaturaiton. This suggests that the crystal growth model underwent a change from surface diffusion control atÏfâ?¤9 to polynucleation control atÏfâ?¥10. In the presence of citrate, the HAP growth rate was markedly reduced. However, the change of crystal growth rate with the citrate concentration is not monotonic; there exists of an inhibition maximum at a specific citrate concentration (8E-5 M). CC data also show that at higher citrate levels (â?¥7.5E-5M), the pH increases in the first 60min indicating citrate involvement in crystal-solution interface layer formation. SEM images showed that by increasing the citrate concentration, the crystal growth mechanism may change from classic step growth to a nucleation growth model. CC data indicate that at lower citrate concentration, the presence of this ion decreases the density of crystal growth sites and surface steps as crystal growth proceeds, thus stabilizing the apatite crystals. This study is a major step forward in our understanding of the model of HAP crystal growth and mechanism of crystal size control and stabilization in vitro. This work was supported by NIH grant DE003223.

Advances in technology demand an ever-increasing degree of control over material structure, properties and function. As the range of properties that can be obtained from monolithic materials is necessarily limited, one potential strategy for the development of new materials is the creation of composites in which two or more dissimilar materials are combined. Biominerals such as bones, teeth and seashells provide a beautiful demonstration that mineral-based composites can be fabricated under ambient conditions and in aqueous solutions. These materials show many unique features which can be attributed to their inorganic/ organic composite structures, which are formed through intimate association of organic molecules with the mineral phase. Further, examination of the ultrastructure of single crystal biominerals has suggested that the organics can be occluded as either isolated particles or a network of gel fibres. In this work we use biominerals, and specifically their occlusion of gel fibres, as an inspiration to generate inorganic/ inorganic composites in which magnetite and zinc oxide nanoparticles are incorporated within single crystals of calcite (CaCO₃). While isolated nanoparticles have been successfully occluded within calcite single crystals, this process relies on the particles being engineered with the appropriate surface chemistries. In contrast, occlusion of gels is subject to far fewer constraints, and effective occlusion can be achieved according to the gel rigidity and the rate of crystal growth. We demonstrate here how we can profit from this behaviour to achieve both efficient and controlled occlusion of inorganic nanoparticles within a single crystal host. By precipitating crystals within a gel formed from bio-polymer coated nanoparticles, we can generate inorganic/inorganic composites in which the nanoparticles are distributed throughout the crystal host. The gel maintains its gross form when entrapped within the crystal, therefore defining the locations of the nanoparticles within the crystal and preventing, for example, the accumulation of nanoparticles at the crystal surface. Further, association of the nanoparticles with the gel serves to significantly increase the quantity of gel that is occluded within the calcite crystals. Finally, measurement of the optical and magnetic properties of the composites shows that the original function of guest nanoparticles can be successfully translated into the composite crystals. This methodology therefore provides a new and flexible method for constructing CaCO₃ â?" based composites, it is envisaged that this methodology could be applied to many other systems, potentially leading to new materials and properties, and further work will investigate the generality of the approach.

Biogenic and synthetic calcite single crystals have recently been reported to have higher hardnesses and fracture toughnesses as compared to geologic calcite. Most investigations to date have focused on the role of macromolecular inclusions in altering the mechanical properties of the inherently brittle single crystals. Other possible origins of the enhanced mechanical properties include an increased defect density as the result of growth from an amorphous precursor phase and solid-solution hardening by magnesium. We have designed a synthetic system that allows us to independently probe the contributions of organic content, growth mechanism, and inorganic impurities on the enhanced mechanical properties of synthetic calcite crystals. In this work, we use nanoindentation to characterize both calcitic prisms from mollusks and synthetic calcite crystals, which were grown in a variety of conditions. Both the biogenic and synthetic composite crystals have increased hardnesses as compared to geologic control crystals, with the biogenic crystals having the highest hardnesses of the group. In all systems, there were significant differences in hardness as a function of tip orientation with respect to the {001} face of calcite. By examining the indent morphologies by SEM, we have identified possible deformation mechanisms, including pressure induced twinning. Insights provided by this work may help to elucidate the intertwined roles of growth mechanism and incorporated organic material in determining the properties of biogenic and synthetic single crystals.

Glucocorticoid therapy is a widespread treatment mostly addressed to the elderly population who are suffering with overactive immune system disorders such as asthma, autoimmune diseases and arthritis. There are several short term (high blood glucose levels, insomnia and euphoria) and long term side effects (Cushingâ?Ts syndrome, glaucoma and cataracts) associate with this particular steroid therapy. One of the most serious long-term side effects of glucocorticoid treatment is secondary (drug-induced) osteoporosis, enhancing fracture risk in bone. The rapid increase in bone fracture risk is indicative both of loss of bone quantity and degradation of bone quality. Reduction in bone quantity can be assessed using bone mineral density (BMD) measurements by clinical tools like DXA and qCT. However, the alterations to the material level properties of bone due to glucocorticoid treatment are not well understood. Understanding the nanostructural origins of increased fracture fragility in drug induced osteoporosis is essential to correlate bone quality alterations in the fibrillar level to mechanical deteriorations. Here we demonstrate alterations in the nanostructural mechanical response of the mineralized fibrillar collagen matrix (quality) and link to an increased fracture risk in glucocorticoid induced osteoporosis. Using bone from a murine model for glucocorticoid induced osteoporosis developed by ethylnitrosurea (ENU) mutagenesis, we measure the deformation of the mineralized fibrils occurring in-situ during external loading, by combining mechanical testing with synchrotron small angle X-ray scattering (SAXS), a form of functional imaging. These provide nano-mechanical parameters of bone quality, including fibril elastic modulus, maximum fibril strain and fibril to tissue strain ratio. A significant reduction (67%) of fibril modulus, enhancement (125%) of maximum fibril strain occurs in osteoporotic mice. We also find a much larger fibril strain/tissue strain ratio in osteoporotic mice compared to the wild type mice. Our study demonstrates the ability of in-situ synchrotron X-ray nanomechanical imaging as a high resolution diagnostic technique to link the changes in steroid-induced osteoporotic bone to local mechanical competence and increases in bone fragility.

Nature provides exquisite examples of hierarchically structured biomineralized composites produced at low temperatures with extreme fidelity. These structures are often controlled by an underlying organic template that serves as a guide for precise nucleation and growth of mineral, which significantly affects its mechanical performance. Here, we examine the mineralization process in an ultrahard tooth found in a mollusk, Cryptochiton stelleri, and uncover the strategy utilizing the organic substructures (i.e., primarily α-chitin) that ultimately arrange the hierarchical design of these abrasion resistant teeth. The dynamic phase and transformation of biomineralization, occurring in multiple stages, has been examined by Synchrotron XRD, μXRF, SEM and TEM. Our analysis shows that 6-line ferrihydrite deposits on the α-chitin fibers distributed within the tooth and subsequently transforms to magnetite. This transformation initiates at the tip of the leading edge and propagates through the tooth to the trailing edge. There is a concurrent increase in particle size increase from 41nm to 74nm between leading and trailing edges of the tooth and is directly related to the spacing between α-chitin fibers. We propose that the smaller particle size and higher particle density at leading edge results from the smaller spacing between fibers, and subsequently results in a higher hardness and modulus, enhancing the performance of the tooth and provides a self-sharpening mechanism.

X-ray diffraction microscopy is a newly developed imaging modality that extends the methodology of X-ray crystallography to allow the structural determination of noncrystalline specimens. Since its first experimental demonstration in 1999, coherent diffraction microscopy has been applied to imaging a wide range of materials science and biological specimens such as nanoparticles, nanocrystals, biomaterials, cells, cellular organelles, viruses and carbon nanotubes by using synchrotron radiation, soft X-ray laser sources, free electron lasers and electrons. Here we applied coherent X-ray diffraction microscopy to the imaging of the mineral crystals inside biological composite materials - intramuscular bone - at different stages of mineralization. Quantitative nanoscale imaging of mineral crystals inside Alewife herring bone at a resolution of 24 nm and 3D spatial analysis of the mineral crystals inside the collagen matrix were performed by using x-ray diffraction microscopy. We identified the spatial relationship of mineral crystals to collagen matrix at different stages of mineralization. Based on the experimental results and our biomineralization analyses, we suggested a dynamic model to account for the nucleation, growth, and orientation of mineral crystals in the collagen matrix at different stages of mineralization. These results indicate the development of three-dimensional architecture of Alewife herring bone, which will not only enable us to obtain a better understanding of the hierarchical structure of bone at the nanometer resolution, but also provide important design principles for hard tissue engineering and the development of biocompatible materials.

Magnetic nanoparticles have been attracting much interest as a labeling material in the fields of biotechnological applications, since they can be conventionally collected with an external magnetic field. Because the size and morphology of magnetic nanoparticles are strongly influence to their magnetic properties, control of these factors is important issue with regard to using them for advanced biological and medical, such as drug delivery, magnetic resonance imaging, magnetic cell separation, and solid-based bioassays. Magnetotactic bacteria provide an ideal model organism for investigating the mechanism of nano-sized magnetite formation including size and morphological regulation, because the cells synthesize highly controlled single crystalline magnetites within the cell. In our previous study, we identified a series of proteins (Mms5, Mms6, Mms7, and Mms13), which are tightly associated with magnetite particles in Magnetospirillum magneticum strain AMB-1. The primary protein function has been suggested to be morphological regulation at the crystallographic level in nano-sized magnetite biomineralization, based on the results of the in vitro experiment. In this study, in order to understand the role of Mms6 protein during magnetite formation, we constructed and analyzed the gene deletion mutant strains of mms5, mms6, mms7, and mms13 by homologous recombination. The crystallographic study of nano-sized magnetite crystals synthesized in vivo was conducted by high-resolution transmission electron microscopy. Surprisingly, the mutant strains were found to synthesize the smaller magnetite particles with uncommon crystal faces, while the wild-type strain synthesized highly ordered cubo-octahedral particles. The average number of particles per cell in the deletion strains was found to be similar to that of the wild type (approximately 20 particles/cell). Moreover, the mutant strains produced elongated particles with high-index faces, which are unable to synthesize by chemical synthetic method. These results suggested that the protein functions in the regulation of surface structure of magnetite during crystal growth. This is the first example of a protein being involved in the regulation of a nano-sized crystallographic structure in in vivo biomineralization. Further elucidation of the mechanism will allow us to design and control size and morphology of magnetic nanomaterials in bacterial cells.

A fundamental characteristic of biological systems is that their organisation and function are based on compartmentalisation. Biomineralisation processes are no exception to this and it has long been recognised that biominerals form within the confines of â?oprivileged environmentsâ? delineated from the organism, where spatial constraints and chemical conditions can be precisely controlled. Despite this, experiments aiming to mimic these processes are generally carried out in bulk solution. This work describes systematic studies investigating the influence of confinement on the crystallisation of calcium carbonate and calcium sulfate. These minerals are each precipitated within the confines of an annular wedge, formed around the contact point of two crossed half-cylinders which were functionalised with self-assembled monolayers (SAMs) of mercaptohexadecanoic acid on gold. This configuration enables a systematic study of the effects of confinement on crystallisation since the surface separation increases continuously from zero at the contact point to macroscopic (mm) separations. The experiments demonstrate that control over the crystal polymorph could be obtained for both crystal systems according to the separation of the cylinders. While oriented rhombohedral calcite crystals formed at 10 μm separations in the calcium carbonate system, amorphous calcium carbonate (ACC) particles were stabilised at submicron separations. Similarly, while the thermodynamically predicted phase gypsum (CaSO₄.2H₂O) was precipitated at large surface separations, the metastable phases calcium sulphate hemihydrate (CaSO₄.0.5H₂O) and amorphous calcium sulfate were observed at decreasing separations. This study therefore shows that the environment in which minerals form can have a significant effect on their stability and demonstrates that metastable phases of calcium carbonate and calcium sulphate can be stabilised by confinement alone. That this can be achieved at such large (micron) separations shows the versatility of this strategy, and its potential value in biological systems.

Biologyâ?Ts unrivalled control over mineral growth is evident in the unique crystal morphologies and remarkable mechanical properties of biominerals. To assert this control over the nucleation and growth, structure, and location of mineral tissue, a widespread strategy in biomineralization is to initiate precipitation in phospholipid membrane-delimitated compartments.[1, 2] These specialized vesicles are typically tens of nanometers to microns in size, and can be used to sequester and transport ions, control nucleation, and confine mineral growth. In vitro studies suggest that crystallization proceeds from a series of intermediates, with the final crystalline polymorph determined by the pH and supersaturation of the medium.[3, 4] Regulation of the chemical environment by the phospholipid bilayer and integral membrane proteins can therefore be used to select the mineralization pathway. How biology uses vesicles to form, stabilize, and shape the mineral precursors prior to crystallization is still not understood. To answer these fundamental questions, we utilize an in vitro model to systematically study the role of vesicles in biomineralization.[5] In this system, aqueous calcium salts are encapsulated within liposomes and calcium carbonate precipitation is initiated by passive diffusion of ammonium carbonate. Using simultaneous small- and wide angle X-ray scattering in conjunction with cryo-electron microscopy, we observe the formation of liposome-encapsulated amorphous calcium carbonate (ACC) nanoparticles up to 200 nm in diameter that do not crystallize for at least 20 h. Similar to strategies that may be employed in biology, we demonstrate that nucleation and growth of the ACC nanoparticles can be regulated by controlling carbon dioxide flux, vesicle size, and the pH of the system. The absence of strong membrane-mineral interaction, observed by cryo-electron microscopy, suggests that ACC is stabilized by confinement in the liposomes, which both limit growth and restrict aggregation. In contrast to bulk precipitation, isolated precipitation inside vesicles offers a unique platform to further study the effects of supersaturation, particle size, membrane chemistry, and co-encapsulated additives on the formation and stability of ACC. An understanding of how organisms use these â?oprivileged environmentsâ? to control crystal growth will inform the synthesis of shaped single crystals, stabilized far from equilibrium. 1. J. H. Collier and P. B. Messersmith, Ann. Rev. Mater. Res., 2001, 31, 237-263. 2. S. Weiner and L. Addadi, Ann. Rev. Mater. Res., 2011, 41, 21-40. 3. D. Gebauer, A. Volkel and H. Colfen, Science, 2008, 322, 1819-1822. 4. E. M. Pouget, P. H. H. Bomans, J. Goos, P. M. Frederik, G. de With and N. Sommerdijk, Science, 2009, 323, 1555-1458. 5. C. C. Tester, R. E. Brock, C.-H. Wu, M. R. Krejci, S. Weigand and D. Joester, CrystEngComm, 2011, 13, 3975-3978.

Organisms produce a large number of minerals in the course of biomineralization. It has been shown that biogenic CaCO₃ is, in fact, a nano-composite mineral containing a very low amount of organic molecules, which is present within the single crystals (intra-crystalline) and in-between them (inter-crystalline). These organic molecules are largely studied for their effect on crystal orientation, morphology and shape. Yet, the understanding of the specific organic-mineral interactions, the mechanisms of incorporation, and the influence of organic molecules on the structure of the inorganic phase is rather poor. In this work, we focus on the effect of the intra-crytalline biomolecules and address the questions of how these organic molecules are incorporated into the lattice of inorganic crystals, and how they affect the crystalline short- and long-range order. For this purpose, CaCO₃ crystals were synthetically grown in the presence of different short peptides. The crystal structure was investigated by means of high-resolution synchrotron powder diffraction techniques (ESRF, APS), single crystal XRD, and ultra-high resolution 3D imaging by TEM tomography. The biomimetic crystals that were grown were found to be structurally similar to the biogenic carbonates. The effect of the different protein domains on the mineral structure is highlighted.

The formation mechanism of the thermodynamically unstable calcite phase, very high Mg calcite, in biological organisms such as sea urchin teeth (SUT) and coralline algae has been an enigma for a very long time. In contrast to conventional methods such as KBr pellet Fourier Transform infrared (FTIR) and X-ray diffraction, FTIR microspectroscopy (FTIRM) provides additional information about local disorder such as an amorphous phase or the occlusion of Mg ions in the calcite lattice. In this work, we characterise for the first time systematically synthetic and biogenic Mg-containing calcium carbonate samples in detail by using two FTIRM instruments and compare these samples with KBr pellet FTIR measurements. Furthermore, we present spectra from geogenic calcite and dolomite minerals, recorded with both FTIRM systems, as well as KBr pellet FTIR spectroscopy as reference. We analyse the spectra by applying multi-peak curve fitting on the in-plane-bending (ν4) and out-of-plane (ν2) bands. Based on the obtained results we attribute the two singlet bands at ~860-865 cm-1 and ~695-704 cm-1 observed in the SUT FTIRM spectra to the existence of amorphous calcium carbonate (ACC), and report for the first time the existence of ACC at the mature end of SUT. In the other three studied biominerals, however, we did not find any ACC. Also, based on the FTIRM results, we observe that not only ν4, but also ν2 shifts to higher wavenumbers if more calcium ions are replaced by magnesium ions in the calcite lattices.

Interplay between soft and hard components in biogenic composites is attracting a great deal of attention of numerous research groups worldwide, who are attempting to deeply understand the source of the improved mechanical characteristics of these natural materials. A large part of bio-composites is built of brittle calcium carbonate crystals (hard ceramic component) surrounded by organic substances (soft component), the latter being primarily consisted of polysaccharides and proteins. Recently, it turned out that some organics are also located within individual crystallites. The function of these intra-crystalline organics is not yet clear. However, we know that they cause anisotropic lattice distortions of crystal unit cells of both aragonite and calcite, the distortions which can be treated in terms of rather high local stresses (about 100-200 MPa). In this paper, we discuss the spatial arrangement of intra-crystalline organics in selected mollusk shells. Specifically, we speak about aragonite crystals in the nacre layer of the Perna canaliculus shell and calcite crystals in the Pinna nobilis shell. Experimental results are obtained by using small-angle X-ray scattering (SAXS) at synchrotron beam lines and scanning transmission electron microscopy (STEM) in the high-angle annular dark field (HAADF) mode. These characterization techniques are based on the so called atomic number contrast (Z-contrast) and are free from diffraction contrast. The Z-contrast between organic and ceramic components is used for visualizing the intra-crystalline organics in both real space (by HAADF) and reciprocal space (by SAXS). Furthermore, by using the electron tomography mode, i.e. taking a series of the HAADF STEM images, when tilting the sample with respect to the incident beam, we were able to reconstruct the 3D-distribution of organic inclusions within individual tablets of the nacre.

Calcium carbonate is the most abundant biogenic mineral, found in geological deposits, marine organisms and is used in various industrial applications. Despite its relevance, a fundamental understanding of the evolution of its crystalline forms is still under debate, in particular since recent reports on non-classical crystallization pathways. In the present study, the existence of stable 0.4-0.6nm calcium carbonate (CaC) clusters in calcium carbonate mineralization is demonstrated, both before and after precipitation, even in undersaturated regimes. For this we used cryo-TEM in combination with a titration set-up, where a dilute calcium source was added to a dilute carbonate/bicarbonate buffer over time, at constant pH and temperature. Although previously characterized as equilibrium species, results from detailed quantification of the titration curve before, during, and after the phase transition instead show that these clusters behave as kinetically trapped, off-equilibrium species. Furthermore, in contrast to the previously proposed neutral CaCO3 clusters, in the present case the chemistry of the CaCO3-cluster is determined to have a Ca/C ratio <1 and to contain at least 50 mol% of bicarbonate. Such a composition implies that the clusters would have a negative charge, which is indeed confirmed by zeta-potential measurements in this work. We further demonstrate that sub-micron sized assemblies of clusters exist already before the nucleation event, which act as a precursor to the vaterite polymorph. We propose a mechanism that explains the transition from clusters via this intermediate cluster-aggregate state into the final calcium carbonate polymorph, including thermodynamical considerations on CaCO3 cluster formation assuming a local supersaturation.

Biopolymers are often found to be occluded in single crystals of biogenic calcite formed through biomineralization by organisms such as sea urchins and mollusks. For example, proteins and cellular tissue networks are occluded in sea urchin spines and plates, which are made of magnesian calcite single crystals, improving their fracture toughness. To gain some insight into the process of biopolymer incorporation on the nano and micrometer scales, we used micelles of a carboxylated block copolymer (PSPMA30-PDPA47) as a model biopolymer and observed their incorporation into growing calcite single crystals in real time by in situ AFM. In calcium carbonate solution, micelles that were otherwise relatively monodisperse in water adsorbed onto the calcite surface in two distinct populations: one consisting of the standard ~20 nm micelles and the other comprised of ~5 nm particles of the block copolymer. The micelles that adsorbed onto the growing calcite surface did not act as common inhibitors or (less common) promoters of growth. Instead the steps passed by the position of the adsorbed micelles without being inhibited, and the continuous passage of steps encapsulated the micelles into the calcite single crystal. In this way, a micelle-calcite single crystal composite was created. However, after the micelles were incorporated, two different outcomes were observed. At the place of incorporation of the ~20 nm micelles, cavities were created that often persisted for the length of the experiment, leading to empty channels in the crystal. Unlike the micelles, these cavities acted as local pinning sites to arrest segments of the advancing steps. In contrast the ~5 nm block copolymer particles left no apparent trace of their incorporation. Companion experiments on mica surfaces in the calcium carbonated solution at pH ~8.5 showed that the negatively charged micelles initially formed at high pH (pH > 9) did not adsorb to the negatively charged (bare) mica surface but adsorbed to the positively charged (polylysine-treated) mica surface. This suggests that the surface- or background solution-calcium ions play a key role in establishing electrostatic adhesion of the micelles to the calcite surface.

Soluble macromolecules are essential to Natureâ?Ts control over biomineral formation and properties. Following early studies where acidic macromolecules rich in aspartic and glutamic acid were extracted from nacre, negatively charged additives have been considered unique in their ability to control calcium carbonate precipitation. In this work, we provide a dramatic demonstration that positively charged additives can cause dramatic changes in calcium carbonate morphologies. It is shown that poly(allylamine hydrochloride) (PAH) can direct the formation of thin films and fibres of CaCO3 analogous to those produced with poly(aspartic acid) via a so-called PILP (polymer induced liquid precursor) phase. The mechanism by which PAH induces these effects is investigated using a range of techniques including cryo-TEM, Raman microscopy and thermogravimetric analysis, and the data obtained shows that hydrated Ca2+/ PAH/ CO32â?" droplets initially form in solution, before coalescing and ultimately crystallizing to give calcite, together with small quantities of vaterite. It is therefore suggested that it is the initial formation of hydrated Ca2+/ PAH/ CO32â?" droplets that is key to this process, rather than a specific polymer/ mineral interaction. These results are discussed in terms of their relevance to biomineralization processes and also highlight the opportunity for using counter-ion induced phase separation of polyelectrolytes as a method for generating minerals with non-crystallographic morphologies.

The formation of calcium carbonate in organisms proceeds in many cases via an amorphous precursor, very often in a confined environment. Due to its low level of long-range order amorphous calcium carbonate (ACC) is extremely moldable and can adapt to a wide range of forms and shapes, which otherwise would not be accessible for crystalline materials in their typical equilibrium morphologies. Hence, a pathway via ACC is vital for many calcium carbonate forming organisms.¹⁾ However, little is known about the influence of the confinement on the microstructure of calcium carbonate or the actual conversion of the amorphous phase into the crystalline form, such as calcite. This is because the in-situ and time-resolved observation of the mineralization processes of single particles is even nowadays a very challenging task; for example, X-ray analysis or Raman spectroscopy methods can only produce data averaged over a large volume. In this work we used electron microscopy based methods, which allow for an in-situ observation of the formation of calcium carbonate crystals and their subsequent growth from a supersaturated solution with a spatial resolution in the nanometer range. The JEOL Clairscopeâ"¢, an instrument combining a scanning electron microscope with a light microscope, enables in-situ study of the precipitation of calcium carbonate from solution, through a 100 nm silicon nitride membrane separating the sample from the SEM vacuum. In these experiments a 10 mM concentration of calcium and carbonate ions resulted in growth rates of 10 nm/s for the exposed CaCO₃ facets. The appearance and disappearance of â?obright spotsâ? was observed in the vicinity of the observed crystals indicating the possible formation and dissolution of amorphous calcium carbonate. To investigate the influence of confinement on the growth of calcium carbonate crystals we analyzed the microstructure of and defects in calcite nano-rods grown in track-etch membranes via an amorphous precursor. Series of electron diffraction patterns along these single crystalline calcite rods revealed that they show twists of up to 4° per µm in rod direction demonstrating a remarkable elastic response of these nanostructure to the growth conditions. Our results help to get a better understanding of the mechanisms involved in the transformation of an amorphous material into the resulting crystals. Reference: 1) L. Addadi, Adv. Mater. 2003, 15, 12

Biomineralization proteins offer biotechnological routes towards multi-functional materials. However, for many well-known proteins and peptides it is difficult to predict their function even in natural mineralization processes. In mollusc shell formation, for example, there exist a couple of regulatory mechanisms involving transmembrane and soluble proteins, carbohydrates and minerals at the interface between the cellular tissue and the structured composite material. We achieved molecular cloning and prokaryotic expression of proteins involved in natural biomineralization processes. These proteins were natively purified and their interaction with calcium carbonate minerals was investigated in vitro using Raman imaging spectroscopy and time-lapse video microscopy. Our data suggest that certain protein domains temporarily interfere with metastable calcium carbonate polymorphs such as vaterite. The structure and function of the organic matrix and its solubility will be discussed in view of potential applications for synthetic mineralization on small and large scales.

The huge diversity of hierarchical micro-/nano- rigid structures existing in biological systems is increasingly becoming a source of inspiration of materials scientists and engineers to create next generation advanced functional materials. Recently, accompanied with the development of nanotechnology, some biologically hierarchical rigid structures have been duplicated and mimicked in artificial materials through hierarchical organization of micro-/nano- building blocks. At first, we will report several facile synthetic protocols for one-pot controlled synthesis of several kinds of unique nano-building blocks, which include ultrathin nanowires, nanoplates, and magnetic nanoparticles, conducting nanocables, nanotubes, and carbon-based nanostructures. Then, we discuss how to assemble these nanobuilding blocks into ordered assemblies as well as their functionalities. Periodic ordered mesostructures of hydrophilic ultrathin nanowire thin films can be produced by Langmuir-Blodgett technique, which show reversibly switched photoelectric properties. In addition, We have fabricated a series of layered double hydroxides (LDH) or montmorillonite (MTM) micro/nano-platelets reinforced free-standing, strong, transparent, and functional layered organic-inorganic hybrid films using series of LDH micro/nano-platelets or MTM as building blocks. These assembled structures based on bio-inspired approaches will find potential applications in different fields. Recent advances have emphasized that it is possible to access a variety of high quality hybrid materials with tunable mechanical property and multifunctionalities.

We are developing several techniques to analyze both create and analyze the adhesive properties of nanoscale fibrillar structures that might be useful as bio-mimetic adhesives. These materials could be useful in surgical tapes, self-cleaning adhesives, and adhesive applications that require samples to be placed under vacuum conditions. A particular interest is the ability of these adhesives to reversibly adhere to other materials. We have fabricated a range of nanoscale fibrils by nanofabrication processes that include molding and directional ion-based etching techniques. We are evaluating the adhesive strength of these structures as a function of process conditions, position within an array of fibrillar structures, and structural variations. These correlations are useful for fine tuning the process conditions for making these adhesive fibrils. Surface modifications are also being pursued to improve the adhesive strength of these fibrils. This presentation will introduce the tools and techniques we are developing to evaluate the adhesive strength of nanoscale fibrillar structures, as well as methods to tune the adhesive properties of these materials.

Co-assembling proteins and polymers holds great potential for creating functional hybrid materials that can be used as platforms for enzymatic purposes, as immunoassays, to tailor cell adhesion and proliferation etc. Implementing functional proteins into block copolymer nano-structures combines the strength and processability of polymer films with the biological function of proteins. In addition, by varying the BCP composition, both the morphology and length-scales can be varied creating tunable long-range ordered nano-structures, which can be used as scaffolds for proteins. The main challenge is however, how to incorporate proteins into polymer films without destroying their functionality such as their enzymatic activity. While the majority of protein/BCP assemblies have been prepared via stepwiseadsorption of proteins from aqueous solution onto prefabricated polymer surfaces, we prepared peptide- and protein-polymer conjugates that could be simultaneously co-assembled with BCPs from organic solutions [1,2]. This co-assembly strategy benefits from enhanced processability, as well as biological activity that is integral to the material rather than appended as a discrete surface layer. We have recently developed two promising methodologies where metalloproteins or enzymes can be readily solution processed with BCP and form long-range ordered arrays in thin films. In the presentation we will review recent results; discuss various strategies, their pros and cons and how they can be used for new exciting biotechnological and nano-medical applications. References: 1. Presley, A. D.; Chang, J. J.; Xu, T. Soft Matter 2010, 7, 172. 2. Presley, A. D.; Chang, J. J.; Xu, T. Submitted

Recombinant proteins have been explored for numerous biotechnological applications. In particular, artificial proteins based on structural repeats of elastin and silk have been investigated for use in tissue engineering and drug delivery applications. Compared to synthetic or natural materials, recombinant proteins have a number of advantages that include: exquisite control over sequence and composition; precise molecular weight; and a modular nature, which allows for the functional domains to be easily altered. We are developing an artificial protein biomaterial based on a structural repeat found in resilin, an elastic protein from insect joints and tendons that contributes to flight and jumping. Resilin has a high strain and can efficiently store energy. In addition, it efficiently recovers after deformation and has a high fatigue lifetime, which makes it ideal for use in repetitive environments. In our work, we used a recursive cloning technique to precisely and easily tune the number of resilin repeats. This technique allowed us to explore the effect of the number of repeats on the protein properties. Our resilin-based proteins display an inverse transition temperature â?" above the temperature, the protein aggregates in solution and below the temperature, the protein is soluble. The temperature can be controlled through protein design â?" increasing either the number of repeats or protein concentration resulted in a lower transition temperature. We are currently investigating the effect of temperature on protein structure and are building a model to predict the inverse transition temperature based on protein characteristics.

Soft materials with a spatially non-uniform composition closely linked to their function are common in nature and often possess a hierarchical architecture with length scales ranging from hundreds of nanometers to several millimeters. Currently available strategies for creating planar soft materials with an organized microstructure mimic natureâ?Ts ability in two ways: by the assembly of predefined building blocks or by planar microfabrication. However, these strategies necessitate a sequence of processing steps and often lack spatiotemporal control. We present a continuous one-step digital approach for the preparation of mosaic and coded planar hydrogels with spatiotemporally varying compositions. The ability to tune the material local composition offers control over its local and bulk properties (elasticity, permeability). This approach is enabled by a scaled-out microfluidic device, composed of 10 PDMS layers that distribute a biopolymer solution (2%w.t. alginate) in the horizontal plane surrounded by streams of a crosslinker fluid (aqueous solutions of CaCl2). The spatially organized biopolymer exited the microfluidic device into a liquid perfused reservoir where the crosslinking reaction took place. Upon gelation, the mosaic planar soft material was wound around a rotating drum, which velocity defined the planar hydrogel thickness from 130-500µm. The precise patterning of various biopolymers onto the base hydrogel was enabled by 7 on-chip reservoirs pressurized by computer-controlled solenoid valves, enabling the incorporation of up to 7 distinct biopolymers. To illustrate the dynamic control over the local composition, we continuously incorporated up to 886Ã-7 bit binary codes. Each bit corresponded to 2nL volume of a secondary biomaterial that locally replaced the base hydrogel. The obtained mosaic hydrogels allowed the local storage or the timed release of colloidal or biomolecular payloads. Elastic moduli and permeability of dextran within mosaic hydrogels composed of a combination of 2%w.t. alginate and 1%w.t. pectin-1%w.t. alginate were measured. We envision the routine fabrication of heterogeneous soft materials with higher structural and compositional complexity. The potential ability to assign to regions of a hydrogel a biomolecular or cellular payload promises the multiplexed incorporation of tailored cellular niches and spatiotemporally controlled molecular release.

Geckos can cling to almost any surface using dense arrays of microscopic hierarchical hairs called setae. The flat, regular, terminal branches of the setae adhere by the van der Waals dispersion force, and the mechanics of the gecko attachment scheme are a current topic among biologists and researchers in smart materials for adhesion. Recent results raise the possibility that gecko setal arrays, and their artificial analogues, can be employed as a ``massively multicontact AFM'' in investigations of surface forces. We studied the adhesion and friction behavior of natural gecko arrays on customized Si/SiO2/octadecyltrichlorosilane (OTS) substrates which can have different van der Waals interactions produced by stratification. Adhesion is significantly stronger when the SiO2 layer is thin (~2 nm), even in the presence of an OTS outer layer. Friction experiments demonstrate the presence of velocity-strengthening state-rate friction over the range of velocities $10^{-2}$ to $10^{2}$ mm/s. The role of setal morphology in determining the overall performance of setal arrays will also be discussed.

Many geometric patterns are seen in the natural world, including patterns that take form spontaneously by self-assembly. Among these, the wrinkle structure is very commonly seen on the surface of a leaf, in a ripple, in sand, on skin, or in the pleats of curtains. At the same time, additional mechanical factors in the outer layer of our cells play a large role in the formation and the morphogenetic process of our bodies and tissues. The concave-convex surface structure of wrinkles in the brain and that of folds in the intestines are both determined by the mechanical balance between the surface and cytoskeleton. In our study, these morphogenetic processes are mimicked and the surface buckling phenomenon is used to create a variety of minute wrinkle structures on the surface of silicon rubber. We discussed the fabrication of unique wrinkle patterns using polydimethylsiloxane (PDMS) as elastomeric substrate covered by a hard layer coating. We have succeeded construction of complex topological wrinkle patterns inspired by the brain wrinkle, using new 3D axial stretching method. These various wrinkle patterns offers the opportunity to creating hierarchically organized surface and various-sized polystyrene (PS) nanoparticle for the fabrication of micro-robot, brain-shaped structure for cell culture and superhydrrophobic surface, and constructing surface-enhanced Raman scattering (SERS) surface with a highly sensitivity.

Collagen is a major structural protein found in a wide variety of connective tissues. It has a broad range of functions, one of which is providing integrity and mechanical strength throughout its hierarchical organization. Instability or weakness of collagen at the molecular and/or microscopic levels of assembly can lead to serious connective tissue diseases. Additionally, collagen is widely used as a biomaterial, including acting as a scaffold for cell growth in tissue engineering. Given that mechanical properties are related to the structure of materials, the main goal of our research is to understand how molecular structure correlates with microscale mechanical properties of collagen solutions and networks. Here, we investigate the mechanical ramifications of one possible sequence change in collagen: the removal of its non-helical ends (so-called telopeptides). Various studies have shown that telopeptides play an important role in fibril formation. Their removal slows down the kinetics of fibril assembly and changes the final fibril morphology. Also, the physical characteristics of gels made from collagen with and without telopeptides are changed at the macroscale. However, it is not yet clear how the existence of these short telopeptides alters mechanical properties at the microscale. In order to address this question, we probe viscous and elastic properties of collagen solutions at the micron scale using optical tweezers and a technique known as microrheology. Microrheology studies the time-dependent viscoelastic response on the micron scale by analyzing the motion of tracer particles within the material of interest. Optical tweezers can be used for microrheology and offer many advantages over conventional particle-tracking approaches. Optical tweezers employ a highly focused beam of laser light to hold and manipulate a micron-sized particle, producing readouts of its position at high bandwidths (~100 kHz). In addition, since the particle is locally confined, a specific region of solution is probed at all time points. In this study, we obtain the local complex shear moduli of collagen solutions using optical-tweezers-based microrheology. Probing the concentration dependence of viscoelastic response, we find that collagen solutions with and without telopeptides exhibit elasticity of comparable strength to viscosity when the concentration reaches ~5mg/ml. This elasticity may arise from physical and/or chemical chain-to-chain interactions, possibilities we explore here. We find that the presence of telopeptides alters the viscoelastic response of collagen solutions, particularly at high frequencies, and we seek the cause for this difference in response. Using frequency-dependent viscoelastic models, we evaluate possible models to address the role telopeptides play in conferring viscoelastic behavior to collagen solutions.

In recent years, it has been found that the activity of gelatinase could be responsible in the proteolytic process of fibronectin (Fn), resulting in the release of the Fn fragments. It is also believed that those fragments contribute to the mineralization of extracellular matrix (ECM) by stabilizing the collagen triple helix, which becomes better mineralized when partially denatured. Therefore we carried out research to understand the molecular mechanisms involved in the regulation of fibronectin proteolysis and their effects on osteoblast-mediated biomineralization. Preliminary results show that a mild 50°C heat treatment induced a normally weakly mineralizing subclone of murine pre-osteoblast cells (subclone 24 MC3T3-E1) to form mineral nodules after 21 days of exposure in simulated body fluid (SBF). This confirms that denaturation plays a critical role in the mineralization process in our system. We then hypothesize that the activity of latent collagenase within the Fn molecule may be the underlying cause of collagen stabilization. We are currently investigating Fn interactions and proteolysis in MC3T3-E1 subclone 24 cells as well as strongly mineralizing subclone 4 cells, using gelatin zymography and radiolytic protein footprinting. Understanding the role of Fn fragments in the mineralizating process of type I collagen can assist in the development of novel pharmacological agents for the treatment of bone-released diseases.

It has long been known that the physical and chemical properties of substrate surfaces can affect the biofunction of adherent cells. However, the processes behind why these properties have such an effect are unknown. The major protein responsible for cell-substrate interactions is the Extracellular Matrix (ECM) protein, fibronectin (Fn). It is also an essential component of the bone ECM and may have regulatory roles in osteoblast-mediated biomineralization. Fn is extremely sensitive to modulations in surface chemistry which can induce different conformations of fibronectin, thus exposing cryptic binding sites to proteases in the microenvironment. The main goal of our research is to understand how proteolytic events in the ECM affect Fn cleaving and the involvement of Fn fragments in cellular functions. Using standard tissue culture polystyrene (TCPS) and plasma-etched polydimethylsiloxane (PDMS), we observed that substrates which impeded Fn fibrillogenesis significantly increased osteoblast motility but impaired mineralization. We hypothesize that the surface chemistry can influence the conformation of adsorbed Fn in such a way as to render it more susceptible for proteolysis by matrix metalloproteinases (MMPs). Currently we are investigating the role of MMP-2 in the proteolysis of Fn, which may regulate osteoblast migration and ECM mineralization. Live cell time-lapse microscopy, gelatin zymography, and radiolytic protein footprinting experiments are being carried out to determine the binding interaction between MMP-2 and Fn. Elucidating the molecular mechanisms involved in the regulation of Fn proteolysis can assist in the understanding of bone-related diseases.

Immediately following vascular injury the blood coagulation system is initiated in an attempt to prevent excessive blood loss. The result of this intricate enzyme-based activation cascade is the formation of a protein polymer network comprised of the protein fibrin. This polymer network, also known as a blood â?~clotâ?T or thrombus, seals the vascular injury site. Because of their vital importance in hemostasis, fibrin fibers have become the subject of significant research in the biomedical engineering field. Individual fibrin fibers provide the structure and integrity of thrombi and must be able to resist physical forces generated by blood flow and cellular invasion. Due to this role, one of the main questions that has arisen in the field is: What gives fibrin fibers their overall elasticity? Several researchers have begun to investigate the overall elasticity of fibrin fibers. However, recent interest has been focused on understanding the mechanical behavior of fibrin fibers with emphasis at the molecular scale. We have developed a new mathematical and steered molecular dynamics (SMD) model that can be used to determine the mechanical response of fibrin fibers under applied load. The model can be used to determine the force vs. strain response of single fibrin fibers considering contributions at the molecular scale from two main sources of the fibrin fiber: 1) the fibrinogen structure and 2) the alpha-C region. The force response of the fibrinogen structure has previously been obtained using steered molecular dynamics simulations in [1]. The alpha-C region, due to its two-component composition of a connector (alpha-C connector) and domain (alpha-C domain) was modeled using a worm-like chain (WLC) model and a steered molecular dynamics simulation, respectively. All three components were combined to create a model that represents the bulk mechanical behavior of the fibrin fiber, and these were compared to AFM experimental results obtained from [2]. Good agreement was obtained for the model when compared to the AFM results. The results from this study suggest that the model can be used to ascertain the various contributions on fiber strain from the different sources (fibrinogen and alpha-C region) and also can be used to determine force vs. strain response for fibers with different porosity values. 1. Lim, B.B.C., et al., Molecular Basis of Fibrin Clot Elasticity. Structure, 2008. 16(3): p. 449-459. 2. Liu, W., et al., The mechanical properties of single fibrin fibers. Journal of Thrombosis & Haemostasis, 2010. 8(5): p. 1030-1036.

Advances in technology demand an ever-increasing degree of control over material structure, properties and function. As the properties of monolithic materials are necessary limited, one route to extending them is to create a composite by combining contrasting materials. The potential of this approach is beautifully illustrated by the formation of biominerals where organic macromolecules are combined with brittle minerals such as calcite to create crystals with considerable fracture toughness. This talk will discuss a number of bio-inspired approaches leading to crystals with composite structures. 200 nm polymer particles have been entrapped within calcite single crystals by a simple one-pot method, using the particles as soluble additives. Very high levels of occlusion can be achieved, generating crystals containing over 20 vol% of particles, according to the particle surface chemistry and reaction conditions. The occlusion of copolymer micelles, which with diameters in the range 20-30 nm fall in the same size regime as intracrystalline proteins, was also investigated. These experiments also provided us with a unique opportunity to perform a systematic and quantitative analysis of the influence of entrapped, soluble macromolecular additives on the microstructure of calcite single crystals from atomic to micron length scales, and therefore to correlate the microscopic structure of our â?oartificialâ? biomineral with its macroscopic properties. A range of techniques including IR spectroscopy, high resolution powder XRD and high resolution TEM were used to compare the structures of these crystals with calcite single crystals of geological and biogenic origin and these â?oartificial biomineralsâ? were shown to exhibit many properties in common with single crystal biominerals. The influence of these changes in the microscopic structure of the calcite lattice on the macroscopic properties of the crystals was also evaluated by measuring the mechanical properties of the nanocomposite crystals using nano-indentation methods and it was shown that the nanocomposite crystals were approximately 16 % harder than their synthetic counterparts. We have also recently extended this strategy to generate inorganic/ inorganic composites by incorporation of inorganic particles such as magnetite and gold within calcite.

Biomineralization of atmospheric or dissolved CO2 by CaCO3 precipitation is a fundamental process in the global carbon cycle. Bacterial Surface layers (S layers) play a central role in biological CO2 capture and storage. However, the mechanistic details of the biomineralization process are only poorly understood. In order to have a platform on which to study this process at the single molecule level, we immobilized S-layer proteins on solid supports (mica, lipid bilayers, silicon nitride, etc) and perform structural characterization by ex-situ and in-situ atomic force microscopy (AFM) and transmission electron microscopy (TEM) in liquid flow cells. Our results show that the self assembly of S layer proteins can be tuned by the degree of hydrophilicity of the exposed solid surface. Moreover, by tracking in-situ the structural properties of CaCO3 nanostructures as they growth on self assembled S layers by AFM (using a liquid cell for the flow of NaHCO3 and CaCl2 to promote CaCO3 nucleation) we shed light on the role of surface S layer proteins in the mechanism of calcium carbonate formation. Finally, by integrating the structural AFM and TEM data with the dynamic chemical characterization achieved by X-ray spectroscopy, we aim to broaden our understanding of the process of CO2 mineralization. This will pave the way to ultimately engineering organisms that can accelerate CO2 sequestration and to explore potential routes to low-carbon emission energy technologies.

The development of â?obottom-upâ? crystallization strategies to fabricate crystalline inorganic materials patterned on the micro- and nanometer scale is of great technological significance. It is well-known that biological systems are good at producing a wide range of crystalline minerals with intricate architectures. Here we report on monolayer colloidal crystal (MCC) templating routes toward CaCO3 crystals with two-dimensional (2D) ordered patterns via a bio-inspired approach. MCCs floating at a solution surface were employed as templates for the facile fabrication of honeycomb-patterned thin films of amorphous calcium carbonate (ACC) and calcite via colloidal lithography at the gas/liquid interface. Honeycomb-patterned ACC (HP-ACC) thin films were fabricated by a polymer-induced ACC coating of the MCC template at the air/water interface and subsequent controlled crystallization of the HP-ACC thin films led to honeycomb-patterned, crystalline films with different microstructures. The highly ordered HP-ACC thin films possessed brilliant structural colors and exhibited typical photonic properties. On the other hand, MCCs covering the surface of calcite single crystals were used as templates for the epitaxial growth of calcite single crystals with tunable surface patterns. The morphology of the patterned surface can be readily adjusted from bowl-arrays to diamond-arrays by controlling the growth parameters. Notably, unique concave micolens arrays of calcite with a single crystal structure were successfully fabricated by this method. Moreover, the obtained 2D patterned calcite surface can be employed as templates for the vapor deposition of plasmonic crystals with controlled patterns.

Objective Modern conservative dentistry preserves tooth structure and minimizes restoration. Dentin forms the bulk of the tooth and it is challenging to remineralize dentin caries. Like bone, dentin is a hydrated biological composite of collagen I fibrils reinforced with apatite within the fibrils (Intrafibrillar) and between them (extrafibrillar). Restoration of the mechanical properties of carious dentin requires appropriate reintroduction of the mineral into both compartments. Recent concepts in biomineralization are providing new approaches to this problem and our research seeks to translate such new approaches to functionally remineralize carious dentin, i.e. restore the mechanical properties of the hydrated tissue as well as the mineral content. Materials and Methods Artificial dentin lesions produced by buffers at pH=5.0, produced two zones: an outer highly demineralized zone and at greater depths, a gradient zone of increasing mineral until normal levels are reached. Lesions were remineralized in calcium and phosphate containing solutions using either 1) constant composition to maintain mineralization conditions at specific degrees of saturation (CC) or a polymer-induced-liquid-precursor (PILP) process using poly-L-aspartic as the precursor inducing agent. Mechanical property profiles were obtained by nanoindentation (E_R and H) and mineral profiles by micro x-ray computed tomography. Cross sections were studied using SEM/ EDS and TEM/SAED at differing depths and remineralization times. Results Successful remineralization for CC and PILP occurred without surface precipitates and proceeded from the gradient toward the outer zone. With CC, indentation modulus (E_R) recovered to at least 60% of normal dentin at all lesion depths, mostly in the first 24 hours. The PILP process promotes mineralization without the presence of remnant nuclei, and gave 50-60% property recovery in the outer zone, and complete recovery in the gradient zone between 14 and 28 days. Interestingly, mineral level was complete in both zones, implying important structural differences. The two remineralized zones had distinct morphologies and TEM/SAED revealed intrafibrillar oriented apatite with crystal growth over time. Conclusions Significant portions of artificial carious lesions can be functionally remineralized by classical crystal growth strategies and/or using new approaches such as the PILP strategy. This may lead to functional remineralization of natural carious lesions. Acknowledgements NIH/NIDCR Grant R01DE016849

Bone grafts are used in orthopaedic reconstructive procedures to provide mechanical support and promote bone regeneration. Bone is organized into two different organizational structures: trabecular and cortical bone. Cortical bone is composed of tightly packed units, called osteons that are 200 µm wide and are composed of concentric layers of mineralized collagen fibers with Haversian canals in the center. Few attempts have been made to fabricate scaffolds that mimic the cortical organization of the bone. The objective of this study was to successfully fabricate three dimensional (3D) electrospun scaffolds that mimic both cortical and trabecular sections of the bone. Osteon-like scaffolds were fabricated by electrospinning PLLA-gelatin mixture onto rotating poly (ethylene) oxide (PEO) micro-fibers, which were then dissolved to create channels inside the osteon-like scaffolds. Individual osteon-like scaffolds were packed together and heat sintered into 3D scaffolds to resemble cortical segments. For the scaffolds mimicking trabecular segments, electrospun mats were cut into 1cm wide strips and wrapped around a 24-gauge needle into 3D cylinders. Complete scaffolds containing both segments were fabricated by creating trabecular core scaffold and surrounding it with osteon-like segments. All the scaffolds were then heated to 54°C to sinter the layers together. Scaffolds were mineralized by incubation in 10X simulated body fluid (SBF) for 6hr and 24hr. We successfully fabricated osteon-like scaffolds that were 200-500µm in diameter and consist of concentric layers of PLLA-gel nanofibers with a channel running along the scaffold length. The individual osteon-like scaffolds were found to support cell attachment, proliferation and mineral production in vitro. The resulting cortical scaffolds consisted of packed osteon-like scaffolds with series of parallel channels along the scaffold length, thus mimicking the native organization of cortical bone. Mineral deposited on the scaffold was identified as mixture of brushite and hydroxyapatite, naturally found in bone. After 24 hr of mineralization in 10XSBF, cortical scaffolds had significantly higher percentage of mineral (20.18%±2.54) than trabecular scaffolds (7.86%±2.67%). This could be due to presence of the channels in cortical segments and exposed surface area. We also found that the mechanical properties increased with increased mineralization, but the mineral distribution was not uniform. Complete scaffolds that contained both segments were successfully fabricated. The resulting scaffolds consisted of a 4 mm diameter trabecular core, surrounded with packed osteon â?"like scaffolds to total a 6 mm diameter, resulting in a 2:1 of trabecular to cortical ratio. Mineralization of scaffolds up to 48 hr resulted in increased compressive mechanical properties. In vitro evaluation of scaffolds is ongoing.

The beauty of butterflies has fascinated humans over centuries. Of the many genera of butterflies, perhaps one of the best known is the genus Morpho from Central and South Americas. From the early days, it was understood that the color of Morpho butterflies is due to the interference within the multilayered ridges on the scales that cover the surface of their wings. However, structural colors due to multilayer interference change as the angle of either illumination or observation is varied. Yet the paradox of Morpho butterflies is that their color appears to remain constant over a wide range of viewing angles while still maintaining the bright, metallic iridescence. Investigation into this paradox has been the subject of much research, and by now, there is a general agreement that Morpho wings need to be understood as a complex collection of structural elements, with both order and disorder in scales ranging from nm to mm, which all work together to produce the overall appearance. Unfortunately, such complexity has so far been challenging to reproduce in a man-made structure. But realizing a more complete model structure that can be easily fabricated would not only help us better understand the photophysics of Morpho butterflies, but also allow us to move beyond mimicry to expand the capacities of the Morpho structure. In this paper, we use a combination of a silica microsphere baselayer and a dielectric multilayer to produce such a structure. To fabricate the structure, we first spin-coat a random mixture of silica microspheres with diameters ranging from ~200 nm to ~400 nm on a Si wafer to form a loosely packed monolayer. A 300 nm thin film of Cr is then deposited to provide a uniform, dark background in order to mimic the melanin-containing scales and base layer of Morpho butterflies. Finally, 8 pairs of TiO2 and SiO2 layers, whose thicknesses ranged from 30 to 200 nm to control the resulting structural color, were then sputter-deposited at low pressure to induce directional deposition and to complete the structure. The resulting structure reproduces nearly all of the major aspects that have been reported to be central for producing the bright, angle-independent iridescence of Morpho butterflies â?" nm-scale multilayers with strict thickness control for color selection, mm-scale disorder among the reflecting elements for angle-independence, and mm-scale disorder on the surface for glittering, non-specular appearance. We find that the fabricated thin film not only reproduces the bright, saturated color of Morpho butterflies, but also provides better color and brightness stability over wider viewing angles and directions. Expanding on this structure, we move beyond mimicry by creating a flexible thin-film color reflector that, unlike real Morpho wings, can be bent and folded freely and yet retains its Morpho-mimetic photonic properties.

Cellulose nanocrystal aerogels are a renewable material with a unique microstructure and impressively high mechanical properties, making them attractive as a potential low cost renewable alternative for fiber reinforced polymers with potential applications such as plastic casings for cell phones and laptops. Unfortunately, the sensitivity of cellulose aerogels to temperature and oxygen limits their ability to be incorporated into polymers which require high temperature processing (>200 °C) in oxygen containing environments. Atomic layer deposition (ALD) allows surface limited deposition of highly uniform and conformal thin films of inorganic oxides over high aspect ratio porous and large surface area structures. Thin ALD Al₂O₃ coatings have been shown to make effective oxygen and water permeation barriers. In this work, we form a hybrid organic/inorganic nanocomposite by conformally coating cellulose aerogel scaffolds with a thin layer of oxide. Using tri-methyl aluminum and water, approximately 8 nm layer of Al₂O₃ was coated onto a cellulose aerogel produced from wood. Energy dispersive x-ray spectroscopy indicates the penetration of Al₂O₃ into the aerogel. Thermogravimetric analysis reveals that cellulose nanocrystal aerogels coated with Al₂O₃ by ALD have improved temperature and oxidation resistance, which should allow for an extended processing window for incorporation of cellulose nanocrystal aerogels into polymers. It is anticipated that the hard, wear-resistant Al₂O₃ coating should result in a nanocomposite with improved mechanical properties, increased allowable processing temperature and improved barrier properties.

Biological minerals have sophisticated morphologies and functions originating from inorganic/organic composite structures. We can observe mesocrystal structures consisting of oriented nanocrystals with organic macromolecules in various biominerals, such as nacreous layers, sea urchin spines, and eggshells. Recently, a number of biomimetic minerals having the mesocrystal structures similar to that of biominerals have been prepared in aqueous solution systems. These mesocrystals have attracted much interest as a nonclassical crystallization behavior and their potential as functional materials. Here, we show the property of the biological and biomimetic mesocrystal structures and its application to the host for organic molecules and inorganic ions. The oriented nanocrystals regarded as mesocrystal are fundamentally observed in various biominerals. The mesocrystal is essential as a building unit for the biological inorganic architectures. We succeeded in in vitro repairing of sea urchin spines and prismatic layers with the mesocrystal structure in the presence of a soluble organic macromolecule. This fact suggests the role of the mesocrystal in construction of the complex architectures. The mesocrystal structures have the amphiphilic nanospace among the nanocrystal units. Thus, various materials were prepared by the combinations of host mesocrystals and guest organic molecules. The specific photoluminescence properties, photochemical reactions, and polymerization were observed on the materials. Moreover, the biomimetic mesocrysals are applicable to electrochemical devices such as electrodes and sensors, because their porous body has both high crystallinity and high specific surface area. The mesocrystal structure was found to exhibit a good performance as electrodes of lithium-ion battery.

The concept of â?~mesocrystalâ?T, abbreviation of Mesoscopically Structured Crystal, developed by Cölfen and Mann, 2003 (see also Cölfen and Antonietti 2008), involves the three-dimensional crystallographic organization of nano or submicrometer-sized particles into a highly ordered mesostructure. Yet, structural properties of this new class of solid materials are not fully described. Biominerals commonly display complex hierarchical mesocrystalline organizations and bring new insights on properties of mesocrystals. Two types of biomineral structures found in the red coral have been studied: the axial skeleton and the sclerites. 1 - In the red coral skeleton, â?~building blocksâ?T are arranged into eight hierarchical levels of similarly (but not identically) oriented modules. Each module is made of an organized array of sub-units, and is at the same time a sub-unit of a larger module. This crystallographic organization is observed down to a few nm. Thus, the concept of â?~mesocrystallineâ?T organization applies to the red coral skeleton; we add to this concept the notion of â?~multilevel modularityâ?T. EBSD and TEM studies show that the degree of crystallographic misorientation between the building blocks decreases with decreasing module size. Thus, the transition from imperfect crystallographic order at mm scale to nearly perfect single crystalline domains at nm scale is progressive. 2 - Sclerites (also called spicules) are small grains of Mg-rich calcite found in the living tissues of Corallium rubrum and other octocorals; they are particularly appropriate to decipher the principles of crystallographic organization in biominerals and explore the relationships between morphology and crystallography. Sclerites are made of well separated submicrometer crystalline units (ca 80 nm); on the other hand, EBSD studies show that these crystalline units are similarly oriented with only a low degree of misorientation between them. Thus, the concept of â?~mesocrystalâ?T applies also to sclerites. High resolution EBSD data show that quite unexpectedly slight misorientations of crystallites are not at random but result from rotations around the three equivalent 'a' axes of the calcite hexagonal unit cell. This observation leads to the concept of â?~misorientation orderingâ?T. Whether the concept applies to other natural or synthetic mesocrystals is expected but remains to be demonstrated. Cölfen, H., and Mann, S. (2003) Higher-order organization by mesoscale self-assembly and transformation of hybrid nanostructures. Angewandte Chemie-International Edition, 42(21), 2350-2365. Cölfen, H., and Antonietti, M. (2008) Mesocrystals and nonclassical crystallization. 276 p. John Wiley & Sons, Ltd.