Biomineralisation relies on the ability of an organism to control the structure and form of a solid phase that it can use for a specific purpose. This control takes place at the boundary between fluid and solid, nearly always mediated by some organic compound. Traditional analytical methods give information about the properties of the bulk solid or the bulk fluid that can be used to make interpretations about what goes on at the interface between the two, which is often only a few molecular layers thick. Computational methods have added considerably to understanding the relationships between components on both sides of the boundary but by combining experiment and theory, we can advance much further in our understanding. Analyses made with nanotechniques, that can "see" at the molecular level, provide data that can be used directly for calibrating simulations. Likewise, theory can provide information that makes it possible to more fully interpret experimental data. By combining the two approaches, we can begin to understand some of the processes that organisms use to tailor their environment.
Atomic force microscopy (AFM) allows us to map the topography and other physical parameters at the nanometre scale. By functionalising the AFM tip with organic compounds, one can map adhesion or elasticity over surfaces in contact with fluids and one can monitor their response as the fluids are changed. X-ray photoelectron spectroscopy (XPS) provides information about the composition and bonding environment in the top 10 nm of a solid and ultrarapid cooling of a wet sample vitrifies the adsorbed water, allowing the solid-fluid interface to be characterised. Field emission scanning electron microscopy (FE-SEM) and energy dispersive X-ray spectroscopy (EDXS) provide images with chemical mapping and using a focussed ion beam (FIB) to sequentially mill through a sample, one can create 3D images that show the internal structure and composition at submicrometer scale. X-ray nanotomography (XNT) also provides 3D images without destroying the sample, allowing us to determine physical properties and how they change with time or exposure to solutions, temperature or pressure. Several other synchrotron radiation methods provide a range of other types of information such as atomic structure, particle size and crystal size. Applying these techniques on calcite and chalk, with complementary modelling using density functional theory (DFT) and molecular dynamics (MD), has provided interesting new insight into the processes that affect coccoliths, the hard parts formed by some species of algae. For example, combining nanoscale observations with computational results has proven that organic molecules bind to calcite through -OH functional groups and this bond is much stronger than water, allowing organisms to block some crystal sites, thus favouring growth in a desired direction. Other work has shown that only a few percent of Mg²⁺ adsorption can change calcite surface tension dramatically.

The interface between soft and hard materials is currently underexplored territory which is critical to underrated structure relationships with the hard/soft materials interface domain. You want high resolution of the hard material, but good contrast (and resolution) of the corresponding soft materials. Low-voltage Electron Microscopy has several advantages: increased cross-sections for inelastic and elastic scattering and hence higher contrast efficiency from each atom and reduced radiation knock-on damage to samples insensitive to other damage mechanisms, which includes most metals, semiconductors and other solid state materials.
Historically, the use of higher TEM voltages were favored since they reduce the spherical and chromatic aberration effects, but the development of spherical aberration correctors has allowed atomic resolution at lower voltages. Although the TEM samples must be significantly thinner for low keV observation, the improvement in contrast for inorganic materials, biological samples and especially nano-biological samples in low-voltage TEM while retaining atomic resolution cannot be understated.
The fundamental aspects of electron microscopy all relate directly to the physics of the interactions between the electron beam and sample. Energetic electrons are described as “ionizing radiation” - the general term used to describe radiation that is able to ionize or remove the tightly bound inner shell electrons from a material. This is obviously an advantage for electron microscopy in that it produces a wide range of secondary signals such as secondary electrons and X-rays, but is also a disadvantage from the perspective that the sample is “ionized” by the electron beam and possibly structurally damaged, which depending on the accelerating voltage happens in a number of different ways.
Low-Voltage High-Resolution Electron Microscopy has several advantages (and of course disadvantages), including increased cross-sections for inelastic and elastic scattering, increased contrast per electron and improved spectroscopy efficiency, decreased delocalization effects and reduced radiation knock-on damage. Together, these often improve the contrast to damage ratio obtained on a large class of samples. 3rd order aberration correction now allows us to operate the TEM at low energies while retaining atomic resolution, which was previously impossible. Using the spherical aberration corrector in conjunction with electron monochromator for example at 40 keV takes the user surprisingly close to the lower bound imposed by fifth-order spherical aberration, and enables imaging with an information limit better than 1Å, and a workable resolution of better than 1.4Å.

Thermal resistance at hard-soft material interfaces has been one of the most important bottlenecks for the improvement of thermal transport properties of nanocomposites which are critical for a wide range of applications like thermal interface materials, nanofluids and nanoparticle-assisted therapeutics. Conventional strategies have focused on improving adhesion of the interface to increase thermal conductance. Here we demonstrate a significant enhancement of thermal transport across the hard-soft material interfaces consisting of gold and amorphous polyethylene (PE) by functionalizing the gold surfaces with self-assembled monolayers (SAM). Our transient thermoreflectance (TTR) measurements show remarkable increases by as much as 7 times in the thermal conductance of gold-hexadecane (HD) and gold-paraffin wax (PW) interfaces. Surprisingly, such significant increases are realized despite the observed decrease in the interface adhesion energy when the gold surface is functionalized. Our molecular dynamics (MD) simulations reveal that the SAMs chemically absorbed on the gold surface act as media to bridge the vibrational mismatch (acoustic mismatch) between gold and polymer and thus enable efficient resonance-like thermal transport. Such a strategy can be generalized to any hard-soft material interfaces to provide a design principle that can be used to synthesize nanocomposites for heat transfer applications.

Novel materials based on various environmentally responsive polymers hold multiple important applications. However, the functionality of these materials alone is often limited, and thus the integration of these, with other functional materials like Silicon, is strongly desired. Here we demonstrate the capability of integrating thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) hydrogels with Silicon nanoribbons. This will enable the stiff silicon ribbons to become adaptive and drivable by the soft environmentally sensitive substrate, such as becoming mechanically stretched and compressed when inducing a temperature change. Furthermore, we investigate how advanced lithographic techniques can be used to generate patterned deformation on the above integrated structures. We also explore bi-layer hydrogel structures formed by the integration of different types of polymers of PNIPAAm. These structures have been determined to achieve tunable curvature under the influence of different stimuli. The integration of soft materials with traditional hard semiconductor materials could have interesting implications for the development of novel “smart” devices especially in bio-medical aplications.

Hard-soft interfaces are ubiquitous in energy-relevant systems. Examples include the subterranean mineral-fluid interfaces that govern carbon sequestration, the electrode-fluid interfaces in batteries and supercapacitors, and the fluid-solid interfaces at which heterogeneous catalysis takes place. For many years, we have studied mineral-fluid interfaces with a combination of molecular (e.g., X-ray reflectivity, quasi-elastic neutron scattering and neutron spin echo) and bulk (e.g., titration) experimental probes closely integrated with molecular dynamics simulations using fluid-solid forcefields derived from ab initio calculations. Recently, as part of the activities of the Fluid Interface Reactions Structure and Transport (FIRST) Engineering Frontier Research Center (EFRC), we have extended this approach to the study of interfaces encountered in batteries, supercapacitors, and heterogeneous catalysis. The FIRST Center performs fundamental research on fluid-solid interfaces based on the premise that the next generation of electrical storage devices with superior performance will require a fundamental knowledge of the nanoscale architecture of the interface, the effect of nanotexture on interfacial properties, and the structural and dynamic changes that occur during charge and discharge cycles. In this presentation, we will provide an overview of our research on the molecular-level modeling and experimental characterization of energy-relevant hard-soft interfaces.

An on-going challenge in materials synthesis is purposeful control over crystallite size, shape, and microstructure. Our research focuses on the mechanisms by which crystals form, grow, crystallize, and transform, with particular focus on particle mediated crystal formation, crystal growth, and phase transformations. Oriented attachment is a non-classical crystal growth mechanism that can result in twinning as well as the incorporation of defects like dislocations and stacking faults. This mechanism can be exploited to produce nanocrystals with unique and symmetry-defying shapes. Generally speaking, crystal growth typically occurs by a combination of mechanisms. For example, oriented attachment is a particle mediated crystal growth mechanism, but experimental data consistently demonstrate that crystals concomitantly grow by dissolution and reprecipitation. We employ cryogenic transmission electron microscopy (TEM) and in situ TEM, in combination with conventional TEM, dynamic light scattering, X-ray diffraction and scattering, UV-visible spectroscopy, as well as kinetic modeling, to elucidate the fundamental processes that govern the kinetics of crystal growth. Each of these techniques has advantages and limitations, and a combination of methods is crucial to push our understanding of nonclassical crystal growth forward.

The non-covalent interaction of biomolecules with materials interfaces and/or nanoparticles is of great interest due to potential applications such as material synthesis, biosensing and nano-medicine.¹ These applications have prompted significant interest in the field, however, to fully realize these applications, a deeper understanding of the interfacial interactions occurring at the molecular level is required. Molecular dynamics (MD) simulations, with the ability to predict and reveal interactions at the atomic level, can play a contributing role in elucidating the structure/property relationships of such systems.²
Here, we investigate the interaction of both individual amino acids and peptides at the aqueous graphene interface using a polarizable force-field specifically developed to model the interaction of biomolecules with graphitic nanostructures.³ The free-energy of adsorption of all twenty naturally-occurring amino acids has been determined from meta-dynamics simulations.⁴ Furthermore, we model the adsorption of peptides known to bind to the basal plane of graphene,^5,6 and mutant analogues. Traditionally, MD simulations of peptides at interfaces have struggled to ensure adequate sampling of the conformational space. We address this challenge via use of the replica exchange with solute tempering (REST) technique.^7,8 Our results allow the binding propensity of the individual amino acids to be compared against the binding propensity of residues within peptides. This provides valuable information regarding how the peptide sequence influences the interfacial binding properties, moving us closer to the ultimate goal of de novo design of materials-selective peptides.
[1] Grey, J.J., The interaction of proteins with solid surfaces, Curr. Opin. Struct. Biol., 2004, 14, 110-115.
[2] Tang, Z., et al., Biomolecular recognition principles for bionanocombinatorics: an integrated approach to elucidate enthalpic and entropic factors, ACS Nano, 2013, 7, 9632-9646.
[3] Hughes, Z.E., Tomásio, S.M. and Walsh, T.R., Efficient simulations of the aqueous bio-interface of graphitic nanostructures with a polarisable model, Nanoscale, 2014, 6, 5438-5448.
[4] Laio, A. and Parrinello, M., Escaping free-nergy minima, Proc. Natl. Acad. Sci. USA, 2002, 99, 12562-12566.
[5] Kim, S.N., et al., Preferential Binding of Peptides to Graphene Edges and Planes, J. Am. Chem. Soc., 2011, 133, 14480-14483.
[6] So, C.R., et al., Controlling Self-Assembly of Engineered Peptides on Graphite by Rational Mutation,ACS Nano, 2012, 5, 1648-1656.
[7] Terakawa, T., et al., On easy implementation of a vairent of the replica exchange with solute tempering in GROMACS, J. Comput. Chem., 2010, 32, 1228-1234.
[8] Wright, L.B. and Walsh, T.R., Efficient conformational sampling of peptides adsorbed onto inorganic surfaves: insights from a quartz binding peptide, Phys. Chem. Chem. Phys.,2013, 15, 4715-4726.

An area of growing interest is the development of noble-metal metamaterials because of their unique properties[1,2]. Development of versatile generation strategies for production of these metamaterials remains challenging; these materials comprise assemblies of nanoparticles of different compositions, arranged in 3-D arrays; fine control over the interparticle spacings in these arrays is essential. It is also highly desirable that these arrays can be dynamically reconfigured. As the first steps towards realizing these goals, we investigate Au- and Ag-binding peptide sequences conjugated with light-switchable azobenzene moieites, for the purpose of designing stimuli-responsive biomolecule linkers that can ultimately facilitate assembly of different types of nanoparticles into 3D arrays. Here, we use advanced sampling molecular dynamics simulations[3] to investigate the molecular conformations and materials-binding properties of these molecules. These peptide sequences have a light sensitive thiol-maleimide azobenzene thiol-maleimide (MAM) unit conjugated onto either the N- or the C-terminus of the peptide. We have also carried out well-tempered meta-dynamics[4] simulations to estimate the free energy of binding, of the MAM unit alone, at the Au and Ag aqueous interfaces. Our results indicate that the MAM unit binds more strongly at Au compared with Ag, with the trans conformation of the MAM binding more strongly than the cis for both metals. Our simulations of the N- and C-conjugated peptides reveal that the presence of the MAM unit can significantly affect the adsorbed structures and conformational dynamics of the surface adsorbed peptide. Our findings provide guidance in the design and development of a stimuli-responsive biomolecule linker for nanoparticle assembly purposes[5].
[1] P.-Y. Chen, J. Soric and A. Alu, Adv. Mater., 2012, 24, OP281-304.
[2] K. L. Young, M. B. Ross, M. G. Blaber, M. Rycenga, M. R. Jones, C. Zhang, A. J. Senesi, B. Lee, G. C. Schatz and C. A. Mirkin, Adv. Mater., 2014, 26, 653-9.
[3] T. Terakawa, T. Kameda and S. Takada, J. Comput. Chem., 2011, 32, 1228-34.
[4] A. Barducci, G. Bussi and M. Parrinello, Phys. Rev. Lett., 2008, 100, 020603.
[5] K. L. M. Drew, Z. Tang, J. P. Palafox-Hernandez, Y. Li, M. T. Swihart, C. -K. Lim, P. N. Prasad, M. R. Knecht and T. R. Walsh, in preparation.

Silica nanostructures are biologically available and find wide applications for drug delivery, catalysts, separation processes, and composites. However, specific recognition of biomolecules on silica surfaces and control in biomimetic synthesis remain largely unpredictable. A silica force field is introduced that resolves numerous shortcomings of prior silica force fields over the last 30 years and reduces uncertainties in computed interfacial properties relative to experiment
from several 100% to less than 5%. In addition, a silica surface model database is introduced for the full range of variable surface chemistry and pH (Q2, Q3, Q4 environments with adjustable degree of ionization) that have shown to determine selective molecular recognition. The force field enables accurate computational predictions of aqueous interfacial properties of all types of silica, which is substantiated by extensive comparisons to experimental measurements. The parameters are integrated into multiple force fields for broad applicability to biomolecules, polymers, and inorganic materials (AMBER, CHARMM, COMPASS, CVFF, PCFF, INTERFACE force fields). We also explain mechanistic details of molecular adsorption of water vapor, as well as significant variations in the amount and dissociation depth of superficial cations at silicaminus;water interfaces that correlate with zeta;-potential measurements and create a wide range of aqueous environments for adsorption and self-assembly of complex molecules.
The systematic analysis of adsorption free energies and binding conformations of distinct peptides to silica surfaces as a function of pH and particle size will be specifically reported as a prime example for validation and specific predictions. Example peptides were positively charged, neutral, and negatively charged, and a variety of silica surfaces were employed. The computed binding affinities agree remarkably with adsorption isotherms and zeta potential measurements for the same systems, and underline the significance of the surface chemistry, pH, and topography for specific binding outcomes. Adsorption free energies and binding residues were quantitatively analyzed, and tunable contributions to binding identified, including ion pairing, hydrogen bonds, hydrophobic interactions, and conformation effects. The resulst show that molecular dynamics simulation with the CHARMM-INTERFACE force field and synthesis can be employed to optimize interactions of all types of silica surfaces with organic and biological molecules under realistic solution conditions at the scale of 1 to 100 nm. Applications include the controlled binding and release of drugs, cell receptors, polymers, surfactants, and gases.

Controlled peptide adsorption at aqueous metallic interfaces is relevant to many bio-inspired applications including self-assembled materials, biosensors, and nano-catalytic materials. To realize these applications, a greater depth of understanding of the underlying principles governing peptide adsorption is required. Recently, a comprehensive study of peptide binding affinity at aqueous gold interfaces identified certain residues as behaving as strong-binding ‘anchor&’ points (1). This study also suggested that the local environment of these anchoring residues could modulate the binding propensity of these residues. Here, we use the known peptide sequence Z1, KHKHWHW (2), to systematically investigate how a small change to the sequence environment, namely the position of the histidine protonation site, impacts on adsorption at the aqueous Au (111) interface, employing state-of-the-art replica-exchange simulation approaches (3). The sequence characteristics of Z1 allow us to methodically probe these positional and environmental effects (4). We find that the position of the protonated histidine drastically modifies the modes of adsorption of this peptide at the aqueous Au interface, producing non-local effects that can modulate the behavior of non-neighboring residues. We also find that the location of the protonated histidine could either promote or disrupt co-operative binding of the peptide as a whole. These findings contribute to the on-going generation of systematic rules that will ultimately facilitate greater control over peptide-materials binding.
(1) Tang Z.; Palafox-Hernandez J. P.; Law C. W.; Hughes Z. E.; Swihart M. T.; Prasad P. N.; Knecht M. R.; Walsh T. R. ACS Nano 2013, 7, 9632
(2) Peelle B. R.; Krauland E. M.; Wittrup K. D.; Belcher A. M. Langmuir, 2005, 21, 6929.
(3) Terakawa, T.; Kameda, T.; Takada, S. J. Comput. Chem.2011, 32, 1228.
(4) Palafox-Hernandez J. P.; Walsh T. R. in submmition, 2014.

Crystallization of inorganic (and organic) matter often proceeds via intermediate stages, rather than by simple nucleation and growth mechanisms.These precursor stages not only comprise crystal modifications that are less stable than the final one, but also amorphous, hydrated (nano-) particles and emulsion-like precursors have been observed. These precursors tend to aggregate or restructure before being dissolved and entering the next structural stage.[1,2]
Structural information on all these intermediates - and by which mechanisms they form - is essential for the development of additives to control crystallization processes - either to achieve particles with a certain size distribution, with certain functionalities or to impede crystallization in water treatment processes.
Data on the structural evolution of precipitating calcium carbonate and other systems obtained by means of X-ray microscopy and quench cryo-transmission electron microscopy will be presented, emphasizing that the respective particle formation processes do not follow classical nucleation and growth mechanisms.
The role of polymers in nucleating, templating, stabilizing and/or preventing these structures is outlined, together with possible applications. Since charged polymers, like polycarboxylates, not only interact with the inorganic precursors and crystals but also with the cations right from the beginning of the crystallization it is essential to understand the details of this interaction. Time-resolved molecular modelling experiments on the complexation mechanisms of calcium to polycarboxylates unravel an unexpected richness in the binding process.
[1] Rieger, J.; Kellermeier, M.; Nicoleau, L.; Angew. Chem. Int. Ed. 2014 in press.
[2] Gebauer, D.; Kellermeier, M.; Gale, J.D.; Bergström, H.; Cölfen, H.; Chem. Soc. Rev. 43, 2014, 2348.

Regulating the ice nucleation has broad implications in a variety of areas such as cryopreservation of cells and tissues, prevention of the freezing of crops, cloud seeding and snow making etc. Although it is well recognized that ice nucleation usually initiates at the liquid-solid interfaces, current fundamental understanding of nucleation in general and ice nucleation in particular is insufficient. Thus it remains a great challenge to tune the ice nucleation.This talk discusses the tuning of ice nucleation via modifying the solid surfaces with antifreeze proteins (AFP), which play an irreplaceable role for the survival of many living organisms in subzero environments. Moreover we found in our experiments that the structure of the interfacial water is crucial in tuning the ice nucleation. Our results not only complements the widely accepted mechanism of AFPs, that is, AFPs inhibit the growth of microscopic ice crystals at subzero temperatures via absorption-inhibition. Also the results shed a new light on the fundamentals of the ice nucleation, which is critical for the promotion or suppression of the ice nucleation.

Bio-composites provide inspiration for the design and generation of new synthetic high-performance composites. For example, in nacre, proteins play an important role in exerting polymorph control of CaCO₃, stabilizing the aragonite polymorph¹ over the more favorable calcite. How this stabilization works at the molecular level is unknown, due to our lack of understanding of how the protein structure(s) relate to the function in this context. Determination of these protein structures particularly when adsorbed at the mineral interface, is now possible, but challenging to accomplish in practice².
A derivative of one such protein known as n16N, an intrinsically disordered peptide, has been experimentally shown to stabilize aragonite at a model tri-layer interface comprising chitin, an n16N overlayer and the mineral³; all of which approximate the interfaces found in nacre. Chitin is chiefly found in two polymorphs, α- and β-chitin. Experiments show that only β-chitin, in the presence of the adsorbed n16N overlayer, is capable of conferring polymorph control (i.e. aragonite formation)³. Experiments also suggest that n16N preferentially binds to β- over α-chitin³. Having previously successfully predicted the ensemble of structures of n16N in solution⁴, here we build on these insights to investigate the structures of n16N adsorbed at the aqueous α- and β-chitin interfaces, to determine the origins of this selectivity and reveal the exposed sites on the peptide potentially responsible for aragonite stabilization.
Using molecular dynamics simulations, namely Replica Exchange with Solute Tempering (REST)⁵, we provide insight into the chitin polymorph binding selectivity of n16N. The interplay between the n16N sequence, structure and function at the chitin interface is further investigated via umbrella sampling simulations of 9 representative amino acids adsorbed at both the (100) and (010) facets of the α- and β-chitin aqueous interfaces. Our findings are not only key to understanding the mechanism by which n16N can facilitate the formation of aragonite, but are also applicable to the potential use of chitin nanoparticles as a drug delivery vehicle.
1. S. Collino and J. S. Evans, Biomacromol., (2008), 9, 1909-1918
2. P. A. Mirau, R. R Naik and P. Gehring, J. Amer. Chem. Soc., (2011), 133, 18243-18248
3. E. C. Keene, et al., Cryst. Growth Des. (2010), 10, 5169-5175.
4. A. H. Brown, P. M. Rodger, J. S. Evans , T. R. Walsh, (2014), In preparation
5. T. Terakawa, et al., J. Comput. Chem., (2011), 32, 1228-1234

Calcium sulfate is a highly abundant natural mineral that finds extensive application as raw material in construction processes. It can exist in three major crystalline polymorphs, or rather hydrates, which differ essentially in their respective water content: gypsum (the dihydrate, CaSO₄#8729;2H₂O), bassanite (the hemihydrate, CaSO₄#8729;0.5H₂O), and anhydrite (the anhydrous form, CaSO₄). All of these three phases are used as key components in products like mortars, plasters, binders or adhesives. Thus, it is desirable to get access to either of the different polymorphs and control their formation in, ideally, straightforward reactions at low temperatures. Under ambient conditions, precipitation of calcium sulfate from aqueous solutions readily affords gypsum, the thermodynamically stable form, while bassanite and in particular anhydrite can only be obtained at significantly higher temperatures. Therefore, the current practice to produce these phases is (partial) dehydration of gypsum by heating at 100-150°C, an energy- and cost-intensive process.
In the present contribution, we show that both bassanite and anhydrite can also be obtained at room temperature by means of a simple precipitation step in organic media. To that end, aqueous solutions of calcium sulfate were reacted with an excess of an organic solvent like ethanol, giving well-defined bassanite nanoparticles. Both the yield and kinetic stability of these particles can be tuned by adjusting certain experimental parameters, such as the final water content in the reaction medium. In this way, either phase-pure bassanite or defined mixtures with gypsum can be prepared. Furthermore, below a certain threshold in the water content, a gradual transition in the product composition from bassanite to anhydrite is observed so that, again, either phase-pure materials or binary mixtures of the two polymorphs are formed. Taken together, the collected data allow for a precise control of calcium sulfate polymorphism under ambient conditions and even enable the formulation of phase diagrams for predicting product composition as a function of distinct process parameters. Our results thus provide an alternative low-temperature route for the synthesis of bassanite and anhydrite, and furthermore highlight that organic solvents - which are often used to quench crystallization reactions - can actually induce the formation of metastable phases rather than freezing any ongoing processes.

Soft matter organization under hard confinement can be fundamentally different from that obtained in thin films or in the bulk. Nanoporous hard templates provide a two-dimensionally confined space in which self-organization processes such as crystallization, protein secondary structure formation, mesophase formation and phase separation can be altered. Herein we employ self-ordered nanoporous aluminum oxide (AAO) made by the electrochemical anodization of aluminum substrates as the inorganic model matrix that provides the required uniformity in diameter/length, thermal stability and resistance to organic solvents. Understanding the self-assembly, thermodynamics and dynamics of soft materials under confinement will allow for their rational design as functional devices with tunable mechanical strength, processability, electronic and optical properties.
A principal focus of this work is finding the basic underlying principles that give rise to directed self-assembly and controlled phase state in a range of soft materials under confinement. This includes the synthesis of hard templates, subsequent infiltration, surface functionalization and surface characterization as well as structural, thermodynamic and dynamical characterization in a number of soft materials with different type of interactions. These encompass crystallizable polymers [1-4], amorphous polymers [5], amphiphilic molecules, liquid crystals [6,7] and biopolymers [8] with important potential applications. These studies addressed the effect of confinement on: (a) the type of nucleation (homogeneous vs. heterogeneous), the size of critical nucleus, crystal orientation and the possibility to control the overall crystallinity; (b) the segmental and global polymer dynamics; (c) the nematic-to-isotropic, crystal-to-nematic and columnar liquid crystal-to-crystal phase transitions in rod- and disk-like liquid crystals, respectively and (d) the self-assembly and dynamics on α-helical polypeptides.
[1] H. Duran, M. Steinhart, H.-J. Butt, G. Floudas, Nano Letters 11,1671, 2011.
[2] Y. Suzuki, H. Duran, M. Steinhart, H.-J. Butt, and G. Floudas, Soft Matter 9, 2769, 2013.
[3] Y. Suzuki, H. Duran, W. Akram, M. Steinhart, G. Floudas and H.-J. Butt, Soft Matter, 9, 9189, 2013.
[4] Y. Suzuki, H. Duran, M. Steinhart, H.-J. Butt and G. Floudas, Macromolecules 47, 1793, 2014.
[5] S. Alexandris, G. Sakellariou, M. Steinhart, G. Floudas, Macromolecules (DOI: 10.1021/ma5006638) 2014.
[6] H. Duran, M. Steinhart, H.-J. Butt, G. Floudas, Nano Letters 11,1671, 2011.
[7] C. Grigoriadis, H. Duran, M. Steinhart, M. Kappl, H.-J. Butt, G. Floudas ACS Nano 11, 9208, 2011.
[8] H. Duran, A. Gitsas, G. Floudas, M. Mondeshki, M. Steinhart, W. Knoll, Macromolecules (Commun.) 42, 2881, 2009.

Interest in the molecular scale processes underlying the onset of mineral formation is on the rise due to the detection of nanoscale ion aggregates in concentrated aqueous solutions. As the archetypal system for “pre-nucleation” clusters, the early stages of calcium carbonate have been intensely scrutinized from both theoretical and experimental perspectives. The definition of a pre-nucleation cluster, initially described as being relatively constrained in size and thermodynamically stable with respect to both dissolution and growth [1], is currently evolving and is now identified with a rather broad distribution of aqueous species whose continued growth is restricted by diffusion limitation [2]. This reinterpretation is due in part to the results of molecular dynamics simulations that suggest an exponentially decaying cluster size distribution rather than monodisperse cluster species [3]. The simulated size distribution is also consistent with the results of cryo-TEM [4] and the predictions of classical nucleation theory (CNT). This work [5] uses atomistic and coarse-grained simulation techniques to explore the formation of clusters from supersaturated solutions. The results of molecular dynamics simulations indicate the accessibility of a metastable liquid-liquid binodal/spinodal. Coalescence and partial dehydration of the dense liquid droplets results in the formation of a solid phase whose structure is consistent with amorphous calcium carbonate. Coarse-grained simulations of fluid-fluid separation in the spinodal regime produce cluster size distributions that are qualitatively similar to those produced from molecular dynamics simulations of spontaneous phase separation in the CaCO₃ system [3]. The presence of a dense liquid phase of CaCO3 is also supported by recent experimental efforts [6], which suggest an entropy driven phase transition may precede solid CaCO₃ formation under certain conditions.
[1] Gebauer et al. (2008) Science322 1819-1822. [2] (2012) Faraday Discuss.155, 139-180. [3] Demichelis et al. (2011) Nat. Commun. 2, 590. [4] Pouget et al. (2009) Science323 1455-1458. [5] Wallace et al. (2013) Science341 885-889. [6] Bewernitz et al. (2012) Faraday Discuss.159 291-312.

Protein crystallisation is a vital process towards the success of rational drug design for treatment of diseases.¹ However, obtaining protein crystals of high quality to determine their complex structure is non-trivial. Therefore, the design of nucleant materials to positively direct protein crystallisation is sought. Here, by tailoring properties through the functionalisation of several nanocarbons including commercial and in house MWNTs and SWNTs, we have systematically assessed how these materials direct and promote protein crystallisation.
Two complementary techniques have been utilised for nanocarbon functionalisation resulting in a library of anionic, cationic, hydrophilic and hydrophobic nucleants. The versatile nature of nanocarbons has enabled us to tailor the nucleant chemistry and as a result several proteins were crystallised including five model proteins: lysozyme, thaumatin, trypsin, haemoglobin and catalase. The crystallisation of target proteins has also been investigated.
References
1. E. Saridakis and N. E. Chayen Trends in Biotechnology, 2009, 27, 99.

We present our recent collaborative studies of colloidal nanoparticles with active ligands that can control the nanoparticle behavior. In particular, we show that 1) nanoparticles of different sizes can have different solvation properties, 2) self-assembly and positioning of nanoparticles at the interfaces of different ionic solvents can be controlled by electric fields, 3) pH can control the self-assembly of nanoparticles into exotic superstructures, and 4) ligands can determine the biological responses or nanoparticles. We use atomistic molecular dynamics simulations to capture the unique characteristics of these novel systems and explain their properties through the physical, chemical, and biological processes taking part at their ligands.

We describe the synthesis of micron-scale patterns of surface-chemical functional groups on thin films of elastomeric polymers and illustrate the use of these soft structures in hard material crystallization. Using elastic deformations, such as those caused by tensile strain, we reconfigure critical features (e.g., geometry and molecular density) of these “soft” surface-chemical patterns enabling dynamic control of surface energy and the arrangement of functional groups. We use this capability to direct the surface nucleation and growth of hard, crystalline materials that possess functional (e.g., electronic or optical) properties. We further modify the morphology and structure-property relationships of these crystals by rationally manipulating their spatial arrangement through reversible deformations of the elastomeric films. Specifically, we report the surface-chemical patterning of silicones, such as polydimethylsiloxane, and the use of these materials as dynamic substrates for the mineralization of metal chalcogenides and oxides. We are inspired by the soft, active materials (e.g., cellular machinery) that produce sophisticated hard-soft composites in biomineralization processes; however, we do not attempt to replicate the mechanistic complexity found in these systems. Instead, our approach focuses on simple analogs—chemically patterned two-dimensional soft surfaces in this case—that mimic important characteristics (e.g., dynamic surface chemistry) found in biology. This approach eliminates many of the constraints presented by biology and simplifies experimental design and interpretation. The hybrid hard-soft materials we report are difficult to fabricate directly using other approaches and their physical properties are potentially applicable to soft sensors and electronics.

Crystallization plays a vital role in many fields like biomineralization, production of pharmaceuticals, scale and gas hydrate formation in oil and gas pipelines etc. Controlling the nucleation and growth aspects of crystallization is of utmost importance to achieve a better understanding of the processes and to improve the efficiency of the systems. Here we suggest a novel way of achieving this by designing a solid-liquid hybrid surface - a solid textured surface imbibed with a liquid. The properties of the impregnating liquid can be modified to alter the nucleation and growth rates of the crystals on the surface. We use gypsum as a model system to demonstrate the advantages of these impregnated surfaces. The use of a lower surface tension liquid that completely spreads on the solid surface results in a lower nucleation rate of the gypsum crystals. The growth of the salt crystals is also dependent on the crystal orientation, which in turn can be controlled by suitably modifying the liquid. The flexibility offered by these surfaces in terms of the solid texture and the impregnating liquid can be used to tune the different aspects of crystallization with high precision.

Calcium carbonates are the most ubiquitous functional biomineral in nature. Microscopic living organisms direct CaCO₃ precipitation from aqueous ions with an unprecedented level of control and this underpins a vast array of Earth system processes, including the global carbon cycle. For example, cocolithophores internally mineralize CaCO₃ into single crystal calcite frustule plates that are structurally highly complex suggesting a complicated, but controlled chemical route between dissolved ions and final calcite morphology. Being able to elucidate and mimic, such biomineralization mechanisms, would not only increase our knowledge about important processes supporting a vast array of marine planktonic life forms, but would also provide us with a powerful tool to nanoengineer complex (bio)inorganic materials.
Here we report on a study that mimiced such processes through the use of water-in-oil microemulsions. Microemulsions increasingly gain interested in colloid chemistry because they offer a confined reaction environment ideal for synthesis of nanoparticles of low polydispersity and well-defined shapes. Water-based micelles stabilised by an interface surfactant are typically 1-10 nm in diameters and each carries dissolved ions and upon mixing and collision the individual micelles exchange their contents. Thus, they are well-suited for mimicking the nanoscale mineral precipitation at the polar/non-polar interface. We used such microemulsions as bioinspired nanoreactor systems and mimiced CaCO₃ mineralization. We followed the reaction upon mixing of two initally clear microemulsions (containing either [Ca²⁺] or [CO₃^2-]) and quantified the gradual development of a white precipitate using ex situ and in situ methods.
Our ex situ data showed stabile nanoparticles (Oslash; ~10 nm) of low-polydispersity that eventually formed large aggregates (Oslash; ~250 nm). Time-resolved, in situ small angle X-ray scattering demonstrated a slow but progressive agglomeration of liquid-like, ion-carrying micelles to larger mass-fractals (Oslash; ~ 100 nm ) of high fractal dimension (D_f >2.8). Our liquid-cell TEM results confirmed that these liquid phase aggregates remain stable and only upon destabilization did a transformation to crystalline phases take place.
By combining these results with the fact that an individual micelle can accommodate in its water core only a limited number of reacting ions, we hypothesize that these micellar mass-fractal-like aggregates likely contain only liquid-like CaCO₃ complexes stabilised by the confinement and interfaces and not CaCO₃ solid particles per se. Only once the micellar large aggregates are destabilized is crystallization induced.

Carbonates are ubiquitous materials that can either have a positive or negative impact on life and the economy. Biomineralisation and carbon sequestration are examples of areas where the formation of carbonate minerals have a positive effect, while the formation of scale in pipes is a global industrial problem, which causes down times and major losses to chemical and oil extraction companies.
In recent years the appearance of calcium carbonate stable clusters and of a dense liquid phase before the onset of nucleation has been proposed and verified, both experimentally and computationally [1-3]. This has led to a new picture of the formation pathway of carbonate minerals emerging. Initially, the ions aggregate to form dynamic chain like structures [1,2] (DOLLOP) that, upon increase in the ion activities, undergo a liquid-liquid phase separation to form a dense liquid phase [2]. This dense liquid phase progressively looses water and precipitates in the form of amorphous calcium carbonate, which eventually transforms into one of the commonly found anhydrous polymorphs.
This phenomenon is believed not to be limited to calcium carbonate, but to be a general growth mechanism for the formation of many minerals [4], such as the other alkaline earth carbonates, sulphates and phosphates. Here large-scale computer simulations can provide a unique opportunity to test this hypothesis . A carefully parameterised force field, which was calibrated against the thermodynamics of the species in solution and in the crystalline phases [2,5], has indeed been already used to study this very phenomenon for the calcium carbonate system [2]. Here we will present our results on the development of new force fields for the study of alkaline earth metal carbonates in aqueous solution and their application to investigate the existence of prenucleation clusters in these systems.
[1] Gebauer et. al. (2008) Science. 322, 1819-1822.
[2] Demichelis et. al. (2011) Nat. Commun. 2, 590.
[3] Wallace et. al. (2013) Science, 341, 885-889.
[4] Gebauer et. al. (2014) Chem. Soc. Rev., DOI: 10.1039/C3CS60451A.
[5] Raiteri, et al. (2010) J. Phys. Chem. C, 114, 5997- 6010.

Many biological minerals are synthesized from metastable precursor nanophases, which are concentrated at the site of mineral deposition and subsequently converted to a stable biomineral composite. These transient precursors are first synthesized within lipid membrane vesicles, which facilitate the formation, stabilization, transport, and deposition of the metastable material. Organisms&’ use of metastable precursors within liposomes is well documented, but the mechanism(s) of stability and the role of the lipid membrane chemistry on the mineralization process remain poorly understood. Using dioleoylphosphotidylcholine (DOPC) liposomes as a model system for intravesicular mineral precursor formation, we have previously shown that membrane confinement alone can stabilize single amorphous calcium carbonate (ACC) nanoparticles for over 24 hours.[1] Recently, we have extended this model to giant liposomes 10-100 mu;m in diameter.[2] These liposomes enclose over six orders of magnitude more volume than biological vesicles, yet ACC particles formed in the absence of any additives do not crystallize for over one week. We attribute the (meta)stability afforded by liposomal confinement to the exclusion of strong heterogeneous nucleators and the resulting high nucleation barriers for crystalline polymorphs.
In nanoscale biological vesicles, the high surface-to-volume ratio of a liposome permits extensive interaction with a mineral phase contained within. Membrane surface chemistry is therefore an important consideration in the pathway from precursor to stable biomineral. In both small and giant liposomes, ACC was distributed throughout the liposome lumen just after formation and was not intimately associated with the DOPC membrane. However, altering the methylation of lipid headgroups, or introducing biologically important lipids like phosphatidylethanolamine and phosphotidylserine drastically alters the degree of membrane interaction, morphology, and stability of ACC. Large populations of thousands of giant liposomes, isolated from highly concentrated suspensions, were observed with polarized light microscopy and characterized with confocal Raman spectroscopy to study the rates of ACC crystallization within liposomes of different lipid compositions. From these rates, kinetic barriers for crystallization and the relevant interfacial energies were determined. These data will help guide experimental and computational investigations of ACC in complex, but biologically relevant conditions.
1. Tester, Chantel C. et al. Faraday Discussions 159 (2012): 345-356.
2. Tester, Chantel C. et al. Chemical Communications 50.42 (2014): 5619-5622.

Acorn barnacles like Balanus amphitrite develop complex, protective shells surrounding their soft tissues. On the sides, the shells are comprised of multiple, interlocking plates enabling vertical and circumferential expansion throughout the life of the organism; on the top, there are two movable plates enabling feeding when open and protecting the barnacle from predation and from dehydration during intertidal cycles when closed. Many acorn barnacles also have a mineralized base plate underneath. The shells are composed primarily of calcium carbonate, and unlike mollusks, in the form of calcite rather than aragonite. We have examined the initial formation of calcified structures in juvenile and adult barnacle base plates using a variety of optical and x-ray spectroscopies including x-ray tomography, high resolution transmission electron microscopy (HR-TEM), atomic force microscopy-based Fourier transform infrared spectroscopy (AFM-IR), micro-Raman spectroscopy, and scanning electron microscopy (SEM). Samples were examined in plan view, both in vivo and ex situ, as well as with focused ion beam milled cross sections ex situ. We find that the mineralized regions of the base plates are comprised of highly oriented, hierarchically structured calcite with nanometer to micron-sized grains. Underneath the calcified structures are cuticular tissues enriched in Ca and Mg, and proteinaceous material. The initial mineralization of barnacle shell begins on the upper shell, while the base plate underneath begins to calcify about two weeks after metamorphosis from cypris larvae. Mineralization proceeds inwards in a thin prismatic layer, as well as outwards from the periphery as the barnacle grows. We will describe the heirarchy, structure and composition of the base plate as it develops, including evidence for amorphous calcium carbonate within the mineralized regions.

The introduction of occluded biomacromolecules in biogenic calcite has been shown to physical properties such as hardness. Inspired by this, synthetic ‘large&’ molecules such as micelles, when incorporated, can also increase hardness with comparable structural effects. Further still, unprecedentedly high incorporation of amino acids has been reported, however the physical ramifications were not investigated. Here, the incorporation of Asp and Gly in calcite is studied extensively by HPLC, high-resolution pXRD and nanoindentation. Initially, quantification of occluded amino acid in calcite single crystals is determined, revealing a quasi-linear dependence on initial [amino acid]/[Ca] ratio. Secondly, a strong relationship between pXRD/Rietveld-derived microstrain and concentration of occluded amino acid; and anisotropic lattice distortion, confirms direct incorporation. Finally, nanoindentation studies revealed hardness measurements comparable to biogenic calcite, commensurate with imposed inhomogeneous microstrain. We propose, therefore, that structural biomacromolecules may have roles beyond that of increasing mineral hardness.

The formation of CaCO₃ has been studied for many years. Much attention has been devoted to amorphous CaCO₃ (ACC) as a singular material, because there is increasing evidence that this phase plays a crucial role in biomineralization. ACC is the least stable form of CaCO₃, and under ambient conditions it transforms quickly into more stable crystalline forms, such as vaterite and calcite. Many mineralization processes are now believed to occur through the transformation of a transient amorphous precursor, which has been shown to act a reactive in intermediate in generating complex functional materials.
We have studied the effect of proteins on the homogeneous formation of the liquid-amorphous CaCO₃ (LACC) precursor, by a combination of complementary methods like in situ WAXS, light scattering, TEM and cryo-TEM. Lysozyme destabilizes the LACC emulsion, whereas the glycoprotein ovalbumin extends the lifetime of the emulsified state. We demonstrate ovalbumin to act as a stabilizer for a polymer-induced liquid precursor (PILP) process. Emulsified LACC carries a negative surface charge and is stabilized electrostatically. We propose that the liquid amorphous CaCO₃ is affected by polymers by depletion stabilization and de-emulsification rather than by acidic proteins and polymers during a polymer-induced liquid precursor process. Thus, the original PILP coating effect appears to be a result of a de-emulsification process of a stabilized LACC phase. Silicatein-a, a protein from marine sponges, guides the self-assembly of calcite “spicules” similar to the spicules of the calcareous sponge Sycon. The spicules, 10-300 µm in length and 5-10 µm in diameter, are composed of aligned calcite nanocrystals. They are initially amorphous but transform into calcite within months, exhibiting unusual growth along [100]. They scatter X-rays like twinned calcite crystals. While natural spicules evidence brittle failure, the synthetic spicules show an elastic response, which greatly enhances bending strength. This feature is linked to a protein content of approx. 10%.
Later stages of nucleation have been studied by “trapping” nuclei from solution. This yields snapshots of the structure formation process at given point. In a first step the full determination of the structure of vaterite, one of the common CaCO₃ polymorphs, was solved on nanometer-sized crystallites by electron crystallography. These results demonstrate that crystals that are too small for single-crystal X-ray diffraction and too difficult to solve by powder diffraction may nevertheless be amenable to accurate structure determination by electron crystallography.

The study of nucleation is of paramount importance because it represents the seminal event in the growth of a solid phase from solution. Organothiol self-assembled monolayers (SAMs) have been utilized as a model system to mimic biological templates, with which to investigate the underlying mechanisms by which organic matrices control nucleation. There exist many long-standing questions surrounding templated nucleation due to a lack of experimental approaches suitable for probing this seminal event in mineral formation. Of particular interest is the formation pathway that takes the mineral from its solvated state to the final, oriented calcite crystal. Whether mineralization pathways on organic templates differ from pathways in purely inorganic calcite formation is currently unknown. Resolving this question is of great importance to understanding the mechanisms by which organic matrices exert control over the organization of minerals.
In recent years developments in transmission electron microscopy (TEM) have produced platforms allowing observation of fluid environments. Here we use fluid cell TEM to investigate calcium carbonate (CaCO₃) nucleation. During nucleation in the absence of organic templates, we observe direct formation of amorphous calcium carbonate (ACC), as well as the three predominant crystalline phases: calcite, vaterite, and aragonite. The direct formation of the crystalline phases is observed under conditions in which ACC readily forms. These observations provide direct evidence that multiple phases of calcium carbonate can form without the intermediate stage of ACC. For all phases measured, radial/edge growth rates following nucleation are constant, showing growth is limit by reaction kinetics.
In addition to direct formation pathways, in a purely inorganic system we observe transformation from ACC to aragonite and vaterite, but, significantly, not to calcite. ACC transforms directly to the crystalline phases starting on or just beneath the surface, rather than via dissolution and reprecipitation through distinct nucleation events. These formation pathways are confirmed by collecting diffraction information of the various phases of calcium carbonate.
We show how this approach can be modified to investigate CaCO₃ nucleation on SAMs of γ-substituted alkanethiols, and compare initial results of our observations on CaCO₃ nucleation pathways on carboxyl-terminated alkanethiol SAMs organized on gold to the formation pathways observed in the absence of a SAM.

Then nucleation of calcium carbonate and calcium phosphaste have been studied by titration experiments with ion selective electrodes and cryoTEM, and molecular dynamics simulations. Both experiments and simulation show that the nucleation process in both cases can be described by classical concepts, involving only ions, ion pairs and small ion-association complexes.

Many different species use a number of crystallisation processes to form supportive, protective, or otherwise useful structures. The wide diversity in chemical and physical properties of the resulting crystals arises from the organisms' exquisite control over the crystal morphology produced. This control comes from the ability an organism possesses to produce tailored environments, including three dimensional structures and extracellular matrices comprising organic molecules, during nucleation and growth of crystals. Minerals, such as calcite, from biological sources often contain proteins/peptides, probably included in the minerals during formation.^1,2 These structures are complex to simulate due to the chirality of a large number of amino acids, the lack of three dimensional structural information and their large size.
In order to simplify this problem, peptoids, or poly N-substituted glycines, are used as biomimetic substitutes for peptides. They can be altered by very simple chemistry involving substitutions at the nitrogen and contai less complexity due to their lack of the normally chiral alpha-carbon present in peptides.^3,4 These molecules are, therefore, very useful as models in experiments studying the impact of the physical and chemical structure of peptides in affecting mineral growth as noted in in the biomineralisation of calcite in aquatic species such as coccolithophores as well as being of interest in promoting the sequestration of carbon dioxide from the atmosphere as calcite⁵. Simulations ahve been performed, using several model peptoids discussed within the work by DeYoreo et al⁵ which contain a number of differences in their chemical and physical properties. In the work done by DeYoreo's group, peptoids of similar molecular weight were found to have significantly different effects upon the growth and morphology of calcite based on their differences in acidity, connectivity and substitution patterns. Aqueous phase simulations show that even in the absence of a surface these molecules act differently, possessing different radii of gyration and diffusion coefficients. Current calculations involving these peptoids at either stepped, kinked, or flat calcite surfaces provide an atomistic level insight into the origins of the variation in growth rate and crystal morphology depending on peptoid composition. This variation in flexibility is maintained on the surface, with the molecules showing the greatest changes in radii of gyration while at the surface corresponding with the molecules showing the greatest increase in the calcite growth rate.
[1] Meldrum, F. and Cölfen, M. Chem. Rev.2008, 10, 4332-4432
[2] Weiner, S. and Addadi, L. Ann. Rev. Mater. Sci.2011, 41, 21-40
[3] Butterfoss, G. et al. J. Am. Chem. Soc.2009, 131, 16798-16807
[4] Yoo, B. and Kirschenbaum. K, Curr. Op. Chem. Bio. 2008, 12, 714-721
[5] Chen C. L., Qi J., Zuckermann R. N., DeYoreo J. J., J. Am. Chem. Soc.2011, 133, 5214-7

Sessile marine organisms such as barnacles uniquely maintain a permanent proteinaceous interface between themselves and the marine environment throughout their adult existence. These proteins play a dual role in adhering to both the native organism and a foreign substratum, which are often crystalline calcium carbonates from other marine invertebrates, cuticular exoskeleton or sedimentary minerals. Though the sequence and composition of several barnacle cement proteins have been reported, little is known about their molecular adhesion mechanism towards marine relevant surfaces such as calcite or polysaccharides. Herein, a previously identified 20 kD protein found in the primary cement of Megabalanus rosa (MRCP20) is expressed recombinantly and observed to inhibit both the dissolution and formation of calcite mineral in vitro through edge-mediated fibril formation. Further, using a comparative modeling approach, we identify significant structural homology to a Wheat Germ lectin and demonstrate selectivity for polysaccharides. We use in situ high resolution atomic force microscopy (AFM), attenuated total reflectance (ATR) infrared spectroscopy (IR), and circular dichroism (CD) to reveal the effect and mechanism of MRCP20 on both calcite dissolution and mineralization. MRCP20 is found to specifically adhere to step edges of freshly cleaved {104} calcite faces, inhibiting edge-mediated dissolution processes in water. During this inhibition, we directly observe proteins to form linear chains that remain pinned on the surface and induce localized defects in the morphology of receding calcite terraces. Alternately, under saturated CaCO₃ conditions, MRCP20 delays nucleation of, and limits the amount of calcite formed. ATR-IR spectra indicate the stabilization of an amorphous calcium carbonate phase during this inhibited growth regime, shedding light on a potential role for MRCP20 in remodeling dynamic calcareous interfaces found along the growing edge of the organism. We propose that the adhesion mechanism of MRCP20 to calcite is two-fold, through both a direct adsorption to step-edges of {104} calcite and also through interaction with an intermediate ACC phase adhering to exposed calcite faces.

Nanowire (NW) arrays offer novel opportunities for parallel, nondestructive intracellular access for biomolecule delivery, intracellular electrical recording, and intracellular biochemical sensing. Spontaneous cell membrane penetration by vertical nanowires is the essential step for these applications, yet how and when the penetration process occurs is still poorly understood, with experimental evidence suggesting that penetration occurs in a geometry-dependent fashion. Previous models of membrane penetration have focused on static mechanical models in ideal geometries, ignoring effects such as cell spreading and motility. In this work, the dynamic NW-cell interface during cell spreading were studied with electronic microscope, and cell penetration was revealed to occur right after cell adheres to substrate based on cobalt ion delivery assay. Inspired by these experimental observations, a mechanical continuum model is presented accounting for the dynamic evolution of the cell membrane profile as the cell spreads through adhesive interactions with a NW array. Penetration was determined by calculating a series of snapshots that conservatively estimate the amount of tension expected in a given membrane configuration. The calculation results reveal that NWs are more likely to induce penetration within a finite window shortly after initial cell contact and adhesion, while NWs that are closely engulfed by the cell membrane lose their capability to induce new penetration. The effects of NW geometry (NW radius, height and spacing) and cell properties (cell stiffness, adhesion and membrane strength) are systematically evaluated to identify the key factors for penetration.

Bamboo is a naturally occurring composite material in which cellulose fibers reinforce lignin carbohydrate complex (LCC) matrix. On the nano- and micro-scales, nanoindentation techniques are used to study the local variations in the Young&’s moduli of moso culm bamboo cross-sections. These are then incorporated into finite element models in which the actual variations in Young&’s moduli are used to model the deformation and fracture of bamboo during fracture toughness experiments. Similarly, the measured gradations in moduli are incorporated into crack bridging models that predict the toughening observed during resistance curve tests. Multiscale atomistic simulations were then used to understand the mechanics of crack bridges in the microstructure of bamboo. On the macro-scale the shear properties are studied using a torsion experiments. The effects of humidity are further studied on the stiffness and strength of bamboo samples. The implications of the results are discussed for the bio-inspired design of structures that mimic the layered, functionally graded structure of bamboo.

Amphiphilic molecules spontaneously self-assemble into ordered nanostructures at the liquid-liquid interface. This presentation will feature how the soft surface of an aqueous microdroplet can be engineered to modulate the key properties of the surface using amphiphilic molecules. The exterior of a water droplet can support monolayers and bilayers that mimic the structure and function of the cell membrane, thereby offering a powerful and controllable model for bilayers, the essential feature of biological membranes. In turn, the interfacial surface of the droplet can be employed to influence the formation of a microparticle within the droplet, through templated nucleation. We will show that the use of self-assembled monolayers of various monoglycerides and other biological lipids having various molecular shapes can influence the surface properties of the monolayer and bilayer. In addition, the subtle characteristic interactions between the droplet content and the self-assembled structure at the monolayer and bilayer will be shown to have a remarkable difference in its packing arrangements and permeability. The successful demonstration of soft surface engineering at the micron size level will provide important insights into a wide variety of biologically relevant phenomena, such as membrane permeability and biomineralization.

Some of the most ubiquitous biological processes are not in equilibrium. Processes such as wound healing, reproduction, and cell division require cell motility, in particular motility through a tissue interface. It is this motility that both pushes the system to a state of nonequilibrium and leads to collective motion or migration of cells. Recently, there has been extensive interest in understanding this mobility induced spontaneous nonequilibrium phase behavior. Particularly in developing theory and simulations, experimental systems being few and far between, in order to gain further insight into these phenomenon. A number of studies have shown phase separation both in systems composed of purely active particles and in systems with active and inactive particles. Yet, in these studies the origin of this aggregation behavior is not clearly characterized, in particular the length scale of this attractive force between active particles. We have developed a novel and versatile artificial system to study the non-equilibrium behavior of hybrid active-inactive systems and characterize this mobility induced attractive force between active particles. The system was composed of active magnetic polystyrene particles (5µm in diameter), and inactive polystyrene particles of the same size. The inactive and active particles were mixed in a surfactant solution, inserted into a microfluidic channel, and allowed to sediment on the substrate of the device, forming a concentrated monolayer of active and passive colloids. For this system the magnetic field was rotated parallel to the substrate, causing the active particles to rotate or spin parallel to the substrate, henceforth referred to as spinners. In the absence of this inactive monolayer the spinners will simply spin in place and interact only if they are close enough so the magnetic dipole interaction is dominant. In the presence of a concentrated monolayer of inactive particles the spinners rotated the inactive particles around them there imparting mobility. Within minutes of the field being applied the spinners began to aggregate. This attractive force was found to be an extremely long range interaction and could be felt beyond distances of 12d (diameter of the particle). This attractive interaction was found to be modulated by both the mobility of the spinners and the ability of the spinners to cooperatively shear the inactive particle interface separating the spinners. Additionally, this attractive force was dependent on the frequency at which the magnetic field was rotated and the concentration of the inactive particles in the monolayer. Understanding this mobility induced attractive force is crucial not only to better understand nonequilibrium phase behavior but to better understand vital biological functions. This system is also easily adaptable and can be used to investigate similar behavior between particles with harder or softer interfaces to better mimic biological systems of interest.

Otoconia are small spindle shaped calcite-based biominerals found in the saccule and utricle of the ear. They are instrumental in detecting vertical and horizontal linear movement. Like many biominerals, otoconia appear to form from precursor particles within a fibrilar matrix, but mature into faceted, single crystal products. We investigated the roles the four major otoconial proteins Fetuin A, Osteopotion (OPN), Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. Atomic force microscopy (AFM) observations of Otolin-1 adsorption onto mica surfaces showed that it formed homomeric protein complexes and self-assembled networks, supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. In situ AFM measurements of the effects of the four proteins on the growth of atomic steps on calcite surfaces showed that Fetuin A, OPN and OC90 are potent inhibitors of calcite growth. For activity products as much as 2.3 times above equilibrium, they completely blocked step propagation at sub-100 nM concentrations. In contrast, Otolin-1 strongly enhanced growth rates, accelerating step speeds by a factor of two to three for the same range of conditions. Inverted optical microscopy was invoked to observe calcite nucleation on films of Fetuin A, OC90 and OPN adsorbed onto mica surfaces. By measuring the nucleation rate as a function of supersaturation, the value of the interfacial energy a that controls the free energy barrier to heterogeneous nucleation was determined for each protein. While OPN and OC90 films led to significantly reduced a as compared to that for homogeneous calcite nucleation in bulk solution (77 and 96 vs. 109 mJ/m²), the value for Fetuin A (117 mJ/m²) was larger. Because an increases with increasing affinity of the protein for the solution (i.e., low liquid-substrate interfacial energy a_ls) and decreases with increasing affinity of the protein for the nucleating crystal (i.e., low crystal-substrate interfacial energy a_cs), the source of the observed variations in a can not be determined without probing one or more of these two terms. To investigate the affinity of the protein films for the solution, we measured the zeta potential, which was previously shown to be a proxy for a_ls, with large negative surface charge corresponding to low a_ls. The results revealed a negative linear relationship between the measured values of a and the zeta potential. Thus the variation in interfacial energy observed here is likely due to the differences in the affinity of the substrates for the aqueous solution (i.e., the hydrophilicity). The consequence is that while Fetuin A presents a higher barrier to nucleation than the bulk solution, OPN and OC90 are potent promoters of calcite nucleation.

Understanding the functionality of the hard-soft matter interface requires suitable characterization techniques. One of many options, which all have to be used complementary, is analytical high resolution electron microscopy. The quantitative and spatially resolved characterization of organic material and of composits consisting of a mixture of light and heavier elements in the electron microscope is a challenge. This is due to sample preparation artefacts, radiation damage effects and signal absorption in mixtures of elements with very different atomic number. Energy dispersive X-ray spectroscopy (EDS) is well-established for composition analysis of bulk and electron transparent samples in the electron microscope (STEM, TEM and SEM). Various multiple detector arrangements simplify the analysis of bulk samples with complex topography and limit the need for bulk sample preparation. Quantitative bulk analysis by EDS has been exploited close to its full potential. The Cliff-Lorimer method, widely used for quantification of electron transparent objects, provides data on the accuracy level of a few at% already, and if using large solid and take-off angles for stable samples even on the level of ppm. In case of beam sensitive samples, high solid angles for signal collection allow very fast data acquisition avoiding extended sample exposure to the damaging electron probe. The results of the Cliff-Lorimer quantification method are only valid relative to a suitable standard of similar thickness and composition though, which is often difficult to obtain. An alternative quantification procedure, the Zeta-factor method, has been developed by M. Watanabe [1]. It additionally includes information on the beam current and can accomodate the use of any standard as long as its composition, thickness and density are well known. This method can deliver absolute quantification data on the composition of an unknown sample of interest while accounting for absorption and fluorescence effects. The implementation, application options and further development of this analysis method will be discussed. [1] Watanabe M. & Williams D.B, J. of Micr. Vol. 221. (2006) 89-109.

Engineering mechanically and biologically compatible regenerative bone implants has proven an arduous and multifaceted pursuit. Most current hip implants employ a titanium alloy as a scaffold for bone remodeling, which involves regenerating a trabecular bone network and profits from bearing load applied through exercise. Titanium is stiffer and stronger than the native hierarchical network of trabecular bone, which causes the latter to carry less of a load and hinders the native tissue remodeling. These types of implants are not biodegradable or porous, which further inhibits osteointegration. To successfully mimic the trabecular bone in creating better prosthetics (or bone replacement), it is essential to understand its properties at the fundamental level.
We investigate the mechanical properties of the native human trabecular bone at each level of hierarchy: from several hundred nanometers to several tens of microns. We fabricate micro- and nano-sized cylinders and dogbone shaped samples from decelluarized trabeculae which contains collagen and hydroxyapatite via Focused Ion Beam milling. Stand-alone cylinders are then tested in compression and tension under prescribed nominal displacement rate. Both monotonic and cyclical experiments were performed, and evolved microstructure was analyzed. We present nano-mechanical behavior of site-specific samples within trabeculae and discuss them in the framework of small-scale mechanical deformation.

Dental pulp stem cells (DPSCs), which are highly proliferative and can be induced to differentiate along several mesenchymal cell lineages, offer the possibility for pulpal regeneration and treatment of injured dentition. The mechanics and topography of cell substrates can direct their differentiation. Polybutadiene (PB) can be spun casted into films of different thicknesses with different moduli. The mechanical properties of these substrates follow an inverse power law dependent on film thickness. DPSCs grown on PB films constantly regulate their modulus, Ec, to execute a power law similar to that of the substrate modulus, G, where Ec=0.5G. A critical value, Go, exists such that when G>Go, the DPSCs biomineralize depositing crystalline calcium phosphate without a requirement for the typical induction factor, dexamethasone (Dex). The moduli of cells track with the moduli of the surface suggesting that mechanics controls mineralization. The purpose of this study was to determine whether the major effect of Dex on biomineralization is the result of its ability to alter cell mechanics or its ability to induce osteogenesis/odontogenesis. DPSCs sense substrate mechanics through the focal adhesions, whose function is in part regulated by the Ras homolog gene (Rho) and its downstream effectors Rho associated kinases (ROCKs). ROCKs control actin filament polymerization and interactions with myosin light chain. Because cells sense substrate mechanics through focal adhesion proteins whose function is regulated by ROCKs, the impact of a ROCK inhibitor, Y-27632, was monitored. Blocking this pathway with Y-27632 suppressed the ability of DPSCs to sense the PB substrate. The cell modulus, plasma membrane stiffness, and cytosol stiffness were all lowered and biomineralization was suppressed in all cultures independent of substrate modulus or the presence of Dex. In other words, the inability of DPSCs to sense mechanical cues suppressed their ability to promote mineralization. On the other hand the expression of osteogenic/odontogenic markers (alkaline phosphatase and osteocalcin) were enhanced, perhaps due to Y-27632 induced changes in Wnt signaling as seen in other mesenchymal stem cells. How mechanical sensing regulates matrix proteins to promote their mineralization remains an open question.

We are studying crystalline materials associated with pathological processes, in particular sodium hydrogen urate monohydrate (MSUM), which accumulates in humans afflicted with gout. According to current knowledge, MSUM deposits by bio-mineralization. Two related aspects of hard-soft interfaces are important to this pathology. One is the extent to which a harder material like MSUM can mechanically damage softer biological tissues. Two is how the biological environment not only influences the nucleation and growth of this bio-mineral, but also its properties. It is known that some bio-minerals are less brittle, but also less hard than their purely mineral counterparts. This has been explained in terms of the hard-soft interfaces in bio-mineralization processes. Comparisons have been presented for other materials produced by normal bio-mineralization, such as teeth and bone, to minerals and materials obtained by bio-mimetic approaches, and for a few pathological cases - but not for gout. Towards that goal, we will present data on growth morphologies, fragmentation patterns, and mechanical properties of synthetic MSUM, and interpret them in terms of physical chemical parameters. Morphologies were examined by optical and electron microscopies, and mechanical properties were obtained by AFM nano-indentation. With this context, we will discuss the potential of embedded MSUM to produce mechanical stresses, tissue damage/injury, and ultimately cell death. Fragmentation may be linked to solubility, secondary nucleation, and inflammation. This will provide a basis for interpreting observations of the corresponding bio-material.

Developing the next generation of advanced biomaterials with specific properties for a particular application is a challenging task. Spider silk is an extremely tough, environmentally friendly biopolymer that can be used as a biocompatible cell scaffold for medicine or a lightweight composite for robotics. We synergistically integrated genetic control of spider silk protein biosynthesis and processing with coarse-grained simulations to design and fabricate mechanically robust biomimetic fibers. Recombinant spider silk block copolymers were prepared based on the assembly of two individual blocks, a hydrophobic poly-alanine rich block (A) and a hydrophilic glycine-rich block (B) in vitro. The A block consisted of one polyalanine/polyglycine repeat (GAGAAAAAGGAG) responsible for β-sheet (crystalline) formation. The B block was composed of four GGX repeats, separated by the GSQGSGR sequence. Simultaneously, the coarse-grained mesoscopic dissipative particle dynamics simulation was performed to establish the optimal A/B ratio, which led to homogenous self-assembled aggregates and connected polymer networks. Based on this optimal domain ratio, we further pursued new design improvements with protein sequences possessing longer chains, which resulted in the dramatic enhancement in mechanical performance. To validate the in silico predictions, the constructs were cloned, expressed and purified and their secondary structures and morphologies assessed by FTIR, SEM, and AFM. H(AB)₁₂ block copolymer with Mw of 43.7kDa self-assembled into fibrils with an average diameter of 25 nm in aqueous media; whereas, H(AB)₂ with an Mw of 11.6 kDa did not form particular morphologies. Moreover, fiber formation of H(AB)₁₂ was induced by wet spinning of a concentrated protein solution while H(AB)₂ only form disordered films, in good agreement with our model predictions. The mechanical properties of spun fibers were characterized by nanoindentation and the H(AB)₁₂ fibers exhibited a higher stiffness modulus of 1 ~ 8 GPa, agreeing well with the simulated results. This integrated experimental - modeling approach demonstrates successful implementation of our previously developed silk block copolymer strategy to regulate material features by manipulation of the block domains in spider silk block copolymers and provides a novel tool to generate advanced biomaterials with well-defined properties.

Nature provides a multitude of examples of multifunctional structural materials. These materials cannot be optimal at all functions, which results in trade-off situations. In this work, we study one such example of the biomineralized armor of the chiton Acanothopleura granulata, which has an integrated sensory system. Synchrotron µCT was used to investigate the 3D morphology of the biomineralized eyes, through which quantitative measurements revealed the lenses (thickness: ~38 µm) have both parabolic top and bottom surfaces. A pear-shaped chamber directly underneath the lens has a depth and width of ~55 µm and 75 µm, respectively, resulting in the chamber volume that is ~5× greater than that of the normal sensory structures without lenses. Moreover, electron backscattered diffraction showed that the aragonite-based lenses have much higher crystallographic alignment and larger grain sizes as compared to non-sensory regions. Transmission electron microscopic imaging of the localized regions prepared using focused ion beam milling technique also suggested that the lens may possess less intracrystalline organic material than the surrounding non-sensor regions. 2D ray-trace simulations incorporated with key elements of the geometry, composition and crystallography of the lens revealed the lenses are able to focus the light within the chamber in both air and seawater (8-28 µm and 25-51 µm below the bottom of the lens, respectively). We, for the first time, experimentally demonstrated that the lenses are able to form clear images. The focal distance determined through this experimental approach (72 ± 17 µm) is comparable to the maximum value calculated from ray-trace simulations (65 µm). Lastly, with instrumented indentation technique, we found that the eyes exhibit catastrophic failure by pushing the entire lens into the chamber with the critical force of 0.84 ± 0.11 N, while the non-lens region are much more resistant to mechanical damage. This study of the structure/property/performance relationships of the shell of A. granulata demonstrates that trade-offs are fundamentally present at the materials level within a single organism due to the requirement of multi-functionality. As the size, complexity, and functionality of the integrated sensory elements increases, the local mechanical performance of the armor decreases.

Carbon nanotubes (CNTs) have strong affinity for amino acids and subsequent conformations of the proteins. Proteins are known to adsorb onto almost all forms of carbon. A crucial role in such an adsorption is played by the interfacial interactions such as van der Waals, electrostatics and hydrophobic attractions. A particular protein called flagellin of approximately 1-2 nm in diameter, which is a primary component in a bacterial flagella is of great interest in studying the bacterial motility and associated dynamics. However, there is very little information on how flagellin responds when in direct contact with CNTs. CNTs being hydrophobic in nature have shown to be a good substrate for protein adsorption and at the same time a very good candidate to influence the protein conformations to disrupt the functional characteristics of the protein. Using molecular dynamics simulation, we demonstrate a chirality-based interaction of secondary structure of the domain 3 (DOM3) of flagellin that predominantly consists of extended beta sheets. It is found that the extended beta sheets are stable during the interaction with the carbon nanotubes compared to the helices. A fragment made up of 12 residues, however, is a rapidly conforming spot that changes back and forth from a turn to a 3-10 helix and α-helix. It is also observed that glycine is favored in the form of helices by the metallic CNT, whereas an area devoid of glycine on DOM3 is favored by the semiconducting CNT. The experimental study of the Magnetospirillum magneticum(AMB-1) motion dynamics utilizing 50 micron diameter magnetic coils in the presence and absence of CNTs indicate that the bacterial flagellum is a potential component for creating a hybrid complex to aid in selective deposition of CNTs.

Bacterial biofilms are typically thought of in the context of infectious diseases, where they provide a protective community for bacterial cells. Many different bacterial species can form biofilms on hard/soft tissues and biomedical devices. However, many biofilms are actually benign and can be useful tools for materials science and nanotechnology. A distinctive property of biofilms is their sticky nature, which is associated with their ability to create 3D extracellular matrices. In addition to cells, bacterial biofilms can contain complex carbohydrates and amyloid proteins, which are functional rather than pathogenic. Under the electron microscope, amyloid nanofibers can be observed within biofilms, where they exist as highly ordered hierarchical nanostructures.
In our studies, we have been especially interested in engineering curli fibers that are found in E. coli biofilms called curli. From an engineering point of view, curli nanofibers have great potential to be utilized in nanotechnology, especially in nanomaterials formation, patterning, and assembly applications.. However, to control the formation of biofilms and engineer novel functionalities, we need to first achieve synthetic control over curli nanofiber formation. To do so, we created a knockout mutant of E. coli that is unable to produce curli nanofibers on its own. Next, we designed a synthetic genetic circuit where the csgA gene is under the control of a riboregulator system. This synthetic riboregulator system can strictly control the production of the CsgA protein under a TetR-regulated promoter. This circuit enabled us to control the synthetic biogenesis of curli nanofibers. In addition, through protein engineering of CsgA, we were able to functionalize the final structure of CsgA-based curli fibers. We first attached a histidine tag to the C-terminus of CsgA and expressed the fusion protein on the cell surface. We observed that this engineering step enabled the creation of functionalized biofilm surfaces. We used this approach to assemble inorganic nanoparticles on curli nanofibers. Following the attachment of gold nanoparticles on curli nanofibers, we used subsequently applied gold salts to create nanowire structures through a gold enhancement process. Finally, we used a conductivity assay to show that the resulting biofilm-based nanowire network was conductive.
Using the tools of synthetic biology, we have engineered a living nanomaterial system whose capabilities can be extended to create nanomaterial systems for many technological applications relevant to biomedicine and nanotechnology. Specifically, we showed that E. coli biofilms and curli nanofibers can be used to form conductive nanofiber networks. Such systems could also be adapted for bioelectricity, biosensing and bioelectrosynthesis uses. We envision that synthetic biology has great potential for programming the bio-nano-interface and for realizing novel materials and nanotechnology applications.

The micro-scale pattern formation on surface is of significant interest to applications such as optics, microfluidics and stretchable electronics. Subject to a compressive surface strain, a film bonded to a soft substrate can develop different morphological phases, transitioning from surface wrinkles into folds. We developed experimental system to observe the wrinkle-to-fold transition of layered elastic materials under compressive stresses. However an understanding of how folds initiate, propagate, and interact under compressive stresses remains lacking. Here, we experimentally find that in the process of folding network formation, dynamic interaction of propagating fold tips under biaxial compression can be observed which are similar to crack tip interactions on the surface fracture. Based on our experimental results, we divide tip interactions into two primary modes of resolution for a pair of approaching tips: bending or coalescing. We also investigate that the final state of interactions is primarily determined by the geometric relation of each tips which induced stress concentration.

The large deformation response of individual, electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers was determined experimentally before and after mineralization to investigate deformation mechanisms at the individual nanofiber level, and to explain and improve the mechanical response of biomimetic scaffolds for tendon-to-bone tissue engineering. The uniaxial extension experiments with single nanofibers were carried out with the aid of MEMS microdevices. The nanofibers demonstrated significant strain-hardening leading to ~500% increase in failure strain and up to 200% increase in failure strength compared to bulk PLGA. The interfacial adhesive strength between the hydroxy apatite mineral coating and PLGA nanofibers as well as the thickness of mineral coatings were shown to be critical in preserving the mechanical response of unmineralized nanofibers. It was found that weakly bonded thin mineral coatings did not compromise the outstanding mechanical behavior of unmineralized PLGA while a minimally damaged surface mineral layer was retained at nearly 100% nanofiber elongation. Increased mineral thickness resulted in small change in the mechanical response of mineralized nanofibers but the mineral coating was not preserved for large nanofiber extensions.

Natural polysaccharides are of significant interest in chemistry and biomedical research because of their structural diversity and essential roles in metabolism and other physiological processes. Carbohydrates have become important therapeutic targets and are scaffolds towards new drug development and tissue engineering opportunities. Therefore developing biomaterials that can mimic or enhance the biochemical and structural properties of natural polysaccharides are highly desired. Currently to obtain carbohydrate polymers, especially of high molecular weights, they must be extracted from natural resources, which can introduce unwanted variation between batch-to-batch samples and require extensive purification protocols. Additionally carbohydrate polymers are challenging synthetic targets due to the high density of repeated functional groups, difficulty to maintain the rigid pyranose backbone, and need to control the stereochemistry at the glycosidic linkages.
We have developed a methodology using on an anionic ring-opening polymerization of a β-lactam sugar monomer to synthesize high-yields of enantiopure carbohydrate-based polymers, termed poly-amido-saccharides (PASs). PASs can be synthesized with batch-to-batch consistency, defined molecular weights, low polydispersity, and the ability to functionalize at the monomer or polymer level. Similar to natural polysaccharides, these synthetic carbohydrate polymers contain the chiral, cyclic main-chain structure and pyranose backbone. However unlike natural polysaccharides, in which monomer units are joined by glycosidic ether linkages, sugar units in PASs are joined by an unnatural α-(1,2)-amide linkage, which affords PASs with unique chemical properties and interesting structural architectures. We anticipate that PASs possess desirable attributes and interesting behaviors that will be beneficial for the development of new carbohydrate-based materials and applications to address current biomedical challenges. The chemical stability of PASs, characterization of their secondary structure, hydrolysis mechanisms, cytotoxicity, and cellular up-take pathway will be discussed.

Controlling the nucleation and growth of CaCO₃ in biominerals is of great interest for the development of new, novel materials. A range of biomolecules, including polysaccharides¹, peptides², peptoids³, proteins⁴, polymers⁵ and dendrimers⁶ have been observed to affect the growth rate, morphology and polymorph selection of CaCO₃ crystals. Recently intrinsically disordered proteins (IDPs) such as AP7^7,8, n16⁹ and the Asprich protein family¹⁰ have experimentally been found to inhibit the growth of the more stable calcite polymorph and promote the formation of other, less stable, polymorphs of CaCO₃. In this work the tripeptide ENG (glutamic acid - asparagine - glycine) is used as a shortened model component of these IDPs, to analyse the configurational behaviour of these species in solution and upon binding to calcite surfaces and to model the binding of multiple molecules to the same surface.
Molecular dynamics simulations were used to determine the energy of adsorption of these species on flat, stepped and kinked calcite surfaces and also the change in configurational behaviour upon binding through the analysis of the backbone dihedral angles of the tripeptide. The simultaneous binding of multiple tripeptides on the calcite surfaces gives insight into the competitive/additive nature of the adsorption of these biomolecules.
We will present the results both from simulations and from matching experimental studies performed in collaboration with the University of Copenhagen. These results will show the quantitative effect of the ENG tripeptide on the growth of calcite at varying peptide and CaCO₃ concentrations
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(3) Chen, C.; Qi, J.; Zuckermann, R. N.; DeYoreo, J. J. J. Am. Chem. Soc.2011, 133, 5214-17.
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Calcium phosphate is the main component of bones and teeth [1]. Understanding how this mineral nucleates and grows is also important for understanding pathological mineralization including that of cartilage and cardiovascular tissues [1]. Previous work has shown that biological calcium phosphate can crystallize via an amorphous precursor phase and Dey et al [2] observed that this amorphous phase formed via the aggregation of pre-nucleation clusters [2, 3]. Habraken et al [4] observed that before nucleation the and ions form complexes which then further aggregate to form pre-nucleation clusters that could be described as an inorganic polymer network [4]. It was suggested that these complexes interacted by hydrogen bonds formed between the complexes involving the phosphate hydroxyl groups. However this could not be experimentally observed and the association of such negatively charged monomers seems unlikely.
Molecular dynamics (MD) simulations have been used during this project to probe the formation, structure, stability and potential mechanism of the interaction of the units. As a means to obtain the energy barrier to the formation of this species and the relative probability of the species existing in a solution, umbrella sampling was used. This technique permits us to sample the configurational space with respect to a specific order parameter and thus to obtain the relative free energy curve of the reaction. Additional MD simulations were also carried out to analyse the aggregation of the units in proximity of additional ions. The results partially agree with previous analysis and give new insights into the molecular mechanisms behind the initial precipitation of amorphous calcium phosphate.
[1] Dorozhkin, S. V.; Epple, M. (2002). Biological and Medical Significance of Calcium Phosphates. Agewandte Chemie International Edition. 41 (17), p3131-3146.
[2] Dey, A. et al. (2010). The role of prenucleation clusters in surface-induced calcium phosphate crystallization. Nature Materials. 9 (1), p1010-1014
[3] Mahamid, J. et al. Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to the bone in zebrafish fin rays. Proc. Natl Acad. Sci.USA 107, 6316#8208;6321 (2010).
[4] Habraken, W. J. E. M. et al. (2013). Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate. Nature Communications. 4 (1)
[5] Torrie, G.; Valleau, J. (1977). Nonphysical Sampling Distributions in Monte Carlo Free Energy Estimation: Umbrella Sampling. J. Comput. Phys. 23 (1), p187-199

INTRODUCTION
Surface modification of endosseous Ti-based implants at the nano-/micron-scale has been shown to enhance bone formation and encourage rapid osseointegration and biomechanical stability [1,2,3]. Plasma Electrolytic Oxidation (PEO) creates a textured TiO₂ surface layer, suitable for orthopaedic applications. Among the three polymorphs of TiO₂ (anatase, rutile and brookite), anatase has been shown to enable rapid precipitation of HA in Simulated Body Fluid [4]; hence, it is desirable to maximize the surface anatase content. Also, in comparison to α- and (α+β)-Ti alloys, β-Ti alloys have a modulus closer to that of natural bone, and thus potentially offer a lower stress shielding effect. This study explores the possibility of extending the application of PEO to novel low-modulus β-Ti alloys, while maintaining similar cellular activity as commercial α- and (α+β)-Ti alloys.
METHODS
Coatings were produced on α-Ti, (α+β)-Ti6Al4V, β-Ti13Nb13Zr and β-Ti45Nb substrates using a 10 kW 50 Hz Keronite rig (Jasymp;20 Amiddot;dm^-2). The physicochemical variations of the coatings were investigated using SEM, XRD, DSA, BET and Optical Interferometry. The influence of coatings on metabolic activity, proliferation and differentiation of foetal human osteoblasts was studied using alamarBlue, CyQuant and ALP, respectively. The effects on cell mineralization (OsteoImage kit), collagen release (immunoblotting) and surface distribution of cells (immunofluorescence) were also investigated.
RESULTS AND DISCUSSION
PEO was shown capable of producing rough, anatase-rich TiO₂ films on α-, (α+β)- and β-Ti alloys. The development of surface physicochemical features during PEO was found to depend on processing time and substrate material, and caused primarily by micro-plasma discharges. These short-term discharges seemed to depend on the initial formation of an electrically insulating oxide layer. Although the anatase:rutile ratios varied, the degree of crystallinity remained constant at all times due to the continuous formation of an initially amorphous oxide layer. At longer processing times, anatase content dropped, while hydrophilicity and roughness increased. Uniform pore size distribution at the nano-/micron-scale was observed for all four alloys. Regardless of the material stiffness, in vitro biological studies showed similar osteoblast metabolic activity, proliferation and differentiation on all four alloys. Cell mineralization appeared to be enhanced after 30 min, which may be associated with increased coating thickness, roughness, hydrophilic properties and/or net substarte weight gain. This suggests that PEO application can be extended to low-modulus β-Ti alloys offering a lower stress shielding effect, without compromising the biological activity.
REFERENCES
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[5] Hanada S. et al., 1284:239-247, 2005

Porous coatings at the surface of living cells have application in human cell transplantation by controlling the transport of biomolecules to and from the cells. Sol gel-derived mesoporous silica materials are good candidates for such coatings, owing to their biocompatibility, facile solution-based synthesis conditions, and thin film formation. Diffusion and transport across the coating correlates to long-range microstructural properties, including pore size distribution, porosity, and pore morphology. Here, we investigated collagen-fibril matrices with known biocompatibility to serve as templating systems for directed silica deposition. Collagen-fibril matrices formed from type I collagen oligomers derived from porcine skin are customizable with respect to their fibril density and interfibril branching character. We show that these matrices template and direct the deposition of mesoporous silica at the level of individual collagen fibrils. We varied the fibril density, silica concentration, and time of exposure to silicifying solution and characterized the resulting hybrid materials by scanning electron microscopy, energy-dispersive x-ray spectroscopy, rheology, and diffusion studies. The collagen-fibril template is capable of regulating silica formation based on relative matrix density such that silica content decreases from the outer surface of the template to the core. Results for matrices with three different fibril densities, corresponding to shear storage moduli of 200 Pa, 1000 Pa, and 1600 Pa indicate that increased fibril density increases the amount of templated silica when all other silica synthesis conditions are kept constant. Additionally, the mechanical properties of the hybrid material are dominated by the presence of the silica coating rather than the amount of silica present or the starting matrix stiffness. We further extend this work towards recombinant Elastin-Like Polymer (ELP) hydrogels with thermoresponsive structures, investigating their potential as biocompatible and tunable templates for directing sol gel silica deposition.

Despite the material selection limitations faced by biological systems, many natural composites achieve superior toughness and strength to weight ratios through the precise ordering of their microscopic building blocks. Structural bio-composites such as bone, nacre, and hexactinellid sponges exhibit mechanical properties far exceeding that of their individual elements due to their hierarchical reinforcement. Understanding the strengthening mechanisms of various structural bio-composites can lead to implementing these designs in high performance man-made composite materials. However, there is currently a gap between understanding these structures and being able to successfully assemble them. This research covers a new approach for obtaining polymer-based composites exhibiting bioinspired, deliberate orientation of reinforcing particles using additive manufacturing. Super-paramagnetic iron oxide nanoparticles are used to coat less than 5% of the surface of each filler element, which can then be manipulated using ultra-low magnetic fields. By applying this method of magnetic assembly to current steriolithography technologies, the 3D printing of composites with discrete 3D reinforcement is achieved. Our ability to tune the position and orientation of reinforcing particles within a polymer matrix that is being printed can lead to heterogeneous structures with novel mechanical properties such as tailored localized wear resistance, hardness, and toughness. Furthermore, this technique can be applied to other types of filler elements, resulting in unique thermal and optical properties for applications in thermal interface materials (TIMs), optical filters, and various multi-functional materials.

New materials are key in the development of advanced tissue and biomedical engineering technologies. Materials such as porous ceramics are important candidates for bone matrices and regenerative medicine applications. Challenges in the improvement of such materials are twofold. First, more accurate and reliable characterization techniques are needed. Second, a stronger control over properties and microstructure of these materials through processing is required. Barium titanate foams have promising biocompatibility and are of significant interest for aforementioned applications. Here, a new measurement technique for characterization of compressive behavior of such highly porous brittle materials is proposed. More specifically a methodology for the reliable characterization of mechanical properties such as crushing strength (collapse stress) and the effective compressive modulus is introduced. We demonstrate that based on the application of an epoxy layer, stress concentrations can be avoided during measurement and much more reliable data can be obtained. The effect of strut microstructure (grain size, porosity, etc.) on mechanical properties will be discussed in the particular case of of barium titanate foams processed via a two-step sintering techniques.

The ability to exploit the structure-function relationship of peptide-decorated metal nanoparticles, suspended in aqueous solution, offers much promise for the production of catalytically active nanomaterials with controlled functionality.¹ Experimental studies have determined the structure of the nanoparticles as well as investigated their catalytic functionality,^1,2 however, making clear connections between the structure of the adsorbed peptide overlayer and the catalytic performance of these systems, under aqueous conditions, is challenging to obtain via experimental techniques alone. Molecular simulation has provided valuable information about the interaction of peptide species known to have a propensity for gold.^3-5 However, the majority of these studies have investigated the behavior of such peptides at periodic surfaces or with ideal faceted nanoparticles rather than surfaces with irregular features occurring on the nanoscale. Recent studies have shown using peptides to cap the growth of metal nanoparticles can lead to nanoparticles that possess a degree of surface irregularity.² Here, we predict the structure and dynamics of an overlayer of multiple peptides at the surface of gold nanoparticles with irregular surface features, employing Hamiltonian replica-exchange^6,7 methods to ensure sufficient sampling of peptide conformational space. A range of different peptide/AuNP systems are considered to elucidate structure/property relationships regarding the different catalytic behaviors of each peptide/AuNP system.
[1] Bhandari, R., et al., Structural Control and Catalytic Reactivity of Peptide-Templated Pd and Pt Nanomaterials for Olefin Hydrogenation, J. Phys. Chem. C, 2013, 117, 18053-18062.
[2] Bedford, N.M., Analysis of 3D structures of platinum nanoparticles by high energy X-ray diffraction and reverse Monte Carlo simulations, Solid State Comm., 2010, 150, 1505-1508.
[3] Feng, J., et al., Influence of the shape of nanostructured metal surfaces on the adsorption of single peptide molecules in solution, Small, 2012, 8, 1049-1059.
[4] Tang, Z., et al., Biomolecular recognition principles for bionanocombinatorics: an integrated approach to elucidate enthalpic and entropic factors, ACS Nano, 2013, 7, 9632-9646.
[5] Palafox-Hernandez, J.P., et al., Comparative Study of Materials-Binding Peptide Interactions with Gold and Silver Sufraces and Nanostructures: A Themodynamic Basis for Biological Selectivity of Inorganic Materials, Chem. Mater., 2014, In Submission.
[6] Terakawa, T., et al., On easy implementation of a vairent of the replica exchange with solute tempering in GROMACS, J. Comput. Chem., 2010, 32, 1228-1234.
[7] Wright, L.B. and Walsh, T.R., Efficient conformational sampling of peptides adsorbed onto inorganic surfaves: insights from a quartz binding peptide, Phys. Chem. Chem. Phys.,2013, 15, 4715-4726.

A fundamental understanding of cell-nanomaterial interaction is essential for biomedical diagnostics, therapeutics, and nanotoxicity. Here we perform a theoretical analysis to investigate the phase diagram and morphological evolution of an elastic rod-shaped nanoparticle wrapped by a lipid membrane in two dimensions. We show that there exist five possible wrapping phases based on the stability of full wrapping, partial wrapping, and no wrapping states. The wrapping phases depend on the shape and size of the particle, adhesion energy, membrane tension, and bending rigidity ratio between the particle and membrane. While symmetric morphologies are observed in the early and late stages of wrapping, in between a soft rod-shaped nanoparticle undergoes a dramatic symmetry breaking morphological change while stiff and rigid nanoparticles experience a sharp reorientation. These results are of interest to the study of a range of phenomena including viral budding, exocytosis, as well as endocytosis or phagocytosis of elastic particles into cells.

Peptoids are a promising class of bioinspired polymers, which have several unique properties that bridge the gap between proteins and bulk polymers, such as sequence specificity, thermal stability, and resistance to protease digestion. Studies have shown that certain sequences can form 1D fibers and 2D sheets, thus they are promising candidates to develop self-assembling protein-like materials. The purpose of this research is to understand the mechanisms and controls on peptoid self-assembly and relate them to the underlying sequence.
Several peptoids with different sequences of hydrophilic and hydrophobic side chains were synthesized and shown to self-assemble into 2D or 3D structures ranging from bi-layer sheets to 2D fiber arrays to 3D porous networks. Their assembly pathways on mica surfaces were characterized in real time using in situ atomic force microscopy (AFM) and related to solution species observed through dynamic light scattering and TEM. During the assembly of porous networks, 3nm-high “proto-fibers” were found to be the basic building blocks. These extended laterally to create an initial 3-fold symmetric array of fibers one molecule in width. These grew outward in 3nm increments to ultimately form a 3D porous network. However, the nucleation of the proto-fibers occurred through transformation of a population of 5Å-high clusters having no apparent order and coexisting with monomers on the surface. Based on these results and XRD analysis of peptoid fibers and sheets, we propose a model to describe self-assembly of this peptoid. Initially, peptoid molecules adsorb onto the mica surface and aggregate to form the 5Å-high clusters in which the molecules are randomly arranged and “lying down”. Over time, these clusters undergo a transformation in which the molecules “stand up” and are joined by others, possibly from solution, to form 3nm-high bi-layer particles. The bi-layer particles then grow in length by addition of molecules to the fiber ends (x direction) and nucleation of new bilayers on the top (z direction).
The kinetics of peptoid assembly was also investigated. We found that the nucleation rate of 3 nm proto-fibers was initially constant, but dramatically increased after an incubation time that increased with peptoid concentration and decreased with calcium concentration. In contrast, formation of the 5Å clusters was initially constant and rapid by comparison, but their number reached a maximum before rapidly decreasing due to their conversion into 3 nm proto-fibers. We constructed a kinetic model that considers the competing rates of the various deposition, nucleation, and transformation processes. The predicted behavior agrees qualitatively with the observations and places bounds on the magnitude of the kinetic terms controlling the assembly pathway.

Seashells are natural nanocomposites with superior mechanical strength and eminent toughness. What is the recipe that Mother Nature uses to fabricate seashells? What roles do the nanoscale structures play in the strengthening and toughening of seashells? Can we learn from this to produce seashell-inspired nanocomposites? The recent discoveries of nanoparticles in seashells are summarized, and the roles these nanoparticles play in seashell&’s strength and toughness are elucidated. It was found that rotation and deformation of aragonite nanoparticles are the two prominent mechanisms contributing to energy dissipation in seashells. The biopolymer spacing between nanoparticles facilitates the particle rotation process. Individual aragonite nanoparticles are deformable. Dislocation formation and deformation twinning were found to play important roles in the plastic deformation of individual nanoparticles, contributing remarkably to the strength and toughness of seashells upon dynamic loading.

Organic molecules in aqueous solutions are good candidates in the inhibition of some biogenic crystals growth. The methanoic acid HCOOH is considered to study the interaction between the carboxyl functional group -COOH and the {10.4} hydrated surface of calcium carbonate, CaCO³, in the form of calcite. In this work the inhibiting effects of protonated -COOH and deprotonated -COO- headgroups ontop of the {10.4} hydrated surface of calcite are investigated by means of ab initio DFT-GGA simulations. The interfacial properties and the trend of adsorption energies for different coverages are given in details and show that the adsorption is favored by the presence of water. The reaction path of the deprotonation mechanism is investigated via the climbing image nudged elastic band (CI-NEB) method.

Xenograft bone substitutes, such as deproteinized bovine bone mineral (DBBM), have been widely employed as osteoconductive structural materials for bone tissue engineering. However, the loss of their particles in defects has been a major limitation, along with a lack of osteoinductive function. Mussel adhesive protein (MAP), a powerful glue in nature, can attach to various substrates, even in wet environments, and its properties are considered to be mainly derived from 3,4-dihydroxyphenylalanine (DOPA) residues. Here, we evaluated the use of DOPA-containing engineered MAP (mMAP) as a functional binder for in vivo bone regeneration. We observed that mMAP was able to bind DBBM particles easily to make an aggregate, and grafted DBBM particles was not lost in a defect in the rat calvaria during the healing period. Importantly, grafting of a mMAP-bound DBBM aggregate resulted in remarkably accelerated in vivo bone regeneration and even bone remodeling. Interestingly, we found that mMAP had osteoinductive ability based on clear observation of the in vivo maturation of new bones with similar bone density as normal bone and of the in vitro osteogenic differentiation. Collectively, mMAP is a promising functional binder material for xenograft bone substitute-assisted bone regeneration with greatly enhanced osteoconductivity and acquired osteoinductivity.

Introduction: Previous mathematical models for the clinical prediction of bone fracture toughness are based on its morphological structures at macro- and micro-scale levels. However, bone is a polycrystalline nanostructured material. Accordingly, our aim is to include the crystallographic ultrastructures at nano-scale level in a mathematical model to describe bone fracture toughness.
Methods: Tibiae of 35 9-week old mice were collected. Information about the mineral density (through DXA), protein content (through Raman spectroscopies), geometrical morphometrics (through micro CT), crystallography nonostructure (through XRD and Raman) and mechanical properties (through 3-point bending and tensile tests) of the bone samples were collected. The results obtained from the above measurements were correlated with each other and used to develop a model for bone fracture mechanics based on Hall-Petch theory.
Results: Bone strength and fracture energy were determined by the size of its nanocrystals and geometric dimensions. Ultimate force to induce bone fracture was controlled by the density of bone mineral. Work to failure relied mainly on the toughness of its collagen.
Conclusion: The mechanical strength and fracture mode/resistance of bone is regulated by its crystallographic structure at the nano-scale level.

Potassium is a key mineral used for fertilizer in world agriculture. However, there is currently a discrepancy in the locations of production and consumption of world potash; it is primarily produced in the northern hemisphere, where potassium ores are abundant, while it is consumed in the southern hemisphere, where the land and climate enable sustained agriculture. With a growing world population, there is an increasing need for both new sources of potassium and more sustainable extraction methods. There exist substantial amounts of potassium in the southern hemisphere, but they are largely unobtainable in the form of potassium feldspar rock formations. Studies have shown that bacteria and fungi produce and secrete molecules which promote mineral weathering. This study takes this inspiration from nature to find a viable processing technique to leach potassium from potassium feldspar using organic ligand solutions, while investigating the organic-inorganic interactions at the mineral surface. Batch leaching experiments with microcline powder were conducted using a variety of organic ligands, both naturally occurring and synthesized. Inductively coupled plasma atomic emission spectroscopy was used to determine the potassium extraction efficiencies of the various ligands. Results suggest that multidentate ligands have higher potassium extraction efficiencies than monodentate or bidentate ligands.

Droplets in a Pickering emulsion can be arrested in non-spherical shapes due to the jamming of colloidal particles adsorbed at the droplet interface. In this jammed state, particles on the interface tend to be nearly close-packed and there is a high degree of hexagonal ordering. The Gaussian curvature of the surface induces defects in this ordering, and because a non-spherical surface has spatially varying Gaussian curvature, it is natural to ask how this variation affects the distribution of defects. In this work we look at the case of hard spheres jammed on an ellipsoidal surface. Simulated jammed configurations are generated with varying particle number and ellipsoid aspect ratio. The relation between defect charge density and Gaussian curvature is determined. Intriguingly, it is found that for prolate ellipsoids of sufficiently high aspect ratio (>3.0) there is, on average, an excess of dislocations between the equator and the pole. The formation of scars, or chains of defects, is also studied. It is found that on nearly spherical surfaces there is minimum particle number for which scars will form, i.e. a “scar transition,” though it is lower than that previously observed in similar systems due to the hard-sphere nature of the particles. As the eccentricity of the surface is increased, this scar transition is softened until there is no longer a minimum particle number required for scar formation. We also performed an exhaustive search of the parameter space to find highly symmetric particle configurations. Finally, we show an experimental realization of arrested ellipsoidal droplets and compare the particle configurations to those created in the simulations.

We develop a computational model to design and characterize multi-component nanostructured soft biomaterials interacting with functionalized and charged nanoparticles using the Molecular Dynamics simulation technique. Our aim is to generate a stable vesicle through the self-assembly of amphiphilic lipid molecules via the use of implicit solvent coarse-grained molecular models in order to investigate biologically important physiological processes occurring on the mesoscopic spatio-temporal scales. The amphiphilic lipid molecules are represented by a hydrophilic head group and two hydrophobic tails. In addition, we use two component lipid mixtures to study the formation of hybrid lipid vesicles through the self-assembly. We find that the degree of phase segregation between the two lipid species is tunable via their distinct effective chemical specificity and molecular architecture. Furthermore, we have extended the model to introduce the charged lipid molecules into the vesicle by using long-range electrostatic interactions between charged molecules. We investigate the interactions of various functionalized nanoparticles with the charged lipid species and effects of these interactions on the dynamics and morphologies of the lipid vesicles. Our findings can be used to design new soft biomaterials for various applications in medicine, sensing and energy.

Bio-inspired adhesives have attracted a lot of attention for robotics and medical applications. Of particular interest is the smart adhesive with the capability of active control of adhesion strength. Here, mimicking the wet adhesion of the microstructured foot of a tree frog, we demonstrate that the combination of a thermo-responsive polymer (poly(N-isopropylacrylamide) (pNIPAM)) and an elastomer (polydimethylsiloxane (PDMS)) microstructures enables a switchable adhesion in response to thermal stimulus. In this smart adhesive pad, the capillary bridges between the pNIPAM coated microstructure arrays and the opposing surface can be actively controlled by swelling or shrinking behavior of pNIPAM in response to the temperature change, resulting in reversible smart adhesion. Through control of temperature, the adhesion strength of smart adhesive pad can be switched between ~59 kPa at high temperature and ~0.3 kPa at low temperature. Furthermore, we show that the switchable adhesive pad can be employed in transfer printing process to heterogeneously integrate silicon and compound semiconductor nanomembranes onto arbitrary substrates. The bio-inspired smart adhesive pad suggested in this study is applicable to heterogeneous integration of arbitrary micro- and nanoscale objects, which have great potentials in high-performance transistors and optoelectronic devices.

The key tenet of bone regeneration is to control the population of cells that are capable of entering into active phases of the cell cycle, complete mitosis, and achieve differentiation into osteoblast phenotype. Such cell cycle control was believed to have been achieved by the controlled release of ionic dissolution products (Ca, Si) from bioactive glasses. However, none of the studies showed the potential osteoinductive effects of soluble silica separated from the effects of Ca, which is known to be a pro-osteoindictive factor. Also the possible mechanisms by which soluble silica may regulate the effects on bone cells were not identified. Here, we demonstrated that soluble silica by itself stimulates osteoblastic differentiation of human mesenchymal stem cells (MSCs) and promotes bone formation, but suppresses osteoclastic differentiation of hematopoietic stem cells (HSCs) and bone resorption. Differential microRNA (miR) microarray analysis revealed that miR-146a is significantly up-regulated by soluble silica. In addition, overexpression of miR-146a antagonizes the activation of NF-κB, a signal transduction pathway required for osteoclastic bone resorption, but inhibitory to osteoblastic bone formation. TNF-α and RANKL increased NF-κB nuclear translocation and DNA binding, which were down-regulated by the addition of Si. Thus, our data revealed for the first time that a major mechanism by which Si promotes osteoblastogenesis and suppresses osteoclastogenesis may center on the antagonism of NF-κB activation via miR-146a.

This work studies the suitability of using Tobacco mosaic virus-like particles (VLPs) as receptors in biosensors. The morphology of VLP on different materials in both macroscale and in microfluidics was investigated using scanning electron and atomic force microscopy. Surface evaporation was explored as means to accelerate the VLP assembly process.
Currently, selective and sensitive sensors or assays are essential to identify pathogens and assist with the treatment. VLPs are promising bioreceptors that overcome existing challenges such as complexity in surface functionalization chemistry and random functional layer density. These macromolecules have high-aspect-ratio and rod-like nanostructures and can be genetically modified with thousands of functional residues on the coat proteins (CP). In this work, a cysteine residue is expressed on the C-terminus of the CP to promote VLP self-assembly and metallization. Meanwhile, a peptide sequence (FLAG-tag) is expressed on the N-terminus of the CP for selective binding to the target antibody. Different from the conventional monolayer of bioreceptor immobilization, the VLP self-assembly transfers a planar surface to a nanoscale 3-dimentional functional surface with densely covered VLP nanorod receptors.
The experimental results show that VLPs are capable of self-assembly on materials that are widely used in sensors such as Au, Si, SiO₂, Si₃N₄, TiN, etc. The VLPs self-assemble to a mesh of close-packed nanorods on the surface when a static VLP solution is used. However, under lateral flow or dehydration conditions, these nanorods tend to assemble in parallel with the surface due to surface tension. In microfludics, higher density of the assembled VLPs is observed close to the edges than the center of microfluidic channels, which may result from the combination of higher surface tension and lower velocity profile at the edge. Evaporation greatly reduces the conventional 18 hour assembly time of VLP to around 15 min at 50°C. The vitality of cysteines and binding peptides on VLPs after evaporation has been verified using metallization and immunoassays, respectively. With evaporation, uniform and dense VLP assembly on the gold surface was observed using scanning electron microscopy (SEM). In addition, after performing immunoassays on chip, the SEM images and the colored precipitate formation show that the evaporation-enhanced VLP assembly on gold surface is stable with no significant VLP migration.
These results combined highlight the feasibility of using VLP as programmable receptors to rapidly functionalize transducer surfaces for biosensing applications. The process of VLP self-assembly can be accelerated through evaporation, and the surface morphology of the VLP functional layer can be manipulated by controlling the surface tension. This work will potentially contribute to the development of macromolecule-based biosensors with rapidly programmable functional layers.

Dental implant prosthesis greatly depends on primary stability and biocompatibility through a correct and stable osseointegration between titanium fixture and bone cells for tissue regeneration. The concept of artificial extracellular matrix (ECM) has been the focus of attention with its significant biological activitiy for the enhancement of target cell behaviors such as proliferation and differentiation in tissue engineering. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on inroganic and organic surfaces even in wet environment. Previously, we developed new cell adhesive, MAP-RGD, by fusion of MAP with RGD peptide, one of the major cell adhesion recognition motifs. In the present work, we investigated in vitro and in vivo osteoinductivity of MAP-RGD on titanium mesh surfaces. We found that cellular behaviors of mouse pre-osteoblast MC3T3-E1 cells such as adhesion, proliferation, spreading, and differentiation were significantly increased on MAP-RGD-coated titanium mesh surface. Also, the mRNA expressions of osteogenic differentiation marker genes were up-regulated in MC3T3-E1 cells on MAP-RGD-coated titanium mesh surface. Furthermore, MAP-RGD-coated titanium mesh surface showed improved new bone formation in rat calvarial defect model compared to non-coated surface as a result of the radiographic analysis and histological evalutations. Collectively, RGD peptide-fused MAP can be successfully employed in medical implant prosthesis and bone tissue engineering applications.

Removal of heavy metals from contaminated sites, soils and water, by natural, low cost and widely available adsorbents has been gaining an increasing relevance in the last decade. Many methods, both in-situ and ex-situ [1], and adsorbent materials have been proposed [2-4]: clay minerals, many different kinds of plants and plant wastes, bacteria. Calcium carbonate, either in the form of calcite or aragonite, e.g. obtained from mollusk shell, has been proposed as suitable heavy metal adsorbent [5,6].
The efficiency of calcium carbonate as filter for metals is now well established, but the entrapment mechanism, also known as coprecipitation, at the atomic level is still debated.
In this work the results of a theoretical study on the interaction between the Cd(II) ion with its first hydration shell and two non-equivalent stepped calcite surfaces, namely the (318) and the (3-1-216) are discussed. The first one has rows of 78 deg steps, the second 102 deg.
We used a first principles method within the framework of the Density Functional Theory (DFT) in the Generalized Gradient Approximation (GGA-PBE) and ultrasoft pseudopotentials.
Cd(II) forms an inner sphere adduct on both surfaces and the adsorption energy is of the order of 350 kJ/mol. The results obtained with 20ps simulation of Car-Parrinello molecular dynamics demonstrated that the favorite adsorption site of Cd(II) is the (3-1-216) surface, whose adsorption energy is 50 kJ/mol higher than the other.
Also, in order to understand the diffusion mechanism of the metal ion in the calcite crystal, we studied, by means of NEB and metadynamics, the migration of Cd from the surface to the bulk of calcite. Preliminary results are illustrated.
[1] F. Fu, Q. Wang, J. Environ. Manage. 92 (2011) 407-418
[2] J.-P. Schwitzguébel, J. Kumpiene, E. Comino, T. Vanek, Agrochimica, LIII - 4 (2009) 1-29
[3] W.S. Wan Ngah, M.A.K.M. Hanafiah, Bioresour. Technol. 99 (2008) 3935-3948
[4] T. Halttunen, M.C. Collado, H. El-Nezami, J. Meriluoto and S. Salminen, Lett. Appl. Microbiol. 46 (2008) 160-165
[5] Y. Liu, C. Sun, J. Xu, Y. Li, J. Hazard. Mater. 168 (2009) 156-162
[6] Y. Du, L. Zhu, G. Shan, J. Colloid Interface Sci. 367 (2012) 378-382

Gold and silver nanostructures are at the centre of plasmonics and applications in plasmon enhanced spectroscopies. In particular, gold nanoparticles present fascinating aspects involving daily life and because of their facile synthesis, easy surface functionalization, chemical stability and biocompatibility. Silver nanoparticles, on the other hand, have been studied mostly due their antimicrobial activity against different bacteria, fungi, protozoa and certain viruses. These noble metal nanoparticles also offer enhanced size-dependent catalytic, electronic and optical properties. For this reason, they become one of the most commonly used materials in plasmonics and ultrasensitive detection or surface-enhanced plasmon spectroscopy. Recently, hybrid structures of microorganisms with inorganic elements received great interest, showing to be versatile templates for the organization of nanostructured functional materials in large scale. The combination of living structures with advanced materials using small biological structures as templates is becoming suitable to produce functional materials for advanced and original applications. There are several examples of the use of living templates for noble metal nanoparticles such as fungus, yeast, viruses or simply supramolecular structures of biological origin. In this work, we used fungi as biotemplating to obtain self-assembled systems of gold and gold-silver nanoparticles forming stable mesostructures with potential use as biosensors via plasmon enhanced Raman spectroscopy. In our work the spores of the filamentous fungus Cladosporium sphaerospermum were inoculated in a suspension of gold nanoparticles, forming stable microtubules of gold nanoparticles during the fungus growth. These materials were exposited to a second suspension of silver nanoparticles, resulting in a complex multilayer structure of gold and silver nanoparticles, which were evaluated as surface enhanced Raman scattering (SERS) substrate using small amounts of thiophenol as probe molecules directly on the microtubules. Our strategy showed to be an innovative method to fabricate stable self-assembled structures of gold and gold-silver nanoparticles using the filamentous fungus Cladosporium sphaerospermum as biotemplates with potential use as biosensors via plasmon enhanced Raman spectroscopy. The FG microtubules, fabricate only with AuNP, showed a uniform morphology and a smooth surface that could be reproduced in large scale, with controlled thickness adjusting the correct initial concentration of AuNP. On the other hand, the FGS microtubules, which were obtained in two steps by deposition of AgNP on the surface of FS microtubules, exhibited a much rougher surface and superior potential for using as substrate for surface-enhanced Raman spectroscopy, with the SERS signal of thiophenol, which was used as probe molecule, 10 times more intense than the FG substrate.

Graphene is a new material, which has much potential for biomedical applications. It has unique properties such as high electrical and thermal conductivity, mechanical elasticity, and has been shown to induce differentiation of mesenchymal stem cells. Polyisoprene (PI) is a major component of Gutta Percha, the material used for obdurating root canals. Recently, we have shown that this material may enhance dental pulp cell differentiation, which opens up the opportunity for its use in regenerative dental therapy. Currently, in order to achieve the appropriate mechanical and anti-bacterial properties for use in root obduration, PI is compounded with zinc oxide nanoparticles. As these particles are cytotoxic when not encased in PI, we sought a replacement material more appropriate for regenerative therapies. Here we show that graphene may be an appropriate substitute. It is biocompatible, non-toxic, and provides the desired mechanical and anti-bacterial response. For these experiments 50% graphene and PI suspensions were spun cast from toluene solutions onto Si substrates and annealed in vacuum at 130C. Dental pulp cells were plated on the films and incubated in for 28-days in growth media with and without dexamethasone as an inducer of differentiation. Biomineralization and cell differentiation were compared to cells plated on 120 nm PI films and tissue culture plastic controls. In the absence of dexamethasone, no biomineralization was observed on the PI film or on tissue culture plastic. However, copious amounts were observed on the graphene containing PI films. All samples biomineralized in the presence of dexamethasone. Expression of the differentiation marker osteocalcin by immunohistochemistry followed the biomineralization results, suggesting that graphene promoted both differentiation and biomineralization. In order to understand whether the induction is due to mechanical or electrical stimuli, experiments are being conducted where DPSC are incubated on TCP with graphene in the media and with PI containing non-conducting clay platelets.

Biominerals have intricate nanocomposite structures, which affect their elastic properties, hardness and, in particular, their fracture toughness, which is often orders of magnitude higher than that of inorganic minerals. The origin of the exceptional mechanical properties of biominerals is complex and currently poorly understood and this lack of understanding is inhibiting the development of man-made composites with comparable properties.
A recent study used nanoindentation experiments to demonstrate that biogenic calcite exoskeletons achieve high penetration resistance through deformation twinning [1]. The biogenic calcite underwent twinning in three crystallographic directions in response to indentation whereas only one twin orientation was activated in single crystal calcite. Understanding how twinning, and other energy dissipating mechanisms, are affected by organic inclusions is key to improving toughness and penetration resistance in man-made ceramic composites. The aim of our study is to use advanced modelling techniques to investigate the effects of amino acid inclusions on the mechanical properties and deformation mechanisms of calcite. We investigate the process of inclusion of amino acids in calcite (CaCO₃) during crystallization, using metadynamics, and we examine the structural arrangement of the amino acids in the calcite crystal. We calculate the full elastic constant tensors of calcite at ambient conditions and we investigate how amino acid inclusions alter the elastic properties. Finally, we model deformation twinning in calcite using molecular dynamics. We observe the formation of pairs of (1 0 4) twin boundaries in response to uniaxial tensile strain and the kink mediated twin boundary motion, which results in grain growth. We evaluate the effect of amino-acid inclusions on the creation and motion of twin boundaries.
[1] Ling, L and Ortiz, C., Nature Materials 13, 501-507 (2014)

Many of the physical properties of biogenic crystals originate from the presence of intracrystalline organic molecules within individual inorganic crystalline hosts. The presence of these molecules has been shown to strongly influence the crystalline host microstructure and structure to (anisotropic lattice distortions). Recently, by applying a bio-inspired approach, we have shown that similar microstructures and lattice distortions can be achieved in synthetic calcium carbonate crystals grown in the presence of organic molecules¹. However, no similar approach has been performed on non-calcium carbonate crystals. In this work, we utilize this bio-inspired approach so as to modify the crystal properties of functional semiconductor material^2,3.
We will show that amino acids can get incorporated into the crystal lattice of semiconductor hosts, similar to the process observed for calcium carbonate. Moreover, not only that such incorporation exists; the resulting lattice distortions are accompanied by a significant band-gap energy shift of the semiconductor host.
We will discuss possible mechanisms for this phenomenon and show that such bio-inspired organic/inorganic interfaces have much potential for the manipulation of not only structural properties of different crystalline hosts but rather of a variety of functional properties. Moreover, we believe that this research may open a new bio-inspired route for tuning the band-gaps of semiconductors in addition to the ones known and utilized to date.
¹ Borukhin S, Bloch L, Radlauer T, Hill AH, Fitch AN, Pokroy B. Screening the Incorporation of Amino Acids into an Inorganic Crystalline Host: the Case of Calcite. Advanced Functional Materials 2012;22:4216.
² Brif A, Ankonina G, Drathen C, Pokroy B. Bio-inspired Band Gap Engineering of Zinc Oxide by Intracrystalline Incorporation of Amino Acids. Advanced Materials 2014;26: 477.
³ Brif A, Pokroy B. Bio-inspired Engineering of Zinc Oxide/Amino Acid composite: Synchrotron Microstructure Study. CrystEngComm 2014;16: 3268.

Outstanding mechanical properties of biological multilayered materials are strongly influenced by nano-scale features in their structure. In this study, mechanical behavior and toughening mechanisms of abalone nacre-inspired multilayered materials are explored. In nacre's structure, the organic matrix, pillars and the roughness of the aragonite platelets play important roles in its overall mechanical performance. A micromechanics model for multilayered biological materials is proposed to simulate their mechanical deformation and toughening mechanisms. The fundamental hypothesis of the model is that nano-scale pillars and asperities have near theoretical strength. It has previously shown that organic matrix behaves stiffer in proximity of mineral platelets and short molecules in organic matrix behave stiffer. The proposed model assumes that pillars and the asperities confine the organic matrix to the proximity of the platelets and hence increase their stiffness. The modeling results are in excellent agreement with the experimental results for abalone nacre. The results show that the stiffness of the organic matrix affects the stiffness of material while platelets' aspect ratio determines the ultimate strength. The pillars and the asperities are responsible for the ductility of multilayered material. In the proposed model, while all the components of the bioinspired structure have brittle behavior, the overall structure has a ductile response. The highly nonlinear behavior of the suggested multilayered material is a result of a distributed deformation in the nacre-like structure due to existence of nano-asperities and nano-pillars with near theoretical strength.

This study aims to determine the impact of ocean acidification on Southern Ocean pteropods via a detailed understanding of the structural and mechanical properties of their shells.
Many marine organisms build their external skeletons and shells from biogenic calcium carbonate (CaCO_3shy;). As both the ocean pH and the availability of dissolved CO₃^2- decreases, it is likely that these calcifying organisms will find it increasingly difficult to form and maintain their shells.
Pteropods, also known as sea butterflies, are small planktonic free-swimming marine molluscs that are common in polar waters. They produce shells of aragonite, a form of calcium carbonate, and are expected to be adversely affected by ocean acidification. Pteropods are an integral part of the marine food chain of the Southern Ocean with many organisms, from zooplankton to whales, relying on them as a food source [2]. Consequently, changes in their abundance or distribution could have a substantial flow-on effect for the whole Southern Ocean ecosystem.
In this work, two sets of Southern Ocean pteropod shells collected in 1998 and 2007 were analyzed. Shells were mounted in epoxy resin and polished to reveal a cross-section. Nanoindentation was used to measure the mechanical properties (hardness and modulus), and Raman microspectroscopy identified the material of the shells. Focused Ion Beam (FIB) methods were used to prepare sections for transmission electron microscopy (TEM). Scanning electron microscopy (SEM) was used to examine both the surfaces of the shells (prior to mounting in epoxy resin) and cross-sections (after polishing).
The average hardness from each shell ranged between 0.5 GPa to 4.5 GPa and the modulus ranged between 12 GPa to 64 GPa. The mechanical properties were not position sensitive with respect to the region of the shell indented. There was no statistically significant difference in the average modulus between the two sets of shells, but some weak evidence of a difference in average hardness. TEM revealed information on the multi-layered structure of the shells and with Raman microspectroscopy, identified the shell material as aragonite.
[1] J. Orr et al, “Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms”, Nature, vol. 437, pp. 681-686, September 2005.
[2] Hunt et al, “Pteropods in Southern Ocean ecosystems”, Progress in Oceanography, vol 78, pp. 193-221, 2008.

Collagen is the principal load-bearing protein in both mineralized and unmineralized tissues. In tendon, where the collagen is usually unmineralized, an interface is formed with the solvent (most commonly water, but other organic liquids are also considered in this study) which significantly alters its tensile modulus. In bone, an interface may also form with the mineral phase, which can result in an enormous increase in tensile modulus and stability. For example, the collagen in fossils can resist degradation for periods up to 1 million years. In this work, we present strain-realistic molecular dynamics simulations combined with thermodynamic integration to investigate the interfacial energy of collagen systems. Direct scaling of the interfacial energy reveals the importance of solvent-induced surface stress, which stabilizes the triple helix structure via a circumferential (not radial) contraction. Results are corroborated by small- and wide-angle X-ray scattering experiments, and the confining effect of solvent is attributed mainly to the entropy change of solvent ordering around the triple helix. We present Raman spectroscopy evidence for a mineralization-induced peak shift in the vicinity of 855 cm^-1. Using density functional calculations, we demonstrate the connection between proline ring pseudorotation and stress transfer within the mineralized collagen structure

Biominerals produces by living organisms show very diverse composition and structure. Several of these biominerals are complex composite materials, where organic matrix is closely linked together with crystalline components forming unique materials with excellent physical and mechanical properties [1, 2].
Rodents possess opposing long pairs of continuously growing incisors. The front surface of the incisors is enamel that is composed of 96 wt% of inorganic material; the inner part is softer dentine that forms the bulk of the teeth [3]. The surface of incisors of many different rodent species shows characteristic orange-brown color and is identified with the presence of iron [4].
In our study incisors from the feral coypu (Myocastor coypus Molina) were investigated using imaging and analytical transmission electron microscopy (TEM) methods. The microstructure and the chemical composition of these continuously growing teeth were investigated at high spatial and high energy resolution. Our investigations uncovered the layer with variable thickness positioned on the outer surface of the teeth which has not been observed before in rodent teeth. Based on our analytical measurements the amount of iron in this surface layer is much higher compared to the concentration values reported in the literature by now. Studies of electronic structure suggest that iron is present as in predominantly 3+ valence state. Within the iron-rich surface layer we surprisingly detected multiple iron containing varieties. In addition, the interfaces between the iron-rich surface layer and iron-rich enamel, as well as between enamel and dentine were investigated. Despite of wide occurrence of iron in many living organisms, its function in hard dental tissues is not unambiguously understood. Present discoveries will endorse the understanding and function of iron incorporation in the hard dental tissues at the nanoscale level.
[1] UGK Wegst and MF Ashby, Philos Mag 84 (2004), 2167.
[2] AP Jackson and JFV Vincent, J Mater Sci 25 (1990), 3173.
[3] BA Niemec in “Small animal dental, oral & maxillofacial disease” (2010), Manson Publishing Ltd, London.
[4] EV Pindborg JJ Pindborg and CM Plum, Acta Pharmacol 2 (1946), 294.

Polymer-nanoparticle (NP) composites have attracted renewed attention due to their enhanced mechanical strength combined with potential advantageous electrical, optical or magnetic properties. The interfacial interaction of NPs with the polymer matrix is crucial for the material&’s mechanical behavior, however controlling this interface remains a major challenge. Inspired by the adhesion chemistry of mussel fibers, we have investigated a novel approach to incorporate iron oxide nanoparticles (Fe₃O₄ NPs) into a hydrogel matrix as crosslinkers. A polyethylene glycol polymer terminated by catechol groups is designed to form a hydrogel network crosslinked via coordination bonding at the surfaces of Fe₃O₄ NPs, yielding a stiff and self-healing hydrogel. Due to the reversible nature and unique crosslinking geometry of coordination bonding, the composite hydrogel presents a very different relaxation behavior compared to conventional gel networks. The interfacial coordination interaction can be well-engineered by controlling the inorganic surfaces, polymer ligands and environment pH, thereby determining the material&’s relaxation kinetics. In addition, the superparamagnetic property of Fe₃O₄ NP is preserved after gelation, allowing for design of responsive functional materials. Finally, we have demonstrated that this gelation motif is applicable in various inorganic nanomaterials, which can open up a versatile approach for designing polymer nanocomposite materials with controlled mechanical properties and functionalities.

Chitosan is a naturally derived polymer. It represents one of the most technologically important classes of active materials with applications in a variety of industrial and biomedical fields. However, they are used in limited applications because of disadvantages such as poor electromechanical properties, high brittleness with a low strain at break, and sensitivity to water. In certain critical applications, the need arises to modify the physical, mechanical and electrical properties of the polymer. When blends of polymer films with other materials are used for medical devices, as is commonly the case, device performance directly depends on the nanoscale morphology and phase separation of the blend components. Here, chitosan reinforced bio-nanocomposite films with varying concentrations of gold nanoparticles were prepared through a solution casting method. Gold nanoparticles (~ 32 nm diameter) were synthesized via a citrate reduction method from chloroauric acid and incorporated in the prepared Chitosan solution. Uniform distribution of gold nanoparticles was achieved throughout the chitosan matrix and was confirmed by SEM images. Synthesis outcomes and prepared nanocomposites were characterized using TEM, SAED, SEM, EDX, XRD, UV-vis, particle size analysis, zeta potential and FT-IR for their physical, morphological and structural properties. Nanoscale mechanical properties of the nanocomposite films were characterized at room temperature, human body temperatures and higher temperatures using instrumented nanoindentation techniques. The obtained films were confirmed to be biocompatible by their ability to support the growth and proliferation of human cells in vitro. Statistical analysis on mechanical properties and biocompatibility results, were conducted. Results revealed significant enhancement on both the mechanical properties and cell adherence and proliferation. The results will enhance our understanding of the effect of nanostructures reinforcement on these important functional polymeric thin films for potential biomedical applications.

Bio-inspired synthesis of inorganic nanomaterials has received much attention due to the ability to generate technologically relevant materials. Optimized surface features of noble metal nanoparticles can lead to a variety of super-functional materials as sensors, electrodes, catalysts, and biomedical diagnosis and therapeutic agents. By exploiting the complexity found in materials binding peptides, an ultimate goal in peptide-derived nanotechnology is to control a material&’s properties by rational design of the amino acids sequence of the peptide; however, a thorough understanding of biotic/abiotic interactions and atomistic scale probe of the structures are prerequisites for accomplishing this goal. In this contribution, atomic pair distribution function (PDF) analysis of high-energy X-ray diffraction (HE-XRD) patterns are used to obtain an atomic scale structure details of peptide-derived Pd nanocatalysts. The atomic PDFs are modeled using Reverse Monte Carlo (RMC) simulations, which result in a size averaged configurations that are highly disordered at the nanoparticle surface. Molecular dynamics (MD) simulations of the particles using a monolayer of the corresponding peptides in aqueous solution were then performed using the INTERFACE-CHARMM force field to obtain thermodynamically stable morphologies of the particles as well as configuration of the peptides on the surface. The functionality of Pd nanoparticles in the model systems of carbon-carbon coupling and allyl alcohol hydrogenation reactions with different catalytic mechanisms were examined computationally and experimentally. A strong correlation was found between the measured TOFs in experiment and calculations, indicating that structure of the particles resemble the actual morphologies in the experiment. Taken together, the combination of PDF, RMC, MD analysis reveals sequence-dependent atomic-scale structural differences in Pd nanocatalysts that were previously assumed to be structurally identical. This technique can be applied to optimize the catalytic activity of similar nanoparticles and alloys, and more broadly to predict structure/property relationships of nanomaterials at the 1 to 100 nm scale.

Bone is a hierarchical organic-inorganic composite which at the nanostructural level consists of an assembly of collagen fibrils that are embedded with uniaxially-aligned nanocrystals of hydroxyapatite. Our in vitro model system has shown that bone&’s nanostructure can be reproduced using polymeric additives to mimic the action of the non-collagenous proteins found in bone. The high charge density of the polyanionic additive (e.g. polyaspartate or osteopontin) sequesters ions/clusters such that nanodroplets/particles of a hydrated amorphous mineral precursor are formed, and these can infiltrate into the interstices of collagen fibrils, leading to an interpenetrating organic-inorganic composite that emulates bone&’s nanostructure. Through optimization of reaction parameters, compositions matching bone (60-70 wt% mineral) have been achieved in a variety of collagen scaffolds, including both reconstituted type-I collagen and biogenic (bone, dentin, tendon) matrices. We believe that by mimicking the hierarchical structure of bone from the nano- to micro-structural level, it will be possible to match the mechanical properties of bone. Therefore, our current studies are directed at assembling parallel-fibered collagen which can be mineralized in the form of laminated composites, mimicking the lamellar microstructure of bone. With respect to bioactivity, we have found that cell signaling mechanisms provided by osteopontin additive may provide a means for modulating osteoclast activity, thus providing a means for tailoring the resorption rate of these bone-like composites. The long-range goal of these studies is to prepare bioresorbable load-bearing bone substitutes that can be remodeled through the natural bone remodeling unit (BRU). In contrast to the more common approach of biodegradable implants, which require careful matching of the degradation rate to bone ingrowth, we hypothesize that a biomimetic approach will enable synchronization of the cellular activity within the multicellular BRU, which could then enable load-bearing capacity to be maintained throughout the remodeling of the implant, as occurs during natural bone remodeling.

The mechanism behind the assembly of the mineral hydroxyapatite (HA) phase and organic collagen presents one of the greatest challenges in the understanding of biochemical processes in bone formation. It is widely believed that collagen fibrils act as template for the formation of the hydroxyapatite mineral. To shed light on the nanoscale details of this mineral/organic composite high-resolution electron microscopy based characterization techniques in conjunction with µ-Raman spectroscopy were employed to investigate the nanoscale structure of human femur bone. For this purpose focused ion beam milling was applied to produce less than 100 nm thick electron transparent lamellae of untreated bone. Our studies show that the hydroxyapatite phase in human bone is constituted of needle shaped crystals elongated in the <001> direction with base diameters of 5 - 10 nm and several 100 nm length. Moreover, a plywood type of stacking could be observed in accordance with previous reports studying the collagen arrangement in demineralized bone. If imaged edge-on along the <001> direction the HA needles show a regular spiral type of arrangement with spiral diameters of approximately 100 - 150 nm and decreased mineral density at the centers of the spirals. Based on these observations we speculate that spiral crystalline areas represent areas of originally similarly oriented collagen which acted as template for the subsequent HA growth. This also indicates that there is significantly less mineral deposited in the collagen fibril centers.

Tooth enamel is the hardest tissue in vertebrates. Optimized to withstand the forces of mastication, it is composed of hydroxylapatite nanowires, thousands of which are bundled into rods that are organized in a three-dimensional weave.¹ The outstanding fracture resistance of enamel and its long fatigue life are the consequence of this hierarchical architecture. Tooth enamel is also the target of the most prevalent infectious disease in humans: dental caries. It is an infectious disease that has extremely high morbidity, with 60-90% of children and nearly 100% of adults worldwide having or having had caries.² Caries, simply put, is the destruction of tooth biominerals by dissolution and commonly begins with the demineralization of enamel by acids produced in plaque biofilms.³ It has long been known that the susceptibility of enamel to acid dissolution is greatly dependent on the presence of magnesium, carbonate, and fluoride ions. However, imaging the distribution of these impurities in enamel has remained a great challenge. Here we show that the majority of Mg and majority of residual biomacromolecules in enamel are present at grain boundaries and in inter-crystalline precipitates of Mg-substituted amorphous calcium phosphate (Mg-ACP). We find evidence that Mg-ACP is responsible for the rapid and highly anisotropic acid etching of enamel. The very architecture that makes enamel mechanically robust may thus be its Achilles heel. Remarkably, inspiration on how to overcome this weakness comes from pigmented rodent enamel. Here, a mixture of metastable ferrihydrite and amorphous calcium/iron phosphate replaces the much more soluble Mg-ACP. This substitution not only renders pigmented enamel mechanically harder, but also hardens it to acid attack to a greater extent than topical fluoride treatment. This discovery will pave the way for bio-inspired engineering of enamel grain boundaries and for developing next generation, “post fluoride” dental care products.
[1] Nanci, A., Ten Cate's Oral Histology: Development, Structure, and Function, 8 ed., C.V. Mosby Co., 2012.
[2] World Health Organization, 2014, http://www.who.int/mediacentre/factsheets/fs318/en/.
[3] Robinson, C., Shore, R., Brookes, S., Strafford, S., Wood, S., Kirkham, J., Critical Reviews in Oral Biology & Medicine 2000, 11, 481. "The chemistry of enamel caries."

Collagen biomineralization is a complex process for which the controlling factors, at the molecular level, are still not well understood. A particularly high level of spatial control over collagen mineralization is evident in the anchorage of teeth to the jawbone by the periodontal ligament: unmineralized collagen fibrils of the ligament become a fully-mineralized part of the cementum layer of the tooth at an unusually abrupt interface (~200 nm). The close juxtaposition of mineralized and unmineralized tissues provides an excellent model in which to study the molecular factors that control collagen biomineralization, and in particular the relative roles of collagen fibril structure vs. non-collagenous proteins. This knowledge will be critical in the design of collagen-based scaffolds for tissue engineering of hard-soft interfaces such as the periodontal ligament-cementum junction, or for the reattachment of tendons and ligaments to bone.
Here we present a two-pronged approach to study control of mineralization at the periodontal ligament-cementum junction. By applying cryogenic electron microscopy techniques (cryo-TEM) to these tissues for the first time, we were able to study collagen structure at high resolution under near-native conditions. We show that ligament fibrils increase in width by 40% in cementum, while fibril axial periodicity decreases by ~ 3 nm, representing a significant structural change. We also developed a novel in vitro model, in which we demonstrate that the demineralized extracellular matrix of periodontal tissues contains sufficient information to reproduce with high fidelity the pattern of mineralization in native tissues in metastable calcium and phosphate-containing solutions. This model provides a unique platform with which to probe which extracellular matrix molecules are responsible for this control through selective enzymatic removal of different components.

One of the challenges of probing the bioimineralisation process is the ability to identify mineral compositions at the nanometre scale during tissue formation and tissue disease. Acquisition of this information is the first step in facilitating characterisation of different phases present in tissues and bioceramics. Since mineralisation events frequently occur at the length scale of the collagen fibrils, it is critical that compositional information be acquired with nanometre scale spatial resolution. A second challenge is to characterise the organic-inorganic interface using methods that combine nanometre spatial resolution with the ability to detect and identify chemical bonding environments.
The modern analytical electron microscope is the only characterisation tool that can address both of these challenges. However, the heavy metal stains commonly used to provide contrast in biological materials directly mask the local chemistry and bonding so cannot be used. Aberration corrected scanning transmission electron microscopy (STEM) combined with detector geometries such as annular bright field (ABF) and high angle annular dark field (HAADF) imaging allow us to obtain contrast in images of unstained samples. Analytical techniques such as electron energy-loss spectroscopy (EELS) and energy dispersive X-ray (EDX) analysis provide information about chemistry and composition. More importantly, developments in spectrometer and detector technology that have taken place in the last five years mean that we can not only identify the type of atom at specific locations, we can also probe how it is bonded to the other atoms around it.
In this contribution we will demonstrate the use of optimized focused ion beam (FIB) methods to produce site-specific samples from dentin and other biomineralised tissues. We will show how STEM-EDX mapping using a high solid angle quadrant detector allows for fast chemical mapping to identify compositional fluctuations arising from biomineralisation processes. Monochromated STEM-EELS has been used to investigate hydroxyapatite and related bioceramics and we will show how this can provide information on substitutional groups in the apatite phases and that this can be achieved with high spatial resolution. Finally, we will report how combining these methods for the study of healthy and abnormal (osteogenesis imperfecta) mice tissues can be used to reveal defects in the fibril architecture and mineral chemistry in the OI model.

Composite materials and multilayered systems are widely used in civilian and military applications. However, composite materials tend to be brittle, and are consequently vulnerable to delamination when impacted. Delamination at the interface is the primary mechanism that leads to the failure of composite materials. The interface is an important factor controlling the stiffness, strength, and fracture properties for composite materials at the macro scale. Biological materials possess a combination of properties that are not found in man-made engineered materials. These naturally occurring biocomposite structures rely upon their biochemistry to produce biological scaffolds that lead to the formation of hierarchical structures. These hierarchical structures provide exceptional mechanical properties such as high stiffness, toughness and crack/delamination resistance. Stiffness and toughness are examples of mutually exclusive properties for man-made materials. Whereas nature uses limited constituent components to produce remarkable functional materials that possess high toughness and crack resistance characteristics. Substantial efforts have been spent investigating the mechanical response of biological systems. However, very little research has focused on studying the fundamental principles of how nature uses biochemistry to form bio-laminate structures comprised of hard-soft matter interfaces that are delamination resistant.
The Atractosteus spatulas (Alligator gar) was used as the model bio-laminate structure. Alligator gar possess a flexible dermal armor consisting of overlapping ganoid scales. Each scale is a bilayer hydroxyapatite (Hard Construct) and collagen (Soft Construct) bio-laminate and is thought to be used for protection against its predators. The exoskeleton fish scale interface was modeled using the finite element method. Preliminary results were based on an idealized elastic-perfectly plastic mathematical model. The mathematical model was used to infer the effects of the nonlinear material response in terms of in terms of energy dissipation and stress redistribution at the ganoine-bone interface. The results indicate that at the ganoine-bone interfaces stress is reduced and energy is dissipated across the saw tooth junction points.
Keywords: Biological Materials, Delamination Resistance, Alligator gar, Finite Elements

Intrinsically disordered peptides (IDPs) defy conventional views of the protein/peptide structure-property paradigm, by showing the ability to confer function in the absence of a well-defined secondary or tertiary structure. The suggested function of IDPs such as n16N in the bio-mineralization of calcium carbonate materials throughout Nature highlights the potential use of IDPs for the generation of new high-performance composite materials. An interesting sub-class of IDPs comprise proteins/peptides that can reversibly switch conformation from disordered to a well-defined structure, in the presence of an external stimulus, potentially as part of a bio-mineralization process[1].
JAK1 is a de novo designed hydroxyapatite-binding peptide that exhibits IDP-like behaviour[2]. Based on experimental findings, JAK1 is thought to be unstructured when free in NaCl solution. While the addition of aqueous Ca²⁺ appears to induce α-helical folding. Furthermore JAK1 is thought to fold into an α-helical conformation when bound at the calcium-rich aqueous hydroxyapaptite interface. A control mutant known as cJAK1 (where key GLA residues are mutated to GLU) does not appreciably fold or bind to hydroxyapatite.
Molecular simulation can provide complementary understanding about the IDP-like nature of JAK1, granting in-depth molecular-level insights into the properties which may allow for general conformation switching mechanisms as thought to operate in such IDPs, and the importance of this ability to the bio-mineralization process. Here, we present results of large-scale Replica Exchange with Solute Tempering (REST)[3,4] molecular dynamics simulations of JAK1 and cJAK1 in NaCl and CaCl₂ solutions, to provide such insights.
Utilizing a clustering analysis, we predict the most prevalent structures present in solution for JAK1 and cJAK1. Ramachandran plots are utilized to identify dominant secondary structure motifs, and residue-residue contact maps are used to determine key intra-peptide interactions. Utilizing these techniques we provide molecular-level insight into how environmental influences may mediate the conformational switching of IDPs such as JAK1 (in contrast to cJAK1) through side-chain interactions.
References:
[1] Uversky, V. N. Int. J. Biochem., 2011, 43, 1090
[2] Capriotti, L. A.; Beebe, T. P.; Schneider J. P. J. Am. Chem. Soc., 2007, 129, 5281
[3] Terakawa, T.; Kameda, T.; Takada, S. J. Comp. Chem., 2010, 32, 1228
[4] Wright, L. B.; Walsh, T. R.; Phys.Chem. Chem. Phys., 2013, 15, 4715

The amelogenin protein is the dominant matrix protein expressed in enamel-forming tissues and is believed to regulate hydroxyapatite (HAP) mineralization extracellularly. This regulation leads to highly intricate and hierarchical enamel microstructures and unusually hard mechanical properties. The fundamental knowledge of amelogenin&’s behavior on HAP surfaces may lead to applications in treatments for caries, periodontal disease, and enamel regeneration. In this study we try to understand how amelogenin interacts with the HAP (100) face using in-situ atomic force microscopy (AFM). Mouse amelogenins (rp M179 and rp(H)M180, which is a modification of rp M179 with 11 amino acid histidine tag) were characterized by dynamic light scattering (DLS) in pH 8.0 buffered by Tris. The amelogenin solutions contained nanospheres, which were aggregates of amelogenin oligomers 20-25 nm in diameter. The protein solutions were introduced into a liquid AFM cell and images of protein adsorbed on HAP (100) were obtained in tapping mode with a minimal tapping force of 50 pN. When the rp(H)M180 nanospheres interacted with the crystal surface, they became destabilized due to weak oligomer-oligomer binding. The nanospheres disassembled onto the surface, breaking down into oligomeric subunits 6-8 nm in height and thereby reversing the process of nanosphere self-assembly. The oligomers became more monodisperse in height through post-adsorption disassembly followed by re-aggregation. Above a critical protein concentration of 63 µg/mL, rp(H)M180 formed two-layers of oligomers on HAP (100). In contrast, rpM179 adsorbed in the form of a single monolayer of oligomers over a wide range of protein concentrations, but the adsorption rate of rpM179 was 1.9 times higher than that of rp(H)M180. These results indicate that rpM179-HAP (100) binding is stronger than rp(H)M180-HAP while rpM179-rpM179 binding is much weaker than rp(H)M180-rp(H)M180. Thus we conclude that the histidine tag alters the binding of amelogenin to both HAP and to itself. Moreover, these different behaviors of rp(H)M180 and rp M179 on HAP surface may provide a good model for malformation of enamel under control of mutant proteins due to the high sensitivity of amelogenin-HAP interactions to errors in protein sequence.