Self-assembly of organic matrices and subsequent directed nucleation of the mineral constituents is a widespread paradigm in biomineral formation. The architecture of the underlying matrix imposes order on the nucleating mineral species. For example, in bone collagen monomers form intro triple helices, which then self assemble into well-organized fibrils. Within these fibrils highly-oriented hydroxyapatite crystals nucleate and grow with a specific crystal face in contact with the collagen fibril. In order to understand the underlying physical controls governing both matrix self-assembly and biomolecule-directed crystallization, we are using in situ AFM and TEM combined with molecular dynamics (MD) to investigate these processes. I first examine the results of investigations into the assembly of extended protein structures formed from both collagen and microbial S-layer membrane proteins. The findings reveal the key role that is played by conformational transformations associated with assembly in controlling the pathways and kinetics of this process. The larger barriers associated with the transformation renders it the rate-limiting step in forming the ordered structure. Consequently, these systems must be driven to condense into metastable liquid-like or amorphous structures in which protein-protein contact times are large before the ordered state can emerge. Moreover, the emergence of order catalyzes the further transformation and attachment of the monomeric proteins. Studies of mineral nucleation dynamics on organic monolayers and collagen matrices are then used to show that these surfaces promote nucleation through a reduction in interfacial energy. However, nucleation of the amorphous phase in the calcium phosphate-on-collagen system is observed at supersaturations that are too low to be explained by classical nucleation theory (CNT). The existence of pre-nucleation clusters is shown to provide a low-barrier pathway to crystallization that circumvents the large barriers predicted by CNT. Molecular dynamics simulations of clustering in solutions that utilize replica exchange methods to reach large cluster sizes with accurate potentials reveals the common occurrence of multi-ion clusters and, at least in the FeCO₃ and CaCO₃ systems, formation of 3D polyhedra that can be mapped to the hydrated crystal structures. Efforts are currently underway to compute the free energy landscape of the clusters in order to develop a predictive understanding of cluster-driven nucleation.

Tooth decay, also known as dental caries, is the most ubiquitous disease in humans and severely affects quality of life [1]. Enamel, the outermost covering of the tooth is the most susceptible to caries. With billions of dollars being spent on dental services in the US alone, there is no question that a significant demand exists for improved dental treatments . Currently, treatments involve the use of resins or alloys, which replace damaged tissue. This approach is far from ideal, as repairs commonly fail and can damage surrounding healthy tissue. One of the largest challenges of working with tooth is that, unlike other mineralized tissues such as bone, the outermost enamel layer does not regenerate. Despite decades of research on enamel, the complex nanoscale structure and chemistry of the tissue is still not fully understood. As more complete understanding of enamel nanostructure is a fundamental milestone for preventing and properly treating dental caries. Atom-probe tomography (APT) is uniquely capable of providing the necessary structural insight at the atomic scale by directly probing the location and chemical identity of the atoms within a small sample of material. APT consists of a field-ion microscope coupled to a time-of-flight mass spectrometer, providing chemical and three-dimensional positional information of ions successively field evaporated from a sharp sample. We recently demonstrated the remarkable ability of APT to map the structure and chemistry of buried interfaces in biological minerals in the magnetite cusp of the chiton tooth [2]. We report here on the application of APT to the characterization of mouse incisor enamel. We will show three-dimensional reconstructions of the detected ions originating from the mineral and remnant organic molecules within the enamel sample volumes. Such reconstructions clearly show the individual 10-20 nm diameter faceted crystallites. The unique sensitivity of APT revealed numerous (ex. Na+, Mg2+, CO32-) localized primarily at the crystallite boundaries. It is possible that the ions are associated with organic molecules or they might be impurities pushed to the grain boundaries during crystal growth. In addition to the impurity ions which are found natively in enamel, we will report on our investigation of the nature of fluorine incorporation in enamel. Although enamel fluoridation for caries prevention is extremely widespread the exact nature of the fluorine incorporation into the structure of enamel at the nanoscale is not yet understood. Preliminary results suggest that fluorine incorporation differs substantially between systemic exposure and topical fluoridation of enamel surfaces. [1] National Institute of Dental and Craniofacial Research [2] L. Gordon, D. Joester, Nature 2011, 469, 194-197

Nanomechanical characterization of materials has recently attracted significant attention because of the emergence of various novel nanoscale materials and structures over the past decade. Mechanical properties of bone have been studied intensively and the results suggest that interpenetrating structure of the single mineralized collagen fibril makes an important contribution to the toughness and stiffness of bone. However, the complicated structure of bone and limited experimental data at the nanoscale make the role of individual fibrils unclear. Our goal is to build and validate a new and improved structural model of the mineralized collagen fibril to help understand the mechanisms of bone toughness and deformation. In this new model, the mineralized collagen fibril is composed of a bundle of subfibrils that are 10-nm thick and 50 to 150 nm long. These subfibrils are made of collagen microfibrils encapsulated with HA nanocrystals to form core-shell cylinders. Interdigitations exist between the collagen microfibrils through entanglement and cross-linking. Mineralization over the interdigitized collagen network produces two continuous phases, facilitating stress distribution. The mechanical responses of these 3-D structured networks of mineralized collagen fibrils were simulated using finite element modeling (FEM). Several structural parameters, including thickness and length of mineral, interdigitations and tilting angle of the microfibrils with respect to the long axis of the fibril, were analyzed to determine their influence on the mechanical behavior of the mineralized collagen fibrils. In addition, individual fibrils were placed on a Hysitron Push-to-Pullâ"¢ device using a nanomanipulater system, and tensile tests were performed on them inside a Hysitron PI 85 SEM PicoIndenterâ"¢ to allow their deformation during loading to be observed in situ. The modeling results were compared with the experimental tensile tests to validate the model.

Under increasing tensile load, the lifetime of a bond can decrease exponentially (a slip bond) or counterintuitively increase up to a maximum and then decrease exponentially (a catch bond). So far, the characteristics of a single slip or catch bond dissociation have been extensively studied. However, how a cluster of catch or slip bonds behave under non-uniform tensile load has not been fully understood. We develop a mechanistic model and perform computational analysis on Atomic Force Microscope (AFM)-manipulated cell setup to study the effect of slip bond on the cell adhesion and detachment. The simulation results are not only compared favorably with experimental measurements, but also reveal several interesting insights into how the probing speed and depth affect the cell adhesion strength. We further examine the characteristics of clustered catch bonds under three different loading conditions: 1) clusters of catch bonds with equal load sharing, 2) clusters of catch bonds with linear load sharing, and 3) clusters of catch bonds in micropipette-manipulated cell detachment. We identify the critical factors for exhibiting the characteristics of catch-bond mechanism for the multiple-bond system. Our computation reveals that for a multiple-bond cluster, the catch bond behavior could only manifest itself under relatively uniform loading condition and at certain stage of detachment, explaining the difficulties in observing the catch bond mechanism under real biological conditions.

Structure provides fundamental constraints on reaction mechanisms as well as interactions at interfaces. Pair distribution function (PDF) analysis of synchrotron X-ray total scattering data provides insight to structure over short- and medium-range length scales relevant for amorphous calcium carbonate. A structure model developed using reverse Monte Carlo refinements of total scattering data shows a wide distribution of local calcium coordination environments, reflecting local structure as seen in crystalline polymorphs. The model suggests ready nucleation pathways for multiple crystalline phases. PDF results of transformed ACC indicate vaterite and calcite form coevally, with minor aragonite appearing in some instances. In transformation experiments a small fraction of ACC is found to persist for long periods following the onset of crystallization. The role of molecular water in ACC has been studied using NMR spectroscopy and PDF analysis, combined with thermal analysis. NMR spectroscopy shows two distinct populations of molecular water differing in mobility. Characterization of ACC before and after heating suggests that initial water loss has only a minor effect on structure. NMR reveals that the population of both types of water decrease proportionally with heating but that the hydroxyl content remains constant. Significant water loss accompanies transformation to vaterite and calcite. The findings raise important questions concerning reaction pathways in bulk ACC and in the presence of interfaces.

It is now widely recognized that the carbonate mineral constituents of many biomineralized tissues form by a multi-stage crystallization process that involves the nucleation and structural reorganization of transient amorphous phases. Experimental studies of the early stages of carbonate mineralization have suggested that nanometer-sized pre-nucleation clusters may be stable with respect to solution. Whether these cluster species are truly stable or merely longer-lived fluctuations as predicted by classical nucleation theory has not been resolved. Addressing this question is critical both because the details of the crystallization pathway may strongly influence the interpretation of elemental and isotopic climate proxy data obtained from authigenic and biogenic carbonates and because biomimetic strategies for materials synthesis will differ greatly if stable clusters with pre-existing structure can be exploited. Our approach utilizes molecular dynamics simulations to probe the initial formation and onset of order within hydrated CaCO3 and FeCO3 cluster species. To overcome the usual inability of atomistic simulation methods to address events that occur over extended timescales, this research utilizes replica-exchange molecular dynamics (REMD) techniques to enhance sampling of the potential landscape upon which the growing clusters reside. Results to date suggest that growing clusters initiate as short linear ion chains that evolve into two- and three-dimensional structures with continued growth. The planar structures exhibit an obvious 2d lattice, while establishment of a 3d lattice is hindered by incomplete ion desolvation. At a diameter of ~ 1 nm three-dimensional motifs begin to appear in the clusters which are similar to structural elements of both hydrated and anhydrous carbonate mineral phases. The motifs consist of distorted rhombohedral cages of metal atoms, with a single carbonate ion in the interior and additional carbonate ions proximal to the rhombohedron faces. With continued growth the contiguous extent of the ordered domains increases suggesting that formation of these motifs represent critical bottlenecks on the free energy landscape. Current work is in progress to determine the evolution of the free energy as a function of cluster size along the simulated crystallization pathways to identify cluster species that are stable or metastable with respect to solution.

A carbonated form of hydroxyapatite (HA) [Ca10(PO4)6(OH)2] is one of the most abundant materials in mammal bone. However, despite its potential application in bone repair, both, experimentally and theoretically little is known about the HA surface. Using density functional theory, we analyze the vibrational and electronic properties, surface energy and reactivity of HA for different orientations and terminations. We identify the chemically stable low energy surface configurations as function of the OH, PO4 and Ca concentration. In the experimentally relevant OH-rich regime we find only two surfaces competing for the lowest energy. Remarkably, neither one has been considered in previous studies. The surface most stable over almost the entire OH-rich regime is OH-terminated which was never theoretically described before.

We describe an instrumental and methodology development aimed at measuring and imaging by SIMS (secondary ion mass spectrometry) the local trace element or isotopic distribution with 50nm lateral resolution and 10-20nm depth resolution. SIMS is based on the sputtering and ionization of surface atoms by a focused beam of primary ions scanned across a sample. Ejected secondary ions are here mass filtered in a magnetic sector and detected in a multicollection (up to seven masses in parallel). The method requires putting the sample in ultra-high vacuum hence biological samples must be dehydrated using similar methods as used in TEM (transmission electron microscopy). Cells or tissue can be analysed after simple dehydration or more usually after fixation, resin embedding and microtoming. Harder materials (corals, bones, metals,â?¦) must be polished in order to obtain a flat surface. One key characteristic of the technique is to deliver isotopic analysis, permitting the use of analytical strategies based on (stable) isotope labelling. This can be used for biological samples by chaining a sequence of labelling (providing a isotopically-labelled substrate or food for a given period) with a sequence of 2D or 3D mapping of the localized enrichments. The technique can also be used in materials science by following the path of a tracer in the materials (ex: oxidation path from the surface using an 18O-enriched atmosphere). As the technique is destructive (sputtering of atoms), a series of 2D images can be piled to form an image stack for 3D rendering and visualization. Buried interfaces can also be analysed by imaging a perpendicular section of the sample extracted by cutting, FIB method or polishing. We will illustrate the application of the technique in a variety of fields among which: Molecule incorporation in bone and hair. Paleoclimate studies. The past seawater temperature can be deduced from Sr/Ca or Mg/Ca ratio in sediments. The imaging of coral sections perpendicular to coral growth or of foraminferal calcareous shells permits to resolve interfaces and layers linked to the past sea water temperature but also governed by biological factors. Studies on sheet nacre formation in bivalve. SIMS is one of the rare techniques capable of imaging hydrogen in 2D or 3D. Coupling NanoSIMS analysis with FIB-SEM lamella extraction is illustrated by the cross-section analysis of bacillus spores revealing the inner structure of small objects. In biological field NanoSIMS analyses are coupled with stable isotope labelling to measure protein turnover down to intracellular level, or follow and measure the accumulation and trafficking of molecules in cells or tissues. The general method of coupling NanoSIMS isotopic analyses with isotope labelling experiment permits to follow specific molecules in 2D or 3D without perturbing the living species. In environmental microbiology, after labelling microbes with isotopically-labelled substrates, microbes are hybridized with fluorescent probes. Subsequent FISH fluorescent phylogenic imaging permit to identify the individual microbe species. The NanoSIMS isotopic ratio images of the label fixation (ex: 15N, 13C, â?¦) permit then to couple the metabolic activity of individual microbes with their species without the need for cultivation.

The treatment of natural fibers to minimize lignin and hemicellulose content, provide adequate surface modification, and a chemical bonding interface between matrix and natural fiber surface is critical to produce natural fibers composites suitable for medium strength applications. Unsaturated polyester resin (S154-412) and partially bio-renewable polyester resin (Envirez 1807) are used as matrix with various concentrations of potassium hydroxide, acetic acid, and methyl acetate are used to treat and modify the surface of flax fibers. Single droplet/single fiber samples and panels made from untreated and treated loose fiber and woven cloth have been made. Tensile, flexure, and dynamic mechanical analysis tests were conducted to measure variances in modulus and strength among the various treatments and with a sample of e-glass/polyester resin. Post-treatment analysis of the surface energy characteristics, work of adhesion, and interfacial shear strength by Fourier transform infrared spectroscopy, scanning electron microscopy, and x-ray diffraction were performed. The Nardin/Schultz adhesive pressures are studied to determine the level of molecular interactions at the matrix/fiber interface, including the reversible work of adhesion.

The development of highly-sensitive and label-free, semiconductor-based, biomaterial detecting sensors has important applications in areas such as environmental science, biomedical research and medical diagnostics. In the present study, we developed an FET-like Indium Phosphide (InP) semiconductor based resistive biosensor using the change of its electronic properties upon specific biomaterial adsorption as sensing element. To detect biomaterial at low concentrations, the functionalization and covalent biomolecule immobilization procedure was also optimized to guarantee high molecule density, high reproducibility and high surface passivation against non-specific adsorption which are prerequisite for sensitive results. The characterizations, such as biomolecular conjugation efficiency via fluorescence microscopy, the detection concentration limits, receptor:ligand specificity and concentration detection range of the InP biosensor were analyzed by using three different biomolecular systems as test subjects: i) dsDNA and two phytopathogenic diseases ii) Citrus Tristeza Virus (CTV) and iii) Xylella fastidiosa (Xf), which cause great economic loss worldwide. The experimental results show a sensitivity of 1pM for specific ssDNA detection and about 2nM for the specific detection of surface proteins of CTV and Xf phytopathogens. The high detection sensitivity for phytopathogenic diagnosis can be compared with the most sensitive biosensing methods using an electrochemical approach with a concentration detection limit of ~1nM. In contrast to silicon based FET-like operating detection platforms, our InP biosensor shows a desirable increase in detection limits up to four orders of magnitude. The great advantage of the developed biosensor is the effortless application and direct molecular detection without the need of additional labeling. Taken together, the results from this study provide a great approach to highly-sensitive and easy applicable InP based sensors for early diagnosis in environmental and life sciences.

The periodic nanoporosity in ceramic materials can be leveraged towards designing molecular sieve based sensors for detection of small molecules. Nanoporous alumina which is generated through electrochemical anodization comprises of high density arrays of uniform nanopores. The porosity and pore dimensions of the nanopores are engineered to immobilize an important class of small molecules namely pesticides. Trace contamination of ground water sources has been a problem ever since the introduction of high-soil-mobility pesticides, one such example is atrazine. A novel nanoporous portable wireless bio-sensing device has been designed that can identify trace contamination of atrazine through a label-free electrochemical assay. The nanoporous alumina membrane is integrated onto a printed circuit board platform. Atrazine small molecule detection is achieved through electrochemical impedance spectroscopy by evaluating changes to the electrical double layer of the nanoporous alumina membrane due to size based immobilization of atrazine Sensor performance has been evaluated in phosphate buffered saline, river and drinking water. The limit of detection in all the three cases was in the femtogram/mL (fg/mL) (parts-per-trillion) regime with a dynamic range of detection spanning from 10 fg/mL to 1 ng/mL (0.01 parts-per-trillion to 1 parts-per-million). The selectivity of the device was tested using a competing pesticide; malathion and selectivity in detection was observed in the fg/mL regime in all the three cases. We have also performed proof-of-feasibility studies for integration of wireless technology with the biosensor for remote as well as â?oreal-timeâ? monitoring. A MicaZ mote capable of wireless communication using the ZigBeeTM standard at the 2.4 GHz frequency spectrum band was interfaced with the bio-sensor allowing it to collect data samples remotely and also exchange control commands.

Hydroxyapatite (HA) is the main mineral component of mammal bone. It crystallizes in hexagonal and monoclinic phase, the main difference between them being the orientation of the hydroxyl groups. While under ambient conditions the monoclinic phase is lowest in energy, when heated above 470K HA always assumes the hexagonal structure. Within the framework of density functional theory we identify a structural transition path from the hexagonal to monoclinic phase with the activation energy of 0.66 eV per hexagonal cell. At room temperature the transition occurs on a millisecond time scale. We suggest that the phase transition occurs due to a crossover in the vibrational contribution to the free energy of the two phases at the transition temperature. The claim is supported by first principles calculations of the phonon dispersion for both the monoclinic and hexagonal phase. We are able to identify the phonon modes driving the structural transition from monoclinic to hexagonal structure. The phonon density of states and heat capacity are computed and their dependence on HAâ?Ts dielectric constant is clarified. For the widely used value of the dielectric constant of 5 we calculate the theoretical transition temperature of 600K.

Biomaterials are widely used for dental implants, orthopedic devices, cardiac pacemakers and catheters. One of the main concerns on using bio-implants is the risk of infection on the materials used. In this study, our aim is to quantify the effect of surface roughness and patterning on the infection resistance and cell growth promotion of the titanium based bio-materials and alloys which are commonly used for orthopedic devices1. To modify the surface roughness of the surfaces in a controlled manner, Chemical Mechanical Polishing technique, which is extensively used in semiconductor industry for the planarization of the interlayer dielectrics and metals, is utilized. To improve the infection resistance, self- protective oxide films of the titanium alloys are studied and promoted through enhanced biological environments. The antimicrobial activity of the surfaces are measured through Japanese Industrial Standard method (JIS Z2801:2000). Furthermore, the cell growth analyses are in progress to quantify the controlled micro roughness and pattern schemes on cell growth as it has been demonstrated earlier through inducing nano ad micro patterns on various biomaterials 2. 1. M. Shirkhanzadeh, M. Azadegan, G. Q. Liu, Mater. Lett., 24 (1995) 7-12. 2. E. Martinez, E. Engel, J.A. Planell, J. Samiteier, Ann Anat, 191 (2009) 126-135.

While traditional biochemical dogma states that protein function requires the adoption of a well-defined three-dimensional structure, it is increasingly recognized that the function of many proteins derives from the absence of tertiary structure. These intrinsically disordered proteins (IDPs) potentially possess many attractive material properties, yet have been largely unexplored as protein-based building blocks. Here we report the design, development, and characterization of synthetic IDPs based on the C-terminal sidearm domain of the human neurofilament heavy chain (NF-H) protein, a key component of the neuronal intermediate filament. The NF-H sidearm domain contains many charged residues and is highly phosphorylated in vivo, and the degree of phosphorylation has been hypothesized to regulate NF network mechanics by regulating sidearm excluded volume. We therefore sought to develop NF-H-mimetic proteins whose conformational properties could be actuated by controlling these electrostatic interactions. We cloned three constructs based on the human NF-H protein sequence, expressed them in E. coli, and purified them: (1) wild-type NF-H; (2) a phosphomimetic NF-H (S to D substitution in the KSP repeats); and (3) a phospho-null polypeptide (S to A substitution in the KSP repeats). We designed the recombinant proteins to contain four Cys residues at the N-terminus for surface conjugation and a poly-(His-Asn) sequence at the C-terminus to facilitate purification. Dynamic light scattering measurements indicate that the recombinant wild-type protein adopts an extended conformation with a hydrodynamic radius of ~100 nm, far larger than would be expected for a globular protein. Quartz crystal microbalance studies suggest successful adsorption of the recombinant wild type NF-H side arm domain onto gold surfaces. Fitting the data to either the Sauerbrey equation (550 ng/cm²) or the Voigt model (750 ng/cm²) shows a clear increase in mass adsorbed with time. We also demonstrate our ability to microcontact-print these proteins onto a gold-coated silicon wafer using a PDMS stamp, and we verify spatially-patterned protein deposition using immunofluorescence imaging. This study represents among the first efforts to engineer IDPs and systematically characterize their sequence-structure relationships. Further studies on the relationship between IDP phosphorylation state, solvent conditions, and conformational properties should facilitate the exploration of these proteins as a new class of stimulus-responsive biomimetic materials.

Magnetic particles have been used in various biomedical and environmental applications. The major advantage of magnetic particles is its ease of handling which allows the separation of target molecules from reaction mixtures using external magnetic force. Magnetic particles was modified various proteins such as enzyme and antibody. In this study, novel protein-conjugated magnetic particles, which are functionally thermoresponsive proteins for cancer antigen peptide screening, have been developed. It consists of Elastin-like polypeptides (ELP), which has the reversible temperature-driven conformational change property and a Major Histocompatibility Complex class II molecule (MHC II), which consist of heterodimers including α chain and β chain. Afer MHC II specifically binds and present exogenously derived antigen peptide onto cell surfaces, MHC II-peptide complex is recognized by T cell and stimulating T cell-mediated immune response. Construction of thermoresponsive MHC II was generated as single-chain polypeptide, which is engineered constructs composed of a α chain and β chain linked by a ELP linker (VPGVG) _n . The binding of melanoma antigen peptide to ELP-linker MHC II-conjugated magnetic nanoparticles was demonstrated by using biotinyl MAGE-A3 _146-160 peptide and streptavidin-alkaliphosphatase. Furthermore, the releasing efficiency of antigen peptide from ELP linker MHC II-conjugated magnetic nanoparticles was evaluated. The efficiency depended on the length of ELP linker, and high responsive ELP linker to temperature was developed. This result suggested that conformation of peptide binding site of MHC II was collapsed associated with structural change of ELP linker by increase of temperature. The developed technology might be useful for antigen peptide screening from reaction mixtures.

While there are many reported methods to precipitate amorphous calcium carbonate (ACC), these typically suffer from problems of poor reproducibility. As such, the ACC produced can vary considerably in terms of stability and the amount of bound and surface water, which makes characterization of the mechanism of its crystallization extremely challenging. In this work, we have developed a simple synthetic method based on freeze concentration of saturated, counter ion free calcium carbon solutions, and show that this provides a highly reproducible method for the precipitation of highly disordered ACC. The ACC produced was characterized using a range of techniques. Increased disorder was evident from central Raman peak broadening, while thermogravimetric investigations of the obtained mineral phase revealed the absence of surface-bound water and a molecular composition of CaCO3: H2O. The acquired amorphous phase also showed extended atmospheric stability of up to six weeks. The mechanism of crystallization in air of the ACC was investigated using IR spectroscopy, where it was shown that the observed transition proceeds via the dehydration of the initial precursor phase to anhydrous amorphous calcium carbonate, followed by a structural rearrangement to calcite. The freeze concentration method was also successfully extended to the calcium phosphate and calcium oxalate systems, demonstrating its potential generality in obtaining well-defined amorphous mineral precursor phases.

We fabricated crosslinked silk LbL microcapsules with tunable permeability and variable micromechanical properties composed of biocompatible silk fibroin ionomers modified with poly(lysine) and poly(glutamic) acid. The porous shell structures were controlled with varying number of bilayers, deposition conditions, and external pH. Correspondingly, the cut-off molecular weight for the permeation of dextran macromolecules can be tuned. The mechanical properties of silk shells were studied by atomic force spectroscopy both in air and swelling state. In the dry state, the value of the elastic modulus was around 2 GPa, which was much higher than the typical value for hydrogen-bonded LbL shells. Stiffness of silk shells increased with 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide cross-linking. The correlations between the surface morphology and transport properties of shells and mechanical properties are addressed in this study.

The interface between minerals (hard) and organic molecules, arrays and scaffolds (soft) exemplifies problems in soft and hard matter science at several lengthscales. First, there are the issues of binding large molecules on surfaces. These include conformational folding induced by the surface and consequent effects on molecular function. Understanding this binding is in turn essential for understanding the attachment of cells and bacteria (and consequently biofilms) to surfaces. Equally, the presence of soft matter in the form of molecules,arrays or scaffolds can control the nucleation and growth of crystals. The resulting materials have complex structures, often with distinctive features at different lengthscales. We present a number of examples from our recent work showing how a combination of theory and experiment can shed light on the fundamental mechanisms involved: from the atomic to the macroscopic level. We will consider interfaces between soft matter and calcium carbonate, calcium phosphate, titanium dioxide, fused silica, quartz and gold. We demonstrate the important role that local water structure often shows at these interfaces. Also we show the importance of the flexibility of the soft interface in controlling crystal nucleation and growth.

The gene delivery system using â?osmartâ? biomaterials and nano-scale viral vectors offers an efficient and safe approach for enhancing gene delivery for stem cell. In this study, we have developed substrate-mediated gene delivery system using adeno-associated viral (AAV) vectors and elastin-like polypeptide (ELP) for transducing fibroblasts and human neural stem cells. AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired â?osmartâ? biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. ELP investigated in this study are biopolymers of the penta-peptide repeat [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat neural stem cell, was non-specifically bound onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The cellular transduction was manipulated by surface morphology, roughness and wettability as the presence of different ELP quantities on the TCPS. Importantly, ELP-mediated AAV delivery system enhanced delivery efficiency with substantially reduced viral quantities compared to bolus delivery in fibroblasts and human neural stem cells. Therefore this system with capacity to enhance gene delivery efficiency has strong potential for use in tissue engineering applications and neurodegenerative disorder treatments. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

Remote-controlled drug elution activity of antibiotic loaded TiO2 nanotubes Kyung-Suk Moon, Ji-Young Park, Ji-Myung Bae, Kyung-Hee Choi, Seunghan Oh Objectives: We developed antibiotic loaded TiO2 nanotubes releasing antibiotics remotely by UV and visible light irradiation. Also, we evaluated (1) the drug elution behavior of antibiotic loaded TiO2 nanotubes and (2) remote-controlled antibacterial activity of antibiotic loaded TiO2 nanotubes by UV and visible light irradiation. Material and Methods: TiO2 nanotubes having 30 - 100 nm of diameter and 0.1 - 3 μm of length were prepared by anodization. Nitrogen was doped to TiO2 nanotubes to be catalytically active by visible light irradiation. SEM and XPS were used to observe surface morphology and confirm nitrogen incorporation, respectively. Antibiotics (tetracycline and chlorhexidine) were mixed with poly-lactic acid(PLA) and loaded into TiO2 nanotubes. ELISA microplate reader was used to measure the released amount of the antibiotic. The antibacterial activity of antibiotic loaded TiO2 nanotubes were performed by measuring the size of dead Staphylococcus aureus colony after 24 hrs of incubation. The results were analyzed statistically by one-way ANOVA test. Results: SEM observation indicated that antibiotics were loaded well at the surface of short nanotubes (< 1 μm) compared to long nanotubes. The results of XPS confirmed the nitrogen incorporation into TiO2 nanotubes. The results of drug release test showed that antibiotic loaded TiO2 nanotubes showed the behavior of sustained and prolonged drug elution with the help of PLA. Also, the concentration of antibiotics released by light irradiated group was statistically higher than that released by non-light irradiated group (p<0.05). The antibacterial activity test by Staphylococcus aureus resulted the antibacterial zone of light irradiated group was significantly larger than that of non-light irradiated group (p<0.05). Conclusion: In summary, antibiotic loaded TiO2 nanotubes showing the behavior of sustained and prolonged drug elution is expected to be suitable for the resource of remote-controlled drug releasing device. Also, drug elution was controlled remotely by UV and visible light irradiation. Therefore, the remote-controlled drug releasing device based on TiO2 nanotubular structure is expected to be helpful for more effective antibacterial activity of titanium implant.

Polydopamine is an emerging material in surface chemistry, which was inspired by the adhesive properties of marine mussels. Despite the versatile use of polydopamine, the molecular mechanisms explaining the polydopamine formation have not fully investigated. It has been suggested that the polydopamine formation largely shares characteristics of the melanin biosynthesis pathways. Melanin biosynthesis includes oxidative reaction pathway from dopamine to DHI and further polymerization. The exact structure of melanin is also unclear, but the intermediates have been studied in relation to 5,6-dihydroxyindole (DHI) chemistry, forming covalently bonded DHI dimer, trimer and tetramer. Previously reported polydopamine structure followed this DHI chemistry in melanin synthesis, and the brown-black colored polydopamine layer was believed to be totally covalent bonded organic polymer, repeated by DHI and its derivatives. Here we report that the physical self-assembly of free dopamine and its oxidative product, DHI, contribute on forming the polydopamine structure. We found that a significant amount of dopamine is not polymerized during polydopamine formation and the unpolymerized dopamine is involved within the polydopamine as forming the stable DHI-dopamine physical complex. Oxidative polymerization that previously proposed is still occurred resulting polydopamine structure with this physical dimer. This is the first evidence that co-existence of oxidative polymerization and physical self-assembly pathways on polydopamine synthesis. Large amount of free dopamine may cause the cell toxicity, but the physical adsorption of unpolymerized dopamine is very strong so free dopamine is hardly released from the polydopamine-modified substrates. Our study revealed a different picture of polymerization formation in which polydopamine is not a totally covalent bonded high-molecular polymer structure and the contribution of physical interaction is a considerable part in dopamine polymerization pathway.

Sessile marine organisms are very effective at adhering to substrates under wet conditions and in harsh environments. The proteins employed by mussels have very specialized amino acid compositions undoubtedly related to the particular challenges of achieving permanent adhesion in the wet marine environment. Mussel adhesive proteins are known to contain high levels of 3,4-dihydroxy-L-alanine (DOPA), a catecholic amino acid that is believed to confer cohesive and adhesive properties to these proteins. In this talk I will describe the use of mussel-inspired molecules to perform surface modifications. Synthetic mimics of MAPs may take the form of linear or branched polymers with catecholic endgroups or side chains, where the catechols serve the role of a surface anchor for attachment to surfaces- therefore these functional groups end up â?oburiedâ? under a grafted polymer coating. With judicious choice of polymer chemical composition these coatings are able to confer a variety of functional properties to substrates. Several examples of these will be given, with an emphasis on grafting of antifouling polymers onto surfaces via catechol-based chemistry, for the purposes of controlling protein, cell and bacterial adhesion on surfaces, including the surfaces of nanoparticles.

The underwater adhesion of mussel onto the solid substrates has been extensively analyzed due to its versatility and robustness. A high content of 3,4-dihydroxy-L-phenylalanine (DOPA) and lysine (Lys) has been found in the mussel adhesive protein, and the DOPA-Lys motif provided a new insight into the identification of catecholamines as they share chemical functionality with the side chains of DOPA-Lys: catechol from DOPA and amine from lysine. After the identification, catecholamines such as dopamine and norepinephrine have been studied as small molecule mimics of mussel adhesive protein, and it was found that they have the ability to modify many diverse material surfaces. For example, oxidative polymerization of dopamine or norepinephrine modified a wide range of surfaces, and, moreover, the functionalized surfaces were successfully used for preparation of self-assembled monolayers, bioactive surfaces, and bioinert surfaces by additional stepwise reactions. Herein, we present a highly useful surface functionalization method: catecholamine-mediated one-step surface immobilization of functional molecules. We found that molecules co-dissolved with catecholamine (i.e. one-pot) were readily immobilized onto surfaces during oxidative polymerization of catecholamine under alkaline conditions. This approach showed the surface versatility, and the molecules immobilized on surfaces have various chemical functionalities and a wide range of molecular weights. A variety of applications including biomimetic silicification, surface-initiated polymerization followed by the bioconjugation on the polymerized surface, and cell-adhesive interfaces were demonstrated for emphasizing the wide applicability of the method. We believe that this approach could have broad utility for development of multifunctional surfaces for a variety of applications.

One of the most fascinating properties of materials in nature is the superhydrophobic and self-cleaning capabilities of different plant surfaces. This is usually achieved by the hydrophobic cuticles that are made of cutin containing wax crystals both within them and on their surfaces. Herein we have deposited bio-inspired n-hexatriacontane wax films via thermal evaporation and show that the surface evolves in time via self-assembly. This leads to a dramatic change in the wetting properties with a transition from hydrophobic to superhydrophobic characteristics which take place within several days and at room temperature. We investigated this phenomenon and propose a mechanism for it, namely, strain induced recrystallization. We believe that this work could become the basis for the inspiration and production of tuned, time-defendant, temperature-sensitive variable-wettability surfaces.

Changes in molecular conformation are intrinsically coupled to receptor-ligand binding, yet it remains experimentally challenging to probe this relationship directly. As a technological standard in biosensing, surface plasmon resonance (SPR) provides a label-free means of quantifying binding affinity and kinetics but is limited in resolving conformational changes based on difficulties to ascertain the refractive index at the sensor surface. Here, we describe an SPR method that mathematically overcomes this constraint by invoking the signal of non-interacting molecules to â?oprobeâ? conformational changes within a molecular layer. This is demonstrated using bovine serum albumin (BSA) to determine the heights of surface-tethered polyethylene glycol (PEG) chains of different molecular weights. External validation is provided by atomic force microscopy (AFM) measurements, which corroborate the SPR-resolved conformational changes in the PEG caused by subsequent anti-PEG binding. Further relevance is obtained in revealing conformational changes in Nsp1, an intrinsically disordered FG-nucleoporin that is central to the biological function of nuclear pore complexes upon binding importin-beta, a nuclear import receptor. This easy-to-implement SPR methodology provides for a reversible non-destructive means to quantify and correlate conformational changes and relative in-plane molecular arrangements to receptor-ligand binding affinity in situ.

Porous structures with complex three-dimensional (3-D) morphologies and well-controlled micro-to-nanoscale features are assembled with a high degree of fidelity by certain organisms (e.g., the siliceous frustules of diatoms, the calcitic lens arrays of brittlestars, the chitinous scales of Morpho butterflies). Such impressive structure formation has inspired the development of bio-based protocols for synthesizing hierarchical 3-D assemblies for man-made applications. Two bio-enabled approaches for generating intricate, porous 3-D structures with synthetic (non-biological) chemistries will be discussed: i) biomolecule-enabled layer-by-layer deposition of thin, conformal, porous oxide coatings onto self-assembled synthetic templates, and ii) shape-preserving chemical conversion of biological templates into new (non-biological) ceramic and metallic materials. Protamine, a readily-available and inexpensive arginine-rich protein harvested from a variety of fish (e.g., salmon, herring, trout, tuna), is capable of binding to a number of oxides (e.g., titania, silica, alumina) and of inducing the formation of titania from a water- soluble precursor (TiBALDH, Ti[IV] bisammonium-lactato-dihydroxide). Continuous and conformal Ti-O-bearing coatings have been deposited onto self-assembled synthetic templates (e.g., porous anodic alumina membranes) via alternating exposure to protamine-bearing and TiBALDH-bearing solutions. Because the protamine becomes intimately mixed with the deposited titania, subsequent organic pyrolysis yields a highly-porous, but interconnected, crystalline titania coating. Selective removal of the underlying template (via dissolution through the porous coating) then yields a freestanding, porous-wall titania structure that possesses the template morphology. Inorganic and organic 3-D biogenic templates have been chemically converted into porous replicas via the use of shape-preserving displacement reactions and/or synthetic layer-by-layer coating protocols. For example, silica diatom frustules of low specific surface area (SSA = 2 m²/g) have been converted into high SSA Si (>500 m²/g) or C (SSA > 1300 m²/g) replicas via gas/solid reactions. Subsequent loading of such porous frustule replicas with catalysts has yielded highly-active composite materials. The layer-by-layer deposition of conformal oxide coatings onto chitinous butterfly scales, followed by organic pyrolysis, has been used to generate nanocrystalline 3-D oxide replicas with altered optical properties. Conformal gold coating of diatom frustules possessing quasi-periodic pore patterns, followed by selective silica dissolution, has yielded freestanding gold replicas that exhibit extraordinary infrared transmission. The application of such porous bio-enabled structures in gas sensing, fluid purification, and optical/energy-harvesting devices (e.g., solar cells, batteries, fuel cells) will be discussed.

Superhydrophobic surfaces with extremely water repellent properties have received considerable attention as they have various applications including self-cleaning fabrics, anti-fog windows, drag reduction, and droplet manipulation. It has been theoretically and experimentally understood that superhydrophobic surfaces can be achieved by a combination of low surface energy and topographically roughened surface structures. In particular, patterning of extremely hydrophilic regions on superhydrophobic surfaces is crucial in practical applications such as open channel microfluidic systems, water harvesting surfaces, highly efficient biomolecule analysis, bio-specific cell adhesive surfaces, dynamic solubility control of nanoparticles, and dynamic solution transfers. Although several researches have reported on the methods to obtain superhydrophobic surfaces featuring hydrophilic patterns, the methods require additional complex processes and are not suited for many practical applications where the control of the direction and amount of liquid droplets displacement is necessary, in integration with Si based electronics. Here, we demonstrate a facile method to create re-definable hydrophilic patterns on superhydrophobic Si nanowire (NW) arrays as a open-channel fluidic guiding track. Superhydrophobic Si NW arrays with extreme water repellent properties were achieved by a simple dip-coating process in a dodecyltrichlorosilane (DTS) solution. The fabricated superhydrophobic Si NW arrays exhibited very low CA hysteresis and extremely low sliding angles (< 5°). The hydrophilic patterns of liquid-guiding tracks were defined on the superhydrophobic Si NW arrays by a UV-enhanced selective patterning process. Changes of surface wettability from superhydrophobic to hydrophilic are attributed to the photo-decomposition of DTS alkyl chains on superhydrophobic Si NW arrays, thereby locally altering the surface energy. The motions of the liquid droplets, including de-ionized water, rat blood, and CdTe nanoparticle suspension were precisely controlled along the trajectory of the selectively patterned hydrophilic liquid guiding tracks, and the amount of the liquid droplets could be handled by designing the dimension of the guiding tracks. The hydrophilic liquid guiding tracks could be successfully re-defined via the repetition of coating and decomposition of DTS within our experimental ranges.

Organic substrates are known to have a strong influence the crystallization of inorganic materials from solution. The substrates may enhance or inhibit the crystallization rate, modify the morphology of the growing crystals and even induce the crystallization of different polymorphs. Living organisms may use organic substrates to control polymorph and morphology of inorganic crystals in biomineralisation processes and the successful application of such crystallization control could have significant potential for technological applications. However the fundamental mechanisms involved in selective crystallization on substrates are poorly understood. Templating and stereochemical matching are often assumed to be the controlling factors, but it is clear from experimental results that these mechanisms can only partially explain the observations. Self-assembled monolayers (SAMs) are ideal substrates for crystallization studies because they form well-characterised, ordered arrangements of functional head groups. Several experimental groups have used SAMs as substrates for the crystallization of calcium carbonate. One particularly interesting result is the so called â?oodd-even effectâ? observed for alkyl thiols with carboxylic acid head groups1, where changing the length of the alkyl chain by one CH2 unit had a strong influence on the orientation of the resulting crystals. This effect was attributed to the different head group orientation of the two thiol monolayers. In a recent publication2 we used atomistic modelling techniques to simulate crystallization of CaCO3 on 16-mercaptohexadecanioc acid (MHA) and 15-mercaptopentadecanoic acid (MPA) monolayers. The crystallization rate was enhanced using metadynamics and it was found that the MHA monolayer induced crystallization in the (01.2) orientation whereas the crystal orientation on MPA was less well defined, in strong agreement with the experimental studies. We have recently performed further simulations using mercaptodecanebenzoic acid (MDBA) SAMs as substrates. These monolayers have the advantage that the benzene head groups present well defined orientations that can be measured experimentally using X-ray absorption spectroscopy, thereby affording the possibility of direct comparison between modeling and experiment. The results from the simulations will be presented and the insight into the crystallization control that can be gained from such simulations will be discussed. 1. Y. Han and J. Aizenberg, Angew. Chem., Int. Ed. 42, 3668 (2003) 2. D. Quigley, P.M. Rodger, C.L. Freeman, J.H. Harding and D.M. Duffy, J. Chem. Phys. 131, 094703 (2009)

Probing the early events that determine the nucleation pathway and final crystal structure is one of the challenges in understanding templating, aggregation and oriented growth of biominerals. Despite the many advances in knowledge regarding these processes, important aspects remain poorly understood such as the energetics of directed nucleation, the structural evolution of incipient nuclei, and the mechanisms of particle-mediated growth such as oriented attachment. We are using in situ and ex situ TEM to investigate crystal nucleation and oriented attachment in a number of systems. Solution imaging at nanometer scale and video rates is enabled by the combination of a custom designed TEM stage and fluid cell. Significantly, the design of the cell and holder enables temperature and electrochemical control to initiate reactions of interest, such as the onset of crystal nucleation. Here we show how to extend the technique to observe particle-mediated growth. Growth of branched rutile TiO2 nanostructures, obtained under hydro-solvothermal synthesis conditions, was investigated as a function of time using these techniques. Initially, a primary wire forms with subsequent branching occurring via absorption of oriented anatase nanoparticles and their eventual transformation to rutile. We propose that the branching in these nanostructures occurs via (101) twins. Iron oxide nanorods of the akaganeite phase grown hydrothermally self-assemble into high-aspect ratio ellipsoidal mesocrystals of the hematite phase. In situ imaging shows a progression of nanorod aggregation into disordered aggregates followed by gradual ordering into the final mesocrystal. For these systems, current work is focused on understanding the mechanism and progression of the structural transformations.

The compositional analysis of buried interfaces at near-atomic scale is one of the hallmarks of Atom Probe Tomography (APT) [1]. For many materials, controlled field-evaporation results in the removal of atoms from the surface of a sharp needle-shaped specimen that is reconstructed into a three-dimensional composition map with sub-nanometer resolution. Although field evaporation of polymeric materials under pulsed-laser APT conditions has been demonstrated [2], the accuracy of the reconstructions and the potential for this data to impact biological materials development is not well understood. The aim of this presentation is to review the technique by highlighting recent developments in applications across a wide spectrum of inorganic materials (e.g. metals, semiconductors, ceramics) as well as detailing recent progress toward better understanding the information content of analyses from biological materials. [1] M.K. Miller et al., Atom Probe Field Ion Microscopy, Oxford University Press, Oxford, 1996. [2] T.F. Kelly et al., MRS Bulletin, 34 (2009) 744.

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

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

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

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

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

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

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

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

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

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

The interface between biological cells and adjacent materials -- which include other cells, extracellular matrices, and drug-delivery vehicles -- is spatially and temporally dynamic. As the physical connection between the cell microenvironment and internal protein networks, the cell-material interface is also a prime example of chemomechanical coupling: small changes in either local chemical or mechanical cues can result in significant changes in cell organization and function. Here, we will discuss our recent work in understanding how extracellular cues such as macromolecular concentrations, pH, and effective matrix stiffness affect functions of tissue and stem cells ranging from adhesion to migration to differentiation of new biological functions. We will focus on the new experimental, analytical, and computational tools that we have developed to interrogate this interface from the molecular to the cell scale -- particularly in the context of soft-tissue diseases and cancer -- and to design new strategies that manipulate cell behavior directly by engineering of this buried interface.

Effective, easy to use, inexpensive, and rapid selective label-free cell isolation technologies are significantly needed to enable a new era of down-stream processing of stem cells for broad applications in tissue engineering, regenerative medicine, biological research and diagnosis of diseases. Isolation of stem cells (e.g., CD34+ endothelial progenitor cells) from peripheral blood, cord blood, and bone marrow has found significant applications in regenerative medicine and tissue engineering. Fluorescence-activated and magnetic-activated cell sorting are commonly used methods for cell isolation, which require extensive preliminary sample processing and tagging of the cells with fluorophores or magnetic particles conjugated with antibodies. While these methods are powerful and sort cells from heterogeneous mixtures, the cost, complexity and the requirement for infrastructure limit their use at the point-of-care (POC). Here we present a disposable, easy to fabricate microfluidic chip to retrieve label-free viable CD34+ stem cells from peripheral blood without any pre-processing in less than 10 minutes, which can also be used at the POC. We have developed a biotin binding protein (Neutravidin) and biotinylated antibody based surface chemistry on temperature responsive polymer (pNIPAAm) layered microfluidic channels. Whole blood was flown through the channels without any preliminary processing. The CD34+ cells were captured on the channel surface with antibody-antigen interaction. The non-captured cells in the channels were rinsed away with buffer solution. Captured cells were then released from the surface by reducing the temperature to <32C. The released cells were analysed for viability (live/dead assay) and cultured in vitro. The CD34+ cells in the whole blood samples were quantified by flow cytometry by using calibration and counting beads. We have specifically captured and controllably released the rare CD34+ stem cells from peripheral blood, which were quantified to be 19 cells per million blood cells in the patient whole blood samples used in this study. We achieved a capture specificity of 89% (±12%) and a release efficiency of 82% (±15%). Our results indicated that CD34+ cells released from the microchannels were viable (>90%) and amenable for in vitro cultivation. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains as a significant challenge. We have shown that cells can be rapidly, controllably released in microchannels with high viability and specificity after they were selectively label-free captured from blood without any pre-processing. The controlled release of live cells that are selectively captured from heterogeneous cell suspensions, biological and bodily fluids can generate a great impact in areas such as stem cell isolation/purification for regenerative therapies, genomic/proteomic analysis, and circulating rare tumor cell isolation for cancer research.

Pure titanium and titanium alloys are widely used in orthopedic and dental implants because on their desirable mechanical properties and biocompatibility. However, these materials suffer from electrochemical corrosion when implanted in the mouth, which induces release of metallic particles from the titanium surface, promoting inflammation around the implant, and implant failure, which was associated to the presence of macrophages in peri-implant soft tissue [1]. A novel ultrananocrystalline diamond (UNCD) thin film developed and patented at Argonne National Laboratory exhibit excellent bionert/biocompatible properties and strong resistance to chemical attack as coating for implantable biomedical devices, as recently shown for encapsulation of a Si microchip implantable in the eye to restore sight to people blinded by retina degeneration [2]. Therefore, we are investigating UNCD coatings for dental implants, because their high resistance to chemical corrosion, strong mechanical resistance and biocompatibility. The UNCD coating acts as a protective barrier between the implant and the biological environment, which would prevent the release of metallic ions from titanium implants into the body. In order to assess the potential of UNCD as a coating for titanium dental implants, we investigated the osteointegration rate of titanium, UNCD-coated titanium, and UNCD/W-coated titanium implants using the rat diaphyseal tibia as a model to study osteointegration, relevant to dental implants. Titanium laminar implants (Implant-Vel, Buenos Aires, Argentina) were coated with the three type of layers indicated above and used to study the osteointegration rate in the diaphysis of the tibia bone of rats, simulating the maxilar bone of the mouth cavity. All animal experiments were carried out following NIH guidelines for the care and use of laboratory animals. Macroscopic observations revealed that wound healing was occurring satisfactorily in all specimens and all implants remained in situ in the diaphyseal areas. Subsequently, the implants were extracted after 14 days of implantation and histological sections were prepared and analyzed via Optical Microscopy. The optical pictures showed laminar bone formation on the surfaces of the three groups studied. Histological results, from short term experiments, indicated that UNCD and UNCD/W coatings of dental implants are well tolerated by the surrounding tissue and did not elicit any inflammatory reactions, similarly to Ti implants without coating. However, the corrosion observed in long-term Ti dental implants and the total inertness of UNCD coatings, as shown in long term in vivo animal eyes studies, indicate that UNCD-coated dental implants will outperform bare Ti or Ti-alloy implants. Work is in progress to perform long term in vivo studies of UNCD-coated dental implants. 1. D. Olmedo et al, Imp Dent vol 12 (2003) 75. 2. X. Xiao / O. Auciello et al., J. Biomedical Materials 77B (2006) 273.

Development of blood vessel remains a big challenge for vascular tissue engineering. We recently demonstrated that poly(allylamine hydrochloride)/poly(styrene sulfonate) (PAH/PSS) films constitute excellent substrates for vascular progenitor cell differentiation. However, no intact cell sheets could be harvested. Using a spraying procedure, we develop a user friendly technology for the development of small blood vessels based on alginate membranes. The key point of our approach is based on a multilayered PAH/PSS film built on micro-textured alginate gels, about 140 µm thick and which can be peeled off. The alginate, calcium chloride and polyelectrolyte solutions are sprayed on a vertical glass substrate to obtain an oriented micro-textured gel. The PAH/PSS film, built on top of the gel, induces a good proliferation without phenotype alteration of Smooth Muscle Cells (SMC) and Human Gingival Fibroblasts. Parallel running micro-textures on the top of the gel allow the orientation of SMC cells. After peeling off the substrate, the cellularized membrane could be rolled around a mandrel or cultured later on showing a good retention of differentiated cells shapes after 10 days of culture. This work represents the initial stages of a new, original blood vessel reconstruction strategy via tissue engineering.

Interfaces capable of biological recognition and specificity are sought after for conferring bioinspired functionality onto synthetic biomaterials systems. Here, we demonstrate how intrinsic polymerâ?"protein interactions between highly localized polyethylene glycol (PEG) brushes and PEG-binding antibodies (anti-PEG) can be used for sorting specific biomolecules from complex bulk biological fluids to synthetic nanoscale targets. A principal feature lies with the antifouling property of PEG that prevents unspecific binding. As a means to replicate the cellular property of protein targeting, exclusive access is provided by anti-PEG, which acts as a biohybrid molecular adaptor that sifts out and targets a model IgG "cargo" from solution to the PEG. The PEG target sites can be reversibly washed and targeted in blood serum, which suggests potential benefits in technological applications such as biosensing and biomarker detection. Moreover, anti-PEG binding triggers a stimuli-responsive conformational collapse in the PEG brush, thereby imparting an intrinsic "smart" biorecognition functionality to the PEG that can considerably impact its use as an antifouling biomaterial. This stimuli-responsive behavior can be harnessed to create bio-inspired nanoporous membranes, which allow for the exclusive transport of specific molecular species via biochemical recognition.

While many different peptide sequences have now been identified to have strong affinity for a huge range of inorganic materials, the question of why a given peptide sequence binds so well while another does not remains to be properly answered. So too does the question of how the binding structure of the biomolecule impacts on the properties of the interface. To address these questions will enable significant advances in the fabrication of bio-inorganic interfaces with controllable, multi-functional properties. Application of molecular simulation to model these interfaces is one of many complementary techniques that enables us to investigate these phenomena. A key challenge for molecular simulation in this area is to achieve sufficient conformational sampling of the peptide in this aqueous interfacial environment. In this contribution, a summary of our recent work probing the effectiveness of advanced sampling approaches, namely Replica Exchange Molecular Dynamics, and variants of this method, will be presented, with examples taken from the quartz-binding peptide system.

The chiral biasing of proteins towards only L-amino acid configurations from a racemic mixture is a fundamental paradigm in biology. To date, there has been some speculation and recent evidence to suggest that differential hydration, preferential crystallization, chiral selection by minerals, and the presence of right circularly polarized light on earth may have had an impact in the evolutionary selection of L- over D-amino acids. It is also believed that inorganic matrices may have played a substantial role in producing left-handed protein structures in nature beyond simple chiral selection by acting both as a catalytically active and chiral surface for D-amino acid containing peptides and proteins. For example, silver NPs have been implicated in the conversion of right-handed DNA to left-handed DNA; while metal ions have been used to post-translationally modify L-amino acids into D-amino acid peptides. In this study, we show that a right-handed peptide composed of all D-amino acids undergoes an optical and chemical transition to the left-handed L-form in the presence of gold nanoparticles. This chiral conversion activity of nanoparticles will likely expand the tunability and scope of metamaterials as well as provide a platform for chiral sensing.

Peptides that bind inorganic surfaces and template the formation of nanometer-sized inorganic particles are of great interest for the self- or directed assembly of nanomaterials for sensors and diagnostic applications. These surface-recognizing peptides can be identified from combinatorial phage-display peptide libraries, but little experimental information is available for understanding the relationship between the peptide sequence, structure at the nanoparticle surface and function. We have developed NMR methods to determine the structures of peptides bound to inorganic nanoparticles and report on the structure of peptides bound to silica, titania and palladium nanoparticle surfaces. Samples were prepared under conditions leading to rapid peptide exchange at the surface such that solution-based nuclear Overhauser experiments can be used to determine the three-dimensional structure of the bound peptide. The binding motif for the silica and titania binding peptides is defined by a compact "C"-shaped structure for the first six amino acids in the 12-mer. The orientation of the peptide on the nanoparticle surface was determined by magnetization transfer from the nanoparticle surface to the nearby peptide protons. Broader lines are observed for the Pd4 12-mer peptide bound to 2 nm Pd particles. 2D NMR methods are developed to estimate the separation of the amino acid protons from the metal surface in Pd4 and in single and double mutants with alanine in place of histidine at amino acids 6 and 11. The results show that the peptide bulges out from the metal surface at the mutation site. Pulse-field gradient diffusion and x-ray scattering experiments show that the Pd nanoparticle solutions are stable for months and that the peptide is not exchanging on and off the surface of the nanoparticles on the time scale of the NMR experiments. These methods can be applied to a wide variety of abiotic interfaces to provide an insight into the relationship between the primary sequence of peptides and their functionality at the interface.

We report on designing highly permeable and robust nanoscale gel shells for cells, cell assemblies, spherical microparticles, and anisotropic inorganic crystals. These responsive nanoshells are formed through a layer-by-layer assembly based upon hydrogen-bonding, ion pairing, hydrophobic-hydrophobic interactions, and components interdiffusion. These LbL shells can be reinforced via physical or covalent crosslinking by inclusion of nanoparticles, inducing local crystallization, or adding crosslinking agents. These shells are exploited to control and tune the permeability and robustness of the nanoshells. We demonstrated that the nanoshells support high viability of the cells with minor effect of growth and dividing of cells especially if a cationic component is screened or completely removed. We discuss significance of multilayer shell morphologies for designing novel vehicles with tunable loading-unloading properties and enhanced robustness.

A useful strategy emerging from mussel inspired adhesion appears to be the decoration of synthetic polymers with catecholic functionalities such as 3, 4-dihydroxyphenylalanine (Dopa) to make them stickier. The most adhesive proteins from mussels, namely mfp-3 and mfp-5, contain 20 and 30 mol% Dopa, respectively. While dopa abundance in proteins is one predictor of adhesion, the processing history of the adhesive proteins is equally and, perhaps sometimes, more important. Lee et al (PNAS 103:12999 [2006]) first demonstrated how oxidation of Dopa to Dopaquinone compromises adhesion to titania. In a similar vein, investigating the adhesion of Dopa-containing proteins with the surface forces apparatus, we found that titration of Dopa oxidation directly impacts adhesion. Namely, the strength of mfp-3 and -5 adhesion to mica is inversely correlated with the degree of Dopa oxidation to Dopaquinone. Complete oxidation to the quinone abolishes adhesion; reduction to Dopa restores it. Mussels cosecrete Dopa-containing adhesives with thiolate antioxidants to preserve the Dopa-dependent sticking power. A recent survey of â?omussel inspiredâ? adhesive applications, however, reveals that most or all rely on the intentional oxidation of catechol-functionalized polymers for adhesion to bioorganic surfaces. The reason usually given for this is that quinones have a better chance of reacting covalently with the target surface. Although quinones do form in the adhesive plaques and lead to cross-linking between different plaque proteins, we have found no evidence that mussels present quinones to any kind of foreign surface. Indeed, even in the SFA, oxidation of mfp-3 or -5 abolishes its adhesion to another protein layer except after very long contact times (~12h). Failure of such cross-links to form between oxidized protein films as well as evidence of the strongly reducing environments created by mussels during plaque formation argues against the widely assumed position that mussels exploit dopaquinones for covalent adhesion to biopolymeric surfaces. (related literature: Lee, B. P., Messersmith, P. B., Israelachvili, J. N., Waite, J. H. (2011). Mussel inspired wet adhesives and coatings. Annual Review of Materials Research 41: 99-132. Yu, J., Wei, W., Danner, E., Ashley, R.K., Israelachvili, J.N., and Waite, J.H. (2011). Mussel protein adhesion depends on interprotein thiol-mediated redox modulation. Nature Chem. Biol. 7: 588-590. Yu, J. Wei, W. Danner, E. Israelachvili, J. and Waite, J. H. (2011). Effects of interfacial redox in mussel adhesive protein films on mica. Advanced Materials 23: 2362-2366.)

The freshwater Zebra Mussel and the closely-related Quagga Mussel are a major cause of biofouling in North American water bodies due to their ability to adhere strongly to a variety of substrates including boat hulls and water intake pipes. Like the well-studied marine mussels, these freshwater mussels attach to surfaces by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. While marine and freshwater mussels differ in their overall protein compositions, they both have proteins containing the rare amino acid 3,4-dihydroxyphenylalanine (DOPA) that is responsible for both adhesive and cohesive interactions in the byssus. In contrast to marine mussels, the distribution of proteins in the byssus of zebra and quagga mussels remains largely uncharacterized, due to extensive cross-linking which renders the mature structure largely resistant to extraction and immunolocalization. The functions of these proteins thus remain a mystery. Here we report on the byssal distribution of proteins in freshwater mussels based on results from mass spectrometry and extraction from freshly secreted threads and plaques, in which cross-linking is minimized. Additionally, we studied the self-assembly of peptide mimics of the one sequenced zebra mussel protein. We report here on our characterization of aggregate size, peptide secondary structure and DOPA-Fe(III) complexation under varied Fe(III) - peptide mixture conditions. Understanding the mechanism of freshwater mussel adhesion may ultimately lead to targeted strategies for controlling biofouling, and inform the design of medical and dental adhesives.

The underwater adhesion of marine mussels relies on mussel foot proteins (mfps) rich in the catecholic amino acid 3, 4-dihydroxyphenylalanine (dopa). As a side-chain, dopa is capable of strong bidentate interactions with a variety of surfaces, including many minerals and metal oxides. TiO2 is one of the most commonly used materials for medical implants. Studying the binding mechanism of dopa to TiO2 surface is therefore of theoretical and practical interests. Using a surface forces apparatus, we explored the force distance profile and the binding mechanisms of mussel foot protein 3 (mfp-3) to TiO2 surface in three different pHs (pH3, 5.5, and 7.5). At pH3, mfp-3 showed the strongest adhesion force on TiO2, with an adhesion energy of ~ -7.0 mJ/m2. Increasing the pH has two opposing effects: it causes the oxidation of dopa, which eliminates the dopa adhesion; meanwhile, higher pH increases the binding affinity of dopa-Ti coordination bond, leading to the further stabilization of the dopa group and an increase of the adhesion force. The two competing effects give rise to a higher adhesion force of mfp-3 on TiO2 surface at pH 7.5 than the adhesion force measured at pH 5.5. Our results suggest that dopa containing proteins and synthetic polymers have the potential to be used as coating materials for medical implants.

The need for effective biomedical glues spans a wide range of applications, from wound-healing sealants to dental implants and restorations. While the majority of synthetic adhesives lose their bridging abilities in aqueous, saline environments such as the body, nature has developed efficient solutions to this problem. Marine mussels are able to make strong, permanent attachments to nearly any available surface in their ocean habitat, for which the mussel uses an assembly of proteins rich in the amino acid 3,4-dihydroxyphenyl-alanine (DOPA). While the DOPA moiety has been heralded as the entity responsible for mussel adhesion, a repeating decapeptide construct derived from mussel foot protein-1 (mfp-1) has been reported to demonstrate stronger attachment strengths than equivalent amounts of DOPA in isolation, motivating further study into the adhesive mechanisms of this protein. In this work, quasi-static atomic force microscopy (AFM) force-distance measurements were performed on films of crosslinked and non-crosslinked mfp-1 deposited onto mica substrates, and both adhesion and stiffness of the protein were assessed under tensile stress. Surface coverage and distribution of mfp-1 at the surface were also characterized using contact and intermittent-contact modes of AFM imaging. Our data indicate that (i) crosslinking mfp-1 via coordination of DOPA with metal cations significantly decreases adhesion and somewhat decreases stiffness, while (ii) covalent diDOPA crosslinking results in stiffer films with very low adhesion, when compared to non-crosslinked films. Higher homogeneity and film coverage were also observed for covalently crosslinked mfp-1. Finally, detailed analysis of the attractive forces during tip-sample approach reveals that mfp-1 is able to extend over discreet distances to â?~captureâ?T the tip and pull it into contact with the filmâ?Ts surface. These results will be discussed in terms of likely protein conformation, as well as the distance between bridging sites along the protein backbone. Overall, our studies indicate that crosslinking DOPA groups give rise to coarse and fine tuning of both adhesion and stiffness of mfp-1, and support the importance of DOPAâ?Ts role in mussel adhesion and byssus strength.

Mucoadhesive polymers are being extensively investigated for different biomedical applications due to their ability to increase residence time on mucosal surfaces. Chitosan (CH), partially N-deacetylated chitin, is a biodegradable and biocompatible bioadhesive polymer that has shown great potential in drug delivery and tissue engineering applications. However, chitosan hydrogels have limited mucoadhesion due to weak electrostatic interactions with mucin. DOPA (3,4-dihydroxy-L-phenylalanine), a catechol containing molecule naturally present in marine mussel foot proteins, has been shown to increase the mucoadhesion of several polymers. The catechol group (ortho-dihydroxyphenyl) in DOPA is believed to be responsible for this enhanced mucoadhesion, while its oxidation seems to decrease it, although the mechanism for this process is not well known. In this study, we developed a new class of catechol/chitosan hydrogels with superior mucoadhesion, for biomedical applications including drug delivery and tissue engineering. Chitosan hydrogels (MW 190,000~300,000 Da) were prepared with and without compounds containing catechol groups (DOPA; dopamine: DA; hydrocaffeic acid: HCA). The hydrogels were characterized for their swelling, mucoadhesive strength and release kinetics of the catechol containing compounds. The gels were also evaluated for the oxidation of the incorporated catechol-containing compounds as a function of solution pH. The swelling of the gels was pH dependent with maximum swelling being detected at pH 1. In general, the gels showed higher degree of swelling in the presence of the catechol containing compounds. For instance, at pH 6.8 the swelling followed this order CH/DOPA=CH/DA > CH > CH/HCA. Hydrogel swelling was higher in the presence of the catechol compounds presuambly due to the hydrophilic nature of these compounds allowing more water to be retained in the hydrogel matrix. In contrast, the swelling of CH/HCA was less than CH itself most probably due to charge neutralization of CH amino groups by electrostatic interactions with HCA carboxylic acid groups. The presence of the catechol compounds also increased the adhesion strength to rabbit intestine compared to chitosan alone. Thus, after 3 minutes of contact time with rabbit intestine, CH/HCA showed almost as twice adhesion strength as CH alone. The catechol compounds were released from the gels in a sustained fashion where only 10% was released after 24 h. The catchol compounds were stable in aqueous solutions at pHâ?¤6, and rapidly oxidized at pHâ?¥6. Their oxidation was much slower in the presence of chitosan. Catechol/chitosan hydrogels are currently being evaluated for oral delivery of protein and peptide drugs. This new catechol/chitosan hydrogel with enhanced mucoadhesive properties could be useful in drug delivery, tissue engineering and other biomedical applications.

Marine polychaetes in the family sabellariidae live in high-energy shoreside environments in tubular shells cobbled together with sandgrains, the broken skeletons of marine invertebrates, and a protein-based underwater glue. The robust construction of the honeycomb-like colonies reflects the durability of the water-proof adhesive bonds holding the composite tubes together. The sandcastle glue consists of oppositely charged polyelectrolytes; at least 4 polybasic and at least 2 polyacidic phosphoproteins. As products of the regulated secretory system, the glue precursors are condensed through electrostatic interactions into two major types of secretory granules. So-called heterogeneous granules contain two of the polybasic proteins (Pc1/4) plus the polyphosphorylated proteins (Pc3A/B). The polyphosphoproteins are condensed with Mg2+ into heterogeneous subgranules of variable size and number. The homogeneous granules contain the other two polybasic proteins (Pc2/5) and unknown counter polyions. The condensed granules of adhesives precursors are secreted separately and coalesce into an adhesive mass that in turn sets up into a solid foam within 30 s. The co-secreted polyelectrolyte components, initially separated into different granules, remain poorly mixed in the final glue. The sandcastle glue has served as a model for synthetic underwater adhesives based on electrostatically condensed polyelectrolytes. Oppositely charged polyelectrolytes chemically analogous to the sandcastle glue proteins have been synthesized and when mixed they condense into complex coacervates that have ideal properties as the foundation for underwater adhesives. Progress characterizing the natural sandcastle glue and the synthetic adhesive complex coacervates it inspired will be presented.

The plant pathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different types of plants. Understanding the factors which determine bacterial adhesion and biofilm development is a key issue in any effort aiming at the identification of mechanisms to prevent biofilm formation. Within these factors, electrostatic interactions between the bacterial cell membrane and a surface have been proposed as an important step for the initial bacterial adhesion. In this work we have investigated this process using two different approaches. In one of them, we have studied surface potential (SP) distribution on both abiotic (silicon) and biotic (derivatized cellulose) surfaces by Kelvin Probe Force Microscopy. SP variations were observed for acetate and ethyl cellulose surfaces indicating an inhomogeneous charge distribution. Relatively large changes in SP levels were observed for both abiotic and biotic surfaces after interaction with Periwinkle wilt (PW) culture medium. In this case, silicon and ethyl cellulose surfaces show an increase in SP levels while SP becomes spatially more inhomogeneous on acetate cellulose (AC) surfaces. Furthermore, from a molecular point of view, adhesion forces between these same surfaces and an important membrane adhesin from X. fastidiosa (XadA1) were investigated by force spectroscopy. To this end, AFM tips were functionalized with XadA1 adhesin and force-distance curves were acquired. In this case, a different behavior was observed for force-distance curves acquired on AC surfaces suggesting both a lower overall binding affinity of XadA1 to AC and the activation of a different mechanism for X. fastidiosa attachment to AC. Thus our results reveal the interplay between culture medium and substrate as a determinant factor for electrostatic interactions and clearly show the influence of a biopolymer such as cellulose on the surface in determining X.fastidiosa adhesion and further biofilm development.

Synthetic materials are often used for biomedical applications. Interaction of cells with the interfaces and tissue components determine the biological outcome of the device. These interactions are mediated at the molecular and macromolecular level and advances in understanding of them may lead to improved technology in areas such as biomaterials, tissue engineering and cell based therapies. Specific interaction with the extracellular matrix components or macromolecules in the outer membrane of adjacent cells provides signalling and communication pathways. Here we report a model system with functionalizes surfaces presenting the ectodomain of E-cadherins in an oriented fashion, which mimic the cadherin presentation on cell surfaces. The model system has been used to study the development of cellular adhesion complexes. Protein patterns with systematically varied patch size from 100-800 nm have been made by using a nanoscale chemical contrast of Au domains in a background of SiO2 by colloidal lithography. We have studied the effect of variation in ligand patch size on cellular behaviour and demonstrate a systematic change of cell attachment and morphology with a threshold of substantially reduced adhesion to patches below 200nm. Increasing the patch size result in more adhering cells and the same number of attached cells are observed for all samples with protein patch sizes of 200nm or above. Increasing the patch size from 100nm to 800nm will affect both the proportion of spread cells and the mean area of each cell. Most of the adhering cells on the smallest nanopatches were found as single or in pairs and the fraction of cells found in smaller clusters is the same for all sample types. The number for cells found in larger clusters (>4) increases with patch size. A uniform distribution of cellular E-cadherin near the cells edge is observed for cells adhering to 100nm and 200nm patches whereas a punctated distribution is observed for cells attached to 300nm, 800nm and the homogeneous hydrophobic control. The cells ability to form a strong cortical actin ring and discontinuous AJs is affected by the underlying patterns. A strong continuous cortical ring and discontinuous AJs are only observed for cells attached to protein patched above 300nm. On basis of our result we propose that an adhesive area of 0.03 µm2 (200nm in diameter) is a threshold for cellular attachment. Below this threshold the cadherin within the cells are not able to assemble into large clusters for formation of strong adherens junction.

Supramolecular chemistry and fabrication methods provide nowadays an excellent prospect to construct reversible dynamic biological interfaces that can be employed for supramolecular cell manipulation experiments. Making use of supramolecular chemistry is a rewarding task in developing functional materials and devices. Knowing the limitations involved in ordering proteins at different length scales will surely hasten the development of future applications, supramolecular nanobiology being the most prominent. The construction of synthetic supramolecular assemblies of proteins provides an excellent tool to fabricate organized bioactive components in the sub-micron regime at surfaces. I will present new synthetic procedures for site-specific anchoring of proteins to surfaces and polymers aiming at more control over structure and function of the proteins. Special attention is paid to orientational and conformational aspects at the surface and will be demonstrated with a few examples. For example, supramolecular interactions that are sensitive to remote electrochemical stimuli, using cucurbituril (CB) and cyclodextrin (CD)-modified surfaces will be presented. Specifically, site-selectively ferrocene-tagged fluorescent protein (Fc-YFP), which binds to self-assembled surfaces through weak or strong interactions between the ferrocenyl guest and the host surface of beta-CD or CB[7], resp. The methods involved the spontaneous adsorption of beta-CD or CB[7] onto a gold surface, the attachment of the ferrocene moiety to the protein, and the monovalent immobilization of this ferrocene-tagged protein to the surface. Upon reduction of the ferrocene unit, the protein was removed from either surface, with the CD surface showing the fastest disassembly kinetics. In another method viologen-functionalized surfaces were used to capture naphthol-tagged proteins from solution to the surface forming a ternary complex with CB[8]. The complexes were patterned on surfaces by microcontact printing (µCP). Electrochemical switching was studied using surface embedded electrodes. Cell release was studied in detail in the case of cell-adhesive peptides and growth factors. Dynamic linkers were compared to systems that are nonreversible providing insight in the cell receptor signalling pathway. With the development of supramolecular bioactive platforms on surfaces serving as a reversible dynamic interface to cells, improved scaffolds for tissue regeneration will become in hand. First steps into this directions will be introduced.

We report a device called spheroforms, a spheroid-forming superhydrophobic surface. The spheroforms are prepared by photolithographic patterning of catecholamine on superhydrophobic anodized aluminum oxide (AAO) surfaces. On the spheroforms, homogeneous sizes of cell aggregates (i.e. spheroids) are generated with high efficiency, and size of the spheroids is readily controllable by the hanging drop method. It has been reported that the size of cell spheroids is a critical factor to affect cell viability, proliferation, cellular functions and the fate of stem cells, which is primarily due to a different degree of cell-to-cell interactions. Thus, the size of spheroids and the homogeneity in their sizes should be precisely controlled for therapeutic applications. In this study, spheroids originated both from insulin-secreting, primary islet β-cells and mesenchymal stem cells (MSCs) are formed, demonstrating biological versatility of the spheroform surfaces. Both types of cell spheroids showed much improved viability compared to the spheroids produced by the traditional hanging drop methods . The diameter of spheroids was efficiently controlled from 50 μm to 250 μm with high size homogeneity. For islet β-cells spheroids, stimulation index (SI), the indicator of glucose sensitivity of β-cells, was increased when the spheroids were formed on the spheroforms. Also, the size-dependence secretion of vascular endothelial growth factor (VEGF) in MSCs is demonstrated. In addition, the catecholamine micropatterns on the superhydrophobic surfaces are stable enough enabling for one to use the spheroforms for many times. The concept demonstrated by the spherofom clearly opens a new direction of the use of surface superhydrophobicity for biotechnology and cell biology applications.

Self-assembling systems containing supramolecular assemblies and covalent polymers have the potential to integrate order, dynamics, and mechanical robustness into functional structures. This lecture will describe systems discovered when contact between aqueous solutions of self-assembling peptide amphiphiles and polymers of opposite charge form a diffusion barrier leading to ordered membranes in millisecond times scales. This lecture will also describe how formation of these membranes depends critically on the presence in solution of pre-assembled supramolecular nanofibers with strong affinity for polymer chains. In the absence of strong interactions between both components the membrane structure changes dramatically into a cubic phase formed by interdiffusion of both components. The lecture will also show that bioactive membranes, cell-like objects, and novel catalytic systems can be formed by these systems.

Electrically conductive polymers have been developed for applications ranging from solar cells to stimulation that can aid in biological repair. For biological applications, strict mechanical properties are critical to emulate the function of tissue in need of repair and to permit biological tissue to replace implanted materials. Specifically, electrical stimulation across a peripheral nerve gap has been shown to upregulate chemical factors resulting in a three-fold increase in repair rate. We hypothesize that repetition of electric field gradients may overcome the present limitation of nerve gap repair. To achieve repeating electric field gradients, we developed a bioresorbable sheet comprised of alternately high and low electrically conductive polymers. Native nerve tissue has an elastic moduli range of 0.4 - 0.7 MPa and tensile strength of 0.21 - 1.49 N. To achieve appropriate mechanical properties, electrically conductive, bioresorbable elastomers made of poly glycerol sebacate acrylate (PGSA) were synthesized with 25, 30, 35, and 40% acrylation as verified by H1-NMR. Tensile strength, elastic moduli, and elongation to break were evaluated via Instron 5500R mechanical tester to select the acrylation percentage of PGSA that matched the mechanical properties of native nerve tissue. Conductivity limits were calculated based on current density in native nerve and the electric field strength surrounding the nerve yielding a target high conductivity of 1x10^-4 S/cm and a target low conductivity of 1x10^-5 S/cm. Composites of PGSA with polypyrrole or multi walled carbon nanotubes (MWCNT) were prepared with conductivity calculated from current and voltage measurements via 4 point probe. Using a ribbed silicone mold, high and low conductivity polymers were alternated and UV crosslinked to achieve repeated electric field gradients. In future investigations, the materials will be implanted into a rat model for peripheral nerve repair, opening the door for translation of tunable electrically conductive nano-composites in clinically relevant tissue engineering applications.

The integration of viral gene delivery with tissue engineering scaffolds composed of biocompatible biomaterials that can adjust viral delivery in a controlled manner offers a promising strategy for numerous tissue engineering applications. In this present study, adeno-associated virus (AAV), which has been regarded as a safe, efficient and potential gene carriers in a variety of gene therapy applications, was encapsulated within electrospun nanofibers composed of blended mixtures of elastin-like polypeptides (ELP) and poly(ε-caprolactone) (PCL). AAV vectors were released from the scaffolds in a controlled manner, depending on incubating temperatures and weight ratios between ELP and PCL, and efficiently transduced fibroblasts adherent on nanofibrous scaffolds. Combinatorial interactions between ELP and PCL chains by physical blending significantly altered the mechanical properties (i.e. wettability, elastic modulus, strain, etc.) of the ELP/PCL composites, thus providing important means to mediate controlled release of AAV vectors and thriving cellular transduction on the electrospun scaffolds. The ability of ELP/PCL composites to operate the controlled release of AAV and AAV-mediated gene delivery with high transduction efficiency will provide enormous opportunities for various tissue engineering applications.

Scaffolds are porous materials used in tissue engineering as platforms to enhance cell attachment, proliferation and activity, leading to shorter healing time of injured or missing tissue. The surface of the scaffolds is the first region cells get in contact with upon implantation, and determines their reaction to it. Since the surface of synthetic polymeric scaffolds is not ideal for cell adhesion and proliferation because of their hydrophobic nature, surface modification of these materials is crucial for enhancing implant integration in the body. When these scaffolds are used in orthopedic applications, surface modifications can help the formation of hydroxyapatite, the mineral component of bonesâ?"a process known as biomineralization. Many people have succeeded in surface modification of bi-dimensional polymer films using plasma methods, but diffusion is a major obstacle when trying to modify the surface of three-dimension scaffolds by plasma treatment. In this study, we used diazonium chemistry for the first time to functionalize poly(D, L-lactic)acid (PDLLA) scaffolds with pores in the range of 200-350 µm, fabricated with the salt leaching method. By carrying out the modification in solution, we were able to modify both the outer and the inner surface of the porous scaffolds, and we introduced covalently bonded amino groups using para-phenylenediamine as precursor for in-situ generated diazonium cations. By changing parameters such as reaction time and reducing agent concentration we were able to tune the concentration of amino groups present on the scaffolds. The amino-phenyl layer formed on the scaffold surface is â?oself-adhesiveâ?, and can be exploited to bind other biomolecules. Here we present spectroscopic results confirming the successful surface modification of PDLLA scaffolds, and the effect of these modifications on the in-vitro biomineralization of the modified scaffolds.

Wear and corrosion debris from the bearing surfaces of total hip replacements can lead to an adverse local tissue response and implant failure. Selected current designs of metal on metal hip replacements have had a relatively high failure rate based, at least in part, on the quantity of wear and corrosion debris. Our group has been evaluating metal on metal implants retrieved at revision surgery and have observed tribochemical reaction (TCR) layers that may govern the in vivo behavior of these bearing surfaces. In vitro tribological and tribocorrosion testing has suggested that 1) the synergistic component of tribocorrosion exceeds the magnitude of debris generated by either corrosion or wear alone; 2) the TCR layer may be protective against these degradative processes; and 3) the composition and ultrastructure oof the TCR is unique and can provide insights into the basic mechanisms of tribocorrosion in metal on metal bearings.

In the present study, Ti-10Zr-10Nb-5Ta alloy has been elaborated by levitation melting. Mechanical, and microstructural properties of this new bioalloy have been investigated and its corrosion and tribocorrosion resistance in H2SO4 (0.5M) and NaCl (9g/l) solutions has been studied. The results have been compared to those obtained with pure titanium and Ti-6Al-4V alloy. The electrochemical study showed that the passivating film formed on Ti-10Zr-10Nb-5Ta surface is more stable than those obtained on the surface of titanium or Ti-6Al-4V alloy. In addition, the new alloy presents a better resistance to corrosion and tribocorrosion. Additional corrosion and tribocorrosion tests have been conducted in Ringerâ?Ts solution and in presence of bovine serum albumin to reproduce conditions of the human body. The influence of the presence of proteins molecules on corrosion and triboccorrosion behaviour of the studied materials has been particularly analyzed.

The information storage capacity of DNA has been explored in various different applications ranging from sensors and assays and also to build supramolecular architectures. Its use not as a carrier of information but as a biomaterial, however, is essentially unexplored. The favorable non-immunogenic properties of this degradable biopolymer, however, have prompted us to develop biomaterials coating based on DNA. For this we apply Layer-by-Layer self-assembly which allows us to build layered coatings by the repeated sequential deposition of negatively charged ( e.g. DNA) and positively charged components on to substrates of any shape or form.[1] The layered build up of these coatings can be used for the localized entrapment of drug or growth factors released during the degradation process. To ensure a truly layered coating structure we designed a self-assembling surfactant based on the bis-urea motif as the positively charged component. Using cryoTEM imaging we demonstrate that this bis-urea surfactant forms stable highly ordered ribbon-like aggregates.[2,3] In addition, cryo-electron tomography (3D cryoTEM) shows that these not only prevent mixing of the layers, but also allow anchoring of biomolecules in specific layers through supramolecular modification employing the bis-urea unit.[4] The coatings were subsequently applied to substrates and used for in-vitro as wel as in-vivo studies. The in-vitro studies show that the DNA-based coatings enhanced promote cell proliferation, increase cell viability, promote calcium phosphate deposition from SBF and can be combined with growth factors.[5-7] The in-vivo studies show that the coatings are not only biocompatible but also bio-active: they are histocompatible and favor early bone responses, moreover, they increase both early & late peri-implant bone responses similar to conventional calcium phosphate coatings but with the possibility to include (different) bioactive molecules in predefined layers.[8] [1] G. Decher science, 277, 1232 (1997) [2] M.R.J. Vos, N.A.J.M. Sommerdijk, et al J. Am. Chem. Soc, 127, 16768 (2005) [3] M.R.J. Vos, N.A.J.M. Sommerdijk, et al. Chem Commun, 46 6063 (2010). [4] M.R.J.; Vos, N.A.J.M. Sommerdijk, et al. J. Am. Chem. Soc. 130, 12608-12609, (2008). [5] J.J.J.P. van den Beucken, N.A.J.M. Sommerdijk, J.A. Jansen. et al Acta Biomaterialia, 3, 587 (2007). [6] J.J.J.P. van den Beucken, N.A.J.M. Sommerdijk, J.A. Jansen, et al. Tissue. Eng. 13, 711 (2007) [7] J.J.J..P. van den Beucken, N.A.J.M. Sommerdijk, J.A Jansen, et al Key Eng. Mater., 361-363, 605 (2008). [8] C. Schouten, N.A.J.M. Sommerdijk J.A. Jansen, J.of Biomed.Mater ResA, 92A, 931(2010).

The outermost layer of skin, the stratum corneum (SC), protects the body from harmful environmental conditions by serving as a selective barrier. This crucial barrier function is possible due to buried interfaces within the SC in the form of intercellular lipid boundaries between corneocyte cells. These interfaces act as a controlled permeable barrier to the external environment while subject to highly variable conditions. Solar ultraviolet (UV) radiation is one of the most ubiquitous conditions the body encounters and, although necessary for vitamin D production, is responsible for a host of negative skin responses, including inflammation and infection due to compromised barrier function. The biophysical and biochemical aspects of UV damage on skin have been studied, but little is understood regarding its effects on the SCâ?Ts interfacial structure and resulting alterations in the biomechanical barrier function of skin. We explored the effect of UV exposure on cell cohesion and mechanical integrity of SC, as a function of UV wavelength (UVA, UVB and UVC), dosage and tissue depth. We show that UV exposure has dramatic effects on SC interfacial lipids. We found that the uniaxial keratin-controlled stiffness of the SC remained constant with UV exposure, while the SC fracture strength, fracture strain, biaxial modulus, and the energy required to separate intercellular boundaries decreased significantly with increasing UV exposure. Our results indicate that solar UV radiation poses a double threat to skin by increasing the driving force for cracking while simultaneously decreasing skinâ?Ts resistance to cracking by significantly reducing cellular cohesion, largely dominated by the intercellular lipids and corneodesmosomes, thereby impairing the critical barrier function of the skin.

Much of our research is inspired by, and directed at, understanding the formation of novel structures (both relatively static and highly dynamic) with distinct shapes and morphologies observed in charged biological systems. The structures, in turn, often correlate to specific functions. For example, charged nanoscale tubules and rods and their assemblies are of interest in a range of applications, including as templates for hierarchical nanostructures, encapsulation systems, and biosensors. A series of studies will be described on charged biological assemblies exhibiting â?omolecularly-triggeredâ? dynamical shape changes. In particular, we will focus on protein and lipid based nanotubule formation through small molecule stimuli-induced shape remodeling events. The systems include invertible protein nanotubes from two-state tubulin-protein building blocks and lipid nanotubes and nanorods from curvature stabilizing lipids (mimicking membrane curvature generating proteins). Funded by DOE-BES grant number DOE-DE-FG02-06ER46314 (protein and lipid assembly, lipid synthesis, structure-function) and NSF-DMR-1101900 (phase behavior).

Physiological behaviors such as chromosomal segregation rely on the dynamic self-assembly and reorganization of protein polymer filaments in the cellâ?Ts cytoskeleton. Understanding the underlying principles of these processes opens the door for applying dynamic assembly to design and synthesize ex vivo, hybrid materials systems with emergent behaviors (e.g., self-healing). Recently, we discovered a novel mechanism for the growth of cytoskeletal protein filaments, deemed â?omicrotubule fusion,â? that is unique from the classical step-growth polymerization of αβ tubulin dimers. In our new growth mechanism, the interfacial adhesion between microtubule segments (25 nm diameter and 8-10 microns long) drives self-assembly in a head-to-tail manner to produce macromolecular filaments with lengths exceeding 150 microns. The initial step in this process occurs when the plus-end (β subunit-terminated) of one microtubule encounters the minus-end (α subunit-terminated) of another microtubule and interacts to form a junction. While the collision of the ends likely occurs stochastically, attraction and adhesion of the ends is aided by long range interactions (e.g., hydrophobic) between α and β tubulin subunits. The second step of the fusion process involves dynamic rearrangement and/or addition of tubulin dimers near the junction in an â?oannealingâ? process that eliminates defects. This step is critical as the junctions between segments are formed by microtubule ends that may be blunt, tapered, or curved. The annealing process is facilitated by free GTP-tubulin dimers that can fill in defects or imperfections, and stabilize the interfacial junction. This presentation will cover experimental work and molecular dynamics simulations describing the process of microtubule fusion, as well as efforts to apply this assembly mechanism to synthesize heterostructured nanowires. * Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

Self-assembled protein architectures exhibit a wide range of structural motifs with functions that include selective transport, structural scaffolding, mineral templating and propagation of pathogenesis. Although the primary sequences of the individual proteins define their governing interactions, their functions depend on the quaternary architecture that emerges from self-assembly. Proteins that naturally self-assemble into extended structures of oligomers with long-range order often adopt conformations that are distinct from those of the individual monomeric protein. When individual proteins fold from an unstructured state to the final stable state, the concept of a folding funnel with kinetic traps, in which the protein exhibits non-equilibrium structures for extended period of time, is used to describe the pathway. Unfortunately, these transient states are difficult to observe at the single molecular level largely due to the limitations of spatial resolution of conventional optical techniques. Consequently, detailed information about the dynamics and energetics of protein collapsing down the folding funnel is limited. Despite the fact that folding transformations are part and parcel of protein self-assembly, this concept of the folding funnel has not been considered in that context. Here, we investigate the connection between these two phenomena and explore the dynamics and energetics of folding transformations by utilizing the inherent single molecule resolution of in situ AFM to follow 2D crystallization of S-layers on atomically flat mica surfaces at molecular-scale. We demonstrate the emergence of binary states of S-layer organization in 2D. We find this system possesses a kinetic trap associated with a conformational difference between a long-lived transient state and the final stable state. Both ordered tetrameric states emerge from clusters of an amorphous precursor phase, however, they then track along two different pathways. Over time, we show that the trapped state transforms into the stable state. By analyzing the time and temperature dependencies of formation and transformation, we find that the energy barriers to formation of either state are quite similar, differing by a mere 1.6 kJ/mol. However, once the higher energy state forms, the barrier to transformation to the lower energy state is much larger at 94 kJ/mol, leading to the slow transformation process. In conclusion, our findings demonstrate the importance of kinetic traps in determining the pathway of S-layer crystallization and suggest that the concept of the folding funnel for individual proteins can be equally applied to assembly of extended protein superstructures.

Tailoring self-assembly of proteins on solid surfaces remains a great challenge in diverse fields, e.g., enzyme catalysis, molecular biosensing, and biomineralization, where surface functions can be controlled by molecular structures. In these fields, biomolecular self-assembly at liquid-solid interfaces is the key in enabling facile molecular surface coatings for a wide range of applications. Protein function, such as molecular recognition and self-assembly, derive from the rich chemistry and molecular conformations coded within its amino acid sequence. Exquisite 2D organizations of proteins are achieved on surfaces using, e.g., bacterial surface-layer proteins, linear amyloid structures and ordered films of de novo designed proteins. These systems offer highly programmable constructs to tailor structure and chemistry of bio-solid interfaces. A thorough understanding of how a particular amino acid sequence is related to its molecular self-assembly process on a solid surface is the key to control these biomolecular surface nanostructures. Despite considerable efforts in peptide/protein assembly on solids, however, the coupling of a sequence to its detailed molecular ordering has remained largely unknown. Recently developed solid-binding peptides (7-12 amino acids), due to their short length and ease of chemical synthesis, offer potential interrogation and, hence, control over self-assembly in contrast to large proteins. In our work, by rational mutation of a dodecapeptide, we probe molecular interactions and form spatially controlled peptide nanostructures with defined physical and chemical characteristics on atomically flat solid surfaces. Using atomic force microscopy and contact angle studies, we identify three domains of amino acids along the primary sequence that steer peptide binding, surface diffusion and assembly, leading to uniformly displayed residues and sequence-programmable surface chemistries. Using rationally designed mutant peptides, we show that one can steer surface phenomena, including solid binding, surface diffusion, and intermolecular interactions leading to long-range ordered self-assembly through a unique disorder-to-order phase transformation. The relationship established here between peptide sequence and self-assembly enables predictable formation of 0D peptide nanoclusters, 1D nanowires and 2D molecular nanoarchitectures on graphite and graphene in aqueous solutions with programmable surface chemistries. Research supported by NSF-MRSEC, NIH-T32, NSF-BioMat and JST-PRESTO programs.

A broad range of peptides that have been shown to form stable nanowires or nanotubes are based on amyloid proteins attributed to diseases such as Alzheimerâ?Ts and Type II diabetes. Such peptide nanotubes have robust self-assembly and have been used as building blocks for applications including electronics and as templates for growth of inorganic material. In this study, we have examined the ability to sublime different peptides and the subsequent deposition of peptide nanotubes and nanowires using Plasma Enhanced Chemical Vapor Deposition (PECVD). The aromatic dipeptides, diphenylalanine and dityrosine are peptide monomers, which have been used in this study. The morphology of peptide nanostructures and characteristics such as their length, thickness, density have been controlled by varying parameters during the deposition process as well as the effects of using different substrates has been studied. Plasma used in the PECVD process allows control over the composition of the nanotube growth imparting unique surface properties without modifying the bulk material properties of the substrate. The nanoassemblies fabricated were characterized using SEM, TEM, FTIR, AFM and XRD. Some of the synthesized nanotubes show quantum confinement and are fluoresce in the blue region (400 â?" 500 nm) as shown by photoluminescence measurements. Mechanical testing techniques such as nanoindetation have been used to demonstrate the mechanical stability and hardness of the deposited nanotubes. AFM techniques have been used to study the details of the tubular outer shell with tapping and electrostatic force modes, while also probing the mechanical integrity. We have sampled single nanotubes as well as vertically aligned 3D arrays. I-V characterization was carried out via electrical contacts deposited on the nanotube ends by focused ion beam deposition. The electrical conductivity characteristics have been studied using the Scanning Kelvin Probe Microscopy and conductive AFM as well. Thermal responses of the nanotubes to localized tip-side heating using the scanning thermal probe techniques have been studied.

The physical system considered is a stack of many identical homogenous bio-membranes immersed in water; the nominal inter-membrane spacing is uniform. The membranes experience thermal fluctuations and, for a sufficiently small inter-membrane spacing, the fluctuations of neighboring membranes interfere. Symmetry arguments show that behavior under these circumstances can be analyzed by considering fluctuations of a single membrane confined between rigid plane surfaces separated by the mean membrane spacing. A statistical mechanics analysis can lead to an estimate of the mean steric pressure acting between the membranes but there is some variation among the results reported for the spatial dependence of this variation. We describe results which indicate that, for a given inter-membrane spacing, differences in the membrane fluctuation correlation length, determined largely by the elastic bending stiffness, can account for these discrepancies.

Biomimetic crystal growth has been extensively studied over the last decade. One versatile manner in which biomimetic crystal growth has been controlled is via organic/inorganic interfaces. One prominent example is the growth of crystals in alkanthiol SAM surfaces. In order to obtain a well-ordered SAMs on coinage metal supports it is important to obtain ultraflat metal surfaces. Ultra-flat metal surfaces are used, produced via Template Stripping (TS), which is a method for obtaining a metal with an average surface roughness in the order of < 1nm. Here we show for the first time that the TS technique indeed introduces a very high density of surface nano-defects (twinning and stacking faults), which can strongly hinder surface-induced properties such as SAM ordering, despite the seeming overall ultra-high flatness. We have used state of the art characterization techniques such as HRXRD, spherical aberration corrected HRTEM and STM. We also demonstrate how these nano-defects can be completely eliminated¹. 1) Borukhin, S.; Pokroy, B., Formation and Elimination of Surface Nanodefects on Ultraflat Metal Surfaces Produced by Template Stripping. Langmuir 2011, DOI: 10.1021/la203596p.

Collagen represents the major structural protein of bone, dentine and the extracellular matrix and can template growth of numerous mineral phases. Both the molecular-scale conformation and mesoscale architecture of collagen are critical for its activity. Because both are influenced by interactions with substrates, understanding those interactions and the mechanisms of assembly on surfaces, may enable us to direct assembly and hence engineer bioactive surfaces with tailored properties. We studied the dynamics and structure of collagen type I self-assembly on mica surfaces by AFM. At acidic conditions, K+ ions critically affected the collagen-mica interaction leading to ordered structures that varied dramatically with changing ion concentration. The structure evolved from 2D films of randomly oriented fibers to co-aligned fibers and finally to ordered 3D bundles as the K+ concentration increased from 100mM to 300mM. High-resolution AFM images showed the bundles consisted of intertwined single collagen triple-helices bound by lateral interactions. The magnitude of the collagen-mica and collagen-collagen interactions at 200 and 300 mM K+ were measured by dynamic force spectroscopy (DFS). The free energy for collagen-mica and collagen-collagen interaction at 200 mM K+ were 13.7kT and 1.4kT respectively, while the free energies at 300 mM K+ were 5.7kT and 12.3kT, respectively. The observed reversal in the sequence of collagen-collagen and collagen-mica binding energies provides an answer to why the architecture switches from a 2D film to highly organized 3D bundles. Transformations between different assemblies were studied by in-situ AFM. Pre-organized films of ordered fibers transformed into ordered 3D bundles upon incubation in 300mM K+ solution. Because the free energy of collagen-mica binding at 300mM K+ was only 5.7kT, collagen fibers were still partially mobile with a diffusion coefficient D1~4Ã-10-17cm2s-1. The stronger interaction between collagen and collagen (12.3kT) drove bundle nucleation, which occurred through lateral movement and twisting of individual fibers. This result confirmed that the film-substrate interactions are too weak to enforce a direct registry with the substrate lattice, while intrafilm interactions drive reorganization, giving surface templated quasiepitaxial growth. For the 2D co-aligned fibers, the dependence of surface coverage on time followed a simple Langmuir adsorption curve, consistent with the observation that there is no cooperativity between fibers. However, for the bundles, assembly followed a highly non-linear time dependence in which acceleration in assembly rate was correlated with bundle size, Thus bundle assembly on mica proceeds in three steps: (1) adsorption of a poorly ordered fibers, serving as the â?onutrientâ?; (2) surface diffusion of the adsorbed fiber; and (3) nucleation of the ordered bundle through reorganization of aggregated fibers.

Via Dissipative Particle Dynamics (DPD) approach, we study the self-assembly of hybrid structures comprising lipids and end-functionalized nanotubes. Individual lipids are composed of a hydrophilic head group and two hydrophobic tails. Each bare nanotube encompasses an ABA architecture, with a hydrophobic shaft (B) and two hydrophilic ends (A). To allow for regulated transport through the nanotube, we also introduce hydrophilic hairs at one or both ends of the tube. We select the dimensions of the nanotube architecture to minimize its hydrophobic mismatch with the lipid bilayer. We find the amphiphilic lipids and functionalized nanotubes to self-assemble into a stable hybrid vesicle or a bicelle in the presence of a hydrophilic solvent. We show that the equilibrium morphology of the functionalized nanotube-lipid hybrid material is determined by its bending rigidity. We demonstrate that the morphology of the hybrid structures is controlled by temperature, rigidity of the lipid molecules, and concentration of the nanotubes.