Emmanuel Belamie

Fibrillogenesis in Dense Collagen Solutions: A Physicochemical Study

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength=150 mM, 20 degrees C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH approximately 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.

Chitin-silica nanocomposites by self-assembly.

Self-assembly of chemical entities is at the basis of many biological processes and increasingly in materials syntheses. Self-assembled surfactants and block copolymers are widely and successfully used to prepare ordered hybrid and mesoporous materials with outstanding properties. 7–9] A strong interest also rises for energy-saving chemical processes and reactants from renewable resources. 11] Self-assembled biomacromolecules and other biological objects have been used previously as liquid-crystal templates for the formation of mesoporous silica. Herein we present a novel and versatile colloid-based approach for the large-scale synthesis of a new family of hybrid bioorganic–inorganic nanocomposites with an unprecedented control in texture and morphology. This approach combines the self-assembly properties of polysaccharide chitin nanorods, with the flexibility of sol– gel processes involving siloxane oligomers. The resulting optical and mechanical properties of the chitin–silica nanocomposites can be tuned by varying the chitin volume fraction fCHI. Nanorod alignment inside these materials was achieved under moderate magnetic fields (9 T), generating highly oriented textures and providing an alternative method to that developed for surfactant-templated materials. Furthermore, sol–gel chemistry is amenable to a variety of processing techniques such as spray-drying, which allowed us to prepare micrometer-size chitin–silica particles with variable porosity in the calcined replicas. Our approach consists in the formation and processing of a stable suspension containing two colloids, a-chitin nanorods and siloxane precursors, both in a dispersed state. This is a challenging issue owing to differences in stability and reactivity of the colloids. Chitin nanorods purified from shrimp shells (L = 260 80 nm, D = 23 3 nm) are bundles of monocrystals (D = 3.2 0.6 nm) with amino groups at their surface (Figure 1a). They are stably dispersed in water by electrostatic repulsions in slightly acidic conditions. The simple addition of silica precursors would lead to uncontrolled chitin/silica precipitation owing to electrostatic interactions and/or rapid siloxane condensation. To avoid this, we prepared mixed alcoholic suspensions of the chitin nanorods with siloxane oligomers through repeated solvent exchange cycles. The siloxane oligomers were formed by controlled acid-catalyzed hydrolysis with a hydrodynamic diameter of about 4 nm and a degree of siloxane condensation c of 0.75 (Figure 1a). The resulting clear to translucent suspensions (depending on chitin concentration) are stable over a long period of time (several months). Upon evaporation, the colloidal suspension shows a marked increase in viscosity and turbidity, which is accompanied by a gradual rise in optical birefringence. In a standard procedure, the resulting pasty mixture was cast and further dried overnight (348 K), yielding hard bulk materials with condensation degrees c in the expected range of 0.8–0.9. Preliminary compression tests show that the elastic mechanFigure 1. a) Representation of the siloxane oligomers (upper panel), and of the chitin nanorods (core: poly[b-(1!4)-2-acetamido-2-deoxy-dglucopyranose]) as bundles of monocrystals with amino groups at their surface (lower panel). The colloid sizes were estimated by DLS and TEM. b–d) TEM analysis: b) bulk nanocomposite (fCHI = 0.28); spray-dried microparticles (fCHI = 0.28) before (c) and after (d) calcination (porous replicas).

Possible transient liquid crystal phase during the laying out of connective tissues: α-chitin and collagen as models

Morphogenesis of extracellular matrices can be considered from different perspectives. One is that of ontogenesis, i.e., an organisms development, which is mostly concerned with the spatiotemporal regulation of genes, cell differentiation and migration. Complementary to this purely biological point of view, a physico-chemical approach can help in understanding complex mechanisms by highlighting specific events that do not require direct cellular control. Because of a structural similarity between some biological systems and liquid crystals, it was supposed that similar mechanisms could be involved. In this respect, it is important to determine the intrinsic self-assembly properties driving the ordering of biological macromolecules. Here we review in vitro studies of the condensed state of major biological macromolecules from extracellular matrices and related theories describing a mesophase transition in suspensions of rodlike particles. Dilute suspensions of collagen or chitin are isotropic, i.e., the macromolecules can take on any orientation in the fluid. Beyond a critical concentration, an ordered nematic phase appears with a higher volume fraction. The two-phase coexistence can be seen between crossed polarizers since the nematic phase is strongly birefringent and appears bright, whereas the isotropic phase remains dark. A widespread property of these structural macromolecular scaffolds is their chirality. Although the origin of chirality in colloidal suspensions is still a subject of debate, the helical nature of the cholesteric phase can be quantified. Small angle x-ray scattering performed on shear-aligned samples can help demonstrate the cholesteric nature of the anisotropic phase, inferred from optical observations. Liquid-like positional local order is revealed by the presence of broad interference peaks at low angle. The azimuthal profiles of these patterns are fitted to determine the value of the nematic order parameter at the transition. A few physico-chemistry experiments can assess the nature of the transition, and in turn, applying theoretical models can prove useful in predicting and controlling the structure of assemblies of biological macromolecules.

Tunable hierarchical porosity from self-assembled chitin–silica nano-composites

We studied new mesoporous materials with original properties and obtained from self-assembled chitin–silica nano-composites. Our novel synthesis allows the controlled colloidal assembly of α-chitin nanorods (bundles of elongated chitin monocrystals) and siloxane oligomers. Calcination of nano-composites results in mesoporous silica materials. Their pore volume fraction ϕPOR (0–0.52) is strongly correlated to the initial chitin content. Using N2 sorption and TEM data, we identify and characterize primary and secondary textural units related to the imprints of chitin monocrystals (2.5 nm wide) and nanorods (20–30 nm wide) respectively. Primary textural units are preserved over a wide ϕPOR range (linear relationship between pore volume and specific surface area). The coating of monocrystals by siloxane oligomers leads to a siloxane network of fractal nature as deduced from complementary SAXS data. Beyond a critical value ϕPOR′ estimated near 0.2, the coating is partial, and the porosity becomes more open and connected. At larger scales, the arrangements of secondary textural units result in complex textures and long-range ordering, showing similarities with textural features found in natural materials. We discuss the competition between entropy-driven transitions typical of anisotropic particles and kinetic arrest due to colloidal gelation and inorganic condensation. Finally, a schematic model for texture formation is given.

Efficient mesoporous silica–titania catalysts from colloidal self-assembly

Mesoporous silica-titania materials of tunable composition and texture, which present a high catalytic activity in the mild oxidation of sulfur compounds, have been obtained by combining the spray-drying process with the colloidal self-assembly of α-chitin nanorods (biopolymer acting as a template) and organometallic oligomers.

Characterization of phospholipid bilayer formation on a thin film of porous SiO2 by reflective interferometric Fourier transform spectroscopy (RIFTS).

Classical methods for characterizing supported artificial phospholipid bilayers include imaging techniques such as atomic force microscopy and fluorescence microscopy. The use in the past decade of surface-sensitive methods such as surface plasmon resonance and ellipsometry, and acoustic sensors such as the quartz crystal microbalance, coupled to the imaging methods, have expanded our understanding of the formation mechanisms of phospholipid bilayers. In the present work, reflective interferometric Fourier transform spectrocopy (RIFTS) is employed to monitor the formation of a planar phospholipid bilayer on an oxidized mesoporous Si (pSiO(2)) thin film. The pSiO(2) substrates are prepared as thin films (3 μm thick) with pore dimensions of a few nanometers in diameter by the electrochemical etching of crystalline silicon, and they are passivated with a thin thermal oxide layer. A thin film of mica is used as a control. Interferometric optical measurements are used to quantify the behavior of the phospholipids at the internal (pores) and external surfaces of the substrates. The optical measurements indicate that vesicles initially adsorb to the pSiO(2) surface as a monolayer, followed by vesicle fusion and conversion to a surface-adsorbed lipid bilayer. The timescale of the process is consistent with prior measurements of vesicle fusion onto mica surfaces. Reflectance spectra calculated using a simple double-layer Fabry-Perot interference model verify the experimental results. The method provides a simple, real-time, nondestructive approach to characterizing the growth and evolution of lipid vesicle layers on the surface of an optical thin film.

Improved silica–titania catalysts by chitin biotemplating

Silica–titania materials with improved catalytic performance were elaborated as mesoporous microparticles by combining sol–gel and spray-drying processes with the self-assembly properties of α-chitin nanorods acting as biotemplates. Three different synthesis approaches are discussed, leading to materials with varied textural and chemical characteristics studied by SEM, N2 volumetry, TEM, XPS and DR-UV techniques. The use of water or ethanol as initial solvent for chitin nanorod suspensions, as well as the mixing conditions of the precursors, has been shown to have a significant impact on the final properties. Materials of specific surface areas of up to 590 m2 g−1 and porous volumes of up to 0.84 mL g−1, with low surface Si/Ti ratio, could be disclosed. Properties were further investigated by employing the silica–titania materials as heterogeneous catalysts for the sulfoxidation of bulky model compounds. The location of Ti active sites at the pore surface has been maximized and allows for improved productivity.

Electric-Field Alignment of Chitin Nanorod–Siloxane Oligomer Reactive Suspensions

Uniaxially anisotropic chitin-silica nanocomposite solids have been obtained thanks to the electric field-induced macroscopic alignment of liquid-crystalline reactive cosuspensions. We demonstrate how chitin nanorods (260 nm long, 23 nm thick) can be aligned upon the application of an alternating current (ac) electric field, and within water-ethanol suspensions containing reactive siloxane oligomers (D(h) ∼ 3 nm). The alignment at the millimeter length scale is monitored by in situ small-angle X-ray scattering (SAXS) and polarized light optical microscopy. The composition and state (isotropic, chiral nematic) of the cosuspensions are proven to be determining factors. For nematic phases, the alignment is preserved when the electric field is switched off. Further solvent evaporation induces sol-gel transition, and uniaxially anisotropic chitin-silica nanocomposites are formed after complete drying of the aligned nematic suspensions. Here, the collective response of colloidal mesophases to external electric fields and the subsequent formation of ordered nanocomposite solids would represent a new opportunity for materials design.

Thick collagen-based 3D matrices including growth factors to induce neurite outgrowth

Designing synthetic microenvironments for cellular investigations is a very active area of research at the crossroads of cell biology and materials science. The present work describes the design and functionalization of a three-dimensional (3D) culture support dedicated to the study of neurite outgrowth from neural cells. It is based on a dense self-assembled collagen matrix stabilized by 100-nm-wide interconnected native fibrils without chemical crosslinking. The matrices were made suitable for cell manipulation and direct observation in confocal microscopy by anchoring them to traditional glass supports with a calibrated thickness of ∼50μm. The matrix composition can be readily adapted to specific neural cell types, notably by incorporating appropriate neurotrophic growth factors. Both PC-12 and SH-SY5Y lines respond to growth factors (nerve growth factor and brain-derived neurotrophic factor, respectively) impregnated and slowly released from the support. Significant neurite outgrowth is reported for a large proportion of cells, up to 66% for PC12 and 49% for SH-SY5Y. It is also shown that both growth factors can be chemically conjugated (EDC/NHS) throughout the matrix and yield similar proportions of cells with longer neurites (61% and 52%, respectively). Finally, neurite outgrowth was observed over several tens of microns within the 3D matrix, with both diffusing and immobilized growth factors.

Nanostructuration of titania films prepared by self-assembly to affect cell adhesion

Nanostructured and dense titania films prepared by evaporation-induced self-assembly (EISA) are shown to possess tunable topographical nanoscale features on the order 2-12 nm. Thermal treatment (calcination) induces a transition from amorphous titania to crystalline anatase that modifies the chemical and structural properties of the surfaces via the migration of matter. For nanostructured films, the nanoporous network changes from organized ellipsoidal pores, approximately 4 nm x 2 nm, to a grid-like structure with pores on the order of 12 nm, whereas dense films show a slight roughening of the surface. Cells seeded on templated films show measurable, statistically significant differences in morphology compared with cells seeded on dense films. Moreover, although crystallization of templated films results in surfaces that promote less well-spread cells with higher circularities, the opposite trend is observed for dense films. As such, these results represent a new method to tailor interfaces for biomaterial applications, using EISA to control material patterning on the nanoscale. This self-assembly based approach allows the patterning on size scales that are inaccessible by most traditional techniques while offering the added potential to package and control the release of bioactive molecules.