Validation of a new fluidic device for mechanical stimulation and characterization of microspheres: A first step towards cartilage characterization.

Articular cartilage is made of chondrocytes surrounded by their extracellular matrix that can both sense and respond to various mechanical stimuli. One of the most widely used in vitro model to study cartilage growth is the model of mesenchymal stromal cells-derived cartilage micropellet. However, mechanical stimulation of micropellets has never been reported probably because of their small size and imperfect round shape. The objective of the study was to develop an original custom-made device allowing both the mechanical stimulation and characterization of cartilage micropellets. The fluidic-based device was designed for the concomitant stimulation or characterization of six microspheres placed into the conical wells of a tank. In the present study, the device was validated using alginate-, collagen- and crosslinked collagen-based microspheres. Different types and ranges of pressure signals (square, sinusoidal and constant) were applied. The mechanical properties of microspheres were equivalent to those determined by a conventional compression test. Accuracy, repeatability and reproducibility of all types of pressure signals were demonstrated even though square signals were less accurate and sinusoidal signals were less reproducible than the others. The interest of this new device lies in the reliability to mechanically stimulate and characterize microspheres with diameters in the range of 900 to 1500 µm. Mechanical stimulation can be performed on six microspheres in parallel allowing the mechanical and molecular characterization of the same group of cartilage micropellets. The device will be useful to evaluate the growth of cartilage micropellets under mechanical stimuli.

AFM PeakForce QNM mode: Evidencing nanometre-scale mechanical properties of chitin-silica hybrid nanocomposites.

PeakForce Quantitative Nanomechanical Mapping (QNM) AFM mode was used to explore the mechanical properties of textured chitin-silica hybrid films at the nanoscale. The influence of the force applied by the tip on the sample surface was studied for standard homogeneous samples, for chitin nanorods and for chitin-silica hybrid nanocomposites. Thick films of superimposed chitin nanorods showed a monotonous increase of DMT modulus (based on the Derjaguin-Muller-Toporov model) owing to an increase in modulus at the interface between nanorods due to geometrical constraints of the AFM acquisition. A similar variation of DMT modulus was obtained for chitin-silica hybrid thick films related to mechanical strengthening induced by the presence of silica. This work revealed the role of the organic-inorganic interface, at the nanoscale, in the mechanical behaviour of textured materials using PeakForce QNM mode, with optimized analysis conditions.

Induction of mesenchymal stem cell differentiation and cartilage formation by cross-linker-free collagen microspheres.

Because of poor self-healing ability, joint cartilage can undergo irreversible degradation in the course of various diseases or after injury. A promising approach for cartilage engineering consists of using of mesenchymal stem cells (MSC) and a differentiation factor combined with an injectable carrier biomaterial. We describe here a novel synthesis route for native collagen microspheres that does not involve the use of potentially toxic crosslinking agents. An emulsion was formed between a type I collagen solution and perfluorinated oil, stabilised by a biocompatible triblock perfluorinated copolymer surfactant. Spherical microparticles of fibrillar collagen were formed through a sol-gel transition induced by ammonia vapours. Electron microscopy observations showed that these self-cross-linked microspheres were constituted by a gel of striated collagen fibrils. Microspheres that were loaded with transforming growth factor beta (TGF-ß)3 progressively released this differentiation factor over a four weeks period. Human MSC rapidly adhered to TGF-ß3-loaded microspheres and, after 21 d of culture, exhibited typical chondrocyte morphology and produced an uncalcified matrix made of the predominant cartilage components, aggrecan and type II collagen, but devoid of the hypertrophic marker type X collagen. Subcutaneous co-injection of MSC and TGF-ß3-loaded microspheres in mice consistently led to the formation of a cartilage-like tissue, which was however hypertrophic, calcified and vascularised. In conclusion, we developed cross-linker free collagen microspheres that allowed chondrogenic differentiation of MSC in vitro and in vivo.

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.

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.

5-ASA loaded chitosan-Ca-alginate microparticles: Preparation and physicochemical characterization.

The objective of the work was to prepare chitosan-Ca-alginate microparticles that can effectively deliver 5-ASA to the colon after peroral administration. For these requirements, a spray-drying technique was applied to 5-ASA/sodium alginate aqueous solution to obtain spherical particles having a mean diameter less than 10microm. The microparticles formed were cross-linked and coated into solution of CaCl(2) and chitosan to obtain stable microsystem. (1)H NMR and UV-vis spectra of 5-ASA have shown no degradation when working in adequate conditions, such as light protection, freshly prepared solution and use of nitrogen to prevent the oxidative self-coupling of 5-ASA moieties. By imaging with SEM, acceptable spherical morphology was observed, but also flattened, disk-shaped particles of smooth surface and low porosity. CLSM imaging showed dominant localization of chitosan in the particle wall, while for alginate, a homogeneous distribution throughout the particle was observed giving the particles negative charge. In the FTIR spectra of 5-ASA loaded Ca-alginate microparticles the characteristic peaks of 5-ASA were not altered indicating no covalent interaction between the drug and the polymer. DSC and X-ray diffraction studies revealed that 5-ASA was molecularly dispersed within the chitosan-Ca-alginate microparticles during the production process.

Cooperative ordering of collagen triple helices in the dense state.

Extracellular matrixes such as bone, skin, cornea, and tendon have ordered structures comprised for the most part of collagen, an elongated protein of well-defined dimensions and composition. Here we show how the cooperative ordering of collagen triple helices in the dense fluid state is exploited to produce dense ordered collagen matrixes. The spontaneous formation of a birefringent phase occurs at critical concentrations that increase from 50-60 to 80-85 mg/mL as the acetic acid concentration of the solvent increases from 5 to 500 mM. We studied by small-angle X-ray scattering (SAXS) the local liquidlike positional order across the isotropic/anisotropic phase transition by unwinding the cholesteric phase with moderate shearing stress. Interparticle scattering gives rise to a broad interference peak. The average distance between triple helices, dav, is thus estimated and decreases linearly as a function of phi-1/2 from 12.7 +/- 0.9 nm (22.5 mg/mL) to 5.0 +/- 0.6 nm (166.4 mg/mL). Equilibrium concentrations and the order parameter of the nematic phase agree reasonably well with theoretical predictions for semiflexible macromolecules. Striated fibrils with a high degree of alignment were obtained by fine-tuning the delicately balanced electrostatic interactions, which yielded strong elastic gels with a hierarchical organization very similar to that of major biological tissues. Typical Bragg reflections corresponding to the 67 nm period characteristic of collagen fibrils in biological tissues were recorded by SAXS with ordered collagen matrixes reconstituted in vitro.