Second Harmonic Generation imaging of collagen fibrillogenesis

International audienceDevelopment of nonlinear optical microscopy has significantly improved three-dimensional (3D) imaging of biological tissues in recent years. In particular, collagen has been shown to exhibit endogenous Second Harmonic Generation (SHG) signals and SHG microscopy has proved to enable the visualization of collagen architecture in tissues with unequalled contrast and specificity [1, 2]. Type I collagen is a major structural protein in mammals and shows highly structured macromolecular organizations specific to each tissue. It is synthesized by cells as triple helices, which self-assemble outside the cells into fibrils that further form fibers, lamellae or other three-dimensional (3D) networks. This assembly mechanism depends critically on the collagen concentration, as well as on the temperature, pH and ionic strength of the solution in vitro. Thorough characterization of collagen fibrillogenesis is crucial to understand the biological mechanisms of tissue formation and tissue remodeling in response to a variety of pathologies. Booming of tissue engineering furthermore requires advanced in situ quantitative imaging techniques to verify whether the tissue substitutes display appropriate biomimetic 3D organization for cell culture scaffolds or functional implants. In this study, we continuously monitored the formation of collagen fibrils by time-lapse SHG microscopy [3]. Fibrillogenesis was triggered in a controlled way by increasing the pH in a dilute solution of collagen I. The fibril density was measured every 10 to 20 minutes as the number of voxels with significant SHG signal in 3D image stacks [1]. Our results showed reproducible dynamics of fibrillar collagen formation that could be changed by tuning the pH (see figure 1). We also monitored the growth of single fibrils and measured the length increase over time, which had never been reported before using an optical technique. We then correlated these SHG images to TEM images at nanometer-scale resolution by blocking the fibrillogenesis at early stages and drying the samples. It showed that SHG microscopy allows imaging of fibrils with a diameter down to 30-50 nm in our experimental conditions. We finally investigated surface-mediated fibrillogenesis by adding silica nanoparticles to the solution [4]. We used Two-Photon excited fluorescence (2PEF) microscopy to visualize the fluorescently-died nanoparticles and quantify the self-assembly of collagen around these nanoparticles. In conclusion, SHG microscopy enabled sensitive and well contrasted 3D visualization of collagen fibrillogenesis in a non invasive way. This work illustrates the potential of SHG microscopy for the rational design and characterization of collagen-based biomaterials. 0 200 400 600 800 0.0 0.5 1.0 1.5 2.0 2.5 Pixel fraction (%) Time (min) (d) (a) (c) (b) Fig 1. 3D reconstruction of SHG images of collagen fibrillogenesis (at pH=6.5) after a) 170 b) 410 and c) 730 minutes; d) Experimental kinetics of fibril density in the SHG images (black dots) with exponential fitting (red line). References [1] M

Rheological behavior of thermoreversible k-carrageenan/nanosilica gels

The rheological behavior of silica/κ-carrageenan nanocomposites has been investigated as a function of silica particle size and load. The addition of silica nanoparticles was observed to invariably impair the gelation process, as viewed by the reduction of gel strength and decrease of gelation and melting temperatures. This weakening effect is seen, for the lowest particle size, to become slightly more marked as silica concentration (or load) is increased and at the lowest load as particle size is increased. These results suggest that, under these conditions, the particles act as physical barriers to polysaccharide chain aggregation and, hence, gelation. However, for larger particle sizes and higher loads, gel strength does not weaken with size or concentration but, rather, becomes relatively stronger for intermediate particles sizes, or remains unchanged for the largest particles, as a function of load. This indicates that larger particles in higher number do not seem to increasingly disrupt the gel, as expected, but rather promote the formation of stable gel network of intermediate strength. The possibility of this being caused by the larger negative surface charge found for the larger particles is discussed. This may impede further approximation of neighboring particles thus leaving enough inter-particle space for gel formation, taking advantage of a high local polysaccharide concentration due to the higher total space occupied by large particles at higher loads.FCT - PTDC/QUI/67712/2006FEDE

NIRS a potential tool for monitoring harvesting and trade of similar woods: a case study for discrimination of mahogany, cedar, and crabwood.

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Novel Crystalline SiO2 Nanoparticles via Annelids Bioprocessing of Agro-Industrial Wastes

The synthesis of nanoparticles silica oxide from rice husk, sugar cane bagasse and coffee husk, by employing vermicompost with annelids (Eisenia foetida) is reported. The product (humus) is calcinated and extracted to recover the crystalline nanoparticles. X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS) show that the biotransformation allows creating specific crystalline phases, since equivalent particles synthesized without biotransformation are bigger and with different crystalline structure

Hybrid Shell Engineering of Animal Cells for Immune Protections and Regulation of Drug Delivery: Towards the Design of “Artificial Organs”

BACKGROUND: With the progress in medicine, the average human life expectancy is continuously increasing. At the same time, the number of patients who require full organ transplantations is augmenting. Consequently, new strategies for cell transplantation are the subject of great interest. METHODOLOGY/PRINCIPAL FINDINGS: This work reports the design, the synthesis and the characterisation of robust and biocompatible mineralised beads composed of two layers: an alginate-silica composite core and a Ca-alginate layer. The adequate choice of materials was achieved through cytotoxicity LDH release measurement and in vitro inflammatory assay (IL-8) to meet the biocompatibility requirements for medical purpose. The results obtained following this strategy provide a direct proof of the total innocuity of silica and alginate networks for human cells as underscored by the non-activation of immune defenders (THP-1 monocytes). The accessible pore size diameter of the mineralised beads synthesized was estimated between 22 and 30 nm, as required for efficient immuno-isolation without preventing the diffusion of nutrients and metabolites. The model human cells, HepG2, entrapped within these hybrid beads display a high survival rate over more than six weeks according to the measurements of intracellular enzymatic activity, respiration rate, as well as the "de novo" biosynthesis and secretion of albumin out of the beads. CONCLUSIONS/SIGNIFICANCE: The current study shows that active mammalian cells can be protected by a silica-alginate hybrid shell-like system. The functionality of the cell strain can be maintained. Consequently, cells coated with an artificial and a biocompatible mineral shell could respond physiologically within the human body in order to deliver therapeutic agents in a controlled fashion (i.e. insulin), substituting the declining organ functions of the patient

Chemically and thermally stable silica nanowires with a β-sheet peptide core for bionanotechnology

Background: A series of amyloidogenic peptides based on the sequence KFFEAAAKKFFE template the silica precursor, tetraethyl orthosilicate to form silica-nanowires containing a cross-β peptide core. Results: Investigation of the stability of these fibres reveals that the silica layers protect the silica-nanowires allowing them to maintain their shape and physical and chemical properties after incubation with organic solvents such as 2-propanol, ethanol, and acetonitrile, as well as in a strong acidic solution at pH 1.5. Furthermore, these nanowires were thermally stable in an aqueous solution when heated up to 70 °C, and upon autoclaving. They also preserved their conformation following incubation up to 4 weeks under these harsh conditions, and showed exceptionally high physical stability up to 1000 °C after ageing for 12 months. We show that they maintain their β-sheet peptide core even after harsh treatment by confirming the β-sheet content using Fourier transform infrared spectra. The silica nanowires show significantly higher chemical and thermal stability compared to the unsiliconised fibrils. Conclusions: The notable chemical and thermal stability of these silica nanowires points to their potential for use in microelectromechanics processes or fabrication for nanotechnological devices

Cell delivery systems using alginate : carrageenan hydrogel beads and fibers for regenerative medicine applications

The present work was focused on the development and characterization of new hydrogel systems based on natural origin polymers, namely, alginate and carrageenan, into different formats and with adequate properties to sustain the viability of encapsulated cells, envisioning their application as cell delivery vehicles for tissue regeneration. Different formulations of alginate and carrageenan hydrogels and different processing parameters were considered to determine the best conditions required to achieve the most adequate response in terms of the mechanical stability, cell viability, and functionality of the developed systems. The morphology, size, and structure of the hydrogels and their degradation behavior and mechanical properties were evaluated during this study. In addition to cytotoxicity studies, preliminary experiments were carried out to investigate the ability of alginate−carrageenan beads/fibers to encapsulate chondrocytes. The results obtained indicated that the different formulations, both in the form of beads and fibers, have considerable potential as cell-carrier materials for cell delivery in tissue engineering/ regenerative medicine applications.European NoE EXPERTISSUES - NMP3-CT-2004-500283Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/64070/2009