Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics

Abstract

The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects.

Collagen: a network for regenerative medicine

“…When silica nanoparticles were negatively charged, positively charged collagen fibrils associated with the particles, and formed fibers as the pH was lowered. 55 …”

“…87 Another possible route for controlling fibrillogenesis with inorganic materials is to use particles as nucleators, which has been demonstrated with silicate nanoparticles which underwent sulphation. 55 These examples from the field of bionanocomposites, centered on the interactions between organic polymers and inorganic colloids, illustrate the advances in recent years, which are reviewed elsewhere. 88 …”

Generation and Assessment of Functional Biomaterial Scaffolds for Applications in Cardiovascular Tissue Engineering and Regenerative Medicine

“…It has been shown that coupling SHG and TPEF microscopy to a multimodal imaging system can provide qualitatively and quantitatively information on 3D collagen and elastic fiber networks . Multiphoton microscopy can therefore detect damaged ECM structures and represents a possibility to monitor fiber assembly, maturation and remodeling in time lapse . Based on multiphoton signals of cardiac myosin and collagen fibrils, the level of fibrosis in rat hearts after myocardial infarction was quantitatively assessed .…”

Understanding and Tuning Bioinorganic Interfaces for the Design of Bionanocomposites

“…114 The strong acidity of the sulfonate group insures the preservation of significant attractive electrostatic interactions between the particle and the protein during the neutralization step, leading to large, well‐organized fibrils intimately associated with silica colloids. Surface‐mediated growth of the collagen fibrils could be observed and was attributed to the fact that although the protein was initially confined on the particle, it preserves some mobility allowing for its subsequent self‐assembly 115…”