Multimodal non-linear optical approach to assess fibrillogenesis in collagen constructs and spinal cord injury in mice model

Joshua DE LIZARAGA

Following trauma, a dense fibrotic scar rapidly develops at the lesion site. This scar is characterized by extensive formation of extracellular matrix proteins, including type I collagen. Although collagen is widely recognized as a major component of fibrosis, many aspects of its organization remain poorly understood. In particular, the relationship between its fibrillar alignment, and higher-order supramolecular assemblies during fibrillogenesis remains poorly understood. Fibrillar collagen exhibits a highly ordered, non-centrosymmetric structure that makes it detectable by nonlinear optical (NLO) imaging as second harmonic generation (SHG) imaging. This thesis presents a comprehensive investigation into the multiscale structure and organization of collagen through a multimodal strategy combining NLO imaging and spectroscopy. The work was structured around two complementary approaches: an ex vivo study focusing on a murine spinal cord injury SCI model, and an in vitro study addressing the fundamental mechanisms of collagen fibrillogenesis. This dual approach enabled us to analyze collagen formation within fibrotic tissue in a complex biological environment, while also allowing to monitor the evolution of polar order and band-specific orientation in simplified model systems. The ex vivo part of this work follows collagen reorganization along the rostrocaudal axis at different timepoints post-injury. SHG imaging and a custom made, subsequent 2D fingerprint data analysis quantified fiber tortuosity, orientation, and heterogeneity, while 3D variance analysis captured volumetric disorder. The 2D analysis revealed three phases of scar evolution: an initial phase of collagen formation, a disorganized phase with increased fiber density and heterogeneity, and a later phase suggesting fibers’ reorganization. Interestingly, 2D and 3D analyses diverged in the rostral and caudal regions but converged at the lesion core, suggesting a homogenous remodeling across the depth of the spinal cord tissue. In 2D, heterogeneity rose between 2 and 4 weeks before decreasing at 6 weeks, suggesting transient disorganization followed by partial reorganization. In contrast, 3D variance decreased at 4 weeks and rose again at 6 weeks, pointing to the opposite temporal trend. This discrepancy indicates that planar metrics and volumetric descriptors capture distinct facets of scar remodeling: 2D emphasizes surface-level alignment and density of collagen fibers, whereas 3D analysis exhibits depth-dependent fluctuations and organization of fibers in microdomains. Next we adopted a bottom-up approach, to dissect the molecular and supramolecular mechanisms of collagen fibrillogenesis in vitro. Simplified models of collagen peptides and fibrillar assemblies were studied under controlled conditions. A multimodal strategy combining second harmonic scattering (SHS) to probe molecular symmetry and supramolecular arrangement, and vibrational sum frequency generation (VSFG) measurements to analyze molecular orientation and structure, as well as atomic force microscopy (AFM) to visualize fibrillar morphology at the nanoscale was employed. This multimodal approach identified structural signatures of collagen at different stages of fibrillogenesis and demonstrated the crucial role of telopeptides in modulating polar order and supramolecular organization. Our results demonstrate that collagen assembly in fibers is determined not only by the intrinsic properties of the triple helix but also by the presence of the telopeptides, specific molecular domains at the end of the helices with the pH further modulating the supramolecular arrangement. Altogether, this thesis demonstrates the potential of multimodal nonlinear optical methods to characterize collagen organization across different scales, in in vitro models and in ex-vivo fibrotic spinal cord tissue.