Spinal cord injury is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After injury the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier. The scarring process involves microglia, astrocytes and extracellular matrix components, such as collagen, con- structing the fibrotic component of the scar. To investigate the role of collagen, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopy. The second harmonic generation signal exhibited by fibrillar collagen enabled to specifically monitor it as a biomarker of the lesion. An increase in collagen density and the formation of more tortuous fibers over time after injury are observed. Nano-mechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers' formation. These observations indicate the concomitance of important structural and mechanical modifications during the fibrotic scar evolution.

Spinal cord injury (SCI) is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After the injury, the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier[1]. The scarring process involves microglia, astrocytes, and extracellular matrix components, such as collagen, composing the fibrotic part of the scar[2]. To investigate the role of collagen and microglia, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopies. The second harmonic generation signal exhibited by fibrillar collagen-I enables specifically monitoring it as a biomarker of the lesion. An increase in collagen density and the formation of more curved fibers over time after SCI are observed. Whereas 2-photon excitation microscopy (2PEF) showed the appearance and activation of microglia over millimeters in length near the injured area. Nanomechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers’ development. Additionally, we observed that inhibition of microglial proliferation by oral administration of GW2580 decreased the collagen density at the injured area. These observations indicate the concomitance of relevant structural and mechanical modifications during the fibrotic scar evolution.

Introduction: The functionalization of titanium (Ti) and titanium alloys (Ti6Al4V) implant surfaces via material-specific peptides influence host/biomaterial interaction. The impact of using peptides as molecular linkers between cells and implant material to improve keratinocyte adhesion is reported.Results: The metal binding peptides (MBP-1, MBP-2) SVSVGMKPSPRP and WDPPTLKRPVSP were selected via phage display and combined with laminin-5 or E-cadherin epithelial cell specific peptides (CSP-1, CSP-2) to engineer four metal-cell specific peptides (MCSPs). Single-cell force spectroscopy and cell adhesion experiments were performed to select the most promising candidate. In vivo tests using the dental implant for rats showed that the selected bi functional peptide not only enabled stable cell adhesion on the trans-gingival part of the dental implant but also arrested the unwanted apical migration of epithelial cells.Conclusion: The results demonstrated the outstanding performance of the bioengineered peptide in improving epithelial adhesion to Ti based implants and pointed towards promising new opportunities for applications in clinical practice.

Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide, with no current curative treatment. To understand the mechanisms underlying the absence of spontaneous regeneration following injury, we are combining the non-linear multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM), in a mouse model, to monitor the glial scar, a scar that inhibits the axonal regeneration by forming a physical and chemical barrier composed mainly of astrocytes and microglia. We recorded 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues in MPM at 72h, 1week and 6 weeks post-lesion, and further performed polarization dependent measurements of the SHG signal to assess the preferential orientation of the collagen bundles. Our MPM images revealed a strong SHG signal at 1 week post injury, due to the formation of fibrillary collagen fibers (collagen type I) by the injury site. The SHG signal was increased at 6 weeks after injury, and associated with (1) a higher fiber density (2) a shorter fiber length and less fibers oriented in the same direction. AFM based force spectroscopy measurements, performed at the same post-lesion time-points to map the elastic properties of the spared grey and white matters and injured (lesion) parts of the tissue, suggested an increase of the lesion area stiffness over time. These results together indicate the presence of a fibrotic process seven days after injury, that is further increased at later time points. We similarly started to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage proliferation) in mice that underwent SCI. Our preliminary results suggested an increase in fibers length in treated tissues, as well as a reduction of the collagen extension around the injury site.

Background: Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context.Method: To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9.Results: We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (β1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (β1-6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression.Conclusion: MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer.

Aim: With the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following spinal cord injury (SCI) we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature, and the nanobiomechanical behavior of healthy and injured spinal cord tissue we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h, 1week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks’post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 1 week and 6 weeks post injury.

Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide yielding major handicaps and inducing high economical costs. A scar, called glial scar and composed of two main cellular populations i.e. astrocytes and microglia, inhibits axonal regeneration by forming a physical and chemical barrier. Currently, there is no curative treatment on any symptoms associated with SCI. In this context, with the objective to investigate the mechanisms underlying absence of spontaneous axonal regeneration following SCI, we employ a multimodal label-free imaging approach to monitor glial scar in a mice SCI model. Method: To determine the relevant structural signature and the nanobiomechanical behavior of healthy and injured spinal cord tissue, we combine the non-linear, multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM). The glial scar at different key post lesion time-points is investigated with these two techniques to monitor structural and elasticity (Young modulus) changes of the tissue.Results: 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues were recorded in MPM at 72h,1 week and 6 weeks post-lesion. The MPM images revealed the apparition of a strong SHG signal at 1week post injury, due to the formation of fibrillar collagen fibers (collagen type I) by the injury site in the glial scar. At 6 weeks post-injury, the SHG signal is more intense and a higher number of fibers are detected in average. We further assessed the preferential orientation of the collagen bundles performing polarization dependent measurements of the SHG signal. The AFM based force spectroscopy measurements have been performed at the same post-lesion time-points to map the elastic properties of the healthy (grey and white matters) and injured (lesion) parts in the spinal cord tissue. The results suggested an increase of the lesion area stiffness over time that could be correlated with the apparition of fibrillar collagen observed in MPM, indicating the presence of a fibrotic process seven days after injury, that develops in time. As tissue stiffness is a regulator of neuronal growth, such kind of measurements might help to understand why adult mammalian axons do not regenerate after an injury. Our next step is to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage in mice that underwent SCI) on the structure and mechanical properties of the lesion site at 2 weeks and 6 weeks post injury.