Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide (40 000 in France). Handicaps induced by SCI range from minimal sensory motor deficits to complete tetraplegia. Currently, there is no curative treatment on any symptoms associated with SCI. A scar, called glial and composed mainly of astrocytes and microglia, appears at the the injury site and inhibits axonal regeneration. This mechanism is poorly understood. Better monitoring of animal models of SCI in particular through the development of bioimaging translational tools are therefore mandatory.Our main objectives are to pursue and extend a multimodal imaging approach combining multiphoton spectroscopy (MPM), Atomic Force Microscopy (AFM) and Broadband coherent anti-Stokes Raman Spectroscopy (BCARS), a novel technique [1] based on Raman spectroscopy with faster image acquisitions [2], to better understand mechanisms that underlie the glial scar formation and the subsequent absence of spontaneous axonal regeneration following SCI, as well as to evaluate outcomes of therapeutic strategies that favor axonal regeneration in mice and non-human primates. MPM, conceived to specifically reveal non centro-symmetric structures with second harmonic generation (SHG) [3], provided a first evidence of the presence of collagen fibers as a biomarker of the injury site. AFM showed significant differences in Young Modulus between healthy and injured areas. These results must be supplemented with a deeper collagen characterization and axons imaging using MPM, as well as a treatment monitoring by co-localizing drugs within healthy/diseased tissues using BCARS [4].[1]. C. H. Camp Jr, Y. J. Lee, J. M. Heddleston, C. M. Hartshorn, A. R. Hight Walker, J. N. Rich, J. D. Lathia, M. T. Cicerone, Nat. Photonics 2014, 8, 627.[2]. C. M. Hartshorn, Y. J. Lee, C. H. Camp Jr, Z. Liu, J. M. Heddleston, N. Canfield, T. A. Rhodes, A. R. Hight Walker, P. J. Marsac, M. T. Cicerone, Anal. Chem. 2013, 85, 8102[3]. L. Mostaço-Guidolin, N.L. Rosin, T.L. Hackett, Int. J. Mol. 2017, 18(8), 1772. [4]. C. Zhang, D. Zhang, J.-X. Cheng, Annu. Rev. Biomed. Eng. 2015, 17, 415. [4]. C. Zhang, D. Zhang, J.-X. Cheng, Annu. Rev. Biomed. Eng. 2015, 17, 415.

Aim: Our main objective is to implement a multimodal (bio)physical approach to decipher mechanisms that underlie absence of spontaneous axonal regeneration following spinal cord injury (SCI) in mice and nonhuman primates. Method: A multimodal imaging strategy is employed combining state-of-the-art non-linear microscopy techniques (multiphoton - MPM and BCARS microscopy) with force spectroscopy measurements via atomic force microscopy (AFM). This approach enables to assess the relevant structural, morphological and spectral signature as well as the nanobiomechanical behavior of healthy and injured spinal cord tissue at cellular level. Results: We have first imaged excised mice SC tissues in 2-photon excited fluorescence and second harmonic generation (SHG). These MPM studies revealed the apparition of a strong SHG signal due to the formation of collagen I fibers by the injury site in the glial scar. AFM based force spectroscopy measurements have been also performed to map the elastic properties of the healthy and injured spinal cord tissue. Therefore, MPM data could be correlated with Young modulus elasticity results, indicating the presence of a fibrotic process 7 days after injury. As tissue stiffness is a regulator of neuronal growth, such kind of measurements will enable to understand why adult mammalian axons do not regenerate spontaneously after injury. Our next step will be to use a novel, emerging imaging technology that is the broadband coherent anti-Stokes Raman scattering (BCARS) microscopy for fast, label-free, highly selective and specific screening of spinal cord injury and potentially of drugs uptake into excised tissues. Combination of BCARS and MPM will first enable to retrieve information on the biological state of the cells/tissues that may underly the absence of spontaneous axonal regeneration following SCI (Raman neuronal markers: myelin, acetylcholine neurotransmitter, glutamate; MPM markers: collagen, neural axons, mitochondria,…). Second, it will permit to further correlate modification of these structural, morphological and spectral signatures in the context of therapeutic strategies.

Biological activities of cells such as survival and differentiation processes are mainly maintained by a specific extracellular matrix (ECM). Hydrogels have recently been employed successfully in tissue engineering applications. In particular, scaffolds made of gelatin methacrylate-based hydrogels (GelMA) showed great potential due to their biocompatibility, biofunctionality, and low mechanical strength. The development of a hydrogel having tunable and appropriate mechanical properties as well as chemical and biological cues was the aim of this work. A synthetic and biological hybrid hydrogel was developed to mimic the biological and mechanical properties of native ECM. A combination of gelatin methacrylate and acrylamide (GelMA-AAm)-based hydrogels was studied, and it showed tunable mechanical properties upon changing the polymer concentrations. Different GelMA-AAm samples were prepared and studied by varying the concentrations of GelMA and AAm (AAm2.5% + GelMA3%, AAm5% + GelMA3%, and AAm5% + GelMA5%). The swelling behavior, biodegradability, physicochemical and mechanical properties of GelMA-AAm were also characterized. The results showed a variation of swelling capability and a tunable elasticity ranging from 4.03 to 24.98 kPa depending on polymer concentrations. Moreover, the podocyte cell morphology, cytoskeleton reorganization and differentiation were evaluated as a function of GelMA-AAm mechanical properties. We concluded that the AAm2.5% + GelMA3% hydrogel sample having an elasticity of 4.03 kPa can mimic the native kidney glomerular basement membrane (GBM) elasticity and allow podocyte cell attachment without the functionalization of the gel surface with adhesion proteins compared to synthetic hydrogels (PAAm). This work will further enhance the knowledge of the behavior of podocyte cells to understand their biological properties in both healthy and diseased states.

Photobiomodulation (PBM) has been shown to improve cell proliferation and cell migration. Many cell types have been 
investigated, with most studies using deep penetrating red light irradiation. Considering the interest of surface biostimulation 
of oral mesenchymal cells after surgical wound, the present study aimed to assess green light irradiation effects on Dental Pulp 
Stem Cells’ (DPSC) proliferation and migration. To understand the mechanisms underlying these effects, we investigated 
cytoskeleton organization and subsequent cell shape and stiffness. 
A 532-nm wavelength Nd:YAG laser (30 mW) was applied 
between 30 and 600 s on DPSC in vitro. Cell proliferation was analyzed at 24, 48, and 72 h after irradiation, by cell counting and enzymatic activity quantification (paranitrophenylphosphate phosphatase (pNPP) test). A wound healing assay was used to study 
cell migration after irradiation. Effects of PBM on cytoskeleton organization and cell shape were assessed by actin filaments 
staining. Elasticity changes after irradiation were quantified in terms of Young’s modulus measured using Atomic Force 
Microscopy (AFM) force spectroscopy. Green light significantly improved DPSC proliferation with a maximal effect obtained after 300-s irradiation (energy fluence 5 J/cm2). This irradiation had a significant impact on cell migration, improving wound 
healing after 24 h. These results were concomitant with a decrease of cells’ Young’s modulus after irradiation. This cell softening 
was explained by actin cytoskeleton reorganization, with diminution of cell circularity and more abundant pseudopodia. This 
study highlights the interest of green laser PMB for the proliferation and migration of mesenchymal stem cells, with encouraging 
results for clinical application, especially for surgical wound healing procedures. 


Chronic kidney disease (CKD) is characterized by a gradual decline in renal function that progresses toward end-stage renal disease (ESRD). Podocytes are highly specialized glomerular epithelial cells, which form with the glomerular basement membrane (GBM) and capillary endothelium, the glomerular filtration barrier (GFB). GBM is an extracellular matrix (ECM) that acts as a mechanical support and provides biophysical signals that control normal podocytes behavior in the process of glomerular filtration. Thus the modulus of elasticity E or stiffness of “ECM” represents an essential characteristic that controls podocyte functions. The biophysical properties of hydrolyzed Polyacrylamide (PAAm) gels resemble to in vivo ECM and thus provide an opportunity to be applied as ECM-like membranes to study cellular behaviors. Therefore, hydrolyzed PAAm hydrogels were investigated for their potential use as new ECM-like constructs to engineer a basement membrane that form with cultured human podocytes a functional glomerular-like filtration barrier. Such ECM-like polyacrylamide hydrogel construct will provide the unique opportunity of understanding in an in vivo-like setting podocyte cells biological responses by controlling the physical properties of the PAAm membranes. In this work, several PAAm hydrogel layers were prepared by changing the crosslinker concentration. The macromolecular microstructure and stiffness were evaluated by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) techniques respectively. Accordingly, the mechanical properties and the polymeric network porosity can be effectively controlled by modulating the crosslinker concentration as well as the swelling degree. Moreover, modulating gel stiffness significantly influenced podocyte behavior including morphology, actin cytoskeleton reorganization. In conclusion, podocytes response to the variation of the mechanical properties of the membranes correlated with the hydrogels stiffness. This work addresses the complexity of podocytes behavior which will further enhance our knowledge to develop a kidney-on-chip model much needed to study kidney function in both health and disease states.

Microgels specific structural and functional features are attracting high research interest in several appli- cations such as bioactives and drug delivery or functional food ingredients. Whey protein microgels (WPM) are obtained by heat treatment of whey protein isolate (WPI) in order to promote intramolecular cross-linking. In the present work, atomic force microscopy (AFM) was used in contact mode and in liquid to investigate WPM particles topography and mechanical properties at the nanoscale at native pH (6.5) and acid pH (5.5 and 3.0). Prior to AFM, WPM particles were captured on a gold substrate via low energy interactions by means of specific monoclonal antibodies. AFM images clearly showed an increase in the size of WPM particles induced by pH decrease. AFM in force spectroscopy mode was employed to monitor the elasticity of WPMs. The obtained effective Young’s modulus data showed a significant increase in stiffness at pH 5.5 and pH 3.0, over 15-fold compared to native pH. These findings indicate that the mechanical profile of the WPM network varied with the pH decrease. The WPM topographic and nanome- chanical changes induced by acidification were most likely due to substantial changes in the shape and inner structure of WPM particles. Our results suggest that internally cross-linked structures, modified by acidification could display interesting functional properties when used as a food ingredient.