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.

Motivation Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process. Results For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization. Implementation To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes. Supplementary information Supplementary data are available at Bioinformatics online.

With their potent regenerative and protective capacities, stem cell-derived conditioned media emerged as an effective alternative to cell therapy, and have a prospect to be manufactured as pharmaceutical products for tissue regeneration applications. Our study investigates the neuroregenerative potential of human dental pulp cells (DPCs) conditioned medium (CM) and defines an optimization strategy of DPC-CM for enhanced neuronal outgrowth. Primary sensory neurons from mouse dorsal root ganglia were cultured with or without DPC-CM, and the lengths of βIII-tubulin positive neurites were measured. The impacts of several manufacturing features as the duration of cell conditioning, CM storage, and preconditioning of DPCs with some factors on CM functional activity were assessed on neurite length. We observed that DPC-CM significantly enhanced neurites outgrowth of sensory neurons in a concentration-dependent manner. The frozen storage of DPC-CM had no impact on experimental outcomes and 48 h of DPC conditioning is optimal for an effective activity of CM. To further understand the regenerative feature of DPC-CM, we studied DPC secretome by human growth factor antibody array analysis and revealed the presence of several factors involved in either neurogenesis, neuroprotection, angiogenesis, and osteogenesis. The conditioning of DPCs with the B-27 supplement enhanced significantly the neuroregenerative effect of their secretome by changing its composition in growth factors. Here, we show that DPC-CM significantly stimulate neurite outgrowth in primary sensory neurons. Moreover, we identified secreted protein candidates that can potentially promote this promising regenerative feature of DPC-CM.

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.