Chronic kidney disease is characterized by a gradual decline in renal function that progresses toward end-stage renal disease.Podocytes are highly specialized glomerular epithelial cells which form with the glomerular basement membrane (GBM) and capillaryendothelium the glomerular filtration barrier. GBM is an extracellular matrix (ECM) that acts as a mechanical support and providesbiophysical signals that control normal podocytes behavior in the process of glomerular filtration. Thus, the ECM stiffness represents anessential characteristic that controls podocyte function. Hydrolyzed Polyacrylamide (PAAm) hydrogels are smart polyelectrolytes materials.Their biophysical properties can be tuned as desired to mimic the in natural ECM. Therefore, these hydrogels are investigated as new ECM-like constructs to engineer a podocyte-like basement membrane that forms with cultured human podocytes a functional glomerular-like filtration barrier. Such ECM-like PAAm hydrogel construct will provide unique opportunity to reveal podocyte cell biological responses in an in vivo-like setting by controlling the physical properties of the PAAm membranes. In this work, Hydrolyzed PAAm scaffolds having different stiffness ranging between 0.6− 44 kPa are prepared. The correlation between the hydrogel structural and mechanical properties and Podocytes morphology, elasticity, cytoskeleton reorganization, and podocin expression is evaluated. Results show that hydrolyzed PAAm hydrogels promote good cell adhesion and growth and are suitable materials for the development of future 3D smart scaffolds. In addition, the hydrogel properties can be easily modulated over a wide physiological range by controlling the cross-linker concentration. Finally, tuning the hydrogel properties is an effective strategy to control the cells function. This work addressed the complexity of podocytes behavior which will further enhance our knowledge to develop a kidney-on-chip model much needed in kidney function studies on both healthy and diseased states.

Extensive use of porous silicon (PSi) for tissue engineering is due to its convenient properties as it is both non-toxic and bioresorbable. Moreover, PSi surface modification is an important step to enhance cell adhesion and proliferation. In this work, a combination of optical and electrochemical studies is performed to elaborate a suitable PSi multilayer substrate for cell culture. For this study, we modified PSi surface by silanization and antibody grafting (APTES-anti STRO1), the 12-mer specific peptide to silicon p+type coating and the peptide modified with the antibody recognition sequence. Electrochemical characterization of PSi multilayers is performed to investigate its electrical behavior, determine the optimal measuring conditions and reveal the most stable PSi surfaces. Then, the behavior of dental pulp stem cells (DPSC) was investigated on various modified PSi surfaces. An electrochemical method was applied for the first time monitoring the electrical behavior of stem cell adhesion. The cells electrochemical behavior depends on the nature of the surface coating and the peptide-anti STRO1 improved adhesion and cell spreading onto the PSi surface compared to bare surface and the one coated with the peptide. Fluorescent microscopy revealed that all surface modi cation methods enhance cell adhesion compared to the bare PSi surface. An increased cell number is observed on APTES-anti STRO1, peptide and peptide-anti STRO1 coated PSi. The peptide-anti STRO1 provided the best cell proliferation results suggesting the improved accessibility of the recognition fragment of the antibody anti-STRO1.

Cost-effective health care technologies for detection of disease biomarkers at ultralow concentrations can significantly improve the quality of life in resource-constrained countries. However, current techniques require expensive instruments, complex conjugation protocols and tedious laboratory procedures that may not be affordable for the major part of the world population. Here, we propose to sidestep the problem by establishing a simple, relatively inexpensive sensing method employing a photonic substrate, a material affinity peptide, and a smart phone CCD detector to achieve detection of clinically relevant proteins in serum at concentrations much lower than standard enzyme-linked immunosorbent assay (ELISA). Easy to process porous silicon (PSi) micro-cavities were employed as substrates that provide a three-dimensionality, large surface area, and convenient light enhancement properties for molecular detection. Anti-histone H2B antibodies and biomarkers of severe illnesses are detected in whole serum at concentrations as low as 10 fg mL−1 by using the proposed PSi-ELISA protocol. Due to its easy use, cost effectiveness and high sensitivity, the proposed method has potential large- scale applications in ultrasensitive sensing of different other clinically relevant molecules for early stage diagnostics.

Dentin carious lesion is a dynamic process that involves de- mineralization and collagen denaturation. Collagen type I is the major protein in dentin and it has been investigated based on its optical properties. Multiphoton microscopy (MPM) is a nonlinear imaging technique that reveals the car- ies process using the collagen two-photon excitation fluo- rescence (2PEF) and its second-harmonic generation (SHG). Combining the histological and the International Caries De- tection and Assessment System (ICDAS) classifications with nonlinear optical spectroscopy (NLOS), 2PEF and SHG inten- sities of enamel and dentin were highly altered during the caries process. It has been proven that the ratio SHG/2PEF is a relevant indicator of the organic matrix denaturation [Ter- rer et al.: J Dent Res 2016;96:574–579]. In the present study, a series of measurable signals is made to detect early stages of carious lesion according to the ICDAS classification and to explore the relationship between these measures and theICDAS scale. Comparison of the efficiency of nonlinear opti- cal signals for caries detection with the ICDAS classification is essential to evaluate their potential for clinical applica- tion. In our study, the use of the NLOS measured by MPM allowed us to monitor a quantitative parameter (SHG/2PEF ratio) according to the dentin carious lesion state (ICDAS and histological examination). Three coherent new groups were defined (ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6), where the carious process can be clearly described with a statistically significant decrease of the SHG/2PEF ratio.

Multiphoton microscopy (MPM) is a nonlinear high-resolution technique that can be applied to collagen studies such as collagen type I denaturation during caries process. The MPM lies in its simultaneous image creation based on the fluorescence signals produced by a multiphoton excitation (2-photon excited fluorescence [2PEF]) and a coherent nonlinear phenomenon, second harmonic generation (SHG). This study aims to investigate the structural modifications during the dentin caries process by monitoring the variations of the SHG and the SHG/2PEF ratio recorded by nonlinear optical spectroscopy (NLOS). The International Caries Detection and Assessment System (ICDAS) was used as a clinical reference in vitro.To this end, teeth for each ICDAS score were selected and longitudinal sections through the caries lesion were performed. SHG and 2PEF images and spectra were recorded for each ICDAS score (0 to 6).2PEF and SHG images highlighted the microarchitecture and optical properties of the collagen denaturation in dentin during the caries process. The SHG images of carious dentin demonstrated a destruction of the collagen matrix. The variation of intensity of the 2PEF, SHG and the ratio SHG/2PEF allowed the merge of ICDAS scores: ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6. The new groups tend to be similar to the International Caries Classification and Management System (ICCMS). The 2PEF and SHG intensities and the ratio SHG/2PEF are highly altered during the caries process. The ratio SHG/2PEF is a relevant parameter to monitor the caries evolution and it defined three stages: initial lesion (ICDAS 0/1), moderate lesion (ICDAS 2/3) and extensive lesion (ICDAS 4/5/6). The development of a clinical device based on the nonlinear optics will bring a valuable diagnostic tool for caries detection in an everyday practice.

Luminescent nanodiamonds (ND) are attractive tools for nanoscale biologic cellular imaging allowing both photoluminescence (PL) and magnetic resonance imaging [1]. Recent technological developments enable to fabricate bright NDs with high content of nitrogen-vacancy centres [2] that are anticipated to serve as a cell probes. In this work we present novel method of NDs detection in cellular environment. We demonstrate simultaneous visualization of NDs and of the non- labeled nucleus of living cells based on Raman and PL detection as a new tool for the localization of internalized nanoparticles.To this end, NDs of size ranging from ultra-small particles ~ 5 nm to 60 nm were used, prepared from Ib synthetic diamond. Cells used for this experiment were from mammalian breast cancer (MCF7). We report on accomplishing to successfully internalize ND particles in MCF7 cells. We show that ND internalization can be monitored by Raman imaging method using K-mean cluster analysis. Whilst standard Raman imaging methods of NDs make use of the sp3 diamond Raman signal, which limits their use to 100 nm size particles or bigger [3], here we employ Raman imaging in a novel way to detect small near-IR cellular probe. Changes of cells stiffness were detected by force measurement in atomic force microscopy after incubating cells with NDs, suggesting cell membrane hardening upon ND uptake.[1] L. Moore, M. Nesladek et al., Nanoscale (2014)[2] J. Havlik, M. Gulka, M. Nesladek et al., Nanoscale (2013) [3] C.-Y. Cheng et al., Applied Physics Letter (2007)