[Show abstract][Hide abstract] ABSTRACT: Mechanical interaction between cells and their surrounding extracellular matrix (ECM) controls key processes such as proliferation, differentiation and motility. For many years, two-dimensional (2D) models were used to better understand the interactions between cells and their surrounding ECM. More recently, variation of the mechanical properties of tissues has been reported to play a major role in physiological and pathological scenarios such as cancer progression. The 3D architecture of the ECM finely tunes cellular behavior to perform physiologically relevant tasks. Technical limitations prevented scientists from obtaining accurate assessment of the mechanical properties of physiologically realistic matrices. There is therefore a need for combining the production of high-quality cell-derived 3D matrices (CDMs) and the characterization of their topographical and mechanical properties. Here, we describe methods that allow to accurately measure the young modulus of matrices produced by various cellular types. In the first part, we will describe and review several protocols for generating CDMs matrices from endothelial, epithelial, fibroblastic, muscle and mesenchymal stem cells. We will discuss tools allowing the characterization of the topographical details as well as of the protein content of such cell-derived matrices. In a second part, we will report the methodologies that can be used, based on atomic force microscopy, to accurately evaluate the stiffness properties of the CDMs through the quantification of their young modulus. Altogether, such methodologies allow characterizing the stiffness and topography of matrices deposited by the cells, which is key for the understanding of cellular behavior in physiological conditions.
[Show abstract][Hide abstract] ABSTRACT: Mammalian cell culture is the starting point in many research studies focusing on biomedical applications. However, researchers have little control over the standardized cell microenvironment parameters. Here a modular ECM-mimicking surface coating for cell culture environment is designed. This substrate is a new and versatile thin film obtained by spin-coating of concentrated gelatin crosslinked by transglutaminase. It can be modified with respect to the biochemical and biophysical needs of the final cell destination, i.e. it delivers loaded multi-growth factors and serum components and allows for cell culture in a serum-free culture medium. Also, a well-known cell behavior modulator, the substrate stiffness, is controlled exogenously by addition of nanoparticles. In addition to growth factors, antimicrobial agents such as natural peptides are added to the substrate for limiting the repeated addition of antimicrobial agents to the culture medium and to prevent the increase of resistant bacterial strains in the culture environment. Finally, this substrate contains simultaneously ECM components, growth factors, stiffening elements and antimicrobial agents. It provides a favorable microenvironment and sterile conditions. It is a free-of-maintenance system, as cells will grow without addition of serum or antimicrobial cocktails. This low cost and easy-to-use substrate could emerge as a new standard for cell culture.
[Show abstract][Hide abstract] ABSTRACT: The Bell-Evans theory relative to rupture forces between non-covalently interacting molecules predicts that the rupture force increases linearly with the logarithm of the force loading rate. Here we investigate by force spectroscopy Atomic Force Microscopy (AFM) the rupture forces between surfaces covered by [small beta]-cyclodextrin ([small beta]-CD) molecules and AFM tips coated with adamantane (AD) groups. The [small beta]-CD molecules are either deposited through a self-assembled monolayer (SAM) or grafted on poly(allylamine) chains (PAH-CD) that are adsorbed on a substrate. The AD groups are fixed covalently on the AFM tip through either a poly(ethylene oxide) (PEO) linker or a four AD platform linked to the surface though a PEO chain. It is found that while the rupture forces between AFM tips covered with tetravalent AD molecules and SAM-CD surfaces do not exceed twice those found with tips covered by monovalent AD molecules, the rupture forces increase by a factor of 20 on PAH-CD substrates. The
Journal of Materials Chemistry B 02/2015; 3(9):1801-1812. DOI:10.1039/C4TB01261E · 4.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using modified β-galactosidase covalently linked into cross-linked polyelectrolyte multilayers (PEM), catalytically active materials have been designed. Their enzymatic activity can be modulated, partially in a reversible way, simply by stretching. This strategy, based on enzyme conformational changes, constitutes a new tool for the development of biocatalytic mechano-responsive materials.
Chemical Communications 02/2015; 51(26). DOI:10.1039/C5CC00329F · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on
four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with
brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in
1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of
recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development
and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study
of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and
molars, hereby recapitulating the AI phenotype in the human disorder.
Human Molecular Genetics 02/2015; 24(11). DOI:10.1093/hmg/ddv053 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since many years, it is a major challenge to regenerate damaged tissues using synthetic or natural materials. To favor healing processes after tendon, cornea, muscle or brain injuries, aligned collagen-based architectures are of utmost interest. In this study, we define a novel aligned coating based on a collagen/alginate multilayer film. The coating exhibiting a nanofibrillar structure is cross-linked with genipin for stability in physiological conditions. By stretching collagen/alginate-coated polydimethylsiloxane substrates, we developed a versatile method to align the collagen fibrils of the polymeric coating. Assays on cell morphology and alignment were performed to investigate the properties of these films. Microscopic assessments revealed that cells align with the stretched collagen fibrils of the coating. The degree of alignment is tuned by the stretching rate (i.e. the strain) of the collagen/alginate coated elastic substrate. Such coatings are of great interest for strategies that require aligned nanofibrillar biological material as a substrate for tissue engineering.
Tissue Engineering Part C Methods 02/2015; 21(9). DOI:10.1089/ten.TEC.2014.0479 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GFP has been genetically modified at two specific positions of its molecular architecture. These modifications allow its covalent attachment onto PEG brushes grafted on functionalized silicone surfaces. The stretching of this material leads to a reversible decrease of the fluorescence intensity due to stretch-induced forces applying on GFP molecules.
Chemical Communications 11/2014; 51(1). DOI:10.1039/C4CC07486F · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Laminins (LM), basement membrane molecules and mediators of epithelial-stromal communication, are crucial in tissue homeostasis. Inflammatory Bowel Diseases (IBD) are multifactorial pathologies where the microenvironment and in particular LM play an important yet poorly understood role in tissue maintenance, and in cancer progression which represents an inherent risk of IBD. Here we showed first that in human IBD colonic samples and in murine colitis the LMα1 and LMα5 chains are specifically and ectopically overexpressed with a concomitant nuclear p53 accumulation. Linked to this observation, we provided a mechanism showing that p53 induces LMα1 expression at the promoter level by ChIP analysis and this was confirmed by knockdown in cell transfection experiments. To mimic the human disease, we induced colitis and colitis-associated cancer by chemical treatment (DSS) combined or not with a carcinogen (AOM) in transgenic mice overexpressing LMα1 or LMα5 specifically in the intestine. We demonstrated that high LMα1 or LMα5 expression decreased susceptibility towards experimentally DSS-induced colon inflammation as assessed by histological scoring and decrease of pro-inflammatory cytokines. Yet in a pro-oncogenic context, we showed that LM would favor tumorigenesis as revealed by enhanced tumor lesion formation in both LM transgenic mice. Altogether, our results showed that nuclear p53 and associated overexpression of LMα1 and LMα5 protect tissue from inflammation. But in a mutation setting, the same LM molecules favor progression of IBD into colitis-associated cancer. Our transgenic mice represent attractive new models to acquire knowledge about the paradoxical effect of LM that mediate either tissue reparation or cancer according to the microenvironment. In the early phases of IBD, reinforcing basement membrane stability/organization could be a promising therapeutic approach.
PLoS ONE 10/2014; 9(10):e111336. DOI:10.1371/journal.pone.0111336 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Poly(lactic acid) (PLA) represents one of the most promising and attractive biobased polymer for the industrial development of environmentally sustainable packaging. However, oxygen and water barrier properties of PLA based films cannot compete with those of commercially available composite multilayers. To fill this gap, we used the layer-by-layer deposition technique on commercially used PLA thin films (30 pm thick) in order to increase their barrier properties to oxygen and water vapor. Nanometric films were grown by alternating branched poly(ethylene imine) (BPEI), hydrophobic fluorinated polymer (Nafion), and montmorillonite clay (MMT) layers with the aim of obtaining low gas permeability in both dry and moist conditions as well as low water vapor permeability. Two different kinds of architectures were designed and successfully prepared, based on a 4 layer repeating unit (BPEI/MMT/BPEI/Nafion), represented here as quadlayer (QL), and on a 6 layer repeating-unit ((BPEI/Nafion)(2)/BPEI/MMT), hexalayer (HL). Reduction in oxygen and water permeabilities is observed for films based on both types of repeat units. The reduction of the permeabilities increases with the number of quad and hexalayers achieving reductions in terms of oxygen permeability in both dry and humid conditions up to 98% and 97% respectively for 10 HL and QL. Furthermore, a reduction of 78% of water vapor transmission rate through the functionalized film was obtained for these films. As far as oxygen permeability is concerned, HL films are more efficient than QL films for smaller numbers of deposition units. These properties are shown to result from the complementarity between the presence of BPEI/Nafion and MMT layers.
Chemistry of Materials 10/2014; 26(19):5459-5466. DOI:10.1021/cm501359e · 8.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The formation of polydopamine under mild oxidation conditions from dopamine solutions with mechanical
agitation leads to the formation of films that can functionalize all kinds of materials. In the absence of stirring of the solution, we report the formation of polydopamine films at the air/water interface (PDA A/W) and suggest that it arises from an homogeneous nucleation process. These films grow two times faster than in solution and can be deposited on hydrophilic or hydrophobic substrates by the Langmuir−Schaeffer technique. Thanks to this new method, porous and hydrophobic materials like polytetrafluoroethylene (PTFE) membranes can be completely covered with a 35 nm thick PDA A/W film after only 3h of reaction. Finally the oxidation of a monomer followed by a polymerization in water is not exclusive to polydopamine since we also transferred polyaniline functional films from the air/water interface to solid substrates. These findings suggest that self-assembly from a solution containing hydrophilic monomers undergoing a chemical transformation (here oxidation and oligomerization) could be a general method to produce films at the liquid/air interface.
[Show abstract][Hide abstract] ABSTRACT: The fine influence of several key parameters onto the recently reported 2-in-1 step-by-step construction of PEDOT-PSS nanofilms by spin-coating is investigated by laser ellipsometry, UV–vis–NIR spectrometry, tapping-mode AFM and 4-point probe conductimetry following Van der Pauw geometry. First, the thickness of the film increases when deposited under good ventilation. Then, the linearity of film thickness with respect to the PEDOT-PSS deposition step number is maintained by thermal treatment at 423 K during 30 min, showing that the 2-in-1 deposition method is compatible with the thermal annealing steps used in electronic devices containing PEDOT-PSS. Moreover, the concentration of the PEDOT-PSS suspension used for the deposition exerts major influence on the film buildup rate, with a minimum one needed for the method to process at reasonable pace. Finally, analogously to what is known for films obtained by a sole deposition step, the conductivity of 2-in-1 PEDOT-PSS nanofilms is shown to behave differently at ambient to high temperature (373 K) than at the lower temperatures where the conductivity studies are usually made. All these results will be precious for the construction of devices containing a PEDOT-PSS film with a thickness needing to be controlled reproducibly at the nanoscale.
[Show abstract][Hide abstract] ABSTRACT: Herein, we investigate human single hydroxyapatite crystals (enamel and dentine) by convergent‐beam electron diffraction (CBED) and automated electron‐diffraction tomography (ADT). The CBED pattern shows the absence of the mirror plane perpendicular to the c axis leading to the P63 space group instead of the P63/m space group considered for larger‐scale crystals, this is confirmed by ADT. This experimental evidence is of prime importance for understanding the morphogenesis and the architectural organization of calcified tissues. Human single hydroxyapatite crystals (enamel and dentine; see figure) were investigated by convergent‐beam electron diffraction (CBED) and automated electron‐diffraction tomography (ADT). The CBED pattern shows the absence of the mirror plane perpendicular to the c axis leading to the P63 space group instead of the P63/m space group considered for larger‐scale crystals. This is confirmed by ADT. This experimental evidence is of prime importance for understanding the morphogenesis and the architectural organization of calcified tissues.
[Show abstract][Hide abstract] ABSTRACT: PEDOT-PSS 2-In-1 step-by-step film buildup Thermal annealing Nanometric thickness control Electronic conductivity Inelastic cut-offs of scaling a b s t r a c t The fine influence of several key parameters onto the recently reported 2-in-1 step-by-step construction of PEDOT-PSS nanofilms by spin-coating is investigated by laser ellipsometry, UV–vis–NIR spectrometry, tapping-mode AFM and 4-point probe conductimetry following Van der Pauw geometry. First, the thick-ness of the film increases when deposited under good ventilation. Then, the linearity of film thickness with respect to the PEDOT-PSS deposition step number is maintained by thermal treatment at 423 K dur-ing 30 min, showing that the 2-in-1 deposition method is compatible with the thermal annealing steps used in electronic devices containing PEDOT-PSS. Moreover, the concentration of the PEDOT-PSS suspen-sion used for the deposition exerts major influence on the film buildup rate, with a minimum one needed for the method to process at reasonable pace. Finally, analogously to what is known for films obtained by a sole deposition step, the conductivity of 2-in-1 PEDOT-PSS nanofilms is shown to behave differently at ambient to high temperature (373 K) than at the lower temperatures where the conductivity studies are usually made. All these results will be precious for the construction of devices containing a PEDOT-PSS film with a thickness needing to be controlled reproducibly at the nanoscale.
[Show abstract][Hide abstract] ABSTRACT: Simultaneous spraying of polyelectrolytes and small multi-charged molecules of opposite charges onto a vertical substrate leads to continuous buildups of organic films. Here, we investigate the rules governing the buildup of two such systems: poly(allylamine hydrochloride)/sodium citrate (PAH/citrate) and PAH/sulfated alpha-cyclodextrin (PAH/CD-S). Special attention is paid to the film growth rate as a function of the spraying rate ratio of the two constituents. This parameter was varied by increasing the spraying rate of one of the constituents while maintaining constant that of the other. For PAH/CD-S systems, whatever the constituent (PAH or CD-S) whose spraying rate was kept fixed, the film growth rate first increases, passes through a maximum before decreasing when the spraying rate of the other constituent is increased. For PAH/citrate, the film growth rate reaches a plateau value when the spraying rate of citrate is increased while that of PAH is maintained constant whereas when the spraying rate of citrate is maintained constant and that of PAH is increased, a behavior similar to that of PAH/CD-S is observed. The composition of PAH/CD-S sprayed films determined by X-ray photoelectron spectroscopy is independent of the spraying rate ratio of the two constituents and corresponds to one allylamine for one sulfate group. For PAH/citrate, by increasing the PAH/citrate spraying rate ratio, the carboxylic/nitrogen ratio in the film increases and tends to 1. There is thus always a deficit of carboxylic groups (COO- + COOH) with respect to amines (NH2 + NH3+). Yet, the ratio (COO-/NH3+) is always close to 1 ensuring exact charge compensation. The film morphology determined by atomic force microscopy is granular for PAH/CD-S and is smooth and liquid-like for PAH/citrate. A model based on strong (respectively weak) interactions between PAH and CD-S (respectively citrate) is proposed to explain these features.
[Show abstract][Hide abstract] ABSTRACT: Proper staining of grids is critical for transmission electron microscopy (TEM). Staining must be done as quickly as possible using minimal reagents and with consideration for the environment. We developed a new device for efficient staining of multiple TEM grids. We studied reagent evaporation, rinsing volume, flow rate and re-use of uranyl acetate, and provide here a procedure for efficient staining using the new device. Our device permits TEM grids to be stained with less reagent than alternative staining apparatuses; staining requires a total volume of 260 μl for five grids. Reagent evaporation is less than 6% even if used at 37° C. Moreover, our staining apparatus reduces chemical waste and shortens experiment time by staining several grids simultaneously. Our staining device is a compromise between time-consuming single grid processing and expensive commercial devices that consume large amounts of reagents.
[Show abstract][Hide abstract] ABSTRACT: In this article, we focus on hypomaturation autosomal-recessive-type amelogenesis imperfecta (type IIA2) and describe 2 new causal Matrix metalloproteinase 20 (MMP20) mutations validated in two unrelated families: a missense mutation p.T130I at the expected homozygous state, and a compound heterozygous mutation having the same mutation combined with a nucleotide deletion, leading to a premature stop codon (p.N120fz*2). We characterized the enamel structure of the latter case using scanning electron microscopy analysis and microanalysis (Energy-dispersive X-ray Spectroscopy, EDX) and confirmed the hypomaturation-type amelogenesis imperfecta as identified in the clinical diagnosis. The mineralized content was slightly decreased, with magnesium substituting for calcium in the crystal structure. The anomalies affected enamel with minimal inter-rod enamel present and apatite crystals perpendicular to the enamel prisms, suggesting a possible new role for MMP20 in enamel formation.
Journal of dental research 04/2013; 92(7). DOI:10.1177/0022034513488393 · 4.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We propose a simple method to elaborate a filler-free stretchable PDMS surface strong enough to resist to successive elongation/retraction cycles even at high degree of stretching. It consists in creating free radicals on a filler-containing PDMS surface by argon plasma exposure and reacting them with a filler-free PDMS resin during the crosslinking step. Changes of physical and chemical properties upon plasma modification are monitored by FTIR and XPS spectroscopies, contact angle measurements and atomic force microscopy. Electron spin resonance (ESR) is used to identify the nature of radicals involved in interfacial bonding. Although a brittle silica-like layer is created on the filler-containing PDMS surface after plasma treatment, an increase in the PDMS/PDMS interfacial strength is observed and a high interfacial resistance has been found under elongation/retraction (stretching/relaxation) cycles.