Arnaud Ponche

Cell/Material Interfaces: Influence of Surface Chemistry and Surface Topography on Cell Adhesion

The need to control the adhesion of cells to material surfaces plays an important role in determining the design of biomaterial substrates for biotechnology and tissue-engineering applications. As a the first step in a cascade of cellular events, adhesion affects many aspects of cell function, including spreading, migration, proliferation and differentiation. After a short description of cell adhesion and essential molecules involved in, the present knowledge on the influence of surface topography on cell behavior will be described by considering not only the amplitude of the surface topography but also its organization at all scales (micro- and nano-scale). The biological mechanisms underlying the cell response to topography will be evoked. Secondly, the influence of surface chemistry as well as surface energy on cell adhesion will be described. Thirdly, as the cells never interact with a bare material but with materials on which the proteins from biological fluids have adsorbed, some studies on the role of proteins in cell adhesion will be used to illustrate this point. Finally, the influence of substrate mechanics on cell differentiation will be described.

Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties

Most of the implant-associated infections are attributed to bacteria adhering to biomaterial surfaces as “biofilm” communities. Bacterial transport, first contact with the surface as well as some of the further developments can be considered and can be described using physical–chemical concepts. However, far from simple colloidal particles, bacteria have various macromolecular structures at their cell wall surface for interacting with their surroundings through specific and non-specific bindings. They are also able to modify composition and features of their cell wall in response to specific surrounding conditions. Therefore, bacteria/surface material interface is a complex topic, involving chemical and physical–chemical characteristics of both material surface and bacterial cell wall, as well as biological characteristics of bacteria. Furthermore, proteins and other biomolecules coming from surrounding medium influence the bacteria/material interface by adsorbing onto the material surface prior to any adhesion of bacteria. Finally, bacterial adhesion and biofilm formation phenomena occur at the same time as eukaryotic cell adhesion in an acute competition for adhering to and colonising the biomaterial surface. Therefore, developing biomaterials able to favour cell adhesion without promoting also bacterial adhesion appears still to be a challenge. In this article, we describe briefly the common development and particularities of biofilms before focusing on what for and whether bacterial features and material surface properties are likely to be involved in bacterial adhesion, the first step in biofilm formation. The influence of adsorbed biomolecules at the bacteria/material interface is finally addressed, as well as the current knowledge about the competition between bacteria and eukaryotic cells.

Opposite responses of cells and bacteria to micro/nanopatterned surfaces prepared by pulsed plasma polymerization and UV-irradiation.

Chemically and topographically patterned surfaces have high potential as model surfaces for studying cell and bacteria responses to surface chemistry and surface topography at a nanoscale level. In this work, we demonstrated the possibility to combine pulsed plasma polymerization and UV-irradiation to obtain topographical patterns and chemical patterns perfectly controlled at microlateral resolution and sub-micrometer depth level. Biological experiments were conducted using human osteoprogenitor cells and Escherichia coli K12. Proliferation and orientation of cells and bacteria were analyzed and discussed according to the size and the chemistry of the features. This work showed interesting opposite behavior of bacteria compared to eukaryotic cells, in response to the surface chemistry and to the surface topography. This result may be particularly useful on medical implants. From a methodological point of view, it highlighted the importance of working with versatile and well-characterized surfaces before and after sterilization. It also points out the relevance and the necessity of analyzing eukaryotic cell and bacteria adhesion in parallel way.

Phosphonate monolayers functionalized by silver thiolate species as antibacterial nanocoatings on titanium and stainless steel

Titanium and stainless steel substrates were modified by grafting with mercaptododecylphosphonic acid (MDPA) followed by reaction with silver nitrate (AgNO3), in order to investigate the potential of phosphonate self-assembled monolayers functionalized by silver thiolate species as antibacterial nanocoatings for inorganic biomaterials. The samples were characterized by Fourier transform infrared (FTIR) spectroscopy in grazing-incidence mode, water contact angle measurements, and X-ray photoelectron spectroscopy (XPS). The influence of the surface modification on bacterial adhesion and biofilm growth was investigated in vitro using Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus strains. The stability of the monolayer in blood-mimicking medium was examined. Despite their very low silver content, MDPA + AgNO3 monolayers strongly decreased bacterial adhesion (>99.9% reduction in the number of viable adherent bacteria) and biofilm formation in comparison to the bare substrates.

Polymer Multilayer Films Obtained by Electrochemically Catalyzed Click Chemistry

We report the covalent layer-by-layer construction of polyelectrolyte multilayer (PEM) films by using an efficient electrochemically triggered Sharpless click reaction. The click reaction is catalyzed by Cu(I) which is generated in situ from Cu(II) (originating from the dissolution of CuSO(4)) at the electrode constituting the substrate of the film. The film buildup can be controlled by the application of a mild potential inducing the reduction of Cu(II) to Cu(I) in the absence of any reducing agent or any ligand. The experiments were carried out in an electrochemical quartz crystal microbalance cell which allows both to apply a controlled potential on a gold electrode and to follow the mass deposited on the electrode through the quartz crystal microbalance. Poly(acrylic acid) (PAA) modified with either alkyne (PAA(Alk)) or azide (PAA(Az)) functions grafted onto the PAA backbone through ethylene glycol arms were used to build the PEM films. Construction takes place on gold electrodes whose potentials are more negative than a critical value, which lies between -70 and -150 mV vs Ag/AgCl (KCl sat.) reference electrode. The film thickness increment per bilayer appears independent of the applied voltage as long as it is more negative than the critical potential, but it depends upon Cu(II) and polyelectrolyte concentrations in solution and upon the reduction time of Cu(II) during each deposition step. An increase of any of these latter parameters leads to an increase of the mass deposited per layer. For given buildup conditions, the construction levels off after a given number of deposition steps which increases with the Cu(II) concentration and/or the Cu(II) reduction time. A model based on the diffusion of Cu(II) and Cu(I) ions through the film and the dynamics of the polyelectrolyte anchoring on the film, during the reduction period of Cu(II), is proposed to explain the major buildup features.

Characterization of Carbon Surface Chemistry by Combined Temperature Programmed Desorption with in Situ X-ray Photoelectron Spectrometry and Temperature Programmed Desorption with Mass Spectrometry Analysis

The analysis of the surface chemistry of carbon materials is of prime importance in numerous applications, but it is still a challenge to identify and quantify the surface functional groups which are present on a given carbon. Temperature programmed desorption with mass spectrometry analysis (TPD-MS) and X-ray photoelectron spectroscopy with an in situ heating device (TPD-XPS) were combined in order to improve the characterization of carbon surface chemistry. TPD-MS analysis allowed the quantitative analysis of the released gases as a function of temperature, while the use of a TPD device inside the XPS setup enabled the determination of the functional groups that remain on the surface at the same temperatures. TPD-MS results were then used to add constraints on the deconvolution of the O1s envelope of the XPS spectra. Furthermore, a better knowledge of the evolution of oxygen functional groups with temperature during a thermal treatment could be obtained. Hence, we show here that the combination of these two methods allows to increase the reliability of the analysis of the surface chemistry of carbon materials.

Direct ArF laser photopatterning of metal oxide nanostructures prepared by the sol-gel route.

We developed specific negative tone resists suitable for preparing periodic inorganic nanostructures by ArF photolithography. This approach is based on the sol-gel chemistry of modified metal alkoxides followed by DUV laser irradiation. Patterning at the nanoscale was demonstrated by using an achromatic interferometer operating at 193 nm. In a second step, thermal treatment could be used to obtain metal oxide nanostructures (ZrO(2), TiO(2)). Such thermal treatment did not affect the integrity of the nanostructures. The DUV-induced modifications of the physico-chemical properties of the sol-gel thin film were followed by ellipsometry, XPS and AFM. The crystalline structure of the material after thermal treatment was proved by DRX analysis. Examples of periodic nanostructures are given in order to illustrate the possibilities opened by this new route that provides a convenient method to create transparent, robust, high refractive index nanostructures compatible with a wide variety of substrates.

Surface transformation of silicon-doped hydroxyapatite immersed in culture medium under dynamic and static conditions

A comparative study of in vitro bioactivity of hydroxyapatite (HA) and silicon-doped hydroxyapatite (SiHA) has been carried out by immersion in a cell culture medium with or without fetal bovine serum during 14 days in static and dynamic conditions. A specific bioreactor was developed for the experiments in dynamic conditions. Ceramic surface transformations were characterized by electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy before and after immersion. The monitoring of calcium, phosphate and proteins in immersion medium was also done during the experiment. The two hydroxyapatite surfaces immersed in cell culture medium under dynamic conditions were found to be more probably covered by a new Mg-enriched Ca-deficient apatite layer than surfaces immersed under static conditions. These results suggest that dynamic procedure and medium with serum macromolecules seem to be more adequate to predict the in vivo activity of bioceramics. Moreover, SiHA presented a higher capacity of protein adsorption.

Oligonucleotide nanostructured surfaces: effect on Escherichia coli curli expression.

Oligonucleotide model surfaces allowing independent variation of topography and chemical composition were designed to study the adhesion and biofilm growth of E.coli. Surfaces were produced by covalent binding of oligonucleotides and immobilization of nucleotide-based vesicles. Their properties were confirmed through a combination of fluorescence microscopy, XPS, ellipsometry, AFM and wettability studies at each step of the process. These surfaces were then used to study the response of three different strains of E.coli quantified in a static biofilm growth mode. This study led to convincing evidence that oligonucleotide-modified surfaces, independent of the topographical feature used in this study, enhanced curli expression without an increase in the number of adherent bacteria.

In vitro and in vivo characterization of antibacterial activity and biocompatibility: A study on silver-containing phosphonate monolayers on titanium

Infections associated with implanted medical devices are a major cause of nosocomial infections, with serious medical and economic repercussions. A variety of silver-containing coatings have been proposed to decrease the risk of infection by hindering bacterial adhesion and biofilm formation. However, the therapeutic range of silver is relatively narrow and it is important to minimize the amount of silver in the coatings, in order to keep sufficient antibacterial activity without inducing cytotoxicity. In this study, the antibacterial efficiency and biocompatibility of nanocoatings with minimal silver loading (∼0.65 nmol cm(-2)) was evaluated in vitro and in vivo. Titanium substrates were coated by grafting mercaptododecylphosphonic acid (MDPA) monolayers followed by post-reaction with AgNO3. The MDPA/AgNO3 nanocoatings significantly inhibited Escherichia coli and Staphylococcus epidermidis adhesion and biofilm formation in vitro, while allowing attachment and proliferation of MC3T3-E1 preosteoblasts. Moreover, osteogenic differentiation of MC3T3 cells and murine mesenchymal stem cells was not affected by the nanocoatings. Sterilization by ethylene oxide did not alter the antibacterial activity and biocompatibility of the nanocoatings. After subcutaneous implantation of the materials in mice, we demonstrated that MDPA/AgNO3 nanocoatings exhibit significant antibacterial activity and excellent biocompatibility, both in vitro and in vivo, after postoperative seeding with S. epidermidis. These results confirm the interest of coating strategies involving subnanomolar amounts of silver exposed at the extreme surface for preventing bacterial adhesion and biofilm formation on metallic or ceramic medical devices without compromising their biocompatibility.