1-3

Abstracts English

Despite strict operative procedures to minimize microbial contaminations, bacterial infection of implants significantly raises postoperative complications of surgical procedures. One of the promising approaches is to adjust and control antimicrobial properties of the implant surface.

New types of antibacterial coatings prepared via plasma polymer functionalization step have been developed. These coatings contain and release in a control way a bioactive agent. Controlled release was achieved by the fabrication of plasma polymer multilayer systems, which consist of two layers of Maleic Anhydride Plasma Polymer deposited on the surface of Polypropylene made surgical mesh. In between plasma polymer layers, silver nanoparticles are trapped as an antibacterial agent reservoir.

Owing to differences between mechanical properties of the plasma-polymer thin films and the elastic bulk substrates, tensile strengths generate cracks within the plasma polymer, which might be used as diffusive channels for bioactive substances, here silver ions. The cracks can be controlled mechanically in a reversible way (Figure 01).

The tailoring of the spontaneous release of bioactive agent is achieved by the modification of the second plasma polymer deposition conditions. In addition, during mechanical stimulation of the designed material, control over silver ion release is achieved through an elongation-dependent releasing process allowed by the reversible control of the cracks. In the field of textiles and other soft Abstracts 1-4 biomaterials, this strategy is promising due to the mechanical stresses that naturally occur at the implant location. In regard of possible application of the developed system as a future biomaterial, the impact of different types of commonly used sterilization procedures on the properties of developed material was studied. The effects of autoclaving and electron beam sterilization methods on the surface chemistry, the dispersion of embedded silver nanoparticles in the plasma polymer and the cracks formation of the developed material was verified. Results showed the compatibility of the developed system with electron beam sterilization method.

The antibacterial properties of the new material have been evaluated. The effect of developed system on planktonic bacteria, bacterial adhesion and biofilm formation on stretched and unstretched system was studied. The membrane integrity of the adhered bacteria and bacteria in biofilms was followed during the study as an indicator of the physiologic state of bacteria. Results suggested that the sensitivity of bacteria to low concentrations of released silver ions resulted The results give a strong base on the future of intelligent, silver containing materials that control the release at the site of infection. Our results show that low doses of silver may be sufficient to control infection by acting on the structure of bacterial biofilms.

Français

Les infections bactériennes lorsqu'elles se développent à partir d'implants sont très difficiles à traiter, l'issue courante étant un retrait pur et simple de l'implant incriminé. Dans ce cadre, les revêtements des biomatériaux ont un rôle important à jouer pour, d'une part, prévenir l'adhésion bactérienne et d'autre part, éliminer les bactéries présentes. Ces revêtements antibactériens doivent par ailleurs permettre une intégration tissulaire des biomatériaux aux cellules rencontrées sur le site de l'implantation.

Dans ce travail une nouvelle famille de revêtements antibactériens a été développée. Ils contiennent et libèrent de manière contrôlée un agent bioactif.

Ils sont constitués de multicouches de polymère plasma d'anhydride maléique déposées à la surface de fibres de polypropylène tressées et constituant le matériau à implanter. Entre chaque dépôt de polymère plasma (agissant comme couche barrière), des nanoparticules d'argent sont piégées formant ainsi des réservoirs d'agent antibactérien. En raison des différences de propriétés mécaniques entre les films minces plasma et le substrat massique élastique (i.e. tissu de fibre de polypropylène), la résistance à la traction génère des fissures dans les couches polymère plasma, qui sont utilisées comme More special thanks for Nelly Hobeika for all the discussions we had, for spending a lot of her free time with me and for being good friend.

Besides all my friends from Mulhouse and other places I thank my family and Nicolas Ducruet, who provided support and mental backup whenever it was needed.

Funding for this project from Region Alsace is also gratefully acknowledged.

General Introduction

Bioactive materials: toward new mechanoresponsive polymeric materials

Implantation of biomaterials during surgery is a common and often lifesaving procedure. However the use of implants is also one of the most important risk factor of biofilm-associated nosocomial infections and may expose the recipient to a lifelong risk and health problems. Some of these infections are extremely resistant to antibiotics and host defenses. Therefore, the most useful and standard therapy usually consists of removing implant which causes tissue damage, further increases the susceptibility to infections, activates host defenses and stimulates the generation of inflammatory mediators [START_REF] Gristina | Biomaterial-Centered Infections: Microbial Adhesion versus Tissue Integration[END_REF]. So, new solutions have to be found as alternative methods of control, such as infection-resistant materials.

Bioengineering field of implant materials is undergoing rapid new developments and one of the possible strategies to prevent infections is incorporation of bioactive agents (i.e. drugs) at the surface of implant coatings. The efficiency and durability of such bioactive coatings are strongly related to the control of the rate of drug release. For this purpose, researchers are focusing on the development of adaptive surfaces, known as stimuli-responsive materials, and able to release bioactive agent in a controlled way. Today, triggered release is possible through artificial stimuli [START_REF] Kost | Responsive polymeric delivery systems[END_REF] such as light-irradiation [START_REF] Katz | Light-Responsive Biomaterials: Development and Applications[END_REF][START_REF] Nicoletta | Light Responsive Polymer Membranes: A Review[END_REF], magnetic [START_REF] Thevenot | Magnetic responsive polymer composite materials[END_REF][START_REF] Filipcsei | Magnetic Field-Responsive Smart Polymer Composites[END_REF], ultrasonic [START_REF] Lentacker | Ultrasound-Responsive Polymer-Coated Microbubbles That Bind and Protect DNA[END_REF], thermal [START_REF] Yamato | Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity[END_REF][START_REF] Chilkoti | Targeted drug delivery by thermally responsive polymers[END_REF][START_REF] Yoshizako | Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer[END_REF], electric [START_REF] Moroder | Material properties and electrical stimulation regimens of polycaprolactone fumarate-polypyrrole scaffolds as potential conductive nerve conduits[END_REF] and, as well, natural stimuli like pH changes [START_REF] Gillies | pH-Responsive Copolymer Assemblies for Controlled Release of Doxorubicin[END_REF], redox gradients [START_REF] Saito | Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities[END_REF] and enzymatic stimuli [START_REF] Miyata | Biomolecule-sensitive hydrogels[END_REF]. Surprisingly, only [START_REF] Gristina | Biomaterial-Centered Infections: Microbial Adhesion versus Tissue Integration[END_REF][START_REF] Kost | Responsive polymeric delivery systems[END_REF][START_REF] Katz | Light-Responsive Biomaterials: Development and Applications[END_REF][START_REF] Nicoletta | Light Responsive Polymer Membranes: A Review[END_REF][START_REF] Thevenot | Magnetic responsive polymer composite materials[END_REF][START_REF] Filipcsei | Magnetic Field-Responsive Smart Polymer Composites[END_REF][START_REF] Lentacker | Ultrasound-Responsive Polymer-Coated Microbubbles That Bind and Protect DNA[END_REF][START_REF] Yamato | Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity[END_REF][START_REF] Chilkoti | Targeted drug delivery by thermally responsive polymers[END_REF][START_REF] Yoshizako | Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer[END_REF][START_REF] Moroder | Material properties and electrical stimulation regimens of polycaprolactone fumarate-polypyrrole scaffolds as potential conductive nerve conduits[END_REF][START_REF] Gillies | pH-Responsive Copolymer Assemblies for Controlled Release of Doxorubicin[END_REF][START_REF] Saito | Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities[END_REF][START_REF] Miyata | Biomolecule-sensitive hydrogels[END_REF][START_REF] Del Rio | Stretching single talin rod molecules activates vinculin binding[END_REF][START_REF] Zhong | Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly[END_REF][START_REF] Arm | Effects of cyclical mechanical stress on the controlled release of proteins from a biodegradable polymer implant[END_REF][START_REF] Hemmerlé | Mechanically Responsive Films of Variable Hydrophobicity Made of Polyelectrolyte Multilayers[END_REF][START_REF] Mertz | Mechanically Responding Nanovalves Based on Polyelectrolyte Multilayers[END_REF][START_REF] Mertz | Mechanotransductive surfaces for reversible biocatalysis activation[END_REF] few study report developments of adaptive surfaces which respond to mechanical stimuli [START_REF] Del Rio | Stretching single talin rod molecules activates vinculin binding[END_REF][START_REF] Zhong | Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly[END_REF][START_REF] Arm | Effects of cyclical mechanical stress on the controlled release of proteins from a biodegradable polymer implant[END_REF][START_REF] Hemmerlé | Mechanically Responsive Films of Variable Hydrophobicity Made of Polyelectrolyte Multilayers[END_REF]. The benefit of developing such mechanoresponsive surfaces is that mechanical constraints naturally exists in the human body e.g., in the muscles, joints, arteries.

Mechanical stimuli responsive material was used for the first time in the work of Arm et al. [17], in which the authors controlled the release of proteins from biodegradable polymer implant. Another example has been described in the work of Mertz et al. [START_REF] Mertz | Mechanically Responding Nanovalves Based on Polyelectrolyte Multilayers[END_REF], which was based on deposition of exponentially and linearly growing polyelectrolyte multilayers. The system, consisting in nanometer-sized multilayer barriers, was deposited on or between multilayer compartments and was designed to respond to mechanical stimuli and to act as nanovalves. The diffusion of drugs through the barrier from one compartment to another was switched on/off by tuning the mechanical stretching and thereby opening or closing nanopores in the barrier. Recent work of the same group [START_REF] Mertz | Mechanotransductive surfaces for reversible biocatalysis activation[END_REF] has reported a study on nanoassemblies, which consisted of a first polyelectrolyte multilayer stratum loaded with enzymes and capped with a second polyelectrolyte multilayer acting as a mechanically sensitive nanobarrier. The biocatalytic activity of the film is reversibly switched on/off by mechanical stretching, which exposes enzymes through the capping barrier, similarly to mechanisms involved in proteins like fibronectin during mechanotransduction.

Finally, study of Bacharouche et al. [START_REF] Bacharouche | Biomimetic Cryptic Site Surfaces for Reversible Chemo and Cyto-Mechanoresponsive Substrates[END_REF] reported the first example of synthetic surfaces allowing specific interactions with proteins or cells promoted by mechanical action in a fully reversible manner. Silicone sheets were first treated with plasma and then functionalized by grafting sequentially under stretching poly(ethyleneglycol) (PEG) chains and biotin or arginine-glycine-aspartic acid (RGD) peptides. At rest position, these ligands were not accessible for their receptors. Under stretching, the surface became specifically interactive to streptavidin, biotin-antibodies or adherent for cells and the interactions were 1-21 fully reversible by stretching/unstretching both for proteins and cells, revealing a reversible exposure process of the ligands.

In this work, our objective was to develop a new antibacterial mechanoresponsive coating for textile material. As mentioned before, the need of implants with controlled antibacterial properties is increasing due to the risk of infections which follows the implantation of biomaterials. Here, the main idea is to use the mechanical stimuli already present in the implantation site to reversibly switch on/off the release of the antimicrobial agent.

For this purpose, polypropylene mesh material is used as the substrate and the antibacterial coating consists in a multilayer plasma polymer system and a silver nanoparticles reservoir (Figure 01). Silver nanoparticles as silver ions source are used as the antibacterial agent. System is designed to release only silver in an ionic form, since studies have shown that silver nanoparticles may induce cytotoxicity when in contact with human cells [START_REF] Park | The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles[END_REF]. Tailoring of the release of silver ions is achieved by mechanical stimulation of the designed material and these results are presented in terms of elongation-dependent release measurements. Results show that taking advantages of different mechanical properties between the different layers, cracks are induced under elongation in the plasma polymer overlayer. The diffusion of silver ions through the plasma overlayer is switched on/off by tuning the mechanical stretching and thereby opening and closing cracks. Antibacterial properties and efficiency of open/close crack system of the material are verified. Bacterial colonization and biofilm formation on the developed systems are studied. This thesis consists of four chapters:

1-22

The first chapter is a literature review on the relevant concepts of this work:

biofilm growth and development (Figure 0-2), implant-related infections, preventive applications used nowadays and research strategies. Focus is put on antibacterial polymers and antibacterial surfaces. Nanosilver as a product for biomedical use is also described. Plasma polymerization is introduced, as method for deposition of thin layer.

1-23

Figure 0-2: Biofilm formation process. Illustration from Ploux et al. [START_REF] Ploux | Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties[END_REF].

The second chapter describes the polypropylene mesh used and the different steps for elaborating the antimicrobial multilayer plasma system. It consists of a first plasma polymer layer, which is enriched in silver nanoparticles and covered with a second plasma polymer overlayer (Figure 01). Effect of different crosslink densities in the overlayer on the release properties is studied.

Then, the release of the active agent in a controlled way through the presence of mechanically reversible fragmentations in the top-layer is discussed. For example, the possibility to reversibly switch on/off the release of Ag + ions is shown in Figure 0-3. The third chapter describes the influence of sterilization procedures on plasmadeposited thin films. This topic is very rarely discussed in literature. Due to a growing interest of using maleic anhydride plasma polymer-based coatings for biomedical application, there is a need to investigate the impact of different types of sterilization on the properties of such surfaces. In this chapter, we examine the effects of autoclaving and electron beam sterilization on surface chemistry and the impact on the cracks formation.

1-24

Finally, the fourth chapter focus is put on antibacterial properties of the material.

We discuss the impact of the material properties on planktonic bacteria and show that, taking advantage of open/close cracks systems, it is possible to modulate the colonization of the implant by bacteria (Figure 01234). We discuss the changes in biofilm structure triggered by small doses of silver. At the end, a general conclusion is given with some ideas for future works.

Biofilm

Biofilms are very complex ecosystems consisting of microorganisms attached to the surface and enclosed in polysaccharide matrix, generally defined as "microbial cells immobilized in a matrix of extracellular polymers acting as an independent functioning ecosystem, homeostatically regulated" [START_REF] Percival | Bacterial biofilms: from the Natural environment to infectious diseases[END_REF]. Aside from microbial components and depending on the surface composition and its environment, non-cellular materials like minerals, crystals, corrosion particles, clay particles, blood components may also be found in the polymer matrix. For example, in natural environments, biofilms are very complex due to the high variety of species in an ecosystem [25]. On the other hand, on medical devices, they may be simpler and be composed of a single species, for example coccoid or rod-shape organisms [25].

Biofilms play a role in a variety of industrial and medical environments. They have beneficial contribution in the wastewater treatment plants, denitrification of the drinking water, hazardous xenobiotic detoxification, heavy metal removal from the contaminated waters [START_REF] Schmidt | New concepts of microbial treatment processes for the nitrogen removal in wastewater[END_REF][START_REF] Nicolella | Wastewater treatment with particulate biofilm reactors[END_REF]. They protect the plants against soil borne diseases, improve crop productivity by regulating nutrient and water flow across the roots [START_REF] Alami | Rhizosphere soil aggregation and plant growth promotion of sunflowers by an exopolysaccharide-producing Rhizobium sp. strain isolated from sunflower roots[END_REF] and stimulate plant growth under salt stress [START_REF] Ashraf | Inoculating wheat seedlings with exopolysaccharide-producing bacteria restricts sodium uptake and stimulates plant growth under salt stress[END_REF]. They can improve the physico-chemical characteristics of the soils [START_REF] Alami | Rhizosphere soil aggregation and plant growth promotion of sunflowers by an exopolysaccharide-producing Rhizobium sp. strain isolated from sunflower roots[END_REF]. In aquatic industrial environments, biofilms cause corrosion, reduce heat transfer, liberate noxious gases, and can serve as a reservoir for pathogens [START_REF] Keevil | Biofilms in the aquatic environment[END_REF]. Biofilms also naturally occur in the human body such as dental plaque [START_REF] Marsh | Dental plaque as a biofilm[END_REF] or in intestine [START_REF] Macfarlane | Mucosal biofilm communities in the human intestinal tract[END_REF].

Unbeneficial existence of biofilms include for example periodontal diseases associated with caries and infections of urogenital systems [START_REF] Tenke | Update on biofilm infections in the urinary tract[END_REF].

Because biofilm formation and device-related infections are an issue of growing importance in our daily life, the understanding of biofilm growth mechanisms and factors that influence this process are essential.

How does biofilm grow?

Formation of biofilm and its maturation is a multistage process and is dependent on a number of variables. The environment conditions such as temperature, humidity, pH of medium, atmospheric conditions, nutrition conditions have a strong influence on the biofilm development [START_REF] Goller | Environmental influences on biofilm development[END_REF]. The type of microorganism and the nature of surface on which the attachment starts plays also a crucial role [START_REF] Teughels | Effect of material characteristics and/or surface topography on biofilm development[END_REF].

Classical biofilm formation is described in [START_REF] Ploux | Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties[END_REF]. It starts with surface conditioning by the medium (protein, ions etc.). This process can strongly modify surface properties and therefore influence biofilm formation. Further, the transport of bacteria from the medium to the surface occurs (Figure 1-1, step 1) and is followed by the bacterial adhesion (Figure 1-1, step 2).

Figure 1-1: Biofilm formation process. Illustration from Ploux et al. [START_REF] Ploux | Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties[END_REF].

Factors that influence planktonic process are the gravitational (sedimentation), hydrodynamic (fluid flow, cell mobility), thermodynamic forces (Brownian motion), Van der Waals forces, electrical double layer forces and also steric hindrance, temperature. Brownian motion plays a major role in the diffusive transport closer to the surface of material. When bacterium is close enough to the surface, with a distance depending on the compositions of cell surface, substrate and environmental medium, direct interactions take place between cell surface and the substrate through attractive and repulsive forces (Van der Waals and double layer interactions). When cell attaches to the substrate, the distance is less than 1.5 nm. Then, the strength of the attachment is governed by short range interactions (hydrogen bonding, Van der Waals forces, electrostatic, ionic and dipole interactions [START_REF] Bos | Physico-chemistry of initial microbial adhesive interactions -its mechanisms and methods for study[END_REF]), which provokes the resistance to detachment [START_REF] Melo | Biofilms--science and Technology[END_REF][START_REF] Percival | [END_REF]. Bacterial adhesion is then qualified as irreversible.

Irreversible adhesion can be strengthened when bacteria express adhesion proteins like curli or fimbriae to enhance the possibility of adhesion on the surfaces.

The reversible part of bacterial adhesion can be described as a physicochemical process [START_REF] Rijnaarts | DLVO and steric contributions to bacterial deposition in media of different ionic strengths[END_REF][START_REF] Bayoudh | Assessing bacterial adhesion using DLVO and XDLVO theories and the jet impingement technique[END_REF][START_REF] Boks | Forces involved in bacterial adhesion to hydrophilic and hydrophobic surfaces[END_REF] using the Derjaguin-Landau-Verwey-Overbeek

Bibliographic synthesis (DLVO) theory [START_REF] Hermansson | The DLVO theory in microbial adhesion[END_REF]. In this approach the bacterial cells are considered as colloids and only physicochemical parameters as surface tension, surface charge are considered to explain adhesion. It is now well accepted in the literature that this vision is too simple to completely describe the process and studies have shown the influence of other important parameters such as surface wetness [START_REF] Wang | Sensing wetness: a new role for the bacterial flagellum[END_REF], local pH variation [START_REF] Ponsonnet | Local pH variation as an initial step in bacterial surface-sensing and biofilm formation[END_REF], and osmolarity variation [START_REF] Booth | Mechanosensitive channels in bacteria: signs of closure?[END_REF].

The third part of biofilm development (Figure 1-1, step 3) starts when bacteria develop intercellular connections and polymeric matrix [START_REF] Costerton | Bacterial Biofilms: A Common Cause of Persistent Infections[END_REF][START_REF] Katsikogianni | CONCISE REVIEW OF MECHANISMS OF BACTERIAL ADHESION TO BIOMATERIALS AND OF TECHNIQUES USED IN ESTIMATING BACTERIAMATERIAL INTERACTIONS[END_REF][START_REF] Prigent-Combaret | Developmental pathway for biofilm formation in curliproducing Escherichia coli strains: role of flagella, curli and colanic acid[END_REF]. The growth of biofilm continues and colonies are spreading until biofilm structures become complex and contain several layers of microorganisms [START_REF] Flemming | Relevance of biofilms for the biodeterioration of surfaces of polymeric materials, in Polymer Degradation and Stability[END_REF]. Microorganisms are producing extracellular polymer substances (EPS) [START_REF] Costerton | Bacterial Biofilms: A Common Cause of Persistent Infections[END_REF], which contain nutrients, surfactants, proteins, nucleic acids, lipids and polysaccharides (colonic acid, alginate, dextran, chitosan) [50]. Microbial cells are enclosed in this complex matrix. EPS influence the adhesion of microbial biofilm to the surface and play for bacteria a role as a protective matrix from the environment [START_REF] Fux | Survival strategies of infectious biofilms[END_REF]. At this step of development, coagregation can also occur. This is a process of cell to cell adherence [START_REF] Kolenbrander | Bacterial interactions and successions during plaque development[END_REF] which can be highly specific and governs the development of multi species biofilms in many different environments (e.g. dental plaque [START_REF] Rickard | Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria[END_REF] urogenital systems [START_REF] Drinka | Complications of chronic indwelling urinary catheters[END_REF]).

The last step of biofilm development is the detachment of biofilm parts and the release of free bacteria (Figure 1-1, step 4). This process can be enhanced by survival mechanisms caused for example in response to stresses like a lack of nutrition [START_REF] Kaplan | Enzymatic detachment of Staphylococcus epidermidis biofilms[END_REF]. It promotes the genetic diversity of bacteria in biofilms due to cell communication by gene transfer [START_REF] Molin | Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure[END_REF]. It has also important implications on medical aspects and public health due to increased resistance against antibiotics or antimicrobial agents and the host immune system attacks of biofilm-associated infections [START_REF] Costerton | Bacterial Biofilms: A Common Cause of Persistent Infections[END_REF].

Finally, biofilms can reach a thickness of several tens of micrometers. For example the thickness of the biofilm recovered from indwelling bladders catheters was varied between 3 and 490 µm [START_REF] Percival | Introduction to Biofilms[END_REF]. They have high stability and can have high species diversity. Bacteria in mature biofilm show unique physiological characteristics that are significantly different from planktonic cultured phenotypes [START_REF] Sauer | Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[END_REF]. Microbial gene expression is altered in biofilm. For example Sauer et al. [START_REF] Sauer | Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[END_REF] used gene analysis together with protein identification to observe changes in biofilm physiology of biofilm-grown populations. They observed that the average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins).

Additionally, when planktonic cells were compared with maturation stage of biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level. Brozel et al. [START_REF] Brözel | A method for the study of de novo protein synthesis in pseudomonas aeruginosa after attachment[END_REF] have shown global gene expression changes in attached Pseudomonas aeruginosa cells and found more than 11 proteins whose levels were altered during various stages of attachment. Microbial cells in biofilm are also able to communicate by gene transfer [START_REF] Molin | Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure[END_REF] and by secretion of diffusible signaling molecules through a biochemical system which is known as Quorum Sensing [START_REF] Marsh | Dental plaque: biological significance of a biofilm and community lifestyle[END_REF][START_REF] Miller | Quorum sensing in bacteria[END_REF]. As a result, biofilms offer bacteria an increased resistance against antimicrobial agents (antibiotics, detergents), host immune system attacks and unfavorable environmental conditions.

Methods of characterization of biofilm

As mentioned before, biofilms are complex systems, which are highly hydrated and consist of many microorganisms. A classical method to characterize biofilm is the visualization under electron microscopy. In this case biofilm often needs a dehydratation step with various graded solvents [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF]. This process distorts the real thickness of biofilm and creates artefacts in the structure determination 1-34 [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF]. Another possibility is the use of specific stains. For example, after labeling of matrix [START_REF] Sutherland | Biofilm Exopolysaccharides[END_REF], the nature of polysaccharides in biofilms and their association with the cells can be investigated using high magnification transmission electron microscopy (TEM) [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF]. But here again, it is needed to dry samples which also cause modifications in biofilms characteristics.

The aforementioned disadvantages have been overcome with the development of Confocal Laser Scanning Microscopy (CLSM) and Epifluorescence

Microscopy. After staining procedure, the examination of biofilm can be determined in the native, liquid environments, with the possibility of investigation of both its structure and its dimensions [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF][START_REF] Lawrence | Confocal laser scanning microscopy for analysis of microbial biofilms[END_REF]. The possibility of inserting genes expressing fluorescent proteins, like green fluorescent protein (GFP), in the genome of bacteria have also been developed. In this approach, bacteria produce fluorescent molecules either constantly or initiated by antibiotics for instance [START_REF] Burton | Molecular Gradients of Bioinertness Reveal a Mechanistic Difference between Mammalian Cell Adhesion and Bacterial Biofilm Formation[END_REF][START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context[END_REF][START_REF] Bateman | Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation[END_REF], allowing the observations of living bacteria. This approach allows following the biofilm development together with metabolic state of bacterial cells.

In general, images obtained by CLSM, can be treated and analyzed by a large number of specific or unspecific softwares (CellC [START_REF] Selinummi | Software for quantification of labeled bacteria from digital microscope images by automated image analysis[END_REF], ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] for example). For treatment of three-dimensional biofilm image stacks obtained by CLSM microscopy novel computer programs such as Comstat [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF], Comstat2 [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF][START_REF] Heydorn | Experimental reproducibility in flow-chamber biofilms[END_REF] or bioImage_L [START_REF] Chavez De Paz | Image analysis software based on color segmentation for characterization of viability and physiological activity of biofilms[END_REF] have been specifically developed. Their use allows to describe the structural organization of microbial communities by the quantification of mean biofilm thickness and substratum coverage for example.

Comstat and Comstat2 also provide parameters like roughness and surface to biovolume ratio [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF][START_REF] Heydorn | Experimental reproducibility in flow-chamber biofilms[END_REF][START_REF] Chavez De Paz | Image analysis software based on color segmentation for characterization of viability and physiological activity of biofilms[END_REF].

Aside from the aforementioned methods of biofilm visualization, it is possible to measure the total biomass (i.e. the quantity of microbial cells in the biofilm) accumulation by optical measurements after adequate staining with crystal 1-35 violet. Total amount of proteins or staining intensity of bacteria is assessed by measurements of the absorbance at 490 nm or examination by bright light optical microscopy directly on surfaces [START_REF] Ziebuhr | Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates[END_REF][START_REF] Pratt | Genetic analyses of bacterial biofilm formation[END_REF] or after detaching bacteria from the surface by sonication, vortexing or with a brush or a swab [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF]. In the second case, cells are then plated and counted [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF][START_REF] Katsikogianni | Interactions of bacteria with specific biomaterial surface chemistries under flow conditions[END_REF][START_REF] Olofsson | N-acetyl-L-cysteine affects growth, extracellular polysaccharide production, and bacterial biofilm formation on solid surfaces[END_REF]. However, with this method the modification of the biofilm characteristics are possible. In this approach, it is also possible to weight the detached bacteria and thus determine the biomass with, however, a weak sensitivity of measurements. These techniques are usually used for characterization and quantification of biofilms formed on infected implants [START_REF] Donlan | Biofilms: microbial life on surfaces[END_REF]. The disadvantages of this method are the modification of biofilm characteristics and the absence of information about localization of bacteria on the implant. However, localization of bacteria on the implant surface can be crucial in case of studies on complex surfaces, for example polymeric meshes, due to the existence of preferable areas of bacterial adhesion on implant surface [START_REF] Engelsman | Morphological aspects of surgical meshes as a risk factor for bacterial colonization[END_REF]. Additionally, information about architecture of the biofilms cannot be retrieved using these methods of biofilm investigation.

Biofilms-related infections on biomaterials 1.1.4.1 Introduction to biomaterials

Use of biomaterials dates as far as ancient civilizations: Artificial noses, ears or teeth were found in Egyptian's mummies [START_REF] Thomas | Fossilized Biomaterials Must Be Young[END_REF]. Through the centuries advancement, using artificial materials to replace or restore body functions of degenerated tissues or to assist in healing is progressing. Surgical techniques and sterilization methods now allow the use of biomaterials in many biomedical applications [START_REF] Ramakrishna | Biomedical applications of polymer-composite materials: a review[END_REF]. Biomaterials in form of sutures, bone implant, vascular graft, heart valves, lenses or dental implants are used nowadays [START_REF] Ramakrishna | Biomedical applications of polymer-composite materials: a review[END_REF]. In spite of the prefix bio-which is usually related to life or living objects, the use of prefix bioin biomaterial implies that the material derives from life or is beneficial for life [START_REF] Donaruma | Definitions in biomaterials[END_REF]. The consensus in definition of biomaterial was firstly achieved on Consensus Conference on Definitions in Biomaterials Science, in 1987, by the European Society for Biomaterials [START_REF] Donaruma | Definitions in biomaterials[END_REF]. Biomaterial was then defined as 'a non viable material used in a medical device, intended to interact with biological systems'. However, further debates on this subject took place few years later.

The expression "non viable" was removed from the definition [START_REF] Williams | On the nature of biomaterials[END_REF]. Finally, in 1999, the definition of biomaterial was established as "a material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body" [START_REF] Williams | On the nature of biomaterials[END_REF]. Under further developments of new types of substances and medical technologies, in 2009, Williams [START_REF] Williams | On the nature of biomaterials[END_REF] suggested to redefine the biomaterial definition as "a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure, in human or veterinary medicine".

Biomaterials are chosen based on their properties, to match them with the surrounding tissues. The choice of biomaterial based on its ability to match with the host tissue is essential to produce the desired tissue remodeling.

Considering the structural or mechanical compatibility with tissues, metals are often chosen for hard tissue applications and polymers for soft tissue applications. Commonly used low modulus materials are polymers, mainly polypropylene (PP), polyethylene (PE), polyurethane (PUE), poly(methyl methacrylate) (PMMA), polyethylene(terephthalate) (PET), poly(tetrafluoroethylene) (PTFE), hydrogels and some biodegradable polymers such as polyglycolide acid (PGA), polylactic acid (PLA). Their advantages are the large variety of chemical compositions, properties and forms such as solids, gels, films, fibers and the easiness of polymer fabrication into complex shape and structures (see Figure 12). Disadvantages of polymers as biomaterials are their low resistance against sterilization processes (ethylene oxide, gamma irradiation, electron beam irradiation) and insufficient mechanical properties to use them as orthopedic implants. These disadvantages are overcome by using metallic implants such as stainless steel, titanium or titanium alloys, tantalum, or gold [START_REF] Ramakrishna | Biomedical applications of polymer-composite materials: a review[END_REF][START_REF] Ramsden | The Design and Manufacture of Biomedical Surfaces[END_REF]. These materials have high mechanical strength, resistance and they are very stiff. However, mismatch of stiffness between bone and metallic implant is often causing implant loosening. Other disadvantages of metals as biomaterials are insufficient biocompatibility and tendency to corrode, or provoke allergic tissue reactions due to release of metal ions [START_REF] Ramakrishna | Biomedical applications of polymer-composite materials: a review[END_REF][START_REF] Ramsden | The Design and Manufacture of Biomedical Surfaces[END_REF]. These disadvantages can be overcome by the use of bioceramic materials such as diamond like carbon, alumina, zirconia, hydroxyapatites and bioglasses. These materials are biocompatible and resistant to corrosion. Some properties of bioceramic materials, which are similar to human bones in terms of mineral composition, make this material especially adequate for bone implants. In addition, bioceramics are resistant to various methods of sterilizations. Another type of biomaterials is composite materials. They consist of mixture of above mentioned monolithic materials, for example metal-polymer composites or hydroxyapatite-polymer composite for bone tissue replacement. Controlling the volume fractions and local arrangements of the reinforcement phase, it is possible to design materials with appropriate structures and to tailor the properties to suit the requirements of mechanical properties for the material. It is concluded by Ramakrishna et al. [79] that composite materials offer greater potential of structural biocompatibility than the homogenous monolithic materials.

In case of biomaterials for abdominal wall reconstruction, pelvic floor reconstruction and stress urinary incontinence treatment, the use of polymeric meshes is accepted. Biomaterials in this case should be resistant, have good tissue tolerance, be easily manipulated, flexible, and elastic in more than one

Bibliographic synthesis

1-38 dimension. They should correspond to tissue dynamics as closely as possible and should resist degradation over time [START_REF] Zogbi | The Use of Biomaterials to Treat Abdominal Hernias[END_REF]. However, the resistance to the infection should be improved in case of polymeric meshes biomaterials.

Therefore, composite materials associating polymer and antibacterial agent which would prevent infection on the site, should be a good perspective.

In conclusion, physical, chemical and mechanical properties have all to be considered for determining the most appropriate biomaterial for a specific application. However, additional factors must also be taken into account, e.g. bio-integration, biocompatibility and capacity to resist to biomaterial-related infections. For this reason, to develop a composite material made of polymer which provides desired mechanical characteristics corresponding to surrounded tissue dynamics and an antibacterial agent e.g. silver nanoparticles would be a promising strategy for mesh implants.

Biomaterial-related infections

Infections are the most common and serious cause of postoperative complications in patients surgically-treated with implants. Infections associated with surgical implants are generally difficult to cure and have a larger adverse impact on quality of life [START_REF] Arciola | Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials[END_REF][START_REF] Lorenz | In Vivo Detection of Staphylococcus aureus in Biofilm on Vascular Prostheses Using Non-invasive Biophotonic Imaging[END_REF][START_REF] Rohde | Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections[END_REF]. Common sites of primary infection are the mouth (through bleeding gums), catheter entries (such as the subvenous catheter), and implanted medical devices (such as an artificial hip joint) (Figure 1-3) [START_REF] Donlan | Biofilms and device-associated infections[END_REF]. Infections of urinary and vascular catheters then account for the majority of device-related infections. Once bacteria enter the circulatory system, they can reach all parts of the body and cause secondary infections. Their most common sites include brain, kidneys, intervertebral spaces, and bones around implanted medical devices.

Microorganisms which cause contaminations and infections after surgical procedures can be residents of already existing infections in the patient body (appendicitis, deep organ abscesses). They also often originate from the patient's skin [START_REF] Arciola | Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinical isolates from orthopaedic implant infections[END_REF] or can come from the hospital environment. In these last cases, adhesion of bacteria to biomaterial surfaces is usually initiated at the place where biomaterial touches the skin [START_REF] Vuong | Staphylococcus epidermidis infections[END_REF] or other hospital strains [START_REF] Bagnall | Surgical-site infection[END_REF]. The origin of infection is then related to inadequate cleaning of the patient before the operation, inadequate care of decontamination of operated area or inadequate care of operated tissue after surgery. Common skin pathogens like staphylococci (Staphylococcus epidermidis and Staphylococcus aureus) are frequently detected microorganisms in device-related infections [START_REF] Vuong | Staphylococcus epidermidis infections[END_REF][START_REF] Mccann | Staphylococcus epidermidis devicerelated infections: pathogenesis and clinical management[END_REF]. Other environmental pathogens detected on infected implants are E.coli, Pseudosomonas, Klebsiella, Enterobacter, Bacteroides, Candida albicans [START_REF] Weigelt | Surgical site infections: Causative pathogens and associated outcomes[END_REF].

In this context, biofilms formed in later stages of infection protect bacterial cells against defensive mechanisms of the host organism, hamper phagocytosis, cause disturbances in chemotaxis and reduce antibiotic and antibody penetration. Furthermore, when multi-species biofilm are formed on biomaterials, [START_REF] Costerton | Bacterial Biofilms: A Common Cause of Persistent Infections[END_REF] interspecies transfer of antimicrobial resistance traits, symbiotic interactions and sequential colonization patterns can enhance the virulence of the infection [START_REF] Wargo | Fungal-bacterial interactions: a mixed bag of mingling microbes[END_REF]. Aside from the consequences related to infections, the presence of biofilm can lead to implant biodegradation. This is potentially the case with polymeric biomaterials, which can lose their functionality due to this process [START_REF] Peter Kaali | Prevention of Biofilm Associated Infections and Degradation of Polymeric Materials Used in Biomedical Applications[END_REF]. Factors that influence the rate of degradation are pH, environment, oxygen, salts, redox potential and temperature. The exoenzymes from microorganisms may accelerate material degradation by breaking down complex polymers yielding smaller molecules or short chains e.g., oligomers, dimers and monomers, that are the molecules small enough to pass the semi-permeable outer bacterial membranes and be utilized as a carbon and energy sources [START_REF] Gu | Microbiological deterioration and degradation of synthetic polymeric materials: recent research advances[END_REF].

The cost of treating device-related infections is generally high and together with long term antibiotic treatment, prolonged operation times, size of the operated area and surgical techniques applied, put at risk patient health. For these reasons, there is a growing interest in the field of biomaterial-related research to provide new coatings and/or materials able to prevent bacterial adhesion and biofilm formation [START_REF] De Boer | Risk assessment for surgical-site infections in orthopedic patients. Infection control and hospital epidemiology : the official journal of the Society of Hospital Epidemiologists of America[END_REF][START_REF] Sheng | Comparative impact of hospital-acquired infections on medical costs, length of hospital stay and outcome between community hospitals and medical centres[END_REF]. tomography, magnetic resonance imaging or nuclear scans) can be helpful in diagnosis of inflammatory post-surgical changes [START_REF] Darouiche | Treatment of Infections Associated with Surgical Implants[END_REF]. Hence, the implantrelated infections can be distinguished and confirmed only upon surgical exploration. Because this approach compromises the health of patient, different preventive strategies are used. They are listed below.

Cleaning

To prevent biofilm formation a thorough cleaning is need using water, oxidizers, enzymatic products or detergents, which weakens the physical stability of the biofilm, making it easier to remove by brushing, flashing or other physical treatment [START_REF] Simões | A review of current and emergent biofilm control strategies[END_REF][100][101]. the surface, but it is not efficient enough to kill them [START_REF] Simões | A review of current and emergent biofilm control strategies[END_REF]. Additionally, it may not be appropriate for medical devices e.g. porous materials since disinfectant can be absorbed and the residuals can irritate the tissues after implantation [101].

Therefore, other methods are necessary (e.g., sterilization) to inhibit bacterial development on biomaterials.

Sterilization

Sterilization is a process that destroys or eliminates all forms of microbial life

1-44

The use of ethylene oxide gas as a sterilization method is widely applied in the medical device industry and in hospitals especially for polymer materials.

Ethylene oxide gas is used as a sterilizing agent due to its capability of efficiently killing all the species and forms of micro-organisms in a short time.

Ethylene Oxide is bactericidal, virucidal, and sporicidal [108]. The advantage of this method of sterilization is its compatibility with majority of plastic materials. [111,113,114]. Other modifications can take place such as crosslinking [115], branching and degradation of additives often used in medical polymers [116]. These processes may cause decrease in stability, promote the release of volatile fractions and cause toxicity [116,117].

As previously mentioned, electron beam irradiation induces crosslinking in polymers. This can be used to improve the properties of a final product by improving tensile and impact strength and chemical resistance through irradiation. In case of plasma polymers, by choosing the level of e-beam exposure, irradiation can advantageously influence the mechanical properties of plasma polymers.

These approaches which are physical and chemical treatments applied directly on material surfaces decrease the possibility of contamination. Other ways to reduce the possibility of biofilm formation and avoid the degradation of material are antibiotic treatments applied to patient.

Antimicrobial therapies

Antimicrobial approaches are used to prevent implant-associated infections by decreasing bacterial adherence to the implant surface and/or reducing the concentration of bacteria in the immediate vicinity of the implant. These approaches are applied after placement of surgical implant [118]. Systemic antibiotic prophylaxis provides protection from organisms which disseminate through bloodstream from a different site that the site of surgery [119].

However, biofilms are often resistant to antibiotic treatment due to low antibiotic penetration into biofilm matrix because of sorption to or reaction with the components of biofilm matrix [START_REF] Fux | Survival strategies of infectious biofilms[END_REF]120]. Additional protection from implant associated infections is local application of antimicrobial agents (antibiotics or antiseptics). It provides higher drug concentration at the crucial implantation site Bibliographic synthesis 1-46 [118]. Antimicrobial agents on the skin (skin antisepsis), irrigation of the surgical wound [121] or dipping of surgical implant before implantation in antimicrobial solution are strategies applied for that purpose [118,122]. These strategies have been shown to be efficient in practical cases. For example, dipping the implant in the antimicrobial solution reduces bacterial adherence to the implant surface and/or minimizes the concentration of bacteria in the immediate vicinity of the implant [123]. However, analysis of the existing clinical practices indicates that, although some antimicrobial strategies constitute the standard of care for preventing infections associated with surgical implants, such strategies are applied based on clinical experience, often in "nonstandardized fashion and without clear evidence of clinical efficacy" [124]. Furthermore, the consequence of antibiotic and other antimicrobial therapies, systematically and in high doses, is the reason of dramatic increase of bacterial resistance [125]. Therefore, these treatments should be limited.

In conclusion there is a need of a new approach based on local delivery (at the implant location) of antibacterial agent. For example, antibacterial agent delivered in a control way from the implant surface to the environment is a promising approach. Control release would also allow to prolong the lifetime of an implant with antibacterial properties.

Research

Influence of the surface properties on bacterial adhesion

The material surface properties are crucial factors that influence the process of biofilm formation. They are therefore relevant candidates for controlling the bacterial adhesion preventive properties of a material. Surface topography and surface chemistry are considered the parameters the best known for their action on bacterial adhesion. Other rarely mentioned parameters are mechanical properties, which can as well have a strong impact on bacterial adhesion.

Surface topography

In spite of numerous studies in the literature, the exact mechanisms of the impact of topographical features on bacterial adhesion and biofilm formation are still poorly understood. Reviews on this subject have been published [START_REF] Ploux | Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties[END_REF]134].

Usually it is considered that the size and orientation of topographical features, can influence the arrangement and the resulting behavior of cells on the surfaces and may affect biofilm development [135]. However, several studies suggested that the influence of surface roughness is much less important than chemistry of the surface [136][137][138]. Additionally, it should be noted that the importance of the surface topography in bacterial behavior on the surface, especially bacterial localization according to topographical features, is often overvalued, due to observations realized in non-aqueous condition of culture [134,135].

Bacterial responses to topographical features of surface are species and strain depended [134]. However, some general trends have been highlighted.

Bacterial responses to surface topography with surface features higher that bacterial cell size are supposed to be similar to smooth surfaces, except that transport of bacteria to the surface can be modified due to the topographical features. Furthermore, preferential bacterial adhesion at the bottom of surface Bibliographic synthesis 1-49 crevices has been demonstrated for concave features with sizes similar or higher than bacterial size [139]. However, considering feature size, shape, distance between features has not been taken under consideration. Several works have also demonstrated that greatest bacterial cell retention occurred on material roughness R a value close to bacterial size (e.g., 0.9 µm of Ra for 1 µm diameter) [140][141][142], suggesting that bacteria preferentially adhere on or are retained in features with similar sizes. This result is illustrated in Figure 123456.

When the characteristic dimensions of confined spaces approach those of bacterial cells, their interactions with surface change. This would be due to the contact area between cell and substratum, which is greater, enhancing therefore binding energy and the propensity for the microorganisms to be retained on the surface [140]. Then, tuning the periodicity of structures within relevant cellular scale, lead to distinctive differences in bacterial assembly [135],

but roughness and surface topographical features do not always result in bacterial localization or alignment on the surface, especially when topographical features are at the nanometric scale [134,143]. Nevertheless, it has been proposed, and recently showed, that macromolecular structures of the bacterial cell wall, such as flagella or pili, may allow bacteria to sense and/or attach to topographical features of nanometric size [144].

Bibliographic synthesis Experimentally, influence of nanotopography has been demonstrated. For example the statistical difference in initial deposition rate of bacteria was observed on surfaces modified at a nanoscale level (unworn and overworn contact lenses), where the surface roughness R a was equal to 4 nm and 10 nm respectively [145]. Correlation between surface nanoroughness and bacterial adhesion was shown by Bakker et al. [146] using linear regression analysis.

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Their results showed positive relation between mean surface nanoroughness (which ranges from 2.3 nm up to 12.9 nm) and the deposition of micron-sized bacteria. Results of have shown an influence of small topographical changes of etched glass surfaces on bacterial adhesion.

Contrarily to so far mentioned studies, decrease of surface roughness R a of 1 nm after etching, resulted in increase in the number of attached bacteria.

Surface nanotopographical features influence on bacterial adhesion and cell orientation was also studied by Ploux et al. [148]. In this study, surface consisting of 160 nm period modulation and 50 nm deep grooves were Bibliographic synthesis 1-51 prepared by plasma polymerization and UV-Irradiation. Authors observed statistically significant higher number of adherent bacteria on patterned surface.

However in case of bacterial cell orientation, these surface grooves had no influence. Authors suggested that this can be explained by small depth and the sharpness of the grooves, which could prevented energetically favorable cellsurface binding [148].

Surface chemistry

Chemistry of the material surface have been already shown to have influence on the bacterial adhesion. In particular, parameters such as surface hydrophobicity and charge are influencing bacterial adhesion.

In the study of Tegoulia et al. [149], bacterial adhesion of S. aureus was studied on self-assembled monolayers (SAMs). These surfaces had methyl, hydroxyl carboxylic acid and tri(ethylene oxide) terminated groups. Authors observed that bacteria adhered the most to carboxylic acid and methyl terminated surfaces.

The lowest adhesion of bacteria was observed in case of tri(ethylene oxide)

surfaces. Authors suggested that stable interfacial water layer on the material surface, may play a role in this case, preventing from direct contact between bacteria and surface. Park et al. [150] surface. Their results showed that, all bacteria adhered most rapidly to the positively charged surface. However, there was no surface growth of the Gramnegative strains on positively charged surface. On the negatively charged surfaces, bacteria were adhering in a slower manner, but surface growth of the adhering bacteria was exponential for both Gram-positive and Gram-negative strains. These results demonstrate that the influence of surface charge is complex.

Surface chemistry has also been shown to influence biofilm formation. Ploux et al. [159] focused on differences in biofilm morphology and long-term variations in growth dynamics of biofilm. By using NH 2 and CH 3 terminated self assembled monolayers (SAM) on silicon wafers, the authors have shown low impact of surface chemistry on quantity of adhered bacteria but a strong impact on biofilm morphology, i.e., spatial distribution of bacteria and kinetics of biofilm formation.

Bacteria on CH 3 -terminated surfaces were organized in clusters while bacteria on NH 2 terminated surfaces formed tree-like biofilm morphology. Furthermore, the biofilm detachment was also dependent on the substrate, suggesting that the surface wettability influences the signal controlling the detachment of biofilm parts.

Surface mechanical properties

As previously mentioned the third crucial parameter that influences bacterial Bibliographic synthesis 1-57 flexible to be able to penetrate the bacterial cell walls, their antibacterial properties are preserved and expressed even in a dry state. This gives the possibility to use such molecules to coat the surfaces, rendering the surface antibacterial.

Another example of "nonrelease" approach to making materials bactericidal by covalently attaching certain hydrophobic polycations to their surfaces was shown in the work of Lin et al. [176]. Alkylated polyethylenimine (PEIs) chains attached to flat surfaces, by rupture of bacterial cell membranes, made these surfaces bactericidal toward Gram-positive and Gram-negative pathogenic bacteria.

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Antimicrobial polymer (tertiary amine 2-(dimethylamino)ethyl methacrylate)

which was grown directly on the surfaces of glass and paper using atom transfer radical polymerization (ATRP) was synthesized by Lee et al. [177].

Following the polymerization, the tertiary amino groups were quaternized using an alkyl halide to produce a large concentration of quaternary ammonium groups on the polymer-modified surfaces. Incubation of Escherichia coli or Bacillus subtilis on these surfaces have reveled the antimicrobial properties of the surfaces. Using the same surface repeatedly did not show decreased antibacterial activity of modified glass surfaces. In this case, quaternary amines are the biocides which are believed to cause cell death by disrupting cell membranes and allowing release of the intracellular content [177].

In study of Jampala et al. [178] et al. [181] showed biocidal activity against Gram+ and Gram-bacteria. From 3 to 6 log 10 reductions of both Gram+ and Gram-bacterial strains adhesion were obtained to the coated surfaces compared to uncoated stainless steel substrates and depending on the particular antibacterial peptide immobilized.

Additionally, the antibacterial surfaces were resistant to several cleaning conditions. This is an important concern due to leaching, de-lamination, rearrangement and ageing of the coating, which can lead to insufficient long term biofilm resistance of the surface [181].

Using plasma polymer pre-treatment, polyethylene surface was modified by Conte et al. [182]. Further, lysozyme in its active form was immobilized on the surface of polymer. Lysozyme in its active form damages bacterial cell wall.

Antimicrobial activity of modified polyethylene surface was verified by observations of death rate of Micrococcus lysodeikticus cells which were in contact with the substrate. Results demonstrated the antibacterial activity of this new system, which was coming from the activity of the immobilized enzyme.

Antimicrobial activity was related to the concentration of immobilized lysozyme. [194]. This gives the opportunity to functionalize and tailor the surfaces of implants by applying various polymers onto it and achieving antibacterial properties. For example, local delivery of antibiotics has been applied for a long time in materials like cements, for orthopedic and orthodontic implants [195]. Upon hardening, in situ release of antibiotics has been shown to prevent adhesion and growth of significant numbers of bacteria.

Plasma-based approaches include also materials with grafted antibiotics. Aumsuwan et al. [196,197] used a microwave plasma of maleic anhydride to graft commonly used antibiotics such as penicillin and ampicillin. It was firstly achieved by a hydrolysis process, which formed carboxylic acid groups on the surface followed by the immobilization of the antibiotic molecule through a polyethylene glycol spacer. The length of the spacer was not long enough to penetrate the entire bacterial cell. However it was providing mobility to antibiotic molecules, leading to inhibition of bacterial wall synthesis. Moreover, a release of antibiotic molecules from these surfaces was shown. 10% in case of ampicillin and 32% in case of penicillin have been released after 24 h of immersion in Phosphate buffered saline (PBS) solution. The loss of ampicillin and penicillin from the surface did not alter the effectiveness of the surfaces against gram-positive and gram-negative bacteria. Nablo et. al. [171,198,199] have synthesized antibacterial coatings based on release of nitric oxide (NO). It was shown that NO destroy colonies of plated bacteria [200]. Mechanism this of antibacterial action is coming from oxidative and nitrosative stress generated by reactive intermediates of NO (e.g., peroxynitrite and dinitrogen trioxide) that may result in damage to DNA, proteins, and/or cells [201]. Coating showed excellent material stability and antibacterial efficiency. However, the use of NO in the implant coating is questionable. It has been found that NO is an important mediator in numerous mammalian biological processes, including neural transmission, septic shock, and apoptosis [202,203]. This reveals high risk of cytotoxicity of this product as a potential antibacterial agent released from implant surface [204]. Numerous examples of coatings with silver (nanoparticles or silver salts) are found in literature [173,194,205,206]. Antibacterial action of these coatings is often based on releasing properties of silver ions. These examples will be discussed in further sections.

The choice of antibacterial agent 1.2.2.3.1 Antibiotics and antiseptics

Antibiotics have been used against bacterial infection since their discovery by Fleming in 1928 [207]. The treatments using antibiotics are also used against medical device related infections [START_REF] Darouiche | Treatment of Infections Associated with Surgical Implants[END_REF]208]. To accomplish a sustained level of antibiotic at the site of infection and to overcome the protection that biofilms provide for bacteria, controlled local release has been proposed [195,209].

Strategy based on delivery of gentamycin from biodegradable surface coatings 1-63 made of poly D,L-Lactide (PDLLA) [210] or mixtures of antibiotics like rifampicin and fusidic acid in PLLA matrix have been used [211]. The effectiveness of this approach was shown in a rabbit tibia infection model, resulting in effective killing of Staphylococcus aureus infection. In another study, approach using chlorohexadine diacetate release from coating made of Polyvinylopyrrolidone (PVP) was shown to be efficient against infection only upon fast release [212]. Darouiche et al. [213] have used coating with minocycline and rifampin to prevent colonization of a grit-blasted titanium implant. In vivo results showed that these devices significantly protected medical device from colonization and infection of Staphylococcus aureus. Another approach using coatings containing combinations of antibiotics and antiseptics like minocycline and rifampin or chlorohexidin and silver-sulfadiazine have been applied to the internal and external surface of catheters [214,215]. Results showed reduction of catheter colonization and catheter related blood-stream infections.

Disadvantage of using antibiotics or antiseptics loaded medical devices is that release is finite. For long term release, the concentration of the antimicrobial agent will eventually drop, below minimal inhibitory concentration [216]. This is general characteristics of coatings where there is certain reservoir of an antibacterial agent. It is disadvantageous when release cannot be fully controlled. However once the release is controlled and antibacterial agent released in suitable doses, the lifetime of this kind of coatings can be prolonged together with antibacterial properties. However, the major disadvantage of using antibiotics as a infection preventive strategy is that more and more multi-drug resistant bacterial strains are developing [217,218].

Antibacterial polymers

Antibacterial macromolecular systems are known since more than three The advantages of using antimicrobial polymers e.g. for coatings include reduction of residual toxicity comparing to low molecular weight antimicrobial agents, increase of their selectivity, and prolongation of the lifetime of the antimicrobial coating. Antimicrobial polymers can render the surface of biomaterial resistant to microbial colonization, and can provide better protection for materials exposed to the atmosphere after sterilization procedure and before implantation. The polymers which action is based on contact active principle could be advantageous in case of permanent implants, providing them protection from infection. However, disadvantages of antimicrobial polymers often include expensive process of synthesis, decomposition or partial solubility in some cases, loss of activity upon use or toxicity to mammalian cells.

Degradation due to elevated temperatures could also be a limiting factor to processing of these materials into desired forms. For these reasons, better control of the infection at the implantation site would be achieved with a polymeric coating embedding an antibacterial agent active against a broad spectrum of bacterial species e.g. silver nanoparticles.

Silver as a product for antibacterial purposes

Since ancient times, silver is known to be a good antimicrobial, antifungal and antiviral agent. In recent years, the use of silver as a biocide in solution, suspension, and especially in nanoparticles form has increased. Silver has been widely used in many bactericidal applications as well as in medical field to

Mechanism of silver ion antibacterial action

The silver in the form of Ag + have been shown to be effective against a large number of bacteria and fungi, which absorb and concentrate silver ions from dilute solutions (1ppm). Using solution with a concentration of Ag + of 60 ppm should be sufficient to control the majority of bacterial and fungal pathogens

Toxicity of silver ions for human cells

The use of silver in composite materials, polymers, metal coatings and scaffolds affects bacteria and disturbs their biological functions but the question arises about the toxicity of silver ions for the human cells. Due to the similarities in the structural elements of human and bacterial cells, important is to know the way of interference of silver ions and human tissue. A disruption of cell and tissue functions coming from silver ions were reported by Schrand et al. [240] including the induction of ROS, degradation of mitochondrial membrane integrity, disruption of actin cytoskeleton and reduction of proliferation after stimulation with nerve growth factor when exposing neuroblastoma cells to silver nanoparticles. In case of high release rate of silver, the cytotoxic effect appears. It was reported that in case of the use of biomedical products for wound treatment (silver wound dressing), some patients showed hepatotoxicity and argyria-like symptoms, and the silver levels in plasma and urine were elevated as well as liver enzymes [241]. Hardes et al. [242] used silver-coated orthopedic implants in humans, which proved their effectiveness on reducing infections. Results showed no side-effects of this medical device towards human. The silver-levels in the blood did not exceed 56.4 parts per billion (ppb)

and were considered as non-toxic. Additionally authors excluded significant changes in liver and kidney functions. Materials with well-controlled release of

Bibliographic synthesis

1-68 silver ions seem therefore to be reasonable as a next step toward infection preventive medical devices.

Role of silver nanoparticles in the antibacterial properties

Usually, it is assumed that the silver nanoparticles (AgNPs) antibacterial action is mainly based on the ability of silver ion to release from NPs. However, it was also shown that AgNPs can have their own antimicrobial effect without the release of ion forms [228,243] by penetration of the cell membrane [234].

Nanoparticles may attach to the cell membrane that contains electron donor groups like sulfur, oxygen, and nitrogen. Electrostatic interaction may take place between negatively charged electron donor groups and positively charged AgNPs, which brings structural and functional changes in the microbial cell membrane, disturbing the permeability by modifying the cell potential and inhibiting cell respiration [244]. In addition, AgNPs may bind to thiol groups of bacteria proteins, disturbing the activity of cell and causing death [234,245].

Some studies also show that silver nanoparticles express antiviral action as well [246,247]. It has been shown that silver nanoparticles exert an action against

HIV-1 at concentrations nontoxic for human cells. The action involves virion binding, inhibition of replication and inactivation [247].

Silver nanoparticles incorporated in materials as a research strategies for antibacterial materials

With rapid development of nanotechnology, applications where silver is used as an engineered nanomaterial are increasing. Different forms of silver in different kind of materials are incorporated to exploit the antimicrobial properties of silver.

Nanosilver was incorporated in various materials such as engineering polymers like nylon 6,6 [248], polyurethanes [249][250][251], poly(methyl methacrylate [252,253] and as well in carbohydrates and biopolymers [254][255][256][257][258]. Silver Factors, which influence the efficiency of silver incorporated materials, are:

 Rate of release which is usually fast. This may limit the time of protection,

 Silver needs to be oxidized in order to exhibit high efficiency against bacteria in case of silver nanoparticles-loaded coatings.

Recent literature reports have explored antibacterial properties of silvercontaining materials. Examples based on relevancy for the use in biomedical purposes are presented below [172,243,[264][265][266][267][268][269][270][271][272]. .2.2.3.3.4.1 Silver in inorganic systems (bioceramics) Silver nanoparticles antimicrobial properties in bulk materials have been explored in the field of bone tissue regeneration, mainly with bioceramics. By using silver nanoparticles with the size in range 1-2 nm, the authors have [268] have developed a series of triglyceride oil based polymer-silver nanocomposites using electron transfer reaction and free radical polymerization processes. These nanocomposite materials exhibited an antibacterial effect against gram-positive, gram-negative, and spore forming bacteria. Furno et al.

1
[274] have used supercritical carbon dioxide to impregnate silicone with nanoparticles of metallic silver. It was found that efficiency of those systems on bacteria killing depended on the silver ion release rates from the coating.

Silver in organic polymer systems

Polymeric hydrogels have been used for the incorporation of antibacterial agents, including silver, with good results [270,271,275,276]. The advantage of these systems is their physicochemical properties and good mechanical properties, also suitable for coatings. These systems have ability to absorb water due to hydrophilic functional groups attached to polymer backbone.

Moreover, hydrogels are resistant to dissolution due to cross-links between chains. Hydrogels can serve as matrix, which allows the diffusion of some Bibliographic synthesis 1-71

molecules. An example is shown by Vachon et al. [270] who proposed a novel dressing material based on a sulfonated triblock polymer. This polymer possesses an ion-exchange capability that allows binding and controlled release of a variety of therapeutic agents. The polymer was coated onto a polyester fabric and then modified by ion exchange to prepare sodium, silver, or doxycycline salts. Results showed that polymer hydrogel which had an ionexchange capability allowed to control the release of silver and other therapeutic agents. Jones et al. [172] have determined the antimicrobial activity of a silver-containing Hydrofiber ® dressing (AQUACEL ® Ag) on aerobic and anaerobic microorganisms, using the zone-of-inhibition method. The results showed that the material makes silver available at the dressing-agar interface at a concentration that is effective against a broad range of aerobic, anaerobic, and antibiotic-resistant microorganisms. Varaprasad et al. [271] have also synthesized hydrogel matrices by polymerizing acrylamide in the presence of poly(vinyl sulfonic acid sodium salt) and a trifunctional crosslinker (2,4,6-triallyloxy 1,3,5-triazine, TA) using redox initiating system (ammonium persulphate/ Tetramethylethylenediamine (TMEDA)). Silver nanoparticles were generated throughout the hydrogel networks using in situ method by incorporating the silver ions and doing a subsequent reduction with sodium borohydride. Results showed an interesting arrangement of silver nanoparticles in the hydrogel matrices such as a shining sun shape (ball) (∼ 5 nm) with apparent smaller grown nanoparticles (∼ 1 nm). The developed hydrogels showed also antibacterial properties, which was evaluated by the zone-ofinhibition method. In another work, hydrogel networks with cross-linked polymers like poly(acryl amide), carboxymethyl cellulose, starch and gum acacia were used as matrix. Silver nanoparticles were loaded in situ using silver nitrate solution followed by reduction with borohydride [277].

Another antibacterial Ag/polymer nanocomposite was developed by Zaporojtchenko et al. [272]. Uniform dispersion of nanoparticles in polymer matrix was obtained using co-sputtering of metal and poly(tetrafluoroethylene)

approach. The bactericidal activity due to silver release from the surface was tested against S. aureus and S. epidermidis bacteria. The efficiency of the coatings against different bacteria species was demonstrated at extremely small metal consumption: Ag: ~0.1 g m -2 . Composite films containing silver nanoparticles and organic molecules were also synthesized by researchers films. These films as well have shown antibacterial activity related to the release of silver ions from the film. Silver-loaded hollow microcapsules made of hydrogen bonded poly(acrylic acid)/polyacrylamide multilayer thin films using LbL method was developed by Lee et al. [281]. By the number of loading cycles researchers were able to control the size and amount of loaded silver in the films. The concept of developing drug delivery system composed of liposome-AgNO 3 aggregatesembedded in polyelectrolyte multilayer films using LbL method was proposed by Malcher et al. [283]. These films were build using poly(L-lysine)/hyaluronic acid polyelectrolytes. Coating showed strong antibacterial properties revealed under thermal stimuli, however spontaneous release of silver ions and/or erosion of AgNO 3 -liposome aggregates have led to loss of the functionality of the coating.

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Polyelectrolyte multilayers films prepared by LbL technique have emerged as a simple, multistep approach to functionalize surfaces for biomedical applications however some disadvantage can be named such as need of several step in the material development; not sufficient stability of the films caused by processes as interdiffusion, echange of polyelectrolytes in the LbL assemblies, diffusion of external molecules within the films [285]; use of chemical solvents (tetrahydrofuran, toluene [283]) which potentially leave the residues on the medical device and can be toxic to human cells. Some of these disadvantages can be overcome by the use of another approach to develop Ag/polymer coatings such as plasma polymerization [194,[286][287][288].

Plasma polymer acts as a matrix for the out-diffusion of the silver ions. As we have seen before, nanoparticles and ions have been incorporated in release matrices, by many ways, but the major advantage of plasma polymers for this purpose is the good adhesion to the substrate and the possibility to deposit polymer coating with very low thickness. Mechanical properties of substrate material are not significantly affected in this case. Additionally, plasma polymer can be used to control the diffusion rate via plasma polymer overlayer. The part describing plasma polymer incorporated with silver nanoparticles will be detailed in section 2.2.

Plasma Polymerization 1.3.1 What is plasma?

"There are more things between anode and cathode that dreamt of in our philosophy".

H. Raether (1909Raether ( -1986) ) 1-74

Plasma is considered as fourth state of matter and it is the most abundant state in the universe. Plasma is present in nature in the sun, stars, in the tails of comets and as flashes of lightning. With the application of sufficient heat, solid materials transform into liquid state and, then, with further application of heat it transfer into gas. Once the electrons receive sufficient energy to separate from the atoms or molecules of gas and become electrically conductive, the gas undergoes a phase transition to a partially or completely ionized gas (Figure

1-8).
Plasmas are composed of positively and negatively charged ions, electrons, radicals, neutral atoms and also atoms and molecules in excited state, which exhibit collective behavior. Plasmas can be divided into two main categories:

hot plasma (near equilibrium plasma) and cold plasma (non-equilibrium plasma). Hot plasmas are in thermodynamical equilibrium and are strongly ionized with high density of energy. The temperatures of ions and electrons are equal. Cold plasmas are in thermodynamical disequilibrium, with low level of ionization (magnitude of 10 -4 ), with a density of energy lower than hot plasmas.

Cold plasmas are characterized by three temperatures (T celectron temperature, T i -ionic temperature and T n -neutral temperature).

1-75

In this work, we have used low temperature plasma (cold plasma) to treat the substrate materials at low temperatures, which limits the problem related to degradation of material.

Low temperature plasmas

Plasma can be obtained using different forms of energy (thermal, electric current, electromagnetic radiations, light from laser). Properties of plasma such as electron density or temperature can change depending on the type of energy supplied and the amount of energy transferred to the plasma. Low temperature plasmas are characterized by non-equilibrium between electron temperature and gas temperature. Hence, the main parameters, which define the characteristics of plasmas are its temperatures, types and densities of radicals In an alternating current (ac) glow discharge, the mechanism depends on the frequency of excitation. At low frequencies, the system can be looked upon a direct current (DC) glow discharge with alternating polarity. By increasing the frequency of the applied voltage, positive ions become immobile, because they can no longer follow the periodic changes in the field polarity, and only respond to time-averaged fields. At frequencies above 500 kHz, the half cycle is so short that all electrons and ions stay within the interelectrode volume. This reduces the loss of charged particles from the system. Regeneration of electrons and ions occurs within the plasma body, through collisions of electrons with gas molecules. In radiofrequency plasma (13.56 MHz) A variety of precursors can be used for the formation of plasma polymer thin film deposits. This method of polymerization presents the advantage of demanding lower quantities of precursors and limitating the number of side reactions that can happen in other media (solvent termination in liquid medium for instance). The plasma technology is used to deposit films on various substrates (polymers, metals and ceramics) of various geometries without degrading it. The resulting good adhesion between the plasma film and the underneath material allows the use of the plasma technology in a broad spectrum of applications, from protecting layer to adhesion primers. The main drawback comes from the cost of used gases during the process and the fact that formation mechanisms are not to date perfectly understood.

The formation mechanisms

In a given plasma, the electrons distribution of energy is often described by the Druyvesteyn approximation (Figure 1 After activation of the precursor by the plasma phase, the growth of polymer film can happen both in the plasma phase and onto the substrate surface. The rate of polymerization in the plasma phase and onto the substrate surface is highly dependent on the process conditions. For example, plasma polymerization at atmospheric pressure tends to favorite the growth in the plasma phase due to higher collision rate between species.

Growth kinetics

At low pressure the precursor activation leads to the creation of a single or bi free radical(s) in the structure of the precursors by mechanisms such as dehydrogenation, ring opening or bond dissociation and these radicals randomly react with the surrounding species. This polymerization mechanism is illustrated by the 3 step models proposed by H. Yasuda [294,295] 1-80 The influence of the discharge's power has been widely studied [304,305].

The power used has a high influence on the kinetics of forming reactive species in the plasma, especially on the density of electrons, the radiation and the ionic bombardment. The plasma power influences both the growth rate of the polymer film and its chemical structure [306].

To control the power of the plasma's discharge, the control of electric The curve can be decomposed into 3 areas. The first (on the left) corresponds to a growth regime where the energy is insufficient. In this area, the precursor dissociation is a process limited by the quantity of matter brought in the plasma reactor. When increasing the W/FM parameter, the kinetics of film formation increases linearly until reaching a plateau. The last area (on the right) corresponds to a growth regime where the energy is in excess, the growth of the film is limited by the high dissociation occurring and the ablation process. The stable area, is a region where, speed of deposit is maximized, due to optimized conditions of monomer flow and power of discharge.

Additionally to the study of the film growth, several studies related the influence of the W/FM parameter on the structure of plasma film polymer.

These studied were done at low pressure with different molecules such as carboxylates [308,309] natrium based [310], fluoro [311] or siloxanes [312].

In the first regime, the deficiency in energy allows to keep intact the 1-84 reactivity of the monomers. In certain cases, the formation of oligomer structure close to conventional polymer has been observed [313]. In the last regime, the high dissociation of the monomer leads to an increase of the functionality and the obtention of a highly cross-linked polymers. To prevent this high dissociation, the use of plasma in pulsed mode allows the preservation of monomer functionality [294,314].

c. The pulsed mode

This mode is principally described by its Duty Cycle (DC) time, as shown on Equation 1, Equation 1t on -time where precursor's monomer is excited by the plasma phase t off -time where precursor's monomer is not excited by the plasma phase

The use of low DC times allows to diminish the effective power (P eq ) of the plasma to limit the precursor's dissociation. During the t on period, the precursor's monomer is excited by the plasma phase. The radicals formed in the precursor's monomer will then recombine during the t off period. Several studies showed that the use of pulsed mode allows a smoother polymerization where the retention rate of chemical functions is higher [315,316].

The actual power received by the substrate during the pulse duration (P eq ) is a result of DC and the input power value (P peak ) The general characteristics of the plasma reactor, such as volume, shape, oscillation frequency of the electric field, temperature, influence also the film growth [328]. The position of the substrate in regards to the gas injection allows the recuperation of condensed species in the plasma phase or in "post-discharge" [329]. In the latter case, the substrate is placed in the gas stream to collect ionized species of the plasma without modifying the substrate surface. Form in which precursor is introduced to the reactor, also influence the plasma polymer film. For example, the use of precursors in the state of vapor or aerosols allows obtaining dense films. Borra [330] and Badyal [331] groups developed ultrasonic injections systems of precursors to insert the precursor in nano-drops. Their results showed that the resulting fragmentation of the precursor was then lower. Pressure is also playing an important role in the process of plasma polymer film growth. In this case, according to Debye's equation, the rise of the pressure in the reactor increases the density of plasma's particles, decreasing the average free paths of the electrons, leading ultimately in the rise of collisions. Thereby, kinetic energy of plasma species is also decreasing, which does not favor generation of inelastic collisions, which are responsible for generating new plasma species. Above mentioned parameters have influence on physicochemical properties and architecture of plasma polymer films.

Conclusions

In this frame, the goal of the present work is to develop an antibacterial coating, especially design for soft biomaterial such as polypropylene mesh, which will exhibit antibacterial properties, allowing the control over bioactive agent releasing properties. The final material should exhibit anti-biofilm formation properties to prevent the common problems of mesh-related infections after surgical operations.

The following chapter will be focused on describing the elaboration of mechanically-sensitive materials made of plasma polymer multilayers and bioactive agents. These systems are developed on modified polypropylenemade mesh substrate using solvent-free steps based on plasma polymerization.

Tailored release of an active agent is achieved by mechanical stimulation of the designed material. Silver, stored as AgNPs in the plasma polymer multilayer system and released in the material surroundings in its bioactive ionic form process, is described. One of the main features of this system is that, taking advantage of different mechanical properties between the different layers, a system of cracks in the plasma polymer overlayer allows control of the diffusion of silver ions. The antibacterial properties are evaluated and described in the following part. The stability of the properties of the film under the stress of elongation is also shown.

The emphasis of this research is the use of control release of silver ions from mechanoresponsive plasma polymeric coating deposited on the biomaterial as a tool for non-antibiotic and anti-biofilm strategies for implant related infections.

CHAPTER II

Introduction

Use of a mesh-biomaterial in abdominal wall reconstruction, pelvic floor reconstruction and stress urinary incontinence is a common procedure to decrease morbidity, decrease the rate of recurrences and improve the outcome of patient [START_REF] Zogbi | The Use of Biomaterials to Treat Abdominal Hernias[END_REF][332][333][334][335].

Introduction of a polypropylene (PP) mesh during surgical procedure to increase the strength of abdominal wall was an important breakthrough in 1962. PP is non-toxic, cannot be absorbed, and can be cut and modeled without deforming.

It displays good mechanical characteristics such as durability and flexibility

[336] and can be sterilized. Positive effects of the use of such mesh has stimulated the research for the optimal mesh. Meshes are available in a variety of geometrical designs, pore size and morphology of monofilament fiber [START_REF] Zogbi | The Use of Biomaterials to Treat Abdominal Hernias[END_REF]336,337]. Surgical meshes available on the market can be made of absorbable synthetic, non-absorbable synthetic or organic material. Absorbable meshes contain glycolic acid (GA) and poly(lactic acid) (PLA) in various ratios. In the case of non-absorbable meshes, the most common is expanded polytetrafluoroethylene (ePTFE), PTFE, PP. Organic meshes have human or porcine origin. Meshes are available as monofilament or multifilament yarns and a knitted or woven structure. Ideal mesh has following characteristics: it should be flexible; it should have elasticity in more than one dimension, allowing it to stretch in more than one direction and then return to its original shape. In this way, mesh should match the tissue dynamics as closely as possible [START_REF] Zogbi | The Use of Biomaterials to Treat Abdominal Hernias[END_REF]. In regard to these requirements, existing polymeric mesh materials fulfill the above mentioned criteria. However the infection preventive feature of a mesh is not achieved yet.

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Meshes show different characteristics concerning tissue ingrowths, rate of recurrence and rate of infection according to their morphological, chemical, and physical-chemical properties [332,337].

Therefore, there is a need of development of a mesh which specifically prevents the infection on the implantation site.

In this regard, surface modification of existing PP mesh material can be promising way to render the surface of polymeric mesh implants as antibacterial. For this purpose, in this work, plasma polymerization is used as a fast, solventless method to change surface properties of PP mesh by deposition of plasma polymer nano-thick layer. Silver nanoparticles as a reservoir of antibacterial agent, which can be released to the environment, are enclosed in this plasma polymer and protected from spontaneous release by deposition of second plasma polymer layer. Due to particular characteristic of a second plasma polymer layer, control of the release properties of silver ions from the coating is possible under mechanical stimulation.

In this chapter, detailed description of elaboration process and characterization of a multilayer plasma polymer systems, which consist of two layers of maleic anhydride plasma polymer deposited on the surface of polypropylene-made surgical mesh is given. AgNPs as an antibacterial agent reservoir were trapped between the two plasma polymer layers. Owing to differences between the mechanical properties of the plasma polymer thin films and the elastic bulk substrate, tensile stresses generate cracks within the plasma polymer overlayer, which might be used as diffusive channels for Ag + species. The aperture of the cracks is shown to be mechanically controlled in a reversible way, allowing the control over releasing properties. Moreover, first investigations of the antibacterial properties of the materials are presented.

This chapter consists of a scientific paper that has been recently published in a peer-reviewed journal.

Introduction

Hospital infections are a major cause of morbidity and mortality [338]. Today, more than 1 million people all over the world suffer from nosocomial infectious complications [338]. Most frequently, these infections are associated with surgical wounds, the urinary tract or respiratory tract and directly involve invasive medical devices such as catheters or implants [339][340][341]. Medical devices thus act as passive surfaces prone to bacterial adhesion and biofilm formation [START_REF] Ploux | Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties[END_REF]342]. Nowadays, preventive strategies are used to fight bacterial colonization of such devices, in addition to oral treatments with antibiotics. They directly involve the biomaterial, which is modified to specifically exhibit antiadhesive or antibacterial chemical surface properties [343][344][345][346][347]. In hospital environments, more and more devices with biopassive (in this case, the surface chemistry prevents bacterial adhesion without any antibacterial effect) or bioactive (here, the surface chemistry has a biological i.e., antibacterial effect) surfaces are exploited [348][349][350]. Nevertheless, they are mainly dedicated to external use and are rarely employed for implants. For internal use, drug delivery systems are preferred, aimed at fighting both planktonic and attached bacterial populations, and at controlling the dose of antibacterial agent delivered to the patient, the lifespan of the implant and the lifespan of its antibacterial effect [351,352]. Until now, however, these objectives have remained a challenge for most surgical situations. Solutions need to be specifically developed for each of them according to their specific constraints.

Typically, polymer meshes used as implants for abdominal and gynecological repairs are favorable to bacterial infections due to the uneven topography of meshes and, in the specific case of gynecological repairs, the large quantity of bacteria present at the implantation location. Antibacterial smart coatings dedicated to such soft and deformable materials are therefore needed.

Nevertheless, aside from the difficulty in controlling drug delivery dose and kinetics, coatings on soft and deformable materials are highly sensitive to 2-96 damage that may be induced by movements of the patient, affecting the lifespan of the biomaterial.

In addition, bacterial resistance to antibiotics continues to increase, while antibiotics are the only (and thus essential) treatment to fight infections [353][354][355][356]. This implies the necessity of reducing the use of antibiotics in systems dedicated to local and long-term drug delivery. Therefore, the use of alternative antibacterial agents is now highly desired in the medical field. In this framework, silver species that have long been known to have antimicrobial activity are used in more and more biomedical sectors [165,279,357,358] Recently, we proposed a new concept for a drug delivery system dedicated to soft materials, providing control of duration and doses of delivery [194]. Based on Ag + release driven by mechanical stimuli, the concept was proved for PDMS model materials. In the present work we propose a new generation of bioactive biomaterials that are able to adjust their release of bioactive agent under mechanical stimuli thanks to the same concept. This was achieved by the fabrication of multilayer plasma polymer systems, which consisted of two layers of maleic anhydride plasma polymer deposited on the surface of polypropylenemade surgical mesh. AgNPs were trapped between the two plasma polymer layers and acted as an antibacterial agent reservoir. Owing to differences between the mechanical properties of the plasma polymer thin films and the elastic bulk substrate, tensile stresses generate cracks within the plasma polymer overlayer, which might be used as diffusive channels for Ag + species.

The originality of the system is that the aperture of the cracks can be mechanically controlled in a reversible way.

Antimicrobial Coatings for Soft Materials

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This new system makes it possible to tailor doses of the antibacterial agent released over time and thus to prolong the lifetime of the antibacterial properties of the material. In the case of surgical polypropylene meshes and more generally in the field of textiles for implants and other soft biomaterials, this strategy is promising. Cracks will be created in a controlled way before implantation, and crack opening and subsequent Ag + release will be induced by mechanical stresses naturally occurring at the location of the implant.

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Experimental

Materials

Undyed, knitted, pure polypropylene (PP) mesh was provided by Cousin Biotech (Biomesh ® P1). Briquettes of maleic anhydride (99.5% purity), silver nitrate solution (0.1 M) and NaBH 4 (98% purity) were obtained from Sigma-Aldrich and used as received.

Plasma reactor

Plasma treatments were carried out in an electrodeless cylindrical glass reactor (6 cm diameter, 680 cm 3 volume, base pressure of 5 x 10 -4 mbar and leak rate better than 1.0 x 10 -10 kg.s -1 ) enclosed in a Faraday cage (see Figure 2-16 in supporting information). The chamber was fitted with a gas inlet, a Pirani pressure gauge, a two-stage rotary pump (Edwards) connected to a liquid nitrogen cold trap and an externally wound copper coil (4 mm diameter, 5 turns). All joints were grease-free. An L-C matching network (Dressler, VM 1500 W-ICP) was used to match the output impedance of a 13.56 MHz radiofrequency (R.F.) power supply (Dressler, Cesar 133) to the partially ionized gas loaded by minimizing the standing wave ratio of the transmitted power. During electrical pulsing, pulse shape was monitored with an oscilloscope. The average power <P> delivered to the system was calculated using the following expression: <P> = P p [t on / (t on + t off )], where P p is the average continuous wave power output and t on / (t on + t off ) is defined as the duty cycle.

Antibacterial coating

PP substrates (2 x 4 cm²) were placed in the center of the plasma reactor chamber followed by evacuation back down to the initial pressure ( 5 x 10 -4 mbar). It is worth noticed here that the PP mesh was at floating potential. They were first activated by argon plasma during 1 min at 60 W (PP/Ar).

Subsequently, maleic anhydride vapor was introduced into the reaction chamber (pressure of 0.2 mbar) with a flow rate of approximately 1.6 x 10 -9 kg.s - Importantly, the results presented in the next section, which demonstrate that the release of Ag + and its control could be perfectly controlled by playing with mechanical stresses, was confirmed for both procedures of sterilization

Elongation of the bioactive materials

Elongation of the coated meshes was performed on a homemade stretching device (see Figure 2-17 in supporting information) with a speed of 24 µm/s.

Samples were subjected to stretching/unstretching cycles where the elongation rates were set to 20%, 40% or 80% of the initial sample length (100% of elongation means twice the initial length).

Electron Microscopy

Scanning electron microscopy (SEM) images were acquired using an FEI environmental microscope (Quanta 400 model) working at 30 keV. Samples were observed using high vacuum mode.

Transmission electron microscopy (TEM) images were obtained using a Philips CM20 instrument with a Lab6 cathode, at an acceleration voltage of 200 kV.

Cross-sections of samples were obtained using a microtome (LKB model 8800).

Infrared Analysis

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra were recorded with a Brucker IFS 66 FTIR spectrometer equipped with an ATR accessory from Specac. The spectrometer was equipped with a nitrogen cooled MCT detector. Spectra were the result of the sum of 256 scans and were recorded in the range of 4000-650 cm -1 with a spectral resolution of 4 cm -1 . A single reflection Ge crystal (refractive index ~ 4.0) with a 65 ° beam incidence was used. The spectrum of a freshly cleaned Ge crystal was used as a background. solution with a concentration of 2.0 x 10 -3 M was then added to reduce silver from the ionic form to zero valent AgNPs. After 5 min, these samples were analyzed and the surface plasmon absorbance peak around 400 nm was observed. The corresponding peak area was measured. Then, the stretching ratio was changed and the sample was immersed in distilled water for another 24 h period. The UV/Vis absorbance of the new solution was measured by repeating the aforementioned procedure.

UV-Visible analysis

XPS analysis.

XPS spectra of PP and PP-functionalized surfaces were recorded with a VG SCIENTA SES-200 spectrometer equipped with a concentric hemispherical analyzer. The incident radiation was generated by a monochromatic Al K α X-ray source (1486.6 eV) operating at 420 W (14 kV; 30 mA). Photo-emitted electrons were collected at a take-off angle of 90  from the sample with electron detection in the constant analyzer energy mode. Signals for survey spectra were recorded with a pass energy of 500 eV. For high-resolution spectra of C1s, O1s and Ag3d components, the pass energy was set to 100 eV. The analyzed surface area of PP mesh sample was approximately 3 mm 2 and the base pressure in the analysis chamber during experimentation was about 10 -9

mbar. Charging effects on these isolating samples were compensated for by using a Flood Gun. The spectrometer energy scale was calibrated using the Ag 3d 5/2 , Au 4f 7/2 and Cu 2p 3/2 core level peaks, set respectively at binding energies of 368.2, 84.0 and 932.7 eV. Peak fitting was performed with mixed Gaussian-Lorentzian (30%) components with equal full-width-at-half-maximum (FWHM) using CASAXPS software.

Differential Scanning Calorimetry (DSC) measurements

A METTLER TOLEDO DSC 1 was used for thermal analysis and crystallinity determination. The polypropylene sample was heated from -100 °C to 250 °C, cooled to -100 °C and re-heated to 250 °C. The heating and cooling rates were +/-10 °C/min. Crystallinity was calculated during the heating cycles by comparing experimental peak areas to the theoretical one obtained in the case of 100% crystalline polypropylene (207.1 J/g).

Mechanical tests

An

Antibacterial assay: On-plate diffusion test

Bacterial culture experiments were conducted with the Escherichia coli (E.coli) K12 (PHL628) strain [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context[END_REF]380] This strain expresses curli and produces exocellular polymeric substances (EPS). One milliliter of bacteria stock culture (stored at -80 °C) was used to inoculate 10 ml of selective growth medium (M63G [380], pH = 6.8), resulting in a pre-culture that was grown further overnight at 30 °C. This culture was then used to inoculate a second pre-culture (10 vol.-% of the last pre-culture), which was grown for 4 h under the same conditions. One hundred microliters of this fresh bacterial suspension was 2-106 spread on Luria-Bertani (LB) agar-supplemented growth medium (15 g.L -1 ) in order to form a thin bacterial film.

The sterilized samples were placed in contact with agar plates previously homogeneously inoculated (with the top side of the samples in contact with the agar). After overnight incubation at 30 °C, bacterial growth around the samples was observed in order to identify the potential presence of zones without bacterial growth (growth-inhibition areas).

Antibacterial assay: Analysis of biofilm development

Experiments Images so extracted were then analyzed with the COMSTAT2 ® software to determine the quantity of biofilm (biomass) [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF].

Antibacterial analyses were conducted on PP/Ar/MAPP/Ag/MAPP samples with and without stretching at 60% of their initial length as well as on PP and PP/Ar/MAPP/Ag samples, used as negative and positive controls, respectively.

Two samples of each material type were used for each experiment. The experiment was repeated at least 3 times.

Results and discussion

The antibacterial coatings, consisting of multi-layer plasma polymer systems, were fabricated by following a multi-step procedure schematized in A first step of Ar plasma treatment applied to the polypropylene mesh (step b)

was followed by deposition of a minimally cross-linked plasma polymer thin film with an anhydride-rich structure (step c). Subsequent immersion in water led to the opening of cyclic anhydride groups and the formation of carboxylic acids.

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Successive immersion in solutions of silver ions and reductive agent readily triggered the immobilization of silver nanoparticles onto the plasma polymer layer (step d). The last step in the procedure consisted of depositing a highly cross-linked plasma polymer overlayer.

The originality of this work derives from the fact that the aforementioned procedure led to a multi-layered plasma polymer system in which cracks in the overlayer were anticipated under mechanical strains and in a controlled manner, aimed at creating diffusive channels for better control of Ag + species release.

Polypropylene mesh substrates (PP)

Mechanical properties

The polypropylene implant used in this work was biomesh ® P1 (parietal Antimicrobial Coatings for Soft Materials

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Contact angle measurements taken at each stage of this reaction scheme were found to be consistent with the aforementioned description (Table 2-2

Nanoparticle loading within the plasma polymer layer

The pKa values of maleic anhydride plasma polymers films have previously been found to be pKa 1 = 3.5 and pKa 2 = 10.5 [371]. So, according to the experimental conditions (pH  6-7), a portion of the carboxylic acid groups was deprotonated. These carboxylate groups allowed the attachment of silver ions via electrostatic interactions by immersing the coated substrate into a silver nitrate solution. It is interesting to recall here that hydrolysis of a plasma 2-2). The increase in thickness compared to the first plasma polymer layer alone can be explained by the formation of silver nanoparticles at the top of the plasma polymer layer. The change may also have been induced by the growth of silver nanoparticles inside the polymer matrix. Thus, tensile stress resulting from particle growth should have been transferred to the polymer matrix leading to expansion of the plasma polymer layer. The work of Vasilev et al. [287] clearly shows that this quantity can be tuned by playing with the Ag + loading time, time of reduction and thickness of the plasma polymer thin film. Here, the thickness of the plasma polymer layer was set to  10 nm and the Ag + loading time to 30 min, keeping in mind that the quantity of silver particles could be easily adjusted if necessary.

Concerning the reducing step, we examined the effect of the reducing time on the quantity of embedded silver nanoparticles and found that most of the Ag + species had been consumed after 30 min of reduction (see 2-4 in supporting information).

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Plasma polymer overlayer.

The last step of the elaboration procedure consisted of depositing a highly cross-linked plasma polymer overlayer. This was done by using a high duty cycle (50%) during deposition. Indeed, it is well known that a less selective chemistry occurs during plasma "on" excitation periods, where the predominance of fragmentation of monomer molecules to atoms or fragments leads to more cross-linked and less 'polymer-like' structures at relatively short off-times (e.g., high duty cycle) [326,387]. XPS results confirmed this tendency.

The contribution of the binding energy peak at 289.4 eV corresponding to O=C-

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O-C=O/HO-C=O groups decreased, relatively, to 6.2 at.%, accompanied by a significant increase of the contribution of the peak at 285.0 eV associated with cross-linked carbon/hydrocarbon (C x H y ) species (Figure 2.3 -spectra f and Table 2-4 in supporting information). This was also confirmed by infrared analysis (Figure 234-spectra e), with significant enhancement of carboxylic acid bands at the expense of cyclic anhydride stretching and cyclic unconjugated anhydride stretching vibrations. Once again, the final thickness of the second plasma layer was strongly dependent on the complex structure of the PP mesh. The thickness of this cross-linked overlayer was estimated by ellipsometry to be 125  4 nm (Table 2-2). By XPS analysis, the Ag 3d 3/2 and Ag 3d 5/2 photoemission X-ray lines of silver were also detected at 367. By depositing a plasma polymer overlayer, a barrier against diffusion of silver ions was created. In this section, we explored the effect of the crosslink density within the second plasma polymer layer as a parameter influencing the release behavior of silver ions from the coating. Increasing the crosslink density improved the barrier properties of the developed multilayer system and allowed control of the time (t 0 ) after which ions started to be released to the environment (the lowest concentration of silver detectable with the UV spectrometer used in this study is  3 ppm). The crosslink density of the second plasma polymer layer was modified by changing the conditions of plasma deposition according to 2-3) significantly reduced the rate of Ag + release in comparison with the system without a second layer. The time, t 0 , needed to detect any release of silver ions in the immersion solution increased to 24 hours. By increasing the crosslink density of the plasma polymer overlayer (MAPP I, Table 2-3), the rate of Ag + release was further reduced and t 0 increased to 1 week. Further increases in the crosslink density (MAPP II, Table 2-3) blocked the release of silver ion up to 16 weeks. The conditions of deposition of the overlayer are thus able to determine the time, t 0 , needed to start the release of Ag + into the environment. The rate of Ag + release decreases when cross-linking of the overlayer increases.

To evaluate the influence of the surrounding liquid medium used during the release assays, similar experiments were undertaken in PBS buffer solution or M63G bacterial culture medium. In each case, NaBH 4 was used as a reducing agent and the integrity of the plasma polymer multilayer was checked by SEM analysis after immersion in the solution. Then, release experiments were repeated with increasing densities of crosslinking in the overlayer. Similar tendencies as those obtained in water were observed, independent of the nature of the solution used for the release. Antimicrobial Coatings for Soft Materials A remarkable result was the closure of the cracks (e.g., closure of the diffuse channels) when the material was switched back to its initial length (the rate of retraction was also 24 µm/s) (Figure 2-11 -images d, e and f). SEM images clearly show that the mechanism of closure was a collapsing rather than a selfhealing process. The cracks were not completely closed, allowing silver ions to continue to diffuse. Therefore, the flux of diffusion of silver ions can be tailored by playing with the mechanical stimulation (here, elongation cycles) and crack opening/closing cycles. The effect of the degree of stretching on the diffusion of silver ions was investigated. The results showed a regular increase in the amount of released silver ions when the degree of stretching was increased up to 80 %. This can be due to a continuous opening of the cracks during stretching.

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Antimicrobial Coatings for Soft Materials

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The release of silver ions was measured under a series of stretching/unstretching cycles applied to the plasma polymer multilayer systems. The evolution of the UV absorbance is shown in In the absence of any cracks, a background signal was obtained, originating from the diffusion of silver species through the pores of the native plasma polymer layers or of silver species located at the near surface region of the plasma polymer overlayer. When this substrate was stretched at 60%, a strong increase in UV absorbance was observed due to the aperture of the diffusive channels, allowing an increase in the flux of silver ions from the material to the solution. When the surface was switched back to its initial position, a drop in the intensity of UV absorbance was observed, indicating that the flux of silver ions was reduced. This cycle was repeated three times and appeared fully reversible.

0% 60% 0% 60% 0% Absorbance [a.u.]
Stretching ratio [%]

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These results clearly show that a multilayer plasma polymer system allowing the release of a bioactive agent (here, silver ions as an antibacterial agent)

under stretching was successfully created and that the releasing process was fully reversible.

Antimicrobial activity of the multilayer system

The barrier role of the plasma-polymer overlayer and recovery of its antibacterial action through the elongation-related release were investigated by preliminary microbiological experiments. The diffusion of silver ions through the plasma polymer overlayer, with or without stretching, was evaluated with samples placed on semi-solid nutritive agar that was previously inoculated with a thin film of bacterial suspension.

Conclusions

Mechanically sensitive materials made of plasma polymer multilayers and bioactive agents can be developed on modified polypropylene substrate using solvent-free steps based on plasma polymerization. Tailored release of an active agent can be achieved by mechanical stimulation of the designed material. This has been demonstrated for silver, stored as AgNPs in the plasma polymer multilayer system and released in the material surroundings in its bioactive ionic form. Taking advantage of different mechanical properties between the different layers, a system of cracks in the plasma polymer overlayer allowed control of the diffusion of silver ions. Its antibacterial properties have been confirmed. The stability of the properties of the film under the stress of elongation has been also proven (no delamination).

PP

Ag MAPP 60%

Acknowledgements

The authors gratefully acknowledge the financial support from Region Alsace in

France. The authors would like to thank Cousin Biotech for providing polypropylene mesh substrates. Florent Kuntz and Ludovic Frechard from Aerial are kindly acknowledged for their expertise and assistance in the electron beam sterilization process.

Keywords

Maleic anhydride; plasma polymerization; antibacterial coating; silver particles; controlled release; responsive materials. 3 Effect of ageing and sterilization on plasma multilayer system

Introduction

In the previous chapter, we have seen the details of the multistep procedure to elaborate a new generation of bioactive coating, dedicated to soft implant and able to adjust the release of bioactive agent under mechanical stimuli (see section 2). A plasma multilayer coating was deposited on the surface of polypropylene made surgical mesh. Silver nanoparticles were trapped between two plasma polymer layers and acted as an antibacterial reservoir. Owing to the differences between mechanical properties of the plasma polymer thin films and the polypropylene fiber, tensile strength generates cracks within the plasma polymer overlayer which might be used as diffusive channels for Ag + species.

Since the biomaterial device has to be sterile before the implantation in the body, the sterilization step is considered as the last step of the surface modification during elaboration. But ageing is also an important step to be considered as it can dramatically affect the properties of the plasma polymer coating in combination with the sterilization step.

Concerning ageing, it is well known that plasma deposited films undergo certain chemical reactions when they are exposed to atmosphere. For example, freshly deposited plasma polymers typically contain radicals, which rapidly react with in-diffusing oxygen during exposure to air. This ageing process is well known as Predicting what would be the outcome after a sterilization process on a specific material like coated substrates is very complicated, if not impossible. The best way to determine how a specific sterilization process affects the surface properties is a comprehensive test of the coating properties before and after sterilization, and as well the tests during the ageing period. In general manner, it is known that the use of heat (steam), radiation ( β/ electrons or γ) or chemicals (mostly ethylene oxide) can affect the mechanical and optical properties of polymer [392,393]. Radiation, in particular the effect of γ-rays, has shown to induce chain scission leading to mechanical and thermal degradation effects So, depending on material properties the method used for a particular device has to be carefully selected.

The present chapter focuses on the stability of the plasma polymer multilayer coating on polypropylene mesh when undergoing ageing and standard sterilization procedures. More precisely, we examine the effects of i) ageing and ii) autoclaving and electron beam sterilization on surface properties of our system made of plasma polymerized layers of maleic anhydride. This study has important impact due to the recent growing interests of plasma modified surfaces used for a biomedical applications [133,179,194,374,[401][402][403][404].

Materials and methods

Plasma multilayer systems

Details of the elaboration steps and characterization of the plasma multilayer system are fully described in section 2.

Briefly, PP substrates were placed in the center of the plasma reactor chamber followed by evacuation back down to the initial pressure ( 5 x 10 -4 mbar). They were first activated by argon plasma during 1 min at 60 W (PP/Ar).

Subsequently, maleic anhydride vapor was introduced into the reaction chamber (pressure of 0.

Ageing

Before elongation

After completion of deposition of the second plasma polymer layer, coated substrates were stored in polystyrene box in room temperature and ambient atmosphere. Time of ageing before elongation was 1h, 10 days and 20 days for respective samples.

Before sterilization

After completion of deposition of the second plasma polymer layer, coated substrates were stored in polystyrene box in room temperature and ambient atmosphere for 3 days.

After sterilization

After sterilization, samples were kept in polystyrene sealed box in a room temperature and ambient atmosphere. Analysis of samples was conducted i)

the same day after sterilization, ii) after 3 days of ageing.

Techniques of sterilization

Autoclave

Samples were put into glass Petri dishes and autoclaved (Varioklav 400, H + P Labortechnik, Germany) for 15 min at 121⁰C and high pressure (100 kPa).

Samples were cooled to room temperature and kept in Petri dish prior to analysis.

Electron beam irradiation

Electron beam radiation was performed by the Center of Irradiation Aérial (CRITT Aérial, Illkirch). The radiation station contains a Van de Graaf electrostatic generator with a tension ranging from 0.5 to 2.5 MV, a current intensity ranging from 0.01 to 200 μA and a maximum power around 500 W.

This apparatus is known for its excellent tension's stability (fluctuation around 0.5 % of the nominal tension) and intensity's stability (fluctuation around 1% of the nominal intensity)

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To perform sterilization, substrates were packed in the polystyrene box and set for sterilization with doses of 25 kGy. The samples were left sealed until being used for XPS, SEM, TEM investigations and antibacterial assays.

Surface characterization

Scanning Electron Microscopy

SEM observations were performed using a FEI environmental microscope (Quantana 400 model) working at 30 keV. Samples were stretched on a homemade stretching device with a speed of 24 µm/s and the elongation was set to 20%, 40% or 80% of the initial sample size respectively (see details in section 2.3.4).

X-ray Photoelectron Spectroscopy

XPS spectra of PP-functionalized surfaces were recorded with a VG SCIENTA SES-200 spectrometer equipped with a concentric hemispherical analyzer. The incident radiation was generated by a monochromatic Al K α X-ray source (1486.6 eV) operating at 420 W (14 kV; 30 mA). Photo-emitted electrons were collected at a take-off angle of 90 from the sample with electron detection in the constant analyzer energy mode. Signals for survey spectra were recorded with pass energy of 500 eV. For high-resolution spectra of C1s, O1s and Ag3d components, the pass energy was set to 100 eV. The analyzed surface area of PP mesh sample was approximately 3 mm 2 and the base pressure in the analysis chamber during experimentation was about 10 -9 mbar. Charging effects on these isolating samples were compensated for by using a Flood Gun.

The spectrometer energy scale was calibrated using the Ag 3d 5/2 , Au 4f 7/2 and Cu 2p 3/2 core level peaks, set respectively at binding energies of 368.2, 84.0 and 932.7 eV. Peak fitting was performed with mixed Gaussian-Lorentzian 3-151 (30%) components with equal full-width-at-half-maximum (FWHM) using CASAXPS software

Transmission Electron Microscopy

TEM observations were performed using Philips CM20 instrument with Lab6 cathode at an acceleration voltage 200 kV. For the investigations of influence of sterilization methods on silver nanoparticles immobilized in the multilayer system (see section 3.3.2.3), plasma polymer multilayer system was deposited on copper grids, instead of polypropylene surface, to facilitate the study.

Antibacterial assay

Experiments were conducted with the PHL628-based E.coli SCC1 strain [380].

Bacteria taken from frozen stock (-80 °C) were inoculated on an agar plate of Luria-Bertani (LB, LB purchased from Sigma-Aldrich) medium for an overnight culture. One colony of bacteria was inoculated in 10 mL of LB medium and cultivated at 30 °C overnight for a first pre-culture. A second pre-culture was inoculated with the first pre-culture (10 vol-%) in 10 mL of selective M63G medium and cultivated at 30 °C overnight. A third pre-culture was inoculated from the second pre-culture (10 vol-% of the second pre-culture) for 4 h at 30

°C. The final culture used to perform the experiment was inoculated from the 4h pre-culture (10 vol-%). Its absorbance at 600 nm (Abs 600 ) was adjusted to 0.01 (i.e, 10 6 bacteria/mL). Coated-mesh samples, previously sterilized by autoclave

Results and discussion

Effect of ageing

Experiment was designed to show the effect of ageing on plasma polymer multilayer system deposited on polypropylene mesh. Physical and topographical changes of the surface of plasma polymer multilayer system deposited on the mesh were investigated by means of SEM microscopy observations. Changes in the surface chemical composition were investigated by means of XPS spectroscopy measurements. The adhesion between plasma polymer multilayer system and polypropylene substrate and/or between second and first plasma polymer deposited layer seems also to change during ageing process. This can be already seen at 60% or 80% of elongation (Figure 3 In conclusion, after ageing, plasma polymer matrix becomes softer and the intensity of internal tensions in plasma polymer interfacial tensions are lower.

Scanning Electron

This is most likely due to the influence of humidity. Because of high concentration of oxygen, plasma polymer coating can act as a sponge adsorbing water molecules from the environment via hydrolysis bounding. This increase in water concentration inside the plasma polymer matrix plays the role of plasticizer, tuning polymer more flexible and less crackable. Tension at the interface between polypropylene and plasma polymer is lower, since delamination does not occur after ageing process. 3-1).

XPS

Table 3-1: Surface atomic composition of (a) plasma polymer multilayer system freshly prepared (b) plasma polymer multilayer system after aging. * Gold and copper elements are from sample holder, possible to detect due to mesh pores. 3-2). visible after deposition of plasma polymer multilayer system, only small circular irregularities can be detected.

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After autoclave sterilization

After autoclave sterilization of PP (Figure 3 in the steam autoclave showed some changes comparing to plasma modified multilayer system deposited on PP mesh without sterilization (Figure 3456).

Beside paralleled lengthways grooves and small circular irregularities visible on the fiber, a number of undirected tears and small bubbles are also noticeable.

On the Figure 3-6 D, dark spots are observed, which are very similar to ones found on PP surface after autoclave sterilization. Antimicrobial Coatings for Soft Materials SEM micrographs of plasma polymer multilayer system sterilized by electron beam irradiation with the 25 kGy dose showed no changes in the physical and topographical shape of the meshes. Paralleled lengthways grooves and very few small circular irregularities can be observed on the fiber (Figure 3-6 E, F).

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Results showed no differences or signs of thread-like or dark spot-like degradations after using this method of sterilization.

To conclude, Scanning Electron Microscopy observations suggest that preferable method of sterilization for multilayer system is electron beam irradiation with the dose of 25 kGy. Autoclave treatment influence surface structure unfavorably.

XPS analysis

Surface atomic concentration of plasma polymer multilayer system and plasma polymer multilayer system after sterilization by means of autoclave and electron beam irradiation is presented in Table 3-3. Table 3- was observed (see Table 3-3). Atomic concentration of silver has decreased from 2.5 % before sterilization (Table 2-1) to 0.6 % in case of autoclave sterilization and 0.4 % after electron beam sterilization (Table 3-3). No other significant changes were observed after autoclave sterilization. 34). Decrease of carbon atoms belonging to O-C-O/-CO group from 12.9% (see Table 3-4) to 9.39% (see Table 3- XPS analysis allowed also the detection of Ag 3d 3/2 and Ag 3d 5/2 photoemission X-ray lines at 367.7 eV and 373.8 eV, respectively (Table 34 3-4). At the same time migration of short carbon chains from the bulk of plasma polymer to the surfaces takes place.

To conclude, effect of autoclave sterilization did not affect the main chemical character of coating. However, chemistry of surface was influenced by electron beam irradiation method.

TEM analysis

Before sterilization

Particles size and particles distribution in the plasma polymer matrix before sterilization are described and commented in details in section 2.4.3. The differences in particles distribution can be seen on Figure 3-8 A and Figure 3456789A.

3-169 Because it is well known that thermal treatment has an impact on silver nanoparticles nucleation [411,412], synthesis [413], shape and size [413,414] influence of autoclave sterilization on silver nanoparticles embedded in the induces. Furthermore these modifications can change the release of silver ions kinetics, antibacterial properties and toxicity of the final material [START_REF] Park | The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles[END_REF]416,417]. The effect of electron beam method of sterilization was also verified. any other modification of silver nanoparticles was observed using TEM microscopy.

After autoclave sterilization

After electron beam sterilization

Antibacterial assay

As we have seen in the previous sections, autoclave sterilization method has a major impact on the surface properties of plasma polymer multilayer system. It causes physical degradation of plasma polymer film and aggregation and changes in distribution of silver nanoparticles. Electron beam sterilization however, has an impact only on the chemistry of the PP/Ar/MAPP/Ag/MAPP surface. Further we have verified how these changes influence the bacterial response to the multilayer plasma polymer system. For this reason, effect of the plasma polymer multilayer system samples sterilized by autoclave and electron beam (with the dose of 25 kGy), on bacterial growth in the planktonic state after 4 h of incubation was tested. Results are expressed in terms of inhibition rate compared to bacterial colonization on PP reference sample.

Combining effect of ageing and sterilization

Preliminary results from tests of combined effects of ageing and sterilization on plasma polymer multilayer system deposited on polypropylene mesh were obtained. Tests were performed in the same manner as tests described in section 3.3.1.1.

For the Scanning Electron Microscopy observations first type of sample was freshly prepared and sterilized by means of electron beam (25 kGy) at the same day. Further, the sample was stretched and images were taken. Second type of sample was left to age for three days and next, sterilization with electron beam (25 kGy) was performed.

SEM

The combined effect of aging of sample and sterilization was verified by observation of physical features formed during stretching of the sample. The apparition and regularity of cracks, their size of aperture were observed and compared. In both cases cracks appear after 20% of elongation (see Figure Figure 3-12 20%). For both types of samples, between 40% and 80% of elongation, appeared cracks are regular and propagate along the fiber.

3-11 20% and

There is no distinction for cracks to appear between different places on the fiber in the mesh structure (close to weave or along the fiber between two weaves).

Interestingly, comparing to the results from previous section, cracks appear with the same distance from one to another along the whole length of the fibers, (without differences between places closer to weave or in between two weaves on the fiber). Antimicrobial Coatings for Soft Materials

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In both cases no delamination occurred during the stretching even up to 80 % of elongation. This indicates that ageing combined with sterilization have a beneficial influence on plasma polymer multilayer system properties, which is related to changes in crosslink density after sterilization and absorption of water molecules which acts as plasticizer for plasma polymer. Ageing and sterilization allows to tailor the "crackability" of plasma polymer and thus it allows to achieve the desired properties of plasma polymer multilayer system.

Conclusions

Result of studies of the effects of sterilization and ageing on plasma polymer multilayer system suggests that during ageing some identifiable changes occur in the high resolution C(1s) envelope indicating hydrolysis of anhydride groups and migration of short chains of plasma polymer to the extreme surface Preferable method of sterilization for these systems is electron beam irradiation.

Autoclave treatment influence surface structure unfavorably. Also, tears-like shaped features, dark spots suggesting degradation and irregularities were observed on the surface of plasma deposited layers, after autoclave treatment in 121°C for 15 min. Electron beam irradiation with the dose 25 kGy did not induce surface physical or topographical changes. Autoclave sterilization did not significantly affect the main chemical character of coatings. However, electron beam sterilization induces changes in chemistry of the surface causing decrease in O/C elemental concentration ratio. Resistance against sterilization procedures of surface which is intended to undergo sterilization has to be carefully analyzed. Engineered surfaces designed for biomedical application which consist of silver nanoparticles have to be verified after undergoing the sterilization procedures. Humid environment and high temperature during autoclave sterilization have affected the size and distribution of silver nanoparticles in the multilayer system. Visually compared microphotographs 3-175 which show nanoparticles, prove that autoclave sterilization induces changes in nanoparticles size and distribution. However, electron beam irradiation chosen as a method of sterilization for plasma polymer multilayer system did not induce any aggregation or any other modification of silver nanoparticles. Additionally, plasma multilayer system after electron beam irradiation showed better antibacterial efficiency towards planktonic bacteria. This is related to preserved size and distribution of silver nanoparticles after electron beam sterilization procedure.

Observations of stretched samples after ageing and electron beam sterilization suggest that electron beam method of sterilization has beneficial effect on final properties of the material.

CHAPTER IV

4 Bacterial colonization and biofilm formation on plasma polymer modified surgical polypropylene mesh

Introduction

Synthetic surgical meshes are widely used for the tension free repair (does not put tension on muscles) of abdominal wall and gynecological defects. Use of this material has proven their effect in reduction of recurrence rate of abdominal hernia to less than 1.5 percent [418]. Structural design of yarn types of materials is nowadays used routinely for repair of abdominal wall defects in more than 50 percent of cases [START_REF] Engelsman | Morphological aspects of surgical meshes as a risk factor for bacterial colonization[END_REF]. In case of gynecological procedures, for example management of stress urinary incontinence, synthetic meshes are now the gold standard [419]. However, the use of implant exposes the patient to a lifelong risk of infection. The infection caused by bacterial colonization may occur even up to 39 months [420,421]. In case of gynecological procedures with such mesh, complications appear in 33-45% [419], including mesh exposure into the vagina or even the bladder or bowel, infection and pain syndrome [421]. This has impact on the recurrence of abdominal wall or gynecological defect by interfering with the recovery process after the operation and can cause the reoperation with increased patient morbidity, hospital costs and mortality rates [START_REF] Zogbi | The Use of Biomaterials to Treat Abdominal Hernias[END_REF]419].

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Among complications, mesh-related infections are difficult to treat and can induce morbidity recurrence [419]. Unfortunately, anti-infection strategies using antibiotics are often failing. This is due to microorganisms which adhere to the surface of implant, and start to form biofilm. In the biofilm, microorganisms develop higher resistance to environmental stresses, antibiotic treatments and they effectively protect themselves from host's immune system.

For this reason, it is important to understand the meshes-related bacterial colonization and biofilm formation. For example, it was shown by Burger et al.

that the mesh characteristics have impact on infection rates [422,423]. Study of Engelsman et al. [77] on the bacterial colonization on different meshes materials has revealed the existence of niches in the morphology of the meshes where the bacteria tend to grow. The niches were found between the filaments in case of multifilament meshes providing increased surface area and more contact points permitting the adhesion between microorganisms and implant.

This has influenced the biofilm properties, resulting in massive production of slime and increased biofilm density on the multifilament meshes compared to monofilament ones [START_REF] Engelsman | Morphological aspects of surgical meshes as a risk factor for bacterial colonization[END_REF]. Engelsman et al. [332] have shown that consequently, different rates of infection were associated to the different types of surgical meshes.

Polypropylene mesh, which is used in this study, was reported to be associated with an infection rate between 2.5% -5.9% in case of abdominal wall implants [424][425][426], similarly to polyester meshes. It was shown that both gram-negative and gram-positive bacteria form biofilms on these types of materials [START_REF] Engelsman | Morphological aspects of surgical meshes as a risk factor for bacterial colonization[END_REF]427].

However, it is dependent on the surgical technique used (open or laparoscopic implantation) and patient characteristics. The material structure (PP or expanded Polytetrafluoroethylene (ePTFE), multifilament or monofilament mesh) influenced the infection rate. Changes in material composition and textile properties influenced bacterial adherence as well [START_REF] Engelsman | Morphological aspects of surgical meshes as a risk factor for bacterial colonization[END_REF]332,427].

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One of the strategies to reduce the rate of infection on mesh materials was introduced by Scheibach et al. [428]. Authors rendered the surface of mesh anti-adherent for bacteria. S. epidermidis and S. aureus adherence on titanium coating on polypropylene mesh surface was evaluated in vitro. Results showed the biocompatibility of modified polypropylene and the reduction of bacterial adhesion. Similar study on coatings for mesh material was performed by Saygun et al. [429]. Authors have used similar anti-adhesive coatings for bacteria using gold and gold/ palladium coatings. Their study revealed significant reduction of bacterial colonization on gold/ palladium coated meshes.

Another strategy to reduce mesh related infection is proposed in commercially available surgical meshes such as the GORE ® DUALMESH ® PLUS. This strategy includes presence of biocide in the coating, which can be released to the environment. In this case, material is made of polytetrafluoroethylene (ePTFE) which contains silver carbonate and chlorhexidine diacetate. It is claimed to resist to initial biofilm formation on the device up to 14 days postimplantation.

Before studying the development of biofilms on mesh materials, some aspects of bacterial biofilm should be noted. Tolker-Nielsen and Molin [430] have noted that every microbial biofilm community is unique, however some structural attributes can be generally considered universal. In particular, biofilms are not always continuous monolayers. They can be very heterogeneous, containing microcolonies of bacterial cells organized in various ways. Heterogeneity of biofilms exists not only for mixed culture biofilms (these can be found in environmental biofilms) but also for pure culture biofilms common on medical devices and those associated with infectious diseases. Heterogeneity can be a result of chemical gradients, adaptation to local environmental conditions, e.g. nutrition, temperature. Stoodley et al. [431] defined some criteria and characteristics that could be considered for biofilms in general. In particular, biofilms are thin flat film, ranging 4-182 from a monolayer of cells to a film consisting of several layers, which contain water channels. The microorganisms, which compose the biofilm also have an impact on the biofilm structure. For example, it was showed that biofilm thickness can be affected by the number of component organisms [432]. Pure cultures of K. pneumoniaeor and P. aeruginosa biofilms in a laboratory reactor were thinner than biofilm composed of both species (thickness of 15 µm for K.

pneumoniaeor, 30 µm for P. aeruginosa and around 40 µm for mixed biofilm).

Biofilm architecture is heterogeneous both in space and time, changing due to external and internal processes and conditions. Structure of biofilm depends also on the environmental conditions such as temperature of cultivation, growth media compositions and diluted or rich compositions of the same kind of growth medium [433][434][435][436]. Differences in bacterial biofilm architectures result in different susceptibility of bacteria towards antibacterial agents [390,437]. It was determined by Teitzel et al. [366] that biofilms of P. aeruginosa were more resistant between 2 to 600 times to heavy metal stress than planktonic bacteria cells. Only bacteria at the border of the biofilm structure were killed after exposure to elevated concentrations of copper. Near the substratum, cells were still alive. EPS matrix was suggested to protect cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.

Differences in the susceptibility of planktonic and biofilm cell populations of P. However, in their opinion, the quantitative analysis using these parameters does not necessarily predict the differences in biofilm architecture. Apart from surface coverage parameter which differed significantly, other analyzed structural parameters did not show significant differences in biofilm architectures although visual observations from CLSM micrographs clearly showed morphological differences [436]. This is probably due to implemented CVF parameter which removed parts of biofilm from analysis. Heydorn et al. [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF] In this chapter, we present study of the bacterial colonization, biofilm development together with biofilm structure analysis on plasma polymer mechanoresponsive antibacterial coating deposited on PP mesh substrate.

Additionally the study of the impact of Ag ions released from the coating on planktonic bacteria living in the surrounding of the developed material is presented. Moreover, the preliminary study of the biocompatibility of the developed plasma polymer multilayer coating deposited on PP mesh is evaluated using eukaryotic cells and can be found in the annex (section 6).

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Materials and Methods

Bacterial strain

Experiments were conducted with the laboratory strain Escherichia coli (E. coli) SCC1. This bacteria strain is autofluorescent through a genetic insertion, expresses curli and produces exo-cellular polymeric substances (EPS). E coli was purchased by Prof. Miao [START_REF] Miao | Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context[END_REF] from the Technological Institute, Singapore.

E. coli bacterial strain was long term stored either in liquid nitrogen at -180°C or in freezer at -80°C.

Cultivation

Bacteria were taken from frozen stock (-80°C) and were spread on an agar plate (Luria-Bertani medium) for an overninght culture.

One colony of bacteria was inoculated in 10 mL of Luria-Bertani (LB) (LB purchased from Sigma-Aldrich) medium and cultivated at 30°C, overnight. A second preculture was inoculated from the first preculture (10 vol-% of the first culture) in 10 mL of selective M63G-B1 medium [380] (see Table 4-1) and cultured overnight. A third preculture was inoculated from the second preculture in M63G-B1 (10 vol-% of the second preculture) and cultured for 4 h at 30°C.

The final culture used to perform the experiments was inoculated from 4 h preculture (10 vol-%). The optical density at 600 nm (Abs 600 ) was adjusted to 0.01 (i.e. 10 6 bacterial/mL).

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Substrates

The substrates developed during this research and described in details in the

Sample Identification Description

PP

Polypropylene mesh

PP/Ar/MAPP(2 nd )

Polypropylene mesh after argon plasma treatment and deposition of plasma polymer layer with the conditions used for 2 nd plasma polymer layer (overlayer) in the multilayer plasma polymer system, described in details in section 2.3.3

PP/Ar/MAPP/Ag

Polypropylene mesh after argon plasma treatment and deposition of first plasma polymer layer with the synthesis of silver nanoparticles afterwards PP/Ar/MAPP/Ag/MAPP Complete plasma polymer multilayer system deposited on polypropylene mesh PP/Ar/MAPP/Ag/MAPP-60%

Complete plasma polymer multilayer system deposited on polypropylene mesh, stretched with the 60% of elongation compared to initial length of the sample

Sterilization

Sterilization of the samples used for the experiments was performed in the

Analysis of the effect on planktonic bacteria

In order to test the potential toxicity of the coating and/or Ag + ions releasing from the surface on bacteria, cell growth in planktonic state was measured. For this purpose, substrates were placed into small Petri dishes (Ø 35 mm) on the previously placed and sterilized in oven (180°C for 1 h) silicon wafers. Stainless steel wire was used as a support for keeping the substrate immersed in solution. 4 mL of the bacterial culture (Abs 600 = 0.01) was added to the substrates. Bacteria were grown for 4 h and 168 h at 30°C on the samples.

After incubation, the samples were thoroughly and carefully rinsed with NaCl ( 9g/L in distilled water), to eliminate non-adherent or loosely attached bacteria from the substrate surfaces and remove media from the Petri dish. For this purpose, 2 mL of supernatant i.e., bacterial suspension for the first rinsing and NaCl solution for further rinsing, were removed and replaced by fresh NaCl solution (9 g/L in water). The rinsing step was performed without direct flushing, with the stream directed to the walls of a Petri dish and with the attention to the force of rinsing fluid. The substrate surface remained immersed in liquid during the rinsing steps (i.e., adhered bacteria and biofilm were not dried by air-surface interface creation). The effect of the new coatings on bacterial growth in the planktonic state was tested by measuring the absorbance at 600 nm (A 600 ) of a culture in which the substrate was immersed.

Analysis of bacterial colonization and the biofilm formation

Substrates were placed into small Petri dishes (Ø 35 mm) on the previously placed and sterilized in oven (180°C for 1 h) silicon wafers. Stainless steel wire was used as a support for keeping the substrate immersed in solution.

4 mL of the bacterial culture (Abs 600 = 0.01) was added to the substrates.

Bacteria were grown for 4 h and 168 h at 30°C on the samples. After incubation, the samples were thoroughly and carefully rinsed with NaCl (9 g/L in distilled water), to eliminate non-adherent or loosely attached bacteria from the 4-189 substrate surfaces and remove media from the Petri dish. For this purpose, 2 mL of supernatant i.e., bacterial suspension for the first rinsing and NaCl solution for further rinsing were removed and replaced with fresh NaCl solution Syto ® 9 stains cells with intact membranes and propidium iodide stains cells with compromised membranes [440,441]. The green Syto ® 9 dye enters all cells, while the red propidium iodide enters only cells with damaged membranes. This approach was used to expand the information obtained from CLSM images for qualitative and quantitative analysis of bacteria in connected and nonconnected biomasses.

Microscopy

Confocal microscopy 3D images with the constant interval in z-direction equal to 2 µm were taken. Experiments were conducted using an upright confocal V4.0g plug-ins [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]381] and Comstat2 [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF]438].

CLSM images of biofilms on mesh materials can be considered to be not always suitable for quantifying the number of adhered bacteria on the meshes, due to the observer's bias toward "nice" images as it was pointed by Engelsman et al. [START_REF] Engelsman | Morphological aspects of surgical meshes as a risk factor for bacterial colonization[END_REF]. In present study, the investigated area was chosen randomly, even if the bacteria or biofilm were not present on the mesh surface in particular place.

Biofilms never cover 100% of the surface of a biomaterial, and biofilm-surface free area was not considered out of the scope of interests. Due to this approach the variation of number of bacteria or biomass in case of biofilm, may differ greatly on this type of material. However, variability represents the reality of the bacterial adhesion and biofilm development on mesh monofilament material.  Biovolume is definied as the number of biomass pixels in all images of a stack multiplied by the voxel size and divided by the substratum area of the image stack. The resulting value is the biomass volume divided by substrate area unit. Biovolume represents the overall volume of the biofilm, and also provides an estimate of the biomass in the biofilm. In the present study, calculation of biomass was performed after using CVF function (biomass named "connected") and as well without activating this function (biomass named "total biomass"). Further, the "non-connected" part of biofilm was calculated by subtraction of "connected" biomass from "total biomass".

 Another parameter, which was calculated, was the surface to biovolume ratio, defined as surface area of biofilm divided by the biovolume. The surface to biovolume can be considered as reflecting what fraction of the biofilm is in fact exposed to the nutrient flow, and thus may indicate how the biofilm adapts to the environment. For instance, it could be speculated that in the environments of low nutrient concentration, the surface to biovolume ratio would increase in order to optimize access to the limited supply of nutrients [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF]. In our study, it may reflect also the response of bacteria to low concentration of silver ions release from the substrate.

 The average diffusion distance has been also calculated. The diffusion distance is defined as the minimum distance from a given living cell to The results are expressed in terms of mean values and standard deviations for two samples of each substrate including positive and negative controls.

Significant differences between sample sets were determined using the classical parametric test for mean comparison, so-called Student test (t-test)

(n-1 degree of freedom).

To test the reproducibility, bacterial colonization, biofilm formation and the effect on the planktonic bacteria experiments were repeated several times. The number of repetition for effect on planktonic bacteria experiment was 2 times.

The number of reproduction for bacterial colonization after 4h of incubation and biofilm formation after 168 h of incubation was 3 times using Live/Dead dye.

Additional two times repetition were performed for those studies using only Syto ® 9 dye. Graphs presented in following results are average of the experiment repetitions.

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The potential growth inhibition of planktonic bacteria free-living in liquid medium surrounding immersed samples was evaluated.

Effect on bacterial colonization of the substrates

We have quantified the number of adherent bacteria on the different surfaces in order to examine the bacterial adhesion, biofilm growth, biofilm structure and is found to be significantly different with p>99.9%. This result suggests that barrier effect of plasma polymer overlayer, which was not pronounced at 4h, is playing a role for longer incubation time. For bacteria in planktonic state also, the barrier effect of PP/Ar/MAPP/Ag/MAPP sample was found to have a significant efficiency at 168 h, while it was inefficient at 4h to block the antibacterial inhibition effect of Ag + released from the coating. A slightly significantly higher colonization was observed on PP/Ar/MAPP/Ag/MAPP than on PP/Ar/MAPP(2 nd ), which may be attributed to the presence of silver in PP/Ar/MAPP/Ag/MAPP samples. Ag + released from the unstretched material resulted in a significant decrease of the colonization at 4h, due to the antibacterial effect of Ag + . In contrast, this weak quantity of Ag + may have been too low to lead to antibacterial effect at 168h, but may have stimulate the growth of biofilm as a protection environment for bacteria [454,455]. Parameters that can be modified to increase the bacterial killing of this system is the quantity of AgNPs which serves as a reservoir, as well as the size distribution, which is an important issue to optimize surface to volume ratio of AgNPs and therefore available surface for oxidation for subsequent Ag + release. The quantity of silver in the plasma polymer layer can also be modified by changing the thickness of plasma polymer. In this way, the efficiency of the system could be modulated. ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP respectively. Probability is noted with the number of symbols: 1 symbol for p>95.0%, 2 symbol for p>99,0%, 3 symbols for p>99,9%. Abbreviations MAPP [START_REF] Kost | Responsive polymeric delivery systems[END_REF] the bacterial viability on surfaces containing amino groups was found between 70-90%. In the work of Kostenko et al. [457] who studied silver-containing dressings on bacterial biofilm viability, the value of bacterial viability observed after 24h was found between 2.6% and 99.8%, depending on the type of bacteria and material used. In our study, viability on the silicon wafer placed during experiment at the bottom of the Petri dish, was also of about 80%. Thus, in this study, 80% viability is considered as a reference for further discussion.

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The lowest viability was observed for the bacteria adhered on PP surface.

However, the highest number of bacteria was observed for the same surface.

This suggests that here, bacterial membrane changes may not be an indication of viability but rather the result of a more advanced step for the formation of biofilm. Adherent bacteria adapt to the biofilm phenotype. They change their sensitivity to the environment and their activity (integrity of membrane for better uptake of nutrient and oxygen) by adjusting the gene expression pattern when they are included in a biofilm [458]. In particular, it was already shown that a part of the bacteria of a biofilm have membranes allowing bacterial staining by propidium iodide, the molecule used for indicating compromised membranes [390,439,459]. Therefore, the highest bacterial adhesion but highest permeability of the bacterial membrane on PP surfaces compared to MAPPcovered surfaces may suggest that biofilm formation is in a more advanced stage. @@@ *** @@@ *** @@@ *** The rate of connected biomass for polypropylene and polypropylene modified substrates, corresponding to the ratio between connected and total biomass, is Results of the study on connected and non-connected bacterial population can be divided into two groups. In the first group, the rate of connected biomass value is above 50% (Figure 4 suggest that the synergistic effect is principally due to the "structuration" of the biofilm resulting from the Ag + treatment.

Membrane of the bacteria in biofilms

For 168 hours of bacterial culture, the state of bacterial membrane was investigated for both bacteria populations in connected biofilms and in nonconnected populations. In general, state of bacteria with compromised membranes are similar on all the substrates (between ~50% and 65% for non-connected biofilms and between ~30% and ~40% for connected biofilms) except on the PP/Ar/MAPP/Ag substrate (~50% for both connected and non-connected biofilms). To understand these results, it must be considered that, in the case of biofilms, red staining of bacteria with Live/Dead ® mixture is the result of increase of membrane permeability due to the balance of different effects: 1) the fluidity of the membrane, which allows permeability, decreases to prevent entrance of toxic agent i.e., here Ag + , and then to protect bacteria from biocide effect. This protection may be further enhanced by the specific biofilm-related conditions of life. Especially starvation, due to nutrient limitation in micro-environments of the biofilm, has been shown to increase antibiotic tolerance [START_REF] Fux | Survival strategies of infectious biofilms[END_REF]; 2) the membrane is compromised by the incorporation of Ag + ions, leading to increase in the membrane permeability [237]; 3) the specific physiology of the bacteria in biofilms can modify the membrane fluidity.

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It was shown that in the depths of a biofilm, bacteria showed similarities to stationary-phase planktonic cells [START_REF] Fux | Survival strategies of infectious biofilms[END_REF]. Both are affected by nutrient limitation and high cell densities, which result in an accumulation of metabolic waste products and/or extracellular signaling molecules [START_REF] Fux | Survival strategies of infectious biofilms[END_REF]. Therefore, bacteria in high-density biofilms are differentiated from low-density cultures which results

for example in different antibiotic tolerance. Population density is a factor which determines bacterial physiology. For example, in rhizobium, more cells survived stationary phase if starved at high density [461]. In E.coli, high-density (6x10 8 bacteria/ml) cultures expressed stationary-phase characteristics and reduced outer membrane permeability while going through the exponential phase of growth [459].

In the present study, therefore, differences in biomass (Figure 45678) measured for each type of the samples indicate that cell densities are different allowing to expect associated differences in the bacterial membrane permeability.

However, the other factors may counter-balance this effect due to the opposite influences on membrane permeability. Therefore, the reasons of weak differences between biofilms formed on the different substrates cannot be elucidated. Hence, the bacterial susceptibility may vary, which results also in differences in the permeability of the bacterial membrane.

Only, the rates of bacteria with compromised membrane in connected and nonconnected biofilms for samples with silver nanoparticles without second plasma polymer overlayer (PP/Ar/MAPP/Ag) follow a significantly different trend as on the other substrates. In this case, the rate of bacteria with compromised membrane in connected biofilm has very similar value to the one in nonconnected colonies (Figure 4 -14). This is in agreement with the faster release of silver ions (already after 30 min of sample immersion Ag + is detected; section

Relation between biofilm structure and bacterial membrane integrity in biofilms

To better understand the differences in biofilm structure on the various modified substrates, two additional parameters were calculated for bacteria with noncompromised and compromised membrane living in the connected biofilms: (i)

the "surface to biovolume ratio" (S/V). It reflects the fraction of the biofilm which is exposed to the surrounding (potentially nutrient, toxic, etc.) medium. It gives indications about the shape of the biofilm outer surface and may also indicate how the biofilm adapts to the environment (e.g., in environments of low nutrient concentration, the surface to volume ratio may increase in order to optimize access to the limited supply of nutrients [438]); (ii) the "average diffusion distance". It is the distance from living cell to the "void", that potentially contains nutrients, oxygen etc. The average diffusion distance gives therefore indications about compactness of the biofilm structure. In this study, "red" and "green" bacteria and or biofilm parts have been distinguished. The average diffusion distance of red part (ADD R ) is therefore the distance to void or green parts, while the average diffusion distance of green part (ADD G ) is the distance to void or red parts. The so-called G/R ratio was also calculated. biomass is distributed uniformly, intimately mixed with the "green" bacteria. All together, they form small clusters of bacteria. In case of biofilms developed on PP/Ar/MAPP/Ag/MAPP and PP/Ar/MAPP/Ag/MAPP-60%, "red" biomass has higher "specific surface" than "green" biomass, suggesting multiple clusters of "red" biomass distributed uniformly in a "green" biofilm matrix. Average diffusion distance of "green" biomass (ADD G ) is the average of diffusion distances among biomass pixels. Finally, results of ADD G show that, in the presence of Ag + , bacteria have difficulties to build thick complex structures. At the same time, the release of Ag + from PP/Ar/MAPP/Ag and PP/Ar/MAPP/Ag/MAPP-60% samples leads to the smallest "red" bacteria clusters and/or even single cells, present in large number in the biofilms developed on these substrates.

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gives the indication about roughness of outer surface of biofilm, G/R and ADD is sum up below in Table 4-3  Biofilm presents a non-well-cohesive structure i.e., small clusters of bacteria (lower value of "connected biomass" with higher value of S/V total parameter) at 168 h of culture  Bacteria with compromised membrane are in the form of small clusters or single cells (ADD R ), spread between bacteria with "non-compromised" membrane (G/R) at 168 h of culture

General conclusion and Perspectives

The objective of this work was to develop a new antibacterial mechanoresponsive coating for textile material. The need of implants with controlled antibacterial properties is increasing due to the risk of infections which follows the implantation of biomaterials.

In the first part of experimental section of this work, detailed description of low biomass value and with important amount of colonies which were not part of a biofilm. This is in agreement with a recent study that showed that even sublethal concentration of silver ions can remarkably increase the efficiency of antibiotics against gram-negative bacteria. Our study suggest that this may be due to the less cohesive structure (therefore less protective for bacteria) resulting from the action of Ag + .

These results give therefore new argument to use silver as antibacterial agent in implant materials. Even in low quantity, it may reduce the infection on site, and, additionally, in case of higher risk of infection, may enhance the efficiency of an antibiotic therapy.

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Results from this work, give a strong base for the future of intelligent silver containing materials that controls the release at the site of infection. For PP mesh materials, this way of preventing infection is very promising due to the low quantity of silver release sufficient to act on site against the infection.

Additionally, in case of higher risk of infection, the antibiotic therapy together with silver containing implant material may prevent the infection development and biofilm formation on implanted biomaterial.

The investigations included in this thesis give a strong base for the further development of antimicrobial polymer mesh-like materials and biomaterials surfaces that will eventually be used as implants. The new concept proposed in this thesis has been proved.

Further investigations should include the optimization of the developed system.

Plasma polymer second layer barrier properties should be improved by changing the crosslink density of plasma polymer, to provide efficient barrier effect for Ag + release. The quantity of silver nanoparticles which serves as an Ag + reservoir should be increased to prolong the antibacterial effect. This can be performed by changing the thickness of the first plasma polymer layer, which serves as a matrix for embedded nanoparticles. Control over size of nanoparticles should be improved, since this affects releasing properties of the coating. Better control over size of nanoparticles embedded in the plasma polymer could be achieved by changing the method of synthesis of silver nanoparticles to an in situ photochemically assisted synthesis. Further, the effects of ethylene oxygen sterilization method on the developed system could be studied, since this method of sterilization is also often used in the health care facilities and may only weakly affect the initial properties of the material.

Antibacterial efficiency of the system should be further investigated under flow culture conditions by real time observation. This will allow to study bacterial attachment and detachment involved during bacterial adhesion and biofilm 5-235 growth on these substrates. Additional studies could involve antibacterial properties study conducted in a modular bioreactor where it is possible to apply series of stretching and retraction cycles to the material ("similarly" to [462]).

Further fundamental investigations of influence of Ag + release on biofilm structure are also possible using developed system and Comstat 2 software.

Parameters such as "thickness distribution", "area occupied in layers", "substratum coverage", "microcolonies at substratum", "volume of microcolonies at substratum", "fractal dimension" or "dimensionless roughness coefficient" can serve to provide profound study on the influence of low concentration of Ag + release on biofilm development and structure. For example, "volume of microcolonies at substratum" parameter can serve to investigate how microcolony volumes vary under different Ag + release kinetics, or to establish whether there exists an "optimal micro-colony volume" depending on the quantity of Ag + released from the material, so that micro-colony growth ceases when a certain micro-colony size is reached.

Further investigations using clinically relevant cell lines such as fibroblasts and better simulation of the in vivo conditions should be the next step towards confirmation of the absence of cytotoxicity of developed system.

ANNEX

6.1 Cytotoxicity of plasma polymer modified mesh substrates

Introduction

Abdominal surgery is associated with significant risk of incisional hernia (10-20%) (see Figure 6-1) and gynecological defects operation [421,463,464]. The best and often used method to repair hernia is use of synthetic mesh that bridges the abdominal wall defect in a tension-free way [418] (see Figure 6-2).

Mesh implants are also used in vaginal prolapse repair of the anterior vaginal wall. One of the complications after mesh implantation is infection. This is described in details in the previous section -4.1. Although for the gynecological operations the cellular adhesion on meshes is not a problem, in case of abdominal wall defect operation adhesions between the visceral side of the mesh and adjacent organs can take place [465][466][467] and become a serious complication [428,467]. Such adhesions between the implanted material and organs can lead to chronic pain, recurrence of the hernia, intestinal obstruction or enterocutaneous fistulas. The ideal mesh for vaginal prolapse repair should prevent infection but favor cell adhesion. For abdominal wall defect operation, ideal mesh should also show integration with the tissue of the abdominal wall (primarily to prevent mesh migration and hernia recurrence), but no adhesions on the visceral side. This approach was explored by Van't Riet et al. [465] to develop non adhesive coatings. They performed an in vivo study, in which they implanted meshes that were coated with collagen on their visceral side. There was a clear positive effect of the collagen coating, with less visceral adhesions. Other studies have shown less successful results with different coatings from a biological origin such as sodium hyaluronate /carboxymethylcellulose [465,468] or an absorbable layer of oxidized regenerated cellulose [465,469,470]. However the long term prevention should be also provided by this type of coatings. Indeed, considering that biological coatings are susceptible to hydrolytic or enzymatic degradation, it can be expected that these coatings will not provide long-term non-adhesiveness [471]. In the study of Emans et al. [471], a synthetic and 6-241 biocompatible hydrophilic coating on the PP mesh was tested. The coating was based on N-vinylpyrrolidinone (NVP) and N-butylmethacrylate (BMA) as hydrophilic and hydrophobic building blocks, respectively. The coated mesh was compared in a series of in vivo experiments (using a rat model) with the uncoated PP meshes. The histological evaluation after 30 days follow-up, have

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shown that in case of PP-coated meshes no adhesions were observed and in case of PP-uncoated meshes adhesion and tissue granulations were present.

At 30 days follow-up the implanted material is expected to not form any new adhesions. Thus, the results of this study showed that stable hydrophilic polymer coating has a beneficial effect on adhesion formation after implantation of a mesh.

The authors suggested also that "A hydrophilic coating (degradable or stable) may be exploited in another manner such as a reservoir for controlled release of an antibiotic agent. A hydrophilic surface per se may play a role in the prevention of bacterial adhesion [332], while controlled swelling of the coating after implantation may provide a mean to engineer and control the release kinetics". Bringman et al [472] in an article which sum up over 50 years of advances in hernia repair techniques, concluded that ideal mesh prostheses would be a monofilament mesh that would prevent adhesions, yet still enable growth of the adjacent tissue for optimal augmentation.

As it was showed in the section 4.3.2.1.2, the presence of plasma polymer coating and presence of plasma polymer multilayer system deposited on PP mesh has a beneficial influence for preventing the bacterial colonization on the mesh material. In the following section the preliminary study of the biocompatibility of the developed plasma polymer multilayer coating deposited on PP mesh is evaluated.

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In-vitro cytotoxicity test by direct and indirect (extracts) contact

Firstly, the eventual cytotoxicity of products that could be released from substrates was evaluated. Extracts were prepared in accordance with

International Standard ISO 10993-12 (1996). Substrates were totally immersed in cell culture DMEM medium (Sigma-Aldrich, D5546) containing penicillinstreptomycin (Sigma-Aldrich, P0781) and L-glutamine (Sigma-Aldrich, G7513) and incubated at 37°C for 24h under static condition. Two conditions were tested: extraction in culture medium with or without addition of foetal bovine serum (VWR, BWSTS1830). Control groups included Phenol (dilution at 25% of a solution at 6.4 gL -1 in culture medium) as a positive cytotoxicity control and

Thermanox ® coverslips extraction medium as negative cytotoxicity controls. In parallel, cells were seeded in 24-well culture plates (80000 cells/ml of medium)

and incubated for 24h to allow attachment. Then, the culture medium was replaced with 500 µl of test extract or control extract. Cytotoxicity was assessed after a further 24h of incubation. After this time, viability was evaluated using MTT test (Sigma-Aldrich, M5655). Three samples were used for each experiment. For each sample, measurements of the optical density using 570nm and 620nm for reference were obtained on three aliquots.

Secondly, the cytotoxicity of substrates directly in contact with cells was evaluated. Four types of substrates were used: PP, P/Ar/MAPP(2 nd ), PP/Ae/MAPP/Ag/MAPP, PP/Ae/MAPP/Ag (see Table 6-1). Substrates were immersed overnight in complete DMEM medium (Sigma-Aldrich, D5546) containing penicillin-streptomycine (Sigma-Aldrich, P0781) and L-glutamine (Sigma-Aldrich, Sigma G7513). Further, cells were seeded at 80000 cells on each sample and 35000 cells on control Thermanox ® coverslips in a 24-well polystyrene plate coated with a layer of 2% agar in the bottom of each well to avoid adhesion and proliferation of cells on polystyrene around or below the substrates. Cells were incubated for 24 hours in 37°C and 5% of CO 2 containing 6-244 atmosphere . After this time, viability using PrestoBlue (Lifetechnologies, A-13261) was evaluated.

Cell adhesion on substrates was also observed by confocal microscopy. For that, cells were fixed with a solution of 2% (v/v) paraformaldehyde in NaK2P buffer for 30 min and permeabilized with 0.1% (v/v) of Triton X-100 for 15 min.

Actin filaments were labeled during 1 hour using phalloidin-FITC at a concentration of 0.4 µg/ml -1 .

Results

The extraction test with culture medium without serum demonstrated that all test specimens were non-toxic compared with the negative control (see Figure 6-3).

Indeed the viability of all tested samples was higher than 75% of the negative control (Thermanox ® ) that is considered as the threshold of cytotoxicity. The PP/Ar/MAPP(2 nd ) sample was shown to elicit significantly higher cell viability compared to negative control. Additionally, PP/Ar/MAPP/Ag substrate has shown significantly higher (p>95.0%) toxicity compared to PP sample. The least cell viability was logically found for the positive control (p<0.001).

T h e rm a n o x ® p h e n o l P P M A P P (2 ) A g M A P P The response of STRO1A+ cells in direct contact with the tested materials was studied. However, it appears that the absorbance measured by UV-Vis spectroscopy for PP samples and PP modified samples was extremely low representing less than 500 cells per sample. Therefore, the cell adhesion on above mentioned substrates was observed by confocal microscopy (Figure 6-6). From these images, the conclusions can be drawn that cells are effectively not able to adhere on the tested substrates. Very few adhering cells were visible 6-247 on each mesh fiber and they were less spread than on Thermanox ® negative control. An auto-fluorescence due to PP/Ar/MAPP/Ag/MAPP coating was observed.

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Discussion

Differences in sensitivities were observed from the results of the three tests performed, despite the fact that factors such as type of cells used and endpoint viability assay were standardized. The ranking order of substrates in the extract tests performed with and without serum was found to be slightly different. In the test with medium without serum, PP/Ar/MAPP/Ag substrate was found to be significantly different compared to negative control while it was not the case in the test with medium containing serum. In this last case, the leaching of components (silver ions) from PP/Ar/MAPP/Ag substrate may be insufficient to Moreover, although using extracts in a culture medium at 37°C for 24 h is a common procedure according ISO guidelines, the importance of the duration of extraction may play a role and need to be optimized to ensure complete elution of the leached components. Dilution of extracts must be also used to definitively conclude for PP/Ar/MAPP(2 nd ) and PP/Ar/MAPP/Ag indirect cytotoxicity.

However, as the testing method using medium containing serum is closer to the in vivo conditions, we can already conclude that silver release from samples should not have any cytotoxic effect on cells of the surrounding tissues.

The direct observations using confocal microscopy of cell adherence on PP and PP modified substrates revealed a very low affinity of the cells for these substrates (see Figure 6-6). This can be explained either by an unfavorable surface chemistry or surface morphology. Concerning the surface chemistry, PP is known to be unfavorable to cell adhesion due to its hydrophobic surface.

Efforts are made to tailor polypropylene surface more favorable to cell attachment, often using plasma discharge surface treatments [475][476][477][478].

Increase of surface energy, roughness and incorporation of various functional groups on PP surface often results in improved cell adhesion [479] even after only one day [476]. In spite that polypropylene surface is not favorable for cell attachment, in vivo study of polypropylene lightweight meshes revealed adverse effects of this material, resulting in high risk of adhesions [465,471]. Nevertheless, as the cell adhesion was low on mesh fibers whatever their chemistry, it can be concluded that it may be more probably the topography of the fibers that is unfavorable for cell adhesion than surface chemistry. Indeed, Bigerelle et al. [480] who studied the adhesion of mesenchymal stem cells on electro-eroded titanium surfaces presenting peaks and valleys morphology, showed that when the distance between surface features (peaks or valleys) was equal to 110 µm, the number of adherent cells drop drastically to 50 %. With this level of roughness, the peaks and valleys were slightly wider than the cell size but not enough that the cells will see a flat surface. The surface curvatures at this level are detected by the cells and may constrain the cell body impeding Hence, from both of the studies [480,481] it can be concluded that in case of PP mesh and PP modified mesh substrates, cell adhesion may be impeded because of the curvature of fibers. Additionally since the adhesion on fiber was not favorable, the manipulation during preparation of samples before examination under confocal microscopy (move of the plate when removing from incubator) could strongly influence the number of cells adhered on the surface of the fiber and must be better controlled.

Conclusions

In summary, plasma polymer coatings deposited on polypropylene mesh substrate have not shown cytotoxic effects compared to controls. The effects of silver ions release from samples are minor in conditions close to the in vivo ones i.e. in presence of serum. The adhesion of cells on meshes is limited probably because of the morphology of fibers. Considering chemistry of the surface, the application of a modified mesh material should serve well in gynecological defects operations. Also, the objective to produce meshes able to prevent cell adhesion for abdominal wall operations but without leaching cytotoxic molecules, is also possible to be reached.

However, the cytotoxicity tests performed herein using an established cell line would serve only as a general and preliminary study. Selection of appropriate testing method, as well as the use of more clinically relevant cell lines should be 6-252 the next step towards confirmation of the absence of cytotoxicity of plasma polymer modified multilayer systems. Use of flat surfaces to facilitate this study must be performed. Moreover, further research should concentrate on better simulation of the in vivo conditions within cell cultures.

Figure 0 - 1 :

 01 Figure 0-1: Plasma multilayer system consisting in two plasma polymer layers and a silver nanoparticles reservoir.

  of different types of structures of the biofilms on the studied materials.

  pour les substances bioactives (dans notre cas les ions argent). Avant étirement, la libération spontanée des ions argent par simple diffusion aux travers des couches barrières peut être contrôlée en jouant sur le taux de réticulation des couches plasma. Au cours de l'étirement, le contrôle réversible de l'ouverture des fissures permet une libération maîtrisée des ions argent (Figure 0-1bis). Dans le domaine des textiles et d'autres biomatériaux souples, cette stratégie est prometteuse en raison des contraintes mécaniques qui se produisent naturellement sur le site de l'implantation.

Figure 0 -

 0 Figure 0-1bis: Système multicouche plasma constitué de deux couches de polymère plasma autour d'un réservoir de nanoparticules d'argent.

Figure 0 - 1 :

 01 Figure 0-1: Plasma multilayer system consisted of two plasma polymer layers and silver nanoparticles reservoir.

Figure 0 - 3 :

 03 Figure 0-3: Release of Ag species followed by UV-Vis spectroscopy sample during elongation (60%) /retraction (0%) cycles (100% of elongation means twice the initial length).

Figure 0 - 4 :

 04 Figure 0-4: Biofilm formation study of E. coli SCC1 bacteria, conducted on the neat polypropylene and plasma/silver nanoparticles-modified surfaces. Typical images illustrating differences in biofilm quantity after 168 hours of incubation in static conditions.

Figure 1 - 2 :

 12 Figure 1-2: Various applications of polymer biomaterials as implants. Illustration from Ramakrishna et al. [79].

Figure 1 - 3 :

 13 Figure 1-3: Three examples of the many possible points of entry for bacteria are shown here, along with common areas of secondary infection (highlighted in green). Adapted from Center of Biofilm Engineering, with permission from the authors.

  and is carried out in health-care facilities by physical or chemical methods[101].Sterilization of biomaterials is needed due to exposure of these materials to microorganisms during the production step. The current methods include dry heating, autoclaving, irradiation (Gamma ray or Electron beam) and gaseous chemicals (Figure1-4). The dry heat and autoclaving in high temperature (120-180°C) and high pressure (100 kPa) sterilization methods are efficient, but the exposure of material to these conditions, especially polymeric material, may induce changes in material properties e.g., mechanical, chemical and structural changes[103][104][105]. In case of hydrophilic polymers, using heat and steam as methods of sterilization render the implant mechanically weaker [106] or less biocompatible[107].

Figure 1 - 4

 14 Figure 1-4 Sterilization methods used for biomedical polymers [94].

  Polymeric materials offer great possibilities in the materials design and properties.Antibacterial (i.e., preventive and bactericidal) properties can be obtained through various strategies. From the exploitation of intrinsic properties of a material or a coating surface, the most common strategies for the incorporation of biocidal agent in the material possibly leading to the release of 1-47 the agent, are presented below. In the first part of this section, the influences of topography, chemistry and mechanical properties of the surface on bacterial adhesion and bacterial colonization are described as potential ways to provide bacterial colonization preventive properties to the material. In the second part, antibacterial strategies based either on the release of bioactive agent or on the contact-active principle are shown (Figure1-5).Contact active polymers exhibit biocidal action through contact with bacteria [126, 127] (Figure 1-7 a). For inert polymers, surface modifications such as chemical grafting [128, 129], layer-by-layer deposition [130, 131] or plasma polymerization [132] can render polymer surface able to exhibit contact active antibacterial properties (Figure 1-7 b) for example by preventing bacterial attachment [133].

Figure 1 - 5 :

 15 Figure 1-5: Principles for antibacterial properties of polymers through, releasing or contactactive principle.

Figure 1 - 6

 16 Figure 1-6 The shape of surface features is important in determining the number of cells retained at any directionality. A) surface grooves close together; b) surface grooves of microbial size; c) wider than cell size surface features. Illustration adapted from ref. [140]

Figure 1 - 7 :

 17 Figure 1-7: Antimicrobial polymeric materials. (a) antibacterial strategy expressed by bulk material, (b) biocidal macromolecule attached to the polymeric material surface, (c) antibacterial agent released from polymer matrix (d) antibacterial agent released from coating.

  decades[219] when firstly biologically active polymers of 2methacryloxytroponones were described[220]. These polymers and polymethacrylate containing side biguanide groups and polyvinylbenzyl ammonium chloride) or N-halamine groups in different areas of the polymer structure[221] have been reviewed considering their process of synthesis[222], chemical diversity of antimicrobial systems[174], and different approaches of preparation[174] (synthesis of novel monomers, attaching bioactive substrates such as quaternary ammonium or phosphonium groups, attaching cyclic N-halamine compounds to synthetic monomers or polymers, or modification of naturally occurring macromolecules). Their antimicrobial activity have been considered as well[174]. In particular, mechanisms of action and applications have been reviewed recently[223].Tew et al. [224] were focused on antimicrobial polymers that that can mimic the biological activity of the antimicrobial natural host-defense peptides. Biological activity of these systems is considered to be influenced by the amphiphilicity of the polymer or oligomer as a whole, rather than the activity of an antimicrobial moiety either embedded or covalently attached. Majority of antimicrobial polymers are designed as cationic hydrophilic-hydrophobic macromolecular systems[223]. Target for those system to reveal antibacterial action is cytoplasmic membrane which regulates the transfer of solutes and metabolites between cytoplasm of bacterial cell and external environment[225]. Bacterial membrane has selective permeability properties which gives the opportunity to damage the structural organization and integrity of this membrane. Polymers are designed to have a hydrophilic polar functional block bearing cationic charge and a hydrocarbon non-polar hydrophobic block or are formed with random copolymers formed by a hydrophobic monomer and a hydrophilic comonomer with a functional group[174, 186, 222, 226]. These structures then, have high binding affinity for bacterial cells[226]. For example, in case of N-halamines, polymers with Nhalamine functional groups have been developed to stabilize the antimicrobial properties of free halogens such as chlorine or bromine. In N-halamines, one or more halogen atoms are covalently bonded to the nitrogen atoms of the Bibliographic synthesis 1-65 compounds providing stability and slow release of free active halogen species into the environment. The main biocidal impact of the N-halamines is coming from a specific action of oxidative halogen rather than polymer action itself. Cl + or Br + halogen targeted at a biological receptor such as thiol groups or amino groups in proteins upon direct contact with a cell, leads to cell inhibition or cell inactivation[223].

  variety of infections[227]. Metallic silver is inert in the presence of human tissues but ionizes in the presence of moisture, body fluids, and secretions to release the biologically active Ag +[228]. The release of Ag + depends on the size of metallic nanoparticles[229]. For example release is accelerated by particle size reduction[230]. In consequence, size-dependent interaction and properties of nanoparticles insight researchers to explore potential benefits from implementing disinfecting filters, drug delivery systems and coating materials with nanosilver[194, 231].

[

  228]. However the level of concentration needed to control the bacterial growth is most probably depending on the type of bacteria and experimental conditions (e.g., type of medium). The mechanisms of this efficiency are only partially understood so far. One possible mechanism is based on the fact that silver ions have the capability to exchange with the ions in the environment (e.g. Ca + , Zn + ) and attach to bacterial cells [232]. Ag ions bind protein residues [233] on cell membranes of sensitive bacteria and are adsorbed intracellulary by pinocytosis [228]. The consequence of the ions binding to cell membrane is the damage of it, the disturbance of bacterial electron transport and of the production of cell's energy by impeding the ATP production. Ag + species also interact with the oligonucleotide bases in DNA and affect DNA ability to replicate [227]. Oxidative stress can also play a role in the mechanisms of DNA damage [234]. Another potential way of antimicrobial properties of silver ion can come from inhibition of respiratory enzymes through reactive oxygen species (ROS) formation [235, 236]. Also, Ag + indirectly leads to Fe 2+ leakage by stimulating the production of Bibliographic synthesis 1-67 superoxide, disturbing internal iron homeostasis and by directly or indirectly disrupting intracellular Fe-S clusters [237]. It was found also that attachment of silver ions (or nanoparticles) to the cell wall caused accumulation of envelope protein precursors which resulted in immediate dissipation of the proton motive force [238]. Mechanisms of action are based also on interaction between Ag + and phosphorous-and sulfur-containing compounds found in variety of proteins [239]. Ag + disrupts disulfide bond formation in vivo, which affect protein shape, functionality and stability [237].

  types of materials like silver-impregnated zeolite powders and activated carbon materials[259, 260], dendrimer-silver complexes and composites[261][262][263] were also synthesized. Today, the possibility to impregnate or coat alginates, polyurethane, silicones and textile fibers with ionisable silver compounds provides clinicians with efficacious means of prevention from infections in wound care and device-related infections. The particles or other forms of silver can be incorporated into the bulk of the material by mixing (composite) or by a surface modification procedure (coating).

  increased the antimicrobial effect by two-to-three orders of magnitude comparing to antimicrobial activity of a silver-containing surface[264]. This approach was based on decorating bigger 20-50 nm carrier particles consisting of a phosphate-based, biodegradable ceramic, with silver nanoparticles. When growing, microorganisms were dissolving the carrier particles and the release of silver was triggered. Electrospun, fibrous and porous poly(lactide-co-glyclolide) (PLGA) was used as a matrix for composite consisting in silver (0.5 wt%) on Bibliographic synthesis 1-70 amorphous tricalcium phosphate nanoparticles. Authors performed antibacterial tests which showed a prolonged effect of the scaffolds with silver uniformly dispersed on the tricalcium phosphate, comparing to current clinically used methods, based on soaking the scaffolds with a tetracycline solution prior to implantation [265]. Hydroxyapatite (HA) coatings with incorporated silver species have also shown decreased probability of infection and decrease of the biofilm growth [266, 267]. The results showed that, HA coatings expressing antimicrobial properties and releasing Ag ions killed methicillin-resistant Staphylococcus aureus in vivo. Multilayer approach has been also investigated for the development of silver embedded films adherent on SiOx surface. Ag nanoparticle-embedded synthetic catecholamine polymer films showed enhanced antibacterial effects compared to the films without Ag and the bare SiOx surface [273]. Eksik et al.

  using layer-by-layer (LbL) deposition method[278][279][280][281][282][283]. Interactions used to fabricate such films include ligand -silver ionligand bridges [278] and covalent bonding. Often the deposition is based on alternating adsorption of oppositely charged polyelectrolytes[278, 284]. These multilayer films are used as reservoirs for silver.Dai et al. [278] have used Polyethylene-imine (PEI) stabilized Ag colloids in solution to synthesize antibacterial films by performing alternative adsorption of polyacrylic acid and PEI stabilized colloids using LbL technique. The reduction of silver ions was performed by addition of NaBH 4 solution. Prepared LbL films which contained silver nanoparticles or silver ions have shown to inhibit bacterial growth. Similarly Li et al. [280] have used LbL method for synthesis of poly(acrylic acid) -Ag 0 /poly(allyl-amine hydrochloride)

Figure 1 - 8 :

 18 Figure 1-8: Different states of matter.

1 . 3 . 1 . 1 . 1

 13111 therefore, no contact between the electrodes and the plasma is required. The plasma can be initiated and sustained by external electrodes, at much lower voltage that is required for maintaining a direct current glow discharge[290, 291] Plasma polymerizationThe term « plasma polymer » usually describes a polymer film obtained by the activation of an organic precursor (often a gas) with electric impulses. The polymer film obtained often presents a molecular structure different from the ones obtained by other conventional techniques. As shown on Figure1-9, a plasma polymer presents a heterogeneous chemical structure due to the high rate of the activated monomer recombination during the film formation, leading to a highly cross-linked and amorphous film, while conventional polymers are composed of a single unit of monomer repeated regularly. The structure of plasma film can be tailored by the experimental conditions during the process: using strong dissociating conditions for the monomer results in a film being highly cross-linked, while smoother conditions often lead to a structure close to films obtained by conventional methods (degree of retention).

Figure 1 - 9

 19 Figure 1-9 Schematic representation of monomer, conventional polymer and plasma polymer.

Figure 1 - 79 Figure 1 - 11

 179111 Figure 1-10 (a) Electron density distribution in function of their energy [292]. (b)Table with values of often studied bonding energies.

  First step: Activation of the precursor (creation of free radicals) by collisions between the precursor molecules and the active species (electrons, ions, radicals) in the plasma phase. Second step: The polymerization starts by chemical reactions between the activated and non-activated molecules of precursor, in the plasma phase or onto the substrate surface. Third step: Polymerization stops with the recombination of two activated molecules (two molecules of activated precursor and/or a molecule of activated precursor and an activated specie from plasma phase (electrons, ions, radicals)) Even after the termination and the obtainment of a stable polymer film, the latter can still be re-activated by the surrounding plasma phase, as represented by cycle n° I and II shown on Figure 1-12 (cycle I: reactivation by mono radical / cycle II: activation by bi-radicals).

Figure 1 - 12 :

 112 Figure 1-12: General mechanism of plasma step growth.

  parameters and the precursor's flux is necessary. To allow standardization in the comparison between plasma depositions, H. Yasuda introduced the W/FM parameter [307] (W is the power of the discharge, F the precursor's stream and M the molecular weight of the precursor). The W/FM parameter corresponds to an energy brought to a unit of precursor mass. The Figure1-13 shows the deposition kinetics as a function of the W/FM parameter.

Figure 1 - 13

 113 Figure 1-13 Polymer deposition kinetics as a function of W/FM parameter, (W is the power of the discharge, F the precursor's stream and M the molecular weight of the precursor), according to the theory of H. Yasuda [307].

Equation 2

 2 Better functional retention can be achieved using low DC values and high values of input power. During the whole cycle, the equivalent power received by substrate is lower than input power. Use of pulsed plasma over continuous plasma has advantages like increased operational stability, reduction of trapped radicals in the film, lower deposition surface temperatures and decreased highenergy ion bombardment and UV flux to the surface [317]. Control over the chemistry of plasma deposited film can be achieved with better efficiency by use of radio frequency plasma and variation of plasma-on and plasma-off times, instead of continuous wave plasma. With relatively long plasma-off time, the domination of radical-monomer type processes leads to the structures which reassemble standard polymer-like structures [317-320]. Varying plasma-on time and plasma off-time it is possible to obtain plasma polymer film through free radical polymerization (a chain polymerization in which the kinetic-chain carriers are radicals [321]). For monomers, which have in structure double or triple bonds, the growth mechanisms take place during the off-time, retaining the original chemical structure of the monomer with the preservation of desired functionalities. Pulsed plasma improves the chance to achieve more regular structures using classic monomers with great tendency of chemical polymerization [322-326]. Possible modifications of surface properties such as electrochemical charge or amount of oxidation as well as attachment or modification of surface-bound chemical groups can be achieved by plasma processes. Properties like hardness, resistance to chemical corrosion or physical abrasion, wettability, water absorption capacity and as well affinity to specific molecules can be tailored by choosing specifically and precisely the plasmas [327]. 1-86 d. Other parameters

1 .

 1 At this stage, the plasma was ignited and run for 30 min. The deposition conditions corresponded to: power output = 20 W, duty cycle = 2% and frequency = 816 Hz and deposition rate = ~0.3  0.2 nm.min -1 . Upon completion of deposition, the R.F. generator was switched off and the flow of monomer was maintained in the system for a further 2 min period prior to venting up to atmospheric pressure. Prior to use, plasma polymer modified substrates were immersed in deionised water to promote the hydrolysis of maleic anhydride groups into dicarboxylic acid groups (PP/Ar/MAPP). Then, silver was loaded directly on the PP/Ar/MAPP surface by immersion of the coated substrate into a 1.0 mM silver nitrate solution for 30 min. Silver cations diffused into the reservoir and formed electrostatic pairs with carboxylate groups. Ag + ions bound to carboxylate were reduced to zero-valent Ag nanoparticles using an aqueous solution of NaBH 4 (2.0 mM). Then, the PP/Ar/MAPP/Ag material was placed in the chamber of the plasma reactor and the system was pumped down to base pressure. Maleic anhydride vapor was introduced into the reaction chamber at a constant pressure of 0.2 mbar. The plasma was ignited and run for 10 min according to the following conditions: power output = 20 W, duty cycle = 50% and frequency = 816 Hz The deposition rate was ~12.5  0.5 nm.min -1 . Upon completion of deposition, the R.F. generator was switched off and the flow of monomer was maintained in the system for a further 2 min period prior to venting up to atmospheric pressure. The integrity of the plasma polymer layers was checked after immersion in a phosphate buffered saline (PBS) solution or 2-102 in the bacterial culture medium by scanning electron microscopy. Then, the final bioactive coating (PP/MAPP/Ag/MAPP) was sterilized either with an electron beam (Aerial, Illkirch, France) or dry heating (1 h, 100 °C). Modifications in the bioactive coating properties potentially induced by changing the procedure of sterilization have been studied and will be the subject of a future paper.

  photomultipliers. The light sources were deuterium (DL) and tungsten-halogen (THL) lamps (12V) working in the range of 190-3300 nm. Spectra were the result of the sum of 100 scans. Microsoft Windows based Perkin-Elmer UV Winlab software was used for data acquisition and analysis. Spectra were recorded between 250 and 800 nm. Two types of measurements were done using a UV-Visible spectrophotometer: i) UV-Visible absorbance of samples coated with the plasma polymer multilayer system and ii) UV/Vis absorbance of released Ag + and AgNPs. For the first type of measurement, the PP/Ar/MAPP/Ag/MAPP sample was placed in the holder of the instrument and the measurement was performed. For the second type, the release of Ag + and AgNPs from the plasma polymer multilayer system into distilled water was followed during stretching of the material. In this case, the limit of detection was 3 ppm AgNO 3 (see Figure2-18 in supporting information). For testing the effect of elongation on the release of silver, the sample was stretched under ambient atmosphere and immediately immersed in 9 ml of distilled water. After

  INSTRON 4505 (Zwick upgraded) dynamometer was used to perform tensile tests at room temperature. In all experiments, the initial length between jaws was 40 mm. During conventional tests, the rate was set to 5 mm/min. Each curve corresponds to the average of five experiments. During cyclic tensile tests, the rate was set to 10 mm/min for both loading and unloading steps. The number of cycles was 50 and the elongation was 20% or 60% of the initial length.

  were conducted with the PHL628-based E.coli SCC1 strain[380].This strain is auto-fluorescent through a GFP-based genetic insertion. Bacteria taken from frozen stock (-80 °C) were inoculated on an agar plate of LB medium for an overnight culture. One colony of bacteria was inoculated in 10 mL of LB medium and cultivated at 30 °C overnight for a first pre-culture. A second preculture was inoculated with the first pre-culture (10 vol-%) in 10 mL of selective M63G medium and cultivated at 30 °C overnight. A third pre-culture was inoculated from the second pre-culture (10 vol-% of the second pre-culture) for 4 h at 30 °C. The final culture used to perform the experiment was inoculated from the 4 h pre-culture(10 vol-%). Its absorbance at 600 nm (Abs 600 ) was adjusted to 0.01 (i.e, 10 6 bacteria/mL). Coated-mesh samples, previously sterilized by e-beam with a dose of 25 kGy, were placed into small Petri dishes (Ø 35 mm) on sterilized-in-oven (180 °C for 1 h) silicon wafers. Stainless steel wire was used to maintain the sample on the dish bottom. Samples were inoculated with 4 mL of the final bacterial culture and incubated for 168 h (i.e., one week) at 30 °C. Samples were then thoroughly and carefully rinsed to eliminate non-adherent or loosely attached bacteria from the substrate surfaces.For this purpose, 2 mL of supernatant (i.e. bacterial suspension for the first rinse and NaCl solution for further rinsing steps) were removed, kept for further bacterial concentration measurement and replaced by 2 mL of fresh NaCl 2-107 solution. The rinsing step was performed without direct flushing, with the stream directed toward the walls of a petri dish and with attention to the force and content of the rinsing fluid. The material samples remained immersed in liquid during the rinsing steps, without creating any air-sample interface. The rinsing step was repeated 16 times. Material samples were kept immersed in the last rinsing solution. They were then observed in situ by using an upright Confocal Laser Scanning Microscope (CLSM; Carl Zeiss, LSM700) equipped with a long working distance objective (LD EC Epiplan Neofluar 50x/0.55 DIC M27; working distance of 9.1 mm).Three-dimensional pictures were taken of at least at five different, randomly chosen locations on each sample. Image processing was performed using the ImageJ ® V.1.44a software with the LSM toolbox V4.0g plugin[381], allowing separation of fluorescence and reflection images from Zeiss LSM raw data.

Figure

  reinforcement implant) from Cousin Biotech. Images of this substrate are shown in Figure 2-1a. The diameter of each fiber was about 200 µm. The nominal aperture was estimated to 0.60 ± 0.05 mm 2 with 8 threads per centimeter and an open area of 47.7%. Such a mesh design can be elongated almost twice its initial length (100% elongation) before failure. It is obvious that the strength and extensibility of the mesh differed considerably according to the direction of stretching.

Figure 2 - 1 :

 21 Figure 2-1: a) Photograph (© CNRS Photothèque / Hubert Raguet) and scanning electron microscopy of Biomesh®P1 used in this work (Cousin Biotech) and b) typical loaddisplacement curves during transverse and longitudinal elongation.

Figure 2 - 1 (

 21 Figure 2-1 (b) shows typical loading curves recorded during uniaxial tensile tests using transversal or longitudinal elongation. In both cases, two differentregimes in the load-elongation curves were observed, evolving from a nonlinear to a quasi-linear regime. During transverse stretching, the material displayed an initially, relatively high stiffness (defined as F/ where F is the applied force and  the displacement produced by F) that gradually increased, changing to a high, linear stiffness above 30% of the failure strain. When the tensile was applied longitudinally, the same behavior was observed but the stiffness was much less and the transition between the non-linear and quasilinear regimes occurred at about 70% of maximal elongation. The corresponding failure strain was 3-fold less compared to transverse elongation.The origin of different regimes during stretching is well-explained in the literature[382]. The non-linear regime is mainly driven by the mesh design,

Figure 2 - 2 :

 22 Figure 2-2: X-ray diffraction pattern of a) polypropylene substrate, b) (a) coated with the first plasma polymer layer, c) (b) loaded with silver nanoparticles, d) (c) coated with a second plasma polymer layer.

  mesh, b) (a) expose to Ar plasma, c) (b) coated with a first maleic anhydride plasma polymer layer, d) (c) hydrolyzed, e) (d) loaded with silver nanoparticles, f) (e) coated with second plasma polymer layer; * The presence of gold is due to detection of the gold sample holder during analysis because of the mesh design.An anhydride containing plasma polymer layer was deposited onto the polypropylene substrate and subsequently hydrolyzed to form carboxylic acid groups. An activation step using Ar plasma was used in order to strengthen the interface between the polypropylene surface and the plasma polymer layer. XPS results revealed that exposure to Ar plasma led to an increase in oxygen content accompanied by the appearance of two new peaks at 288.0 eV and 289.4 eV belonging to C=O and O-C=O groups, respectively (see Figure 2-3spectrum b,

Figure 2 - 3 :

 23 Figure 2-3: High resolution C(1s) XPS spectra of a) polypropylene mesh substrate, b) (a) exposed to Ar plasma, c) (b) coated with a first maleic anhydride plasma polymer layer, d) (c) hydrolyzed, e) (d) loaded with silver nanoparticles, f) (e) coated with a second maleic anhydride plasma polymer layer.

Figure 2 - 4 : 4 -

 244 Figure 2-4: Infrared spectra of a) polypropylene mesh substrate, b) (a) exposed to Ar plasma, c) (b) coated with a first maleic anhydride plasma polymer layer and hydrolyzed, d) (c) loaded with silver nanoparticles, e) (d) coated with a second maleic anhydride plasma polymer layer.

Table 2 - 2 :

 22 Contact angle and thickness values measured on a) polypropylene mesh substrate, b) (a) exposed to Ar plasma, c) (b) coated with a first maleic anhydride plasma polymer layer, d) (c) hydrolyzed, e) (d) loaded with silver nanoparticles, f) (e) coated with a second maleic anhydride plasma polymer layer. * The overlayer thickness was in addition to the thickness for the Ag-loaded first layer.

  ). A change in the water contact angle (99 ° to 64 °) occurred upon argon plasma treatment, indicating increased interactions with water. Maleic anhydride plasma polymer deposition produced a slight increase in the contact angle (up to 66 °, a typical value reported in the literature for such plasma polymers [385]) followed by a decrease after hydrolysis (14 °) indicating here again increased interactions with water due to high surface coverage with carboxylic acid groups. A negligible change in thickness of the plasma polymer layer occurred after hydrolysis (Table2-2). The aforementioned treatment did not affect the crystalline character of the polypropylene fibers or the mechanical properties of the polypropylene mesh (see Figure 2-20 and Figure 2-19 of supporting information respectively).

  polymer thin film led to extraction of low molecular weight water soluble fractions of the film, thus creating a nanoporous structure which also helps the loading of Ag + ions [386]. Silver ions were therefore anticipated to diffuse within the entire thickness of the first plasma polymer layer. Then, Ag + ions were reduced to zero-valent AgNPs using aqueous NaBH 4 solution as a reducing agent. TEM images indicated that particles were generated (Figure 2-5a) with a broad size distribution centered on 38 nm (Figure 2-5b), and EDX analysis confirmed the presence of silver particles (Figure 2-21 in supporting 2-117 information). The thickness of the plasma polymer thin film loaded with nanoparticles was estimated to 92 ± 2 nm (Table

Figure 2

 2

Figure 2 - 5 :

 25 Figure 2-5: a) TEM images of the nanoparticle-loaded plasma polymer thin film and b) size distribution of the silver nanoparticles (inner graph shows results for particle diameters in the range of 2-20 nm).

Figure 2 - 6 :

 26 Figure 2-6: a) Optical images of untreated PP (top) and PP covered with the plasma polymer multilayer system (bottom). b) UV-visible absorbance spectra of I) polypropylene mesh substrate, II) (a) exposed to Ar plasma and then coated with a first maleic anhydride plasma polymer layer.

Figure 2 - 7 :

 27 Figure 2-7: High resolution Ag(3d) XPS spectra of a) polypropylene mesh substrate exposed to Ar plasma, coated with a first maleic anhydride plasma polymer layer, hydrolyzed and loaded with silver nanoparticles, b) (a) coated with a second maleic anhydride plasma polymer layer.

  7 eV and 373.8 eV, respectively (Figure 2-7spectrum b). Plasma polymerization results from the well-known competition between deposition and etching during process [388]. Both the coating and the surface were bombarded by ions from the plasma leading mainly to fragmentation during the deposition. Due to surface mobility, a fraction of the silver species could migrate to the nearsurface regions of the plasma polymer layer. TEM images of a cross-section of the plasma polymer system are shown in figure 8 (Figure 2-8a). They clearly show silver nanoparticles embedded in the first plasma polymer layer and covered by a second thick plasma polymer layer (Figure 2-8b). They also confirm the presence of nanoparticles in the near surface region of the plasma polymer overlayer. Careful survey of Figure 2-6b indicates a single absorption band at 480 nm before plasma deposition (Figure 2-6b -spectrum b), while this band was shifted to a lower wavelength (~ 460 nm) after plasma deposition (Figure 2-6b -spectrum c). It is well known that SPR absorption is sensitive to many factors, including the geometric parameters, microstructure and aggregated structure of the particles as well as the nature of the surrounding matrixes [389]. Physical and/or chemical changes in the interactions between particles and the polymer matrix during plasma deposition may induce changes in the electronic band structure and optical inter-band transitions of the particle 2-121 surface, which, as a consequence, affect the optical absorbance of the composite [386].

Figure 2 - 8 : 5

 285 Figure 2-8: TEM images of cross-sections of the plasma polymer multilayer system at two different magnifications.

Figure 2 - 9 :

 29 Figure 2-9: Schematic illustration of the oxidation process of silver nanoparticles.

Figure 2 -

 2 Figure 2-10 shows that the deposition of a second plasma polymer overlayer (MAPP, Table 2-3) significantly reduced the rate of Ag + release in comparison

Figure 2 - 10 :

 210 Figure 2-10: Duration of immersion until the presence of released silver was detected. In the case of MAPP II, no signal was detected even after 16 weeks.

Figure 2 -

 2 Figure2-11 shows SEM images of polypropylene mesh substrate coated with the plasma polymer multilayer system while mechanical strain was applied (e.g., 40%, 60% and 80% of elongation from the initial length, Figure2-11images a, b and c, respectively). The rate of elongation was 24 µm/s and the elongation was applied in the direction shown by the white arrows. As already mentioned, cracks in the overlayer were desired to create diffusive channels for Ag + species. Under stretching, plasma polymer multilayer coatings revealed remarkable fractures at their surfaces. The lengths of these fractures were perpendicular to the direction of elongation. This behavior was explained by the nature of the composite system that was strained: it was formed by the superposition of stiff layers at the top of a flexible polypropylene fiber. The fragmentation was thus relied on the transfer of the tensile stress of the substrate to the surface plasma polymer layer, as well as on the tensile properties and thickness of this layer[372]. In order to obtain accurate details on the density of the potential channels so created, image and statistical analysis were performed on 0.1 x 0.1 mm² area samples of polypropylene fibers. The density of the cracks and their size were found to be strongly correlated with the elongation state. The density of cracks and the total area of the apertures increased with the stretching ratio (Figure2-24a in supporting information), while the mean value of the apertures did not vary (Figure 2-24b in supporting information), indicating a uniform growth in crack size with increasing elongation. Interestingly, most of the apertures in the plasma polymer layer were between 40 µm² and 150 µm², independent of the elongation state (Figure 2-12).

Figure 2 - 11 :

 211 Figure 2-11: SEM images of the plasma polymer multilayer system undergoing stretching/unstretching cycles at (a)-(d) 40% of elongation, (b)-(e) 60% of elongation and (c)-(f) 80% of elongation.100% of elongation means twice the initial length.

Figure 2 - 12 :

 212 Figure 2-12: Size and number of cracks as a function of the stretching ratio.

Figure 2

 2

Figure 2 - 13 :

 213 Figure 2-13: Release of Ag species followed by UV-Visible spectroscopy in the case of a PP/Ar/MAPP/Ag/MAPP sample during a series of elongation/retraction cycles. Absorbance values correspond to mesh elongation or retraction maintained during 24 hours.

  After 24 hours of incubation, a film of bacteria was visible on the agar gel, indicating normal growth of the previously inoculated bacteria. Nevertheless, bacterial growth was inhibited, as expected, in the near surroundings of PP/Ar/MAPP/Ag samples, in agreement with silver delivery from the sample (Figure 2-14-image a). In contrast, bacterial colonization was not inhibited around the sample covered by a plasma polymer overlayer (PP/Ar/MAPP/Ag/MAPP), and was even observed between fibers of the mesh, in contact with the silver-containing coating (Figure 2-14-image b). The efficiency of the barrier effect of the plasma polymer overlayer was thus clearly confirmed. Importantly, the antibacterial effect was recovered under mechanical stimulation (Figure 2-14 -image c), which is related to the opening of cracks in the plasma polymer overlayer, providing diffusive channels for the antibacterial agent. This result thus highlighted the relevance of the open/close crack system to control bacterial colonization in the near surroundings of the biomaterial. The antibacterial efficiency of the open/close crack system on the growth of a biofilm was also evaluated.Figure 2-15 shows the quantity of biofilm (i.e., biomass) developed after 168 hours of incubation. 2-130 Biofilm development on PP/Ar/MAPP/Ag/MAPP was similar to on PP samples, indicating that Ag + species were not present in a sufficient quantity to affect the growth of the bacteria, in agreement with the barrier role of the second plasma polymer overlayer. In contrast, the biofilm quantity was significantly reduced on PP/Ar/MAPP/Ag and stretched PP/Ar/MAPP/Ag/MAPP samples, due to the greater release of Ag + . Inhibitions of biofilm development on stretched samples were moderate (around 50% compared to uncoated PP samples), but clearly demonstrate that the open/close crack system allows modulation of the bacterial colonization of the biomaterial.

Figure 2 - 14 :

 214 Figure 2-14: Bacterial growth on nutritive agar plates in the surroundings of a) a PP/Ar/MAPP/Ag system, b) a PP/Ar/MAPP/Ag/MAPP system and c) a PP/Ar/MAPP/Ag/MAPP system undergoing stretching.

  that, taking advantage of open/closed crack systems, it is possible to control the flux of an antibacterial agent and to modulate bacterial colonization of the biomaterial. This original approach offers a great opportunity to readdress the persistent challenge of obtaining effective and long-lasting antibacterial coatings.

Figure 2 -Figure 2 - 16 :(Figure 2 - 18 :

 2216218 Figure 2-17: a) Photograph (IS2M) and b) schematic illustration of the homemade stretching device.

Figure 2 - 19 :

 219 Figure 2-19: Typical loading-displacement curves of polypropylene meshes according to the direction of elongation and the stretching ratio (100% of elongation means twice the initial length). a) Transverse and 20% of elongation, b) transverse and 60% of elongation

Figure 2 - 20 :

 220 Figure 2-20: DSC analysis of polypropylene mesh: amelting peak , bcrystallization peak(first heating), c -crystallization peak (second heating). The degree of crystallinity was estimated to be 47 ± 5%. For this purpose, the samples of polypropylene were analyzed over the temperature range from -100 °C to 250 °C. A heating rate of 10 °C/min was used. The thermal history of the sample was erased by thermal treatments consisting of cooling the sample from 250 °C to -100 °C at 10 °C/min. The integrated area under the melting curve was calculated to obtain the enthalpy, which was further divided by the thermodynamic heating of fusion of 100 % crystalline polypropylene (207 J/g according to Vander Wal A, Mulder JJ and Gaymans RJ. Facture of polypropylene: 2. The effect of crystallinity.Polymer. 1998, 39, 5477).

Figure 2 - 21 :Figure 2 - 22 :

 221222 Figure 2-21: EDX analysis of the first plasma polymer layer after loading with silver particles (PP/Ar/MAPP/Ag).

Figure 2 - 23 :

 223 Figure 2-23: UV-Visible spectra of the release solution after immersion of the plasma polymer multilayer system in water, recorded before and after reduction with NaBH4 solution.

  auto-oxidation[391]. It consist in two steps[391]. The primary ageing step is that molecular oxygen reacts with C-radical sites. This results in the formation of 3-146 peroxy radicals (C-O-O•). Secondary reaction step is the decay of these peroxy radicals and formation of different oxygen functionalities like C=O and COO.Besides, there are many different sterilization methods available; each with different mode of action can contribute to modification of surface properties.

[

  394]. Steam sterilization, carried out in a temperature range of 120-130°C, does not affect molecular structure of polymer, however it leads to postcrystallization and physical aging effects[395]. Chemical sterilizing agents like ethylene oxide have a limited effect on polymer[392, 393]. However, an evidence of toxic potential of medical grade plastics sterilized using ethylene was showed[396]. Ethylene oxide diffusion in complex substrates is also an issue which has to be considered for sterilization and aeration conditions[397].Results of modern alternative sterilization processes such as electron irradiation, UV irradiation and the application of ozone is limited. All of these three methods are capable of forming radicals in polymers, which might result in similar degradation effects as the γ -radiation[392].Few authors have study more specifically the influence of sterilization step on plasma polymer coatings.Haddow et al. [398] demonstrated that plasma polymer modified surfaces containing carboxylic acid groups can function successfully after a standard ethylene oxide sterilization procedure. Study ofCalderon et al. [399] performed on plasma-polymerized allylamine films have shown good stability during autoclaving process. The influence of autoclaving, 3-147 γ-irradiation, ethylene oxide exposure and Ar/H 2 low pressure plasma treatment have been also investigated on the surface chemistry of thin films of plasma polymerized diethylene glycol dimethyl ether[400]. This study revealed that all types of sterilization methods influenced the surface chemistry.

1 .

 1 2 mbar) with a flow rate of approximately 1.6 x 10 -9 kg.s - At this stage, the plasma was ignited and run for 30 min. The deposition conditions corresponded to: power output = 20 W, duty cycle = 2% and frequency = 816 Hz. Upon completion of deposition, the R.F. generator was 3-148 switched off and the flow of monomer was maintained in the system for a further 2 min period prior to venting up to atmospheric pressure. Prior to use, plasma polymer modified substrates were immersed in deionised water to promote the hydrolysis of maleic anhydride groups into dicarboxylic acid groups (PP/Ar/MAPP). Then, silver was loaded directly on the PP/Ar/MAPP surface by immersion of the coated substrate into a 1.0 mM silver nitrate solution for 30 min. Silver cations diffused into the reservoir and formed electrostatic pairs with carboxylate groups. Ag + ions bound to carboxylate were reduced to zero-valent Ag nanoparticles using an aqueous solution of NaBH 4 (2.0 mM). Then, the PP/Ar/MAPP/Ag material was placed in the chamber of the plasma reactor and the system was pumped down to base pressure. Maleic anhydride vapor was introduced into the reaction chamber at a constant pressure of 0.2 mbar. The plasma was ignited and run for 10 min according to the following conditions: power output = 20 W, duty cycle = 50% and frequency = 816 Hz. Upon completion of deposition, the R.F. generator was switched off and the flow of monomer was maintained in the system for a further 2 min period prior to venting up to atmospheric pressure. Samples were then stored until use in ambient environment.

  or e-beam (25 kGy), were placed into small Petri dishes (Ø 35 mm) on sterilized-in-oven (180 °C for 1 h) silicon wafers. Stainless steel wire was used to maintain the sample on the dish bottom. Samples were inoculated with 4 mL of the final bacterial culture and incubated for 4 h at 30 °C. Samples were then thoroughly and carefully rinsed to eliminate non-adherent or loosely attached bacteria from the substrate surfaces. For this purpose, 2 mL of supernatant (i.e.3-152bacterial suspension for the first rinse and NaCl solution for further rinsing steps) were removed, kept for further bacterial concentration measurement and replaced by 2 mL of fresh NaCl solution. The rinsing step was performed without direct flushing, with the stream directed toward the walls of a Petri dish.Samples remained immersed in liquid during the rinsing steps, without creating any air-sample interface. The rinsing step was repeated 6 times. The effect of the samples on bacterial growth in the planktonic state was tested by measuring the absorbance of each supernatant at 600 nm (A 600 ). Results are expressed in terms of Inhibition rate compared to bacterial colonization on PP reference sample.

Figure 3 - 1

 31 Figure 3-1 shows SEM images of PP/Ar/MAPP/Ag/MAPP while mechanical strain was applied (e.g., 40%, 60% and 80% of elongation comparing to the initial length of the sample). Observation experiments were performed 1 hour after preparation of the samples. The rate of elongation was 24 µm/s.

Figure 3 - 1 :

 31 Figure 3-1: Plasma polymer multilayer system deposited on polypropylene mesh substrate. Stretching and observation experiment was performed one hour after preparation of the sample.

Figure 3 - 2 :

 32 Figure 3-2: Plasma polymer multilayer system deposited on polypropylene mesh substrate. Stretching and observation experiment was performed 10 days after preparation of the sample.

Figure 3 - 2 Figure 3 - 2

 3232 Figure 3-2 shows, that plasma polymer and/or interface between deposited layer and polypropylene material undergo changes during ageing process which can be seen by different fracturation processes of the surface during stretching. When experiment is conducted on freshly prepared sample (1 h of ageing) the apparition of cracks takes place immediately at ~20 % of elongation (Figure 3-1 20%). For similar stretching ratio and for samples aged during 10 days (see Figure 3-2 20%) or 20 days (see Figure 3-3 20%), cracks are not likely to appear neither on the fiber nor on the weave. When the stretching ratio was increasing, cracks are well seen along the fiber but more likely to appear first at the weave where tensile stresses are higher (Figure 3-1 40%). After 10 days (Figure 3-2 40%) or 20 days (see Figure 3-3 40%) of ageing, cracks are only visible close to weave.

- 1 )

 1 , where partial delamination of the layer close to cracks occurs (Figure3-1). After ageing process (both 10 and 20 days) this is no longer the case. Cracks are formed regularly with similar sizes of aperture and no delamination occurs.

Figure 3 - 3 :

 33 Figure 3-3: Plasma polymer multilayer system deposited on polypropylene mesh substrate. Stretching and observation experiment was performed 20 days after preparation of the sample.

  Surface atomic concentration measured by means of XPS spectroscopy on PP/Ar/MAPP/Ag/MAPP before and after ageing (in this case ageing time is 7 days) revealed increase in O/C ratio after ageing. Besides the decrease of 1% in oxygen atomic concentration in case of aged sample, detection of 1.3% of Antimicrobial Coatings for Soft Materials 3-157 sodium element was observed which come from NaBH 4 solution which is used during synthesis of silver nanoparticles. Presence of sodium element may indicate process of migration of substrate species during ageing time due to surface mobility (see Table

Figure 3 - 4 :

 34 Figure 3-4: High resolution spectra of C(1s) recorded for plasma polymer multilayer system after preparation and after ageing for 7 days.

  Studies using SEM microscopy observation allow to visually compare the surface morphology of PP before and after autoclave and E-beam sterilization procedures. Figure 3-5 (A and B) shows fragment of fiber of virgin PP mesh. Paralleled lengthways grooves (fibrillar strands) are visible due to polymer processing (extrusion) [405, 406]. On the Figure 3-6 (A and B) PP/Ar/MAPP/Ag/MAPP is shown. No significant changes of the surface were

- 5 (

 5 C and D)), number of small dark color bubbles and several areas with pieces of torn biomaterial are found. Also an undirected tears like deformations on the surface are visible (Figure3-5 (D)). Defects of the surface observed on PP and PP modified surfaces after autoclave sterilization (see Figure 3-5 (C and D) and Figure 3-6 (C and D)suggest degradation of the material caused by hot steam. Similar results were obtained for polypropylene mesh alone subjected to autoclave treatment[407] and to polypropylene mesh subjected to resterilization using autoclave repeated treatments[408]. It is known that polypropylene material can be heated up to 140°C for short periods of time. The melting point of polypropylene is 160°C which is about 40 degrees higher than autoclaving temperature. However the humid environment caused by steam and 121°C temperature seems to be 3-161 enough to induce physical changes in the surface of polypropylene and plasma modified polypropylene meshes. Result of autoclave sterilization treatment on PP/Ar/MAPP/Ag/MAPP is shown on Figure 3-6 (C and D). Surface of the substrate which was sterilized at 121°C

Figure 3 - 5 :

 35 Figure 3-5: SEM micrograph of polypropylene mesh (A and B), Polypropylene mesh after autoclave sterilization (C and D) and Polypropylene mesh after electron beam irradiation (E and F). Microphotographs A, C and E are taken under 500 x magnification and microphotographs B,D and F are taken under 1000 x magnification.

Figure 3 - 6 :Figure 3 - 5 (

 3635 Figure 3-6: SEM micrograph of plasma polymer multilayer system deposited on Polypropylene mesh (A and B), plasma polymer multilayer system after autoclave sterilization (C and D) and plasma polymer multilayer system after electron beam irradiation (E and F). Microphotographs A, C and E are taken under 500 x magnification and microphotographs B,D and F are taken under 1000 x magnification.

3 :

 3 Surface atomic composition of (a) plasma polymer multilayer system. (b) plasma polymer multilayer system after autoclaving and (c) plasma polymer multilayer system after electron beam irradiation. * Gold and copper elements are from sample holder, possible to detect due to mesh pores. No significant change was observed in O/C elemental concentration ratio for PP/Ar/MAPP/Ag/MAPP before and after autoclave sterilization (0.45 and 0.44 respectively). However, decrease in O/C elemental concentration ratio from 0.45 before electron beam sterilization to 0.36 after electron beam sterilization

Table 3 - 4 :

 34 Results of C(1s), O(1s) and Ag(3d) peak decomposition of high resolution spectra recorded for a) plasma polymer multilayer system, b) plasma polymer multilayer system after autoclave sterilization, c) plasma polymer multilayer system after electron beam irradiation with the dose of 25 kGy.

Figure 3 - 7 :

 37 Figure 3-7: High resolution spectra of C(1s) recorded for plasma polymer multilayer system after a) autoclave sterilization, b) electron beam irradiation with the dose of 25 kGy.

4 )

 4 was recorded after electron irradiation sterilization (on the basis of the C(1s) envelope peak fitting) while no change is observed after autoclave sterilization. The contribution of O=C-O-C=O/COOH, groups have increased after electron beam sterilization at the expense of C-C/C-H, (associated with cross-linked carbon/hydrocarbon (C x H y ) species) and O-C-O/-CO groups.

  ). As previously explained, plasma polymerization results from the well-known competition between deposition and etching during process[388]. Both the coating and the surface were bombarded by ions from the plasma leading mainly to 3-168 fragmentation during the deposition. Due also to surface mobility, a fraction of the silver species could migrate to the near-surface regions of the plasma polymer layer. Decrease of Ag concentration in the near surface region was observed after autoclave sterilization. This can be explained by dissolution of silver nanoparticles due to swelling of plasma polymer and washing off nanoparticles during autoclave process. However, in case of electron beam bombardment the decrease in silver concentration might be explained by modifications of plasma polymer such as initiation of further crosslinking that quenches any remaining radicals or chain scission due to electron displacement and generation of free radicals[409, 410]. These free radicals react with oxygen from atmosphere and form acid groups (increase of O=C-O-C=O/COOH groups, Table

Figure 3 - 8 :

 38 Figure 3-8: TEM micrograph of plasma multilayer system A) before sterilization with autoclave, B) after sterilization with autoclave.

3 - 8 .

 38 multilayer system was verified by means of Transmission Electron Microscopy. TEM microphotographs of PP/Ar/MAPP/Ag/MAPP before and after autoclave sterilization are shown in Figure Before sterilization, silver nanoparticles seen as dark spots are uniformly distributed (see also section 2.4.3). Since the temperature is one of the key parameters that can allow to control the particle growth, the particles shape and size distribution during the synthesis, the autoclave treatment (121°C) should influence the silver nanoparticles embedded in the multilayer plasma polymer system [415]. This is due to the fact that during the synthesis stabilizer was not used [415].Figure 3-8 represents PP/Ar/MAPP/Ag/MAPP after autoclave treatment. Humid environment and high temperature during sterilization have affected the size and distribution of silver nanoparticles in the multilayer system. Visually compared microphotographs prove that autoclave sterilization used for thin polymeric film systems with embedded nanoparticles should be carefully considered due to changes that it 3-170

Figure 3 - 9 :

 39 Figure 3-9: TEM micrograph of plasma multilayer system A) before sterilization with electron beam using 25kGy dose, B) after sterilization with electron beam using 25 kGy.

Figure 3 - 9

 39 (A) shows a microphotograph of PP/Ar/MAPP/Ag/MAPP before the electron beam irradiation sterilization. After electron beam sterilization no aggregation or

Figure 3 - 10 :

 310 Figure 3-10: Inhibition rate of planktonic bacteria compared to bacterial colonization on PP sample after 4h of bacterial culture.

Figure 3 -

 3 Figure 3-10 shows that bacteria proliferate easier on the sample which undergo autoclave sterilization comparing to sample that undergo electron beam sterilization. Comparing to bacterial proliferation on PP sample, the inhibition rate of bacterial proliferation on plasma multilayer system in case of autoclave sterilization is 32%. In case of electron beam sterilization inhibition rate value reaches 89%. This can be caused by dissolution of silver nanoparticles in the plasma polymer layer during autoclave treatment resulting in the lower concentration of silver ion release during antibacterial tests which can result in higher bacterial survival and proliferation.

Figure 3 - 11 :

 311 Figure 3-11: Plasma polymer multilayer system deposited on polypropylene mesh. Sample freshly prepared and sterilized by means of electron beam (25 kGy) the same day.

Figure 3 - 12 :

 312 Figure 3-12: Plasma polymer multilayer system deposited on polypropylene mesh. Sample aged for three days and sterilized by means of electron beam (25 kGy).

  aeruginosa were also showed by for various antibiotics. It was suggested, that matrix prevents access of antibiotics to the bacterial cells embedded in the community.Sommerfeld et al. [390] have studied susceptibility of bacteria to ciprofloxacin hydrochloride, in two bacterial populations such as bacteria in connected biofilm structures (bacteria that grow in close proximity inside the biofilm) and planktonic bacteria, or unconnected bacteria (bacteria outside of biofilm). The study revealed that bacteria in unconnected population were more susceptible to antibiotics.4-183To visualize biofilms, Confocal Laser Scanning Microscopy (CLSM) technique is often used. This technique allows fluorescence imaging at different planes within the biofilm, which allows to generate 3D images with high resolution. For the quantification and further analysis of such biofilms micrographs, diverse softwares have been developed. In particular, detailed quantification of biofilm using parameters such as biomass, average thickness, and roughness coefficient, surface to biovolume ratio, maximum thickness, surface coverage is possible with COMSTAT software[START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF]. In the software function called Connected Volume Filtering (CVF) bacterial elements that are not part of a biofilm are removed from the stack of images, when quantifying the biofilm parameters. Then the removed part is not included in the further calculations [438]. For example, Sommerfeld et. al [390] used the function CVF of Comstat2 software to analyze the susceptibility of bacteria in connected biofilms and unconnected sections. Sanchez et al. [436] have used Comstat software for the quantification of the biomass, average thickness, roughness coefficient, surface to biovolume ratio, maximum thickness and surface coverage to follow the changes in biofilm architecture caused by different nutrition concentrations.

  analyzed biofilm structures of four Pseudomonas sp. strains which formed different patterns. The analysis of biofilm structures in terms of roughness coefficient, substratum coverage and surface to volume ratio by Comstat was able to show very weak differences. This allows to consider that the use of CLSM technique together with Comstat2 software gives an opportunity to analyze in details the architecture of biofilms.

Figure 4 - 1 :

 41 Figure 4-1: Three-dimensional reconstructions of GFP-labeled P. aeruginosa PAO1 biofilms show round, dispersed cellular aggregates when grown in LB medium (A), and flat-compact, sheet-like structures in 1/3-diluted LB medium (B) at 72 h. Each image consists in thin optical sections collected at 1-mm intervals in z-section. Image resolution: 512x512 pixel; size: 488.52mm x488.52mm. Images obtained from Sanchez et al. [436].

chapter 1

 1 have been used for bacterial adhesion (4 hours culture) and bacterial biofilm development (168 hours culture) experiments. Their toxicity towards bacterial cells has been evaluated. Untreated PP substrate and PP substrate with deposition of the only second layer of plasma polymer (PP/Ar/MAPP(2 nd )) were used as controls. Substrate toxicity experiments were performed two times, each time in a double determination (i.e. two samples of each condition). Bacterial colonization and biofilm development experiments were performed three times, each time in a double determination.

  Center of Irradiation Aérial (CRITT, Aérial, Illkirch). Samples were sterilized by electron beam with the electrostatic generator of Van Graff type with maximal voltage of 2.5 MV and maximal power 500W. Voltage can be varied between 0.5 and 2.5 MV and intensity of electric current between 0.01 à 200 μA. Dose of irradiation received by samples was set to 25 kGy. Sterilization of the samples was performed two days before each experiment. Silicon wafers and stainless steel wires used during experiments were sterilized in dry oven for 1 hour at 180°C.

( 9 g

 9 /L in water). The rinsing step was performed without direct flushing, with the stream directed to the walls of a Petri dish and with the attention to the force of rinsing fluid. The substrate surface remained immersed in liquid during the rinsing steps. The rinsing step was repeated six times for the inoculation time of 4 h and 16 times for the inoculation time of 168 h. In order to visualize cells and monitor their viability, bacteria were stained after last rinsing step with NaCl, with 1 µL/mL of Live/Dead ® bacterial viability stain kit (Molecular Probes) for 15 minutes. The kit employs two nucleic acid stainsgreen-fluorescent Syto ® 9 and red-fluorescent propidium iodide stain [439].

  Zeiss, LSM 700) with a 9.1 mm working distance focal objective (Zeiss LD EC "epiplan neofluar" 50X/0.55 DIC M27). Fluorescence with Syto ® 9 labeling was exploited for imaging with laser wavelength of 488 nm for excitation with pinhole size of 1 AU. The collection of emission fluorescence was performed in the range of 420 -550 nm. In case of propidium iodide (PI), collection of emission fluorescence was collected in the range of 560 -700 nm with the excitation wavelength of 555 nm. In addition, excitation at 405 nm and pinhole size 0.3 AU was used in reflection mode allowing to perform image of the surface of the substrate. Confocal 3D images were taken for 5 randomly chosen locations of each sample. CLSM were treated with the Zen2009 software provided by Carl Zeiss, Image J V1.46d software with LSM toolbox

4. 2 . 6 . 1 . 3

 2613 Image analysis of bacterial colonization after 4h of incubation timeNumber of adherent bacteria on each location observed on the surface was quantified in this experiment. For this purpose, the stacks of images were treated with the Image Processing Software -ImageJ 1.46[START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] to perform the counting of bacteria on the three dimensional surface of a fiber. Firstly, separation of fluorescence images and reflection images was performed.Secondly, summing up of the slices corresponding to the fluorescence images was executed. Further, Particle Detector &Tracker plug-in was used to count the number of adherent bacteria [442].4.2.6.1.4 Analysis of biofilm micrographs after 168 h of incubation timeFor the quantification of biofilms after 168h of incubation time, a novel computer program Comstat 2 was used. This is a development from its original Comstat 1 version[START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF][START_REF] Heydorn | Experimental reproducibility in flow-chamber biofilms[END_REF] and was developed by Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S. It was provided by Claus Sternberg. The software was analyzing the native confocal LSM Zeiss format images. Manual thresholding was performed and fixed threshold value was kept for all analyzed images The total biomass was measured.Parameters that are extracted:  The parameter named "Connected Volume Filtering" (CVF) is used to separate the CLSM image pixels of connected biofilms from unconnected bacteria, or to remove elements that are not part of the biofilm. The algorithm extracts the bacteria that are not part of a biofilm from an image stack[438]. In addition, "if an element in the image stack is not in some way connected to the substratum (first layer of the detected biofilm), it is not considered part of biofilm". It can be piece of torn biomaterial but it might be also the part of biofilm connected to the substratum outside the scope of the image stack under observation[438]. The bacteria retained after using this function to calculate biomass are those in the connected biofilm. The bacteria which are eliminated are considered to be outside of the biofilm. Due to the geometrical shape of the substrate, use of this function was explored to investigate the "connectivity" of observed biofilms, i.e. if biofilm is likely in forms of separated colonies and/or single bacteria or as a one "connected" (cohesive) compact structure. By using this parameter, our aim was to extract additional information about biofilm architecture, growth and viability. This feature of biofilm structure is crucial in the antibiotic or other biocides therapies due to the efficiency of penetration[443] and the protection of bacterial cells by structural organization of the biofilm[444].

  distance from all points in the image stack to the nearest void (background)). Since the void can potentially contain nutrients, oxygen etc, the diffusion distance plays a serious role in the development and understanding of biofilms. In the present study we have also calculated all above mentioned parameters for the PI stained bacteria (red) in biofilms.Parameters that are further calculated on the basis of the previous four parameters are:  Inhibition rate compared to reference sample o OD ref -Optical density of reference sample o OD i Optical density of measured sample  Rate of connected biomass o Biomass CVF (i) -Biomass calculated with CVF parameter o Biomass Total (i) -Biomass calculated without CVF parameter  "Viability" (rate of cells with non-compromised membrane) o -Average number of bacteria with non-compromised membrane o -Average Total number of bacteria o -Standard deviation of number of bacteria with noncompromised membrane o -Standard deviation of number of total number of bacteria  Rate of bacteria with compromised membrane in connected biofilms o BiomassRed CVF (i) -Biomass of bacteria with compromised membrane calculated with CVF parameter o BiomassTotal CVF(i) -Total Biomass calculated with CVF parameter

Figure 4 - 4 ,Figure 4 - 4 ,Figure 4 - 4 :

 444444 Figure 4-4, even concentration of silver ion as low as 3.0 µM has weak impact on short term of bacterial growth.

Figure 4 - 5 :Figure 4 - 5 ).Figure 4 - 5 ). 4 . 3 . 2 . 1 . 2 4 - 7 . 4 - 8 .

 454545432124748 Figure 4-5: Number of bacteria on the surface of substrates recorded after 4 hours of incubation time. *, @, # and § symbols indicate significant differences to PP, PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP respectively. Probability is noted with the number of symbols: 1 symbol for p>95.0%, 2 symbol for p>99.0%, 3 symbols for p>99.9%. Abbreviations MAPP(2), Ag, MAPP, 60% on the graph represent PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP, 60% stretched PP/Ar/MAPP/Ag/MAPP samples respectively.

Figure 4 - 7 :

 47 Figure 4-7: Confocal 3D images (50x magnification), transformed to 2D using ZEN software, representing biofilm development of Escherichia coli after 168 hours of incubation on A) polypropylene B) polypropylene with only second plasma polymer deposited layer, PP/Ar/MAPP(2 nd ) C) polypropylene with plasma deposited layer with loaded silver nanoparticles, PP/Ar/MAPP/Ag D) plasma multilayer system deposited on polypropylene substrate, PP/Ar/MAPP/Ag/MAPP E) plasma multilayer system on PP mesh under stretching with elongation 60%, PP/Ar/MAPP/Ag/MAPP-60%.

Figure 4 - 8 :

 48 Figure 4-8: Total biomass value calculated from the CLSM images using Comstat 2 software. *, @, # and § symbols indicate significant differences to PP, PP/Ar/MAPP(2 nd

Figure 4 - 9 :

 49 Figure 4-9: "Viability" of bacteria on the surface of substrates after 4 hours of incubation time. *,@,# and § symbols indicate significant differences (probability p> minimum 95,0%) to PP, PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP respectively. Abbreviations MAPP(2), Ag, MAPP, 60% on the graph represent PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP, stretched PP/Ar/MAPP/Ag/MAPP samples respectively.

Figure 4 - 10 :

 410 Figure 4-10: Bacterial adhesion on PP/Ar/MAPP(2 nd ) substrate after 4h of incubation. Surface irregularities are the results of swelling properties of plasma polymer.

shown on Figure 4 - 11 .

 411 Using this approach, it is possible to distinguish if biofilm is in the form of one connected biofilm or in form of colonies or bacteria not connected one to another (Figure4-12).

Figure 4 - 12 :

 412 Figure 4-12: Schematic representation of "connected" biofilm and "non-connected" bacteria or colonies. Green and red colors represent alive bacteria and bacteria with compromised membrane respectively.

Figure 4 - 13 :

 413 Figure 4-13: Two examples of confocal 3D images (50x magnification), transformed to 2D using ZEN software, representing biofilm development of Escherichia coli after 168 hours of incubation on polypropylene (PP) sample. Left and right images show respectively that there are places where majority of bacteria are alive (green color) or have compromised membrane.

Figure 4 - 14 :

 414 Figure 4-14: Rate of bacteria with compromised membrane in connected biofilm and nonconnected colonies after 168 h of incubation. Abbreviations MAPP(2), Ag, MAPP, 60% on the graph represent PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP, stretched PP/Ar/MAPP/Ag/MAPP samples respectively.

2. 4 . 5 ;

 45 Figure 2-10 compared with the other silver containing coatings). The bacterial metabolism is then similarly influenced if bacteria are in biofilms as if they are in smaller colonies. This is probably the result from the size of the 4-221 bacterial colonies composing the connected part of the biofilm, since, in the presence of silver ions, bacteria have difficulty to form biofilm structures. As discussed previously, this is due to the growth delay which probably involves both short-term cell killing [445] and inhibition of the biofilm development.

Figure 4 - 15 :

 415 Figure 4-15: Surface to biovolume ratio for total and green to red ratio in biofilm. G/R represents ratio of bacteria with non-compromised membrane to bacteria with compromised membrane. Abbreviations MAPP(2), Ag, MAPP, 60% on the graph represent PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP, stretched PP/Ar/MAPP/Ag/MAPP samples respectively. *, # symbols indicate significant differences (probability p> 95,0%) to PP, Ag respectively.

Figure 4 - 16 :

 416 Figure 4-16: Average diffusion distance calculated for bacteria with non-compromised membrane (green on the graph) and for bacteria with compromised cell membrane (red on the graph). Values are calculated for the biofilms after 168 hours of incubation. Abbreviations MAPP(2), Ag, MAPP, 60% on the graph represent PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP, stretched PP/Ar/MAPP/Ag/MAPP-60% samples respectively. *, @, § symbols indicate significant differences (probability p> 95.0%) to PP, MAPP(2) and MAPP respectively.

.

  The thickness of biofilm is deducted from total biomass (shown in Figure 4-8) which is expressed in . Between the brackets, values of the parameter which confirm the given hypothesis are written.

  development and characterization of mechanically sensitive materials made of plasma polymer multilayers and silver nanoparticles was given. It has been demonstrated that tailoring of the release of an active agent can be achieved by mechanical stimulation of the designed material. Silver ions as a biocide are stored as AgNPs in the plasma polymer multilayer system and released in the material surroundings. Taking advantage of different mechanical properties between the different layers, a system of cracks in the plasma polymer overlayer allowed control of the diffusion of silver ions. Antibacterial properties of the developed system have been confirmed by disk diffusion method.Due to the fact that biomaterial device has to be sterile before the implantation in the body, the sterilization step was studied as the last step of the material modification during elaboration. Influence of ageing and two types of sterilization procedures (autoclave and electron beam irradiation) on the material surface chemistry and morphology was analyzed. Results showed that autoclave treatment influences surface structure unfavorably, causing degradation of the material without significantly changing the surface chemistry.Electron beam sterilization did not influence surface physical or topographical changes. However, it induced changes in chemistry of the surface causing decrease in O/C elemental concentration ratio. It was also shown that autoclave 5-232 sterilization should be carefully considered for biomedical devices containing silver nanoparticles due to changes that it induces in nanoparticles size and distribution. In contrast, electron beam sterilization did not affect particles size or distribution.Further investigations were focused on the antibacterial properties. In this study, the effects of PP and PP-modified mesh substrates on planktonic bacteria, bacterial adhesion and biofilm formation were studied. The antibacterial efficiency of the mechanoresponsive coating was verified under stretching conditions.Plasma polymer itself have shown to influence bacteria proliferation in planktonic state for short time of incubation (4h) suggesting that elution of low molecular weight water soluble fractions of plasma polymer takes place.Nevertheless, there is no difference in planktonic bacteria proliferation between PP/Ar/MAPP(2 nd ) and PP sample for 168 h of incubation time, suggesting that this low molecular fraction is in small amount and is easily extracted during the first time of immersion. In contrast, no similar effect was observed with eukaryotic cells (results are shown in Annex). Substrates without plasma polymer overlayer (PP/Ar/MAPP/Ag) have shown to inhibit bacterial proliferation in planktonic state after 4 and 168 h. Plasma polymer multilayer system and the same system under stretching conditions have exhibited antibacterial properties for short time of incubation (4h), inhibiting the bacterial proliferation in planktonic state, despite of the presence of the second polymer layer in the case of PP/Ar/MAP/Ag/MAPP. This was proposed to be due to presence of silver nanoparticles close to the surface region which may provide a fast release of silver ions when in contact with liquid environment and due to the insufficient barrier effect of second plasma polymer overlayer. For longer time of incubation, the barrier effect of plasma polymer overlayer was playing a role, showing only a weak inhibition rate.5-233Quantitative and qualitative evaluations of bacterial adhesion after 4 hours of incubation and biofilm formation after 168 hours of incubation for Polypropylene and plasma polymer modified polypropylene substrates have been done using of Confocal Laser Scanning Microscopy. Such comprehensive study of biofilm characteristics on coatings with loaded silver nanoparticles, especially on mesh materials, have not been performed before. In the case of surfaces which were releasing silver ions, bacteria adhered after 4h in lower quantity than on polypropylene and polypropylene modified with plasma polymer layer without silver nanoparticles. Bacterial behaviors have shown similar trends when incubation time increased to 168 h: Biomasses calculated for polypropylene (PP) and plasma multilayer system (PP/Ar/MAPP/Ag/MAPP) have shown similar values. For substrates with higher and/or faster release of silver ions (PP/Ar/MAPP/Ag and PP/Ar/MAPP/Ag/MAPP-60%), the biomass value have significantly decreased. More detailed study based on rate of connected biomass, surface to biovolume ratio, average diffusion distance and bacterial rates with compromised membrane have shown structural differences of biofilms developed on the different substrates: coatings releasing silver ions favored the formation of a weakly connected biofilm, composed of frequent bacterial clusters of small size. The biofilm was in mushroom-like structures with

Figure 6 - 1 :

 61 Figure 6-1: (A) Schematic drawing of a normal abdominal wall and its layers: Skin (S); Fat Tissue (F); Aponeurosis (A); Pre-peritoneal Fat Tissue (F); Peritoneum (P); and the abdominal viscera (V). (B) Schematic drawing of a hernia. Illustration adopted from Zogbi et al.[START_REF] Zogbi | The Use of Biomaterials to Treat Abdominal Hernias[END_REF].

Figure 6 - 2 :

 62 Figure 6-2:Possible way of mesh implantation. Illustration adopted from Zogbi et al.[START_REF] Zogbi | The Use of Biomaterials to Treat Abdominal Hernias[END_REF]

Figure 6 - 3 :Figure 6 - 4 :

 6364 Figure 6-3: Cytotoxicity of plasma polymerized coatings deposited on PP mesh evaluated using the extraction test method. Medium used in this experiment did not contain serum. Abbreviations MAPP(2), Ag, MAPP on the graph represent PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag,

Figure 6 - 5

 65 Figure 6-5 illustrates the influence of the serum addition in the extraction medium on the indirect contact test. The results are presented as a rate of cell viability on PP and PP modified substrates, compared to cell viability on Thermanox ® negative control. No significant influence of serum was observed except for PP/Ar/MAPP(2 nd ) samples for which the cytotoxicity of the extraction medium increased in presence of serum.

Figure 6 - 5 :

 65 Figure 6-5: Influence on the indirect toxicity test of serum addition in the extraction medium. The results are presented as a rate of cell viability on PP and PP modified substrates compared to cell viability on Thermanox ® negative control. Dashed line represents 75 % of Thermanox ® control which represents the threshold of cytotoxicity. Abbreviations MAPP(2), Ag, MAPP on the graph represent PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP samples

Figure 6 - 6 :

 66 Figure 6-6: Confocal images representing cell adhesion on PP and PP modified substrates after 24 hours of incubation. (-) Th represents Thermanox ® negative control. Arrows: Cells adhering on mesh fibers. MAPP(2), Ag, MAPP on the images represent PP/Ar/MAPP(2 nd ),

  give a similar response as in test where serum was not used. This can be explained by the presence of adsorbed proteins on the surface lowering down the diffusion of silver ions. It has been effectively reported that the composition and the solution parameters of the solvent used for extraction influence the kinetics and mechanism of the elution process[474]. Similarly, 6-249 PP/Ar/MAPP(2 nd ) substrates have given an opposite response in presence or in absence of serum comparing to negative control. A slight increase in viability was obtained without serum while a slight decrease in viability was observed with serum containing medium. Leaching of low molecular weight water soluble fractions of plasma polymer takes place[386] and it can differ according to the medium content. How the presence of serum could increase the leaching of cytotoxic molecules or decrease the leaching of favorable ones is unclear.

  Additionally in case of gynecological surgeries such as in pelvic reconstructive surgery the use of mesh is based on substitution or replacement of lacking supportive tissue, reinforcement of weak or defective supportive tissues or 6-250 induction of new supportive tissue by mesh material[419]. Thus, as for the meshes used in the pelvic reconstructive surgery the adhesion of cells is required, in case of meshes for abdominal wall surgeries efforts are made to render surface of mesh anti-adhesive, yet still favorable for growth of the adjacent tissue. For this reason the developed PP/Ar/MAPP/Ag/MAPP substrate might be a promising way to achieve this property of meshes materials. This hypothesis is supported by work ofPloux et al [148]. In their study, cell behavior on maleic anhydride plasma polymer deposited thin films with chemical and topographical patterns was verified. Results showed that adherent human osteoprogenitor cells (HOP) presented normal morphology at different times of incubation. The differences in number of cells between the substrates were correlated with the differences in surface charge. It appeared that the negatively charged surfaces favored the formation of conditioning layer on the surface and consequently cell adhesion. Negative surface charge of these surfaces was related to presence of -C(=O)-O -groups. Similar chemistry of the surface was observed for the PP modified meshes, including the presence of -C(=O)-O -groups. However it is important to note that differences in substrate, condition of deposition and sterilization procedures may play a role and more detailed study have to be performed to prove the biocompatibility of developed PP modified materials.

  Similarly, Park et al. [481] used surface features on PMMA substrates with dimensions very similar with the mesh fiber diameter used in our study. Cell culture on concave and convex hemispheric microstructures measuring 200 µm in diameter and 50 µm in depth/height have shown greater adhesion of fibroblasts and hMSC cells on convex structures compared with concave structures. The researchers concluded that the concave micro-wells provided unfavorable conditions for cell attachment, cell growth and that it stimulated cell mobility and migration of cells to a more favorable location.

1.2 Inhibition of biofilm formation and prevention of related infections 1.2.1 Prevention of biofilm formation

Prevention of biofilm formation is necessary because it is almost impossible to remove biofilm without removing the implant from patient. However, the technique that is able to fully prevent or control the formation of unwanted biofilm without causing side effects is not known so far. Another problem is the accurate diagnosis. Infection can be suspected based on clinical observations like swelling, warmth, erythema, tenderness or microbiological data (positive culture blood culture in a patient with prosthetic heart valve, imaging findings such as echocardiogram). Also advanced imaging studies (e.g., computed

.2.2 Different strategies to achieve antibacterial materials

	evidence of the possibility of controlling bacterial adhesion by tailoring the material mechanical properties [160, 161]. Bakker et al. [160] were focused on determining the importance of surface tension and elastic modulus on bacterial that with increasing elastic modulus values (between 1 MPa and 100 MPa) for the polyelectrolyte multilayered film, the adhesion of S. epidermidis and E. coli bacteria were increasing. It was shown that S. epidermidis exhibits 1.2Releasing of bioactive agent strategy can be expressed by the bulk or only by
	adhesion. Authors have worked with marine bacterial strains Halomonas mechanoselective adhesion and authors have proved that this correlation was the surface (Figure 1-7). Bioactive agent release strategy can come from
	pacifica, Psychrobacter and Marinobacter hydrocarbonoclasticus and independent from other surface properties such as posited physicochemical polymers that consist of bioactive repeating units (interconnected bioactive
	polyurethane coatings deposited on glass. Coatings had different surface regulators of bacterial adhesion, surface roughness, surface interaction energy monomers), that exhibit bactericidal action through release of antibacterial
	tensions values, achieved by changing the amount of fluorine, whereas using and surface charge density. Additionally, it was found that this correlation is not agent (Figure 1-7 c) e.g., antibiotic molecules (Penicilin V, cephradine) which is
	more or less branched polymers, different elastic moduli values were obtained. limited to a single type or shape of bacteria. Authors have concluded that attached during synthesis to the polymer chain via hydrolytically labile ester
	Study was performed under different mass transport conditions, in a parallel mechanical properties of the surface are parameters, which allow to modulate bonds [164]. Another strategy is to release antibacterial agent, which is
	plate and stagnation point flow chamber. Results showed that bacterial bacterial colonization on the surfaces. embedded in the polymer matrix (e.g., silver [165], Cu [166], ZnO particles
	adhesion in the parallel plate flow chamber was not influenced by the elastic [167], chloroxidine or active chlorine [168]). As previously mentioned, the Recently, our group demonstrated for the first time that the surface mechanical modulus of the coatings. However, influence of surface tension exists bioactive agent release principle can be applied also for material coating properties is an approach to control not only bacterial adhesion but even predominantly for deposition in the parallel plate flow chamber. Authors chamber were assumed to cause a rebound effect which was due to the compared to control. elastic modulus values. High energy collisions in the stagnation point flow viscosity (0,0038 kg/m 2 ). Bacterial number was reduced by a factor 20-fold were adhering in higher numbers to hard surfaces, than to coatings of lower surfaces. These surfaces possessed high shear modulus (6.10 5 Pa) and low surface tension surfaces. In case of stagnation point flow chamber, bacteria adhesive effect of surface to visco-elastic properties of oligonucleotide coated concluded that low surface tension bacteria should adhere preferentially to low proliferation of already adhering bacteria [162]. Authors attributed the anti-(Figure 1
	dissipation of elastic energy during or after collision. This was the reason for Influence of surface stiffness on bacterial adhesion was also very recently
	decrease in deposition rates. shown by Saha et al. [163]. Authors have prepared polyelectrolyte films from
	poly(l-lysine) (PLL) and a hyaluronan derivative modified with photoreactive Influence of mechanical properties of material surfaces on controlling the vinylbenzyl groups (HAVB). Further nanomechanical properties of these films adhesion of Staphylococcus epidermidis bacteria and its colony growth was were modified by UV light illumination. Bacterial behavior of Gram positive studied by Lichter et al. [161]. Authors used polyelectrolyte multilayer (PEM) Lactococcus lactis and Gram negative E. coli was studied on non-cross-linked thin films made of poly(allylamine) hydrochloride (PAH) and poly(acrylic acid) and cross-linked films. Results showed that E. coli exhibited a more rapid (PAA). The chemical functionality and mechanical properties of the films were growth on non-cross-linked softer films compared to the stiffer ones. However L. adjusted by variations of the layer-by-layer assembly conditions (choice of polyanion/polycation) or by changes of pH. Especially the elastic modulus or lactis was shown to grow slowly on both films, independently of their rigidity.
	stiffness values for these polymeric films can be adjusted by manipulation of pH
	adhesion is mechanical properties of the surface. Few studies have shown the values, independently of physicochemical characteristics. Authors have found
	1-55

-7 d). The

incorporation of bioactive agent, which can be e.g., organic antibiotics [169, 170], nitric oxide [171] or silver [172, 173] can be achieved by different techniques of deposition such as dip coating, spin coating, Langmuir-Blodgett, Layer by Layer and plasma polymerization.

1.2.2.2.1 Release of bioactive agent strategy

	effect of the material. For instance, polymerization of antibiotics was performed 1.2.2.2.1.1 Release of antimicrobial agent from coatings
	as a way to prolong activity and reduce the toxicity [164]. The work of Nathan
	et al. showed that antibiotics Penicillin V and Cephradine exhibited full In many cases, there is no possibility to embed the antibacterial agent such as
	antimicrobial activity if the biocides were tethered via hydrolytically labile bond. antibiotic molecule into the bulk due to technical limitations e.g. thermal
	Their activity was retained under release [164]. Another strategy, resistance of these molecules. For this reason, coatings, particularly polymeric
	copolymerization of methacrylate modified Norfloxacin and PEG-methacrylates coatings, are the optimal solution when deposited after the medical device is
	lead to bioactivity of the final product [183]. Following the same idea, direct manufactured. The strategy is based on the use of coated surfaces able to
	modification of Vancomycin with PEG-methacrylate and subsequent release bioactive agent.
	polymerization allowed to preserve antibacterial activity of the final product Promising approach is to use controlled release, where antibacterial agent is
	[184]. However, the antibacterial action was in this case coming from partial embedded in polymer coating and released in a control way. Various
	solubility of the copolymer in water [184]. Further, penicillin molecule attached antibacterial compounds have been studied and loaded into polymer bulk or
	to polyacrylate nanoparticles, showed higher antibacterial activity than free composite film such as organic antibiotics [169, 170], nitric oxide [171] and
	antibiotics in solution [185]. Interesting results were achieved by mimicking the silver [172, 173]. Different techniques of deposition can be used: dip coating
	natural polymers-polyphenols. Synthesis resulted with activity pattern based on [190], spin coating [191], Langmuir-Blodgett [192], Layer by Layer [193] and
	phenol release in order to achieve killing of microbial cells [186]. plasma polymerization
	Releasing of active agent can be also achieved by fabrication of materials
	loaded with biocide. Biocide in this case is not chemically bounded to polymer
	chain. These systems are often achieved by polymer matrices loaded with
	biocides like silver, Cu, ZnO particles, active chlorine. For biomedical
	application concepts of self-assembled structures from amphiphilic have also
	been developed in the field of drug delivery. These macromolecules are soluble
	in dilute aqueous media and their ability to assemble into micelles and vesicles
	Some biocides can be polymerized to provide antibacterial materials. Then, the are used to carry drugs and genes [187, 188]. A class of antimicrobial polymers
	material consists of bioactive repeating units, i.e. polymers are just was developed by Worley et al. using N-halamine groups attached to polymer
	interconnected biocides. In other cases, biocide is tethered via hydrolytically backbone [168]. The design of these molecules allowed the long-term storage
	labile bond to main polymer chain. Releasing of monomer which is biocide in of active chlorine which kills microbial cells by oxidizing the phospholipids of
	the first case or biocide molecule previously tethered to main polymer chain in the cell membrane [189].
	the second case, provides antibacterial effect based on releasing properties.
	The purpose of these structures is to prolong and/or control the antibacterial
	1-60 1-61

2 Mechanically responsive antibacterial plasma polymer coatings for textile biomaterials

	2.1 Abstract
	We have developed a new type of mechanically responsive material on the
	basis of a plasma-modified soft biomaterial substrate (polypropylene surgical
	mesh). A first plasma deposited layer, enriched in a bioactive agent (silver
	Emilia Kulaga, Lydie Ploux, Lavinia Balan, Gautier Schrodj nanoparticles), has been covered by a second plasma polymer layer that acts
	and Vincent Roucoules
	INSTITUT DE SCIENCE DES MATERIAUX DE MULHOUSE
	IS2M -C.N.R.S. -UMR 7361 -UHA
	15, RUE JEAN STARCKY
	68057 MULHOUSE Cedex
	FRANCE
	Kulaga, E., Ploux, L., Balan, L., Schrodj, G. and Roucoules, V. (2013),
	Mechanically Responsive Antibacterial Plasma Polymer Coatings for Textile
	Biomaterials. Plasma Processes Polym. doi: 10.1002/ppap.201300091
	2-94

as a barrier to spontaneous release. The release of the active agent in a controlled manner was achieved by the presence of mechanically-reversible fragmentations in the top-layer of deposited plasma polymer, induced by mechanical stimuli. Characterization of the material was performed by Scanning Electron Microscopy, Transmission Electron Microscopy, Attenuated Total Reflexion-Fourier Tranform Infra-Red, Xray Photoelectron and Ultra Violet-Visible Spectroscopies. The antibacterial properties of the material have been verified.

  films loaded with AgNPs. The quantity of nanoparticles loaded was controlled by the time of loading with AgNO 3 and by the thickness of the plasma polymer layer. The authors also demonstrated control over the release of Ag + by depositing a thin (< 20 nm) plasma polymer overlayer.Very interestingly, the works reported byKörner et al. and Vasilev et al. clearly demonstrated that the amount of AgNPs and the rate of release of Ag + can be tuned by playing with the properties of the plasma polymer. However, these adjustments are only possible during fabrication of the biomaterials. Once the

	unbalanced magnetron operating in a Ar/n-hexane or nitrogen/hydrocarbon
	working gas mixture to form Ag/C:H or Ag/C:H:N composite films. The authors
	demonstrated the possibility of controlling the size of the silver particles by
	changing the power during the deposition step, but no information on the
	release of Ag + was given. Later, Zanna et al. [378] reported the synthesis of
	nanocomposite thin films composed of silver particles enclosed in an
	organosilicon matrix. The film deposition was carried out by combining
	radiofrequency glow discharge fed with argon (Ar) and hexamethyldisiloxane
	In this context, recent efforts have been made to develop Ag-containing
	coatings and plasma polymerization appears as a good candidate offering great mixture of CO 2 /C2H 4 yielding a functional hydrocarbon matrix. Argon was flexibility. One of the main advantages is that plasma coatings can be deposited simultaneously used to sputter Ag atoms from the Ag electrode, forming onto practically any type of material and device [328, 369-375]. Several nanoparticles within the growing polymer matrix. By adjusting the process examples are reported in the literature based on the use of plasma polymerization deposition and co-sputtering of a silver target under low parameters, the coating could be tailored (e.g., Ag content, Ag particle's
	2-97

. No toxicity to human cells has been highlighted for silver ions (Ag + ) up to micromolar concentrations

[359, 360]

. Besides aiming to avoid potential toxicity due to the specific nanoparticle form, AgNPs can be the source of Ag+ delivery, without release in the patient body. As with other inorganic nanoparticles, AgNPs exhibit inherent and unique characteristics such as large specific surface areas, controlled surface composition and reactivity, which endow them with remarkable physical, chemical and biological properties

[361][362][363]

. They can constitute efficient reservoirs for delivery of Ag + through their oxidation in contact with body fluids. Furthermore, despite undisputable bacterial resistance to heavy metals

[364- 366]

resistance to silver remains low compared to antibiotics

[367, 368]

. The risk of significantly reducing silver's antibacterial efficiency through increasing its use as an antibacterial agent in medical devices is still low, due to the rare exploitation of silver for human health. pressure plasma conditions.

Boldyryeva et al. [376]

and Hlidek et al.

[377]

investigated the potential of using DC glow discharge deposition and an with simultaneous silver sputtering. The authors focused their work on the stability of such coatings. Aging was studied under saline conditions and different mechanisms were suggested depending on the initial concentration of silver particles in the film. In particular, for films with high silver content, the coating thickness decreased with immersion time, due to significant silver release and matrix erosion assigned to a percolation-like effect. However, a strongly-bonded polymer layer remained even after long periods of immersion. Recently, Beier et al. improved the resistance of Ag-rich-organosilicon plasma films to abrasion by working at atmospheric pressure and directly spraying a silver nitrate solution within the plasma [379].

In other works, more effort has been focused on controlling the amount of AgNPs loaded in the film as well as controlling the rate of release of Ag + in order to avoid cytotoxic overdoses.

Körner et al. [288]

showed that plasma polymer coatings with embedded AgNPs can be deposited in a low pressure radio frequencies plasma reactor using an asymmetrical setup with an Ag electrode. The plasma polymer was deposited from a reactive gas/monomer morphology and distribution) to different requirements for specific applications and act as efficient reservoirs for Ag + . In addition,

Vasilev et al. [287]

used 2-98 amine plasma polymer Ag-containing coating is implanted, the release process cannot be adjusted anymore. It can also be strongly affected by arbitrary variations occurring in the surrounding medium. In other words, the biomaterial is bioactive but the rate of the release process still passive. The challenge of creating materials with full control of the release process after implantation thus remains totally open.

Table 2 - 1 :

 21 Surface atomic composition at each step of material development, a) polypropylene

	5	66.3	69.8	62.8

Table 2 -1 and Table 2-4 in supporting information). The

 2

concentration of C-O groups increased to 4.2% after plasma treatment while the concentration of C-C or C-H groups decreased to 87.3%.

Table 2 -

 2

	3. The silver release was studied by immersion of samples of the
	multilayer system in distilled H 2 O and subsequent evaluation of the solution
	concentration of silver by measuring its content in AgNPs before and after
	reduction with NaBH

4 (see Figure 2-23 in supporting information).

Table 2 - 3 :

 23 Conditions of plasma polymerization chosen for testing the barrier properties of the plasma polymer overlayer.

Table 3 - 2

 32

	increase	in	C-C/C-H	functionalities	associated	with	cross-linked
	carbon/hydrocarbon (C x H

: Results of C(1s), O(1s) peak decomposition of high resolution spectra recorded for freshly prepared and aged for 7 days plasma polymer multilayer system deposited of PP mesh substrate.

After ageing, a decrease of carbon atoms belonging to OC-O/OC-O-CO groups from 12.06% to 10.87% (see Table

3

-2) and a decrease of C-O functionality from 11.21% to 8.21% (see Table

3

-2) was accompanied by an y ) species (on the basis of the C(1s) envelope peak fitting). An increase in O-C-O/-C=O groups is also observed. These changes include hydrolysis of anhydride groups and migration of short chains of plasma polymer to the extreme surface.

Table 4 - 1 :

 41 M63G-B1 medium composition [380].

	M63G-B1
	KH 2 PO 4	0.1 M
	(NH 4 )SO 4	20 % wt
	FeSO 4	0.1 % wt
	MgSO 4	20 % wt
	KOH	6 M
		1.5 % wt
		optional, adjust
	agar	to pH 6,8
		Autoclave for 20
		min at 120°C
	Vitamine B1	0.05 % wt
	glucose	10 % wt

Table 4 - 2 :

 42 Samples identification and description.

  Ag sample, the high level of inhibition rate is due to sufficient release of antibacterial agent in the adequate concentration/time rate, which blocks and/or inhibits the bacterial proliferation in planktonic state. In case of PP/Ar/MAPP/Ag/MAPP samples, the decrease in inhibition rate to 43% is observed, which implies the efficiency of the barrier effect for a part of the samples, blocking Ag + release from the nanoparticles which are enclosed in the plasma polymer overlayer. The release kinetics of the ionic form of silver from the plasma polymer containing silver nanoparticles is influenced by several factors, as discussed in the section 2.4.5 of this work. The difference in may not be high enough to lead to bacteria growth inhibition on the contrary to those obtained with PP/Ar/MAPP/Ag substrate. Nevertheless, as shown by

	results suggest that effect of slowing down the growth may also exist after 4 By stretching samples, a similar inhibition rate to those obtained with
	Results, that are expressed as hours although after longer time of incubation (in this case 24 hours), bacterial PP/Ar/MAPP/Ag samples was obtained in the first 4 h after stretching and
	referred to bacterial growth around uncoated PP samples showed that growth is not affected any more. inoculation with bacteria. Nevertheless, inhibition of bacteria growth could not
	PP/Ar/MAPP(2 nd ) substrates have influence on the bacterial proliferation in the planktonic state, reaching the inhibition rate equal 38% for incubation time 4 h, comparing to bacterial colonization on PP samples (Figure 4-2 A). This suggests that PP/Ar/MAPP(2 nd ) surface may exert antibacterial properties on planktonic bacteria in the first 4 h of incubation time. This may be due to the elution of low molecular weight water soluble fractions of plasma polymer [386]. Nevertheless, there is no difference in planktonic bacteria proliferation between PP/Ar/MAPP(2 nd ) and PP sample for 168 h of incubation time, suggesting that this low molecular fraction is in small amount and is easily extracted during the first time of immersion. Such effect of MAPP was never highlighted in previous studies so far. Importantly, the results also showed that the concentration of silver ions that is released in the liquid medium from PP/Ar/MAPP/Ag samples at concentration as low as 0.5 ppm. With the concentration lower than 0.5 ppm, inhibition of bacterial growth) [445]. bacterial growth of Escherichia coli is inhibited by presence of silver ions, even activity of the silver ions (e.g. the ratio of silver ions per cell plays a role in the sensitive to concentration of silver under this level. As it is shown on Figure 4-4, addition, the microbial population size may be a limiting factor in the inhibition spectroscopy (i.e., 1.5 ppm (8,8 µM) -Figure 4-3). However, bacteria can be phase ([446]) and decreases specific growth rate in the log phase [447]. In sample before this time was under the limit of detection with UV-Vis growth [445]. The growth delay involves short-term cell killing, prolongs lag samples The concentration of Ag + released from PP/Ar/MAPP/Ag/MAPP behavior such as growth delay with later re-growth or complete inhibition of the 2.4.5) only after 24 hours of immersion in case of PP/Ar/MAPP/Ag/MAPP susceptibility of bacterial organisms can give different outcomes of microbial presence of silver ions in the supernatant solution was detected (see section term bacteria proliferation. If the release of silver is relatively low, the when in contact with liquid environment). As it is shown in the chapter 2, words, this Ag + quantity is probably not high enough to efficiently block long-PP/Ar/MAPP/Ag/MAPP samples (which may provide a fast release of silver ions surface is probably too small to perform long-term release of Ag + . In other presence of silver nanoparticles close to the surface region in case of overlayer. The reservoir of Ag + constituted by these AgNPs at the extreme of inhibition rates, in contrast to expected. This can be explained by the release from nanoparticles distributed in the near surface plasma polymer PP/Ar/MAPP/Ag/MAPP samples for 4 h of incubation time lead to similar values PP/Ar/MAPP/Ag/MAPP samples without stretching is coming from the ion and 168 hours (Figure 4-2 A). Surprisingly, of PP/Ar/MAPP/Ag and explained as followed: the "short" time (4h) antibacterial effect of was high enough to significantly inhibit planktonic bacteria growth in case of 4 be achieved for long incubation time. This is probably the consequence of a too In contrast, inhibition rate of PP/Ar/MAPP/Ag/MAPP samples is significantly less low quantity of silver ions that released from samples. The surface area covered than for PP/Ar/MAPP/Ag samples, for 168 hours of bacterial incubation. In case by cracks compared to the total area of fibers in a sample was estimated at 1%, which may reduce concentrations of Ag + released in liquid surroundings of a factor around 10 -2 for stretched PP/Ar/MAPP/Ag/MAPP samples compared to PP/Ar/MAPP/Ag ones. Concentrations in silver ions of the bacterial suspensions in contact with PP/Ar/MAPP/Ag and PP/Ar/MAPP/Ag/MAPP samples are therefore estimated at 10 -6 M and 10 -8 M respectively. Hence, concentration obtained with stretched PP/Ar/MAPP/Ag/MAPP referred as "60%" on the Figure of PP/Ar/MAPP/inhibition rate for PP/Ar/MAPP/Ag/MAPP samples in case of 4 and 168 h can be 4-2,
	4-199 4-201

3.2.2 Bacterial membrane integrity and biofilm structure 4.3.2.2.1 Viability of bacteria adhered after 4h of incubation

  shows the viability of bacteria adherent on the polypropylene and polypropylene modified substrates. In all of the cases, the viability of bacterial cells is above 50%. The viability on PP/Ar/MAPP/Ag/MAPP samples is considered "normal". It was already shown in literature that ~80% of viability can be found for control samples. For example, in the work ofTerada et al. [456],

	[439]. Further in the text, terms "compromised" and "non-compromised"
	membrane are used together with viability term, indicating the state of bacterial
	membrane.
	Figure 4-9
									, Ag,
	MAPP,	60%	on	the	graph	represent	PP/Ar/MAPP(2	nd ),	PP/Ar/MAPP/Ag,
	PP/Ar/MAPP/Ag/MAPP, stretched PP/Ar/MAPP/Ag/MAPP samples respectively.
	"Viability" of bacteria adhered after 4h of incubation was monitored as a
	function of the cell membrane integrity. Measurements of viability provide an
	indicator of the physiological state of individual bacteria in mono-culture or in
	consortia with low numbers of species. Cells with a compromised membrane,
	that are considered to be dead or with depleted "viability" are stained red,
	whereas cells with an intact (non-compromised) membrane are stain green
									4-212

4.

  Sommerfeld et al. [390] observed that unconnected bacteria populations were more susceptible to antibiotics. Since they are less encased within the biofilm, but they may be mobile and may be therefore able to recolonize elsewhere.

	results indicate that even low concentration (1.5-6.0 ppm -8,8-35,3 µM) of
	released silver ions can influence the biofilm development. This shows in
	addition that the bacterial susceptibility to slightly toxic conditions can be
	highlighted when biofilm structure is investigated (Figure 4-11). This specific
	impact of low quantity of silver ions on the biofilm structure is here
	demonstrated for the first time. Consequences for treating biofilms may be
	important since "unconnected" parts of biofilm may be more accessible to
	antibiotics or other biocides administrated by the classical systemic way.
	-11). PP and PP/Ar/MAPP(2 nd ) substrates are in study revealed that silver enhances antibiotic activity against gram negative
	bacteria. They have used lethal (10 ppm; 60 µM) and sublethal (5 ppm; 30µM) this group. Second group includes PP/Ar/MAPP/Ag, PP/Ar/MAPP/Ag/MAPP and PP/Ar/MAPP/Ag/MAPP-60% substrates and the rate of connected biomass concentrations of Ag + to demonstrate that Ag + can potentiate the activity of
	broad range of antibiotics against gram-negative bacteria in distinct metabolic value in this group is between 20% and 40%. These results show that the silver states. The synergistic effect between Ag + and the different antibiotics was ions released from the substrates have significantly influenced the biofilm structure. achieved by adding 2.5 ppm (15 µM) and 5 ppm (30 µM) of Ag + (as AgNO 3 ) for
	cells metabolically active and dormant cells, respectively [237]. Our results
	More specifically, biofilm in form of "connected" colonies was mainly found on
	PP substrates. On the average, only ~30% of biomass was considered not-
	connected. In case of biofilms developed on PP/Ar/MAPP(2 nd ) substrates, this
	value increased to 46%. The difference between biofilm structures observed on
	both substrates confirms that biofilms are more developed on PP than on
	PP/Ar/MAPP(2 nd ), in agreement with the hypothesis of a more advanced biofilm
	formation on PP than on the other substrates. In the case of the samples
	containing silver nanoparticles, rate of connected biomass was significantly
	lower than values measured for PP and PP/Ar/MAPP(2 nd ) substrates. These
	4-217

Susceptibility of bacteria in smaller population was also showed by

. They showed that bacteria in the form of smaller populations, can be more susceptible to additional antibacterial treatment. Their

  "specific surface" of the bacteria with compromised membrane is higher than for the one with non-compromised membrane. For example, bacteria with compromised membrane may be inside a smooth biofilm as small clusters or as separate cells in the biofilm matrix, increasing in this way the specific surface of the red part of the biofilm. For PP and PP/Ar/MAPP(2 nd ), the value was superior to 1 probably due to a rough outer surface of the biofilm (less rough for

	those of bacteria with compromised membrane. This G/R ratio gives indication
	about the distribution of both types of bacteria (i.e., with non-compromised and
	compromised membrane) inside the biofilms. If G/R parameter is close to 1,
	then surface to biovolume ratio (in other words "specific surface") of bacteria
	with compromised and non-compromised membrane are similar. If G/R is below
	1, the
	It represents the surface to
	biovolume ratio of bacteria with non-compromised membrane and compared to
	4-223

PP/Ar/MAPP

(2 nd

) than for PP) with a smoother outer surface of the red biofilm part. For the samples containing AgNPs, the G/R value is equal 1 in case of PP/Ar/MAPP/Ag sample and below 1 in case of PP/Ar/MAPP/Ag/MAPP and PP/Ar/MAPP/Ag/MAPP-60%. For PP/Ar/MAPP/Ag sample, this indicates that parts of biomass with compromised membrane and non-compromised membrane respectively have similar "specific surfaces", This suggests that "red"

  The highest value of this parameter is observed for biofilm developed on PP substrates. Average diffusion distance of "green" biomass in this case is significantly different from the same parameter calculated for all other samples. ADD G for PP/Ar/MAPP/Ag sample has the lowest value and is significantly different from PP and PP/Ar/MAPP(2 nd ).

	highest biomass. For PP/Ar/MAPP/Ag, biofilm is the least thick, which agrees
	with the least biomass and the antibacterial action of Ag + released from the
	substrate in an efficient concentration. Tendency observed for ADD G parameter
	which follow the order (from the highest to the lowest): PP/Ar/MAPP(2 nd )>
	PP/Ar/MAPP/Ag/MAPP > PP/Ar/MAPP/Ag/MAPP-60% are also in agreement
	with the measured biomasses and it suggests that variations in the thicknesses
	of biofilms follow variations in Ag + releasing.
	Average diffusion distance of "red" bacteria (ADD R ) parameter calculated for
	biofilms shows the following tendency (from the highest to the lowest):
	PP/Ar/MAPP/Ag/MAPP > PP > PP/Ar/MAPP(2 nd ) > PP/Ar/MAPP/Ag/MAPP-
	60% > PP/Ar/MAPP/Ag. Significant difference between ADD R calculated for
	PP/Ar/MAPP/Ag/MAPP and PP/Ar/MAPP/Ag/MAPP-60% is observed. These
	results are in agreement with "red" biomass in form of big clusters for
	PP/Ar/MAPP/Ag/MAPP	and	PP	samples.	On	PP/Ar/MAPP(2 nd ),
	PP/Ar/MAPP/Ag and PP/Ar/MAPP/Ag/MAPP-60% substrates, red biomass
	have lower thickness compared to PP and PP/Ar/MAPP/Ag/MAPP suggesting
	flat or small clusters of "red" biomasses or even single bacteria.
	ADD G parameter calculated for other substrates shows slight tendency following
	the order from the higher value: PP/Ar/MAPP(2 nd )>PP/Ar/MAPP/Ag/MAPP>
	PP/Ar/MAPP/Ag/MAPP-60%.
	In case of ADD G calculated for PP substrates, results show that biofilm
	developed on this substrate is the thickest, which is in agreement with the
						4-225

Table 4 - 3 :

 43 Characteristics of the biofilms

	4.4 Conclusions
	For short time of bacterial culture (4h), plasma modified substrates
	(PP/Ar/MAPP/Ag/MAPP and PP/Ar/MAPP/Ag) showed antibacterial properties,
	inhibiting between 90% and 100% of bacterial growth. Small antibacterial effect
	was also observed for substrate with only second plasma polymer
	(PP/Ar/MAPP(2 nd )). For long time of bacterial culture (168 h), high antibacterial
	effect was preserved in case of PP/Ar/MAPP/Ag substrate. For complete
	plasma polymer modified system, antibacterial effect decreased to ~40% due to
	barrier effect of the plasma polymer second layer. The antibacterial efficiency of
	the PP/Ar/MAPP/Ag/MAPP-60% against planktonic bacteria could be only
	demonstrated for 4h of bacterial culture.
	Bacterial adhesion tested after 4h of bacterial culture showed antibacterial
	effect of PP/Ar/MAPP/Ag and PP/Ar/MAPP/Ag/MAPP substrates. Comparing to
	PP surface, lower number of bacteria was also observed for PP/Ar/MAPP(2 nd )
	substrates due to different chemistry of the surface for this substrate. Study of
	bacterial membrane integrity after 4h of bacterial culture also showed lower
	"viability" of bacteria which adhered on the substrates containing silver
	nanoparticles (PP/Ar/MAPP/Ag and PP/Ar/MAPP/Ag/MAPP) comparing to the
	ones adhered on the substrate without AgNPs (PP/Ar/MAPP(2 nd )).
	At longer culture time (168h), total biomass measurements revealed
	significantly lower bacterial retention on PP/Ar/MAPP/Ag compared to PP
	substrates, due to released silver ions which inhibit bacterial growth. Similarly to
	the antibacterial effect on planktonic bacteria, in case of PP/Ar/MAPP/Ag/MAPP
	sample, bacterial growth was only slightly inhibited due to the barrier effect of
	the plasma polymer second layer.
	More detailed study of the biofilm structure revealed differences in biofilms
	developed	on	PP,	PP/Ar/MAPP(2 nd ),	PP/Ar/MAPP/Ag	and

"Red" bacteria form small clusters inside the biofilm.

Finally, antibacterial efficiency of the system under stretching (PP/Ar/MAPP/Ag/MAPP-60%) is also superior to the efficiency of PP/Ar/MAPP/Ag/MAPP after 168h of culture time. Biomass value for stretched samples after 168 h of culture is significantly lower than biomass measured for PP/Ar/MAPP/Ag/MAPP samples. Study of the biofilm structure on PP/Ar/MAPP/Ag/MAPP-60% compared to PP/Ar/MAPP/Ag/MAPP also indicates that:

CHAPTER III

Results and discussion

The following section consists of the study of the anti-bacterial properties of the PP and PP modified mesh substrates. The effect of these materials on planktonic bacteria growth is described as first. Secondly, bacterial colonization and biofilm formation is discussed, together with the viability of bacterial cells.

The biofilm architecture on the studied substrates is also analyzed.

Effect on planktonic bacteria tests

The effect of the new coatings on bacterial growth in the planktonic state was tested by measuring the absorbance at 600 nm (A 600 ) of a culture in which the substrate was immersed. Results are expressed in terms of "Inhibition rate" compared to bacterial colonization on PP reference sample for the set of PP/Ar/MAPP(2 nd ), PP/Ar/MAPP/Ag and PP/Ar/MAPP/Ag/MAPP substrates. The effect of stretching on the Ag + release and subsequent anti-bacterial property was expressed in terms of "Inhibition rate" of stretched PP/Ar/MAPP/Ag/MAPP-60% samples compared to not stretched PP/Ar/MAPP/Ag/MAPP samples.

Antimicrobial Coatings for Soft Materials

4-197

4-205

cavities, groove-like features, which correspond to their size, where they can be protected from shear forces [135]. This type of behavior was observed on the PP sample, where bacteria showed preference to adhere in groove-like features of the surface (see Figure 456). However, quantitative investigations of the modified surface roughness was not performed to support this hypothesis.

Numbers of bacteria on the substrates containing silver nanoparticles (PP/Ar/MAPP/Ag/MAPP, PP/Ar/MAPP/Ag/MAPP-60%) were found to be significantly lower (p>99.9%) than the number of bacteria on PP sample (see adhesion may be considered to increase as well. As previously mentioned, the size and orientation of topographical features can influence the retention of cells on the surfaces [START_REF] Ploux | Bacteria/Material Interfaces: Role of the Material and Cell Wall Properties[END_REF]134,135]. The greatest bacterial cell retention is usually found to occur on the materials with size of features value close to the bacterial size [140,143,451,452]. In our study, at 4h of incubation, some bacteria that adhered on PP/Ar/MAPP/Ag/MAPP-60% stretched samples are in this situation.

Therefore, stretching at 60% rate of initial length of the coated PP may not provide the expected increase of bacterial colonization inhibition, compared to unstretched samples. Nevertheless, the number of bacteria on stretched samples remains lower than bacteria numbers on substrates without silver (PP and PP/Ar/MAPP substrates). As discussed in this section and in section 2.4.5, significant increase of the polymer overlayer crosslinking could allow to 4-226 4.3.2.2.2.3 Summary Characteristics of the biofilms on the basis of four calculated parameters such as "connected" biomass, thickness of biofilm, surface to biovolume ratio, which