Pascale Chevallier

Characterization of Multilayer Anti-Fog Coatings

Fog formation on transparent substrates constitutes a major challenge in several optical applications requiring excellent light transmission characteristics. Anti-fog coatings are hydrophilic, enabling water to spread uniformly on the surface rather than form dispersed droplets. Despite the development of several anti-fog coating strategies, the long-term stability, adherence to the underlying substrate, and resistance to cleaning procedures are not yet optimal. We report on a polymer-based anti-fog coating covalently grafted onto glass surfaces by means of a multistep process. Glass substrates were first activated by plasma functionalization to provide amino groups on the surface, resulting in the subsequent covalent bonding of the polymeric layers. The anti-fog coating was then created by the successive spin coating of (poly(ethylene-maleic anhydride) (PEMA) and poly(vinyl alcohol) (PVA) layers. PEMA acted as an interface by covalently reacting with both the glass surface amino functionalities and the PVA hydroxyl groups, while PVA added the necessary surface hydrophilicity to provide anti-fog properties. Each step of the procedure was monitored by XPS, which confirmed the successful grafting of the coating. Coating thickness was evaluated by profilometry, nanoindentation, and UV visible light transmission. The hydrophilic nature of the anti-fog coating was assessed by water contact angle (CA), and its anti-fog efficiency was determined visually and tested quantitatively for the first time using an ASTM standard protocol. Results show that the PEMA/PVA coating not only delayed the initial period required for fog formation but also decreased the rate of light transmission decay. Finally, following a 24 hour immersion in water, these PEMA/PVA coatings remained stable and preserved their anti-fog properties.

Polydopamine as an intermediate layer for silver and hydroxyapatite immobilisation on metallic biomaterials surface

Hydroxyapatite (HA) coated implant is more susceptible to bacterial infection as the micro-structure surface which is beneficial for osseointegration, could also become a reservoir for bacterial colonisation. The aim of this study was to introduce the antibacterial effect of silver (Ag) to the biomineralised HA by utilising a polydopamine film as an intermediate layer for Ag and HA immobilisation. Sufficient catechol groups in polydopamine were required to bind chemically stainless steel 316 L, Ag and HA elements. Different amounts of Ag nanoparticles were metallised on the polydopamine grafted stainless steel by varying the immersion time in silver nitrate solution from 12 to 24 h. Another polydopamine layer was then formed on the metallised film, followed by surface biomineralisation in 1.5 Simulated Body Fluid (SBF) solution for 3 days. Several characterisation techniques including X-Ray Photoelectron Spectroscopy, Atomic Force Microscopy, Scanning Electron Microscopy and Contact Angle showed that Ag nanoparticles and HA agglomerations were successfully immobilised on the polydopamine film through an element reduction process. The Ag metallisation at 24 h has killed the viable bacteria with 97.88% of bactericidal ratio. The Ag was ionised up to 7 days which is crucial to prevent bacterial infection during the first stage of implant restoration. The aged functionalised films were considered stable due to less alteration of its chemical composition, surface roughness and wettability properties. The ability of the functionalised film to coat complex and micro scale metal make it suitable for dental and orthopaedic implants application.

Multidentate block-copolymer-stabilized ultrasmall superparamagnetic iron oxide nanoparticles with enhanced colloidal stability for magnetic resonance imaging.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) with diameters <5 nm hold great promise as T1-positive contrast agents for in vivo magnetic resonance imaging. However, control of the surface chemistry of USPIOs to ensure individual colloidal USPIOs with a ligand monolayer and to impart biocompatibility and enhanced colloidal stability is essential for successful clinical applications. Herein, an effective and versatile strategy enabling the development of aqueous colloidal USPIOs stabilized with well-defined multidentate block copolymers (MDBCs) is reported. The multifunctional MDBCs are designed to consist of an anchoring block possessing pendant carboxylates as multidentate anchoring groups strongly bound to USPIO surfaces and a hydrophilic block having pendant hydrophilic oligo(ethylene oxide) chains to confer water dispersibility and biocompatibility. The surface of USPIOs is saturated with multiple anchoring groups of MDBCs, thus exhibiting excellent long-term colloidal stability as well as enhanced colloidal stability at biologically relevant electrolyte, pH, and temperature conditions. Furthermore, relaxometric properties as well as in vitro and in vivo MR imaging results demonstrate that the MDBC-stabilized USPIO colloids hold great potential as an effective T1 contrast agent.

Blood protein adsorption on sulfonated chitosan and κ-carrageenan films.

Many strategies have been reported to improve compatibility of biopolymers using chemical and physical modifications. One possibility is the introduction of sulfonate groups (R-SO3(-)) in the chitosan chain. Another biopolymer with similar characteristics to those of heparin is κ-carrageenan. This study proposed to investigate the application of these two polymers, based on their potential for globular protein adsorption (BSA and fibrinogen). Polymeric films of chitosan and κ-carrageenan were prepared; all films were characterized by elemental analyses, FTIR, XPS and SEM. Characterization techniques showed that the chitosan chain was modified and confirmed the existence of sulfonate groups, as well as in the κ-carrageenan chain, indicating surfaces with similar chemical properties to those of heparin. The effect of charge density was observed for each adsorption condition (BSA at pH 5.0 and 7.4). A more pronounced adsorption rate was observed at pH 5.0 than at pH 7.4 and equilibrium adsorption was achieved, in both cases, after approximately 20 min. The equilibrium data indicate a lower adsorption rate for the sulfonated chitosan film, in comparison to the other films. These results confirm the potential of modified chitosan for use in applications in which globular protein adsorption should be avoided.

RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells.

UNLABELLED Human bone marrow mesenchymal stem cells (hBMSCs) commitment and differentiation are dictated by bioactive molecules sequestered within their Extra Cellular Matrix (ECM). One common approach to mimic the physiological environment is to functionalize biomaterial surfaces with ECM-derived peptides able to recruit stem cells and trigger their linage-specific differentiation. The objective of this work was to investigate the effect of RGD and BMP-2 ligands crosstalk and density on the extent of hBMSCs osteogenic commitment, without recourse to differentiation medium. RGD peptide promotes cell adhesion via cell transmembrane integrin receptors, while BMP-2 peptide, corresponding to residues 73-92 of Bone Morphogenetic Protein-2, was shown to induce hBMSCs osteoblast differentiation. The immobilization of peptides on aminated glass was ascertained by X-ray Photoelectron Spectroscopy (XPS), the density of grafted peptides was quantified by fluorescence microscopy and the surface roughness was evaluated using Atomic Force Microscopy (AFM). The osteogenic commitment of hBMSCs cultured on RGD and/or BMP-2 surfaces was characterized by immunohistochemistry using STRO-1 as specific stem cells marker and Runx-2 as an earlier osteogenic marker. Biological results showed that the osteogenic commitment of hBMSCs was enhanced on bifunctionalized surfaces as compared to surfaces containing BMP-2, while on RGD surfaces cells mainly preserved their stemness character. These results demonstrated that RGD and BMP-2 mimetic peptides act synergistically to enhance hBMSCs osteogenesis without supplementing the media with osteogenic factors. These findings contribute to the development of biomimetic materials, allowing a deeper understanding of signaling pathways that govern the transition of stem cells towards the osteoblastic lineage. STATEMENT OF SIGNIFICANCE For a long time, scientists thought that the differentiation of Mesenchymal Stem Cells (MSCs) into bone cells was dictated by growth factors. This manuscript shed light on other ligands that play a crucial role in regulating MSCs fate. In concrete terms, it was demonstrated that the osteoinductive effect of BMP-2 peptide is 2 folds improved in the presence of adhesive RGD peptide. Compared to previous works highlighting this synergistic cooperation between RGD and BMP-2 peptides, the main strength of this work lies to the use of primitive human cells (hMSCs) and well-defined biomimetic material surfaces (controlled surface roughness and peptide densities). This work provides valuable insights to develop custom-designed in vitro cell culture models, capable of targeting the desired cell response.

A fluorophore-tagged RGD peptide to control endothelial cell adhesion to micropatterned surfaces.

The long-term patency rates of vascular grafts and stents are limited by the lack of surface endothelialisation of the implanted materials. We have previously reported that GRGDS and WQPPRARI peptide micropatterns increase the endothelialisation of prosthetic materials in vitro. To investigate the mechanisms by which the peptide micropatterns affect endothelial cell adhesion and proliferation, a TAMRA fluorophore-tagged RGD peptide was designed. Live cell imaging revealed that the micropatterned surfaces led to directional cell spreading dependent on the location of the RGD-TAMRA spots. Focal adhesions formed within 3 h on the micropatterned surfaces near RGD-TAMRA spot edges, as expected for cell regions experiencing high tension. Similar levels of focal adhesion kinase phosphorylation were observed after 3 h on the micropatterned surfaces and on surfaces treated with RGD-TAMRA alone, suggesting that partial RGD surface coverage is sufficient to elicit integrin signaling. Lastly, endothelial cell expansion was achieved in serum-free conditions on gelatin-coated, RGD-TAMRA treated or micropatterned surfaces. These results show that these peptide micropatterns mainly impacted cell adhesion kinetics rather than cell proliferation. This insight will be useful for the optimization of micropatterning strategies to improve vascular biomaterials.

Metal chelate grafting at the surface of mesoporous silica nanoparticles (MSNs): physico-chemical and biomedical imaging assessment

Mesoporous silica nanoparticles (MSNs) are being developed as drug delivery vectors. Biomedical imaging (MRI and PET) enables their tracking in vivo, provided their surface is adequately grafted with imaging probes (metal chelates). However, MSNs are characterized by huge specific surfaces, and high-quality metal chelate anchoring procedures must be developed and validated, to demonstrate that their detection in vivo is associated to the presence of nanoparticles and not to detached metal chelates. MCM-48 nanospheres (M48SNs, 150 nm diam., 3-D pore geometry) were synthesized and functionalized with diethylenetriaminepentaacetic acid (DTPA). The strong grafting of DTPA was confirmed by 29Si MAS-NMR, XPS, FTIR and TGA. The particles were labeled with paramagnetic ions Gd3+ (for MRI) as well as radioactive ions 64Cu2+ (for PET; half-life: 12.7 h). Gd3+-DTPA-M48SNs formed a stable colloid in saline media for at least 6 months, without any sign of aggregation. The relaxometric properties were measured at various magnetic fields. The strength of DTPA binding at the surface of MSNs was also assessed in vivo, by injecting mice (i.v.) with Gd3+/64Cu2+-DTPA-M48SNs. Vascular retention and urinary clearance were monitored by MRI, whereas the PET modality provided dynamic and quantitative assessment of biodistribution and blood/organ clearance. No significant 64Cu activity was detectable in the bladder. The study confirmed the very limited detachment of DTPA from M48SNs cores once injected in vivo. The transit of MSNs through the liver and intestinal tract, does not lead to evidence of Gd3+/64Cu2+-DTPA in the urine. This physico-chemical and biodistribution study confirms the quality of DTPA attachment at the surface of the particles, necessary to allow further development of PET/MRI-assisted MSN-vectorized drug delivery procedures.

Plasma functionalization of poly(vinyl alcohol) hydrogel for cell adhesion enhancement

Tailoring the interface interactions between a biomaterial and the surrounding tissue is a capital aspect to consider for the design of medical devices. Poly(vinyl alcohol) (PVA) hydrogels present suitable mechanical properties for various biological substitutes, however the lack of cell adhesion on their surface is often a problem. The common approach is to incorporate biomolecules, either by blending or coupling. But these modifications disrupt PVA intra- and intermolecular interactions leading therefore to a loss of its original mechanical properties. In this work, surface modification by glow discharge plasma, technique known to modify only the surface without altering the bulk properties, has been investigated to promote cell attachment on PVA substrates. N2/H2 microwave plasma treatment has been performed, and the chemical composition of PVA surface has been investigated. X-ray photoelectron and Fourier transform infrared analyses on the plasma-treated films revealed the presence of carbonyl and nitrogen species, including amine and amide groups, while the main structure of PVA was unchanged. Plasma modification induced an increase in the PVA surface wettability with no significant change in surface roughness. In contrast to untreated PVA, plasma-modified films allowed successful culture of mouse fibroblasts and human endothelial cells. These results evidenced that the grafting was stable after rehydration and that it displayed cell adhesive properties. Thus plasma amination of PVA is a promising approach to improve cell behavior on contact with synthetic hydrogels for tissue engineering.

Influence of the 316 L stainless steel interface on the stability and barrier properties of plasma fluorocarbon films.

Coatings are known to be one of the more suited strategies to tailor the interface between medical devices and the surrounding cells and tissues once implanted. The development of coatings and the optimization of their adhesion and stability are of major importance. In this work, the influence of plasma etching of the substrate on a plasma fluorocarbon ultrathin coating has been investigated with the aim of improving the stability and the corrosion properties of coated medical devices. The 316 L stainless steel interface was subjected to two different etching sequences prior to the plasma deposition. These plasma etchings, with H(2) and C(2)F(6) as gas precursors, modified the chemical composition and the thickness of the oxide layer and influenced the subsequent polymerization. The coating properties were evaluated using flat substrates submitted to deformation, aging into aqueous medium and corrosion tests. X-ray photoelectron spectroscopy (XPS), time of flight-secondary ion mass spectrometry (ToF-SIMS), ellipsometry, and atomic force microscopy (AFM) were performed to determine the effects of the deformation and the aging on the chemistry and morphology of the coated samples. Analyses showed that plasma etchings were essential to promote reproducible polymerization and film growth. However, the oxide layer thinning due to the etching lowered the corrosion resistance of the substrate and affected the stability of the interface. Still, the deformed samples did not exhibited adhesion and cohesion failure before and after the aging.

Toward High-Performance Coatings for Biomedical Devices: Study on Plasma-Deposited Fluorocarbon Films and Ageing in PBS

High performance coatings tailored to medical devices represent a recognised approach to modulate surface properties. Plasma-deposited fluorocarbon films have been proposed as a potential stent coating. Previous studies have shown promising adhesion properties: the 35 nm-thick film sustained plastic deformation up to 25% such as induced during the clinical implantation. In this study, the compositional and morphological changes of plasma-deposited fluorocarbon films were examined during ageing in a pseudo-physiological medium, a phosphate buffer solution (PBS), by angle-resolved XPS, FT-IR data and AFM images. The evolution of the ageing process is discussed: defluorination and crosslinking yielded an oxidized protective top layer onto the films, which showed further degradation.