In the last decades, there is an increased interest in developing strategies for improving bio-interfaces in the field of orthopedic or dental implants. Various coating materials were used as nano and micro-structured interfaces to fine-tune the cellular response. Among these, ceria (CeO2) nanostructures are thought to improve the biointerface mechanical properties, but also to stimulate the regenerating tissue biochemical activity by neutralizing the oxidative stress and stimulating cells proliferation. Within this context, this work presents for the first-time pyramid-shaped nanostructured ceria films obtained by Pulsed Laser Deposition (PLD) targeting the early response of human osteosarcoma cells (SaOs-2). SEM and AFM images of the nanostructured surfaces revealed shapes from the quasi-pyramidal, with rounded edges and dimensions of 90-120 nm, to the sharp edges and dimensions up to 350 nm, obtained by varying the number of pulses. The transition from hydrophobic to moderate hydrophilic behavior, as well as the increase of the polar component for the samples with more prominent features, were correlated to the topographical characteristics and further to cell behaviour. The influence of pyramidal-shaped ceria on the in vitro biological performance of SaOs-2 cells was demonstrated by the differences in early adhesion and distribution of phenotype SaOs-2 cells. Nevertheless, as the adhesion and spreading characteristics are cell line specific, mesenchymal stem cells (MSCs) were used for comparing the different cytoskeleton response as response to the ceria nanostructure type. In perspective, the behavior of mesenchymal progenitor cells is foreseen to be analyzed in view of establishing an osteogenic response on these biomaterials and the best-suited processing approach in order to use them as substrates for future bone regeneration applications.

We report on the synthesis by PLD of simple and lithium-doped biological-origin hydroxyapatite (HA) films. The role of doping reagents (Li2CO3, Li3PO4) on the morphology, structure, chemical composition, bonding strength and cytocompatibility of the films was investigated. SEM investigations of the films evidenced a surface morphology consisting of particles with mean diameters of (5-7) mu m. GIXRD analyses demonstrated that the synthesized structures consisted of HA phase only, with different degrees of crystallinity, mainly influenced by the doping reagent type. After only three days of immersion in simulated body fluid, FTIR spectra showed a remarkable growth of a biomimetic apatitic film, indicative of a high biomineralization capacity of the coatings. EDS analyses revealed a quasi-stoichiometric target-to-substrate transfer, the values inferred for the Ca/P ratio corresponding to a biological apatite. All synthesized structures displayed a hydrophilic behavior, suitable for attachment of osteoblast cells. In vitro cell viability tests showed that the presence of Li2CO3 and Li3PO4 as doping reagents promoted the hMSC growth on film surfaces. Taking into consideration these enhanced characteristics, corroborated with a low fabrication cost generated by sustainable resources, one should consider the lithium-doped biological-derived materials as promising prospective solutions for a next generation of coated implants with rapid osteointegration. (C) 2018 Elsevier B.V. All rights reserved.

In this study, coatings based on lysozyme embedded into a matrix of polyethylene glycol (PEG) and polycaprolactone (PCL) were fabricated by two different methods (Matrix Assisted Pulsed Laser Evaporation -MAPLE and Dip Coating) for obtaining antimicrobial coatings envisaged for long term medical applications. Coatings with different PEG: PCL compositions (3: 1; 1: 1; 1: 3) were synthesized in order to evaluate the antimicrobial activity of lysozyme embedded into the polymeric matrix. The main surface features, such as roughness and wettability, with impact on the microbial adhesion as well as on the eukaryote cell function were measured. The obtained composite coatings exhibited a significant antibacterial activity against Escherichia coli, Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus strains. As well, specific blended coatings showed appropriate viability, good spreading and normal cell morphology of SaOs2 human osteoblasts and mesenchymal stem cells (MSCs). These investigations highlight the suitability of biodegradable composites as implant coatings for decreasing the risk of bacterial contamination associated with prosthetic procedures. (C) 2016 Elsevier B.V. All rights reserved.

The purpose of this study was to obtain, characterize and evaluate the cytotoxicity and antimicrobial activity of coatings based on poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) - Lysozyme (P(3HB-3HV)/Lys) and P(3HB-3HV) - Polyethylene glycol - Lysozyme (P(3HB-3HV)/PEG/Lys) spheres prepared by Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique, in order to obtain functional and improved Ti-based implants. Morphological investigation of the coatings by Infrared Microscopy (IRM) and SEM revealed that the average diameter of P(3HB-3HV)/Lys spheres is around 2μm and unlike the drop cast samples, IRM recorded on MAPLE films revealed a good distribution of monitored functional groups on the entire scanned surface. The biological evaluation of MAPLE structured surfaces revealed an improved biocompatibility with respect to osteoblasts and endothelial cells as compared with Ti substrates and an enhanced anti-biofilm effect against Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) tested strains. Thus, we propose that the fabricated P(3HB-3HV)/PEG/Lys and P(3HB-3HV)/Lys microspheres may be efficiently used as a matrix for controlled local drug delivery, with practical applications in developing improved medical surfaces for the reduction of implant-associated infections.

Pulsed Laser Deposition (PLD) and Matrix Assisted Pulsed Laser Evaporation (MAPLE) techniques were applied for growing hydroxyapatite (HA) thin films on titanium substrates. All experiments were conducted in a reaction chamber using a KrP* excimer laser source (lambda = 248 nm, TEWHM approximate to 25 ns). Half of the samples were post-deposition thermally treated at 500 degrees C in a flux of water vapours in order to restore crystallinity and improve adherence.Coating surface morphologies and topographies specific to the deposition method were evidenced by scanning electron, atomic force microscopy investigations and profilometry. They were shown to depend on deposition technique and also on the post-deposition treatment. Crystalline structure of the coatings evaluated by X-ray diffraction was improved after thermal treatment. Biocompatibility of coatings, cellular adhesion, proliferation and differentiation tests were conducted using human mesenchymal stem cells (MSCs). Results showed that annealed MAPLE deposited HA coatings were supporting MSCs proliferation, while annealed PLD obtained films were stimulating osteogenic differentiation. (C) 2016 Elsevier B.V. All rights reserved.

In this work, we report the synthesis of functional biocompatible piezoelectric (1 - x)Ba(Ti0.8Zr0.2)TiO3-x(Ba0.7Ca0.3)TiO3, x = 0.45 (BCZT45), thin films with high piezoelectric properties. Pulsed-laser-based techniques, classical pulsed-laser deposition and matrix-assisted pulsed-laser evaporation, were used to synthesize the BCZT45 thin films. The second technique was employed in order to ensure growth on polymer flexible Kapton substrates. The BCZT45 thin films grown by both techniques show similar structural properties and high piezoelectric coefficient coupling between the mechanical loading and electrical potential. While it has long been shown that the electrical potential favors biological processes like osteogenesis, the assessment of cell adhesion and osteogenic differentiation onto BCZT materials has not yet been demonstrated. We prove here for the first time that BCZT 45 coatings on Kapton polymer substrates provide optimal support for osteogenic differentiation of mesenchymal stem cells in the bone marrow.

Silicon substrates were irradiated at normal incidence with a femtosecond Ti:sapphire laser (Quatronix, 90 fs pulse duration, 1 kHz repetition rate, M(2) ~ 1.2, maximum energy peak 350 mJ ) operating at a wavelength of 400 nm and focused via a microscope objective (Newport; UV Objective Model, 37x 0.11 N.A.). The laser scanning was assisted by liquids precursors media such as methanol and 1,1,2-trichlorotrifluoroethane. By altering the processing parameters, such as incident laser energy, scanning speed, and different irradiation media, various surface structures were produced on areas with 1 mm(2) dimensions. We analyzed the dependence of the surface morphology on laser pulse energy, scanning speed and irradiation media. Well ordered areas are developed without imposing any boundary conditions for the capillary waves that coarsens the ripple pattern. To assess biomaterial-driven cell adhesion response we investigated actin filaments organization and cell morphological changes following growth onto processed silicon substrates. Our study of bone cell progenitor interaction with laser nanoprocessed silicon lines has shown that cells anchor mainly to contact points along the nanostructured surface. Consequently, actin filaments are stretched towards the 15 µm wide parallel lines increasing lateral cell spreading and changing the bipolar shape of mesenchymal stem cells.

Active protein and bioceramic calcium hydroxyapatite (HA) bilayers were grown by combining conventional pulsed laser deposition (PLD) and matrix-assisted pulsed laser evaporation (MAPLE) techniques. A pulsed UV KrF* excimer laser was used for the irradiations. The HA layers were grown by PLD. Proteins with antimicrobial action were attached to the bioceramic layers using MAPLE. The composite MAPLE targets were obtained by dissolving the proteins powder in distilled water. The crystalline status and chemical composition of the obtained structures were studied by X-ray diffractometry and Fourier transform infrared spectroscopy. The layers were grown for the design of advanced future metal implants coatings, ensuring both enhanced bone formation and localized antimicrobial therapy. Our results demonstrated that protein coatings improve bone cell proliferation in vitro. Immunofluorescence experiments show that actin filaments stretch throughout bone cells and sustain their optimal spreading.

The diagnosis and treatment of malignant melanoma by means of the formulation of active principles with dendrimeric nanoparticles is an area of great current interest. The identification and understanding of molecular mechanisms which ensure the integration of particular dendrimeric nanostructures in tumor cellular environment can provide valuable guidance in their coupling strategies with antitumor or diagnostic agents. Two structurally distinct maltose-shell modified 5th generation (G5) poly(propylene imine) (PPI) glycodendrimers fluorescently labeled, (a) with open maltose shell, cationic charged G5-PPI-OS and (b) with dense maltose shell and nearly neutral G5-PPI-DS, were tested in relation with several melanoma cell lines. We found that three melanoma cell lines internalize G5-PPI-DS structure more efficiently than non tumoral HEK297T cells. Furthermore, the internalization pathways of G5-PPI-OS and G5-PPI-DS are characteristic for each tumor cell phenotype and include more than one mechanism. As a general trend, large amounts of both G5-PPI-OS and G5-PPI-DS are internalized on cholesterol-dependent pathway in MJS primary melanoma cells and on non conventional pathways in SK28 metastatic melanoma cells. G5-PPI-OS, temporarily retained at plasma membrane in both cell lines, is internalized slower in metastatic than in primary phenotype. Unlike G5-PPI-OS, G5-PPI-DS is immediately endocytosed in both cell lines. The unconventional internalization pathway and trafficking, exclusively used by G5-PPI-DS in metastatic cells, is described at molecular level. The decay kinetics of fluorescent labeled G5-PPI-OS and G5-PPI-DS is distinct in the two cellular phenotypes. Both cationic and neutral maltose G5-PPI glycodendrimeric structures represent molecules based on which designing of new formulations for therapy or/and diagnosis of melanoma can be further developed.

We introduce a combinatorial approach for the fabrication of organic biopolymer thin films. Structures with compositional gradient are obtained by simultaneous laser vaporization of two distinct targets. Matrix-assisted pulsed laser evaporation deposition method was applied to obtain a compositional library of levan and oxidized levan in form of thin film. The gradient of film composition and structure was demonstrated by infrared spectroscopy while in vitro cell culture assays illustrated characteristic responses of cells to specific surface regions. The method can rapidly generate discrete areas of organic film compositions with improved properties than starting materials.

Several dermal substitutes for skin grafting are now commercially available, although their performance still needs improvement. Most artificial dermises have a lower take rate than autologous grafts and require more time for sufficient vascular ingrowth to overlay the skin graft. Herein we characterize new two-dimensional scaffolds for tissue-engineering applications, which were fabricated by two-photon polymerization (2PP) of ormosils hybrid materials. For the 2PP experiments, a Ti:sapphire laser was used to induce the photopolymerization. In this study we showed that the polymeric structures with controlled architectures produced via 2PP could be used as scaffolds for the in vitro culture and proliferation of human dermal fibroblasts. Fluorescence microscopy revealed that the fibroblasts' orientation was guided by the scaffold geometry, consisting of ormosils lines or grids. This 'dermal equivalent' was investigated for its ability to accommodate epidermal cells. To evaluate this interaction, two experimental approaches were hence used: (a) fibroblastmelanocyte co-cultures; and (b) fibroblastkeratinocyte organotypic cultures. During their growth on ormosil scaffolds, productive interaction of fibroblasts with both epidermal cell types was found. Moreover, this pseudo-dermis was shown to support the growth of keratinocytes for up to 8days after their seeding. Copyright (c) 2011 John Wiley & Sons, Ltd.

Hybrid methacrylates based on silane derivates (ormosils) have been considered for applications in electronics, microtechnology, corrosion resistant coatings, dentistry, and biomedical implants. The presence of both inorganic chains, responsible for chemical and thermal stability, hardness, and transparency, and organic groups, which bring new advantages such as the possibility of functionalization and easy, low temperature processing, can result in the appearance of unique properties. 2D structures of hybrid polymers were produced by Two Photon Polymerization (2PP). A Ti: Sapphire laser having 200 fs pulse duration and 2 kHz repetition rate, working at a wavelength of 775 nm, was used for the 2PP experiments. The biocompatibility of the obtained structures (scaffolds) was tested in different cell cultures, which is a first step toward exploring their potential for applications in tissue engineering. Cells morphology, adhesion, and alignment were studied on polymeric structures with different shapes, obtained in various experimental conditions. Their interaction with normal human epidermal melanocytes (NHEM) and dysplastic oral keratinocytes (DOK) was investigated, with the aim of obtaining an epidermal graft.

The deposition of fibronectin (FN) from saline buffer-based cryogenic targets by matrix-assisted pulsed laser evaporation (MAPLE) onto silicon substrates is reported. A uniform distribution of FN was revealed by Ponceau staining after control experiments on nitrocellulose paper. Well-organized particulates with heights from hundreds of nanometers up to more than 1 μm packed in homogeneous layers were evidenced by optical microscopy and profilometry on Si substrates. Atomic force microscopy images showed regions composed of buffer and FN aggregates forming a compact film. Comparison of infrared spectra of drop-cast and MAPLE-deposited FN confirmed the preservation of composition and showed no degradation of the protein. The protein deposition on Si was confirmed by antibody staining. Small aggregates and fluorescent fibrils were visualized by fluorescence microscopy. Superior attachment of human osteoprogenitor cells cultivated for 3 h proved the presence of stable and intact FN molecules after transfer.

We report on the synthesis of 700 nm thick bioglass thin films by magnetron sputtering from 45S5 bioglass targets in the argon atmosphere. The adhesion of films to Ti substrates increased 1 75 times when introducing an similar to 70 nm thick mixed glass-Ti buffer layer (BG(1-x)Ti(x) (x = 0-1)) with gradient of composition by co-sputtering. The morphological and structural properties of the films were studied by XRD, ETIR and microscopic techniques, showing an improvement after the two-hour thermal air treatment at 650 degrees C. We investigated in vitro the viability and proliferation of human osteoblast cells cultivated onto the surface of the films The osteoblasts spread over the surface adopting typical polyhedral shapes with numerous focal adhesion points and protrusions infiltrating deep into the films. We assigned this evolution to the improved mechanical properties and enhanced bioactivity due to the prevalent formation of combeite and wollastonite crystalline phases in the heat treated bioglass films (C) 2010 Elsevier B.V All rights reserved

The aim of this study was to obtain biomimetic inorganic–organic thin films as coatings for metallic medical implants. These contain hydroxyapatite, the inorganic component of the bony tissues, and a natural biopolymer – silk fibroin – added in view to induce the surface functionalization. Hydroxyapatite (HA), silk fibroin (FIB) and composite HA–FIB films were obtained by Matrix Assisted Pulsed Laser Evaporation (MAPLE) in order to compare their physical and biological performances as coatings on metallic prostheses. We used an excimer laser source (KrF*, λ=248nm, τ=25ns) operated at 10Hz repetition rate. Coatings were deposited on quartz, Si and Ti substrates and then subjected to physical (FTIR, XRD, AFM, SEM) analyses, correlated with the results of the cytocompatibility in vitro tests. The hybrid films were synthesized from frozen targets of aqueous suspensions with 3:2 or 3:4 weight ratio of HA:FIB. An appropriate stoichiometric and functional transfer was obtained for 0.4–0.5J/cm2 laser fluence. FTIR spectra of FIB and HA–FIB films exhibited distinctive absorption maxima, in specific positions of FIB random coil form: 1540cm−1 amide II, 1654cm−1 amide I, 1243cm−1 amide III, while the peak from 1027cm−1 appeared only for HA and composite films. Osteosarcoma SaOs2 cells cultured 72h on FIB and HA–FIB films showed increased viability, good spreading and normal cell morphology. The well-elongated, flattened cells are a sign of an appropriate interaction with the MAPLE FIB and composite HA–FIB coatings.