Laser deposition of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) - lysozyme microspheres based coatings with anti-microbial properties.

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.

Lead-Free Piezoelectric (Ba,Ca)(Zr,Ti)O3 Thin Films for Biocompatible and Flexible Devices.

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.

Cell adhesion response on femtosecond laser initiated liquid assisted silicon surface.

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 calcium hydroxyapatite bilayers grown by laser techniques for therapeutic applications.

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.

Dermal cells distribution on laser-structured ormosils.

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) fibroblast-melanocyte co-cultures; and (b) fibroblast-keratinocyte 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 8 days after their seeding.

Internalization and intracellular trafficking of poly(propylene imine) glycodendrimers with maltose shell in melanoma cells.

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.

Fibronectin layers by matrix-assisted pulsed laser evaporation from saline buffer-based cryogenic targets.

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.

Specific biofunctional performances of the hydroxyapatite-sodium maleate copolymer hybrid coating nanostructures evaluated by in vitro studies.

The nanohybrid structures consisting of hydroxyapatite (HA) and sodium maleate-vinyl acetate copolymer (MP) deposited by Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique on Ti surfaces were investigated for specific biological qualities required in bone implantology. The data from in vitro studies demonstrated that human primary osteoblasts (OBs) firmly adhered to Ti coated with HA-MP as indicated by cytoskeleton and vinculin dynamics. OBs spread onto biomaterial surface and formed groups of cells which during their biosynthetic activity expressed OB phenotype specific markers (collagen and non-collagenous proteins) and underwent controlled proliferation.