PLASMATIC LEVELS OF NEUROPEPTIDES, INCLUDING OXYTOCIN, IN CHILDREN WITH AUTISM SPECTRUM DISORDER, CORRELATE WITH THE DISORDER SEVERITY.

CONTEXT: Oxytocin has been investigated as a potential medication for psychiatric disorders. OBJECTIVE AND DESIGN: This study prospectively investigates correlations between oxytocin and other neuropeptides plasma levels in patients with autism spectrum disorders (ASD) according to severity and treatment, as compared to controls. SUBJECTS AND METHODS: Thirty-one children (6 neurotypical as control) participated in this study. The patients were classified into mildly and severely-affected, according to Autism Diagnostic Observation Schedule (ADOS) scores. Oxytocin, orexin A and B, α-MSH, ß-endorphins, neurotensin and substance P were investigated using a quantitative multiplex assay or a competitive-ELISA method. RESULTS: Plasma oxytocin levels differed between the groups (F (2, 24) =6.48, p=0.006, η2=0.35, observed power=86%): patients with the mild ASD had higher values of plasma oxytocin than those with the severe form (average difference=74.56±20.74pg/mL, p=0.004). CONCLUSIONS: These results show a negative correlation between plasma levels of oxytocin and the severity of ASD and support the involvement of oxytocinergic mechanisms in ASD.

Updated European Consensus Statement on diagnosis and treatment of adult ADHD.

Background Attention-deficit/hyperactivity disorder (ADHD) is among the most common psychiatric disorders of childhood that often persists into adulthood and old age. Yet ADHD is currently underdiagnosed and undertreated in many European countries, leading to chronicity of symptoms and impairment, due to lack of, or ineffective treatment, and higher costs of illness. Methods The European Network Adult ADHD and the Section for Neurodevelopmental Disorders Across the Lifespan (NDAL) of the European Psychiatric Association (EPA), aim to increase awareness and knowledge of adult ADHD in and outside Europe. This Updated European Consensus Statement aims to support clinicians with research evidence and clinical experience from 63 experts of European and other countries in which ADHD in adults is recognized and treated. Results Besides reviewing the latest research on prevalence, persistence, genetics and neurobiology of ADHD, three major questions are addressed: (1) What is the clinical picture of ADHD in adults? (2) How should ADHD be properly diagnosed in adults? (3) How should adult ADHDbe effectively treated? Conclusions ADHD often presents as a lifelong impairing condition. The stigma surrounding ADHD, mainly due to lack of knowledge, increases the suffering of patients. Education on the lifespan perspective, diagnostic assessment, and treatment of ADHD must increase for students of general and mental health, and for psychiatry professionals. Instruments for screening and diagnosis of ADHD in adults are available, as are effective evidence-based treatments for ADHD and its negative outcomes. More research is needed on gender differences, and in older adults with ADHD.

Optimized silicon reinforcement of carbon coatings by pulsed laser technique for superior functional biomedical surfaces fabrication.

We report on the fabrication of silicon-reinforced carbon (C:Si) structures by combinatorial pulsed laser deposition to search for the best design for a new generation of multi-functional coated implants. The synthesized films were characterized from the morphological, structural, compositional, mechanical and microbiological points of view. Scanning electron microscopy revealed the presence, on top of the deposited layers, of spheroid particulates with sizes in the micron range. No micro-cracks or delaminations were observed. Energy dispersive x-ray spectroscopy and grazing incidence x-ray diffraction pointed to the existence of a C to Si compositional gradient from one end of the film to the other. Raman investigation revealed a relatively high sp3 hybridization of up to 80% at 40-48 mm apart from the edge with higher C content. Si addition was demonstrated to significantly increase C:Si film bonding to the substrate, with values above the ISO threshold for coatings to be used in high-loading biomedical applications. Surface energy studies pointed to an increase in the hydrophilic character of the deposited structures along with Si content up to 52 mN m-1. In certain cases, the Si-reinforced C coatings elicited an antimicrobial biofilm action. The presence of Si was proven to be benign to HEp-2 cells of human origin, without interfering with their cellular cycle. On this basis, reliable C:Si structures with good adherence to the substrate and high efficiency against microbial biofilms can be developed for implant coatings and other advanced medical devices.

Combinatorial MAPLE deposition of antimicrobial orthopedic maps fabricated from chitosan and biomimetic apatite powders.

Chitosan/biomimetic apatite thin films were grown in mild conditions of temperature and pressure by Combinatorial Matrix-Assisted Pulsed Laser Evaporation on Ti, Si or glass substrates. Compositional gradients were obtained by simultaneous laser vaporization of the two distinct material targets. A KrF* excimer (λ=248nm, τFWHM=25ns) laser source was used in all experiments. The nature and surface composition of deposited materials and the spatial distribution of constituents were studied by SEM, EDS, AFM, GIXRD, FTIR, micro-Raman, and XPS. The antimicrobial efficiency of the chitosan/biomimetic apatite layers against Staphylococcus aureus and Escherichia coli strains was interrogated by viable cell count assay. The obtained thin films were XRD amorphous and exhibited a morphology characteristic to the laser deposited structures composed of nanometric round shaped grains. The surface roughness has progressively increased with chitosan concentration. FTIR, EDS and XPS analyses indicated that the composition of the BmAp-CHT C-MAPLE composite films gradually modified from pure apatite to chitosan. The bioevaluation tests indicated that S. aureus biofilm is more susceptible to the action of chitosan-rich areas of the films, whilst the E. coli biofilm proved more sensible to areas containing less chitosan. The best compromise should therefore go, in our opinion, to zones with intermediate-to-high chitosan concentration which can assure a large spectrum of antimicrobial protection concomitantly with a significant enhancement of osseointegration, favored by the presence of biomimetic hydroxyapatite.

Structural, compositional, mechanical characterization and biological assessment of bovine-derived hydroxyapatite coatings reinforced with MgF2 or MgO for implants functionalization.

Hydroxyapatite (HA) is a consecrated biomaterial for bone reconstruction. In the form of thin films deposited by pulsed laser technologies, it can be used to cover metallic implants aiming to increase biocompatibility and osseointegration rate. HA of animal origin (bovine, BHA) reinforced with MgF2 (2wt.%) or MgO (5wt.%) were used for deposition of thin coatings with improved adherence, biocompatibility and antimicrobial activity. For pulsed laser deposition experiments, a KrF* (λ=248nm, τFWHM≤25ns) excimer laser source was used. The deposited structures were characterized from a physical-chemical point of view by X-Ray Diffraction, Fourier Transform Infra-Red Spectroscopy, Scanning Electron Microscopy in top- and cross-view modes, Energy Dispersive X-Ray Spectroscopy and Pull-out adherence tests. The microbiological assay using the HEp-2 cell line revealed that all target materials and deposited thin films are non-cytotoxic. We conducted tests on three strains isolated from patients with dental implants failure, i.e. Microccocus sp., Enterobacter sp. and Candida albicans sp. The most significant anti-biofilm effect against Microcococcus sp. strain, at 72h, was obtained in the presence of BHA:MgO thin films. For Enterobacter sp. strain a superior antimicrobial activity at 72h was noticed, in respect with simple BHA or Ti control. The enhanced antimicrobial performances, correlated with good cytocompatibility and mechanical properties recommend these biomaterials as an alternative to synthetic HA for the fabrication of reliable implant coatings for dentistry and other applications.

Stainless steel surface biofunctionalization with PMMA-bioglass coatings: compositional, electrochemical corrosion studies and microbiological assay.

A solution is proposed to surpass the inconvenience caused by the corrosion of stainless steel implants in human body fluids by protection with thin films of bioactive glasses or with composite polymer-bioactive glass nanostructures. Our option was to apply thin film deposition by matrix-assisted pulsed laser evaporation (MAPLE) which, to the difference to other laser or plasma techniques insures the protection of a more delicate material (a polymer in our case) against degradation or irreversible damage. The coatings composition, modification and corrosion resistance were investigated by FTIR and electrochemical techniques, under conditions which simulate their biological interaction with the human body. Mechanical testing demonstrates the adhesion, durability and resistance to fracture of the coatings. The coatings biocompatibility was assessed by in vitro studies and by flow cytometry. Our results support the unrestricted usage of coated stainless steel as a cheap alternative for human implants manufacture. They will be more accessible for lower prices in comparison with the majority present day fabrication of implants using Ti or Ti alloys.

Fabrication of magnetite-based core-shell coated nanoparticles with antibacterial properties.

We report the fabrication of biofunctionalized magnetite core/sodium lauryl sulfate shell/antibiotic adsorption-shell nanoparticles assembled thin coatings by matrix assisted pulsed laser evaporation for antibacterial drug-targeted delivery. Magnetite nanoparticles have been synthesized and subsequently characterized by transmission electron microscopy and x-ray diffraction. The obtained thin coatings have been investigated by FTIR and scanning electron microscope, and tested by in vitro biological assays, for their influence on in vitro bacterial biofilm development and cytotoxicity on human epidermoid carcinoma (HEp2) cells.

Structural and biological evaluation of lignin addition to simple and silver-doped hydroxyapatite thin films synthesized by matrix-assisted pulsed laser evaporation.

We report on thin film deposition by matrix-assisted pulsed laser evaporation of simple hydroxyapatite (HA) or silver (Ag) doped HA combined with the natural biopolymer organosolv lignin (Lig) (Ag:HA-Lig). Solid cryogenic target of aqueous dispersions of Ag:HA-Lig composite and its counterpart without silver (HA-Lig) were prepared for evaporation using a KrF* excimer laser source. The expulsed material was assembled onto TiO2/Ti substrata or silicon wafers and subjected to physical-chemical investigations. Smooth, uniform films adherent to substratum were observed. The chemical analyses confirmed the presence of the HA components, but also evidenced traces of Ag and Lig. Deposited HA was Ca deficient, which is indicative of a film with increased solubility. Recorded X-ray Diffraction patterns were characteristic for amorphous films. Lig presence in thin films was undoubtedly proved by both X-ray Photoelectron and Fourier Transform Infra-Red Spectroscopy analyses. The microbiological evaluation showed that the newly assembled surfaces exhibited an inhibitory activity both on the initial steps of biofilm forming, and on mature bacterial and fungal biofilm development. The intensity of the anti-biofilm activity was positively influenced by the presence of the Lig and/or Ag, in the case of Staphylococcus aureus, Pseudomonas aeruginosa and Candida famata biofilms. The obtained surfaces exhibited a low cytotoxicity toward human mesenchymal stem cells, being therefore promising candidates for fabricating implantable biomaterials with increased biocompatibility and resistance to microbial colonization and further biofilm development.

Multi-layer haemocompatible diamond-like carbon coatings obtained by combined radio frequency plasma enhanced chemical vapor deposition and magnetron sputtering.

Radio-frequency Plasma Enhanced Chemical Vapour Deposition (in different methane dilutions) was used to synthesize adherent and haemocompatible diamond-like carbon (DLC) films on medical grade titanium substrates. The improvement of the adherence has been achieved by interposing a functional buffer layer with graded composition TixTiC1-x (x = 0-1) synthesized by magnetron co-sputtering. Bonding strength values of up to ~67 MPa have been measured by pull-out tests. Films with different sp(3)/sp(2) ratio have been obtained by changing the methane concentration in the deposition chamber. Raman spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction were employed for the physical-chemical characterization of the samples. The highest concentration of sp(3)-C (~87 %), corresponding to a lower DLC surface energy (28.7 mJ/m(2) ), was deposited in a pure methane atmosphere. The biological response of the DLC films was assayed by a state-of-the-art biological analysis method (surface enhanced laser desorption/ionization-time of flight mass spectroscopy), in conjunction with other dedicated testing techniques: Western blot and partial thromboplastin time. The data support a cause-effect relationship between sp(3)-C content, surface energy and coagulation time, as well as between platelet-surface adherence properties and protein adsorption profiles.

Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots.

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.

Functionalized magnetite silica thin films fabricated by MAPLE with antibiofilm properties.

We report on the fabrication of magnetite/salicylic acid/silica shell/antibiotics (Fe(3)O(4)/SA/SiO(2)/ATB) thin films by matrix-assisted pulsed laser evaporation (MAPLE) to inert substrates. Fe(3)O(4)-based powder have been synthesized and investigated by XRD and TEM. All thin films were studied by FTIR, SEM and in vitro biological assays using Staphylococcus aureus and Pseudomonas aeruginosa reference strains, as well as eukaryotic HEp-2 cells. The influence of the obtained nanosystems on the microbial biofilm development as well as their biocompatibility has been assessed. For optimum deposition conditions, we obtained uniform adherent films with the composition identical with the raw materials. Fe(3)O(4)/SA/SiO(2)/ATB thin films had an inhibitory activity on the ability of microbial strains to initiate and develop mature biofilms, in a strain- and antibiotic-dependent manner. These magnetite silica thin films are promising candidates for the development of novel materials designed for the inhibition of medical biofilms formed by different pathogenic agents on common substrates, frequently implicated in the etiology of chronic and hard to treat infections.

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.

Immobilization of urease by laser techniques: synthesis and application to urea biosensors.

Urease thin films have been immobilized using matrix-assisted pulsed laser evaporation for biosensor applications in clinical diagnostics. The targets exposed to laser radiation were made of frozen composites that had been manufactured by dissolving urease in distilled water. An UV KrF* (lambda = 248 nm, tauFWHM congruent with 30 ns, nu = 10 Hz) excimer source was used for the multipulse laser irradiation of the targets that were cooled down to solidification using Peltier elements. The incident laser fluence was set at 0.4 J/cm2. The surface morphology and chemical bonding states of the laser immobilized urease thin films were investigated by atomic force microscopy and Fourier transform infrared spectroscopy. The enzymatic activity and kinetics of the immobilized urease were assayed by the Worthington method, which monitors urea hydrolysis by coupling ammonia production to a glutamate dehydrogenase reaction. Decreased absorbance was found at 340 nm and correlated with the enzymatic activity of urease.

Strontium-substituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response.

The increasing interest in strontium incorporation into biomaterials for hard tissue repair is justified by the growing evidence of its beneficial effect on bone. We successfully synthesized hydroxyapatite (HA) thin films with different extents of strontium substitution for calcium (0, 1, 3 or 7 at.%) by pulsed-laser deposition. The coatings displayed a granular surface and a good degree of crystallinity, which slightly diminished as strontium content increased. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin films up to 21 days. MG63 cells grown on the strontium-doped HA coatings displayed normal morphology, good proliferation and increased values of the differentiation parameters, whereas the number of osteoclasts was negatively influenced by the presence of strontium. The positive effect of the ion on bone cells was particularly evident in the case of coatings deposited from HA at relatively high strontium contents (3-7%), where significantly increased values of alkaline phosphatase activity, osteocalcin, type I collagen and osteoprotegerin/TNF-related activation-induced cytokine receptor ratio, and considerably reduced values of osteoclast proliferation, were observed.

Creatinine biomaterial thin films grown by laser techniques.

Creatinine thin films were synthesised by matrix assisted pulsed laser deposition (PLD) techniques for enzyme-based biosensor applications. An UV KrF* (lambda=248 nm, tau approximately 10 ns) excimer laser source was used for the irradiation of the targets at incident fluence values in the 0.3-0.5 J/cm2 range. For the matrix assisted PLD the targets consisted on a frozen composite obtained by dissolving the biomaterials in distilled water. The surface morphology, chemical composition and structure of the obtained biomaterial thin films were investigated by scanning electron microscopy, Fourier transform infrared spectroscopy, and electron dispersive X-ray spectroscopy as a function of the target preparation procedure and incident laser fluence.

Human osteoblast response to pulsed laser deposited calcium phosphate coatings.

Octacalcium phosphate (OCP) and Mn(2+)-doped carbonate hydroxyapatite (Mn-CHA) thin films were deposited on pure, highly polished and chemically etched Ti substrates with pulsed laser deposition. The coatings exhibit different composition, crystallinity and morphology that might affect their osteoconductivity. Human osteoblasts were cultured on the surfaces of OCP and Mn-CHA thin films, and the cell attachment, proliferation and differentiation were evaluated up to 21 days. The cells showed a normal morphology and a very good rate of proliferation and viability in every experimental time. Alkaline phosphatase activity was always higher than the control and Ti groups. From days 7 to 21 collagen type I production was higher in comparison with control and Ti groups. The level of transforming growth factor beta 1 (TGF-beta1) was lower at 3 and 7 days, but reached the highest values during following experimental times (14 and 21 days). Our data demonstrate that both calcium phosphate coatings favour osteoblasts proliferation, activation of their metabolism and differentiation.

Biocompatible Mn2+-doped carbonated hydroxyapatite thin films grown by pulsed laser deposition.

Mn(2+)-doped carbonated hydroxyapatite (Mn-CHA) thin films were obtained by pulsed laser deposition on Ti substrates. The results of the performed complementary diagnostic techniques, X-ray diffraction, infrared spectroscopy, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy investigations indicate that the films are crystalline with a Ca/P ratio of about 1.64-1.66. The optimum conditions, when nearly stoichiometric crystalline thin films were deposited, were found to be 10 Pa oxygen pressure, 400 degrees C substrate temperature, and postdeposition heat treatment in water vapors at the same substrate temperature. The films were seeded with L929 fibroblast and hFOB1.19 osteoblast cells and subjected to in vitro tests. Both fibroblast and osteoblast cells have a good adherence on the Mn-CHA film and on the Ti or polystyrene references. Proliferation and viability tests showed that osteoblast cells growth on Mn-CHA-coated Ti was enhanced as compared to uncoated pure Ti surfaces. Caspase-1 activity was not affected significantly by the material, showing that Mn-CHA does not induce apoptosis of cultured cells. These results demonstrate that Mn-CHA films on Ti should provoke a faster osteointegration of the coated implants as compared to pure Ti. (c) 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 353-358, 2004.

Biocompatible nanocrystalline octacalcium phosphate thin films obtained by pulsed laser deposition.

We extended for the first time pulsed laser ablation to the deposition of octacalcium phosphate Ca8H2(PO4)6.5H2O (OCP) thin films. The depositions were performed with a pulsed UV laser source (lambda=248 nm, tau> or =20 ns) in a flux of hot water vapors. The targets were sintered from crystalline OCP powder and the laser ablation fluence was set at values of 1.5-2 J/cm2. During depositions the collectors, Si or Ti substrates, were maintained at a constant temperature within the range 20-200 degrees C. The resulting structures were submitted to heat treatment in hot water vapors for up to 6 h. The best results were obtained at a substrate temperature of 150 degrees C during both deposition and post-deposition treatment. High-resolution electron microscopy and XRD at grazing incidence indicated that the coatings obtained were made of nanocrystalline OCP. Cross-section TEM investigations showed that the coatings contained droplets stacked on Ti substrates as well as distributed across the entire thickness of the arborescence-like structure layers. The results of WST-1 assay, cell adherence, DNA replication, and caspase-1 activity confirmed the good biocompatibility of the coatings.