Every year a large part of western countries' medical charges is assigned to medical care of patients suffering from tissues and/or organs alterations. Health centers spend for this purposes more than 10 million euro/year and this expense grow continuously due to the population mean age's increase. Tissue engineering (TE) represents a valid alternative to organ/tissue transplantation. A fundamental step for TE is the cell seeding directly on porous tridimensional artificial structures called scaffolds. However on artificial polymer scaffolds made of PCL or PLA as an example often higher cell colonization at the periphery and inadequate colonization at the center of the scaffold were noted due to the hydrophobic character of the materials [1,2]. In this work a single and multistep innovative approaches based on plasma technology are proposed as an interesting alternative. A plasma deposition of Polyethylene oxide like (PEO-like) coatings is proposed alone and in combination with O2 plasma treatment to stimulate cell ingrowth.

Atmospheric pressure plasmas are acquiring more interest in biomedical applications where synthetic biodegradable polymers modified to impart cells adhesion properties play a crucial role. This paper shows a new approach for the bio functionalization of such materials: inclusion of a biomolecule during the plasma deposition. A dielectric barrier discharge system was used for the coatings deposition, coupled with an atomizer for the lactic acid/elastin aerosol feeding. ATR-FTIR, XPS, and UV-VIS were used to investigate the chemical composition of the coatings. By properly tuning plasma parameters, a good retention of the monomer chemical structure could be obtained in the coatings, as well as the inclusion of elastin in its structure.

Two different dielectric barrier discharge processes are presented, fedwith the aerosol of the organic precursor, todeposit -CHO containing coatings from lactic acid (pdLA) and tetraethylene glycol dimethyl ether (PEO-like) ofpossible interest in biomedical applications as biodegradable and non-fouling polymers, respectively.

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.

Atmospheric pressure air dielectric barrier discharges were applied to phosphate-buffered saline and complete Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum to investigate their oxidative chemical modifications induced in liquids that are relevant for cell culture experiments in the field of Plasma Medicine. The study mainly identified long-living reactive species. Hydrogen peroxide, superoxide anion radicals, and nitrite and nitrate ions were detected in DMEM. The density of the reactive species was correlated with the energy dose delivered to the liquids by the discharges.

Advances in the synthesis of porous microspheres and in their functionalization are increasing the interest in applications of alumina. This paper deals with coatings plasma deposited from 3-aminopropyltriethoxysilane by means of dielectric barrier discharges on alumina porous microspheres, shaped by a vibrational droplet coagulation technique. Aims of the work are the functionalization of the particles with active amino groups, as well as the evaluation of their surface coverage and of the penetration of the coatings into their pores. A multi-diagnostic approach was used for the chemical/morphological characterization of the particles. It was found that 5min exposure to plasma discharges promotes the deposition of homogeneous coatings onto the microspheres and within their pores, down to 1?m.

The atmospheric pressure plasma deposition of hydroxyl functionalized hydrocarbon films is reported in this work, with a reactor fed with water aerosol and ethylene. The effects of power and feed flow rates onto film chemistry have been investigated. Coatings produced with this approach can find application in the biomedical field, among others, as platforms for cell adhesion and proliferation. Results show that operating at 4 kHz provides a much higher amount of hydroxyl group in the coating compared with samples obtained at 11 kHz. After water immersion, the stability of the films and their amount of hydroxyl groups remain high. A simplified deposition mechanism is proposed.

Bio-composite coatings, consisting of an organic matrix embedding a bioactive molecule, have been deposited by means of atomizer-assisted atmospheric pressure plasma. Ethylene was chosen as the precursor of the matrix, while the atomizer was fed with a water solution of lysozyme. Coatings chemical composition was investigated by XPS, FTIR and MALDI-TOF spectroscopies, and it has been proved that the one-step inclusion of protein domains in the composite coatings is successful and lysozyme chemical structure is only slightly altered. The amount of embedded lysozyme is as high as 14?g/cm² as evaluated from water release test. Finally, the activity of the plasma-embedded protein is close to that of pure lysozyme as verified against Micrococcus lysodeikticus ATCC 4698 through an agar plate diffusion test.

The present study investigates the effect of a silver (Ag)-containing nanocomposite coating on Staphylococcus epider-midis adhesion and icaA gene expression. Bacterial interactions with organic coatings with and without Ag nanoclusters were assessed through a combination of both conventional phenotypic analysis, using microscopy, and genotypic analy-sis, using the relative reverse transcription Real-Time Polymerase Chain Reaction (RT-PCR). The results suggest that the incorporation of Ag in organic coatings can significantly decrease bacterial adhesion and viability with time, in comparison to the organic coating alone. The initial Ag release though at concentrations lower than the bactericidal, significantly increased icaA gene expression for the bacteria interacting with the Ag containing coating two hours post adhesion, especially under the higher shear rate. Stress-inducing conditions such as sub-bactericidal concentrations of Ag and high shear rate can therefore increase icaA expression, indicating that analysis of gene expression can not only refine our knowledge of bacterial-material interactions, but also yield novel biomarkers for potential use in assessing biomaterials antimicrobial performance.

In this work a genuine combination of a bottom-up approach, which is based on synthesis andfunctionalization of emitting nanocrystals (NCs), with a top-down strategy, which relies on aflexible and versatile cold plasma process, is shown. Luminescent semiconducting colloidalNCs consisting of a CdSe core coated with a ZnS shell (CdSe@ZnS) are directly assembledonto micro-patterned substrates previously functionalized by means of glow dischargesperformed through physical masks. The NC assembly is driven by electrostatic interactionsthat led to their successful organization into spatially resolved domains. Two distinct protocolsare tested, the former using a plasma deposition process combined with an electrostaticlayer-by-layer procedure, the latter based on a two-step plasma deposition/treatment process.The procedures are thoroughly monitored with fluorescence microscopy, atomic forcemicroscopy, x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy,transmission electron microscopy and scanning electron microscopy. The two-step plasmaprotocol is demonstrated to be more efficient in directing a uniform and specific assembly ofluminescent NCs with respect to the hybrid procedure. The presented 'mix and match'approach offers great potential for integrating NCs, with their unique size-dependentproperties, into microstructures, providing a universal platform for the fabrication of sensors,biochips, displays and switches.

Stainless steel surfaces were processed by means of plasma enhanced chemical vapor deposition (PE-CVD) fed with acrylic acid vapors in order to functionalize them with carboxyl groups, which were subsequently activated for covalent immobilization of heparin-loaded (HEP) NH2 group-functionalized (Fun) nanoliposomes (NLs). Empty Fun or HEP non-functionalized (control) NLs were used as controls. NLs were characterized for mean diameter, surface charge and heparin encapsulation/release. Different lipid compositions were used for NL construction; PC/Chol (2:1 mol/mol) or PC/Chol (4:1 mol/mol) (fluid type vesicles) [ which allow gradual release of heparin] and DSPC/Chol (2:1 mol/mol) (rigid type vesicles). Surface haemocompatibility was tested by measuring blood clotting time. Platelet adhesion on surfaces was evaluated morphologically by SEM and CLSM. The haemocompatibility of plasma-processed surfaces was improved (compared to untreated surfaces); Fun-HEP NL-coated surfaces demonstrated highest coagulation times. For short surface/blood incubation periods, surfaces coated with Fun-HEP NLs consisting of PC/Chol (2:1) had higher coagulation times (compared to DSPC/Chol NLs) due to faster release of heparin. Heparin release rate from the various NL types and surface platelet adhesion results were in agreement with the corresponding blood coagulation times. Concluding, covalent immobilization of drug entrapping NLs on plasma processed surfaces is a potential method for preparation of controlled-rate drug-eluting metallic stents or devices.

Plasma Processes and Polymers

Non-equilibrium plasmas offer several strategies for developing antibacterial surfaces that are able to repel and/or to kill bacteria. Due to the variety of devices, implants, and materials in general, as well as of bacteria and applications, plasma assisted antibacterial strategies need to be tailored to each specific surface. Nano-composite coatings containing inorganic (metals and metal oxides) or organic (drugs and biomolecules) compounds can be deposited in one step, and used as drug delivery systems. On the other hand, functional coatings can be plasma-deposited and used to bind antibacterial molecules, for synthesizing surfaces with long lasting antibacterial activity. In addition, non-fouling coatings can be produced to inhibit the adhesion of bacteria and reduce the formation of biofilm. This paper reviews plasma-based strategies aimed to reduce bacterial attachment and proliferation on biomedical materials and devices, but also onto materials used in other fields. Most of the activities described have been developed in the lab of the authors.

The behavior of cells in terms of cell-substrate and cell-cell interaction is dramatically affected by topographicalcharacteristics as shape, height, and distance, encountered in their physiological environment. The combinationof chemistry and topography of a biomaterial surface influences in turns, important biological responses asinflammatory events at tissue-implant interface, angiogenesis, and differentiation of cells. By disentangling theeffect of material chemistry from the topographical one, the possibility of controlling the cell behavior can beprovided. In this paper, surfaces with different roughness and morphology were produced by radiofrequency (RF,13.56 MHz) glow discharges, fed with hexafluoropropylene oxide (C3F6O), in a single process. Coatings withdifferent micro/nanopatterns and the same uppermost chemical composition were produced by combining twoplasma deposition processes, with C3F6O and tetrafluoroethylene (C2F4), respectively. The behavior of osteoblastlikecells toward these substrates clearly shows a strict dependence of cell adhesion and proliferation on surfaceroughness and morphology.

In this paper, we describe the deposition of PEO-like coatings using dielectric barrier discharges (DBDs) fed with aerosols of the TEGDME organic precursor in helium. By properly tuning plasma parameters such as aerosol/carrier flow ratio, frequency of the electric field applied and input power, the deposition process could be modulated to obtain coatings with variable PEO character, from 50% (cell adhesive) to 70% (nonfouling), which are interesting for surface modification of biomaterials and biomedical devices.

Herein, plasma deposited thermally responsive thin polymer films from N-vinylcaprolactam (NVCL) is reported for the first time by using a low pressure RF plasma process. While FT-IR and XPS analyses highlight the film chemistry, ToF-SIMS combined with MALDI-MS analyses allow to accurately identify different oligomer distributions in the deposited film. The switching behavior of these smart surfaces is confirmed with water contact angle measurements at low and high temperatures, allowing also to estimate the Lower Critical Solution Temperature.

Polymers are commonly used in biomedical applications because of their excellent bulk properties, such as strength and good resistance to chemicals. However, their surface properties are for most biomedical applications inadequate due to their low surface energy. A surface modification is often needed, and plasma surface modification has been used with success in the past decades. A recent application of plasma processing is devoted to 3D scaffolds for tissue engineering [1,2]. In this field, the surface properties of porous scaffolds need to be altered to promote a good cell adhesion, growth and proliferation in order to make them suitable for implants and tissue regeneration. In this study, we utilized N2 and H2O vapor plasmas to modify PCL porous scaffolds and looked at their interaction with cells.

The improvement of scaffold performances as cell carriers in a tissue implant is still a challenge in tissue engineering. Since cells in contact with a scaffold firstly sense its top surface before interacting with its macro-/micro-porous structure, the insertion of chemical motifs within the body of the scaffold could improve cell colonization through its entire structure. In this study, combinations of plasma deposition and treatment processes have been employed to create chemical gradients inside polycaprolactone porous scaffolds, whose micro-morphology has been finely characterized with synchrotron radiation computed micro-tomography. The graded chemical composition of these scaffolds has successfully allowed the increase of cell viability with respect to untreated materials.

The interplay between plasma processes and the biological environment is a long and intriguing story that spans different applications, from surface modification of biomaterials to the direct interaction of plasma with cells. This makes plasma processes very powerful tools in such biomedical fields as tissue engineering and sterilization, which are much different than the typical field in which plasmas are used. In vitro cell culture experiments represent the best way to fully understand the more subtle and fundamental interactions between the chemical species produced by glow discharge and cells. Among the different kind of cells that can be used, cell lines allow high reproducibility and control of results. This article reviews 3 main items, ranging from low-pressure plasma modifications of 2-and 3-dimensional materials to dielectric barrier discharges directly on cells, with respect to the authors' scientific work.

In this work, different techniques are proposed to realize ammonia (NH3) sensors working at room temperature and a preliminary electrical characterization under water vapor and in NH3 atmospheres is presented. Three families of ceramic planar sensors based on a zinc oxide (ZnO) layer overlapped by screen-printed Pd-doped carboxyl groups functionalized multi-walled carbon nanotubes (Pd-COOH-MWCNTs) or by blocks of vertically aligned MWCNTs or by graphite as such and functionalized with fluorinated or nitrogenous functional groups were studied. These sensors were almost insensitive to humidity, while all of them gave a good response in NH3 atmosphere, starting from about 45 ppm in the case of zinc oxide with fluorinated or nitrogenous MWC-NTs and graphite or 50 ppm for Pd-COOH-MWCNTs sensors. These results are not actually as good as those reported in the literature, but this preliminary work proposes simpler and cheaper processes to realize NH3 sensor for room temperature applications.

In this Letter, a solution-based approach has been used for chemically immobilising oleic acid (OLEA)-capped TiO2 nanocrystals (NCs) on the surface of microcantilevers formed of SU-8, a negative tone epoxy photoresist. The immobilisation has been carried out at room temperature, under visible light, in ambient atmosphere and without applying any external driving force or chemical activation of the epoxy photoresist surface. Atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy investigation demonstrate the spontaneous chemical anchoring of the organic-coated TiO2 NCs on the microcantilevers, which resulted in a highly interconnected nanoporous multilayer structure. The chemical and morphological characterisation shows that the immobilised NCs do not change either their pristine morphology or the chemical structure after binding. Spectroscopic investigation infers that the TiO2 NCs chemically bind through the free and highly reactive epoxy groups located on the epoxy photoresist surface by means of the OLEA capping molecules. Finally, the results show that the fabrication procedure of the microcantilevers has not been affected by the immobilisation protocol. The capability of the immobilised TiO2 NCs to generate surface-reactive hydroxyl radicals under UV-light irradiation has a good potential for detecting families of organic compounds when integrating the modified microcantilevers in electronic noses.

In order to improve the dispersion of multi-walled carbon nanotubes (MWCNTs) in aqueous media, their surface functionalization was carried out in O2-fed low-pressure plasmas. Differently from what can be found in the literature of this field, homogeneous functionalization was achieved by generating the plasma inside vials containing the nanotube powders properly stirred. Experimental parameters, such as input power, treatment time and pressure, were varied to investigate their influence on the process efficiency. A detailed characterization of the plasma treated nanotubes, dry and in aqueous suspension, was carried out with a multi-diagnostic analytical approach, to evaluate their surface chemical properties, morphology, structural integrity and stability in the colloidal state. The plasma grafting of polar ionizable (e.g. acid) groups has been proved to successfully limit the agglomeration of MWCNTs and to produce nanotubes suspensions that are stable for one month and more in water.

Cold plasma processes for surface engineering of biomaterials and biomedical devices are traditionallyperformed at low pressure; more and more, though, surface modification plasma processes at atmospheric pressure are also gaining popularity. This short review is aimed to list briefly atmospheric pressure plasma processes reported, in the last decade, for adapting the surface of materials to the best interactions with cells, bacteria and biomolecules.

Processo per la produzione mediante deposizione plasmochimica di un film di spessore nanometrico, eventualmente multistrato, che consente di eseguire in modo controllato, uniforme e duraturo, il rilascio di sostanze di interesse in un terreno circostante contenente liquidi, da un substrato che include la sostanza a essere rilasciati come particelle micro / nano o da uno strato depositato sul substrato, compresa la sostanza da rilasciare sotto forma di particelle micro / nano, o da uno strato della sostanza da rilasciare depositato sul substrato o da un substrato che è il sostanza da rilasciare facoltativamente sotto forma di particelle. Le sostanze da rilasciare possono essere metalli, composti aventi proprietà anti-batteriche, molecole biologicamente attive come farmaci, ormoni, estratti vegetali, peptidi, lipidi, protidi e glucidi. Lo strato con la sostanza da rilasciare, sia essa organica o inorganica, è ottenuto mediante deposizione plasmochimica facoltativamente avente una struttura simile al polietilenossido (PEO) o polietilenglicole (PEG), detti polimeri PEO-simili, costituiti, in una percentuale variabile da unità di ossido di etilene (-CH2CH2O-, EO); il film barriera è ottenuto depositando per plasma almeno uno strato organico o inorganico, opzionalmente con una struttura simile a PEO, in cui composizione chimica, grado di reticolazione e spessore sono regolabili dai parametri del processo di deposizione chimica plasmo e consentono di regolare il rilascio di la sostanza attiva in base alle esigenze specifiche. Le strutture su cui possono essere depositati i suddetti film sono: dispositivi medico-chirurgici, lavori manuali comuni, strutture note come scaffold e le sostanze sopra definite da rilasciare loro stesse. L'invenzione riguarda anche dispositivi medico-chirurgici, lavori manuali e scaffold rivestiti da un substrato e strato barriera, nonché a sostanze biologicamente attive rivestite da almeno uno strato barriera.