E. De Giglio

Electropolymerization of pyrrole on titanium substrates for the future development of new biocompatible surfaces

Titanium and its alloys are widely used in load-bearing implants as a result of their excellent mechanical properties and corrosion resistance. In order to improve their performances with respect to osseointegration, the use of bioactive coatings has been suggested. Polypyrrole (PPy) has been chosen as coating polymer because of its ability to be electrochemically grown directly onto metallic substrates, of any shape and dimension, leading to remarkably adherent overlayers. This polymer, in addition to protecting the metal implant against corrosion, could be surface modified with biologically active molecules able to stimulate positive interactions with bone tissue. In this work, PPy electrosynthesis on both titanium and Ti-Al-V substrates has been investigated. The chemical composition and the morphology of the polymeric films, deposited under different conditions, were evaluated by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), respectively.

An innovative, easily fabricated, silver nanoparticle-based titanium implant coating: development and analytical characterization

AbstractMicrobial colonization and biofilm formation on implanted devices represent an important complication in orthopaedic and dental surgery and may result in implant failure. Controlled release of antibacterial agents directly at the implant site may represent an effective approach to treat these chronic complications. Resistance to conventional antibiotics by pathogenic bacteria has emerged in recent years as a major problem of public health. In order to overcome this problem, non-conventional antimicrobial agents have been under investigation. In this study, polyacrylate-based hydrogel thin coatings have been electrosynthesised on titanium substrates starting from poly(ethylene glycol diacrylate)–co–acrylic acid. Silver nanoparticles (AgNPs) with a narrow size distribution have been synthesized using a “green” procedure and immobilized on Ti implant surfaces exploiting hydrogel coatings’ swelling capabilities. The coatings have been characterized by XPS and SEM/EDX, while their silver release performances have been monitored by ICP–MS. The antibacterial activity of these AgNP-modified hydrogel coatings was tested evaluating in vitro inhibition growth of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, among the most common pathogens in orthopaedic infections. Moreover, a preliminary investigation of the biocompatibility of silver-loaded coatings versus MG63 human osteoblast-like cells has been performed. An important point of strength of this paper, in fact, is the concern about the effect of silver species on the surrounding cell system in implanted medical devices. Silver ion release has been properly tuned in order to assure antibacterial activity while preserving osteoblasts’ response at the implant interface. FigureSilver nanoparticles-loaded PEGDA-AA hydrogel coatings for inhibition of titanium implants associated infections

Ciprofloxacin-modified electrosynthesized hydrogel coatings to prevent titanium-implant-associated infections

New promising and versatile materials for the development of in situ sustained release systems consisting of thin films of either poly(2-hydroxyethyl methacrylate) or a copolymer based on poly(ethylene-glycol diacrylate) and acrylic acid were investigated. These polymers were electrosynthesized directly on titanium substrates and loaded with ciprofloxacin (CIP) either during or after the synthesis step. X-ray photoelectron spectroscopy was used to check the CIP entrapment efficiency as well as its surface availability in the hydrogel films, while high-performance liquid chromatography was employed to assess the release property of the films and to quantify the amount of CIP released by the coatings. These systems were then tested to evaluate the in vitro inhibition of methicillin-resistant Staphylococcus aureus (MRSA) growth. Moreover, a model equation is proposed which can easily correlate the diameter of the inhibition haloes with the amount of antibiotic released. Finally, MG63 human osteoblast-like cells were employed to assess the biocompatibility of CIP-modified hydrogel coatings.

The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii

Implant-related infection of biomaterials is one of the main causes of arthroplasty and osteosynthesis failure. Bacteria, such as the rapidly-emerging Multi Drug Resistant (MDR) pathogen Acinetobacter Baumannii, initiate the infection by adhering to biomaterials and forming a biofilm. Since the implant surface plays a crucial role in early bacterial adhesion phases, titanium was electrochemically modified by an Anodic Spark Deposition (ASD) treatment, developed previously and thought to provide osseo-integrative properties. In this study, the treatment was modified to insert gallium or silver onto the titanium surface, to provide antibacterial properties. The material was characterized morphologically, chemically, and mechanically; biological properties were investigated by direct cytocompatibility assay, Alkaline Phosphatase (ALP) activity, Scanning Electron Microscopy (SEM), and Immunofluorescent (IF) analysis; antibacterial activity was determined by counting Colony Forming Units, and viability assay. The various ASD-treated surfaces showed similar morphology, micrometric pore size, and uniform pore distribution. Of the treatments studied, gallium-doped specimens showed the best ALP synthesis and antibacterial properties. This study demonstrates the possibility of successfully doping the surface of titanium with gallium or silver, using the ASD technique; this approach can provide antibacterial properties and maintain high osseo-integrative potential.

Biocompatibility of Poly(Acrylic Acid) Thin Coatings Electro-synthesized onto TiAlV-based Implants

The protection of metal orthopedic implants against corrosion is a crucial medical problem. It was found that electrochemical polymerization of thin, passive poly(acrylic acid) (PAA) films on titanium and TiAlV substrates provides good anti-corrosion properties. In this work, an investigation of anti-corrosion features was carried out to clarify the hypothesis of the presence of an electrostatic contribution to the performance of a PAA coating. Ion release tests were performed at three different pHs; the pH dependence of the polymer swelling was examined by quartz crystal microbalance with dissipation monitoring, to establish the role of this phenomenon on the polymer barrier properties. The potential application of these PAA thin films as biocompatible protective coatings for metal implants and compatibility towards MG-63 human osteoblast-like cells was assessed.

Electrosynthesised thin polymer films: the role of XPS in the design of application oriented innovative materials

The paper reviews some significant pieces of work carried out in the authors’ laboratory in the course of about three decades and concerned with the development of market oriented devices, which exploit the singular characteristics of electrosynthesised polymers. The strategic role of X-Ray Photoelectron Spectroscopy is underlined not only as a powerful technique for the characterization of these thin films, but mainly as an unvaluable tool to feedback optimization procedures both in film synthesis and modification. Case studies relevant to the development of permselective membranes for biosensors, of biocompatible coatings and active layers for gas sensors have been selected and reviewed. Future trends and prospects of the work in progress are also described. Preface During electrochemical experiments electrode materials often undergo more or less pronounced modifications of their surface composition, for example by chemical interaction with the media and/or as a consequence of the applied potential. These phenomena, should they be promoted deliberately or completely undesired, can influence the behaviour of the electrode systems particularly when surface species are directly involved in the electrochemical processes. This can lead to a lack of molecular specificity in electrochemical measurements so that the knowledge of the chemical composition of the electrode surface becomes of paramount importance. In this context, surface analysis techniques have played, and still play, a key-role in the characterization of surface chemically-modified electrodes. In the course of a systematic investigation performed in our laboratory in the period 1960–1975, aimed at the development of oxygen and hydrogen electrodes to be employed in medium temperature fused salt fuel cells, irreversible potentiometric behaviours, largely dependent on the working temperature, and Nernstian behaviours different from those expected on the basis of the overall electrode processes, were observed. These results were rationalised by hypothesizing potential-determining steps involving solid species (metal oxides) present on the electrode surface [1–4]. In the following years, combined potentiometric and XPS studies were performed on those (and others) electrode systems [5–9] and the results obtained well supported the proposed mechanistic models through the detection and identification of solid surface species. As an example of this research activity, results obtained on the system (Ni)CO2,O2/CO3= in a (Na,K)NO3 equimolar mixture will be briefly reviewed. Potentiometric measurements performed in the temperature range 507<T<623 K in molten nitrates containing a carbonate ion concentration in the range 10−5<[CO3=]<10−2 mol kg−1 and fluxed with a mixture of CO2 and O2 at variable partial pressures indicated that at the highest tested temperature the system was irreversible and the potential was independent of oxygen concentration. This was interpreted on the basis of the following mechanistic model Mechanism I: (1)2NiO+CO3= ⇔ NiO-O-NiO+CO2 +2e(fast) (2)Ni2O3 ⇒ 2NiO+1/2 O2(slow) ----------------------------------------------------- (3)CO3= ⇔ CO2+1/2 O2+2e Where reaction 1 represents the potential-determining step and reaction 3 the overall [10,3] electrode process. On the other hand a dependence of the potential on O2 (other than CO2) was seen at lower temperatures. At the same time a large irreversibility of the system was still present. To explain this behavior, the presence of a second parallel mechanism, increasingly competitive with the first one as the temperature decreases, was proposed. Mechanism II: (4)2NiO+CO3=⇔ NiO–CO2–NiO +1/2 O2 +2e(fast) (5)NiO–CO2–NiO ⇒ 2NiO +CO2(slow) ------------------------------------------------------------ CO3= ⇔ CO2+1/2 O2+2e(3) As is apparent, both mechanisms involve, in the potential determining step, solid species present on the electrode surface. Due to the strong oxidising power of nitrate melts, the following reactions can occur when nickel electrodes are immersed in such a solvent Ni +NO3−⇔ NiO +NO2− 2NiO +NO3− ⇔ Ni2O3+NO2− Ni2O3+Ni⇔ 3NiO An XPS study was carried out on this system in order to verify the reliability of the proposed mechanistic models. Spectra recorded on nickel foils maintained in contact with the melt at two different temperatures and for different lapses of time are reported in Figs. 1 and 2 Download high-res image (149KB) Download full-size image Fig. 1. Typical photoelectron spectra relevant to the nickel 2p3/2 level recorded at the following metal-melt times of contact (in min): (a)0.0, (b)5.0, (c)30.0, (d)90.0, (e)120.0. T=511 K. The arrows define the range in which binding energies of some nickel compounds (NiO, Ni2O3, Ni(OH)2, NiCO3) fall. Download high-res image (87KB) Download full-size image Fig. 2. Typical photoelectron spectra relevant to the nickel 2p3/2 level recorded at the following metal-melt times of contact: (a’) 0.0, (b’) 5.0 s, (c’) 5.0 min. T=609 K. The arrows define the range in which binding energies of some nickel compounds (NiO, Ni2O3, Ni(OH)2, NiCO3) fall. . The presence of oxidised species is clear; Ni2O3 and Ni(OH)2 species seem to be present in the first tract of the experiment, while, for longer periods of contact, NiO becomes the predominant one. This is consistent with the fact that Ni2O3 is the primary species formed on nickel-oxygen surfaces [11,12], since kinetically favoured, and with the observation that NiO is the only known stable “bulk” oxide. In order to test for the presence of carbonate species [involved in the proposed mechanism II] XPS spectra were recorded on nickel foils maintained in contact with the melt for different lapses of time, under two different gas atmospheres, both at low (511 K) and high (609 K) temperature. The C1s signals reported in Figs. 3 and 4 Download high-res image (82KB) Download full-size image Fig. 3. Typical photoelectron spectra relevant to C1s region recorded on nickel samples under the following working conditions. Metal-melt times of contact (in min): (a’) 10.0, (b’) 30.0, (c’) 120.0. T=511 K; gas atmosphere: CO2=0.9 atm, O2=0.1atm. Download high-res image (62KB) Download full-size image Fig. 4. Typical photoelectron spectra relevant to C1s region recorded on nickel samples under the following working conditions. Metal-melt times of contact (in min): (a’) 10.0, (b’) 30.0, (c’) 120.0. T=609 K; gas atmosphere: CO2=0.9 atm, O2=0.1atm. clearly show that at low temperature a carbonate peak (ca. 290 eV) is present, whose intensity depends on carbon dioxide partial pressure and on the time of contact between nickel substrate and molten nitrates. At high temperatures, whatever the composition of the gas mixture and the time of immersion of the nickel foils, no carbonate peak could be recorded. As expected, at this temperature the formation of a nickel carbonate species is strongly prevented because of its thermal decomposition. In conclusion, XPS investigation unequivocally showed that solid species (carbonate and/or oxides) are formed on the surface of nickel electrode, thus confirming the hypothesis that the large potentiometric irreversibility as well as the marked dependence of the potentiometric behaviour on the temperature were consequences of the electrode chemical corrosion and could be explained by the establishment of two competitive mechanistic models (mechanisms I and II). As a more general conclusion, the research activity carried out by our group in the field of fuel cells contributed further evidence of the great potential of XPS in providing definitely the “chemical situations” existing at modified electrode surfaces.

Electrosynthesis of hydrogel films on metal substrates for the development of coatings with tunable drug delivery performances.

Novel polyacrylates-based hydrogel thin films were prepared by electrochemical polymerization, a new method to obtain hydrogels directly onto metal substrates. 2-Hydroxy-ethyl-methacrylate (HEMA), a macromer poly (ethylene-glycol diacrylate) (PEGDA) and PEGDA copolymerized with acrylic acid (AA) were used to obtain hydrogels. The electrosynthesized coatings were characterized by X-ray photoelectron spectroscopy, to assess their surface chemical composition, and by water content determination measurements, to characterize the swelling behavior. In particular, quartz crystal microbalance with dissipation monitoring was used to evaluate the pH-dependency of the swelling for AA-containing hydrogels. Moreover, a model protein (bovine serum albumin) and a model drug (caffeine) were entrapped within the hydrogel coatings during electrosynthesis, to examine the release performances and mechanisms of the electrosynthesized hydrogels. It was observed that all the examined polymers showed significant release properties and, in particular, AA-containing hydrogel films confirmed a strong pH-dependence as expected. These coatings seem to be promising in orthopedic field for in situ drug delivery applications.

Evaluation of in vitro degradation of PCL scaffolds fabricated via BioExtrusion. Part 1: Influence of the degradation environment

One of the most promising approaches in tissue engineering (TE) comprises the development of 3D porous scaffolds which are able to promote tissue regeneration. Biocompatible and biodegradable poly(ϵ-caprolactone) (PCL) structures are increasingly used as temporary extra-cellular matrices for bone tissue engineering. To ensure an appropriate bone restoration over the long term, the selected material must have a degradation rate that match the in-growth of new bone. The in vivo process, by which the scaffold degrades and is resorbed transferring the load and function back to the host tissue, is complex. Consequently, an appropriate preliminary in vitro study is required. A novel extrusion-based technology called BioExtruder was used to produce PCL porous scaffolds made with layers of directionally aligned microfilaments. The in vitro degradation behaviour in both simulated body fluid (SBF) and phosphate buffer solution (PBS) were investigated over 6 months. The characterization of the degradation behaviour of the structures was performed at specific times by evaluating changes in the average molecular weight, the weight loss and its thermal properties. Morphological and surface chemical analyses were also performed using a Scanning Electron Microscopy (SEM) and an X-ray Photoelectron Spectroscopy (XPS), respectively.

PHEMA-based thin hydrogel films for biomedical applications

Poly(2-hydroxyethyl methacrylate) based thin coatings were electro-synthesized by cyclic voltammetry on Au-coated quartz crystal surfaces to study different solid—liquid interfacial processes. By varying the electrochemical parameters and the presence or not of a crosslinking agent, films were obtained with thicknesses ranging from 5 to 90 nm. Surface characterization was performed by X-ray photoelectron spectroscopy, atomic force microscopy, and static contact angle measurements. Using quartz crystal microbalance with dissipation monitoring to investigate the relationship between the film thickness and the swelling behavior, it was found that these characteristics can be modulated by varying either the number of voltammetric cycles or the presence of the crosslinker. Cell adhesion and biocompatibility tests indicate that these film coatings were suitable for biomedical applications.

Evaluation of in vitro degradation of PCL scaffolds fabricated via BioExtrusion – Part 2: Influence of pore size and geometry

The in vivo degradation processes by which scaffolds degrade and are replaced by neo-tissue are complex and may be influenced by many factors, including environmental conditions, material properties, porosity and 3D architecture. The present study is focused on the influence of design parameters, filament distance (FD) and lay-down pattern, on the degradation kinetics of Polycaprolactone (PCL) scaffolds obtained via BioExtrusion. Through the variation of design parameters it was possible to obtain two groups of scaffolds with distinct pore geometry and size. The in vitro degradation was performed in simulated body fluid (SBF) and in phosphate buffer solution (PBS) for six months. Our results highlight a more complex degradation pattern of the scaffolds in SBF than in PBS, probably related to a mineral deposition. Significant statistical differences in weight loss values at month 6, allowed us to conclude that degradation kinetics of PCL scaffolds is strongly influenced by the pore size.