Development of polymeric coatings with combined antifouling/antibacterial properties for titanium dental implants

Titanium dental implants are a commonly used solution for the replacement of lost teeth. Even though the success rate is high, the number of infections related to the placement of the implant is still remarkable and may impair the proper function of the device, leading to health and economic costs. The infections related to medical devices start with a bacterial adhesion and proliferation on the material surface, leading to the formation of a complex biofilm able to protect the bacteria from the host immune response and the treatment with antibiotic. Due to the difficulty of treatment of the implant site one the biofilm is settled, one of the strategies to avoid the infection is to deal with the initial bacterial adhesion. This PhD thesis deals with the development of polymeric antibacterial coatings on titanium for dental implants, focusing on the achievement of fast and cost-effective procedure. With this aim, different coating strategies have been developed, tested and compared. A pre-treatment of the titanium surface was optimized in the first part of the thesis in order to achieve a clean surface and to enhance the chemical reactivity of the titanium oxide. With this aim, low pressure plasma activation was the selected method. The use of plasma activation allows for the removal of organic contaminants while increasing the surface energy of the treated surfaces. For the preparation of the polymeric antibacterial coatings, two different antifouling polymers have been used, namely, polyethylene glycol (PEG) and poly-2-hydroxyethylmetacrylate (PHEMA). PEG coatings were prepared by three different techniques, a wet chemical technique (silanization), a plasma enhanced chemical vapor deposition and an electrochemical process (electrodeposition). The three methods rendered an ultra-thin coating able to resist the bacterial adhesion. On the other hand, PHEMA-like coatings were prepared in a novel set-up by treating the liquid monomer by a plasma jet. Moreover, the different coatings were biofunctionalized in order to achieve multifunctionality and enhance the performance of the coating. For instance, the combination of PEG with a cell adhesion peptide (RGD) reported a better human fibroblast adhesion while maintaining the antifouling properties of the coating. PEG was also used as a platform for the immobilization of antimicrobial peptides (AMP). The bonding of the polymer with the AMP was optimized, achieving a surface able to reduce the bacterial adhesion and to kill the bacteria still able to adhere to the surface. Finally, the combination of two different plasma polymerized coatings with antibiotics (either Doxycycline or Vancomycin) was used as a drug delivery system.Els implants dentals de titani són la solució més estesa per substituir peces dentals. Tot i que les taxes d'èxit són elevades, el nombre d'infeccions relacionades amb la col·locació de l'implant és elevat, i influeix en el mal funcionament de l'implant, amb un elevat cost tan a nivell econòmic com de salut. Les infeccions associades als dispositius sanitaris comencen amb una adhesió i proliferació dels bacteris a la superfície del material, que comporta la formació d'un biofilm capaç de protegir els bacteris de l'acció del sistema immunitari de l'hoste i del tractament amb antibiòtics. Aquesta tesi doctoral es basa en el desenvolupament de recobriments polimèrics antibacterians en titani per aplicacions dentals, buscant aconseguir mètodes ràpids i econòmics. Per tal d'assolir aquest objectiu, s'han desenvolupat, provat i comparat diferents estratègies per obtenir els recobriments. En la primera part de la tesi s'ha optimitzat un pretractament de la superfície del titani, per tal d'obtenir una superfície neta i millorar la reactivitat química de l'òxid de titani. El mètode seleccionat per l'activació ha estat l'activació per plasma, que permet eliminar els contaminants orgànics i augmentar l'energia superficial de les mostres tractades. Els polímers seleccionats per als recobriments han estat el polietilenglicol (PEG) i el 2-hidroxietilmetacrilat (PHEMA), que tenen propietats antifouling. Per preparar els recobriments de PEG s'han utilitzat tres mètodes diferents, la silanització, la polimerització per plasma i l'electrodeposició. Els tres mètodes han donat com a resultat una capa fina capaç de resistir l'adhesió bacteriana. Per altra banda, els recobriments amb PHEMA s'han preparat amb una nova metodologia, tractant el líquid amb un plasma jet. Els diversos recobriments s'han biofuncionalitzat per tal d'aconseguir una multifuncionalitat i millorar el seu funcionament. La combinació del PEG amb un pèptid d'adhesió cel·lular ha permès millorar l'adhesió de fibroblasts i mantenir les propietats antifouling del recobriment. La immobilització de pèptids antibacterians al PEG permet obtenir una superfície resistent a l'adhesió bacteriana i amb efecte antibacterià sobre els bacteris capaços d'adherir-se al recobriment. Per últim, la combinació de dos recobriments preparats per polimerització per plasma amb dos antibiòtics (vancomicina o doxiciclina) permet obtenir un sistema d'alliberació de fàrmacs a la superfície del titani.Postprint (published version

Modulation of release kinetics by plasma polymerization of ampicillin-loaded ß-TCP ceramics

Beta-tricalcium phosphate (ß-TCP) bioceramics are employed in bone repair surgery. Their local implantation in bone defects puts them in the limelight as potential materials for local drug delivery. However, obtaining suitable release patterns fitting the required therapeutics is a challenge. Here, plasma polymerization of ampicillin-loaded ß-TCP is studied for the design of a novel antibiotic delivery system. Polyethylene glycol-like (PEG-like) coating of ß-TCP by low pressure plasma polymerization was performed using diglyme as precursor, and nanometric PEG-like layers were obtained by simple and double plasma polymerization processes. A significant increase in hydrophobicity, and the presence of plasma polymer was visible on the surface by SEM and quantified by XPS. As a main consequence of the plasma polymerisation, the release kinetics were successfully modified, avoiding burst release, and slowing down the initial rate of release leading to a 4.5¿h delay in reaching the same antibiotic release percentage, whilst conservation of the activity of the antibiotic was simultaneously maintained. Thus, plasma polymerisation on the surface of bioceramics may be a good strategy to design controlled drug delivery matrices for local bone therapiesPeer ReviewedPostprint (author's final draft

Desenvolupament d’un tractament antiadherent de superfícies de titani per a aplicacions biomèdiques

L’ús d’implants de titani per a traumatologia i odontologia és una pràctica molt estesa actualment. El seu èxit, però, es veu afectat pels casos de infeccions que pateixen. En el cas concret dels implants dentals, el 14% dels implants pateixen infeccions bacterianes i entre el 5 i el 8% presenten infecció bacteriana que desenvolupa una peri-implantitis i una conseqüent pèrdua de l’implant. Per aquest motiu, en els darrers anys s’ha estudiat el desenvolupament de superfícies antibacterianes. En aquest treball s’optimitza un tractament antiadherent per a superfícies de titani amb l’objectiu de minimitzar l’adhesió bacteriana que pugui produir una infecció posterior. S’ha desenvolupat un tractament de la superfície de titani per formar un recobriment amb un polímer antiadherent. S’han estudiat les característiques fisicoquímiques de la superfície que en resulta. També s’ha estudiat la seva biocompatibilitat i capacitat per evitar l’adhesió de proteïnes i bacteris.The use of titanium implants for orthopedic and dental practice is currently widespread. Its success, however, is affected by implant-related infections. In the case of dental implants, 14% of implants suffered bacterial infections and between 5 and 8% have a bacterial infection that develops peri-implantitis and consequent loss of the implant. For this reason, in the recent years the development of antibacterial surfaces has been thoroughly studied. In this paper, a treatment has been optimized for creating antifouling titanium surfaces with the objective to minimize bacterial adhesion that may result in an infection. The titanium surface treatment produces a coating with an antifouling polymer. The physicochemical characteristics of the resulting surface have been studied, as well as the biocompatibility and the ability to prevent the adhesion of proteins and bacteria.Peer Reviewe

On the plasma deposition of vancomycin-containing nano-capsules for drug-delivery applications

Aerosol-assisted atmospheric pressure plasma allows for a one-step synthesis of vancomycin-containing nano-capsules. Morphological and chemical analyses are carried out to estimate how different discharge parameters affect the plasma deposition process. Nano-capsules size and abundance largely depend on the shell precursor content in the gas feed and on the drug concentration in the aerosol solution. Based on these results a deposition mechanism is proposed, where, interestingly, the key step is the formation of the nano-capsules in the plasma phase. Furthermore, the related antibacterial activity is proved against Staphylococcus aureus. Preliminary release tests indicate the possible exploitation of the plasma-deposited vancomycin-containing nano-capsules in the drug delivery field, and systems based on other bioactive molecules can be expected.Peer ReviewedPostprint (published version

Polyethylene glycol pulsed electrodeposition for the development of antifouling coatings on titanium

Titanium dental implants are widely used for the replacement of damaged teeth. However, bacterial infections at the interface between soft tissues and the implant can impair the functionality of the device and lead to failure. In this work, the preparation of an antifouling coating of polyethylene glycol (PEG) on titanium by pulsed electrodeposition was investigated in order to reduce Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) adhesion while maintaining human fibroblast adhesion. Different pulsed conditions were prepared and characterized by contact angle, Focused Ion Beam (FIB), Fourier Transformed Infrared Spectroscopy in the Attenuated Total Reflectance mode (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). XPS tested fibronectin adsorption. S. aureus, E. coli and human fibroblast adhesion was tested in vitro in both mono and co-culture settings. Physicochemical characterization proved useful for confirming the presence of PEG and evaluating the efficiency of the coating methods. Fibronectin adsorption decreased for all of the conditions, but an adsorption of 20% when compared to titanium was maintained, which supported fibroblast adhesion on the surfaces. In contrast, S. aureus and E. coli attachment on coated surfaces decreased up to 90% vs. control titanium. Co-culture studies with the two bacterial strains and human fibroblasts showed the efficacy of the coatings to allow for eukaryotic cell adhesion, even in the presence of pre-adhered bacteria.Peer ReviewedPostprint (published version

Antibacterial properties of triethoxysilylpropyl succinic anhydride silane (TESPSA) on titanium dental implants

Infections related to dental implants are a common complication that can ultimately lead to implant failure, and thereby carries significant health and economic costs. In order to ward off these infections, this paper explores the immobilization of triethoxysilylpropyl succinic anhydride (TESPSA, TSP) silane onto dental implants, and the interaction of two distinct monospecies biofilms and an oral plaque with the coated titanium samples. To this end, titanium disks from prior machining were first activated by a NaOH treatment and further functionalized with TESPSA silane. A porous sodium titanate surface was observed by scanning electron microscopy and X-ray photoelectron spectroscopy analyses confirmed the presence of TESPSA on the titanium samples (8.4% for Ti–N-TSP). Furthermore, a lactate dehydrogenase assay concluded that TESPSA did not have a negative effect on the viability of human fibroblasts. Importantly, the in vitro effect of modified surfaces against Streptococcus sanguinis, Lactobacillus salivarius and oral plaque were studied using a viable bacterial adhesion assay. A significant reduction was achieved in all cases but, as expected, with different effectiveness against simple mono-species biofilm (ratio dead/live of 0.4) and complete oral biofilm (ratio dead/live of 0.6). Nevertheless, this approach holds a great potential to provide dental implants with antimicrobial properties.Peer ReviewedPostprint (published version

Implementation of bactericidal topographies on biomimetic calcium phosphates and the potential effect of its reactivity

Since the discovery that nanostructured surfaces were able to kill bacteria, many works have been published focusing on the design of nanopatterned surfaces with antimicrobial properties. Synthetic bone grafts, based on calcium phosphate (CaP) formulations, can greatly benefit from this discovery if adequate nanotopographies can be developed. However, CaP are reactive materials and experience ionic exchanges when placed into aqueous solutions which may in turn affect cell behaviour and complicate the interpretation of the bactericidal results. The present study explores the bactericidal potential of two nanopillared CaP prepared by hydrolysis of two different sizes of a-tricalcium phosphate (a-TCP) powders under biomimetic or hydrothermal conditions. A more lethal bactericidal response toward Pseudomonas aeruginosa (~75% killing efficiency of adhered bacteria) was obtained from the hydrothermally treated CaP which consisted in a more irregular topography in terms of pillar size (radius: 20–60 nm), interpillar distances (100–1500 nm) and pillar distribution (pillar groups forming bouquets) than the biomimetically treated one (radius: 20–40 nm and interpillar distances: 50–200 nm with a homogeneous pillar distribution). The material reactivity was greatly influenced by the type of medium (nutrient-rich versus nutrient-free) and the presence or not of bacteria. A lower reactivity and superior bacterial attachment were observed in the nutrient-free medium while a lower attachment was observed for the nutrient rich medium which was explained by a superior reactivity of the material paired with the lower tendency of planktonic bacteria to adhere on surfaces in the presence of nutrients. Importantly, the ionic exchanges produced by the presence of materials were not toxic to planktonic cells. Thus, we can conclude that topography was the main contributor to mortality in the bacterial adhesion tests.Peer ReviewedPostprint (published version

Funcionalización de superficies anti-fouling sobre titanio para mejora de sus propiedades

Currently, implant and prosthetic infections are a serious problem, due to the increased use of these prostheses and the presence of bacteria multiresistant to antibiotics. These infections originate, in most cases, from planktonic bacteria. A possible strategy to avoid infections is to develop anti-fouling surfaces that prevent bacterial adhesion. Another strategy focuses on conferring bactericidal properties to the surfaces of the implants with the use of antimicrobial peptides. In both cases, it is necessary to maintain the excellent union to the tissues that titanium presents. The ideal surface for prostheses would combine anti-fouling, bactericidal and osseointegration properties, achieving an excellent synergic effect on the surfaces of the implants, improving their stability and functionality. To achieve this goal it is necessary to solve a critical point, which is to functionalize the anti-fouling layer with other biomolecules that can improve its properties. The objective of the present work is to deposit an anti-fouling layer on a metallic biomaterial, which can be further functionalized with other biomolecules. Titanium has been coated with functionalized polyethylene glycol (PEG) to which the Arg-Gly-Asp (RGD) peptide sequence has been attached. The treated titanium surfaces have shown an excellent combination of anti-fouling properties and good cellular response.Actualmente las infecciones de implantes y prótesis son un problema grave, debido al incremento de uso de dichas prótesis y a la cada vez mayor presencia de bacterias resistentes a los antibióticos. Estas infecciones tienen su origen, en la mayoría de casos, en bacterias planctónicas. Una posible estrategia para evitar las infecciones es desarrollar superficies anti-fouling que eviten la adhesión bacteriana. Otra estrategia se centra en conferir propiedades bactericidas a las superficies de los implantes en el uso de péptidos antimicrobianos. En ambos casos, es necesario mantener la excelente unión a los tejidos que presenta el titanio. La superficie ideal para prótesis combinaría los efectos anti-fouling, bactericida y osteointegrativo, logrando un excelente efecto sinérgico en las superficies de los implantes, mejorando su estabilidad y funcionalidad. Para conseguir este objetivo es necesario solventar un punto crítico, que es funcionalizar la capa anti-fouling con otras biomoléculas que permitan dotarla de las propiedades mencionadas. El objetivo del presente trabajo es depositar una capa anti-fouling sobre un biomaterial metálico con un grupo funcional que permita enlazar a la superficie tratada otras biomoléculas. Se ha conseguido depositar una capa de (poli)etilenglicol (PEG) funcionalizado sobre titanio, al cual se ha unido la secuencia tripeptídica Arg-Gly-Asp (RGD). Las superficies tratadas han mostrado una excelente combinación de propiedades anti-fouling y de buena respuesta celular

Recobriments antiadherents sobre titani: mètodes de deposició de poli(etilenglicol)

Els implants dentals són àmpliament utilitzats per a la substitució de peces dentals perdudes. Tot i la seva elevada taxa d’èxit, es produeixen fallades en un 5-10% dels implants col•locats. Per evitar les fallades per infecció cal evitar l’adhesió bacteriana inicial, fita que es pot assolir amb els recobriments antiadherents. En aquest estudi es realitza la comparativa de tres mètodes diferents per l’obtenció de recobriments de poli(etilenglicol) (PEG) sobre titani per aplicacions en implants dentals. Els mètodes estudiats han estat la polimerització per plasma, l’electrodeposició i la silanització. Els tres mètodes permeten obtenir un recobriment de PEG a la superfície del titani, amb composició química semblant i una reducció de l’adhesió bacteriana. L’adhesió cel•lular no es veu significativament afectada en cap dels casos.Peer Reviewe