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Aim: To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). Materials & methods:Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. Results: A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. Conclusion: Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential., Peer Reviewed, Postprint (author's final draft)

Titanium and its alloys are widely used in medical implants because of their excellent properties. However, bacterial infection is a frequent cause of titanium-based implant failure and also compromises its osseointegration. In this study, we report a new simple method of providing titanium surfaces with antibacterial properties by alternating antibacterial chitosan domains with titanium domains in the micrometric scale. Surface microgrooves were etched on pure titanium disks at intervals of 60 μm using a modified 3D printer and were then coated with chitosan antibacterial polysaccharide. The dimensions of the patterned microgrooves made it possible to fix the chitosan domains to the titanium substrate without the need for covalent bonding. These domains were stable after 5 days of immersion in water and reduced the surface contact angle. Preliminary cell adhesion assays demonstrated that MC3T3-E1 pre-osteoblasts preferentially adhered to the titanium regions, while C2C12 myoblasts were uniformly distributed over the whole surface., Peer Reviewed, Postprint (author's final draft)

The objective of the study is to characterise the mechanical properties of Ti-15Zr binary alloy dental implants and to describe their biomechanical behaviour as well as their osseointegration capacity compared with the conventional Ti-6Al-4V (TAV) alloy implants. The mechanical properties of Ti-15Zr binary alloy were characterised using Roxolid© implants (Straumann, Basel, Switzerland) via ultrasound. Their biomechanical behaviour was described via finite element analysis. Their osseointegration capacity was compared via an in vivo study performed on 12 adult rabbits. Young’s modulus of the Roxolid© implant was around 103¿GPa, and the Poisson coefficient was around 0.33. There were no significant differences in terms of Von Mises stress values at the implant and bone level between both alloys. Regarding deformation, the highest value was observed for Ti-15Zr implant, and the lowest value was observed for the cortical bone surrounding TAV implant, with no deformation differences at the bone level between both alloys. Histological analysis of the implants inserted in rabbits demonstrated higher BIC percentage for Ti-15Zr implants at 3 and 6 weeks. Ti-15Zr alloy showed elastic properties and biomechanical behaviours similar to TAV alloy, although Ti-15Zr implant had a greater BIC percentage after 3 and 6 weeks of osseointegration., Peer Reviewed, Postprint (published version)

In dentistry and orthopedics, it is well accepted that implant fixation is a major goal. However, an emerging concern is bacterial infection. Infection of metallic implants can be catastrophic and significantly reduce patient quality of life. Accordingly, in this work, we focus on multifunctional coatings to simultaneously address and mitigate both these problems. We have developed a tailor-made peptide-based chemical platform that integrates the well-known RGD cell adhesive sequence and the lactoferrin-derived LF1-11 antimicrobial peptide. The platform was covalently grafted on titanium via silanization and the functionalization process characterized by contact angle, XPS, and QCM-D. The presence of the platform statistically improved the adhesion, proliferation and mineralization of osteoblast-like cells compared to control surfaces. At the same time, colonization by representative bacterial strains was significantly reduced on the surfaces. Furthermore, the biological potency of the multifunctional platform was verified in a co-culture in vitro model. Our findings demonstrate that this multifunctional approach can be useful to functionalize biomaterials to both improve cell integration and reduce the risk of bacterial infection., Peer Reviewed, Postprint (published version)

Metals are widely employed for many biological artificial replacements, and it is known that the quality and the physical/chemical properties of the surface are crucial for the success of the implant. Therefore, control over surface implant materials and their elastic moduli may be crucial to avoid undesired effects. In this study, surface modification upon cleaning and activation of a low elastic modulus Ti alloy (Ti25Hf21Nb) was investigated. Two different methods, oxygen plasma (OP) cleaning and piranha (PI) solution, were studied and compared. Both surface treatments were effective for organic contaminant removal and to increase the Ti-oxide layer thickness rather than other metal-oxides present at the surface, which is beneficial for biocompatibility of the material. Furthermore, both techniques drastically increased hydrophilicity and introduced oxidation and hydroxylation (OH)-functional groups at the surface that may be beneficial for further chemical modifications. However, these treatments did not alter the surface roughness and bulk material properties. The surfaces were fully characterized in terms of surface roughness, wettability, oxide layer composition, and hydroxyl surface density through analytical techniques (interferometry, X-ray photoelectron spectroscopy (XPS), contact angle, and zinc complexation). These findings provide essential information when planning surface modifications for cleanliness, oxide layer thickness, and surface hydroxyl density, as control over these factors is essential for many applications, especially in biomaterials., Peer Reviewed, Postprint (published version)

Titanium and its alloys are widely used in medical implants because of their excellent properties. However, bacterial infection is a frequent cause of titanium-based implant failure and also compromises its osseointegration. In this study, we report a new simple method of providing titanium surfaces with antibacterial properties by alternating antibacterial chitosan domains with titanium domains in the micrometric scale. Surface microgrooves were etched on pure titanium disks at intervals of 60 µm using a modified 3D printer and were then coated with chitosan antibacterial polysaccharide. The dimensions of the patterned microgrooves made it possible to fix the chitosan domains to the titanium substrate without the need for covalent bonding. These domains were stable after 5 days of immersion in water and reduced the surface contact angle. Preliminary cell adhesion assays demonstrated that MC3T3-E1 pre-osteoblasts preferentially adhered to the titanium regions, while C2C12 myoblasts were uniformly distributed over the whole surface., Peer Reviewed, Postprint (author's final draft)

Immobilization of bioactive peptide sequences on CoCr surfaces is an effective route to improve endothelialization, which is of great interest for cardiovascular stents. In this work, we explored the effect of physical and covalent immoblization of RGDS, YIGSR and their equimolar combination peptides on endothelial cells (EC) and smooth muscle cell (SMC) adhesion and on thrombogenicity. We extensively investigated using RT-qPCR, the expression by ECs cultured on functionalised CoCr surfaces of different genes. Genes relevant for adhesion (ICAM-1 and VCAM-1), vascularization (VEGFA, VEGFR-1 and VEGFR-2) and anti-thrombogenicity (tPA and eNOS) were over-expressed in the ECs grown to covalently functionalized CoCr surfaces compared to physisorbed and control surfaces. Pro-thrombogenic genes expression (PAI-1 and vWF) decreased over time. Cell co-cultures of ECs/SMCs found that functionalization increased the amount of adhered ECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. SMCs adhered less compared to ECs in all surfaces. All studied peptides showed a lower platelet cell adhesion compared to TCPS. Covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represented prevailing strategy to enhance the early stages of ECs adhesion and proliferation, while preventing SMCs and platelet adhesion., Postprint (author's final draft)

Biomaterial-associated infection is a serious complication of modern implantation surgery. Thus, the improvement of implant surfaces is required to avoid the first stage for biofilm formation, bacterial adhesion. The current research addresses this issue by developing drug delivery systems (DDS) consisting of antibiotic-loaded polyhydroxyalkanoates (PHAs) coatings on titanium implants. Dip-coating technique was used to achieve optimal coatings with biodegradable biopolyesters, polyhydroxybutyrate (PHB) and its copolymer, polyhydroxybutyrate-co-hydroxyvalerate (PHBV). The coatings were completely characterized (wettability, topography, thickness and roughness), and studies of drug delivery, toxicity, antibacterial effect, and cell adhesion were performed. For both of biopolymers, surfaces were partially covered with 1 and 3 immersions, while with 6, they were completely covered. Although both antibiotic-loaded biopolymer coatings assure the protection against bacteria populations, PHBV coatings are closer to the desired release profile; its faster degradation provides for a greater and more stable drug release for a given period of time compared to PHB coatings. The use of coatings with different drug concentration per layer results in more controlled and homogeneous releases. The DDS designed not only assure to avoid the first stage of bacterial adhesion, but also their proliferation and biofilm formation, since the coatings degrade with time under physiological conditions, guaranteeing a prolonged drug release., Preprint

Versió amb diverses seccions encriptades, per drets a'autor, Cobalt-chromium (CoCr) alloys are widely used as biomaterials for coronary stents due to their excellent mechanical properties, biocompatibility and corrosion resistance. However, these materials are bioinert, retarding the complete endothelialization and resulting in a higher risk of restenosis, narrowing of the artery, and late-stent thrombosis. Therefore, the improvement of implants surface endothelialization has acquired importance in the last years. Immobilization of cell adhesive biomolecules onto biomaterials surface is a well-known strategy to control cell response. However, the strategy of immobilization, the optimal combination or the appropriate spatial presentation of the bioactive sequences to enhance endothelialization for cardiovascular applications, remains to be elucidated. The present PhD thesis focused on the development of a new biofunctionalized CoCr alloy surfaces in order to improve the endothelialization. To that end, elastin-like recombinamers (ELR) genetically modified with an REDV (Arg-Glu-Asp-Val) sequence and short synthetized peptides RGDS (Arg-Gly-Asp-Ser), REDV, YIGSR (Tyr-Ile-Gly-Ser-Arg) and their equimolar combination, were attached by physisorption and covalent bonding onto CoCr alloy surfaces and thoroughly characterized physico-chemically and evaluated in vitro with human umbilical vein endothelial cells (HUVECs), coronary artery smooth muscle cells (CASMCs) and platelets from blood donors. First, biofunctionalized surfaces with ELR were developed and optimized by evaluating different surface activation treatments, oxygen plasma and sodium hydroxide etching, and different binding strategies, physisorption and covalent bonding. The functionalized surfaces demonstrated a higher cell adhesion and spreading of HUVEC cells, this effect is emphasized as increases the amount of immobilized biomolecules and directly related to the immobilization technique: covalent bonding. Nevertheless, the silanization process was not completely effec, Las aleaciones de cobalto-cromo (CoCr) son ampliamente utilizadas como biomateriales para stents coronarios debido a sus excelentes propiedades mecánicas, biocompatibilidad y resistencia a la corrosión. Sin embargo, estos materiales son bio-inertales, retardando la completa endotelialización y resultando en un mayor riesgo de reestenosis, estrechamiento de la arteria y trombosis tardía. Por lo tanto, la mejora de la endotelización de superficie de los implantes ha adquirido importancia en los últimos años. La inmovilización de biomoléculas adhesivas celulares sobre la superficie de los biomateriales es una estrategia bien conocida para controlar la respuesta celular. Sin embargo, queda por aclararse la estrategia de inmovilización, la combinación óptima o la presentación espacial apropiada de las secuencias bioactivas para potenciar la endotelización en aplicaciones cardiovasculares. La presente tesis doctoral se centró en el desarrollo de una nueva superficie biofuncionalizada de la aleación CoCr con el fin de mejorar la endotelialización. Para ello, los recombinameros tipo elastina (ELR) modificados genéticamente con una secuencia REDV (Arg-Glu-Asp-Val) y péptidos cortos sintetizados RGDS (Arg-Gly-Asp-Ser), REDV, YIGSR (Tyr-Ile-Gly -Ser-Arg) y su combinación equimolar, fueron anclados por fisisorción y unión covalente a las superficies de la aleación, se caracterizaron físicamente-químicamente y evaluó in vitro con células endoteliales de vena umbilical humana (HUVECs), células de músculo liso de arteria coronaria (CASMCs) y plaquetas de donantes de sangre. En primer lugar, se desarrollaron y optimizaron las superficies biofuncionalizadas con ELR mediante la evaluación de diferentes tratamientos de activación superficial: plasma de oxígeno y ataque con hidróxido de sodio, y diferentes estrategias de unión: fisisorción y unión covalente. Las superficies funcionalizadas demostraron una mayor adhesión celular y propagación de células HUVEC, este efecto se enfatiza a, Postprint (published version)

Engineering the interface between biomaterials and tissues is important to increase implant lifetime and avoid failures and revision surgeries. Permanent devices should enhance attachment and differentiation of stem cells, responsible for injured tissue repair, and simultaneously discourage bacterial colonization; this represents a major challenge. To take first steps towards such a multifunctional surface we propose merging topographical and biochemical cues on the surface of a clinically relevant material such as titanium. In detail, our strategy combines antibacterial nanotopographical features with integrin selective synthetic ligands that can rescue the adhesive capacity of the surfaces and instruct mesenchymal stem cell (MSC) response. To this end, a smooth substrate and two different high aspect ratio topographies have been produced and coated either with an avß3-selective peptidomimetic, an a5ß1-selective peptidomimetic, or an RGD/PHSRN peptidic molecule. Results showed that antibacterial effects of the substrates could be maintained when tested on pathogenic Pseudomonas aeruginosa. Further, functionalization increased MSC adhesion to the surfaces and the avß3-selective peptidomimetic-coated nanotopographies promoted osteogenesis. Such a dual physicochemical approach to achieve multifunctional surfaces represents a first step in the design of novel cell-instructive biomaterial surfaces., Peer Reviewed, Postprint (published version)

El titanio es un material muy utilizado en el campo de la biomedicina por sus buenas propiedades mecánicas y su gran biocompatibilidad. Como todos los metales, presenta un problema para actuar como biomaterial: el apantallamiento de tensiones. Para reducir el módulo elástico del titanio se han construido muestras porosas con la técnica de impresión 3D. El titanio no presenta bioactividad, por eso se ha biofuncionalizado con 4 biopolímeros diferentes tipo elastina (ELPs). Estos biopolímeros con fragmentos de elastina y cada uno tiene diferentes motivos celulares para caracterizar funciones biológicas distintas. 1K no tiene ningún motivo celular, H3 tiene el motivo celular DDDEE4 que favorece la mineralización, HRGD contiene el motivo celular RGD que promueve la adhesión y H4R4 contiene los dos motivos. La adhesión de los polímeros a las estructuras de titanio se ha realizado mediante la silanización, creando así un enlace covalente entre el biopolímero y el titanio. Se han realizado ensayos in vitro de adhesión tras 4h de incubación, de proliferación, mineralización y diferenciación para 4h, 7 días, 14 días, 21 días y 28 días de incubación. La caracterización celular del ensayo de adhesión se ha hecho mediante microscopía electrónica de barrido (SEM), donde se ha observado que las células con una mejor adhesión se encontraban en la muestra funcionalizada con HRGD. El ensayo de proliferación se ha realizado midiendo la actividad de la enzima lactato deshidrogenasa (LDH) y se ha observado que la muestra que más ha proliferado ha sido la funcionalizada con HRGD. El ensayo de diferenciación se ha realizado midiendo la actividad de la enzima fosfato alcalina (ALP) y se ha observado que las células que han diferenciado más a los 14 días de incubación han sido las que se encontraban en las muestras funcionalizadas con H3. El ensayo de mineralización se ha realizado con Alizarin Red - S (AR-S) y la muestra dónde las células han mineralizado más han sido las funcionalizadas c

Biomimetic surface modification with peptides that have specific cell-binding moieties is a promising approach to improve endothelialization of metal-based stents. In this study, we functionalized CoCr surfaces with RGDS, REDV, YIGSR peptides and their combinations to promote endothelial cells (ECs) adhesion and proliferation. An extensive characterization of the functionalized surfaces was performed by XPS analysis, surface charge and quartz crystal microbalance with dissipation monitoring (QCM-D), which demonstrated the successful immobilization of the peptides to the surface. Cell studies demonstrated that the covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represents the most powerful strategy to enhance the early stages of ECs adhesion and proliferation, indicating a positive synergistic effect between the two peptide motifs. Although these peptide sequences slightly increased smooth muscle cells (SMCs) adhesion, these values were ten times lower than those observed for ECs. The combination of RGDS with the REDV sequence did not show synergistic effects in promoting the adhesion or proliferation of ECs. The strategy presented in this study holds great potential to overcome clinical limitations of current metal stents by enhancing their capacity to support surface endothelialization., Peer Reviewed, Postprint (author's final draft)

Tesi per compendi de publicacions, Despite being biocompatible and with adequate mechanical properties for application as a bone replacement material, titanium lacks osteoinductive capacity, i.e. it supports new bone growth on its surface but does not foster its formation. This may lead to failure of the implant due to poor osseointegration. Together with infection, this is in fact the main cause of failure of orthopedic and dental implants. Therefore, this thesis explores the possibility to convert titanium surface into a bioactive substrate, which is actively capable of influencing cell fate in vitro and enhance implant osteointegration in vivo. To install such bioactivity, surface chemical functionalization was chosen, since it allows for the modification of the external layer of the material, which is responsible for the interactions with the surrounding tissues, while leaving the bulk properties unaffected. Two families of extracellular matrix (ECM)-inspired integrin-binding biomolecules were tested. Integrins are the major cell surface receptor, whose main role is to mediate cell-surface interactions; thus, addressing these receptors could be beneficial to tune cell response to the surface. One type of biomolecule tested is a double-branched peptidic ligand that allows for the simultaneous presentation of the cell-adhesive RGD (Arg-Gly-Asp) motif and the synergic PHSRN (Pro-His- Ser-Arg-Asn) motif, which synergizes the RGD-mediated binding to integrin a5ß1. Alternatively, non peptidic integrin-selective ligands were tested as surface coating molecules. These highly stable ligands were designed by the group of Prof. Kessler at the Technische Universität München (TUM, Munich, Germany) to be selective for either integrins a5ß1 or avß3 and are introduced in a review paper included in the thesis. The role of both of these receptor subtypes in several bone biology events is currently matter of discussion in literature. Grafting of the ligands on titanium was either carried out via physisorption or ch, El objetivo principal de este proyecto de tesis es la instalación de bioactividad en la superficie de titanio para implantes ortopédicos y dentales a través de un proceso de funcionalización superficial. A pesar de ser biocompatible y con buenas propiedades mecánicas para substituir el tejido óseo dañado o ausente, el titanio carece de capacidades osteoinductivas, es decir, soporta pero no favorece los procesos de formación de hueso. Esto puede llevar a la fallida del implante debido a una falta de osteointegración. Las modificaciones superficiales permiten transformar la superficie bioinerte del titanio en una superficie bioactiva que estimula la producción de hueso, sin afectar las propiedades mecánicas del material. Para lograr dicha bioactividad, en este trabajo de tesis se inmovilizaron dos familias de moléculas peptidicas inspiradas en la matriz extracelular de las células que interactúan con las integrinas, los receptores celulares más importantes que transmiten la información entre las células y su matriz. El primer tipo de biomolécula testada es una molécula ramificada, cuyas dos extremidades llevan dos motivos peptídicos distintos: el motivo de adhesión celular RGD (Arg-Gly-Asp) y el motive de sinergia PHSRN (Pro-His-Ser-Arg-Asn), que incrementa la afinidad para la integrina a5ß1, muy relevante en procesos de crecimiento óseo. La segunda familia de biomoléculas abarca dos peptidomiméticos selectivos para la integrina a5ß1 o la integrina avß3, cuyo rol en la formación de hueso es también objeto de discusión en la literatura, y que fueron sintetizados por el grupo del Prof. H. Kessler de la Technische Universität München. Para la inmovilización de las moléculas en el titanio se utilizaron dos técnicas distintas: la fisisorción, que sólo está basada en la formación de enlaces débiles electrostáticos, puentes hidrogeno, etc., y la unión covalente, más estable, mediante la silanización de la superficie metálica. Las superficies de titanio modificadas se testaro, Postprint (published version)

Novel researches are focused on the prevention and management of post-operative infections. To avoid this common complication of implant surgery, it is preferable to use new biomaterials with antibacterial properties. Therefore, the aim of this work is to develop a method of combining the antibacterial properties of antibiotic-loaded poly(3-hydroxybutyrate) (PHB) nano-and micro-spheres and poly(ethylene glycol) (PEG) as an antifouling agent, with titanium (Ti), as the base material for implants, in order to obtain surfaces with antibacterial activity. The Ti surfaces were linked to both PHB particles and PEG by a covalent bond. This attachment was carried out by firstly activating the surfaces with either Oxygen plasma or Sodium hydroxide. Further functionalization of the activated surfaces with different alkoxysilanes allows the reaction with PHB particles and PEG. The study confirms that the Ti surfaces achieved the antibacterial properties by combining the antibiotic-loaded PHB spheres, and PEG as an antifouling agent., Postprint (author's final draft)

A bioactive layer, free of nickel, has been performed for its greater acceptability and reliability in clinical applications for NiTi shape memory alloys. In the first step, a safe barrier against Ni release has been produced on the surface by means of a thicker rutile/anastase protective layer free of nickel. In the second step, a sodium alkaline titanate hydrogel, which has the ability to induce apatite formation, has been performed from oxidized surface. An improvement of host tissue–implant integration has been achieved in terms of Ni ions release and the bioactivity of the treated NiTi alloys has been corroborated with both in vitro and in vivo studies. The transformation temperatures (As, Af, Ms, and Mf), as well as the critical stresses (sß¿M), have been slightly changed due to this surface modification. Consequently, this fact must be taken into account in order to design new surface modification on NiTi implants., Postprint (published version)

The aim of this study was to evaluate the in vivo effect of antibacterial modified dental implants in the first stages of peri-implantitis. Thirty dental implants were inserted in the mandibular premolar sites of 5 beagle dogs. Sites were randomly assigned to Ti (untreated implants, 10 units), Ti_Ag (silver electrodeposition treatment, 10 units), and Ti_TSP (silanization treatment, 10 units). Coated implants were characterized by scanning electron microscopy, interferometry and X-ray photoelectron spectroscopy. Two months after implant insertion, experimental peri-implantitis was initiated by ligature placement. Ligatures were removed 2 months later, and plaque formation was allowed for 2 additional months. Clinical and radiographic analyses were p¬erformed during the study. Implant-tissue samples were prepared for micro computed tomography, backscattered scanning electron microscopy, histomorphometric and histological analyses and ion release measurements. X-ray, SEM and histology images showed that vertical bone resorption in treated implants was lower than in the control group (P < 0.05). This effect is likely due to the capacity of the treatments to reduce bacteria colonization on the implant surface. Histological analysis suggested an increase of peri-implant bone formation on silanized implants. However, the short post-ligature period was not enough to detect differences in clinical parameters among implant groups. Within the limits of this study, antibacterial surface treatments have a positive effect against bone resorption induced by peri-implantitis., Peer Reviewed, Postprint (author's final draft)

Surface modification stands out as a versatile technique to create instructive biomaterials that are able to actively direct stem cell fate. Chemical functionalization of titanium has been used in this work to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage, by covalently anchoring a synthetic double-branched molecule (PTF) to the metal that allows a finely controlled presentation of peptidic motifs. In detail, the effect of the RGD adhesive peptide and its synergy motif PHSRN is studied, comparing a random distribution of the two peptides with the chemically-tailored disposition within the custom made synthetic platform, which mimics the interspacing between the motifs observed in fibronectin. Contact angle measurement and XPS analysis are used to prove the efficiency of functionalization. We demonstrate that, by rationally designing ligands, stem cell response can be efficiently guided towards the osteogenic phenotype: In vitro, PTF-functionalized surfaces support hMSCs adhesion, with higher cell area and formation of focal contacts, expression of the integrin receptor a5ß1 and the osteogenic marker Runx2, and deposition a highly mineralized matrix, reaching values of mineralization comparable to fibronectin. Our strategy is also demonstrated to be efficient in promoting new bone growth in vivo in a rat calvarial defect. These results highlight the efficacy of chemical control over the presentation of bioactive peptides; such systems may be used to engineer bioactive surfaces with improved osseointegrative properties, or can be easily tuned to generate multi-functional coatings requiring a tailored disposition of the peptidic motifs., Peer Reviewed, Postprint (author's final draft)

Since metallic biomaterials used for bone replacement possess low bioactivity, the use of cell adhesive moieties is a common strategy to improve cellular response onto these surfaces. In recent years, the use of recombinant proteins has emerged as an alternative to native proteins and short peptides owing to the fact that they retain the biological potency of native proteins, while improving their stability. In the present study, we investigated the biological effect of two different recombinant fragments of fibronectin, spanning the 8–10th and 12–14th type III repeats, covalently attached to a new TiNbHf alloy using APTES silanization. The fragments were studied separately and mixed at different concentrations and compared to a linear RGD, a cyclic RGD and the full-length fibronectin protein. Cell culture studies using rat mesenchymal stem cells demonstrated that low to medium concentrations (30% and 50%) of type III 8–10th fragment mixed with type III 12–14th fragment stimulated cell spreading and proliferation compared to RGD peptides and the fragments separately. On the other hand, type III 12–14th fragment alone or mixed at low volume percentages =50% with type III 8–10th fragment increased alkaline phosphatase levels compared to the other molecules. These results are significant for the understanding of the role of fibronectin recombinant fragments in cell responses and thus to design bioactive coatings for biomedical application, Peer Reviewed, Postprint (author's final draft)

Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria-cell co-cultures.; Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. (C) 2015 Elsevier B.V. All rights reserved., Peer Reviewed, Postprint (author's final draft)

La consulta íntegra de la tesi, inclosos els articles no comunicats públicament per drets d'autor, es pot realitzar prèvia petició a l'Arxiu de la UPC, Premi Extraordinari de Doctorat, promoció 2014-2015. Àmbit d'Enginyeria Industrial, Tanto el titanio como sus aleaciones son usados como biomateriales por sus excelentes propiedades mecánicas, biocompatibilidad y resistencia a la corrosión. Sin embargo, el fracaso a medio y largo plazo de los implantes dentales fabricados en titanio sigue siendo significativo, debido principalmente a infecciones peri-implantarias. A pesar de que los antibióticos son altamente eficientes en el control de enfermedades infecciosas, la aparición de resistencias por parte de cepas bacterianas es un hecho preocupante para la salud pública. Por lo tanto, el estudio de nuevos tratamientos antibacterianos para implantes biomédicos no basados en antibióticos ha adquirido una especial importancia en los últimos años. La presente tesis doctoral se focaliza en el desarrollo de recubrimientos antibacterianos en superficies de titanio para aplicación en implantes dentales. Así pues, este trabajo puede dividirse en tres partes principales: (i) desarrollo de recubrimientos antibacterianos. En particular, han sido estudiados cinco tratamientos basándose en agentes antimicrobianos inorgánicos y orgánicos (electrodeposición de plata, funcionalización del péptido lactoferrina (hLf1-11) mediante silanización, inmovilización de hLf1-11 mediante polimerización radical controlada por trasferencia de átomo (ATRP) y silanización de 3-(trietoxisilil)propil anhídrido succínico (TESPSA)) (Capítulo 4, 5, 6 y Anexo I); (ii) evaluación de las propiedades antimicrobianas de las superficies optimizadas en un modelo in vitro de placa dental (Anexo II); (iii) comparación in vivo de las propiedades antibacterianas y de osteointegración de dos de las superficies desarrolladas durante la tesis doctoral en un modelo animal (Anexo III). Inicialmente, se describe el desarrollo y optimización de las cinco superficies, incluyendo las características físico-químicas. Además, las propiedades antibacterianas y/o antifouling de los recubrimientos han sido estudiadas in vitro a partir de dos cepas bucales represen, Award-winning, Postprint (published version)

Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins alpha v beta 3 and alpha 5 beta 1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials. (c) 2015 Elsevier B.V. All rights reserved., Peer Reviewed, Postprint (published version)

ObjectivesThe aim of this study was to determine the antibacterial properties of silver-doped titanium surfaces prepared with a novel electrochemical anodizing process.; Material and methodsTitanium samples were anodized with a pulsed process in a solution of silver nitrate and sodium thiosulphate at room temperature with stirring. Samples were processed with different electrolyte concentrations and treatment cycles to improve silver deposition. Physicochemical properties were determined by X-ray photoelectron spectroscopy, contact angle measurements, white-light interferometry, and scanning electron microscopy. Cellular cytotoxicity in human fibroblasts was studied with lactate dehydrogenase assays. The in vitro effect of treated surfaces on two oral bacteria strains (Streptococcus sanguinis and Lactobacillus salivarius) was studied with viable bacterial adhesion measurements and growth curve assays. Nonparametric statistical Kruskal-Wallis and Mann-Whitney U-tests were used for multiple and paired comparisons, respectively. Post hoc Spearman's correlation tests were calculated to check the dependence between bacteria adhesion and surface properties.; ResultsX-ray photoelectron spectroscopy results confirmed the presence of silver on treated samples and showed that treatments with higher silver nitrate concentration and more cycles increased the silver deposition on titanium surface. No negative effects in fibroblast cell viability were detected and a significant reduction on bacterial adhesion in vitro was achieved in silver-treated samples compared with control titanium.; ConclusionsSilver deposition on titanium with a novel electrochemical anodizing process produced surfaces with significant antibacterial properties in vitro without negative effects on cell viability., Peer Reviewed, Postprint (author's final draft)

A biocompatible new titanium alloy Ti-16Hf-25Nb with low elastic modulus (45 GPa) and the use of short bioadhesive peptides derived from the extracellular matrix have been studied. In terms of cell adhesion, a comparative study with mixtures of short peptides as RGD (Arg-Gly-Asp)/PHSRN (Pro-His-Ser-Arg-Asn) and RGD (Arg-Gly-Asp)/FHRRIKA (Phe-His-Arg-Arg-Ile-Lys-Ala) have been carried out with rat mesenchymal cells. The effect of these mixtures of short peptides have already been studied but there are no comparative studies between them. Despite the wide variety of silane precursors available for surface modification in pure titanium, the majority of studies have used aminosilanes, in particular 3-minopropyltriethoxysilane (APTES). Nevertheless, the 3-chloropropyltriethoxysilane (CPTES) is, recently, proposed by other authors. Unlike APTES, CPTES does not require an activation step and offers the potential to directly bind the nucleophilic groups present on the biomolecule (e.g., amines or thiols). Since the chemical surface composition of this new alloy could be different to that pure titanium, both organosilanes have been compared and characterized by means of a complete surface characterization using contact angle goniometry and X-ray photoelectron spectroscopy. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B:495-502, 2015., Peer Reviewed, Postprint (author's final draft)

Postprint (published version)

Postprint (published version)

Los biomateriales se hacen cada vez más importante en la medicina moderna y se utilizan para curar a millones de personas, gracias al avance de la investigación. Estos materiales están diseñados para interactuar beneficiosamente con los sistemas biológicos. Después de varias décadas de investigación, uno de los mayores problemas sigue siendo la biocompatibilidad, que es la capacidad del material para llevar a cabo su función(es) con una respuesta apropiada del huésped en una situación específica. Este proyecto tiene como objetivo participar en la investigación global de mejorar la biocompatibilidad de biomateriales estudiando particularmente una propiedad de la superficie, que puede tener una gran influencia, llamado "mojabilidad". Traduce como un fluido va a reaccionar en contacto con la superficie del material y depende de las interacciones intermoleculares entre los dos. Más específicamente, hemos trabajado en el control de la mojabilidad haciendo microestructuras en placas de acero inoxidable, después de estudiar los tres modelos de mojabilidad descritos por los científicos Young, Wenzel y ambos Cassie y Baxter. Esta investigación contiene tres partes. En la primera, intentamos producir estructuras de micro pilares con un Focused Ion Beam (FIB) con el fin de probar las capacidades y los límites de este proceso y teniendo en cuenta que tal vez le usaremos en un proceso de estampación de microestructuras. En una segunda parte se utilizó un dispositivo de micro-­‐corte, equipado con una herramienta de diamante, para hacer estructuras de micro ranuras y luego, estructuras de micro pilares sobre áreas suficientemente grandes para que sea posible probar su mojabilidad. Al mismo tiempo, un trabajo teórico se ha hecho con el fin de comparar las mojabilidades teorética y experimental. Por último, hemos diseñado la geometría y el proceso con el FIB de dos herramientas de diamante "borde múltiples" que optimizarán el proceso de micro corte en las siguientes obras. El FIB n

In the present study, production of tantalum porous scaffolds using the space holder technique was performed. The effect of size and content of sodium chloride particles, used as space holder, as well as compacting pressure on foam structure and mechanical properties have been investigated. The morphological characterization was carried out by means of scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP) and micro-CT technique. The relationship between the elastic modulus and yield strength of the tantalum porous scaffold and the pore structure was evaluated. Space holder technique allows obtaining tantalum open-cell structure (70% of porosity) and modulus of elasticity similar to cancellous bone, with reproducible processability into three-dimensional structures and reasonable manufacturing costs., Peer Reviewed, Postprint (author’s final draft)

Nowadays, one of the main challenges in metal implants for bone substitution is the achievement of an elastic modulus close to that of human cortical bone as well as to provide an adequate interaction with the surrounding tissue avoiding in vivo foreign body reaction. From this perspective, a new Ti-based alloy has been developed with Nb and Hf as alloying elements which are known as non-toxic and with good corrosion properties. The microstructure, mechanical behaviour and the physicochemical properties of this novel titanium alloy have been studied. Relationship of surface chemistry and surface electric charge with protein adsorption and cell adhesion has been evaluated due to its role for understanding the mechanism of biological interactions with tissues. The Ti25Nb21Hf alloy presented a lower elastic modulus than commercial alloys with a superior ultimate strength and yield strength than CP-Ti and very close to Ti6Al4V. It also exhibited good corrosion resistance. Furthermore, the results revealed that it had no cytotoxic effect on rat mesenchymal stem cells and allowed protein adsorption and cell adhesion. The experimental results make this alloy a promising material for bone substitution or for biomedical devices., Peer Reviewed, Postprint (author’s final draft)

Dental plaque is a biofilm that Causes dental caries, gingivitis, and periodontitis. Most of the studies in antibacterial coatings have been conducted by in vitro Single-species biofilm formation, but oral biofilm involves more than 700 different bacterial Species that are able to interact. Therefore, new studies are focused on in vitro multispecies biofilm models that mimic in vivo biofilms. The aim Of the present work was to study different antibacterial coatings onto titanium surfaces and evaluate the in vitro antimicrobial properties of the surfaces on two different bacterial species and an oral biofilm. The lactate dehydrogenase assay determined that treated samples did not affect fibroblast viability. In addition, the viability of microorganisms on modified samples was evaluated by a LIVE/DEAD BacLight bacterial viability kit. Although a decrease in viable bacteria onto, treated samples was obtained, the results Showed differences in effectiveness when single-biofilm and oral plaque were tested. It confirms, as we expected, the distinct sensitivities that bacterial strains have. Thus, this multispecies biofilins model holds a great potential to assess antibacterial properties Onto samples for dental purposes., Postprint (author’s final draft)

Dental implant failure can be associated with infections that develop into peri-implantitis. In order to reduce biofilm formation, several strategies focusing on the use of antimicrobial peptides (AMPs) have been studied. To covalently immobilize these molecules onto metallic substrates, several techniques have been developed, including silanization and polymer brush prepared by surface-initiated atom transfer radical polymerization (ATRP), with varied peptide binding yield and antibacterial performance. The aim of the present study was to compare the efficiency of these methods to immobilize the lactoferrin-derived hLf1-11 antibacterial peptide onto titanium, and evaluate their antibacterial activity in vitro. Smooth titanium samples were coated with hLf1-11 peptide under three different conditions: silanization with 3-aminopropyltriethoxysilane (APTES), and polymer brush based coatings with two different silanes. Peptide presence was determined by X-ray photoelectron spectroscopy, and the mechanical stability of the coatings was studied under ultrasonication. The LDH assays confirmed that HFFs viability and proliferation were no affected by the treatments. The in vitro antibacterial properties of the modified surfaces were tested with two oral strains (Streptococcus sanguinis and Lactobacillus salivarius) showing an outstanding reduction. A higher decrease in bacterial attachment was noticed when samples were modified by ATRP methods compared to silanization. This effect is likely due to the capacity to immobilize more peptide on the surfaces using polymer brushes and the nonfouling nature of polymer PDMA segment., Peer Reviewed, Postprint (author’s final draft)

Implant materials require optimal biointegration, including strong and stable cell-material interactions from the early stages of implantation. Ti-based alloys with low elastic modulus are attracting a lot of interest for avoiding stress shielding, but their osseointegration potential is still very low. In this study, we report on how cell adhesion is influenced by linear RGD, cyclic RGD, and recombinant fibronectin fragment III8–10 coated on titanium versus a novel low-modulus TiNbHf alloy. The bioactive molecules were either physisorbed or covalently coupled to the substrates and their conformation on the surfaces was investigated with atomic force microscopy (AFM). The influence of the different bioactive coatings on the adhesion of rat mesenchymal stem cells was evaluated using cell culture assays and quantitatively analyzed at the single cell level by AFM-based single-cell force spectroscopy. Our results show that bioactive moieties, particularly fibronectin fragment III8–10, improve cell adhesion on titanium and TiNbHf and that the covalent tethering of such molecules provides the most promising strategy to biofunctionalize these materials. Therefore, the use of recombinant protein fragments is of high importance for improving the osseointegration potential of implant materials, Peer Reviewed, Postprint (author's final draft)

A novel class of polyurethane-polyurea nanoparticles (PUUa NPs) to install multifunctionality on biomaterials is presented. Biofunctionalization of titanium with roxithromycin loaded RGD-decorated PUUa NPs results in an outstanding improvement of osteoblast adhesion and strong suppression of bacterial attachment. This strategy represents a powerful approach to enhance the osseointegration of implant materials., Peer Reviewed, Postprint (published version)

NiTi alloy is the only practical shape memory alloy (SMA) in biomedical use because of its excellent mechanical stability and functionality. However, it is estimated that between 4.5% and 28.5% of the population are hypersensitive to nickel metal, with a higher prevalence in females. Therefore, developing nickel-free low modulus beta-type titanium alloys showing shape memory or super elastic behavior would have a great interest in the biomaterials field. Homogeneous 127 mu m diameter Ti25Hf21Nb wires were produced and compared to straight annealed Ti-50.8 at% Ni (Nitinol) and 90% cold-drawn 316L wires. Microstructural changes taking place during the heat treatment of cold-worked Ti25Hf21Nb wires were investigated. Large plastic deformation during wire drawing and subsequent annealing led to nano-crystallization and amorphization which may contribute to the observed superelasticity. Mechanical properties were characterized using cyclic uniaxial tension and rotary beam fatigue test modes. A modulus of elasticity of less than 60 GPa and axial recoverable strain of greater than 3% were observed with stress hysteresis resembling a reversible stress-induced martensitic transformation at higher temperatures. The new Ti25Hf21Nb alloy is an important candidate for developing Ni-free SMAs in the future. (C) 2015 Elsevier B.V. All rights reserved., Peer Reviewed, Postprint (author's final draft)

To improve cardiovascular implant success, metal-based stents are designated to modulate endothelial cells adhesion and migration in order to prevent restenosis and late thrombosis diseases. Biomimetic coatings with extra-cellular matrix adhesive biomolecules onto stents surfaces are a strategy to recover a healthy endothelium. However, the appropriate bioactive sequences to selective promote growth of endothelium and the biomolecules surface immobilization strategy remains to be elucidated. In this study, biofunctionalization of cobalt chromium, CoCr, alloy surfaces with elastin-like recombinamers, ELR, genetically modified with an REDV sequence, was performed to enhance metal surfaces endothelialization. Moreover, physical adsorption and covalent bonding were used as biomolecules binding strategies onto CoCr alloy. Surfaces were activated with plasma and etched with sodium hydroxide previous to silanization with 3-chloropropyltriethoxysilane and functionalized with the ELR. CoCr alloy surfaces were successfully biofunctionalized and the use of an ELR with an REDV sequence, allows conferring bioactivity to the biomaterials surface, demonstrating a higher cell adhesion and spreading of HUVEC cells on the different CoCr surfaces. This effect is emphasized as increases the amount of immobilized biomolecules and directly related to the immobilization technique, covalent bonding, and the increase of surface charge electronegativity. Our strategy of REDV elastin-like recombinamers immobilization onto CoCr alloy surfaces via covalent bonding through organosilanes provides a bioactive surface that promotes endothelial cell adhesion and spreading. (C) 2015 Elsevier B.V. All rights reserved., Peer Reviewed, Postprint (author’s final draft)

Examen final

With the evolution of human civilization, the field of biomaterials evolved relating different materials with a simple focus: to extend human life and improve the quality of life. Materials used for biomedical applications must exhibit specific properties. Titanium is well-known to be a material used for implants because of its high strength, biocompatibility and high resistance corrosion. It can be used as hard tissue implant, such as artificial bone, hip joint, dental and bone plate. Nevertheless, a relevant aspect of the use of biomaterials is linked with medical device-related and post-operative infections. A promising approach for reducing the occurrences of infections is to provide the surfaces of biomedical devices and implants with features that are unfavourable for bacterial attachment and proliferation. Therefore, the aim of the present work is focused to develop a new method to combine the antibacterial properties of antibiotic-loaded Poly(3-hydroxybutyrate) (PHB) nano- and microspheres, and poly(ethylene glycol) (PEG) as an antifouling agent, with titanium, as the base material for implants, in order to obtain biomimetic surfaces with antibacterial activity. The titanium surfaces were linked to both PHB particles and PEG by covalent bond. This attachment was carried out by firstly activating the titanium surfaces with either plasma or sodium hydroxide. Further functionalization of the activated surfaces were carried out with two different alkoxysilanes which provided the titanium surfaces different organofunctional groups that allowed the reaction with PHB particles or with PEG. A difunctionalized PEG was used in one last step, which allowed the covalent attachment with the PHB particles. Therefore, the antifouling properties of PEG were related with the antibacterial ones of the PHB spheres. Finally, the different steps of the process were evaluated. The success of the surfaces functionalization as well as the different superficial linkages was confirme

En este trabajo se estudia el efecto de la inclusión de micropartículas y nanopartículas de distintos tipos de cerámicas (dióxido de titanio con formas critalinas de rutilo y anatasa, y carburo de silicio)en láminas de polietilentereftalato, teniendo en cuenta el efecto de la concentración de cerámicas y dispersante, el tamaño y tipo de partícula. Las láminas se someten a ensayos mecánicos de tracción y flexión. Se realiza la caracterización térmica a través de calorimetría diferencial de barrido y análisis termomecánico. Se aplican las técnicas de diseño de experimentos y de análisis estadístico de resultados para modelizar la influencia de las variables del proceso en las respuestas, y determinar valores óptimos. Una vez optimizadas las propiedades (valores máximos de características mecánicas, máxima estabilidad térmica) se derivan conclusiones prácticas para futuras aplicaciones que se puedan hacer sobre estos materiales compuestos de polietilentereftalato en aplicaciones tanto textiles como de materiales técnicos de aplicación industrial, además de indicar futuras líneas de interés para este tipo de materiales.

A new thermoplastic polymer for orthodontic applications was obtained and extruded into wires with round and rectangular cross sections. We evaluated the potential of new aesthetic archwire: tensile, three point bending, friction and stress relaxation behaviour, and formability characteristics were assessed. Stresses delivered were generally slightly lower than typical beta-titanium and nickel-titanium archwires. The polymer wire has good instantaneous mechanical properties; tensile stress decayed about 2% over 2 h depending on the initial stress relaxation for up to 120 h. High formability allowed shape bending similar to that associated with stainless steel wires. The friction coefficients were lower than the metallic conventional archwires improving the slipping with the brackets. This new polymer could be a good candidate for aesthetic orthodontic archwires., Peer Reviewed, Postprint (published version)

Bacterial infection represents a major cause of implant failure in dentistry. A common approach to overcoming this issue and treating pen-implant infection consists in the use of antibiotics. However, the rise of multidrug-resistant bacteria poses serious concerns to this strategy. A promising alternative is the use of antimicrobial peptides due to their broad-spectrum activity against bacteria and reduced bacterial resistance responses. The aim of the present study was to determine the in vitro antibacterial activity of the human lactoferrin-derived peptide hLf1-11 anchored to titanium surfaces. To this end, titanium samples were functionalized with the hLf1-11 peptide either by silanization methods or physical adsorption. X-ray photoelectron spectroscopy analyses confirmed the successful covalent attachment of the hLf1-11 peptide onto titanium surfaces. Lactate dehydrogenase assay determined that hLf1-11 peptide did not affect fibroblast viability. An outstanding reduction in the adhesion and early stages of biofilm formation of Streptococcus sanguinis and Lactobacillus salivarius was observed on the biofunctionalized surfaces compared to control non-treated samples. Furthermore, samples coated with the hLf1-11 peptide inhibited the early stages of bacterial growth. Thus, this strategy holds great potential to develop antimicrobial biomaterials for dental applications. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved., Peer Reviewed, Postprint (published version)

This study investigated the effect of two different activation methods on the surface chemical composition of a CoCrMo-alloy. The activation was performed with oxygen plasma (OP) or nitric acid (NA). The surface physical– chemical properties were thoroughly characterized by means of several analytical techniques: X-ray photoelectron spec- troscopy (XPS), time-of-flight secondary ion mass spec- trometry (ToF-SIMS), zinc-complex substitution technique, contact angle, and interferometry. The surface modification was evaluated by assessing contamination removal, the ‘‘active’’ hydroxyl groups (OH-act) present at the surface, the metal oxide ratio (Co y O x - /Cr y O x - ) and changes in the chem- ical composition and topography of the oxide layer. XPS experimental data showed for both methods (OP and NA) a significant decrease of the carbon contents (C 1 s ) associated with contaminants and at the same time changes in the atomic composition of the oxide layer (O 1 s ). In addition, the O 1 s XPS spectra showed differences between the percentage of OH - before and after OP or NA treatment, leading to the conclusion that both methods are effective for surface ‘‘cleaning’’ and activation. These results were further inves- tigated and corroborated by ToF-SIMS analysis and zinc complex substitution technique. The general conclusion was that NA is more efficient in terms of contaminants removal and generation of accessible OH-act present at the surface and without altering the native metal oxide ratio (Co y O x - /Cr y O x - ) considered to be essential for biocompatibility., Peer Reviewed, Postprint (published version)