Functionalization of titanium with integrin-selective ligands for orthopedic and dental applications

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 chemical anchoring. Silanization was used to create a covalent bond between the synthetic molecules and the metallic oxide. Two cell types were used for the in vitro testing of the functionalization system: human osteoblast-like cells (SaOS-2) and mesenchymal stem cells. The testing of different combinations of biomolecule, grafting technique and cell type is the subject of the four full-papers reported in the thesis. Two of these papers also include the in vivo study of the effect of the chemical functionalization in an animal model. The thesis also includes a work focused on the merging of two surface modification techniques, namely chemical functionalization and topographical modification, to create a multifunctional titanium substrate that simultaneously addresses the problem of infection and poor osseointegration: the nanostructure of the topography acts as the bactericidal element, while the surface-grafted biomolecules give eukaryotic cell-instructive properties to the material. This work was carried out during a stay at the Centre for Cell Engineering at the University of Glasgow (Prof. Dalby, UoG, Glasgow, UK). Overall, the collection of works presented in the thesis delineates a comprehensive scenario of how chemical functionalization with ECM-inspired ligands can act as a powerful tool to tune cell behavior and, ultimately, guide the biological response at the peri-implant site.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 testaron con dos tipos celulares relevantes en el contexto de la substitución de material óseo: células de osteosarcoma (SaOS-2) y células mesenquimales (hMSCs), ambas de procedencia humana. La combinación de esos factores originó cuatro estudios, tres de los cuales son reportados como trabajos publicados. Dos de esos estudios incluyen también un análisis in vivo en un modelo animal, que permitió comprobar el efecto de la funcionalización en un escenario clínico real. Finalmente, se llevó a cabo un estudio en colaboración con el Prof. M. Dalby del Centre for Cell Engineering de la University of Glasgow para combinar la funcionalización química y la nanotopografia, obteniendo así una superficie de titanio multifuncional: las nanoestructuras superficiales son bactericidas, pero carecen de propiedades adhesivas, que pueden ser proporcionadas a través de la inmovilización de motivos peptídicos. En conjunto, los resultados de esta tesis demuestran que la funcionalización química es una herramienta poderosa para optimizar la respuesta celular en la superficie del biomaterial e inducir la respuesta biológica deseada

Blocking methods to prevent non-specific adhesion of mesenchymal stem cells to titanium and evaluate the efficiency of surface functionalization: albumin vs poly(ethylene glycol) coating

Premio SIBB 2014Surface modification of biocompatible materials with biologically-active molecules is a well-known strategy to enhance the osteointegration of implantable devices. In order to evaluate the efficiency of these treatments, an in vitro study of cell behavior on the modified surface is usually carried out. A key point to evaluate the efficacy of this strategy is to avoid non specific protein adsorption by creating a non-fouling background. This blocking step ensures that the observed response of cells can be exclusively ascribed to the modification treatment applied. Several techniques are available to create this neutral background. Thus, the aim of this study is to compare two different blocking methods, namely adsorption of albumin from bovine serum (BSA) and grafting of small polyethylene glycol (PEG) chains to titanium, which is the gold standard metal for orthopedics and dentistry. To this end, titanium surfaces were coated with a recently synthesized cell adhesive peptide-based molecule and subjected to a blocking procedure with either BSA or PEG.  Non-functionalized titanium samples were also blocked and used as controls. The biological response of human mesenchymal stem cells was evaluated by measuring the number of attached cells and studying the degree of cell spreading on the substrate. Both aspects of cell behavior are not affected significantly by the blocking method: cells adhere and spread significantly more on the functionalized samples, regardless of the blocking method used. This confirms that the surface feature that defines cell response is the presence/absence of the biomolecule, and not the anti-fouling layer. These results, together with the reduction of variability of results observed in presence of a blocking layer, demonstrate the efficacy and necessity of blocking the surface. PEG grafting is demonstrated as effective as BSA coating in reducing non-specific interactions and not hindering the effect of the biomolecule. However, taken into account the numerous advantages of a synthetic and customizable polymer chain over a complex natural protein, PEG blocking stands out as a very good alternative to albumin adsorption.Peer ReviewedAward-winnin

Surface guidance of stem cell behavior: Chemically tailored co-presentation of integrin-binding peptides stimulates osteogenic differentiation in vitro and bone formation in vivo

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 ReviewedPostprint (author's final draft

Co-localization of RGD and PHSRN peptides on titanium to guide stem cell behaviour and enhance Implant osteointegration

Postprint (published version

Novel peptide-based strategies to install osteointegrative properties on metallic biomaterials

Postprint (published version

Towards the cell-instructive bactericidal substrate:exploring the combination of nanotopographical features and integrin selective synthetic ligands

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 ReviewedPostprint (published version

Functional cortical source connectivity of resting state electroencephalographic alpha rhythms shows similar abnormalities in patients with mild cognitive impairment due to Alzheimer's and Parkinson's diseases

Objective: This study tested the hypothesis that markers of functional cortical source connectivity of resting state eyes-closed electroencephalographic (rsEEG) rhythms may be abnormal in subjects with mild cognitive impairment due to Alzheimer's (ADMCI) and Parkinson's (PDMCI) diseases compared to healthy elderly subjects (Nold). Methods: rsEEG data had been collected in ADMCI, PDMCI, and Nold subjects (N = 75 for any group). eLORETA freeware estimated functional lagged linear connectivity (LLC) from rsEEG cortical sources. Area under receiver operating characteristic (AUROC) curve indexed the accuracy in the classification of Nold and MCI individuals. Results: Posterior interhemispheric and widespread intrahemispheric alpha LLC solutions were abnormally lower in both MCI groups compared to the Nold group. At the individual level, AUROC curves of LLC solutions in posterior alpha sources exhibited moderate accuracies (0.70-0.72) in the discrimination of Nold vs. ADMCI-PDMCI individuals. No differences in the LLC solutions were found between the two MCI groups. Conclusions: These findings unveil similar abnormalities in functional cortical connectivity estimated in widespread alpha sources in ADMCI and PDMCI. This was true at both group and individual levels. Significance: The similar abnormality of alpha source connectivity in ADMCI and PDMCI subjects might reflect common cholinergic impairment. (C) 2018 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved