Interactions between titanium surfaces and biological components

El conocimiento de las interacciones entre célula/proteína/biomaterial es fundamental para la ingeniería de superficies debido a las numerosas aplicaciones biomédicas y biotecnológicas que se están desarrollando así como al éxito clínico que han alcanzado muchos implantes. La respuesta biológica final inducida por los implantes está fuertemente influenciada por las interacciones superficiales entre los componentes biológicos y el material sintético. Las propiedades físicas y químicas de la superficie de un biomaterial, en lugar de las propiedades en su masa, influyen directamente en la capa de proteínas que se adsorben sobre el biomaterial y, como consecuencia de ello, en la respuesta celular a la misma, tanto in vitro como in vivo.El objetivo de esta tesis doctoral es profundizar en el conocimiento de las interacciones material-biosistema, con el énfasis en el descubrimiento de relaciones entre las propiedades superficiales de las superficies de titanio y su respuesta biológica in vitro.El titanio comercialmente puro (Ti c.p.) está siendo ampliamente utilizado con éxito durante muchos años como biomaterial para implantes en cirugía ósea. Su excelente biocompatibilidad se basa en sus adecuadas propiedades mecánicas y, con mayor importancia, en su excelente resistencia a la corrosión. Esta última se debe principalmente a la formación espontanea de una fina película de óxido de titanio que le confiere protección natural contra los ataques degradativos. La modificación de la topografía de la superficie del titanio ha sido objeto de investigación en el pasado con el fin de mejorar la osteointegración. El granallado de partículas es una de las tecnologías más utilizadas para conferir rugosidad a las superficies del titanio. La rugosidad óptima y el tipo de partículas abrasivas del granallado para una respuesta óptima in vitro e in vivo fue previamente determinada en nuestro laboratorio. Sin embargo, todavía están por determinar cuáles son las causas últimas que llevan al biomaterial a su exitosa respuesta biológica.En este trabajo se han estudiado superficies pulidas y rugosas de Ti c.p. obtenidas mediante el granallado con partículas abrasivas de diferente composición química(Al2O3 y SiC) y diferentes tamaños (212-300μm; 425-600μm; 1000-1400μm). La completa caracterización de las propiedades física y química de la superficie, incluyendo la rugosidad, la composición química, la mojabilidad/energía libre y la carga eléctrica de las superficies ensayadas ha llevado a una serie de relevantes conclusiones. Entre ellas, cabe destacar que a) la composición química de las partículas de granallado, así como el método de esterilización fueron los principales factores que influyeron en la mojabilidad y la energía libre superficial de las superficies de titanio estudiadas, b) el método de esterilización cambió en la energía superficial el carácter de donante de electrones de las superficies mediante el cambio de la cantidad y la naturaleza de las sustancias adsorbidas, y c) la composición química de las partículas de granallado no influyó en la carga eléctrica a pH fisiológico ni en el punto isoeléctrico de las superficies.Un segundo paso consistió en el uso de una microbalanza de cristal de cuarzo con monitorización de la energía de disipación, para el estudio de la cinética de adsorción (cantidad y conformación) y de los procesos de adsorción competitiva de tres proteínas de especial interés en los procesos de curación del hueso - la albúmina de suero bovino (BSA), el fibrinógeno (Fbg), y la fibronectina (Fn)- en sensores lisos recubiertos de TiO2. Se determinaron diferentes modelos de procesos de adsorción con una, dos o múltiples pasos distinguibles en función de las proteínas en solución. La capa adsorbida de BSA mostró los cambios más significativos en sus propiedades mecánicas, de conformación y de incorporación de agua hasta que se alcanzaron las condiciones estables de adsorción de proteínas. La BSA, la más pequeña de las proteínas ensayadas, desplazó la Fn y el Fbg cuando se ensayó en condiciones de la competencia por la adsorción, indicando su mayor afinidad por las superficies de TiO2. También se emplearon técnicas de marcaje fluorescente para el estudio de la adsorción proteica en superficies rugosas granalladas. En este estudio, por un parte, se pudo determinar que la cantidad de Fn y BSA adsorbidas en las superficies granalladas está directamente correlacionada con su energía superficial. Por otra parte, se visualizó la adsorción de fibronectina en solución sobre muestras granalladas rugosas de Ti. La Fn formó un patrón irregular de adsorción con una mayor cantidad de proteína adsorbida en los picos que en los valles de la topografía.También se evaluó la organización espacial de la matriz extracelular de los osteoblastos, ECM, sobre superficies de Ti lisas y rugosas por medio de la visualización de las fibrillas de Fn teñidas con marcador fluorescente. Las células osteoblásticas depositaron las fibrillas de Fn con un determinado patrón organizado dentro de la matriz total secretada. Aparecen como una película que cubre la parte superior de las diferentes superficies rugosas de titanio. Un resultado relevante es que el espesor de esta capa aumentó con la rugosidad de la topografía subyacente. Sin embargo no más de la mitad de la máxima distancia pico-valle se cubrió con la proteína secretada y/o reorganizada.Por último, teniendo en cuenta las diferencias en la organización de la ECM y laadsorción de Fn en las superficies ensayadas de Ti, se realizó un estudio de qRT-PCR para determinar la influencia de las propiedades superficiales del titanio, con y sin preadsorción de Fn, en la respuesta osteoblástica. La expresión génica de la subunidad 5 de la integrina celular, como marcador de la adhesión celular, se incrementó en las superficies granalladas con SiC en comparación con las granalladas con alúmina. Este resultado fue correlacionado con la mayor cantidad de Fn adsorbida debido a la mayor energía superficial de las superficies granalladas con SiC. El aumento de la rugosidad, así como la presencia de partículas de alúmina en las superficies rugosas incrementó la actividad de ALP y la expresión génica de ALP mRNA por los osteoblastos, y por lo tanto su diferenciación.The understanding of cell/protein/biomaterial interactions is critical to the engineering of substrates for numerous biomedical and biotechnological applications and to the clinical success of implants. The final biological response induced by implants is strongly influenced by the biological-components/synthetic-material surface interactions. It is well accepted that the physical and chemical surface properties of a biomaterial rather than its bulk properties will influence the protein adlayer and then the cell response to it, both in vitro and in vivo.The aim of this PhD thesis is to gain an increased understanding of the materialbiosystem interactions, with an emphasis on establishing correlations between surface properties of titanium surfaces and its in vitro biological response.Commercially pure titanium (c.p. Ti) is being widely and successfully used implant biomaterial in bone surgery over many years. Its excellent biocompatibility is based in its appropriate mechanical properties and, more importantly, in its excellent corrosion resistance, which is mainly due to the presence of a naturally-occurring thin protective titanium oxide film. Modification of titanium surface topography has been a subject of research in the past with the purpose of improving its osseointegration. Grit blasting is one of the most used technologies to roughen titanium surfaces for this purpose. The optimal roughness and type of abrasive blasting-particles for a better in vitro and in vivo response was previously determined in our lab. However, which and how different relevant surface properties of the blasted titanium surfaces induce that optimal biological behavior is still poorly understood.Smooth/polished and rough c.p. Ti surfaces obtained by blasting with abrasiveparticles of different chemical composition (Al2O3 and SiC) and different sizes (212-300μm; 425-600μm; 1000-1400μm) were studied. The comprehensive characterization of physical and chemical surface properties, including roughness, chemical composition, wettability/free energy and electrical charge of the tested surfaces led to a series of relevant conclusions. Among them, it is worth noting that a) the chemical composition of the grit-blasting particles as well as the method of sterilization were found the main factors influencing wettability and surface free energy of the titanium surfaces; b) the sterilization method changed the electron donor character of the surfaces by changing the amount/nature of physisorbed substances on the surfaces, and c) the chemical composition of the blasting particles did not influence on the electrical charge at physiological pH and the isoelectric point of the surfaces.A second step consisted in the use of a quartz crystal microbalance with monitoring of the energy dissipation to study the adsorption kinetics (amount and conformation) and adsorption competition processes of three proteins of special interest in the healing processes of bone -bovine serum albumin (BSA), fibrinogen (Fbg), and fibronectin (Fn)-on smooth TiO2-coated sensors. Different patterns of adsorption with processes in one, two or multiple distinguishable steps were determined depending of the protein in solution. The BSA adlayers showed the most significant changes in their mechanical properties/conformation/incorporation of water until steady protein-adsorption conditions were reached. BSA, the smallest of the tested proteins, displaced Fn and Fbg when in competition for adsorption, which is an indication of its higher affinity for TiO2 surfaces. Fluorescent labelling techniques where used to study protein adsorption on blasted rough surfaces. Most significantly, the amount of Fn and BSA adsorbed on blasted surfaces was positively correlated with their surface energy. The adsorption of fibronectin from solution on shot-blasted rough titanium surfaces resulted in an irregular pattern of adsorption with a higher amount of protein adsorbed on peaks than on valleys of the topography.Further, the spatial organization of the osteoblast extracellular matrix, ECM, on smooth and rough Ti surfaces was evaluated by visualizing fluorescently-stained Fn-fibrils. Osteoblast-like cells deposited Fn- fibrils in a specific facet-like pattern that was organized within the secreted total matrix. It appeared as a film overlying the top of the different rough titanium surfaces. Interestingly, the thickness of this layer increased with the roughness of the underlying topography, but no more than half of the total maximum peak-to-alley distance was covered.Finally, taking into consideration the differences in ECM organization and Fn adsorption on the tested Ti surfaces a qRT-PCR study was carried out to elucidate the influence of titanium surface properties with and without Fn-precoatings on the osteoblast response. The expression of 5 integrin subunit gene, as a marker for cell adhesion, was increased in SiC-blasted surfaces compared to alumina-blasted surfaces. This was related to the higher amount of adhesive-protein Fn adsorbed caused by the higher surface energy of SiC-blasted surfaces. The increase of roughness as well as the presence of alumina particles on blasted surfaces increased ALP activity and ALP mRNA gene expression by osteoblasts, and so their differentiation.This research work contribute to increase our knowledge on the interactions taking place at the bio/non-bio interface between different biological components -water, proteins, cells- and materials of clinical relevance, such as rough titanium. Theintertwined effects of the different properties of the synthetic surfaces appear as a challenge to unravel the ultimate causes that determine the fate of cells on synthetic biomaterials

Binder jetting additive manufacturing of biodegradable Zn

Biodegradable materials avoid second surgeries and long-term associated risks of conventional inert implants. Zn arose as a potential candidate for bioresorbable implants due to its proper degradation behaviour and biocompatibility [1]. However, its low melting point induces uncontrolled porosity in LPBF, promoting the future cracking of the implant. Therefore, new fabrication techniques need to be explored. In this work, binder jetting 3d printing (BJ3P) was studied for Zn powders. The samples were printed and sintered under different conditions. It is concluded that, the increase the temperature almost up to melting point leads to higher densification, at the same time, the rise of temperature provokes the formation and growth of oxidized layer on the surface of the powders

Micropatterned 3D-printed PLLA/PLCL bioresorsable stents: degradation and influence of sterilization

Bioresorbable stents (BRS) are cylindrical scaffolds designed to provide a temporary support to the vessel wall while the structure slowly degrades until completely resorbed [1]. Current stent fabrication technology hinders local modification of the surface topography. This work presents a novel solvent-cast direct-write (SC-DW) 3D printing system to manufacture inner patterned BRS. Poly-L-lactic acid (PLLA) and poly(lactic-co-caprolactone) (PLCL) stents were obtained by cylindrical printing onto a Ø 3 mm rotating mandrel (Figure 1a) [2]. The ink consisted in a solution of high Mw PLLA or PLCL copolymer (95:5) in chloroform at 10% w/v and 12.5% w/v, respectively. Steel mandrels were modified by direct laser interference patterning with a femtosecond laser to obtain a linear micropatterning with a periodicity of 10 μm, which was transferred onto stents' luminal surface (Figure 1b). Stents biodegradation was characterized by an accelerated degradation assay in PBS at 50oC over 4 months and characterized in terms of mass loss, SEM, DSC, mechanical tests, GPC and 1 H-NMR. PLLA and PLCL stents underwent bulk degradation, with a sustained decrease in molecular weight and an increase in crystallinity as degradation proceeded. PLCL stents degraded 1.5 times faster than PLLA stents due to higher water penetration in amorphous regions. Finally, two sterilization methods were evaluated: γ-irradiation (8 kGy) and ethylene oxide (EtO). Whereas γ- irradiation induced chain scission and a marked decrease in molecular weight, no structural or chemical alterations were found after EtO sterilization (Figure 1c). In conclusion, customizable PLLA and PLCL BRS were successfully fabricated through SC-DW technique, showing luminal micropatterning for enhanced endothelialization and adequate degradation timeframe for resorption

Estudio histológico e histomorfométrico de la respuesta ósea de implantes dentales en cerdos minipigs

Preprin

Biodegradable metallic zinc alloys for biomedical applications

Biodegradable metals, such as zinc (Zn) appear to overcome some of the drawbacks of permanent metallic implants. However, the uncontrolled biodegradation of Zn-alloyed materials is still a concern for biomedical applications compromising biocompatibility and mechanical properties. In this work, two strategies based on severe plastic deformation or polymeric coatings are evaluated to overcome degradation drawbacks. Cold-rolled Zn-0.5Mg and Zn-2Ag bars (Goodfellow, UK) were modified as follows: (1) ECAP was performed to the bars, supplying an equivalent strain of 0.76 each pass; (2) PCL was dissolved in chloroform and spin-coated onto the surfaces. The microstructure was observed by SEM/EDS and EBSD. Tensile and nanoindentation tests were performed. The corrosion was studied by PDP and EIS. Fig. 1 shows the microstructure of the as-received alloys. Ultra-fine grain structure was achieved after ECAP (Fig. 2), providing superplastic behavior to the Zn-2Ag alloy (elongation over 200 %). Nanoindentation maps showed similar hardness distribution after ECAP. PCL-coated samples presented a noteworthy decrease in current density (from 15 A/cm2 down to 0.5 A/cm2), and EIS confirmed the effect of the PCL layer with a higher impedance modulus. The influence of the secondary phases on the mechanical reinforcement of Zn was previously studied [1]. However, their presence also forms galvanic pairs and favors localized corrosion, which could provoke the future cracking of the implant. Regarding this, our study showed that PCL coating delays early degradation, while the refined microstructure obtained after ECAP homogenizes further corrosion. Both approaches can be used to control corrosion at different degradation timepoints, fundamental for the proper biointegration of the Zn-based implants

Numerical simulation of the micro-extrusion process of printable biomaterials

This work aims to gain a better understanding of how the rheological properties of printable materials affect their processability, as well as the quality of the final product, which at the end can lead to reducing time and costs of the process and increase product development. As the first step, the proper rheological non-Newtonian models are extracted from experimental studies. Later, three-dimensional numerical simulation of extrusion process is performed in the context of Direct Numerical Simulation (DNS) of governing equations, where the whole physics of fluid motion is taken into account. A finite-volume fractional step approach is used to solve the Navier-Stocks equations on collocated arbitrary meshes. Geometrical volume-of-fluid (GVOF) interface capturing approach is used to resolve the topological changes of the moving interface. The governing equations are solved using High-Performance Computing (HPC) parallel approaches. Besides the contribution of this work to the advancement of numerical techniques applied to multiphase complex flows, obtained results will shed light on the nature of non-Newtonian extrusion process with vast applications in the 3D printer industrial sectors.This work was developed in the context of a research project (BASE3D 001-P-001646) co-financed by the European Union Regional Development Fund within the framework of the ERDF Operational Program of Catalonia 2014-2020 with a grant of 50% of total cost eligible.Postprint (published version

Solvent-cast direct-writing and electrospinning as a dual fabrication strategy for drug-eluting polymeric bioresorbable stents

Bioresorbable stents (BRS) are conceived to retain sufficient radial strength after implantation while releasing an antiproliferative drug in order to prevent vessel restenosis until complete resorption. Ongoing research trends involve the use of innovative manufacturing techniques to achieve thinner struts combined with optimized local drug delivery. This work presents a combination of solvent-cast direct-writing (SC-DW) and electrospinning (ES) using poly-l-lactic acid (PLLA) and poly(l-lactic-co-¿-caprolactone) (PLCL) as a new approach to generate everolimus-eluting BRS for cardiovascular applications. A Design of Experiment (DoE) was conducted to determine the optimal parameters to obtain a homogeneous coating with high specific surface. Manufactured stents were characterized by means of mechanical tests and scanning electron microscopy (SEM), with everolimus release in accelerated conditions quantified through High Performance Liquid Chromatography (HPLC). Drug loading was achieved either encapsulated in the struts of the stent or in an electrospun PLCL membrane covering the stent. In the former case, everolimus release was found to be insufficient, less than 3% of total drug loading after 8 weeks. In the latter, everolimus release considerably increased with respect to drug-loaded 3D-printed stents, with over 50% release in the first 6 hours of the test. In conclusion, everolimus release from PLCL-coated 3D-printed stents would match the dose and timeframe required for in vivo applications, while providing thinner struts than SC-DW drug-loaded stents.Peer ReviewedPostprint (published version

Biofunctionalization of REDV elastin-like recombinamers improves endothelialization on CoCr alloy surfaces for cardiovascular applications

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 ReviewedPostprint (author’s final draft

RGD mutation of the heparin binding II fragment of fibronectin for guiding mesenchymal stem cell behavior on titanium surfaces

Installing bioactivity on metallic biomaterials by mimicking the extracellular matrix (ECM) is crucial for stimulating specific cellular responses to ultimately promote tissue regeneration. Fibronectin is an ECM protein commonly used for biomaterial functionalization. The use of fibronectin recombinant fragments is an attractive alternate to the use of full-length fibronectin because of the relatively low cost and facility of purification. However, it is necessary to combine more than one fragment, for example, the cell attachment site and the heparin binding II (HBII), either mixed or in one molecule, to obtain complete activity. In the present study, we proposed to install adhesion capacity to the HBII fragment by an RGD gain-of-function DNA mutation, retaining its cell differentiation capacity and thereby producing a small and very active protein fragment. The novel molecule, covalently immobilized onto titanium surfaces, maintained the growth factor-binding capacity and stimulated cell spreading, osteoblastic cell differentiation, and mineralization of human mesenchymal stem cells compared to the HBII native protein. These results highlight the potential capacity of gain-of-function DNA mutations in the design of novel molecules for the improvement of osseointegration properties of metallic implant surfaces.Peer ReviewedPostprint (author's final draft

Enhanced osteoconductivity on electrically charged titanium implants treated by physicochemical surface modifications methods

Biomimetic design is a key tenet of orthopedic device technology, and in particular the development of responsive surfaces that promote ion exchange with interfacing tissues, facilitating the ionic events that occur naturally during bone repair, hold promise in orthopedic fixation strategies. Non-bioactive nanostructured titanium implants treated by shot-blasting and acid-etching (AE) induced higher bone implant contact (BIC=52% and 65%) compared to shot-blasted treated (SB) implants (BIC=46% and 47%) at weeks 4 and 8, respectively. However, bioactive charged implants produced by plasma (PL) or thermochemical (BIO) processes exhibited enhanced osteoconductivity through specific ionic surface-tissue exchange (PL, BIC= 69% and 77% and BIO, BIC= 85% and 87% at weeks 4 and 8 respectively). Furthermore, bioactive surfaces (PL and BIO) showed functional mechanical stability (resonance frequency analyses) as early as 4 weeks post implantation via increased total bone area (BAT=56% and 59%) ingrowth compared to SB (BAT=35%) and AE (BAT=35%) surfaces.Peer ReviewedPostprint (author's final draft