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Hydroxyapatite-based materials have been widely used in countless applications, such as bone regeneration, catalysis, air and water purification or protein separation. Recently, much interest has been given to controlling the aspect ratio of hydroxyapatite crystals from bulk samples. The ability to exert control over the aspect ratio may revolutionize the applications of these materials towards new functional materials. Controlling the shape, size and orientation of HA crystals allows obtaining high aspect ratio structures, improving several key properties of HA materials such as molecule adsorption, ion exchange, catalytic reactions, and even overcoming the well-known brittleness of ceramic materials. Regulating the morphogenesis of HA crystals to form elongated oriented fibres has led to flexible inorganic synthetic sponges, aerogels, membranes, papers, among others, with applications in sustainability, energy and catalysis, and especially in the biomedical field., This project has received funding from the European Research Council (ERC), grant agreement number 101055053 (BAMBBI). Maria-Pau Ginebra acknowledges the support received through the ICREA Academia Award for excellence in research, funded by the Generalitat de Catalunya. Montserrat Espanol acknowledges the support from Generalitat de Catalunya through the Serra Hunter program, and Anna Diez-Escudero acknowledges the Spanish Ministry of Universities for the support received through the Maria Zambrano fellowship., Peer Reviewed, Postprint (published version)

Human bone is a composite material composed of a mineral and organic phase. It is a living tissue that, when damaged, regenerates spontaneously and naturally to allow reconstruction or replacement of lost or damaged bone tissue. In cases of critical-sized bone defects in the maxillofacial region, spontaneous regeneration becomes impossible, necessitating guided bone regeneration (GBR) to reconstruct the defect surrounding dental implants. This surgical procedure involves the use of a bone graft and a barrier membrane to create a suitable environment for new bone cell formation. Despite this widely used process, commercially available GBR membranes exhibit numerous disadvantages and fail to be active in the bone regeneration process. This research project aims to develop an innovative GBR membrane utilizing hydroxyapatite doped with therapeutic ions to enhance its bioactivity. The production of this membrane involves the synthesis of hydroxyapatite ultra-long nanofibres doped with therapeutic ions in order to obtain a flexible and bioactive GBR membrane. The primary focus of the project is to establish a synthesis mechanism for doping hydroxyapatite ultralong nanofibres with strontium, zinc and magnesium. Two distinct mechanisms were employed, both derived from the synthesis of hydroxyapatite ultra-long nanofibres through a hydrothermal treatment of a calcium oleate precursor. The parameters studied during this project encompassed the synthesis time, the content of doping ions and the type of therapeutic ions. The first mechanism, the one-step synthesis, involves mixing all reagents to form a calcium oleate precursor with therapeutic ions, followed by high-temperature and high-pressure hydrothermal treatment. This process successfully yielded Sr-doped hydroxyapatite ultra-long nanofibres. However, further studies are required to assess the extent of doping of the fibres with magnesium and zinc as the introduction of the doping ion into the hydroxyapatite crystal latt, El hueso humano es un material constituido por una fase mineral y otra orgánica. Es un tejido vivo que, cuando se daña, se regenera de forma espontánea y natural para permitir la reconstrucción o sustitución del tejido óseo perdido o dañado. En casos de defectos óseos de tamaño crítico en la región maxilofacial, la regeneración espontánea resulta imposible, por lo que es necesaria la regeneración ósea guiada para reconstruir el defecto que rodea a los implantes dentales. Este procedimiento quirúrgico implica el uso de un injerto óseo y una membrana de barrera para crear un entorno adecuado para la formación de nuevas células óseas. A pesar de tratarse de un proceso ampliamente utilizado, las membranas GBR disponibles en el mercado presentan numerosos inconvenientes y no consiguen ser activas en el proceso de regeneración ósea. Este proyecto de investigación pretende desarrollar una membrana GBR innovadora que utilice hidroxiapatita dopada con iones terapéuticos para mejorar su bioactividad. La producción de esta membrana implica la síntesis de nanofibras ultralargas de hidroxiapatita dopadas con iones terapéuticos para obtener una membrana GBR flexible y bioactiva. El objetivo principal del proyecto es establecer un mecanismo de síntesis para dopar nanofibras ultralargas de hidroxiapatita con estroncio, zinc y magnesio. Se emplearon dos mecanismos distintos, ambos derivados de la síntesis de nanofibras ultralargas de hidroxiapatita mediante un tratamiento hidrotérmico de un precursor de oleato de calcio. Los parámetros estudiados durante este proyecto involucraron el tiempo de síntesis, el contenido de iones dopantes y el tipo de iones terapéuticos. El primer mecanismo, la síntesis en un solo paso, consiste en mezclar todos los reactivos para formar un precursor de oleato de calcio con iones terapéuticos, seguido de un tratamiento hidrotérmico a alta temperatura y alta presión. Este proceso produjo con éxito nanofibras ultralargas de hidroxiapatita dopada con Sr. Si, L'os humain est un matériau composite composé d'une phase minérale et organique. C'est un tissu vivant qui, lorsqu'il est endommagé, se régénère spontanément et naturellement pour permettre la reconstruction ou le remplacement du tissu osseux perdu ou endommagé. Dans les cas de défauts osseux de taille critique dans la région maxillo-faciale, la régénération spontanée devient impossible, ce qui nécessite une régénération osseuse guidée pour reconstruire le défaut afin de pouvoir visser un implant dentaire. Cette procédure chirurgicale implique l'utilisation d'un greffon osseux et d'une membrane barrière afin de créer un environnement propice à la formation de nouvelles cellules osseuses. Malgré ce processus de régénération osseuse guidée, les membranes actuellement disponibles dans le commerce pour la régénération osseuse guidée présentent de nombreux inconvénients et ne parviennent pas à être actives dans le processus de régénération osseuse. Ce projet de recherche vise ainsi à développer une membrane GBR innovante utilisant de l'hydroxyapatite dopée avec des ions thérapeutiques pour améliorer sa bioactivité. Ainsi, la fabrication de cette membrane implique la synthèse de nanofibres d’hydroxyapatite ultra-longues dopées avec des ions thérapeutiques afin d’obtenir une membrane flexible et bioactive. L'objectif principal du projet est d'établir un mécanisme de synthèse pour le dopage des nanofibres ultra-longues d'hydroxyapatite avec du strontium, du zinc et du magnésium. Deux mécanismes distincts ont été utilisés, tous deux dérivés de la synthèse de nanofibres ultra-longues d'hydroxyapatite par traitement hydrothermique d'un précurseur d'oléate de calcium. Les paramètres étudiés au cours de ce projet comprennent le temps de synthèse, la teneur en ions dopants et le type d'ions thérapeutiques. Le premier mécanisme, correspondant à une synthèse en une étape, implique le mélange de tous les réactifs pour former un précurseur d'oléate de calcium avec des ions thérapeuti, Incoming

L'os és un òrgan amb una alta capacitat de regeneració, aquesta regeneració ve donada per l'activitat coordinada de les cèl·lules òssies, però quan el defecte generat és molt gran o les cèl·lules tenen alguna patologia és necessari usar un implant que les cèl·lules puguin utilitzar com a suport on moure's i crear el nou teixit. Entre tots els tipus d'implants que hi ha disponibles el que presenta menys problemes és l'ús d'un material sintètic, això ha fet que l'ús de biomaterials sintètics capaços d'imitar la matriu extracel·lular dels ossos es vegi potenciat, un exemple és la hidroxiapatita. En aquest Treball de fi de màster (TFM), l'objectiu principal és aconseguir formar implants ossis a partir de les nanofibras de hidroxiapatita, ja que amb aquesta morfologia s'aconsegueix un material ceràmic amb unes propietats elàstiques mai vistes. Aquestes nanofibras s'han sintetitzat mitjançant un procés hidrotèrmic amb l'ajuda d'una molècula orgànica, en aquest cas oleat sòdic, que ajuda a direccionar el creixement del material. Durant aquest treball de fi de màster s'estudiaran diversos paràmetres principals en la síntesi de les nanofibras; el temps de síntesi, l'efecte de la pressió i la concentració dels components. Per a observar les diferències entre síntesis, les fibres seran observades en el TEM i el SEM. També seran analitzades amb FTIR, DSC i DRX per a veure la composició de les fibres i es calculés la superfície especifica mitjançant la tècnica ASAP. A més, també s'establirà un valor per a comparar el rendiment de cada síntesi. També s'ha avaluat la capacitat de consolidar les fibres en bastides tridimensionals. Per a això s'estudiaran dues vies “Ice templating” i impressió 3d per extrusió., El hueso es un órgano con una alta capacidad de regeneración, esta regeneración viene dada por la actividad coordinada de las células óseas, pero cuando el defecto generado es muy grande o las células tienen alguna patología es necesario usar un implante que las células puedan utilizar como soporte donde moverse y crear el nuevo tejido. Entre todos los tipos de implantes que hay disponibles el que presenta menos problemas es el uso de un material sintético, esto ha hecho que el uso de biomateriales sintéticos capaces de imitar la matriz extracelular de los huesos se vea potenciado, un ejemplo es la hidroxiapatita. En este Trabajo de fin de máster (TFM), el objetivo principal es conseguir formar implantes óseos a partir de las nanofibras de hidroxiapatita, ya que con esta morfología se consigue un material cerámico con unas propiedades elásticas nunca vistas. Estas nanofibras se han sintetizado mediante un proceso hidrotérmico con la ayuda de una molécula orgánica, en este caso oleato sódico, que ayuda a direccionar el crecimiento del material. Durante este trabajo de fin de máster se estudiarán varios parámetros principales en la síntesis de las nanofibras; el tiempo de síntesis, el efecto de la presión y la concentración de los compuestos. Para observar las diferencias entre síntesis, las fibras serán observadas en el TEM y el SEM. También serán analizadas con FTIR, DSC y DRX para ver la composición de las fibras y se calculara la superficie especifica mediante la técnica ASAP. Además, también se establecerá un valor para comparar el rendimiento de cada síntesis. También se ha evaluado la capacidad de consolidar las fibras en andamios tridimensionales. Para ello se estudiarán dos vías “Ice templating” y impresión 3d por extrusión., Bone is an organ with a high capacity for regeneration, this regeneration is given by the coordinated activity of bone cells, but when the defect generated is very large or the cells have some pathology, it is necessary to use an implant that the cells can use as a support where they can move and create new tissue. Among all the types of implants available, the one that presents the fewest problems is the use of a synthetic material. This has led to the use of synthetic biomaterials capable of imitating the extracellular matrix of bones, one example being hydroxyapatite. In this Master's thesis (TFM), the main objective is to form bone implants from hydroxyapatite nanofibres, as this morphology results in a ceramic material with unprecedented elastic properties. These nanofibres have been synthesised by means of a hydrothermal process with the help of an organic molecule, in this case sodium oleate, which helps to direct the growth of the material. During this master's thesis, several main parameters in the synthesis of nanofibres will be studied; the synthesis time, the effect of pressure and the concentration of the reagents. To observe the differences between syntheses, the fibres will be observed in TEM and SEM. They will also be analysed by FTIR, DSC and XRD to see the composition of the fibres and the specific surface area will be calculated using the ASAP technique. In addition, a value will also be established to compare the performance of each synthesis. The ability to consolidate the fibres into three-dimensional scaffolds has also been evaluated. For this purpose, two routes will be studied: Ice templating and 3D printing by extrusion.

The ability of the high-temperature polymorph of tricalcium phosphate, a-TCP, to hydrolyse to calcium-deficient hydroxyapatite underlies many developments in the field of synthetic bone grafts, including calcium phosphate cements, foams, and self-setting inks. The objective of the present work was to investigate the effect of humidity on a-TCP powder reactivity. The results showed that a 3-week incubation at high relative humidity (80%) had no impact on reactivity, but, as the incubation was prolonged, the powder started to hydrolyse. This reactivity was associated to the presence of defects and to an amorphous phase induced during powder milling. Moisture studies performed under static/dynamic conditions gave comparable water adsorption percentages. The dynamic studies further proved irreversible water sorption, indicating that some water molecules reacted with the powder after short incubation times. Taken together, these results demonstrate that, although a-TCP powder adsorbs water from moisture immediately, the impact on reactivity appears only after several weeks of storage., Peer Reviewed, Postprint (published version)

El següent projecte tracta sobre el disseny i desenvolupament d’una línia de microcargols d’ortodòncia. Des del propi disseny del microcargol, fins al planteig dels accessoris necessaris, de la caixa quirúrgica que s’oferirà i del procés de fabricació que es seguirà. Per a la realització d’aquest, inicialment es desenvoluparà una comparativa (Benchmarking) del mercat, es realitzaran diferent reunions amb doctors referents del sector per detectar les necessitats actuals del mercat i, finalment s’utilitzarà el programa SolidWorks pel disseny del propi microcargol. Addicionalment, es realitzarà una revisió bibliogràfica de diferents estudis d’elements finits i, es plantejaran un seguit de propostes futures per la possible futura realització d’un anàlisi d’aquesta tipologia. Finalment, s’han pogut dissenyar onze referències diferents de microcargols, juntament amb el planteig dels accessoris necessaris, la caixa quirúrgica i el procés de fabricació (mecanitzar, neteges, envasat individual i esterilització). Amb aquest treball, s’han pogut conèixer i entendre les diferents fases per les quals ha de passar un projecte d’enginyeria i, també s’han pogut definir tres propostes futures per realitzar anàlisis d’elements finits sobre els microcargols, El siguiente proyecto trata sobre el diseño y desarrollo de una línea de microtornillos de ortodoncia. Desde el propio diseño del microtornillo, hasta el planteo de los accesorios necesarios, de la caja quirúrgica que ser ofrecerá y del proceso de fabricación que se seguirá. Para la realización de este, inicialmente se desarrollará una comparativa (Benchmarking) del mercado, se realizarán diferentes reuniones con doctores referentes del sector para detectar las necesidades actuales del mercado y, finalmente se utilizará el programa SolidWorks para el diseño del propio microtornillo. Adicionalmente, se realizará una revisión bibliográfica de diferentes estudios de elementos finitos y, se planteará un seguido de propuestas futuras para la posible realización de un análisis de esta tipología. Finalmente, se han podido diseñar once referencias diferentes de microtornillos, juntamente con el planteo de los accesorios necesarios, la caja quirúrgica y el proceso de fabricación (mecanizado, limpiezas, envasado individual y esterilización). Con este trabajo, se han podido conocer y entender las diferentes fases por las que debe pasar un proyecto de ingeniería y, también se ha podido definir tres propuestas futuras para realizar análisis de elemento finitos sobre los microtornillos, The following project is about the design and development of a line of orthodontic microscrews. It includes the design of the microscrew itself, the identification of necessary accessories, the development of a surgical kit, and the manufacturing process to be followed. To carry out this project, a market benchmarking will be initially conducted, followed by meetings with key doctors in the field to identify current market needs. The SolidWorks software will be used for the design of the microscrew. In addition, a literature review of various finite element studies will be conducted, and a series of future proposals will be presented for the possible implementation of an analysis of this type. Ultimately, eleven different references of microscrews have been designed, along with the approach of the necessary accessories, the surgical kit, and the manufacturing process (machining, cleaning, packaging, and sterilization). This project has provided insights into the different phases involved in an engineering project, and it has also defined three future proposals for conducting finite element analysis on the microscrews

Tesi amb menció de Doctorat Internacional, (English) Substantial research efforts in the field of tissue engineering have been done in the recent years to overcome the bone regeneration limitations of the human body and to treat bone-related pathologies like osteosarcoma. Nanotechnology offers limitless ways to improve bone regeneration and to fight bone cancer by means of hydroxyapatite (HA) nanoparticles (NPs) and through the design of small organic molecules with capacity to trigger specific biological reactions. In the present thesis, the implementation of these molecules in cancer therapy and in the bone regeneration field is studied, and their interaction with biological systems is investigated. Chapter 1 offers a general perspective of the topic and introduces the state of the art with the main strategies for bone cancer therapy and bone regeneration reported in the literature. Chapter 2 explores the use of hydroxyapatite nanoparticles for bone cancer therapies, with a view to elucidate the underlying cytotoxic mechanism. Moreover, the techniques commonly used to study the cellular uptake of these materials are compared with more novel characterisation alternatives. Indeed, implementation of cryo-soft X-ray tomography is presented as a key tool for tracking and monitoring the fate of internalised HA NPs, as it allows visualising both solid and liquid calcium deposits resulting from the dissolution of the nanomaterials. Chapter 3 investigates the use of gelatine microspheres (MS) as carriers of HA NPs for bone regeneration. Additionally, the ionic doping of these NPs with Mg2+, Zn2+ and Sr2+, with recognised benefits in the bone remodelling process, is studied and their release kinetics in cell culture medium is further quantified. Although dissolution and precipitation events take places simultaneously, MS demonstrate to be an effective method to deliver the therapeutical loaded ions. Chapter 4 and Chapter 5 focus on the design of bioinks as a strategy for bone tissue engineering. Alginate is chosen a, (Català) En els últims anys s'han fet un gran esforç de recerca en el camp de l'enginyeria de teixits per tal de superar les limitacions de regeneració òssia del cos humà, això com per tractar patologies relacionades amb l'os com és l'osteosarcoma. La nanotecnologia ofereix múltiples maneres de millorar la regeneració òssia i de lluitar contra el càncer d'os, gràcies a l'ús de nanopartícules d'hidroxiapatita (NPs HA) i a través del disseny de molècules orgàniques molt petites amb capacitat per desencadenar reaccions biològiques específiques. En aquesta tesi, s'estudia la implementació d'aquestes molècules en la teràpia anti-càncer i en el camp de la regeneració òssia, així com la seva interacció amb els sistemes biològics. El Capítol 1 ofereix una perspectiva general del tema i introdueix l'estat de l'art amb les estratègies pel tractament del càncer d'os i la regeneració òssia més comunes reportades en la literatura. En el Capítol 2 s'explora l'ús de les nanopartícules d'hidroxiapatita com a teràpia pel càncer d'os amb la intenció d'investigar-ne el mecanisme de citotoxicitat subjacent. A més, les tècniques més utilitzades per a l'estudi de la internalització cel·lular d'aquests materials es comparen amb alternatives de caracterització més innovadores. D'aquesta manera, la implementació de la crio-tomografia de raigs X tous es presenta com a una eina clau pel seguiment i monitorització del destí d'aquestes NPs internalitzades, ja que permet la visualització de dipòsits de calci en estat sòlid i líquid, que deriven de la dissolució del nanomaterial. En el Capítol 3 s'investiga l'ús de microesferes (MS) de gelatina com a portadores de NPs HA per a regeneració òssia. S'estudia el dopatge iònic de les NPs amb Mg2+, Zn2+ i Sr2+, que són ions molt implicats en el procés de remodelació òssia, i es quantifica la seva cinètica d'alliberació en medi de cultiu cel·lular. Tot i que hi ha processos de dissolució i precipitació simultanis, les MS demostren ser un mètode efectiu, Postprint (published version)

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form., Peer Reviewed, Postprint (published version)

The version of record is available online at: http://dx.doi.org/10.1007/s10237-022-01577-2, Scaffolds are microporous biocompatible structures that serve as material support for cells to proliferate, differentiate and form functional tissue. In particular, in the field of bone regeneration, insertion of scaffolds in a proper physiological environment is known to favour bone formation by releasing calcium ions, among others, triggering differentiation of mesenchymal cells into osteoblasts. Computational simulation of molecular distributions through scaffolds is a potential tool to study the scaffolds’ performance or optimal designs, to analyse their impact on cell differentiation, and also to move towards reduction in animal experimentation. Unfortunately, the required numerical models are often highly complex and computationally too costly to develop parametric studies. In this context, we propose a computational parametric reduced-order model to obtain the distribution of calcium ions in the interstitial fluid flowing through scaffolds, depending on several physical parameters. We use the well-known Proper Orthogonal Decomposition (POD) with two different variations: local POD and POD with quadratic approximations. Computations are performed using two realistic geometries based on a foamed and a 3D-printed scaffolds. The location of regions with high concentration of calcium in the numerical simulations is in fair agreement with regions of bone formation shown in experimental observations reported in the literature. Besides, reduced-order solutions accurately approximate the reference finite element solutions, with a significant decrease in the number of degrees of freedom, thus avoiding computationally expensive simulations, especially when performing a parametric analysis. The proposed reduced-order model is a competitive tool to assist the design of scaffolds in osteoinduction research., The authors acknowledge the financial support from the Ministerio de Ciencia e Innovación (MCIN/ AEI/10.13039/501100011033) through the grants PID2020-113463RBC32, PID2020-113463RB-C33, CEX2018-000797-S and PID2019-103892RB-I00, and the Generalitat de Catalunya for the Serra Hunter Fellowship of ME and the ICREA Academia Award of MPG., Peer Reviewed, Postprint (published version)

Although alginate is widely used as a matrix in the formulation of cell-laden inks, this polymer often requires laborious processing strategies due to its lack of cell adhesion moieties. The main objective of the present work was to explore the incorporation of microspheres into alginate-based bioinks as a simple and tuneable way to solve the cell adhesion problems, while adding extra biological functionality and improving their mechanical properties. To this end, three types of microspheres with different mineral contents (i.e. gelatine with 0% of hydroxyapatite, gelatine with 25 wt% of hydroxyapatite nanoparticles and 100 wt% of calcium -deficient hydroxyapatite) were synthesised and incorporated into the formulation of cell-laden inks. The results showed that the addition of microspheres generally improved the rheological properties of the ink, favoured cell proliferation and positively affected osteogenic cell differentiation. Furthermore, this differentiation was found to be influenced by the type of microsphere and the ability of the cells to migrate towards them, which was highly dependent on the stiffness of the bioink. In this regard, Ca2+ supplementation in the cell culture medium had a pronounced effect on the relaxation of the stiffness of these cell-loaded inks, influencing the overall cell performance. In conclusion, we have developed a powerful and tuneable strategy for the fabrication of alginate-based bioinks with enhanced biological characteristics by incorporating microspheres into the initial ink formulation., Peer Reviewed, Postprint (published version)

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

Incoming

Un dels materials més rellevants en les últimes dècades és la zircònia (ZrO2) degut a les seves excel·lents propietats mecàniques. Degut a les seves propietats, aquest material s’ha utilitzat àmpliament en aplicacions dentals i ortopèdiques. Avui en dia continua sent un material àmpliament estudiat, ja que es volen trobar noves maneres per millora l’osteointegració i prevenir els problemes relacionats amb la degradació hidrotèrmica. En aquest projecte final de grau (TFG), l’objectiu principal és aconseguir conformar un implant dental. S’ha intentat conformar amb tres materials base zircònia; 3Y-ZrO2, 12Ce-ZrO2 i un composite de 3Y/12Ce-ZrO2. Per tal d’aconseguir aquest objectiu s’han utilitzat dues tècniques, la impressió 3D, mitjançant la tècnica del Robocasting també coneguda com Direct Ink Writing (DIW), i el fresat. En el transcurs del TFG, s’ha hagut de caracteritzar la pols de partida, en termes de mida de gra i cristal·linitat, així com les propietats tant microestructurals com mecàniques mitjançant tècniques avançades de caracterització (com per exemple microscòpia electrònica de rastreig, difracció de rajos X, entre altres) dels diferents materials tant processats mitjançant tècniques de processat tradicional, Cold Isostatic Pressing (CIP) com mitjançant impressió 3D. Finalment, s’han investigat les propietats tant microestructurals com mecàniques en condicions de servei, per això les mostres han estat degradades hidrotèrmicament. La caracterització de la pols així com la seva cristal·linitat ha permès observar que els diferents materials d’estudi en aquest TFG poden ser impresos en 3D, i que les seves propietats mecàniques resultants no es veuen molt afectades en comparació amb les propietats obtingudes quan els materials han estat conformats mitjançant tècniques tradicionals. A més s’ha pogut comprovar com el material composite 85/15-ZrO2 no ha patit degradació hidrotèrmica en concordança amb el que hi ha reportat a la literatura. Pel que fa a la impressi, Uno de los materiales más relevantes en las últimas décadas es la circonia (ZrO2) debido a sus excelentes propiedades mecánicas. Debido a sus propiedades, este material se ha utilizado ampliamente en aplicaciones dentales y ortopédicas. Hoy en día continúa siendo un material ampliamente estudiado, puesto que se quiere encontrar nuevas maneras para mejorar la osteointegración y prevenir problemas relacionados con la degradación hidrotérmica. En este proyecto final de grado (TFG), el objetivo principal es conseguir conformar un implante dental. Se ha intentado conformar con tres materiales base circonia; 3Y-ZrO2, 12Ce-ZrO2 y un composite 3Y/12Ce-ZrO2. Para conseguir este objetivo se han utilizado dos técnicas, la impresión 3D, mediante la técnica del Robocasting también conocida como Direct Ink Writing (DIW), y el fresado. En el transcurso del TFG, se ha tenido que caracterizar el polvo de partida, en términos de tamaño de grano y cristalinidad, así como las propiedades tanto microestructurales como mecánicas mediante técnicas avanzadas de caracterización (como por ejemplo microscopia electrónica de barrido, difracción de rayos X, entre otros) de los diferentes materiales tanto procesados mediante técnicas de procesado tradicional, Cold Isostatic Pressing (CIP) como mediante impresión 3D. Finalmente, se han investigado las propiedades tanto microestructurales como mecánicas en condiciones de servicio, por eso las muestras han sido degradadas hidrotermalmente. La caracterización del polvo así como su cristalinidad ha permitido observar que los diferentes materiales estudiados en este TFG pueden ser imprimidos en 3D, y que sus propiedades mecánicas resultantes no se ven muy afectadas en comparación con las propiedades obtenidas cuando los materiales han sido conformados mediante técnicas tradicionales. Además, se ha podido comprobar como el material composite 85/15-ZrO2 no ha sufrido degradación hidrotérmica en concordancia con lo que hay reportado en la literatura. En, One of the most important materials in the last decades is the zirconia (ZrO2) due to its excellent mechanical properties. Due to its properties, this material has been widely used in dental and orthopaedic applications. Nowadays it remains under investigation, as new ways are to be found to improve the osseointegration and prevent the issues related to the hydrothermal degradation. In this Bachelor’s degree project, the main objective is to achieve the shaping of a dental implant. Three zirconia-based materials have been used; 3Y-ZrO2, 12Ce-ZrO2 and a composite of 3Y/12Ce-ZrO2. To achieve this objective, two techniques have been used, 3D printing, using the Robocasting technique also known as Direct Ink Writing (DIW), and milling. In the course of this Bachelor’s degree project, it has been necessary to characterise the starting powders, in terms of grain size and crystallinity, as well as the microstructural and mechanical properties by means of advanced characterization techniques (such as scanning electron microscopy, X-Ray Diffraction, among others) of the different materials both processed by traditional processing techniques, Cold Isostatic Pressing (CIP) and 3D printing. Finally, both microstructural and mechanical properties have been investigated under service conditions, so the samples have been hydrothermally degraded. The characterization of the powder as well as its crystallinity has allowed to observe that the different materials studied in this Bachelor’s degree project can be 3D printed, and that their resulting mechanical properties are not much affected in comparison with the properties obtained when the materials have been made by traditional techniques. Furthermore, it has been verified that the 85/15- ZrO2 composite materials has not suffered hydrothermal degradation as accordance in the literature. As for the 3D printing of implants, it has been possible to print the implant with different lengths, 24, 17 and 12 mm, which implies 600%, 400% an

In the present study, amorphous tricalcium phosphate (TCP) has been synthetized by a wet route to obtain low temperature a-TCP at 650 °C (LT-aTCP) and compare its structural, physical–chemical and thermal properties with those of a-TCP obtained by the conventional solid-state reaction method at 1400 °C (HT-aTCP). Even if no significant differences were observed concerning the values of lattice parameters measured by Rietveld refinement, LT-aTCP presented lower crystallinity and higher crystal strains than HT-aTCP. The reactivity in water of the a-TCP obtained by the two different routes was assessed. Both raw samples appeared relatively inert in solution and did not favour the nucleation of calcium deficient apatite (CDA); the LT-aTCP and HT-aTCP were converted into apatite only after milling. The mechanical process leads to a decrease in crystallinity and the formation of an amorphous phase, which is supported in this work by Raman spectroscopy. The faster rate of conversion of milled LT-aTCP compared to HT-aTCP can be assigned to its higher specific surface area, lower crystallinity and higher residual crystal strain; these favour the dissolution of the a-TCP phase. Finally, the setting properties of a-TCP-based bone cements were compared regarding their synthesis route. Although the synthesis route does not significantly affect the setting times, the kinetic of conversion into CDA was faster for LT-aTCP than for HT-aTCP. Thus, the modulation of the dissolution rate of a-TCP-based cement determined by the preparation route and the grinding process allows control of the overall setting reaction., Peer Reviewed, Postprint (author's final draft)

Porosity plays a key role on the osteogenic performance of bone scaffolds. Direct Ink Writing (DIW) allows the design of customized synthetic bone grafts with patient-specific architecture and controlled macroporosity. Being an extrusion-based technique, the scaffolds obtained are formed by arrays of cylindrical filaments, and therefore have convex surfaces. This may represent a serious limitation, as the role of surface curvature and more specifically the stimulating role of concave surfaces in osteoinduction and bone growth has been recently highlighted. Hence the need to design strategies that allow the introduction of concave pores in DIW scaffolds. In the current study, we propose to add gelatin microspheres as a sacrificial material in a self-setting calcium phosphate ink. Neither the phase transformation responsible for the hardening of the scaffold nor the formation of characteristic network of needle-like hydroxyapatite crystals was affected by the addition of gelatin microspheres. The partial dissolution of the gelatin resulted in the creation of spherical pores throughout the filaments and exposed on the surface, increasing filament porosity from 0.2 % to 67.9 %. Moreover, the presence of retained gelatin proved to have a significant effect on the mechanical properties, reducing the strength but simultaneously giving the scaffolds an elastic behavior, despite the high content of ceramic as a continuous phase. Notwithstanding the inherent difficulty of in vitro cultures with this highly reactive material an enhancement of MG-63 cell proliferation, as well as better spreading of hMSCs was recorded on the developed scaffolds., Peer Reviewed, Postprint (published version)

Biomimetic calcium-deficient hydroxyapatite (CDHA) as a bioactive material exhibits exceptional intrinsic osteoinductive and osteogenic properties because of its nanostructure and composition, which promote a favorable microenvironment. Its high reactivity has been hypothesized to play a relevant role in the in vivo performance, mediated by the interaction with the biological fluids, which is amplified by its high specific surface area. Paradoxically, this high reactivity is also behind the in vitro cytotoxicity of this material, especially pronounced in static conditions. The present work explores the structural and physicochemical changes that CDHA undergoes in contact with physiological fluids and to investigate its interaction with proteins. Calcium-deficient hydroxyapatite discs with different micro/nanostructures, coarse (C) and fine (F), were exposed to cell-free complete culture medium over extended periods of time: 1, 7, 14, 21, 28, and 50 days. Precipitate formation was not observed in any of the materials in contact with the physiological fluid, which would indicate that the ionic exchanges were linked to incorporation into the crystal structure of CDHA or in the hydrated layer. In fact, CDHA experienced a maturation process, with a progressive increase in crystallinity and the Ca/P ratio, accompanied by an uptake of Mg and a B-type carbonation process, with a gradual propagation into the core of the samples. However, the reactivity of biomimetic hydroxyapatite was highly dependent on the specific surface area and was amplified in nanosized needle-like crystal structures (F), whereas in coarse specimens the ionic exchanges were restricted to the surface, with low penetration in the material bulk. In addition to showing a higher protein adsorption on F substrates, the proteomics study revealed the existence of protein selectivity toward F or C microstructures, as well as the capability of CDHA, and more remarkably of F-CDHA, to concentrate specific protein, Peer Reviewed, Postprint (published version)

The combination of the direct ink writing (DIW) manufacturing technique, also named robocasting, with the use of self-setting calcium phosphate inks based on α-tricalcium phosphate opens new possibilities in the field of bone regeneration: i) On one hand, the DIW fabrication process allows a precise control on the external shape and internal porosity of the scaffold. The porosity allows the colonization of the bone tissue and the shape control opens new perspectives in personalised medicine; ii) On the other hand, the use of self-setting α-TCP inks provide a micro/nano porosity and a high specific surface area (SSA) to the bone graft. Both factors have been identified as crucial for the bioactivity of the material. Since the fabrication time is a crucial factor for the successful translation of these technologies to the clinical field, and the hardening reaction of conventional self-setting inks is slow, recent investigations have developedan alternative setting procedure (hydrothermal) that considerably reduces the hardening step from 7 days to 30 minutes. Regarding the role of scaffold architecture in bone regeneration, it has been recently proved that the presence of concave surfaces enhances osteogenesis. However, since DIW is based on the extrusion of a paste through a needle, conventional DIW scaffolds are composed of extruded filaments with convex surfaces. Hence the interest in developing scaffolds with non-cylindrical strands, which have concave surfaces. The aim of this study was to assess the in-vivo performance of calcium phosphate scaffolds, analysing on one side the effect of the setting treatment, i.e., comparing the biomimetic setting with the hydrothermal setting treatment, and on the other side comparing cylindrical vs.non-cylindrical strands. The characterization of the scaffolds obtained with the two different treatments revealed that whereas the biomimetic treatment resulted in calcium deficient hydroxiapatatite (CDHA), the hydrothermal treatmen, Incoming

La esperanza de vida del ser humano ha aumentado considerablemente a lo largo de los años y esto requiere del aumento en la durabilidad de los materiales implantables con el fin de sustituirlos el menor número de veces posibles a lo largo de la vida del individuo. Esto motiva una línea de investigación científica basada en el proceso de recubrir el titanio utilizado para prótesis de cadera e implantes dentales con hidroxiapatita, el material que forma la parte mineral del hueso. En esta línea se basa el siguiente TFG con el objetivo de modificar superficialmente una lámina de Ti6Al-7Nb con el fin de inducir rugosidad sobre la misma. Esta modificación superficial se lleva a cabo mediante dos técnicas diferentes, una técnica mecánica (desbaste) y otra térmica (ablación láser). Posteriormente, se recubre dicha superficie mediante la tecnología de deposición térmica por plasma atmosférico y posteriormente se caracteriza microestructuralmente y se ensaya el conjunto recubrimiento/sustrato con el fin de conocer qué sustrato proporciona una mayor resistencia a la adherencia. Este proceso permite llegar a la conclusión, de que es necesaria una rugosidad de alrededor de 60 µm para poder conseguir unos resultados aceptables., L'esperança de vida de l'ésser humà ha augmentat considerablement al llarg dels anys i això requereix de l'augment en la durabilitat dels materials implantables amb la finalitat de substituir-los el menor nombre de vegades possibles al llarg de la vida de l'individu. Això motiva una línia de recerca científica basada en el procés de recobrir el titani utilitzat per a pròtesi de maluc i implants dentals amb hidroxiapatita, el material que forma la part mineral de l'os. En aquesta línia es basa el següent TFG amb l'objectiu de modificar superficialment una làmina de Ti6Al-*7Nb amb la finalitat d'induir rugositat sobre aquesta. Aquesta modificació superficial es duu a terme mitjançant dues tècniques diferents, una tècnica mecànica (desbastament) i una altra tèrmica (ablació làser). Posteriorment, es recobreix aquesta superfície mitjançant la tecnologia de deposició tèrmica per plasma atmosfèric i posteriorment es caracteritza microestructuralment i s'assaja el conjunt recobriment/substrat amb la finalitat de conèixer quin substrat proporciona una major resistència a l'adherència. Aquest procés permet arribar a la conclusió, que és necessària una rugositat al voltant de 60 µm per a poder aconseguir uns resultats acceptables., Human life expectancy has increased considerably over the years and this requires an increase in the durability of implantable materials in order to replace them as few times as possible throughout the individual's life. This motivates a line of scientific research based on the process of coating the titanium used for hip prostheses and dental implants with hydroxyapatite, the material that forms the mineral part of bone. The following TFG is based on this line with the aim of superficially modifying a Ti-6Al-7Nb sheet in order to induce roughness on it. This surface modification is carried out using two different techniques, a mechanical technique (grinding) and a thermal technique (laser ablation). Subsequently, said surface is coated using atmospheric plasma thermal deposition technology and subsequently it is microstructurally characterized and the coating/substrate assembly is tested in order to know which substrate provides greater resistance to adhesion. This process allows us to reach the conclusion that a roughness of around 60 µm is necessary to achieve acceptable results.

Outgoing

Outgoing

Due to the increase of bone diseases caused by the ever-aging population, new biomaterials destined for bone regeneration need to be developed. New methods, such as robocasting are gaining interest as they allow printing of self-setting calcium phosphate (CaP) scaffolds. The control of the architecture by robocasting together with the control of composition using CaPhave resulted in the development of materials very similar to the structure and composition of natural bone. However, the mineral phase in bone is a calcium phosphate (hydroxyapatite) enriched with many ionic substitutions fundamental for bone growth. This project will focus on the incorporation of four different ions (Mg, Sr, Zn and Si) in hydroxyapatite scaffolds produced by robocasting. The incorporation of these ions is explored using two strategies, by incorporating the ion of interest during a post-printing treatment and by incorporation the ion of interest directly in the ink formulation. Different characterization techniques have been used to determine ion incorporation: X-ray powder diffraction, elemental analysis by energy-dispersive X-ray, Raman spectroscopy and electron diffraction by transmission electron microscopy (TEM). Among them, the most successful technique has been TEM. The results show that, in general, ion doping post printing is more successful than changing the ink formulation. In addition, ion concentration had a strong effect on scaffold purity and the degree of ion-doping.

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium-deficient hydroxyapatite (CDHA). The effect of heparin-functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (ß-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered ß-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and ß-TCP present OC at 21–28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption., Peer Reviewed, Postprint (author's final draft)

Tissue engineering is an emerging technology whose efforts are aimed towards repairing and regenerating injured tissues by developing bioactive scaffolds. Recently, scaffolds made of hydrogels have become interesting candidates for tissue engineering due to their tunable structures that allow to obtain constructs with a wide range of properties. However, incorporating cells into them and allowing cell proliferation and differentiation has been a challenge for tissue engineers in the past years. In this context, the aim of this project was to develop biocompatible bioinks capable of forming self-supportive 3D scaffold able to host cells. To achieve this challenge, different sets of bioinks were tested. Firstly, different general formulations based on alginate pluronic and gelatin were tested. In a second group of formulations, pluronic was removed to assess cell viability. The last group of formulations was based on a mixture of alginate, collagen and hydroxyapatite nanoparticles, focusing on a putative application in bone tissue. All formulations were characterized in terms of morphology, degradation and cell viability. In conclusion, gelatin-containing bioinks tended to be weaker than collagen-hydroxyapatite ones. However, there was no significant difference in cell viability studies between all of them

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation., Peer Reviewed, Postprint (author's final draft)

The geometry, arrangement and size of the topographic elements of the surface of a biomaterial has a very important impact on bacterial adhesion. This project aims to investigate the effect of nano and microtopography of calcium phosphate substrates on the adhesion of bacteria., Incoming

Inspired by nature, poly(acrylic acid) (PAA) and other polyelectrolytes have been used as noncollagenous proteins (NCPs) surrogates for biomimetic intrafibrillar mineralization of collagen fibrils and, thus, to model the ultrastructure of bone, to study the mechanism of bone mineralization, and more scarcely to fabricate scaffolds for hard tissue engineering. The objective of this study was to systematically investigate the effect of the molecular weight (MW) and the concentration of PAA on the rate and pattern of biomineralization of collagen matrices. Densified type I collagen films were mineralized in supersaturated PAA-stabilized amorphous calcium-phosphate (PAA-ACP) solutions containing increasing MW (2 kDa, 50 kDa, 450 kDa) and concentrations (10, 25, 50 mg/L) of PAA up to 7 days. The stability and physical properties of collagen-free PAA-ACP solutions were also investigated. In our system, lowering PAA MW and increasing PAA concentration resulted in solutions with increasing stability. Overstable PAA-ACP solutions that fully inhibited mineralization of the collagen matrices were achieved using PAA 2kDa-50mg/L. Conversely, unstable solutions were obtained using high PAA MW at low concentrations. Nucleation and growth of a significant amount of extrafibrillar minerals on the collagen fibrils was obtained using these solutions. In a wide range of combined MW and concentration of PAA, we obtained intrafibrillar mineralization of collagen with hydroxyapatite crystals aligned parallel to the collagen fibril as in natural tissues. Intrafibrillar mineralization was correlated with PAA-ACP stability and growth of the PAA-ACP particles in solution. Our results support using PAA to act as a surrogate to NCPs function as selective inhibitors or promoters of biological mineralization and provide parameters to manufacture new biomimetic scaffolds and constructs for bone and dentin tissue engineering., Peer Reviewed, Preprint

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12¿weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation., Peer Reviewed, Postprint (author's final draft)

Calcium phosphates have long been used as synthetic bone grafts. Recent studies have shown that the modulation of composition and textural properties, such as nano-, micro- and macro-porosity, is a powerful strategy to control and synchronize material resorption and bone formation. - Biomimetic calcium phosphates, which closely mimic the composition and structure of bone mineral, can be produced using low-temperature processing routes, and offer the possibility to modulate the material properties to a larger extent than conventional high temperature sintering processes. - Advanced technologies open up new possibilities in the design of bioceramics for bone regeneration; 3D-printing technologies, in combination with the development of hybrid materials with enhanced mechanical properties, supported by finite element modelling tools, are expected to enable the design and fabrication of mechanically competent patient-specific bone grafts. - The association of ions, drugs and cells allows leveraging of the osteogenic potential of bioceramic scaffolds in compromised clinical situations, where the intrinsic bone regeneration potential is impaired., Postprint (published version)

Biomaterials can interact with cells directly, that is, by direct contact of the cells with the material surface, or indirectly, through soluble species that can be released to or uptaken from the surrounding fluids. However, it is difficult to characterise the relevance of this fluid-mediated interaction separately from the topography and composition of the substrate, because they are coupled variables. These fluid-mediated interactions are amplified in the case of highly reactive calcium phosphates (CaPs) such as biomimetic calcium deficient hydroxyapatite (CDHA), particularly in static in vitro cultures. The present work proposes a strategy to decouple the effect of ion exchange from topographical features by adjusting the volume ratio between the cell culture medium and biomaterial (VCM/VB). Increasing this ratio allowed mitigating the drastic ionic exchanges associated to the compositional changes experienced by the material exposed to the cell culture medium. This strategy was validated using rat mesenchymal stem cells (rMSCs) cultured on CDHA and beta-tricalcium phosphate (ß-TCP) discs using different VCM/VB ratios. Whereas in the case of ß-TCP the cell response was not affected by this ratio, a significant effect on cell adhesion and proliferation was found for the more reactive CDHA. The ionic exchange, produced by CDHA at low VCM/VB, altered cell adhesion due to the reduced number of focal adhesions, caused cell shrinkage and further rMCSs apoptosis. This was mitigated when using a high VCM/VB, which attenuated the changes of calcium and phosphate concentrations in the cell culture medium, resulting in rMSCs spreading and a viability over time. Moreover, rMSCs showed an earlier expression of osteogenic genes on CDHA compared to sintered ß-TCP when extracellular calcium fluctuations were reduced. Statement of Significance Fluid mediated interactions play a significant role in the bioactivity of calcium phosphates. Ionic exchange is amplified in the case of, Peer Reviewed, Postprint (author's final draft)

"This is the pre-peer reviewed version of the following article: A. Diez-Escudero, M. Espanol, M. Bonany, X. Lu, C. Persson, M.-P. Ginebra, Adv. Healthcare Mater. 2018, 7, 1700867, which has been published in final form at https://doi.org/10.1002/adhm.201700867. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.", Immune cells play a vital role in regulating bone dynamics. This has boosted the interest in developing biomaterials that can modulate both the immune and skeletal systems. In this study, calcium phosphates discs (i.e., beta-tricalcium phosphate, ß-TCP) are functionalized with heparin to investigate the effects on immune and stem cell responses. The results show that the functionalized surfaces downregulate the release of hydrogen peroxide and proinflammatory cytokines (tumor necrosis factor alpha and interleukin 1 beta) from human monocytes and neutrophils, compared to nonfunctionalized discs. The macrophages show both elongated and round shapes on the two ceramic substrates, but the morphology of cells on heparinized ß-TCP tends toward a higher elongation after 72 h. The heparinized substrates support rat mesenchymal stem cell (MSC) adhesion and proliferation, and anticipate the differentiation toward the osteoblastic lineage as compared to ß-TCP and control. The coupling between the inflammatory response and osteogenesis is assessed by culturing MSCs with the macrophage supernatants. The downregulation of inflammation in contact with the heparinized substrates induces higher expression of bone-related markers by MSCs, Peer Reviewed, Postprint (author's final draft)

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and ß-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics., Peer Reviewed, Postprint (author's final draft)

The capacity of calcium phosphates to be replaced by bone is tightly linked to their resorbability. However, the relative importance of some textural parameters on their degradation behavior is still unclear. The present study aims to quantify the effect of composition, specific surface area (SSA), and porosity at various length scales (nano-, micro- and macroporosity) on the in vitro degradation of different calcium phosphates. Degradation studies were performed in an acidic medium to mimic the osteoclastic environment. Small degradations were found in samples with interconnected nano- and micropores with sizes below 3 µm although they were highly porous (35–65%), with maximum weight loss of 8 wt%. Biomimetic calcium deficient hydroxyapatite, with high SSA and low crystallinity, presented the highest degradation rates exceeding even the more soluble ß-TCP. A dependence of degradation on SSA was indisputable when porosity and pore sizes were increased. The introduction of additional macroporosity with pore interconnections above 20 µm significantly impacted degradation, more markedly in the substrates with high SSA (>15 m2/g), whereas in sintered substrates with low SSA (<1 m2/g) it resulted just in a linear increase of degradation. Up to 30 % of degradation was registered in biomimetic substrates, compared to 15 % in ß-TCP or 8 % in sintered hydroxyapatite. The incorporation of carbonate in calcium deficient hydroxyapatite did not increase its degradation rate. Overall, the study highlights the importance of textural properties, which can modulate or even outweigh the effect of other features such as the solubility of the compounds. Statement of Significance The physicochemical features of calcium phosphates are crucial to tune biological events like resorption during bone remodeling. Understanding in vitro resorption can help to predict the in vivo behavior. Besides chemical composition, other parameters such as porosity and specific surface area have a strong inf, Peer Reviewed, Postprint (author's final draft)

Although calcium phosphate cements (CPCs) are used for bone regeneration in a wide range of clinical applications, various physicochemical phenomena are known to hinder their potential use in minimally invasive surgery or in highly vascularized surgical sites, mainly because of their lack of injectability or their low washout resistance. The present work shows that the combination of CPCs with an inverse-thermoresponsive hydrogel is a good strategy for finely tuning the cohesive and rheological properties of CPCs to achieve clinical acceptable injectability to prevent phase separation during implantation and cohesion to avoid washout of the paste. The thermoresponsive CPC developed combines alpha-tricalcium phosphate with an aqueous solution of pluronic F127, which exhibits an inverse thermoresponsive behaviour, with a gelling transformation at around body temperature. These novel CPCs exhibited temperature-dependent properties. Addition of the polymer enhanced the injectability of the paste, even at a low liquid-to-powder ratio, and allowed the rheological properties of the cement to be tuned, with the injection force decreasing with the temperature of the paste. Moreover, the cohesion of the paste was also temperature-dependent and increased as the temperature of the host medium increased due to gelling induced in the paste. The thermoresponsive cement exhibited excellent cohesion and clinically acceptable setting times at 37 °C, irrespective of the initial temperature of the paste. The addition of pluronic F127 slightly delayed the setting reaction in the early stages but did not hinder the full transformation to calcium-deficient hydroxyapatite. Moreover, the frozen storage of premixed thermoresponsive cement pastes was explored, the main physicochemical properties of the cements being maintained upon thawing, even after 18 months of frozen storage. This avoids the need to mix the cement in the operating theatre and allows its use off-the-shelf. The reverse ther, Peer Reviewed, Postprint (author's final draft)

This study describes the effects of nitrilotriacetic acid (NTA) and ethylenediaminotetraacetic acid (EDTA) on the mineralization of calcium phosphate from bulk aqueous solution. Mineralization was performed between pH 6 and 9 and with NTA or EDTA concentrations of 0, 5, 10, and 15 mM. X-ray diffraction and infrared spectroscopy show that at low pH, mainly brushite precipitates and at higher pH, mostly hydroxyapatite forms. Both additives alter the morphology of the precipitates. Without additive, brushite precipitates as large plates. With NTA, the morphology changes to an unusual rod-like shape. With EDTA, the edges of the particles are rounded and disk-like particles form. Conductivity and pH measurements suggest that the final products form through several intermediate steps, Peer Reviewed, Postprint (published version)

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low- and high-temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize., Peer Reviewed, Postprint (author's final draft)

Premi Extraordinari de Doctorat, promoció 2016-2017. Àmbit d’Enginyeria Industrial, Bone is the most transplanted tissue after blood. As pointed out by the World Health Organization, musculoskeletal diseases can potentially rise as the fourth largest cause of disability within the next years. Unfortunately, despite the natural ability of bone to self-heal it cannot bridge large bone defects without the help of a material. Still today the gold standard to restore bone function remains the use of natural bone grafts. However, they have several limitations that need to be overcome to accommodate the high demands of a global ageing population. Calcium phosphate (CaP) bone grafts have been known since the 1970s and stand as excellent synthetic candidates due to their composition, similar to the mineral phase of bone which consists of approximately 70 wt% of hydroxyapatite (HA). CaPs, and in particular HA, possess outstanding intrinsic properties such as biocompatibility, bioactivity and the ability to support bone growth. However, HA is too stable and once implanted it hardly degrades. Ideal synthetic bone grafts should integrate in the bone remodelling cycle, balancing implant resorption with its progressive replacement by new bone. This can be achieved either by modulating the material¿s physicochemical properties, or by combining the substrate with biological molecules capable of adequately orchestrating the various cells involved in the bone healing process. The present thesis seeks to explore, on the one hand, the feasibility of modulating the physicochemical properties of CaPs towards improving its degradation behavior, and, on the other hand, to investigate the potential CaP functionalization with heparin as a strategy to improve their biological performance at the various stages of bone healing: during the initial phase of inflammation, and during the stages of bone resorption and bone growth. The first part of the present thesis deals with the in vitro degradation of CaPs in a solution mimicking the osteoclastic environment, focusing specifical, L'os és el teixit més trasplantats desprès de la sang. L'organització Mundial de la Salut ha posat de relleu l'increment de les malalties musculoesquelètiques, les quals esdevindran la quarta causa mundial de discapacitat en el següents anys. Malgrat la capacitat natural de l'ós per autoregenerar-se, els defectes ossis de grans dimensions necessiten l'ajuda de materials per restaurar-se completament. Actualment, l'ús d'empelts naturals és l'alternativa més emprada clínicament. Tot i això, els autoempelts comporten certes limitacions que requereixen ser adreçades per tal de fer front a les elevades demandes d'una població mundial amb un grau d'envelliment creixent. Els empelts basats en fosfats càlcics (CaPs) són coneguts des de la dècada del 1970 i són uns excel·lents candidats per a la regeneració òssia donada la seva composició, similar a la fase mineral de l'os, que consisteix en aproximadament un 70% d'hidroxiapatita (HA). Els CaPs, en particular l'HA, posseeixen unes propietats intrínseques excepcionals com ara la biocompatibilitat, bioactivitat o la capacitat de suportar el creixement de nou os. Malgrat la seva semblança amb l'os, l'HA és massa estable químicament, i un cop implantada es degrada molt lentament. L'empelt ossi sintètic idealment s'hauria d'integrar en el cicle de remodelació òssia, el que requereix d'un balanç entre la seva reabsorció i la progressiva substitució per os nou. Aquesta capacitat es pot modular mitjançant propietats inherents del material o per mitjà de la combinació de substrats amb molècules capaces d'orquestrar adequadament les respostes de les diverses cèl·lules implicades en la restauració o regeneració òssia. La present tesis cerca explorar, per una banda, la possibilitat de modular les propietats físico-químiques dels CaPs per tal de millorar la seva degradació, així com investigar el potencial de funcionalitzar els CaPs amb heparina, amb la finalitat de millorar les interaccions biològiques a les diferents etapes de la resta, Award-winning, Postprint (published version)

© Copyright 2017, Mary Ann Liebert, Inc.The fabrication of calcium phosphates using biomimetic routes, namely, precipitation processes at body temperature, results in distinct features compared to conventional sintered calcium phosphate ceramics, such as a high specific surface area (SSA) and micro-or nanometric crystal size. The aim of this article is to analyze the effects of these parameters on cell response, focusing on two bone cell types: rat mesenchymal stem cells (rMSCs) and human osteoblastic cells (SaOS-2). Biomimetic calcium-deficient hydroxyapatite (CDHA) was obtained by a low temperature setting reaction, and a-Tricalcium phosphate (a-TCP) and ß-Tricalcium phosphate were subsequently obtained by sintering CDHA either at 1400°C or 1100°C. Sintered stoichiometric hydroxyapatite (HA) was also prepared using ceramic routes. The materials were characterized in terms of SSA, skeletal density, porosity, and pore size distribution. SaOS-2 cells and rMSCs were seeded either directly on the surfaces of the materials or on glass coverslips subsequently placed on top of the materials to expose the cells to the CaP-induced ionic changes in the culture medium, while avoiding any topography-related effects. CDHA produced higher ionic fluctuations in both cell culture media than sintered ceramics, with a strong decrease of calcium and a release of phosphate. Indirect contact cell cultures revealed that both cell types were sensitive to these ionic modifications, resulting in a decrease in proliferation rate, more marked for CDHA, this effect being more pronounced for rMSCs. In direct contact cultures, good cell adhesion was found on all materials, but, while cells were able to proliferate on the sintered calcium phosphates, cell number was significantly reduced with time on biomimetic CDHA, which was associated to a higher percentage of apoptotic cells. Direct contact of the cells with biomimetic CDHA resulted also in a higher alkaline phosphatase activity for both cel, Peer Reviewed, Postprint (author's final draft)

Peer Reviewed, Postprint (author's final draft)

Peer Reviewed, Postprint (author's final draft)

The self-assembling properties of thermally-sensitive amphiphilic elastin-like multiblock recombinamers have been combined with the capacities of calcium phosphate binding of the SN(A)15 epitope inspired by the salivary protein statherin. In this regard, the interaction between calcium and phosphate ions was examined in the presence of two hybrid recombinamers. The first recombinamer comprised a simple amphiphilic diblock in which the SN(A)15 epitopes were combined, at the gene level, to the hydrophilic end. This recombinamer can self-assemble into nanoparticles that can control the transformation of amorphous calcium phosphate (ACP) into a fibre-like hydroxyapatite structure. In the other recombinamer, the SN(A)15 domains are distributed along the monomer chain, with the hydrophilic blocks being distributed amongst the hydrophobic ones. In this case, the resulting nanohybrid ACP/recombinamer organises into neuron-like structures. Thus, combining the amphiphilic elastin-like recombinamers to the SN(A)15 functionality is a powerful mean to tune the formation of different complex calcium phosphate nanostructures., Peer Reviewed, Postprint (author's final draft)

Although it is widely acknowledged that ionic substitutions on bulk hydroxyapatite substrates have a strong impact on their biological performance, little is known of their effect on nanoparticles (NPs) especially when used for gene transfection or drug delivery. The fact that NPs would be internalized poses many questions but also opens up many new possibilities. The objective of the present work is to synthesize and assess the effect of a series of hydroxyapatite-like (HA) NPs doped with various ions on cell behavior, i.e. carbonate, magnesium and co-addition. We synthesized NPs under similar conditions to allow comparison of results and different aspects in addition to assessing the effect of the doping ion(s) were investigated: (1) the effect of performing the cell culture study on citrate-dispersed NPs and on agglomerated NPs, (2) the effect of adding/excluding 10% of foetal bovine serum (FBS) in the cell culture media and (3) the type of cell, i.e. MG-63 versus rat mesenchymal stem cells (rMSCs). The results clearly demonstrated that Mg-doping had a major effect on MG-63 cells with high cytotoxicity but not to rMSCs. This was a very important finding because it proved that doping could be a tool to modify NP internalization. The results also suggest that NP surface charge had a large impact on MG-63 cells and prevents their internalization if it is too negative—this effect was less critical for rMSCs, Peer Reviewed, Postprint (author's final draft)

From a chemical and structural point of view, hydroxyapatite (HA) is a strong candidate in biomedical applications owing to its similarity to the inorganic components of bones and teeth. HA nanoparticles (NPs) as colloidal suspensions are becoming a popular tool in biomedical applications such as gene/drug delivery, bio-imaging etc. Although it is widely acknowledged that ionic substitutions on bulk HA substrates have a strong impact on their biological performance, little is known of their effect on NPs with potential use in gene transfection or drug delivery. In the third chapter carbonate (CO3) and magnesium (Mg) ions, which are the major substitutions in biological apatite, have been explored in the synthesis of ion-doped HA NPs under similar reaction conditions to allow comparison of results. CO3 and Mg ion were incorporated in the crystal lattice of HA and caused various changes mainly in the morphology and solubility of the different nanoparticles. In addition, the impact of ion doping on the interaction of HA NPs with cells was also evaluated under various cell culture conditions: 1) performing the cell culture study on citrate-dispersed NPs and on agglomerated NPs, 2) adding/excluding 10 % of foetal bovine serum (FBS) in the cell culture media and 3) using different types of cells, i.e. osteosarcoma MG-63 cells versus rat mesenchymal stem cells (rMSCs). The in vitro results indicated that Mg-doped HA NPs induced a profound impact on MG63 cells and, in the absence of citrate and FBS these nanoparticles were clearly cytotoxic. However, Mg-doped HA NPs did not alter cell viability in rMSCs under the same conditions. In the fourth chapter, Sr, Zn, Si and Fe(III) ions, which are minor ionic substitutions in biological apatite, were introduced to synthesize additional ion-doped HA NPs. Physicochemical characterization demonstrated that as-synthesized NPs were phase pure and doped ions had little influence on the morphology of NPs as in all cases they kept needle, Desde el punto de vista químico y estructural la hidroxiapatita (HA) se considera un gran candidato para aplicaciones biomédicas por su similitud con la fase mineral del hueso y los dientes. Aunque el efecto del dopaje se ha investigado con detalle en la fabricación de implantes viéndose que su presencia tiene un gran impacto en el comportamiento celular, poco se sabe de su efecto en nanopartículas para su uso en terapia génica y liberación de fármacos. En concreto, el tercer capítulo se centra en el dopaje de la apatita con iones carbonato (CO3) y magnesio (Mg) por ser éstas las sustituciones más importantes en la apatita biológica. Para ello todas las reacciones de síntesis se realizan bajo las mismas condiciones con la finalidad de poder comparar resultados. Los resultados muestran que ambas sustituciones acaban incorporando los iones dentro de la estructura del cristal causando diferentes impactos principalmente a nivel de morfología y solubilidad. Con respecto al impacto del dopaje en la caracterización celular, se llevaron a cabo diferentes ensayos: 1) utilizando suspensiones dispersadas con citrato o sin él, 2) en medio de cultivo con o sin 10 % v/v de suero fetal bovino y 3) utilizando dos tipos de células diferentes, células de osteosarcoma (MG63) y células de rata mesenquimales (rMSCs). Los resultados in vitro mostraron que las nanopartículas dopadas con Mg eran claramente citotóxicas a las células MG63 en ausencia de FBS. Sin embargo, las mismas NPs no alteraron la viabilidad celular en rMSCs bajo las mismas condiciones. El capítulo cuarto se centra en la síntesis y caracterización de NPs dopadas con iones Sr, Zn, Si y Fe(III), que representan sustituciones minoritarias en la apatita biológica. Su caracterización fisicoquímica mostró que todas las NPs eran puras con morfología acicular. Los estudios de citotoxicidad con células MG63 y rMSCs, con y sin FBS, mostraron viabilidad en presencia de FBS para todas las NPs. Además, para las NPs dopadas con Zn y F, Postprint (published version)

The success or failure of a material when implanted in the body is greatly determined by the surface properties of the material and the host tissue reactions. The very first event that takes place after implantation is the interaction of soluble ions, molecules and proteins from the biological environment with the material surface leading to the formation of an adsorbed protein layer that will later influence cell attachment. In this context, the particular topography and surface charge of a material become critical as they influence the nature of the proteins that will adsorb. However, very limited information is available on the surface charge of porous substrates. Only until very recently was the determination of the zeta potential on porous membranes accurately determined. The goal of this work was to implement the previous findings for the determination of the zeta potential of a series of porous hydroxyapatite (HA) substrates and to assess how porosity affects the measurements. In addition, studies using various electrolytes were also performed to prove how the specific affinity of certain ions for HA can further impact surface charge. The results showed that all materials exhibited very similar external surface charge (approximately -23 mV), consistent with their almost identical topographies. However, the presence of interconnected pores underneath the sample surface resulted in an additional internal zeta potential that varied with the porosity content. Measurements with different electrolytes confirmed the selectivity of divalent ions for HA underlying the importance of testing biomaterials using relevant electrolytes, Peer Reviewed, Postprint (author's final draft)

The proliferation and activation of leukocytes upon contact with a biomaterial play a crucial role in the degree of inflammatory response, which may then determine the clinical failure or success of an implanted biomaterial. The aim of this study was to evaluate whether nano-and microstructured biomimetic hydroxyapatite substrates can influence the growth and activation of macrophage-like cells. Hydroxyapatite substrates with different crystal morphologies consisting of an entangled network of plate-like and needle-like crystals were evaluated. Macrophage proliferation was evaluated on the material surface (direct contact) and also in extracts i.e. media modified by the material (indirect contact). Additionally, the effect of supplementing the extracts with calcium ions and/or proteins was investigated. Macrophage activation on the substrates was evaluated by quantifying the release of reactive oxygen species and by morphological observations. The results showed that differences in the substrate's microstructure play a major role in the activation of macrophages as there was a higher release of reactive oxygen species after culturing the macrophages on plate-like crystals substrates compared to the almost non-existent release on needle-like substrates. However, the difference in macrophage proliferation was ascribed to different ionic exchanges and protein adsorption/retention from the substrates rather than to the texture of materials., Peer Reviewed, Postprint (published version)

The use of hydroxyapatite (HA) based materials is widely spread in the repair of bone tissue and their great acceptance arises from the close similarity of HA to the mineral phase of bone. In the last years HA has been explored in a new area of application where it, in the form of nanoparticles (NPs), has been used as a carrier for the delivery of drugs and genes inside targeted cells. In spite of the promising results, which have been reported up to day, there are fundamental questions that remain unanswered. The goal of this project is to address one specific aspect: if the addition of doping ions in the formulation of hydroxyapatite NPs affects cell behaviour. For this purpose the first objective was to prepare HA NPs with various types and degree of ion substitution: carbonate (CO3)-, magnesium (Mg)- and CO3-Mg-co-substituted HA NPs. They were prepared via a wet precipitation route involving neutralization of calcium hydroxide with phosphoric acid and were characterised by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), inductively coupled plasma (ICP) elemental analysis, transmission electron microscopy (TEM) and nitrogen (N2) absorption. The next objective was to obtain stable aqueous suspensions of the NPs. This was achieved by the simultaneous use of a natural dispersant (sodium citrate) and with the help of a probe sonicator. The dispersibility was evaluated visually and with the help of a particle size analyser. The ultimate objective was to assess the cytotoxicity of the well dispersed and non-dispersed ion-substituted HA NPs in a cell culture. Two types of cells were used for this purpose: osteoblast-like cancer cells (MG63) and rat mesenchymal stem cells (rMSC). The results showed that although the incorporation of the doping ions (e.g. carbonate) changed substantially the morphology and specific surface area of some NPs, such alterations did not seem to have any effect on cell behaviour. Yet, the presence of Mg at percentages o