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  • Assessment of protein entrapment in hydroxyapatite scaffolds by size exclusion chromatography

     Español Pons, Montserrat; Casals, Isidre; Lamtahri, Sarah; Valderas, Maite; Ginebra Molins, Maria Pau
    Biointerphases: an open access journal for the biomaterials interface community
    Date of publication: 2012-12
    Journal article

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  • Calcium phosphate cements as drug delivery materials

     Ginebra Molins, Maria Pau; Canal Barnils, Cristina; Español Pons, Montserrat; Pastorino, David; Montufar Jimenez, Edgar Benjamin
    Advanced drug delivery reviews
    Date of publication: 2012-01-25
    Journal article

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    Calcium phosphate cements are used as synthetic bone grafts, with several advantages, such as their osteoconductivity and injectability. Moreover, their low-temperature setting reaction and intrinsic porosity allow for the incorporation of drugs and active principles in the material. It is the aim of the present work to: a) provide an overview of the different approaches taken in the application of calcium phosphate cements for drug delivery in the skeletal system, and b) identify the most significant achievements. The drugs or active principles associated to calcium phosphate cements are classified in three groups, i) low molecular weight drugs; ii) high molecular weight biomolecules; and iii) ions.

  • Ceramics

     Español Pons, Montserrat; Perez Antoñanzas, Roman; Montufar Jimenez, Edgar Benjamin; Ginebra Molins, Maria Pau
    Date of publication: 2012-04-10
    Book chapter

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  • Novel phosphate-based cements for clinical applications  Open access

     Mestres Bea, Gemma
    Defense's date: 2012-07-09
    Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya
    Theses

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    This Thesis aims at the development of two novel families of inorganic phosphate cements with suitable characteristics for clinical applications in hard tissue regeneration or replacement. It is organized in two distinct parts. The first part focuses at the development of silicon-doped a-tricalcium phosphate and the subsequent preparation of a silicon-doped calcium phosphate cement for bone regeneration applications. For this purpose, silicon-doped a-tricalcium phosphate was synthesized by sintering a calcium-deficient hydroxyapatite at 1250ºC with different amounts of silicon oxide. The high temperature polymorph a-tricalcium phosphate was stabilized by the presence of silicon, which inhibited reversion of the b-a transformation, whereas in the Si-free a-tricalcium phosphate completely reverted to the b-polymorph. It was observed that the presence of Si did not alter the b-a transformation temperature. Both the Si-doped a-tricalcium phosphate and its Si-free counterpart were used as reactants in the formulation of calcium phosphate cements. While Si-doped a-tricalcium phosphate showed faster hydrolysis to calcium deficient hydroxyapatite, the composition, morphology and mechanical properties of both cements were similar upon completion of the reaction. When the samples were immersed in simulated body fluid, the Si-doped cement exhibited a faster deposition of an apatite layer on its surface than its Si-free counterpart, suggesting an enhanced bioactivity of the doped-cement. An in vitro cell culture study, in which osteoblast-like cells were exposed to a medium modified by the materials, showed a delay in cell proliferation and a stimulation of cell differentiation, the differentiation being more marked for the Si-containing cement. These results were attributed to the Ca depletion from the medium by both cements and to the continuous Si release detected for the Si-containing cement. The second part of this Thesis is focused on the development of a new family of inorganic phosphate-based cements for biomedical applications, namely magnesium phosphate cements. The magnesium phosphate cements have been extensively used in civil engineering due to their fast setting, early strength acquisition and adhesive properties, properties that can be also of use for biomedical applications. However, there are some aspects that should be improved before they can be used in the human body, namely their high exothermic setting reaction and the release of potentially harmful ammonium ions. Therefore, a new family of magnesium phosphate cements was explored as candidate biomaterials for hard tissue applications. These cements were prepared by mixing magnesium oxide with either sodium dihydrogen phosphate, ammonium dihydrogen phosphate or an equimolar mixture of both. The exothermia and the setting kinetics of the new cement formulations were tailored. The ammonium-containing magnesium phosphate cements resulted in struvite as the major reaction product, whereas the magnesium phosphate cement prepared with sodium dihydrogen phosphate resulted in an amorphous product. The magnesium phosphate cements studied showed an early compressive strength substantially higher than that of conventional apatitic calcium phosphate cements. Moreover, they showed antimicrobial properties against bacteria present in dental infections, which were attributed to the synergistic effect of a high osmolarity and high pH of the cement extracts. These properties make magnesium phosphate cements good candidates for endodontic applications. It is with this latter point in mind that some of the most relevant physico-chemical properties were further optimized and characterized. Particularly, their radiopacity was enhanced by the addition of bismuth oxide. The sealing efficiency of the magnesium phosphate cements and their adhesion to dentin were shown to be comparable or even higher than those presented by other inorganic cements used for endodontic treatments.

    Aquesta Tesi té com a objectiu el desenvolupament de dues noves famílies de ciments inorgànics de base fosfat amb propietats adequades per a aplicacions clíniques en regeneració o substitució de teixits durs. La Tesi està organitzada en dues parts. La primera part està centrada en el desenvolupament de fosfat tricàlcic a dopat amb silici i la subseqüent preparació de ciments de fosfat de calci dopats amb silici. Per a aquest objectiu, es va obtenir fosfat tricàlcic a dopat amb silici mitjançant la sinterització d’una hidroxiapatita deficient en calci amb diferents quantitats d’òxid de silici a 1250°C. La presència de silici va estabilitzar el polimorf d’alta temperatura (fosfat tricàlcic a), inhibint-se la reversió de la transformació b-a, mentre que el fosfat tricàlcic a sense silici va revertir completament a polimorf b. La presència de silici no va alterar la temperatura de la transformació b-a. Tant el fosfat tricàlcic a dopat amb silici com el seu homòleg sense silici van ser utilitzats com a reactius en la formulació de ciments de fosfat de calci. Si bé el fosfat tricàlcic a dopat amb silici va mostrar en les fases inicials una hidròlisi més ràpida a hidroxiapatita deficient en calci, un cop completada la reacció, la composició, morfologia i propietats mecàniques d’ambdós ciments van ser similars. L’estudi de bioactivitat mitjançant la immersió de les mostres en fluid corporal simulat va donar com a resultat la formació d’una capa d’apatita a la superfície del ciment dopat amb silici, més ràpida que al seu homòleg sense silici, fet que va suggerir una bioactivitat millorada del ciment dopat. L’estudi in vitro, en el qual cèl·lules osteoblàstiques es van exposar a un medi de cultiu que havia estat prèviament en contacte amb els ciments estudiats, va mostrar un retràs en la proliferació cel·lular i un estímul de la diferenciació cel·lular, aquest últim més marcat pel ciment que contenia silici. Aquests resultats es van atribuir a la reducció de calci en els medis en els quals estaven introduïts els ciments i a l’alliberament continu d’ions silici per part del ciment que en contenia.

  • Hydroxyapatite foams with tuned macro- and microstructure

     Español Pons, Montserrat; Valderas, Maite; Ginebra Molins, Maria Pau
    Symposium and Annual Meeting of International Society for Ceramics in Medicine
    Presentation's date: 2011-11-10
    Presentation of work at congresses

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  • Sensors for in vitro Bone Tissue Engineering Applications  Open access

     Gustavsson, Johan Mikael
    Defense's date: 2011-07-27
    Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya
    Theses

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    This doctoral thesis explores how ion sensors can provide spatial and temporal control of specific cellular and biomaterial activity related to bone tissue engineering applications. First it was investigated the influence of different osteoblast-like cell models on the ionic extracellular environment (IEE) in vitro. Rat-derived mesenchymal stem cells (rMSCs) and SAOS-2 cells were observed to express high alkaline phosphatase (ALP) activity, and as a consequence they increased the concentration of inorganic phosphorus (Pi) in culture medium containing B-glycerophosphate. On the contrary, MG63 cells showed low ALP activity and did not influence [Pi]. Moreover, cell-induced calcium deposition in the extracellular matrix was observed both in mature SAOS-2 and rMSC layers but not in MG63 layers, and coincided with decreased [Ca2+] of the cell culture medium. Fluctuations in the IEE with respect to Pi and Ca2+ may therefore be indicative of specific osteoblast activity. Second, the ion reactivity of calcium-deficient hydroxyapatite (CDHA), a scaffold candidate material for bone TE, was systematically investigated in vitro by exposing it during different time periods to culture media of varying chemical composition. Traditional sorption models described experimental data well, revealing both significant sorption of Ca2+ onto CDHA and acidification of all culture media. Interestingly, different chemical composition of culture medium provoked opposite ion reactivity of CDHA with respect to Pi. Consequently, cellular sensitivity to dynamic IEEs may cause different cellular response using different culture media. Third, the effects of the dynamic IEE induced by CDHA on cellular behaviour were evaluated by growing SAOS-2 cells in semi-permeable inserts and in close proximity to CDHA. Cells proliferated well and their ALP-activity was modified mainly in time rather than in absolute levels. While cellular ALP-activity created conditions for Ca2+-deposition in the extracellular matrix in absence of CDHA, presence of CDHA caused competition between cells and material for Ca2+ and Pi which initially impeded cell-induced Ca2+-deposition. However, as sorption of Pi onto CDHA gradually decreased with time, conditions for bone mineralisation were created also in presence of CDHA. Obtained results indicate that sorption of Pi rather than sorption of Ca2+ was the main limiter for bone mineralisation in presence of CDHA. Fluctuations in the IEE of bone TE applications awoke interest in developing a generic setup for potentiometric ion and pH measurements online. Traditional Ca2+-selective electrodes for measurements in small volumes (easily down to 0.1 mL) were fabricated. They exhibited a Nernstian response to Ca2+, and were little influenced by other major extracellular ions. Moreover, the electrodes resisted sterilisation through UV radiation, did not induce any cytotoxic effects in contact with osteoblasts, and the electrode potential was subject only to minor drift during longer measurements in cell culture medium. The electrodes were used to successfully monitor sorption of Ca2+ onto CDHA when immersed in culture medium, as well as osteoblast-induced Ca2+-deposition in mature extracellular matrix during time frames of 24 hours. Also, all-solid-state potentiometric microelectrodes based on iridium oxide were prepared for real-time monitoring of pH in traditionally inaccessible bone TE environments. Specifically, pH was measured inside curing bone cement (a-tri calcium phosphate) as well as at its immediate interface with extracellular fluid. In both cases the developed pH microelectrodes indicated how the material initially provoked an alkaline environment, which gradually acidified with time. Absolute pH variations caused by the material were of such magnitude that they should be considered upon drug loadings and/or implantation.

    Esta tesis doctoral pretende explorar cómo los sensores iónicos permiten controlar a nivel espacial y temporal la actividad específica de células y biomateriales dentro del contexto de la bioingeniería del tejido óseo. En primer lugar, se investigó la influencia de diferentes modelos celulares de osteoblastos (osteoblast-like cells) cultivados in vitro en el ambiente iónico extracelular (ionic extracellular environment = IEE). Observamos que las células mesenquimales de rata (rMSCs = Rat-derived mesenchymal stem cells) y las células de tipo SAOS-2 expresan altos niveles de actividad fosfatasa alcalina (ALP = alkaline phosphatase), y en consecuencia, causan un incremento de la concentración de fósforo inorgánico (Pi) por hidrólisis del -glicerofosfato presente en el medio de cultivo. Por el contrario, las células MG63 mostraron baja actividad ALP y no modificaron la [Pi]. Es más, la deposición de calcio en la matriz extracelular inducida por las células se observó tanto en los cultivos maduros de células SAOS-2 y rMSC, pero no en las de tipo MG63, coincidiendo con una menor [Ca2+] en el medio de cultivo. Por lo tanto, las fluctuaciones de los iones Pi y Ca2+ en el IEE pueden ser posibles indicadores de la actividad mineralizadora de los osteoblastos. En segundo lugar, la reactividad iónica de la hidroxiapatita deficiente en calcio (CDHA = calcium-deficient hydroxyapatite) - un material candidato a ser utilizado como matriz en ingeniería del tejido óseo – fue investigada sistemáticamente in vitro exponiéndola durante diferentes periodos de tiempo a medios de cultivo con distinta composición química. Los modelos de sorción clásicos describen correctamente los datos experimentales, revelando procesos paralelos de sorción del ión Ca2+ en la CDHA y acidificación del medio. Cabe destacar que la distinta composición química del medio de cultivo puede provocar una reactividad iónica totalmente opuesta del CDHA respecto al anión Pi. En consecuencia, la sensibilidad celular a un entorno iónico dinámico puede dar lugar a respuestas celulares distintas al usar diferentes medios de cultivo. En tercer lugar, los efectos en el comportamiento celular provocados por la actividad de la CDHA en el entorno iónico dinámico se evaluaron realizando un cultivo de células SAOS-2 en un inserto semi-permeable colocado cercano pero separado de CDHA. Las células proliferaron bien y su actividad ALP sólo se alteró en el tiempo, pero no en términos absolutos. Mientras que, en ausencia de CDHA, la actividad ALP crea las condiciones para la deposición del ión Ca2+ en la matriz extracelular, presencia de CDHA causa competición entre las células y el biomaterial por los iones Ca2+ y Pi , que inicialmente bloquean la deposición celular de Ca2+. Sin embargo, a medida que la sorción del anión Pi disminuye con el tiempo, aparecen condiciones favorables para la mineralización incluso en presencia de CDHA. Los resultados obtenidos indican que la sorción de Pi más que la de Ca2+ es el principal factor limitante para la mineralización del hueso en presencia de CDHA. Las fluctuaciones del IEE en la bioingeniería del hueso han promovido un enorme interés en desarrollar un sistema genérico que permita monitorizar las medidas potenciométricas tanto de iones como del pH online. Fabricamos electrodos Ca2+-selectivos para medidas en volúmenes pequeños (hasta 0.1 mL). Dichos eléctrodos exhiben una respuesta Nernstiana al Ca2+, y son poco afectados por la presencia de los principales iones extracelulares. Asimismo, resisten la esterilización con UV, no presentan efectos citotóxicos en contacto con osteoblastos, y los potenciales presentan un “drift” despreciable en/durante medidas de gran duración en el medio de cultivo. Los electrodos se utilizaron con éxito para monitorizar la sorción de Ca2+ en CDHA dentro del medio de cultivo, así como la deposición de Ca2+ inducida por osteoblastos en la MEC madura, en intervalos de tiempo de hasta 24 horas.

  • Preparación de una propuesta para la convocatoria NMP-2010-LARGE-4 del FP7

     Rodriguez Rius, Daniel; Canal Barnils, Cristina; Español Pons, Montserrat; Manero Planella, Jose M.; Gil Mur, Francisco Javier; Pegueroles Neyra, Marta; Ginebra Molins, Maria Pau
    Participation in a competitive project

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  • NOVEL BIOMIMETIC STRATEGY FOR BONE REGENERATION

     Canal Barnils, Cristina; Gil Mur, Francisco Javier; Manero Planella, Jose M.; Español Pons, Montserrat; Ginebra Molins, Maria Pau
    Participation in a competitive project

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  • New processing approaches in calcium phosphate cements and their applications in regenerative medicine

     Ginebra Molins, Maria Pau; Español Pons, Montserrat; Montufar Jimenez, Edgar Benjamin; Mestres, Gemma; Perez Antoñanzas, Roman
    International Congress on Biomaterials
    Presentation's date: 2010-03-17
    Presentation of work at congresses

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  • New processing approaches in calcium phosphate cements and their applications in regenerative medicine

     Ginebra Molins, Maria Pau; Español Pons, Montserrat; Montufar Jimenez, Edgar Benjamin; Pérez, Ricardo A.; Mestres Bea, Gemma
    Acta biomaterialia
    Date of publication: 2010-08
    Journal article

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    The key feature of calcium phosphate cements (CPCs) lies in the setting reaction triggered by mixing one or more solid calcium phosphate salts with an aqueous solution. Upon mixture, the reaction takes place through a dissolution–precipitation process which is macroscopically observed by a gradual hardening of the cement paste. The precipitation of hydroxyapatite nanocrystals at body or room temperature, and the fact that those materials can be used as self-setting pastes, have for many years been the most attractive features of CPCs. However, the need to develop materials able to sustain bone tissue ingrowth and be capable of delivering drugs and bioactive molecules, together with the continuous requirement from surgeons to develop more easily handling cements, has pushed the development of new processing routes that can accommodate all these requirements, taking advantage of the possibility of manipulating the self-setting CPC paste. It is the goal of this paper to provide a brief overview of the new processing developments in the area of CPCs and to identify the most significant achievements.

  • Investigation of the hydroxyapatite obtained as hydrolysis product of alpha-tricalcium phosphate by transmission electron microscopy

     Español Pons, Montserrat; Portillo, Joaquim; Manero Planella, Jose M.; Ginebra Molins, Maria Pau
    Crystengcomm
    Date of publication: 2010
    Journal article

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  • Espumas inyectables de hidroxiapatita obtenidas por el método de espumado de la fase líquida de un cemento de fostato tricálcico alfa  Open access  awarded activity

     Montufar Jimenez, Edgar Benjamin
    Defense's date: 2010-07-26
    Department of Automatic Control, Universitat Politècnica de Catalunya
    Theses

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    Actualmente existe un gran número de lesiones y enfermedades que afectan el tejido óseo. El origen de estos problemas es diverso y su tratamiento también es diferente en cada caso. Uno de los principales problemas es la pérdida de masa ósea, que puede ser causada por la reabsorción de hueso, fracturas o extirpación de tumores; en estos casos el tratamiento requiere de un material de relleno óseo. Se espera que este material de relleno sea capaz de promover la regeneración de hueso; los materiales que cumplen con este requisito se les denomina injertos óseos. Otras aplicaciones de los injertos óseos son el aumento de hueso para corregir deformaciones o para fijar implantes dentales u ortopédicos. Se estima que en Europa los sustitutos óseos se utilizan en un millón de cirugías anualmente. El mercado mundial de estos materiales se estima en cinco billones de Euros con una tasa de crecimiento anual de 8-15 %.La hidroxiapatita, que corresponde a la fase mineral de hueso y dientes, es uno de los mejores materiales utilizados en el desarrollo de injertos óseos sintéticos. Las bioactividad y osteoconductividad de la hidroxiapatita son la razón de su éxito. Sin embargo, la hidroxiapatita es relativamente estable, por ello, los bloques sólidos no son reabsorbidos a largo plazo. Para incrementar la reabsorción de la hidroxiapatita, y permitir la formación de nuevo hueso hacia su interior, se incorporan macroporos abiertos en el material, mejorando también la osteointegración del implante. Un método para conseguir lo anterior es el espumado de los cementos de fosfatos de calcio; este enfoque tiene la ventaja de que la hidroxiapatita obtenida después del fraguado del cemento es más similar a la hidroxiapatita biológica que las hidroxiapatitas sinterizadas. Además, debido a que los cementos son inyectables cuando son una pasta, es posible obtener injertos óseos inyectables, macroporosos y autoconsolidables. El objetivo de la presente tesis es desarrollar espumas sólidas de hidroxiapatita a través del método de espumado de la fase líquida de un cemento de fosfato de calcio. Se espera que las espumas obtenidas puedan ser utilizadas como injertos óseos inyectables y autoconsolidables bajo condiciones fisiológicas. A lo largo de la tesis se estudian dos tipos de agentes espumantes. En primer lugar, el surfactante no iónico Tween 80 se estudia como agente espumante sintético, en segundo lugar, la gelatina, el alginato de sodio y el extracto de soja se estudian como agentes espumantes naturales. Estos polímeros se proponen como agentes espumantes con la intención de obtener espumas compuestas que imiten la composición y la estructura del nanocompuesto natural que es el tejido óseo.Los resultados mostraron que es posible obtener espumas inyectables de hidroxiapatita empleando contenidos de Tween 80 por debajo de la dosis máxima recomendada en administración parenteral. También se comprobó que la gelatina y el extracto de soja permiten obtener espumas inyectables y autofraguables in situ. Por el contrario, el alginato de sodio no mostró ser un buen agente espumante. Al comparar los dos tipos de agentes espumantes estudiados (sintético y natural) se puede concluir que el espumante sintético Tween 80 es más eficiente que los espumantes naturales; no obstante, la gelatina y el extracto de soja mejoran la Inyectabilidad y/o la cohesión de las espumas, ambas propiedades indispensables para utilizar estos materiales como injertos óseos inyectables y autoconsolidables.Los estudios in vitro e in vivo mostraron que las espumas obtenidas de hidroxiapatita no son toxicas, que pueden ser preparadas bajo condiciones estériles dentro del quirófano sin equipos especiales, y que las espumas seleccionadas tienen cohesión para endurecer in situ bajo condiciones fisiológicas, adaptando la forma y rellenando perfectamente el defecto cuando son implantadas por inyección.

    Nowadays, there are great number of injuries and illnesses that affect bone tissue. The origin of these diseases is diverse and their treatment is also different in each case. A great problem is bone loss, which can be caused by bone resorption, fractures or bone tumor extirpation; in these cases, a bone filler material will be required. It is expected that the filling material should be able to promote bone healing; materials that meet this properties are known as bone grafts. Other applications of bone grafting materials are the bone mass augmentation to correct deformations or to fix dental and orthopedic implants. It is estimated that in Europe bone substitutes are used in one million surgical procedures annually. The worldwide market of these materials is an estimated of five billion Euros with annual growth rate of 8-15%.One of the most successful materials used in the development of synthetic bone grafting materials is hydroxyapatite, which correspond to the mineral phase of bone and teeth. The bioactivity and the osteoconductivity properties of hydroxyapatite are the reasons of its success. Nevertheless, hydroxyapatite is relatively stable; therefore, solid blocks are not resorbed in the long term. In order to increase the hydroxyapatite resorption, and allow new bone ingrowth, open macropores are incorporated to the material, improving also implant osteointegration. One method to do this is by foaming calcium phosphate cements; this approach has the advantage that the resulting hydroxyapatite after cement setting is more similar to the biological apatite than sintered hydroxyapatite. In addition, since bone cements are injectable when they are a paste, it is possible to develop an injectable, macroporous, and self-setting bone graft.The objective of this thesis was to develop solid hydroxyapatite foams through the liquid phase foaming of a calcium phosphate bone cement. It is expected that the developed foams can be applied as injectable, self-setting, synthetic bone grafts under physiological conditions. Along with this thesis, two types of foaming agent were studied. In first place, non ionic surfactant Tween 80 was tested as syntactic foaming agent; and second, gelatin, sodium alginate and soy extract were tested as natural foaming agents. These biopolymers are proposed as foaming agents with the objective of obtaining composite foams that mimic the composition and structure of the natural nanocomposite that is bone tissue.The results showed that is possible to obtain injectable hydroxyapatite foams using Tween 80 amounts below the maximum dosage recommended for parenteral administration. Also, it was validated that gelatin and soy extract allow obtaining injectable, in situ self-setting foams. In contrast, sodium alginate did not show a good foaming capacity. Comparing the two types of studied foaming agents (synthetic and natural) it can be concluded that the synthetic foaming agent Tween 80 is more efficient than the natural foaming agents; however, gelatin and soy extract improved the injectability and/or the cohesion of the foams, both essential properties to use these materials as injectable and self-setting grafts.The in vitro and in vivo studies showed that the hydroxyapatite foams obtained were not toxic, they could be prepared under sterile conditions in the operating room without special staff, and the selected foams maintained their cohesion to set in situ under physiological conditions, adapting and filling perfectly the shape of the defect when implanted through injection.

  • Máquina de preparación de muestras para Histologías de Tejidos con Biomateriales Implantados

     Pegueroles Neyra, Marta; Canal Barnils, Cristina; Rodriguez Rius, Daniel; Español Pons, Montserrat; Manero Planella, Jose M.; Gil Mur, Francisco Javier; Montufar Jimenez, Edgar Benjamin; Ginebra Molins, Maria Pau
    Participation in a competitive project

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  • Preparación de propuestas para la convocatoria HEALTH 2009 del FP7

     Rodriguez Rius, Daniel; Gil Mur, Francisco Javier; Salvagni, Emiliano; Español Pons, Montserrat; Ginebra Molins, Maria Pau
    Participation in a competitive project

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  • REGENERATING BONE DEFECTS USING NEW BIOMEDICAL ENGINEERING APPROACHES

     Manero Planella, Jose M.; Salvagni, Emiliano; Español Pons, Montserrat; Canal Barnils, Cristina; Ginebra Molins, Maria Pau
    Participation in a competitive project

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  • Intrinsic porosity of calcium phosphate cements and its significance for drug delivery and tissue engineering applications

     Español Pons, Montserrat; Pérez, Ricardo A.; Montufar Jimenez, Edgar Benjamin; Marichal, C.; Sacco, A.; Ginebra Molins, Maria Pau
    Acta biomaterialia
    Date of publication: 2009-09
    Journal article

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  • Occurrence of Pasteuria spp. in the northeastern Spain.

     Verdejo Lucas, Soledad; Español Pons, Montserrat; Ornat Longarón, César; Sorribas Royo, Francisco Javier
    Nematologia Mediterranea
    Date of publication: 1997
    Journal article

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