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  • Enhanced cell-material interactions through the biofunctionalization of polymeric surfaces with engineered peptides

     Punet, Xavier; Mauchauffé, Rodolphe; Giannotti, Marina I.; Rodriguez Cabello, Jose Carlos; Sanz Carrasco, Fausto; Engel Lopez, Elisabet; Mateos Timoneda, Miguel Angel; Planell Estany, Josep Antón
    Biomacromolecules
    Date of publication: 2013-08
    Journal article

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    Research on surface modification of polymeric materials to guide the cellular activity in biomaterials designed for tissue engineering applications has mostly focused on the use of natural extracellular matrix (ECM) proteins and short peptides, such as RGD. However, the use of engineered proteins can gather the advantages of these strategies and avoid the main drawbacks. In this study, recombinant engineered proteins called elastin-like recombinamers (ELRs) have been used to functionalize poly(lactic) acid (PLA) model surfaces. The structure of the ELRs has been designed to include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a nonfouling canvas able to restrict unspecific adsorption of proteins. Moreover, AFM analysis reveals the conformation and disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and compared with the use of short peptides. Cell response has been assessed for different functionalization conditions in the presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface-cell interaction by adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a more efficient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces.

    Research on surface modi fi cation of polymeric materials to guide the cellular activity in biomaterials designed for tissue engineering applications has mostly focused on the use of natural extracellular matrix (ECM) proteins and short peptides, such as RGD. However, the use of engineered proteins can gather the advantages of these strategies and avoid the main drawbacks. In this study, recombinant engineered proteins called elastin- like recombinamers (ELRs) have been used to functionalize poly(lactic) acid (PLA) model surfaces. The structure of the ELRs has been designed to include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a nonfouling canvas able to restrict unspeci fi c adsorption of proteins. Moreover, AFM analysis reveals the conformation and disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and compared with the use of short peptides. Cell response has been assessed for di ff erent functionalization conditions in the presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface − cell interaction by adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a more e ffi cient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces

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    Hierarchically engineered fibrous scaffolds for bone regeneration  Open access

     Sachot, Nadège; Castaño Linares, Oscar; Mateos Timoneda, Miguel Angel; Engel Lopez, Elisabet; Planell Estany, Josep Antón
    Journal of the Royal Society Interface
    Date of publication: 2013-08-28
    Journal article

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    Surface properties of biomaterials play a major role in the governing of cell functionalities. It is well known that mechanical, chemical and nanotopographic cues, for example, influence cell proliferation and differentiation. Here, we present a novel coating protocol to produce hierarchically engineered fibrous scaffolds with tailorable surface characteristics, which mimic bone extracellular matrix. Based on the sol-gel method and a succession of surface treatments, hollow electrospun polylactic acid fibres were coated with a silicon-calcium-phosphate bioactive organic-inorganic glass. Compared with pure polymeric fibres that showed a completely smooth surface, the coated fibres exhibited a nanostructured topography and greater roughness. They also showed improved hydrophilic properties and a Young's modulus sixfold higher than non-coated ones, while remaining fully flexible and easy to handle. Rat mesenchymal stem cells cultured on these fibres showed great cellular spreading and interactions with the material. This protocol can be transferred to other structures and glasses, allowing the fabrication of various materials with well-defined features. This novel approach represents therefore a valuable improvement in the production of artificial matrices able to direct stem cell fate through physical and chemical interactions. © 2013 The Author(s).

    Surface properties of biomaterials play a major role in the governing of cell functionalities. It is well known that mechanical, chemical and nanotopo- graphic cues, for example, influence cell proliferation and differentiation. Here, we present a novel coating protocol to produce hierarchicallyengineered fibrous scaffolds with tailorable surface characteristics, which mimic bone extracellular matrix. Based on the sol–gel method and a succession of surface treatments, hollow electrospun polylactic acid fibres were coated with a silicon–calcium–phosphate bioactive organic–inorganic glass. Compared with pure polymeric fibres that showed a completely smooth surface, the coated fibres exhibited a nanostructured topography and greater roughness. They also showed improved hydrophilic properties and a Young’s modulus sixfold higher than non-coated ones, while remaining fully flexible and easy to handle. Rat mesenchymal stem cells cultured on these fibres showed great cellular spreading and interactions with the material. This protocol can be transferred to other structures and glasses, allowing the fabrication of var- ious materials with well-defined features. This novel approach represents therefore a valuable improvement in the production of artificial matrices able to direct stem cell fate through physical and chemical interactions

  • Ion-selective electrodes to monitor osteoblast-like cellular influence on the extracellular concentration of calcium

     Gustavsson, Johan; Planell Estany, Josep Antón; Engel Lopez, Elisabet
    Journal of tissue engineering and regenerative medicine
    Date of publication: 2013-04-27
    Journal article

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  • High-resolution PLA-based composite scaffolds via 3-D printing technology

     Serra, Tiziano; Planell Estany, Josep Antón; Navarro Toro, Melba Eugenia
    Acta biomaterialia
    Date of publication: 2013-03
    Journal article

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    Fabrication of new biodegradable scaffolds that guide and stimulate tissue regeneration is still a major issue in tissue engineering approaches. Scaffolds that possess adequate biodegradability, pore size, interconnectivity, bioactivity and mechanical properties in accordance with the injured tissue are required. This work aimed to develop and characterize three-dimensional (3-D) scaffolds that fulfill the aforementioned requirements. For this, a nozzle-based rapid prototyping system was used to combine polylactic acid and a bioactive CaP glass to fabricate 3-D biodegradable scaffolds with two patterns (orthogonal and displaced double layer). Scanning electron microscopy and micro-computer tomography showed that 3-D scaffolds had completely interconnected porosity, uniform distribution of the glass particles, and a controlled and repetitive architecture. Surface properties were also assessed, showing that the incorporation of glass particles increased both the roughness and the hydrophilicity of the scaffolds. Mechanical tests indicated that compression strength is dependent on the scaffold geometry and the presence of glass. Preliminary cell response was studied with primary mesenchymal stem cells (MSC) and revealed that CaP glass improved cell adhesion. Overall, the results showed the suitability of the technique/materials combination to develop 3-D porous scaffolds and their initial biocompatibility, both being valuable characteristics for tissue engineering applications.

  • A bioactive elastin-like recombinamer reduces unspecific protein adsorption and enhances cell response on titanium surfaces.

     Salvagni, Emiliano; Berguig, Geoffrey Yehuda; Engel Lopez, Elisabet; Rodriguez Cabello, Jose Carlos; Coullerez, Geraldine; Textor, Marcus; Planell Estany, Josep Antón; Gil Mur, Francisco Javier; Aparicio, Conrado
    Colloids and Surfaces B. Biointerfaces
    Date of publication: 2013-10-17
    Journal article

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    We present the immobilization on synthetic substrates of elastin-like recombinamers (ELR) that combine a bioactive motif for cell adhesion with protein antifouling properties. Physical adsorption of the recombinamers and covalent-grafting through organosilane chemistry were investigated. The biochemically-modified surfaces were thoroughly characterized and tested for protein absorption in serum by fluorescence-labelling, XPS, Ellipsometry, and OWLS. The ELR were successfully grafted and stable, even upon mechanical stresses; being the covalent bonding favourable over physical adsorption. The coated metal surfaces exhibited excellent reduction of serum protein adsorption (9 ng/cm2) compared to the bare metal surface (310 ng/cm2). Non-specific protein adsorption may mask the introduced bioactive motifs; therefore, the bioactivated surfaces should display serum-protein antifouling properties. Finally, improved hMSCs response was assessed on the bioactivated substrates. In summary, the coatings simultaneously displayed anti-fouling and bioactive properties. These studies investigated key factors to enhance tissue material interactions fundamental for the design of bioactive devices and future biomedical applications

  • The effect of the composition of PLA films and lactate release on glial and neuronal maturation and the maintenance of the neuronal progenitor niche

     Álvarez Pinto, Zaída; Mateos Timoneda, Miguel Angel; Hýro¿¿ová, Petra; Castaño Linares, Oscar; Planell Estany, Josep Antón; Perales Losa, José Carlos; Engel Lopez, Elisabet; Alcántara Horrillo, Soledad
    Biomaterials
    Date of publication: 2013-03-06
    Journal article

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    To develop tissue engineering strategies useful for repairing damage in the central nervous system (CNS) it is essential to design scaffolds that emulate the NSC niche and its tight control of neural cell genesis, growth, and differentiation. In this study we tested two types of poly l/dl lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. Both PLA were slightly hydrophobic and negatively charged but differed in crystallinity, stiffness and degradation rate. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the one-month period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the culture medium. PLA70/30 performs better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro.

  • Inducing functional radial glia-like progenitors from cortical astrocyte cultures using micropatterned PMMA.

     Mattotti, Marta; Álvarez Pinto, Zaída; Ortega, Juan A.; Planell Estany, Josep Antón; Engel Lopez, Elisabet; Alcántara Horrillo, Soledad
    Biomaterials
    Date of publication: 2012-02-01
    Journal article

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    Radial glia cells (RGC) are multipotent progenitors that generate neurons and glia during CNS development, and which also served as substrate for neuronal migration. After a lesion, reactive glia are the main contributor to CNS regenerative blockage, although some reactive astrocytes are also able to dedifferentiate in situ into radial glia-like cells (RGLC), providing beneficial effects in terms of CNS recovery. Thus, the identification of substrate properties that potentiate the ability of astrocytes to transform into RGLC in response to a lesion might help in the development of implantable devices that improve endogenous CNS regeneration. Here we demonstrate that functional RGLC can be induced from in vitro matured astrocytes by using a precisely-sized micropatterned PMMA grooved scaffold, without added soluble or substrate adsorbed biochemical factors. RGLC were extremely organized and aligned on 2 mm line patterned PMMA and, like their embryonic counterparts, express nestin, the neuron-glial progenitor marker Pax6, and also proliferate, generate different intermediate progenitors and support and direct axonal growth and neuronal migration. Our results suggest that the introduction of line patterns in the size range of the RGC processes in implantable scaffolds might mimic the topography of the embryonic neural stem cell niche, driving endogenous astrocytes into an RGLC phenotype, and thus favoring the regenerative response in situ.

  • In silico evaluation of a new composite disc substitute with a L3-L5 lumbar spine finite element model

     Noailly, Jerome; Ambrosio, Luigi; K. Elizabeth, Tanner; Planell Estany, Josep Antón; Lacroix, Damien
    European spine journal
    Date of publication: 2012-06-21
    Journal article

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    When the intervertebral disc is removed to relieve chronic pain, subsequent segment stabilization should restore the functional mechanics of the native disc. Because of partially constrained motions and the lack of intrinsic rotational stiffness ball-on-socket implants present many disadvantages. Composite disc substitutes mimicking healthy disc structures should be able to assume the role expected for a disc substitute with fewer restrictions than ball-on-socket implants. A biomimetic composite disc prototype including artificial nucleus fibre-reinforced annulus and endplates was modelled as an L4–L5 disc substitute within a L3–L5 lumbar spine finite element model. Different device updates, i.e. changes of material properties fibre distributions and volume fractions and nucleus placements were proposed. Load- and displace- ment-controlled rotations were simulated with and without body weight applied. The original prototype reduced greatly the flexibility of the treated segment with signifi- cant adjacent level effects under displacement-controlled or hybrid rotations. Device updates allowed restoring large part of the global axial and sagittal rotational flexibility predicted with the intact model. Material properties played a major role, but some other updates were identified to potentially tune the device behaviour against specific motions. All device versions altered the coupled interseg- mental shear deformations affecting facet joint contact through contact area displacements. Loads in the bony endplates adjacent to the implants increased as the implant stiffness decreased but did not appear to be a strong limi- tation for the implant biomechanical and mechanobiolog- ical functionality. In conclusion, numerical results given by biomimetic composite disc substitutes were encouraging with greater potential than that offered by ball-on-socket implants

  • Osteoblast-like cellular response to dynamic changes in the ionic extracellular environment produced by calcium-deficient hydroxyapatite

     Gustavsson, Johan; Ginebra Molins, Maria Pau; Planell Estany, Josep Antón; Engel Lopez, Elisabet
    Journal of materials science. Materials in medicine
    Date of publication: 2012-10
    Journal article

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  • Modular polylactic acid microparticle-based scaffolds prepared via microfluidic emulsion/solvent displacement process: Fabrication, characterization, and in vitro mesenchymal stem cells interaction study

     Salerno, Aurelio; Levato, R.; Mateos Timoneda, Miguel Angel; Engel Lopez, Elisabet; Netti, Paolo Antonio; Planell Estany, Josep Antón
    Journal of biomedical materials research. Part A
    Date of publication: 2012-08-31
    Journal article

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    The present study reports a novel approach for the design and fabrication of polylactic acid (PLA) microparticle- based scaffolds with microstructural properties suitable for bone and cartilage regeneration. Macroporous PLA scaffolds with controlled shape were fabricated by means of a semicon- tinuous process involving (1) microfluidic emulsification of a PLA/ethyl lactate solution (5% w/v) in a span 80/paraffin oil so- lution (3% v/v) followed by (2) particles coagulation/assembly in an acetone/water solution for the development of a continu- ous matrix. Porous scaffolds prepared from particles with monomodal or bimodal size distribution, overall porosity ranges from 93 to 96%, interparticles porosity from 41 to 54%, and static compression moduli from 0.3 to 1.4 MPa were man- ufactured by means of flow rate modulation of of the continu- ous phase during emulsion. The biological response of the scaffolds was assessed in vitro by using bone marrow-derived rat mesenchymal stem cells (MSCs). The results demonstrated the ability of the scaffolds to support the extensive and uni- form three-dimensional adhesion, colonization, and prolifera- tion of MSCs within the entire construct

  • Preparation of biodegradable polylactide microparticles via a biocompatible procedure

     Levato, R.; Mateos Timoneda, Miguel Angel; Planell Estany, Josep Antón
    Macromolecular bioscience
    Date of publication: 2012-04
    Journal article

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    PLA MPs are prepared via a novel and toxic-chemical-free fabrication route using ethyl lactate, a green solvent and FDA-approved aroma. MPs are obtained by a solution jet break-up and solvent displacement method. Adjusting flow parameters allows the tuning of MPs size between 60 and 180 mm, with reduced polydispersity. Morphological analysis shows microporous particles with Janus-like surface. A fluorophore is successfully loaded into the MPs during their formation step. This versatile green solvent-based procedure is proven to be suitable for drug encapsulation and delivery applications. The method may be extended to different droplet generation techniques.

  • Adsorption of fibronectin, fibrinogen and albumin on TiO2: A kinetics, structural changes, and competition study

     Pegueroles Neyra, Marta; Tonda Turo, Chiara; Planell Estany, Josep Antón; Gil Mur, Francisco Javier; Aparicio, Conrado
    Journal of the Royal Society Interface
    Date of publication: 2012-12
    Journal article

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  • Dynamic behavior of type IV collagen at cell-biomaterial interface

     Miranda Guerra coelho, Nuno
    Defense's date: 2012-05-25
    Department of Automatic Control, Universitat Politècnica de Catalunya
    Theses

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  • Biological response to structured and functionalized substrates for nerve tissue regeneration  Open access

     Mattotti, Marta
    Defense's date: 2012-10-29
    Department of Automatic Control, Universitat Politècnica de Catalunya
    Theses

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    After a lesion in the CNS, glial cells play a fundamental role, being the mediators of both the inhibitory and the beneficial response for neural regeneration. The tissue engineering approach consists in the use of biomaterials to help the regeneration and guide the regenerative capable cells to create a permissive environment. The main working hypothesis of this thesis is that we can promote a favourable environment for CNS regeneration identifying material properties which can modulate neuronal cells behaviour. In a first place we analyzed glial and neuronal response to two very different biopolymers, PMMA and chitosan. Wettability, surface and mechanical properties were characterized for both materials. Then line pattern of different dimensions in the micrometrical range were introduced. The response of glial cell and neurons were analyzed in terms of cell adhesion, morphology and differentiation state. Finally, we studied the behaviour of glial cells on glass model surfaces functionalized by self assembling monolayers with different wettability (OH, COOH, NH2, CH3), in order to identify the specific role that wettability plays in determining cell response. The dates suggest that the adhesion, the morphology and the differentiation state of neuron and glial cells can be controlled by choosing the proper combination of material properties and physical patterns. Overall, line patterns resulted to be a suitable tool to use in biomaterial design for nerve regeneration. However, the performance of each material must be analyzed with attention, since the combination of material properties, which most of the time is not predictable, play important roles in the biological activity.

  • Evaluación computacional en andamios por prototipado rápido para la ingeniería de tejidos  Open access

     Olivares Miyares, Andy Luis
    Defense's date: 2012-02-10
    Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya
    Theses

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    In tissue engineering, the ultimate goal of the development of degradable porous scaffolds integrated with cells is the regeneration of new living tissue to repair biological defects. Scaffolds have certain requirements depending on the application, since they have to provide cells physiological conditions for specific tissue regeneration. In vitro analysis of scaffolds efficiency for tissue engineering is normally very expensive and it is generally difficult to control the processes that occur within the scaffolds. In this thesis numerical simulation were used in order to understand the influence of the morphology of scaffolds and culture conditions on different cellular processes, simulating in vitro experiments. Due to the need to optimize some of those cellular processes such as, cell seeding and cell culture different methodologies and scaffolds pores design were proposed to control the cellular responses.Physic-chemical culture conditions can be reproduced using bioreactor systems since testing these factors using the dynamic environment of bioreactors has been proved to be more efficient that testing the same factors experimentally under static conditions. Also testes done under static conditions uses small scaffolds but some tissue defects happen in a larger scale. Thus, in this thesis a method to analyze a perfusion bioreactor at a larger scale is proposed. The goal is to apply the results from an experimental level to a clinical application, where a larger graft is necessary. This methodology can be used to create an articular cartilage capable to fully cover the knee (diameter = 50 mm). One method that ensures the implementation of the macroscopic properties (permeability) of the biomaterial was developed. Using fluid dynamics simulations, the bioreactor design to provide better distribution of cells in the scaffold (seeded) and the right amount of stimulus (culture) was determined. Briefly, simulation predictions for the articular cartilage formation showed that cartilage growth is more uniform for a more homogeneous fluid-flow distribution.Different pores morphologies from rapid-prototyping fabrication were numerically evaluated. Two scaffold designs were proposed, one with gyroid morphology and the other with hexagonal prism shape. For both designs porosities of 55% and 70% were tested. In addition, different mechanical stimuli were computed to study the effect on cell adhesion. Mechanical stimuli are known to determine cell differentiation through a mechano-regulation theory. According to this theory, optimization of external conditions for cell differentiation were discussed comparing the effect of fluid-flow and axial compression. Fluid-flow was demonstrated to be more effective for cell differentiation on scaffolds that axial compression. The gyroid architectures lead to a better accessibility of the fluid into the scaffold, which can be related with higher efficiency during cell seeding.Based on the fact that the gyroid structure leads to a most effective distribution of the fluid, a system to control cell seeding was developed. The parameters of the system were: 1) the prediction of mechanical stimuli (numerically), 2) the distribution of gyroid pore (fabrication) and 3) the experimental verification. The scaffolds were reconstructed based on micro CT images. Two type of scaffold were studied; one with isotropic pore size distribution (412 ± 13 µm) and another with a gradual variation of pore size (250-500 µm). The results showed that on the isotropic scaffolds, the stimuli calculated were more uniform (15s-1 y 24 s-1)and varied gradually (12s-1 -38 s-1) in the second type of scaffold. The distribution of the seeded cells also follows the pattern of distribution of stimuli and pores.Finally, a new methodology to evaluate cell seeding under perfusion conditions was proposed. The entire process for cell seeding was simulated using a Lagrangian multiphase model.

  • 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.

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    Control of microenvironmental cues with a smart biomaterial composite promotes endothelial progenitor cell angiogenesis  Open access

     Aguirre, Adrián; Gonzalez, Arlyng; Navarro, Melba; Castaño Linares, Oscar; Planell Estany, Josep Antón; Engel Lopez, Elisabet
    European cells and materials
    Date of publication: 2012-09
    Journal article

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    Smart biomaterials play a key role when aiming at successful tissue repair by means of regenerative medicine approaches, and are expected to contain chemical as well as mechanical cues that will guide the regenerative process. Recent advances in the understanding of stem cell biology and mechanosensing have shed new light onto the importance of the local microenvironment in determining cell fate. Herein we report the biological properties of a bioactive, biodegradable calcium phosphate glass/polylactic acid composite biomaterial that promotes bone marrowderived endothelial progenitor cell (EPC) mobilisation, differentiation and angiogenesis through the creation of a controlled bone healing-like microenvironment. The angiogenic response is triggered by biochemical and mechanical cues provided by the composite, which activate two synergistic cell signalling pathways: a biochemical one mediated by the calcium-sensing receptor and a mechanosensitive one regulated by non-muscle myosin II contraction. Together, these signals promote a synergistic response by activating EPCs-mediated VEGF and VEGFR-2 synthesis, which in turn promote progenitor cell homing, differentiation and tubulogenesis. These findings highlight the importance of controlling microenvironmental cues for stem/progenitor cell tissue engineering and offer exciting new therapeutical opportunities for biomaterialbased vascularisation approaches and clinical applications

  • Injectable calcium-phosphate-based composites for skeletal bone treatments

     Ambrosio, Luigi; Guarino, V.; Torricelli, P.; Fini, M.; Ginebra Molins, Maria Pau; Planell Estany, Josep Antón; Giardino, R.
    Biomedical materials
    Date of publication: 2012
    Journal article

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  • Biopolímeros Biodegradabes Funcionalizados con colágeno que comprenden células de epitelio del limbo esclerocorneal para la reconstrucción de la superficie ocular

     Planell Estany, Josep Antón; Mateos Timoneda, Miguel Angel; Engel Lopez, Elisabet; Calonge Cano, Margarita; de la Mata Sampedro, Ana; Lopez Paniagua, Marina; Galindo de la Rosa, Sara; Nieto Miguel, Teresa
    Date of request: 2012-06-28
    Invention patent

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  • Fabrication and characterization of biodegradable composite scaffolds for tissue engineering

     Serra, Tiziano; Navarro, Melba; Planell Estany, Josep Antón
    International Conference on Advanced Research in Virtual and Rapid Prototyping
    Presentation's date: 2011-09
    Presentation of work at congresses

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  • On the collagen criss-cross angles in the annuli fibrosi of lumbar spine finite element models

     Planell Estany, Josep Antón; Lacroix, Damien Jerome; Noailly, Jerome
    Biomechanics and modeling in mechanobiology
    Date of publication: 2011-04
    Journal article

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    In the human lumbar spine, annulus fibrosus fibres largely contribute to intervertebral disc stability. Detailed annulus models are therefore necessary to obtain reliable predictions of lumbar spine mechanics by finite element modelling. However, different definitions of collagen orientations coexist in the literature for healthy human lumbar annuli. Therefore, four annulus fibre-induced anisotropy models were built from reported anatomical descriptions, and inserted in a L3–L5 lumbar bi-segment finite element model. Annulus models were, respectively, characterized by radial, tangential, radial and tangential, and no fibre orientation gradients. The effect of rotational and axial compressive loadings was simulated and first, predictions were compared to experimental data. Then, intervertebral disc local biomechanics was studied under axial rotation and axial compression. A new parameter, i.e. the fibre contribution quality parameter, was computed in the anterior, lateral, postero-lateral, and posterior annuli of each model, in function of fibre stresses, radial load distributions, and matrix shear strains. Locally, each annulus model behaved differently, affecting intervertebral disc biomechanics and segmentalmotions. The fibre contribution quality parameter allowed establishing direct links between local annulus fibre organization and local annulus loadings, while other kinematical and biomechanical data did not. It was concluded that functional relations should exist between local annulus fibre orientations and overall segment morphology. The proposed fibre contribution quality parameter could be used to examine such relations and calibrate lumbar spine finite element models by locally adjusting the annulus bundle criss-cross angles. Conclusions of this study are particularly relevant to patient-specific models or artificial disc designs.

  • Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media

     Gustavsson, Johan Mikael; Ginebra Molins, Maria Pau; Engel Lopez, Elisabet; Planell Estany, Josep Antón
    Acta biomaterialia
    Date of publication: 2011-12
    Journal article

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  • Effect of blasting treatment and Fn coating on MG63 adhesion and differentiation on titanium: a gene expression study using real-time RT-PCR.

     Pegueroles Neyra, Marta; Aguirre Cano, Aitor; Engel Lopez, Elisabet; Pavon-Djavid, Graciela; Gil Mur, Francisco Javier; Planell Estany, Josep Antón; Migonney, Veronique; Aparicio, C
    Journal of materials science. Materials in medicine
    Date of publication: 2011-03-02
    Journal article

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    Biomaterial surface properties, via alterations in the adsorbed protein layer, and the presence of specific functional groups can influence integrin binding specificity, thereby modulating cell adhesion and differentiation processes. The adsorption of fibronectin, a protein directly involved in osteoblast adhesion to the extracellular matrix, has been related to different physical and chemical properties of biomaterial surfaces. This study used blasting particles of different sizes and chemical compositions to evaluate the response of MG63 osteoblast-like cells on smooth and blasted titanium surfaces, with and without fibronectin coatings, by means of real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays. This response included (a) expression of the a5, av and a3 integrin subunits, which can bind to fibronectin through the RGD binding site, and (b) expression of alkaline phosphatase (ALP) and osteocalcin (OC) as cell-differentiation markers. ALP activity and synthesis of OC were also tested. Cells on SiC-blasted Ti surfaces expressed higher amounts of the a5 mRNA gene than cells on Al2O3-blasted Ti surfaces. This may be related to the fact that SiC-blasted surfaces adsorbed higher amounts of fibronectin due to their higher surface free energy and therefore provided a higher number of specific cell-binding sites. Fn-coated Tisurfaces decreased a5 mRNA gene expression, by favoring the formation of other integrins involved in adhesion over a5b1. The changes in a5 mRNA expression induced by the presence of fibronectin coatings may moreover influence the osteoblast differentiation pathway, as fibronectin coatings on Ti surfaces also decreased both ALP mRNA expression and ALP activity after 14 and 21 days of cell culture.

  • Modifying biomaterial surfaces for the repair and regeneration of nerve cells

     Planell Estany, Josep Antón; Engel Lopez, Elisabet; Mateos Timoneda, Miguel Angel
    Date of publication: 2011-09-01
    Book chapter

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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.

  • Desarrollo y Caracterización de Andamios Biodegradables para Sustitución Ósea mediante Ingeniería Tisular

     Márquez Pérez, Lucia
    Defense's date: 2011-11-29
    Department of Automatic Control, Universitat Politècnica de Catalunya
    Theses

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  • Desarrollo de un cemento de fosfato de calcio macroporoso: influencia de la macroporosidad y de la microestructura en el comportamiento biológico  Open access

     Del Valle Fresno, Sergio
    Defense's date: 2011-05-27
    Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya
    Theses

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    Los cementos de fosfato de calcio son biomateriales utilizados para la regeneración ósea. En esta tesis doctoral se estudia la influencia de la microestructura y la macroporosidad en el comportamiento biológico de un cemento de fosfato de calcio. En la primera parte, se caracterizaron las propiedades físico-químicas y superficiales de dos sustratos de apatita deficiente en calcio con diferente microestructura obtenidos a partir de un cemento de fosfato de calcio (CPC) cuyo principalmente componente es el fosfato tricálcico en fase $\alpha$. % con dos distribuciones de tamaño de partícula distintos. Se estudió la influencia de la microestructura en el comportamiento celular y la adsorción de proteínas como la albúmina, la fibronectina y la lisozima. El uso de dos agentes desorción (EDTA y SDS) permitió evidenciar la influencia de la morfología y tamaño de los cristales de apatita en la fuerza de adhesión de las proteínas estudiadas. Los cultivos celulares con la línea MG-63 mostraron el efecto de la microestructura y el intercambio iónico sobre la proliferación celular. Se observó asimismo una estimulación en la diferenciación celular en uno de los sustratos que fue atribuido a un efecto de la topografía. En la segunda parte, se desarrolló un cemento macroporoso inyectable para aplicaciones médicas, utilizando como agente espumante una solución proteica de albumen. Se optimizó la obtención de la macroporosidad a partir de variables como: la distribución del tamaño de partícula, la relación entre la fase sólida y la fase líquida, la cantidad de agente espumante y la adición de un agente de cohesión como el alginato de sodio para posibilitar su inyección directa en el lugar de implantación. Por último, se demostró mediante estudios \emph{in vivo} en fémur de conejos un mayor potencial de regeneración ósea por parte del cemento macroporoso en comparación con su homólogo microporoso, al obtenerse una mayor cantidad de tejido óseo y una mayor reabsorción en el cemento macroporoso.

  • Comparison of a low molecular weight and a macromolecular surfactant as foaming agents for injectable self setting hydroxyapatite foams: Polysorbate 80 versus gelatine

     Montufar Jimenez, Edgar Benjamin; Traykova, Tania; Planell Estany, Josep Antón; Ginebra Molins, Maria Pau
    Materials science and engineering C. Biomimetic and supramolecular systems
    Date of publication: 2011-10-10
    Journal article

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    Hydroxyapatite foams are potential synthetic bone grafting materials or scaffolds for bone tissue engineering. A novel method to obtain injectable hydroxyapatite foams consists in foaming the liquid phase of a calcium phosphate cement. In this process, the cement powder is incorporated into a liquid foam, which acts as a template for macroporosity. After setting, the cement hardens maintaining the macroporous structure of the foam. In this study a low molecular weight surfactant, Polysorbate 80, and a protein, gelatine, were compared as foaming agents of a calcium phosphate cement. The foamability of Polysorbate 80 was greater than that of gelatine, resulting in higher macroporosity in the set hydroxyapatite foam and higher macropore interconnectivity. Gelatine produced less interconnected foams, especially at high concentrations, due to a higher liquid foam stability. However it increased the injectability and cohesion of the foamed paste, and enhanced osteoblastic-like cell adhesion, all of them important properties for bone grafting materials.

  • Electrochemical microelectrodes for improved spatial and temporal characterization of aqueous environments around calcium phosphate cements.

     Gustavsson, Johan Mikael; Ginebra Molins, Maria Pau; Planell Estany, Josep Antón; Engel Lopez, Elisabet
    Acta biomaterialia
    Date of publication: 2011-10-07
    Journal article

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    Calcium phosphate compounds can potentially influence cellular fate through ionic substitutions. However, to be able to turn such solution-mediated processes into successful directors of cellular response, a perfect understanding of the material-induced chemical reactions in situ is required. We therefore report on the application of home-made electrochemical microelectrodes, tested as pH and chloride sensors, for precise spatial and temporal characterization of different aqueous environments around calcium phosphate- based biomaterials prepared from a-tricalcium phosphate using clinically relevant liquid to powder ratios. The small size of the electrodes allowed for online measurements in traditionally inaccessible in vitro environments, such as the immediate material–liquid interface and the interior of curing bone cement. The kinetic data obtained has been compared to theoretical sorption models, confirming that the proposed setup can provide key information for improved understanding of the biochemical environment imposed by chemically reactive biomaterials.

  • Development of provisional extracellular matrix on biomaterials interface: lessons from in vitro cell culture

     Altankov, George Petrov; Groth, Thomas; Gustavsson, Johan Mikael; Engel Lopez, Elisabet; Pegueroles Neyra, Marta; Aparicio, Conrado; Gil Mur, Francisco Javier; Ginebra Molins, Maria Pau; Planell Estany, Josep Antón
    Date of publication: 2010-08-20
    Book chapter

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  • Injectable hydroxyapatite foams for bone regeneration

     Montufar Jimenez, Edgar Benjamin; Planell Estany, Josep Antón; Ginebra Molins, Maria Pau
    TOPEA
    Presentation's date: 2010-07-01
    Presentation of work at congresses

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  • Gene expression of integrins alpha 5, alpha V, alpha 3, beta 1 and beta 3 by osteoblasts adhered on plane and fibronectin coated cp Ti. Effect of physical, chemical and topographical surface properties

     Gil Mur, Francisco Javier; Pegueroles Neyra, Marta; Aparicio, Conrado; Aguirre Cano, Aitor; Engel Lopez, Elisabet; Planell Estany, Josep Antón
    European Conference on Biomaterials
    Presentation's date: 2010-09
    Presentation of work at congresses

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  • Foamed surfactant solution as a template for self-setting injectable hydroxyapatite scaffolds for bone regeneration

     Montufar Jimenez, Edgar Benjamin; Traykova, Tania Valentinova; Gil, C.; Harr, I.; Almirall Gonzalo, Arturo; Aguirre Cano, Aitor; Engel Lopez, Elisabet; Planell Estany, Josep Antón; Ginebra Molins, Maria Pau
    Acta biomaterialia
    Date of publication: 2010-03
    Journal article

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  • Perfusion cell seeding on large porous PLA/calcium phosphate composite scaffolds in a perfusion bioreactor system under varying perfusion parameters

     Koch, Martin Andreas; Vrij, E. J; Engel Lopez, Elisabet; Planell Estany, Josep Antón; Lacroix, Damien Jerome
    Journal of biomedical materials research. Part A
    Date of publication: 2010-12-15
    Journal article

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    A promising approach to bone tissue engineering lies in the use of perfusion bioreactors where cells are seeded and cultured on scaffolds under conditions of enhanced nutrient supply and removal of metabolic products. Fluid flow alterations can stimulate cell activity, making the engineering of tissue more efficient. Most bioreactor systems are used to culture cells on thin scaffold discs. In clinical use, however, bone substitutes of large dimensions are needed. In this study, MG63 osteoblast-like cells were seeded on large porous PLA/glass scaffolds with a custom developed perfusion bioreactor system. Cells were seeded by oscillating perfusion of cell suspension through the scaffolds. Applicable perfusion parameters for successful cell seeding were determined by varying fluid flow velocity and perfusion cycle number. After perfusion, cell seeding, the cell distribution, and cell seeding efficiency were determined. A fluid flow velocity of 5 mm/s had to be exceeded to achieve a uniform cell distribution throughout the scaffold interior. Cell seeding efficiencies of up to 50% were achieved. Results suggested that perfusion cycle number influenced cell seeding efficiency rather than fluid flow velocities. The cell seeding conducted is a promising basis for further long term cell culture studies in large porous scaffolds.

  • Spatial organization of osteoblast fibronectin matrix on titanium surfaces: Effects of roughness, chemical heterogeneity and surface energy

     Pegueroles Neyra, Marta; Aparicio Bádenas, Conrado José; Bosio, M.; Engel Lopez, Elisabet; Gil Mur, Francisco Javier; Planell Estany, Josep Antón; Altankov, George Petrov
    Acta biomaterialia
    Date of publication: 2010-01
    Journal article

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  • Injectable and fast resorbable calcium phosphate cement for body-setting bone grafts

     Rajzer, I.; Castaño Linares, Oscar; Engel Lopez, Elisabet; Planell Estany, Josep Antón
    Journal of materials science. Materials in medicine
    Date of publication: 2010
    Journal article

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  • Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis

     Aguirre Cano, Aitor; Planell Estany, Josep Antón; Engel Lopez, Elisabet
    Biochemical and biophysical research communications
    Date of publication: 2010-09-17
    Journal article

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  • Materials surface effects on biological interactions

     Planell Estany, Josep Antón; MELBA, NAVARRO TORO; Altankov, George Petrov; Aparicio, Conrado; Engel Lopez, Elisabet; Gil Mur, Francisco Javier; Ginebra Molins, Maria Pau; Lacroix, D.
    Date of publication: 2010-08-20
    Book chapter

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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.

  • Principles of Vascularization in Tissue Engineering using Endothelial Progenitors and Bioactive Composite Biomaterials

     Aguirre Cano, Aitor
    Defense's date: 2010-12-20
    Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya
    Theses

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  • Development of in vitro and in vivo Bioreactors for Bone Tissue Engineering  Open access

     Koch, Martin Andreas
    Defense's date: 2010-04-23
    Department of Automatic Control, Universitat Politècnica de Catalunya
    Theses

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    Grandes defectos óseos constituyen un reto para el campo clínico, ya que no puede ser reparado por el propio organismo, sino que requieren la implantación de injertos de hueso adecuado. Para superar los inconvenientes de los injertos procedentes de fuentes autólogas o allogeneicas, la ingeniería de tejidos óseos pretende sustituir el tejido perdido utilizando el cultivo de células in vitro sobre biomateriales porosos. El cultivo de células en grandes andamios porosos ha demostrado ser difícil, que requiere bioreactores, que se utilizan para el cultivo de tejidos y el estudio del comportamiento de células en 3D de los andamios. De interés especial es el condicionamiento mecánico de los tejidos cultivados por bioreactor de la ingeniería del tejido óseo, que es capaz de aumentar el potencial osteogénico de los injertos sintéticos.En este trabajo, dos sistemas de bioreactores fueron desarrollados para permitir comprender las propiedades bioactivas de andamios de diferentes materiales y la mecanoregulación del comportamiento de células o tejidos. Un sistema de bioreactor de perfusión in vitro fue desarrollado para el sembrado y cultivo de células incorporadas en cilindros de un biomaterial poroso. Varios estudios para la determinación de los parámetros del sembrado de células aplicable se llevaron a cabo, así como experimentos de cultivo de células bajo flujo de fluido constante con una estimulación mecánica adicional por alternancia del flujo.Un sistema de cámara ósea fue desarrollado como un bioreactor in vivo. El sistema produjo un defecto óseo grande en tibias de perros y permitió la implantación repetida de grandes andamios porosos de materiales diferentes. El tejido creciendo en los andamios permite extraer conclusiones sobre las propiedades de osteoconductividad u osteinductividad de los andamios. Además, un dispositivo de compresión se ha desarrollado para aplicar cargas cíclicas en los andamios en vivo para estudiar el efecto de la estimulación mecánica en el desarrollo de los tejidos.Los estudios con el sistema de perfusión desarrollado han demostrado que el sembrado de células en grandes andamios porosos es posible, lo que se considera crucial para el cultivo celular. El largo tiempo de cultivo de células mostró la proliferación de las células madre mesenquimales hasta dos semanas. El patrón de estimulación utilizado en el estudio aumentó la expresión de la osteocalcina, lo que indica una mayor actividad de las células, pero la ausencia de expresión de RunX2 y colágeno I impidió la determinación concluyente de la diferenciación.El sistema desarrollado de la cámara ósea demostró su funcionalidad en el entorno quirúrgico durante los experimentos in vivo. Complicaciones durante los experimentos no permitieron la aplicación de las cargas cíclicas de los andamios implantados. La formación de hueso retrasada debido al defecto óseo creado y material de andamios restantes no permitieron conclusiones definitivas acerca de las propiedades del material del andamio. Sin embargo, el estudio proporciona datos para el desarrollo futuro del dispositivo y protocolo clínico.Los estudios realizados constituyen una novedad en respecto a la creación de bioreactores para el estudio de la andamios porosos sintéticos de grandes dimensiones in vitro e in vivo. Los sistemas desarrollados constituyen la base para otros estudios en mecanobiología de las células óseas y los tejidos.

    Large bone defects constitute a challenge for the clinical field, because they cannot be repaired by the body itself, but require the implantation of suitable bone grafts. To overcome the drawbacks of grafts from autologous or allogous sources, modern bone tissue engineering aims to replace lost tissue by cultivating cells in vitro on porous biomaterials. The cell culture on large porous scaffolds has shown to be difficult, requiring bioreactors, which are used for tissue culture and the study of cell behaviour in 3D scaffolds. Of special interest is the mechanical conditioning of the cultured tissue for bioreactor-based bone tissue engineering, which is able to enhance the osteogenic potential of the synthetic grafts.In this work two bioreactor systems were developed to allow insight into bioactive properties of different scaffold materials and the mechanoregulation of cell or tissue behaviour. An in vitro perfusion bioreactor system was developed for the cell seeding and culture on porous biomaterial cylinders. Several studies for the determination of applicable cell seeding parameters were conducted, as well as experiments of cell culture under steady fluid flow with additional mechanical stimulation by alternating fluid flow. A bone chamber system was developed as an in vivo bioreactor. The system produced a large bone defect in dog tibia and allowed the repeated implantation of large porous scaffolds of different material compositions.The ingrowing tissue was observed to allow conclusions about osteoconductive or osteinductive properties of the scaffolds. Additionally a compression device was developed to apply cyclic loading on the scaffolds in vivo to study the effect of mechanical stimulation on tissue development.The studies with the developed in vitro perfusion bioreactor system have shown that it is possible to seed cells throughout large porous scaffolds, which is deemed crucial for the further cell culture. The long time cell culture showed the proliferation of mesenchymal stem cells up to two weeks. The stimulation pattern used in the study enhanced the expression of osteocalcin, indicating an enhanced cell activity, but the absence of RunX2 and collagen I expression rendered the determination of differentiation inconclusive.The developed bone chamber system proved to be functional in the surgical environment during the in vivo experiments. Occurring complications during the experiments did not allow the application of the cyclic loading of implanted scaffolds. Delayed bone formation due to created bone defect and remaining scaffold material did not allow final conclusions about the scaffold material properties. Nevertheless the study provides input for further development of the device and clinical protocol.The conducted studies constitute a novelty regarding the creation of bioreactors for the study of synthetic porous scaffolds of large dimensions in vitro and in vivo. The developed systems form the basis for further studies in mechanobiology of bone cells and tissue.

  • 8è premi de valorització de la recerca, modalitat 2

     Ginebra Molins, Maria Pau; Gil Mur, Francisco Javier; Planell Estany, Josep Antón
    Award or recognition

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  • Extracellular calcium modulates in vitro bone marrow-derived Flk-1282B29 CD34282B29 progenitor cell chemotaxis and differentiation through a calcium-sensing receptor

     Aguirre Cano, Aitor; Planell Estany, Josep Antón; Gonzalez Colas, Antonio Maria; Engel Lopez, Elisabet
    Biochemical and biophysical research communications
    Date of publication: 2010-02-26
    Journal article

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  • Materials Surface Effects on Biological Interactions

     Planell Estany, Josep Antón; Navarro, M; Altankov, George Petrov; Aparicio, Conrado; Engel Lopez, Elisabet; Gil Mur, Francisco Javier; Ginebra Molins, Maria Pau; Lacroix, Damien Jerome
    NATO Science for Peace and Security Series A. Chemistry and Biology
    Date of publication: 2010
    Journal article

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  • Different assembly of type IV collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction

     Coelho, Nuno Miranda Guerra; Gonzalez Garcia, C.; Planell Estany, Josep Antón; Salmeron Sánchez, M; Altankov, George Petrov
    European cells and materials
    Date of publication: 2010-06
    Journal article

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    Considering the structural role of type IV collagen (Col IV) in the assembly of the basement membrane (BM) and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass) and hydrophobic trichloro(octadecyl)silane (ODS) surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50μg/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine – nearly single molecular size – network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC) attach less efficiently to the aggregated Col IV (on ODS), as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both α1 and α2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.

  • Simulation of bone tissue formation within a porous scaffold under dynamic compression

     Milan, Jean-Louis; Planell Estany, Josep Antón; Lacroix, Damien Jerome
    Biomechanics and modeling in mechanobiology
    Date of publication: 2010-10
    Journal article

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  • Self-hardening calcium deficient hydroxyapatite/gelatine foams for bone regeneration

     Montufar Jimenez, Edgar Benjamin; Traykova, Tania; Schacht, Etienne; Ambrosio, Luigi; Santin, Matteo Alfredo Giovanni; Planell Estany, Josep Antón; Ginebra Molins, Maria Pau
    Journal of materials science. Materials in medicine
    Date of publication: 2010-03
    Journal article

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    In this work gelatine was used as multifunctional additive to obtain injectable self-setting hydroxyapatite/ gelatine composite foams for bone regeneration. The foaming and colloidal stabilization properties of gelatine are well known in food and pharmaceutical applications. Solid foams were obtained by foaming liquid gelatine solutions at 50 C, followed by mixing them with a cement powder consisting of alpha tricalcium phosphate. Gelatine addition improved the cohesion and injectability of the cement paste. After setting the foamed paste transformed into a calcium deficient hydroxyapatite. The final porosity, pore interconnectivity and pore size were modulated by modifying the gelatine content in the liquid phase.

  • Novel soybean/gelatine-based bioactive and injectable hydroxyapatite foam: material properties and cell response

     Perut, Francesca; Montufar Jimenez, Edgar Benjamin; Ciapetti, Gabriela; Santin, Matteo Alfredo Giovanni; Salvage, J; Traykova, Tania; Planell Estany, Josep Antón; Ginebra Molins, Maria Pau; Baldini, Nicola
    Acta biomaterialia
    Date of publication: 2010-12-10
    Journal article

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    Despite their known osteoconductivity, clinical use of calcium phosphate cements is limited both by their relatively slow rate of resorption and by rheological properties incompatible with injectability. Bone in-growth and material resorption have been improved by the development of porous calcium phosphate cements. However, injectable formulations have so far only been obtained through the addition of relatively toxic surfactants. The present work describes the response of osteoblasts to a novel injectable foamed bone cement based on a composite formulation including the bioactive foaming agents soybean and gelatine. The foaming properties of both defatted soybean and gelatine gels were exploited to develop a self-hardening soy/gelatine/hydroxyapatite composite foam able to retain porosity upon injection. After setting, the foamed paste produced a calcium-deficient hydroxyapatite scaffold, showing good injectability and cohesion as well as interconnected porosity after injection. The intrinsic bioactivity of soybean and gelatine was shown to favour osteoblast adhesion and growth. These findings suggest that injectable, porous and bioactive calcium phosphate cements can be produced for bone regeneration through minimally invasive surgery.

  • Computational modelling of the mechanical environment of osteogenesis within a polylactic acid¿calcium phosphate glass scaffold

     Milan, J-L; Planell Estany, Josep Antón; Lacroix, Damien Jerome
    Biomaterials
    Date of publication: 2009-09
    Journal article

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  • Model developments for in silico studies of the lumbar spine biomechanics.  awarded activity

     Noailly, Jerome
    Defense's date: 2009-06-22
    Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya
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