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by Keyword: Hydroxyapatite


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Ciapetti, G., Di Pompo, G., Avnet, S., Martini, D., Diez-Escudero, A., Montufar, E. B., Ginebra, M. P., Baldini, N., (2017). Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates Acta Biomaterialia 50, 102-113

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. Statement of Significance The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors’ differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.

Keywords: Bone resorption, Differentiation, Hydroxyapatite, Ionic exchange, Osteoclasts, Topography


Maazouz, Y., Montufar, E. B., Malbert, J., Espanol, M., Ginebra, M. P., (2017). Self-hardening and thermoresponsive alpha tricalcium phosphate/pluronic pastes Acta Biomaterialia 49, 563-574

Although calcium phosphate cements (CPCs) are used for bone regeneration in a wide range of clinical applications, various physicochemical phenomena are known to hinder their potential use in minimally invasive surgery or in highly vascularized surgical sites, mainly because of their lack of injectability or their low washout resistance. The present work shows that the combination of CPCs with an inverse-thermoresponsive hydrogel is a good strategy for finely tuning the cohesive and rheological properties of CPCs to achieve clinical acceptable injectability to prevent phase separation during implantation and cohesion to avoid washout of the paste. The thermoresponsive CPC developed combines alpha-tricalcium phosphate with an aqueous solution of pluronic F127, which exhibits an inverse thermoresponsive behaviour, with a gelling transformation at around body temperature. These novel CPCs exhibited temperature-dependent properties. Addition of the polymer enhanced the injectability of the paste, even at a low liquid-to-powder ratio, and allowed the rheological properties of the cement to be tuned, with the injection force decreasing with the temperature of the paste. Moreover, the cohesion of the paste was also temperature-dependent and increased as the temperature of the host medium increased due to gelling induced in the paste. The thermoresponsive cement exhibited excellent cohesion and clinically acceptable setting times at 37 °C, irrespective of the initial temperature of the paste. The addition of pluronic F127 slightly delayed the setting reaction in the early stages but did not hinder the full transformation to calcium-deficient hydroxyapatite. Moreover, the frozen storage of premixed thermoresponsive cement pastes was explored, the main physicochemical properties of the cements being maintained upon thawing, even after 18 months of frozen storage. This avoids the need to mix the cement in the operating theatre and allows its use off-the-shelf. The reverse thermoresponsive cements studied herein open up new perspectives in the surgical field, where the sequential gelling/hardening of these novel cements could allow for a better and safer clinical application. Statement of Significance Calcium phosphate cements are attractive bone substitutes due to their similarity to the bone mineral phase. Although they can be injectable, cohesion and stability of the paste are crucial in terms of performance and safety. A common strategy is the combination with hydrogels. However, this often results in a decrease of viscosity with increasing temperature, which can lead to extravasation and particle leakage from the bone defect. The preferred evolution would be the opposite: a low viscosity would enhance mixing and injection, and an instantaneous increase of viscosity after injection would ensure washout resistance to the blood flow. Here we develop for the first time a calcium phosphate cement exhibiting reverse thermoresponsive properties using a poloxamer featuring inverse thermal gelling.

Keywords: Calcium phosphate cement, Cohesion, Hydroxyapatite, Injectability, Pluronic, Thermoresponsive


Vila, M., García, A., Girotti, A., Alonso, M., Rodríguez-Cabello, J. C., González-Vázquez, A., Planell, J. A., Engel, E., Buján, J., Garcíaa-Honduvilla, N., Vallet-Regí, M., (2016). 3D silicon doped hydroxyapatite scaffolds decorated with Elastin-like Recombinamers for bone regenerative medicine Acta Biomaterialia 45, 349-356

The current study reports on the manufacturing by rapid prototyping technique of three-dimensional (3D) scaffolds based on silicon substituted hydroxyapatite with Elastin-like Recombinamers (ELRs) functionalized surfaces. Silicon doped hydroxyapatite (Si-HA), with Ca10(PO4)5.7(SiO4)0.3(OH)1.7h0.3 nominal formula, was surface functionalized with two different types of polymers designed by genetic engineering: ELR-RGD that contain cell attachment specific sequences and ELR-SNA15/RGD with both hydroxyapatite and cells domains that interact with the inorganic phase and with the cells, respectively. These hybrid materials were subjected to in vitro assays in order to clarify if the ELRs coating improved the well-known biocompatible and bone regeneration properties of calcium phosphates materials. The in vitro tests showed that there was a total and homogeneous colonization of the 3D scaffolds by Bone marrow Mesenchymal Stromal Cells (BMSCs). In addition, the BMSCs were viable and able to proliferate and differentiate into osteoblasts. Statement of Significance Bone tissue engineering is an area of increasing interest because its main applications are directly related to the rising life expectancy of the population, which promotes higher rates of several bone pathologies, so innovative strategies are needed for bone tissue regeneration therapies. Here we use the rapid prototyping technology to allow moulding ceramic 3D scaffolds and we use different bio-polymers for the functionalization of their surfaces in order to enhance the biological response. Combining the ceramic material (silicon doped hydroxyapatite, Si-HA) and the Elastin like Recombinamers (ELRs) polymers with the presence of the integrin-mediate adhesion domain alone or in combination with SNA15 peptide that possess high affinity for hydroxyapatite, provided an improved Bone marrow Mesenchymal Stromal Cells (BMSCs) differentiation into osteoblastic linkage.

Keywords: Bone marrow Mesenchymal Stromal Cells (BMSCs), Bone repair, Elastin-like Recombinamers (ELRs), Rapid prototyped 3D scaffolds, Silicon doped hydroxyapatite (Si-HA), Tissue engineering


Castaño, Oscar, Planell, Josep A., (2014). Cements Bio-Ceramics with Clinical Applications (ed. Vallet-Regí, M.), John Wiley & Sons, Ltd (Chichester, UK) , 193-247

Calcium phosphate cements (CPCs) were meant to produce hydroxyapatite (HA), which is the calcium phosphate that usually results when the cements are mixed with or immersed in aqueous media. The golden age of CPCs was in the late 1990s and the beginning of the 21st century, when they were presented as promising bone substitutes and drug delivery systems. The different reactions that take part in the cement self-setting process depend on many experimental factors – the composition of the cement, the stability of the different components, pH, liquid-to-powder ratio (LPR), and temperature, among others. CPCs have demonstrated fair efficiency for bone regeneration. Cements have gradually been embraced in the wider field of composites by hybridizing their compositions in order that they may adapt to the new trends.

Keywords: Calcium phosphate cements (CPCs), Cements, Hydroxyapatite (HA), Liquid-to-powder ratio (LPR)


Montufar, E. B., Maazouz, Y., Ginebra, M. P., (2013). Relevance of the setting reaction to the injectability of tricalcium phosphate pastes Acta Biomaterialia 9, (4), 6188-6198

The aim of the present work was to analyze the influence of the setting reaction on the injectability of tricalcium phosphate (TCP) pastes. Even if the injection was performed early after mixing powder and liquid, powder reactivity was shown to play a significant role in the injectability of TCP pastes. Significant differences were observed between the injection behavior of non-hardening β-TCP pastes and that of self-hardening α-TCP pastes. The differences were more marked at low liquid-to-powder ratios, using fine powders and injecting through thin needles. α-TCP was, in general, less injectable than β-TCP and required higher injection loads. Moreover, clogging was identified as a mechanism hindering or even preventing injectability, different and clearly distinguishable from the filter-pressing phenomenon. α-TCP pastes presented transient clogging episodes, which were not observed in β-TCP pastes with equivalent particle size distribution. Different parameters affecting powder reactivity were also shown to affect paste injectability. Thus, whereas powder calcination resulted in an increased injectability due to lower particle reactivity, the addition of setting accelerants, such as hydroxyapatite nanoparticles, tended to reduce the injectability of the TCP pastes, especially if adjoined simultaneously with a Na2HPO4 solution. Although, as a general trend, faster-setting pastes were less injectable, some exceptions to this rule were found. For example, whereas in the absence of setting accelerants fine TCP powders were more injectable than the coarse ones, in spite of their shorter setting times, this trend was inverted when setting accelerants were added, and coarse powders were more injectable than the fine ones.

Keywords: Calcium phosphate cement, Hydroxyapatite, Injectability, Setting reaction, Tricalcium phosphate


Perez, R. A., Altankov, G., Jorge-Herrero, E., Ginebra, M. P., (2013). Micro- and nanostructured hydroxyapatite-collagen microcarriers for bone tissue-engineering applications Journal of Tissue Engineering and Regenerative Medicine 7, (5), 353-361

Novel hydroxyapatite (HA)-collagen microcarriers (MCs) with different micro/nanostructures were developed for bone tissue-engineering applications. The MCs were fabricated via calcium phosphate cement (CPC) emulsion in oil. Collagen incorporation in the liquid phase of the CPC resulted in higher MC sphericity. The MCs consisted of a porous network of entangled hydroxyapatite crystals, formed as a result of the CPC setting reaction. The addition of collagen to the MCs, even in an amount as small as 0.8wt%, resulted in an improved interaction with osteoblast-like Saos-2 cells. The micro/nanostructure and the surface texture of the MCs were further tailored by modifying the initial particle size of the CPC. A synergistic effect between the presence of collagen and the nanosized HA crystals was found, resulting in significantly enhanced alkaline phosphatase activity on the collagen-containing nanosized HA MCs.

Keywords: Bone regeneration, Calcium phosphate cement, Cell response, Collagen, Hydroxyapatite, Microcarrier


Ginebra, M. P., Canal, C., Espanol, M., Pastorino, D., Montufar, E. B., (2012). Calcium phosphate cements as drug delivery materials Advanced Drug Delivery Reviews 64, (12), 1090-1110

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.

Keywords: Antibiotic, Bioceramic, Biomaterial, Bone regeneration, Calcium phosphate cement, Ceramic matrix, Growth factor, Hydroxyapatite, Ions, Protein


Gustavsson, J., Ginebra, M. P., Planell, J., Engel, E., (2012). Electrochemical microelectrodes for improved spatial and temporal characterization of aqueous environments around calcium phosphate cements Acta Biomaterialia 8, (1), 386-393

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 α-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.

Keywords: Calcium phosphate, Hydroxyapatite, Ion sorption, Iridium oxide, Sensors, Animals, Biocompatible Materials, Bone Cements, Calcium Phosphates, Cells, Cultured, Chlorides, Electrochemical Techniques, Gold, Hydrogen-Ion Concentration, Hydroxyapatites, Iridium, Materials Testing, Microelectrodes, Powders, Silver, Silver Compounds, Water


Montufar, Edgar B., Traykova, Tania, Planell, Josep A., Ginebra, Maria-Pau, (2011). Comparison of a low molecular weight and a macromolecular surfactant as foaming agents for injectable self setting hydroxyapatite foams: Polysorbate 80 versus gelatine Materials Science and Engineering: C 31, (7), 1498-1504

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.

Keywords: Hydroxyapatite, Porosity, Calcium phosphate cement, Scaffolds, Foaming, Bone regeneration


Gustavsson, J., Ginebra, M. P., Engel, E., Planell, J., (2011). Ion reactivity of calcium-deficient hydroxyapatite in standard cell culture media Acta Biomaterialia 7, (12), 4242-4252

Solution-mediated surface reactions occur for most calcium phosphate-based biomaterials and may influence cellular response. A reasonable extrapolation of such processes observed in vitro to in vivo performance requires a deep understanding of the underlying mechanisms. We therefore systematically investigated the nature of ion reactivity of calcium-deficient hydroxyapatite (CDHA) by exposing it for different periods of time to standard cell culture media of different chemical composition (DMEM and McCoy medium, with and without osteogenic supplements and serum proteins). Kinetic ion interaction studies of principal extracellular ions revealed non-linear sorption of Ca2+ (∼50% sorption) and K+ (∼8%) as well as acidification of all media during initial contact with CDHA (48 h). Interestingly, inorganic phosphorus (Pi) was sorbed from McCoy medium (∼50%) or when using osteogenic media containing

Keywords: Hydroxyapatite, Bioactive materials, Cell culture medium, Ion exchange, Sorption models


Comelles, J., Estevez, M., Martinez, E., Samitier, J., (2010). The role of surface energy of technical polymers in serum protein adsorption and MG-63 cells adhesion Nanomedicine: Nanotechnology Biology and Medicine 6, (1), 44-51

Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. However, novel applications in the biosensor field require materials to be compatible with cell growth and at the same time be suitable for technological processing. Technological polymers are key materials in the fabrication of disposable parts and other sensing elements. As such, it is essential to characterize the surface properties of technological polymers, especially after processing and sterilization. It is also important to understand how technological polymers affect cell behavior when in contact with polymer materials. Therefore, the aim of this research was to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly (methyl methacrylate), polystyrene, and poly(dimethylsiloxane). Glass was used as the control material. From the Clinical Editor: Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. The aim of this research is to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly(methylmethacrylate) (PMMA), polystyrene (PS), and poly(dimethylsiloxane) (PDMS).

Keywords: Thin-films, Poly(methyl methacrylate), Osteoblast adhesion, Electron-microscopy, Fibronectin, Polystyrene, Oly(dimethylsiloxane), Biocompatibility, Hydroxyapatite, Behavior


Montufar, E. B., Traykova, T., Gil, C., Harr, I., Almirall, A., Aguirre, A., Engel, E., Planell, J. A., Ginebra, M. P., (2010). Foamed surfactant solution as a template for self-setting injectable hydroxyapatite scaffolds for bone regeneration Acta Biomaterialia 6, (3), 876-885

The application of minimally invasive surgical techniques in the field of orthopaedic surgery has created a growing need for new injectable synthetic materials that can be used for bone grafting In this work a novel fully synthetic injectable calcium phosphate foam was developed by mixing alpha-tricalcium phosphate (alpha-TCP) powder with a foamed polysorbate 80 solution Polysorbate 80 is a non-ionic surfactant approved for parenteral applications The foam was able to retain the porous structure after injection provided that the foamed paste was injected shortly after mixing (typically 2 5 min), and set through the hydrolysis of alpha-TCP to a calcium-deficient hydroxyapatite, thus producing a hydroxyapatite solid foam in situ The effect of different processing parameters on the porosity. microstructure, injectability and mechanical properties of the hydroxyapatite foams was analysed, and the ability of the pre-set foam to support osteoblastic-like cell proliferation and differentiation was assessed. Interestingly, the concentration of surfactant needed to obtain the foams was lower than that considered safe in drug formulations for parenteral administration The possibility of combining bioactivity, injectability, macroporosity and self-setting ability in a single fully synthetic material represents a step forward in the design of new materials for bone regeneration compatible with minimally invasive surgical techniques.

Keywords: Calcium phosphate cement, Hydroxyapatite foam, Scaffold, Surfactant, Injectable material


Manara, S., Paolucci, F., Palazzo, B., Marcaccio, M., Foresti, E., Tosi, G., Sabbatini, S., Sabatino, P., Altankov, G., Roveri, N., (2008). Electrochemically-assisted deposition of biomimetic hydroxyapatite-collagen coatings on titanium plate Inorganica Chimica Acta 361, (6), 1634-1645

A biomimetic bone-like composite, made of self-assembled collagen fibrils and carbonate hydroxyapatite nanocrystals, has been performed by an electrochemically-assisted deposition on titanium plate. The electrolytic processes have been carried out using a single type I collagen molecules suspension in a diluted Ca(NO3)(2) and NH4H2PO4 solution at room temperature and applying a constant current for different periods of time. Using the same electrochemical conditions, carbonate hydroxyapatite nanocrystals or reconstituted collagen. brils coatings were obtained. The reconstituted collagen. brils, hydroxyapatite nanocrystals and collagen fibrils/apatite nanocrystals coatings have been characterized chemically, structurally and morphologically, as well as for their ability to bind fibronectin (FN). Fourier Transform Infrared microscopy has been used to map the topographic distribution of the coating components at different times of electrochemical deposition, allowing to single out the individual deposition steps. Moreover, roughness of Ti plate has been found to affect appreciably the nucleation region of the inorganic nanocrystals. Laser scanning confocal microscopy has been used to characterize the FN adsorption pattern on a synthetic biomimetic apatitic phase, which exhibits a higher affinity when it is inter-grown with the collagen fibrils. The results offer auspicious applications in the preparation of medical devices such as biomimetic bone-like composite-coated metallic implants.

Keywords: Hydroxyapatite-collagen coating, Electrochemically-assisted deposition, Micro-imaging FTIR spectroscopy, Laser scanning confocal microscopy, Biomimetic crystal growth, Fibronectin binding


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