Publications

by Keyword: Regeneration


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Oliveira, H., Catros, S., Castano, O., Rey, S., Siadous, R., Clift, D., Marti-Munoz, J., Batista, M., Bareille, R., Planell, J., Engel, E., Amédée, J., (2017). The proangiogenic potential of a novel calcium releasing composite biomaterial: Orthotopic in vivo evaluation Acta Biomaterialia 54, 377-385

Insufficient angiogenesis remains a major hurdle in current bone tissue engineering strategies. An extensive body of work has focused on the use of angiogenic factors or endothelial progenitor cells. However, these approaches are inherently complex, in terms of regulatory and methodologic implementation, and present a high cost. We have recently demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate (CaP) ormoglass particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. Here we have devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a (Hydroxypropyl)methyl cellulose (HPMC) matrix, with the capacity to release calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. The bone regeneration kinetics was dependent on the Ca2+ release rate, with the faster Ca2+ release composite gel showing improved bone repair at 3 weeks, in relation to control. In the same line, improved angiogenesis could be observed for the same gel formulation at 6 weeks post implantation. This methodology allows to integrate two fundamental processes for bone tissue regeneration while using a simple, cost effective, and safe approach. Statement of Significance In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, we have shown that calcium ions, released by the degradation of calcium phosphate ormoglasses (CaP), are effective angiogenic promoters, in both in vitro and in a subcutaneous implantation model. Here, we devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a HPMC matrix, enabling the release of calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. This simple and cost effective approach holds great promise to translate to the clinics.

Keywords: Angiogenesis, Bone regeneration, Calcium phosphate ormoglasses


Diez-Escudero, A., Espanol, M., Montufar, E. B., Di Pompo, G., Ciapetti, G., Baldini, N., Ginebra, M. P., (2017). Focus ion beam/scanning electron microscopy characterization of osteoclastic resorption of calcium phosphate substrates Tissue Engineering Part C: Methods 23, (2), 118-124

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

Keywords: Bone Regeneration, Calcium Phosphate, Focus Ion Beam, Osteoclast, Resorption, Scanning Electron Microscopy


Oliveira, Hugo, Catros, Sylvain, Boiziau, Claudine, Siadous, Robin, Marti-Munoz, Joan, Bareille, Reine, Rey, Sylvie, Castano, Oscar, Planell, Josep, Amédée, Joëlle, Engel, Elisabeth, (2016). The proangiogenic potential of a novel calcium releasing biomaterial: Impact on cell recruitment Acta Biomaterialia 29, 435-445

Abstract In current bone tissue engineering strategies the achievement of sufficient angiogenesis during tissue regeneration is still a major limitation in order to attain full functionality. Several strategies have been described to tackle this problem, mainly by the use of angiogenic factors or endothelial progenitor cells. However, when facing a clinical scenario these approaches are inherently complex and present a high cost. As such, more cost effective alternatives are awaited. Here, we demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate ormoglass (CaP) particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. We show that the current approach elicited the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial. As both PLA and CaP are currently accepted for clinical application these off-the-shelf novel membranes have great potential for guided bone regeneration applications. Statement of significance In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, our group has found that calcium ions released by the degradation of calcium phosphate ormoglasses (CaP) are effective angiogenic promoters. Based on this, in this work we successfully produced hybrid fibrous mats with different contents of CaP nanoparticles and thus with different calcium ion release rates, using an ormoglass – poly(lactic acid) blend approach. We show that these matrices, upon implantation in a subcutaneous site, could elicit the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial, in a CaP dose dependent manner. This off-the-shelf cost effective approach presents great potential to translate to the clinics.

Keywords: Angiogenesis, Bone regeneration, Calcium phosphate ormoglass


Kovtun, A., Goeckelmann, M. J., Niclas, A. A., Montufar, E. B., Ginebra, M. P., Planell, J. A., Santin, M., Ignatius, A., (2015). In vivo performance of novel soybean/gelatin-based bioactive and injectable hydroxyapatite foams Acta Biomaterialia Elsevier Ltd 12, (1), 242-249

Major limitations of calcium phosphate cements (CPCs) are their relatively slow degradation rate and the lack of macropores allowing the ingrowth of bone tissue. The development of self-setting cement foams has been proposed as a suitable strategy to overcome these limitations. In previous work we developed a gelatine-based hydroxyapatite foam (G-foam), which exhibited good injectability and cohesion, interconnected porosity and good biocompatibility in vitro. In the present study we evaluated the in vivo performance of the G-foam. Furthermore, we investigated whether enrichment of the foam with soybean extract (SG-foam) increased its bioactivity. G-foam, SG-foam and non-foamed CPC were implanted in a critical-size bone defect in the distal femoral condyle of New Zealand white rabbits. Bone formation and degradation of the materials were investigated after 4, 12 and 20 weeks using histological and biomechanical methods. The foams maintained their macroporosity after injection and setting in vivo. Compared to non-foamed CPC, cellular degradation of the foams was considerably increased and accompanied by new bone formation. The additional functionalization with soybean extract in the SG-foam slightly reduced the degradation rate and positively influenced bone formation in the defect. Furthermore, both foams exhibited excellent biocompatibility, implying that these novel materials may be promising for clinical application in non-loaded bone defects.

Keywords: Bone regeneration, Calcium phosphate cement, Gelatine, Rabbit model, Soybean


Álvarez, Z., Castaño, O., Castells, A. A., Mateos-Timoneda, M. A., Planell, J. A., Engel, E., Alcántara, S., (2014). Neurogenesis and vascularization of the damaged brain using a lactate-releasing biomimetic scaffold Biomaterials 35, (17), 4769-4781

Regenerative medicine strategies to promote recovery following traumatic brain injuries are currently focused on the use of biomaterials as delivery systems for cells or bioactive molecules. This study shows that cell-free biomimetic scaffolds consisting of radially aligned electrospun poly-l/dl lactic acid (PLA70/30) nanofibers release l-lactate and reproduce the 3D organization and supportive function of radial glia embryonic neural stem cells. The topology of PLA nanofibers supports neuronal migration while l-lactate released during PLA degradation acts as an alternative fuel for neurons and is required for progenitor maintenance. Radial scaffolds implanted into cavities made in the postnatal mouse brain fostered complete implant vascularization, sustained neurogenesis, and allowed the long-term survival and integration of the newly generated neurons. Our results suggest that the endogenous central nervous system is capable of regeneration through the invivo dedifferentiation induced by biophysical and metabolic cues, with no need for exogenous cells, growth factors, or genetic manipulation.

Keywords: Lactate, Nanofibers, Neural stem cells, Neurogenesis, Regeneration, Vascularization


Vila, O. F., Martino, M. M., Nebuloni, L., Kuhn, G., Pérez-Amodio, S., Müller, R., Hubbell, J. A., Rubio, N., Blanco, J., (2014). Bioluminescent and micro-computed tomography imaging of bone repair induced by fibrin-binding growth factors Acta Biomaterialia 10, (10), 4377-4389

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.

Keywords: Angiogenesis, Bioluminescence imaging, Bone regeneration, Fibrin, Mesenchymal stem cell


Pérez-Amodio, Soledad, Engel, Elisabeth, (2014). Bone biology and Regeneration Bio-Ceramics with Clinical Applications (ed. Vallet-Regí, M.), John Wiley & Sons, Ltd (Chichester, UK) , 315-342

Each bone of the skeleton constantly undergoes modeling during life to help it to adapt to changing biomechanical forces as well as remodeling to remove old bone and replace it with new, mechanically stronger bone to help preserve bone strength. Bone remodeling involves the removal of mineralized bone by osteoclasts, followed by the formation of bone matrix through the osteoblasts that subsequently become mineralized. All these assets make bone a suitable model for regeneration. Bone tissue can be grossly divided into inorganic mineral material (mostly HA), and organic material from cells and the extracellular matrix. This chapter outlines some of the bone diseases such as osteoporosis and Paget's disease. Bone can be considered as a biphasic composite material, with two phases: one the mineral and the other collagen. This combination confers better mechanical properties on the tissue than each component itself.

Keywords: Bone biology, Bone cells, Bone diseases, Bone extracellular matrix, Bone mechanics, Bone remodeling, Bone tissue regeneration, Skeleton


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


Riggio, C., Nocentini, S., Catalayud, M. P., Goya, G. F., Cuschieri, A., Raffa, V., del Río, J. A., (2013). Generation of magnetized olfactory ensheathing cells for regenerative studies in the central and peripheral nervous tissue International Journal of Molecular Sciences 14, (6), 10852-10868

As olfactory receptor axons grow from the peripheral to the central nervous system (CNS) aided by olfactory ensheathing cells (OECs), the transplantation of OECs has been suggested as a plausible therapy for spinal cord lesions. The problem with this hypothesis is that OECs do not represent a single homogeneous entity, but, instead, a functionally heterogeneous population that exhibits a variety of responses, including adhesion and repulsion during cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. In this paper, we report a system based on modified OECs carrying magnetic nanoparticles as a proof of concept experiment enabling specific studies aimed at exploring the potential of OECs in the treatment of spinal cord injuries. Our studies have confirmed that magnetized OECs (i) survive well without exhibiting stress-associated cellular responses; (ii) in vitro, their migration can be modulated by magnetic fields; and (iii) their transplantation in organotypic slices of spinal cord and peripheral nerve showed positive integration in the model. Altogether, these findings indicate the therapeutic potential of magnetized OECs for CNS injuries.

Keywords: Magnetic nanoparticle, Nerve regeneration, Olfactory ensheathing cell, Organotypic culture


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


Castaño, O., Eltohamy, M., Kim, H. W., (2012). Electrospinning technology in tissue regeneration Nanotechnology in Regenerative Medicine - Methods and Protocols (Methods in Molecular Biology) (ed. Navarro, M., Planell, J. A.), Springer (New York, USA) 811, 127-140

Electrospinning is one of the most versatile and effective tools to produce nanostructured fibers in the biomedical science fields. The nanofibrous structure with diameters from tens to hundreds of nanometers largely mimics the native extracellular matrix (ECM) of many tissues. Thus far, a range of compositions including polymers and ceramics and their composites/hybrids have been successfully applied for generating electrospun nanofibers. Different processing tools in electrospinning set-ups and assemblies are currently developed to tune the morphology and properties of nanofibers. Herein, we demonstrate the electrospinning process and the electrospun biomaterials for specific use in tissue regeneration with some examples, involving different material combinations and fiber morphologies.

Keywords: Ceramic, Composites, Electrospinning, Nanofi bers, Nanostructured fi bers, Polymer, Tissue regeneration


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


Byrne, Damien P., Lacroix, Damien, Prendergast, Patrick J., (2011). Simulation of fracture healing in the tibia: Mechanoregulation of cell activity using a lattice modeling approach Journal of Orthopaedic Research 29, (10), 1496-1503

In this study, a three-dimensional (3D) computational simulation of bone regeneration was performed in a human tibia under realistic muscle loading. The simulation was achieved using a discrete lattice modeling approach combined with a mechanoregulation algorithm to describe the cellular processes involved in the healing process namely proliferation, migration, apoptosis, and differentiation of cells. The main phases of fracture healing were predicted by the simulation, including the bone resorption phase, and there was a qualitative agreement between the temporal changes in interfragmentary strain and bending stiffness by comparison to experimental data and clinical results. Bone healing was simulated beyond the reparative phase by modeling the transition of woven bone into lamellar bone. Because the simulation has been shown to work with realistic anatomical 3D geometry and muscle loading, it demonstrates the potential of simulation tools for patient-specific pre-operative treatment planning.

Keywords: Tissue differentiation, Computational analysis, Mechanical conditions, Bone regeneration, Weight-bearing, Proliferation, Osteoblast, Stiffness, Ingrowth, Scaffold


Ginebra, M. P., Espanol, M., Montufar, E. B., Perez, R. A., Mestres, G., (2010). New processing approaches in calcium phosphate cements and their applications in regenerative medicine Acta Biomaterialia 6, (8), 2863-2873

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

Keywords: Bone regeneration, Calcium phosphate cements, Granules, Microcarriers, Scaffolds


Messeguer, J., Masip, I., Montolio, M., del Rio, J. A., Soriano, E., Messeguer, A., (2010). Peptoids bearing tertiary amino residues in the n-alkyl side chains: synthesis of a potent inhibitor of Semaphorin 3A Tetrahedron 66, (13), 2444-2454

A study on the preparation of N-alkylglycines (peptoids) that contain tertiary amino residues on the N-alkyl side chains is reported. The appropriate combination of the submonomer strategy with N-alkylglycine monomer couplings depending upon the structure of the N-alkyl side chain that must be incorporated into the peptoid is determinant for the efficiency of the synthetic pathway. The application of this strategy to the preparation of SICHI, an N-alkyglycine trimer containing tertiary amino residues in the three N-alkyl branches, and that has been identified as a potent Semaphorin 3A inhibitor, is presented.

Keywords: Peptoids, N-Alkylglycine monomers, Solid-phase synthesis, Semaphorin inhibition, Axonal regeneration


Rajzer, I., Castano, O., Engel, E., Planell, J. A., (2010). Injectable and fast resorbable calcium phosphate cement for body-setting bone grafts Journal of Materials Science-Materials in Medicine 21, (7), 2049-2056

In this work a calcium phosphate (CPC)/polymer blend was developed with the advantage of being moldable and capable of in situ setting to form calcium deficient hydroxyapatite under physiological conditions in an aqueous environment at body temperature. The CPC paste consists in a mix of R cement, glycerol as a liquid phase carrier and a biodegradable hydrogel such as Polyvinyl alcohol, which acts as a binder. Microstructure and mechanical analysis shows that the CPC blend can be used as an injectable implant for low loaded applications and fast adsorption requirements. The storage for commercial distribution was also evaluated and the properties of the materials obtained do not significantly change during storage at -18A degrees C.

Keywords: Clinical-applications, Composites, Regeneration, Behavior, Scaffold, Repair


Sanzana, E. S., Navarro, M., Macule, F., Suso, S., Planell, J. A., Ginebra, M. P., (2008). Of the in vivo behavior of calcium phosphate cements and glasses as bone substitutes Acta Biomaterialia 4, (6), 1924-1933

The use of injectable self-setting calcium phosphate cements or soluble glass granules represent two different strategies for bone regeneration, each with distinct advantages and potential applications. This study compares the in vivo behavior of two calcium phosphate cements and two phosphate glasses with different composition, microstructure and solubility, using autologous bone as a control, in a rabbit model. The implanted materials were alpha-tricalcium phosphate cement (cement H), calcium sodium potassium phosphate cement (cement R), and two phosphate glasses in the P2O5-CaO-Na2O and P2O5-CaO-Na2O-TiO2 systems. The four materials were osteoconductive, biocompatible and biodegradable. Radiological and histological studies demonstrated correct osteointegration and substitution of the implants by new bone. The reactivity of the different materials, which depends on their solubility, porosity and specific surface area, affected the resorption rate and bone formation mainly during the early stages of implantation, although this effect was weak. Thus, at 4 weeks the degradation was slightly higher in cements than in glasses, especially for cement R. However, after 12 weeks of implantation all materials showed a similar degradation degree and promoted bone neoformation equivalent to that of the control group.

Keywords: Calcium phosphates, Calcium phosphate cements, Phosphate glasses, Bone grafts, Bone regenerations


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