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by Keyword: yap

Elyaderani AK, De Lama-Odría MDC, Valle LJD, Puiggalí J, (2022). Multifunctional Scaffolds Based on Emulsion and Coaxial Electrospinning Incorporation of Hydroxyapatite for Bone Tissue Regeneration International Journal Of Molecular Sciences 23, 15016

Tissue engineering is nowadays a powerful tool to restore damaged tissues and recover their normal functionality. Advantages over other current methods are well established, although a continuous evolution is still necessary to improve the final performance and the range of applications. Trends are nowadays focused on the development of multifunctional scaffolds with hierarchical structures and the capability to render a sustained delivery of bioactive molecules under an appropriate stimulus. Nanocomposites incorporating hydroxyapatite nanoparticles (HAp NPs) have a predominant role in bone tissue regeneration due to their high capacity to enhance osteoinduction, osteoconduction, and osteointegration, as well as their encapsulation efficiency and protection capability of bioactive agents. Selection of appropriated polymeric matrices is fundamental and consequently great efforts have been invested to increase the range of properties of available materials through copolymerization, blending, or combining structures constituted by different materials. Scaffolds can be obtained from different processes that differ in characteristics, such as texture or porosity. Probably, electrospinning has the greater relevance, since the obtained nanofiber membranes have a great similarity with the extracellular matrix and, in addition, they can easily incorporate functional and bioactive compounds. Coaxial and emulsion electrospinning processes appear ideal to generate complex systems able to incorporate highly different agents. The present review is mainly focused on the recent works performed with Hap-loaded scaffolds having at least one structural layer composed of core/shell nanofibers.

JTD Keywords: bone tissue, coaxial electrospinning, composite nanofibers, drug-release behavior, emulsion electrospinning, hydroxyapatite, in-vitro evaluation, mechanical-properties, osteogenic differentiation, pickering emulsions, protein adsorption, structured scaffolds, surface-initiated polymerization, tissue regeneration, Bone tissue, Coaxial electrospinning, Emulsion electrospinning, Hydroxyapatite, Multifunctional scaffolds, Poly(3-hydroxybutyrate) phb patches, Tissue regeneration


Bonany M, Pérez-Berná AJ, Dučić T, Pereiro E, Martin-Gómez H, Mas-Moruno C, van Rijt S, Zhao Z, Espanol M, Ginebra MP, (2022). Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles Biomaterials Advances 142, 213148

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.

JTD Keywords: adsorption, cryo-soft x-ray tomography, cytotoxicity, expression, flow cytometry, internalisation, intracellular calcium, magnesium, nano, nanomaterials, nanoparticles, proliferation, protein corona, ultrastructure, Calcium-phosphate nanoparticles, Cryo-soft x-ray tomography, Flow cytometry, Hydroxyapatite, Internalisation, Intracellular calcium, Nanoparticles


Widhe, M, Diez-Escudero, A, Liu, YL, Ringstrom, N, Ginebra, MP, Persson, C, Hedhammar, M, Mestres, G, (2022). Functionalized silk promotes cell migration into calcium phosphate cements by providing macropores and cell adhesion motifs Ceramics International 48, 31449-31460

Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive and osteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano-and microporosity that resembles natural bone and is paramount for biological processes such as protein adhesion, which can later enhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allow cell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions through their membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity and cell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell binding motif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds with multiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features of HA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration and differentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA in the absence of silk, and importantly, the presence of silk greatly enhances cell migration into the scaffold. Additionally, cell proliferation and osteogenic differentiation are achieved in the scaffolds.

JTD Keywords: Bioceramics, Bone, Bone regeneration, Composites, Degradation, Fabrication, Hydroxyapatite, Hydroxyapatite scaffolds, Injectability, Porosity, Recombinant spider silk, Rgd motifs, Silk, Stem-cells


Mochi F, Scatena E, Rodriguez D, Ginebra MP, Del Gaudio C, (2022). Scaffold-based bone tissue engineering in microgravity: potential, concerns and implications Npj Microgravity 8, 45

One of humanity's greatest challenges is space exploration, which requires an in-depth analysis of the data continuously collected as a necessary input to fill technological gaps and move forward in several research sectors. Focusing on space crew healthcare, a critical issue to be addressed is tissue regeneration in extreme conditions. In general, it represents one of the hottest and most compelling goals of the scientific community and the development of suitable therapeutic strategies for the space environment is an urgent need for the safe planning of future long-term manned space missions. Osteopenia is a commonly diagnosed disease in astronauts due to the physiological adaptation to altered gravity conditions. In order to find specific solutions to bone damage in a reduced gravity environment, bone tissue engineering is gaining a growing interest. With the aim to critically investigate this topic, the here presented review reports and discusses bone tissue engineering scenarios in microgravity, from scaffolding to bioreactors. The literature analysis allowed to underline several key points, such as the need for (i) biomimetic composite scaffolds to better mimic the natural microarchitecture of bone tissue, (ii) uniform simulated microgravity levels for standardized experimental protocols to expose biological materials to the same testing conditions, and (iii) improved access to real microgravity for scientific research projects, supported by the so-called democratization of space.© 2022. The Author(s).

JTD Keywords: biomaterials, collagen/hydroxyapatite, composite scaffolds, in-vitro, mineralization, proliferation, regenerative medicine, stem-cells, vivo, Hydroxyapatite scaffolds


Sans, J, Arnau, M, Sanz, V, Turon, P, Aleman, C, (2022). Fine-tuning of polarized hydroxyapatite for the catalytic conversion of dinitrogen to ammonium under mild conditions Chemical Engineering Journal 446, 137440

Polarized hydroxyapatite (p-HAp), a calcium phosphate catalyst obtained at high temperature under intense electric field, has been used for the synthesis of ammonium starting from N2 and liquid water at low pressure (<6 bar) and temperatures below 120 C. The success of the nitrogen fixation process has been demonstrated by isotope labelling experiments using 15N2. Considering the optimal reaction conditions for the production of ammonium, the yield is as high as 154.6 +/- 25.8 mu mol/g of catalyst. The proposed synthesis exhibits three important advantages for its utilization in green chemistry environmental processes related to the recycling of polluted air. These are: i) the catalysts converts CO2 into valuable chemical products in addition of transforming N2 in ammonium; ii) the final energy balance is very favorable since no external electrical field is necessary to promote nitrogen and carbon fixation reactions; and iii) products are easily transferred to water favoring their extraction and avoiding the saturation of the catalyst.

JTD Keywords: Adsorbed nitrogen, Air pollution, Amino-acids, Electrophotosynthesis, Environmental process, Facile synthesis, Fixation, Functionalization, Hydroxyapatite, Nitride, Nitrogen reduction, Polarized catalyst


De Lama-Odría, María del Carmen, del Valle, Luis J., Puiggalí, Jordi, (2022). Hydroxyapatite Biobased Materials for Treatment and Diagnosis of Cancer International Journal Of Molecular Sciences 23, 11352

Great advances in cancer treatment have been undertaken in the last years as a consequence of the development of new antitumoral drugs able to target cancer cells with decreasing side effects and a better understanding of the behavior of neoplastic cells during invasion and metastasis. Specifically, drug delivery systems (DDS) based on the use of hydroxyapatite nanoparticles (HAp NPs) are gaining attention and merit a comprehensive review focused on their potential applications. These are derived from the intrinsic properties of HAp (e.g., biocompatibility and biodegradability), together with the easy functionalization and easy control of porosity, crystallinity and morphology of HAp NPs. The capacity to tailor the properties of DLS based on HAp NPs has well-recognized advantages for the control of both drug loading and release. Furthermore, the functionalization of NPs allows a targeted uptake in tumoral cells while their rapid elimination by the reticuloendothelial system (RES) can be avoided. Advances in HAp NPs involve not only their use as drug nanocarriers but also their employment as nanosystems for magnetic hyperthermia therapy, gene delivery systems, adjuvants for cancer immunotherapy and nanoparticles for cell imaging.

JTD Keywords: antitumoral, cell imaging, controlled-release, drug-carrier, efficient drug-delivery, fatty-acid-metabolism, fe3o4 nanoparticles, gene delivery, hydroxyapatite, hyperthermia, immunotherapy, in-vitro, magnetic hydroxyapatite, nano-hydroxyapatite, protein adsorption, tumor-growth, Calcium-phosphate nanoparticles, Cancer


Bonany, M, del-Mazo-Barbara, L, Espanol, M, Ginebra, MP, (2022). Microsphere incorporation as a strategy to tune the biological performance of bioinks Journal Of Tissue Engineering 13,

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

JTD Keywords: 3d bioprinting, Alginate, Behavior, Bioink, Cell-culture, Gelatin, Gelatine, Hydrogels, Hydroxyapatite, Laden, Microspheres, Mineralization, Scaffolds


Sans, J, Arnau, M, Roa, JJ, Turon, P, Alernan, C, (2022). Tailorable Nanoporous Hydroxyapatite Scaffolds for Electrothermal Catalysis Acs Applied Nano Materials 5, 8526-8536

Polarized hydroxyapatite (HAp) scaffolds with customized architecture at the nanoscale have been presented as a green alternative to conventional catalysts used for carbon and dinitrogen fixation. HAp printable inks with controlled nanoporosity and rheological properties have been successfully achieved by incorporating Pluronic hydrogel. Nanoporous scaffolds with good mechanical properties, as demonstrated by means of the nanoindentation technique, have been obtained by a sintering treatment and the posterior thermally induced polarization process. Their catalytic activity has been evaluated by considering three different key reactions (all in the presence of liquid water): (1) the synthesis of amino acids from gas mixtures of N-2, CO2, and CH4; (2) the production of ethanol from gas mixtures of CO2 and CH4; and (3) the synthesis of ammonia from N-2 gas. Comparison of the yields obtained by using nanoporous and nonporous (conventional) polarized HAp catalysts shows that both the nanoporosity and water absorption capacity of the former represent a drawback when the catalytic reaction requires auxiliary coating layers, as for example for the production of amino acids. This is because the surface nanopores achieved by incorporating Pluronic hydrogel are completely hindered by such auxiliary coating layers. On the contrary, the catalytic activity improves drastically for reactions in which the HAp-based scaffolds with enhanced nanoporosity are used as catalysts. More specifically, the carbon fixation from CO2 and CH4 to yield ethanol improves by more than 3000% when compared with nonporous HAp catalyst. Similarly, the synthesis of ammonia by dinitrogen fixation increases by more than 2000%. Therefore, HAp catalysts based on nanoporous scaffolds exhibit an extraordinary potential for scalability and industrial utilization for many chemical reactions, enabling a feasible green chemistry alternative to catalysts based on heavy metals.

JTD Keywords: Amino acids, Amino-acids, Ammonium production, Bone, Carbon fixation, Composites, Constitutive phases, Decarbonization, Dinitrogen, Ditrogen fixation, Elastic-modulus, Electrophotosynthesis, Ethanol production, Hardness, Indentation, Nanoindentation, Pluronic hydrogel, Polarized hydroxyapatite


Sans, Jordi, Arnau, Marc, Turon, Pau, Alemán, Carlos, (2022). Permanently polarized hydroxyapatite, an outstanding catalytic material for carbon and nitrogen fixation Materials Horizons 9, 1566-1576

Permanently polarized hydroxyapatite is a new material with electrical enhanced properties. This review discusses the advances in this material in terms of structure, properties and catalytic activity of green processes.

JTD Keywords: ammonia, bone, copper hydroxyapatite, electrophotosynthesis, nanoparticles, oxidation, phase-transition, reduction, Amino-acids


Bohner, Marc, Maazouz, Yassine, Ginebra, Maria-Pau, Habibovic, Pamela, Schoenecker, Jonathan G., Seeherman, Howard, van den Beucken, Jeroen, Witte, Frank, (2022). Sustained local ionic homeostatic imbalance caused by calcification modulates inflammation to trigger heterotopic ossification Acta Biomaterialia 145, 1-24

Heterotopic ossification (HO) is a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues. Despite being a frequent complication of orthopedic and trauma surgery, brain and spinal injury, the etiology of HO is poorly understood. The aim of this study is to evaluate the hypothesis that a sustained local ionic homeostatic imbalance (SLIHI) created by mineral formation during tissue calcification modulates inflammation to trigger HO. This evaluation also considers the role SLIHI could play for the design of cell-free, drug-free osteoinductive bone graft substitutes. The evaluation contains five main sections. The first section defines relevant concepts in the context of HO and provides a summary of proposed causes of HO. The second section starts with a detailed analysis of the occurrence and involvement of calcification in HO. It is followed by an explanation of the causes of calcification and its consequences. This allows to speculate on the potential chemical modulators of inflammation and triggers of HO. The end of this second section is devoted to in vitro mineralization tests used to predict the ectopic potential of materials. The third section reviews the biological cascade of events occurring during pathological and material-induced HO, and attempts to propose a quantitative timeline of HO formation. The fourth section looks at potential ways to control HO formation, either acting on SLIHI or on inflammation. Chemical, physical, and drug-based approaches are considered. Finally, the evaluation finishes with a critical assessment of the definition of osteoinduction.

JTD Keywords: beta-tricalcium phosphate, bone, bone graft, bone morphogenetic protein, demineralized bone-matrix, experimental myositis-ossificans, extracellular calcium, heterotopic ossification, in-vitro, inflammation, multinucleated giant-cells, osteoinduction, spinal-cord-injury, total hip-arthroplasty, traumatic brain-injury, Apatite, Calcium-sensing receptor


Marhuenda, Esther, Villarino, Alvaro, Narciso, Maria Leonor, Camprubí-Rimblas, Marta, Farré, Ramon, Gavara, Núria, Artigas, Antonio, Almendros, Isaac, Otero, Jorge, (2022). Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells International Journal Of Molecular Sciences 23, 4888

One of the main limitations of in vitro studies on lung diseases is the difficulty of maintaining the type II phenotype of alveolar epithelial cells in culture. This fact has previously been related to the translocation of the mechanosensing Yes-associated protein (YAP) to the nuclei and Rho signaling pathway. In this work, we aimed to culture and subculture primary alveolar type II cells on extracellular matrix lung-derived hydrogels to assess their suitability for phenotype maintenance. Cells cultured on lung hydrogels formed monolayers and maintained type II phenotype for a longer time as compared with those conventionally cultured. Interestingly, cells successfully grew when they were subsequently cultured on a dish. Moreover, cells cultured on a plate showed the active form of the YAP protein and the formation of stress fibers and focal adhesions. The results of chemically inhibiting the Rho pathway strongly suggest that this is one of the mechanisms by which the hydrogel promotes type II phenotype maintenance. These results regarding protein expression strongly suggest that the chemical and biophysical properties of the hydrogel have a considerable impact on the transition from ATII to ATI phenotypes. In conclusion, culturing primary alveolar epithelial cells on lung ECM-derived hydrogels may facilitate the prolonged culturing of these cells, and thus help in the research on lung diseases.

JTD Keywords: adhesion, alveolar cells, expression, hydrogels, pathway, surfactant, type ii phenotype, yap, Extracellular matrix, Transplantation


Iglesias-Fernandez, M, Buxadera-Palomero, J, Sadowska, JM, Espanol, M, Ginebra, MP, (2022). Implementation of bactericidal topographies on biomimetic calcium phosphates and the potential effect of its reactivity Biomaterials Advances 136, 212797

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

JTD Keywords: Adhesion, Antibacterial, Bactericidal, Biomaterials, Calcium deficient hydroxyapatite, Calcium phosphates, Hydroxyapatite, In-vitro, Infections, Nanopillars, Pseudomonas aeruginosa, Pseudomonas-aeruginosa, Reactivity, Recent progress, Silver, Topography, Transmission


Sans J, Arnau M, Sanz V, Turon P, Alemán C, (2022). Hydroxyapatite-based biphasic catalysts with plasticity properties and its potential in carbon dioxide fixation Chemical Engineering Journal 433, 133512

The design of catalysts with controlled selectivity at will, also known as catalytic plasticity, is a very attractive approach for the recycling of carbon dioxide (CO2). In this work, we study how catalytically active hydroxyapatite (HAp) and brushite (Bru) interact synergistically, allowing the production of formic acid or acetic acid depending on the HAp/Bru ratio in the catalyst. Raman, wide angle X-ray scattering, X-ray photoelectron spectroscopy, scanning electron microscopy and electrochemical impedance spectroscopy studies, combined with an exhaustive revision of the crystalline structure of the catalyst at the atomic level, allowed to discern how the Bru phase can be generated and stabilized at high temperatures. Results clearly indicate that the presence of OH– groups to maintain the crystalline structural integrity in conjunction with Ca2+ ions less bonded to the lattice fixate carbon into C1, C2 and C3 molecules from CO2 and allow the evolution from formic to acetic acid and acetone. In this way, the plasticity of the HAp-Bru system is demonstrated, representing a promising green alternative to the conventional metal-based electrocatalysts used for CO2 fixation. Thus, the fact that no electric voltage is necessary for the CO2 reduction has a very favorable impact in the final energetic net balance of the carbon fixation reaction. © 2021

JTD Keywords:

ethanol production & nbsp, brushite, co2 reduction, conversion, electrocatalytic reduction, electrode, formate, heterogeneous catalysis & nbsp, hydrogen evolution, insights, monetite, polarized hydroxyapatite,

, Acetic acid, Acetone, Biphasic catalyst, Brushite, Calcium phosphate, Carbon dioxide, Carbon dioxide fixation, Catalysis, Catalyst selectivity, Co 2 reduction, Co2 reduction, Electrocatalysts, Electrochemical impedance spectroscopy, Electrochemical reduction, Electrochemical-impedance spectroscopies, Ethanol production, Formic acid, Heterogeneous catalysis, Hydroxyapatite, Ph, Polarized hydroxyapatite, Property, Reduction, Scanning electron microscopy, Temperature programmed desorption, Wide angle x-ray scattering, X ray photoelectron spectroscopy, X ray scattering, ]+ catalyst


Raymond, Yago, Johansson, Linh, Thorel, Emilie, Ginebra, Maria-Pau, (2022). Translation of three-dimensional printing of ceramics in bone tissue engineering and drug delivery Mrs Bulletin 47, 59-69

Sans, Jordi, Arnau, Marc, Sanz, Vanesa, Turon, Pau, Alemán, Carlos, (2022). Polarized Hydroxyapatite: New Insights and Future Perspectives Through Systematic Electrical Characterization at the Interface Advanced Materials Interfaces 9, 2101631

Kadkhodaie-Elyaderani A, de Lama-Odría MC, Rivas M, Martínez-Rovira I, Yousef I, Puiggalí J, Del Valle LJ, (2022). Medicated Scaffolds Prepared with Hydroxyapatite/Streptomycin Nanoparticles Encapsulated into Polylactide Microfibers International Journal Of Molecular Sciences 23, 1282

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanopar-ticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaf-folds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concen-trations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: antibiotics, antimicrobial activity, behavior, cytotoxicity, delivery, drug, drug delivery, hydroxyapatite nanoparticles, in-vitro, mechanisms, mitochondria, polylactide, release, streptomycin, Antimicrobial activity, Cancer stem-cells, Cytotoxicity, Drug delivery, Hydroxyapatite nanoparticles, Polylactide, Streptomycin


Boda, SK, Aparicio, C, (2022). Dual keratinocyte-attachment and anti-inflammatory coatings for soft tissue sealing around transmucosal oral implants Biomaterials Science 10, 665-677

Unlike the attachment of soft epithelial skin tissue to penetrating solid natural structures like fingernails and teeth, sealing around percutaneous/permucosal devices such as dental implants is hindered by inflammation and epidermal down growth. Here, we employed a dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium to promote oral epithelial tissue attachment. For minimizing inflammation-triggered epidermal down growth, we coated pristine and oxygen plasma pre-treated polished titanium (pTi) with conjugated linoleic acid (CLA). Further, in order to aid in soft tissue attachment via the formation of hemidesmosomes, adhesive structures by oral keratinocytes, we coated the anionic linoleic acid (LA) adsorbed titanium with cationic cell adhesive peptides (CAP), LamLG3, a peptide derived from Laminin 332, the major extracellular matrix component of the basement membrane in skin tissue and Net1, derived from Netrin-1, a neural chemoattractant capable of epithelial cell attachment via alpha 6 beta 4 integrins. The dual CLA-CAP coatings on pTi were characterized by X-ray photoelectron spectroscopy and dynamic water contact angle measurements. The proliferation of human oral keratinocytes (TERT-2/OKF6) was accelerated on the peptide coated titanium while also promoting the expression of Col XVII and beta-4 integrin, two markers for hemidesmosomes. Simultaneously, CLA coating suppressed the production of inducible nitric oxide synthase (anti-iNOS); a pro-inflammatory M1 marker expressed in lipopolysaccharide (LPS) stimulated murine macrophages (RAW 264.7) and elevated expression of anti-CD206, associated to an anti-inflammatory M2 macrophage phenotype. Taken together, the dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium can help reduce inflammation and promote permucosal/peri-implant soft tissue sealing.

JTD Keywords: Adhesives, Animal, Animals, Anti-inflammatories, Anti-inflammatory agents, Antiinflammatory agent, Biomolecules, Bone, Cell adhesion, Cell-adhesives, Coatings, Conjugated linoleic acid, Conjugated linoleic-acid, Contact angle, Hemidesmosome, Hemidesmosomes, Human, Humans, Hydroxyapatite, Inflammation, Integrins, Keratinocyte, Keratinocytes, Linoleic acid, Macrophages, Mice, Mouse, Nitric oxide, Oral implants, Pathology, Peptides, Skin tissue, Soft tissue, Supplementation, Surface properties, Surface property, Tissue, Titania, Titanium, X ray photoelectron spectroscopy


Sans, Jordi, Sanz, Vanesa, Turon, Pau, Alemán, Carlos, (2021). Enhanced CO2 Conversion into Ethanol by Permanently Polarized Hydroxyapatite through C-C Coupling Chemcatchem 13, 5025-5033

Raymond Y, Pastorino D, Ginebreda I, Maazouz Y, Ortiz M, Manzanares M-C, Ginebra M-P, (2021). Computed tomography and histological evaluation of xenogenic and biomimetic bone grafts in three-wall alveolar defects in minipigs Clinical Oral Investigations 25, 6695-6706

Objectives This study aimed to compare the performance of a xenograft (XG) and a biomimetic synthetic graft (SG) in three-wall alveolar defects in minipigs by means of 3D computerised tomography and histology. Materials and methods Eight minipigs were used. A total of eight defects were created in the jaw of each animal, three of which were grafted with XGs, three with SGs, and two were left empty as a negative control. The allocation of the different grafts was randomised. Four animals were euthanised at 6 weeks and four at 12 weeks. The grafted volume was then measured by spiral computed tomography to assess volume preservation. Additionally, a histological analysis was performed in undecalcified samples by backscattered scanning electron microscopy and optical microscopy after Masson's trichrome staining. Results A linear mixed-effects model was applied considering four fixed factors (bone graft type, regeneration time, anatomic position, and maxilla/mandible) and one random factor (animal). The SG exhibited significantly larger grafted volume (19%) than the XG. The anterior sites preserved better the grafted volume than the posterior ones. Finally, regeneration time had a positive effect on the grafted volume. Histological observations revealed excellent osseointegration and osteoconductive properties for both biomaterials. Some concavities found in the spheroidal morphologies of SGs were associated with osteoclastic resorption. Conclusions Both biomaterials met the requirements for bone grafting, i.e. biocompatibility, osseointegration, and osteoconduction. Granule morphology was identified as an important factor to ensure a good volume preservation.

JTD Keywords: bone graft, bone regeneration, in vivo, miniature swine, synthetic graft, 3-dimensional changes, Anorganic bovine bone, Autogenous bone, Bio-oss, Biomaterials, Bone graft, Bone regeneration, Calcium-phosphate, Hydroxyapatite, In vivo, Miniature swine, Sinus floor augmentation, Substitute, Synthetic graft, Volume, Xenograft


Brennan M, Monahan DS, Brulin B, Gallinetti S, Humbert P, Tringides C, Canal C, Ginebra MP, Layrolle P, (2021). Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects Acta Biomaterialia 135, 689-704

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

JTD Keywords: beta-tricalcium phosphate, bone regeneration, calcium deficient hydroxyapatite, differentiation, engraftment, human bone marrow mesenchymal stromal cells, hydroxyapatite scaffolds, in-vitro, inhibition, osteogenesis, osteoinduction, stem-cells, surface-topography, tissue, Beta-tricalcium phosphate, Bone regeneration, Calcium deficient hydroxyapatite, Engraftment, Human bone marrow mesenchymal stromal cells


Raymond Y, Bonany M, Lehmann C, Thorel E, Benítez R, Franch J, Espanol M, Solé-Martí X, Manzanares MC, Canal C, Ginebra MP, (2021). Hydrothermal processing of 3D-printed calcium phosphate scaffolds enhances bone formation in vivo: a comparison with biomimetic treatment Acta Biomaterialia 135, 671-688

Hydrothermal (H) processes accelerate the hydrolysis reaction of α-tricalcium phosphate (α-TCP) compared to the long-established biomimetic (B) treatments. They are of special interest for patient-specific 3D-printed bone graft substitutes, where the manufacturing time represents a critical constraint. Altering the reaction conditions has implications for the physicochemical properties of the reaction product. However, the impact of the changes produced by the hydrothermal reaction on the in vivo performance was hitherto unknown. The present study compares the bone regeneration potential of 3D-printed α-TCP scaffolds hardened using these two treatments in rabbit condyle monocortical defects. Although both consolidation processes resulted in biocompatible scaffolds with osseointegrative and osteoconductive properties, the amount of newly formed bone increased by one third in the hydrothermal vs the biomimetic samples. B and H scaffolds consisted mostly of high specific surface area calcium-deficient hydroxyapatite (38 and 27 m2 g-1, respectively), with H samples containing also 10 wt.% β-tricalcium phosphate (β-TCP). The shrinkage produced during the consolidation process was shown to be very small in both cases, below 3%, and smaller for H than for B samples. The differences in the in vivo performance were mainly attributed to the distinct crystallisation nanostructures, which proved to have a major impact on permeability and protein adsorption capacity, using BSA as a model protein, with B samples being highly impermeable. Given the crucial role that soluble proteins play in osteogenesis, this is proposed to be a relevant factor behind the distinct in vivo performances observed for the two materials. Statement of significance: The possibility to accelerate the consolidation of self-setting calcium phosphate inks through hydrothermal treatments has aroused great interest due to the associated advantages for the development of 3D-printed personalised bone scaffolds. Understanding the implications of this approach on the in vivo performance of the scaffolds is of paramount importance. This study compares, for the first time, this treatment to the long-established biomimetic setting strategy in terms of osteogenic potential in vivo in a rabbit model, and relates the results obtained to the physicochemical properties of the 3D-printed scaffolds (composition, crystallinity, nanostructure, nanoporosity) and their interaction with soluble proteins.

JTD Keywords: 3d printing, behavior, biomimetic, bone scaffolds, calcium phosphate, deficient hydroxyapatite, design, graft, hydrothermal, in vivo, morbidity, osteoinduction, porosity, standard, tricalcium phosphate, 3d printing, Biomimetic, Bone scaffolds, Calcium phosphate, Fibula free-flap, Hydrothermal, In vivo


Konka J, Buxadera-Palomero J, Espanol M, Ginebra M-P, (2021). 3D printing of hierarchical porous biomimetic hydroxyapatite scaffolds: Adding concavities to the convex filaments Acta Biomaterialia 134, 744-759

Porosity plays a key role on the osteogenic performance of bone scaffolds. Direct Ink Writing (DIW) allows the design of customized synthetic bone grafts with patient-specific architecture and controlled macroporosity. Being an extrusion-based technique, the scaffolds obtained are formed by arrays of cylindrical filaments, and therefore have convex surfaces. This may represent a serious limitation, as the role of surface curvature and more specifically the stimulating role of concave surfaces in osteoinduction and bone growth has been recently highlighted. Hence the need to design strategies that allow the introduction of concave pores in DIW scaffolds. In the current study, we propose to add gelatin microspheres as a sacrificial material in a self-setting calcium phosphate ink. Neither the phase transformation responsible for the hardening of the scaffold nor the formation of characteristic network of needle-like hydroxyapatite crystals was affected by the addition of gelatin microspheres. The partial dissolution of the gelatin resulted in the creation of spherical pores throughout the filaments and exposed on the surface, increasing filament porosity from 0.2 % to 67.9 %. Moreover, the presence of retained gelatin proved to have a significant effect on the mechanical properties, reducing the strength but simultaneously giving the scaffolds an elastic behavior, despite the high content of ceramic as a continuous phase. Notwithstanding the inherent difficulty of in vitro cultures with this highly reactive material an enhancement of MG-63 cell proliferation, as well as better spreading of hMSCs was recorded on the developed scaffolds. Statement of significance: Recent studies have stressed the role that concave surfaces play in tissue regeneration and, more specifically, in osteoinduction and osteogenesis. Direct ink writing enables the production of patient-specific bone grafts with controlled architecture. However, besides many advantages, it has the serious limitation that the surfaces obtained are convex. In this article, for the first time we develop a strategy to introduce concave pores in the printed filaments of biomimetic hydroxyapatite by incorporation and partial dissolution of gelatin microspheres. The retention of part of the gelatin results in a more elastic behavior compared to the brittleness of hydroxyapatite scaffolds, while the needle-shaped nanostructure of biomimetic hydroxyapatite is maintained and gelatin-coated concave pores on the surface of the filaments enhance cell spreading. © 2021 The Authors

JTD Keywords: 3d printing, bioceramics, biomimetic, bone, bone regeneration, concavity, concavity, bone regeneration, gelatin, hydrogel, hydroxyapatite, microspheres, osteoinduction, porosity, porous filament, substitutes, tissue-growth, 3d printing, Biomimetic, Calcium-phosphate scaffolds, Concavity, bone regeneration, Gelatin, Hydroxyapatite, Porous filament


Konka, J, Espanol, M, Bosch, BM, de Oliveira, E, Ginebra, MP, (2021). Maturation of biomimetic hydroxyapatite in physiological fluids: a physicochemical and proteomic study Materials Today Bio 12,

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

JTD Keywords: calcium phosphates, ion exchange, nanostructure, protein adsorption, Biological-systems, Biomaterials, Biomimetic hydroxyapatites, Biomimetics, Bone-formation, Calcium deficient hydroxyapatite, Calcium phosphate, Calcium phosphates, Cell proliferation, Crystal structure, Crystallinity, Crystals structures, Culture medium, Growth, High reactivity, Hydroxyapatite, In-vitro, Ion exchange, Ionic exchange, Molecular biology, Nanocrystalline apatites, Nanostructure, Nanostructures, Octacalcium phosphate, Physicochemical studies, Physiological fluids, Physiology, Protein adsorption, Proteins, Proteomic studies, Raman spectroscopy, Serum-albumin, Specific surface area


Andreu, I, Falcones, B, Hurst, S, Chahare, N, Quiroga, X, Le Roux, AL, Kechagia, Z, Beedle, AEM, Elosegui-Artola, A, Trepat, X, Farre, R, Betz, T, Almendros, I, Roca-Cusachs, P, (2021). The force loading rate drives cell mechanosensing through both reinforcement and cytoskeletal softening Nature Communications 12,

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat


Olmo C, Franco L, Vidal A, Del Valle LJ, Puiggalí J, (2021). Ultrasound micromolding of porous polylactide/hydroxyapatite scaffolds Express Polymer Letters 15, 389-403

© BME-PT. Ultrasound micromolding (USM) preparation of hybrid scaffolds based on polylactide (PLA) and hydroxyapatite (HAp) particles has been evaluated. PLA was stable under the applied ultrasound source since a minimum degradation was detected. Porous materials were achieved using polyethylene glycol (PEG) and NaCl salts to the initial PLA and the subsequent leaching of the micromolded specimens. To avoid cavitation and decomposition problems during micromolding, it was necessary to use HAp free of typical synthesis impurities like carbonate and nitrate compounds. Compact PLA/HAp pieces allowed a maximum HAp load of 60 wt%, while porous specimens could be obtained with a maximum load of 38 wt%. Physical characterization of new scaffolds was performed by X-ray diffraction, spectroscopic and calorimetric techniques, stress-strain tests and contact angle measurements. Results indicated that a degree of porosity of 35% and relatively good mechanical properties could be achieved (i.e., 580 MPa, 4%, and 15.6 MPa for the Young modulus, elongation at break, and tensile strength, respectively). Scaffolds showed the positive effect of HAp and porosity on cell proliferation; this latter was 40% higher than that detected for non-porous PLA specimens.

JTD Keywords: apatite, conformation, fabrication, hydroxyapatite, micropieces, polymers, porous scaffolds, proliferation, tissue, ultrasound micromolding, vibration, Composite scaffolds, Hydroxyapatite, Micropieces, Porous scaffolds, Processing technologies, Ultrasound micromolding


Sans, J, Sanz, V, del Valle, LJ, Puiggali, J, Turon, P, Aleman, C, (2021). Optimization of permanently polarized hydroxyapatite catalyst. Implications for the electrophotosynthesis of amino acids by nitrogen and carbon fixation Journal Of Catalysis 397, 98-107

The enhanced catalytic activity of permanently polarized hydroxyapatite, which is achieved using a thermally stimulated polarization process, largely depends on both the experimental conditions used to prepare crystalline hydroxyapatite from its calcium and phosphate precursors and the polarization process parameters. A mineral similar to brushite, which is an apatitic phase that can evolve to hydroxyapatite, is found at the surface of highly crystalline hydroxyapatite. It appears after chemical precipitation and hydrothermal treatment performed at 150 degrees C for 24 h followed by a sinterization at 1000 degrees C and a polarization treatment by applying a voltage of 500 Vat high temperature. Both the high crystallinity and the presence of brushite-like phase on the electrophotocatalyst affect the nitrogen and carbon fixation under mild reaction conditions (95 degrees C and 6 bar) and the synthesis of glycine and alanine from a simple gas mixture containing N-2, CO2, CH4 and H2O. Thus, the Gly/Ala ratio can be customized by controlling the presence of brushite on the surface of the catalyst, enabling to develop new strategies to regulate the production of amino acids by nitrogen and carbon fixation. (C) 2021 Elsevier Inc. All rights reserved.

JTD Keywords: Amino acids, Brushite, Carbon, Carbon dioxide fixation, Catalyst activity, Catalytic apatites, Chemical precipitation, Crystalline hydroxyapatite, Crystallinity, Decomposition, Enhanced catalytic activity, Experimental conditions, Heterogeneous catalysis, High crystallinity, Hydrothermal synthesis, Hydrothermal treatments, Hydroxyapatite, Lactic-acid, Mild reaction conditions, Molecular nitrogen fixation, Nitrogen, Nitrogen fixation, Phosphate, Polarization, Precipitation (chemical), Process parameters, Thermally stimulated polarization


Vidal, E, Guillem-Marti, J, Ginebra, MP, Combes, C, Ruperez, E, Rodriguez, D, (2021). Multifunctional homogeneous calcium phosphate coatings: Toward antibacterial and cell adhesive titanium scaffolds Surface & Coatings Technology 405,

Implants for orthopedic applications need to be biocompatible and bioactive, with mechanical properties similar to those of surrounding natural bone. Given this scenario titanium (Ti) scaffolds obtained by Direct Ink Writing technique offer the opportunity to manufacture customized structures with controlled porosity and mechanical properties. Considering that 3D Ti scaffolds have a significant surface area, it is necessary to develop strategies against the initial bacterial adhesion in order to prevent infection in the early stages of the implantation, while promoting cell adhesion to the scaffold. The challenge is not only achieving a balance between antibacterial activity and osseointegration, it is also to develop a homogeneous coating on the inner and outer surface of the scaffold. The purpose of this work was the development of a single-step electrodeposition process in order to uniformly cover Ti scaffolds with a layer of calcium phosphate (CaP) loaded with chlorhexidine digluconate (CHX). Scaffold characterization was assessed by scanning electron microscopy, Energy dispersive X-ray spectroscopy, X-ray diffraction, micro-Raman microscopy and compressive strength tests. Results determined that the surface of scaffolds was covered by plate-like and whisker-like calcium phosphate crystals, which main phases were octacalcium phosphate and brushite. Biological tests showed that the as-coated scaffolds reduced bacteria adhesion (73 +/- 3% for Staphylococcus aureus and 70 +/- 2% for Escherichia coli). In vitro cell studies and confocal analysis revealed the adhesion and spreading of osteoblast-like SaOS-2 on coated surfaces. Therefore, the proposed strategy can be a potential candidate in bone replacing surgeries.

JTD Keywords: Antibacterial, Bacterial, Behavior, Biocompatibility, Calcium phosphate coating, Chlorhexidine, Chlorhexidine digluconate, Deposition, Electrodeposition, Hydroxyapatite coatings, Implants, One-step pulse electrodeposition, Plasma-spray, Release, Surface, Titanium scaffolds


Revilla-López, G., Sans, J., Casanovas, J., Bertran, O., Puiggalí, J., Turon, P., Alemán, C., (2020). Analysis of nitrogen fixation by a catalyst capable of transforming N2, CO2 and CH4 into amino acids under mild reactions conditions Applied Catalysis A: General 596, 117526

The processes related to the fixation of nitrogen in a catalyst able to produce glycine and alanine from a N2, CO2 and CH4 gas mixture at mild reaction conditions have been studied by combining experimental and theoretical investigations. Results have allowed to understand the role of different elements of the catalyst, which is constituted by permanently polarized hydroxyapatite (p-HAp), zirconia, and aminotris(methylenephosphonic acid) (ATMP). ATMP attracts N2 molecules towards the surface, maintaining them close to the zirconia and p-HAp components that are the most active from a catalytic point of view. On the other hand, the associative mechanism is thermodynamically favoured under mild reaction conditions with respect to the dissociative one, which is limited by the barrier associated to the Nsingle bondN bond cleavage. Because this reaction mechanism is similar to that employed in the nitrogen fixation by nitrogenase enzymes, these findings provide an opportunity to design new bioinspired catalysts.

JTD Keywords: Artificial photosynthesis, Carbon fixation, Hydroxyapatite, N[sbnd]N bond cleavage


Sans, J., Armelin, E., Sanz, V., Puiggalí, J., Turon, P., Alemán, C., (2020). Breaking-down the catalyst used for the electrophotosynthesis of amino acids by nitrogen and carbon fixation Journal of Catalysis 389, 646-656

The electrophotocatalytic synthesis of Glycine and Alanine from a simple gas mixture containing N2, CO2, CH4 and H2O under mild reaction conditions (95 °C and 6 bar) was recently developed using a catalyst formed by permanently polarized hydroxyapatite, which is achieved using a thermally stimulated polarization process, coated with two layers of aminotris(methylenephosphonic acid) (ATMP) separated by an intermediate layer of zirconyl chloride (ZC). This work reports the optimization of the ATMP- and ZC-coating content by examining the influence of their concentration of each component in each layer on the structural and electrochemical properties of the catalyst. After exhaustive analyses, such properties have been related with the efficiency of the catalysts prepared using different ATMP- and ZC-concentrations to yield Gly and Ala amino acids by fixing nitrogen from N2 and carbon from CO2 and CH4. Results show that, although the concentrations of ATMP and ZC in the first and the intermediate layers are important, the third layer plays a predominant role as is responsible of the apparition of supramolecular structures on the surface and the capacitive behavior of the coating

JTD Keywords: Carbon dioxide fixation, Electrocatalyst, Heterogeneous catalysis, Phosphonic acid, Photocatalyst, Polarized hydroxyapatite, Surface chemistry, Zirconyl chloride


Casanellas, Ignasi, Lagunas, Anna, Vida, Yolanda, Pérez-Inestrosa, Ezequiel, Andrades, J. A., Becerra, J., Samitier, Josep, (2020). The Janus role of adhesion in chondrogenesis International Journal of Molecular Sciences 21, (15), 5269

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell–cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell–substrate adhesion in the tissue engineering strategies for cartilage repair.

JTD Keywords: Dendrimer, Nanopatterning, RGD, Mesenchymal cell condensation, Cell–cell interactions, YAP, Chondrogenesis


Diez-Escudero, A., Torreggiani, E., Di Pompo, G., Espanol, M., Persson, C., Ciapetti, G., Baldini, N., Ginebra, M. P., (2019). Effect of calcium phosphate heparinization on the in vitro inflammatory response and osteoclastogenesis of human blood precursor cells Journal of Tissue Engineering and Regenerative Medicine 13, (7), 1217-1229

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

JTD Keywords: Biomaterial, Heparin, Hydroxyapatite, Inflammation, Osteoclastogenesis


Sadowska, Joanna M., Wei, Fei, Guo, Jia, Guillem-Marti, Jordi, Ginebra, Maria-Pau, Xiao, Yin, (2018). Effect of nano-structural properties of biomimetic hydroxyapatite on osteoimmunomodulation Biomaterials 181, 318-332

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7 cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines.

JTD Keywords: Calcium phosphates, Biomimetic hydroxyapatite, Osteoimmunomodulation, Inflammation, Osteogenesis, Osteoclastogesis


Raymond, Santiago, Maazouz, Yassine, Montufar, Edgar B., Perez, Roman A., González, Borja, Konka, Joanna, Kaiser, Jozef, Ginebra, Maria-Pau, (2018). Accelerated hardening of nanotextured 3D-plotted self-setting calcium phosphate inks Acta Biomaterialia 75, 451-462

Direct ink writing (DIW) techniques open up new possibilities for the fabrication of patient-specific bone grafts. Self-setting calcium phosphate inks, which harden at low temperature, allow obtaining nanostructured scaffolds with biomimetic properties and enhanced bioactivity. However, the slow hardening kinetics hampers the translation to the clinics. Different hydrothermal treatments for the consolidation of DIW scaffolds fabricated with an α-tricalcium phosphate /pluronic F127 ink were explored, comparing them with a biomimetic treatment. Three different scaffold architectures were analysed. The hardening process, associated to the conversion of α-tricalcium phosphate to hydroxyapatite was drastically accelerated by the hydrothermal treatments, reducing the time for complete reaction from 7 days to 30 minutes, while preserving the scaffold architectural integrity and retaining the nanostructured features. β-tricalcium phosphate was formed as a secondary phase, and a change of morphology from plate-like to needle-like crystals in the hydroxyapatite phase was observed. The binder was largely released during the treatment. The hydrothermal treatment resulted in a 30% reduction of the compressive strength, associated to the residual presence of β-tricalcium phosphate. Biomimetic and hydrothermally treated scaffolds supported the adhesion and proliferation of rat mesenchymal stem cells, indicating a good suitability for bone tissue engineering applications. Statement of Significance: 3D plotting has opened up new perspectives in the bone regeneration field allowing the customisation of synthetic bone grafts able to fit patient-specific bone defects. Moreover, this technique allows the control of the scaffolds’ architecture and porosity. The present work introduces a new method to harden biomimetic hydroxyapatite 3D-plotted scaffolds which avoids high-temperature sintering. It has two main advantages: i) it is fast and simple, reducing the whole fabrication process from the several days required for the biomimetic processing to a few hours; and ii) it retains the nanostructured character of biomimetic hydroxyapatite and allows controlling the porosity from the nano- to the macroscale. Moreover, the good in vitro cytocompatibility results support its suitability for cell-based bone regeneration therapies.

JTD Keywords: Calcium phosphate, Hydroxyapatite, Biomimetic, Bone regeneration, 3D plotting, Direct ink writing, Bone graft


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.

JTD 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.

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


Sadowska, J. M., Guillem-Marti, J., Montufar, E. B., Espanol, M., Ginebra, M. P., (2017). Biomimetic versus sintered calcium phosphates: The in vitro behavior of osteoblasts and mesenchymal stem cells Tissue Engineering Part A , 23, (23-24), 1297-1309

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

JTD Keywords: Biomimetic hydroxyapatite, Calcium phosphate, Mesenchymal stem cell, Osteoblast


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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

JTD 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 β-glycerophosphate, but not from DMEM medium. Non-linear sorption data could be perfectly described by pseudo-first-order and pseudo-second-order sorption models. At longer contact time (21 days), and with frequent renewal of culture medium, sorption of Ca2+ remained constant throughout the experiment, while sorption of Pi gradually decreased in McCoy medium. In great contrast, CDHA began to release Pi slowly with time when using DMEM medium. Infrared spectra showed that CDHA exposed to culture media had a carbonated surface chemistry, suggesting that carbonate plays a key role in the ion reactivity of CDHA. Our data show that different compositions of the aqueous environment may provoke opposite ion reactivity of CDHA, and this must be carefully considered when evaluating the osteoinductive potential of the material.

JTD 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).

JTD 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.

JTD 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.

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