Publications by authors named "Maria-Pau Ginebra"

98 Publications

Cold Atmospheric Plasma: A New Strategy Based Primarily on Oxidative Stress for Osteosarcoma Therapy.

J Clin Med 2021 Feb 23;10(4). Epub 2021 Feb 23.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Engineering, Escola d'Enginyeria Barcelona Est (EEBE), Universitat Politècnica de Catalunya (UPC), 08930 Barcelona, Spain.

Osteosarcoma is the most common primary bone tumor, and its first line of treatment presents a high failure rate. The 5-year survival for children and teenagers with osteosarcoma is 70% (if diagnosed before it has metastasized) or 20% (if spread at the time of diagnosis), stressing the need for novel therapies. Recently, cold atmospheric plasmas (ionized gases consisting of UV-Vis radiation, electromagnetic fields and a great variety of reactive species) and plasma-treated liquids have been shown to have the potential to selectively eliminate cancer cells in different tumors through an oxidative stress-dependent mechanism. In this work, we review the current state of the art in cold plasma therapy for osteosarcoma. Specifically, we emphasize the mechanisms unveiled thus far regarding the action of plasmas on osteosarcoma. Finally, we review current and potential future approaches, emphasizing the most critical challenges for the development of osteosarcoma therapies based on this emerging technique.
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http://dx.doi.org/10.3390/jcm10040893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926371PMC
February 2021

Quantification of Plasma-Produced Hydroxyl Radicals in Solution and their Dependence on the pH.

Anal Chem 2021 03 17;93(8):3666-3670. Epub 2021 Feb 17.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), c. Eduard Maristany 16, 08019 Barcelona, Spain.

HO radicals are the most important reactive species generated during water treatment by non-thermal plasma devices. In this letter, we report the first quantification of the steady-state concentration and lifetime of plasma-produced hydroxyl radicals in water solutions at pH 3 and 7, and we discuss the differences based on their reactivity with other plasma-generated species. Finally, we show to what extent the use of chemical probes to quantify short-lived reactive species has an influence on the results and that it should be taken into account.
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http://dx.doi.org/10.1021/acs.analchem.0c04906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931173PMC
March 2021

Plasma-Conditioned Liquids as Anticancer Therapies In Vivo: Current State and Future Directions.

Cancers (Basel) 2021 Jan 25;13(3). Epub 2021 Jan 25.

Biomaterials, Biomechanics and Tissue Engineering Group, Department Materials Science and Engineering, Escola d'Enginyeria Barcelona Est (EEBE), Universitat Politècnica de Catalunya (UPC), 08930 Barcelona, Spain.

Plasma-conditioned liquids (PCL) are gaining increasing attention in the medical field, especially in oncology, and translation to the clinics is advancing on a good path. This emerging technology involving cold plasmas has great potential as a therapeutic approach in cancer diseases, as PCL have been shown to selectively kill cancer cells by triggering apoptotic mechanisms without damaging healthy cells. In this context, PCL can be injected near the tumor or intratumorally, thereby allowing the treatment of malignant tumors located in internal organs that are not accessible for direct cold atmospheric plasma (CAP) treatment. Therefore, PCL constitutes a very interesting and minimally invasive alternative to direct CAP treatment in cancer therapy, avoiding surgeries and allowing multiple local administrations. As the field advances, it is progressively moving to the evaluation of the therapeutic effects of PCL in in vivo scenarios. Exciting developments are pushing forward the clinical translation of this novel therapy. However, there is still room for research, as the quantification and identification of reactive oxygen and nitrogen species (RONS) in in vivo conditions is not yet clarified, dosage regimens are highly variable among studies, and other more relevant in vivo models could be used. In this context, this work aims to present a critical review of the state of the field of PCL as anticancer agents applied in in vivo studies.
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http://dx.doi.org/10.3390/cancers13030452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865855PMC
January 2021

Electroresponsive Alginate-Based Hydrogels for Controlled Release of Hydrophobic Drugs.

ACS Biomater Sci Eng 2020 11 8;6(11):6228-6240. Epub 2020 Oct 8.

Departament d'Enginyeria Química, EEBE, Universitat Politècnica de Catalunya, C/ Eduard Maristany, 10-14, Barcelona 08019, Spain.

Stimuli-responsive biomaterials have attracted significant attention for the construction of on-demand drug release systems. The possibility of using external stimulation to trigger drug release is particularly enticing for hydrophobic compounds, which are not easily released by simple diffusion. In this work, an electrochemically active hydrogel, which has been prepared by gelling a mixture of poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) and alginate (Alg), has been loaded with curcumin (CUR), a hydrophobic drug with a wide spectrum of clinical applications. The PEDOT/Alg hydrogel is electrochemically active and organizes as segregated PEDOT- and Alg-rich domains, explaining its behavior as an electroresponsive drug delivery system. When loaded with CUR, the hydrogel demonstrates a controlled drug release upon application of a negative electrical voltage. Comparison with the release profiles obtained applying a positive voltage and in the absence of electrical stimuli indicates that the release mechanism dominating this system is complex because of not only the intermolecular interactions between the drug and the polymeric network but also the loading of a hydrophobic drug in a water-containing delivery system.
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http://dx.doi.org/10.1021/acsbiomaterials.0c01400DOI Listing
November 2020

A versatile click chemistry-based approach for functionalizing biomaterials of diverse nature with bioactive peptides.

Chem Commun (Camb) 2021 Jan 13;57(8):982-985. Epub 2021 Jan 13.

Department of Materials Science and Engineering, Biomaterials, Biomechanics and Tissue Engineering Group (BBT), Universitat Politècnica de Catalunya, Spain.

A novel and versatile toolkit approach for the functionalization of biomaterials of different nature is described. This methodology is based on the solid-phase conjugation of specific anchoring units onto a resin-bound azido-functionalized peptide by using click chemistry. A synergistic multifunctional peptidic scaffold with cell adhesive properties was used as a model compound to showcase the versatility of this new approach. Titanium, gold and polylactic acid surfaces were biofunctionalized by this method, as validated by physicochemical surface characterization with XPS. In vitro assays using mesenchymal stem cells showed enhanced cell adhesion on the functionalized samples, proving the capacity of this strategy to efficiently bioactivate different types of biomaterials.
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http://dx.doi.org/10.1039/d0cc07463bDOI Listing
January 2021

Osteosarcoma tissue-engineered model challenges oxidative stress therapy revealing promoted cancer stem cell properties.

Free Radic Biol Med 2021 Feb 2;164:107-118. Epub 2021 Jan 2.

Biomaterials, Biomechanics and Tissue Engineering Group, Department Materials Science and Metallurgy, Technical University of Catalonia (UPC), Escola D'Enginyeria Barcelona Est (EEBE), C/Eduard Maristany 14, 08019, Barcelona, Spain; Barcelona Research Center in Multiscale Science and Engineering, UPC, 08019, Barcelona, Spain; Research Centre for Biomedical Engineering (CREB), UPC, 08019, Barcelona, Spain. Electronic address:

The use of oxidative stress generated by Cold Atmospheric Plasma (CAP) in oncology is being recently studied as a novel potential anti-cancer therapy. However, the beneficial effects of CAP for treating osteosarcoma have mostly been demonstrated in 2-dimensional cultures of cells, which do not mimic the complexity of the 3-dimensional (3D) bone microenvironment. In order to evaluate the effects of CAP in a relevant context of the human disease, we developed a 3D tissue-engineered model of osteosarcoma using a bone-like scaffold made of collagen type I and hydroxyapatite nanoparticles. Human osteosarcoma cells cultured within the scaffold showed a high capacity to infiltrate and proliferate and to exhibit osteomimicry in vitro. As expected, we observed significantly different functional behaviors between monolayer and 3D cultures when treated with Cold Plasma-Activated Ringer's Solution (PAR). Our data reveal that the 3D environment not only protects cells from PAR-induced lethality by scavenging and diminishing the amount of reactive oxygen and nitrogen species generated by CAP, but also favours the stemness phenotype of osteosarcoma cells. This is the first study that demonstrates the negative effect of PAR on cancer stem-like cell subpopulations in a 3D biomimetic model of cancer. These findings will allow to suitably re-focus research on plasma-based therapies in future.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.12.437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921834PMC
February 2021

An Engineered Biomimetic Peptide Regulates Cell Behavior by Synergistic Integrin and Growth Factor Signaling.

Adv Healthc Mater 2021 Apr 18;10(7):e2001757. Epub 2020 Dec 18.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Engineering, Universitat Politècnica de Catalunya (UPC), Barcelona, 08019, Spain.

Recreating the healing microenvironment is essential to regulate cell-material interactions and ensure the integration of biomaterials. To repair bone, such bioactivity can be achieved by mimicking its extracellular matrix (ECM) and by stimulating integrin and growth factor (GF) signaling. However, current approaches relying on the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entail clinical risks. Here, a biomimetic peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif derived from the wrist epitope of BMP-2 is presented. The approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors. Such multifunctional platform is designed to incorporate 3,4-dihydroxyphenylalanine to bind metallic oxides with high affinity in a one step process. Functionalization of glass substrates with the engineered peptide is characterized by physicochemical methods, proving a successful surface modification. The biomimetic interfaces significantly improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. These effects are not observed on surfaces displaying only one bioactive motif, a mixture of both motifs or soluble DWIVA. These data prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics.
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http://dx.doi.org/10.1002/adhm.202001757DOI Listing
April 2021

Chemically Diverse Multifunctional Peptide Platforms with Antimicrobial and Cell Adhesive Properties.

Chembiochem 2021 Mar 20;22(5):839-844. Epub 2020 Nov 20.

Department of Materials Science and Engineering, Biomaterials Biomechanics and Tissue Engineering Group (BBT), Universitat Politècnica de Catalunya (UPC), Diagonal, 647, 08019, Barcelona, Spain.

Bacterial infections and incomplete biomaterial integration are major problems that can lead to the failure of medical implants. However, simultaneously addressing these two issues remains a challenge. Here, we present a chemical peptide library based on a multifunctional platform containing the antimicrobial peptide LF1-11 and the cell-adhesive motif RGD. The scaffolds were customized with catechol groups to ensure straightforward functionalization of the implant surface, and linkers of different length to assess the effect of peptide accessibility on the biological response. The peptidic platforms significantly improved the adhesion of mesenchymal stem cells and showed antimicrobial effects against Staphylococcus aureus. Of note is that peptides bearing spacers that were too long displayed the lowest efficiency. Subsequently, we designed a platform replacing linear RGD by cyclic RGD; this further enhanced eukaryotic cell adhesion while retaining excellent antimicrobial properties, thus being a suitable candidate for tissue engineering applications.
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http://dx.doi.org/10.1002/cbic.202000670DOI Listing
March 2021

Enhanced Generation of Reactive Species by Cold Plasma in Gelatin Solutions for Selective Cancer Cell Death.

ACS Appl Mater Interfaces 2020 Oct 6;12(42):47256-47269. Epub 2020 Oct 6.

Biomaterials, Biomechanics and Tissue Engineering Group, Universitat Politècnica de Catalunya, Av. Eduard Maristany 10-14, 08019 Barcelona, Spain.

Atmospheric pressure plasma jets generate reactive oxygen and nitrogen species (RONS) in liquids and biological media, which find application in the new area of plasma medicine. These plasma-treated liquids were demonstrated recently to possess selective properties on killing cancer cells and attracted attention toward new plasma-based cancer therapies. These allow for local delivery by injection in the tumor but can be quickly washed away by body fluids. By confining these RONS in a suitable biocompatible delivery system, great perspectives can be opened in the design of novel biomaterials aimed for cancer therapies. Gelatin solutions are evaluated here to store RONS generated by atmospheric pressure plasma jets, and their release properties are evaluated. The concentration of RONS was studied in 2% gelatin as a function of different plasma parameters (treatment time, nozzle distance, and gas flow) with two different plasma jets. Much higher production of reactive species (HO and NO) was revealed in the polymer solution than in water after plasma treatment. The amount of RONS generated in gelatin is greatly improved with respect to water, with concentrations of HO and NO between 2 and 12 times higher for the longest plasma treatments. Plasma-treated gelatin exhibited the release of these RONS to a liquid media, which induced an effective killing of bone cancer cells. Indeed, in vitro studies on the sarcoma osteogenic (SaOS-2) cell line exposed to plasma-treated gelatin led to time-dependent increasing cytotoxicity with the longer plasma treatment time of gelatin. While the SaOS-2 cell viability decreased to 12%-23% after 72 h for cells exposed to 3 min of treated gelatin, the viability of healthy cells (hMSC) was preserved (∼90%), establishing the selectivity of the plasma-treated gelatin on cancer cells. This sets the basis for designing improved hydrogels with high capacity to deliver RONS locally to tumors.
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http://dx.doi.org/10.1021/acsami.0c12930DOI Listing
October 2020

Inflammation and biomaterials: role of the immune response in bone regeneration by inorganic scaffolds.

J Mater Chem B 2020 10;8(41):9404-9427

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Engineering, Universitat Politècnica de Catalunya, Av. Eduard Maristany 16, 08019 Barcelona, Spain. and Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028 Barcelona, Spain.

The regulatory role of the immune system in maintaining bone homeostasis and restoring its functionality, when disturbed due to trauma or injury, has become evident in recent years. The polarization of macrophages, one of the main constituents of the immune system, into the pro-inflammatory or anti-inflammatory phenotype has great repercussions for cellular crosstalk and the subsequent processes needed for proper bone regeneration such as angiogenesis and osteogenesis. In certain scenarios, the damaged osseous tissue requires the placement of synthetic bone grafts to facilitate the healing process. Inorganic biomaterials such as bioceramics or bioactive glasses are the most widely used due to their resemblance to the mineral phase of bone and superior osteogenic properties. The immune response of the host to the inorganic biomaterial, which is of an exogenous nature, might determine its fate, leading either to active bone regeneration or its failure. Therefore, various strategies have been employed, like the modification of structural/chemical features or the incorporation of bioactive molecules, to tune the interplay with the immune cells. Understanding how these particular modifications impact the polarization of macrophages and further osteogenic and osteoclastogenic events is of great interest in view of designing a new generation of osteoimmunomodulatory materials that support the regeneration of osseous tissue during all stages of bone healing.
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http://dx.doi.org/10.1039/d0tb01379jDOI Listing
October 2020

Use of three-dimensionally printed β-tricalcium phosphate synthetic bone graft combined with recombinant human bone morphogenic protein-2 to treat a severe radial atrophic nonunion in a Yorkshire terrier.

Vet Surg 2020 Dec 8;49(8):1626-1631. Epub 2020 Jul 8.

Biomaterials, Biomechanics, and Tissue Engineering Group, Materials Science and Metallurgical Engineering Department, Universitat Politècnica de Catalunya, Barcelona, Spain.

Objective: To describe a novel surgical approach to treat a critical-sized bone defect due to severe, radial atrophic nonunion in a miniature dog.

Study Design: Case report ANIMAL: A 1-year-old Yorkshire terrier with a critical-sized left radial defect after failed internal fixation of a transverse radial fracture.

Methods: Computed tomographic (CT) images of the radius were imported for three-dimensional (3D) printing of a custom-designed synthetic 3D-printed β-tricalcium phosphate (β-TCP) scaffold. The radius was exposed, and the β-TCP scaffold was press-fitted in the bone gap underneath the plate. Recombinant human bone morphogenic protein-2 (RhBMP-2) collagen sponges were squeezed to soak the scaffold with growth factor and then placed on both sides of the synthetic graft. Two additional cortical screws were also placed prior to routine closure of the surgical site.

Results: Radiographic examination was consistent with complete healing of the radius defect 4 months after surgery. The bone plate was removed 10 months after surgery. According to CT examination 18 months after surgery, there was no evidence of the synthetic graft; instead, complete corticalization of the affected area was noted. Complete functional recovery was observed until the last clinical follow-up 36 months postoperatively.

Conclusion: Screw fixation and use of a 3D-printed ceramic scaffold augmented with rhBMP-2 resulted in excellent bone regeneration of the nonunion and full recovery of a miniature breed dog.

Clinical Significance: The therapeutic approach used in this dog could be considered as an option for treatment of large-bone defects in veterinary orthopedics, especially for defects affecting the distal radius of miniature dogs.
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http://dx.doi.org/10.1111/vsu.13476DOI Listing
December 2020

Regeneration of segmental defects in metatarsus of sheep with vascularized and customized 3D-printed calcium phosphate scaffolds.

Sci Rep 2020 04 27;10(1):7068. Epub 2020 Apr 27.

Inserm, UMR 1238, PHY-OS, Bone sarcomas and remodelling of calcified tissues, Faculty of Medicine, University of Nantes, Nantes, 44035, France.

Although autografts are considered to be the gold standard treatment for reconstruction of large bone defects resulting from trauma or diseases, donor site morbidity and limited availability restrict their use. Successful bone repair also depends on sufficient vascularization and to address this challenge, novel strategies focus on the development of vascularized biomaterial scaffolds. This pilot study aimed to investigate the feasibility of regenerating large bone defects in sheep using 3D-printed customized calcium phosphate scaffolds with or without surgical vascularization. Pre-operative computed tomography scans were performed to visualize the metatarsus and vasculature and to fabricate customized scaffolds and surgical guides by 3D printing. Critical-sized segmental defects created in the mid-diaphyseal region of the metatarsus were either left empty or treated with the 3D scaffold alone or in combination with an axial vascular pedicle. Bone regeneration was evaluated 1, 2 and 3 months post-implantation. After 3 months, the untreated defect remained non-bridged while the 3D scaffold guided bone regeneration. The presence of the vascular pedicle further enhanced bone formation. Histology confirmed bone growth inside the porous 3D scaffolds with or without vascular pedicle inclusion. Taken together, this pilot study demonstrated the feasibility of precised pre-surgical planning and reconstruction of large bone defects with 3D-printed personalized scaffolds.
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http://dx.doi.org/10.1038/s41598-020-63742-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184564PMC
April 2020

Effect of Allogeneic Cell-Based Tissue-Engineered Treatments in a Sheep Osteonecrosis Model.

Tissue Eng Part A 2020 09 27;26(17-18):993-1004. Epub 2020 Mar 27.

Musculoskeletal Tissue Engineering Group, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.

Osteonecrosis of the femoral head (ONFH) is defined as a tissue disorder and successive subchondral bone collapse resulting from an ischemic process, which may progress to hip osteoarthritis. Cell therapy with multipotent bone marrow mesenchymal stromal cells (BM-MSC) of autologous origin appears to be safe and has shown regenerative potential in previous preclinical and clinical studies. The use of allogeneic cells is far more challenging, but may be a promising alternative to use of autologous cells. Moreover, an optimized dosage of cells from an allogeneic source is needed to obtain off-the-shelf tissue engineering products (TEPs). The purpose of this study was to evaluate the efficacy of a TEP composed of undifferentiated expanded BM-MSC of allogeneic origin, combined with bone matrix particles in variable doses. A comparative analysis of TEP's bone regenerative properties against its autologous counterpart was performed in an early-stage ONFH preclinical model in mature sheep. Allogeneic BM-MSC groups demonstrated bone regeneration capacity in osteonecrotic lesions equivalent to autologous BM-MSC groups 6 weeks after treatment. Likewise, stimulation of bone regeneration by a low cell dose of 0.5 × 10 BM-MSC/cm was equivalent to that of a high cell dose, 5 × 10 BM-MSC/cm. Neither local nor systemic immunological reactions nor tumorigenesis were reported, strengthening the safety profile of allogeneic BM-MSC therapy in this model. Our results suggest that low-dose allogeneic BM-MSC is sufficient to promote bone regeneration in femoral head osteonecrotic lesions, and should be considered in translation of new allogeneic cell-based TEPs to human clinics. Impact statement Cell therapy and tissue engineering hold promise as novel regenerative therapies for musculoskeletal diseases, and particularly in bone regeneration strategies. In this article, we report the evaluation of the efficacy of an allogeneic cell-based tissue engineering product (TEP) in an early-stage osteonecrosis of the femoral head preclinical model in skeletally mature sheep. Moreover, we demonstrate its bone regeneration capacity and safety and its equivalence to autologous counterparts. These findings have important implications for the translation of new allogeneic cell-based TEPs to human clinics.
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http://dx.doi.org/10.1089/ten.TEA.2019.0339DOI Listing
September 2020

Cold Plasma-Treated Ringer's Saline: A Weapon to Target Osteosarcoma.

Cancers (Basel) 2020 Jan 17;12(1). Epub 2020 Jan 17.

Biomaterials, Biomechanics and Tissue Engineering Group, Department Materials Science and Metallurgy, Technical University of Catalonia (UPC), Escola d'Enginyeria Barcelona Est (EEBE), c/Eduard Maristany 14, 08019 Barcelona, Spain.

Osteosarcoma (OS) is the main primary bone cancer, presenting poor prognosis and difficult treatment. An innovative therapy may be found in cold plasmas, which show anti-cancer effects related to the generation of reactive oxygen and nitrogen species in liquids. In vitro models are based on the effects of plasma-treated culture media on cell cultures. However, effects of plasma-activated saline solutions with clinical application have not yet been explored in OS. The aim of this study is to obtain mechanistic insights on the action of plasma-activated Ringer's saline (PAR) for OS therapy in cell and organotypic cultures. To that aim, cold atmospheric plasma jets were used to obtain PAR, which produced cytotoxic effects in human OS cells (SaOS-2, MG-63, and U2-OS), related to the increasing concentration of reactive oxygen and nitrogen species generated. Proof of selectivity was found in the sustained viability of hBM-MSCs with the same treatments. Organotypic cultures of murine OS confirmed the time-dependent cytotoxicity observed in 2D. Histological analysis showed a decrease in proliferating cells (lower Ki-67 expression). It is shown that the selectivity of PAR is highly dependent on the concentrations of reactive species, being the differential intracellular reactive oxygen species increase and DNA damage between OS cells and hBM-MSCs key mediators for cell apoptosis.
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http://dx.doi.org/10.3390/cancers12010227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017095PMC
January 2020

Production of reactive species in alginate hydrogels for cold atmospheric plasma-based therapies.

Sci Rep 2019 11 6;9(1):16160. Epub 2019 Nov 6.

Biomaterials, Biomechanics and Tissue Engineering Group, Dpt. Materials Science and Metallurgy, Universitat Politècnica de Catalunya (UPC), Escola d'Enginyeria Barcelona Est (EEBE), c/Eduard Maristany 14, 08019, Barcelona, Spain.

In the last years, great advances have been made in therapies based in cold atmospheric plasmas (CAP). CAP generate reactive oxygen and nitrogen species (RONS) which can be transferred to liquids. These CAP activated liquids display the same biological efficacy (i.e. on killing cancer cells) as CAP themselves, opening the door for minimally invasive therapies. However, injection of a liquid in the body results in fast diffusion due to extracellular fluids and blood flow. Therefore, the development of efficient vehicles which allow local confinement and delivery of RONS to the diseased site is a fundamental requirement. In this work, we investigate the generation of RONS (HO, NO, short-lived RONS) in alginate hydrogels by comparing two atmospheric pressure plasma jets: kINPen and a helium needle, at a range of plasma treatment conditions (time, gas flow, distance to the sample). The physic-chemical properties of the hydrogels remain unchanged by the plasma treatment, while the hydrogel shows several-fold larger capacity for generation of RONS than a typical isotonic saline solution. Part of the RONS are quickly released to a receptor media, so special attention has to be put on the design of hydrogels with in-situ crosslinking. Remarkably, the hydrogels show capacity for sustained release of the RONS. The plasma-treated hydrogels remain fully biocompatible (due the fact that the species generated by plasma are previously washed away), indicating that no cytotoxic modifications have occurred on the polymer. Moreover, the RONS generated in alginate solutions showed cytotoxic potential towards bone cancer cells. These results open the door for the use of hydrogel-based biomaterials in CAP-associated therapies.
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http://dx.doi.org/10.1038/s41598-019-52673-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834627PMC
November 2019

The effect of biomimetic calcium deficient hydroxyapatite and sintered β-tricalcium phosphate on osteoimmune reaction and osteogenesis.

Acta Biomater 2019 09 30;96:605-618. Epub 2019 Jun 30.

Institute of Health and Biomedical Innovation and the Australia-China Centre for Tissue Engineering and Regenerative Medicine (ACCTERM), Queensland University of Technology, Brisbane, QLD 4059, Australia. Electronic address:

Biomaterial implantation triggers inflammatory reactions. Understanding the effect of physicochemical features of biomaterials on the release of inflammatory cytokines from immune cells would be of great interest in view of designing bone graft materials to enhance the healing of bone defects. The present work investigated the interactions of two chemically and texturally different calcium phosphate (CaPs) substrates with macrophages, one of the main innate immune cells, and its further impact on osteogenic differentiation of bone forming cells. The behaviour of macrophages seeded on biomimetic calcium deficient hydroxyapatite (CDHA) and sintered β-tricalcium phosphate (β-TCP) was assessed in terms of the release of inflammatory cytokines and osteoclastogenic factors. The osteogenic differentiation of bone progenitor cells (bone marrow stromal cells (BMSCs) and osteoblastic cell line (SaOS-2)) were subsequently studied by incubating with the conditioned medium induced by macrophage-CaPs interaction in order to reveal the effect of immune cell reaction to CaPs on osteogenic differentiation. It was found that the incubation of macrophages with CaPs substrates caused a decrease of pro-inflammatory cytokines, more pronounced for β-TCP compared with CDHA showing significantly decreased IL-6, TNF-a, and iNOS. However, the macrophage-CDHA interaction resulted in a more favourable environment for osteogenic differentiation of osteoblasts with more collagen type I production and osteogenic genes (Runx2, BSP) expression, suggesting that osteogenic differentiation of bone cells is not only determined by the nature of biomaterials, but also significantly influenced by the inflammatory environment generated by the interaction of immune cells and biomaterials. STATEMENT OF SIGNIFICANCE: The field of osteoimmunology highlights the importance of the cross-talk between immune and bone cells for effective bone regeneration. This tight interaction opens the door to new strategies that encompass the development of smart cell-instructive biomaterials which performance covers the events from early inflammation to osteogenesis. The present work links the anti-inflammatory and osteoimmunomodulatory features of synthetic bone grafts to their chemistry and texture, focussing on the cross-talk between macrophages and two major orchestrators of bone healing, namely primary mesenchymal stem cells and osteoblasts. The results emphasize the importance of the microenvironment created through the interaction between the substrate and the immune cells as it can stimulate osteogenic events and subsequently foster bone healing.
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http://dx.doi.org/10.1016/j.actbio.2019.06.057DOI Listing
September 2019

Injectable calcium phosphate foams for the delivery of Pitavastatin as osteogenic and angiogenic agent.

J Biomed Mater Res B Appl Biomater 2020 04 12;108(3):760-770. Epub 2019 Jun 12.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya (UPC), Barcelona, Spain.

Apatitic bone cements have been used as a clinical bone substitutes and drug delivery vehicles for therapeutic agents in orthopedic applications. This has led to their combination with different drugs with known ability to foster bone formation. Recent studies have evaluated Simvastatin for its role in enhanced bone regeneration, but its lipophilicity hampers incorporation and release to and from the bone graft. In this study, injectable calcium phosphate foams (i-CPF) based on α-tricalcium phosphate were loaded for the first time with Pitavastatin. The stability of the drug in different conditions relevant to this study, the effect of the drug on the i-CPFs properties, the release profile, and the in vitro biological performance with regard to mineralization and vascularization were investigated. Pitavastatin did not cause any changes in neither the micro nor the macro structure of the i-CPFs, which retained their biomimetic features. PITA-loaded i-CPFs showed a dose-dependent drug release, with early stage release kinetics clearly affected by the evolving microstructure due to the setting of cement. in vitro studies showed dose-dependent enhancement of mineralization and vascularization. Our findings contribute towards the design of controlled release with low drug dosing bone grafts: i-CPFs loaded with PITA as osteogenic and angiogenic agent.
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http://dx.doi.org/10.1002/jbm.b.34430DOI Listing
April 2020

Effect of calcium phosphate heparinization on the in vitro inflammatory response and osteoclastogenesis of human blood precursor cells.

J Tissue Eng Regen Med 2019 07 31;13(7):1217-1229. Epub 2019 May 31.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Universitat Politècnica de Catalunya (UPC), Barcelona, Spain.

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.
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http://dx.doi.org/10.1002/term.2872DOI Listing
July 2019

A Dual Molecular Biointerface Combining RGD and KRSR Sequences Improves Osteoblastic Functions by Synergizing Integrin and Cell-Membrane Proteoglycan Binding.

Int J Mol Sci 2019 Mar 21;20(6). Epub 2019 Mar 21.

Biomaterials, Biomechanics and Tissue Engineering Group (BBT), Department of Materials Science and Metallurgical Engineering, Universitat Politècnica de Catalunya (UPC), 08019 Barcelona, Spain.

Synergizing integrin and cell-membrane heparan sulfate proteoglycan signaling on biomaterials through peptidic sequences is known to have beneficial effects in the attachment and behavior of osteoblasts; however, controlling the exact amount and ratio of peptides tethered on a surface is challenging. Here, we present a dual molecular-based biointerface combining integrin (RGD) and heparin (KRSR)-binding peptides in a chemically controlled fashion. To this end, a tailor-made synthetic platform (PLATF) was designed and synthesized by solid-phase methodologies. The PLATF and the control linear peptides (RGD or KRSR) were covalently bound to titanium via silanization. Physicochemical characterization by means of contact angle, Raman spectroscopy and XPS proved the successful and stable grafting of the molecules. The biological potential of the biointerfaces was measured with osteoblastic (Saos-2) cells both at short and long incubation periods. Biomolecule grafting (either the PLATF, RGD or KRSR) statistically improved ( < 0.05) cell attachment, spreading, proliferation and mineralization, compared to control titanium. Moreover, the molecular PLATF biointerface synergistically enhanced mineralization ( < 0.05) of Saos-2 cells compared to RGD or KRSR alone. These results indicate that dual-function coatings may serve to improve the bioactivity of medical implants by mimicking synergistic receptor binding.
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http://dx.doi.org/10.3390/ijms20061429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470513PMC
March 2019

Impact of Biomimicry in the Design of Osteoinductive Bone Substitutes: Nanoscale Matters.

ACS Appl Mater Interfaces 2019 Mar 21;11(9):8818-8830. Epub 2019 Feb 21.

Institute for Bioengineering of Catalonia (IBEC) , Barcelona Institute of Technology (BIST) , 08028 Barcelona , Spain.

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation.
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http://dx.doi.org/10.1021/acsami.8b20749DOI Listing
March 2019

The Effect of the Thermosensitive Biodegradable PLGA⁻PEG⁻PLGA Copolymer on the Rheological, Structural and Mechanical Properties of Thixotropic Self-Hardening Tricalcium Phosphate Cement.

Int J Mol Sci 2019 Jan 17;20(2). Epub 2019 Jan 17.

CEITEC ⁻ Brno University of Technology, Purkynova 656/123, 612 00 Brno, Czech Republic.

The current limitations of calcium phosphate cements (CPCs) used in the field of bone regeneration consist of their brittleness, low injectability, disintegration in body fluids and low biodegradability. Moreover, no method is currently available to measure the setting time of CPCs in correlation with the evolution of the setting reaction. The study proposes that it is possible to improve and tune the properties of CPCs via the addition of a thermosensitive, biodegradable, thixotropic copolymer based on poly(lactic acid), poly(glycolic acid) and poly(ethylene glycol) (PLGA⁻PEG⁻PLGA) which undergoes gelation under physiological conditions. The setting times of alpha-tricalcium phosphate (α-TCP) mixed with aqueous solutions of PLGA⁻PEG⁻PLGA determined by means of time-sweep curves revealed a lag phase during the dissolution of the α-TCP particles. The magnitude of the storage modulus at lag phase depends on the liquid to powder ratio, the copolymer concentration and temperature. A sharp increase in the storage modulus was observed at the time of the precipitation of calcium deficient hydroxyapatite (CDHA) crystals, representing the loss of paste workability. The PLGA⁻PEG⁻PLGA copolymer demonstrates the desired pseudoplastic rheological behaviour with a small decrease in shear stress and the rapid recovery of the viscous state once the shear is removed, thus preventing CPC phase separation and providing good cohesion. Preliminary cytocompatibility tests performed on human mesenchymal stem cells proved the suitability of the novel copolymer/α-TCP for the purposes of mini-invasive surgery.
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http://dx.doi.org/10.3390/ijms20020391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359562PMC
January 2019

RGD Mutation of the Heparin Binding II Fragment of Fibronectin for Guiding Mesenchymal Stem Cell Behavior on Titanium Surfaces.

ACS Appl Mater Interfaces 2019 Jan 16;11(4):3666-3678. Epub 2019 Jan 16.

Institute for Bioengineering of Catalonia (IBEC) , Barcelona Institute of Science and Technology (BIST) , 08028 Barcelona , Spain.

Installing bioactivity on metallic biomaterials by mimicking the extracellular matrix (ECM) is crucial for stimulating specific cellular responses to ultimately promote tissue regeneration. Fibronectin is an ECM protein commonly used for biomaterial functionalization. The use of fibronectin recombinant fragments is an attractive alternate to the use of full-length fibronectin because of the relatively low cost and facility of purification. However, it is necessary to combine more than one fragment, for example, the cell attachment site and the heparin binding II (HBII), either mixed or in one molecule, to obtain complete activity. In the present study, we proposed to install adhesion capacity to the HBII fragment by an RGD gain-of-function DNA mutation, retaining its cell differentiation capacity and thereby producing a small and very active protein fragment. The novel molecule, covalently immobilized onto titanium surfaces, maintained the growth factor-binding capacity and stimulated cell spreading, osteoblastic cell differentiation, and mineralization of human mesenchymal stem cells compared to the HBII native protein. These results highlight the potential capacity of gain-of-function DNA mutations in the design of novel molecules for the improvement of osseointegration properties of metallic implant surfaces.
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http://dx.doi.org/10.1021/acsami.8b17138DOI Listing
January 2019

Effects of Molecular Weight and Concentration of Poly(Acrylic Acid) on Biomimetic Mineralization of Collagen.

ACS Biomater Sci Eng 2018 Aug 29;4(8):2758-2766. Epub 2018 Jun 29.

MDRCBB, Minnesota Dental Research Center for Biomaterials and Biomechanics, University of Minnesota, 16-212 Moos Tower, 515 Delaware St. SE, Minneapolis, MN, USA.

Inspired by nature, poly(acrylic acid) (PAA) and other polyelectrolytes have been used as noncollagenous proteins (NCPs) surrogates for biomimetic intrafibrillar mineralization of collagen fibrils and thus, to model the ultrastructure of bone, to study the mechanism of bone mineralization and, more scarcely to fabricate scaffolds for hard tissue engineering. The objective of this study was to systematically investigate the effect of the molecular weight (MW) and the concentration of PAA on the rate and pattern of biomineralization of collagen matrices. Densified type I collagen films were mineralized in supersaturated PAA-stabilized amorphous calcium-phosphate (PAA-ACP) solutions containing increasing MW (2 kDa, 50 kDA, 450 kDa) and concentrations (10, 25, 50 mg/L) of PAA up to 7 days. The stability and physical properties of collagen-free PAA-ACP solutions were also investigated. In our system, lowering PAA MW and increasing PAA concentration resulted in solutions with increasing stability. Over stable PAA-ACP solutions that fully inhibited mineralization of the collagen matrices were achieved using PAA 2k-50. Conversely, unstable solutions were obtained using high PAA MW at low concentrations. Nucleation and growth of significant amount of extrafibrillar minerals on the collagen fibrils was obtained using these solutions. In a wide range of combined MW and concentration of PAA we obtained intrafibrillar mineralization of collagen with hydroxyapatite crystals aligned parallel to the collagen fibril as in natural tissues. Intrafibrillar mineralization was correlated with PAA-ACP stability and growth of the PAA-ACP particles in solution. Our results support using PAA to surrogate NCPs function as selective inhibitors or promoters of biological mineralization and provide parameters to manufacture new biomimetic scaffolds and constructs for bone and dentin tissue engineering.
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http://dx.doi.org/10.1021/acsbiomaterials.8b00512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298758PMC
August 2018

Vertical Bone Regeneration with Synthetic Biomimetic Calcium Phosphate onto the Calvaria of Rats.

Tissue Eng Part C Methods 2019 01;25(1):1-11

1 Inserm, UMR 1238, PHY-OS, Laboratory of Bone Sarcomas and Remodelling of Calcified Tissues, Faculty of Medicine, University of Nantes, Nantes, France.

Impact Statement: This work reports a new bone substitute made of precipitated apatite crystals that resemble in composition and crystallinity to the mineral phase of bone. The bone regeneration capacity of this synthetic biomimetic calcium phosphate (SBCP) was studied by using an original model of vertical bone regeneration with cups on the calvaria of rats. After 4 weeks, a significantly higher bone growth was found with SBCP compared with deproteinized bovine bone matrix and empty controls. This rapid vertical bone regeneration indicated that this new biomaterial is particularly interesting for filling bone defects in oral surgery.
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http://dx.doi.org/10.1089/ten.TEC.2018.0260DOI Listing
January 2019

The Influence of Physicochemical Properties of Biomimetic Hydroxyapatite on the In Vitro Behavior of Endothelial Progenitor Cells and Their Interaction with Mesenchymal Stem Cells.

Adv Healthc Mater 2019 01 5;8(2):e1801138. Epub 2018 Dec 5.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Universitat Politècnica de Catalunya (UPC), EEBE, Av. Eduard Maristany 10-14, 08019, Barcelona, Spain.

Calcium phosphate (CaP) substrates are successfully used as bone grafts due to their osteogenic properties. However, the influence of the physicochemical features of CaPs in angiogenesis is frequently neglected despite it being a crucial process for bone regeneration. The present work focuses on analyzing the effects of textural parameters of biomimetic calcium deficient hydroxyapatite (CDHA) and sintered beta-tricalcium phosphate (β-TCP), such as specific surface area, surface roughness, and microstructure, on the behavior of rat endothelial progenitor cells (rEPCs) and their crosstalk with rat mesenchymal stem cells (rMSCs). The higher reactivity of CDHA results in low proliferation rates in monocultured and cocultured systems. This effect is especially pronounced for rMSCs alone, and for CDHA with a fine microstructure. In terms of angiogenic and osteogenic gene expressions, the upregulation of particular genes is especially enhanced for needle-like CDHA compared to plate-like CDHA and β-TCP, suggesting the importance not only of the chemistry of the substrate, but also of its textural features. Moreover, the coculture of rEPCs and rMSCs on needle-like CDHA results in early upregulation of osteogenic modulator, i.e., protein deglycase 1 might be a possible cause of overexpression of osteogenic-related genes on the same substrate.
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http://dx.doi.org/10.1002/adhm.201801138DOI Listing
January 2019

Osteogenesis by foamed and 3D-printed nanostructured calcium phosphate scaffolds: Effect of pore architecture.

Acta Biomater 2018 10 6;79:135-147. Epub 2018 Sep 6.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Universitat Politècnica de Catalunya, Av. Eduard Maristany 10-14, 08019 Barcelona, Spain; Barcelona Research Center in Multiscale Science and Engineering, Universitat Politècnica de Catalunya, Av. Eduard Maristany 10-14, 08019 Barcelona, Spain; Institut for Bioengineering of Catalonia (IBEC), Barcelona Institute of Technology (BIST), 08028 Barcelona, Spain. Electronic address:

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12 weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.
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http://dx.doi.org/10.1016/j.actbio.2018.09.003DOI Listing
October 2018

Effect of nano-structural properties of biomimetic hydroxyapatite on osteoimmunomodulation.

Biomaterials 2018 10 31;181:318-332. Epub 2018 Jul 31.

Institute of Health and Biomedical Innovation and the Australia-China Centre for Tissue Engineering and Regenerative Medicine (ACCTERM), Queensland University of Technology, Brisbane, QLD 4059, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biomaterials.2018.07.058DOI Listing
October 2018

Bioceramics and bone healing.

EFORT Open Rev 2018 May 21;3(5):173-183. Epub 2018 May 21.

Mimetis Biomaterials, Spain.

Calcium phosphates have long been used as synthetic bone grafts. Recent studies have shown that the modulation of composition and textural properties, such as nano-, micro- and macro-porosity, is a powerful strategy to control and synchronize material resorption and bone formation.Biomimetic calcium phosphates, which closely mimic the composition and structure of bone mineral, can be produced using low-temperature processing routes, and offer the possibility to modulate the material properties to a larger extent than conventional high temperature sintering processes.Advanced technologies open up new possibilities in the design of bioceramics for bone regeneration; 3D-printing technologies, in combination with the development of hybrid materials with enhanced mechanical properties, supported by finite element modelling tools, are expected to enable the design and fabrication of mechanically competent patient-specific bone grafts.The association of ions, drugs and cells allows leveraging of the osteogenic potential of bioceramic scaffolds in compromised clinical situations, where the intrinsic bone regeneration potential is impaired. Cite this article: Open Rev 2018;3 DOI: 10.1302/2058-5241.3.170056.
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http://dx.doi.org/10.1302/2058-5241.3.170056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994622PMC
May 2018

In vitro response of mesenchymal stem cells to biomimetic hydroxyapatite substrates: A new strategy to assess the effect of ion exchange.

Acta Biomater 2018 08 20;76:319-332. Epub 2018 Jun 20.

Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgical Engineering, Universitat Politècnica de Catalunya (UPC), Av. Eduard Maristany 16, 08019 Barcelona, Spain; Barcelona Research Centre in Multiscale Science and Engineering, Universitat Politècnica de Catalunya (UPC), Av. Eduard Maristany 16, 08019 Barcelona, Spain; Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028 Barcelona, Spain. Electronic address:

Biomaterials can interact with cells directly, that is, by direct contact of the cells with the material surface, or indirectly, through soluble species that can be released to or uptaken from the surrounding fluids. However, it is difficult to characterise the relevance of this fluid-mediated interaction separately from the topography and composition of the substrate, because they are coupled variables. These fluid-mediated interactions are amplified in the case of highly reactive calcium phosphates (CaPs) such as biomimetic calcium deficient hydroxyapatite (CDHA), particularly in static in vitro cultures. The present work proposes a strategy to decouple the effect of ion exchange from topographical features by adjusting the volume ratio between the cell culture medium and biomaterial (V/V). Increasing this ratio allowed mitigating the drastic ionic exchanges associated to the compositional changes experienced by the material exposed to the cell culture medium. This strategy was validated using rat mesenchymal stem cells (rMSCs) cultured on CDHA and beta-tricalcium phosphate (β-TCP) discs using different V/V ratios. Whereas in the case of β-TCP the cell response was not affected by this ratio, a significant effect on cell adhesion and proliferation was found for the more reactive CDHA. The ionic exchange, produced by CDHA at low V/V, altered cell adhesion due to the reduced number of focal adhesions, caused cell shrinkage and further rMCSs apoptosis. This was mitigated when using a high V/V, which attenuated the changes of calcium and phosphate concentrations in the cell culture medium, resulting in rMSCs spreading and a viability over time. Moreover, rMSCs showed an earlier expression of osteogenic genes on CDHA compared to sintered β-TCP when extracellular calcium fluctuations were reduced.

Statement Of Significance: Fluid mediated interactions play a significant role in the bioactivity of calcium phosphates. Ionic exchange is amplified in the case of biomimetic hydroxyapatite, which makes the in vitro characterisation of cell-material interactions especially challenging. The present work proposes a novel and simple strategy to explore the mechanisms of interaction of biomimetic and sintered calcium phosphates with mesenchymal stem cells. The effects of topography and ion exchange are analysed separately by modifying the volume ratio between cell culture medium and biomaterial. High ionic fluctuations interfered in the maturation of focal adhesions, hampering cell adhesion and leading to increased apoptosis and reduced proliferation rate.
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http://dx.doi.org/10.1016/j.actbio.2018.06.025DOI Listing
August 2018

Accelerated hardening of nanotextured 3D-plotted self-setting calcium phosphate inks.

Acta Biomater 2018 07 26;75:451-462. Epub 2018 May 26.

Dept. Materials Science and Metallurgical Engineering, Group of Biomaterials, Biomechanics and Tissue Engineering, Universitat Politècnica de Catalunya (UPC), Barcelona, Spain; Barcelona Research Centre for Multiscale Science and Engineering, Universitat Politècnica de Catalunya (UPC), Barcelona, Spain; Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028 Barcelona, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.actbio.2018.05.042DOI Listing
July 2018