Borax induces osteogenesis by stimulating NaBC1 transporter via activation of BMP pathway

[EN] The intrinsic properties of mesenchymal stem cells (MSCs) make them ideal candidates for tissue engineering applications. Efforts have been made to control MSC behavior by using material systems to engineer synthetic extracellular matrices and/or include soluble factors in the media. This work proposes a simple approach based on ion transporter stimulation to determine stem cell fate that avoids the use of growth factors. Addition of borax alone, transported by the NaBC1-transporter, enhanced MSC adhesion and contractility, promoted osteogenesis and inhibited adipogenesis. Stimulated-NaBC1 promoted osteogenesis via the BMP canonical pathway (comprising Smad1/YAP nucleus translocation and osteopontin expression) through a mechanism that involves simultaneous NaBC1/BMPR1A and NaBC1/alpha (5)beta (1)/alpha (v)beta (3) co-localization. We describe an original function for NaBC1 transporter, besides controlling borate homeostasis, capable of stimulating growth factor receptors and fibronectin-binding integrins. Our results open up new biomaterial engineering approaches for biomedical applications by a cost-effective strategy that avoids the use of soluble growth factors. Rico et al. propose a simple approach based on borax stimulation of NaBC1 transporter, which enhances FN-binding integrin-dependent mesenchymal stem cell adhesion and contractility, promotes osteogenesis and inhibits adipogenesis. Osteogenic differentiation depends on activation of the BMP pathway through a mechanism that involves simultaneous co-localization of NaBC1 with FN-binding integrins and BMPR1A.P.R. acknowledges support from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096794), and Fondo Europeo de Desarrollo Regional (FEDER). 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Simultaneous boron ion-channel/growth factor receptor activation for enhanced vascularization

[EN] Boron ion is essential in metabolism and its concentration is regulated by ion-channel NaBC1. NaBC1 mutations cause corneal dystrophies such as Harboyan syndrome. Here we propose a 3D molecular model for NaBC1 and show that simultaneous stimulation of NaBC1 and vascular growth factor receptors (VEGFR) promote angiogenesis in vitro and in vivo with ultra-low concentrations of VEGF. We show Human Umbilical Vein Endothelial Cells (HUVEC) organization into tubular structures indicative of vascularization potential. Enhanced cell sprouting was found only in the presence of VEGF and boron, effect abrogated after blocking NaBC1. We demonstrate that stimulated NaBC1 promotes angiogenesis via PI3k-independent pathways and that ¿5ß1/¿vß3-integrin binding is not essential to enhanced HUVEC organization. We describe a novel vascularization mechanism that involves the crosstalk and colocalization between NaBC1/VEGFR receptors. This has important translational consequences: just by administering boron, taking advantage of endogenous VEGF, in vivo vascularization is shown in a chorioallantoic membrane assay.P.R. acknowledges support from the Ministerio de Economia, Industria y Competitividad, Gobierno de Espana (MINECO) (MAT2015-69315-C3-1-R), and European Regional Development Fund (FEDER). CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. M. S. S. acknowledges support from the European Research Council (ERC-HealInSynergy 306990) and the UK Engineering and Physical Sciences Research Council (EPSRC-EP/P001114/1). 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A Mechanism for Modulation of Cellular Responses to VEGF. Molecular Cell, 6(4), 851-860. doi:10.1016/s1097-2765(05)00076-

Engineered 3D hydrogels with full-length fibronectin that sequester and present growth factors

[EN] Extracellular matrix (ECM)-derived matrices such as Matrigel are used to culture numerous cell types in vitro as they recapitulate ECM properties that support cell growth, organisation, migration and differentiation. These ECM-derived matrices contain various growth factors which make them highly bioactive. However, they suffer lot-to-lot variability, undefined composition and lack of controlled physical properties. There is a need to develop rationally designed biomaterials that can also recapitulate ECM roles. Here, we report the development of fibronectin (FN)-based 3D hydrogels of controlled stiffness and degradability that incorporate full-length FN to enable solid-phase presentation of growth factors in a physiological manner. We demonstrate, in vitro and in vivo, the effect of incorporating vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) in these hydrogels to enhance angiogenesis and bone regeneration, respectively. These hydrogels represent a step-change in the design of well-defined, reproducible, synthetic microenvironments for 3D cell culture that incorporate growth factors to achieve functional effects.This study was supported by the UK Regenerative Medicine Platform (MRC grant MR/L022710/1), the UK Engineering and Physical Sciences Research Council (EPSRC EP/P001114/1) and a programme grant from the Sir Bobby Charlton Foundation. mu CT work was supported by the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013) (grant agreement No. [615030]). S.T. acknowledges support from the University of Glasgow through their internal scholarship funding program. We thank the support of David Adams (Anatomy lab, University of Glasgow) and the Biological Services and Veterinary Research Facility (University of Glasagow) for their assistance.Trujillo-Muñoz, S.; González-García, C.; Rico Tortosa, PM.; Reid, A.; Windmill, J.; Dalby, MJ.; Salmerón Sánchez, M. (2020). Engineered 3D hydrogels with full-length fibronectin that sequester and present growth factors. Biomaterials. 252:1-15. https://doi.org/10.1016/j.biomaterials.2020.120104S115252Jarad, M., Kuczynski, E. A., Morrison, J., Viloria-Petit, A. M., & Coomber, B. L. (2017). Release of endothelial cell associated VEGFR2 during TGF-β modulated angiogenesis in vitro. BMC Cell Biology, 18(1). doi:10.1186/s12860-017-0127-ySpence, J. R., Mayhew, C. N., Rankin, S. A., Kuhar, M. F., Vallance, J. E., Tolle, K., … Wells, J. M. (2010). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 470(7332), 105-109. doi:10.1038/nature09691Yang, S.-J., Son, J. 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Development of a Ta/TaN/TaNx(Ag)y/TaN nanocomposite coating system and bio-response study for biomedical applications

[EN] TaN(Ag) composited coatings are being investigated to improve biocompatibility of different biomedical devices due to the mechanical and chemical stability of TaN and bactericidal effect of silver nanoparticles. However, controlling the size, density, shape and especially the release of silver ions (Ag) into the surrounding medium becomes a challenge, since elevated levels of Ag could be cytotoxic. The aim of this work is to design and develop a new Ta/TaN/TaNx(Ag)y/TaN coating system, deposited by unbalanced DC magnetron sputtering technique, presenting an adequate balance between biocompatibility and bactericidal effect for potential applications in biomedical field. For this purpose, four different coating systems were deposited on 316 L stainless steel and silicon (100) samples applying a bias voltage of ¿30, ¿60, ¿90 and ¿120 V during the deposition of the top layer of TaN to vary its density. This manufacturing strategy allowed controlling the diffusion of silver nanoparticles to the coating surface and the release kinetics of silver ions in simulated body fluid (SBF). Biologic characterization has been performed with MC3T3-E1 pre-osteoblastic cells in terms of cell adhesion and long-term differentiation. Additionally, the adhesion and biofilm formation of the bacteria Streptococcus sanguinis strain in the deposited coating systems of Ta/TaN/TaNx(Ag)y/TaN were analyzed. The results indicated an improvement of cell adhesion and differentiation of the composited coating deposited with a bias of ¿30 V compared to other coatings. Concordantly, this coating showed the lowest bacterial adhesion and biofilm formation, representing an attractive and suitable composited material for biomedical applications.The technical support from the Spanish Ministry of Economy and Competitiveness (MINECO) (through the MAT2015-69315-C3-1-R) and FEDER funds project are acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Echavarria, AM.; Rico Tortosa, PM.; Gómez Ribelles, JL.; Pacha-Olivenza, MA.; Fernandez-Calderon, M.; Bejarano-G, G. (2017). Development of a Ta/TaN/TaNx(Ag)y/TaN nanocomposite coating system and bio-response study for biomedical applications. Vacuum. 145:55-67. https://doi.org/10.1016/j.vacuum.2017.08.020S556714

Structure, morphology, adhesion and in vitro biological evaluation of antibacterial multi-layer HA-Ag/SiO2/TiN/Ti coatings obtained by RF magnetron sputtering for biomedical applications

[EN] Biocompatible and antibacterial multi-layer coatings of hydroxyapatite (HA)-Ag/SiO2/TiN/Ti were obtained on the Ti-6Al-4V alloy, by means of the magnetron sputtering technique. During characterization of the coatings, the chemical composition was evaluated by energy dispersive X-ray spectroscopy and the phase analysis was carried out by X-ray diffraction. The morphology of the coatings was observed by field emission scanning electron microscopy, while transmission electron microscopy was used to appreciate their structure. The adhesion of the coatings to the substrate was evaluated by micro scratch test. The in vitro biological response was evaluated in terms of cytotoxicity, adhesion and differentiation of mouse mesenchymal stem cells, as well as adhesion and bacterial viability of Staphylococcus aureus strain. Through the compositional study carried out, the deposition of the HA phase was verified, with a Ca/P ratio close to 1.67 and the characteristic diffraction peaks of this compound. The structural study of the coatings evidenced the obtention of multi-layer architectures. The use of an intermediate SiO2/TiN/Ti trilayer was found to improve adhesion between HA-Ag and the substrate by 84%. Finally, the in vitro biological tests carried out indicated a potentially non-toxic character in the coatings. Additionally, an antibacterial effect was registered at low concentrations of Ag (3.0.co;2-3Lalueza, P., Monzón, M., Arruebo, M., & Santamaría, J. (2011). Bactericidal effects of different silver-containing materials. Materials Research Bulletin, 46(11), 2070-2076. doi:10.1016/j.materresbull.2011.06.041Peetsch, A., Greulich, C., Braun, D., Stroetges, C., Rehage, H., Siebers, B., … Epple, M. (2013). Silver-doped calcium phosphate nanoparticles: Synthesis, characterization, and toxic effects toward mammalian and prokaryotic cells. Colloids and Surfaces B: Biointerfaces, 102, 724-729. doi:10.1016/j.colsurfb.2012.09.040Yanovska, A. A., Stanislavov, A. S., Sukhodub, L. B., Kuznetsov, V. N., Illiashenko, V. Y., Danilchenko, S. N., & Sukhodub, L. F. (2014). Silver-doped hydroxyapatite coatings formed on Ti–6Al–4V substrates and their characterization. Materials Science and Engineering: C, 36, 215-220. doi:10.1016/j.msec.2013.12.011DeVasConCellos, P., Bose, S., Beyenal, H., Bandyopadhyay, A., & Zirkle, L. G. (2012). Antimicrobial particulate silver coatings on stainless steel implants for fracture management. Materials Science and Engineering: C, 32(5), 1112-1120. doi:10.1016/j.msec.2012.02.020VALLETREGI, M. (2004). Calcium phosphates as substitution of bone tissues. Progress in Solid State Chemistry, 32(1-2), 1-31. doi:10.1016/j.progsolidstchem.2004.07.001Surmenev, R. A., Surmeneva, M. A., Evdokimov, K. E., Pichugin, V. F., Peitsch, T., & Epple, M. (2011). The influence of the deposition parameters on the properties of an rf-magnetron-deposited nanostructured calcium phosphate coating and a possible growth mechanism. Surface and Coatings Technology, 205(12), 3600-3606. doi:10.1016/j.surfcoat.2010.12.039Surmeneva, M. A., Sharonova, A. A., Chernousova, S., Prymak, O., Loza, K., Tkachev, M. S., … Surmenev, R. A. (2017). Incorporation of silver nanoparticles into magnetron-sputtered calcium phosphate layers on titanium as an antibacterial coating. Colloids and Surfaces B: Biointerfaces, 156, 104-113. doi:10.1016/j.colsurfb.2017.05.016Mohseni, E., Zalnezhad, E., Bushroa, A. R., Abdel Magid Hamouda, Goh, B. T., & Yoon, G. H. (2015). Ti/TiN/HA coating on Ti–6Al–4V for biomedical applications. Ceramics International, 41(10), 14447-14457. doi:10.1016/j.ceramint.2015.07.081Ghasemi, S., Shanaghi, A., & Chu, P. K. (2017). Nano mechanical and wear properties of multi-layer Ti/TiN coatings deposited on Al 7075 by high-vacuum magnetron sputtering. Thin Solid Films, 638, 96-104. doi:10.1016/j.tsf.2017.07.049Hamdi, D. A., Jiang, Z.-T., No, K., Rahman, M. M., Lee, P.-C., Truc, L. N. T., … Dlugogorski, B. Z. (2019). Biocompatibility study of multi-layered hydroxyapatite coatings synthesized on Ti-6Al-4V alloys by RF magnetron sputtering for prosthetic-orthopaedic implant applications. Applied Surface Science, 463, 292-299. doi:10.1016/j.apsusc.2018.08.157Qi, J., Yang, Y., Zhou, M., Chen, Z., & Chen, K. (2019). Effect of transition layer on the performance of hydroxyapatite/titanium nitride coating developed on Ti-6Al-4V alloy by magnetron sputtering. Ceramics International, 45(4), 4863-4869. doi:10.1016/j.ceramint.2018.11.183Lenis, J. A., Hurtado, F. M., Gómez, M. A., & Bolívar, F. J. (2019). Effect of thermal treatment on structure, phase and mechanical properties of hydroxyapatite thin films grown by RF magnetron sputtering. Thin Solid Films, 669, 571-578. doi:10.1016/j.tsf.2018.11.045Lenis, J. A., Gómez, M. A., & Bolívar, F. J. (2019). Effect of deposition temperature and target-substrate distance on the structure, phases, mechanical and tribological properties of multi-layer HA-Ag coatings obtained by RF magnetron sputtering. Surface and Coatings Technology, 378, 124936. doi:10.1016/j.surfcoat.2019.124936Sofronia, A. M., Baies, R., Anghel, E. M., Marinescu, C. A., & Tanasescu, S. (2014). Thermal and structural characterization of synthetic and natural nanocrystalline hydroxyapatite. Materials Science and Engineering: C, 43, 153-163. doi:10.1016/j.msec.2014.07.023Lenis, J. A., Bejarano, G., Rico, P., Ribelles, J. L. G., & Bolívar, F. J. (2019). Development of multilayer Hydroxyapatite - Ag/TiN-Ti coatings deposited by radio frequency magnetron sputtering with potential application in the biomedical field. Surface and Coatings Technology, 377, 124856. doi:10.1016/j.surfcoat.2019.06.097Lenis, J. A., Toro, L. J., & Bolívar, F. J. (2019). Multi-layer bactericidal silver - calcium phosphate coatings obtained by RF magnetron sputtering. Surface and Coatings Technology, 367, 203-211. doi:10.1016/j.surfcoat.2019.03.038Valverde, A., Pérez-Álvarez, L., Ruiz-Rubio, L., Pacha Olivenza, M. A., García Blanco, M. B., Díaz-Fuentes, M., & Vilas-Vilela, J. L. (2019). Antibacterial hyaluronic acid/chitosan multilayers onto smooth and micropatterned titanium surfaces. Carbohydrate Polymers, 207, 824-833. doi:10.1016/j.carbpol.2018.12.039Surmeneva, M. A., Chaikina, M. V., Zaikovskiy, V. I., Pichugin, V. F., Buck, V., Prymak, O., … Surmenev, R. A. (2013). The structure of an RF-magnetron sputter-deposited silicate-containing hydroxyapatite-based coating investigated by high-resolution techniques. Surface and Coatings Technology, 218, 39-46. doi:10.1016/j.surfcoat.2012.12.023Nelea, V., Morosanu, C., Iliescu, M., & Mihailescu, I. N. (2003). Microstructure and mechanical properties of hydroxyapatite thin films grown by RF magnetron sputtering. Surface and Coatings Technology, 173(2-3), 315-322. doi:10.1016/s0257-8972(03)00729-1Ding, S.-J., Ju, C.-P., & Lin, J.-H. C. (1999). Characterization of hydroxyapatite and titanium coatings sputtered on Ti-6Al-4V substrate. Journal of Biomedical Materials Research, 44(3), 266-279. doi:10.1002/(sici)1097-4636(19990305)44:33.0.co;2-4Ivanova, A. A., Surmeneva, M. A., Tyurin, A. I., Pirozhkova, T. S., Shuvarin, I. A., Prymak, O., … Surmenev, R. A. (2016). Fabrication and physico-mechanical properties of thin magnetron sputter deposited silver-containing hydroxyapatite films. Applied Surface Science, 360, 929-935. doi:10.1016/j.apsusc.2015.11.087Contreras Romero, E., Cortínez Osorio, J., Talamantes Soto, R., Hurtado Macías, A., & Gómez Botero, M. (2019). Microstructure, mechanical and tribological performance of nanostructured TiAlTaN-(TiAlN/TaN)n coatings: Understanding the effect of quaternary/multilayer volume fraction. Surface and Coatings Technology, 377, 124875. doi:10.1016/j.surfcoat.2019.07.086Deligianni, D. (2001). Effect of surface roughness of the titanium alloy Ti–6Al–4V on human bone marrow cell response and on protein adsorption. Biomaterials, 22(11), 1241-1251. doi:10.1016/s0142-9612(00)00274-xZareidoost, A., Yousefpour, M., Ghaseme, B., & Amanzadeh, A. (2012). The relationship of surface roughness and cell response of chemical surface modification of titanium. Journal of Materials Science: Materials in Medicine, 23(6), 1479-1488. doi:10.1007/s10856-012-4611-9Surmeneva, M. A., Tyurin, A. I., Mukhametkaliyev, T. M., Pirozhkova, T. S., Shuvarin, I. A., Syrtanov, M. S., & Surmenev, R. A. (2015). Enhancement of the mechanical properties of AZ31 magnesium alloy via nanostructured hydroxyapatite thin films fabricated via radio-frequency magnetron sputtering. Journal of the Mechanical Behavior of Biomedical Materials, 46, 127-136. doi:10.1016/j.jmbbm.2015.02.025Quirama, A., Echavarría, A. M., Meza, J. M., Osorio, J., & Bejarano G, G. (2017). Improvement of the mechanical behavior of the calcium phosphate coatings deposited onto Ti6Al4V alloy using an intermediate TiN/TiO2 bilayer. Vacuum, 146, 22-30. doi:10.1016/j.vacuum.2017.09.024Surmeneva, M. A., Kleinhans, C., Vacun, G., Kluger, P. J., Schönhaar, V., Müller, M., … Surmenev, R. A. (2015). Nano-hydroxyapatite-coated metal-ceramic composite of iron-tricalcium phosphate: Improving the surface wettability, adhesion and proliferation of mesenchymal stem cells in vitro. Colloids and Surfaces B: Biointerfaces, 135, 386-393. doi:10.1016/j.colsurfb.2015.07.057Chen, Y., Zheng, X., Xie, Y., Ji, H., Ding, C., Li, H., & Dai, K. (2010). Silver release from silver-containing hydroxyapatite coatings. Surface and Coatings Technology, 205(7), 1892-1896. doi:10.1016/j.surfcoat.2010.08.073Actis, L., Gaviria, L., Guda, T., & Ong, J. L. (2013). Antimicrobial surfaces for craniofacial implants: state of the art. Journal of the Korean Association of Oral and Maxillofacial Surgeons, 39(2), 43. doi:10.5125/jkaoms.2013.39.2.43Parvizi, J., Aggarwal, V., & Rasouli, M. (2013). Periprosthetic joint infection: Current concept. Indian Journal of Orthopaedics, 47(1), 10. doi:10.4103/0019-5413.106884Redey, S. A., Nardin, M., Bernache-Assolant, D., Rey, C., Delannoy, P., Sedel, L., & Marie, P. J. (2000). Behavior of human osteoblastic cells on stoichiometric hydroxyapatite and type A carbonate apatite: Role of surface energy. Journal of Biomedical Materials Research, 50(3), 353-364. doi:10.1002/(sici)1097-4636(20000605)50:33.0.co;2-cSpriano, S., Bosetti, M., Bronzoni, M., Vernè, E., Maina, G., Bergo, V., & Cannas, M. (2005). Surface properties and cell response of low metal ion release Ti-6Al-7Nb alloy after multi-step chemical and thermal treatments. Biomaterials, 26(11), 1219-1229. doi:10.1016/j.biomaterials.2004.04.026Vogler, E. A. (1999). Water and the acute biological response to surfaces. Journal of Biomaterials Science, Polymer Edition, 10(10), 1015-1045. doi:10.1163/156856299x00667Lim, J. Y., Liu, X., Vogler, E. A., & Donahue, H. J. (2004). Systematic variation in osteoblast adhesion and phenotype with substratum surface characteristics. Journal of Biomedical Materials Research, 68A(3), 504-512. doi:10.1002/jbm.a.20087Sengstock, C., Diendorf, J., Epple, M., Schildhauer, T. A., & Köller, M. (2014). Effect of silver nanoparticles on human mesenchymal stem cell differentiation. Beilstein Journal of Nanotechnology, 5, 2058-2069. doi:10.3762/bjnano.5.214Sahuquillo Arce, J. M., Iranzo Tatay, A., Llácer Luna, M., Sanchis Boix, Y., Guitán Deltell, J., González Barberá, E., … Gobernado Serrano, M. (2011). Estudio in vitro de las propiedades antimicrobianas de una espuma de poliuretano que libera iones de plata. Cirugía Española, 89(8), 532-538. doi:10.1016/j.ciresp.2011.02.015Jamuna-Thevi, K., Bakar, S. A., Ibrahim, S., Shahab, N., & Toff, M. R. M. (2011). Quantification of silver ion release, in vitro cytotoxicity and antibacterial properties of nanostuctured Ag doped TiO2 coatings on stainless steel deposited by RF magnetron sputtering. Vacuum, 86(3), 235-241. doi:10.1016/j.vacuum.2011.06.011Chernousova, S., & Epple, M. (2012). Silver as Antibacterial Agent: Ion, Nanoparticle, and Metal. Angewandte Chemie International Edition, 52(6), 1636-1653. doi:10.1002/anie.20120592

Lithium Directs Embryonic Stem Cell Differentiation Into Hemangioblast-Like Cells

[EN] Definitive hematopoietic stem cells (HSCs) derive from specialized regions of the endothelium known as the hemogenic endothelium (HE) during embryonic developmental processes. This knowledge opens up new possibilities for designing new strategies to obtain HSCs in vitro from pluripotent stem cells (PSCs). Previous advances in this field show that the Wnt/beta-catenin signaling pathway plays a crucial role in PSC-derived HSC formation. In this work, lithium, a GSK3 inhibitor, is identified as an element capable of stabilizing beta-catenin and inducing embryonic stem cells (ESCs) differentiation in hemangioblast-like cells, highly consistent with the role of Wnt agonists on ESC differentiation. ESCs treated with 10 mm lithium express CD31+, SCA-1+, Nkx2-5+, CD34+, and FLK1+ cells characteristic of the hemangioblast cells that precede HE development. However, 10 mm Li treated cells remain arrested in a hemangioblast-like phase, which switched into the expression of HE markers after stimulation with maturation medium. The ability of lithium-treated ESCs to further derive into HE is confirmed after defined maturation, resulting in a rapid increase in cells positive for the HE markers RUNX1 and SOX17. The results represent a novel strategy for generating HSC precursors in vitro as a multipotent source of stem cells for blood disease therapies.P.R. acknowledges support from the Spanish Ministry of Science, Innovation, and Universities (RTI2018-096794), and Fondo Europeo de Desarrollo Regional (FEDER). CIBER-BBN was an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. M.S.-S. acknowledges support from the UK Engineering and Physical Sciences Research Council (EPSRC - EP/P001114/1Mnatsakanyan, H.; Salmerón Sánchez, M.; Rico Tortosa, PM. (2021). Lithium Directs Embryonic Stem Cell Differentiation Into Hemangioblast-Like Cells. Advanced Biosystems. 5(8):1-10. https://doi.org/10.1002/adbi.202000569S1105

In Situ Hydroxyapatite Content Affects the Cell Differentiation on Porous Chitosan/Hydroxyapatite Scaffolds

Highly porous chitosan/hydroxyapatite composite structures with different weight ratios (100/0; 90/10; 80/20; 70/30; 60/40; 50/50; 40/60) have been prepared by precipitation method and freeze-gelation technique using calcite, urea phosphate and chitosan as starting materials. The composition of prepared composite scaffolds was characterized by X-ray diffraction analysis and Fourier transformed infrared spectroscopy, while morphology of scaffolds was imaged by scanning electron microscopy. Mercury intrusion porosimetry measurements of prepared scaffolds have shown different porosity and microstructure regarding to the HA content, along with SEM observations of scaffolds after being immersed in physiological medium. The results of swelling capacity and compressive strength measured in Dulbecco’s phosphate buffer saline (DPBS) have shown higher values for composite scaffolds with lower in situ HA content. Viability, proliferation and differentiation of MC3T3-E1 cells seeded on different scaffolds have been evaluated by live dead assay and confocal scan microscopy. Our results suggest that the increase of HA content enhance osteoblast differentiation confirming osteogenic properties of highly porous CS/HA scaffolds for tissue engineering applications in bone repair.The financial support of the Croatian Science Foundation (project: "Development of Biocompatible Hydroxyapatite Based Materials for Bone Tissue Engineering Applications") and L'Oreal-UNESCO Foundation 'For Women in Science' is gratefully acknowledged. The financial support from the Spanish Ministry of Economy and Competitiveness and the Feder funds through the MAT2013-46467-C4-1-R project is acknowledged by the Spanish co-authors. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The authors want to acknowledge Pilar Gomez Tena and Sergio Mestre Beltran from Instituto de Tecnologia Ceramica, Castellon, Spain, for theirs assistance with porosity measurements.Rogina, A.; Rico Tortosa, PM.; Gallego Ferrer, G.; Ivankovic, M.; Ivankovic, H. (2016). In Situ Hydroxyapatite Content Affects the Cell Differentiation on Porous Chitosan/Hydroxyapatite Scaffolds. Annals of Biomedical Engineering. 44(4):1107-1119. https://doi.org/10.1007/s10439-015-1418-0S11071119444Azzaoui, K., A. Lamhamdi, E. M. Mejdoubi, M. Berrabah, B. Hammouti, A. Elidrissi, M. M. G. Fouda, and S. S. Al-Deyab. Synthesis and characterization of composite based on cellulose acetate and hydroxyapatite application to the absorption of harmful substances. Carbohydr. Polym. 111:41–46, 2014.Bacakova, L., E. Filova, M. Parizek, T. Ruml, and V. Svorcik. 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Kakar, K. A. Jacobsen, and T. A. Einhorn. Osteogenic growth factors and cytokines and their role in bone repair. In: Engineering of Functional Skeletal Tissues, in Topics in Bone Biology, edited by M. C. Farach-Carson, A. G. Mikos, and F. Bronner. London: Springer, 2005, pp. 17–44.Harada, S.-I., and G. A. Rodan. Control of osteoblast function and regulation of bone mass. Nature 423:349–355, 2003.Ishihara, S., T. Matsumoto, T. Onoki, T. Sohmura, and A. Nakahira. New concept bioceramics composed of octacalcium phosphate (OCP) and dicarboxylic acid-intercalated OCP via hydrothermal hot-pressing. Mater. Sci. Eng. C 29:1885–1888, 2009.Karageorgiou, V., and D. Kaplan. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26:5474–5491, 2005.Kirkham, G.R., Cartmell, S.H. Genes and proteins involved in the regulation of osteogenesis. In: Topics in Tissue Engineering, edited by N. Ashammakhi, R.L. Reis, and E. Chiellini, R.R.E.C., 2007. pp. 1–22.Lee, H., and G. H. Kim. Cryogenically fabricated three-dimensional chitosan scaffolds with pore size-controlled structures for biomedical applications. Carbohydr. Polym. 85:817–823, 2010.Lewandowska, K. Miscibility and interactions in chitosan acetate/poly(Nvinylpyrrolidone) blends. Thermochim. Acta 517:90–97, 2011.Li, J., D. Zhu, J. Yin, Y. Liu, F. Yao, and K. Yao. Formation of nano-hydroxyapatite cristal in situ in chitosan-pectin polyelectrolyte complex network. Mater. Sci. Eng. C 30:795–803, 2010.Martel-Estrada, S. A., C. A. Martínez-Pérez, J. G. Chacón-Nava, P. E. García-Casillas, and I. Olivas-Armendariz. Synthesis and thermo-physical properties of chitosan/poly(dl-lactide-co-glycolide) composites prepared by thermally induced phase separation. Carbohydr. Polym. 81:775–783, 2010.Martins, A. M., R. C. Pereira, I. B. Leonor, H. S. Azevedo, and R. L. Reis. Chitosan scaffolds incorporating lysozyme into CaP coatings produced by a biomimetic route: a novel concept for tissue engineering combining a self-regulated degradation system with in situ pore formation. Acta Biomater. 5:3328–3336, 2009.Martins, A. M., M. I. Santos, H. S. Azevedo, P. B. Malafaya, and R. L. Reis. Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications. Acta Biomater. 5:1637–1645, 2008.Mohamed, K. R., Z. M. El-Rashidy, and A. A. Salama. In vitro properties of nanohydroxyapatite/chitosan biocomposites. Ceram. Int. 37:3265–3271, 2011.O’Brien, F. J. Biomaterials & scaffolds for tissue engineering. Mater. Today 14:88–95, 2011.Osborn, J. F., and H. Newesely. The material science of calcium phosphate ceramics. Biomaterials 1:108–111, 1980.Rogina, A., M. Ivanković, and H. Ivanković. Preparation and characterization of nano-hydroxyapatite within chitosan matrix. Mater. Sci. Eng. C 33:4539–4544, 2013.Rogina, A., P. Rico, G. Gallego Ferrer, M. Ivanković, and H. Ivanković. Effect of in situ formed hydroxyapatite on microstructure of freeze-gelled chitosan-based biocomposite scaffolds. Eur. Polym. J. 68:278–287, 2015.Sarem, M., F. Moztarzadeh, and M. Mozafari. How can genipin assist gelatin/carbohydrate chitosan scaffolds to act as replacements of load-bearing soft tissues? Carbohydr. Polym. 93:635–643, 2013.Seibel, M. J. Biochemical markers of bone turnover part I: biochemistry and variability. Clin. Biochem. Rev 26:97–122, 2005.Shaltout, A. A., M. A. Allam, and M. A. Moharram. FTIR spectroscopic, thermal and XRD characterization of hydroxyapatite from new natural sources. Spectrochim. Acta A 83:56–60, 2011.Silva, S. S., S. M. Luna, M. E. Gomes, J. Benesch, I. Paskuleva, J. F. Mano, and R. L. Reis. Plasma surface modification of chitosan membranes: characterization and preliminary cell response studies. Macromol. Biosci. 8:568–576, 2007.Stein, G. S., J. B. Lian, A. J. van Wijnen, J. L. Stein, M. 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Lin, D.-M. Wang, and H.-J. Hsieh. Fabrication of a novel porous PGA-chitosan hybrid matrix for tissue engineering. Biomaterials 24:1047–1057, 2003.Yuan, N. Y., Y. A. Lin, M. H. Ho, D. M. Wang, J. Y. Lai, and H. J. Hsieh. Effect of the cooling mode on the structure and strength of porous scaffolds made of chitosan, alginate and carboxymethyl cellulose by freeze-gelation method. Carbohydr. Polym. 78:349–356, 2009

Effect of in situ formed hydroxyapatite on microstructure of freeze-gelled chitosan-based biocomposite scaffolds

New in situ highly-porous chitosan/hydroxyapatite (CS/HA) biocomposite scaffolds have been prepared via freeze-gelation technique. Different content of in situ synthesized hydroxyapatite within chitosan solution was obtained by changing the amount of calcium and phosphate precursors. The composition of precipitated inorganic phase was characterized by X-ray diffraction analysis (XRD) and Fourier transformed infrared spectroscopy (FTIR), while morphology of scaffolds was imaged by scanning electron microscopy (SEM). SEM observations of cross section and surface area of prepared scaffolds have shown different microstructure and topography regarding to the HA content, which plays an important role in cell adhesion and proliferation, and nutrient transport. The MIT assay of scaffolds with different content of hydroxyapatite has shown no toxicity which is one of the main requirements for potential biomedical application. Likewise, the presented synthesis allows preparing the scaffolds with large and very well interconnected pores without obtaining toxic intermediate products.CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Rogina, A.; Rico Tortosa, PM.; Gallego Ferrer, G.; Ivankovic, M.; Ivankovic, H. (2015). Effect of in situ formed hydroxyapatite on microstructure of freeze-gelled chitosan-based biocomposite scaffolds. European Polymer Journal. 68:278-287. https://doi.org/10.1016/j.eurpolymj.2015.05.004S2782876

Determination of synergistic effect between roughness and surface chemistry on cell adhesion of a multilayer Si- Hydroxyapatite coating on Ti6Al4V obtained by magnetron sputtering

[EN] Ti alloys are widely used in the biomedical field because they have an adequate balance between mechanical properties, corrosion resistance and biocompatibility. However, when this material is incorporated in the human body, unfavorable reactions can be obtained which do not allow a good osseointegration due to the formation of a fibrous and non-adherent layer between the biomaterial and the bone, which can lead to failure or rejection of the implant. In this study, the influence of surface modification of Ti6Al4V alloy with a hydroxyapatite (HA) and silicon (Si) coating on its biological response in vitro was evaluated. The deposition process was performed by sputtering on two Ti6Al4V surfaces with different root mean square roughness values, the first one with 3.8 nm and the second one with 48.7 nm. The surface morphology of the coatings was observed by Scanning Electron Microscopy and Atomic Force Microscopy, the chemical composition was evaluated by X-ray Energy Dispersive Spectrometry and micro-Raman Spectroscopy. The in vitro biological response of the coatings was evaluated by cell metabolic activity assay and cell adhesion tests, using mouse mesenchymal stem cells. The control of the process parameters allowed obtaining a multilayer HA-Si coating with good compositional balance (Ca/P ratio very close to 1.67 and characteristic vibrations of the HA phase). Roughness values of 27 +/- 5 nm and 52 +/- 6 nm were obtained for the multilayer coatings obtained on Ti6Al4V (smooth surface) and Ti6Al4V (rough surface), respectively. Biological assays indicated a potentially non-toxic character of the coatings, moreover, the cell adhesion of Ti6Al4V was favored both by the incorporation of the HA-Si multilayer coating and by the increase in the roughness of the substrate.We thank the University of Antioquia, the Centro de Investigacion, Innovacion y Desarrollo de materiales (CIDEMAT) group, the Departamento Administrativo de Ciencia, Tecnologia e Innovacion (COLCIEN-CIAS) for financing the Project 15-1696, the scholarship program of Enlazamundos, the Agencia de Educacion Superior de Medellin (Sapiencia), JLGR acknowledges financial support from the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-1-R and MAT 2015-63974-C4-3-R (AEI/FEDER, UE) (including the FEDER financial support). PR acknowledges support from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096794), and Fondo Europeo de Desarrollo Regional (FEDER). CIBER-BBN is an initiative funded by the VI National R & D & I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the EuropeanPérez Zapata, K.; Lenis, J.; Rico Tortosa, PM.; Gómez Ribelles, JL.; Bolívar, F. (2022). Determination of synergistic effect between roughness and surface chemistry on cell adhesion of a multilayer Si- Hydroxyapatite coating on Ti6Al4V obtained by magnetron sputtering. Thin Solid Films. 760:1-8. https://doi.org/10.1016/j.tsf.2022.1394891876

Fibronectin distribution on demixed nanoscale topographies

[EN] Purpose: It is known that surface nanotopography influences cell adhesion and differentiation. Our aim is to analyze the effect of nanoscale topography on fibronectin adsorption and, afterwards, on cell adhesion in order to rationalize the cell-material interaction by focusing on the state of the intermediate layer of adsorbed fibronectin at the material interphase. Methods: Nanotopographic surfaces were produced by demixing of thin film polymer blends - PLLA and PS - during a high speed spin-casting process. Fibronectin (FN) was adsorbed on the different nanotopographies and the protein distribution was directly observed by atomic force microscopy (AFM). The fraction of the surface covered by the protein was quantified by image analysis, as well as the distribution of FN between peaks and valleys. Focal adhesion protein -vinculin- was immunostained and quantified by image analysis on the different nanoscale surfaces. Results: Different nanoscale domains were obtained by changing the composition of the system within a height range of 3 nm to 30 nm. FN tends to adsorb on the peaks of nanoisland topographies, especially in compositions that did not enhance cell adhesion. Moreover, protein distribution between valleys and peaks alters the size of focal adhesion plaques, which grew larger on surfaces with an even distribution of fibronectin. Conclusions: Our results suggest that the surface nanotopography is a key material property capable of influencing protein adsorption. Additionally, the distribution of the protein on the different samples was correlated to the initial ability of cells to adhere in terms of the size of the focal plaques. © 2011 Wichtig Editore.This studied was funded by the Spanish Ministry of Science and Innovation through MAT2009-14440-C02-01 and TEC2009-14128 grants. CIBER-BBN is an initiative funded by the VI National R&D&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was supported by funds for research in the field of Regenerative Medicine through the collaboration agreement with the Conselleria de Sanidad (Generalitat Valenciana), and the Instituto de Salud Carlos III.Perez Garnes, M.; González García, C.; Moratal Pérez, D.; Rico Tortosa, PM.; Salmerón Sánchez, M. (2011). Fibronectin distribution on demixed nanoscale topographies. International Journal of Artificial Organs. 34(1):54-63. https://doi.org/10.5301/ijao.2011.6316S546334