Dencho Milkov Gugutkov | Research Assistant
We are interested in cell–biomaterials interaction, and more specifically, on the dynamic formation of the provisional extracellular matrix (ECM) – the thin protein layer that cells recognize, produce, and remodel at the materials interface.
We aim to learn how this process affects the biocompatibility of materials, and if it can be controlled by engineering the surface properties of materials. For this purpose, we perform systematic studies in the following directions:
Remodelling of ECM proteins at cell-biomaterials interface
ECM remodelling is a dynamic process that occurs in various physiological and pathological conditions, such as normal development, wound healing and angiogenesis, but also in atherosclerosis, fibrosis, ischemic injury and cancer. It consists of two fundamental processes: assembly and degradation. The organization of ECM is fundamental for biology and medicine, and its proteolytic degradation is a physiological mechanism for the removal of excess ECM. Although matrix remodelling is a subject of extensive biomedical research, the way it is related to the biocompatibility of materials is poorly understood and is therefore a hot topic of our research.
Fig. 1: Dynamic behaviour of ECM proteins at cell-biomaterials interface: Fibroblast remodelling of adsorbed collagen IV (green) depend on a2 integrin (red) function. Colocalization is in orange. Dark zones represents the mechanical removal of adsorbed protein followed by fibri-like organization (arrow).
ECM organization at the biomaterial interface depends on the allowance of cells to rearrange adsorbed matrix proteins – a process strongly dependent on proper functioning of integrin receptors. We anticipate that materials that bind proteins loosely will support the arrangement of a provisional ECM, while stronger binding provokes its degradation.
Biomaterials surface-driven assembly of ECM proteins at the nanoscale
Upon adsorption at material interfaces, proteins may assemble spontaneously and this interaction has significant consequences for their biological response. Recently we have employed distinct silane-inspired chemistries and polymer compositions to create model substrates with tailored densities of -OH, -COOH, -NH2 and -CH3 groups, thus varying the chemistry, charge and hydrophilic/ hydrophobic balance. In a series of communications combining AFM and other nanoindentation techniques, we have described a novel phenomenon of substratum-driven protein assembly depicting the fate of various matrix proteins such as fibronectin, collagen IV, vitronectin and fibrinogen at the above model biomaterials interfaces.
Specifically, we show that by varying the density of chemical functions one can tailor both the assembly and degradation of proteins. Following those findings we aim to control ECM remodelling by engineering specific material properties. Understanding the behavior of ECM proteins on flat biomaterials interface further boosts an important bioengineering target – the biohybrid organ technologies based on two-dimensional protein layers that mimic the arrangement of the natural basement membrane.
Development of artificial basement membrane
This project aims to develop a synthetic basement membrane (BM) to be used as a supportive lining for cellularized implants, with specific focus on the design of a bioengineered blood vessel. Taking advantage of the self-assembly properties of the two principal components of the BM, laminin and collagen IV, composite matrices of these molecules are produced by mixing them before or during the polymerization of laminin under acidic conditions.
Selected composites will be deposited on scaffolds produced using electronspun nanofibers preferentially made of polyethyl acrilate (PEA), which additionally favour networking of laminin and collagen IV. The resemblance to natural BM will be evaluated in terms of their morphological features and ability to properly induce the formation of biomimetic monolayers of endothelial cells. This project is driven involving joint efforts of Dr Coelho-Sampaio’s Lab from the Federal University of Rio de Janeiro, Brazil.
Electrospinning of nanofibers from natural and synthetic polymers for guiding cellular behaviour
In solution, proteins can form structures of various shapes, including fibers with a diameter of only a few nanometers and with lengths up to centimeters. A fascinating possibility to mimic similar ECM structures is to engineer protein-like or matrix protein-containing nanofibers via electrospinning technology. For this purpose we are developing electrospun nanofibers from natural (e.g., fibrinogen) and synthetic polymers (e.g. PLA, PEA) in order to direct the desired cellular response via spatially organized cues (e.g. fiber size and geometrical organization) as well as by tailoring their chemical and mechanical properties.
Fig. 4: Hybrid PLA/fibrinogen nanofibers deposited in random (A) and aligned (B) configurations. Human mesenchymal stem cells adhere to the fibers and acquire a stellate-like (C & E) or elongated (D & F) morphology, depending on the fiber orientations (staining: vinculin in red and actin in green).
Nanofibers-based 3D constructs providing stem cells with spatially organized stimuli
Examining hierarchical biology in only two dimensions (i.e., cells confined to a monolayer) is in most cases insufficient as cells typically exhibit unnatural behavior if excised from native three-dimensional (3D) tissues. Therefore, within the European FIBROGELNET project (under our coordination) we are developing 3D biohybrid constructs that combine the structural and biological properties of electrospun nanofibers with the optimized mechanical properties of specific hydrogels in order to provide stem cells with relevant spatial orientation in three dimensions.
Creating dynamic stem cell niches using stimuli-responsive biomaterials
In addition to engineering the spatial configuration of cellular microenvironments, we are also interested in addressing the dynamic (i.e., temporal) aspects of the stem cell niche. To do that we take advantage of stimuli-responsive polymers to obtain control over an artificial cell-adhesive environment via dynamically altering either cell-cell (using cadherin-like ligands) or cell-matrix (using ECM proteins) interactions. By modulating the strength of adhesive protein-to-substratum interactions we aim to control the stem cell adhesive machinery, and which allows us to mimic the dynamic conditions of the stem cell niche.
Fig. 6: Reversible attachment/detachment of human mesenchymal stem cells from thermo-responsible PNIPAM substrata: Cells were cultured at 37ºC for 5 h on PNIPAM (A) and left to detach at room temperature for 2 hours (B), then switched again to 37ºC overnight (C).
The two IBEC-led CIBER-BBN tissue regeneration projects that were earmarked for funding by the EU’s ERA-NET EuroNanoMed initiative last year (see here) have both received the national support they need to get started.
A multidisciplinary research project coordinated at IBEC by group leader George Altankov has been selected for funding by the EU as part of the European-Latin American Network for Science and Technology (EULANEST).
|STRUCTGEL Nanostructured Gel for Cellular Therapy of Degenerative Skeletal Disorders||EURONANOMED||George Altankov|
|FIBROGELNET Network for Development of Soft Nanofibrous Construct for Cellular Therapy of Degenerative Skeletal Disorders||FP7-PEOPLE-2012-IAPP||George Altankov|
|Materiales que inducen la fibrilogénesis de la fibronectina para producir microambientes sinérgicos en los factores de crecimiento||I+D-Investigación fundamental no orientada||George Altankov|
|MYOREM Remodelación por mioblastos de la matriz extracelular en la interfaz celula-biomaterial (2016-2018)||MINECO, Retos investigación: Proyectos I+D||George Altankov|
|MYOHEAL Muscle regeneration after injury. Engineered biodegradable ion–loaded scaffolds to promote muscle regeneration (2015-2017)||MINECO, MAT 2015 – 69315 –C3||George Altankov|
Kristova-Panusheva, K., Keremidarska-Markova, M., Altankov, G., Krasteva, N., (2017). Age-related changes in adhesive phenotype of bone marrow-derived mesenchymal stem cells on extracellular matrix proteins Journal of New Results in Science 6, (1), 11-19
Nedjari, Salima, Awaja, Firas, Altankov, George, (2017). Three dimensional honeycomb patterned fibrinogen based nanofibers induce substantial osteogenic response of mesenchymal stem cells Scientific Reports 7, (1), 15947
Bianchi, M. V., Awaja, F., Altankov, G., (2017). Dynamic adhesive environment alters the differentiation potential of young and ageing mesenchymal stem cells Materials Science and Engineering C 78, 467-474
Gugutkov, D., Gustavsson, J., Cantini, M., Salmeron-Sánchez, M., Altankov, G., (2017). Electrospun fibrinogen-PLA nanofibres for vascular tissue engineering Journal of Tissue Engineering and Regenerative Medicine 11, (10), 2774-2784
Gugutkov, D., Awaja, F., Belemezova, K., Keremidarska, M., Krasteva, N., Kuyrkchiev, S., GallegoFerrer, G., Seker, S., Elcin, A. E., Elcin, Y. M., Altankov, G., (2017). Osteogenic differentiation of mesenchymal stem cells using hybrid nanofibers with different configurations and dimensionality Journal of Biomedical Materials Research - Part A 105, (7), 2065-2074
Zhao, M., Altankov, G., Grabiec, U., Bennett, M., Salmeron-Sanchez, M., Dehghani, F., Groth, T., (2016). Molecular composition of GAG-collagen I multilayers affects remodeling of terminal layers and osteogenic differentiation of adipose-derived stem cells Acta Biomaterialia 41, 86-99
Forget, J., Awaja, F., Gugutkov, D., Gustavsson, J., Gallego Ferrer, G., Coelho-Sampaio, T., Hochman-Mendez, C., Salmeron-Sánchez, M., Altankov, G., (2016). Differentiation of human mesenchymal stem cells toward quality cartilage using fibrinogen-based nanofibers Macromolecular Bioscience 16, (9), 1348-1359
Coelho, N. M., Llopis-Hernández, V., Salmerón-Sánchez, M., Altankov, G., (2016). Dynamic reorganization and enzymatic remodeling of type IV collagen at cell–biomaterial interface Advances in Protein Chemistry and Structural Biology (ed. Christo, Z. Christov), Academic Press (San Diego, USA) 105, 81-104
Toromanov, Georgi, Gugutkov, Dencho, Gustavsson, Johan, Planell, Josep, Salmerón-Sánchez, Manuel, Altankov, George, (2015). Dynamic behavior of vitronectin at the cell-material interface ACS Biomaterials Science & Engineering 1, (10), 927-934
Keremidarska, M., Gugutkov, D., Altankov, G., Krasteva, N., (2015). Impact of electrospun nanofibres orientation on mesenchymal stem cell adhesion and morphology Comptes Rendus de L'Academie Bulgare des Sciences 68, (10), 1271-1276
Rico, P., Cantini, M., Altankov, G., Sanchez, M. , (2015). Matrix-protein interactions with synthetic surfaces Polymers in Regenerative Medicine: Biomedical Applications from Nano- to Macro-Structures (ed. Monleon Pradas, M., Vicent, M.J.), John Wiley & Sons Inc (Hoboken, USA) , 91-146
Groth, T., Guduru, D., Altankov, George, Zenoby-Wong, M., Millan, C., Cavalli, E., Kesti, M., (2014). Procedure for making scaffold structures for tissue engineering applications, implants and surgery grafts by means of layer-by-layer method Martin Lutter University-Halle-Wittenberg; Fundació Institut de Bioenginyeria de Catalunya; Eidgenoessische Technische Hochschule Zuerich , (DE 10 2014 017 950.8)
Perez, Roman A., Riccardi, Kiara, Altankov, George, Ginebra, Maria-Pau, (2014). Dynamic cell culture on calcium phosphate microcarriers for bone tissue engineering applications Journal of Tissue Engineering 5, 2041731414543965
Gugutkov, Dencho, Gustavsson, Johan, Ginebra, Maria Pau, Altankov, George, (2013). Fibrinogen nanofibers for guiding endothelial cell behavior Biomaterials Science 1, (10), 1065-1073
Coelho, Nuno Miranda, Salmeron-Sanchez, Manuel, Altankov, George, (2013). Fibroblasts remodeling of type IV collagen at a biomaterials interface Biomaterials Science 1, (5), 494-502
Cantini, M., Sousa, M., Moratal, D., Mano, J. F., Salmerón-Sánchez, M., (2013). Non-monotonic cell differentiation pattern on extreme wettability gradients Biomaterials Science 1, (2), 202-212
Llopis-Hernández, V., Rico, P., Moratal, D., Altankov, George, Salmeron-Sanchez, Manuel, (2013). Role of material-driven fibronectin fibrillogenesis in protein remodeling BioResearch Open Access 2, (5), 364-373
Perez, R. A., Altankov, G., Jorge-Herrero, E., Ginebra, M. P., (2013). Micro- and nanostructured hydroxyapatite-collagen microcarriers for bone tissue-engineering applications Journal of Tissue Engineering and Regenerative Medicine 7, (5), 353-361
González-García, C., Cantini, M., Moratal, D., Altankov, G., Salmerón-Sánchez, M., (2013). Vitronectin alters fibronectin organization at the cell-material interface Colloids and Surfaces B: Biointerfaces 111, 618-625
Cantini, M., Rico, P., Moratal, D., Salmerón-Sánchez, M., (2012). Controlled wettability, same chemistry: Biological activity of plasma-polymerized coatings Soft Matter 8, (20), 5575-5584
Pecheva, E., Pramatarova, L., Hikov, T., Hristova, K., Altankov, G., Montgomery, P., Hanawa, T., (2012). Electrodeposition of hydroxyapatite-nanodiamond composite coating on metals, interaction with proteins and osteoblast-like cells Electrodeposition: Properties, processes and applications (ed. Udit Surya Mohanty), Nova Publishers (Hauppauge, USA) Electrical Engineering Developments, 233-253
Miranda Coelho, Nuno, Gonzalez-Garcia, Cristina, Salmeron-Sanchez, Manuel, Altankov, George, (2011). Arrangement of type IV collagen and laminin on substrates with controlled density of -OH groups Tissue Engineering Part A 17, (17-18), 2245-2257
Miranda Coelho, Nuno, Gonzalez-Garcia, Cristina, Salmeron-Sanchez, Manuel, Altankov, George, (2011). Arrangement of type IV collagen on NH(2) and COOH functionalized surfaces Biotechnology and Bioengineering 108, (12), 3009-3018
Gugutkov, Dencho, Gonzalez-Garcia, Cristina, Altankov, George, Salmeron-Sanchez, Manuel, (2011). Fibrinogen organization at the cell-material interface directs endothelial cell behavior Journal of Bioactive and Compatible Polymers 26, (4), 375-387
Hristova, K., Pecheva, E., Pramatarova, L., Altankov, G., (2011). Improved interaction of osteoblast-like cells with apatite-nanodiamond coatings depends on fibronectin Journal of Materials Science: Materials in Medicine 22, (8), 1891-1900
Perez, R. A., Del Valle, S., Altankov, G., Ginebra, M. P., (2011). Porous hydroxyapatite and gelatin/hydroxyapatite microspheres obtained by calcium phosphate cement emulsion Journal of Biomedical Materials Research - Part B: Applied Biomaterials 97B, (1), 156-166
Coelho, N. M., Gonzalez-Garcia, C., Planell, J. A., Salmeron-Sanchez, M., Altankov, G., (2010). Different assembly of type iv collagen on hydrophilic and hydrophobic substrata alters endothelial cells interaction European Cells & Materials 19, 262-272
Pegueroles, M., Aparicio, C., Bosio, M., Engel, E., Gil, F. J., Planell, J. A., Altankov, G., (2010). Spatial organization of osteoblast fibronectin matrix on titanium surfaces: Effects of roughness, chemical heterogeneity and surface energy Acta Biomaterialia 6, (1), 291-301
Toromanov, Georgi, González-García, Cristina, Altankov, George, Salmerón-Sánchez, Manuel, (2010). Vitronectin activity on polymer substrates with controlled -OH density Polymer 51, (11), 2329-2336
Gugutkov, D., Altankov, G., Hernandez, J. C. R., Pradas, M. M., Sanchez, M. S., (2010). Fibronectin activity on substrates with controlled -OH density Journal of Biomedical Materials Research - Part A 92A, (1), 322-331
Krasteva, N. A., Toromanov, G., Hristova, K. T., Radeva, E. I., Pecheva, E. V., Dimitrova, R. P., Altankov, G. P., Pramatarova, L. D., (2010). Initial biocompatibility of plasma polymerized hexamethyldisiloxane films with different wettability Journal of Physics: Conference Series 16 ISCMP: Progress in Solid State and Molecular Electronics, Ionics and Photonics , IOP Publishing Ltd. (Varna, Bulgaria) 253, (1), 012079 (7 pp.)
Pramatarova, L. D., Krasteva, N. A., Radeva, E. I., Pecheva, E. V., Dimitrova, R. P., Hikov, T. A., Mitev, D. P., Hristova, K. T., Altankov, G., (2010). Study of detonation nanodiamond - Plasma polymerized hexamethildisiloxan composites for medical application Journal of Physics: Conference Series 16 ISCMP: Progress in Solid State and Molecular Electronics, Ionics and Photonics , IOP Publishing Ltd. (Varna, Bulgaria) 253, (1), 012078 (7 pp.)
Salmeron-Sanchez, M., Altankov, G., (2010). Cell-Protein-Material interaction in tissue engineering Tissue Engineering (ed. Eberli, D.), Intech (Vukovar, Croatia) , 77-102
Altankov, George, Groth, Thomas, Engel, Elisabeth, Gustavsson, Jonas, Pegueroles, Marta, Aparicio, Conrado, Gil, Francesc J., Ginebra, Maria-Pau, Planell, Josep A., (2010). Development of provisional extracellular matrix on biomaterials interface: Lessons from in vitro cell culture NATO Science for Peace and Security Series A: Chemistry and Biology Advances in Regenerative Medicine: Role of Nanotechnology, and Engineering Principles (ed. Shastri, P., Altankov, G., Lendlein, A.), Springer Netherlands (Dortrecht, The Netherlands) , 19-43
Planell, Josep A., Navarro, Melba, Altankov, George, Aparicio, Conrado, Engel, Elisabeth, Gil, Javier, Ginebra, Maria Pau, Lacroix, Damien, (2010). Materials surface effects on biological interactions NATO Science for Peace and Security Series A: Chemistry and Biology Advances in Regenerative Medicine: Role of Nanotechnology, and Engineering Principles (ed. Shastri, P., Altankov, G., Lendlein, A.), Springer Netherlands (Dortrecht, The Netherlands) , 233-252
Gugutkov, Dencho, Gonzalez-Garcia, Cristina, Rodriguez Hernandez, Jose Carlos, Altankov, George, Salmeron-Sanchez, Manuel, (2009). Biological activity of the substrate-induced fibronectin network: insight into the third dimension through electrospun fibers Langmuir 25, (18), 10893-10900
Kostadinova, A., Seifert, B., Albrecht, W., Malsch, G., Groth, T., Lendlein, A., Altankov, G., (2009). Novel polymer blends for the preparation of membranes for biohybrid liver systems Journal of Biomaterials Science, Polymer Edition 20, (5-6), 821-839
Rico, P., Rodriguez Hernandez, J. C., Moratal, D., Altankov, G., Monleon Pradas, M., Salmeron-Sanchez, M., (2009). Substrate-induced assembly of fibronectin into networks. Influence of surface chemistry and effect on osteoblast adhesion Tissue Engineering Part A 15, (00), 1-11
Kirchhof, K., Hristova, K., Krasteva, N., Altankov, G., Groth, T., (2009). Multilayer coatings on biomaterials for control of MG-63 osteoblast adhesion and growth Journal of Materials Science: Materials in Medicine 20, (4), 897-907
Engel, E., Del Valle, S., Aparicio, C., Altankov, G., Asin, L., Planell, J. A., Ginebra, M. P., (2008). Discerning the role of topography and ion exchange in cell response of bioactive tissue engineering scaffolds Tissue Engineering Part A 14, (8), 1341-1351
Maneva-Radicheva, L., Ebert, U., Dimoudis, N., Altankov, G., (2008). Fibroblast remodeling of adsorbed collagen type IV is altered in contact with cancer cells Histology and Histopathology 23, (7), 833-842
Manara, S., Paolucci, F., Palazzo, B., Marcaccio, M., Foresti, E., Tosi, G., Sabbatini, S., Sabatino, P., Altankov, G., Roveri, N., (2008). Electrochemically-assisted deposition of biomimetic hydroxyapatite-collagen coatings on titanium plate Inorganica Chimica Acta 361, (6), 1634-1645
Gustavsson, J., Altankov, G., Errachid, A., Samitier, J., Planell, J. A., Engel, E., (2008). Surface modifications of silicon nitride for cellular biosensor applications Journal of Materials Science-Materials in Medicine 19, (4), 1839-1850
- Laser scanning confocal microscope equiped for performing dynamic
studies with living cells
- Full facilities for cell culturing
- Electrospinning device designed for the production of nanofibers from natural and synthetic polymers
- Laboratory freeze-dryer (Telstar Cryodos)
- Spectrofluorometer Fluormax 4 (Horiba, Jobin Yvon)
- Complete chromatographic and electrophoretic equipment
- Flow chamber setup for measuring the strength of cell adhesion
- Programmable compact spin coater
- Center for Biomaterials
Technical University of Valencia, Spain
- Institute of Pharmacy
Martin Luther University, Halle, Germany
- Institute of Biomedical Science
Federal University of Rio de Janeiro, Brazil
- Institute for Biophysics and Biomedical Engineering
Bulgarian Academy of Sciences, Sofia, Bulgaria
- Institute of Solid State Physics
Bulgarian Academy of Sciences, Sofia, Bulgaria
- Division of Biomedical Engineering, School of Engineering
University of Glasgow, United Kingdom