Staff member

George Altankov

Group Leader / ICREA Research Professor
Molecular Dynamics at Cell-Biomaterial Interface
+34 934 039 709
CV Summary

George Altankov received his MD in 1974 from the Medical University of Varna (Bulgaria) where he also started his research career as Assistant Professor of Physiology. After obtaining his PhD he joined a research team at the Institute of Biophysics, Bulgarian Academy of Sciences, advancing to Associate Professor (1995) and Full Professor (2004) as well as Head of the Cell Adhesion Department (2004-2007) and Research Director of the institute (2004-2006). In 2003 he obtained a Doctor of Sciences degree in Cell Biology. In 1991-1993 he joined the group of Fred Grinnell in Southwestern Medical Center at Dallas and later visited several leading laboratories in Europe, including Humboldt University, GKSS Research Center (Berlin), Technical University of Barcelona and others, working on joint projects. He was awarded a Marie Curie senior EU fellowship and a Sabbatical Grant of the Spanish Ministry (2006). He has published more than 100 original papers and filed several patents in the field of cell-biomaterials interaction and tissue engineering. Since April 2007 he has been an ICREA Research Professor at the Institute for Bioengineering of Catalonia (IBEC), continuing his research in the field with emphasis on the organization of extracellular matrix at biomaterials interfaces.

Staff member publications

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

Engineering dynamic stem cell niche-like environment offers opportunity to obtain better control of the fate of stem cells. We identified, for the first time, that periodic changes in the adhesive environment of human adipose derived mesenchymal stem cells (ADSCs) alters dramatically their asymmetric division but not their ability for symmetric renewal. Hereby, we used smart thermo-responsive polymer (PNIPAM) to create a dynamic adhesive environment for ADSCs by applying periodic temperature cycles to perturb adsorbed adhesive proteins to substratum interaction. Cumulative population doubling time (CPDT) curves showed insignificant decline in the symmetric cell growth studied for up to 13th passages accompanied with small changes in the overall cell morphology and moderately declined fibronectin (FN) matrix deposition probably as a functional consequence of ADSCs ageing. However, a substantial alteration in the differentiation potential of ADSCs from both early and late passages (3rd and 14th, respectively) was found when the cells were switched to osteogenic differentiation conditions. This behavior was evidenced by the significantly altered alkaline phosphatase activity and Ca deposition (Alizarin red) assayed at 3, 14 and 21 day in comparison to the control samples of regular TC polystyrene processed under same temperature settings.

Keywords: Cell ageing, Dynamic adhesive environment, Extracellular matrix, Mesenchymal stem cells, PNIPAM, Stem cell niche, Symmetric and asymmetric cell growth, Thermo-cycling, Thermo-responsive polymer

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

Here we report on the development of a new type of hybrid fibrinogen-polylactic acid (FBG-PLA) nanofibres (NFs) with improved stiffness, combining the good mechanical properties of PLA with the excellent cell recognition properties of native FBG. We were particularly interested in the dorsal and ventral cell response to the nanofibres' organization (random or aligned), using human umbilical endothelial cells (HUVECs) as a model system. Upon ventral contact with random NFs, the cells developed a stellate-like morphology with multiple projections. The well-developed focal adhesion complexes suggested a successful cellular interaction. However, time-lapse analysis shows significantly lowered cell movements, resulting in the cells traversing a relatively short distance in multiple directions. Conversely, an elongated cell shape and significantly increased cell mobility were observed in aligned NFs. To follow the dorsal cell response, artificial wounds were created on confluent cell layers previously grown on glass slides and covered with either random or aligned NFs. Time-lapse analysis showed significantly faster wound coverage (within 12 h) of HUVECs on aligned samples vs. almost absent directional migration on random ones. However, nitric oxide (NO) release shows that endothelial cells possess lowered functionality on aligned NFs compared to random ones, where significantly higher NO production was found. Collectively, our studies show that randomly organized NFs could support the endothelization of implants while aligned NFs would rather direct cell locomotion for guided neovascularization.

Keywords: Electrospun nanofibers, Endothelial cells, Fibrinogen, Guided cellular behavior, Polylactic acid, Vascular tissue engineering

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

Novel hybrid, fibrinogen/polylactic acid (FBG/PLA) nanofibers with different configuration (random vs. aligned) and dimensionality (2D vs.3D environment) were used to control the overall behaviour and the osteogenic differentiation of human Adipose Derived Mesenchymal Stem Cells (ADMSCs). Aligned nanofibers in both the 2D and 3D configurations are proved to be favoured for osteo-differentiation. Morphologically we found that on randomly configured nanofibers, the cells developed a stellate-like morphology with multiple projections, however, time-lapse analysis showed significantly diminished cell movements. Conversely, an elongated cell shape with advanced cell spreading and extended actin cytoskeleton accompanied with significantly increased cell mobility were observed when cells attached on aligned nanofibers. Moreover, a clear tendency for higher alkaline phosphatase activity was also found on aligned fibres when ADMSCs were switched to osteogenic induction medium. The strongest accumulation of Alizarin red (AR) and von Kossa stain at 21 day of culture in osteogenic medium were found on 3D aligned constructs while the rest showed lower and rather undistinguishable activity. Quantitative reverse transcription-polymerase chain reaction analysis for Osteopontin (OSP) and RUNX 2 generally confirmed this trend showing favourable expression of osteogenic genes activity in 3D environment particularly in aligned configuration.

Keywords: Mesenchymal stem cells, Nanofibers, Osteogenic, Fibrinogen, Cell movements

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

The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2β1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment. Statement of Significance: Natural polymer derived polyelectrolyte multilayers (PEMs) have been recently applied to adjust biomaterials to meet specific tissue demands. However, the effect of molecular composition of multilayers on both surface properties and cellular response, especially the fate of human adipose derived stem cells (hADSCs) upon osteogenic differentiation has not been studied extensively, yet. In addition, no studies exist that investigate a potential cell-dependent remodeling of PEMs made of extracellular matrix (ECM) components like collagens and glycosaminoglycans (GAGs). Furthermore, there is no knowledge whether the ability of cells to remodel PEM components may provide an added value regarding cell growth and differentiation. Finally, it has not been explored yet, how intrinsic cross-linking of ECM derived polyelectrolytes that improve the stability of PEMs will affect the differentiation potential of hADSCs. The current work aims to address these questions and found that the type of GAG has a strong effect on properties of multilayers and osteogenic differentiation of hADSCs. Additionally, we also show for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal layers as completely new finding, which allows cells to form an ECM-like environment supporting differentiation upon osteogenic lineage. The finding of this work may open new avenues of application of PEM systems made by layer by layer (LbL) technique in tissue engineering and regenerative medicine.

Keywords: Collagen reorganization, Glycosaminoglycans, Layer-by-layer technique, Mesenchymal stem cells, Osteogenic differentiation

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

Mimicking the complex intricacies of the extra cellular matrix including 3D configurations and aligned fibrous structures were traditionally perused for producing cartilage tissue from stem cells. This study shows that human adipose derived mesenchymal stem cells (hADMSCs) establishes significant chondrogenic differentiation and may generate quality cartilage when cultured on 2D and randomly oriented fibrinogen/poly-lactic acid nanofibers compared to 3D sandwich-like environments. The adhering cells show well-developed focal adhesion complexes and actin cytoskeleton arrangements confirming the proper cellular interaction with either random or aligned nanofibers. However, quantitative reverse transcription-polymerase chain reaction analysis for Collagen 2 and Collagen 10 genes expression confirms favorable chondrogenic response of hADMSCs on random nanofibers and shows substantially higher efficacy of their differentiation in 2D configuration versus 3D constructs. These findings introduce a new direction for cartilage tissue engineering through providing a simple platform for the routine generation of transplantable stem cells derived articular cartilage replacement that might improve joint function.

Keywords: Cartilage, Chondrogenic response, Collagen, FBG/PLA nanofibers, Mesenchymal stem cells

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

Abstract Vascular basement membrane remodeling involves assembly and degradation of its main constituents, type IV collagen (Col IV) and laminin, which is critical during development, angiogenesis, and tissue repair. Remodeling can also occur at cell–biomaterials interface altering significantly the biocompatibility of implants. Here we describe the fate of adsorbed Col IV in contact with endothelial cells adhering on positively charged NH2 or hydrophobic CH3 substrata, both based on self-assembly monolayers (SAMs) and studied alone or mixed in different proportions. AFM studies revealed distinct pattern of adsorbed Col IV, varying from single molecular deposition on pure NH2 to network-like assembly on mixed SAMs, turning to big globular aggregates on bare CH3. Human umbilical endothelial cells (HUVECs) interact better with Col IV adsorbed as single molecules on NH2 surface and readily rearrange it in fibril-like pattern that coincide with secreted fibronectin fibrils. The cells show flattened morphology and well-developed focal adhesion complexes that are rich on phosphorylated FAK while expressing markedly low pericellular proteolytic activity. Conversely, on hydrophobic CH3 substrata HUVECs showed abrogated spreading and FAK phosphorylation, combined with less reorganization of the aggregated Col IV and significantly increased proteolytic activity. The later involves both MMP-2 and MMP-9, as measured by zymography and FITC-Col IV release. The mixed SAMs support intermediate remodeling activity. Taken together these results show that chemical functionalization combined with Col IV preadsorption provides a tool for guiding the endothelial cells behavior and pericellular proteolytic activity, events that strongly affect the fate of cardiovascular implants.

Keywords: Type IV collagen, Adsorption, Remodeling, Pericellular proteolysis, Reorganization, Substratum chemistry, CH3 and NH2 groups, Self-assembly monolayers

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

Considering that vitronectin (VN) can promote both cell adhesion and matrix degradation, it is likely to play a dual role at the cell-biomaterial interface. In this paper we therefore describe details of the dynamic interplay between matrix adhesion and pericellular proteolysis in endothelial cells adhered to glass model substratum. Initially we show that coating concentration determines protein organization at the surface. When the protein coating density approached saturation (63 ng cm?2), VN spontaneously organized itself in multimeric aggregates at the surface (30?50 nm in diameter). At subsaturation protein density (17 ng cm?2) VN molecules were present predominantly as single entities, indicating that a minimum coating density was required for VN multimerization. By fluorescent visualization of surface-associated VN in different ways, we provide the first evidence of significant proteolytic remodelling of VN by endothelial cells (HUVECs) at the sites of αv integrin clusters. The degree of proteolysis was estimated using a novel approach relying on dequenching of FITC-labeled VN upon proteolytic activity, showing that about one-third of the surface-associated VN was proteolytically altered by adhering HUVECs. In addition, we demonstrate that HUVECs can internalize surface-associated VN and deposit it in a linear pattern along longitudinal actin filaments. Deposited VN was partly colocalized with urokinase receptors. Taken altogether, we elucidate the complex and dynamic behavior of VN during initial cell?biomaterials interactions, the equilibrium if which could have a significant impact on the biocompatibility of any blood contacting implants. Considering that vitronectin (VN) can promote both cell adhesion and matrix degradation, it is likely to play a dual role at the cell-biomaterial interface. In this paper we therefore describe details of the dynamic interplay between matrix adhesion and pericellular proteolysis in endothelial cells adhered to glass model substratum. Initially we show that coating concentration determines protein organization at the surface. When the protein coating density approached saturation (63 ng cm?2), VN spontaneously organized itself in multimeric aggregates at the surface (30?50 nm in diameter). At subsaturation protein density (17 ng cm?2) VN molecules were present predominantly as single entities, indicating that a minimum coating density was required for VN multimerization. By fluorescent visualization of surface-associated VN in different ways, we provide the first evidence of significant proteolytic remodelling of VN by endothelial cells (HUVECs) at the sites of αv integrin clusters. The degree of proteolysis was estimated using a novel approach relying on dequenching of FITC-labeled VN upon proteolytic activity, showing that about one-third of the surface-associated VN was proteolytically altered by adhering HUVECs. In addition, we demonstrate that HUVECs can internalize surface-associated VN and deposit it in a linear pattern along longitudinal actin filaments. Deposited VN was partly colocalized with urokinase receptors. Taken altogether, we elucidate the complex and dynamic behavior of VN during initial cell?biomaterials interactions, the equilibrium if which could have a significant impact on the biocompatibility of any blood contacting implants.

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

Electrospun nanofibrous materials mimicking the architecture of native extracellular matrix (ECM) hold great promise as scaffolds in tissue engineering. In order to optimize the properties of nanofibrous scaffolds it is important to understand the impact of fibres’ organization on cell behaviour. Herein, we investigated the effect of nanofibres (NFs) alignment on human adipose-derived mesenchymal stem cells (hAD-MSCs) adhesion and morphology. Electrospun composite fibrinogen/poly-lactic acid (FNG/PLA) NF scaffolds with same composition and comparable fibre size were fabricated into randomly oriented and aligned configuration and stem cells adhesion was characterized by the meaning of overall cell morphology, actin cytoskeleton organization and expression of molecules, involved in the development of focal adhesion complexes. We found that hAD-MSCs altered their morphology, actin cytoskeleton and cell attachment in accordance with nanofibre orientation while cell spreading, focal adhesions and expression of β1 and αNintegrin receptors were not influenced significantly by fibre orientation. These results confirmed that fibre alignment of scaffold guide cellular arrangement and could be beneficial for stem differentiation and therefore for the successful scaffolds development if its contact guidance coincided with the cell shape and cytoskeletal tension.

Keywords: Electrospinning, Fibrinogen/polylactic acid hybrid nanofibres, Human adipose-derived stem cells

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

The ability of polymers to span wide ranges of mechanical properties and morph into desired shapes makes them useful for a variety of applications, including scaffolds, self-assembling materials, and nanomedicines. With an interdisciplinary list of subjects and contributors, this book overviews the biomedical applications of polymers and focuses on the aspect of regenerative medicine. Chapters also cover fundamentals, theories, and tools for scientists to apply polymers in the following ways: Matrix protein interactions with synthetic surfaces Methods and materials for cell scaffolds Complex cell-materials microenvironments in bioreactors Polymer therapeutics as nano-sized medicines for tissue repair Functionalized mesoporous materials for controlled delivery Nucleic acid delivery nanocarriers Concepts include macro and nano requirements for polymers as well as future perspectives, trends, and challenges in the field. From self-assembling peptides to self-curing systems, this book presents the full therapeutic potential of novel polymeric systems and topics that are in the leading edge of technology.

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

Developing appropriate cell culturing techniques to populate scaffolds has become a great challenge in tissue engineering. This work describes the use of spinner flask dynamic cell cultures to populate hydroxyapatite microcarriers for bone tissue engineering. The microcarriers were obtained through the emulsion of a self-setting aqueous α-tricalcium phosphate slurry in oil. After setting, hydroxyapatite microcarriers were obtained. The incorporation of gelatin in the liquid phase of the α-tricalcium phosphate slurry allowed obtaining hybrid gelatin/hydroxyapatite-microcarriers. Initial cell attachment on the microcarriers was strongly influenced by the speed of the dynamic culture, achieving higher attachment at low speed (40 r/min) as compared to high speed (80 r/min). Under moderate culture speeds (40 r/min), the number of cells present in the culture as well as the number of microcarrier-containing cells considerably increased after 3 days, particularly in the gelatin-containing microcarriers. At longer culture times in dynamic culture, hydroxyapatite-containing microcarriers formed aggregates containing viable and extracellular matrix proteins, with a significantly higher number of cells compared to static cultures.

Gugutkov, Dencho, Gustavsson, Johan, Ginebra, Maria Pau, Altankov, George, (2013). Fibrinogen nanofibers for guiding endothelial cell behavior Biomaterials Science 1, (10), 1065-1073

This paper describes the biological consequences of presenting electrospun fibrinogen (FBG) to endothelial cells as a spatially organized nanofibrous matrix. Aligned and randomly oriented FBG nanofibers with an average diameter of less than 200 nm were obtained by electrospinning of native FBG solution. Electrophoretic profiling confirmed that the electrospun FBG resembled the native protein structure, and fluorescent tracing of FITC-labeled FBG showed that electrospun fibers withstood immersion in physiological solutions reasonably well for several days. With respect to cellular interactions, the nanofibrous FBG matrix provided better conditions for initial recognition by human umbilical vein endothelial cells compared to pre-adsorbed FBG on a flat surface. Furthermore, the spatial organization of electrospun FBG fibers presented opportunities for guiding the cellular behavior in a way that is not possible when the protein is presented in another form (e.g. adsorbed or soluble). For example, on aligned FBG fibers, cells rapidly oriented themselves along the fibers, and time-lapse recordings revealed pronounced cellular movements restricted to the fiber direction. In great contrast, on randomly deposited fibers, cells acquired a stellate-like morphology and became locally immobilized by the fibers. We also show that the FBG fiber orientation significantly influenced both the cytoskeleton organization in confluent cell layers and the orientation of the extracellular fibronectin matrix secreted by the cells. In conclusion, this study demonstrates that electrospun FBG nanofibers can be a promising tool for guiding endothelial cell behavior for tissue engineering applications.

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

This paper describes the fate of adsorbed type IV collagen (Col IV) in contact with fibroblasts on model biomaterial surfaces, varying in wettability, chemistry and charge. We found that fibroblasts not only interact but also tend to remodel differently adsorbed Col IV employing two distinct mechanisms: mechanical reorganization and proteolytic degradation. Apart from the trend of adsorption -NH2 > CH3 > COOH > OH- the cells interact better with NH2 and OH surfaces - i.e. independently of the amount of adsorbed Col IV - evident from the quantitative measurements of cell adhesion and spreading and the improved recruitment of alpha 1 and alpha 2 integrins as well as p-FAK in focal adhesions. The linearly arranged Col IV co-localize with FN fibrils formed from either secreted, or exogenously added protein, which confirms their interdependence during a reorganization process. We further found that this reorganization is better pronounced on hydrophilic OH and positively charged NH2 surfaces correlating with the improved cellular interaction. Conversely, the fibroblasts tend to round on COOH and CH3 surfaces in compliance with the altered integrin signaling and also the increased pericellular proteolysis activity quantified by the increased de-quenching of adsorbed FITC-Col IV and zymography. Taken together, these results show that remodeling of Col IV at a cell-biomaterial interface depends strongly on the surface properties of a material and affects significantly its biological performance.

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

Protein remodeling at the cell–material interface is an important phenomenon that should be incorporated into the design of advanced biomaterials for tissue engineering. In this work, we address the relationship between fibronectin (FN) activity at the material interface and remodeling, including proteolytic cascades. To do so, we studied FN adsorption on two chemically similar substrates, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA), which resulted in different distribution and conformation of the protein at the material interface: FN organized spontaneously upon adsorption on PEA into physiological-like fibrils, through a process called material-driven FN fibrillogenesis. The amount of adsorbed FN and its conformation were investigated in two different coating concentrations (2 and 20 

Perez, R. A., Altankov, G., Jorge-Herrero, E., Ginebra, M. P., (2013). Micro- and nanostructured hydroxyapatite-collagen microcarriers for bone tissue-engineering applications Journal of Tissue Engineering and Regenerative Medicine 7, (5), 353-361

Novel hydroxyapatite (HA)-collagen microcarriers (MCs) with different micro/nanostructures were developed for bone tissue-engineering applications. The MCs were fabricated via calcium phosphate cement (CPC) emulsion in oil. Collagen incorporation in the liquid phase of the CPC resulted in higher MC sphericity. The MCs consisted of a porous network of entangled hydroxyapatite crystals, formed as a result of the CPC setting reaction. The addition of collagen to the MCs, even in an amount as small as 0.8wt%, resulted in an improved interaction with osteoblast-like Saos-2 cells. The micro/nanostructure and the surface texture of the MCs were further tailored by modifying the initial particle size of the CPC. A synergistic effect between the presence of collagen and the nanosized HA crystals was found, resulting in significantly enhanced alkaline phosphatase activity on the collagen-containing nanosized HA MCs.

Keywords: Bone regeneration, Calcium phosphate cement, Cell response, Collagen, Hydroxyapatite, Microcarrier

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

Cells assemble fibronectin (FN) into fibrils in a process mediated by integrins. For this process to occur, it is known that the presence of other serum proteins is necessary. However, the individual effect of these proteins on FN fibrillogenesis has not been addressed so far. In this study, the effect of vitronectin (VN), an ECM adhesion protein, on material-driven FN fibrillogenesis and cell-mediated FN reorganization is investigated. Poly(ethyl acrylate), PEA, which has previously shown the ability to induce the organization of FN into well-developed physiological-like networks upon adsorption, was employed as a material substrate. FN adsorption, cell adhesion and cellular FN reorganization in the presence or absence of VN were studied. Both FN surface density, quantified via western blot, and its distribution on PEA surfaces, determined via atomic force microscopy, were altered when FN was adsorbed competitively with VN at certain compositions. Moreover, the presence of VN on the material surfaces enhanced cell-mediated FN reorganization and secretion, in comparison with the process which took place in the presence of serum proteins.

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

Hydroxyapatite (HA) is the main component of human bones, a highly bioactive and biocompatible material; however, it has poor mechanical properties. Carbon-based coatings are found to significantly improve the mechanical properties of apatite, increase its adhesion, prevent metal ion release from metal implants and inhibit the formation of fibrous tissue and blood clotting upon implantation. In this chapter, homogeneous nanodiamond-reinforced hydroxyapatite (HA-ND) composite coating with improved mechanical strength and ductility was developed to enhance the biological properties of deposited by electrodeposition from simulated body fluid with dispersed nanodiamond particles. Study of the initial interaction of osteoblast-like MG-63 cells revealed that cells attached well on all plain samples (HA-ND, pure HA and stainless steel). However, precoating with fibronectin (FN) even at low adsorption concentrations (1mg/ml) strongly improved cell adhesion and preferentially spreading on the HA-ND samples as indicated by the flattened cell morphology and pronounced vinculin positive focal adhesions. This effect correlates with the observed higher affinity for FN. Moreover, osteoblasts tended to rearrange both adsorbed and secreted FN in a fibril-like pattern, suggesting improved FN matrix organization on HA-ND samples.

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

Collagen IV (Col IV) and laminin (Lam) are the main structural components of the basement membrane where they form two overlapping polymeric networks. We studied the adsorption pattern of these proteins on five model surfaces with tailored density of -OH groups obtained by copolymerization of different ratios ethyl acrylate (EA) and hydroxyl EA (HEA): X(OH) = 0, X(OH) = 0.3, X(OH) = 0.5, X(OH) = 0.7, and X(OH) = 1 (where X refers the ratio of HEA). Atomic force microscopy revealed substratum-specific adsorption patterns of Col IV and Lam, ranging from single molecules deposition on more hydrophilic substrata to the formation of complex networks on hydrophobic ones. Human umbilical endothelial cells were used to study the biological performance of adsorbed proteins, following the overall cell morphology, the quantities for cell adhesion and spreading, and the development of focal adhesion complexes and actin cytoskeleton. Surprisingly, two optima in the cellular interaction were observed-one on the most hydrophilic X(OH) = 1 and other on the relatively hydrophobic X(OH) = 0.3 substrate-valid for both Col IV and Lam. When the proteins were adsorbed consecutively, a hydrophobic shift to X(OH) = 0 substratum was obtained. Collectively, these data suggest that varying with the density of -OH groups one can tailor the conformation and the functional activity of adsorbed basement membrane proteins.

Keywords: Atomic-force microscopy, Fibronectin adsorption, Basement-membranes, Polymer surfaces, Cell-adhesion, Biomaterials, Wettability, Fibrinogen

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

Apart from the paradigm that cell-biomaterials interaction depends on the adsorption of soluble adhesive proteins we anticipate that upon distinct conditions also other, less soluble ECM proteins such as collagens, associate with the biomaterials interface with consequences for cellular response that might be of significant bioengineering interest. Using atomic force microscopy (AFM) we seek to follow the nanoscale behavior of adsorbed type IV collagen (Col IV)-a unique multifunctional matrix protein involved in the organization of basement membranes (BMs) including vascular ones. We have previously shown that substratum wettability significantly affects Col IV adsorption pattern, and in turn alters endothelial cells interaction. Here we introduce two new model surfaces based on self-assembled monolayers (SAMs), a positively charged - NH(2), and negatively charged -COOH surface, to learn more about their particular effect on Col IV behavior. AFM studies revealed distinct pattern of Col IV assembly onto the two SAMs resembling different aspects of network-like structure or aggregates (suggesting altered protein conformation). Moreover, the amount of adsorbed FITC-labeled Col IV was quantified and showed about twice more protein on NH(2) substrata. Human umbilical vein endothelial cells attached less efficiently to Col IV adsorbed on negatively charged COOH surface judged by altered cell spreading, focal adhesions formation, and actin cytoskeleton development. Immunofluorescence studies also revealed better Col IV recognition by both alpha(1) and alpha(2) integrins on positively charged NH(2) substrata resulting in higher phosphorylated focal adhesion kinase recruitment in the focal adhesion complexes. On COOH surface, no integrin clustering was observed. Taken altogether these results, point to the possibility that combined NH(2) and Col IV functionalization may support endothelization of cardiovascular implants.

Keywords: Collagen type IV, SAMs, AFM, Surface-induced protein assembly, Endothelial cells, Vascular grafts

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

Fibrinogen (FG) adsorption on surfaces with controlled fraction of -OH groups was investigated with AFM and correlated to the initial interaction of primary endothelial cells (HUVEC). The -OH content was tailored making use of a family of copolymers consisting of ethyl acrylate (EA) and hydroxyl ethyl acrylate (HEA) in different ratios. The supramolecular distribution of FG changed from an organized network-like structure on the most hydrophobic surface (-OH(0)) to dispersed molecular aggregate one as the fraction of -OH groups increases, indicating a different conformation by the adsorbed protein. The best cellular interaction was observed on the most hydrophobic (-OH(0)) surface where FG assembled in a fibrin-like appearance in the absence of any thrombin. Likewise, focal adhesion formation and actin cytoskeleton development was poorer as the fraction of hydroxy groups on the surface was increased. The biological activity of the surface-induced FG network to provide 3D cues in a potential tissue engineered scaffold, making use of electrospun PEA fibers (-OH(0)), seeded with human umbilical vein endothelial cells was investigated. The FG assembled on the polymer fibers gave rise to a biologically active network able to direct cell orientation along the fibers (random or aligned), promote cytoskeleton organization and focal adhesion formation.

Keywords: Fibrinogen, Cell-material interactions, HUVEC, Electrospun fibers, Fibrinogen organization, Cell-material interface, Endothelial cell behavior, Ethyl acrylate, Hydroxyl ethyl acrylate

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

New apatite (AP)/nanodiamond (ND) coating has been developed to improve physical and biological properties of stainless steel (SS) versus single AP coating. Homogeneously electrodeposited AP-ND layer demonstrates increased mechanical strength, interlayer cohesion and ductility. In the absence of serum, osteoblast-like MG63 cells attach well but poorly spread on both AP and AP-ND substrata. Pre-adsorption with serum or fibronectin (FN) improves the cellular interaction-an effect that is better pronounced on the AP-ND coating. In single protein adsorption study fluorescein isothiocyanate-labeled FN (FITC-FN) shows enhanced deposition on the AP-ND layer consistent with the significantly improved cell adhesion, spreading and focal adhesions formation (in comparison to SS and AP), particularly at low FN adsorption concentrations (1 mu g/ml). Higher FN concentrations (20 mu g/ml) abolish this difference suggesting that the promoted cellular interaction of serum (where FN is low) is caused by the greater affinity for FN. Moreover, it is found that MG63 cells tend to rearrange both adsorbed and secreted FN on the AP-ND layer suggesting facilitated FN matrix formation.

Keywords: Extracellular-matrix, Protein adsorption, Integrins, Adhesion, Biomaterials, Surfaces, Polymerization, Composite, Implants, Titanium

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

Hydroxyapatite and hybrid gelatine/hydroxyapatite microspheres were obtained through a water in oil emulsion of a calcium phosphate cement (CPC). The setting reaction of the CPC, in this case the hydrolysis of alpha-tricalcium phosphate, was responsible for the consolidation of the microspheres. After the setting reaction, the microspheres consisted of an entangled network of hydroxyapatite crystals, with a high porosity and pore sizes ranging between 0.5 and 5 mu m. The size of the microspheres was tailored by controlling the viscosity of the hydrophobic phase, the rotation speed, and the initial powder size of the CPC. The incorporation of gelatin increased the sphericity of the microspheres, as well as their size and size dispersion. To assess the feasibility of using the microspheres as cell microcarriers, Saos-2 cells were cultured on the microspheres. Fluorescent staining, SEM studies, and LDH quantification showed that the microspheres were able to sustain cell growth. Cell adhesion and proliferation was significantly improved in the hybrid gelatin/hydroxyapatite microspheres as compared to the hydroxyapatite ones.

Keywords: Calcium phosphate(s), Bone graft, Microspheres, Composite/hard tissue, Hydroxy(1)lapatite

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

Considering the structural role of type IV collagen (Col IV) in the assembly of the basement membrane (BM) and the perspective of mimicking its organization for vascular tissue engineering purposes, we studied the adsorption pattern of this protein on model hydrophilic (clean glass) and hydrophobic trichloro(octadecyl) silane (ODS) surfaces known to strongly affect the behavior of other matrix proteins. The amount of fluorescently labeled Col IV was quantified showing saturation of the surface for concentration of the adsorbing solution of about 50 mu g/ml, but with approximately twice more adsorbed protein on ODS. AFM studies revealed a fine-nearly single molecular size-network arrangement of Col IV on hydrophilic glass, which turns into a prominent and growing polygonal network consisting of molecular aggregates on hydrophobic ODS. The protein layer forms within minutes in a concentration-dependent manner. We further found that human umbilical vein endothelial cells (HUVEC) attach less efficiently to the aggregated Col IV (on ODS), as judged by the significantly altered cell spreading, focal adhesions formation and the development of actin cytoskeleton. Conversely, the immunofluorescence studies for integrins revealed that the fine Col IV network formed on hydrophilic substrata is better recognized by the cells via both alpha 1 and alpha 2 heterodimers which support cellular interaction, apart from these on hydrophobic ODS where almost no clustering of integrins was observed.

Keywords: Collagen type IV, Adsorption, Assembly, Hydrophilic, Hydrophobic, Surfaces

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

We investigated the early events of bone matrix formation, and specifically the role of fibronectin (FN) in the initial osteoblast interaction and the subsequent organization of a provisional FN matrix on different rough titanium (Ti) surfaces. Fluorescein isothiocyanate-label led FN was preadsorbed on these surfaces and studied for its three-dimensional (3-D) organization by confocal microscopy, while its amount was quantified after NaOH extraction. An irregular pattern of adsorption with a higher amount of protein on topographic peaks than on valleys was observed and attributed to the physicochemical heterogeneity of the rough Ti surfaces. MG63 osteoblast-like cells were further cultured on FN-preadsorbed Ti surfaces and an improved initial cellular interaction was observed with increasing roughness. 3-D reconstruction of the immunofluorescence images after 4 days of incubation revealed that osteoblasts deposit FN fibrils in a specific facet-like pattern that is organized within the secreted total matrix overlying the top of the samples. The thickness of this FN layer increased when the roughness of the underlying topography was increased, but not by more than half of the total maximum peak-to-valley distance, as demonstrated with images showing simultaneous reconstruction of fluorescence and topography after 7 days of cell culture.

Keywords: Fibronectin, Extracellular matrix organization, Titanium, Surface topography, Surface energy

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

Vitronectin (VN) adsorption on a family of model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios (to obtain a controlled surface density of -OH groups) was investigated by Atomic Force Microscopy (AFM). It is shown that the fraction of the substrate covered by the protein depends strongly on the amount of hydroxyl groups in the sample and it monotonically decreases as the -OH density increases. Isolated globular-like VN molecules are observed on the surfaces with the higher OH density. As the fraction of hydroxyl groups decreases, aggregates of 3-5 VN molecules are observed on the sample. Overall cell morphology, focal adhesion formation and actin cytoskeleton development are investigated to assess the biological activity of the adsorbed VN on the different surfaces. Dermal fibroblast cells show excellent material interaction on the more hydrophobic samples (OH contents lower than 0.5), which reveals enhanced VN activity on this family of substrates as compared with other extracellular matrix proteins (e.g., fibronectin and fibrinogen).

Keywords: Copolymers, Vitronectin, AFM, Self-assembled monolayers, Cell-adhesion, Thermal transitions, Protein adsorption, Surfaces, Fibronectin, Biomaterials, Attachment, Fibrinogen

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

Adhesion of human fibroblast to a family of fibronectin (FN) coated model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios to obtain a controlled surface density of -OH groups was investigated. Cell adhesion and spreading surprisingly decreased as the fraction of -OH groups on the Surface increased. AFM studies of FN conformation revealed formation of a protein network on the more hydrophobic surfaces. The density of this network diminished as the fraction of -OH groups in the sample increased, up to a maximal -OH concentration at which, instead of the network, only IN aggregates were observed. The kinetics of network development was followed at different adsorption times. Immunofluorescence for vinculin revealed the formation of well-developed focal adhesion complexes on the more hydrophobic surface (similar to the control glass), which became less defined as the fraction of -OH groups increased. Thus, the efficiency of cell adhesion is enhanced by the formation of FN networks on the substrate, directly revealing the importance of the adsorbed protein conformation for cell adhesion. However, cell-dependent reorganization of substrate-associated FN, which usually takes place on more hydrophilic substrates (as do at the control glass slides), was not observed in this system, suggesting the increased strength of protein-to-substrate interaction. Instead, the late FN matrix formation-after 3 days of culture-was again better pronounced on the more hydrophobic substrates and decreased as the fraction of -OH groups increase, which is in a good agreement with the results for overall cell morphology and focal adhesion formation.

Keywords: Cell adhesion, Fibronectin, Fibroblast, Extracellular matrix, AFM

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.)

Understanding the relationships between material surface properties, behaviour of adsorbed proteins and cellular responses is essential to design optimal material surfaces for tissue engineering. In this study we modify thin layers of plasma polymerized hexamethyldisiloxane (PPHMDS) by ammonia treatment in order to increase surface wettability and the corresponding biological response. The physico-chemical properties of the polymer films were characterized by contact angle (CA) measurements and Fourier Transform Infrared Spectroscopy (FTIR) analysis.Human umbilical vein endothelial cells (HUVEC) were used as model system for the initial biocompatibility studies following their behavior upon preadsorption of polymer films with three adhesive proteins: fibronectin (FN), fibrinogen (FG) and vitronectin (VN). Adhesive interaction of HUVEC was evaluated after 2 hours by analyzing the overall cell morphology, and the organization of focal adhesion contacts and actin cytoskeleton. We have found similar good cellular response on FN and FG coated polymer films, with better pronounced vinculin expression on FN samples while. Conversely, on VN coated surfaces the wettability influenced significantly initial celular interaction spreading. The results obtained suggested that ammonia plasma treatment can modulate the biological activity of the adsorbed protein s on PPHMDS surfaces and thus to influence the interaction with endothelial cells.

Keywords: -----

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.)

The present study reports on how detonation nanodiamond (DND) - plasma poly(hexamethyldisiloxane) composites (PPHMDS) affect osteoblast cell behavior. It has been established that various modified DND nanoparticles (Ag-DND and Si-DND) can be readily integrated into virtually all polymer matrices. In particular, PPHDMS composites have been developed over the past few years because of the variety of their application as medical devices and implants. By incubation of MG-63 osteoblast-like cells on the surface of DND (Ag-DND and Si-DND) - PPHMDS composite, we tested the hypothesis that DND-based polymer composites can influence the adhesion behavior of MG-63 osteoblast-like cells. Morphological and structural characterization of DND, Ag-DND and Si-DND powders was carried out by XRD, HRTEM and EDS. For the study of the composite layers, deposited on cover glass (CG), FTIR spectroscopy has been performed in order to determine if the DND nanofiller can potentially modify the structural and chemical dynamics of the polymer matrix. The kinetic of static water contact angle of composite surfaces as a function of the as-used nanofiller DND's in polymer matrix was measured The results with MG-63 osteoblast-like cells suggest the potential of using DND-based polymer composites for application in engineering implantable scaffolds and devices.

Keywords: -----

Salmeron-Sanchez, M., Altankov, G., (2010). Cell-Protein-Material interaction in tissue engineering Tissue Engineering (ed. Eberli, D.), Intech (Vukovar, Croatia) , 77-102

The initial cellular events that take place at the biomaterials interface mimic to a certain extent the natural adhesive interaction of cells with the extracellular matrix (ECM) (Spie, 2002; Griffin & Naughton, 2002; Grinnell, 1986). In fact, the living cells cannot interact directly with foreign materials, but they readily attach to the adsorbed layer of proteins (upon contact with physiological fluids in vivo or culture medium in vitro) such as fibronectin (FN), vitronectin (VN), fibrinogen (FG), representing the so-called soluble matrix proteins in the biological fluids (Grinnell 1986).

Keywords: Tissue Engineering, Protein-material interaction, ECM, Biomaterials

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

The initial cellular events that take place at the biomaterials interface mimic to a certain extent the natural interaction of cells with the extracellular matrix (ECM). The cells adhering to the adsorbed soluble matrix proteins, such as fibronectin (FN) and fibrinogen (FNG) tend to re-arrange them in fibril-like pattern. Using model surfaces we have demonstrated that this cellular activity is abundantly dependent on the surface properties of materials, such as wettability, surface chemistry, charge and topography. This raises the possibility that tissue compatibility of materials is connected with the allowance of cells to remodel substratum associated proteins presumably to form provisional ECM. We have further shown that antibodies which bind β1 and αv integrins (subunits of the FN and FNG receptors respectively) may induce their linear rearrangement on the dorsal surface of living cells – a phenomenon presumably related to the same early molecular events of fibrillar matrix assembly. Because the quantitative measurements revealed that this receptor dynamics is strongly altered on the low compatible (hydrophobic) substrata we hypothesized that in order to be biocompatible, materials need to adsorb matrix proteins loosely, i.e. in such a way that the cells can easily remove and organize them in matrix-like fibrils via coordinated functioning of integrins. More recent studies on the fate of FN on some real biomaterial surfaces, including different rough titanium (Ti) and hydroxyapatite (HA) cements and the surface of biosensors confirmed this point of view. They also show that quantitative measurements of adsorbed matrix proteins and their dynamic rearrangement at cell-material interface might provide insight to the biocompatibility of given material and even predict its tissue integration.

Keywords: Materials Science

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

At present it is well accepted that different surface properties play a strong role in the interaction between synthetic materials and biological entities. Surface properties such as surface energy, topography, surface chemistry and crystallinity affect the protein adsorption mechanisms as well as cell behaviour in terms of attachment, proliferation and differentiation. The aim of this chapter is to show the most relevant processes and interactions that take place during the first stages of contact between the material and the physiological environment. Some examples show that the modification of different biomaterials surfaces affects both protein adsorption and cell behaviour.

Keywords: Materials Science

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

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network. Atomic force microscopy images of single FN molecules, at the early stages of adsorption on plane PEA, allow one to rationalize the process. Further, the role of the spatial organization of the FN network on the cellular response is investigated through its adsorption on electrospun fibers. Randomly oriented and aligned PEA fibers were prepared to mimic the three-dimensional organization of the extracellular matrix. The formation of the FN network on the PEA fibers but not on the supporting coverglass was confirmed. Fibroblasts aligned with oriented fibers, displayed extended morphology, developed linearly organized focal adhesion complexes, and matured actin filaments. Conversely, on random PEA fibers, cells acquired polygonal morphology with altered actin cytoskeleton but well-developed focal adhesions. Late FN matrix formation was also influenced: spatially organized FN matrix fibrils along the oriented PEA fibers and an altered arrangement on random ones.

Keywords: AFM, Cell-adhesion, Dependent conformations, Hydrophobic surfaces, Extracellular-matrix, Bound fibronectin, Polymer surfaces, Integrin binding, Biocompatibility, Adsorption

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

It was found previously that membranes based on co-polymers of acrylonitrile (AN) and 2-acrylamido-2-methyl-propansulfonic acid (AMPS) greatly stimulated the functionality and survival of primary hepatocytes. In those studies, however, the pure AN-AMPS co-polymer had poor membrane-forming properties, resulting in quite dense rubber-like membranes. Hence, membranes with required permeability and optimal biocompatibility were obtained by blending the AN-AMPS co-polymer with poly(acrylonitrile) homopolymer (PAN). The amount of PAN (P) and AN-AMPS (A) in the blend was varied from pure PAN (P/A-100/0) over P/A-75/25 and P/A-50/50 to pure AN-AMPS co-polymer (P/A-0/100). A gradual decrease of molecular cut-off of membranes with increase of AMPS concentration was found, which allows tailoring membrane permeability as necessary. C3A hepatoblastoma cells were applied as a widely accepted cellular model for assessment of hepatocyte behaviour by attachment, viability, growth and metabolic activity. It was found that the blend P/A-50/50, which possessed an optimal permeability for biohybrid liver systems, supported also the attachment, growth and function of C3A cells in terms of fibronectin synthesis and P-450 isoenzyme activity. Hence, blend membranes based on a one to one mixture of PAN and AN-AMPS combine sufficient permeability with the desired cellular compatibility for application in bioreactors for liver replacement.

Keywords: Bioartificial liver, C3A cells, Fibronectin, P-450, Synthetic membrane

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

The influence of surface chemistry -substrates with controlled surface density of -OH groups- on fibronectin conformation and distribution is directly observed by Atomic Force Microscopy (AFM). FN fibrillogenesis, which is known to be a process triggered by interaction with integrins, is shown in our case to be induced by the substrate (in absence of cells), which is able to enhance FN-FN interactions leading to the formation of a protein network on the material surface. This phenomenon depends both on surface chemistry and protein concentration. The level of the FN fibrillogenesis was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The total amount of adsorbed FN is obtained by making use of a methodology which employs western-blotting combined with image analysis of the corresponding protein bands, with the lowest sensitivity threshold equal to 15 ng of adsorbed protein. Furthermore, FN adsorption is correlated to human osteoblast adhesion through morphology and actin cytoskeleton formation. Actin polymerization is in need of the formation of the protein network on the substrate's surface. Cell morphology is more rounded (as quantified by calculating the circularity of the cells by image analysis) the lower the degree of FN fibrillogenesis on the substrate.

Keywords: Cell-adhesion, Conformational-changes, Electron-microscopy, Protein adsorption, Fractal dimension, Integrin binding, Biocompatibility, Monolayers, Matrix, Fibrillogenesis

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

Here, the layer-by-layer technique (LbL) was used to modify glass as model biomaterial with multilayers of chitosan and heparin to control the interaction with MG-63 osteoblast-like cells. Different pH values during multilayer formation were applied to control their physico-chemical properties. In the absence of adhesive proteins like plasma fibronectin (pFN) both plain layers were rather cytophobic. Hence, the preadsorption of pFN was used to enhance cell adhesion which was strongly dependent on pH. Comparing the adhesion promoting effects of pFN with an engineered repeat of the FN III fragment and collagen I which both lack a heparin binding domain it was found that multilayers could bind pFN specifically because only this protein was capable of promoting cell adhesion. Multilayer surfaces that inhibited MG-63 adhesion did also cause a decreased cell growth in the presence of serum, while an enhanced adhesion of cells was connected to an improved cell growth.

Keywords: Cell-adhesion, Polyelectrolyte multilayers, Substratum chemistry, Surface-properties, Fibroblast-growth, Fibronectin, Polymers, Chitosan, Polysaccharides, Wettability

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

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.

Keywords: Alkaline Phosphatase/metabolism, Bone Cements/pharmacology, Calcium/metabolism, Calcium Phosphates/pharmacology, Cell Adhesion/drug effects, Cell Differentiation/drug effects, Cell Proliferation/drug effects, Cell Shape/drug effects, Cells, Cultured, Culture Media, Durapatite/pharmacology, Humans, Interferometry, Ion Exchange, Materials Testing, Osteoblasts/ cytology/drug effects/enzymology/ultrastructure, Phosphorus/metabolism, Powders, Tissue Engineering, Tissue Scaffolds

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

A series of co-culture experiments between fibroblasts and H-460 human lung carcinoma cells were performed to learn more about the fate of adsorbed type IV collagen (Coll IV). Fibroblasts were able to spatially rearrange Coll IV in a specific linear pattern, similar but not identical to the fibronectin (FN) fibrils. Coll IV partly co-aligns with fibroblast actin cytoskeleton and transiently co-localize with FN, as well as with beta 1 and a 2 integrin clusters, suggesting a cell-dependent process. We further found that this Coll IV reorganization is suppressed in contact with H460 cells. Zymography revealed strongly elevated MMP-2 activity in supernatants of co-cultures, but no activity when fibroblasts or cancer cells were cultured alone. Thus, we provide evidence that reorganization of substrate associated Coll IV is a useful morphological approach for in vitro studies on matrix remodeling activity during tumorigenesis.

Keywords: Adsorbed collagen IV reorganization, Fibroblasts and cancer cells co-culture, MMP-2

Manara, S., Paolucci, F., Palazzo, B., Marcaccio, M., Foresti, E., Tosi, G., Sabbatini, S., Sabatino, P., Altankov, G., Roveri, N., (2008). Electrochemically-assisted deposition of biomimetic hydroxyapatite-collagen coatings on titanium plate Inorganica Chimica Acta 361, (6), 1634-1645

A biomimetic bone-like composite, made of self-assembled collagen fibrils and carbonate hydroxyapatite nanocrystals, has been performed by an electrochemically-assisted deposition on titanium plate. The electrolytic processes have been carried out using a single type I collagen molecules suspension in a diluted Ca(NO3)(2) and NH4H2PO4 solution at room temperature and applying a constant current for different periods of time. Using the same electrochemical conditions, carbonate hydroxyapatite nanocrystals or reconstituted collagen. brils coatings were obtained. The reconstituted collagen. brils, hydroxyapatite nanocrystals and collagen fibrils/apatite nanocrystals coatings have been characterized chemically, structurally and morphologically, as well as for their ability to bind fibronectin (FN). Fourier Transform Infrared microscopy has been used to map the topographic distribution of the coating components at different times of electrochemical deposition, allowing to single out the individual deposition steps. Moreover, roughness of Ti plate has been found to affect appreciably the nucleation region of the inorganic nanocrystals. Laser scanning confocal microscopy has been used to characterize the FN adsorption pattern on a synthetic biomimetic apatitic phase, which exhibits a higher affinity when it is inter-grown with the collagen fibrils. The results offer auspicious applications in the preparation of medical devices such as biomimetic bone-like composite-coated metallic implants.

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

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

Thin films of silicon nitride (Si3N4) can be used in several kinds of micro-sized biosensors as a material to monitor fine environmental changes related to the process of bone formation in vitro. We found however that Si3N4 does not provide optimal conditions for osseointegration as osteoblast-like MG-63 cells tend to detach from the surface when cultured over confluence. Therefore Si3N4 was modified with self-assembled monolayers bearing functional end groups of primary amine (NH2) and carboxyl (COOH) respectively. Both these modifications enhanced the interaction with confluent cell layers and thus improve osseointegration over Si3N4. Furthermore it was observed that the NH2 functionality increased the adsorption of fibronectin (FN), promoted cell proliferation, but delayed the differentiation. We also studied the fate of pre-adsorbed and secreted FN from cells to learn more about the impact of above functionalities for the development of provisional extracellular matrix on materials interface. Taken together our data supports that Si3N4 has low tissue integration but good cellular biocompatibility and thus is appropriate in cellular biosensor applications such as the ion-sensitive field effect transistor (ISFET). COOH and NH2 chemistries generally improve the interfacial tissue interaction with the sensor and they are therefore suitable substrates for monitoring cellular growth or matrix deposition using electrical impedance spectroscopy.

Keywords: Adsorption, Amines/chemistry, Biocompatible Materials/ chemistry, Biosensing Techniques, Cell Differentiation, Cell Line, Cell Proliferation, Electric Impedance, Extracellular Matrix/metabolism, Fibronectins/chemistry, Humans, Materials Testing, Osteoblasts/ cytology, Silicon Compounds/ chemistry, Surface Properties

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