by Keyword: Fibronectin
Casanellas, I, Lagunas, A, Vida, Y, Perez-Inestrosa, E, Rodriguez-Pereira, C, Magalhaes, J, Andrades, JA, Becerra, J, Samitier, J, (2022). Nanoscale ligand density modulates gap junction intercellular communication of cell condensates during chondrogenesis Nanomedicine 17, 775-791
Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.
JTD Keywords: Actin, Adhesion, Arginine-glycine-aspartic acid, Cell adhesion, Collagen, Condensation, Connexin-43, Dendrimer-based nanopatterning, Dynamics, Extracellular-matrix, Fibronectin, Gap junction intercellular communication, Mesenchymal stem cells, Permeability, Phenotype, Vinculin
Almici E, Chiappini V, López-Márquez A, Badosa C, Blázquez B, Caballero D, Montero J, Natera-de Benito D, Nascimento A, Roldán M, Lagunas A, Jiménez-Mallebrera C, Samitier J, (2022). Personalized in vitro Extracellular Matrix Models of Collagen VI-Related Muscular Dystrophies Frontiers In Bioengineering And Biotechnology 10, 851825
Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients’ ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments. Copyright © 2022 Almici, Chiappini, López-Márquez, Badosa, Blázquez, Caballero, Montero, Natera-de Benito, Nascimento, Roldán, Lagunas, Jiménez-Mallebrera and Samitier.
JTD Keywords: alpha-3 chain, binding, collagen vi related muscular dystrophy, decellularisation, decellularized matrices, deficiency, expression, fibroblasts, fibronectin, in vitro model, patient-derived ecms, skeletal-muscle, ullrich, Cell-derived matrices, Collagen, Collagen vi related muscular dystrophy, Decellularisation, Decellularization, Extracellular matrices, Extracellular matrix, Genes, In vitro model, In-vitro, In-vitro models, Matrix, Matrix model, Muscular dystrophy, Pathology, Patient-derived ecm, Patient-derived ecms, Pre-clinical
Rubí-Sans G, Nyga A, Rebollo E, Pérez-Amodio S, Otero J, Navajas D, Mateos-Timoneda MA, Engel E, (2021). Development of Cell-Derived Matrices for Three-Dimensional in Vitro Cancer Cell Models Acs Applied Materials & Interfaces 13, 44108-44123
Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.
JTD Keywords: 3d cell-derived matrices, adipose mesenchymal stem cells, collagen matrix, colorectal adenocarcinoma, cytotoxicity assay, deposition, expansion, extracellular microenvironment, extracellular-matrix, fibronectin, growth, macromolecular crowders, microcarriers, scaffolds, tissue, 3d cell-derived matrices, Adipose mesenchymal stem cells, Cytotoxicity assay, Extracellular microenvironment, Macromolecular crowders, Mesenchymal stem-cells, Microcarriers
Andreu, I, Falcones, B, Hurst, S, Chahare, N, Quiroga, X, Le Roux, AL, Kechagia, Z, Beedle, AEM, Elosegui-Artola, A, Trepat, X, Farre, R, Betz, T, Almendros, I, Roca-Cusachs, P, (2021). The force loading rate drives cell mechanosensing through both reinforcement and cytoskeletal softening Nature Communications 12, 4229
Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.
JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat
Guillem-Marti, J., Gelabert, M., Heras-Parets, A., Pegueroles, M., Ginebra, M. P., Manero, J. M., (2019). RGD mutation of the heparin binding II fragment of fibronectin for guiding mesenchymal stem cell behavior on titanium surfaces ACS Applied Materials and Interfaces 11, (4), 3666-3678
Installing bioactivity on metallic biomaterials by mimicking the extracellular matrix (ECM) is crucial for stimulating specific cellular responses to ultimately promote tissue regeneration. Fibronectin is an ECM protein commonly used for biomaterial functionalization. The use of fibronectin recombinant fragments is an attractive alternate to the use of full-length fibronectin because of the relatively low cost and facility of purification. However, it is necessary to combine more than one fragment, for example, the cell attachment site and the heparin binding II (HBII), either mixed or in one molecule, to obtain complete activity. In the present study, we proposed to install adhesion capacity to the HBII fragment by an RGD gain-of-function DNA mutation, retaining its cell differentiation capacity and thereby producing a small and very active protein fragment. The novel molecule, covalently immobilized onto titanium surfaces, maintained the growth factor-binding capacity and stimulated cell spreading, osteoblastic cell differentiation, and mineralization of human mesenchymal stem cells compared to the HBII native protein. These results highlight the potential capacity of gain-of-function DNA mutations in the design of novel molecules for the improvement of osseointegration properties of metallic implant surfaces.
JTD Keywords: Fibronectin, Growth factor, Mutation, Osseointegration, Recombinant protein, Titanium
González-García, C., Cantini, M., Ballester-Beltrán, J., Altankov, G., Salmerón-Sánchez, M., (2018). The strength of the protein-material interaction determines cell fate Acta Biomaterialia 77, 74-84
Extracellular matrix (ECM) proteins are key mediators of cell/material interactions. The surface density and conformation of these proteins adsorbed on the material surface influence cell adhesion and the cellular response. We have previously shown that subtle variations in surface chemistry lead to drastic changes in the conformation of adsorbed fibronectin (FN). On poly(ethyl acrylate) (PEA), FN unfolds and displays domains for cell adhesion and FN-FN interaction, whereas on poly(methyl acrylate) (PMA) â€“ with only one methyl group less â€“ FN remains globular as it is in solution. The effect of the strength of the protein/material interaction in cell response, and its relation to protein density and conformation, has received limited attention so far. In this work, we used FN-functionalized AFM cantilevers to evaluate, via force spectroscopy, the strength of interaction between fibronectin and the underlying polymer which controls FN conformation (PEA and PMA). We found that the strength of FN/PEA interaction is significantly higher than FN/PMA, which limits the mobility of FN layer on PEA, reduces the ability of cells to mechanically reorganize FN and then leads to enhanced proteolysis and degradation of the surrounding matrix with compromised cell viability. By contrast, both PEA and PMA support cell adhesion when FN density is increased and also in the presence of serum or other serum proteins, including vitronectin (VN) and bovine serum albumin (BSA), which provide a higher degree of mobility to the matrix. Statement of Significance: The identification of parameters influencing cell response is of paramount importance for the design of biomaterials that will act as synthetic scaffolds for cells to anchor, grow and, eventually, become specialised tissues. Cells interact with materials through an intermediate layer of proteins adsorbed on the material surface. It is known that the density and conformation of these proteins determine cell behaviour. Here we show that the strength of protein/material interactions, which has received very limited attention so far, is key to understand the cellular response to biomaterials. Very strong protein/material interactions reduce the ability of cells to mechanically reorganize proteins at the material interface which results in enhanced matrix degradation, leading ultimately to compromised cell viability.
JTD Keywords: Fibronectin adsorption, Fibronectin remodeling, Protein mobility, Protein-material interaction strength
Guillem-Marti, J., Boix-Lemonche, G., Gugutkov, D., Ginebra, M.-P., Altankov, G., Manero, J.M., (2018). Recombinant fibronectin fragment III8-10/polylactic acid hybrid nanofibers enhance the bioactivity of titanium surface Nanomedicine 13, (8), 899-912
Aim: To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). Materials & methods: Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. Results: A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. Conclusion: Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.
JTD Keywords: Electrospinning, Fibronectin, Hybrid nanofibers, Osseointegration, PLA, Recombinant protein
Hristova-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
Mesenchymal stem cells (MSCs) are a promising cell source for cell-based therapies because of their self-renewal and multi-lineage differentiation potential. Unlike embryonic stem cells adult stem cells are subject of aging processes and the concomitant decline in their function. Age-related changes in MSCs have to be well understood in order to develop clinical techniques and therapeutics based on these cells. In this work we have studied the effect of aging on adhesive behaviour of bone marrow-derived MSC and MG- 63 osteoblastic cells onto three extracellular matrix proteins: fibronectin (FN), vitronectin (VN) and collagen I (Coll I). The results revealed substantial differences in adhesive behaviour of both cell types during 21 days in culture. Bone-marrow derived MSCs decreased significantly their adhesive affinity to all studied proteins after 7th day in culture with further incubation. In contrast, MG-63 cells, demonstrated a stable cell adhesive phenotype with high affinity to FN and Coll I and low affinity to vitronectin over the whole culture period. These data suggest that adhesive behaviour of MSCs to matrix proteins is affected by aging processes unlike MG-63 cells and the age-related changes have to be considered when expanding adult stem cells for clinical applications.
JTD Keywords: Cell morphology, Cell attachment and spreading, Fibronectin, Vitronectin, Collagen I
Won, J. E., Mateos-Timoneda, M. A., Castaño, O., Planell, J. A., Seo, S. J., Lee, E. J., Han, C. M., Kim, H. W., (2015). Fibronectin immobilization on to robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone tissue engineering Biotechnology Letters , 37, (4), 935-342
Bioactive nanocomposite scaffolds with cell-adhesive surface have excellent bone regeneration capacities. Fibronectin (FN)-immobilized nanobioactive glass (nBG)/polycaprolactone (PCL) (FN-nBG/PCL) scaffolds with an open pore architecture were generated by a robotic-dispensing technique. The surface immobilization level of FN was significantly higher on the nBG/PCL scaffolds than on the PCL scaffolds, mainly due to the incorporated nBG that provided hydrophilic chemical-linking sites. FN-nBG/PCL scaffolds significantly improved cell responses, including initial anchorage and subsequent cell proliferation. Although further in-depth studies on cell differentiation and the in vivo animal responses are required, bioactive nanocomposite scaffolds with cell-favoring surface are considered to provide promising three-dimensional substrate for bone regeneration.
JTD Keywords: Bone scaffolds, Cell response, Fibronectin, Nanobioactive glass, Nanocomposites, Polycaprolactone, Bone, Cell proliferation, Cells, Cytology, Glass, Nanocomposites, Polycaprolactone, Robotics, Bone scaffolds, Bone tissue engineering, Cell response, Fibronectin, Fibronectin immobilizations, Nano bioactive glass, Nanocomposite scaffolds, Three-dimensional substrates, Scaffolds (biology)
Pegueroles, M., Tonda-Turo, C., Planell, J. A., Gil, F. J., Aparicio, C., (2012). Adsorption of fibronectin, fibrinogen, and albumin on TiO2: Time-resolved kinetics, structural changes, and competition study Biointerphases , 7, (48), 13
An understanding of protein adsorption process is crucial for designing biomaterial surfaces. In this work, with the use of a quartz-crystal microbalance with dissipation monitoring, we researched the following: (a) the kinetics of adsorption on TiO2 surfaces of three extensively described proteins that are relevant for metallic implant integration [i.e., albumin (BSA), fibrinogen (Fbg), and fibronectin (Fn)]; and (b) the competition of those proteins for adsorbing on TiO2 in a two-step experiment consisted of sequentially exposing the surfaces to different monoprotein solutions. Each protein showed a different process of adsorption and properties of the adlayer-calculated using the Voigt model. The competition experiments showed that BSA displaced larger proteins such as Fn and Fbg when BSA was introduced as the second protein in the system, whereas the larger proteins laid on top of BSA forming an adsorbed protein bi-layer when those were introduced secondly in the system.
JTD Keywords: QCM, Human plasma fibronectin, Induced conformational-changes, Von-willebrand-factor, BSA, Protein adsortion, Polymer surfaces, Solid-surfaces, Viscoelastic properties, Globular-proteins
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.
JTD Keywords: Atomic-force microscopy, Fibronectin adsorption, Basement-membranes, Polymer surfaces, Cell-adhesion, Biomaterials, Wettability, Fibrinogen
Comelles, J., Estevez, M., Martinez, E., Samitier, J., (2010). The role of surface energy of technical polymers in serum protein adsorption and MG-63 cells adhesion Nanomedicine: Nanotechnology Biology and Medicine , 6, (1), 44-51
Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. However, novel applications in the biosensor field require materials to be compatible with cell growth and at the same time be suitable for technological processing. Technological polymers are key materials in the fabrication of disposable parts and other sensing elements. As such, it is essential to characterize the surface properties of technological polymers, especially after processing and sterilization. It is also important to understand how technological polymers affect cell behavior when in contact with polymer materials. Therefore, the aim of this research was to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly (methyl methacrylate), polystyrene, and poly(dimethylsiloxane). Glass was used as the control material. From the Clinical Editor: Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. The aim of this research is to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly(methylmethacrylate) (PMMA), polystyrene (PS), and poly(dimethylsiloxane) (PDMS).
JTD Keywords: Thin-films, Poly(methyl methacrylate), Osteoblast adhesion, Electron-microscopy, Fibronectin, Polystyrene, Oly(dimethylsiloxane), Biocompatibility, Hydroxyapatite, Behavior
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.
JTD 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).
JTD 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.
JTD Keywords: Cell adhesion, Fibronectin, Fibroblast, Extracellular matrix, AFM
Roca-Cusachs, P., Gauthier, N. C., del Rio, A., Sheetz, M. P., (2009). Clustering of alpha(5)beta(1) integrins determines adhesion strength whereas alpha(v)beta(3) and talin enable mechanotransduction Proceedings of the National Academy of Sciences of the United States of America 106, (38), 16245-16250
A key molecular link between cells and the extracellular matrix is the binding between fibronectin and integrins alpha(5)beta(1) and alpha(v)beta(3). However, the roles of these different integrins in establishing adhesion remain unclear. We tested the adhesion strength of fibronectin-integrin-cytoskeleton linkages by applying physiological nanonewton forces to fibronectin-coated magnetic beads bound to cells. We report that the clustering of fibronectin domains within 40 nm led to integrin alpha(5)beta(1) recruitment, and increased the ability to sustain force by over six-fold. This force was supported by alpha(5)beta(1) integrin clusters. Importantly, we did not detect a role of either integrin alpha(v)beta(3) or talin 1 or 2 in maintaining adhesion strength. Instead, these molecules enabled the connection to the cytoskeleton and reinforcement in response to an applied force. Thus, high matrix forces are primarily supported by clustered alpha(5)beta(1) integrins, while less stable links to alpha(v)beta(3) integrins initiate mechanotransduction, resulting in reinforcement of integrin-cytoskeleton linkages through talin-dependent bonds.
JTD Keywords: Cell-adhesion, Mechanical force, Vinculin-binding, Fibronectin, Activation, Dynamics, Domain, Alpha-v-beta-3, Translocation, Bonds
Niepel, M. S., Peschel, D., Sisquella, X., Planell, J. A., Groth, T., (2009). pH-dependent modulation of fibroblast adhesion on multilayers composed of poly(ethylene imine) and heparin Biomaterials 30, (28), 4939-4947
Adhesion of tissue cells is a prerequisite for their growth and differentiation but prevents also apoptosis. Here the layer-by-layer technique (LbL) was used to design multilayer structures of poly(ethylene imine) (PEI) and heparin (HEP) on glass as model biomaterial to control the adhesion of primary human dermal fibroblasts. Distinct surface features like wettability, charge and lateral structures were controlled by changing the pH value of the HEP solution during multilayer assembly to acidic neutral or alkaline, values. While plain terminal layers were rather cytophobic, the pre-adsorption of serum or fibronectin (FN) caused a distinct change in cell morphology in dependence on the pH setup. The effect of serum was more prominent on PEI layers probably due to their positive surface charge, whereas the effect of FN was more pronounced on HEP terminated multilayers possibly due to its ability to bind FN specifically. Those layers which hampered cell adhesion also inhibited growth of human fibroblasts under serum conditions. Conversely, on layers where cell adhesion was increased also an elevated growth and, thus, metabolic activity was observed.
JTD Keywords: Surface modification, Layer-by-layer, Poly(ethylene imine), Heparin, Serum, Fibronectin
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.
JTD 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.
JTD Keywords: Bioartificial liver, C3A cells, Fibronectin, P-450, Synthetic membrane
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.
JTD Keywords: Cell-adhesion, Polyelectrolyte multilayers, Substratum chemistry, Surface-properties, Fibroblast-growth, Fibronectin, Polymers, Chitosan, Polysaccharides, Wettability
Navarro, M., Benetti, E. M., Zapotoczny, S., Planell, J. A., Vancso, G. J., (2008). Buried, covalently attached RGD peptide motifs in poly(methacrylic acid) brush layers: The effect of brush structure on cell adhesion Langmuir 24, (19), 10996-11002
Iniferter-mediated surface-initiated photopolymerization was used to graft poly(methacrylic acid) (PMAA) brush layers obtained from surface-attached iniferters in self-assembled monolayers to a gold surface. The tethered chains were subsequently functionalized with the cell-adhesive arginine-glycine-aspartic acid (RGD) motif. The modified brushes were extended by reinitiating the polymerization to obtain an additional layer of PMAA, thereby burying the peptide-functionalized segments inside the brush structure. Contact angle measurements and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the wettability and the chemical properties of these platforms. Time of flight secondary ion mass spectroscopy (TOF-SIMS) measurements were performed to monitor the chemical composition of the polymer layer as a function of the distance to the gold surface and obtain information concerning the depth of the RGD motifs inside the brush structure. The brush thickness was evaluated as a function of the polymerization (i.e.. UV-irradiation) time with atomic force microscopy (AFM) and ellipsometry. Cell adhesion tests employing human osteoblasts were performed on substrates with the RGD peptides exposed at the surface as well as covered by a PMAA top brush layer. Immunofluorescence studies demonstrated a variation of the cell morphology as a function of the position of the peptide units along the grafted chains.
JTD Keywords: Ion mass-spectrometry, Transfer radical polymerization, Asymmetric diblock copolymers, Arg-gly-asp, Swelling behaviour, Endothelial-cells, Thin-films, fibronectin, Surfaces, SIMS
Manara, S., Paolucci, F., Palazzo, B., Marcaccio, M., Foresti, E., Tosi, G., Sabbatini, S., Sabatino, P., Altankov, G., Roveri, N., (2008). Electrochemically-assisted deposition of biomimetic hydroxyapatite-collagen coatings on titanium plate Inorganica Chimica Acta 361, (6), 1634-1645
A biomimetic bone-like composite, made of self-assembled collagen fibrils and carbonate hydroxyapatite nanocrystals, has been performed by an electrochemically-assisted deposition on titanium plate. The electrolytic processes have been carried out using a single type I collagen molecules suspension in a diluted Ca(NO3)(2) and NH4H2PO4 solution at room temperature and applying a constant current for different periods of time. Using the same electrochemical conditions, carbonate hydroxyapatite nanocrystals or reconstituted collagen. brils coatings were obtained. The reconstituted collagen. brils, hydroxyapatite nanocrystals and collagen fibrils/apatite nanocrystals coatings have been characterized chemically, structurally and morphologically, as well as for their ability to bind fibronectin (FN). Fourier Transform Infrared microscopy has been used to map the topographic distribution of the coating components at different times of electrochemical deposition, allowing to single out the individual deposition steps. Moreover, roughness of Ti plate has been found to affect appreciably the nucleation region of the inorganic nanocrystals. Laser scanning confocal microscopy has been used to characterize the FN adsorption pattern on a synthetic biomimetic apatitic phase, which exhibits a higher affinity when it is inter-grown with the collagen fibrils. The results offer auspicious applications in the preparation of medical devices such as biomimetic bone-like composite-coated metallic implants.
JTD Keywords: Hydroxyapatite-collagen coating, Electrochemically-assisted deposition, Micro-imaging FTIR spectroscopy, Laser scanning confocal microscopy, Biomimetic crystal growth, Fibronectin binding
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.
JTD 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