Fibronectin Conformation Research Articles

Fibronectin Conformations after Electrodeposition onto 316L Stainless Steel Substrates Enhanced Early-Stage Osteoblasts' Adhesion but Affected Their Behavior.

The implantation of metallic orthopedic prostheses is increasingly common due to an aging population and accidents. There is a real societal need to implement new metal implants that combine durability, good mechanical properties, excellent biocompatibility, as well as affordable costs. Since the functionalization of low-cost 316L stainless steel substrates through the successive electrodeposition of a polypyrrole film (PPy) and a calcium phosphate deposit doped with silicon was previously carried out by our labs, we have also developed a bio-functional coating by electrodepositing or oxidating of fibronectin (Fn) coating. Fn is an extracellular matrix glycoprotein involved in cell adhesion and differentiation. Impacts of either electrodeposition or oxidation on the structure and functionality of Fn were first studied. Thus, electrodeposition is the technique that permits the highest deposition of fibronectin, compared to adsorption or oxidation. Furthermore, electrodeposition seems to strongly modify Fn conformation by the formation of intermingled long fibers, resulting in changes to the accessibility of the molecular probes tested (antibodies directed against Fn whole molecule and Fn cell-binding domain). Then, the effects of either electrodeposited Fn or oxidized Fn were validated by the resulting pre-osteoblast behavior. Electrodeposition reduced pre-osteoblasts' ability to remodel Fn coating on supports because of a partial modification of Fn conformation, which reduced accessibility to the cell-binding domain. Electrodeposited Fn also diminished α5 integrin secretion and clustering along the plasma membrane. However, the N-terminal extremity of Fn was not modified by electrodeposition as demonstrated by Staphylococcus aureus attachment after 3 h of culture on a specific domain localized in this region. Moreover, the number of pre-osteoblasts remains stable after 3 h culture on either adsorbed, oxidized, or electrodeposited Fn deposits. In contrast, mitochondrial activity and cell proliferation were significantly higher on adsorbed Fn compared with electrodeposited Fn after 48 h culture. Hence, electro-deposited Fn seems more favorable to pre-osteoblast early-stage behavior than during a longer culture of 24 h and 48 h. The electrodeposition of matrix proteins could be improved to maintain their bio-activity and to develop this promising, fast technique to bio-functionalize metallic implants.

Conformational changes of exogenous deposited fibronectin on decellularized aortic conduits after implantation

Fibronectin is an abundant and ubiquitous extracellular matrix component that regulates a wide range of cell functions by directly interacting with cell surface integrin receptors. In a prior study, we observed that fibronectin can accelerate the autologous cellular repopulation of implanted decellularized grafts, enhancing their adhesion capacity and biocompatibility, through fibronectin fibrillogenesis. The supportive effect of deposited fibronectin was shown to be dissimilar in different regions of implanted decellularized grafts. This led us to question whether there is any difference in conformation of fibronectin deposited on the luminal and adventitial surfaces of decellularized aortic conduits after implantation. In this study, we doubly labelled fibronectin with a fluorescent resonance energy transfer (FRET) pair and use this to analyse differences in conformational changes of adsorbed fibronectin at different sites of the decellularized aortic conduits.

Surface Free Energy of Titanium Disks Enhances Osteoblast Activity by Affecting the Conformation of Adsorbed Fibronectin

This study evaluated the influence of surface free energy (SFE) of titanium disks on the adsorption and conformation of fibronectin (FN) and the biological behavior of osteoblasts cultured on the FN-treated modified surfaces. High [H]-SFE titanium disks were irradiated by a 30 W UV light, while low (L)-SFE titanium disks received no treatment. The surface characteristics of the titanium disks were examined using scanning electron microscope, optical surface profilometer, X-ray photoelectron spectroscopy, and contact angle measurements. Adsorbed FN on different groups was investigated using attenuated total reflection-Fourier transform infrared spectroscopy. MG-63 cells were cultured on FN-treated titanium disks to evaluate the in vitro bioactivity. The experiment showed H-SFE titanium disks adsorbed more FN and acquired more ß-turn content than L-SFE group. MG-63 cells cultured on FN-treated H-SFE titanium disks showed better osteogenic responses, including adhesion, proliferation, alkaline phosphatase activity and mineralization than that on FN-treated L-SFE titanium disks. Compared to L-SFE titanium disks, integrin-β1, integrin-α5 and Rac-1 mRNA levels were significantly higher in MG-63 cells on FN-treated H-SFE after 3 h of culture. These findings suggest that the higher SFE of H-SFE compared to L-SFE titanium disks induced changes in the conformation of adsorbed FN that enhanced the osteogenic activity of MG-63 cells.

Correlating degradation of functionalized polycaprolactone fibers and fibronectin adsorption using atomic force microscopy

In previous studies, a bioactive ligament prosthesis made of poly (ethylene terephthalate) and functionalized with sulfonate groups was proposed by our group for the reconstruction of injured ligaments. In vitro and in vivo experiments allowed elucidating the mechanism at the origin of the bioactivity of those prostheses, which involves fibronectin adsorption. As the subject evolves the next challenge was to elaborate a bioactive and biodegradable ligament able to maintain the bioactivity along with the degradation process. Polycaprolactone is the biodegradable polymer of choice for such application, due to its ability to degrade in the body environment without releasing cytotoxic by-products. To follow the evolution of the bioactivity of polycaprolactone, fibronectin adsorption, morphology, and mechanical properties on unmodified and functionalized surfaces were assessed using AFM techniques after real-time degradation under physiological conditions. The results showed that the hydrolytic degradation strongly occurred on functionalized fiber with 8.9 × 10−3w − 1 of degradation rate and half-life time 78 weeks. The degradation also affected to the conformation of fibronectin with the presence of protein aggregation on 72 weeks of degraded functionalized fiber.

Cross-linking and modification of fibronectin by peroxynitrous acid: Mapping and quantification of damage provides a new model for domain interactions

Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where it is exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitrotyrosine, di-tyrosine, and 6-nitrotryptophan. We have shown previously that human plasma FN is readily modified by ONOOH, but the extent and location of modifications, and the role of FN structure (compact versus extended) in determining these factors is poorly understood. Here, we provide a detailed LC-MS analysis of ONOOH-induced FN modifications, including the extent of their formation and the sites of intramolecular and intermolecular cross-links, including Tyr-Tyr, Trp-Trp, and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of ECM modification and turnover in the many pathologies associated with inflammation-associated fibrosis.

Monitoring of Extracellular Matrix Protein Conformations in the Presence of Biomimetic Bone Tissue Regeneration Scaffolds

Tissue scaffolds can be designed to mimic the native extracellular matrix (ECM), making them attractive for the development for a range of regenerative medicine applications. The macromolecules present in the ECM are critical for the provision of structural support to surrounding cells and signalling cues for the modulation of diverse processes including cell migration, proliferation and healing activation. Here, conformational and transitional behaviour of the ubiquitous ECM protein, fibronectin (Fn), in the presence of bone tissue regeneration scaffolds and living C2C12 myoblast cells is reported. Spectral monitoring of Fn functionalised high plasmonic resonance responsive gold-edge-coated triangular silver nanoplates (AuTSNP) is used to distinguish between compact and extended fibronectin conformations. Large spectral red shifts of ~20 to ~59 nm indicate Fn unfolding and fibril formation on incubation with C2C12 cells. The label-free nature, excellent sensitivity and straightforward application of the AuTSNP within cellular environments presents them as a powerful new tool to signature protein conformational activity in living cells and monitor essential protein activity for the assisted development of improved tissue scaffolds promoting enhanced tissue repair.

Nanoscale Surface-Induced Unfolding of Single Fibronectin Is Restricted by Serum Albumin Crowding

Understanding nanoscale protein conformational changes at solid-liquid interfaces is critical for predicting how proteins will impact the performance of biomaterials in vivo. Crowding is an important contributor to conformational stability. Here we apply single-molecule high resolution imaging with photobleaching to directly measure dye-conjugated fibronectin's unfolding in varying conditions of crowding with human serum albumin on aminosilanized glass. Using this approach, we identify serum albumin's crowding mechanism. We find that fibronectin achieves larger degrees of unfolding when not crowded by coadsorbed serum albumin. Serum albumin does not as effectively constrict fibronectin's conformation if it is sequentially, rather than simultaneously, introduced, suggesting that serum albumin's crowding mechanism is dependent on its ability to sterically block fibronectin's unfolding during the process of adsorption. Because fibronectin's conformation is dependent on interfacial macromolecular crowding under in vitro conditions, it is important to consider the role of in vivo crowding on protein activity.

Effect of linking arm hydrophilic/hydrophobic nature, length and end-group on the conformation and the RGD accessibility of surface-immobilized fibronectin.

In order to stimulate the cellular response to implant materials, extracellular matrix (ECM) proteins, such as collagen and fibronectin (FN), are immobilized on the implant surface. Amongst all ECM proteins used for biomimetic materials for medical applications, FN is one of the most investigated proteins thanks to its ability to promote cell adhesion and its contribution to important physiological processes. However, its conformation and hence its bioactivity strongly depend on the hydrophilic/hydrophobic nature of the surface as well as on immobilization strategies. This work investigates the effect of these two parameters, as well as the effect of the crosslinker length. FN was grafted onto silicon wafers using eights different linking arms presenting different lengths, hydrophilic/hydrophobic characters and binding sites. The protein was linked through either its amino groups (lysine amino acids) or sulfhydryl functionalities (cysteine amino acids). The grafting of each crosslinker and subsequent FN conjugation onto the surfaces was evidenced by X-ray photoelectron spectroscopy, while the surface hydrophilicity was determined by contact angle measurements. Moreover, atomic force microscopy images revealed that the conformation of surface conjugated FN only depends on the hydrophilicity of the linking arm. The FN conformation was also probed by enzyme-linked immunosorbent assays (ELISA). ELISA data demonstrated that all of the three investigated parameters linking arm parameter (length, hydrophobic/hydrophilic character, and terminal end-group) somewhat influence the RGD accessibility.

The strength of the protein-material interaction determines cell fate

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) – with only one methyl group less – 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 SignificanceThe 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.

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from −3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. Statement of SignificanceThe ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5β1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation.

Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants.

To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FNΔI1-5, FNΔIII1-3, FNΔIII4-8, and FNΔIII11-14. A monomeric form, FNmono, which lacked the C-terminal dimerization region, was also created. FNtnA-D was generated by swapping FNIII domains 1-8 in FNΔIII11-14 with seven FNIII domains from tenascin-C. The conformations of these mutants were analyzed by glycerol gradient sedimentation under low-salt (20 mM NaCl) and high-salt (200 mM NaCl) conditions. Surprisingly, most of the mutants showed a compact conformation under low-salt conditions, except for FNtnA-D. When we tested these mutants in cell culture, FNΔI1-5, FNΔIII1-3, and FNtnA-D were unable to form a matrix. Interestingly, FNΔIII1-3 and FNtnA-D were capable of co-assembly with full-length FN, while FNΔI1-5 was not. This indicates that the segment I1-5 is crucial for matrix assembly and segment III1-3 is also important. Mutations in FN are associated with glomerulopathy, but when we studied mutant proteins, the single-nucleotide mutations had only minor effects on conformation and matrix assembly. The mutations may destabilize their FNIII domains or generate dimers of dimers by disulfide cross-linking.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2-0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections.

Activation mechanisms of αVβ3 integrin by binding to fibronectin: A computational study.

Integrin αVβ3 plays an important role in regulating cellular activities and in human diseases. Although the structure of αVβ3 has been studied by crystallography and electron microscopy, the detailed activation mechanism of integrin αVβ3 induced by fibronectin remains unclear. In this study, we investigated the conformational and dynamical motion changes of Mn2+ -bound integrin αVβ3 by binding to fibronectin with molecular dynamics simulations. Results showed that fibronectin binding to integrin αVβ3 caused the changes of the conformational flexibility of αVβ3 domains, the essential mode of motion for the domains of αV subunit and β3 subunit and the degrees of correlated motion of residues between the domains of αV subunit and β3 subunit of integrin αVβ3. The angle of Propeller domain with respect to the Calf-2 domain of αV subunit and the angle of Hybrid domain with respect to βA domain of β3 subunit significantly increased when integrin αVβ3 was bound to fibronectin. These changes could result in the conformational change tendency of αVβ3 from a bend conformation to an extended conformation and lead to the open swing of Hybrid domain relative to βA domain of β3 subunit, which have demonstrated their importance for αVβ3 activation. Fibronectin binding to integrin αVβ3 significantly decreased the relative position of α1 helix to βA domain and that to metal ion-dependent adhesion site, stabilized Mn2+ ions binding in integrin αVβ3 and changed fibronectin conformation, which are important for αVβ3 activation. Results from this study provide important molecular insight into the "outside-in" activation mechanism of integrin αVβ3 by binding to fibronectin.

Protein-crystal interface mediates cell adhesion and proangiogenic secretion.

The nanoscale materials properties of bone apatite crystals have been implicated in breast cancer bone metastasis and their interactions with extracellular matrix proteins are likely involved. In this study, we used geologic hydroxyapatite (HAP, Ca10(PO4)6(OH)2), closely related to bone apatite, to investigate how HAP surface chemistry and nano/microscale topography individually influence the crystal-protein interface, and how the altered protein deposition impacts subsequent breast cancer cell activities. We first utilized Förster resonance energy transfer (FRET) to assess the molecular conformation of fibronectin (Fn), a major extracellular matrix protein upregulated in cancer, when it adsorbed onto HAP facets. Our analysis reveals that both low surface charge density and nanoscale roughness of HAP facets individually contributed to molecular unfolding of Fn. We next quantified cell adhesion and secretion on Fn-coated HAP facets using MDA-MB-231 breast cancer cells. Our data show elevated proangiogenic and proinflammatory secretions associated with more unfolded Fn adsorbed onto nano-rough HAP facets with low surface charge density. These findings not only deconvolute the roles of crystal surface chemistry and topography in interfacial protein deposition but also enhance our knowledge of protein-mediated breast cancer cell interactions with apatite, which may be implicated in tumor growth and bone metastasis.

Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Full-Length Fibronectin Drives Fibroblast Accumulation at the Surface of Collagen Microtissues during Cell-Induced Tissue Morphogenesis.

Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is vital. In a 3D model system of tissue morphogenesis, a fibronectin-FRET sensor recently revealed the existence of two separate fibronectin populations with different conformations in microtissues, i.e. ‘compact and adsorbed to collagen’ versus ‘extended and fibrillar’ fibronectin that does not colocalize with the collagen scaffold. Here we asked how the presence of fibronectin might drive this cell-induced tissue morphogenesis, more specifically the formation of gradients in cell density and ECM composition. Microtissues were engineered in a high-throughput model system containing rectangular microarrays of 12 posts, which constrained fibroblast-populated collagen gels, remodeled by the contractile cells into trampoline-shaped microtissues. Fibronectin’s contribution during the tissue maturation process was assessed using fibronectin-knockout mouse embryonic fibroblasts (Fn-/- MEFs) and floxed equivalents (Fnf/f MEFs), in fibronectin-depleted growth medium with and without exogenously added plasma fibronectin (full-length, or various fragments). In the absence of full-length fibronectin, Fn-/- MEFs remained homogenously distributed throughout the cell-contracted collagen gels. In contrast, in the presence of full-length fibronectin, both cell types produced shell-like tissues with a predominantly cell-free compacted collagen core and a peripheral surface layer rich in cells. Single cell assays then revealed that Fn-/- MEFs applied lower total strain energy on nanopillar arrays coated with either fibronectin or vitronectin when compared to Fnf/f MEFs, but that the presence of exogenously added plasma fibronectin rescued their contractility. While collagen decoration of single fibronectin fibers enhanced the non-persistent migration of both Fnf/f and Fn-/- MEFs, the migration speed was increased for Fn-/- MEFs on plasma fibronectin fibers compared to Fnf/f MEFs. In contrast, the average speed was the same for all cells on collagen-coated Fn fibers. A Fn-FRET sensor revealed that fibronectin on average was more extended on the microtissue surface compared to fibronectin in the core. Gradients of collagen-to-fibronectin ratios and of the fraction of collagen-adsorbed to stretched fibrillar fibronectin conformations might thereby provide critical cell migration cues. This study highlights a dominant role for fibronectin in tissue morphogenesis and the development of tissue heterogeneities.

Fibronectin adsorption on surface modified polyetherurethanes for blood-contacting applications

Event Abstract Back to Event Fibronectin adsorption on surface modified polyetherurethanes for blood-contacting applications Ludivine Hugoni1, 2, Vanessa Montaño-Machado1, 2, Emmanuel Pauthe2, Diego Mantovani1 and J Paul Santerre3 1 CHU de Québec Research Center, Laval University, Lab. Biomaterials and Bioengineering, CRC-I, Dept Min-Met-Materials Eng, Canada 2 University of Cergy Pontoise, Equipe de Recherche sur les Relations Matrice Extracellulaire-Cellules, France 3 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada Introduction: After the implantation of a medical device, adhesive proteins such as fibronectin (FN) are adsorbed to the surface of the material[1]. These are the biomolecules that monocytes (MCs) will interact with, to adopt a pro-inflammatory or pro-wound healing phenotype[2]. Hence, it was desired to study FN coatings on two surface modified polyetherurethanes (PEUs) with potential blood-contacting applications, to mimic the biological environment. A fluorinated surface modifying oligomer (PPO212L), known to reduce platelet adhesion[3] and an anionic/dihydroxy oligomeric (ADO) additive, known to enable cell adhesion[4] were used to modify surface blood cell compatibility of a commercial PEU. FN adsorption and the subsequent MC inflammatory response was investigated on the two modified PEUs. Materials and Methods: The PEU was prepared by dissolving Tecoflex® 65D in 10 % (wt/v) chloroform. PPO212L or ADO was added at 0.5 % (wt/wt). Solutions were casted on PTFE for 24 h followed by 2 h in vacuum (30 mmHg), both at room temperature. Protein adsorption was performed with a FN solution (50 μg.mL-1 in HEPES 10 mM, pH 7.4) at 37 °C for 30 min. Surfaces were characterized by water contact angle (WCA), atomic force microscopy (AFM) and immunofluorescence staining (IFS). Adsorbed FN was quantified by a bicinchoninic acid assay. MCs, isolated from blood of healthy donors (University of Toronto ethics protocol #22203) were seeded on PEUs (106 cells.mL-1). After 72 h of culture, the viability of adhered MCs was evaluated by Alamar blue® assay and cell morphology was analyzed by scanning electron microscopy (SEM). Results were given as mean ± standard deviation (SD) and were statistically analyzed by one-way analysis of variance (ANOVA), with significant differences reported for p<0.05. Results and Discussion: On all PEUs, FN adsorption significantly decreased the WCA whereas surface roughness increased approximately 2 fold (Table 1). No statistically significant difference was observed for the amount of adsorbed FN (Table 1) corroborating the presence of a protein monolayer to surfaces[5]. The difference in WCA prior vs after FN adsorption was greatest for the base PEU and PEU-ADO. This result suggested a different FN conformation on PEU-PPO212L than on the two other surfaces. The availability of FN cell binding domains for all coated PEUs was observed after IFS (data not shown). Preliminary tests with MCs showed that all FN-coated PEUs significantly increased MC viability, without affecting cell morphology except for PEU-ADO (Figure 1). These results suggested that FN activated MCs more extensively on PEU-ADO than on the base PEU and PEU-PPO212L, which showed the least activated morphology of all surfaces. Conclusions: A comparative study of two surface modified PEUs, PEU-PPO212L and PEU-ADO, intended for blood-contacting applications was performed. Investigations suggested that FN-coated PEU-PPO212L has less influence on MC activation than FN-coated PEU-ADO. Further investigations are ongoing to determine the effect of FN coating on cell production of cytokines such as pro-inflammatory tumor necrosis factor-α (TNF-α) and anti-inflammatory interleukin 10 (IL-10). This work was supported by a MSc Scholarship from NSERC CREATE Program in Regenerative Medicine (www.ncprm.ulaval.ca), NSERC, CIHR, Fonds Samuel de Champlain, FQRS, and FQRNT; The authors would like to thank Pascale Chevallier and Lucie Levesque for their technical assistance

Engineering plasma-polymerised surfaces for synergistic integrin/growth factor signalling to promote mesenchymal stem cell adhesion and differentiation

Event Abstract Back to Event Engineering plasma-polymerised surfaces for synergistic integrin/growth factor signalling to promote mesenchymal stem cell adhesion and differentiation Annie Zhe Cheng1, 2*, Andrés Alba Perez1*, Nikolaj Gadegaard1, Mathis Riehle2, Matthew Dalby2 and Manuel Salmeron-Sanchez1* 1 University of Glasgow, Biomedical Engineering, United Kingdom 2 University of Glasgow, Centre for Cell Engineering, United Kingdom Introduction: The effective presentation of proteins and growth factors (GFs) is a requirement in synthetic materials for tissue engineering. Fibronectin (FN), an extracellular matrix protein that regulates cell adhesion, binds GFs via specific structural domains[1], exhibiting synergistic effects on cells. Polymers have the ability to facilitate conformational changes in protein structure, in turn altering protein-GF interactions[2]. Here we used an inductively coupled plasma system to modify 2D surfaces with thin coatings of poly(ethyl acrylate) (PEA)[3], a polymer known to induce FN organisation into nanonetworks[4] by exposing its integrin and GF-binding domains. The efficiency of plasma polymerisation as well as FN adsorption and interaction with bone morphogenetic protein-2 (BMP-2) on PEA-coated surfaces were evaluated. Human mesenchymal stem cell (hMSC) adhesion and differentiation were assessed at the protein-GF interface to determine the overall efficiency of the system. Materials and Methods: Plasma polymerisation: A custom-built inductively coupled plasma chamber was used to deposit EA onto glass coverslips via plasma polymerisation. Spin-coated PEA surfaces were used as controls. Surface characterisation: Surfaces coated with plasma-polymerised PEA (pPEA), FN, and BMP-2 were characterised by XPS, WCA, AFM, immunogold staining, and ELISA to verify elemental composition, wettability, protein adsorption, and FN/BMP-2 binding. Cell culture: hMSCs were seeded on PEA-modified surfaces coated with FN and/or BMP-2 for up to 4 weeks to study adhesion, integrin/GF receptor co-localisation, and differentiation. Results and Discussion: The successful coating of pPEA was confirmed by XPS, where carbon moieties corresponding to those in PEA were observed. Increasing water contact angles on pPEA-coated surfaces indicated changes in surface chemistry. AFM revealed differences in the conformation of FN adsorbed on thin pPEA coatings compared with that on spin-coated PEA. BMP-2 localisation was visualised by immunogold labelling, and ELISA for BMP-2 showed comparable FN/BMP-2 binding between surfaces modified with spin-coated PEA and pPEA, an evidence of efficient protein/GF presentation. hMSC adhesion and spreading were enhanced on surfaces coated with pPEA and FN compared with controls. Integrin/GF receptor co-localisation was observed, suggesting that changes in FN conformation on pPEA led to increased interactions with BMP-2 with the potential to promote differentiation. Conclusions: Plasma polymerisation results in thin PEA coatings on 2D surfaces, which enables the effective presentation of FN and low doses of GFs for synergistic interactions. Consequently, hMSC fate can be directed by such surfaces. The originality of this study lies in expanding the utility of PEA and similar polymers as unique materials that trigger FN organisation into nanonetworks. These materials have the potential to be applied as coatings in biodegradable systems. Medical Research Council; National EPSRC XPS Users' Service

Effect of hydroxyapatite materials properties on fibronectin-mediated breast cancer cell adhesion and pro-angiogenic secretion

Event Abstract Back to Event Effect of hydroxyapatite materials properties on fibronectin-mediated breast cancer cell adhesion and pro-angiogenic secretion Fei Wu1, Weisi Chen2, Brian Gillis1, Claudia Fischbach2, 3, Lara Estroff1, 3 and Delphine Gourdon1, 2 1 Cornell University, Materials Science and Engineering, United States 2 Cornell University, Biomedical Engineering, United States 3 Kavli Institute at Cornell for Nanoscale Science, United States Introduction: Breast cancer preferentially metastasizes to bone and induces pathological remodeling. There has been evidence suggesting that the materials properties of bone mineral may play an important role in breast cancer metastasis[1],[2]. Hydroxyapatite (HAP, Ca10(PO4)6(OH)2) is a calcium phosphate mineral with structural and mechanical properties closely related to bone apatite. In this study, we seek to understand: (i) whether HAP materials properties, such as surface chemistry and roughness, alter the mineral/extracellular matrix (ECM) interface in bone, and (ii) how the altered ECM impacts breast cancer cell functions. Specifically, we investigated the conformation of fibronectin (Fn, a major ECM protein), breast cancer cell adhesion and pro-angiogenic secretion as a function of underlying HAP materials properties and associated Fn conformation. Materials and Methods: Geologic HAP crystals were bought from Etsy. Fibronectin (Fn) was obtained from Life Technologies, NY, and labeled for intramolecular Fӧrster resonance energy transfer (FRET) as previously described[3]. Smooth HAP facets (100)S and (001)S and rough HAP facets (100)R and (001)R were obtained by polishing HAP slabs to roughnesses either below 1 nm, i.e., smaller than Fn radius of gyration, or around 200 nm. Then, we incubated Fn (10% FRET-labeled) on HAP facets and analyzed Fn conformation via FRET. Finally, MDA-MB-231 breast cancer cells (ATCC, VA) were seeded onto Fn coated HAP facets to investigate cell adhesion and secretion of vascular endothelial growth factors (VEGF). Student’s t-tests were used to determine statistical significance between conditions in GraphPad Prism (GraphPad Software Inc., CA). Results and Discussion: We first quantified Fn conformation adsorbed onto HAP via FRET (Figure 1). FRET ratios of Fn adsorbed onto (100) were higher than those onto (001) for both smooth and rough HAP, indicating that Fn adsorbed in more compact conformations onto (100) facets. Interestingly, Fn FRET ratios were not significantly different between smooth and rough HAP, suggesting that Fn conformation was independent of roughness within the range of interest. We then assessed MDA-MB-231 cells behavior. Our preliminary results showed (i) higher cell adhesion onto (001) than onto (100) HAP facets, (ii) overall higher cell adhesion onto rough HAP than onto smooth HAP, and (iii) lower VEGF secretion associated with higher adhesion. Conclusions: Our FRET analysis indicated that Fn conformation (% of unfolded Fn) depends exclusively on HAP surface chemistry. In contrast, our cells study revealed enhanced adhesion and decreased pro-angiogenic secretion of cancer cells linked with both (001) HAP facets (coated with more unfolded Fn) and rougher HAP. These findings deconvolute the roles of mineral surface chemistry and roughness in interfacial ECM deposition and have important implications for our understanding of breast cancer bone metastasis. Karin Wang; Jin Ho Chang; Young Hye Song; Siyoung Choi; Maura Weathers

Adsorption force of fibronectin on various surface chemistries and its vital role in osteoblast adhesion.

The amount, type, and conformation of proteins adsorbed on an implanted biomaterial are believed to influence cell adhesion. Nevertheless, only a few research works have been dedicated to the contribution of protein adsorption force. To verify our hypothesis that the adsorption force of protein on biomaterial is another crucial mediator to cell adhesion, fibronectin (FN) adsorbed on self-assembled monolayers (SAMs) with terminal -OH, -CH3, and -NH2 was quantified for FN adsorption force (F(ad)) by utilizing a sphere/plane adsorption model and parallel plate flow chamber. As revealed, F(ad) on SAMs followed a chemistry-dependence of -NH2 > -CH3 ≫ -OH. It is further demonstrated that F(ad) together with FN conformation could regulate the late osteoblast adhesion and the consequent reorganization of the adsorbed FN and fibrillogenesis of the endogenous FN. Our study suggests that protein adsorption force plays a key role in cell adhesion and should be involved for better biomaterial design.