Immobilization Of Fibronectin Research Articles

Comprehensive study of plasma polymerization parameters on thiol-coated LDPE films for effective fibronectin adsorption targeting biomedical applications

In the realm of biomedical applications, biocompatible materials with optimized surface properties are crucial for facilitating cellular interactions. To attain the perfect balance of these properties, surface modification of non-toxic and stable bulk materials is often required. Within this context, this research aimed to improve the physiochemical and cell-responsive properties of a low-density polyethylene film. This was achieved by depositing thiol-rich coatings using a dielectric barrier discharge plasma reactor operating at medium pressures, with 1-propanethiol serving as polymerization precursor. The study systematically investigated the impact of key plasma polymerization parameters, including DBD chamber pressure, treatment time, and the combination of precursor flow rate and discharge power represented by the Yasuda parameter, to identify optimal plasma processing conditions for the formation of thiol-rich coatings. To characterize the coating thickness, hydrophilicity and surface chemical composition, atomic force microscopy, water contact angle measurements, and X-ray photoelectron spectroscopy were employed. The findings indicated that reducing the chamber pressure to 10 kPa led to more hydrophilic and thicker deposits possessing a higher sulphur content. Deposition time (5 to 15 min) also significantly impacted coating thickness and surface chemistry, where long times increased thickness, but also led to a reduced sulphur and increased oxygen content because of more pronounced etching. The optimal deposition time was therefore set at 10 min resulting in the deposition of dense coatings possessing a high number of sulphur-containing functionalities. The Yasuda parameter (W/FM) analysis demonstrated optimal thiol incorporation in combination with high coating stability at an intermediate W/FM value of 72 MJ/kg. Following the determination of the optimal plasma polymerization parameters, the effectiveness of thiol plasma polymerization and subsequent fibronectin immobilization for enhancing cell adhesion and proliferation was investigated. The thiol-coated substrates were found to exhibit superior protein immobilization, because of the high affinity for protein binding of the available thiol groups. To assess cellular responses, Schwann cells were cultured on uncoated and coated samples before and after fibronectin immobilization. The results revealed a superior cellular response of the thiol-coated samples after fibronectin immobilization, showing the highest cell viability and significantly enhanced cell adhesion and proliferation. Collectively, these results underscore the synergistic effect of thiol plasma polymerization and fibronectin immobilization in promoting the cellular response of LDPE substrates, thus highlighting their potential as a surface modification strategy of biomaterials.

Influence of Fibronectin Immobilization on the Orientation of Collagen Bundles in Soft Tissue around Zirconia Implants

Influence of Fibronectin Immobilization on the Orientation of Collagen Bundles in Soft Tissue around Zirconia Implants

The concentration-independence cellular effects of fibronectin adsorbed on material surfaces with different hydrophobicities

In tissue engineering, coating biomaterial’s surface with extracellular matrix (ECM) proteins can promote many biological activities, including cellular responses, wound healing, and rejection reduction. Several interactions occur upon binding proteins onto the surfaces, leading to changes in the protein structural conformation, directly affecting the cell-host interactions. Therefore, this study investigates the impacts of surfaces’ wettability on protein conformation. In order to get the insights, organosilicate (OGS) was utilized to modify the tissue culture plate, resulting in surfaces with different wettability, followed by fibronectin (FN) immobilization. Then, the surfaces were used to study the fibrinogen interaction, cell attachment, and spreading. The results showed that OGS-modified surfaces produced four different wettability, ranging from super-hydrophilic (OGS150), hydrophilic (OGS100), hydrophobic (OGS60), to super-hydrophobic (OGS5). Each surface possessed particular nature, resulting in the variation of FN molecules' structural change. The amount of FN adsorbed on the OGS-coated surfaces was shown not to be perfectly proportional to the results of fibrinogen interaction, cell attachment, and spreading. The super-hydrophobic surfaces (OGS5) were highest in the amount of immobilized FN and the efficiency in subsequent experiments among the OGS-coated surfaces group. Notably, the hydrophobic surface adsorbed the lowest amount of FN but achieved remarkable results in the following experiments. Thus, this study holds a promising potential in producing biocompatible materials in tissue engineering.

Fibronectin-Functionalized Fibrous Meshes as a Substrate to Support Cultures of Thymic Epithelial Cells.

Thymic epithelial cells (TECs) are the main regulators of T lymphocyte development and selection, requiring a three-dimensional (3D) environment to properly perform these biological functions. The aim of this work was to develop a 3D culture substrate that allows the survival and proliferation of TECs. Thus, electrospun fibrous meshes (eFMs) were functionalized with fibronectin, one of the major extracellular matrix (ECM) proteins of the thymus. For that, highly porous eFMs were activated using oxygen plasma treatment followed by amine insertion, which allows the immobilization of fibronectin through EDC/NHS chemistry. The medullary TECs presented increased proliferation, viability, and protein synthesis when cultured on fibronectin-functionalized eFMs (FN-eFMs). These cells showed a spread morphology, with increased migration toward the inner layers of FN-eFMs and the production of thymic ECM proteins, such as collagen type IV and laminin. These results suggest that FN-eFMs are an effective substrate for supporting thymic cell cultures.

The immobilization of fibronectin- and fibroblast growth factor 2-derived peptides on a culture plate supports the attachment and proliferation of human pluripotent stem cells.

Pluripotent stem cells (PSCs) offer a promising tool for regenerative medicine. The clinical application of PSCs inevitably requires a large-scale culture in a highly defined environment. The present study aimed to devise defined coating materials for the efficient adhesion and proliferation of human PSCs (hPSCs). We tested the activity of seven fibronectin-derived peptides and three laminin-derived peptides for the attachment and proliferation of hPSCs through their immobilization on the bottom of culture dishes by creating a fusion protein with the mussel adhesion protein. Among the extracellular matrix (ECM) mimetics tested, one fibronectin-derived peptide, PHSRN-GRGDSP, significantly promoted adhesion, enhanced alkaline phosphatase activity, and increased pluripotency-related gene expression in hPSCs compared to Matrigel. Furthermore, co-immobilization of a particular canofin peptide derived from fibroblast growth factor 2 increased pluripotency marker expression, which may offer the possibility of culture without growth factor supplementation. Our findings afford a novel defined condition for the efficient culture of hPSCs and may be utilized in future clinical applications.

The Effect of Fibronectin-Immobilized Microgrooved Titanium Substrata on Cell Proliferation and Expression of Genes and Proteins in Human Gingival Fibroblasts.

We aimed to determine the effect of fibronectin (FN)-immobilized microgrooved titanium (Ti) on human gingival fibroblast proliferation, gene expression and protein expression. Photolithography was used to fabricate the microgrooved Ti, and amine funtionalization (silanization) was used for FN immobilization on titanium surfaces. Cell proliferation, gene expression and protein expression were analyzed, followed by multiple regression analysis for determining the influential factors on cell proliferation. FN-immobilized microgrooved Ti significantly enhanced the fibroblast proliferation in various timelines of culture, among which a burst of fivefold increase is induced at 96h of culture compared to that on the control smooth Ti. We suggest a presence of the synergistic promotion effect of microgrooves and FN immobilization on fibroblast proliferation. Through a series of analyses on the expression of various genes and proteins involved in cell adhesion and proliferation, cyclin-dependent kinase 6, cyclin D1, integrin α5, oncogene c-Src, osteonectin, paxillin and talin-2 were determined as influential factors on promoting fibroblast proliferation induced by FN-immobilized microgrooved Ti. FN-immobilized microgrooved Ti can act as an effective surface for enhancing fibroblast proliferation, and can be used for promoting soft tissue response on the connective tissue attachment zone of biomaterial surfaces.

Fibronectin modified TiO2 nanotubes modulate endothelial cell behavior.

Cardiovascular disease has become a great threat to the health of mankind; current titanium (Ti) stents fail due to late stent thrombosis caused by the lack of re-endothelialization of the Ti stent. The objective of this study was to design a novel cardiovascular Ti implant with improved surface biocompatibility. TiO2 nanotubes with a diameter of 110 nm were anodized at a constant voltage of 30 V, and fibronectin was immobilized onto the TiO2 nanotubes using polydopamine. The element composition, morphology, and wettability of the different substrate surfaces were characterized by x-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), and contact angle measurements, respectively, confirming the successful immobilization of fibronectin. In vitro experiments including immunofluorescence staining, Cell Counting Kit-8 (CCK-8), and nitric oxide (NO) and prostacyclin (PGI2) release demonstrate that fibronectin modified TiO2 nanotubes supported cell adhesion, proliferation, and normal cellular functions of human umbilical vein endothelial cells (HUVECs). These methodologies can be applied for future fabrication of cardiovascular stents.

Multilayered membranes with tuned well arrays to be used as regenerative patches.

Flexible multilayered membranes containing multiple micro-reservoirs are found as potential regenerative patches. Layer-by-layer (LbL) methodology over a featured PDMS substrate is used to produce patterned membranes, composed only by natural-based polymers, that can be easily detached from the PDMS substrate. The combination of nano-scale control of the polymeric organization along the thickness of the chitosan/alginate (CHT/ALG) membranes, provided by LbL, together with the geometrical micro-scale features of the patterned membranes offers a uniqueness system that allows cells to colonize 3-dimensionally. This study provides a promising strategy to control cellular spatial organization that can face the region of the tissue to regenerate.

Interactions of human embryonic stem cell‐derived cardiovascular progenitor cells with immobilized extracellular matrix proteins

Human embryonic stem cell-derived cardiovascular progenitor cells (hESC-CVPCs) hold great promise for cell-based therapies of heart diseases. However, little is known about their niche microenvironment and in particular the required extracellular matrix (ECM) components. Here we screened combinations of surface-immobilized ECM proteins to identify substrates that support the attachment and survival of hESC-CVPCs. Covalent immobilization of ECM proteins laminin (Lm), fibronectin (Fn), collagen I (CI), collagen III (CIII), and collagen IV (CIV) in multiple combinations and concentrations was achieved by reductive amination on transparent acetaldehyde plasma polymer (AAPP) interlayer coatings. We identified that CI, CIII, CIV, and Fn and their combinations were important for hESC-CVPC attachment and survival, while Lm was dispensable. Moreover, for coatings displaying single ECM proteins, CI and CIII performed better than CIV and Fn, while coatings displaying the combined ECM proteins CIII + CIV and Fn + CIII + CIV at 100 µg/mL were comparable to Matrigel in regard to supporting hESC-CVPC attachment and viability. Our results identify ECM proteins required for hESC-CVPCs and demonstrate that coatings displaying multiple immobilized ECM proteins offer a suitable microenvironment for the attachment and survival of hESC-CVPCs. This knowledge contributes to the development of approaches for maintaining hESC-CVPCs and therefore to advances in cardiovascular regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1094-1104, 2017.

Corrosion resistance and biocompatibility of magnesium alloy modified by alkali heating treatment followed by the immobilization of poly (ethylene glycol), fibronectin and heparin

In recent years, magnesium alloys are attracting more and more attention as a kind of biodegradable metallic biomaterials, however, their uncontrollable biodegradation speed in vivo and the limited surface biocompatibility hinder their clinical applications. In the present study, with the aim of improving the corrosion resistance and biocompatibility, the magnesium alloy (AZ31B) surface was modified by alkali heating treatment followed by the self-assembly of 3-aminopropyltrimethoxysilane (APTMS). Subsequently, poly (ethylene glycol) (PEG) and fibronectin or fibronectin/heparin complex were sequentially immobilized on the modified surface. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed that the above molecules were successfully immobilized on the magnesium alloy surface. An excellent hydrophilic surface was obtained after the alkali heating treatment while the hydrophilicity decreased to some degree after the self-assembly of APTMS, the surface hydrophilicity was gradually improved again after the immobilization of PEG, fibronectin or fibronectin/heparin complex. The corrosion resistance of the control magnesium alloy was significantly improved by the alkali heating treatment. The self-assembly of APTMS and the following immobilization of PEG further enhanced the corrosion resistance of the substrates, however, the grafting of fibronectin or fibronectin/heparin complex slightly lowered the corrosion resistance. As compared to the pristine magnesium alloy, the samples modified by the immobilization of PEG and fibronectin/heparin complex presented better blood compatibility according to the results of hemolysis assay and platelet adhesion as well as the activated partial thromboplastin time (APTT). In addition, the modified substrates had better cytocompatibility to endothelial cells due to the improved anticorrosion and the introduction of fibronectin. The substrates modified by fibronectin or fibronectin/heparin complex can significantly promote endothelial cell adhesion and proliferation. Taking all these results into consideration, the method of the present study can be used for the surface modification of the magnesium alloy to simultaneously impart it better corrosion resistance, favorable blood compatibility and good cytocompatibility to endothelial cells.

The importance of the interface for improving adhesion and stability of substrate/nanocoating systems

Event Abstract Back to Event The importance of the interface for improving adhesion and stability of substrate/nanocoating systems Maxime Cloutier1, Vanessa Montaño-Machado1, Mahrokh Dorri1, Livia Angeloni1, Farid Anooshehpour1, Sergio Díaz-Rodríguez1, Ludivine Hugoni1, Eléonore C. Michel1, Ranna Tolouei1, Carlo Paternoster1, Pascale Chevallier1, Lucie Lévesque1, Stéphane Turgeon1 and Diego Mantovani1 1 CHU de Québec Research Center, Laval University, Lab. Biomaterials and Bioengineering, CRC-I, Dept Min-Met-Materials Eng, Canada Introduction: Coatings are known to be one of the best suited strategies to tailor interactions between medical surfaces and the surrounding cells and tissues. They offer the possibility to modify the surface properties with functionalization and film deposition without affecting the bulk mechanical properties of a material. With the increased role of nanotechnology in the biomaterial field and the experimental evidence gathered in recent years, the need to precisely engineer film/substrate interfaces has become increasingly acknowledged within the biomedical community [1],[2]. Interfaces play a major role on coatings’ adhesion and stability and can impact several other properties. This work presents applications of interface engineering for stability and adhesion improvement, with a focus on two particular applications: bioactive and protective coatings for cardiovascular stents and antibacterial coatings for hospital settings. Methods: The substrate/nanocoating systems investigated in this work were: 1) 316L stainless steel (SS316L) /fluorocarbon (CFx) [3], 2) CFx/biomolecules [4] and 3) SS316L/Diamond-like carbon (DLC) [5]. All interface modifications and coating deposition were performed using low-pressure plasma-based processes. Complementary characterization techniques have been used to assess surface modifications, including X-Ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and scanning electron microscopy (SEM). The systems stability was investigated through electrochemical (potentiodynamic and galvanostatic) and dynamic flow tests. The adhesion of nanocoatings was also studied using small punch (deformation) and pull-off tests. Results and Discussion: The stability of the 316LSS/CFx system was shown to be highly correlated with the crystallinity and thickness of the interfacial oxide layer. A plasma oxidation process was used to produce a thicker, amorphous oxide layer at the SS316L/CFx interface (Figure 1). The corrosion rate was consequently reduced 11-fold compared with the control sample. In the CFx/biomolecule system, different fibronectin immobilization strategies were assessed for stability. According to AFM analysis, skewness (Rsk) values suggested a more homogenous surface in fibronectin grafted (FNg) rather than adsorbed (FNa) (Figure 2). After dynamic tests under flow, the roughness (Rq) value for FNa was similar, while it decreased for FNg, suggesting changes in conformation of the protein leading to a denser coating. For the SS316L/DLC system, the presence of an interfacial carbidic layer was demonstrated to increase the adhesion of DLC on stainless steel, with an observed increase in bonding strength and resistance to delamination (Figure 3). The formation of the thin interfacial carbide layer was achieved using implantation of carbon ions by plasma. Conclusions: Plasma-based surface modifications can be successfully exploited to engineer substrate/nanocoating interfaces and improve the stability and adhesion of several biomaterials. They represents an interesting, polyvalent and powerful tool for tuning the surface and the interface properties for coatings and surfaces. This work was partially funded by NSERC-Canada, FRQ-NT-Québec, CFI-Canada, the Canadian Space Agency, MRI-Québec and with the support of Wallonie-Bruxelles International (WBI); MC was awarded a prestigious NSERC Vanier Scholarship; VMM was awarded a doctoral scholarship from CONACYT – National Council of Science and Technology, Mexico; CP and DM were recipients of the Linkage Grant from Quebec/Italy sub-commission of the Quebec Ministry of Intl Relations

(Invited) Development of Fibronectin-Modified Gold Egfet Sensor for the Detection of S. Epidermidis Biofilm and Staphylococcal 16S rRNA

Prolonged catheter implantation and placement expose patients to the risk of microbial infection and biofilm formation could lead to extensive hospitalization and mortality increase. Among frequent isolated pathogens (gram-positive bacteria (45-65%), gram-negative bacteria (25-40%) and fungi (3-6%), Staphylococcus epidermidis is the most common microbial agents, while Staphylococcus aureus is associated with more severe cases, a higher risk of hospitalization, catheter removal, and finally, death [1]. The pathogenesis of bacteria is mainly driven by their ability to colonize and form a thick multilayer biofilm on the polymer surface (plastics) leading to mild to sepsis that typically binds human matrix proteins such as fibrinogen or fibronectin [2]. Biofilm mostly consists of partially de-acetylated polymer of beta-1-6-linked N-acetylglucosamine polysaccharide intercellular adhesion (PIA) that acts as glue to stick and wrap negatively charged bacterial surface. Substantial quantity of extracellular DNA from the lysed cells is also reported to play a major role in biofilm formation [3]. Within various developed biosensor, field effect transistor (FET) sensor may offer several benefits, in particular EGFET structure with its easy fabrication, small influence of optical illumination and operation temperature, and disposable gate. The application of a commercial metal oxide semiconductor field effect transistor (MOSFET) also reduces the complexity and cost in its fabrication processes [4]. In this opportunity, we would like to demonstrate a novel protocol for bacterial biofilm detection determined by fibronectin immobilization onto Au-EGFET sensing membrane, altogether with the measurement of cellular responses toward antibiotic treatment. Besides, the detection of electronic signals from 16S rRNA of S. epidermidis and S. aureus on Au-wereconducted using new distinctive designed probe. Charge characteristics were shown by a responsive shift of Au-EGFET gate voltage. Not only effective for biofilm detection, we have also discovered that the whole gate surface modification on Au-EGFET is a powerful tool to detect specific bacteria species involved in the infection. In biofilm test, layer upon layer had been built to provide a robust adhesion of biofilm molecules to Au surface. The immersion of Au-electrode in SAMs solution and the graft of fibronectin afterwards successfully increased the surface roughness of bare Au. Fibronectin mostly hosts the adhesion of bacterial cells in human body. The structure of Au-SAMs-fibronectin gradually improved the surface topology of Au as expressed in the root mean square roughness (Rq) value at 3.22 nm, 3.82 nm and 6.32 nm for bare Au, with SAMs and with fibronectin, respectively. Subsequently, Staphylococcus epidermidis biofilm was attached onto Self Assembled Monolayers (SAMs) coated Au substrate of an extended-gate field-effect-transistor (EGFET) sensor with immobilized extracellular matrix protein, fibronectin. Biofilm mostly composed of positively charged Polysaccharide Intercellular Adhesin (PIA) significantly contributed threshold voltage (VTH) shift from -6.67 to -94 mV for biofilm yielded by 3.8x106 to 3.8x108 CFU/ml S. epidermidis and obtained Limit of Detection (LOD) of 9×105 CFU/ml S. epidermidis. SAMs-Au surface functionalization and its properties after fibronectin immobilization showed an optimal surface roughness from atomic force microscope (AFM) view [5]. The presence of biofilm on the surface was validated by (Scanning Electron Microscope) SEM screening after 1 hour incubation at 37oC. Cellular response to antibiotic treatment against biofilm achieved as contaminated dialysate was treated with and without Vancomycin. The hybridization of a highly conserved gene, 16S rRNA of S. epidermidis and S. aureus, the most leading pathogenic bacteria in peritoneal dialysis patients, was also successfully recorded. This finding does not only provide insights into aetiologies of infectious disease but also may guide the clinicians in prescribing antibiotics and determining the treatment duration and infection control procedures when pathogenic species is well identified. In the future work, the functionalities of other SAMs and nanoparticles can be applied to improve the Debye-length issue and uniformity control of biomolecules density onto the surface. Overall, Au-EGFET sensor with its advantages including its simple passivation, packaging and fabrication, and flexibility of extended area gate, opens a new niche in bacterial biochip technology providing a comprehensive biofilm and pathogenic bacteria species identification.

Biochemical surface modifications to titanium implants using the tresyl chlorideactivated method.

Tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride)-activated method is an easy and simple method for immobilizing biological molecules onto a titanium surface. Tresyl chloride was directly dropped onto a titanium surface without any solvent. Tresylated titanium disks were immersed in a protein or peptide solution to give protein- or peptide-immobilized titanium. Frontier molecular orbital calculations suggested that tresylation of the OH compound improved its reactivity towards the amine groups of protein compounds. Quartz-crystal microbalance-dissipation measurements suggested that ionic interaction between the oxygen of the terminal hydroxyl groups of titanium and the nitrogen of fibronectin was important for fibronectin immobilization. Initial cell attachment was enhanced by fibronectin or collagen immobilization. Fibronectin and fibronectin-derived peptide immobilization improved the expression of specific genes related to bone formation. The immobilization of cytokine onto titanium enhanced bone formation following its implantation into rat femur defects. The tresyl chloride-activated method is useful for immobilizing biological molecules onto titanium surfaces.

Fibronectin immobilization on to robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone tissue engineering.

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.

Sensing performance of fibronectin-functionalized Au-EGFET on the detection of S. epidermidis biofilm and 16S rRNA of infection-related bacteria in peritoneal dialysis

This study proposed novel methods for Staphylococcus epidermidis biofilm and 16S rRNA of infection-related bacteria detection. S. epidermidis biofilm was attached onto self assembled monolayers (SAMs) coated Au substrate of an extended-gate field-effect-transistor (EGFET) sensor with immobilized extracellular matrix protein, fibronectin. Biofilm mostly composed of positively charged polysaccharide intercellular adhesin (PIA) significantly contributed threshold voltage (VTH) shift from −6.67 to −94mV for biofilm yielded by 3.8×106 to 3.8×108CFU/ml S. epidermidis and obtained limit of detection (LOD) of 9×105CFU/ml S. epidermidis. SAMs-Au surface functionalization and its properties after fibronectin immobilization showed an optimal surface roughness from atomic force microscope (AFM) view. The presence of biofilm on the surface was validated by (scanning electron microscope) SEM screening after 1h incubation at 37°C. Cellular response to antibiotic treatment against biofilm achieved as contaminated dialysate was treated with and without Vancomycin. The hybridization of a highly conserved gene, 16S rRNA of S. epidermidis and Staphylococcus aureus, the most leading pathogenic bacteria in peritoneal dialysis patients, was also successfully recorded. Findings suggested that Au-EGFET is a promising sensing biochip for detection of bacterial biofilm and determination of species involved in the infection.

Umbilical-cord-blood-derived mesenchymal stem cells seeded onto fibronectin-immobilized polycaprolactone nanofiber improve cardiac function

Stem cells seeded onto biofunctional materials have greater potency for therapeutic applications. We investigated whether umbilical-cord-blood-derived mesenchymal stem cell (UCB-MSC)-seeded fibronectin (FN)-immobilized polycaprolactone (PCL) nanofibers could improve cardiac function and inhibit left ventricle (LV) remodeling in a rat model of myocardial infarction (MI). Aligned nanofibers were uniformly coated with poly(glycidyl methacrylate) by initiated chemical vapor deposition followed by covalent immobilization of FN proteins. The degree of cell elongation and adhesion efficacy were improved by FN immobilization. Furthermore, genes related to angiogenesis and mesenchymal differentiations were up-regulated in the FN-immobilized PCL nanofibers in comparison to control PCL nanofibers in vitro. 4weeks after the transplantation in the rat MI model, the echocardiogram showed that the UCB-MSC-seeded FN-immobilized PCL nanofiber group increased LV ejection fraction and fraction shortening as compared to the non-treated control and acellular FN-immobilized PCL nanofiber groups. Histological analysis indicated that the implantation of UCB-MSCs with FN-immobilized PCL nanofibers induced a decrease in MI size and fibrosis, and an increase in scar thickness. This study indicates that FN-immobilized biofunctional PCL nanofibers could be an effective carrier for UCB-MSC transplantation for the treatment of MI.

Chemically grafted fibronectin for use in QCM-D cell studies

Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using spectroscopic ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics.

A layer-by-layer approach to natural polymer-derived bioactive coatings on magnesium alloys

The development of polyelectrolyte multilayered coatings on magnesium alloy substrates that can be used for controlled delivery of growth factors and required biomolecules from the surface of these degradable implants could have a significant impact in the field of bone tissue regeneration. The current work reports on the fabrication of multilayered coatings of alginate and poly-l-lysine on alkaline- and fluoride-pretreated AZ31 substrates using a layer-by-layer (LbL) technique under physiological conditions. Furthermore, these coatings were surface functionalized by chemical cross-linking and fibronectin immobilization, and the resultant changes in surface properties have been shown to influence the cellular activity of these multilayered films. The physicochemical characteristics of these coated substrates have been investigated using attenuated total reflectance Fourier transform infrared spectroscopy, atomic force microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Cytocompatibility studies using MC3T3-E1 osteoblasts show that the fluoride-pretreated, cross-linked and fibronectin-immobilized LbL-coated substrates are more bioactive and less cytotoxic than the hydroxide-pretreated, cross-linked and fibronectin-immobilized LbL-coated samples. The in vitro degradation results show that the multilayered coatings of these natural polysaccharide- and synthetic polyamino acid-based polyelectrolytes do not alter the degradation kinetics of the substrates; however, the pretreatment conditions have a significant impact on the overall coating degradation behavior. These preliminary results collectively show the potential use of LbL coatings on magnesium-based degradable scaffolds to improve their surface bioactivity.

Influence of nanometer smoothness and fibronectin immobilization of titanium surface on MC3T3‐E1 cell behavior

The aim of the present study was to evaluate the influence of mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm) and sandblasting (Ra: approximately 1.0 μm), as well as biochemical treatment, namely, fibronectin immobilization, of a titanium surface on osteoblast-like cell behavior. Cell proliferation was monitored by measurements of DNA content and ALP activity; osteocalcin production and mineralization behavior were also evaluated, in addition to morphological observation of attached cells. Fibronectin could be immobilized by the tresyl chloride-activation method. A sandblasted surface resulted in significantly more DNA than a nanometer-smooth surface, but fibronectin immobilization did not result in a significant increase of DNA at 52 days of cell culture. The nanometer-smooth surface showed highest ALP activity and osteocalcin production. FN immobilization decreased ALP activity for the nanometer-smooth surface, but increased it for the sandblasted surface. The nanometer-smooth surface also showed the highest osteocalcin production. Scanning electron microscopy showed interesting phenomena of the attached cells. Attached cell area was more rapidly increased on the nanometer-smooth surface than on the sandblasted surface. It was suggested that cultured cells on the nanometer-smooth surface began to spread earlier and that the proportion of spreading cells among total attached cells increased sooner on the nanometer-smooth surface than on the sandblasted rough surface. It appeared that FN immobilization influenced the arrangement of attached cells. In conclusion, the nanometer-smooth surface employed in the present study was beneficial for the differentiation of MC3T3-E1 cells. FN immobilization influenced the morphologies of attached cells.

Solution composition impacts fibronectin immobilization on carboxymethyl-dextran surfaces and INS-1 insulin secretion

It is shown that solution composition during immobilization plays a critical role in the properties of fibronectin (FN) surfaces and their bioactivity towards insulinoma (INS-1) cell function. X-ray photoelectron spectroscopy revealed FN grafting onto low-fouling carboxymethyl-dextran (CMD) surfaces was successful with solutions composed of 10μM CaCl2, 10μM MgCl2, 10μM MnCl2, and 10μM and 1mM NaCl, but unsuccessful with those made of 150mM NaCl or 1× PBS. Circular dichroism and photon correlation spectroscopy revealed that regardless of solution composition, no measurable differences in free FN conformation prevail. AFM imaging of FN-CMD revealed, while there are no quantitative differences in surface roughness, there are some subtle qualitative differences in topography. FN surface immobilization scheme does not influence INS-1 cell growth after 3 and 7 days regardless of the underlying substrate or solution composition. INS-1 cell insulin secretion in response to glucose is affected by the substrate and solution composition during FN immobilization.