Effect of topological cues on material-driven fibronectin fibrillogenesis and cell differentiation

Fibronectin (FN) assembles into fibrillar networks by cells through an integrin-dependent mechanism. We have recently shown that simple FN adsorption onto poly(ethyl acrylate) surfaces (PEA), but not control polymer (poly(methyl acrylate), PMA), also triggered FN organization into a physiological fibrillar network. FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules. In the present study, we investigate the influence of topological cues on the material-driven FN assembly and the myogenic differentiation process. Aligned and random electrospun fibers were prepared. While FN fibrils assembled on the PEA fibers as they do on the smooth surface, the characteristic distribution of globular FN molecules observed on flat PMA transformed into non-connected FN fibrils on electrospun PMA, which significantly enhanced cell differentiation. The direct relationship between the fibrillar organization of FN at the material interface and the myogenic process was further assessed by preparing FN gradients on smooth PEA and PMA films. Isolated FN molecules observed at one edge of the substrate gradually interconnected with each other, eventually forming a fully developed network of FN fibrils on PEA. In contrast, FN adopted a globular-like conformation along the entire length of the PMA surface, and the FN gradient consisted only of increased density of adsorbed FN. Correspondingly, the percentage of differentiated cells increased monotonically along the FN gradient on PEA but not on PMA. This work demonstrates an interplay between material chemistry and topology in modulating material-driven FN fibrillogenesis and cell differentiation.     

Investigation of effects of Argenine–Glycine–Aspartate (RGD) and nano-scale titanium coatings on cell spreading and adhesion

This paper examines the effects of physical and chemical surface modifications on the biocompatibility of silicon surfaces that are relevant to implantable silicon Bio-micro-electro-mechanical systems (BioMEMS). Two types of surface modifications were explored. The first involved the deposition of nano-scale biocompatible layers of pure titanium on silicon, while the second explored the covalent attachment of the binding peptide Argenine–Glycine–Aspartic acid (RGD) for improved cell adhesion. Improvements in biocompatibility were assessed through examination of cell areas after culture, as well as the measurements of adhesion strengths, as determined by shear assay techniques. The titanium nanolayers and the RGD coating resulted in improvements in biocompatibility. Increased cell spreading areas and improved adhesion strength were obtained from short and long-term studies of Human Osteosarcoma (HOS) cells cultured on the coated surfaces. RGD functionalization resulted in the greatest improvement in cell spreading area and adhesion strength for short culture times. The effects of the titanium, while less than those of RGD for short culture times, appeared to be greater after 48 h of culture.     

The impact of the RGD peptide on osteoblast adhesion and spreading on zinc-substituted hydroxyapatite surface

The incorporation of zinc into the hydroxyapatite structure (ZnHA) has been proposed to stimulate osteoblast proliferation and differentiation. Another approach to improve cell adhesion and hydroxyapatite (HA) performance is coating HA with adhesive proteins or peptides such as RGD (arginine–glycine–aspartic acid). The present study investigated the adhesion of murine osteoblastic cells to non-sintered zinc-substituted HA disks before and after the adsorption of RGD. The incorporation of zinc into the HA structure simultaneously changed the topography of disk’s surface on the nanoscale and the disk’s surface chemistry. Fluorescence microscopy analyses using RGD conjugated to a fluorescein derivative demonstrated that ZnHA adsorbed higher amounts of RGD than non-substituted HA. Zinc incorporation into HA promoted cell adhesion and spreading, but no differences in the cell density, adhesion and spreading were detected when RGD was adsorbed onto ZnHA. The pre-treatment of disks with fetal bovine serum (FBS) greatly increased the cell density and cell surface area for all RGD-free groups, overcoming the positive contribution of zinc to cell adhesion. The presence of RGD on the ZnHA surface impaired the effects of FBS pre-treatment possibly due to competition between FBS proteins and RGD for surface binding sites.     

Rationally Designed Peptide Interface for Potential Modulated Cell Adhesion and Migration

Conformational changes of peptides are critically important in the control of their biological activities. Here, a quaternary ammonium group‐terminated RGD‐containing peptide (RGD‐NMe₃) is designed, which may undergo reversible conformational switch upon different electrochemical potentials. Potential responsive peptide interfaces are constructed on gold substrates with RGD‐NMe₃ in a tetra (ethylene glycol) background. It is demonstrated that by applying positive and negative potentials, the RGD peptide can be reversibly switched between linear and cyclic conformation, which can be used in reversible controlling of cell adhesion/migration on the interface. Furthermore, by combining microfluidics, adhesion of the cells in specific areas on the surface and subsequent directional migration of the cells can be controlled. It is believed that this straightforward potential modulation mechanism for peptide conformation control may find a wide use in design responsive peptide interfaces.     

Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment

Arginine-glycine-aspartic acid (RGD) dendrimer-based nanopatterns on poly(L-lactic acid) were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells.During chondrogenic commitment,active extracellular matrix (ECM) remodeling takes place,playing an instructive role in the differentiation process.Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation,these cultures are rather limited with respect to their ability to interrogate cells in cell-ECM interactions.In the present study,the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration.The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance,constructed from the corresponding atomic force microscopy images,and correlated with the cell adhesion and differentiation response.The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment,and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation.     

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.     

磷灰石-硅灰石生物活性玻璃陶瓷表面接枝多肽改性研究

为在磷灰石-硅灰石生物活性玻璃陶瓷(Apatite-Wollastonite Bioactive Glass-Ceramic, AW)表面引入能够促进细胞粘附的RGD(精氨酸-甘氨酸-天冬氨酸)多肽以提高其生物活性, 采用低温等离子法在材料的表面引入活性氨基基团, 并通过浸渍法使氨基基团与多肽发生反应。采用XRD、XPS、ATR-FTIR对AW的相组成及表面改性特性进行表征, 确认通过低温等离子法在AW表面接上氨基, RGD多肽分子与氨基反应以化学键合的形式接枝到材料表面(RGD-AW), 实现了在AW表面接枝生物大分子的改性。将改性前后的材料分别与类成骨细胞(MG63细胞)混合培养并使用荧光显微镜、SEM及MTT等测试方法对材料的细胞生物学性能进行了表征。细胞实验结果表明: 接枝RGD多肽分子的材料在细胞培养的早期阶段比AW更有利于细胞的粘附及铺展。     

TiO<Subscript>2</Subscript> type influences fibronectin adsorption

Human fibronectin (FN) plays a key role in the biointegration of implants as the success depends on adsorption of proteins like FN [1]. Indeed FN can be an intermediary between the biomaterial surface and cells. The adsorption of human fibronectin (FN) on commercially pure titanium with a titanium oxide layer formed in a H₂O₂ solution (TiO₂ cp) and TiO₂ sputtered on Si (TiO₂ sp) was studied. Adsorption isotherms and the work of adhesion were assessed by wettability studies, X-ray photoelectron spectroscopy (XPS), and by radiolabelling of FN with ¹²⁵I, ¹²⁵I-FN. Exchangeability of bound FN by free FN, was also evaluated by the radiolabelling technique. Contact angle determinations have shown that FN displays higher affinity for the TiO₂ cp surface than for the TiO₂ sp. As expected from the surface free energy values, the work of adhesion of FN is higher for the TiO₂ cp substrate, the more hydrophilic one, and lower for the TiO₂ sp substrate, the more hydrophobic one. The adsorption isotherms were evaluated by two different techniques: radiolabelling of FN (¹²⁵I-FN) and XPS. TiO₂ cp adsorbs more FN than the TiO₂ sp surfaces as shown by the radiolabelling data. FN molecules are also more strongly attached to the former surface as indicated by the work of adhesion and by the exchangeability studies. Results using ¹²⁵I-FN also suggests that FN adsorbs as a multilayer for FN concentrations in solution higher than 100 μg/mL.     

Relationship between the fibroblastic behaviour and surface properties of RGD-immobilized PCL membranes

In this study, poly-ε-caprolactone (PCL) membranes were modified with the cell adhesive peptide RGD by chemical immobilization technique. The roughness and hydrophilicity were increased after RGD immobilization and an improved cell attachment was observed.     

Three-dimensional mesoporous gold film to enhance the sensitivity of electrochemical detection

Cell-cell and cell-extracellular matrix (ECM) adhesion are fundamental and important in the development of a cell-based chip. In this study, a novel, simple, rapid, and one-step technique was developed for the fabrication of a uniform three-dimensional mesoporous gold thin film (MPGF) onto a gold (Au) coated glass plate based on an electrochemical deposition method. Scanning electron microscopy images demonstrated that the resulting MPGF electrode had uniformly distributed pores with diameters of about 20 nm. The cyclic voltammetric behavior of [Fe(CN)(6)](4-/3-) coupled onto MPGF and Au electrodes demonstrated that the MPGF electrode had a higher electrocatalytic sensitivity and reversibility than the bare Au electrode. The Arg-Gly-Asp (RGD) sequence containing the peptide was immobilized on the MPGF and bare Au substrates. HeLa cancer cells were then cultured on the RGD peptide layer. The successful immobilization of the peptide and cells was confirmed by atomic force microscopy. The cell proliferation and viability were evaluated by cyclic voltammetry and Trypan blue dyeing assay. These results indicated that the RGD/MPGF modified electrodes showed an electrochemical sensitivity in the detection of cancer cells which is approximately three times higher, especially at low cell density, than RGD/Au electrodes. This much improved sensitivity of the MPGF modified electrode demonstrates the potential for the fabrication of a highly sensitive and low-cost cell-based chip for rapid cancer detection.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

生物材料细胞粘附肽仿生修饰的研究进展

RGD(Arg-G1y-Asp)短肽序列是一种细胞粘附肽,能被细胞膜上的整合素识别,参与细胞与基质间的粘附.为改善合成生物材料缺乏细胞识别信号的缺点,可将含RGD序列肽经本体或表面修饰引入材料,使材料具有良好的细胞亲和性.综述了采用含RGD肽对各种合成生物材料进行仿生修饰的研究进展.     

The covalent grafting of fibronectin at the surface of poly(ethylene terephthalate) track-etched membranes improves adhesion of rat hepatocytes

[³H]methylated fibronectin (FN) has been immobilized on the surface of poly(ethylene terephthalate) track-etched membranes (PET), carboxylated and then activated or not with water-soluble carbodiimide (WSC). Upon washing in 10% SDS or in the nutritive medium used for hepatocytes cultivation (supplemented with 15% newborn calf serum), respectively, 76% and 43% of [³H]FN are released from the unactivated PET membranes and those WSC activated. This difference is almost totally abolished when the-NH₂ functions of FN have been fully acetylated, to impair their reaction with activated-COOH groups. These results strongly suggest that part of FN is covalently grafted to the activated-COOH of PET but that, in addition, FN is also adsorbed on this surface. Rat hepatocytes were inoculated on PET membranes on which FN had been adsorbed and/or grafted. Image analysis clearly indicates that, during the first hours of culture, the FN immobilization on WSC activated, carboxylated PET membranes, significantly favours the adhesion of hepatocytes. After 24 h, the difference between these substrates decreases, probably due to the reconditioning of the PET surface by extracellular matrix constituents secreted by the hepatocytes. Our results confirm that the nature of the protein-polymer interaction strongly affects cell behaviour.This paper was accepted for publication after the 1995 conference of the European Society of Biomaterials, Oporto, Portugal, 10–13 September.     

SPA用于丝素膜的生物改性研究

用生长因子RGD半抗原（GLY-ARG-GLY-ASP-SER-PRO-LYS)连接到卵清蛋白Ovalbumin(OVA)载体上诱发出了抗RGD抗体IgG，并用丝素溶液包埋SPA(Staphylo-coccal protein A，简称A蛋白或SPA)制成不溶性SPA丝素膜为材料，然后用诱发出的RGD抗体IgG结合到不溶性SPA丝素膜的表面，制成IgG-SPA丝素膜，再在其上结合粘附生长因子RGD，制成RGD-IgG-SPA丝素膜。利用这一丝素膜培养血管内皮细胞(Vas-cular Endothelial Cell，简称EC细胞)，用四甲基偶氮些盐比色方法(MTT法)检测细胞的生长增殖情况。结果表明，RGD-IgG-SPA丝素膜能有效促进EC细胞的生长。对不溶性SPA丝素膜和IgG以及IgG和RGD之间的生物结合力测定，表明其结合力远大于离解力。同时在细胞培养液中没有检测到丝素膜中SPA的渗漏。RGD-IgG-SPA丝素膜为其作具有的这些优良性质为血管支架打下了基础。     

Remodeling of fibrinogen by endothelial cells in dependence on fibronectin matrix assembly. Effect of substratum wettability

The endothelization of cardiovascular implants is desirable to improve their blood compatibility. The capacity of the endothelial cells to attach, migrate, proliferate and function on the implant surface depends on the presence of matrix proteins such as fibronectin (FN) and fibrinogen (FNG). In this study, we show that the deposition of fibrinogen into extracellular matrix-like structures by human umbilical vein endothelial cells (HUVEC) is dependent on FN matrix formation. We found further that the process of organization of both adsorbed and soluble FN and FNG is dependent on the wettability of materials since it was observed only on a hydrophilic and not on a hydrophobic model surface. ₃ integrin was involved in the process of cell attachment to adsorbed FNG, while the mechanism of FNG fibrillogenesis required the activity of the ₁ integrin. Studies of EC morphology showed the predominant peripheral organization of actin filaments and the formation of distinct leading and trailing cell edges suggesting a motile phenotype of cells when they are seeded on FNG. In summary, we concluded that adsorbed fibrinogen may enhance the motility of HUVEC and that soluble FNG requires FN matrix assembly to be organized in fibrilar structures.     

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant’s biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO₃ or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO₃-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.     

Evaluation of cell proliferation and differentiation on a poly(propylene fumarate) 3D scaffold treated with functional peptides

Synthetic polymers were used to fabricate a three-dimensional (3D) porous scaffold of poly(propylene fumarate)/diethyl fumarate (PPF/DEF). PPF-based materials are good candidates for bone regeneration, because of their non-toxic, biodegradable byproducts, and excellent mechanical properties. However, they exhibit hydrophobic surface properties that have negative effects on cell adhesion. To change the surface properties of a PPF/DEF scaffold, the authors used three peptide modifications (RGD, cyclo RGD, and RGD-KRSR mixture) to the scaffold and tested the effects on MC3T3-E1 pre-osteoblast adhesion, proliferation, and differentiation. The results indicated that peptide modification (particularly the RGD-KRDR mixture) altered the hydrophobic surface properties of the PPF/DEF scaffold, and promoted cell adhesion. Thus, it was suggest that peptide modification is a useful method for changing the properties of the PPF/DEF scaffold surface and may be applicable in bone tissue engineering.     

A novel strategy to graft RGD peptide on biomaterials surfaces for endothelization of small-diamater vascular grafts and tissue engineering blood vessel

To improve the performance of small-diamater vascular grafts, endothelization of biomaterials surfaces and tissue engineering are more promising strategies to fabricate small-diamater vascular grafts. In this study, a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide was grafted on the surfaces of poly(carbonate urethane)s (PCUs), with photoactive 4-benzoylbenzoic acid (BBA) by UV irradiation. The photoactive peptides (BBM-GRGDSP) were synthesized with classical active ester of peptide synthesis. The modified surfaces of PCU with the photoactive RGD peptides were characterized by water contact angle measurement and X-ray Photoelectron Spectroscopy (XPS), which results suggested that the peptides were successfully grafted on the PCU surfaces. The effect of these modified surfaces on endothelial cells (ECs) adhesion and proliferation was examined over 72 h. PCU surfaces coupled with the synthetic photoactive RGD peptides, as characterized with phase contrast microscope and the metabolic activity (MTT) assay enhanced ECs proliferation and spreading with increasing concentration of RGD peptides grafted on their surfaces. Increased retention of ECs was also observed on the polymers surfaces under flow shear stress conditions. The results demonstrated that GRGDSP peptides grafted on the surfaces of polymers with photoactive 4-benzoylbenzoic acids could be an efficient method of fabrication for artificial small-diamater blood vessels. The modified polymer is expected to be used for small-diamater vascular grafts and functional tissue engineered blood vessels to improve ECs adhesion and retention on the polymer surfaces under flow shear stress conditions.     

Arg-Gly-Asp (RGD) Modified Biomimetic Polymeric Materials

The new generation of biomaterials focuses on the design of biomimetic polymeric materials that are capable of eliciting specific cellular responses and directing new tissue formation. Since Arg-Gly-Asp (RGD) sequences have been found to promote cell adhesion in 1984, numerous polymers have been functionalized with RGD peptides for tissue engineering applications. This review gave the advance in RGD modified biomimetic polymeric materials, focusing on the mechanism of RGD, the surface and bulk modification of polymer with RGD peptides and the evaluation in vitro and in vivo of the modified biomimetic materials.     

MicroRNA conjugated gold nanoparticles and cell transfection

While the importance of microRNAs (miRNAs) in cancer treatment or manipulation of genetic expression has been increasingly recognized for developing miRNA-based therapies, the controlled delivery of miRNAs into specific cells constitutes a challenging task. This report describes preliminary findings from an investigation of the conjugation of gold nanoparticles with miRNAs (miRNA-AuNPs) and their cell transfection. The immobilization of miRNAs on the AuNPs was detected, and the surface stability was substantiated by gel electrophoresis assessment of the highly charged characteristics of miRNA-AuNPs and their surface-exchange inactivity with a highly charged surfactant. The miRNA-AuNPs were tested in cell transfection using multiple myeloma cells, demonstrating efficient knockdown in the functional luciferase assay. The findings have important implications for understanding the mechanistic details of cell transfection involving miRNA-conjugated nanoparticles as biosensing or targeting probes.