Acrylic Acid-RGD as a Biomimetic Surface Modifier for Poly(ethylene terephthalate)

INTRODUCTION　Biomaterials play an importantrole in human disease- treatmentand healing〔1,2〕.Due to the good mechanical property,PET is used to the coating of artificial heartvalve,the film of mending hearts and artificial vessel etc〔3〕.But the imperfection isthe low capability of surface hydrophile leading to the high static and low water ad-sorption〔4〕.In the application,traditional artificial cardiovascular materials( e.g.PET) have blood coagulation,alexin- activation and other…     

The effect of RGD density on osteoblast and endothelial cell behavior on RGD-grafted polyethylene terephthalate surfaces

Hybrid materials combining polyethylene terephthalate and different types of cells (endothelial and osteoblastic cells) have been developed thanks to the covalent grafting of different densities of RGD containing peptides onto the polymer surface. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions, coupling agent grafting and the immobilization of the RGDC peptides. High resolution μ-imager was used to evaluate RGD densities (varying between 0.6 and 2.4 pmol/mm²) and has exhibited the stability of the surface grafted peptides when treated in harsh conditions. The efficiency of this route for biomimetic modification of a PET surface was demonstrated by measuring the adhesion of MC3T3 and HSVEC cells and by focal adhesion observation. Results obtained prove that a minimal RGDC density of 1 pmol/mm² is required to improve MC3T3 and HSVEC cells responses. Indeed, cells seeded onto a RGDC-modified PET with a density higher than 1 pmol/mm² were able to establish focal adhesion as visualized by fluorescence microscope compared to cells immobilized onto unmodified PET and RGDC-modified PET with densities lower than 1 pmol/mm². Moreover, the number of focal contacts was enhanced by the increase of RGDC peptide densities grafted onto the material surface. With this study we proved that the density of peptides immobilized on the surface is a very important parameter influencing osteoblast or endothelial cell adhesion and focal contact formation.     

生物活性短肽RGD在PET表面接枝方法的研究

在高分子材料表面共价引入人工合成的精氨酰-甘氨酰-天冬氨酸三肽(Arg-Gly-Asp peptides，RGD)，以达到让内皮细胞与之特异结合并更加牢固的目的。实验用紫外辐照法将活性基团羧基(-COOH)接枝到聚对苯二甲酸乙二醇酯(Poly(ethylene terephtalate)，PET)膜的表面，将液相合成的RGD三肽耦合接枝到处理过的材料表面，光电子能谱对以羧基为活性基团的接肽反应结果进行分析，光学、电子显微镜观察内皮细胞生长情况以检测接枝短肽的生物活性。内皮细胞生长实验结果表明，成功接枝的RGD序列对材料内皮细胞种植起到了促进作用。本实验成功运用紫外接枝与化学耦合，将生物活性短肽RGD接枝到膜表面，探索了一种新的接枝生物活性短肽的方法。     

The biological response of poly(L-lactide) films modified by different biomolecules: role of the coating strategy

The interactions between the surface of synthetic scaffolds and cells play an important role in tissue engineering applications. To improve these interactions, two strategies are generally followed: surface coating with large proteins and surface grafting with small peptides. The proteins and peptides more often used and derived from the extracellular matrix, are fibronectin, laminin, and their active peptides, RGD and SIKVAV, respectively. The aim of this work was to compare the effects of coating and grafting of poly(L-lactide) (PLLA) films on MRC5 fibroblast cells. Grafting reactions were verified by X-ray photoelectron spectroscopy. Cell adhesion and proliferation on coated and grafted PLLA surfaces were measured by cell counting. Vinculin localization and distribution were performed on cell cultured on PLLA samples using a fluorescence microscopy technique. Finally, western blot was performed to compare signals of cell adhesion proteins, such as vinculin, Rac1, and RhoA, as well as cell proliferation, such as PCNA. These tests showed similar results for fibronectin and laminin coated PLLA, while RGD grafting is more effective compared with SIKVAV grafting. Considering the overall view of these results, although coating and grafting can both be regarded as effective methods for surface modification to enhance cell adhesion and proliferation on a biomaterial, RGD grafted PLLA show better cell adhesion and proliferation than coated PLLA, while SIKVAV grafted PLLA show similar adhesion but worse proliferation. These data verified different biological effects depending on the surface modification method used.     

Influence de la densité de peptides RGD greffés en surface de polyéthylène téréphtalate sur l’attachement des MC3T3

The aim of this study was to evaluate the impact of different densities on MC3T3 cells attachment onto polyethylene terephthalate (PET) film surfaces. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions onto PET surface, coupling agent grafting and finally immobilization of peptides. The originality of this work consist, in one hand on quantifying RGD peptides densities grafted onto PET, and on the other hand on studying MC3T3 cells responses after seeding on such biomimetic surfaces. After each functionnalization step, modifications were validated by several physicochemical techniques: X-Ray Photoelectron Spectroscopy permitted to prove the grafting and high-resolution β-imager coupled with use of radiolabelled amino acids served in evaluation of peptides densities. Moreover, this last technique permit us to ensure stability of binding between peptides and polymer. The efficiency of this new route for biomimetic modification of PET surface was demonstrated by measuring the adhesion at 15 hours of osteoblast like cells. Study of cellular comportment was realized by means of focal contact proteins (vinculin, actin) immunostaining.     

Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine–glycine–aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell–biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.     

Additive effect of RGD coating to functionalized titanium surfaces on human osteoprogenitor cell adhesion and spreading

Titanium-based biomaterials for endosseous implants have found widespread applications in the orthopedic, maxillofacial, and dental domains. Indeed, the surface characteristics such as their chemical modification control considerably the cellular response and, subsequently, the quality and the quantity of new-formed bone around the implant. In this study, human osteoprogenitor (HOP) cell adhesion on different titanium surfaces functionalized with hydroxyapatite (HA), type I collagen, or Arg-Gly-Asp (RGD)-containing peptides is investigated by the quartz crystal resonators and by confocal laser scanning microscopy (CLSM) for the imaging of focal contact formation. Data obtained by quartz crystal resonator technique revealed that RGD-containing peptides alone increase HOP cell adhesion in early time period of culture. Moreover, association of RGD-containing peptides with either type I collagen or with HA layers induces an additive effect on HOP cell adhesion compared to Ti-Coll or Ti-HA. CLSM shows both the area of focal contact by cell unit and the cytoskeleton network organization to differ according to the surfaces. Interestingly, association of RGD-containing peptides with HA layers induces an additive effect on focal contact formation on HOP cells compared to Ti-HA alone. These data confirm that an RGD peptide effect occurs in the early time of culture, which is beneficial for osteoblast to spreading, differentiation, and survival.     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

Synthesis, analytical characterization, and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin (RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.     

Synthesis,analytical characterization,and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin(RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.     

Biomimicking extracellular matrix: cell adhesive RGD peptide modified electrospun poly(D,L-lactic-co-glycolic acid) nanofiber mesh

A cell adhesive peptide, Arg-Gly-Asp (RGD), was immobilized onto the surface of electrospun poly(D,L-lactic-co-glycolic acid) PLGA nanofiber mesh in an attempt to mimic an extracellular matrix structure. A blend mixture of PLGA and PLGA-b-PEG-NH(2) di-block copolymer dissolved in a 1:1 volume mixture of dimethylformamide and tetrahydrofuran was electrospun to produce a nanofiber mesh with functional primary amino groups on the surface. Various electrospinning parameters, such as polymer concentration and the blend ratio, were optimized to produce a nanofiber mesh with desirable morphology, surface characteristics, and fiber diameter. A cell adhesive peptide, GRGDY, was covalently grafted onto the aminated surface of the electrospun mesh under a hydrating condition. The amounts of surface primary amino groups and grafted RGD peptides were quantitatively determined. Cell attachment, spreading, and proliferation were greatly enhanced in the RGD modified electrospun PLGA nanofiber mesh compared with that of the unmodified one.     

Development of RGD peptides grafted onto silica surfaces: XPS characterization and human endothelial cell interactions.

The attachment of human umbilical vein endothelial cells (HUVECs) on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) was investigated. This approach was used to provide substrates that are adhesive to cells even in the absence of serum proteins and to cells that have had no prior treatment of the surface with proteins that promote cell adhesion. We wanted to improve control of cellular interactions with cell-adhesive materials by providing fixedly bound adhesion ligands. Silica was examined as a model surface. The peptides were grafted using three different steps: grafting of aminosilane molecules; reaction with a maleimide molecule; and immobilization of cell-binding peptides containing the RGD sequence. The RGD-grafted surface was characterized by X-ray photoelectron spectroscopy (XPS) and contact-angle measurements.     

不同多肽修饰方法对纯钛微弧氧化膜层性能的影响

背景：理论上推测钛基-微弧氧化陶瓷膜-精氨酸-甘氨酸-天冬氨酸(RGD)序列多肽的结合模式应具较好的力学和生物学性能。 目的：观察不同修饰方法固定RGD多肽后,钛基体微弧氧化膜层表面的微观结构和细胞增殖。 方法：取纯钛与微弧氧化纯钛试件共90枚,分3种方法固定RGD多肽,分别为RGD多肽物理吸附修饰纯钛组、RGD多肽物理吸附修饰微弧氧化组与RGD多肽化学偶联修饰微弧氧化组,每组30枚。应用荧光显微镜观察3组试件表面接枝效果,采用X射线光电子能谱扫描检测试样表面的RGD多肽含量。将3组试件分别与小鼠骨髓基质细胞培养,光镜观察各时间点的细胞黏附及增殖情况。 结果与结论：3组试样表面有大小不一、数量不等的绿色荧光亮点,在单位视野中,RGD多肽化学偶联修饰微弧氧化组荧光最强,提示此组试件接枝了更多的多肽。RGD多肽物理吸附修饰纯钛组试样表面仅含少量或微量多肽,RGD多肽物理吸附修饰微弧氧化组含多肽量居中,RGD多肽化学偶联修饰微弧氧化组含肽量最高。3组试件均无明显的细胞毒性,但RGD多肽化学偶联修饰微弧氧化组细胞生长情况最好。表明化学偶联法可以较好地将RGD多肽固定在含微弧氧化膜层的纯钛试样表面,无明显细胞毒性,有利于细胞的生长增殖。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns

With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast, one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDS-OH peptide. We successfully applied our coupling method to functionalize surface patterns created by microcontact printing (μCP) and chemical etching. Cells recognize areas selectively coated with RGD-containing peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide – even for the preparation of peptide-functionalized small surface areas.     

Covalent immobilization of biomolecules onto polystyrene MicroWells for use in biospecific assays

Modification of polystyrene for higher binding capacity and/or for specific covalent immobilization of biomolecules is discussed. The benefit of covalent coupling of biomolecules onto a new commercially available surface type for covalent immobilization, CovaLink NH, is illustrated. The CovaLink NH solid phase has spacer arms covalently grafted onto the polystyrene solid phase, approximately 10(14) groups/cm2. Coupling procedures for covalent immobilization of biotin and peptides are demonstrated, and the advantage of using carbodiimide for coupling of carboxylic acid containing compounds is shown.     

精氨酸甘氨酸天冬氨酸多肽表面修饰促进生物支架材料对骨髓来源 种子细胞的黏附

背景：精氨酸甘氨酸天冬氨酸多肽具有较强的黏附性和生物支架材料可接枝结合,且不会改变材料的表面理化性质。 目的：观察应用精氨酸甘氨酸天冬氨酸多肽表面修饰猪主动脉瓣去细胞支架材料对骨髓干细胞黏附性的影响。 方法：采用胰蛋白酶+TritonX-100法制备猪主动脉瓣去细胞支架材料,用YGRGDSP多肽(酪氨酸-甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸)进行处理,按照精氨酸甘氨酸天冬氨酸多肽的质量浓度(0.5,1.0,1.5,2.0 g/L)、反应时间(4,8,12,24 h)、反应pH值(7.0,7.4,8.0)分为不同实验组。 结果与结论：茚三酮显示精氨酸甘氨酸天冬氨酸多肽可很好的交联到猪主动脉瓣去细胞支架材料,最佳反应条件为：室温、1.5 g/L精氨酸甘氨酸天冬氨酸、pH 7.4、持续振荡12 h。提示利用YGRGDSP多肽对猪主动脉瓣去细胞支架材料进行表面修饰可显著改善骨髓来源种子细胞的黏附性。     

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.     

Polyelectrolyte multilayer films functionalized with peptides for promoting osteoblast functions

Layer-by-layer deposition of polyelectrolyte multilayer (PEM) thin films has recently been applied to biomaterial applications. This simple and versatile technique provides a wide variety of potential utilization by insertion of biomolecules such as cell adhesion peptides. In this work dual peptides containing RGD (a cell-binding domain) and LHRRVKI (a heparin-binding domain) were immobilized onto polystyrene by the PEM technique and the effects on osteoblast cell culture were investigated. These peptides were conjugated to the amino groups of poly(allylamine hydrochloride) and then adsorbed onto the top of a 10 layer poly(allylamine hydrochloride)/poly(acrylic acid) film assembled at either pH 2.0 or pH 6.5. Osteoblasts, isolated from neonatal rat calvariae, were then seeded and cultured on the peptide-conjugated surfaces. We found that the cells adhered and grew better on the RGD-conjugated PEM films. The osteoblasts exhibited a better differentiated phenotype on the pH 2.0 films than the pH 6.5 films with respect to calcium deposition. The incorporation of LHRRVKI did not support cell adhesion, growth and matrix mineral deposition. Our results showed that the efficacy of RGD conjugation on osteoblast behavior was affected by the base PEM film.     

Generation of cell adhesive substrates using peptide fluoralkyl surface modifiers

Previous studies reported on the delivery of vitamin E to the surface of a polycarbonate polyurethane (PCNU) to produce antioxidant surfaces, using a bioactive fluorinated surface modifer (BFSM). In the current report, a cell adhesive peptide sequence was coupled to the BFSM, and when blended into PCNU, generated a cell adhesive substrate. An NH2-GK*GRGD-CONH2 peptide sequence (referred to as RGD) with a dansyl label (*) on the lysine residue was coupled via the N-terminal to a BFSM precursor molecule. The resulting RGD BFSM was purified and the pmol peptide/mg BFSM value was assayed by amino acid quantification. The migration of the RGD BFSM in a PCNU blend was confirmed by X-ray photoelectron spectroscopy analysis. U937 macrophage-like cells and human monocytes were seeded onto the PCNU and blends of PCNU with non-bioactive fluorinated surface modifier or the RGD BFSM, in order to study the cell response. Both U937 cells and human monocytes adhered in greater numbers to the RGD BFSM substrate when compared to unmodified PCNU or the blend of PCNU with the non-bioactive fluorinated surface modifying macromolecule substrate. The study demonstrated a novel approach for the introduction of peptides onto the surface of polymers by modifying the surface from within the polymer as opposed to the use of cumbersome post-surface modification techniques. The generation of a peptide substrate points to the possibility of producing complex bioactive surfaces using various peptide BFSMs or pharmaceuticals simultaneously to manipulate cell functions.     

Surface modification of biomedical grade polyurethane to enable the ordered co-immobilization of two proteins

Surface modifications of polyurethane (PU)-based implantable materials have the potential to enhance or improve hemo- or cellular-biocompatibility. In general, surface modification methods of PU have included surface treatments, physio-adsorption of desired biomolecules, and the covalent immobilization of reactive or therapeutic biomolecules. When multi-protein immobilizations are desired to mimic the enzymatic reactions found on cells and tissues, it is often necessary to design and develop surface modification strategies that will allow the co-immobilization of proteins. In this study, a surface modification strategy is presented that enables the sequential additional of proteins to a bi-dentate moiety grafted onto the PU surface. The modifications were confirmed via IR and XPS signatures. While the strategy presented is applicable to a wide variety of biomolecules, bovine serum albumin (BSA) and human immunoglobulin (hIgG) were selected as model proteins. A total immobilized protein density of 0.298 ± 0.037 μg/cm2 was obtained, with nearly equal amounts of protein on each arm of the bi-dentate moiety. Proteins immobilizations were also visualized with immunofluorescent staining. Finally, the method proposed in this study was used to demonstrate a significant increase (P < 0.05) in the catalytic conversion of protein C (PC) to activated PC (APC) using sequentially immobilized thrombomodulin (TM) and endothelial PC receptor (EPCR) as compared to the two proteins immobilized onto a surface in random order.