结合RGD肽的聚酯材料表面粘附内皮细胞的抗剪切力研究

精氨酸-甘氨酸-天门冬氨酸(RGD)是许多粘附蛋白的高度保守氨基酸序列.生物材料表面结合RGD肽有助于内皮细胞在材料上的粘附、迁移和增殖.本研究在体外流动条件下观察结合RGD肽或纤维粘连蛋白的聚酯材料表面粘附内皮细胞的抗剪切能力,并通过观察肌动蛋白和踝蛋白的表达初步探讨影响细胞粘附稳定性的机制.结果显示材料表面结合RGD或纤维粘连蛋白可以增加细胞的粘附强度,提高抗剪切能力;而RGD和纤维粘连蛋白导致的细胞抗剪切能力增加可能与细胞内肌动蛋白和踝蛋白的表达增加有关.     

人脐静脉内皮细胞在RGD肽聚酯材料表面的黏附稳定性研究

研究RGD肽对内皮细胞(Endothelialcell,EC)在生物材料表面黏附稳定性的影响。实验材料(聚酯)分为三组:RGD组(表面共价接枝人工合成的RGD三肽)、对照组(表面预衬纤维粘连蛋白)和空白组(表面未作任何处理),然后在三组材料表面种植体外培养的人脐静脉内皮细胞,并在定常流条件下观察比较RGD肽和纤维粘连蛋白对材料表面细胞黏附稳定性的影响。结果显示随着剪切力作用时间延长和剪切力加大,三组材料表面黏附的细胞脱落逐渐增多。空白组PET表面细胞脱落最为明显,8.19dyne/cm2作用4h后,材料表面细胞残留率仅为13.73%。PET表面结合RGD或纤维粘连蛋白后,细胞残留率明显增加,同样剪切力作用下细胞残留率分别为43.33%和40.75%,两组之间无显著性差异。由此得出结论,RGD可以提高EC在材料表面的黏附稳定性。本结果仅是一个体外实验的初步结果,需要进一步的体内实验加以证实。     

RGD肽与生物材料的内皮化

生物材料的表面内皮化可以明显改善材料的血液相容性,促进各种生物材料,尤其是心血管移植物的临床应用,种植内皮细胞在生物材料表面的贴附及生长状况是决定内皮化能否成功的关键因素,RGD是许多粘附蛋白所共有的高度保守氨基酸序列,这一序列在介导细胞粘附,迁移及生长方面起重要作用,我们综述了RGD与整合素受体的相互作用,RGD生物活性的影响因素以及它在生物材料内皮化方向的应用。     

表面修饰对羟基磷灰石修复骨缺损的影响

精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp,RGD）多肽是介导种子细胞与支架材料黏附的多肽链．RGD序列可被固有黏附蛋白受体特异性结合．在生物材料表面自发形成一分子层．为与受体介导的种子细胞提供特异性位点而促进细胞的黏附和分化。笔者旨在应用RGD多肽对羟基磷灰石（hydroxyapatite．HA）材料进行表面修饰处理．以促进骨髓基质干细胞（marrow stromal cells．MSC）在其表面的黏附和生长．为骨组织工程提供一种支架材料的表面修饰手段。     

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：     

骨组织工程材料的表面修饰和细胞粘附

由于人工合成高分子聚合物材料亲水性差，细胞吸附力差，在材料表面包被，铰链某种蛋白或短肽则有利于细胞在材料上的粘附，并影响细胞的增殖和分化。本重点介绍了与成骨细胞有关的蛋白或短肽，并描述了细胞在材料上的粘附过程。     

表面修饰对羟基磷灰石细胞相容性的影响

探讨精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)多肽表面修饰对羟基磷灰石(hydroxyapatite,HA)细胞相容性的影响。以骨髓基质干细胞(marrow stromal stem cells,MSCs)复合精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石或单纯材料培养制备组织工程骨,观察骨髓基质干细胞的粘附和生长情况,检测细胞活力和碱性磷酸酶(alkaline phosphatase,ALP)活性,流式细胞仪分析细胞周期。结果表明:骨髓基质干细胞在材料表面和孔隙内均可粘附和生长,粘附于RGD多肽修饰羟基磷灰石的细胞活力和碱性磷酸酶活性明显高于未经RGD多肽修饰组(P<0.01,P<0.05)。各组细胞周期未见明显变化,未见异倍体细胞。说明RGD多肽表面修饰对HA材料的细胞相容性有明显的优化作用。     

层粘连蛋白和纤维粘连蛋白在正常绒毛膜组织中的表达

层粘连蛋白和纤维粘连蛋白是细胞外基质的重要成分 ,在正常组织中有广泛分布。目前认为层粘连蛋白有 10条不同源的多肽链 ,构成 11种不同的蛋白分子。纤维粘连蛋白的结构具有多形性 ,主要由 ED-A、ED- B、 CS三个结构区不同的拼接方式所决定。层粘连蛋白和纤维粘连蛋白不仅在支持、连接和维持组织形态 ,调节细胞的粘附、生长、分化、增殖等方面起重要作用 ,而且与胚泡着床及胎盘的形成也有密切关系     

血浆蛋白对生物材料细菌粘附影响研究进展

防止生物材料细菌粘附是防治生物材料为中心感染(biomaterial centered infect，BCI)的重要环节。研究发现，人体血浆蛋白对生物材料细菌粘附有重要影响。因此，研究血浆蛋白与生物材料细菌粘附关系为防治：BCI有重要的意义。本综述了与生物材料细菌粘附相关的血浆蛋白、血浆蛋白对生物材料细菌粘附影响的有关机制及如何提高血浆蛋白抗细菌粘附作用的展望。     

RGD肽表面修饰聚苯乙烯及其细胞相容性研究

目的以聚苯乙烯二维平面为模板研究了蛋白表面修饰技术,构建具有生物活性的生物材料表面。方法采用物理包被法依靠疏水作用在PS表面架构明胶、胶原和RGD（精氨酸-甘氨酸-天冬氨酸）多肽的生物活性层。通过光电子能谱（XPS）分析修饰表面的元素含量变化,N元素含量显著提高,说明蛋白分子在表面存在。Bradford方法定量分析明胶、胶原和RGD多肽的表面吸附量。结果XPS证实了表面N原子的引入,存在酰胺键,确定蛋白分子存在于PS表面。结论动态接触角下降显著,证明修饰表面的亲水性得到提高。并在明胶、胶原和RGD多肽修饰表面接种人表皮细胞,对比考察其对细胞行为的影响,提高了细胞的黏附和增殖能力。     

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.     

Immobilization of RGD to < 1 1 1 > silicon surfaces for enhanced cell adhesion and proliferation

The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and microstructure. One of the greatest challenges with silicon-based biomedical microdevices such as those recently developed for neural stimulation, implantable encapsulation, biosensors, and drug delivery, is to improve biocompatibility and tissue integration. This may be achieved by modifying the exposed silicon surface with bioactive peptides. In this study, Arg-Gly-Asp (RGD) peptide conjugated surfaces were prepared and characterized. The effect of these surfaces on fibroblast adhesion and proliferation was examined over 4 days. Silicon surfaces coupled with a synthetic RGD peptide, as characterized with X-ray photoelectron spectroscopy and atomic force microscopy, display enhanced cell proliferation and bioactivity. Results demonstrate an almost three-fold greater cell attachment! proliferation on RGD immobilized surfaces compared to unmodified (control) silicon surfaces. Modulating the biological response of inorganic materials such as silicon will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.     

Human endothelial cell interaction with biomimetic surfactant polymers containing Peptide ligands from the heparin binding domain of fibronectin

Biomimetic materials that mimic the extracellular matrix (ECM) provide a means to control cellular functions such as adhesion and growth, which are vital to successful engineering of tissue-incorporated biomaterials. Novel "ECM-like" biomimetic surfactant polymers consisting of a poly(vinyl amine) backbone with pendant cell-adhesive peptides derived from one of the heparin-binding domains of fibronectin were developed to improve endothelial cell adhesion and growth on vascular biomaterials. Heparin-binding peptide (HBP) sequences, alone and in combination with RGD peptides, were examined for their ability to promote human pulmonary artery endothelial cell (HPAEC) adhesion and growth (HBP1, WQPPRARI; HBP2, SPPRRARVT; HBP1:RGD; and HBP2:RGD) and compared with cell adhesion and growth on fibronectin and on negative control polymer surfaces in which alanines were substituted for the positively charged arginine residues in the two peptides. The results showed that HPAECs adhered and spread equally well on all HBP-containing polymers and the positive fibronectin control, showing similar stress fiber and focal adhesion formation. However, the HBP alone was unable to support long-term HPAEC growth and survival, showing a loss of focal adhesions and cytoskeletal disorganization by 24 h after seeding. With the addition of RGD, the surfaces behaved similarly or better than fibronectin. The negative control polymers showed little to no initial cell attachment, and the addition of soluble heparin to the medium reduced initial cell adhesion on both the HBP2 and HBP2:RGD surfaces. These results indicate that the HBP surfaces promote initial HPAEC adhesion and spreading, but not long-term survival.     

Dynamic cell-adhesive microenvironments and their effect on myogenic differentiation

Integrin-mediated cell adhesion plays a central role in cell behavior on biomaterial surfaces and influences various cell functions. Photoactivatable RGD adhesive peptides were used to investigate the effect of the density and time point of bioadhesive ligand presentation on cell adhesion, proliferation and differentiation. PEGylated self-assembled monolayers were functionalized with RGD and caged RGD ligands and seeded with C2C12 myoblasts. The cultures were irradiated at various time points between 1 and 48 h after cell seeding in order to increase RGD surface concentration at defined time points. Attachment, spreading and myogenic differentiation of C2C12 myoblasts strongly varied with the density of RGD at the surface. Proliferation and myogenesis were further regulated by the time point at which RGD was presented to the cell, reaching highest levels when RGD exposure occurred ≤6 h after cell seeding. These results provide fundamental insights in cell–biomaterial interactions of C2C12 myoblasts in terms of temporal integrin-mediated cell responses.     

Cardiomyocytes in vitro adhesion is actively influenced by biomimetic synthetic peptides for cardiac tissue engineering

Scaffolds for tissue engineering must be designed to direct desired events such as cell attachment, growth, and differentiation. The incorporation of extracellular matrix-derived peptides into biomaterials has been proposed to mimic biochemical signals. In this study, three synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 were investigated for the first time as potential adhesive sequences for cardiomyocytes (CMs) compared to smooth muscle cells. CMs are responsive to all peptides to differing degrees, demonstrating the existence of diverse adhesion mechanisms. The pretreatment of nontissue culture well surfaces with the (Arginine-Glycine-Aspartic Acid) RGD sequence anticipated the appearance of CMs' contractility compared to the control (fibronectin-coated well) and doubled the length of cell viability. Future prospects are the inclusion of these sequences into biomaterial formulation with the improvement in cell adhesion that could play an important role in cell retention during dynamic cell seeding.     

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H(2)N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG(2)PLA(20)) and the thiol-reactive derivative (MP-NH-PEG(2)PLA(40)) were modified with cyclic alphavbeta3/alphavbeta5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell-polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD-integrin-interactions.     

Peptide modification of polyethersulfone surfaces to improve adipose-derived stem cell adhesion

Polyethersulfone (PES) is a nondegradable, biocompatible, synthetic polymer that is commonly utilized as a membrane material for applications such as hemodialysis, ultrafiltration and bioreactor technology. Various studies have shown surface modification to be a valuable tool in the development of nondegradable materials which promote cell adhesion. Cells of interest include adipose-derived stem cells (ASCs). ASCs are multipotent mesenchymal stem cells that are useful for various regenerative medicine applications. In this study, we hypothesized that PES surfaces modified with a peptide sequence based from fibronectin, such as Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser and Gly-Arg-Gly-Asp-Ser, would increase ASC adhesion compared to unmodified PES surfaces. The synthetic peptides were covalently bonded to amine-modified PES surfaces using 1-ethyl-3-(dimethylaminopropyl) carbodiimide. The surfaces were characterized using a ninhydrin assay and contact angle measurements. The ninhydrin assay confirmed the presence of amine groups on the surface of peptide-treated PES disks. Advancing water contact angles were analyzed to detect changes in the hydrophilicity of the polymer surfaces, and results indicated our PES membranes had excellent hydrophilicity. The attachment and proliferation of human ASCs was assessed and RGD-treated surfaces resulted in a higher number of attached ASCs after 6 and 48 h, as compared to unmodified PES surfaces. Additionally, varying concentrations of the RGD peptide sequence concentration were examined. These results indicate that PES membranes modified with the RGD peptide sequence can be utilized for enhanced ASC attachment in biomedical applications.     

Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34+ cells

The interaction between integrins and extracellular matrix proteins play an important role in the regulation of hematopoiesis. Human hematopoietic progenitor cells express very late antigen-4 (VLA-4) and VLA-5, which mediate their interaction with fibronectin by recognizing the connecting segment-1 (CS-1 and RGD motifs, respectively. In this study, we investigated the ex vivo expansion of human umbilical cord blood (UCB) CD34+ cells on synthetic substrates surface-immobilized with peptides containing the CS-1 binding motif (EILDVPST) and the RGD motif (GRGDSPC). These peptides were covalently conjugated to poly(ethylene terephthalate) (PET) film at a surface density of 2.0-2.3 nmol/cm2. UCB CD34+ cells were cultured for 10 days in serum-free medium supplemented with recombinant human thrombopoietin, stem cell factor, flt3-ligand and interleukin 3. The highest cell expansion fold was observed on the CS-1 peptide-modified surface, where total nucleated cells, total colony forming unit, and long-term culture initiating cells were expanded by 589.6+/-58.6 (mean+/-s.d.), 76.5+/-8.8, and 3.2+/-0.9-fold, respectively, compared to unexpanded cells. All substrates surface-immobilized with peptides, including the control peptides, were more efficient in supporting the expansion of CD34+, CFU-GEMM and LTC-ICs than tissue culture polystyrene surface. Nevertheless, after 10-days of ex vivo expansion from 600 CD34+ cells, only cells cultured on CS-1-immobilized surface yielded positive engraftment, even though the frequency was low. PET surface immobilized with RGD peptide was less efficient than that with CS-1 peptide. Our results suggest that covalently immobilized adhesion peptides can significantly influence the proliferation characteristics of cultured UCB CD34+ cells.