RGD修饰钛表面对人牙龈成纤维细胞初期黏附和铺展的影响

用羰基二咪唑(1,1'-carbonyldiimidazole,CDI)将含RGD的短肽共价连接到纯钛表面,研究接枝后的钛表面对原代培养的人牙龈成纤维细胞(human gingival fibroblasts,HGF)初期黏附和铺展的影响.结果表明,RGD修饰的纯钛表面粘附的细胞数比未修饰钛表面多,细胞铺展面积比钛表面的大,应力纤维的形成比钛表面早.RGD接枝钛表面更有利于人牙龈成纤维细胞的粘附,改善了纯钛的生物相容性.     

RGD修饰纯钛表面对人牙龈成纤维细胞生物学行为的影响

用羰基二咪唑(1,1′-carbonyldiimidazole,CDI),将含RGD的短肽共价连接到纯钛的表面,将HGFs分别接种到涂层和未涂层材料表面并培养一定时间后,定量对比接枝和未接枝材料表面的细胞纤粘连蛋白(fibronectin,FN)和粘着斑的形成情况,研究接枝后的纯钛表面对原代培养的人牙龈成纤维细胞(Human Gingival Fibroblasts,HGF)生物学行为的影响.结果表明,RGD修饰的纯钛表面粘着斑的形成比钛表面早.RGD接枝钛表面有利于人牙龈成纤维细胞的粘附,改善了纯钛的生物相容性.     

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。     

聚乳酸材料的仿生修饰研究进展

聚乳酸是生物可降解、生物相容性材料,但由于存在亲水性差、缺乏细胞识别位点等缺陷,限制了其在生物医学工程中的应用.模拟细胞与基质相互作用的特点以及细胞外基质的特性,通过表面修饰、本体改性和复合加工的方法在聚乳酸中引入蛋白胶原、活性肽、多糖以及羟基磷灰石等生物活性分子,实现聚乳酸的仿生修饰,能够有效提高聚乳酸材料的生物学功能.综述了利用这些生物活性分子对聚乳酸进行仿生修饰的研究进展.     

海藻酸钠水凝胶中粘附位点及空间构建促进MG-63细胞铺展及成骨分化

细胞粘附与铺展是三维水凝胶基质中贴壁依赖型细胞存活所必须的两个条件,将细胞粘附位点的引入和凝胶中细胞铺展空间的构建相结合,提出了同时含有RGD多肽和明胶微球的粘附型大孔水凝胶模型,以促进细胞在其中的铺展与分化。该模型采用光交联海藻酸钠水凝胶为基础,同时引入RGD多肽和明胶微球,通过RGD多肽的共价接枝为细胞粘附提供前提,利用明胶微球在37℃下的快速降解性,为细胞的进一步增殖和铺展以及分化提供所需空间。结果显示,明胶微球的加入提高了凝胶的力学性能,同时降低了凝胶的溶胀率。RGD和明胶微球的引入能够很好地支持MG-63细胞在其中的增殖、粘附与铺展,并显著提高其碱性磷酸酶活性,上调成骨相关基因(BMP-2,COL-I和OCN)的表达。而在不含微球的RGD-ALG和ALG凝胶中,细胞铺展及成骨分化均受到很大抑制。     

图案化与生物功能化聚3,4-乙烯二氧噻吩生物界面

本工作制备了具有可调控蛋白/细胞作用且低阻抗的功能化聚3,4-乙烯二氧噻吩(PEDOT)的图案化生物界面,并在空间上引导细胞的粘附行为.功能化PEDOT共聚物由具有抗非特异性粘附的磷酸胆碱功能化的EDOT(EDOT-PC)和可进行生物耦合反应的羧基功能化的EDOT(EDOT-COOH)两种单体共聚而成.本工作研究了不同组分共聚物的电化学阻抗性能及其对蛋白、细胞的抗粘附性能,同时通过精氨酸-甘氨酸-天冬氨酸(RGD)多肽的引入实现了在抗非特异性粘附背景下对细胞的特异性粘附.在此基础上,通过光刻及电化学沉积技术制备了由细胞特异性粘附区与抗细胞粘附区组成的图案化PEDOT生物界面,可有效地在空间上控制细胞粘附行为.该工作为研究细胞在材料表面的其他行为提供了可能性,在组织修复、再生工程中有着潜在的应用价值.     

胶原模拟多肽对L929细胞粘附的影响

设计合成了3种模拟胶原三螺旋结构或/和整合素识别位点的胶原模拟多肽(CMP),对其进行体外细胞相容性评价,研究其对小鼠成纤维细胞(L929)生长、粘附的影响。实验证实,3种CMP对成纤维细胞生长无明显的细胞毒性反应;3种包被胶原模拟多肽的基底均能在一定程度上促进细胞粘附、生长,具有良好的细胞粘附率和细胞附着形态,其中包含三螺旋结构和整合素识别位点的CMP27具有更好的促粘附效果,细胞粘附数量和形态与胶原接近。初步研究结果证实,胶原三螺旋结构与整合素识别位点共同作用促进L929细胞粘附。因此,CMP可以有效促进细胞粘附,有望作为粘附剂应用于生物医学领域,可为设计以多肽为基础的生物活性材料提供新的研究思路。     

RGD修饰载喜树碱微粒的合成和性能研究

采用含二硫键的二甲基丙烯酸酯(CL)为交联剂,以呋喃保护的N-羟乙基马来酰亚胺的甲基丙烯酸酯、聚乙二醇甲基醚甲基丙烯酸酯(PEGMA)、N-乙烯基己内酰胺(VCL)、甲基丙烯酸(MAA)为共聚单体,均相聚合合成微粒。在高温条件下脱保护后,通过巯基和马来酰亚胺的加成反应制得含精氨酸-甘氨酸-天冬氨酸短肽(RGD)修饰的脱保护微粒(TMP)(RGD-TMP)。通过傅里叶红外光谱(FT-IR)、扫描电子显微镜(SEM)表征微粒结构,以喜树碱(CPT)为模型药物分析其载药能力,评价药物细胞毒性。实验结果表明:RGD-TMP粒径为100~150nm,对CPT的载药量为18.56%,在药物浓度10μg/mL的条件下,CPT的细胞存活率为91%,而RGD-TMP载CPT后的细胞存活率降至72%。在控制CPT浓度为10μg/mL情况下,RGD修饰后的载药粒子的细胞存活率由CPT的91%下降为72%,可有效提高药物的细胞毒性。     

聚(D,L-乳酸)基仿生聚合物材料的合成与表征

探索一种新型聚乳酸基仿生聚合物材料的制备新方法.具体实验步骤是:利用聚乳酸上叔碳原子的自由基反应活性,在过氧化二苯甲酰的催化作用下,将马来酸酐引入聚乳酸侧链上,以此提供高反应活性的酸酐键;然后利用酸酐基团与-NH2发生N-酰化反应这一特点,将脂肪族二胺引入聚乳酸侧链上,从而克服聚乳酸降解产物的体液环境呈酸性的缺陷;再用碳二亚胺作缩合剂,在二胺改性聚乳酸材料中共价引入一种细胞粘附肽段Arg-Gly-Asp-Ser(RGDS),赋予材料生物活性和生物特异性,这样就制备了一种新型聚乳酸基仿生材料.采用MALLS、FTIR和XPS对仿生材料进行结构表征;采用罗丹明比色法、茚三酮显色法和氨基酸分析仪检测法对仿生材料中的马来酸酐、二胺和粘附肽RGDS进行定量测定.结果表明,按文中所述之制备技术,在不改变聚乳酸材料主链结构的前提下,该仿生材料中粘附肽RGDS的含量是5.12μmol/g.这就形成了一种具有类似细胞外基质的新型仿生材料.     

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。     

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.     

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.     

Modulation of Lumen Formation by Microgeometrical Bioactive Cues and Migration Mode of Actin Machinery

How endothelial cells (ECs) express the particular filopodial or lamellipodial form of the actin machinery is critical to understanding EC functions such as angiogenesis and sprouting. It is not known how these mechanisms coordinately promote lumen formation of ECs. Here, adhesion molecules (RGD peptides) and inductor molecules (BMP‐2 mimetic peptides) are micropatterned onto polymer surfaces by a photolithographic technique to induce filopodial and lamellipodial migration modes. Firstly, the effects of peptide microgeometrical distribution on EC adhesion, orientation and morphogenesis are evaluated. Large micropatterns (100 μm) promote EC orientation without lumen formation, whereas small micropatterns (10–50 μm) elicit a collective cell organization and induce EC lumen formation, in the case of RGD peptides. Secondly, the correlation between EC actin machinery expression and EC self‐assembly into lumen formation is addressed. Only the filopodial migration mode (mimicked by RGD) but not lamellipodial migration mode (mimicked by BMP‐2) promotes EC lumen formation. This work gives a new concept for the design of biomaterials for tissue engineering and may provide new insight for angiogenesis inhibition on tumors.     

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.     

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.     

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.     

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.     

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.     

Layer-by-layer assembly of chitosan and recombinant biopolymers into biomimetic coatings with multiple stimuli-responsive properties

In this work, biomimetic smart thin coatings using chitosan and a recombinant elastin-like recombinamer (ELR) containing the cell attachment sequence arginine-glycine-(aspartic acid) (RGD) are fabricated through a layer-by-layer approach. The synthetic polymer is characterized for its molecular mass and composition using mass spectroscopy and peptide sequencing. The adsorption of each polymeric layer is followed in situ at room temperature and pH 5.5 using a quartz-crystal microbalance with dissipation monitoring, showing that both polymers can be successfully combined to conceive nanostructured, multilayered coatings. The smart properties of the coatings are tested for their wettability by contact angle (CA) measurements as a function of external stimuli, namely temperature, pH, and ionic strength. Wettability transitions are observed from a moderate hydrophobic surface (CAs approximately from 62° to 71°) to an extremely wettable one (CA considered as 0°) as the temperature, pH, and ionic strength are raised above 50 °C, 11, and 1.25 M, respectively. Atomic force microscopy is performed at pH 7.4 and pH 11 to assess the coating topography. In the latter, the results reveal the formation of large and compact structures upon the aggregation of ELRs at the surface, which increase water affinity. Cell adhesion tests are conducted using a SaOs-2 cell line. Enhanced cell adhesion is observed in the coatings, as compared to a coating with a chitosan-ending film and a scrambled arginine-(aspartic acid)-glycine (RDG) biopolymer. The results suggest that such films could be used in the future as smart biomimetic coatings of biomaterials for different biomedical applications, including those in tissue engineering or in controlled delivery systems.