基于电聚多巴胺技术一锅法快速构建生物活性界面的研究

通过精确控制电化学参数采用循环伏安法在中性无氧水环境中制备得到膜厚可控的电聚多巴胺膜,并将这种电聚多巴胺技术与生物活性分子的负载相结合,通过一锅法电聚得到含REDV活性短肽的聚多巴胺活性膜,快速便捷地构建了具有促内皮细胞粘附的生物活性界面.椭圆偏振仪、扫描电子显微镜证实了材料界面上形成了均一的聚多巴胺膜;X射线光电子能谱以及荧光分析结果证实了REDV短肽已负载于电聚多巴胺涂层中.内皮细胞体外黏附实验证实REDV短肽保持了良好活性,可有效促进内皮细胞黏附、铺展及粘着斑的形成.这种一锅法快速制备具有生物活性的电聚多巴胺涂层技术有望为复杂的导电生物材料和装置的多功能界面修饰提供新的途径.     

表面接枝嵌段共聚物刷实现内皮细胞的选择性黏附

利用表面引发原子转移自由基聚合(SI-ATRP)技术将聚(甲基丙烯酸寡聚乙二醇酯)和聚(甲基丙烯酸缩水甘油酯)的双嵌段共聚物刷(POEGMA-b-PGMA)接枝在材料表面,并通过PGMA中丰富的环氧基团开环固定可特异性黏附内皮细胞的多肽GREDVY.静态水接触角、接枝层厚度、X射线光电子能谱(XPS)以及原子力显微镜(AFM)的测试结果证明了各步接枝反应的成功性.细胞培养研究表明这种连接有GREDVY的双嵌段共聚物表面能够有效的促进血管内皮细胞的黏附,同时排斥成纤维细胞的黏附,从而实现了内皮细胞的选择性黏附.     

基于聚乙二醇和不同多肽构建的生物涂层及其内皮细胞选择性研究

利用自由基聚合反应将甲基丙烯酸聚乙二醇酯(PEGMA)和甲基丙烯酸缩水甘油酯(GMA)的二元共聚物接枝在基材表面,并通过开环反应分别固定精氨酸-甘氨酸-天冬氨酸(RGD)、精氨酸-谷氨酸-天冬氨酸-缬氨酸(REDV)和酪氨酸-异亮氨酸-甘氨酸-丝氨酸-精氨酸(YIGSR)3种可特异性黏附内皮细胞的多肽.通过核磁共振检测合成的聚合物分子结构,并进一步通过X射线光电子能谱(XPS))以及原子力显微镜(AFM)的测试结果证明聚合物成功接枝在基材表面.利用紫外-可见吸收光谱(UV-Vis)对表面固定的3种多肽进行了定量表征.体外内皮细胞和平滑肌细胞黏附结果表明,3种不同多肽修饰的共聚物表面均能够有效阻抗平滑肌细胞的黏附,同时不同程度地促进内皮细胞的黏附,从而实现了基材表面内皮细胞的选择性黏附.其中与RGD和YIGSR多肽修饰的表面相比,REDV多肽修饰的表面呈现出更优异的内皮细胞选择性.这种具有内皮细胞特异选择性的界面在心血管支架涂层原位内皮化方面具有良好的应用前景.     

pH诱导聚多巴胺表面质子化作用机理及其细胞黏附行为

通过电化学聚合在钛基材表面构建聚多巴胺膜层,探讨了pH诱导聚多巴胺膜表面质子化/去质子化及其电势电荷的变化特性,并研究了其对骨髓间充质干细胞(BMSCs)的早期黏附行为的影响.利用拉曼光谱和场发射扫描电镜证实,通过电化学方法在钛表面可构建均匀致密的聚合多巴胺膜层;电化学及动力学分析表明,不同pH介质条件下,聚多巴胺膜的界面电阻不同,且其随着pH的增大而增大,质子化的聚多巴胺有利于电荷累积;原子力显微镜结果表明,质子化和去质子化的聚多巴胺膜表面电势,在不同p H环境下具有可逆周期性变化的特性;探讨了膜层质子化作用对BMSCs的黏附作用机理,表明质子化的聚多巴胺钛表面带正电荷更有利于细胞的黏附和铺展,且具有良好的细胞相容性.     

含悬挂羧基手臂聚乳酸材料的内皮细胞黏附及其细胞活性

为了考察内皮化材料表面的细胞活性, 在前期工作的基础上, 分别在聚乳酸(PLA)、乳酸-苹果酸共聚物(PLMA), 以及含悬挂羟基或羧基的乳酸-苹果酸共聚物膜(PLMAHE,PLMACA)表面种植人脐静脉内皮细胞(HUVEC), 成功地制备了内皮化表面. 通过测定内皮化材料表面内皮细胞释放的内皮型一氧化氮合酶(eNOS)以及一氧化氮的释放量, 间接考察了内皮细胞的抗凝血活性; 另外, 通过内皮化表面的血小板黏附实验, 直接观察了血小板在内皮细胞上的黏附情况. 实验结果表明, 含羧基材料表面的内皮细胞活性比PLA和PLMAHE的高; 相对其它材料PLMACA能更有效地保留黏附于其表面内皮细胞的活性, 其单位内皮细胞的eNOS以及NO的释放量分别为(41.8±8.1) μmol/104 cells和(0.76±0.16) U/104 cells. 电镜照片(SEM)显示, 各种材料表面的内皮细胞均能有效地减少血小板的黏附与聚集; 在内皮细胞脱落的区域, PLMACA仍能较好地实现其抑制血小板黏附的功能, 有望成为新型血管修复(替代)材料.     

基于多巴胺自聚合及肝素固定改善钛的血液相容性

利用多巴胺自聚合及肝素固定的方法对纯钛进行表面修饰, 以改善其血液相容性. 采用水接触角测量、 X射线光电子能谱(XPS)和甲苯胺蓝法(TBO)等方法对所修饰的材料进行了表征. 采用溶血实验检测了材料的溶血性能, 并结合活化部分凝血活酶时间(APTT)测试和血小板黏附实验对所修饰材料的血液相容性进行了评价. 结果表明, 多巴胺能够在钛表面实现自聚合, 肝素可以共价接枝在聚多巴胺层上, 经肝素修饰后的材料的表面亲水性显著提高, 而且具有较低的溶血率, APTT时间显著延长, 血小板的黏附数量和被激活程度也显著降低. 因此, 纯钛经多巴胺自聚合以及肝素接枝修饰后的血液相容性得到了显著改善, 有望成为具有抗凝血功能的新型心血管植入材料.     

人工细胞外基质对血管内皮细胞生存的影响

通过物理吸附方法, 利用胶原、 聚赖氨酸和融合蛋白VEGF-Fc对聚苯乙烯培养板表面进行改性, 以研究细胞外基质材料对血管内皮细胞的影响. 结果表明, 3种蛋白显著提高了聚苯乙烯表面的亲水性. 内皮细胞的黏附、 增殖、 细胞骨架蛋白染色和血管性血友病因子(vWF)免疫染色实验结果表明, 胶原、 聚赖氨酸和VEGF-Fc基质均能有效提高血管内皮细胞的黏附, 其中胶原可与VEGF协同作用促进内皮细胞分化表型的表达; VEGF-Fc基质兼具了VEGF的生物学活性, 可促进内皮细胞的黏附和增殖以及vWF功能性蛋白的表达. 本研究为诱导材料表面内皮化和血管新生的生物活性材料的设计开发提供了新思路.     

基于多巴胺表面改性技术研究现状

海洋贻贝类生物通过足丝分泌的黏附蛋白,在潮湿环境中可以短时间固化,并紧固黏附在基质表面.研究表明,3,4-二羟基-L-苯基丙氨酸(多巴,DOPA)是这种黏附蛋白中的重要组成部分.多巴胺(DA)作为DOPA的一种衍生物,拥有同样的强黏附性.且多巴胺的自聚合产物聚多巴胺(PDA)存在许多官能团,如邻苯二酚、胺、亚胺,这些官...     

聚多巴胺涂层的研究与应用进展

多巴胺在有氧化剂且弱碱性环境下会发生自聚合形成聚多巴胺,聚多巴胺能够在多种基底材料(包括贵金属、金属氧化物、无机及有机高分子材料等)表面实现黏附形成聚多巴胺涂层,基于聚多巴胺涂层中含有大量可以参与反应官能团的特点,聚多巴胺涂层表面可以进行二次修饰从而制备功能性材料表面,也可以基于多巴胺/聚多巴胺与功能性物质之间相互作用,一步法制备功能性材料表面。近些年来,基于聚多巴胺涂层发展起来的两步修饰法和多巴胺一步共混沉积法在诸多领域得到了应用。本文主要综述了聚多巴胺涂层的最新研究和应用进展。     

基于贻贝仿生化学的分离功能材料

贻贝仿生的表面化学是近年来材料学、化学、生物医学等领域的交叉研究热点。多巴胺可以作为贻贝足丝蛋白(Mfp)超强黏附特性的模型分子,通过复杂的氧化-自聚和组装,形成多种功能的聚多巴胺(PDA)纳米涂层和纳米粒子,在分离膜、吸附材料、生物医用材料、生物黏结剂等领域有着广阔的应用前景。本研究小组近年来持续开展了基于贻贝仿生化学的分离功能材料制备与结构调控的研究工作,率先将多巴胺表面沉积方法应用于多孔分离膜表面的构建与功能化,提出了多巴胺的自聚-沉积过程模型,进而验证了PDA沉积层的纳滤分离特性,建立了一条简单方便的膜表面功能化与纳滤膜制备新途径。本文主要对基于贻贝仿生化学的分离功能材料,特别是分离膜的研究进展进行综述,并对将来的发展趋势进行展望。     

Nitrogen-rich plasma-polymerized coatings on PET and PTFE surfaces improve endothelial cell attachment and resistance to shear flow

Low seeding efficiency and poor cell retention under flow-induced shear stress limit the effectiveness of in vitro endothelialization strategies for small-diameter vascular grafts. Primary-amine-rich plasma-polymerized coatings (PPE:N) deposited using low- and atmospheric-pressure plasma discharges on PET and PTFE are evaluated for their ability to improve endothelial cells' kinetics and strength of attachment. PPE:N coatings increase cell adhesion and adhesion rate, spreading, focal adhesion, and resistance to flow-induced shear compared with bare and gelatin-coated PET and PTFE. In particular, about 90% of the cells remain on coated surfaces after 1 h exposure to shear. These coatings, therefore, appear as a promising versatile approach to improve cell seeding strategies for vascular grafts.     

Antithrombogenicity of cultured endothelial cell-detached surface

To examine the antithrombogenicity of cultured endothelial cell-detached surface, a simple hybrid vascular model tube consisting of a glass tube and endothelial cells was constructed. To detach the endothelial cells from the inner surface of the model tube, a steady shear stress of 2 or 8 N m(-2) was imposed onto the surface of endothelial cell monolayer by means of a coaxial double cylinder rotational-type apparatus. Coagulation of blood in contact with the endothelial cell-detached surface was examined using a damped oscillation rheometer. Coagulation of whole blood in the cell-detached tube occurred at about 40 min, which was almost the same as that in the endothelial cell-coated tube. A few platelets without shape change adhered to the endothelial cell-detached surface. These data suggest that the endothelial cell-detached surface may exhibit antithrombogenic and anticoagulant surfaces. Biochemical analyses showed that the glass surface, where endothelial cell was detached, was covered with components such as collagen type IV that is considered to be produced from the endothelial cells on the glass surface.     

Renal Epithelial Monolayer Formation on Monomeric and Polymeric Catechol Functionalized Supramolecular Biomaterials

Induction of a functional, tight monolayer of renal epithelial cells on a synthetic membrane to be applied in a bioartificial kidney device requires for bio‐activation of the membrane. The current golden standard in bio‐activation is the combination of a random polymeric catechol (L‐DOPA) coating and collagen type IV (Col IV). Here the possibility of replacing this with defined monomeric catechol functionalization on a biomaterial surface using supramolecular ureido‐pyrimidinone (UPy)‐moieties is investigated. Monomeric catechols modified with a UPy‐unit are successfully incorporated and presented in supramolecular UPy‐polymer films and membranes. Unfortunately, these UPy‐catechols are unable to improve epithelial cell monolayer formation over time, solely or in combination with Col IV. L‐DOPA combined with Col IV is able to induce a tight monolayer capable of transport on electrospun supramolecular UPy‐membranes. This study shows that a random polymeric catechol coating cannot be simply mimicked by defined monomeric catechols as supramolecular additives. There is still a long way to go in order to synthetically mimic simple natural structures.     

Surface characterization of the extracellular matrix remaining after cell detachment from a thermoresponsive polymer

The temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) directly affects the attachment and detachment of cells cultured on these surfaces. At culture temperatures, cells behave similarly to those on tissue culture polystyrene (TCPS), while at room temperature, cells cultured on pNIPAM spontaneously detach as a confluent sheet. In comparison, cells grown on TCPS remain attached indefinitely after the temperature drop, requiring enzymatic or mechanical removal. In this work, we present an examination of the response of bovine aortic endothelial cells (BAECs) and extracellular matrix (ECM) proteins to plasma polymerized NIPAM (ppNIPAM) surfaces using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and immunostaining. Immunoassay results reveal that, although fibronectin, laminin, and collagen closely associate with the cell sheet, some collagen may be associated with the surface, as well. Our XPS results indicate that ppNIPAM surfaces after cell liftoff differ from their blank counterparts, the primary distinction being the presence of amide and alcohol species on ppNIPAM surfaces used for cell culture, possibly owing to the presence of a proteinaceous film. Finally, a comparison between ppNIPAM-treated surfaces used for cell culture versus control surfaces by principal component analysis of the ToF-SIMS data confirms that the surfaces differ; the presence of molecular ion fragments from amino acids (e.g., alanine, glycine, and proline) is the chief reason for this difference. Therefore, from our surface characterization of ppNIPAM-coated TCPS after cell liftoff, we conclude that although low-temperature liftoff of the BAEC monolayer is accompanied by the majority of the components of the ECM, some of the ECM proteins still remain at the surface.     

Adsorption and conformation behavior of biotinylated fibronectin on streptavidin-modified TiO(X) surfaces studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.     

Formation and recovery of a cell sheet by a particle monolayer with the surface roughness

We studied the topographical effect of roughness displayed by a closely packed particle monolayer on formation of a cell monolayer (cell sheet). Particle monolayers were prepared by Langmuir-Blodgett deposition using particles, which were 527nm (SA053) and 1270nm (SA127) in diameter. Human umbilical vein endothelial cells (HUVECs) were seeded at a high density (2.0 x10(5)cells/cm(2)) onto particle monolayers. It was found that cells gradually became into contact with adjacent cells on the SA053 monolayer and the formed cell sheet could be readily detached from the particle monolayer by gentle pipetting. On the other hand, cells adhering onto the tissue culture polystyrene (TCPS) and the SA127 particle monolayer were difficult to peel off. At a low cell seeding density (5.0x10(4)cells/cm(2)), pre-coating with bovine plasma fibronectin (FN) allowed cell growth on an SA053 particle monolayer, and a confluent monolayer was able to be peeled as a cell sheet from the particle monolayer just by pipetting. By immunostaining of human fibronectin, we found that fibronectin was secreted and concentrated onto the substrate side of a cell sheet. The obtained cell sheet adhered and grew on the TCPS again within 20min.     

Subcellular topological effect of particle monolayers on cell shapes and functions

We studied topological effects of subcellular roughness displayed by a closely packed particle monolayer on adhesion and growth of endothelial cells. Poly(styrene-co-acrylamide) (SA) particles were prepared by soap-free emulsion copolymerization. Particle monolayers were prepared by Langmuir–Blodgett deposition using particles, which were 527 (SA053) and 1270 nm (SA127) in diameter. After 24-h incubation, cells tightly adhered on a tissue culture polystyrene dish and randomly spread. On the other hand, cells attached on particle monolayers were stretched into a narrow stalk-like shape. Lamellipodia spread from the leading edge of cells attached on SA053 monolayer to the top of the particles and gradually gathered to form clusters. This shows that cell–cell adhesion became stronger than cell–substrate interaction. Cells attached to SA127 monolayer extended to the reverse side of a particle monolayer and engulfed particles. They remained immobile without migration 24 h after incubation. This shows that the inhibition of extensions on SA127 monolayer could inhibit cell migration and cell proliferation. Cell growth on the particle monolayers was suppressed compared with a flat TCPS dish. The number of cells on SA053 gradually increased, whereas that on SA127 decreased with time. When the cell seeding density was increased to 200,000 cells cm⁻², some adherent cells gradually became into contact with adjacent cells. F-actin condensations were formed at the frame of adherent cells and the thin filaments grew from the edges to connect each other with time. For the cell culture on SA053 monolayer, elongated cells showed a little alignment. Cells showed not arrangement of actin stress fibers but F-actin condensation at the contact regions with neighboring cells. Interestingly, the formed cell monolayer could be readily peeled from the particle monolayer. These results indicate that endothelial cells could recognize the surface roughness displayed by particle monolayers and the response was dependent on the pitch of particle monolayers.     

Endothelin-1 and angiotensin II secretion at different lengths of endothelial cell monolayer in the view of tensile stress accumulation in the upper endothelial cell membrane

Theoretical analysis and experimental observations have shown that tensile stress inside an endothelial cell membrane is capable of growing in the direction opposite to blood flow and can accumulate to a level that is three or more orders of magnitude higher than flow-induced shear stress on the membrane surface. This phenomenon is called cell membrane tension accumulation (CMTA). We hypothesize that correlation may exist between the endothelial cell monolayer length or CMTA and secretory function of endothelial cells. To verify this hypothesis, a paired experimental study was devised to measure the secretion of endothelin (ET-1) and angiotensin II (Ang II) by two monolayers of cultured human glomerular vascular endothelial cell (HGVEC) monolayers subjected an identical steady shear stress. After replicate cultured HGVEC monolayer with two kinds of length of 6 cm and 10 cm were subjected to the same steady laminar shear stress of 0.45 N/m² for 24 h, the average secretion rates of ET-1 and Ang II in 6 cm long increased l.7- and 0.5-fold (n=26, P<0.00l) over 10 cm long, respectively. Over 10 h of exposure to 0.65 N/m², the average secretion rate of both ET-1 and Ang II by HGVEC monolayer of 6 cm in length exceeded 0.5-fold (n=26, P<0.0001) over 10 cm in length. All these demonstrated that the close relationship may exist between length of endothelial cell monolayer and secretion of ET-1 and Ang II by endothelial cells, indicating the possible existence of the cumulative effect of the tensile stress in the upper endothelial cell membrane under the shear flow field.     

Surface presentation of bioactive ligands in a nonadhesive background using DOPA-tethered biotinylated poly(ethylene glycol)

We have developed surfaces for the selective presentation of biotinylated peptides and proteins in a background that resists nonspecific protein adsorption; controlled amounts of biotinylated poly(ethylene glycol) (MW 3400 Da; PEG3400) anchored to titanium-dioxide-coated surfaces via an adhesive tri-peptide sequence of L-3,4-dihydroxyphenylalanine (DOPA3-PEG3400-biotin; DPB) were incorporated within a DOPA3-PEG2000 background. Using optical waveguide lightmode spectroscopy, we found that the amounts of sequentially adsorbed NeutrAvidin and singly biotinylated molecules increased proportionally with the amount of DPB in the surface. Biotinylated peptides (MW approximately 2000 Da) were able to fill all three of the remaining avidin-binding sites, while only one molecule of biotinylated PEG5000 or stem cell factor bound to each avidin. The resulting biotin-avidin-biotin linkages were stable for prolonged periods under continuous perfusion, even in the presence of excess free biotin. Hematopoietic M07e cells bound to immobilized peptide ligands for alpha5beta1 (cyclic RGD) and alpha4beta1 (cylic LDV) integrins in a DPB-dose-dependent manner, with near-maximal binding to cylic LDV for surfaces containing 1% DPB. Multiple ligands were adsorbed in a controlled manner by incubating NeutrAvidin with the respective ligands in the desired molar ratio and then adding the resulting complexes to DPB-containing surfaces. Cell adhesion to surfaces containing both cylic LDV and cyclic RGD increased in an additive manner compared to that for the individual ligands. The bioactivity of adsorbed biotinylated stem cell factor was retained, as demonstrated by DPB-dose-dependent M07e cell adhesion and ERK1/2 activation.     

Reinforced and self-humidifying composite membrane for fuel cell applications

A novel preparation method for a composite proton exchange membrane with reinforced strength and self-humidifying property was developed. Using self-assembly method, highly dispersed poly(diallyldimethylammonium chloride) (PDDA) stabilized Pt nanoparticles were mounted onto the pores of poly(tetrafluoroethylene) (PTFE) porous film to serve the self-humidifying purpose. With Pt nanoparticles fixed on the PTFE pores, the potential problem of any short circuit because of the use of metal nanoparticles can be prevented. Pt-PDDA/PTFE substrate in the composite membrane can enhance the mechanical strength of the membrane and distribute self-humidifying layer adjacent to the anode side. Compared with the cells fabricated with conventional Nafion^® and PTFE/Nafion membranes, the performance of the cells with this composite membrane is dramatically improved under dry conditions. Electrochemical impedance spectroscopy technique revealed that these self-humidifying composite membranes could minimize membrane conductivity loss under dry conditions.