具有内皮细胞选择性的细胞膜仿生支架材料的研究

利用AIBN引发自由基反应,由单体2-(甲基丙烯酰氧基)乙基-2-(三甲基氨基)乙基磷酸酯(MPC)、甲基丙烯酸十八酯(SMA)、对硝基苯氧羰基聚乙二醇甲基丙烯酸酯(MEONP)合成了一种新型类细胞膜仿生涂层材料.MPC可以阻抗非特异性吸附;MEONP可以结合抗体或多肽促进特异性识别.通过表面固定的方法引入多肽序列Arg-Glu-Asp-Val(REDV),使涂层具有内皮细胞选择性.核磁、紫外吸收、红外光谱表征证实聚合物的组成以及REDV多肽在表面的固定;并通过血浆复钙化实验表征涂层的血液相容性.细胞黏附与增殖实验反映REDV多肽构建的涂层表面具备良好的特异性识别并结合内皮细胞的能力.     

Microfluidic depletion of endothelial cells, smooth muscle cells, and fibroblasts from heterogeneous suspensions

Interactions between ligands and cell surface receptors can be exploited to design adhesion-based microfluidic cell separation systems. When ligands are immobilized on the microfluidic channel surfaces, the resulting cell capture devices offer the typical advantages of small sample volumes and low cost associated with microfluidic systems, with the added benefit of not requiring complex fabrication schemes or extensive operational infrastructure. Cell-ligand interactions can range from highly specific to highly non-specific. This paper describes the design of an adhesion-based microfluidic separation system that takes advantage of both types of interactions. A 3-stage system of microfluidic devices coated with the tetrapeptides arg-glu-asp-val (REDV), val-ala-pro-gly (VAPG), and arg-gly-asp-ser (RGDS) is utilized to deplete a heterogeneous suspension containing endothelial cells, smooth muscle cells, and fibroblasts. The ligand-coated channels together with a large surface area allow effective depletion of all three cell types in a stagewise manner.     

Different complex surfaces of polyethyleneglycol (PEG) and REDV ligand to enhance the endothelial cells selectivity over smooth muscle cells

Arg-Glu-Asp-Val (REDV) peptide with endothelial cells (ECs) selectivity was immobilized onto PEG based polymeric coating via the active p-nitrophenyloxycarbonyl group. The adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) onto surface modified either by REDV end-tethered polyethylene glycol (PEG) or by the complex of free PEG and REDV were investigated to understand the synergic action of nonspecific resistance of PEG and specific recognitions of REDV. Cell culture results indicated that the surfaces end tethered by REDV peptide via PEG "spacer" (n=1, 6, 10) exhibited slight EC selectivity and showed small difference between different lengths of PEG chain. Both separate-culture and co-culture of HUVECs and HASMCs indicated that the introducing of free PEG into REDV tethered surface inhibited HASMCs adhesion significantly and remained a high level of HUVECs growth. Furthermore, the surface with short free PEG chain (n=6) was much more effective to enhance ECs selectivity than long EG chain (n=23). The combination of nonspecific resistance of short free PEG and the ECs selectivity of REDV peptide presents much better ability to enhance the competitive adhesion of HUVECs over HASMCs.     

Study of cell transmigration using a co-culture microsystem under shear stress

This study reports a novel cell co-culture technique using micro-molding in capillaries (MIMIC) technology that was utilized to observe the transmigration conditions of two types of cells with and without fluidic shear stress. Besides, the gap size of co-culture device could achieve shortest and not mixture. Endothelial cells (ECs) and smooth muscle cells (SMCs) were used in our experiment. In addition, concentrations of two cell are 8000 cells/μL (ECs) and 9000 cells/μL (SMCs), respectively, the shear stress is 7 dyne/cm₂, and the isolation distance between two types of cell are 50 and 200 μm. It is found that in the smaller culture space (50 μm) condition, ECs and SMCs would induce mutually, which would further make cell migration; in larger culture space (200 μm) condition, no inducing reaction took place between ECs and SMCs. It will have more advantages in bio-manipulation and tissue repair engineering.     

Selective Adhesion and Directional Migration of Endothelial Cells Guided by Cys‐Ala‐Gly Peptide Density Gradient on Antifouling Polymer Brushes

Selective adhesion and directional migration of endothelial cells (ECs) on biomaterials is critical to realize the rapid endothelialization. In this study, a Cys‐Ala‐Gly (CAG) peptide density gradient is generated on homogeneous cell‐resisting poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) brushes by immersing the brushes in a complementary gradient solution of CAG and competitive mercapto‐terminated methoxyl poly(ethylene glycol). The adhesion and spreading of smooth muscle cells (SMCs) is impaired effectively on the gradient surface. About six folds of adherent ECs over SMCs are achieved at the position (10 mm) of highest CAG density on the gradient surface in a co‐culture condition. Due to the gradient cues, ECs migrate fastest with the best directionality of 86.7% at the middle of the gradient, leading to the maximum net displacement as well.     

Using a co-culture microsystem for cell migration under fluid shear stress

We have successfully developed a microsystem to co-cultivate two types of cells with a minimum defined gap of 50 μm, and to quantitatively study the impact of fluid shear stress on the mutual influence of cell migration velocity and distance. We used the hydrostatic pressure to seed two different cells, endothelial cells (ECs) and smooth muscle cells (SMCs), on opposite sides of various gap sizes (500 μm, 200 μm, 100 μm, and 50 μm). After cultivating the cells for 12 h and peeling the co-culture microchip from the culture dish, we studied the impacts of gap size on the migration of either cell type in the absence or presence of fluid shear stress (7 dyne cm(-2) and 12 dyne cm(-2)) influence. We found that both gap size and shear stress have profound influence on cell migration. Smaller gap sizes (100 μm and 50 μm) significantly enhanced cell migration, suggesting a requirement of an effective concentration of released factor(s) by either cell type in the gap region. Flow-induced shear stress delayed the migration onset of either cell type in a dose-dependent manner regardless of the gap size. Moreover, shear stress-induced decrease of cell migration becomes evident when the gap size was 500 μm. We have developed a co-culture microsystem for two kinds of cells and overcome the conventional difficulties in observation and mixed culture, and it would have more application for bio-manipulation and tissue repair engineering.     

REDV/雷帕霉素复合涂层构建内皮细胞选择性功能界面

运用复合涂层的概念构建了兼具药物洗脱和内皮促进作用的载药涂层. 以载雷帕霉素(Rapamycin, RAP)的聚乙二醇甲基丙烯酸酯(PEGMA)-甲基丙烯酸丁酯(BMA)(PEGMA-BMA, PEGB)为内层, Arg-Glu-Asp-Val(REDV)多肽修饰的PEGBN为外层包裹载药涂层. 体外药物释放结果表明, 雷帕霉素可以维持缓慢稳定的长效释放, 释放过程中没有出现暴释现象. 表面细胞生长行为表明, 雷帕霉素可以有效地阻抗内皮细胞和平滑肌细胞的黏附, 抑制细胞活性; 随着药物释放的进行, 雷帕霉素浓度逐渐减低, 但涂层依然维持对平滑肌细胞的非特异性阻抗; 而REDV修饰的外涂层开始呈现内皮细胞的选择性黏附, 随着释放时间延长, 内皮细胞特异选择性也逐渐加强. 雷帕霉素和REDV多肽协同构建的复合涂层能够有效抑制平滑肌细胞的增殖, 获得内皮细胞选择性黏附.     

基于聚多巴胺辅助自组装单分子层技术的PTFE表面修饰及其内皮细胞选择性黏附研究

利用聚多巴胺技术对PTFE进行表面改性,X射线光电子能谱(XPS)、椭偏、接触角以及石英晶体微天平(QCM-D)证实DOPA分子可以在PTFE表面自聚形成反应性的超薄膜功能涂层,并通过聚多巴胺辅助自组装单分子层(SAM)技术构建了活性多肽链段CGREDVDY的界面.细胞黏附实验反映活性链段CGREDVDY的修饰表面具备良好的内皮细胞选择性黏附能力.这种具有内皮细胞选择性黏附能力的界面有望实现材料在复杂生理环境中对内皮细胞的原位诱导,为制备具有血管内皮原位快速愈合功能的新型血液相容性人造血管提供新途径.     

Recent Advances on Surface-modified Biomaterials Promoting Selective Adhesion and Directional Migration of Cells

The surfaces and interfaces of biomaterials interact with the biological systems in multi-scale levels, and thereby influence the biological functions and comprehensive performance in vitro and in vivo. In particular, a surface promoting the selective adhesion and directional migration of desired types of cells in complex environment is extremely important in the repair and regeneration of tissues such as peripheral nerve and blood vessel, and long-term application of intracorporal devices such as intravascular implants. Therefore, surface modification of biomaterials is a facile and effective method to achieve the desired cell-biomaterials interactions. In this short review, recent advances on the surface modification of biomaterials to regulate selective cell adhesion and migration are briefly summarized. In particular, the surface properties of biomaterials are manipulated via the convenient introduction of amino groups to the ester-based polymers, the formation of polyelectrolyte multilayers, and the fabrication of topology and gradient cues, etc., followed by the association of chemical and biological signals such as collagen, heparin, hyaluronic acid, peptides and cell growth factors. The selective adhesion and directional migration of various types of cells such as endothelial cells(ECs), smooth muscle cells(SMCs), hepatocytes and Schwann cells(SCs) are achieved over the competitive counterpart cells by the use of cell-resisting substances and cell-selective motifs on gradient substrates in most cases. Recent advances on cell behaviors in three-dimensional(3D) cell-extracellular matrix(ECM)-mimicking substrates are also reviewed.     

Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.     

Culture and leukocyte adhesion assay of human arterial endothelial cells in a glass microchip.

Cells are frequently exploited as processing components for integrated chemical systems, such as biochemical reactors and bioassay systems. By culturing vascular endothelial cells (ECs) in integrated chemical devices, vascular models have also been fabricated. Here, we utilized a thermally fused-glass microchip which is chemically and physically stable and favorable for optical detections, and cultured human arterial ECs (HAECs) in it. HAECs reached confluence within 4 days. Survival and tolerance for high shear stress (25 dyn/cm2) of the HAECs were confirmed. Furthermore, HAECs responded to inflammatory cytokine, tumor necrosis facor-alpha (TNF-alpha) and attached to more leukocyte cell line, HL-60 cells than unstimulated HAECs. Our developed device can be applied as a human arterial model, and we propose it as a new method for vascular studies.     

Surface Modification of Multiple Bioactive Peptides to Improve Endothelialization of Vascular Grafts

Endothelialization is an effective approach to prevent thrombus formation and enhance vascular graft survival. Surface modification of biomolecules has been proved to be effective in regulating endothelial cell behaviors. In this study, several peptides including YIGSR, RGD, and REDV sequences are covalently immobilized on the surface of electrospun silk fibroin scaffolds and the effects of combined application of these peptides on cell behaviors are studied. The results show that, compared with the scaffolds modified with single peptides, the scaffolds modified with dual peptides (YIGSR+RGD) could significantly enhance the proliferation of human umbilical vein endothelial cells (HUVECs). However, the combination of REDV+RGD or YIGSR+REDV does not promote the adhesion or proliferation of HUVECs. Notably, YIGSR‐modified scaffolds improved HUVEC migration significantly in comparison to REDV‐ or RGD‐modified groups. Moreover, its combination with either of these two peptides also presents excellent effect on cell migration. Thus, all the data suggest that the combined application of peptides might be a promising method to enhance the endothelialization of small‐diameter vascular grafts.     

Synthesis of potent and selective inhibitors against the proliferation of human coronary artery smooth muscle cells

A series of diarylamide urea derivatives were synthesized and evaluated for their inhibitory activities against human coronary artery smooth muscle cells (SMCs) and human coronary artery endothelial cells (ECs). Compound 2h was much superior to Tranilast, in terms of both the potency of its inhibitory activity toward the proliferation of SMCs and the cell selectivity.     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels

The interactions between endothelial cells and the underlying extracellular matrix regulate adhesion and cellular responses to microenvironmental stimuli, including flow-induced shear stress. In this study, we investigated the adhesion properties of primary porcine aortic endothelial cells (PAECs) and valve endothelial cells (PAVECs) in a microfluidic network. Taking advantage of the parallel arrangement of the microchannels, we compared adhesion of PAECs and PAVECs to fibronectin and type I collagen, two prominent extracellular matrix proteins, over a broad range of concentrations. Cell spreading was measured morphologically, based on cytoplasmic staining with a vital dye, while adhesion strength was characterized by the number of cells attached after application of shear stresses of 11, 110, and 220 dyn cm(-2). Results showed that PAVECs were more well spread on fibronectin than on type I collagen (P < 0.0001), particularly for coating concentrations of 100, 200, and 500 microg mL(-1). PAVECs also withstood shear significantly better on fibronectin than on collagen for 500 microg mL(-1). PAECs were more well spread on collagen compared to PAVECs (P < 0.0001), but did not have significantly better adhesion strength. These results demonstrate that cell adhesion is both cell-type and matrix dependent. Furthermore, they reveal important phenotypic differences between vascular and valvular endothelium, with implications for endothelial mechanobiology and the design of microdevices and engineered tissues.     

Study of bioadhesion on a flat plate with a yeast/glass model system

The attachment of microorganisms to a surface is a critical first step of biofilm fouling in membrane processes. The shear-induced detachment of baker's yeast in adhesive contact with a plane glass surface was thus experimentally studied, using a specially designed shear stress flow chamber. The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast). A complete series of experiments for measuring the shear stress necessary to detach a given proportion of individual yeast cells of type I or II was performed under different environmental conditions (ionic strength, contact time). In parallel, the surface physicochemical properties of the cells (surface charge, hydrophobicity, and electron donor and electron acceptor components) were determined. For the first type of yeast cells, which were rather hydrophilic, adhesion to the glass plate was weak. This was due to both electrostatic effects and hydrophilic repulsion. Furthermore, adhesion was not sensitive to any variation of the ionic strength. For yeast of the second type, adhesion was drastically increased. This could be explained by their physicochemical surface properties and especially their hydrophobic and electron acceptor components, which caused strong attractive van der Waals and Lewis acid-base interactions, counterbalancing the electrostatic repulsion. For increasing ionic strengths, adhesion was greater, due to lower electrostatic repulsion. The results were quantified through the definition of a critical wall shear stress ( tau w 50% ) required to detach 50% of the yeast cells initially deposited on the glass surface. The influence of the contact time was also evaluated and it was shown that, whatever the type of yeast, macromolecules such as proteins were released into the extracellular medium due to cell lysis and could contribute to the formation of a conditioning film. As a result, the cells were more strongly stuck to the glass plate.     

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.     

Influence of poly(ethylene oxide) brushes on the rheological properties of MgO colloidal suspensions in water

A combined experimental and multiscale simulation study of the influence of polymer brush modification on interactions of colloidal particles and rheological properties of dense colloidal suspensions has been conducted. Our colloidal suspension is comprised of polydisperse MgO colloidal particles modified with poly(ethylene oxide) (PEO) brushes in water. The shear stress as a function of shear rate was determined experimentally and from multiscale simulations for a suspension of 0.48 volume fraction colloids at room temperature for both bare and PEO-modified MgO colloids. Bare MgO particles exhibited strong shear thinning behavior and a yield stress on the order of several Pascals in both experiments and simulations. In contrast, simulations of PEO-modified colloids revealed no significant yielding or shear thinning and viscosity only a few times larger than solvent viscosity. This behavior is inconsistent with results obtained from experiments where modification of colloids with PEO brushes formed by adsorption of PEO-based comb-branched chains resulted in relatively little change in suspension rheology compared to bare colloids over the range of concentration of comb-branch additives investigated. We attribute this discrepancy in rheological properties between simulation and experiment for PEO-modified colloidal suspensions to heterogeneous adsorption of the comb-branch polymers.     

Non-adsorbing macromolecules in plasma induce erythrocyte adhesion to the endothelium

Red blood cell (RBC) adhesion to the endothelium is usually insignificant. However, an enhanced adhesion can be observed in various pathological conditions such as diabetes mellitus or sickle cell disease, which is often accompanied by elevated levels of pro-adhesive plasma proteins such as fibrinogen. In the past, these proteins have only been considered to act as ligands, cross-linking the corresponding receptors on adjacent cells, but the detailed underlying mechanism often remained obscure. This work demonstrates that the presence of non-adsorbing polymers in plasma can also enhance the adhesion efficiency of RBCs to endothelial cells (ECs) through depletion interaction. Furthermore, adhesion of RBCs to ECs may be likewise promoted by the protein fibrinogen through depletion interaction. We propose an alternative mechanism for the pro-adhesive effects of plasma proteins and indicate that depletion interaction might play a significant role for the stabilization and destabilization of blood flow in health and disease.     

ICAM-1/LFA-1 interaction contributes to the induction of endothelial cell-cell separation: implication for enhanced leukocyte diapedesis

The basic route and mechanism for diapedesis has not yet to be fully defined. Here we present evidence that "cell-cell separation" between endothelial cells (ECs) may provide a route for leukocyte diapedesis. We unexpectedly found that extensive interaction between peripheral blood leukocytes and ECs that were activated by TNF-α induced the opening of EC contacts and, surprisingly, resulted in cell-cell separation. This event was specific to the intercellular adhesion molecules-1 (ICAM-1)/leukocyte function-associated antigen-1 interaction, as demonstrated by the following: (1) ICAM-1 expression correlated with increased EC contraction; and (2) the blocking of ICAM-1 selectively inhibited EC separation. Thus, we suggest that "cell-cell separation" could be a mechanism for diapedesis in situations that may require massive leukocyte infiltration.