Ansha Zhao

Research of smooth muscle cells response to fluid flow shear stress by hyaluronic acid micro-pattern on a titanium surface

The morphology of vascular smooth muscle cells (SMCs) in the normal physiological state depends on cytoskeletal distribution and topology beneath, and presents vertical to the direction of blood flow shear stress (FFSS) although SMCs physiologically are not directly exposed to the shear conditions of blood flow. However, this condition is relevant for arteriosclerotic plaques and the sites of a vascular stent, and little of this condition in vitro has been studied and reported till now. It is unclear what will happen to SMC morphology, phenotype and function when the direction of the blood flow changed. In this paper, the distribution of SMCs in a specific area on Ti surface was regulated by micro-strips of hyaluronic acid (HA). Cell morphology depended on the distribution of the cytoskeleton extending along the micrographic direction. Simulated vascular FFSS was perpendicular or parallel to the direction of the cytoskeleton distribution. Based on investigating the morphology, apoptotic number, phenotypes and functional factors of SMCs, it was obtained that SMCs of vertical groups showed more apoptosis, expressed more contractile types and secreted less TGF-β1 factor compared with SMCs of parallel groups, the number of ECs cultured by medium from SMCs of parallel groups was larger than vertical groups. This study could help to understand the effect of direction change of FFSS on patterned SMC morphology, phenotype and function.

Cell adhesion on supported lipid bilayers functionalized with RGD peptides monitored by using a quartz crystal microbalance with dissipation.

Supported lipid bilayer (SLB) is one of the most widely used structures to mimic cell membranes. To study the cell-cell, cell-matrix and cell-material interactions, supported lipid bilayers (SLBs) functionalized with RGD peptides (SLBs-RGD) were prepared by vesicle fusion on a SiO2 quartz crystal, and subsequently bone mesenchymal stem cells (BMSCs) adhesion was analyzed. A quartz crystal microbalance with dissipation (QCM-D) was utilized to detect the dynamic adsorption behavior of lipid vesicles and BMSCs in real time. Observations obtained by QCM-D signals are confirmed by conducting fluorescence microscopy. QCM-D measurements showed the SLB formation starts at the critical concentration of the vesicles. More BMSCs adhered on SLBs-RGD than on SLBs. With the presence of SLBs, the adhesion cells on SLBs surfaces had a rounded morphology, and cells on SLBs-RGD will take long time to rearrange their cytoskeleton, which led to incomplete spreading compared with SiO2. Differences in adhesion density and adhesion properties of the cells on the different substrates could be traced at the dissipation versus frequency (ΔD/Δf) plots. These results indicate that RGD in/on SLBs could provide anchorage sites for more cells adhesion. QCM-D is demonstrated to be a useful tool for evaluating the interactions between various biological and non-biological systems in situ and in real-time.

Controlling Molecular Weight of Hyaluronic Acid Conjugated on Amine-rich Surface: Toward Better Multifunctional Biomaterials for Cardiovascular Implants

The molecular weights (MWs) of hyaluronic acid (HA) in extracellular matrix secreted from both vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) play crucial roles in the cardiovascular physiology, as HA with appropriate MW influences important pathways of cardiovascular homeostasis, inhibits VSMC synthetic phenotype change and proliferation, inhibits platelet activation and aggregation, promotes endothelial monolayer repair and functionalization, and prevents inflammation and atherosclerosis. In this study, HA samples with gradients of MW (4 × 103, 1 × 105, and 5 × 105 Da) were prepared by covalent conjugation to a copolymerized film of polydopamine and hexamethylendiamine (PDA/HD) as multifunctional coatings (PDA/HD-HA) with potential to improve the biocompatibility of cardiovascular biomaterials. The coatings immobilized with high-MW-HA (PDA/HD-HA-2: 1 × 105 Da; PDA/HD-HA-3: 5 × 105 Da) exhibited a remarkable suppression of platelet activation/aggregation and thrombosis under 15 dyn/cm2 blood flow and simultaneously suppressed the adhesion and proliferation of VSMC and the adhesion, activation, and inflammatory cytokine release of macrophages. In particular, PDA/HD-HA-2 significantly enhanced VEC adhesion, proliferation, migration, and functional factors release, as well as the captured number of endothelial progenitor cells under dynamic condition. The in vivo results indicated that the multifunctional surface (PDA/HD-HA-2) created a favorable microenvironment of endothelial monolayer formation and functionalization for promoting reendothelialization and reducing restenosis of cardiovascular biomaterials.

Controlling mesenchymal stem cells differentiate into contractile smooth muscle cells on a TiO2 micro/nano interface: Towards benign pericytes environment for endothelialization

Building healthy and oriented smooth muscle cells (SMCs) environment is an effective method for improving the surface endothelialization of the cardiovascular implants. However, a long-term and stable source of SMCs for implantation without immune rejection and inflammation has not been solved, and mesenchymal stem cells (MSCs) differentiation may be a good choice. In this work, two types of TiO2 micro/nano interfaces were fabricated on titanium surface by photolithography and anodic oxidation. These TiO2 micro/nano interfaces were used to regulate the differentiation of the MSCs. The X-ray diffraction (XRD) detection showed that the TiO2 micro/nano interfaces possessed the anatase crystal structure, suggesting good cytocompatibility. The CCK-8 results indicated the TiO2 micro/nano interfaces improved MSC proliferation, further immunofluorescence staining and calculation of the cell morphology index proved the micro/nano surfaces also elongated MSCs and regulated MSCs oriented growth. The specific staining of α-SMA, CNN-1, vWF, CD44 and CD133 markers revealed that the micro/nano surfaces induced MSCs differentiation to contractile SMCs, and the endothelial cells (ECs) culture experiment indicated that the MSCs induced by micro/nano interfaces contributed to the ECs attachment and proliferation. This method will be further studied and applied for the surface modification of the cardiovascular implants.

The effect of full/partial UV-irradiation of TiO2 films on altering the behavior of fibrinogen and platelets

Titanium oxide (TiO2) thin film is a potential candidate for the surface modification of blood-contacting devices. It has previously been reported that ultraviolet light (UV) irradiation could alter the biocompatibility of TiO2 films. However, the effect of UV-irradiated TiO2 films on blood compatibility has rarely been reported. This study attempts to determine: (1) whether UV-irradiation of TiO2 films enhances their blood compatibility, (2) the interaction between UV-irradiated TiO2 films, fibrinogen (Fgn), and platelets, especially how Fgn and platelets respond to the geometry of the partially UV-irradiated TiO2 film surface. Anatase TiO2 films were subjected to full and partial UV-irradiation. Full UV-irradiation improved the blood compatibility of TiO2 films by almost completely inhibiting the adhesion and activation of platelets, strongly suppressing the adsorption and conformational change of Fgn, and preventing the formation of fibrin fibers. Additionally, hemolysis was not observed. After partial UV-irradiation, the regions where Fgn adsorption was reduced (Fgn-dark regions) were formed at regions where UV-irradiation had occurred, but were extended in comparison with the UV-irradiated regions, which could be related to the generation and diffusion of reactive oxygen species (ROS) on the UV-irradiated TiO2 surface. It is worthwhile to study how ROS altered the nature of TiO2 films, thereby enhancing their blood compatibility. Furthermore, platelets were found adhering to the Fgn-adsorbed regions (Fgn-bright regions) selectively, suggesting that the inhibition of platelet adhesion could be related to the suppression of Fgn adsorption on the UV-irradiated TiO2 surface. It was also noted that platelet surface coverage (Sp) was not linearly correlated with Fgn-bright region surface coverage (Sf), which indicated that the adhesion and spreading of platelets were regulated by both Sf and the geometry of Fgn.

The effect of anti-CD133/fucoidan bio-coatings on hemocompatibility and EPC capture

Abstract Surface modification by immobilizing biomolecules has been widely proved to enhance biocompatibility of cardiovascular implanted devices. Here, we aimed at developing a multifunctional surface that not only provides good hemocompatibility but also functions well in capturing circulating endothelial progenitor cells (EPCs) in the blood flow to improve the surface endothelialization. In the present work, we preferred to chemically co-immobilize (Michael addition and Schiff base reaction) the anti-CD133 (EPC-specific antibody) and fucoidan (EPC-mobilization factor, which also contribute to better hemocompatibility) onto a polydopamine (PDA) film which is famous for its stability and endothelial cell (EC) compatibility. The quantality of anti-CD133 and other surface characterization (X-ray photoemission spectroscopy, atomic force microscopy and water contact angle measurement) demonstrated successful preparation of the CD133/fucoidan coating. The platelets adhesion/activation test suggested improved hemocompatibility of this bio-coating. The ex vivo experiment on New Zealand white rabbits showed that the anti-CD133/fucoidan coating had good ability on capture the circulating EPC. In addition, the quartz crystal microbalance-D indicated that the EPC behaviors, including adhesion, spreading and extracellular matrix re-molding, were related to the density of anti-CD133 in the coating. We hope this anti-CD133/fucoidan multi-functional coating may provide potential application on surface modification of cardiovascular biomaterials.

Tailoring of TiO2 films by H2SO4 treatment and UV irradiation to improve anticoagulant ability and endothelial cell compatibility

Surfaces with dual functions that simultaneously exhibit good anticoagulant ability and endothelial cell (EC) compatibility are desirable for blood contact materials. However, these dual functions have rarely been achieved by inorganic materials. In this study, titanium dioxide (TiO2) films were treated by sulphuric acid (H2SO4) and ultraviolet (UV) irradiation successively (TiO2H2SO4-UV), resulting in good anticoagulant ability and EC compatibility simultaneously. We found that UV irradiation improved the anticoagulant ability of TiO2 films significantly while enhancing EC compatibility, though not significantly. The enhanced anticoagulant ability could be related to the oxidation of surface-adsorbed hydrocarbons and increased hydrophilicity. The H2SO4 treatment improved the anticoagulant ability of TiO2 films slightly, while UV irradiation improved the anticoagulant ability strongly. The enhanced EC compatibility could be related to the increased surface roughness and positive charges on the surface of the TiO2 films. Furthermore, the time-dependent degradation of the enhanced EC compatibility and anticoagulant ability of TiO2H2SO4-UV was observed. In summary, TiO2H2SO4-UV expressed both excellent anticoagulant ability and good EC compatibility at the same time, which could be desirable for blood contact materials. However, the compatibility of TiO2H2SO4-UV with smooth muscle cells (SMCs) and macrophages was also improved. More effort is still needed to selectively improve EC compatibility on TiO2 films for better re-endothelialization.

Immobilization of biological macromolecule on titanium oxide film to improve the biocompatibility

Summary form only given. Ti-O film was prepared by magnetron sputtering. The surface of Ti-O film was pretreated with H/sub 2/O/sub 2/ and HCl mixed solution to get a surface with -OH group. The aminopropyltriethoxysilane (APTE) was covalently interacted with -OH group on the surface of Ti-O film. Then biological macromolecule such as albumin and heparin was immobilized through covalent chemical attachment between the -NH/sub 2/ group of the APTE and the -OH group of Ti-O film. X-ray photoelectron spectroscopy (XPS), Atomic Force Microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR) was used to investigate the characteristic of the film of APTE, albumin and heparin on the surface of Ti-O film. The number and the morphology of blood platelet were studied by blood platelet adhesion test in vitro. Endothelial cell culture test was used to investigate the function and influence between materials and the endothelial cells. The results showed that the surface of Ti-O film could be coated covalently with the albumin and heparin. Blood platelet adhesion test proved that the number of blood platelet decreased 50%, meanwhile the morphology of blood platelet was better than the untreated Ti-O film surface. Endothelial cell test showed that the surface coated with albumin and heparin influenced the number and status of endothelial cells compared with the untreated Ti-O film surface. This investigation showed that inorganic surface immobilized with biologic molecular is feasible and effective to improve the biocompatibility of bio-inert materials.