Exosome-Loaded Pro-efferocytic Vascular Stent with Lp-PLA2-Triggered Release for Preventing In-Stent Restenosis.

The efferocytosis defect is regarded as a pivotal event of atherosclerosis. The failure to clear apoptotic cells in atherosclerotic plaques under vascular stents causes a failure to resolve the inflammation underneath. However, efferocytosis repair is still confined to nonstenting therapeutics. Here, we identified a pro-efferocytotic agent and accordingly developed a bioresponsive pro-efferocytotic vascular stent aimed for poststenting healing. Exosomes derived from mesenchymal stem cells were found to be able to regulate efferocytosis via SLC2a1, STAT3/RAC1, and CD300a pathways and modulate foam cell formation processes through a CD36-mediated pathway. Pro-efferocytotic exosomes were encapsulated into liposome-based multivesicular chambers and grafted onto vascular stents. The multivesicular vesicles were able to release exosomes under the Lp-PLA2 environment. Compared to bare metal stents, exosome-stents in the presence of Lp-PLA2 enhanced the ratio of apoptotic cell clearance and reduced the neointimal thickness in the mal-efferocytotic rat model. Overall, we identified a pro-efferocytic agent─exosomes that are able to regulate target cells via multiple signaling pathways and are good candidates to serve complex pathological environments, and this bioresponsive pro-efferocytotic vascular stent is an attractive approach for prevention of poststenting complications.

Comparison of in Vascular Bioreactors and In Vivo Models of Degradation and Cellular Response of Mg-Zn-Mn Stents.

Traditional in vitro evaluation criteria of magnesium (Mg)-based stents cannot reflect the degradation process in vivo, due to the interdependence and interference between biodegradable properties and bioenvironment. The current direct and indirect evaluation approaches of in vitro biocompatibility do not have a hydrodynamic environment and vascular biological structure existing in vivo. Herein, we designed a vascular bioreactor to provide an ex vivo culture environment for vessels, which reveals the degradation behavior of Mg-Zn-Mn stent and the effect of its degradation on cells. We reported that rabbit carotid arteries could maintain native morphology and viability in the bioreactor under the best condition within a flow rate of 5.4 mL min-1 and a culture time of one week. With this culture condition, Mg-Zn-Mn stents were implanted into the arteries in the bioreactors and compared with in vivo rabbit models. The arteries maintained cell survival in the bioreactor, but the cell attachment was absent on the stent struts, associated with a fast degradation. Conversely, the stents achieved a rapid and complete endothelialization in vivo for two weeks. This study could provide a correlation and difference of the degradation behavior and cellular response to the degradation of Mg-based stent between ex vivo and in vivo approaches.

In vitro performance of 3D printed PCL-ß-TCP degradable spinal fusion cage.

Spinal fusion cages are commonly used to treat spinal diseases caused by degenerative changes, deformities, and trauma. At present, most of the main clinical spinal fusion cage products are non-degradable and still cause some undesirable side effects, such as the stress shielding phenomenon, interference with postoperative medical imaging, and obvious foreign body sensation in patients. Degradable spinal fusion cages have promising potential with extensive perspectives. The purpose of this study was to fabricate a degradable spinal fusion cage from both polycaprolactone (PCL) and high-proportion beta-tricalcium phosphate (ß-TCP), using the highly personalised, accurate, and rapid fused deposition modelling 3 D printing technology. PCL and ß-TCP were mixed in three different ratios (60:40, 55:45, and 50:50). Both in vitro degradation and cell experiments proved that all cages with the different PCL:ß-TCP ratios met the mechanical properties of human cancellous bone while maintaining their structural integrity. The biological activity of the cages improved with higher amounts of the ß-TCP content. This study also showed that a spinal fusion cage with high ß-TCP content and suitable mechanical properties can be manufactured using extruding rods and appropriate models, providing a new solution for the design of degradable spinal fusion cages.

Cell-friendly photo-functionalized TiO2 nano-micro-honeycombs for selectively preventing bacteria and platelet adhesion.

Titanium dioxide (TiO2) is a widely used biomaterial. It is a great challenge to confer antibacterial and antithrombotic properties to TiO2 while maintaining its cell affinity. Here, we developed a new strategy to achieve the above goal by comprehensively controlling the chemical cues and geometrical cues of the surface of TiO2. Using colloidal etching technology and UV irradiation treatment, we obtained the photofunctionalized nano-micro-honeycomb structured TiO2. The honeycomb structured increased the photocatalytic activity of TiO2, which endowed TiO2 with photo-induced superhydrophilicity to inhibit bacterial adhesion. The high photocatalytic activity also induced the strong photocatalytic oxidation of TiO2 surface organic adsorbates to suppress fibrinogen and platelet attachment. In addition, owing to the micropore trapping-isolation effect on the bacteria and the nano-frames' contact guidance effect on the growth and spreading of platelet pseudopods, the honeycomb structure also shows a considerable inhibiting effect on bacterial and platelet adhesion. Therefore, due to the controlled chemical and geometrical cues' synergistic effect, the photo-functionalized TiO2 honeycomb structure shows excellent bacterial-adhesion resistance and antithrombotic properties. More importantly, the photo-functionalized TiO2 honeycomb did not inhibit the adhesion and growth of endothelial cells (ECs) after culturing for 3 d, indicating a good cell affinity that the traditional antifouling surfaces do not possess.

The co-deposition coating of collagen IV and laminin on hyaluronic acid pattern for better biocompatibility on cardiovascular biomaterials.

Construct a coating to repair the endothelium function is the ordinary and effective method to get out of the troubles which introduced by the cardiovascular implant devices. It indeed has plenty works on function construction which could inhibit the hyperplasia or accelerate the endothelialization with different functional proteins or molecules. However, a complete and healthy endothelium couldn't survive without the environment around. Thus, a logical biomimetic reconstruction with structure and function factors which using hyaluronic acid patterns to imitate the blood flow shear stress and co-depositing collagen type IV and laminin to achieve the biofunction of basement membrane had been proposed and realized in this work. After the tests of hemocompatibility, cytocompatibility and tissue compatibility, it had been indicated that this biomimetic coating could inhibit the adhesion of platelets, promote the proliferation and biofunction of endothelium cells, regulate smooth muscle cells with contractile phenotype and have much lower inflammatory response which might be a meaningful strategy on reconstruction and repairing of endothelium.

Photofunctionalized and Drug-Loaded TiO2 Nanotubes with Improved Vascular Biocompatibility as a Potential Material for Polymer-Free Drug-Eluting Stents.

Implantation of a drug-eluting stent is the most common treatment method for patients with cardiovascular atherosclerosis. However, this treatment may delay re-endothelialization, and the drug polymer-coated stent may induce thrombosis months after a stent implantation. The development of polymer-free drug-eluting stents is a promising approach to overcome these shortcomings. Titanium dioxide nanotubes (TiO2-NTs) are excellent drug carriers and have been considered as a potential material for polymer-free drug-eluting stents. However, TiO2-NTs reportedly induce severe blood clotting, which is a significant shortcoming for use as a stent. Vascular stents must be compatible with blood and must have antibacterial, anti-inflammatory, and selective inhibitory activities in the abnormal hyperplasia of smooth muscle cells, instead of delaying the re-endothelialization of endothelial cells. To meet these requirements, we presented a composite material that featured ultraviolet (UV) irradiation of TiO2-NTs-containing silver nanoparticles (AgNPs). The AgNPs were loaded in the lumen of TiO2-NTs as a representative compound to suppress the inflammatory response and hyperplasia. UV irradiation was performed as a novel method to improve the anticoagulant ability of the AgNP-loaded TiO2-NTs. The chemical state and biocompatibility of the UV-TiO2-NTs@AgNPs were evaluated. UV irradiation strongly improved the anticoagulant ability of the TiO2-NTs and moderated the release of Ag+ from AgNPs, which selectively suppressed the inflammatory response and hyperplasia. Furthermore, the UV-TiO2-NTs@AgNPs-2 displayed enhanced biocompatibility evidenced by the inhibition of platelet adhesion, bactericidal activity, selective suppression of the smooth muscle cell proliferation, and inhibition of the adhesion of macrophages. The collective findings indicate the potential of the photofunctionalized TiO2-NTs loaded with AgNPs as a material for polymer-free drug-eluting stents.

[An experimental study on the cytokine expression of macrophage influenced by biomaterials].

This experiment was designed to investigate the influence of two biomaterials, titanium oxide (Ti-O) and stainless steel (SS), on the cytokine expression of macrophage, and further, to evaluate their biocompatibility. After being co-cultured with Ti-O and SS for 72 h, the cell number and morphology of macrophages attached on materials were detected by fluorescent microscope and SEM. Nitride oxide (NO) and monocyte chemoattractant protein 1 (MCP-1) released by the macrophages co-cultured with different materials were also examined and compared. We found that the cell number of macrophages attached to Ti-O was smaller than that attached to SS. The levels of NO and MCP-1 released by the macrophages co-cultured with Ti-O were lower when compared with those released by macrophages co-cultured with SS. These results demonstrate that Ti-O has better biocompatibility than does SS.

[Anti-restenosis study on a new drug eluting stent].

The objective of this experimental study was to assess the effects of a new kind of drug eluting coronary stent. Fourteen mini-pigs were used; seven normal stainless stents and seven new drug eluting stents were implanted in their normal coronary arteries, respectively. Angiography was performed and followed by pressure-fixation of the coronary arteries for light and electron microscopic examinations at the end of three months after implantation. Repeated angiography showed that all the stented coronary segments were open. With no additional antithrombotic treatment, there was no thrombus formed in the stented coronary segments. Scanning electron microscopy analysis showed the implanted stent surface was covered by endomembrane without thrombus formation. The endothelial cell in the membrane was clear and lined by the direction of blood flow. Histomorphological analysis revealed the neointima in normal stainless stent group was thicker than that in new drug eluting stent group, and the neointima was composed of smooth muscle cell and extracellular matrix. The result of this study shows that this kind of stent could reduce the rate of the re-stenosis and occlusion of PTCA. This stent can be used in clinical trials.

[Study on a novel vascular stent material (titanium oxide, Ti-O) coated with albumin and heparin: is it hemocompatible with fibrinogen].

The functional hemocompatibility between fibrinogen (FIG) and a novel vascular stent material (Ti-O film fixed with albumin and heparin) was investigated as follows: (1) Preparing the new biologic material (Ti-O) film; (2) Coating albumin and heparin on the Ti-O film; (3) Testing platelets (PL) adsorption; (4) Determining FIG adhesion number by use of enzyme linked immunoassay (ELISA); (5) Implanting the films from the test group of Ti-O film and from the comparison group of stainless steel (SS) film into the left and right femoral arteries respectively in 4 dogs. It was proved that albumin and heparin were fixed on Ti-O film. After 6 months, the femoral arteries of the dogs were resected. In the test group of Ti-O film coated with albumin and heparin, few PL adhered to the coat, their form did not change, and no thrombus was found by scanning electron microscopy; the result was better than that of plain Ti-O film, and was much better than that of SS film. Ti-O maintained normal transformation condition of FIG, and no C terminal of gamma chain in FIG was revealed. As it is known whether the hemocompatibility of a biomaterial is good depends upon its adsorption of FIG, and Ti-O has excellent reaction on albumin and heparin by chemical processes. In this study, the Ti-O film coated with albumin and heparin further reduced the absorption of FIG and PL when compared against the plain Ti-O film. So the Ti-O film coated with albumin and heparin has the insistent and permanent anticoagulant character.

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

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.

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.

A novel coculture model of HUVECs and HUASMCs by hyaluronic acid micropattern on titanium surface.

Orientation smooth muscle cell environment plays a positive role in the development of a functional, adherent endothelium. Therefore, building an orientation coculture model of endothelial cells (ECs) and smooth muscle cells (SMCs) on biomaterials surface may provide more help for understanding the interaction between the two cells in vitro. In the present study, a "SMCs-ColIV-ECs" coculture model was built on the hyaluronic acid (HA) patterned titanium (Ti) surface, and compared with the previous "SMCs-HAa-ECs" model on endothelial cell number, morphology index, nitric oxide (NO), and prostacyclin2 (PGI2) release, anticoagulation property, human umbilical artery smooth muscle cells (HUASMCs) inhibition property and retention under fluid flow shear stress. The result indicated that "SMCs-ColIV-ECs" model could enhance the number, spreading area, and major/minor index of human umbilical vein endothelial cells (HUVECs), which contributed to the retention of HUVECs on the surface. Greater major/minor index may produce more NO and PGI2 release, contributing to the anticoagulation property and HUASMCs inhibition property. In summary, this novel "SMCs-ColIV-ECs" coculture model improved the previous "SMCs-HAa-ECs" model, and may provide more inspiration for the human vascular intima building on the biomaterials in vitro.

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.