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Co-immobilization of two or more molecules with different and complementary functions to prevent thrombosis, suppress smooth muscle cell (SMC) proliferation, and support endothelial cell (EC) growth is generally considered to be promising for the re-endothelialization on cardiovascular stents. However, integration of molecules with distinct therapeutic effects does not necessarily result in synergistic physiological functions due to the lack of interactions among them, limiting their practical efficacy. Herein, we apply heparin and nitric oxide (NO), two key molecules of the physiological functions of endothelium, to develop an endothelium-mimetic coating. Such coating is achieved by sequential conjugation of heparin and the NO-generating compound selenocystamine (SeCA) on an amine-bearing film of plasma polymerized allylamine. The resulting surface combines the anti-coagulant (anti-FXa) function provided by the heparin and the anti-platelet activity of the catalytically produced NO. It also endows the stents with the ability to simultaneously up-regulate α-smooth muscle actin (α-SMA) expression and to increase cyclic guanylate monophosphate (cGMP) synthesis of SMC, thereby significantly promoting their contractile phenotype and suppressing their proliferation. Importantly, this endothelium-biomimetic coating creates a favorable microenvironment for EC over SMC. These features impressively improve the antithrombogenicity, re-endothelialization and anti-restenosis of vascular stents in vivo.

Nitric oxide (NO) is a highly potent, yet short-lived bioactive molecule with a broad spectrum of physiological functions. Continuous and controllable in situ generation of NO from vascular stent surface can effectively prevent restenosis and thrombosis after its implantation. In this study, inspired by the adhesion and protein cross-linking in the mussel byssus, through immersing the stents into an aqueous solution with dopamine (DA) and copper ions (Cu II), we developed a one-step metal-catecholamine assembled strategy to prepare a durable in situ NO-generating biomimetic coating (DA-Cu II). Due to the high NO catalytic efficacy and robust chelation of CuII into the DA-CuII network, the coated stents exhibited excellent hemocompatibility . The coating also catalytically decomposed endogenous S-nitrosothiols (RSNOs) from fresh blood, and locally generated NO for over 30 days with a flux comparable to its physiological range (0.5–4 × 10 −10 mol × cm−2 × min−1 ). Moreover, the optimized biomimetic coatings displayed specific cell selectivity to significantly enhance endothelial cell (EC) growth while substantially inhibit smooth muscle cell (SMC) growth and migration. This feature impressively promoted regeneration of a new endothelial cell layer after stent implantation , hence improved the anti-thrombogenic and anti-restenosis qualities of vascular stents in vivo. We envision that our long-term in situ NO-generating coatings could serve as biosurfaces for long-term prevention of stent thrombosis and restenosis.

The development of a nitric oxide (NO)-generating surface with long-term, stable and controllable NO release improves the therapeutic efficacy of cardiovascular stents . In this work, we developed a “one-pot” method inspired by mussel adhesive proteins for copolymerization of selenocystamine (SeCA) and dopamine (Dopa) to form a NO-generating coating on a 316 L stainless steel (SS) stent. This “one-pot” method is environmentally friendly and easy to popularize, with many advantages including simple manufacturing procedure, high stability and no involvement of organic solvents . Such SeCA/Dopa coatings also enabled us to develop a catalytic surface for local NO-generation by reaction of endogenously existing S-nitrothiol species from fresh blood. We found that the developed SeCA/Dopa coatings could release NO in a controllable and stable manner for more than 60 days. Additionally, the released NO significantly inhibited smooth muscle cell (SMC) proliferation and migration, as well as platelet activation and aggregation through the up-regulation of cyclic guanosine monophosphate synthesis. Moreover, such NO generation enhanced the adhesion, proliferation and migration of endothelial cells (ECs), and achieved rapid in vivo re-endothelialization, effectively reducing in-stent restenosis and neointimal hyperplasia . We envision that the SeCA/Dopa-coated 316 L SS stent could be a promising platform for treatment of cardiovascular diseases.

Specific selectivity of vascular cells and antithrombogenicity are crucial factors for the long-term success of vascular implants. In this work, a novel concept of mussel-inspired "built-up" surface chemistry realized by sequential stacking of a copper-dopamine network basement, followed by a polydopamine layer is introduced to facilitate the combination of nitric oxide (NO) catalysis and vascular cell selectivity. The resultant "built-up" film allowed easy manipulation of the content of copper ions and the density of catechol/quinone groups, facilitating the multifunctional surface engineering of vascular devices. For example, the chelated copper ions in the copper-dopamine network endow a functionalized vascular stent with a durable release of NO via catalytic decomposition of endogenous S-nitrosothiol. Meanwhile, the catechol/quinone groups on the film surface allow the facile, secondary grafting of the REDV peptide to develop a selectivity for vascular cells, as a supplement to the functions of NO. As a result, the functionalized vascular stent perfectly combines the functions of NO and REDV, showing excellent antithrombotic properties and competitive selectivity toward the endothelial cells over the smooth muscle cells, hence impressively promotes re-endothelialization and improves anti-restenosis in vivo. Therefore, the first mussel-inspired "built-up" surface chemistry can be a promising candidate for the engineering of multifunctional surfaces.

Antithrombogenicity, anti-inflammation, and rapid re-endothelialization are central requirements for the long-term success of cardiovascular stents. In this work, a plant-inspired phenolic-amine chemistry strategy was developed to combine the biological functions of a plant polyphenol, tannic acid (TA), and the thrombin inhibitor bivalirudin (BVLD) for tailoring the desired multiple surface functionalities of cardiovascular stents. To realize the synergistic modification of TA and BVLD on a stent surface, an amine-bearing coating of plasma polymerized allylamine was firstly prepared on the stent surface, followed by the sequential conjugation of TA and BVLD in alkaline solution based on phenolic-amine chemistry (i.e., Michael addition reaction). TA and BVLD were successfully immobilized onto the stent surface with considerable amounts of 330 ± 12 and 930 ± 80 ng/cm2, respectively. The abundant phenolic hydroxyl groups of TA imparted the stent with ability to suppress inflammation. Meanwhile, BVLD provided an antithrombogenic and endothelial-friendly microenvironment. As a result, the combined functions of the TA and BVLD facilitate the rapid stent re-endothelialization for reduced intimal hyperplasia in vivo, and may be a promising strategy to address the clinical complications associated with restenosis and late stent thrombosis.

Regarding the high requirement of cardiac and vascular implants in tissue engineering, a novel concept of surface chemistry strategy featuring multiple functions is proposed in this study, which provides glutathione peroxidase (GPx)-like catalytic activity and allows secondary reactions for grafting functional biomolecules. The suggested strategy is the fabrication of a metal-catechol-(amine) network (MCAN) containing copper ions with GPx-like activity, amine-bearing hexamethylenediamine (HD) and wet adhesive catechol dopamine (DA). With a simple one-step molecular/ion co-assembly, the developed copper-DA-HD (CuII-DA/HD) network can be used to catalyze the generation of therapeutic nitric oxide (NO) gas in a durable and dose-controllable manner. The primary amine groups in the CuII-DA/HD network facilitate the secondary immobilization of bivalirudin (BVLD) to further provide an antithrombotic activity as supplement to the functions of NO. The CuII-DA/HD + BVLD coating functionalized on cardiovascular stents successfully improved thromboresistance, anti-restenosis, and promotes re-endothelialization in vivo. With regard to the ease of operation and low cost, the synergetic modification using MCAN strategy is of great potential for developing multifunctional blood-contacting materials/devices.

Nitric oxide (NO) is a highly potent, yet short-lived bioactive molecule with a broad spectrum of physiological functions. Continuous and controllable in situ generation of NO from vascular stent surface can effectively prevent restenosis and thrombosis after its implantation. In this study, inspired by the adhesion and protein cross-linking in the mussel byssus, through immersing the stents into an aqueous solution with dopamine (DA) and copper ions (Cu

Immobilization of selenocystamine on TiO(2) film deposited on silicon wafer and 316 stainless steel stents for catalytic generation of nitric oxide was described. Polydopamine was used as the linker for immobilization of selenocystamine to the TiO(2) surface. In vitro stability of the immobilized selenocystamine was investigated and the result shows surface selenium loss occurs mostly in the first four weeks. The selenocystamine immobilized surface possesses glutathione peroxidase (GPx) activity, and the activity increases with the amount of grafted polydopamine. Such selenocystamine immobilized surfaces show the ability of catalytically decomposing endogenous S-nitrosothiols (RSNO), generating NO; thus the surface displays the ability to inhibit collagen-induced platelet acitivation and aggregation. Additionally, smooth muscle cells are inhibited from adhering to the selenocystamine immobilized sample when RSNO is added to the culture media. ELISA analysis reveals that cGMP in both platelets and smooth muscle cells significantly increases with NO release on selenocystamine immobilized samples. Two months in vivo results show that selenocystamine immobilized stents are endothelialized, and show significant anti-proliferation properties, indicating that this is a favorable method for potential application in vascular stents.

For an improved hemocompatibility of 316L stainless steel (SS), we develop a facile and effective approach to fabricating a pulsed-plasma polymeric allylamine (P-PPAm) film that possesses a high cross-linking degree and a high density of amine groups, which is used for subsequent bonding of heparin. The P-PPAm film as a stent coating shows good resistance to the deformation behavior of compression and expansion of a stent. Using deionized water as an aging medium, it is demonstrated that the heparin-immobilized P-PPAm (Hep-P-PPAm) surface has a good retention of heparin. The systematic in vitro hemocompatibility evaluation reveals lower platelet adhesion, platelet activation and fibrinogen activation on the Hep-P-PPAm surface, and the activated partial thromboplastin time prolongs for about 15 s compared with 316L SS. The P-PPAm surface significantly promotes adhesion and proliferation of endothelial cells (ECs). For the Hep-P-PPAm, although EC adhesion and proliferation is slightly suppressed initially, after cultivation for 3 days, the growth behavior of ECs is remarkably improved over 316L SS. In vivo results indicate that the Hep-P-PPAm surface successfully restrain thrombus formation by growing a homogeneous and intact shuttle-like endothelium on its surface. The Hep-P-PPAm modified 316L SS shows a promising application for vascular devices.

The continuous release of nitric oxide (NO) by the native endothelium of blood vessels plays a substantial role in the cardiovascular physiology, as it influences important pathways of cardiovascular homeostasis, inhibits vascular smooth muscle cell (VSMC) proliferation, inhibits platelet activation and aggregation, and prevents atherosclerosis. In this study, a NO-catalytic bioactive coating that mimics this endothelium functionality was presented as a hemocompatible coating with potential to improve the biocompatibility of vascular stents. The NO-catalytic bioactive coating was obtained by covalent conjugation of 3,3-diselenodipropionic acid (SeDPA) with glutathione peroxidase (GPx)-like catalytic activity to generate NO from S-nitrosothiols (RSNOs) via specific catalytic reaction. The SeDPA was immobilized to an amine bearing plasma polymerized allylamine (PPAam) surface (SeDPA-PPAam). It showed long-term and continuous ability to catalytically decompose endogenous RSNO and generate NO. The generated NO remarkably increased the cGMP synthesis both in platelets and human umbilical artery smooth muscle cells (HUASMCs). The surface exhibited a remarkable suppression of collagen-induced platelet activation and aggregation. It suppressed the adhesion, proliferation and migration of HUASMCs. Additionally, it was found that the NO catalytic surface significantly enhanced human umbilical vein endothelial cell (HUVEC) adhesion, proliferation and migration. The in vivo results indicated that the NO catalytic surface created a favorable microenvironment of competitive growth of HUVECs over HUASMCs for promoting re-endothelialization and reducing restenosis of stents in vivo.

Galectin-1 (GAL1), a beta-galactoside-binding protein, functions in cell adhesion, development, and growth regulation. A number of studies suggest that GAL1 play an important role in enhancing cell adhesion to extracellular matrix and inducing cell proliferation. Chitosan is a derivative of chitin extracted from lobsters, crabs and shrimps' exoskeletons. In clinical medicine, chitosan membrane had been used as a semi-permeable biological dressing. Although chitosan membranes show no cytotoxicity, some cell types (e.g. 3T3 cells) fail to attach and proliferate on their surface. In these studies, we show that over-expression of GAL1 does not enhance 3T3 cell proliferation on chitosan membranes. However, coating the chitosan membrane with recombinant GAL1 proteins significantly expedites 3T3 cells proliferation. The enhanced cell growth was inhibited by thiodigalactoside (TDG, a potent inhibitor of beta-galactoside binding) and GAL1 monoclonal antibodies, suggesting GAL1's specific effect on the proliferation of 3T3 cells upon chitosan membranes. Moreover, immunoblotting detected a markedly suppressed tyrosine phosphorylation in several proteins on 3T3 cell growths upon GAL1-coated chitosan membrane. Pretreating the cells with sodium fluoride (NaF, a phosphatase inhibitor) inhibits the attachment and proliferation of 3T3 cells. These findings support a proposed role for altered levels of protein phosphorylation in GAL1-mediated cell attachment and proliferation on chitosan membranes.

Amorphous carbon films have attracted much attention recently due to their good biocompatibility. Diamond-like carbon (DLC), one form of amorphous carbon that is widely used in many kinds of industries, has been proposed for use in blood contacting medical devices. However, the blood coagulation mechanism on DLC in a biological environment is not well understood. Platelet adhesion and activation are crucial events in the interactions between blood and the materials as they influence the subsequent formation of thrombus. In this work, the behavior of platelets adhered onto hydrogenated amorphous carbon films (a-C:H) is investigated. Hydrogenated amorphous carbon films with different hydrogen contents, structures, and chemical bonds were fabricated at room temperature using plasma immersion ion implantation-deposition (PIII-D). The wettability of the films was investigated by contact angle measurements using several common liquids. Platelet adhesion experiments were conducted to examine the interaction of blood with the films in vitro and the activation of adherent platelets. The results show that the behavior of the platelets adhered on the a-C:H films is influenced by their structure and chemical bond, and it appears that protein interaction plays a key role in the activation of the adherent platelets.

A novel asymmetric chitosan membrane has been prepared by immersion-precipitation phase-inversion method and evaluated as wound covering. This new type of chitosan wound dressing which consists of skin surface on top-layer supported by a macroporous sponge-like sublayer was designed. The thickness of the dense skin surface and porosity of sponge-like sublayer could be controlled by the modification of phase-separation process using per-evaporation method. The asymmetric chitosan membrane showed controlled evaporative water loss, excellent oxygen permeability and promoted fluid drainage ability but could inhibit exogenous microorganisms invasion due to the dense skin layer and inherent antimicrobial property of chitosan. Wound covered with the asymmetric chitosan membrane was hemostatic and healed quickly. Histological examination confirmed that epithelialization rate was increased and the deposition of collagen in the dermis was well organized by covering the wound with this asymmetric chitosan membrane. The results in this study indicate that the asymmetric chitosan membrane thus prepared could be adequately employed in the future as a wound dressing.

The study was designed to characterize the surface properties (including water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility) of a biological patch fixed with genipin, a naturally occurring crosslinking agent. Fresh and glutaraldehyde-fixed counterparts were used as controls. It was found that both glutaraldehyde and genipin are effective crosslinking agents for biological tissue fixation. Fixation of biological tissue with glutaraldehyde or genipin significantly increased its hydrophilicity and surface tension and reduced its mol ratio of adsorbed fibrinogen to adsorbed albumin as well as the amount of adhered platelet. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues. However, the cellular compatibilities of fresh and the genipin-fixed tissues were significantly superior to the glutaraldehyde-fixed tissue.

Multidentate ligands are expected to improve the performance of inorganic nanoparticles (NPs) in biological application. Designing robust multidentate ligands by a facile way and understanding the impact of ligand composition on NP's property are greatly important. We report the effective synthesis of hydrophilic copolymers containing pendent thiol groups along a polyethylene glycol (PEG) methacrylate backbone by classical free radical copolymerization. Gold nanoparticles (AuNPs) coated by these multidentate ligands with two different ratios of thiols to PEG segment (∼1:1 and 1:2) showed much higher colloidal stability in the presence of dithiothreitol (DTT) than AuNPs coated by monothiol-anchored PEG, and AuNPs coated by ligands with higher fraction of thiol groups showed slightly better resistance to DTT competition than did AuNPs coated by ligands with lower thiol fraction, but both of them exhibited excellent stabilities in biological media without obvious difference. In vitro study of uptake by macrophages did not showed significant difference between the two AuNPs with very low endocytosis. However, AuNPs coated by ligands with higher PEG content were found to accumulate in liver with a significantly lower level but a higher level in spleen than AuNPs coated by ligands with lower PEG contents. Moreover, the AuNPs coated with by ligands with higher PEG content showed higher tumor uptake. Additionally, AuNPs coated with both ligands demonstrated good biocompatibility as evaluated by cytotoxicity assays and histological analysis. Together, the composition of multidentate ligands will not only affect the stability of NPs under extreme conditions but also result in quite different fate of NPs in vivo, which can be tailored case by case.

The direct thrombin inhibitor of bivalirudin (BVLD), a short peptide derived from hirudin, has drawn an increasing attention in clinical application because it is safer and more effective than heparin for diabetic patients with moderate- or high-risk for acute coronary syndromes (ACS). In this study, BVLD was covalently conjugated on plasma polymerized allylamine (PPAam) coated 316L stainless steel (SS) to develop an anticoagulant surface. QCM-D real time monitoring result shows that 565±20 ng/cm2 of BVLD was bound to the PPAam surface. Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of BVLD. The conjugation of BVLD onto the PPAam coating led to enhanced binding of thrombin, and the activity of the thrombin adsorbed on its surface was effectively inhibited. As a result, the BVLD immobilized PPAam (BVLD-PPAam) substrate prolonged the clotting times, and exhibited inhibition in adhesion and activation of platelets and fibrinogen. We also found that the BVLD-PPAam coating significantly enhanced endothelial cell adhesion, proliferation, migration and release of nitric oxide (NO) and secretion of prostaglandin I2 (PGI2). In vivo results indicate that the BVLD-PPAam surface restrained thrombus formation by rapidly growing a homogeneous and intact endothelium on its surface. These data suggest the potential of this multifunctional BVLD-PPAam coating for the application not only in general vascular devices such as catheters, tubes, oxygenator, hemodialysis membranes but also vascular grafts and stents.

In this work, the effects of a heparin-functionalized coating on the growth behavior of vascular cells were studied. To retain its functionality, heparin was bound to a cationic plasma polymerized allylamine coating through electrostatic interaction. The heparin binding surface significantly inhibited human umbilical artery smooth muscle cell (HUASMC) adhesion and proliferation. In contrast, human umbilical vein endothelial cells (HUVECs) showed significant enhancement in cell adhesion, proliferation and migration, release of nitric oxide (NO) and secretion of prostaglandin I 2 (PGI 2 ). The test of acute thrombogenicity assessed using human blood exhibited an excellent antithrombotic performance of the heparin grafted surface. The heparinized surface significantly promoted in vivo re-endothelialization and effectively inhibited thrombosis formation. These observations form an important framework for further deciphering the biological functions of heparin. It is highlighted that these striking findings may serve as a guide for the design of multifunctional vascular devices.

Currently available cardiovascular implants, such as heart valves and stents, exhibit suboptimal biocompatibility because of the incomplete endothelialization and sequential thrombosis formation especially after a long-term implantation. To improve the blood compatibility and endothelialization simultaneously and ensure the long-term effect of the cardiovascular implants, a technique of combining electrostatic interaction and coimmobilization was developed to form heparin and fibronectin (Hep/Fn) films on aminosilanized titanium (Ti) surfaces. The Hep/Fn coimmobilized films were stable after immersion in PBS for five days, probed by wettability studies and by the release kinetics of heparin and fibronectin. Blood compatibility tests showed that the coimmobilized Hep/Fn films displayed lower hemolysis rate, prolonged blood coagulation time, higher AT III binding density, less platelets activation and aggregation, and less fibrinogen conformational change compared with Ti surface. Endothelial cells (ECs) seeding and fibronectin bioactivity results showed more attached and proliferated ECs and exposed cell-binding sites on the Hep/Fn immobilized samples than that on Ti surfaces. Thus, the Hep/Fn coimmobilized films kept excellent bioactivity even after immersion in PBS for five days. Systemic evaluation suggests that the coimmobilization of Hep/Fn complex improves the blood compatibility and promotes the endothelialization simultaneously. We envisage that this method will provide a potential and effective selection for biomaterials surface modification of cardiovascular implants.

There is an increasing interest in developing new methods to reduce bacteria adhesion onto polymeric materials that are used in biomedical implants. The antibacterial behavior on polyethylene terephthalate (PET) treated by acetylene (C2H2) plasma immersion ion implantation-deposition (PIII-D) is investigated. The surface structure of the treated PET is determined by laser Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The results show that a thin amorphous polymer-like carbon (PLC) layer is formed on the PET surface. Atomic force micrographs (AFM) show that C2H2 PIII-D significantly changes the surface morphology of PET. The capacities of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) to adhere onto PET are quantitatively determined by plate counting and Gamma-ray counting of 125I radio labeled bacteria in vitro. The results indicate that the adhesion of the two kinds of bacteria to PET is suppressed by PLC. The adhesion efficiency of SE on the coated surface is only about 14% of that of the untreated PET surface, and that of SA is about 35% of that of the virgin surface. The electrokinetic potentials of the bacterial cells and substrates are determined by zeta potential measurement. All the substrates as well as the bacterial strain have negative zeta potentials, and it means that bacterial adhesion is not mediated by electrostatic interactions. The surface energy components of the various substrates and bacteria are calculated based on measurements in water, formamide and diiodomethane. The surface free energies obtained are used to calculate the interfacial free energies of adhesion ( deltaFAdh ) of SA and SE onto various substrates, and it is found that bacterial adhesion is energetically unfavorable on the PLC deposited on PET by C2H2 PIII-D.

Hemocompatibility is a key property of biomaterials that come in contact with blood. Surface modification has shown great potential for improving the hemocompatibility of biomedical materials and devices. In this paper, we describe our work of improving hemocompatibility with Ti-O thin films prepared by plasma immersion ion implantation and deposition and by sputtering. The structure and surface chemical and physical properties of the films were characterized by X-ray diffraction, Auger electron spectroscopy, atomic force microscopy (AFM), contact angle measurement, and Hall effect measurement. The behavior of fibrinogen adsorption was investigated by 125I radioactive isotope labeling and AFM. Systematic evaluation of hemocompatibility, including in vitro clotting time, thrombin time, prethrombin time, platelet adhesion, and in vivo implantation into dog's ventral aorta or right auricle from 17 to 90 days, proved that Ti-O films have excellent hemocompatibility. It is suggested that the significantly lower interface tension between Ti-O films and blood and plasma proteins and the semiconducting nature of Ti-O films give them their improved hemocompatibility.

Recent improvements in the antithrombogenic properties of blood contacting biomaterials permit a hybrid design of layers for biomedical applications such as artificial heart valves and stents. Using magnetron sputtering and thermal oxidation, titanium oxide thin films containing tantalum. Ti(Ta(+5))O2, are fabricated to meet the challenge of enhanced hemocompatibility. The blood compatibility is evaluated in vitro by clotting time and platelet adhesion measurement, and in vivo experiments are also conducted. The Ti(Ta(+5))O2 films exhibit attractive blood compatibility exceeding that of low isotropic pyrolytic carbon. Physical properties such as surface energy and semiconductivity are found to play important roles. Our calculated results reveal that the smaller surface force gamma(s) of the film and the smaller blood film interfacial tension gamma(c,blood) are partially responsible for the enhancement of the blood compatibility. Based on the optical bandgap model, the film possesses better hemocompatibility because its optical bandgap of 3.2 eV is wider than that of fibrinogen having a bandgap of 1.8 eV. These factors result in thinner protein layers on the film surface, less protein denaturing, and overall excellent antithrombogenic properties.