Jialong Chen

Collagen/heparin coating on titanium surface improves the biocompatibility of titanium applied as a blood-contacting biomaterial

Thrombosis and restenosis are the main causes leading to failure of cardiovascular and other blood-contacting biomedical devices. It is recognized that rapid re-endothelialization is a promising method for preventing these complications. This article deals with improving the endothelial progenitor cell (EPC) compatibility and hemocompatibility of titanium by coating an extracellular matrix-like film with heparin(hep) and collagen(col) by a layer-by-layer (LBL) self-assembly technique. In the work described here, LBL-produced col/hep coating growth is initialized by deposition of a layer of poly-L-lysine on a titanium surface, which is negatively charged after treatment with NaOH, followed by formation of a multilayer film formed by alternating deposition of negatively charged heparin and positively charged collagen using electrostatic interaction. The X-ray photoelectron spectroscopy results and fluorescence staining of collagen show that collagen is predominant on the surface and that collagen interpenetrates the heparin layer. In vitro EPC attachment and proliferation increase greatly on the col/hep coating. Immunofluorescent staining of cytoskeleton actin reveals that cells on the col/hep coating form a compact confluent cell layer after culture for 3 days. After culture for 5 days, cell viability on the col/hep increases persistently and on titanium the cell viability begins to decrease, showing that the coating possesses the ability to maintain cell viability. Platelet adhesion under dynamic conditions in vitro implies that the hemocompatibility of the col/hep coating is superior to that of titanium. The col/hep coating improves the biocompatibility of titanium and has good potential for application in blood-contacting biomaterials.

Endothelial Cell and Platelet Behavior on Titanium Modified with a Mutilayer of Polyelectrolytes

Endothelial cell seeding, a promising method for improving the performance of vascular grafts, often requires immobilizing biological molecules on the surface of the substrate material. In this study, chitosan (CS) and sulfated chitosan (SCS) multilayers were coated on pure titanium using a layer-by-layer self-assembly technique. The CS—SCS multilayer growth was carried out by first depositing a single layer of positively charged poly(L-lysine) (PLL) on the NaOHtreated titanium substrate, followed by alternate deposition of negatively charged SCS and positively charged CS, and terminated by an outermost layer of SCS. Platelet-rich plasma (PRP) and endothelial cells were seeded on NaOH treated titanium and CS—SCS coated titanium samples, respectively, to evaluate the adhesion and activation of platelets and the behavior of endothelial cells in vitro. The multilayer processed surfaces displayed reduced platelet adhesion and activation, and promoted endothelial cell attachment and growth in vitro. This approach may be used for the fabrication of titanium-based vascular implant surfaces for endothelial promotion.

Anticoagulant surface modification of titanium via layer‐by‐layer assembly of collagen and sulfated chitosan multilayers

Extracellular matrix (ECM)-like biomimetic surface modification of cardiovascular implants is a promising method for improving hemocompatibility. In the present work, collagen (Col) and sulfated chitosan (SCS) multilayers were coated on pure titanium using a layer-by-layer (LBL) self-assembly technique. The Col-SCS multilayer growth was carried out by first depositing a single layer of positively charged poly-L-lysine (PLL) on the NaOH-treated titanium substrate (negatively charged surface), followed by alternate deposition of negatively charged SCS and positively charged Col, and terminated by an outermost layer of SCS. Platelet adhesion in vitro, partial activated thromboplastin time (APTT) and prothrombin time (PT) assays were used to evaluate the hemocompatibility of the Col-SCS multilayer coated titanium. The multilayer processed surfaces displayed reduced platelet adhesion and activation, and prolonged clotting time of APTT and PT compared with untreated titanium. Thus, the approach described here may provide a basis for the preparation of modified titanium surfaces for application in cardiovascular implants.

Biofunctionalization of titanium with PEG and anti-CD34 for hemocompatibility and stimulated endothelialization

Thrombosis and restenosis are the main causes of failure of cardiovascular and other blood-contacting biomedical devices. It is recognized that rapid endothelialization is a promising method for preventing these complications. Convincing evidence in vivo has further emerged that the vascular homing of endothelial progenitor cells (EPCs) contributes to rapid endothelial regeneration. This study deals with improving the hemocompatibility and enhancing EPC colonization of titanium by covalently bonding PEG(600) or PEG(4000), then end-grafting of an anti-CD34 antibody. For this, a chemically hydroxylated titanium surface was aminosilanized, which was further used for covalent grafting of polyethylene glycol and the antibody. The grafting efficiency was verified in each step. In vitro platelet adhesion analysis confirmed superior hemocompatibility of the modified surface over the control. Affinity of EPC to the surface and inhibition of smooth muscle cell adhesion, two prerequisites for endothelialization, are demonstrated in in vitro cell culture. While the coating selectively stimulates EPC adhesion, its antifouling properties prevent formation of an extracellular matrix and proliferation of the cells. Additional affinity for matrix proteins in the coating is considered for further studies. Potent inhibitory effect on macrophage activation and the relative stability of the coating render this technique applicable.

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.

Guidance of Stem Cells to a Target Destination in Vivo by Magnetic Nanoparticles in a Magnetic Field

Stem cells contribute to physiological processes such as postischemic neovascularization and vascular re-endothelialization, which help regenerate myocardial defects or repair vascular injury. However, therapeutic efficacy of stem cell transplantation is often limited by inefficient homing of systemically administered cells, which results in a low number of cells accumulating at sites of pathology. In this study, anti-CD34 antibody-coated magnetic nanoparticles (Fe3O4@PEG-CD34) are shown to have high affinity to stem cells. The results of hemolysis rate and activated partial thromboplastin time (APTT) tests indicate that such nanoparticle may be used safely in the blood system. In vitro studies showed that a nanoparticle concentration of 100 μg/mL gives rise to a significant increase in cell retention using an applicable permanent magnet, exerting minimal negative effect on cell viability and migration. Subsequent in vivo studies indicate that nanopartical can specifically bind stem cells with good magnetic response. Anti-CD34 antibody coated magnetic nanoparticle may be used to help deliver stem cells to a lesion site in the body for better treatment.

Immobilization of the direct thrombin inhibitor-bivalirudin on 316L stainless steel via polydopamine and the resulting effects on hemocompatibility in vitro†

Bivalirudin (BV), a peptidic direct thrombin inhibitor, derived from hirudin, has gained increasing interest in clinical anticoagulant therapy in the recent years. In this work, a hemocompatible surface was prepared by immobilization of BV on 316L stainless steel (SS) using a bonding layer of polydopamine (DA). X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces to characterize polydopamine intermediate layer and the immobilized BV. The quantity of bound BV was measured by quartz crystal microbalance (QCM). The hemocompatibility in vitro was evaluated by coagulating time of activated partial thromboplastin time (aPTT) and prothrombin time (PT) assay, platelet adhesion and activation, fibrinogen adsorption, and activation and whole blood test. The effect of sterilizing method on the bioactivity of immobilized BV was also evaluated. The results showed that BVs were successfully immobilized on SS surface with the DA interlayer at a density of 98 ng/cm(2) . BV coating surface prolonged aPTT and PT, inhibited the activation of platelet and fibrinogen significantly. Sterilization by ultraviolet radiation was possible with only marginal loss of activity. Thus, the approach described here may provide a basis for the preparation of 316L SS surface modification for use in cardiovascular implants.

Oriented immobilization of anti-CD34 antibody on titanium surface for self-endothelialization induction.

Inducing spontaneous endothelialization of synthetic cardiovascular implant in vivo is thought to be a promising approach to solve the surface-induced thrombosis and restenosis problem. In the present study, anti-CD34 antibody, a kind of special marker of EPC, was oriented immobilized on titanium surface by means of a layer-by-layer self-assembly coating technique. The multilayer coating was prepared by first depositing one layer of avidin on the NaOH-treated titanium substrate, then depositing a layer of biotinylated protein A binding to the avidin, and finally anti-CD34 antibody was oriented immobilized by protein A binding to the Fc fragment (COOH-terminal of a antibody molecule, which has no antigen binding sites) of the anti-CD34 antibody with its antigen binding fragment (Fab) away from the titanium surface. The coated titanium was exposed to EPC derived from mouse bone marrow in vitro, and implanted into dog femoral arteries. The results suggested that the anti-CD34 antibody immobilized surfaces could increase EPC attachment and capture, and induce rapid complete endothelialization of the lumenal surface of the implant in vivo. It suggests that the approach described here may be used for fabrication of titanium-based vascular implant surfaces for inducing endothelialization in vivo.

Immobilization of serum albumin and peptide aptamer for EPC on polydopamine coated titanium surface for enhanced in-situ self-endothelialization

Restenosis and thrombosis are two major complications associated with vascular stents and grafts. The homing of circulating endothelial progenitor cells (EPCs) onto implant surfaces brings a new strategy to solve these problems by accelerating self -endothelialization in situ. Peptide aptamers with high affinity and specific recognition of EPCs can be immobilized to capture EPCs from the circulating blood. In this study, a biotinylated peptide aptamer (TPSLEQRTVYAK-GGGC-K-Biotin) for EPC, and bovine serum albumin (BSA) were co-immobilized onto titanium surface through avidin-biotin recognition to endow the surface with specific affinity for EPC and anti-platelet adhesion properties. Quartz crystal microbalance with dissipation (QCM-D), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and water contact angle measuring were adopted for coating characterization. EPC affinity and hemocompatibility of the coating were also investigated in vitro. The results demonstrated that aptamer and BSA co-immobilized surface significantly reduced platelet adhesion and fibrinogen adsorption/activation. Besides, such functional surface could remarkably enhance EPC adhesion, without affecting the behavior of endothelial cells (ECs) and smooth muscle cells (SMCs) obviously. The result shows the possibility of utilizing such a multifunctional surface in cardiovascular implants.

New strategies for developing cardiovascular stent surfaces with novel functions (Review)

In this review, the authors summarize the developments in surface modification of cardiovascular materials especially in authors laboratory. The authors focus on three different strategies to construct multifunctional surfaces including coimmobilization of various biomolecules on stent surfaces, stem cell based therapy systems, and a single-molecule multipurpose modification strategy in vascular interventional therapy. The roles of various molecules like heparin, gallic acid, various aptamers, and nitric oxide are highlighted in the new strategies for developing cardiovascular stent surfaces with novel functions including excellent hemocompatibility, inhibiting smooth muscle cells proliferation, and native endothelium regeneration. The success of these multifunctional surfaces provides the tremendous potential in designing the next generation of vascular stents.