Hao Chang

Direct Adhesion of Endothelial Cells to Bioinspired Poly(dopamine) Coating Through Endogenous Fibronectin and Integrin α5β1

Mussel-inspired poly(dopamine) (PDA) coating is proven to be a simple, versatile, and effective strategy to promote cell adhesion onto various substrates. In this study, the initial adhesive behavior of human umbilical vein endothelial cells (HUVECs) is evaluated on a PDA coating under serum-free conditions. It is found that HUVECs can attach directly to and spread with well-organized cytoskeleton and fibrillar adhesions on the PDA surface, whereas cells adhere poorly to and barely spread on the control polycaprolactone surface. Endogenous fibronectin and α5 β1 integrin are found to be involved in the cell adhesion process. These findings will lead to a better understanding of interactions between cells and PDA coating, paving the way for the further development of PDA.

Facile fabrication of robust superhydrophobic multilayered film based on bioinspired poly(dopamine)-modified carbon nanotubes

Thin organic films containing carbon nanotubes (CNTs) have received increasing attention in many fields. In this study, a robust thin superhydrophobic film has been created by using layer-by-layer assembly of the carbon nanotubes wrapped by poly(dopamine) (CNT@PDA) and poly(ethyleneimine) (PEI). UV-vis spectroscopy, ellipsometry, and quartz crystal microbalance with dissipation (QCM-D) measurements confirmed that the sequential deposition of PEI and CNT@PDA resulted in a linear growth of the (PEI-CNT@PDA) film. This thin film contained as much as 77 wt% CNTs. Moreover, a very stable and flexible free-standing (PEI-CNT@PDA) film could be obtained by employing cellulose acetate (CA) as a sacrificial layer. The film could even withstand ultrasonication in saturated SDS aqueous solution for 30 min. SEM observations indicated that the ultrathin film consisted of nanoscale interpenetrating networks of entangled CNTs and exhibited a very rough surface morphology. The (PEI-CNT@PDA) film turned superhydrophobic after being coated with a low-surface-energy compound. The superhydrophobic films showed excellent resistance against the adhesion of both platelets and Escherichia coli (E. coli). The (PEI-CNT@PDA) films and the proposed methodology may find applications in the area of medical devices to reduce device-associated thrombosis and infection.

Surface-mediated functional gene delivery: an effective strategy for enhancing competitiveness of endothelial cells over smooth muscle cells.

The non-biorecognition of general biomaterials and inherent biospecificity of biological systems pose key challenges to the optimal functions of medical devices. In this study, we constructed the surface-mediated functional gene delivery through layer-by-layer self-assembly of protamine sulfate (PrS) and plasmid DNA encoding hepatocyte growth factor (HGF), aiming at specific enhancing endothelial cells (EC) compeititiveness over smooth muscle cells (SMC). Characterizations of the (PrS/HGF-pDNA) multilayered films present the linear buildup with homogeneous and flat topographical feature. The amount of DNA can be easily controlled. By using these multilayered films, both human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (HUASMC) can be directly transfected when they contact with the multilayered films. On transfection, increasing secretion of HGF has been detected in both HUVEC and HUASMC culture, which leads to selective promotion of HUVEC proliferation. In the co-culture experiment, we also exhibit the promoted and hindered growth of HUVEC and HUASMC, respectively, which could be attributed to the inverse influence of HUVEC on HUASMC. These results collectively demonstrate that our system can be served as a powerful tool for enhancing competitiveness of EC over SMC, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy.

Effect of Polyelectrolyte Film Stiffness on Endothelial Cells During Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndMT), during which endothelial cells (ECs) transdifferentiate into mesenchymal phenotype, plays a key role in the development of vascular implant complications such as endothelium dysfunction and in-stent restenosis. Substrate stiffness has been confirmed as a key factor to influence EC behaviors; however, so far, the relationship between substrate stiffness and EndMT has been rarely studied. Here, ECs were cultured on the (poly(L-lysine)/hyaluronate acid) (PLL/HA) multilayer films with controlled stiffness for 2 weeks, and their EndMT behaviors were studied. We demonstrated that ECs lost their markers (vWf and CD31) in a stiffness-dependent manner even without supplement of growth factors, and the softer film favored the maintaining of EC phenotype. Further, induced by transforming growth factor β1 (TGF-β1), ECs underwent EndMT, as characterized by losing their typical cobblestone morphology and markers and gaining smooth muscle cell markers (α-smooth muscle actin and calponin). Interestingly, stronger EndMT was observed when ECs were cultured on the stiffer film. Collectively, our findings suggest that substrate stiffness has significant effects on EndMT, and a softer substrate is beneficial to ECs by keeping their phenotype and inhibiting EndMT, which presents a new strategy for surface design of vascular implant materials.

Substrate Stiffness Combined with Hepatocyte Growth Factor Modulates Endothelial Cell Behavior

Endothelial cells (ECs) play a crucial role in regulating various physiological and pathological processes. The behavior of ECs is modulated by physical (e.g., substrate stiffness) and biochemical cues (e.g., growth factors). However, the synergistic influence of these cues on EC behavior has rarely been investigated. In this study, we constructed poly(l-lysine)/hyaluronan (PLL/HA) multilayer films with different stiffness and exposed ECs to these substrates with and without hepatocyte growth factor (HGF)-supplemented culture medium. We demonstrated that EC adhesion, migration, and proliferation were positively correlated with substrate stiffness and that these behaviors were further promoted by HGF. Interestingly, ECs on the lower stiffness substrates showed stronger responses to HGF in terms of migration and proliferation, suggesting that HGF can profoundly influence stiffness-dependent EC behavior correlated with EC growth. After the formation of an EC monolayer, EC behaviors correlated with endothelial function were evaluated by characterizing monolayer integrity, nitric oxide production, and gene expression of endothelial nitric oxide synthase. For the first time, we demonstrated that endothelial function displayed a negative correlation with substrate stiffness. Although HGF improved endothelial function, HGF was not able to change the stiffness-dependent manner of endothelial functions. Taken together, this study provides insights into the synergetic influence of physical and biochemical cues on EC behavior and offers great potential in the development of optimized biomaterials for EC-based regenerative medicine.

Dynamic stiffness of polyelectrolyte multilayer films based on disulfide bonds for in situ control of cell adhesion

The stiffness of the substrates has been found to have a strong effect on cell behaviors, especially on cell adhesion, which is the first cellular event when cells contact materials. Much effort has been made to develop the materials with controlled stiffness for regulating cell adhesion. However, most available strategies for controlling the stiffness of material surfaces are generally limited to be static, which means that the stiffness is fixed during cell adhesion. Herein, we developed polyelectrolyte multilayer films (PEMs), and their stiffness can be dynamically modulated by mild stimuli. The PEMs were made by alternative deposition of poly-l-lysine (PLL) and thiol group modified hyaluronan (HA-SH) using the layer-by-layer assembly technique. The (PLL/HA-SH) multilayers can be cross-linked via oxidation of thiol groups. After crosslinking, the stiffness was increased and the adhesion of fibroblast cells was promoted. The stiffness of the multilayer films can be down-regulated dynamically by adding glutathione (GSH) in the medium, leading to in situ reduction of cell adhesion. Our study provides a promising strategy for the development of material surfaces with dynamically changeable stiffness, which is of great potential in the field of cell-based biomaterials.

The (PrS/HGF‐pDNA) multilayer films for gene‐eluting stent coating: Gene‐protecting, anticoagulation, antibacterial properties, and in vivo antirestenosis evaluation

Vascular gene-eluting stents (GES) is a promising strategy for treatment of cardiovascular disease. Very recently, we have proved that the (protamine sulfate/plasmid DNA encoding hepatocyte growth factor) (PrS/HGF-pDNA) multilayer can serve as a powerful tool for enhancing competitiveness of endothelial cell over smooth muscle cell, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. However, before the gene multilayer films could be used in vascular stents for real clinical application, the preservation of gene bioactivity during the industrial sterilization and the hemocompatibility of film should be taken into account. Actually, both are long been ignored issues in the field of gene coating for GES. In this study, we demonstrate that the (PrS/HGF-pDNA) multilayer film exhibits the good gene-protecting abilities, which is confirmed by using the industrial sterilizations (gamma irradiation and ethylene oxide) and a routine storage condition (dry state at 4°C for 30 days). Furthermore, hemocompatible measurements (such as platelet adhesion and whole blood coagulation) and antibacterial assays (bacteria adhesion and growth inhibition) indicate the good anticoagulation and antibacterial properties of the (PrS/HGF-pDNA) multilayer film. The in vivo preliminary data of angiography and histological analysis suggest that the (PrS/HGF-pDNA) multilayer coated stent can reduce the in-stent restenosis. This work reveals that the (PrS/HGF-pDNA) multilayer film could be a promising candidate as coating for GES, which is of great potential in future clinic application.

Stiffness of polyelectrolyte multilayer film influences endothelial function of endothelial cell monolayer

Endothelialization has proved to be critical for maintaining long-term success of implantable vascular devices. The formation of monolayer of endothelial cells (ECs) on the implant surfaces is one of the most important factors for the endothelialization. However, endothelial function of regenerated EC monolayer, which plays a much more important role in preventing the complications of post-implantation, has not received enough attention. Here, a vascular endothelial growth factor (VEGF)-incorporated poly(l-lysine)/hyaluronan (PLL/HA) polyelectrolyte multilayer film was fabricated. Through varying the crosslinking degree, stiffness of the film was manipulated, offering either soft or stiff film. We demonstrated that ECs were able to adhere and proliferate on both soft and stiff films, subsequently forming an integrated EC monolayer. Furthermore, endothelial functions were evaluated by characterizing EC monolayer integrity, expression of genes correlated with the endothelial functions, and nitric oxide production. It demonstrated that EC monolayer on the soft film displayed higher endothelial function compared to that on the stiff film. Our study highlights the influence of substrate stiffness on endothelial function, which offers a new criterion for surface design of vascular implants.

Improved Endothelial Function of Endothelial Cell Monolayer on the Soft Polyelectrolyte Multilayer Film with Matrix-Bound Vascular Endothelial Growth Factor.

Endothelialization on the vascular implants is of great importance for prevention of undesired postimplantation symptoms. However, endothelial dysfunction of regenerated endothelial cell (EC) monolayer has been frequently observed, leading to severe complications, such as neointimal hyperplasia, late thrombosis, and neoatherosclerosis. It has significantly impeded long-term success of the therapy. So far, very little attention has been paid on endothelial function of EC monolayer. Bioinspired by the microenvironment of the endothelium in a blood vessel, this study described a soft polyelectrolyte multilayer film (PEM) through layer-by-layer assembly of poly(l-lysine) (PLL) and hyaluronan (HA). The (PLL/HA) PEM was chemically cross-linked and further incorporated with vascular endothelial growth factor. It demonstrated that this approach could promote EC adhesion and proliferation, further inducing formation of EC monolayer. Further, improved endothelial function of the EC monolayer was achieved as shown with the tighter integrity, higher production of nitric oxide, and expression level of endothelial function related genes, compared to EC monolayers on traditional substrates with high stiffness (e.g., glass, tissue culture polystyrene, and stainless steel). Our findings highlighted the influence of substrate stiffness on endothelial function of EC monolayer, giving a new strategy in the surface design of vascular implants.

Dynamic spongy films to immobilize hydrophobic antimicrobial peptides for self-healing bactericidal coating

A constant increase of nosocomial infections that are caused by adhesion and colonization of pathogenic microorganisms, especially drug-resistant bacteria, on the surfaces of healthcare devices has received considerable attention worldwide. In this study, bioinspired by antimicrobial skins of natural living beings, we developed a self-healing bactericidal coating through the immobilization of hydrophobic antimicrobial peptides (AMPs) into a multilayer film, which was constructed through the enhanced exponential layer-by-layer assembly of polyethylenimine (PEI) and poly(acrylic acid) (PAA). The (PEI/PAA) film shows particular dynamic properties from the as-prepared thin solid film to a spongy microporous structure via acid solution treatment, and then back to the thin solid film by eliminating micropores via the treatment of saturated humidity. Consequently, the loading and integration of hydrophobic AMPs such as gramicidin A (GA) into the (PEI/PAA) film were achieved via simple wicking action with GA solution and subsequent humidity treatment, respectively. The GA loading densities can be precisely controlled by using different concentrations of GA solution. We demonstrated that the GA immobilized (PEI/PAA) film has rapid self-healing properties, and that Gram-positive bacteria S. aureus including the methicillin-resistant type were efficiently killed through the contact-killing mode. Collectively, this self-healing bactericidal coating shows practical potential in a variety of healthcare applications.