Hsiu-Wen Chien

Surface Modification with Poly(sulfobetaine methacrylate-co-acrylic acid) To Reduce Fibrinogen Adsorption, Platelet Adhesion, and Plasma Coagulation

Zwitterionic sulfobetaine methacrylate (SBMA) polymers were known to possess excellent antifouling properties due to high hydration capacity and neutral charge surface. In this study, copolymers of SBMA and acrylic acid (AA) with a variety of compositions were synthesized and were immobilized onto polymeric substrates with layer-by-layer polyelectrolyte films via electrostatic interaction. The amounts of platelet adhesion and fibrinogen adsorption were determined to evaluate hemocompatibility of poly(SBMA-co-AA)-modified substrates. Among various deposition conditions by modulating SBMA ratio in the copolymers and pH of the deposition solution, poly(SBMA(56)-co-AA(44)) deposited at pH 3.0 possessed the best hemocompatibility. This work demonstrated that poly(SBMA-co-AA) copolymers adsorbed on polyelectrolyte-base films via electrostatic interaction improve hemocompatibility effectively and are applicable for various substrates including TCPS, PU, and PDMS. Furthermore, poly(SBMA-co-AA)-coated substrate possesses great durability under rigorous conditions. The preliminary hemocompatibility tests regarding platelet adhesion, fibrinogen adsorption, and plasma coagulation suggest the potential of this technique for the application to blood-contacting biomedical devices.

Poly(dopamine) coating of scaffolds for articular cartilage tissue engineering

A surface modification technique based on poly(dopamine) deposition developed from oxidative polymerization of dopamine is known to promote cell adhesion to several cell-resistant substrates. In this study this technique was applied to articular cartilage tissue engineering. The adhesion and proliferation of rabbit chondrocytes were evaluated on poly(dopamine)-coated polymer films, such as polycaprolactone, poly(L-lactide), poly(lactic-co-glycolic acid) and polyurethane, biodegradable polymers that are commonly used in tissue engineering. Cell adhesion was significantly increased by merely 15 s of dopamine incubation, and 4 min incubation was enough to reach maximal cell adhesion, a 1.35-2.69-fold increase compared with that on the untreated substrates. Cells also grew much faster on the poly(dopamine)-coated substrates than on untreated substrates. The increase in cell affinity for poly(dopamine)-coated substrates was demonstrated via enhancement of the immobilization of serum adhesive proteins such as fibronectin. When the poly(dopamine)-coating technique was applied to three-dimensional (3-D) polyurethane scaffolds, the proliferation of chondrocytes and the secretion of glycosaminoglycans were increased compared with untreated scaffolds. Our results show that the deposition of a poly(dopamine) layer on 3-D porous scaffolds is a simple and promising strategy for articular cartilage tissue engineering, and may be applied to other types of tissue engineering.

Tunable micropatterned substrates based on poly(dopamine) deposition via microcontact printing.

A simple technique was developed to fabricate tunable micropatterned substrates based on mussel-inspired surface modification. Polydopamine (PDA) was developed on polydimethylsiloxane (PDMS) stamps and was easily imprinted to several substrates such as glass, silicon, gold, polystyrene, and poly(ethylene glycol) via microcontact printing. The imprinted PDA retained its unique reactivity and could modulate the chemical properties of micropatterns via secondary reactions, which was illustrated in this study. PDA patterns imprinted onto a cytophobic and nonfouling substrates were used to form patterns of cells or proteins. PDA imprints reacted with nucleophilic amines or thiols to conjugate molecules such as poly(ethylene glycol) for creating nonfouling area. Gold nanoparticles were immobilized onto PDA-stamped area. The reductive ability of PDA transformed silver ions to elemental metals as an electroless process of metallization. This facile and economic technique provides a powerful tool for development of a functional patterned substrate for various applications.

Spatial control of cellular adhesion using photo-crosslinked micropatterned polyelectrolyte multilayer films.

Cellular patterning on biomaterial surfaces is important in fundamental studies of cell-cell and cell-substrate interactions, and in biomedical applications such as tissue engineering, cell-based biosensors, and diagnostic devices. In this study, we combined the layer-by-layer polyelectrolyte multilayer deposition and photolithographic technique to create an easy and versatile technique for cell patterning. Poly(acrylic acid) (PAA) conjugated with 4-azidoaniline was interwoven in PAA/polyacrylamide (PAM) multilayer films. After UV irradiation through a photo mask, the UV-exposed areas were crosslinked and the unexposed areas were rinsed away by alkaline water, resulting in micropatterns. Cell patterns were formed when the cell adhesion was limited to the base substrate, but not on the multilayer films. The stability of cell patterns could be modulated by simply modification of the surface chemistry of base substrate and PEM films with conjugation of bioactive macromolecules. This technique can be also applied to other PEM systems with proper rinsing protocol, and many types of substrates. Cell co-culture systems can be also achieved by this technique.

Direct cell encapsulation in biodegradable and functionalizable carboxybetaine hydrogels.

Hydrogels provide three-dimensional (3D) frames with tissue-like elasticity and high water content for tissue scaffolds. They were commonly prepared from macromers such as poly(ethylene glycol) diacrylate (PEGDA) via free radical polymerization and used to encapsulate cells. Here, we report the direct encapsulation of cells into hydrogels using a low-toxic and water-soluble monomer, carboxybetaine methacrylate (CBMA), via redox polymerization. A disulfide-containing crosslinker was added to form a biodegradable carboxybetaine (CB) hydrogel, which can be self-degraded as cells grow or degraded in an accelerating way via adding of a cysteine-contained medium NIH-3T3 cells encapsulated in the CB hydrogel formed spherical aggregates that were recovered from hydrogel erosion. Furthermore, an RGD-containing peptide was also added to improve cell adhesion on the two-dimensional (2D) hydrogel surface and promote cell proliferation in the 3D hydrogel. The non-cytotoxic and biodegradable CB hydrogel with additional cell-adhesion moieties provides an excellent 3D environment for cell growth as tissue scaffolds.

Surface conjugation of zwitterionic polymers to inhibit cell adhesion and protein adsorption

Non-fouling surfaces that resist non-specific protein adsorption and cell adhesion are desired for many biomedical applications such as blood-contact devices and biosensors. Therefore, surface conjugation of anti-fouling molecules has been the focus of many studies. In this study, layer-by-layer polyelectrolyte deposition was applied to create an amine-rich platform for conjugation of zwitterionic polymers. A tri-layer polyelectrolyte (TLP) coating representing poly(ethylene imine) (PEI), poly(acrylic acid)-g-azide and PEI was deposited on various polymeric substrates via layer-by-layer deposition and then crosslinked via UV irradiation. Carboxyl-terminated poly(sulfobetaine methacrylate) p(SBMA) or poly(carboxybetaine methacrylate) p(CBMA) was then conjugated onto TLP coated substrates via a carbodiimide reaction. Our results demonstrate that the zwitterionic polymers could be easily conjugated over a wide pH range except under alkaline conditions, and almost completely block protein adsorption and the attachment of L929 cells and platelets. Therefore, this method has outstanding potential in biomedical applications that require low-fouling surfaces.

Fabrication of tunable micropatterned substrates for cell patterning via microcontact printing of polydopamine with poly(ethylene imine)-grafted copolymers.

Cell patterning is an important tool for biomedical research. In this work, we modified a technique combining mussel-inspired surface chemistry and microcontact printing (μCP) to modulate surface chemistry for cell patterning. Polymerized dopamine on poly(dimethylsiloxane) stamps was transferred to several cell-unfavorable substrates via μCP. Since cells only attached to the polydopamine (PDA)-imprinted areas, cell patterns were formed on a variety of cell-unfavorable surfaces. The stability of PDA imprints was proved under several harsh conditions. The cell affinity of PDA was modulated by co-deposition with several poly(ethylene imine) (PEI)-based copolymers, such as PEI, PEI-g-PEG (poly(ethylene glycol)) and PEI-g-galactose. The imprints of PDA/PEI-g-PEG provide the formation of cell patterns on cell-favorable substrates. Neuronal PC12 cells were patterned via imprinting of PDA/PEI, while HepG2/C3A cells were arranged on the imprint of PDA/PEI-g-galactose. Finally, co-culture of HepG2/C3A cells and L929 fibroblasts was accomplished by our micropatterning approach. This study demonstrated this simple and economic technique provides a powerful tool for development of functional patterned substrates for cell patterning. This technique should profit the preparation of cell patterns to study fundamental cell biology and to apply to biomedical engineering such as cell-based biosensors, diagnostic devices and tissue engineering.

Poly(dopamine) coating to biodegradable polymers for bone tissue engineering

In this study, a technique based on poly(dopamine) deposition to promote cell adhesion was investigated for the application in bone tissue engineering. The adhesion and proliferation of rat osteoblasts were evaluated on poly(dopamine)-coated biodegradable polymer films, such as polycaprolactone, poly(l-lactide) and poly(lactic-co-glycolic acid), which are commonly used biodegradable polymers in tissue engineering. Cell adhesion was significantly increased to a plateau by merely 15 s of dopamine incubation, 2.2–4.0-folds of increase compared to the corresponding untreated substrates. Cell proliferation was also greatly enhanced by poly(dopamine) deposition, indicated by shortened cell doubling time. Mineralization was also increased on the poly(dopamine)-deposited surfaces. The potential of poly(dopamine) deposition in bone tissue engineering is demonstrated in this study.

Collaborative Cell-Resistant Properties of Polyelectrolyte Multilayer Films and Surface PEGylation on Reducing Cell Adhesion to Cytophilic Surfaces

Both poly(ethylene glycol) (PEG) grafting and layer-by-layer polyelectrolyte multilayer (PEM) deposition for surface modification of biomaterials have been shown to decrease cell adhesion. The aim of this study was to investigate the synergic efficacy of PEGylated PEM films on reducing cell adhesion. PEG grafted to poly(ethylene imine) (PEI) was deposited onto the top of PEI/PAA (poly(acrylic acid)) multilayer films which were deposited onto cytophilic substrates, including tissue culture polystyrene and collagen-based substrate. The efficacy of the PEGylated PEM films in blocking adhesion of L929 cells was investigated by varying the amount of conjugated PEG and the layer numbers of PEM films. We found that cell adhesion was reduced on the swollen PEM films and further decreased by deposition of PEI-g-PEG as the topmost layer. The ability in cell resistance was enhanced with increasing PEG contents of PEGylated PEM films. PEGylated PEM films were stable for long-term incubation in phosphate-buffered saline. We demonstrated that cell affinity of cytophilic surfaces could be depressed by deposition of PEGylated PEM films.

Modulation of hemocompatibility of polysulfone by polyelectrolyte multilayer films.

Polyelectrolyte multilayer (PEM) films have been recently applied to surface modification of biomaterials. Cellular interactions with PEM films consisted of weak polyelectrolytes are greatly affected by the conditions of polyelectrolyte deposition, such as pH of polyelectrolyte solution. Previous studies indicated that the adhesion of several types of mammalian cells to PAH/PAA multilayer films was hindered by low pH and high layer numbers. The objective of this study is to evaluate whether the hemocompatibility of polysulfone can be modulated by deposition of poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayer films. PAH/PAA multilayer films with different layer numbers were assembled onto polysulfone at either pH 2.0 or pH 6.5. The number of platelet adhesion and the morphology of adherent platelets were determined to evaluate hemocompatibility of modified substrates. Compared to non-treat polysulfone, the PEM films developed at pH 2.0 decreased platelet adhesion, while those built at pH 6.5 enhanced platelet deposition. Platelet adhesion was found positively correlated to polyclonal antibodies binding to surface-bound fibrinogen. The extent of platelet spreading was increased with layer numbers of PEM films, suggesting that the adherent platelets on thick PEM films were prone to activation. In conclusion, PAH/PAA films with few layers developed at pH 2.0 possessed better hemocompatibility compared to other substrates.