New biocompatible polypyrrole-based films with good blood compatibility and high electrical conductivity.

A novel O-butyryl chitosan (OBCS)-grafted polypyrrole (PPy) film was described. The immobilization was accomplished by photocrosslinking the OBCS onto PPy films under ultraviolet light irradiation. The surfaces of OBCS-grafted PPy film were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and electron spectroscopy for chemical analysis (ESCA). The blood compatibility of the OBCS-grafted PPy film was evaluated by platelet-rich plasma (PRP) contacting experiments and protein adsorption experiments in vitro. These results have demonstrated that the surface with immobilized OBCS shows much less platelet adhesive and fibrinogen adsorption compared to the control surface. The bulk conductivity values of PPy films were measured by a modified four-probe method. The composite films have both good blood compatibility and high electrical conductivity that make them suitable for using as potential biomaterials, such as electrically conducting blood vessel and functionally haemocompatible substrate of biosensor used directly in whole blood.

Patterning of a random copolymer of poly[lactide-co-glycotide-co-(epsilon-caprolactone)] by UV embossing for tissue engineering.

The random copolymer, poly[lactide-co-glycotide-co-(epsilon-caprolactone)] (PLGACL) diacrylate was prepared by ring-opening polymerization of L-lactide, glycolide, and epsilon-caprolactone initiated with tetra(ethylene glycol). The diacrylated polymers were extensively characterized. With a UV embossing method, these copolymers were successfully fabricated into microchannels separated by microwalls with a high aspect (height/width) ratio. The PLGACL network films showed good cytocompatibility. Varieties of microstructures were fabricated, such as 10 x 40 x 60, 10 x 80 x 60, 25 x 40 x 60, or 25 x 80 x 60 microm(3) structures (microwall width x microchannel width x microwall height). The results demonstrated that smooth muscle cells (SMCs) can grow not only on the microchannel surfaces but also on the surfaces of the microwall and sidewall. The SMCs aligned along the 25 microm wide microwall with an elongated morphology and proliferated very slowly in comparison to those on the smooth surface with a longer cell-culture term. Few cells could attach and spread on the surface of the 40 microm wide microchannel, while the cells flourished on the 80 microm, or more than 80 microm, wide microchannel with a spindle morphology. The biophysical mechanism mediated by the micropattern geometry is discussed. Overall, the present micropattern, consisting of biodegradable and cytocompatible PLGACL, provides a promising scaffold for tissue engineering.

Adhesion contact dynamics of fibroblasts on biomacromolecular surfaces.

Biomacromolecules like gelatin and chitosan have emerged as highly versatile biomimetic coatings for applications in tissue engineering. The elucidation of the interfacial kinetics of cell adhesion on biomacromolecular surfaces will pave the way for the rational design of chitosan/gelatin-based systems for cell regeneration. Biomacromolecular ultra-thin films, chemically immobilized on fused silica are ideal experimental models for determining the effect of surface properties on the biophysical cascades following cell seeding. In this study, confocal reflectance interference contrast microscopy (C-RICM), in conjunction with phase contrast microscopy and fluorescence confocal microscopy, was applied to detect the adhesion contact dynamics of 3T3 fibroblasts on chitosan and gelatin ultrathin films. X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of chitosan or gelatin on the silanized glass surface. Both the initial cell deformation rate and the change of two-dimensional spread area of the 3T3 fibroblasts are higher on gelatin-modified surfaces than on chitosan surfaces. The steady-state adhesion energy of 3T3 fibroblasts on gelatin film is three times higher than that on chitosan film. Immuno-staining of actin further demonstrates the different organization of cytoskeleton, likely induced by the change in cell signaling mechanism on the two biomacromolecular surfaces. The better attachment of 3T3 fibroblast to gelatin is postulated to be caused by the presence of adhesive domains on gelatin.

The aggregation behavior of O-carboxymethylchitosan in dilute aqueous solution.

O-Carboxymethylchitosan (OCMCS) is a kind of biocompatible derivatives of chitosan whose water solubility is strongly dependent on the degree of carboxymethylation. The OCMCS with 100 carboxymethyl groups and 75 amino groups per 100 anhydroglucosamine units of OCMCS was synthesized by the reaction of chitosan and monochloroacetic. When OCMCS was dissolved in water, its solution was neutral and OCMCS behaved like a weak polyanionic polyeclectrolyte because most of carboxylic groups were not dissociated in neutral aqueous solution. The aggregation behavior of OCMCS in aqueous solution was studied by surface tensiometry, steady-state fluorescence spectroscopy and viscometry. The critical aggregation concentration (cac) of OCMCS was determined to be between 0.042 mg/ml and 0.050 mg/ml. The possible aggregation mechanism of OCMCS in water was elucidated.

Covalent immobilization of chitosan/heparin complex with a photosensitive hetero-bifunctional crosslinking reagent on PLA surface.

Chitosan (CS) was covalently immobilized onto polylactic acid (PLA) film surface using the photosensitive hetero-bifunctional crosslinking reagent, 4-azidobenzoic acid, which was previously bonded to chitosan by reaction between an acid group of the crosslinking reagent and a free amino group of chitosan. The immobilization was accomplished by irradiating with ultraviolet light, the modified chitosan being coated on the film surface to photolyze azide groups, thus crosslinking chitosan and PLA together. The hydroxyl and amino groups of chitosan may provide the opportunity for them being further derived by chemically immobilizing a variety of functional groups. These modifications could be carried out to tailor PLA biomaterial to meet the specific needs of different biomedical applications. For example, chitosan molecules immobilized on the PLA could be modified by heparin (Hp) solution to form a polyelectrolyte complex on the PLA surface. Platelet adhesion assay showed that PLA surface modified by chitosan/heparin complex could inhibit platelet adhesion and activation. Cell culture assay indicated that PLA surface with CS/Hp complex showed enhanced cell adhesion.

Various approaches to modify biomaterial surfaces for improving hemocompatibility.

In this paper, the mechanism of thrombus formation on the surface of polymeric materials and the various approaches of modifying biomaterial surfaces to improve their hemocompatibility are reviewed. Moreover, the blood compatibility of the cellulose membrane grafted with O-butyrylchitosan (OBCS) by using a radiation grafting technique was studied. Surface analysis of grafted cellulose membrane was verified by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and electron spectroscopy for chemical analysis (ESCA), which confirmed that OBCS was successfully grafted onto the cellulose membrane surfaces. Blood compatibility of the grafted cellulose membranes was evaluated by platelet rich plasma (PRP) contacting experiments and protein adsorption experiments using blank cellulose membranes as the control. The blood compatibility of OBCS grafted cellulose membranes is better than that of blank cellulose membranes. These results suggest that the photocrosslinkable chitosan developed here has the potential of serving in blood-contacting applications in medical use.

[Development of the vascular prosthesis research].

The search for a nonthrombogenic material with the potential for use in small diameter vascular graft applications continues to be a field of extensive investigation. This article describes the choice of biomaterials used as vascular prosthesis, the innovation of construction of tissue-engineered blood vessels, the indispensability, methods and the effect produced by surface modification of vascular prosthesis. The article also points out that research achievements of vascular prosthesis must be made with the exploitation of new nonthrombogenic biomaterial and the development of tissue engineering.

Novel surfactant for preparation of poly(L-lactic acid) nanoparticles with controllable release profile and cytocompatibility for drug delivery.

Poly(L-lactic acid) nanoparticles loaded with a hydrophobic drug were prepared by an emulsion-evaporation process (oil in water) with a novel, effective and biocompatible surfactant butanedioic acid, 2-sulfo-1,4-butanedioic acid ditridecyl ester (sodium salt, 1:1) (BASDE). The particles are spherical in morphology and their diameters are controllable from 50 to 550nm with poly-dispersity indexes within the range of 0.122-0.340. The drug entrapment efficiency and drug content were measured by spectrophotometry. The drug release rate is affected by both the size of the particles and the drug content in the particles. In vitro cytotoxicity data indicate that these drug-loaded PLA nanoparticles are safe for hypodermic injection regard to the toxicological acceptance. This study demonstrates that using BASDE surfactant, the size of PLA nanoparticles can be controlled at the nanoscale with a narrow size distribution, and the drug release is controllable with excellent in vitro cytocompatibility. This may be due to efficient emulsification capability and biocompatibility of BASDE.

Synthesis, biocompatible, and self-assembly properties of poly (ethylene glycol)/lactobionic acid-grafted chitosan.

Polymers with targeted ligands are widely used as the anti-cancer drug delivery materials. For applications of chitosan as an anti-liver cancer drug delivery, poly (ethylene glycol)/lactobionic acid-grafted chitosan (PEG/LA-CS) was prepared and investigated since lactobionic acid can be specifically recognized by the hepatocytes. The structure of the PEG/LA-CS was characterized by Fourier transform infrared spectrometry and elemental analysis. The self-assembly behaviors of the PEG/LA-CS were monitored by steady-state fluorescence spectroscopy and electronic transmission microscope. The protein adsorption of the PEG/LA-CS was detected with bovine serum albumin (BSA) by electrochemical impedance spectroscopy. The results showed that the PEG/LA-CS almost did not adsorb protein. To study the effects of PEG/LA-CS on the structure of BSA, the interactions between the PEG/LA-CS and BSA were detected by ultraviolet spectrum, fluorescence spectrum, and circular dichroism. All the data gave one result that BSA maintained its original folded confirmation in PEG/LA-CS solution. The hemocompatibility of PEG/LA-CS was investigated by observing the effects of PEG/LA-CS on the hemolysis rate and the plasma recalcification time (PRT). The results showed that the PRT was prolonged greatly and the hemolysis rate was less than 5%. Furthermore, PEG/LA-CS also showed good cytocompatibility with K562, Hep G2, and LO2 cells. Therefore, the PEG/LA-CS is believed to have great potential for producing injectable anti-liver cancer drug delivery.

Acceleration of aneurysm healing by P(DLLA-co-TMC)-coated coils enabling the controlled release of vascular endothelial growth factor.

Since the introduction of the detachable coil in endovascular treatment of intracranial aneurysms, the in-hospital mortality rate has been significantly decreased. Recurrence of the aneurysm remains the major drawback of using detachable coils. We prepared a bioactive coil coated with poly(d,l-lactide)-7co-(1,3-trimethylene carbonate) (P(DLLA-co-TMC)), a novel copolymer for controlling the release of vascular endothelial growth factor (VEGF). Platinum coils were prepared by successive coating with cationic P(DLLA-co-TMC) and anionic heparin. Then, recombinant human VEGF-165 (rhVEGF) was immobilized by affinity binding to heparin. The morphological characteristics and sustained in vitro release of rhVEGF were examined using scanning electron microscopy and enzyme-linked immunosorbent assay, respectively. The efficacy of these novel coils modified by P(DLLA-co-TMC)/rhVEGF was tested using a common carotid artery aneurysm model in rats. Experimental aneurysms were embolized with unmodified, P(DLLA-co-TMC)/heparin-coated or P(DLLA-co-TMC)/rhVEGF-coated platinum coils (n = 18). The coils were removed on days 15, 30 and 90 after insertion, and the histological and immunohistochemical analysis of factor VIII was performed to confirm the presence of endothelial cells in the organized area. In addition, the controlled in vivo release of VEGF was confirmed by Western blotting analysis. The release of VEGF tended to increase during the whole period and no burst release was observed. In the group treated with P(DLLA-co-TMC)/rhVEGF-coated platinum coils, clot organization and endothelial cell proliferation were accelerated. The immunohistochemistry study showed that the expression of factor VIII was found in the P(DLLA-co-TMC)/rhVEGF-coated coil group but not in the other two groups. Furthermore, Western blotting analysis confirmed that the major released VEGF in the aneurysm sac was from the P(DLLA-co-TMC)/VEGF-coated coil. P(DLLA-co-TMC)/rhVEGF-coated platinum coils can promote clot organization and endothelial cell proliferation in a rat aneurysm model.

The internalization of fluorescence-labeled PLA nanoparticles by macrophages.

Rhodamine B (RhB)-labeled PLA nanoparticles were prepared through surface grafting copolymerization of glycidyl methacrylate (GMA) onto PLA nanoparticles during the emulsion/evaporation process. RhB firstly interacts with sodium dodecyl sulfate (SDS) through electrostatic interaction to form hydrophobic complex (SDS-RhB). Due to the high-affinity of SDS-RhB with GMA, hydrophilic RhB can be successfully combined into PLA nanoparticles. The internalization of RhB-labeled PLA nanoparticles by macrophages was investigated with fluorescence microscope technology. The effects of the PLA nanoparticle surface nature and size on the internalization were investigated. The results indicate that the PLA particles smaller than 200 nm can avoid the uptake of phagocytosis. The bigger PLA particles (300 nm) with polyethylene glycol (PEG) surface showed less internalization by macrophage compared with those with poly(ethylene oxide-propylene oxide) copolymer (F127) or poly(vinyl alcohol) (PVA) surface. The "stealth" function of PEG on the PLA nanoparticles from internalization of macrophages due to the low protein adsorption is revealed by electrochemical impedance technology.

Poly(lactic acid)/N-maleoylchitosan core-shell capsules: preparation and drug release properties.

An oil in water interface radical polymerization was used to prepare felodipine-loaded polymerized-N-maleoylchitosan (p-NMCS) and poly(lactic acid) (PLA)/p-NMCS capsules. Dynamic Light Scattering, Field Emission Scanning Electron Microscopy and Transmission Electron Microscope characterization revealed that both the p-NMCS and PLA/p-NMCS microcapsules had a ~550 nm hydrodynamic diameter, regular spherical morphology and an obvious core-shell structure. The ratio of PLA to p-NMCS in PLA/p-NMCS microcapsules was found affecting the drug loading content and entrapment efficiency. In vitro release kinetic results indicated that the p-NMCS microcapsules had a fast release rate comparing with that of the PLA/p-NMCS core-shell microcapsules, suggesting the release mechanism of the p-NMCS microcapsules was a diffusion-driven process, while the release mechanism of the PLA/p-NMCS microcapsules with high ratio of PLA to p-NMCS (not less than 1/1) was a combined diffusion and degradation-driven process.

Photo-initiated grafting of gelatin/N-maleic acyl-chitosan to enhance endothelial cell adhesion, proliferation and function on PLA surface.

Vascular graft surface properties significantly affect adhesion, growth and function of endothelial cells (ECs). The bulk degradation property of poly(lactic acid) (PLA) makes it possible for it to be replaced by cellular materials and PLA is desirable as a scaffold material for vascular grafts. However, PLA has an unfavorable surface property for EC adhesion and proliferation due to the lack of a selective cell adhesion motif. Photo-initiated surface-grafting polymerization is a promising method for immobilizing certain biomacromolecules on material surfaces without compromising bulk properties. N-Maleic acyl-chitosan (NMCS) is a novel biocompatible amphiphilic derivative of chitosan with double bonds and can be initiated by ultraviolet light. In this study, gelatin was complexed with NMCS via hydrophobic interaction, and gel/NMCS complex thus formed was then grafted on the PLA surface to improve EC biocompatibility. X-ray photoelectron and Fourier transform infrared spectroscopy, and water contact angle measurement confirmed immobilization of the gel/NMCS complex on PLA surface. Moreover, the gel/NMCS modified PLA enhanced human umbilical vein endothelial cell (HUVEC) spreading and flattening, and promoted the expression of more structured CD31 and vWF compared to unmodified PLA film. Compared to the unmodified PLA surface, the HUVECs on the modified PLA surface had elevated uptake of acetylated low-density lipoprotein, and maintained the ability to modulate metabolic activity upon exposure to shear stress at 5dyncm(-2) by up-regulating nitric oxide and prostacyclin production. Cell retention was 1.6 times higher on the gel/NMCS-PLA surface, demonstrating its improved potential for hemocompatibility. These results indicate that photo-initiated surface-grafting of the biomimetic gel/NMCS complex is an effective method to modify material surfaces as vascular grafts.

Synthesis and physicochemical properties of biocompatible N-carboxyethylchitosan.

A novel simple method was developed to successfully synthesize biocompatible N-carboxyethylchitosan (NCECS) with a substitution degree of approx. 41% using the Michael addition reaction. The NCECS structure was characterized by Fourier transform infra-red spectrometry (FT-IR), (1)H-NMR, elemental analysis, X-ray diffraction spectrometry (XRD) and thermogravimetric analysis (TGA). The physicochemical properties of NCECS in solution are found to be strongly dependent on the pH value. In the pH range of 5.0-5.5, NCECS aggregates in dilute aqueous solution as evident by steady-state fluorescence spectroscopy and viscosimetry. The aggregated NCECS shows a spheric morphology from the atomic force microscopy (AFM) study. The possible aggregation mechanism was discussed.

[Preparation and property of platinum microcoil modified by a copolymer-VEGF conjugate].

OBJECTIVE: To prepare a platinum microcoil coated with polymers and vascular endothelial growth factor (VEGF), and evaluate its surface characteristics and property of sustained VEGF release. METHODS: The surface of the platinum microcoils (GDC) were modified by coating P(DLLA-co-TMC) copolymer and immobilizing heparin on the surface of GDC. VEGF was then loaded onto the surface of GDC and the controlled release of VEGF within GDC was achieved. The morphology was observed by scanning electron microscope, and the sustained release of VEGF was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Platinum coils were prepared by successive deposition of P(DLLA-co-TMC) copolymer and anionic heparin, and VEGF was immobilized through affinity interaction with heparin. The accumulative release of VEGF increased obviously during the entire testing period without burst release. CONCLUSION: The use of P(DLLA-co-TMC) copolymer allows immobilization of VEGF on the platinum coils for controlled VEGF release, and improves the biological property of the coils.

Regulation of vascular smooth muscle cells on poly(ethylene terephthalate) film by O-carboxymethylchitosan surface immobilization.

Specifying the chemical environment of cells is a well-established method of controlling cellular behaviors. In this study, poly(ethylene terephthalate) (PET) film was selected as a typical biomaterial to detect the effects of chemical modifications on material surface in controlling cell behaviors. Natural biopolymer chitosan and its biocompatible derivative, O-carboxymethylchitosan (OCMCS) were surface immobilized on PET, respectively, via argon plasma followed by graft copolymerization with acrylic acid (AAc), which was exploited to covalently couple PET with chitosan (CS) and OCMCS molecules. Smooth muscle cells (SMCs) displayed a surface-dependent cell spreading and cytoskeletal organization. The cells spread with a more pronounced elongated spindle shape, smaller cell area, and lower cell shape index (CSI) on OCMCS-modified PET surface than on PET, or the PAA and chitosan-immobilized PET surfaces after 24 h of culture. Cell-culture viability after 5 days showed that all the modified materials possessed good cell proliferation. Our results suggest that cell adhesion, morphology, and growth can be mediated not only by varying the functional groups, electric charge, and wettability of PET surface but also by the specific biological recognition elicited from the biomaterials. These findings strongly support the concept that the microenvironment significantly influences cell behavior, highlighting the importance of environmental material biochemistry in cell-based tissue engineering schemes.

The synthesis and characterization of polymerizable and biocompatible N-maleic acyl-chitosan.

Biocompatible and polymerizable natural macromolecules have been found to provide great advantages in the preparation of hydrogels, which have wide applications in the fields of tissue engineering and polymeric drug delivery systems. To develop a new biocompatible polymerizable chitosan derivative, N-maleic acyl-chitosan (NMCS) was synthesized in this study. This novel biomaterial was designed from the N-acylation of chitosan with maleic anhydride introducing functional carboxyl and vinylated (--C[double bond]C--) groups. The structure of NMCS was characterized by FTIR, (1)H NMR, element analysis, and X-ray diffraction (XRD). NMCS can be dissolved into water because of its decreased crystallinity compared with chitosan. The NMCS's multiporous and microgel morphology was revealed by transmission electron microscope (TEM). Crosslinked hydrogel films can be successfully obtain through the macromolecular polymerization of NMCS. Subsequently, 3T3 fibroblasts were cultured onto the surface of the polymerized NMCS (P-NMCS) films to examine the capability of cell attachment and proliferation. Results from the cell culture demonstrate that P-NMCS films provide significant improvement in cell attachment and proliferation over unmodified chitosan. The improved P-NMCS cytocompatibility is expected to provide substantial contributions to tissue engineering in the future.

Adhesion dynamics, morphology, and organization of 3T3 fibroblast on chitosan and its derivative: the effect of O-carboxymethylation.

Chitosan and O-carboxymethylchitosan (OCMCS) have been proved to have biocompatibility and have been extensively researched in the field of biomaterials. In this study, Confocal-reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast imaging was used to investigate the adhesion contact dynamics of 3T3 fibroblasts on chitoan and OCMCS surface-modified silica coverslips. The C-RICM results demonstrate that the weak cell contact forms on OCMCS surface while a much stronger contact area forms on the chitosan surface. 3T3 fibroblasts are found to spread randomly with spindlelike morphology on the chitosan surface, while they exhibit elongated morphology and align on the OCMCS surface. It is believed that fibroblast behaviors such as migration, spreading with an elongated morphology, and alignment on the OCMCS surface are correlated with the weak cell contact. The mechanisms to form cell adhesion contact on chitosan and OCMCS were discussed.

Covalent immobilization of O-butyrylchitosan with a photosensitive hetero-bifunctional crosslinking reagent on biopolymer substrate surface and bloodcompatibility characterization.

O-Butyrylchitosan (OCS) was covalently immobilized onto a substrate (suture and PTFE) surface using the photosensitive hetero-bifunctional crosslinking reagent 4-azidobenzoic acid, which was previously bonded to OCS by the reaction between an acid group of the crosslinking reagent and a free amino group of OCS. The immobilization was accomplished by irradiating the modified OCS coated on the substrate surface with ultraviolet light to photolyze azide groups, thus the crosslinking OCS was immobilized on the substrate surface. The result indicated that OCS molecules immobilized on the substrate, and significantly reduced the fibrinogen adsorption and the deposition and spreading of platelets, demonstrating superior bloodcompatibility. Therefore, OCS could be developed into bloodcompatible biomaterial that used to modify the biomedical devices, such as PTFE and Dacron vascular prostheses surface.

[Attachment and growth of cultured fibroblast cells on chitosan/PHEA-blended hydrogels].

The chitosan/PHEA-blended hydrogels were prepared from PHEA and chitosan in various blend ratios. The water contents of the hydrogels were in the range of 50%-80% (wt). The attachment and growth of fibroblast cells(L929) on the hydrogels were studied. The results indicated the PHEA content in hydrogels has great effect on cell attachment but has little effect on the growth of L929 cells.