Carboxymethyl Chitosan and Gelatin Hydrogel Scaffolds Incorporated with Conductive PEDOT Nanoparticles for Improved Neural Stem Cell Proliferation and Neuronal Differentiation.

Tissue engineering scaffolds provide biological and physiochemical cures to guide tissue recovery, and electrical signals through the electroactive materials possess tremendous potential to modulate the cell fate. In this study, a novel electroactive hydrogel scaffold was fabricated by assembling poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles on a carboxymethyl chitosan/gelatin (CMCS/Gel) composite hydrogel surface via in situ chemical polymerization. The chemical structure, morphology, conductivity, porosity, swelling rate, in vitro biodegradation, and mechanical properties of the prepared hydrogel samples were characterized. The adhesion, proliferation, and differentiation of neural stem cells (NSCs) on conductive hydrogels were investigated. The CMCS/Gel-PEDOT hydrogels exhibited high porosity, excellent water absorption, improved thermal stability, and adequate biodegradability. Importantly, the mechanical properties of the prepared hydrogels were similar to those of brain tissue, with electrical conductivity up to (1.52 ± 0.15) × 10-3 S/cm. Compared to the CMCS/Gel hydrogel, the incorporation of PEDOT nanoparticles significantly improved the adhesion of NSCs, and supported long-term cell growth and proliferation in a three-dimensional (3D) microenvironment. In addition, under the differentiation condition, the conductive hydrogel also significantly enhanced neuronal differentiation with the up-regulation of ß-tubulin III expression. These results suggest that CMCS/Gel-PEDOT hydrogels may be an attractive conductive substrate for further studies on neural tissue repair and regeneration.

Preparation, characterization and antioxidant activity of protocatechuic acid grafted carboxymethyl chitosan and its hydrogel.

In this study, protocatechuic acid (PCA) was grafted onto carboxymethyl chitosan (CMCS) via EDC/NHS to improve the antioxidant effect. The grafting ratio of PCA-g-CMCS conjugates could be controlled by adjusting the pH value and feed ratio of raw materials. The conjugates exhibited similar pH sensitivity to CMCS and showed dramatic enhancements of DPPH and ABTS radicals scavenging activities, total antioxidant capacity, reducing power, and Fe2+-chelating activity. Three-dimensional porous PCA-g-CMCS hydrogel was prepared by lyophilization and secondary cross-linking. The shaped hydrogel preserved its antioxidant activity, and the sustained release of PCA-containing degraded fragment from biodegradable hydrogel could be achieved with the aid of lysozyme in vitro (15 days). PCA-g-CMCS hydrogel also showed excellent biocompatibility and protective effect on H2O2-induced oxidative damage in SH-SY5Y cells. These results suggested that PCA-g-CMCS conjugates and its hydrogel would appear to be a promising oxidation-resistant material for applications such as drug release and tissue engineering.

Biodegradable and electroconductive poly(3,4-ethylenedioxythiophene)/carboxymethyl chitosan hydrogels for neural tissue engineering.

Electroconductive hydrogels with excellent electromechanical properties have become crucial for biomedical applications. In this study, we developed a conductive composite hydrogel via in-situ chemical polymerization based on carboxymethyl chitosan (CMCS), as a biodegradable base macromolecular network, and poly(3,4-ethylenedioxythiophene) (PEDOT), as a conductive polymer layer. The physicochemical and electrochemical properties of conductive hydrogels (PEDOT/CMCS) with different contents of PEDOT polymer were analyzed. Cell viability and proliferation of neuron-like rat phaeochromocytoma (PC12) cells on these three-dimensional conductive hydrogels were evaluated in vitro. As results, the prepared semi-interpenetrating network hydrogels were shown to consist of up to 1825±135wt% of water with a compressive modulus of 9.59±0.49kPa, a porosity of 93.95±1.03% and an electrical conductivity of (4.68±0.28)×10-3S·cm-1. Cell experiments confirmed that PEDOT/CMCS hydrogels not only had no cytotoxicity, but also supported cell adhesion, viability and proliferation. These results demonstrated that the incorporation of conductive PEDOT component into CMCS hydrogels endowed the hydrogels with enhanced mechanical strength, conductivity and kept the biocompatibility. Thus, the attractive performances of these composite hydrogels would make them suitable for further neural tissue engineering application, such as nerve regeneration scaffold materials.

Synthesis of protocatechuic acid grafted chitosan copolymer: Structure characterization and in vitro neuroprotective potential.

Excessive free radicals can cause oxidative damage to human tissues, which results in a variety of diseases. Therefore, the development of antioxidant materials is one of the great projects in biomedical field. In this work, antioxidant protocatechuic acid (PCA) monomers were grafted onto chitosan (CS) backbones to develop a PCA grafted chitosan (PCA-g-CS) antioxidant copolymer via the method of free radical-induced grafting reaction. The formation of covalent bonds between PCA and CS were confirmed by FTIR, 1H NMR, XRD and UV-vis. The antioxidant activity of PCA-g-CS was analyzed by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging assays. In addition, the cytotoxicity of PCA-g-CS on neuron-like rat phaeochromocytoma (PC12) cells was evaluated by using MTT assay. The neuroprotective effects against hydrogen peroxide (H2O2) and l-glutamic acid (GLU) induced apoptosis in PC12 cells were also investigated. Our results demonstrated that the PCA-g-CS antioxidant copolymer had the ability to scavenge DPPH and hydroxyl radical in vitro. Furthermore, the PCA-g-CS was biocompatible and had neuroprotective effects against free radical-induced apoptosis in PC12 cells. This PCA-g-CS copolymer is firstly synthesized for neuroprotection and the results suggest the PCA-g-CS may be a potential antioxidant material in the treatment of oxidative damage related diseases.

3D culture of neural stem cells within conductive PEDOT layer-assembled chitosan/gelatin scaffolds for neural tissue engineering.

Neural stem cells (NSCs), as a self-renewing and multipotent cell population, have been widely studied for never regeneration. Engineering scaffold is one of the important factors to regulate NSCs proliferation and differentiation towards the formation of the desired cells and tissues. Because neural cells are electro-active ones, a conductive scaffold is required to provide three-dimensional cell growth microenvironments and appropriate synergistic cell guidance cues. In this study, a poly (3,4­ethylenedioxythiophene)/chitosan/gelatin (PEDOT/Cs/Gel) scaffold was prepared via in situ interfacial polymerization, with a nanostructured layer of PEDOT assembling on the channel surface of porous Cs/Gel scaffold. This electrically conductive, three-dimensional, porous and biodegradable PEDOT/Cs/Gel scaffold was used as a novel scaffold for NSCs three-dimension (3D) culture in vitro. It was found that the layer of PEDOT on the channel surface of Cs/Gel scaffolds could greatly promote NSCs adhesion and proliferation. Additionally, under the differentiation condition, the protein and gene analysis suggested that PEDOT/Cs/Gel scaffolds could significantly enhance the NSCs differentiation towards neurons and astrocytes with the up-regulation of ß tubulin-III and GFAP expression. In conclusion, these results demonstrated that the PEDOT/Cs/Gel scaffolds as an electrically conductive scaffold could not only promote NSCs adhesion and proliferation but also enhance NSCs differentiation into neurons and astrocytes with higher protein and gene expression. PEDOT-assembled Cs/Gel scaffold will be a promising conductive substrate for NSCs research and neural tissue engineering.

Hyaluronic acid doped-poly(3,4-ethylenedioxythiophene)/chitosan/gelatin (PEDOT-HA/Cs/Gel) porous conductive scaffold for nerve regeneration.

Conducting polymer, as a "smart" biomaterial, has been increasingly used to construct tissue engineered scaffold for nerve tissue regeneration. In this study, a novel porous conductive scaffold was prepared by incorporating conductive hyaluronic acid (HA) doped-poly(3,4-ethylenedioxythiophene) (PEDOT-HA) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physicochemical characteristics of Cs/Gel scaffold with 0-10wt% PEDOT-HA were analyzed and the results indicated that the incorporation of PEDOT-HA into scaffold increased the electrical and mechanical properties while decreasing the porosity and water absorption. Moreover, in vitro biodegradation of scaffold displayed a declining trend with the PEDOT-HA content increased. About the biocompatibility of conductive scaffold, neuron-like rat phaeochromocytoma (PC12) cells were cultured in scaffold to evaluate cell adhesion and growth. 8% PEDOT-HA/Cs/Gel scaffold had a higher cell adhesive efficiency and cell viability than the other conductive scaffolds. Furthermore, cells in the scaffold with 8wt% PEDOT-HA expressed higher synapse growth gene of GAP43 and SYP compared with Cs/Gel control group. These results suggest that 8%PEDOT-HA/Cs/Gel scaffold is an attractive cell culture conductive substrate which could support cell adhesion, survival, proliferation, and synapse growth for the application in nerve tissue regeneration.

Fabrication and characterization of conductive poly (3,4-ethylenedioxythiophene) doped with hyaluronic acid/poly (l-lactic acid) composite film for biomedical application.

Poly 3,4-ethylenedioxythiophene (PEDOT), a polythiophene derivative, has been proved to be modified by chemical process as biocompatible conductive polymer for biomedical applications. In this study, novel hyaluronic acid (HA)-doped PEDOT nanoparticles were synthesized by the method of chemical oxidative polymerization, then conductive PEDOT-HA/poly(l-lactic acid) (PLLA) composite films were prepared. The physicochemical characteristics and biocompatibility of films were further investigated. FTIR, Raman and EDX analysis demonstrated that HA was successfully doped into PEDOT particles. Cyclic voltammograms indicated PEDOT-HA particles had favorable electrochemical stability. PEDOT-HA/PLLA films showed lower surface contact angle and faster degradation degree compared with PLLA films. Moreover, the cytotoxicity test of PEDOT-HA/PLLA films showed that neuron-like pheochromocytoma (PC12) cells adhered and spread well on the surface of PEDOT-HA/PLLA films and cell viability denoted by MTT assay had a significant increase. PEDOT-HA/PLLA films modified with laminin (LN) also exhibited an efficiently elongated cell morphology observed by fluorescent microscope and metallographic microscope. Furthermore, PEDOT-HA/PLLA films were subjected to different current intensity to elucidate the effect of electrical stimulation (ES) on neurite outgrowth of PC12 cells. ES (0.5 mA, 2 h) significantly promoted neurite outgrowth with an average value length of 122 ± 5 µm and enhanced the mRNA expression of growth-associated protein (GAP43) and synaptophysin (SYP) in PC12 cells when compared with other ES groups. These results suggest that PEDOT-HA/PLLA film combined with ES are conducive to cell growth and neurite outgrowth, indicating the conductive PEDOT-HA/PLLA film may be an attractive candidate with ES for enhancing nerve regeneration in nerve tissue engineering.

Neural stem cell proliferation and differentiation in the conductive PEDOT-HA/Cs/Gel scaffold for neural tissue engineering.

Engineering scaffolds with excellent electro-activity is increasingly important in tissue engineering and regenerative medicine. Herein, conductive poly(3,4-ethylenedioxythiophene) doped with hyaluronic acid (PEDOT-HA) nanoparticles were firstly synthesized via chemical oxidant polymerization. A three-dimensional (3D) PEDOT-HA/Cs/Gel scaffold was then developed by introducing PEDOT-HA nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. HA, as a bridge, not only was used as a dopant, but also combined PEDOT into the Cs/Gel via chemical crosslinking. The PEDOT-HA/Cs/Gel scaffold was used as a conductive substrate for neural stem cell (NSC) culture in vitro. The results demonstrated that the PEDOT-HA/Cs/Gel scaffold had excellent biocompatibility for NSC proliferation and differentiation. 3D confocal fluorescence images showed cells attached on the channel surface of Cs/Gel and PEDOT-HA/Cs/Gel scaffolds with a normal neuronal morphology. Compared to the Cs/Gel scaffold, the PEDOT-HA/Cs/Gel scaffold not only promoted NSC proliferation with up-regulated expression of Ki67, but also enhanced NSC differentiation into neurons and astrocytes with up-regulated expression of ß tubulin-III and GFAP, respectively. It is expected that this electro-active and bio-active PEDOT-HA/Cs/Gel scaffold will be used as a conductive platform to regulate NSC behavior for neural tissue engineering.