Method for in situ polypyrrole coating, and the example of its use for functionalization of polyurethane anisotropic electrospun mats.

The in situ coating of polymer substrate with polypyrrole, described herein with detailed know-how, represents a novel technique of surface functionalization. The choice of oxidizing agent and the polymerization time both affect the properties of the thin polypyrrole layer. The specific conductivity, free surface energy, thickness, topography, and FTIR spectra of polypyrrole layer were determined. The conductive coatings were further used to functionalize both isotropic and anisotropic electrospun polyurethane nanofibrous mats to show their applicability and study the bioactive effect of both the anisotropy and conductivity together. The morphology of composites was studied by means of atomic force microscopy and scanning electron microscopy. A complex cytocompatibility study was performed, including determining cytotoxicity by optical and fluorescence microscopy, the advanced qualification of cell morphology by cell-image analysis, and a study of stem cell behavior. The results clearly showed the significant impact of substrate modification on cells, especially on fibroblasts while the embryonic stem cells were less affected. This study shows not only the effective way to prepare a thin conducting layer based on polypyrrole but also demonstrates its importance for the fabrication of smart biomaterials.

Stimuli-responsive thin film composites of conducting polymers and cellulose nanocrystals for tissue engineering.

Thin composite films comprising two primary representatives of conducting polymers, poly(3, 4-ethylenedioxythiophene) (PEDOT) and polypyrrole (PPy), with eco-friendly cellulose nanocrystals (CNC) were prepared through electrochemical polymerization. The combination of CNC and PEDOT (or PPy) results in the formation of films with highly different surface topography and thickness. Intriguingly, different surface conductivity of PEDOT and PPy was revealed by atomic force microscopy albeit that the electrochemical properties were rather similar. The biological properties of the composites in contact with prospective human induced pluripotent stem cells (hiPSC) and cardiomyocytes derived from hiPSC demonstrated good cytocompatibility of both composites and their potential in engineering of electro-sensitive tissues. The as-prepared conducting, eco-friendly and cytocompatible composites are thus promising candidates for biomedical applications where stimuli-responsivity is a crucial cell-instructive property.

Enzyme-Catalyzed Polymerization Process: A Novel Approach to the Preparation of Polyaniline Colloidal Dispersions with an Immunomodulatory Effect.

A green, nature-friendly synthesis of polyaniline colloidal particles based on enzyme-assisted oxidation of aniline with horseradish peroxidase and chitosan or poly(vinyl alcohol) as steric stabilizers was successfully employed. Physicochemical characterization revealed formation of particles containing the polyaniline emeraldine salt and demonstrated only a minor effect of polymer stabilizers on particle morphology. All tested colloidal particles showed in vitro antioxidation activity determined via scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. In vitro, they were able to reduce oxidative stress and inhibit the production of reactive oxygen species by neutrophils and inflammatory cytokines by macrophages. The anti-inflammatory effect observed was related to their antioxidant activity, especially in the case of neutrophils. The particles can thus be especially advantageous as active components of biomaterials modulating the early stages of inflammation. In addition to the immunomodulatory effect, the presence of intrinsically conducting polyaniline can impart cell-instructive properties to the particles. The approach to particle synthesis that we employed─an original one using environmentally friendly and biocompatible horseradish peroxidase─represents a smart way of preparing conducting particles with unique properties, which can be further modified by the stabilizers used.

Hierarchically Structured Surfaces Prepared by Phase Separation: Tissue Mimicking Culture Substrate.

The pseudo 3D hierarchical structure mimicking in vivo microenvironment was prepared by phase separation on tissue culture plastic. For surface treatment, time-sequenced dosing of the solvent mixture with various concentrations of polymer component was used. The experiments showed that hierarchically structured surfaces with macro, meso and micro pores can be prepared with multi-step phase separation processes. Changes in polystyrene surface topography were characterized by atomic force microscopy, scanning electron microscopy and contact profilometry. The cell proliferation and changes in cell morphology were tested on the prepared structured surfaces. Four types of cell lines were used for the determination of impact of the 3D architecture on the cell behavior, namely the mouse embryonic fibroblast, human lung carcinoma, primary human keratinocyte and mouse embryonic stem cells. The increase of proliferation of embryonic stem cells and mouse fibroblasts was the most remarkable. Moreover, the embryonic stem cells express different morphology when cultured on the structured surface. The acquired findings expand the current state of knowledge in the field of cell behavior on structured surfaces and bring new technological procedures leading to their preparation without the use of problematic temporary templates or additives.

Hierarchically Structured Polystyrene-Based Surfaces Amplifying Fluorescence Signals: Cytocompatibility with Human Induced Pluripotent Stem Cell.

An innovative multi-step phase separation process was used to prepare tissue culture for the polystyrene-based, hierarchically structured substrates, which mimicked in vivo microenvironment and architecture. Macro- (pore area from 3000 to 18,000 µm2; roughness (Ra) 7.2 ± 0.1 µm) and meso- (pore area from 50 to 300 µm2; Ra 1.1 ± 0.1 µm) structured substrates covered with micro-pores (area around 3 µm2) were prepared and characterised. Both types of substrate were suitable for human-induced pluripotent stem cell (hiPSC) cultivation and were found to be beneficial for the induction of cardiomyogenesis in hiPSC. This was confirmed both by the number of promoted proliferated cells and the expressions of specific markers (Nkx2.5, MYH6, MYL2, and MYL7). Moreover, the substrates amplified the fluorescence signal when Ca2+ flow was monitored. This property, together with cytocompatibility, make this material especially suitable for in vitro studies of cell/material interactions within tissue-mimicking environments.

Modulation of Differentiation of Embryonic Stem Cells by Polypyrrole: The Impact on Neurogenesis.

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

Properties of scaffolds prepared by fused deposition modeling of poly(hydroxyalkanoates).

Poly(hydroxyalkanoates) are biodegradable and biocompatible polymers suitable for tissue engineering. Fused deposition modeling (FDM) belongs to modern rapid prototyping techniques for the fabrication of scaffolds. In this work, poly(3-hydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) were tested for FDM. Thermal and rheological properties of industrial PHAs were compared with poly(lactic acid) (PLA), which is a biodegradable polymer commonly used for FDM. The massive decrease in viscosity and loss of molecular weight of PHB and PHBV precluded their use for FDM. On the other hand, the thermal stability of PHBH was comparable to that of PLA. PHBH scaffolds prepared by FDM exhibited excellent mechanical properties, no cytotoxicity and large proliferation of mouse embryonic fibroblast cells within 96 h. The hydrolytic degradation of PHBH and PLA scaffolds tested in synthetic gastric juice for 52 days confirmed a faster degradation of PHBH than PLA. The decrease in molecular weight confirmed the first-order kinetics with a slightly higher (0.0169 day-1) degradation rate constant for PHBH as compared to the value (0.0107 day-1) obtained for PLA. These results indicate that PHBH could be used to produce scaffolds by FDM with application in tissue engineering.

Dual Crosslinked Collagen/Chitosan Film for Potential Biomedical Applications.

The application of polymeric biomaterial scaffolds utilizing crosslinking strategy has become an effective approach in these days. In the present study, the development and characterization of collagen-chitosan hydrogel film has been reported on using dual crosslinking agent's, i.e., tannic acid and genipin simultaneously. Incorporation of genipin imparts a greenish-blue color to the polymeric film. The effect of dual crosslinking and their successful interaction within the matrix was evaluated by infrared analysis spectroscopy. The porosity of the film was examined using scanning electron microscopy (SEM). Results of TGA determine the intermediate thermal degradation. Further, the crosslinking phenomenon has found primary impact on the strength of the films. Enzymatic degradation for the films was performed with lysozyme and lipase. The cell adhesion and proliferation was also accomplished using mouse embryonic cell lines wherein the cells cultured on the dual crosslinked film. The thriving utilization of such dual crosslinked polymeric film finds their applications in ophthalmology especially as an implant for temporary injured cornea and skin tissue regeneration.

In-Vitro Hemocompatibility of Polyaniline Functionalized by Bioactive Molecules.

Hemocompatibility is an essential prerequisite for the application of materials in the field of biomedicine and biosensing. In addition, mixed ionic and electronic conductivity of conducting polymers is an advantageous property for these applications. Heparin-like materials containing sulfate, sulfamic, and carboxylic groups may have an anticoagulation effect. Therefore, sodium dodecylbenzenesulfonate, 2-aminoethane-1-sulfonic acid and N-(2-acetamido)-2-aminoethanesulfonic acid were used for modification of the representative of conducting polymers, polyaniline, and the resulting products were studied in the context of interactions with human blood. The anticoagulation activity was then correlated to surface energy and conductivity of the materials. Results show that anticoagulation activity is highly affected by the presence of suitable functional groups originating from the used heparin-like substances, and by the properties of polyaniline polymer itself.

Exploring the Critical Factors Limiting Polyaniline Biocompatibility.

Today, the application of polyaniline in biomedicine is widely discussed. However, information about impurities released from polyaniline and about the cytotoxicity of its precursors aniline, aniline hydrochloride, and ammonium persulfate are scarce. Therefore, cytotoxicity thresholds for the individual precursors and their combinations were determined (MTT assay) and the type of cell death caused by exposition to the precursors was identified using flow-cytometry. Tests on fibroblasts revealed higher cytotoxicity of ammonium persulfate than aniline hydrochloride. Thanks to the synergic effect, both monomers in combination enhanced their cytotoxicities compared with individual substances. Thereafter, cytotoxicity of polyaniline doped with different acids (sulfuric, nitric, phosphoric, hydrochloric, and methanesulfonic) was determined and correlated with impurities present in respective sample (HPLC). The lowest cytotoxicity showed polyaniline doped with phosphoric acid (followed by sulfuric, methanesulfonic, and nitric acid). Cytotoxicity of polyaniline was mainly attributed to the presence of residual ammonium persulfate and low-molecular-weight polar substances. This is crucial information with respect to the purification of polyaniline and production of its cytocompatible form.