Biologically active artificial scaffolds for cell seeding are developed by mimicking extracellular matrices using synthetic materials. Here, we propose a feasible approach employing biocatalysis to integrate natural components, that is, gelatin and heparin, into a synthetic scaffold, namely a polyethylene glycol (PEG)-based hydrogel. Initiation of horseradish peroxidase-mediated redox reaction enabled both hydrogel formation of tetra-thiolated PEG via disulfide linkage and incorporation of chemically thiolated gelatin (Gela-SH) and heparin (Hepa-SH) into the polymeric network. We found that the compatibility of the type of gelatin with heparin was crucial for the hydrogelation process. Alkaline-treated gelatin exhibited superior performance over acid-treated gelatin to generate dual functionality in the resultant hydrogel originating from the two natural biopolymers. The Gela-SH/Hepa-SH dual functionalized PEG-based hydrogel supported both cellular attachment and binding of basic fibroblast growth factor (bFGF) under cell culture conditions, which increased the proliferation and phenotype transformation of NIH3T3 cells cultured on the hydrogel. Inclusion of bFGF and a commercial growth factor cocktail in hydrogel matrices effectively enhanced cell spreading and confluency of both NIH3T3 cells and HUVECs, respectively, suggesting a potential method to design artificial scaffolds containing active growth factors.

Reactions of N‐(2,4‐dinitrophenyl)pyridinium chloride (salt(Cl)) with H+MCl4−1 (M ≡ Fe and Bi) resulted in an anion exchange between Cl− and MCl4− to yield Zincke salts with metal chloride anions, namely salt(Fe) and salt(Bi), respectively. Reactions of the Zincke salts with piperazine resulted in ring‐opening of the pyridinium ring, yielding ionic polymers with 5‐piperazinium‐2,4‐dienylideneammonium metal chloride units, namely polymer(Fe) and polymer(Bi). The corresponding model compounds were synthesized via reactions using salt(Bi) or salt(Cl) as starting materials. The UV–visible spectra of the polymers had absorption maxima at longer wavelengths than those of the model compounds. This indicated that the π‐conjugation system is expanded along the polymer main chain. Superconducting quantum interference device measurements indicated that polymer(Fe) was paramagnetic. Cyclic voltammetry analysis suggested that the polymers underwent electrochemical oxidation. © 2019 Society of Chemical Industry

Hydrogels possessing the ability to control cell functions have great potential as artificial substrates for cell culture. Herein, we report dual-functionalizable protein–polymer hybrid hydrogels prepared by thiol oxidation catalyzed by horseradish peroxidase and a phenolic molecule. A chimera protein of streptavidin (SA) and the SpyCatcher protein, with a cysteine residue at its N-terminus, (C-SA-SC) was constructed and co-cross-linked with thiol-functionalized four-arm polyethylene glycol (PEG-SH) to obtain hydrogels possessing two orthogonal conjugation moieties. Hydrogel formation using C-SA-SC conjugated with biotinylated or SpyTagged functional molecules (premodification strategy) resulted in the formation of hydrogels with a uniform distribution of the functional molecules. Postmodification of the functional molecules of the C-SA-SC hydrogel with biotin or SpyTag could alter the three-dimensional (3D) spatial distribution of the functional molecules within the hydrogels depending on the mode of conjugation (SA/biotin or SpyCatcher/SpyTag), the size of the functional molecules, and the length of time of the modification. NIH-3T3 cells cultured on a C-SA-SC hydrogel, dual-functionalized with a biotinylated-Arg-Gly-Asp-Ser (RGDS) peptide and a basic fibroblast growth factor (bFGF) with SpyTag, showed cell adhesion to the PEG-SH-based hydrogels and cell morphological changes in response to the immobilized RGDS peptide and the bFGF. Moreover, the cells showed higher proliferation on the dual-functionalized C-SA-SC hydrogel than the cells cultured on hydrogels without either the RGDS peptide or the bFGF, demonstrating the benefits of dual-functionalizable hydrogels. The C-SA-SC hydrogel presented in this study is capable of being orthogonally functionalized by two different functional molecules with different 3D distributions of each molecule within the hydrogel and thus has the potential for use as a cell culturing scaffold for creating artificial cellular microstructures.

No abstract