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.
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.
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