An efficient procedure for chemical initiator-free, in situ synthesis of a functional polyethylene glycol methacrylate (PEG MA) hydrogel on regular glass substrates is reported. It is demonstrated that self-initiated photografting and photopolymerization driven by UV irradiation can yield tens of nanometer-thick coatings of carboxy-functionalized PEG MA on the aldehyde-terminated borosilicate glass surface. The most efficient formulation for hydrogel synthesis contained methyl methacrylic acid (MAA), 2-hydroxyethyl methacrylate (HEMA), and PEG methacrylate (PEG10MA) monomers (1:1:1). The resulting HEMA/PEG10MA/MAA (HPMAA) coatings had a defined thickness in the range from 11 to 50 nm. The physicochemical properties of the synthesized HPMAA coatings were analyzed by combining water contact angle measurements, stylus profilometry, imaging null ellipsometry, and atomic force microscopy (AFM). The latter technique was employed in the quantitative imaging mode not only for direct probing of the surface topography but also for swelling behavior characterization in the pH range from 4.5 to 8.0. The estimated high swelling ratios of the HPMAA hydrogel (up to 3.2) together with its good stability and resistance to nonspecific protein binding were advantageous in extracellular matrix mimetics via patterning of fibronectin (FN) at a resolution close to 200 nm. It was shown that the fabricated FN micropatterns on HPMAA were equally suitable for single-cell arraying, as well as controlled cell culture lasting at least for 96 h.

Chemical and mechanical properties of a tumor microenvironment are essential players in cancer progression, and it is important to precisely control the extracellular conditions while designing cancer in vitro models. The study investigates synthetic hydrogel matrices from multi-arm polyethylene glycol (PEG) functionalized with collagen-like peptide (CLP) CG(PKG) 4 (POG) 4 (DOG) 4 alone and conjugated with either cell adhesion peptide RGD (mimicking fibronectin) or IKVAV (mimicking laminin). Human glioblastoma HROG36, rat C6 glioma cells, and A375 human melanoma cells were grown on the hydrogels and monitored for migration, proliferation, projected cell area, cell shape index, size and number, distribution of focal contacts in individual cells, and focal adhesion number. PEG-CLP-RGD induced migration of both glioma cell lines and also stimulated proliferation (assessed as metabolic activity) of HROG36 cells. Migration of C6 cells were also stimulated by PEG-CLP-IKVAV. These responses strongly correlated with the changes in adhesion and morphology parameters of individual cells-projected cell area, cell shape index, and focal contact number. Melanoma A375 cell proliferation was increased by PEG-CLP-RGD, and this was accompanied by a decrease in cell shape index. However, neither RGD nor IKVAV conjugated to PEG-CLP stimulated migratory capacity of A375 cells. Taken together, the study presents synthetic scaffolds with extracellular matrix (ECM)-mimicking peptides that allow for the exploration of the effect of ECM signaling to cancer cells.

Herein we address, hyaline cartilage regeneration issue by engineering a synthetic biocompatible hydrogel scaffold capable to promote chondrogenic differentiation. In this study, the chemically crosslinked hydrogels consisting of synthetic peptides that have the collagen-like sequence Cys-Gly-(Pro-Lys-Gly)4 (Pro-Hyp-Gly)4 (Asp-Hyp-Gly)4- conjugated with RGD sequence (CLP-RGD) and crosslinked hydrogels of type I collagen (CA) were used. For cartilage formation, we used human skeletal muscle-derived stem/progenitor cells (hMDSPCs) set for differentiation towards a chondrogenic lineage by BMP-7 and TGF-ß3 growth factors.Initially 150, 100 and 75 ng of BMP-7and TGF-ß3 growth factors were inserted in each scaffold and amount of growth factors diffusing out of the scaffolds was observed by ELISA assays. In vitro experiments were performed by seeding hMDSPCs onto hydrogels loaded with growth factors (75ng/scaffold) and cultured for 28 days. Cartilage formation was monitored by ELISA and RT-PCR assays. All experiments were performed in triplicates or quadruplicates.Growth factors incorporation strategy allowed a sustained release of TGF-ß3 growth factor, 6.00.3% of the initially loaded amount diffused out after 4 h and 2.70.5% already at the second time point (24h) from CA and CLP-RGD substrates. For the BMP-7 growth factor, 13.12.3% and 15.751.6% of the initially loaded amount diffused out after 4 h, 1.70.2% and 2.450.3% at the second time point (24 h) from CA and CLP-RGD respectively. In vitro experiments shown that scaffolds with immobilized growth factors resulted in higher collagen type II accumulation when compared to the scaffolds alone. The gene expression on CLP-RGD hydrogels with growth factors has shown lower collagen type I expression and higher aggrecan expression compared to day 0. However, we also report increased collagen X gene expression on CA hydrogels (with growth factors).Our results support the potential of the strategy of combining hydrogels functionalized with differentiation factors toward improving cartilage repair.

Surface immobilization and characterization of the functional activity of fibronectin (Fn) type-III domains are reported. The domains FnIII9-10 or FnIII10 containing the RGD loop and PHSRN synergy site were recombinantly produced and covalently bound to chemically activated PEG methacrylate (MA) hydrogel coatings by microcontact printing. Such fabricated biochip surfaces were 6 mm in diameter and consisted of 190 µm wide protein stripes separated by 200 µm spacing. They were analyzed by imaging null ellipsometry, atomic force microscopy and fluorescence microscopy. Also, the coatings were tested in human foreskin fibroblast and HeLa cultures for at least 96 h, thus evaluating their suitability for controlled cell adhesion and proliferation. However, while HeLa cultures were equally well responsive to the FnIII9-10, FnIII10 and Fn surfaces, the fibroblasts displayed lower cell and lower focal adhesion areas, as well as lower proliferation rates on the Fn fragment surfaces as compared to Fn. Nevertheless, full functional activity of the fibroblasts was confirmed by immunostaining of Fn produced by the cells adherent on the biochip surfaces. The observed interaction differences that were either cell type or surface composition-dependent demonstrate the potential use of specifically engineered Fn and other ECM protein-derived domains in biochip architectures.