The bulk mechanical properties of soft materials have been studied widely, but it is unclear to what extent macroscopic behavior is reflected in nanomechanics. Using an atomic force microscopy (AFM) imaging method called force spectroscopy mapping (FSM), it is possible to map the nanoscopic spatial distribution of Young's modulus, i.e. “stiffness,” and determine if soft or stiff polymer domains exist to correlate nano- and macro-mechanics. Two model hydrogel systems typically used in cell culture and polymerized by a free radical polymerization process, i.e. poly (vinyl pyrrolidone) (PVP) and poly(acrylamide) (PAam) hydrogels, were found to have significantly different nanomechanical behavior despite relatively similar bulk stiffness and roughness. PVP gels contained a large number of soft and stiff nanodomains, and their size was inversely related to crosslinking density and changes in crosslinking efficiency within the hydrogel. In contrast, PAam gels displayed small nanodomains occuring at low frequency, indicating relatively uniform polymerization. Given the responsiveness of cells to changes in gel stiffness, inhomogeneities found in the PVP network indicate that careful nanomechanical characterization of polymer substrates is necessary to appreciate complex cell behavior.

Hydrogels are three-dimensional networks with high water content become soft and elastic mimicking some characteristics of the extracellular matrix. They can be prepared by using either synthetic or natural polymers, therefore, their combination can benefit the overall of the material for a specific biomedical application. In this study, a semi-interpenetrating polymer network hydrogel of polyacrylamide/gelatin (PAM/Gel) with up to 0.3% of gelatin content was synthesized while evidence of gelatin incorporation was obtained from FTIR and Raman spectroscopy. SEM micrographs show uniform porous structure of PAM/gelatin combined hydrogels. According to the micrographs, the amount of pores decrease with the incorporation of gelatin into the PAM network. The combined hydrogel had the maximum swelling at 37 °C (1,800 ± 73%). Results indicate that PAM/Gel hydrogels are biocompatible (fibroblast viability >70%) in the presence of extracts and by direct contact method. These hydrogels can be good candidates for soft tissue engineering applications. GRAPHICAL ABSTRACT

This work shows the synthesis of a polyvinylpyrrolidone (PVP) hydrogel by heat-activated polymerization and explores the production of hydrogels with an open porous network by lyophilisation to allow the three-dimensional culture of human oral mucosa stem cells (hOMSCs). The swollen hydrogel showed a storage modulus similar to oral mucosa and elastic solid rheological behaviour without sol transition. A comprehensive characterization of porosity by scanning electron microscopy, mercury intrusion porosimetry and nano-computed tomography (with spatial resolution below 1 μm) showed that lyophilisation resulted in the heterogeneous incorporation of closed oval-like pores in the hydrogel with broad size distribution (5 to 180 μm, d₅₀ = 65 μm). Human oral mucosa biopsies were used to isolate hOMSCs, expressing typical markers of mesenchymal stem cells in more than 95% of the cell population. Direct contact cytotoxicity assay demonstrated that PVP hydrogel have no negative effect on cell metabolic activity, allowing the culture of hOMSCs with normal fusiform morphology. Pore connectivity should be improved in future to allow cell growth in the bulk of the PVP hydrogel.