Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross‐linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein‐1, macrophage colony‐stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN‐γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN‐γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN‐γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN‐γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN‐γ.
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Abstract Free, publicly-accessible full text available April 18, 2025 -
The utility of active proteases as biomarkers is often limited by overlapping substrate specificity. Here, this feature is leveraged to develop a quantitative pattern-recognition sensing system driven by the degradation patterns of peptide–peptoid hybrid substrates to classify proteases and estimate their concentration by multivariate data analysis.more » « less
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Due to their N -substitution, peptoids are generally regarded as resistant to biological degradation, such as enzymatic and hydrolytic mechanisms. This stability is an especially attractive feature for therapeutic development and is a selling point of many previous biological studies. However, another key mode of degradation remains to be fully explored, namely oxidative degradation mediated by reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS are biologically relevant in numerous contexts where biomaterials may be present. Thus, improving understanding of peptoid oxidative susceptibility is crucial to exploit their full potential in the biomaterials field, where an oxidatively-labile but enzymatically stable molecule can offer attractive properties. Toward this end, we demonstrate a fundamental characterization of sequence-defined peptoid chains in the presence of chemically generated ROS, as compared to ROS-susceptible peptides such as proline and lysine oligomers. Lysine oligomers showed the fastest degradation rates to ROS and the enzyme trypsin. Peptoids degraded in metal catalyzed oxidation conditions at rates on par with poly(prolines), while maintaining resistance to enzymatic degradation. Furthermore, lysine-containing peptide–peptoid hybrid molecules showed tunability in both ROS-mediated and enzyme-mediated degradation, with rates intermediate to lysine and peptoid oligomers. When lysine-mimetic side-chains were incorporated into a peptoid backbone, the rate of degradation matched that of the lysine peptide oligomers, but remained resistant to enzymatic degradation. These results expand understanding of peptoid degradation to oxidative and enzymatic mechanisms, and demonstrate the potential for peptoid incorporation into materials where selectivity towards oxidative degradation is necessary, or directed enzymatic susceptibility is desired.more » « less