Synthetic hydrogels have garnered intense interest as extracellular matrix (ECM) mimics, yet still do not fully replicate the hierarchical nature of biological tissue, which limits their biological efficacy as stem cell culture scaffolds for tissue engineering and regenerative medicine. The native ECM is made up of hierarchical biopolymers with precise sequences and chain structures that yield high molecular rigidity [1], [2], [3], [4]. These structures result in stiff microenvironments at relatively low polymer concentrations. Synthetic hydrogels, meanwhile, typically utilize changes in polymer concentration or crosslinking stoichiometry to modulate mechanics with flexible, disordered polymers [5]. While this approach yields tunable and predictable moduli, it inherently links mechanics to other parameters of the hydrogel system, such as network connectivity and mesh size, and therefore affects permeability to cytokines and growth factors [6], [7], [8], [9], [10]. This inherent coupling may diminish the efficacy of synthetic hydrogels as stem cell culture scaffolds by hindering the concentration or availability of cell signaling factors, as well as disrupting the matrix remodeling processes.

To decouple mechanics from network connectivity, recent efforts have investigated the incorporation of stiff, biomimetic chains into synthetic hydrogel formulations [11], [12], [13], [14], [15]. Helical polypeptides were incorporated into poly(ethylene glycol, PEG)-based hydrogels as crosslinkers, yielding higher elastic moduli than hydrogels with non-helical polypeptide crosslinkers due to the increased persistence length of the helices [11]. In these studies, hydrogels formed with helical crosslinkers facilitated attachment of human mesenchymal stromal cells (hMSCs) [13] and endothelial cells [14] by increasing local stiffness at the nanoscale. In a complementary strategy, collagen mimetic peptides were incorporated into synthetic PEG hydrogels, capturing the multiscale structure of collagen-rich tissue and leading to cell morphologies that resembled those in traditional collagen platforms [15]. While both of the aforementioned systems leveraged biological motifs for stiff secondary structures, it is important to note that they are sensitive to environmental and processing conditions that affect hydrogen bonding interactions. In a non-biological system, it was shown that controlling the stereochemistry of alkene linkages in a PEG hydrogel resulted in control over bulk mechanics, with larger cis contents leading to lower elastic moduli [12]. Again, control over stereochemistry was achieved by adjusting solvent polarity and basicity during hydrogel fabrication [12]. To avoid this dependence, more robust synthetic strategies to control chain rigidity and structure are needed.

Peptoids are a class of non-natural molecules capable of forming biomimetic secondary structures based on their sequence definition, which arises from primary amine submonomers [16], [17], [18]. These secondary structures include helices [19], [20], [21], [22], [23], which have been shown to have higher persistence lengths [24] and shorter end-to-end distances [19,25] than non-helical analogs. Because peptoids form polyproline type I helices through sterics [21,26], they are less sensitive to temperature and solvent conditions than peptide helices, which rely on hydrogen bonding interactions [27]. Previous work has shown helical peptoids can alter the self-assembled nanostructure of block copolymers [28,29], and that they have more local chain stiffness and compact chain conformations compared to racemic analogs [25,30]. In addition, we previously showed that increasing the length of helical peptoid crosslinkers increased the elastic moduli of PEG hydrogels, breaking the trend predicted by rubber elasticity theory for flexible polymer networks [31]. Thus, peptoids are an excellent model system for probing the effect of crosslinker chain structure on bulk mechanics for engineered ECM applications.

In this work, we investigated the impact of peptoid crosslinker secondary structure on hydrogel mechanics and whether the resulting changes in mechanics affected attached cell behavior. Our strategy was to decouple hydrogel storage modulus (G’) from network connectivity using a suite of sequence defined peptoids with different chain structures: a helical sequence, a non-helical but chemically similar sequence, and an unstructured sequence. We show that bulk mechanics can be controlled with the molecular rigidity of peptoids, leveraging hierarchical order for properties in a fashion similar to biopolymers of the native ECM. This series of peptoids enabled a highly tunable crosslinker stiffness, expanding the toolkit of available molecules for synthetic ECM. To determine the applicability of this system for modulating cell-ECM interactions, we seeded human mesenchymal stromal cells (hMSCs) on the hydrogels, since these cells are promising for allogenic cell therapies and exhibit immunomodulatory functions that are sensitive to matrix mechanics [32], [33], [34], [35], [36]. We found that the range of stiffness was sufficient to affect cell morphology, proliferation, and immunomodulatory activity, with softer hydrogels producing more therapeutically potent cells. Altogether, this study highlights a unique way to regulate bulk mechanics using a property that is often overlooked or unachievable in synthetic ECM: molecular rigidity.