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The demand for synthetic soft materials with bioinspired structures continues to grow. Material applications range from in vitro and in vivo tissue mimics to therapeutic delivery systems, where well-defined synthetic building blocks offer precise and reproducible property control. This work examines a synthetic assembling peptide, specifically a multifunctional collagen mimetic peptide (mfCMP) either alone or with reactive macromers, for the creation of responsive hydrogels that capture aspects of soft collagen-rich tissues. We first explored how buffer choice impacts mfCMP hierarchical assembly, in particular, peptide melting temperature, fibril morphology, and ability to form physical hydrogels. Assembly in physiologically relevant buffer resulted in collagen-like fibrillar structures and physically assembled hydrogels with shear-thinning (as indicated through strain-yielding) and self-healing properties. Further, we aimed to create fully synthetic, composite peptide-polymer hydrogels with dynamic responses to various stimuli, inspired by the extracellular matrix (ECM). Specifically, we established mfCMP-poly(ethylene glycol) (PEG) hydrogel compositions that demonstrate increasing non-linear viscoelasticity in response to applied strain as the amount of assembled mfCMP content increases. Furthermore, the thermal responsiveness of mfCMP physical crosslinks was harnessed to manipulate the composite hydrogel mechanical properties in response to changes in temperature. Finally, cells relevant in wound healing, human lung fibroblasts, were encapsulated within these peptide-polymer hydrogels to explore the impact of increased mfCMP, and the resulting changes in viscoelasticity, on cell response. This work establishes mfCMP building blocks as versatile tools for creating hybrid and adaptable systems with applications ranging from injectable shear-thinning materials to responsive interfaces and synthetic ECMs for tissue engineering.

Assembling peptides allow the creation of structurally complex materials, where amino acid selection influences resulting properties. We present a synergistic approach of experiments and simulations for examining the influence of natural and non-natural amino acid substitutions via incorporation of charged residues and a reactive handle on the thermal stability and assembly of multifunctional collagen mimetic peptides (CMPs). Experimentally, we observed inclusion of charged residues significantly decreased the melting temperature of CMP triple helices with further destabilization upon inclusion of the reactive handle. Atomistic simulations of a single CMP triple helix in explicit water showed increased residue-level and helical structural fluctuations caused by the inclusion of the reactive handle; however, these atomistic simulations cannot be used to predict changes in CMP melting transition. Coarse-grained (CG) simulations of CMPs at experimentally relevant solution conditions, showed, qualitatively, the same trends as experiments in CMP melting transition temperature with CMP design. These simulations show that when charged residues are included electrostatic repulsions significantly destabilize the CMP triple helix and that an additional inclusion of a reactive handle does not significantly change the melting transition. Based on findings from both experiments and simulations, the sequence design was refined for increased CMP triple helix thermal stability, and the reactive handle was utilized for the incorporation of the assembled CMPs within covalently crosslinked hydrogels. Overall, a unique approach was established for predicting stability of CMP triple helices for various sequences prior to synthesis, providing molecular insights for sequence design towards the creation of bulk nanostructured soft biomaterials.

Approaches for the creation of soft materials, particularly hydrogels, with hierarchical structure are of interest in a variety of applications owing to their unique properties. In the context of tissue mimics, hydrogels with multiscale structures more accurately capture the complexities of tissues within the body (e.g., fibrous collagen-rich microenvironments). However, cytocompatible fabrication of such materials with hierarchical structures and independent control of mechanical and biochemical properties remains challenging and is needed for probing and directing cell-microenvironment interactions for three-dimensional (3D) cell encapsulation and culture applications. To address this, we have designed innovative multifunctional assembling peptides: these unique peptides contain a core block that mimics the structure of collagen for achieving relevant melting temperatures; 'sticky' ends to promote assembly of long fibrils; and a biocompatible reactive handle that is orthogonal to assembly to allow the formation of desired multiscale structures and their subsequent rapid, light-triggered integration within covalently crosslinked synthetic hydrogels. Nano- to micro-fibrils were observed to form in physiologically-relevant aqueous solutions, where both underlying peptide chemical structure and assembly conditions were observed to impact the resulting fibril sizes. These assembled structures were 'locked' into place and integrated as linkers within cell-degradable, bioactive hydrogels formed with photoinitiated thiol-ene 'click' chemistry. Hydrogel compositions were identified for achieving robust mechanical properties like those of soft tissues while also retaining higher ordered structures after photopolymerization. The utility of these innovative materials for 3D cell culture was demonstrated with human mesenchymal stem cells, where cell morphologies reminiscent of responses to assembled native collagen were observed now with a fully synthetic material. Using a bottom-up approach, a new materials platform has been established that combines the advantageous properties of covalent and assembling chemistries for the creation of synthetic hydrogels with controllable nanostructure, mechanical properties, and biochemical content.

Stem cells reside in complex three-dimensional (3D) environments within the body that change with time, promoting various cellular functions and processes such as migration and differentiation. These complex changes in the surrounding environment dictate cell fate yet, until recently, have been challenging to mimic within cell culture systems. Hydrogel-based biomaterials are well suited to mimic aspects of these in vivo environments, owing to their high water content, soft tissue-like elasticity, and often-tunable biochemical content. Further, hydrogels can be engineered to achieve changes in matrix properties over time to better mimic dynamic native microenvironments for probing and directing stem cell function and fate. This review will focus on techniques to form hydrogel-based biomaterials and modify their properties in time during cell culture using select addition reactions, cleavage reactions, or non-covalent interactions. Recent applications of these techniques for the culture of stem cells in four dimensions (i.e., in three dimensions with changes over time) also will be discussed for studying essential stem cell processes.