Dynamic Biomaterials: Cyclic Stiffness Modulation of Cell‐Laden Protein–Polymer Hydrogels in Response to User‐Specified Stimuli Including Light (Adv. Biosys. 12/2018)

Abstract

There is a growing appreciation for the large role that mechanical signals presented by the local extracellular matrix (ECM) have on cell function. Through direct interaction with physical cues presented in the cellular ECM, these external mechanical signals are translated into internal biochemical responses that govern gene expression and cell fate decisions. [1] Seminal findings in the field of cellular mechanotransduction have demonstrated that matrix stiffness alone can drive changes in essential processes including attachment, cytoskeletal organization , migration, proliferation, and differentiation. [2-4] More recently, it has been observed that cells possess mechanical "memory," storing information about past physical culture conditions to influence future behaviors. [5] These findings represent landmark observations that are rapidly changing standard practices in molecular biology and stem cell culture. Efforts to elucidate the specific effects that ECM stiffness and elasticity have on cell physiology have been performed almost exclusively using static biomaterials. While these studies have provided invaluable insight into the critical roles in which ECM stiffness regulates cellular fate, such simple systems fail to emulate biophysical dynamics known to accompany tissue/organ development, regeneration, and disease progression. Strategies to probe the biological effects of evolving tissue compliance have yielded a variety of synthetic cell culture platforms that can either soften or stiffen over time. [6] Though constructs that undergo spontaneous or cell-mediated transitions have proven beneficial in several applications, those that can be modified on demand are critical for probing biophys-ical responses at well-defined times. [7] Light-mediated material alteration has proven particularly beneficial in this regard as it uniquely grants near-instantaneous and spatiotemporal control over matrix stiffness in a potentially biocompatible manner. [8] Material secondary photo-crosslinking enables one-way stiffening , [9-14] while photodegradation provides for irreversible softening , [15-19] in the presence of live cells. Beyond unidirectional elasticity changes, cells also experience cyclic loading that periodically and reversibly alter local ECM rigidity; pulsatile flow associated with the circulatory system Although mechanical signals presented by the extracellular matrix are known to regulate many essential cell functions, the specific effects of these interactions , particularly in response to dynamic and heterogeneous cues, remain largely unknown. Here, a modular semisynthetic approach is introduced to create protein-polymer hydrogel biomaterials that undergo reversible stiffening in response to user-specified inputs. Employing a novel dual-chemoenzymatic modification strategy, fusion protein-based gel crosslinkers are created that exhibit stimuli-dependent intramolecular association. Linkers based on calmodulin yield calcium-sensitive materials, while those containing the photosensitive light, oxygen, and voltage sensing domain 2 (LOV2) protein give phototunable constructs whose moduli can be cycled on demand with spatiotemporal control about living cells. These unique materials are exploited to demonstrate the significant role that cyclic mechanical loading plays on fibroblast-to-myofibroblast transdifferentiation in 3D space. The moduli-switchable materials should prove useful for studies in mechanobi-ology, providing new avenues to probe and direct matrix-driven changes in 4D cell physiology.