Liquid–Liquid Phase Separation-Mediated Cellular-Scale Compartmentalization of Hydrogel Covalent Cross-Linking Promotes Microtubule-Based Mechanosensing

Abstract

Controlled liquid-liquid phase separation (LLPS) plays an important role in the formation of a heterogeneously structured extracellular matrix (ECM) consisting of densely cross-linked stiff structures compartmentalized in a loosely cross-linked matrix. Moreover, the mechanical cues presented by the cellular-scale structural heterogeneity of the ECM facilitate the mechanotransduction of cells and subsequent cellular development. Therefore, developing ECM-mimetic hydrogels with compartmentalized structural heterogeneity as inductive cell carriers is highly desirable but challenging. Inspired by the ECM formation process, we capitalized on the temperature-assisted LLPS of a custom-designed temperature-responsive macromer (TRM) to concentrate and compartmentalize the TRM in the dense phase of the phase-separated precursor solution while keeping the gelatin comacromer complex in the dilute phase. The subsequent cross-linking produces the cellular (micron)-scale microdomains with dense covalent cross-linking interspersed in the loosely cross-linked cell-adaptable interdomain hydrogel matrix. The obtained ECM-mimetic heterogeneous hydrogel, which is solely cross-linked by covalent bonds, promotes extensive spreading, microtubule-based mechanotransduction, and autophagic flux of encapsulated human mesenchymal stem cells (hMSCs), thereby enhancing osteogenesis and bone regeneration. Our findings not only provide valuable guidance for the fabrication of ECM-mimetic biomaterials via LLPS-mediated assembly but also shed light on the mechanobiological mechanism underlying the regulation of cellular development by mechanical cues of the ECM.