Hydrogel Enhanced Organoid Multidirectional Differentiation via Yap/Tead4 Mechanotransduction for Accelerated Tissue Regeneration.

The repair of multiple organs in motor systems remains a major clinical challenge that necessitates bioactive grafts with a multidirectional differentiation ability. Hydrogel-based organoids implants have emerged as pivotal tools and attracted great attentions. However, strategies to unlock the multipotency of bone marrow mesenchymal stem cells (BMSCs) by precisely modulating the mechanical and structural characteristics of biomimetic extracellular matrix (ECM) during hydrogel-based organoid construction remain underexplored. In this study, a gelatin methacryloyl (GelMA)-based biomimetic ECM mimic hydrogel (HG-2) loaded with BMSCs was developed to construct a multidirectional differentiation organoid, HG-2/3d-BMSC. The hydrogel could provide spatial mechanical stimulation to adherent BMSCs via cell adhesion induced cytoskeleton assembly. RNA sequencing (RNA-Seq) combined with in vitro and in vivo biological experiments reveals that ECM mimic hydrogels deliver adhesion-based spatial mechanical stimulation. This mechanical stimulation specifically unlocks the multipotency of BMSCs during osteogenic differentiation induction. Furthermore, it accelerates and enhances the multidirectional differentiation capacity of BMSCs, simultaneously promoting their commitment to osteogenic, chondrogenic, and tendonogenic tissue lineages. Further investigations prove that adhesion-based spatial mechanical stimulation from the ECM mimic hydrogel enhances multidirectional differentiation of BMSCs-based organoid via Yap/Tead4 (yes-associated protein/TEA domain transcription factor 4) mechanotransduction mediated Kat7 downregulation. The work not only advances the theoretical framework for designing biomaterials that exploit mechanical cues to override biochemical-driven lineage commitment but also establishes a novel paradigm for developing multifunctional organoid constructs to address the clinical challenge of regenerating hierarchically complex tissues in a motor system.