3D in vitro model of skeletal muscle development using stiffening silk biomaterials

含有生物活性多肽的丝蛋白水凝胶已经成功应用 研究骨骼肌分化的细胞过程。但是，以前的 系统由于其静态性质而受到限制--它们的机械性能是固定的。最近， 我们展示了一种新的丝素水凝胶，它与酪胺取代的丝素蛋白交联。 随着时间的推移，以可控的速度变得僵硬。这些水凝胶的可编程硬度使 它们对于模拟与组织水平僵硬相关的变化很有吸引力 肌肉骨骼发育，或受伤后。我们将对这些水凝胶进行修饰，以加入 脱细胞肌肉细胞外基质(ECM)，通过最近建立的 去细胞方案。我们的初步数据表明，将ECM连接到我们的丝绸基质可以 用于进一步微调加劲，实现高度可控和独特的机械 和同一凝胶中的基质蛋白质梯度，通过在空间上改变 ECM与丝绸前体混合在一起。此前还没有开发出丝素-ECM水凝胶。 我们的中心假设是，具有高度可调机械性能的动态硬化水凝胶 生化特征可以更多地概括肌源性环境的关键方面 比现有的工程系统更有效，因此将提高我们对 能够更好地控制肌肉发生分化的治疗潜力的过程 用于再生骨骼肌的IPSCs。我们将开发水凝胶作为新的体外系统来 探讨动态僵硬对IPSCs肌发生的影响。我们将检验我们的假设 使用两个特定的目标。第一个目标将是确定3D水凝胶的硬度如何演变 影响IPSC的肌肉发生。第二个目标将是开发一种生化功能化的 以及机械动态丝素-ECM水凝胶，用于从IPSCs生成骨骼肌。 这些目标的完成将加深我们对骨骼肌调节因子的理解 动态基质硬度和基质组成对干细胞的影响及其发展 分化，最终目标是以这些机制为治疗靶点 使用干细胞再生骨骼肌。3D水凝胶可以进一步用于研究 骨骼肌发育、衰老、损伤和疾病的调节过程。

Hydrogels incorporating silk protein primed with bioactive peptides have been successfully used to study the cellular processes underlying differentiation of skeletal muscle. However, previous systems were limited due to their static nature – their mechanical properties are fixed. Recently, we demonstrated a new silk hydrogel crosslinked with tyramine-substituted silk fibroin that stiffened over time at controllable rates. The programmable stiffness of these hydrogels makes them attractive for modeling the changes in tissue-level stiffness that are associated with musculoskeletal development, or following injury. We will modify these hydrogels to incorporate decellularized muscle extracellular matrix (ECM), obtained through a recently established decellularization protocol. Our preliminary data suggest coupling ECM to our silk matrices can be used to further fine-tune the stiffening, enabling highly controllable and distinct mechanical and matrix protein gradients within the same gel, by spatially varying the amount and type of ECM mixed in with the silk precursors. A silk-ECM hydrogel has not previously been developed. Our central hypothesis is that dynamically stiffening hydrogels with highly tunable mechanical and biochemical characteristics can recapitulate key aspects of the myogenic environment more effectively than existing engineered systems, and as a result, will improve our understanding of the process to enable better control of the therapeutic potential of myogenically differentiating iPSCs for regenerating skeletal muscle. We will develop hydrogels as novel in vitro systems to explore the impacts of dynamic stiffness on myogenesis of iPSCs. We will test our hypothesis using two specific aims. The first aim will be to determine how evolving stiffness in 3D hydrogels impacts iPSC myogenesis. The second aim will be to develop a biochemically functionalized and mechanically dynamic silk-ECM hydrogel for generation of skeletal muscle from iPSCs. Completion of these aims will enhance our understanding of the regulators of skeletal muscle development and the impact of dynamic substrate stiffness and matrix composition on stem cell differentiation, with the ultimate goal of therapeutically targeting these mechanisms to regenerate skeletal muscle using stem cells. 3D hydrogels can be further used to investigate the processes that regulate development, aging, injury, and disease of skeletal muscle.