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

含有生物活性肽引发的丝蛋白的水凝胶已成功地用于 研究骨骼肌分化的细胞过程。但此前的 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， 我们展示了一种与酪胺取代的丝纤蛋白交联的新的丝水凝胶， 随着时间的推移以可控的速率硬化。这些水凝胶的可编程刚度使得 它们对于模拟与以下相关的组织水平刚度的变化是有吸引力的： 肌肉骨骼发育或受伤后。我们将修改这些水凝胶， 脱细胞肌肉细胞外基质（ECM），通过最近建立的 脱细胞方案。我们的初步数据表明，将ECM偶联到我们的丝基质上， 用于进一步微调硬化，实现高度可控和独特的机械性能。 和基质蛋白梯度，通过在空间上改变的量和类型， 细胞外基质和丝的前体混合在一起。丝-ECM水凝胶先前尚未被开发。 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 和生化特征可以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 能够更好地控制肌源性分化的治疗潜力的方法 iPSC用于再生骨骼肌。我们将开发水凝胶作为新的体外系统， 探索动态刚度对iPSCs肌生成的影响。我们将测试我们的假设 有两个具体目标。第一个目标是确定三维水凝胶的刚度如何演变， 影响iPSC肌生成。第二个目标是开发一种生物化学功能化的 和机械动态丝-ECM水凝胶，用于从iPSC产生骨骼肌。 这些目标的完成将增进我们对骨骼肌调节因子的了解 动态基质刚度和基质组成对干细胞的影响 分化，最终目标是治疗靶向这些机制， 用干细胞再生骨骼肌。3D水凝胶可进一步用于研究 调节骨骼肌发育、衰老、损伤和疾病的过程。

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.