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

使用硬化丝生物材料的骨骼肌发育的 3D 体外模型

• 批准号: 10629500
• 负责人: Sophia Katerina Theodossiou
• 金额: $ 13.17万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10629500
• 关键词: 3-Dimensional; Aging; Angiogenic Factor; Biochemical; Biocompatible Materials; Biology; Cell physiology; Cells; Centers of Research Excellence; Characteristics; Coupling; Data; Development; Encapsulated; Endothelial Cells; Engineering; Environment; Extracellular Matrix; FGF2 gene; Fibroins; Gel; Gene Expression; Generations; Goals; Human; Hydrogels; Image; In Vitro; Injury; Insulin-Like Growth Factor I; Mechanics; Mediating; Modeling; Morphology; Mus; Muscle; Muscle Development; Muscle Fibers; Musculoskeletal Development; Myoblasts; Myosin ATPase; Natural regeneration; Nature; Peptides; Phase; Process; Production; Protein Isoforms; Proteins; Protocols documentation; Rattus; Silk; Skeletal Muscle; Stains; System; Testing; Therapeutic; Time; Tissues; Tyramine; Umbilical vein; Vascular Endothelial Growth Factors; Vascularization; crosslink; improved; in vitro Model; induced pluripotent stem cell; mechanical properties; mechanotransduction; myogenesis; novel; programs; skeletal disorder; skeletal muscle differentiation; stem cell differentiation; stem cells; therapeutic target; transcriptome sequencing

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.

已成功使用了与生物活性辣椒一起使用的丝蛋白的水凝胶 研究骨骼肌分化的基础细胞过程。但是，以前 由于其静态性质，系统受到限制 - 其机械性能是固定的。最近， 我们展示了一种新的丝绸水凝胶，与酪胺取代的丝绸纤维交联 随着时间的流逝，以可控制的速度僵硬。这些水凝胶的可编程刚度使得 它们在建模与组织水平刚度的变化的建模方面具有吸引力 肌肉骨骼发育或受伤后。我们将修改这些水凝胶以合并 脱细胞肌肉外基质（ECM），通过最近建立的 脱皮协议。我们的初步数据表明将ECM耦合到我们的丝绸矩阵可以 用于进一步微调僵硬，使高度控制和独特的机械 和基质蛋白梯度在同一凝胶中，通过空间改变数量和类型 ECM与丝绸前体混合。以前尚未开发丝绸ECM水凝胶。 我们的中心假设是，用高度可调的机械进行动态加强水凝胶 生化特征可以概括肌源性环境的关键方面 有效地比现有工程系统有效，结果将提高我们对 能够更好地控制肌遗传学的治疗潜力的过程 IPSC用于再生骨骼肌。我们将开发水凝胶作为新型体外系统 探索动态刚度对IPSC肌发生的影响。我们将检验我们的假设 使用两个具体目标。第一个目的是确定3D水凝胶中如何发展的刚度 影响IPSC肌发生。第二个目的是开发生化功能化 和机械动态的丝绸ECM水凝胶，用于从IPSC产生骨骼肌。 这些目标的完成将增强我们对骨骼肌肉调节因子的理解 动态底物刚度和基质组成对干细胞的发展和影响 分化，其最终目标是将这些机制进行热靶向 使用干细胞再生骨骼肌。 3D水凝胶可进一步研究 调节骨骼肌发育，衰老，损伤和疾病的过程。