Bioengineered skeletal muscle constructs that replicate the architectural, metabolic, and contractile characteristics of native tissue are becoming essential platforms for disease modeling and advancing regenerative medicine. The creation of these constructs relies heavily on cell-mediated gel compaction, a crucial process for facilitating tissue maturation. To ensure myotube alignment, muscle cell-laden hydrogels are typically embedded in 3D-printed molds with anchor structures. However, structural detachment or rupture often occurs during culture, which undermines the stability and functional differentiation of the engineered tissue. To address these challenges, we developed an improved anchor-type mold through a series of structural optimizations. We first compared two anchor geometries—linear and mushroom-shaped pillars—within rectangular frames, finding that the mushroom-shaped design provided better structural retention. However, the rectangular frames led to excessive gel compaction, causing detachment and disrupting cellular alignment, especially in central regions. To alleviate these issues, we introduced a dumbbell-shaped mold with a narrowed midsection to better distribute mechanical stress. This new mold effectively promoted aligned myotube formation, long-term construct maintenance, and functional maturation. Our findings underscore the benefits of structurally optimized molds in creating stable engineered muscle, with significant implications for regenerative therapies and preclinical testing platforms.
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