Casting Environment and Water Retention Modulate β-Sheet Crystallization and Shelf Stability of Native Bombyx mori...

The performance and shelf life of Bombyx mori silk fibroin biomaterials are governed by β-sheet crystallinity, which determines solubility, degradation, and mechanical properties. However, controlling β-sheet crystallinity during processing and storage remains a major challenge because the metastable Silk I structure can convert to the β-sheet-rich, insoluble Silk II form. Here, native silk fibroin dope extracted directly from the gland was dissolved in water and cast into films under ambient or vacuum conditions, then aged and stored under controlled environments to identify the processing variables governing β-sheet formation. Differential scanning calorimetry (DSC) showed that freshly cast films were amorphous and that a crystallization exotherm emerged only after prolonged aging (≥2 months) of ambient-cast films, whereas vacuum-cast films were more resistant to crystallization across concentrations and storage conditions. Thermogravimetric analysis (TGA) revealed that ambient-cast films retained more loosely bound water than vacuum-cast films, while tightly bound water remained comparable, implicating loosely bound water as the plasticizer that enables molecular rearrangement. Quantitative magic angle spinning (MAS) solid-state NMR of 13C-Ala-labeled films confirmed that fresh and aged films remained predominantly amorphous (10-24% Ala β-sheet), demonstrating that the DSC exotherm arises from thermally induced crystallization of a water-plasticized precursor rather than from a pre-existing β-sheet order. Upon thermal annealing, fresh ambient-cast films reached 75% Ala β-sheet compared with only 38% for vacuum-cast films, demonstrating that the casting environment establishes the upper limit of β-sheet conversion. These findings provide a practical processing strategy for tailoring crystallinity and shelf stability: ambient casting promotes subsequent crystallization, vacuum casting preserves a low-crystallinity amorphous state, and dry storage minimizes β-sheet crystallization.