RESEARCH SUMMARY
This study developed calcium ion-mediated silk bulk materials as mechanically adaptable, intrinsically osteogenic biomaterials for bone regeneration. Inspired by calcium-mediated natural silk processing and bone mineralization, the authors incorporated CaCl2 into regenerated silk fibroin, freeze-dried and ground the material into Silk-Ca powders, and thermoplastically molded the powders into dense bulk structures including screws and hollow tubes. Calcium-associated silk-ion-water interactions altered silk molecular assembly, increased glass transition temperature, suppressed beta-sheet crystallization during thermal processing, and introduced humidity-dependent plasticization. Mechanically, increasing CaCl2 reduced flexural strength and modulus but greatly increased toughness, producing bulk materials with adaptable properties and values comparable to proteinaceous biomaterials and cancellous bone-relevant ranges. The embedded calcium ions also acted as intrinsic mineralization reservoirs: SF-95-Ca-5 formed hydroxyapatite-like mineral layers in simulated body fluid, released calcium ions, and enhanced MC3T3-E1 pre-osteoblast viability, alkaline phosphatase activity, mineral deposition, and osteogenic marker expression. In a rat femoral condyle defect model, CaIMS screws improved bone regeneration relative to pure silk controls, with higher BV/TV, trabecular number, bone mineral density, stronger OCN and OPN expression, better osseointegration, reduced fibrous encapsulation, and no visible major-organ toxicity. Overall, the study presents ion-mediated silk regulation as a strategy for producing moldable, bioactive bulk protein implants that combine structural performance with intrinsic bone-regenerative activity.
Flexural mechanical testing was performed using a CellScale UniVert equipped with a 100 N load cell. CaIMS bulk samples with different CaCl2 contents were characterized by three-point bending at a loading speed of 1 mm/min under ambient conditions of 23 ± 2 °C and 50 ± 5% relative humidity. At least ten replicates were tested per composition, and bending modulus was calculated from the initial linear region of each stress-strain curve. The UniVert data showed that CaCl2 incorporation decreased bending strength and modulus, with strength dropping from about 127 MPa for pure silk to roughly 80 MPa below 5% CaCl2 and then leveling near 65 MPa at higher CaCl2 contents. Flexural modulus decreased from about 4.3 GPa to approximately 1.3 GPa at low CaCl2 loading and then stabilized near 1 GPa. In contrast, toughness increased sharply when CaCl2 reached 5% or higher, rising to approximately 8 MJ/m3. Wet-state mechanical testing was also performed after 24 h PBS immersion, showing reduced bending strength and modulus for SF-95-Ca-5 but retained structural integrity. These UniVert measurements were central to defining the composition-dependent balance between strength, stiffness, toughness, humidity-responsive mechanics, and bone-relevant structural performance.
