Enhancing Silk Fibroin Hydrogel Mechanical Properties through Biomimetic Mineralization by Self-Assembled Catalytic Complexes

Protein-based framework hydrogels often exhibit limitations in mechanical strength and biocompatibility, particularly in applications related to medicine, industry, and environmental engineering. To overcome these challenges, the incorporation of natural biological macromolecules has emerged as an effective strategy for enhancing the hydrogel performance. Specifically, mimicking the natural biomineralization process enables the fabrication of tough hydrogels through biomimetic calcium deposition. In this study, we developed a mechanically robust hydrogel by photopolymerizing methacrylated silk fibroin and embedding a recombinant fusion protein, engineered by integrating a chitin-binding domain into Escherichia coli alkaline phosphatase. This fusion protein was successfully immobilized within the hydrogel matrix without leakage, facilitated by the substantial molecular size of nanochitin. Enzyme-mediated mineralization within the hydrogel matrix led to the formation of an organic-inorganic hybrid material characterized by a stable macromolecular network and uniform gel structure. Characterization using Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS), and thermogravimetric analysis (TGA) confirmed the successful in situ formation of hydroxyapatite, with a mineralization content of approximately 60%, homogeneously distributed throughout the silk fibroin network despite some initial brittleness. Notably, a freeze-thaw treatment applied over three cycles significantly improved the mechanical properties of the mineralized hydrogel, increasing its compressive strength by up to 7-fold and enhancing the compressive modulus from 1.1 to 2.2 MPa. Furthermore, cell viability assays demonstrated no significant cytotoxicity toward rat bone-marrow-derived mesenchymal stem cells, underscoring the potential of this composite hydrogel for applications in tissue engineering, particularly for complex bone tissue regeneration.