Marine-Inspired Enzymatic Mineralization of Dairy-Derived Whey Protein Isolate (WPI) Hydrogels for Bone Tissue Regeneration

Whey protein isolate (WPI) is a by-product from the production of cheese and Greek yoghurt comprising β-lactoglobulin (β-lg) (75%). Hydrogels can be produced from WPI solutions through heating; hydrogels can be sterilized by autoclaving. WPI hydrogels have shown cytocompatibility and ability to enhance proliferation and osteogenic differentiation of bone-forming cells. Hence, they have promise in the area of bone tissue regeneration. In contrast to commonly used ceramic minerals for bone regeneration, a major advantage of hydrogels is the ease of their modification by incorporating biologically active substances such as enzymes. Calcium carbonate (CaCO₃) is the main inorganic component of the exoskeletons of marine invertebrates. Two polymorphs of CaCO₃, calcite and aragonite, have shown the ability to promote bone regeneration. Other authors have reported that the addition of magnesium to inorganic phases has a beneficial effect on bone-forming cell growth. In this study, we employed a biomimetic, marine-inspired approach to mineralize WPI hydrogels with an inorganic phase consisting of CaCO₃ (mainly calcite) and CaCO₃ enriched with magnesium using the calcifying enzyme urease. The novelty of this study lies in both the enzymatic mineralization of WPI hydrogels and enrichment of the mineral with magnesium. Calcium was incorporated into the mineral formed to a greater extent than magnesium. Increasing the concentration of magnesium in the mineralization medium led to a reduction in the amount and crystallinity of the mineral formed. Biological studies revealed that mineralized and unmineralized hydrogels were not cytotoxic and promoted cell viability to comparable extents (approximately 74% of standard tissue culture polystyrene). The presence of magnesium in the mineral formed had no adverse effect on cell viability. In short, WPI hydrogels, both unmineralized and mineralized with CaCO₃ and magnesium-enriched CaCO₃, show potential as biomaterials for bone regeneration.