Abstract
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.
Highlights
Natural by-products from industrial processes have tremendous biomimetic properties and are inexpensive as they are infrequently utilized
When Whey protein isolate (WPI) hydrogels containing urease were incubated with calcium and magnesium in equimolar concentrations (WPI_U_MC), calcium content was five-fold that of magnesium, indicating that calcium was preferentially incorporated into the hydrated polymer
The results of this study suggest that magnesium as a dopant of CaCO3 does not provide an appreciable positive biological effect on cell viability, in contrast to reports of a positive effect of magnesium as a dopant of calcium phosphate (CaP)
Summary
Natural by-products from industrial processes have tremendous biomimetic properties and are inexpensive as they are infrequently utilized. WPI in solution has been shown to enhance osteogenic differentiation of bone-forming cells and promote cellular proliferation [1]. The incorporation of a mineral phase may improve the mechanical properties and promote cellular adhesion, proliferation and osteogenic differentiation [5]. It is possible to modify and enhance properties of hydrogels by enriching the solution with a mineral phase either before or after gelation [6]. One explanation may be the presence of residual mineralization medium containing Ca and Mg in the hydrogels, allowing these ions to diffuse out more from unmineralized samples. SubtractedIt from each that the concentrations present the bars extraction medium were similar after 24 h and 72 h
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