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

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.

Keywords: bioinspired; composite; enzyme; hydrogel; mineralization; whey protein isolate.

Figure 1

Mass of elemental calcium (A) and magnesium (B) per unit mass of hydrogel and dry mass percentage (C) of WPI hydrogels without urease (WPI) and with urease (WPI_U) incubated in different media: MA, MB, MC, n = 3, ** p < 0.01, *** p < 0.001.

Figure 2

FTIR (A) and Raman spectra (B) of WPI hydrogels without and with urease WPI_U incubated in different media: MA, MB, MC.

Figure 3

XRD diffractograms of WPI hydrogels incubated in different media MA, MB and MC.

Figure 4

SEM images of WPI and WPI_U hydrogels incubated in media MA (A,D), MB (B,E) and MC (C,F). Scale bar is representative of 1 μm (A,B,C) and 10 μm (D,E,F).

Figure 5

Cell viability of mouse osteoblast (MC3T3-E1 cells) seeded onto hydrogels over 24 h and 72 h. Background fluorescence of AlamarBlue and cell culture media was subtracted from each experimental hydrogel. n = 3, error bars are representative of SD.

Figure 6

Concentrations of elemental Ca and Mg in hydrogel extracts after 24 h and 72 h. n = 3, error bars show standard deviation.