Hydrogen phosphate-mediated acellular biomineralisation within a dual crosslinked hyaluronic acid hydrogel

The creation of hyaluronic acid (HA)-based materials as biomineralisation scaffolds for cost-effective hard tissue regenerative therapies remains a key biomedical challenge. A non-toxic and simple acellular method to generate specific hydrogen phosphate interactions within the polymer network of cystamine-crosslinked HA hydrogels is reported. Reinforced dual crosslinked hydrogel networks were accomplished after 4-week incubation in disodium phosphate-supplemented solutions that notably enabled the mineralisation of hydroxyapatite (HAp) crystals across the entire hydrogel structure. Hydrogen phosphate-cystamine crosslinked HA hydrogen bond interactions were confirmed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and density functional theory (DFT) calculations. Hydrogen phosphate-mediated physical crosslinks proved to serve as a first nucleation step for acellular hydrogel mineralisation in simulated body fluid allowing HAp crystals to be detected by X-ray powder diffraction (2θ = 27°, 33° and 35°) and visualised with density gradient across the entire hydrogel network. On a cellular level, the presence of aggregated structures proved key to inducing ATDC 5 cell migration whilst no toxic response was observed after 3-week culture. This mild and facile ion-mediated stabilisation of HA-based hydrogels has significant potential for accelerated hard tissue repair in vivo and provides a new perspective in the design of dual crosslinked mechanically competent hydrogels.

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