  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 (2{\theta} = 27{\deg}, 33{\deg} and
35{\deg}) 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.
