One-pot fabrication and in vitro characterization of hydroxyapatite-enriched cryogels for bone tissue engineering

Cryogels have emerged as promising biomimetic scaffolds for extracellular matrix emulation in bone tissue engineering (BTE). In this study, we investigated how hydroxyapatite (HA) incorporation influences the physicochemical, mechanical, and biological performance of cryogels based on methacryloyl derivatives of type A gelatin, type B gelatin, and chondroitin sulfate (GelMA_A, GelMA_B, and CSMA, respectively). HA-enriched cryogels were fabricated through a one-pot cryogelation process in which HA particles were dispersed within the polymer solutions prior to crosslinking, without the use of additional dispersing agents. Scaffolds incorporating either commercial spheroidal HA or ultrasound-synthesized needle-to-plate HA were compared with non-mineralized counterparts as well as with each other, to evaluate the effect of the mineral phase on the resulting cryogels properties. The different polymer matrices exhibited distinct capacities to stabilize and homogeneously incorporate HA. Negatively charged polymers (GelMA_B and CSMA) enabled HA incorporation up to 10% w/w, whereas GelMA_A efficiently incorporated only 2.5% w/w. However, increasing mineral content did not result in improved mechanical properties or biological performance, suggesting that alterations in cryogel pore architecture and interconnectivity outweighed the benefits of higher mineral loading. Biological evaluation revealed limited cell response in CSMA-based cryogels, while GelMA-based systems supported cell adhesion and viability, with GelMA_A yielding marginally more favorable outcomes. Furthermore, HA crystal morphology had only a minor influence on scaffold performance, including osteoinductive potential. Overall, the results indicate that the composition of the polymer matrix plays a more critical role than HA loading or crystal morphology in determining the functional performance of mineralized cryogels for BTE.