Development of injectable cuttlebone derived nanohydroxyapatite hydrogel for osteoblast cell encapsulation

This study focused on creating bioactive, injectable hydrogels for bone regeneration, utilizing cuttlebone derived nanohydroxyapatite (CB-nHA) and quercetin (QT), a natural flavonoid, to enhance their capacity. It explored how different ratios of CB-nHA and chitosan/collagen, along with QT, in calcium carbonate (CaCO₃-QT) microcapsules influenced the injectability and osteoconductive properties of thermosensitive chitosan/collagen hydrogels. The hydrogels were prepared with varying concentrations of CB-nHA (0 %, 5 %, 10 %, and 15 % w/v) and chitosan/collagen ratios (7:1 and 14:1 w/w). Their microstructure, mechanical, and physical properties, rheological and injectability tests were examined to identify optimal hydrogel's formulations. Additionally, cytotoxicity and osteoconductivity of the hydrogel were evaluated using encapsulated human fetal osteoblasts (hFOB) obtained from ATCC (USA). It was found that higher concentrations of CB-nHA and chitosan reduced pore size and degradation while increasing complex modulus and injection force. The CaCO₃-QT promoted cell growth by effectively storing and releasing QT. The optimal formulation, a 5 % CB-nHA and 1 % CaCO₃-QT hydrogel with a 7:1 chitosan/collagen ratio, was injectable and exhibited a pore size of 116 ± 47 μm. The hydrogel encapsulated cells demonstrated high viability and extensive intercellular connections, with ALP activity levels significantly higher than those in the groups with 0 % CB-nHA and CaCO₃ alone (p < 0.05). In conclusion, the CB-nHA and CaCO₃-QT enhanced the osteoconductive property of the injectable hydrogel. A thermosensitive hydrogel composed of 5 % CB-nHA, 1 % CaCO₃-QT, and a 7:1 ratio of chitosan to collagen is a bioactive and injectable carrier for osteoblasts and QT to enhance bone regeneration.