Addition of lactoferrin and substance P in a chitin/PLGA-CaSO4 hydrogel for regeneration of calvarial bone defects

Highlights: • Injectable lactoferrin and substance P incorporated chitin-PLGA-CaSO₄ was prepared. • Addition of lactoferrin has modified CaSO₄ crystal structure and Ca²⁺ ion release. • Prepared hydrogel showed sustained release of lactoferrin and substance P. • Addition of substance P has enhanced the stem cell migration. • The optimized hydrogel showed enhanced bone formation. Calcium-based injectable hydrogels with various bioactive active molecules possess a great potential for bone regeneration. Herein, we have synthesized a chitin-PLGA-calcium sulfate hydrogel (CSG) containing bioactive molecules - lactoferrin (LF) and substance P (SP). SEM and XRD analysis revealed that CS crystal growth was altered with the addition of LF. Rheological measurements indicated that the injectability of the hydrogels was maintained after the addition of LF, however, there was a reduction in storage modulus after LF addition. The addition of LF increased stem cell proliferation whereas, SP enhanced the cell migration. Osteogenic gene expression revealed that LF concentration at 25 μg/mg of CSG was optimal for a favourable outcome. To this optimized LF containing CSG, SP was incorporated and 0.05 μg/mg was found to be most effective (CSG-L3S2) in vitro studies. Further, the μ-CT and histological studies confirmed that CSG-L3S2 showed enhanced bone regeneration compared to the controls in critical-sized calvarial defect of mice. Thus the results indicate that a combination of the chemotactic agent (SP), pleiotropic growth protein (LF), and CS in the chitin-PLGA hydrogel could be a promising approach for non-load bearing bone defects.