Osteogenic evaluation of BM-MSCs in thermosensitive chitosan bioinks incorporating gelatin-carrageenan...

Thermosensitive, injectable chitosan-based hydrogels are a promising platform for treating critical bone defects; however, they suffer from poor mechanical properties, printability, stability, and bioactivity. This study addresses these limitations by developing a novel composite bioink. The bioink was incorporated with self-assembled nanofibrous aggregates (SNAs) of gelatin and carrageenan into a thermosensitive chitosan-β-glycerophosphate hydrogel to enhance cell binding and structural integrity. To further enhance the bioink's osteogenic potential, four different types of nanoparticles: silicon dioxide, nanohydroxyapatite, laponite and magnesium whitlockite, were added. The SNAs, with RGD sites (arginine-glycine-aspartic acid) from gelatin, provide essential cell-binding sites. The synergistic effects of the nanoparticles on the bioink's printability, as well as its physicochemical, mechanical, and biological properties, were evaluated. The addition of nanoparticles enhanced the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) encapsulated within the bioprinted hydrogel scaffolds. MTT cytotoxicity analysis showed cell viability of >90 % in both L929 and BM-MSCs. While all the nanoparticles improved the bioink's properties, the samples containing Mg-whitlockite nanoparticles showed the most significant enhancements. They demonstrated superior printability, along with significantly improved mechanical properties and enhanced proliferation and differentiation of BM-MSCs. Overall, bioink incorporating whitlockite nanoparticles shows excellent potential for use in bone tissue engineering.