Injectable in situ forming kartogenin-loaded chitosan hydrogel with tunable rheological properties for cartilage tissue engineering.

Limited regeneration capacity of cartilage can be addressed by tissue engineering approaches including localized delivery of bioactive agents using biomaterials. Although chitosan hydrogels have been considered as appropriate candidates for these purposes, however, their poor mechanical properties limit their real applications. Here, we develop in situ forming chitosan hydrogels with enhanced shear modulus by chemical modification of chitosan using N-(β-maleimidopropyloxy) succinimide ester (BMPS). Moreover, we utilize β-Glycerophosphate (β-GP) in the hydrogels for achieving thermosensitivity. We investigate the effects of BMPS, β-GP and chitosan concentration on rheological and swelling properties of the hydrogels. Accordingly, we generate significant statistical models by response surface method to predict these properties. These models provide us beneficial tools to tune the hydrogel properties depending on the cartilage defect location and properties. Finally, we incorporate a recently discovered small biomolecule, kartogenin (KGN), for promoting chondrogenesis of stem cells into the optimized hydrogel. The hydrogel's shear modulus is 78 ± 5 kPa which covers a wide range of human articular cartilage shear modulus (50-250 kPa). It can be injected to the defects non-invasively at room temperature which gels at 37 °C within minutes. Additionally, it provides a sustained KGN release for ∼40 days that may eliminate the need of multiple injections. In vitro chondrogenic results confirm enhanced chondrogenic differentiation of human adipose mesenchymal stem cells (hAMSCs) treated with KGN-loaded hydrogel, compared to pure KGN. Based on the enhanced hydrogel shear modulus, injectability, gelation behavior, long-term drug release and in vitro results, this thermosensitive hydrogel looks promising for cartilage tissue engineering.