Combinatorial physicochemical stimuli in the three-dimensional environment of a hyaluronic acid hydrogel amplify chondrogenesis by stimulating phosphorylation of the Smad and MAPK signaling pathways

The chondrogenesis of stem cells and cartilage tissue regeneration are more efficient in a three-dimensional (3D) environment than in a two-dimensional (2D) environment. Although extensive studies have examined the effects of biochemical or physical cues alone, it is not fully understood how these biochemical and biophysical cues in the 3D environment are intertwined and orchestrated with chondrogenesis for cartilage tissue regeneration. In this study, we used photocrosslinked hyaluronic acid (HA), the extracellular matrix of cartilage, as a general 3D microenvironment to characterize the effects of dimensionality, localization of biochemical cues, regulation of biophysical cues, and external stimulation on chondrogenic signaling pathways in adipose-derived stem cells (hASCs). TGF-β3 was immobilized in HA hydrogels by ionic or covalent conjugation. The stiffness of the hydrogels was tuned by varying the crosslinking density, and an external stimulus for chondrogenesis was provided by ultrasound. The results revealed that the levels of chondrogenic signals in hASCs cultured in the 3D HA hydrogel depended on the presence of TGF-β3, and a reduction in the stiffness of the TGF-β3 covalent conjugated hydrogel increased the chance of interaction with encapsulated hASCs, leading to an increase in chondrogenic signals. External stimulation with ultrasound increased the interaction of hASCs with HA via CD44, thereby increasing chondrogenesis. Our results present a new understanding of the intertwined mechanisms of chondrogenesis in 3D hydrogels connecting TGF-β3 sequestration, mechanical properties, and ultrasound-based external stimulation. Overall, our results suggest that when designing novel biomaterials for tissue engineering, it is necessary to consider the combinatorial mechanism of action in 3D microenvironments.