Polysaccharides on gelatin-based hydrogels differently affect chondrogenic differentiation of human mesenchymal stromal cells

Highlights: • Developed hybrid hydrogels with tunable anisotropic and stress relaxation characteristics. • The physical-mechanical characteristics of hybrid hydrogels guide chondrogenic differentiation of hBM-MSCs. • Chitosan in hybrid hydrogels is fundamental for the formation of collagen fibrils with dimension closed to healthy articular cartilage. Selection of feasible hybrid-hydrogels for best chondrogenic differentiation of human mesenchymal stromal cells (hMSCs) represents an important challenge in cartilage regeneration. In this study, three-dimensional hybrid hydrogels obtained by chemical crosslinking of poly (ethylene glycol) diglycidyl ether (PEGDGE), gelatin (G) without or with chitosan (Ch) or dextran (Dx) polysaccharides were developed. The hydrogels, namely G-PEG, G-PEG-Ch and G-PEG-Dx, were prepared with an innovative, versatile and cell-friendly technique that involves two preparation steps specifically chosen to increase the degree of crosslinking and the physical-mechanical stability of the product: a first homogeneous phase reaction followed by directional freezing, freeze-drying and post-curing. Chondrogenic differentiation of human bone marrow mesenchymal stromal cells (hBM-MSC) was tested on these hydrogels to ascertain whether the presence of different polysaccharides could favor the formation of the native cartilage structure. We demonstrated that the hydrogels exhibited an open pore porous morphology with high interconnectivity and the incorporation of Ch and Dx into the G-PEG common backbone determined a slightly reduced stiffness compared to that of G-PEG hydrogels. We demonstrated that G-PEG-Dx showed a significant increase of its anisotropic characteristic and G-PEG-Ch exhibited higher and faster stress relaxation behavior than the other hydrogels. These characteristics were associated to absence of chondrogenic differentiation on G-PEG-Dx scaffold and good chondrogenic differentiation on G-PEG and G-PEG-Ch. Furthermore, G-PEG-Ch induced the minor collagen proteins and the formation of collagen fibrils with a diameter like native cartilage. This study demonstrated that both anisotropic and stress relaxation characteristics of the hybrid hydrogels were important features directly influencing the chondrogenic differentiation potentiality of hBM-MSC.