Chitosan-based scaffold counteracts hypertrophic and fibrotic markers in chondrogenic differentiated mesenchymal stromal cells

Cartilage tissue engineering remains problematic because no systems are able to induce signals that contribute to native cartilage structure formation. Therefore, we tested the potentiality of gelatin-polyethylene glycol scaffolds containing three different concentrations of chitosan (CH; 0%, 8%, and 16%) on chondrogenic differentiation of human platelet lysate-expanded human bone marrow mesenchymal stromal cells (hBM-MSCs). Typical chondrogenic (SOX9, collagen type 2, and aggrecan), hypertrophic (collagen type 10), and fibrotic (collagen type 1) markers were evaluated at gene and protein level at Days 1, 28, and 48. We demonstrated that 16% CH scaffold had the highest percentage of relaxation with the fastest relaxation rate. In particular, 16% CH scaffold, combined with chondrogenic factor TGF beta 3, was more efficient in inducing hBM-MSCs chondrogenic differentiation compared with 0% or 8% scaffolds. Collagen type 2, SOX9, and aggrecan showed the same expression in all scaffolds, whereas collagen types 10 and 1 markers were efficiently down-modulated only in 16% CH. We demonstrated that using human platelet lysate chronically during hBM-MSCs chondrogenic differentiation, the chondrogenic, hypertrophic, and fibrotic markers were significantly decreased. Our data demonstrate that only a high concentration of CH, combined with TGF beta 3, creates an environment capable of guiding in vitro hBM-MSCs towards a phenotypically stable chondrogenesis.