Regulation of Chondrogenesis and Hypertrophy in Silk Fibroin-Gelatin-Based 3D Bioprinted Constructs.

To date, the development of phenotypically stable, functionally equivalent engineered cartilage tissue constructs remains elusive. This study explored chondrogenic differentiation and suppression of hypertrophic differentiation in tyrosinase cross-linked silk-gelatin bioink using different cell modalities (dispersed, aggregates) for chondrocytes and mesenchymal progenitor cells (hMSCs) compared against the "gold standard" hMSC spheroids. Chondrogenic differentiation of hMSC spheroids (without silk-gelatin) showed a constant increase in hypertrophy over 21 days (gradual upregulated expression of COL10A1, MMP13). On the contrary, hMSC-laden constructs (both dispersed and aggregates) in bioink showed upregulated hypoxia (HIF1A) which positively regulated the expression of chondrogenic markers (aggrecan, COMP1) over chondrocyte-laden constructs. The gelatin component in the bioink induced MMP2 activity, which degraded the synthesized matrix, creating a pericellular zone for the accumulation of growth factors and newly synthesized matrices. We believe that the combinatorial effect of these accumulated factors as well as the hypoxia-regulated HDAC4 pathway played a pivotal role in stabilizing the chondrogenic phenotype of differentiated hMSCs along with suppressed hypertrophy. Therefore, the results suggest that tyrosinase cross-linked silk-gelatin bioink offers a suitable material composition for 3D bioprinting of cartilage constructs. Further standardization is warranted to investigate the biological mechanisms minimizing hypertrophic differentiation of hMSC/chondrocytes toward development of improved cartilage constructs.