2D Chitosan-Based Films: A Proteomic Mass Spectrometry Study of Chondrocyte Phenotype as a Function of Cell–Biomaterial Interactions

In vitro chondrocyte expansion is key to all tissue engineering (TE) strategies using adult differentiated articular chondrocytes. Unfortunately, high proliferation rates in vitro can cause a progressive loss of chondrocyte phenotype (dedifferentiation) during culture passages. This can impair the quality of newly formed tissue after implantation because dedifferentiated chondrocytes mainly produce fibrocartilage, which hinders successful cartilage repair. Freshly isolated chondrocytes from equine articular cartilage were grown as a primary culture on tissue culture dishes and on 2D chitosan or chitosan/hyaluronic acid films. To evaluate chondrocyte differentiation during in vitro expansion, morphological observations, gene expression of chondrocyte phenotype markers, and LC-MS/MS shotgun proteomics were performed. All types of 2D cultures showed significantly reduced differentiation compared with freshly isolated cells, but chondrocytes grown on biomaterials maintained a rounded morphology and the gene expression of differentiation markers. Interestingly, pairwise proteomics comparison revealed a remarkable number of differentially expressed proteins, highlighting the different dynamics occurring in each experimental condition at the protein level. Based on novel insights into differentiation-dedifferentiation mechanisms, hypotheses were generated to explore new markers implicated in dedifferentiation and the role of biomaterials in this process by investigating the biological pathways associated with the reduced phenotype.