Enhanced Chondrogenic Differentiation Potential of Human Gingival Fibroblasts by Spheroid Formation on Chitosan Membranes

Human gingival fibroblasts (HGF) were recently found to be a source of mesenchymal stem cells. Their behavior on a biomaterial has not been reported so far. The effect of culturing HGF on chitosan membranes on their chondrogenic differentiation was investigated in this study. HGF were first cultured on chitosan membranes and spheroid formation of HGF was observed. Next, HGF on chitosan were induced with chondrogenesis induction medium and their chondrogenic differentiation potential was expressed by assessing the expression of chondrogenesis related genes at both mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. We discovered that the chondrogenic differentiation potential of HGF could be enhanced simply by culturing HGF on chitosan membranes. Expression of neural crest and stemness genes were also analyzed by RT-PCR to evaluate the stemness and self-renewal of HGF spheroids. We found that spheroid formation helped to increase and maintain the expression of stemness genes in HGF. To understand the aspects of the chitosan membranes that induced spheroid formation of HGF, mechanical and physical properties of the chitosan membranes were examined. The migration of HGF on chitosan membranes was also monitored to speculate the process of spheroid formation. In addition, the roles of the Rho/Rho-associated kinase (ROCK) pathway and connexin 43 (Cx43) in spheroid formation were explored. Treatment of HGF cultured on chitosan with the ROCK-activity inhibitor Y27632 clearly inhibited spheroid formation, suggesting that the Rho/ROCK pathway was involved in spheroid formation. The increased Cx43 activity of HGF spheroids on chitosan indicated that the gap junction intercellular communication was regulated by spheroid formation. It was concluded that culturing HGF on chitosan may activate the Rho/ROCK pathway, which led to spheroid formation and gap junction regulation. These changes may contribute to the enhanced chondrogenic differentiation potential of HGF on chitosan.