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

Abstract

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.

Enhanced Chondrogenic Differentiation of Dental Pulp Stem Cells Using Nanopatterned PEG-GelMA-HA Hydrogels

“…35 In the field of cartilage tissue engineering, spheroid formation provides a 3D architecture that enhances chondrogenic differentiation capacity. 36,37 Previous studies have demonstrated that HA and 3D spheroid culture systems using photolithography techniques can promote MSCs to form spheroids. 23,38 Motivated by the urgent need for more efficient cartilage tissue engineering platforms and by the potential of stem cell-based therapies, we sought to assess the combined effects of matrix nanotopography and HAmediated signaling on the chondrogenic differentiation of DPSCs.…”

The Study of Media Effect on Differentiation of Gingival Fibroblast

“…Meretoja [6] reported that the chondrogenesis was enhanced in co-cultures with articular chondrocytes and MSCs (mesenchymal stem cells). The gingival fibroblast was recently found to be a source of mesenchymal stem cells [7]. Recent study reported the condrogenic differentiation potential of human gingival fibroblasts can be enhanced by spheroid formation on chitosan membranes [7].…”

Solid freeform-fabricated scaffolds designed to carry multicellular mesenchymal stem cell spheroids for cartilage regeneration

“…Recent in vitro work has suggested that 3D spheroids of MSCs may possess better differentiation capacity than single cells (Huang et al, 2011;Hsu et al, 2012a). MSC spheroids thus have the potential to replace MSC single-cell suspension for use in tissue engineering.…”

“…MSCs grown on these substrates were self-assembled into 3D cellular spheroids that kept migrating on the substrates. The self-renewal property and chondrogenic differentiation potential were significantly enhanced in these substrate-derived MSC spheroids (Huang et al, 2011;Hsu et al, 2012a). Although MSC spheroids are considered superior to single suspended cells in general, the delivery of relatively bulky spheroids without losing their 3D structure is a challenging task.…”