A thermosensitive chitosan/corn starch/β-glycerol phosphate hydrogel containing TGF-β1 promotes differentiation of MSCs into chondrocyte-like cells

Abstract

© 2014, The Korean Tissue Engineering and Regenerative Medicine Society and Springer Science+Business Media Dordrecht. Our previous study showed that thermosensitive chitosan/corn starch/β-glycerol phosphate (C/S/β-GP) hydrogel was an effective carrier for chondrocytes and their transforming factor, TGF-β1. In the present study, MSCs were grown in C/S/β-GP hydrogels as an effective tool for chondrocyte-like cell differentiation. The MSCs-encapsulated hydrogel was prepared by blending chitosan solution (1.70% w/v in 0.1 M HCl) with pregelatinized corn starch solution (1.70% w/v). The total final concentration of the blended polymers was 1.53% w/v, and the weight ratio of chitosan to corn starch was 4 to 1. The TGF-β1 (final concentration of 25 ng/mL) and 5 × 105 MSCs were added to 500 μL chitosan/starch solution. Finally, β-GP (60% w/v) was added to obtain 6.0% w/v final concentration. The C/S/β-GP hydrogel changed from a liquid at room temperature to a gel at 37 ± 2°C. It converted the fibroblast-like MSCs into spheroid cells. In hydrogels containing TGF-β1, these cells further differentiated into chondrocyte-like cells. This was shown by their expressions of type II collagen and aggrecan mRNA. Type I collagen mRNA was initially expressed but this disappeared by 6 weeks in culture suggesting a complete chondrocyte differentiation by that time. Type II collagen protein production was detected by immunohistochemistry and immunofluorescence, and successively increased after 4–6 weeks in culture. Neither the mRNA nor the collagen expression could be detected in the absence of TGF-β1. The data indicate that MSCs would be an appropriate chondrocyte precursor in conjunction with our hydrogel loading TGF-β1 which is able to sustain chondrocyte function.