Enhanced differentiation of mesenchymal stem cells co-cultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold

The differentiation of mesenchymal stem cells (MSCs) towards fibroblasts is a crucial issue in ligament tissue engineering. This study aims to investigate the feasibility of using co-culture system to induce the differentiation of MSCs for constructing the tissue-engineered ligament in vitro. A kind of silk cable-reinforced gelatin/silk fibroin hybrid scaffold was used to provide three-dimensional (3-D) culture environments for MSCs. The 3-D co-culture system was set up by culturing MSCs/scaffold and ligament fibroblasts in the transwell insert and lower chamber, respectively. The regulatory effects of fibroblasts on MSCs were determined. After 2 weeks of co-culture the MSCs showed faster proliferation and higher DNA content compared with MSCs non-co-cultured. The MSCs were distributed uniformly throughout the scaffold and showed good viability. The collagen production also increased significantly with culture time. The MSCs in co-culture system were proved to differentiate into ligament fibroblasts by expressing ligament extra-cellular matrix (ECM)-specific genes including collagen I, collagen III, and tenascin-C in mRNA and protein level. The immunohistochemistry staining also confirmed the synthesis of key ligament ECM components. This study reveals that specific regulatory signals released from fibroblasts in 3-D co-culture system can enhance the differentiation of MSCs for ligament tissue engineering.