Establishing a Coculture System for Ligament-Bone Interface Tissue Engineering

Abstract

Ligament-bone interface (enthesis) is a complex structure which comprises of ligament, fibrocartilage and bone. The fibrocartilage transformation adds significant insertional strength to the interface and makes it highly resistant to avulsion forces. Many ACL grafts cannot generate native interfacial region, leading to their failure. Co-culture has proved to be an effective way to generate new tissues in tissue engineering. Studies have found important signaling molecules in transduction pathway of chondrogenesis to be transmitted via gap junctions. We hypothesized that stem cells cocultured between ligament and bone cells would enable transmission of chondrogenic factors from bone/ligament cells to bone marrow stem cells (BMSCs) via gap junctions, resulting in their differentiation into fibrocartilage. To test this hypothesis, we studied to establish effective co-culture system. In this study, two set of co-culture (BMSCs and ligament cells; BMSCs and bone cells) were established. Confocal microscopy showed efficient dye transfer from bone/ligament cells into BMSCs. This was further confirmed and quantified by FACS, which showed a gradual temporal increase in the percentage of BMSCs acquiring Calcein. RT-PCR analysis showed that the bone cells-BMSC and the ligament cells-BMSC co-culture systems expressed higher amounts of collagen type-2, as compared to the various monocultures. The results proved the establishment of effective co-culture. The findings provide important information for the development of a more promising ligamentfibrocartilagebone graft.