Role of cell-cell interactions on the regeneration of soft tissue-to-bone interface

Soft tissues such as the anterior cruciate ligament (ACL) connect to bone tissue through a characteristic fibrocartilagenous interface. This interface is essential for load transfer between soft and hard tissues, and its absence is the primary cause of graft failure post ACL reconstruction surgeries. Currently, the mechanism of interface regeneration is not known. Based on in vivo observations that a fibrocartilage-like tissue forms when the graft is in direct contact with bone, we propose here the original hypothesis that fibroblast-osteoblast interactions may lead to the recruitment and differentiation of mesenchymal stem cells or progenitor cells for interface regeneration. To test this hypothesis, a tri-culture model of fibroblasts-osteoblasts and interface-relevant cells was designed. This model mimics the graft-to-bone interface, supports direct cell-to-cell contact, as well as controlled homotypic and heterotypic cell-to-cell interactions. We used this model to determine the effects of fibroblast-osteoblast interaction on the response of interface-relevant cells such as bone marrow stromal cells (BMSCs), chondrocytes and fibroblasts. The response of osteoblasts and fibroblasts in triculture were also assessed. It was found that tri-culture with chondrocytes led to significant changes in cell proliferation and reduced osteoblast-mediated mineralization, accompanied by increased fibroblast mineralization. Interestingly, BMSCs in tri-culture measured higher ALP activity compared to controls. Positive glycosaminoglycan (GAG) production was detected in the chondrocyte tri-culture group. Moreover, expressions of interface-relevant markers such as type II collagen and GAG were detected in tri-culture with BMSCs and fibroblasts. Our results collectively demonstrate that osteoblast-fibroblast interactions modulate cell phenotypes, promote chondrocyte matrix elaboration and may initiate the differentiation of BMSCs into interface relevant phenotype. The findings of thi- s study provide new insight into the mechanisms governing the regeneration of soft tissue-to-bone interfaces.