Connecting pathways between mineralized fibrocartilage and bone at the Achilles tendon insertion

At entheses, tendons and bones are bridged by mineralized fibrocartilage, joined to tissues through dedicated interfaces. Tendons and bones are characterized by cells interconnected thanks to their underlying dense networks. Nanotubes connect tenocytes in tendons, allowing cellular crosstalk and providing biomechanical stability. Osteocytes are involved in bone mechanoresponsiveness and mineralization: they are encased into cavities and their cellular processes run through channels, forming the osteocyte lacunocanalicular network. Here, we explore the structural connectivity between fibrocartilage and bone, exploiting rat enthesis as model system and focusing on two specific regions: the Achilles tendon insertion into calcaneus and the periosteal fibrocartilage, facilitating tendon sliding. Those regions are used to characterize the impact of loading environment on tissue connectivity. Central to our approach is rhodamine staining, employed to trace connections between tissues. This information is interpreted using data on tissue microstructure, organization and composition, acquired combining high-resolution imaging methods. At the enthesis, we observe potential connections between trabecular bone marrow and mineralized fibrocartilage through a subchondral channel network perforating the interface. Direct cellular connections between bone and fibrocartilage cells are rare: canaliculi mostly stop or switch direction at the cement line. Yet, we observed a high density of canaliculi around perforating channels, which reach fibrochondrocyte lacunae. Such connections seem practically absent at the periosteal region. Our findings are preliminary but suggest that inter-tissue connectivity is required to support the enthesis load-bearing function. To understand multi-tissue biochemical cellular crosstalk, the physical infrastructure enabling this communication is also a critical feature to investigate.