Continuous Two-Zone In Vitro Co-culture Model of the Enthesis

Abstract

The enthesis is the interfacial tissue between ligament or tendon, and bone, which connects tissues of distinctly different mechanical properties. Although ligament and enthesis injury is commonplace, the development and healing mechanisms of these tissues are yet unclear. Current models for investigating these mechanisms are primarily in vivo animal models as in vitro models have been limited in their structural and mechanical biomimicry of the native enthesis. In this study, an in vitro enthesis model was developed using a modified gel aspiration-ejection method. Continuous two-zone aligned dense collagen (ADC) hydrogels with an overlapping interface were fabricated within 2 h. The mechanical properties of acellular two-zone ADC confirmed the continuous nature of this model, as the mechanical properties showed no significant difference compared to single-zone ADC and maintained comparable structural and mechanical characteristics of immature ligaments and unmineralized bone. Human anterior cruciate ligament fibroblasts and human spine vertebral osteoblasts were isolated from donor tissues and were seeded to form the enthesis model. These were cultured for 14 days, at which the viability and proliferation was observed to be 85 ± 7.5% and 230 ± 52%, respectively. Histological and immunofluorescence analyses at day 14 revealed extensive cell-driven matrix remodelling, and the seeded fibroblasts and osteoblasts maintained their phenotype within their compartments of the layered co-culture model. These results demonstrate the rapid fabrication of a two-zone co-culture system that can be utilized as an in vitro model to further understand the degenerative and regenerative mechanisms within the enthesis.