Positive impact of pyrocarbon and mechanical loading on cartilage-like tissue synthesis in a scaffold-free process

Aiming to build a tissue analogue engineered cartilage from differentiated chondrocytes, we investigated the potential of a pyrocarbon (PyC)-based and scaffold-free process, under mechanical stimulation. PyC biomaterial has shown promise in arthroplasty and implant strategies, and mechanical stimulation is recognized as an improvement in regeneration strategies. The objective was to maintain the cell phenotype to produce constructs with cartilage-like matrix composition and mechanical properties. Primary murine chondrocytes were deposited in drop form between two biomaterial surfaces expanded to 500 μm and a uniaxial cyclic compression was applied thanks to a handmade tribo-bioreactor (0.5 Hz, 100 μm of amplitude, 17 days). Histology and immunohistochemistry analysis showed that PyC biomaterial promoted expression of cartilage-like matrix components (glycosaminoglycans, type II collagen, aggrecan). Importantly, constructs obtained in dynamic conditions were denser and showed a cohesive and compact shape. The most promising condition was the combined use of PyC and dynamic stimulation, resulting in constructs of low elasticity and high viscosity, thus with an increased damping factor. We verified that no calcium deposits were detectable and that type X collagen was not expressed, suggesting that the cells had not undergone hypertrophic maturation. While most studies focus on the comparison of different biomaterials or on the effect of different mechanical stimuli separately, we demonstrated the value of combining the two approaches to get as close as possible to the biological and mechanical qualities of natural hyaline articular cartilage.