Abstract
Osteocytes are mechanosensitive cells that orchestrate signaling in bone and cartilage across the osteochondral unit. The mechanisms by which osteocytes regulate osteochondral homeostasis and degeneration in response to mechanical cues remain unclear. This study introduces a novel 3D hydrogel bilayer composite designed to support osteocyte differentiation and bone matrix deposition in a bone-like layer and to recapitulate key aspects of the osteochondral unit's complex loading environment. The bilayer hydrogel is fabricated with a soft cartilage-like layer overlaying a stiff bone-like layer. The bone-like layer contains a stiff 3D-printed hydrogel structure infilled with a soft, degradable, cellular hydrogel. The IDG-SW3 cells embedded within the soft hydrogel mature into osteocytes and produce a mineralized collagen matrix. Under dynamic compressive strains, near-physiological levels of strain are achieved in the bone layer (≤ 0.08%), while the cartilage layer bears the majority of the strains (>99%). Under loading, the model induces an osteocyte response, measured by prostaglandin E2, that is frequency, but not strain, dependent: a finding attributed to altered fluid flow within the composite. Overall, this new hydrogel platform provides a novel approach to study osteocyte mechanobiology in vitro in an osteochondral tissue-mimetic environment.