Osteocytes are the most common type of cell in bone, and they reside within the mineralized bone matrix in a three-dimensional (3D) interconnected network of dendritic cells. Osteocytes are mechanosensitive cells that maintain bone homeostasis by directing bone formation or resorption in response to changes in mechanical loading. In the subchondral bone plate, the osteocytes contribute to degeneration during diseases such as osteoarthritis that is accompanied by bone and cartilage deterioration. Yet, it is difficult to study osteocyte mechanobiology in vivo, and existing 2D and 3D in vitro culture methods are insufficient

The goal of this dissertation was to develop a 3D ex vivo culture system to study osteocyte mechanobiology, specifically within the subchondral bone plate. A 3D ex vivo culture system to study osteocyte mechanobiology needs both biological relevance (i.e., mature osteocytes, bone matrix deposition, and 3D interconnected dendritic network) and mechanical relevance (bone-level strains and interstitial fluid flow). This work used the tunable poly(ethylene glycol) (PEG) hydrogel system to develop a 3D culture system. This work developed a degradable PEG hydrogel that enhanced osteocyte differentiation and bone matrix deposition in 3D. Using this degradable PEG hydrogel system, we investigated how osteocyte dendritic network formation is influenced by physical and biochemical factors. The dimensionality (2D culture vs. 3D hydrogel) greatly influenced differentiation and significantly altered the osteocyte response to Prostaglandin E2, a molecule that is rapidly produced by osteocytes in response to mechanical loading Finally, the degradable PEG hydrogel was incorporated into a bilayer composite hydrogel system that was designed to control the bone-level strains and induce interstitial fluid flow, mimicking the loading environment in subchondral bone. This dissertation thus contributes an improved understanding of the physical and biochemical cues that support osteocyte differentiation and dendrite formation in 3D. Further, this system supports the study of osteocyte mechanobiology in a physiologically relevant ex vivo model that captures both biological and mechanical relevance to the subchondral bone plate.