In this thesis, a unique three-dimensional (3D) co-culture trabecular explant model was established for long-term studies of mechanotransduction in bone and to bridge previous in vivo and two-dimensional (2D) in vitro studies. Two mechanotransduction pathways — (1) autocrine/paracrine factor:​ prostaglandin E2-based signaling and (2) gap junctional intercellular communication — were shown to play important roles in trabecular bone adaptation.

A 3D trabecular explant model that allows long-term co-culture of in situ osteocytes and seeded osteoblasts was established, together with the development of a feedbackcontrolled loading system and a medium perfusion system. To study the effects of dynamic deformational loading on trabecular bone adaptation and various mechanotransduction pathways, a custom-made benchtop feedback loading system was used to deliver a precise loading pattern to the explants under sterile conditions. Additionally, a perfusion system was established to allow low-level medium perfusion through the explants to facilitate the delivery of nutrients and oxygen and the removal of metabolic waste so that the viability of osteocytes was maintained up to 4 weeks for longterm mechanotransduction study.

The role of prostaglandin E2-based signaling pathway in mechanotransduction and trabecular bone adaptation was investigated by evaluating the effects of dynamic deformational loading on PGE2 secretion, osteogenic response, and changes in apparent elastic modulus of the 3D co-culture explants with or without NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor that significantly reduced the PGE2 production without affecting the viability of the bone cells. Dynamic deformational loading (initial strain of 2,400 ue, 1Hz, 5 minutes) induced a significant increase in PGE2 secretion compared to the non-loaded explants. In addition, the application of loading for 3 days resulted in significant increases of osteoid surface/bone surface (OS/BS) and apparent elastic modulus after 4 weeks of culture. However, when PGE2 production was inhibited with NS-398, the effects of mechanical loading on PGE2 secretion, OS/BS, and apparent elastic modulus increase were abolished. PGE2 pathway showed an important role in the mechanotransduction of trabecular bone.

The role of gap junctional intercellular communication (GJIC) between osteocytes and osteoblasts in mechanotransduction was investigated using the co-culture explant where the 3D mineralized matrix environment and the intercellular connections of osteocytes and osteoblasts were maintained. By using fluorescent labeling assays and confocal microscopy, direct GJIC was shown, for the first time, to exist in 3D between seeded osteoblasts on the trabecular bone surface and osteocytes embedded in the bone mineralized matrix. To confirm the role of GJIC in mechanotransduction, a gap junction blocker 18a-glycyrrhetinic acid (18a-GA) was used to successfully block the GJIC between the two cell types without affecting their viability. GJIC between osteoblasts and osteocytes was shown to be essential in mechanotransduction when dynamic deformational loading (initial strain of 2,400us at 1Hz for 5 minutes daily for 3 days) induced significant increase in both OS/BS and apparent elastic modulus after 4 weeks, but not when GJIC was blocked with 18a-GA.