Osteocytic PGE2 receptors EP2/4 signaling create a physiological osteogenic microenvironment in polycaprolactone 3D modules

3D bioprinting is a focused field in orthopedics, and its application with physiological osteogenic microenvironments (POMEs) is a prerequisite for (PGE2) in mechanosensory osteocytes, but it remains unclear whether osteocytic PGE2 is a POME. PGE2 is an inducer of osteogenesis by acting on bone marrow stromal cells through its receptors EP2/EP4 to initiate osteogenic differentiation and mineralization. Unfortunately, clinical trials of PGE2 have reported side effects, including fever and drowsiness; targeting the PGE2 receptor in specific tissues can avoid these side effects. Here, we demonstrate that osteocytic cell line MLO-Y4 from murine long bones treated with EP2/EP4 agonists for 24 h enhance osteogenic differentiation and mineralization, inhibit adipogenesis of the stromal cell line ST2, and induce tubule formation and angiogenic marker expression in human umbilical vein endothelial cells (HUVECs). Mechanistically, activation of the PGE2 signaling pathway in osteocytes induces autocrine effects by upregulating the expression of EP2/EP4 receptors and COX-2 (Ptgs2), further amplifying PGE2 signaling. PGE2 produced by treated MLO-Y4 cells appears responsible for osteogenesis, alongside other unidentified factors. MLO-Y4 and ST2 cells, incorporated into POME 3D constructs, maintained over 95% viability over seven days. Treatment of osteocytes with a PGE2 receptor agonist promotes ST2 cell proliferation and enhances osteoblast marker expression and mineralization. As 3D bioprinting closely models in vivo conditions, these data suggest that osteocytic PGE2 receptor signaling is a safe and mild POME with great potential for translational applications.