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

3D bioprinting is a focused field in orthopedics, and its application with physiological osteogenic microenvironments (POME) is a prerequisite for authentic bone reconstruction. Mechanical stimulation produces PGE2 in mechanosensory osteocytes, but it is still 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 shown side effects, including fever and drowsiness, so targeting the PGE2 receptor on specific tissues can avoid side effects. Here, we show that MLO-Y4 osteocytes treated with EP2/EP4 agonists for 24 h develop the functions of promoting osteogenic differentiation and mineralization while inhibiting adipogenesis of the stromal cell line ST2 and inducing tubule formation and angiogenic marker expression in HUVEC cells. Mechanistically, activation of the PGE2 signaling pathway in osteocytes appears to have autocrine effects by inducing the expression of the EP2 and EP4 receptors and COX-2 (Ptgs2), further auto-amplifying PGE2 signaling. PGE2 produced by the treated MLO-Y4 cells appears responsible for osteogenesis in addition to other unknown factors. MLO-Y4 and ST2 cells were incorporated into POME 3D constructs with greater than 95% viability within 7 days. Treatment of osteocytes with a PGE2 receptor agonist lineally proliferates ST2 cells, enhances the expression of osteoblast markers and mineralization. Due to 3D bioprinting being the closest model to in vivo research, these data showed that osteocytic PGE2 receptor signaling is a safe and mild POME with great potential for translational applications.