Soluble Epoxide Hydrolase Inhibition Accelerates Early Bone Repair in Critical-Size Calvarial Defects and Requires ENPP1 Activity for Osteoblast Mineralisation In Vitro

Critical-size craniofacial bone defects heal poorly, and pharmacological strategies capable of accelerating bone repair remain limited. Soluble epoxide hydrolase (sEH) degrades epoxyeicosatrienoic acids (EETs), lipid mediators associated with inflammation resolution; however, the osteoblastic effectors that translate sEH inhibition into matrix mineralisation are unknown. We investigated whether pharmacological sEH inhibition accelerates craniofacial bone repair, and whether ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) a candidate regulator of pyrophosphate metabolism participates in this response. In vivo, bilateral 5-mm critical-size calvarial defects were created in male Wistar rats (n = 6 per group) treated with the sEH inhibitor TPPU (10 mg/kg/day, p.o.) or vehicle for 14 or 28 days. Repair was assessed by micro-computed tomography, histomorphometry, sEH immunohistochemistry and multiplex spatial RNA in situ hybridisation (RNAscope) of a predefined six-gene osteogenic panel ( Spp1, Runx2, Col1a1, Alpl, Bglap, Enpp1 ). In vitro, human SAOS-2 osteoblastic cells were treated with TPPU and/or the ENPP1 inhibitor STF-1084 to assess viability, osteogenic gene expression, ENPP1 expression and enzymatic activity, alkaline phosphatase (ALP) activity and mineralised matrix formation. At 14 days, TPPU increased BV/TV, BS/TV and trabecular number and reduced trabecular separation and total porosity ( p < 0.05); these differences were no longer detected at 28 days, whereas histomorphometry showed a greater newly formed bone area in TPPU-treated animals at both time points. sEH immunolabelling was reduced by TPPU at 14 and 28 days ( p < 0.001), confirming target engagement. Spatially, Enpp1-positive cells were more abundant in TPPU-treated tissue at 14 days, the only panel gene reaching significance at the per-cell level. In vitro, TPPU increased ENPP1 mRNA and protein. STF-1084 dose-dependently reduced ENPP1 activity, ALP activity and mineralised matrix deposition without affecting viability, and co-treatment with TPPU did not restore mineralisation despite preserved or increased OSX, RUNX2 and BSP expression. Pharmacological sEH inhibition accelerates the early phase of craniofacial bone repair and is associated with increased ENPP1 expression in regenerating tissue. In human osteoblastic-SAOS cells, ENPP1 enzymatic activity is necessary for mineralisation and for the pro-mineralising effect of sEH inhibition, identifying ENPP1 as a candidate downstream effector of the sEH/EpFA axis and a target for further genetic validation.