Compression The L vertebral body was tested
Compression: The L4 vertebral body was tested in compression with loads applied along the craniocaudal axis using an Instron 8800 device at a rate of 3mm/min. Load displacement data were captured using Bluehill software. The ultimate load, yield load, stiffness, ultimate stress, yield stress and Young\'s modulus were determined.
Calcein labeling analysis: Two-month old mice, or OVX mice were given two doses of calcein (25mg/kg; Sigma) seven days apart and were sacrificed 7days after the last injection. Calcein labeled tissues were isolated from the calvaria and tibia, fixed in ethanol and embedded in OCT media for frozen sectioning. Tibiae were sectioned longitudinally through the medial bone; the intersection of the anterior cruciate ligament and the posterior cruciate ligament was used as the anatomical maker to signify the middle of the tibia. Calvaria were sectioned coronally through the parietal bones into 5-micron sections and examined by fluorescent microscopy. The following parameters were examined and calculated: single and double labeled surface (sLS, dLS, mm), mineralizing surface (MS=dLS+1/2sLS, mm), mineral apposition rate (MAR=distance between calcein labels/7days, μm/day), bone surface (BS, mm), and bone formation rate (BFR=[MS×MAR]/BS×365/1000, mm/year).
Ovariectomy: Ovariectomy was performed in 4-month-old and WT female mice. Each ovary was removed from the abdominal cavity onto an aseptic field and disconnected at the junction of the oviduct and the uterine body. Mice were sacrificed eight weeks after ovariectomy. Serum estradiol levels were examined using a 17-β-estradiol enzyme immunoassay kit (Assay Designs, Ann Harbour, MI).
EP1 antagonist studies: Isolated bone marrow stromal cells were seeded as 2×106 cells per well of a 12-well plate. Cells were cultured in basal medium (α-MEM, 10% FBS, 1% Penn-Strep) for 5days until cells were ~80% confluent; cells were then cultured in osteogenic media (α-MEM, 10% FBS, 1% Penn-Strep, 10mM beta-glycerophosphate (J. T. Baker) and 50μg/ml ascorbic MDL800 (Sigma) with either vehicle (DMSO) or 10μM SC-19220 (EP1 antagonist) for five days. Media was changed every other day, with addition of fresh SC-19220 or vehicle. Cells were fixed with 10% formalin and stained with alkaline phosphatase. Alkaline phosphatase staining intensity was quantified from triplicate samples per group at an absorbance of 520nm, and normalized to staining in control cells.
Statistical analyses: Results are shown as the mean±SEM. Statistical significance was identified by Student\'s t-tests or two-way ANOVA followed by Dunnett\'s test. p-Values less than 0.05 were considered significant.
Discussion In the present study we identified PGE2/EP1 signaling as a negative regulator of osteoblast differentiation and bone formation, and demonstrate for the first time that the EP1 receptor plays a negative role in regulation of postnatal bone homeostasis. We have previously demonstrated that mice exhibit accelerated fracture healing and that primary bone marrow progenitor cells differentiate into osteoblasts at an increased rate relative to WT cells, while osteoclastogenesis is not affected . Here we show that bones from mice have increased biomechanical strength, and increased bone volume when compared with WT mice. These differences are maintained or even amplified during aging, suggesting that mice are resistant to aging-induced bone loss due to an increase in bone formation rather than a decrease in bone resorption. In vitro experiments using an EP1 antagonist support that EP1 is a negative regulator of osteoblastogenesis. These findings show that in addition to regulating bone formation during injury and repair , activation of the EP1 receptor also has a critical role in the regulation of normal bone metabolism. No significant difference was found between WT and bone marrow progenitor cells in levels of the EP2 or EP4 receptors or of intracellular cAMP activity  suggesting that the bone phenotype in mice is not due to compensation by other EP receptors. These data, taken together with our previous findings of accelerated fracture repair in mice , and enhanced fracture healing with EP4 agonist treatment , , highlight the complex nature of the effects of PGE2 on bone metabolism, which likely involves both stimulatory effects, through EP2 and EP4, and inhibitory effects mediated by EP1.