2.10. OVX induced osteoporosis model

Following the methodology used in classical research [22], we set up an OVX mouse model to assess the effect of tyloxapol on bone loss in vivo. One week after ovariectomy, 8-week-old female C57BL/6 mice were equally randomized into three groups: sham operation (SHAM) group, vehicle (OVX) group, and tyloxapol-treated (OVX ​+ ​Tyloxapol) group. Tyloxapol (200 ​mg/kg) or DMSO (1% in normal saline) was injected intraperitoneally (i.p.) into mice every other day. Twelve weeks after drug injection, the experimental mice were anesthetized by an overdose of avertin. The distal femur bone tissue was de-calcified, paraffin embedded, sliced into sections and TRAP stained to determine whether the osteoclast activity was affected. The third lumbar vertebra was washed, embedded in polymethylmethacrylate, sliced into sections, and von kossa stained to determine whether the bone mass was affected, and Goldner’s stained [23] to determine whether the osteoblast activity was affected. The histomorphometric parameters of the distal femoral metaphysic were evaluated by micro computed tomography (μCT). TRAP level in mouse serum was detected using the mouse TRAP ELISA kit (Cloud-Clone Corp, Houston, USA).

At 3 weeks after ovariectomy, three groups (SHAM, OVX, Tyloxapol) of mice received intraperitoneal injection of 30 ​mg/kg calcein on day 0 and day 7. Mice were sacrificed on day 10, and the isolated third lumbar vertebra were dehydrated and embedded in polymethylmethacrylate. The vertebras were cut into 4 ​μm sections to capture the fluorescence-labelled images. The mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS) were measured to determine whether the bone formation activity was affected by tyloxapol.