Alendronate Promote Apoptosis In Osteoclasts In Vitro And In Vivo

Objectives: To observe the effect of Alendronate(ALN) on osteoclast apoptosis in vitro and in vivo.Methods:There were two groups in the test. One was vitro group ; and the other was vivo group.In vitro. The whole bone marrow cells culture: Bone marrow was obtained from the tibiae and femora of 4-week-old male SD rats. After the cells were centrifuged at 1500rpm for 4 min and washed twice, they were cultured in a-MEM containing 15% heat-inactived fetal calf serum, 1000units/ml penicillin, lOOOug/ml strptomycin, and 10" M l,25(OH)2D3in 5% CO2 atmosphere. 24-well plates and flasks were seeded with approximately 2X 10 cells/ml. The medium was replaced every three days. When it was the sixth day, the cells were tested by special method and proved osteoclasts.The plate andcontrol:Onthesixthday,ALN(10 -10 M)and PBS was added to the medium, then cultured 48 hours. The cells on the culture plates were fixed and stained for TRAP, then counting the numbers of osteoclasts. After a brief rinse in d-H2O, the specimens were subjected to Hoechst 33258 staining (S^ig/ml) for 15 min atroom temperature, and viewed with fluorescence microscopy.The flasks and control: As the same as the plates, after added the ALN and PBS 2 days, the percent was analyzed by flow cytometry with Annexin- V and PI.In vivo: after daily intraperitoneal injection 2mg/kg ALN or PBS for two consecutive days. The rats were sacrificed by cervical dislocation 24hs after the final injection. The hind limbs were fixed and decalcified in 5%EDTA, ph 7.4, for two weeks. The specimens were then processed into paraffin wax according to standard procedures, and 5 um thick sections were cut, deparaffined, stained with TRAP. After incubated 30 min, the slides were put into hematoxylin for nuclear counterstain.Results: In vitro. On the fist day, the cells adhered to the plates or flasks. On the sixth day, there were many cells contained rounded, and intacted nuclei, slim false feet. Cells that had been treated with ALN were found to have markedly different morphology with those of control cells. The addition of ALN induced a dose-dependent decrease in the number of TRAP positive number. Apoptotic osteoclasts were identified as cells that were showed strong expression of TRAP, chromatin condensation, and nuclear fragmentation. The result of Hoechest 33258 was the same as the TRAP'S.Flow cytometry analysis that had been stained with propidium iodide indicated the percent of a population of cells DNA content lower than those of cells in the Gj/G0phase of the cell cycle. The sub-Gj/Go population could be detected in cultures that had been treated for 48hs with ALN. Annexin-V analyzed the percent of earlier apoptosis. The apoptotic percent of treatment was higher than that of control.In vivo. ALN caused significant increase in osteoclasts apoptosis in these rats. Such cells were clearly identifiable as osteoclasts by their size multinuclearity and cytoplasmty staining characteristics. No significant changes in osteoclast number were seen in this short-term experiment.Both in vitro and in vivo were identified with no necrotic osteoclasts.Conclusions: ALN could inhibit osteoclasts and preostoclasts to form, and the main mechanism is by induced apoptosis.