An Osteoclast Bone Resorption Model Established In Vitro And The Effect Of Osteoclast Bone Resorption Supernatants On The Osteogenic Activity Of Mouse MC3T3-E1Cells And Related Signaling Pathway

Objective:To investigate the effect of osteoclast bone resorption supernatants on the osteogenic activity of mouse MC3T3-E1cell line, Combining makes sure by which signaling pathway that it rise a function to find a new therapy target for alveolar bone resorption.Methods:Mouse RAW264.7cell line was induced to osteoclast which was identified with Tartrate resistant acid phosphatase (TRAP) staining and osteoclast specific gene detection. The differentiated RAW264.7osteoclast was co-cultured with bovine milling bone specimen followed by Toluidine blue staining. Then mouse MC3T3-E1cell was cultured with supernatant from the osteoclast bone absorbent model. Methyl thiazolyl tetrazolium (MTT) method, Alizarin Red S staining, ELISA detection of osteocalcin, and RT-PCR detection were adopted to investigate the proliferation, calcification and osteogenic activity of MC3T3-E1cells. Western-Blot is used to examine the expression of related signal molecular after the mouse MC3T3-E1cell was cultured with supernatant and PI3K kinase inhibitor LY294002. Results:TRAP staining, osteoclast specific gene detection and Toluidine blue staining all indicated RAW264.7cell could be differentiated into functioning osteoclast. The supernatant from the osteoclast bone absorbent model could inhibit the proliferation of MC3T3-E1 cells,with the OD value between0.062±0.004and0.405±0.033(P＜O.05). It could also increase the formation of calcification nods, promote the osteocalcin level which peaked with the tenth day's supernatant at a level of2.965±0.047μg/L, as well as enhance the transcription of the Alkaline phosphatase and Runt related transcription factor2gene. The expression of pho-P85and pho-AKT significantly increase when MC3T3-E1cells stimulated by osteoclast bone resorption supernatants (P<0.05). when blocked by PI3K kinase inhibitor LY294002,the expression of pho-P85and pho-AKT are dose-determined decrease, and lower than osteoclast bone resorption supernatants group (P＜O.05).Conclusions:RAW264.7osteoclast bone absorbent supernatant might influence the osteogenic activity of osteoblast-like cell by inhibiting proliferation, promoting differentiation and calcification, and this influence may effect through the PI3K/AKT signaling pathway.