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Published on: June 8, 2014
[Effect of the bone resorption supernatant from RAW264.7 osteoclast on the osteogenic activity of mouse MC3T3-E1
Li-li Chen1, Kai Wang, Jie Zhang
1Department of Oral Medicine, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China.
Objective:
To investigate the effect of osteoclast bone resorption supernatants on the osteogenic activity of mouse MC3T3-E1 cell line.
Methods:
Mouse RAW264.7 cell line was induced to osteoclast which was identified with tartrate resistant acid phosphatase (TRAP) staining and osteoclast specific gene detection. The differentiated RAW264.7 osteoclast was co-cultured with bovine milling bone specimen followed by toluidine blue staining. Then mouse MC3T3-E1 cell was cultured with supernatant from the osteoclast bone absorbent model. Methyl thiazolyl tetrazolium (MTT) method, alizarin red S staining, enzyme-linked immunosorbent assay detection of osteocalcin, and reverse transcriptase polymerase chain reaction detection were adopted to investigate the proliferation, calcification and osteogenic activity of MC3T3-E1 cells.
Results:
TRAP staining, osteoclast specific gene detection and toluidine blue staining all indicated that RAW264.7 cell could be differentiated into functioning osteoclast. The supernatant from the osteoclast bone absorbent model could inhibit the proliferation of MC3T3-E1 cells, with the A value between 0.062 ± 0.004 and 0.405 ± 0.033 (P < 0.05). It could also increase the formation of calcification nods, promote the osteocalcin level which peaked with the tenth day's supernatant at a level of (2.965 ± 0.047) µg/L, as well as enhance the transcription of the alkaline phosphatase and Runt related transcription factor 2 gene.
Conclusions:
RAW264.7 osteoclast bone absorbent supernatant might influence the osteogenic activity of osteoblast-like cell by inhibiting proliferation, promoting differentiation and calcification.
