|
Objective: This study was to: 1, to establish a simple in vitro preparation of the artificial joint metal wear particles; study RANKL (nuclear factor κB receptor activating factor ligand) induced mouse monocyte macrophages The RAW264.7 formation of osteoclasts method. 3, to observe the effects of different concentrations of metal wear particles RAW264.7RANK, TRAP expression and the number of osteoclast formation in RANKL-induced changes. To further elucidate the biological mechanisms of prosthesis loosening. Method: 1, the use of vacuum ball milling vitro preparation of the artificial joint metal wear particles with a chromogenic substrate limulus kit detects endotoxin content in the particle suspension sample, plasma emission spectrometry elemental analysis of wear particles, scanning electron microscopy and wear particle morphology to evaluate the wear, the laser particle analyzer detects wear particle diameter, and particle suspensions of different concentrations (0.1mg / ml, 1.0 mg / ml, 10 mg / ml) to RAW264.7 supernatant lactate dehydrogenase cell toxicity of the particles; 2, tartrate-resistant acid phosphatase (TRAP) staining and scanning electron microscopy to detect induced osteoclast resorption pits identified; 3, wear particle suspensions of different concentrations (0.5 mg / ml, 1.0 mg / ml, 1.5 mg / ml) role in RAW264.7 detected by RT-PCR method RANK expression of TRAPmRNA, again with RANKL-induced TRAP staining under an inverted microscope to count the number of osteoclasts. Results: 1, vacuum milled particles prepared and its elements of the raw material composition substantially the same, particle diameter lt; 5μm consistent, the physicochemical nature of the separation of the artificial joint with the body of metal wear particles in the test concentration (usually much lower than the 10 mg / ml) did not show cytotoxicity. In RAW264.7 induced osteoclast formation in cultured 7 days after the number of cells reached the peak, the osteoclast cell in the body, irregular, jagged around the brush border and pseudopodia multicore; TRAP staining positive resorption has the characteristics of mature osteoclasts. 3 different concentrations of wear particles role After RAW264.7 7d, counted under a microscope the number of osteoclasts, the results show with increasing concentration of wear particles suspension of RANKL induced generated osteoclast number, low, medium and high concentrations The number of osteoclasts were significantly higher than the control group (P lt; 0.05), in the high concentration group number of osteoclasts were significantly higher than the low enriched group (P lt; 0.05), high concentrations of the number of osteoclasts also significantly higher than in concentrated group (P lt; 0.05). 4, with the increase in concentration of wear particles suspension, RANKmRNA expression gradually increased, low, medium, and high concentrations of three-group RANKmRNA expression were significantly higher (P lt; 0.05) in the high concentration group RANKmRNA expression were significantly high In low concentrated group (P lt; 0.05), high concentration the group RANKmRNA expression also significantly higher than in the concentrated group (P lt; 0.05). 5, with the increase in concentration of wear particles suspension, TRAPmRNA increased expression, low, medium and high concentrations the three groups RANKmRNA expression was significantly higher (P lt; 0.05), but low, medium, and high concentrations of the three groups RANKmRNA expression was no significant sex difference (P gt; 0.05). Conclusion: 1, vacuum ball milling vitro preparation of artificial joints metal wear particles is simple, reproducible, and can be used for cytology studies. 2, RAW264.7 a better osteoclast precursor cell model, RAW264.7 method is easy to induce the formation of osteoclast, and the obtained good uniformity of osteoclasts. 3, metal wear particles on mononuclear phagocytes RANK and TRAP expression regulation occurs at the genetic level. Increased expression of RANK and TRAP may RAW264.7 cells into play a positive role in bone absorption function of osteoclasts.
|