Effects Of HU-308 Coating On Osteoclastogenesis In RANKL-stimulated RAW264.7 Cells

ObjectiveTo investigate different concentration of HU-308 coating on formation,differentiation,apoptosis and expression of characteristic genes mRNA of osteoclasts,with a view to provide experimental evidence for its clinical application in dental implants.Method1.Osteoclasts induced culture and identificationRAW264.7 were induced to differentiate into osteoclasts by receptor activator of nuclear factor ? B factor ligand(RANKL),then cells were morphologically observed by light microscopy and tartrate-resistant acid phosphatase(TRAP)stained,bone slice were observed lacunas by toluidine blue staining,expression of CtsK,TRAP,MMP-9 mRNA of osteoclasts were detected by qPCR to identify mature osteoclasts.2.Effects of HU-308 coating on osteoclastogenesis in RANKL-stimulatedRAW264.7 cells(1).Experimental grouping168 Titanium plates were randomly divided into group A,B,C,D,E,F,G,H.As a control group,group A were alkali-heat treated;Group B were prepared Hep/Chi coating by LBL technology after alkali-heat treatment;Group C,D,E,F,G,H were alkali-heat treated and prepared Hep/Chi coating by LBL technology,then they were immersed in a solution containing 0.025mg/L,0.25mg/L,2.5mg/L,25mg/L,100mg/L,250mg/L concentration of HU-308,loading HU-308 on Hep/Chi coating by physical adsorption.In this way,different concentration of HU-308 coating were prepared.(2).Different concentration of HU-308 coating screeningThe titanium plates of group A,B,C,D,E,F,G,H were placed in cell culture plate,RAW264.7 grew on titanium surfaces for 4 days,MTT method was used to calculate the number of viable cells,goup A was the control group.HU-308 coating which had no effects on proliferation of RAW264.7 would be choosed for subsequent experiments.(3).Effects of HU-308 coating on osteoclastogenesis in RANKL-stimulatedRAW264.7 cellsthe titanium plates of group A,B,C,D,E,F were placed in cell culture plate,RAW264.7 cells which grew in the cell culture plates were induced culture by RANKL for 6 days:?Numbers of osteoclasts were counted by TRAP staining;?TRAP activity of osteoclasts were detected by enzyme kinetic method;? expression of CtsK,TRAP,MMP-9 mRNA of osteoclasts were detected by qPCR;? apoptosis rate of osteoclasts were detected by flow cytometry.Result1.Results of osteoclasts induced culture and identificationRAW264.7 were induced culture by RANKL,after TRAP staining,lots of red dye multinucleated giant cells were observed,they liked eggs,numbers of nuclear were from a few to dozens.Round or oval resorption pits were observed from bone slice by toluidine blue-staining,because of fossa Filling and bluish purple dye.Expression of CtsK,TRAP,MMP-9 mRNA of osteoclasts were significantly increased about 23 times,26 times,38 times by RANKL.In conclusion,RAW264.7 could be induced to differentiate into osteoclasts by RANKL.The osteoclasts which have bone resorption fuction could be used for further research.2.Screening results of different concentration of HU-308 coatingComparison group B,C,D,E,F with group A,the difference was not significant(P>0.05).Comparison group G,H with group A,the difference was significant(P<0.05).It indicated that Hep/Chi coating and HU-308 coating(immersing concentration of HU-308 were 0.025mg/L,0.25mg/L 2.5mg/L,25mg/L)had no effects on proliferation of RAW264.7.HU-308 coating(immersing concentration of HU-308 were 100 mg/L,250mg/L)significantly inhibited proliferation of RAW264.7.So we choosed group A,B,C,D,E,F for subsequent experiments.3.Effects of HU-308 coating on osteoclastogenesis in RANKL-stimulated RAW264.7 cells(1)Numbers of osteoclasts:Comparison with each other,group F<group E<group D<group C<group A,group B<group A,the difference was significant(P<0.05),with immersion concentration of HU-308 increasing,osteoclasts became smaller,TRAP staining became lighter,maturation of osteoclasts decreased;The difference between group C and group B was not significant(P>0.05).(2)TRAP activity of osteoclasts:Comparison with each other,group F<group E<group D<group C<group B<group A,the difference was significant(P<0.05),with immersion concentration of HU-308 increasing,TRAP activity gradually reduced by dose dependence.(3)Expression of CtsK,TRAP,MMP-9 mRNA of osteoclasts:?CtsK:Group F<group E<group D<group C<group A,group B<group A,the difference was significant(P<0.05);The difference between group C and group B was not significant(P>0.05);?TRAP:Group F<group E<group D<group C<group B<group A,the difference was significant(P<0.05);?MMP-9:Group E<group D<group C<group A,the difference was significant(P<0.05);The difference between group A and group B,between group E and group F was not significant(P>0.05);(4)Apoptosis rate of osteoclasts:Comparison with each other,group E>group D>group C>group A,the difference was significant(P<0.05);the difference between group E and group F,between group A and group B was not significant(P>0.05).ConclusionA certain concentration of HU-308 coating do not affect proliferation of osteoclast precursor cells in vitro,could directly inhibit formation and differentiation of osteoclasts,decreased expression of CtsK,TRAP,MMP-9 mRNA and promoted apoptosis of osteoclasts in vitro in a dose-dependent manner.