| Objectives Heterodimeric bone morphogenetic protein 2/7(rhBMP2/7)has highly effective osteoinductive activity,while all-trans retinoic acid(ATRA)inhibits cell osteogenic differentiation.In the process of osteogenic differentiation,rhBMP2/7 heterodimer can significantly antagonize the inhibitory effect of ATRA on osteogenic differentiation and promote osteogenic differentiation.The purpose of this study was to examine the combined effects of rhBMP2/7 and/or ATRA on the effect of osteoclast differentiation and osteoclast activity,and then to evaluate the combined effects of rhBMP2/7 and ATRA in the repair of bone defects.Methods 1 Cell proliferation assay.RAW264.7 cells were inoculated with receptor activator of nuclear factor-?B ligand(RANKL)for 24 h,In the presence of RANKL,different concentrations of rhBMP2/7(5 ng/mL or 50 ng/mL)and/or ATRA(1 ?mol/L)were added.After induction culture for 1,3,5 and 7 days,Alamar Blue cell proliferation assay was performed according to the instructions of the kit.2 TRAP staining and morphological analysis.Cells were inoculated with 50ng/mL RANKL for 24 h after inoculation.Then in the presence of RANKL,different concentrations of rhBMP2/7(5 ng/mL or 50 ng/m L)and/or ATRA(1 ?mol/L)were added for stimulation for 7 days.TRAP staining according to the instructions for use of the TRAP staining kit,then the TRAP stained images of each well were randomly collected and recorded.At the same time,semi-quantitative analysis of the total surface area of osteoclasts,the number of osteoclasts,and the average surface area of osteoclasts was performed.3 TRAP activity detection.Cells were inoculated with 50ng/mL RANKL for 24 h after inoculation.Then in the presence of RANKL,different concentrations of rhBMP2/7(5 ng/mL or 50 ng/mL)and/or ATRA(1 ?mol/L)were added to induce culture.TRAP activity was performed according to the TRAP activity test kit on the 1st to the 7th day after induction culture,respectively.4 Osteogenic differentiation-related gene expression detection.Real-time fluorescence quantitative PCR detection of rh BMP2/7 and ATRA on the expression of osteoclast differentiation-specific genes.rhBMP2/7(5 ng/mL or 50 ng/mL)±ATRA(1 ?mol/L)was added to RAW264.7 cell culture medium induced by 50 ng/mL RANKL for 24 h.The total RNA of each group of cells was extracted on the 1st,4th and 7th days.Real-time fluorescence quantitative PCR was performed after reverse transcription.The expression of Calcr,Acp5 and Ctsk genes was detected and analyzed.5 Detection of calcium absorption of osteoclasts.The cells were plated on the bottom of the well plate and covered with a calcium phosphate coated Osteoclast Activity Assay Substrate plates(OAAS,OCT USA,Inc.).After being induced by 50 ng/mL RANKL for 24 h,RANKL containing different concentrations of rhBMP2/7(5 ng/mL or 50 ng/mL)± ATRA(1 ?mol/L)was added to induce culture.After 7 days of continuous culture,the calcium salts in the well plates of the groups absorb the lacunae under the light microscope.And immediately collect pictures of the absorption of calcium salts in the well plates of each group.The area of the lacunae absorbed lacunae on the OAAS plate in the collected photographs was measured with Image-Pro Plus 6.0 software and subjected to semiquantitative analysis.Results 1 Cell proliferation assay.One day after induction of rhBMP2/7(5 ng/mL or 50 ng/mL)and/or ATRA(1 ?mol/L),The rhBMP2/7-induction group,the ATRA-induction group alone,and the combination of the two groups did not significantly affect cell proliferation,and there was no significant difference when compared with the blank control group;On the 3rd day,5ng/mL and 50ng/mL rhBMP2/7 promoted cell proliferation.There was no statistical difference between the 5 ng/mL rhBMP2/7 induction group and the blank control group.However,the cell proliferation of 50 ng/mL rhBMP2/7 induced group was about 1.2 times that of the control group(P<0.0001).The ATRA induction group showed a significant inhibitory effect compared with the blank control group,and there was a statistically significant difference between the two groups(P<0.0001).When rhBMP2/7 was combined with ATRA,ATRA could significantly inhibit cell proliferation regardless of the concentration of rhBMP2/7.And there was no significant difference between the ATRA induction group and the control group.That is,regardless of the presence or absence of rhBMP2/7,1 ?mol/L ATRA can significantly inhibit cell proliferation(P<0.0001).ATRA can inhibit the proliferation of 50ng/mL rhBMP2/7 cells.On the 5th and 7th day,the effects of two concentrations of rhBMP2/7 on cell proliferation were not significant.However,1?mol/L ATRA still significantly inhibited cell proliferation regardless of the addition of BMP2/7(P<0.0001).2 TRAP staining and morphological analysis.After 7 days of rhBMP2/7(5ng/mL or 50ng/mL)and/or ATRA(1?mol/L)induction culture,differentiated mature osteoclasts were observed in the control group and rhBMP2/7 alone.As the concentration of rhBMP2/7 increased,the number of mature osteoclasts increased.In the 1 ?mol/L ATRA induction group,mature osteoclasts were occasionally seen when the rhBMP2/7 concentration was 50 ng/mL.However,mature osteoclasts were almost indistinguishable in the 1 ?mol/L ATRA-induced group and the combination of 5 ng/mL rhBMP2/7 and 1 ?mol/L ATRA.Semi-quantitative analysis of TRAP stained images of each group,The results showed that the mature osteoclast surface area in the 5 ng/mL rhBMP2/7 and 50 ng/mL rhBMP2/7 induced groups was significantly higher than the blank control group,and compared with the blank control group has a statistical difference;At the same time,with the increase of rhBMP2/7 concentration,the surface area of osteoclasts increased significantly.The 1?mol/L ATRA induction group had significant statistical differences with the blank control group and the rhBMP2/7 induction group whether or not rhBMP2/7 was added.The statistical trend of the number of osteoclasts in each experimental group is similar to the statistical trend of the total area of mature osteoclasts in each group.However,the number of cells in the 5 ng/mL rhBMP2/7 induced group was not significantly different from that in the blank control group.Through statistics on the average area of mature osteoclasts in each group,there was no statistical difference between the rhBMP2/7 induction group and the blank control group.3 Detection of TRAP activity.Under rhBMP2/7(5ng/mL or 50ng/mL)and/or ATRA(1?mol/L)induction culture,The TRAP activity in the blank control group and the rhBMP2/7(5 ng/mL or 50 ng/mL)induced group increased linearly from day 1 to day 7.However,starting from the fourth day,the TRAP activity in the 5 ng/mL and 50 ng/mL rhBMP2/7 induced groups was lower than the blank control group.On the 7th day,the TRAP activity of the blank control group was similar to that of the rhBMP2/7 group.In the 1 ?mol/L ATRA induction group,whether or not rhBMP2/7 was added,the TRAP activity of the cells from the 3rd day was significantly lower than that of the non-ATRA group.That is,1?mol/L ATRA can reduce the expression of TRAP activity.4 Osteogenic differentiation-specific gene expression assay.When rhBMP2/7(5ng/mL or 50ng/mL)and/or ATRA(1?mol/L)induced the first day of culture,ATRA and rhBMP2/7 did not significantly affect the expression of Calcr,Acp5 and Ctsk genes;However,on the 4th and 7th day,the expression of the three genes in the blank control group and the rhBMP2/7 alone group was significantly upregulated compared to the 1st day.The expression of three genes in the 1 ?mol/L ATRA induction group was significantly lower than that in the blank control group and the corresponding rhBMP2/7 induced group,and there was a statistically significant difference.5 Osteoclast calcium absorption function test.Compared with the control group,with the increase of rhBMP2/7 concentration,the calcium absorption lacunae in the rhBMP2/7 induced group gradually increased,and the area gradually increased.And the absorption area of calcium salt in the 50ng/mL rhBMP2/7 induced group was significantly higher than that in the 5ng/mL rhBMP2/7 induced group.The 1?mol/L ATRA induction group did not show significant calcium absorption lacunae with or without rhBMP2/7.Conclusions 1 ATRA significantly inhibited proliferation,TRAP activity,osteoclast gene expression,and calcium absorption ability of RAW264.7 cells derived from mouse osteoclasts;2 rhBMP2/7 significantly promoted osteoclast differentiation and osteoclast activity,and increased with increasing rhBMP2/7;3 ATRA significantly down-regulated rhBMP2/7 induced osteoclast proliferation,TRAP activity,osteoclast-related gene expression and calcium absorption capacity.Experiments have shown that ATRA inhibits rhBMP2/7-induced osteoclast differentiation and osteoclast activity.Previous experiments found that rhBMP2/7 can antagonize the inhibitory effect of ATRA on osteoblast differentiation and promote osteogenesis.Therefore,the combination of ATRA and rhBMP2/7 can achieve the effect of inhibiting osteoclast differentiation and promoting osteogenesis in repairing bone loss diseases caused by excessive activity of osteoclasts,thereby achieving a higher net effect of osteogenic bone formation. |
PDF Full Text Request