Effect Of Vitexin And BMP9 On Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective1 To explore the effect of vitexin on i MADs proliferation and osteogenic differentiation at cellular level;2 Explore the synergistic effect of vitexin combined with BMP9 on osteogenic differentiation of i MADs at cellular level;3 In vivo subcutaneous osteogenesis effect of vitexin combined with BMP9 was inspected in mice.Methods1 Cell experiments(1)Effect of vitexin on i MADs proliferation and osteogenic differentiationi MADs are resuscitated,cultured and passed to next generations.CCK8 was used to detect the effect of of vitexin of different concentrations on i MADs proliferation at different time points.A blank control group,an osteogenic induction group and an osteogenic induction plus vitexin group(2,5,10 u M)were set up.Alkaline phosphatase(ALP)activity was quantified and stained to detect early osteogenesis under vitexin stimulation 7 days after induction.Alizarin red staining was used to detect calcium nodule formation 14 days after induction to analyze the impact on late mineralization.The effect of vitexin on osteogenic differentiation-related genes was analyzed by q PCR.(2)Effect of vitexin combined with BMP9 on osteogenic differentiation of i MADsHEK293 cells were infected with Ad-GFP or Ad-BMP9.Repeatedly expanded several times to obtain high-titer Ad-GFP and Ad-BMP9 for subsequent experiments.Four groups were set up: GFP group,BMP9 group and vitexin combined with BMP9 group..Changes of expression levels of osteogenic differentiation markers Runx2,ALP,OSX,OPN and adipogenic differentiation gene PPAR? under different intervention conditions was quantified by q PCR.Seven days after cells seeded in plates were intervened according to their groups,ALP activity and staining were used to analyze the early osteogenesis of i MADs under these conditions of intervention.On day 10,oil red O staining was used to detect fat formation.Alizarin red staining was performed 14 days after intervention to detect the effect of calcium nodule formation on late mineralization2.Animal experimental researchAd-Gluc was used to infect i MADs.Cells were harvested 24 hours after infection and mixed with the scaffold material of thermosensitive macromolecular polymer mixed gelatin(PPCN-G),and dropped into 12 well plates pre-chilled in advance and incubated in a 37 ° incubator.After 30 min,2 ml medium was added,and Gluc activity was measured at different time points to observe the survival of i MADs in the cell scaffold material PPCN-G.Twenty 6-week-old athymic female nude mice were randomly divided into 4 groups,5 in each group.The GFP group,vitexin group,BMP9 group and vitexin combined BMP9 group were infected with adenovirus Ad-GFP,Ad-BMP9 and Polybrene,respectively.After 24 hours of intervention,cells of each group were collected and resuspended in 40 ul PPCN-G.The target concentration of vitexin was add at the same time.Nude mice were inoculated on the back to establish a heterotopic ossification model in nude mice.The growth of subcutaneous masses was monitored.Nude mice were sacrificed 6 weeks after injection.The subcutaneous masses were taken out and fixed.Micro CT scanning and threedimensional reconstruction were adopted to calculate bone volume.Subcutaneous masses were quickly decalcified,dehydrated,embedded,and sectioned.Hematoxylin-eosin(HE)staining and Masson's Trichrome staining were used to evaluate the morphological changes of bone tissue formation within the mass.Results1 Cell experimental research(1)Effect of vitexin on i MADs proliferation and osteogenic differentiation(1)CCK8 was used to determine the effect of vitexin on the activity of i MADs.The results showed that when vitexin was used to intervene i MADs at a concentration between 2-10 ?M for 96 h,the cell growth of the drug-added group was normal,and no cytotoxic effect of vitexin and cell growth inhibition were observed(P>0.05).(2)ALP activity measurement: After 7 days of osteogenic differentiation induction of i MADs,compared with the blank control group,the ALP activity of the osteogenic induction group was significantly increased(P<0.05).within a certain concentration range,the ALP activity in theosteogenic induction plus vitexin group was higher than that in the osteogenesis inducing group alone(P<0.05),and the ALP activity was highest when vitexin concentration was 10 ?M.(3)ALP staining: After 7 days of osteogenic differentiation induction of i MADs,no deep blue staining was observed in the blank control group.ALPpositive blue staining was seen in the osteogenic induction group and the osteogenic induction plus vitexin group.With 10 ?M vitexin,the color is the darkest.(4)Alizarin red staining: After 14 days of osteogenic induction,no obvious calcium nodule formation was observed in the blank control group.Red calcium nodules were formed in some cells of the osteogenic induction group and osteogenesis induction plus vitexin group.At a concentration of 10 ?M,bone mineralization was most pronounced,and the number of red calcium nodules was the largest.(5)Expression of osteogenic differentiation-related genes: On the seventh day of osteogenesis induction,changes in the expression of osteogenesis related genes were detected with or without the intervention of different concentrations vitexin.q PCR results showed that vitexin significantly upregulated the expression of ALP,OPN,Runx2,and OSX in a dose-dependent manner.Therefore,a concentration of 10 ?M was used as the optimal dose for subsequent laboratory studies.(2)Effect of vitexin combined with BMP9 on osteogenic differentiation of i MADs(1)ALP activity measurement: the activity of ALP will first increase and then decrease with the osteogenic differentiation process;at different time points,the ALP activity of the BMP9 group is significantly higher than that of the GFP group(P<0.05),which is also the case for the comparison between vitexin combined with BMP9 group and BMP9 group(P<0.05).(2)ALP staining: the results showed that the blue-violet staining solution of the BMP9 with vitexin group had the darkest color,and showed a stronger positive rate than the BMP9 group,and almost no blue-stained area was observed in the GFP group.The results of ALP staining were consistent with the results of ALP activity measurement.(3)Alizarin red staining: 14 days after the induction of osteogenic differentiation,a large number of calcium nodules were seen in the BMP9 group and the BMP9 with vitexin group,indicating that BMP9 can induce osteogenic differentiation of cells;under the action of 10 u M vitexin,the calcium salt deposition was further enhanced,indicating that vitexin can promote the effect of BMP9-induced osteogenic differentiation.(4)Oil red O staining and expression of osteogenic and adipogenic differentiation-related genes: BMP9 was stained with oil red O 10 days after osteogenic differentiation induction.It was found that cells treated with BMP9 had more oil red O positive cells and intracellular oil red O positive cells were reduced when vitexin was added.Compared with the GFP group,BMP9 significantly up-regulated the expression of osteogenic genes ALP,OPN,Runx2,and OSX.When vitexin was added,osteogenic differentiation-related genes were further up-regulated.In addition,BMP9 can significantly promote the expression of adipogenesis-related genes PPAR?,while adding vitexin effectively inhibited the expression of PPAR?.2.Animal experiment3D culture of cells in PPCN-G scaffolds and determination of Gluc activity showed that PPCN-G scaffolds had good adhesion,vitexin could promote cell differentiation in scaffolds;micro CT results showed that larger masses were formed vitexin combined with BMP9 group.Quantitative analysis found that the vitexin combined with BMP9 group had more bone mass.The HE and Masson staining results showed that both the BMP9 group and BMP9 with vitexin could induce the generation of many mature bones with beam and bone matrix,but vitexin significantly enhances this effect compared with BMP9 alone.Conclusion1 cell partVitexin alone can promote osteogenic differentiation of i MADs cells in vitro,and can enhance the expression of osteogenic related genes.Vitexin plays a synergistic role in BMP9-induced osteogenesis,which not only strengthens the change of osteogenic phenotype,but also improves the expression of osteogenic genes and inhibits the expression of adipogenic genes.Besides,Vitexin can promote i MADs cell proliferation when cultured in PPCNG scaffold.2 Animal experimentVitexin can increase the size of ectopic bone induced by BMP9 in vivo,thicken trabeculae and bone mineralization,and have a good osteoinductive effect.