Mechanism Of Platelet Rich Fibrin/Phytoestrogen Daidzein Complex Promoting Skull Defect Regeneration In Osteoporotic Rats

Objective: To study the effect of Daidzein on the biological behavior of human alveolar bone mesenchymal stem cells(h ABMSCs),and to study the potential mechanism of daidzein promoting the osteogenic differentiation of h ABMSCs by transcriptome sequencing and Western blot.The previous research of our research group showed that platelet rich fibrin(PRF)is a tissue engineering scaffold material derived from itself.Therefore,this experiment further studied whether PRF can carry Daidzein to form a new drug delivery system and promote the osteogenic differentiation of h ABMSCs.Through animal experiments,the practical effect of PRF/Daidzein composite in repairing skull limit bone defect was studied.Methods:1.Isolation,culture and identification of h ABMSCsh ABMSCs were isolated and amplified by tissue block adherent method;The cell surface markers of h ABMSCs were detected by flow cytometry;Alizarin red staining was used to detect the osteogenic differentiation ability of primary cultured cells;The ability of adipogenic differentiation was detected by oil red O staining;Allicin blue staining was used to detect the chondrogenic differentiation ability.2.Effect of phytoestrogen Daidzein on biological behavior of h ABMSCsCCK-8 method was used to observe the effect of phytoestrogen daidzein on the proliferation of h ABMSCs;Alkaline phosphatase(ALP)activity and calcium nodule formation were measured to evaluate the osteogenic induction effect of phytoestrogen Daidzein on h ABMSCs,and the optimal osteogenic induction concentration of phytoestrogen Daidzein was selected;Quantitative real-time polymerase chain reaction(Q-PCR)was used to detect the regulatory effect of phytoestrogen Daidzein on osteogenic differentiation related genes;The effects of phytoestrogen Daidzein on the expression of osteogenic related genes such as type I collagen(COL-I),osteopontin(OPN),runt related transcription factor 2(RUNX2)and ALP were detected by Western blot.3.Effects of PRF/ phytoestrogen Daidzein complex on the biological behavior of h ABMSCsThe ultrastructure of PRF and PRF/Daidzein complex was observed by scanning electron microscope;The sustained-release effect of PRF on phytoestrogen Daidzein was analyzed by liquid chromatography;CCK-8 method was used to observe the effect of PRF/Daidzein complex on the proliferation of h ABMSCs;The effect of PRF/Daidzein complex on the osteogenic differentiation of h ABMSCs was evaluated by alizarin red staining.4.Study on the mechanism of phytoestrogen Daidzein promoting osteogenic differentiation of h ABMSCs0.01% dimethyl sulfoxide(DMSO)and 10μmol/L phytoestrogen Daidzein of the osteogenic induction and differentiation medium was treated with h ABMSCs for 5 days,the total RNA was extracted and the transcriptome was sequenced.According to the sequencing results,two groups of differentially expressed genes were selected,and the protein level was verified by Western blot and the follow-up mechanism pathway was studied.5.In vivo study of PRF/Daidzein complex promoting skull defect regeneration in osteoporotic ratsThe postmenopausal osteoporosis(PMO)model of female SD rats was established by ovariectomy(OVX).After the model was established successfully,the limit bone defect was prepared in the skull.The experimental animals were randomly divided into six groups: blank group,0.01% DMSO/PRF group,estradiol(E2)group and 0.1μmol/L Daidzein/PRF group,1μmol/L Daidzein/PRF group,10μmol/L Daidzein/PRF group.The animals were sacrificed 12 weeks after operation for imaging and histological analysis.Results:1.The primary cultured h ABMSCs grew in spindle and shape,which accorded with the growth characteristics of mesenchymal stem cells;The surface markers CD105,CD29,CD44,CD73 and CD90 of mesenchymal stem cells were positive,while the surface markers CD24,CD34 and CD45 of hematopoietic stem cells were negative;At the same time,the primary cultured h ABMSCs can differentiate into osteoblasts,adipoblasts and chondroblasts under osteogenic induction,adipogenic induction and chondrogenic induction,respectively.2.The best concentration of phytoestrogen daidzein to promote the osteogenic differentiation of h ABMSCs is 10μmol/L;ALP activity in Daidzein group was significantly higher than that in blank group and DMSO group;The number of calcium nodules and CPC activity in the phytoestrogen Daidzein group were significantly higher than those in the control group;After treatment with phytoestrogen Daidzein for 14 days,the m RNA levels of ALP,OCN,RUNX2 and COL-I increased significantly(p<0.05);The expression level of the corresponding protein increased significantly(p<0.05).3.PRF / Daidzein complex can release phytoestrogen Daidzein continuously and slowly in 14 days,and the release amount can reach the peak on the 4th day,with the concentration of(13.57μmol/L),the release amount decreased significantly at 12 days,and tended to end at 14 days;PRF / Daidzein complex can promote the proliferation and osteogenic differentiation of h ABMSCs.4.Transcriptome sequencing showed that phytoestrogen Daidzein could activate extracellular regulated protein kinases 1/2(ERK1/2),and then up regulate hypoxia inducible factors-1α,(HIF-1α),activate the glycolysis pathway and enhance the osteogenic differentiation of h ABMSCs.5.PRF/Daidzein complex group can repair the limit bone defect of skull in osteoporotic rats.Conclusion: Daidzein can activate the glycolytic pathway of h ABMSCs through ERK1/2 and promote the osteogenic differentiation of h ABMSCs.PRF can be used as a sustained-release scaffold of phytoestrogen Daidzein,which makes the local application of phytoestrogen Daidzein possible.PRF/Daidzein complex can repair the limit defect of skull in osteoporotic rats and alleviate osteoporosis.PRF/Daidzein complex is expected to become a new and efficient bone defect repair therapy,which will bring a new opportunity for the treatment of alveolar bone deficiency in patients with osteoporosis.