Genistein loaded by PRF improved bone healing in obese mice
PURPOSE:To clarify the effect of genistein(GEN)on osteogenic differentiation and explore the effect of GEN loaded by platelet-rich fibrin(PRF)on the repair process of bone defects in obese mice.METHODS:In in vitro experi-ments,the effect of GEN(0,0.1,1,10,50 µmol/L)on the proliferation of mouse embryonic osteoblast precursor cells(MC3T3-E1)was determined by CCK 8.Alkaline phosphatase(ALP)staining and quantitative detection of ALP activity were performed to determine the changes of ALP activity in cells;RNA and protein expression levels of ALP,osteopontin(OPN)and osteocalcin(OCN)were detected by quantitative real-time PCR(qRT-PCR)and Western blot.Alizarin red staining was used to define the effect of GEN on mineralization of MC3T3-E1.To verify the feasibility of the PRF drug loading,the ultrastructure of PRF was subsequently observed under SEM.In in vivo experiments,obese C57 mouse models were established by high-fat diet feeding.On this basis,skull defect models with a diameter of 2.8 mm were established,and the prepared GEN/PRF complexes were placed into the bone defect area.The effects of GEN on skull defect repair in obese mice were evaluated by Micro-CT scanning and hematoxylin-eosin(H-E)staining.Statistical analysis was performed with GraphPad Prism 5.0 software package.RESULTS:CCK 8 results showed that 0.1,1 μmol/L GEN promoted cell proliferation within 7 days(P＜0.05);10 μmol/L GEN had no significant effect on the process of cell proliferation.From the second day,50 µmol/L GEN significantly inhibited cell growth and showed cytotoxicity(P＜0.05).These two concentrations had similar effects in promoting cellular osteogenic differentiation.SEM results showed that PRF presented a 3-dimen-sional network structure,providing space for loading drug molecules.In in vivo experiments,the body weight of mice in the high-fat diet(HFD)group was 27.7％greater than that in the normal diet group(P＜0.05)and had abnormal glucose tol-erance(P＜0.05).Micro-CT showed that compared with the normal diet group,the number of bone trabeculae in the femur of obese mice was decreased(P＜0.05),the distance between bone trabeculae was widened(P＜0.05),and the bone density was decreased(P＜0.05).In addition,GEN(0.1,1.0 μmol/L)loaded by PRF increased bone volume fraction in the skull of obese mice(P＜0.05).H-E results showed that GEN/PRF promoted the healing of the bone defects.CONCLUSIONS:GEN promotes osteogenic differentiation of MC3T3-E1,and it can effectively accelerate the healing of cranial bone defects after loading with PRF in obese mice.
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