The Effect Of PPARγ Knockdown In BMP2-Induced Critical-Size Bone Defect Repair

Objective:Bone morphogenetic protein-2(BMP2)is approved by the FDA for clinical use at a concentration of 1.5 mg/ml.At these concentrations,BMP2 can induce both osteoblastogenesis and adipogenesis,producing abnormal,adipose-filled,poor-quality bone.These undesirable complications occur due to increased adipogenesis,at the expense of osteogenesis,through BMP2-mediated increases in the master regulatory gene for adipogenesis,peroxisome proliferator-activated receptor gamma(PPARγ).Inhibiting PPARγ during osteogenesis has been suggested to drive the differentiation of bone marrow stromal/stem cells(BMSCs)toward an osteogenic rather than adipogenic lineage.And due to the limited number of BMSCs,there’s a competitive relationship between cell differentiations,therefore,the result of excessive adipogenesis is at the expense less osteogenesis,leading to dysfunction of new-formed bone structure.To determine if we could improve BMP2 mediated bone healing,we used PPARγ shRNAlentivirus to silence PPARγ gene expression during BMP2 treatment in a mice segmental femoral defect modelMethods:In vitro,primary mouse BMSC and BMSC cell line(M2-10B4)were used to evaluate BMSCs lineage commitment/differentiation induced by BMP2 and PPARγsilence.PPARy shRNA lentivirus were used to silence PPARγ in BMSCs.MTT was used to detect the activity and proliferation of BMSCs.Scratches and Transwell chamber method were used to detect two-dimension and three-dimension migration ability Flowcytometry was used to detect cell apoptosis.Real-time PCR was applied to analyze mRNA expression of osteogenic related gene Ocn,Runx2 and Alp,adipogenic related gene PPARγ,BMP receptor,B-catenin and Smad2/3.In vivo,a 2.5mm defect,which reproducibly leads to nonunion if left untreated,was created in the right femur of each mouse as critical-size bone defect.Three-month-old male mice(n=20)were randomly assigned into 4 treatment groups(n=5 per group):[1]Phosphate buffered saline(PBS),[2]BMP2,[3]BMP2+ Control lentivirus,and[4]BMP2+ PPARγ shRNA lentivirus.The shRNA virus titer was 1×107 pfu/ml and the BMP2 concentration was 0.6 mg/ml.The proteins/virus were loaded onto custom fabricated cylindrical poly lactic-co-glycolic acid(PLGA)scaffolds with hydroxyapatite coating.X-ray was taken every week after surgery Samples were harvested at 8 weeks post-surgery,and analyzed by micro-CT and histology Quantification of bone volume/total volume(BV/TV)bone mineral density(BMD)、trabecular number(Tb.N)and 3D-CT image were generated.The sample were then subjected to histological examination and analyzed by immunohistochemical staining for expression of ONC、RUNX2、PPARγ、BMPR2 and β-cateninResults:In vitro,transient transfected shPPARγ inhibition rate was 60%at 72 h,the stable transfected shPPARγ inhibition rate was 90%,which can be used in further research.At 72 h,for BMSCs proliferation,BMP2 group increased by 46%compared to PBS control group(P<0.05),BMP2+ shPPARγ group was reduced by 50%compared to BMP2+shContorl group(P<0.01).At 21 h,for BMSCs migration,BMP2 group increased by 209%compared to PBS control group(P<0.01),BMP2+ shPPARy was reduced by 57%compared to BMP2+ shContorl group(P<0.01).Cell apoptosis appeared to be significantly higher in the BMP2+shPPARγ group(18.4%)than in the PBS(1.6%)and BMP2(3.2%)groups(P<0.005).Induction after 21 days,calcified nodules from BMSCs osteogenetic differentiation in BMP2 group increased by 532%compared to PBS control group(P<0.01),BMP2+ shPPARγ group was 34%lower than that of BMP2+ shContorl group(P<0.05).Compared with PBS control group,osteogenesis related genes and osteogenesis related genes mRNA expression was significantly increased in BMP2 group Compared with BMP2+ shContorl group,mRNA expression of osteogenesis related gene was significantly reduced in BMP2+ shPPARy group(P<0.01).BMPR2 mRNA expression in BMP2 group increased by 85%compared to PBS control group(P<0.01),BMP2+shPPARy groups showed 44%decrease compared to BMP2+ shContorl group(P<0.01).Beta-catenin expression in BMP2 group increased by 36%compared to PBS control group(P<0.05),BMP2+ shPPARy group showed 24%reduce compared to BMP2+ shContorl group(P<0.05)In vivo,BMP2 ± control-lentivirus groups achieved bone-union with marked adipogenesis,whereas PBS resulted in nonunion.Expectedly,BMP2+ PPARγ shRNA lentivirus application demonstrated less adipogenesis,but also surprisingly,complete nonunion with reduced new bone formation and bone marker expression.BMP2 group showed 106%and 125%higher BV/TV and Tb.N than PBS control group(P<0.01),BMP2+ shPPARy group showed 37%and 54%higher BV/TV and Tb.N than PBS group(P<0.01),but BMP2+ shPPARy group showed 48%and 52%lower BV/TV and Tb.N than BMP2+ shContorl group(P<0.01).Immunohistochemical staining showed that distribution of related proteins such as OCN、RUNX2、PPARy expression were decreased in BMP2+ shPPARy group(P<0.01)Conclusion:shRNA lentivirus transfection is efficiency and stable.PLGA scaffolds have good strength,porosity,biocompatible and biodegradable,non-toxic.In the process of BMP2 induced bone defect repair,PPARy is not only important in adiogenesis,but also one of the important compositions of osteogeneic differentiation.Through the knockout of PPARy to reduce BMP2 induced adipogenesis,then looking forward to promote osteogenesis differentiation hypothesis is not reliable.Therefore,by inhibiting the PPARy to inhibition of BMP2 in the repair of bone into fat side effects is not feasible.