Experimental Research On The Therapeutic Effect Of Aspirin Combined With Adipose Derived Mesenchymal Stem Cells On Type ? Osteogenesis Imperfecta

Objective:Osteogenesis imperfecta(OI),a hereditary disease characterized by bone dysplasia,is commonly caused by mutations COL1A1 or COL1A2 genes,which leads to insufficient type I collagen synthesis or structural abnormalities.Currently,surgery and bisphosphonates is widely used in OI treatment,which can not change the collagen synthesis defect.In recent years,mesenchymal stem cells(MSCs)transplantation has achieved remarkable performance,and is expected to redress the defect of collagen.However,the low homing ratio and osteogenic differentiation of MSCs restrict the therapeutic effect and it is urgent to find a drug to resolve the issue.The present study examined the effects of aspirin on adipose derived mesenchymal stem cells(ADSCs)proliferation,osteogenic differentiation and migration,explored the latent mechanism and evaluate therapeutic benefits of low-dose aspirin combined with ADSCs on osteogenesis imperfecta mice(oim).Methods:(1)ADSCs were isolated from adipose tissue of wild type C57BL/6 mice and the surface marker were identified by flow cytometry.To verify the differentiation potential,ALP staining and Oil red O staining of ADSCs were performed respectively after osteogenesis and lipogenic induction.(2)After treated with different concentrations of aspirin(0,0.5,1,1.5,2,5,10mmol/L),MTS was used to detect the proliferation of ADSCs.The percentage of apoptotic cells was detected by flow cytometry post ADSCs was treated with 1.5mmol/L aspirin.(3)After cultured in control,osteogenesis and osteogenesis+aspirin mediums respectively,the osteogenic differentiation of ADSCs were tested by ALP staining and ALP activity detection.The transcriptional changes of osteoblast associated genes(including Col1a1,Runx2,Bglap,Osterix,?-catenin and Bmp2)were mesured by q-PCR.(4)Cell migration ability was evaluated by scratch test and Transwell assay postADSCs disposed in 1.5mmol/L aspirin,and the change of SDF1-CXCR4 signaling was tested by q-PCR and ELISA.(5)RFP-ADSCs were isolated from red fluorescent protein transgenic mice,and oim mice were randomly divided into 4 groups for treatment: DMSO,aspirin,RFP-ADSCs and RFP-ADSCs+aspirin.3 months later,the total bone mineral density(BMD),bone mineral content(BMC)and femur structure of mice were detected by DXA and Micro-CT.Histological and immunohistochemical staining of femur sections were performed to estimate the therapeutic effect.The osteogenic and osteogenesis-related factors in serum were determined by ELISA.Results:(1)The ADSCs highly expressed mesenchymal stem cells related surface markers(CD29,CD44,CD90 and Scal-1)and barely expressed hemopoietic cell related marker CD45.ALP staining and Oil red O staining showed that ADSCs could differentiate into osteoblasts and adipocytes.(2)MTS assay showed that aspirin could promote the proliferation of ADSCs,and 1.5 mmol/L concentration performed the strongest effect.The percentage of apoptotic cells was significantly lower in aspirin group than control,suggesting that aspirin could inhibit the apoptosis of ADSCs.(3)Both ALP staining and ALP activity detection indicated that the osteogenesis+aspirin group had the strongest ALP expression.The results of q-PCR showed that the expression of osteoblast-associated genes in the osteogenesis+aspirin group was obviously higher than that of osteogenesis group and control group.Also the ?-catenin and Bmp2 level was the highest with aspirin administration,which implied that the enhanced osteogenesis of ADSCs by aspirin treatment might be through Wnt/?-catenin and BMP2 pathways.(4)Scratch test showed a larger cell migration distance in aspirin group than control,and Transwell assay further confirmed that much more cells migrated to the back of the compartment in the aspirin group.q-PCR detection manifested that the transcriptional level of Cxcr4 and Sdf1 significantly increased in aspirin group,and ELISA showed a obviously higher protein content of SDF1 in aspirin group.(5)Compared to oim mice,DXA test indicated the BMD and BMC remarkably increased in the combined treatment group 3 months later.And Micro-CT result showed that the femur structure significant improved in ADSCs+aspirin treated mice.Alizarin red staining indicated that the cortical bone mineralization of femur in the combined treatment group was significantly improved.Much more RFP,BGLAP and CD31 positive signals could be found in ADSCs+aspirin group.The highest concentration of P?NP and SDF1 as well as the lowest level of CTX?and COX2 in serum existed in ADSCs+aspirin group.Conclusions:Aspirin could effectively promote the proliferation of ADSCs at low concentration(0.5-2mmol/L)and 1.5mmol/L dosage obviously inhibited ADSCs apoptosis.Besides,aspirin efficiently facilitated the osteogenic differentiation capacity and migration of ADSCs might by means of Wnt/?-catenin and BMP2 signaling enhancement,and SDF1-CXCR4 signaling activation respectivley.The results of oim mice therapy showed that aspirin combined with ADSCs significantly increased BMD and BMC,improved the femoral microstructure,promote the osteogenesis and vascularization,suggesting a favorable therapeutic effect which provided new idea and method for clinical treatment of OI.