The Research Of Proliferation And Differentiation Of Bone Marrow Mesenchymal Stem Cells (BMSCs) And Bone Micro-structural Changes In Ovariectomized Rats

ObjectiveTo study the function of proliferation and differentiation of bone marrowmesenchymal stem cells(BMSCs) for the changes of bone micro-structure in thepathogenesis of osteoporosis in ovariectomized rats.MethodsAnimal model of osteoporosis(OP) was established by performingovariectomy namely bilateral ovarian resection in10-week-old healthy femaleSprague-Dawley(SD) rats. The sham-operated group was established byresection of bilateral fat tissue near ovarian in the same batch，the same ageand the same weight of healthy female SD rats. The experiment was dividedinto ovx group and sham group.3months after ovariectomy, Micro-CT scannedfemur then we carried out cancellous bone quantitative detection of bonemicrostructure related parameters after3-D reconstruction. BMSCs wereisolated, cultured and purified by the combination of density gradientcentrifugation, adhesion separation and limited dilution method, and cultured invitro to3rd～4th passage in all experiments: The BMSCs phenotype appraisal was studied by Flow Cytometry (FCM);Colony-forming unit (CFU) assaydetected the BMSCs proliferation ability; The MTT method detected the growthcurves of BMSCs; After adipogenic induction(ADI),lipid drops were marked byoil red O dyeing to detect and compare the adipogenic potential between thetwo kind of BMSCs; After osteogenesis induction(OSI),calcium nodules weremarked by alizarin red staining(ARS) to detect and compare the osteogenicpotential between the two kind of BMSCs;The RT-PCR detected the mRNAexpression level of the BMSCs osteogenesis related proteins, forinstance,Runx2、osteocalcin (OCN) and osteopontin (OPN),and the level ofadipogenesis related proteins, for instance, peroxisome proliferator-activatedreceptor gamma (PPARγ) and lipoprotein lipase (LPL).ResultsCompared to the sham group, the bone volume fraction (bone volume/totalvolume，BV/TV), trabecular thickness (Tb. Th) and trabeculae number (Tb.N)decreased in the ovx group rats,and decreased respectively by72.6％,39.0％and56.4％(P＜0.05);The ratio of bone surface area and bone volume (BS/BV),trabecular spacing(Tb.Sp) and trabeculae pattern factor (Tb.Pf) increased in theovx group,and increased respectively by63.7％,354.2％and72.6％(P＜0.05）;The colony-forming ability of BMSCs from ovx group became decreased,the proliferation capacity was declined，the osteogenic potential was decreased,and the adipogenic potential was increased(P＜0.05）; RT-PCR results showedthat compared to sham group, the osteogenesis related protein of Runx2,osteocalcin (OCN) and osteopontin protein (OPN) mRNA expression levels inovx group rats were reduced by87.0%,34.3%,29.7%, and the adipogenesisrelated protein of peroxisome proliferator-activated receptor gamma (PPARgamma) and lipoprotein lipase (LPL) mRNA expression levels in ovx group ratswere increased by39.1%and33.2%. ConclusionsThere was a significant reduction of bone quality in the femoral cancellousbone in ovariectomized rats, and osteoporosis models were established;Theproliferation and osteogenic potential of BMSCs from ovx rats were decreasedand the adipogenic potential was increased;The decreasing potential ofproliferation and osteogenesis and the increasing potential of adipogenesis ofBMSCs can cause significant changes of bone micro-structure inovariectomized rats,and play an important role in the pathogenesis ofosteoporosis in ovariectomized rats.