The Analysis Of Growth Factors In Rabbit Redbone Marrow And The Study On Osteogenic Differentiation Of Rabbit BMSCs

Background: Bone Nonunion pose a difficult problem in the orthopedics field，theclinical effect of autogenous red bone marrow transplantation is effective on bonenonunion. However, the mechanism remains to be investigated.Objective:. To analysis the growth factors in the red bone marrow of New Zealandwhite rabbits and to explore the effect of red bone marrow on differentiation of bonemarrow mesenchymal stem cells (BMSCs) into osteoblasts in vitro.Methods:1. The bone morphogonetic protein-2(BMP-2), insulin-like growth factor-I(IGF-I), basic fibroblastic growth factor (bFGF), platelet-derived growth factor-BB(PDGF-BB), transforming growth factor-beta1(TGF-β1) and vascular endothelialgrowth factor165(VEGF165) in the red bone marrow of the New Zealand white rabbitswere detected by the Enzyme-Linked Immunosorbent Assay (ELI SA).2.BMSCs wereseparated from bone marrow of New Zealand white rabbits by density gradientcentrifugation. The cells were cultivated and sub-cultivated in vitro.3.The growth andmorphology of cells were observed using inverted microscope.4.The proliferation ofthe BMSCs was detected by MTT assay.5. The expression of CD44and CD45onBMSCs were detected by flow cytometry (FCM).6.The differentiation of BMSCs wasinduced in DMEM/F12medium containing15%supernatant of red bone arrow.7.Alkaline phosphatase was identified using alkaline phosphatase staining,8.Theactivity of alkaline phosphatase was detected with the method of enzymekinetics on the3rd,6th,9th,12th and15th day, respectively.9. Calcium nodules were etermined by theARS(Alizarin red S) staining.10.The gene expressions of Osteocalcin mRNA (OCNmRNA）and type I collagen mRNA(Col I mRNA) were determined by RT-PCR assay.Results:1.the concentrations of BMP-2,IGF-I, bFGF, PDGF-BB, TGF-β1and VEGF165were (1.26±0.14) μg/L，(3.87±0.35) μg/L，(4.34±0.22) ng/L，(4.08±0.36) ng/L， (31.45±1.91) ng/L and (14.81±2.01) ng/L in the red bone marrow, and compared withthe whole blood group (0.10±0.02) μg/L,(0.19±0.04) μg/L,(0.28±0.04) ng/L,(0.29±0.08) ng/L,(1.86±0.38) ng/L and (0.99±0.23) ng/L the whole blood, respectively. Theconcentrations of these growth factors were significantly higher than those in the wholeblood (P<0.01).2.The BMSCs were separated and cultured successfully, the expressionratio of CD44and CD45were99.67%and2.34%, respectively.3.The MTT assayshowed the proliferation of BMSCs in the red bone marrow group was significantlyhigher than that in the whole blood group from the3to9days(P <0.05).4.The activityof ALP was significantly higher in the bone marrow group[Days3,6,9,12and15:(4.55±0.15),(8.98±0.20),(12.83±0.21),(15.65±0.23) and (15.23±0.19) U/100ml] thanthat in control group [Days3,6,9,12and15:(2.78±0.08),(3.31±0.13),(3.77±0.13),(4.43±0.16) and (4.23±0.12) U/100ml](P <0.01).5.After14days of osteogenicinduction in the DMEM/F12medium containing15%supernatant of red bone marrow，the BMSCs in the bone marrow group were positive for the Alizarin red S staining andthe Alkaline phosphatase staining. After21days, the BMSCs showed the positiveexpression of Col I mRNA and OCN mRNA. However, there were no detectableexpressions of these mRNA in control group.Conclusions:1. the concentrations of BMP-2, IGF-I,bFGF,PDGF-BB, TGF-β1and VEGF165in thered bone marrow were significantly higher than those in the whole blood.2. BMSCs of New Zealand white rabbits were separated and cultured successfully.3. The red bone marrow can significantly promote the proliferation of BMSCs.4. The supernatant of red bone marrow can significantly increase the ALP activity inBMSCs.5. The supernatant of red bone marrow can effectively induce BMSCs differentiate intoosteoblasts in vitro.