The Study On Osteogenic Differentiation Capacity Of Synovial-derived Mesenchymal Stem Cells And Bone Marrow Mesenchymal Stem Cells

Objective : To investigate chondrogenic and osteogenic differentiation potential and the biological features of synovium-derived mesenchymal stem cells(SMSCs)and bone mesenchymal stem cells(BMSC)in vitro and to observe the osteogenic capability of the construct combined SMSC and BMSC with Hydroxylapatite/Chitosan/Poly L-latic acid(HA/CS/PLLA)composite scaffolds in vivo.To compare the osteogenic differentiation capacity of SMSC and BMSC in vitrol and in vivo.Methods: SMSC and BMSC were obtained by adherent screening method and enzymatic digestion method.Specific phenotype of SMSC and BMSC were detected by flow cytometer after purification.Then,SMSC and BMSC were identified by toluidine blue staining,alizarin red staining and alkaline phosphatase staining after chondrogenic and osteogenic induction,respectively.MTT method was used to detect P1/P2/P3 generation of SMSC and BMSC cell proliferation rate.In vitro experiments,chondrogenic Collagen typeⅡ and Aggrecan,osteogenic related genes Collagen typeⅠ,ALP,OCN and RUNX2 were administrated by real-time PCR after osteogenic induction for 1 day,7 days,14 days and 21 days.ALP activities were also determined by Elisa after osteogenic induction for 1 day,3 days,5 days,7 days,9 days and 11 days.Meanwhile,extracellular matrix calcium mineralization were detected by Alizarin Red S method after osteogenic induction for 7 days,14 days,21 days and 28 days as well.The expression of OCN and RUNX-2 protein were detected by Western-blot method after osteogenic induction for 21 days.In vivo experiments,P3 SMSC and BMSC populations were seeded on HA/CS/PLLA composite scaffolds,after adhesion for 72 h in vitro,the composite scaffolds were implanted into the thigh muscle of SD rats to construct ectopic bone formation models.These rats were sacrificed by anaesthesia overdose at 8 weeks after surgery and the scaffolds were removed for X-ray and histological examination.Results: The positive rates for the molecules of CD147,CD90,CD105 and CD44 were over 95 percents,while rates for CD117,CD34,CD14,CD45 were lower than 10 percents.Toluidine blue staining demonstrated cells stined blue and Alizarin red staining showed flaky red,and cytoplasm was dyed brown in the ALP staining.MTT showed that P1 cells of SMSC and BMSC grow slow with linear relationship,while P2 and P3 cells of SMSC and BMSC grow with “S” relationship.In vitro experiments,Collagen typeⅡ and Aggrecan gene expression were significantly enhanced after chondrogenic induction in SMSC group,compared with BMSC group.While,the expression of Collagen typeⅠ,ALP,OCN and RUNX2 were significantly enhanced after osteogenic induction in BMSC group,compared with SMSC group.ALP activities were significantly increased after osteogenic induction since osteogenic induction for 3 days and reached a maximum at 7 days,and the expression were significantly enhanced in BMSC group compared with SMSC group(P<0.05).Calcium mineralization were significantly enhanced in BMSC group after osteogenic induction for 14 days compared with SMSC group(P<0.05).X-ray and histological examination demonstrated that the new bone tissues formed in both groups,but bone formation content of the BMSC group was significantly more than that of the SMSC group at 8 weeks after implantation.Conclusions: SMSC and BMSC could be induced into osteoblast and Chondrocytes both in vitro and in vivo,while BMSC have higher osteogenic differentiation capacity in vitrol and in vivo.