The Study Of P38and ERK1/2Pathways In BMP9-induced Osteogenesis Of Periodontal Ligament Stem Cells

Part1Isolation and identification of human PDLSCsObjective: To isolate PDLSCs from human periodontium andcomplete their identification.Methods: Healthy premolars were collected from orthodontic patients.We scraped the periodontium from the root, then digested by collagenase Iand dispase II. We got single cell suspension by70μm strainer and selectedhPDLSCs by cloning ring. We identified them by colony-formingunit-Fibroblast (CFU-F) efficiency assay, immunocytochemical analysis,and multipotent differentiation.Results: hPDLSCs exhibited a typical fibroblast-like spindleappearance; the CFU-F was42%; immunocytochemical analysis found thatthey were positive for mesenchymal stem cell markers (STRO-1andCD146), and vimentin, while negative for cytokeratin; they had potency todifferentiate into osteoblasts and adipocytes.Conclusion: Cells isolated by single colony selection were identifiedto be hPDLSCs from such points: appearance, CFU-F, mesenchymal stem cell markers and multipotent differentiation. Part2BMP9-induced osteogenic differentiation of hPDLSCsObjective: To analyze osteogenesis ability of hPDLSCs induced byBMP9.Methods: Ad-BMP9and Ad-GFP were used to infect hPDLSCs aftertesting their infection efficiency. We analyzed the expression of ALP, OPN,OCN, and matrix mineralization at different time points by ALP assays,qPCR and matrix mineralization assay.Results: ALP levels of hPDLSCs infected by Ad-BMP9wereobviously higher than GFP group and blank group (p <0.01), which got thetop at7day, and coincided with ALP staining. The levels of OPN and OCNwere elevated obviously compared to GFP group (p <0.01). The staining ofcalcium mineral deposits was more obvious in BMP9group.Conclusion: After analyzing osteogenic markers at early, intermediateand late periods, we confirmed that BMP9could induce osteogenesis bythe way of Ad-BMP9infection. Part3p38MAPK invovled in BMP9-induced osteogenicdifferentiation of hPDLSCsObjective: To study if p38MAPK is involved in BMP9-inducedosteogenic differentiation of hPDLSCs and its function.Methods: hPDLSCs were infected by Ad-BMP9, and western-blotwas used to analyze the protein levels of MKK3/6, phosphorylatedMKK3/6, p38and phosphorylated p3836h later. After p38inhibitor(SB203580) was added, we analyzed its effect by ALP assays, qPCR andcalcium mineral assay at7,10and21days.Results: Western-blot was found that the protein levels of MKK3/6and p38were not changed while levels of phosphorylated MKK3/6andphosphorylated p38were increased. After adding SB203580, theexpression of ALP was decreased obviously by a dose dependent way at7day (p <0.01), which was most significant at the concentration of10μmol/L. The results of qPCR showed that SB203580reduced the levelsof OPN and OCN obviously after10days (p <0.01), which is same withresults of ELISA (p <0.05). Calcium mineral deposits were also decreased.Conclusion: Western blot exhibited that MKK3/6-p38pathway was involved in BMP9-induced osteogenesis. After adding SB203580, allosteogenic markers (early, intermediate and late periods) were inhibited,which stated that MKK3/6-p38pathway was positive for osteogenesis ofhPDLSCs. Part4ERK1/2MAPK invovled in BMP9-induced osteogenicdifferentiation of hPDLSCsObjective: To study if ERK1/2is involved in BMP9-inducingosteogenesis of hPDLSCs and its function.Methods: hPDLSCs were infected by Ad-BMP9, and western-blotwas used to analyze the protein levels of MEK1/2, phosphorylated MEK1/2,ERK1/2and phosphorylated ERK1/236h later. After ERK1/2inhibitorPD98059was added, we analyzed its effect by ALP assays, qPCR andcalcium mineral assay at7,10and21days.Results: Western-blot was found that the protein levels of MEK1/2and ERK1/2were not changed while levels of phosphorylated MEK1/2andphosphorylated ERK1/2were increased. After adding PD98059, the levelof ALP was elevated obviously at7day (p <0.01), which was most significant at the concentration of25μmol/L. The results of qPCR andELISA showed that PD98059enhanced the levels of OPN and OCNobviously after10days (p <0.05). Calcium mineral deposits were alsoincreased.Conclusion: Western blot exhibited that MEK1/2-ERK1/2pathwaywas involved in BMP9-induced osteogenesis. After adding PD98059, allosteogenic markers (early, intermediate and late periods) were accelerated,which stated that MEK1/2-ERK1/2pathway was negative for osteogenesisof hPDLSCs.