Effects Of PEMF And BMP9 On The Proliferation And Osteogenic Differentiation Of Periodontal Ligament Stem Cells

Research background and objective Chronic periodontitis,tumors,trauma and inappropriate orthodontic therapy may result in the loss of alveolar bone.Recently,tissue engineering has been demonstrated an effective treatment to bone loss and has enormous potentials in the future.Periodontal ligament stem cells(PDLSCs)featured by low-immunity,self-renewal ability,and multi-differentiation potential has been used as primary stem cells in periondontal tissue engineering.Pulsed electromagnetic fields(PEMF),a physical stimulus,and biochemical stimulus bone morphogenetic proteins(BMPs)has been confirmed the ability to promote stem cells proliferation and osteogenic differentiation.Several studies in vitro revealed that PEMF stimulus exerts combined effects with biochemical factors(differentiation media and growth factors)on the cell proliferation,osteogenesis-related factors expression and mineral nodules formation.So far,there in no relevant research on the effect of PEMF stimulation alone or the combined effect with BMP9 in the field of periodontal regeneration.As a result,we investigated the effect of PEMF stimulus of different intensities alone and the synergistic effect with BMP9 on the proliferation,osteogenic differentiation,and mineralization of PDLSCs in vitro,aiming to provide a safe and adjunctive therapy for the regeneration of periondontal bone tissuesMethods1.We collected the extracted healthy premolar(n=20)for orthodontic purpose with the informed consent from subjects.We cultured the primary PDLCs through the tissue block method,then isolated CD146+ PDLSCs by CD146 immunomagnetic isolation,afterthat the percentage of CD146+/STRO-1+ cells was determined via flow cytometry.The origin of PDLSCs was determined via immunofluorescence staining,and then determined the osteogenic and adipogenic differentiation potential of isolated PDLSCs by Alizarin Red S staining and Oil Red O staining.2.Exogenous BMP9 was transduced into PDLSCs using a recombinant adenovirus assay,and detected the BMP9 gene and protein expression in PDLSCs.The cell proliferation ability in BMP9-infected cells was detected through CCK8 assay and draw the cell proliferative curve of successive 10 days.Then,detected the early and intermediate osteogenic genes and proteines expression of runt-related transcription factor 2(Runx2),alkaline phosphatase(ALP),osteopontin(OPN),and late mineralized extracellular matrix formation in h PDLSCs.3.We exposed either h PDLSCs or BMP9-transduced h PDLSCs to PEMF(1 h daily,0.6–3.0 m T at an interval of 0.6 m T)for 10 days as a treatment group,and the sham exposure group was a control group.All groups were measured by CCK8 assay every day for 10 days,and the cell proliferation curve was drawn.4.The cultured h PDLSCs were exposed to PEMF(1 h daily,0.6–3.0 m T at an interval of0.6 m T),then detected the gene expression of ALP?Runx2?OPN in h PDLSCs at indicated time point,in order to determine the proper magnetic intensity of PEMF.Furthermore,BMP9-infected h PDLSCs were exposed to PEMF(1.8 and 2.4 m T)and detected the gene expression of ALP,Runx2,OPN and later excellular calcium deposits induced by two stimuli.Results1.Flow cytometry revealed that the mean percentage of STRO-1+/CD146+cells was32.2% in isolated CD146+ PDLCs.Immunofluorescence staining showed PDLCs were vimentin-positive and cytokeratin-negative.Alizarin Red S and Oil Red O staining determined the multidifferentiation potential of isolated PDLSCs.2.The multiplicities of infection(MOIs)of Adenoviruses infected with PDLSCs was determined a range of 30-50 which had high infective efficiency of 70%-80%.The RT-PCR and Western blot results revealed that BMP9-infected PDLSCs has high BMP9 gene and protein expression contrary to control group.CCK8 results indicated that BMP9 significantly enhanced the PDLSCs proliferation in logarithmic phase.ALP quantitative assay revealed that BMP9 promoted the ALP protein expression of cells in early osteogenic differentiation phrase.q RT-PCR revealed that BMP9 induced the osteogenesis-related genes such as ALP,Runx2 and OPN expression in h PDLSCs.3.CCK8 assay revealed that PEMF had no influence on the h PDLSCs proliferation and did not enhance the proliferative ability of cells stimulated by BMP9.4.q RT-PCR revealed that PEMF(15HZ,1.8 or 2.4 m T)significantly promoted the osteogenesis-related genes expression in h PDLSCs.q RT-PCR,western blot assay and Alizarin Red S staining revealed that the combination of PEMF and BMP9 stimulus induced more ALP,Runx2 and OPN gene and protein expression,even the late mineralized nodules formation than either stimulus alone group.Conclusions This study substantiates that BMP9 has the potential of promoting proliferation and osteogenic differentiation of h PDLSCs;Alone or under the basis of BMP9 existence,PEMF stimulus has no effect on cells proliferation;PEMF can induce the osteogenic differentiation of h PDLSCs,and there also exists the interaction effect when combined with BMP9 stimulus.Therefore,PEMF of proper parameter could enhance the osteo-inducive potential of BMP9 on h PDLSCs.