Preliminary Study On The Mechanism Of Graphene Quantum Dots’ Effect On Differentiation Of Periodontal Ligament Stem Cells And Related Osteogenic Differentiation Mechanism

ObjectiveTo isolate and identify periodontal ligament stem cells（periodontal ligament stem cells,PDLSCs）by tissue culture method,and to further explore the effect of graphene quantum dots（GQDs）on the proliferation,migration and differentiation of PDLSCs.The mechanism of promoting periodontal ligament stem cell into osteogenic differentiation was also studied.Methods1.The PDLSCs was purified by the finite dilution method and the clonal formation rate was determined.The surface specific markers expression of mesenchymal stem calls,including CD90,CD146 and STRO-1 and surface specific molecular marker CD34 of hematopoietic stem cells was detected by flow cytometry.Used purified cells for multiple differentiation culture.Immunofluorescence were used to identify the expression of vimentin and keratin in purified cells.All methods above were used to identify PDLSCs.2.Cell Counting Kit 8（CCK-8）was used to detect the effect of different concentrations of GQDs on PDLSCs proliferation and reflect the toxicity of GQDs.Transwell method was applied to detect the effect of different concentrations of GQDs on PDLSCs migration.The optimal concentrations of GQDs on PDLSCs proliferation and migration was finally determined.3.Culture PDLSCs in control,osteo,5GQD,5GQD+osteo groups by using different culture medium.The osteogenic marker gene expression,including ALP,RUNX2,OCN,fibroblast marker gene,including COL-Ⅰ,Scleraxis and cementum markers gene CEMP-1 of PDLSCs in normal and inflammatory environment were detected by q-PCR.Alizarin red S staining was used to detect the formation of mineralized nodules in different group.Meanwhile,the marker genes（LRP6,β-catenin,LEF1）in Wnt/β-catenin classical pathway were further detected by q PCR for a preliminary study on the mechanism of graphene quantum dots promoting PDLSCs osteogenic differentiation.Results1.PDLSCs isolation and culturePDLSCs was successfully isolated and purified with the ability to form colony units.Flow detection showed high expression of surface specific marker of molecules of mesenchymal stem cells.Osteogenic and adipogenic culture were successfully induced.Vimentin strong positive expression and keratin negative expression were observed after immunofluorescence staining.2.The effect of GQDs on PDLSCs proliferation and migrationNo obvious cell proliferation inhibition was found in the concentration range of 0-30μg/ml.After adding GQDs,the cell migration rate increased as a whole,the most significant increase was observed in 5 or 10μg/ml concentrations;And GQDs with a concentration of 5μg/ml was used in the further study.3.GQDs effect on promoting PDLSCs differentiationALP expression was significantly higher in 5GQD groups in the normal environment than in control group.When cultured in the normal environment,the expression of RUNX2 increased in the osteo and 5GQD+osteo groups;RUNX2 was higher in 5GQD and 5GQD+osteo group than in osteo group.On14^thd,RUNX2 expression was up-reglated in normal environment groups,which did not seen in inflammatory environments.On 21^std,the expression of RUNX2in the 5GQD group was increased significantly in normal and inflammatory environments.The OCN expression was increased in the normal environment in osteo and 5GQD groups on 7^th,14^thd.On 14^thd,the OCN expression in inflammatory environment was lower than the corresponding groups in normal environment.Alizarin red staining showed that the staining effect of 5GQD groups in normal environment were more obvious than that in osteo group,and the mineralized nodules in each group were significantly reduced in inflammatory environment,indicating that the inflammatory environment inhibited the osteogenic properties of the materials. COL-Ⅰexpression increased in each experimental group on 7^th d.The expression of COL-Ⅰin 5GQD group was significantly higher than that in other groups in normal experiment.The expression of COL-Ⅰon 21^st d was higher than that in blank group in normal experiment;In an inflammatory environment,COL-Ⅰexpression in each group was lower than the corresponding group under normal environment.The Scleraxis expression in 5GQD groups in normal environment and groups in inflammatory environment was significantly inhibited on 7^th d.On 14^th d,The Scleraxis expression of 5GQD groups in inflammatory environment was higher than that in control group.On 21^st d,GQDs up-regulated Scleraxis expression in both normal and inflammatory environment.The expression of CEMP-1 markers in normal and inflammatory environments has been inhibited in each group.4.Mechanism of osteogenic differentiation of periodontal ligament stem cells induced by graphene quantum dots:In normal conditions,LRP6,β-catenin relative gene expression in 5GQD group were significantly increased on 7^th,14^th,21^st d,and LEF1 expression was higher in 5GQD group on 14^th d.In the inflammatory environments,LRP6 expression in 5GQD group on14^th d was higher than in osteo group and 5GQD+osteo group,The LRP6expression was increased in the 5GQD,5GQD+osteo group on 21^st d.β-catenin expression in 5GQD group was higher than in osteo group on 14^th d.On 21^st d,β-catenin expression in osteo,5GQD and 5GQD+osteo group were significantly higher than the other groups;the LEF1 expression was higher in the 5GQD group on 21^st d than in the control group.Without osteogenic induce culture medium,GQDs could promote PDLSCs differentiate into osteoblast and fibroblast.Conclusion1.It was confirmed that GQDs is less toxic and has good biocompatibility,which can effectively promote the migration of PDLSCs.2.In both normal and inflammatory environments,GQDs could spontaneously promote PDLSCs osteogenic,fibroblast differentiation,and inhibit cementum differentiation,suggesting that GQDs could be used as a potential scaffold material and grow factor for periodontal tissue engineering.3.GQDs may could upregulate PDLSCs osteogenic differentiation by activating the classical Wnt signaling pathway.