Iron Oxide Nanoparticles Promotes Osteogenic Differentiation Of Human Periodontal Stem Cells And Repair Periodontal Bone Defects In Rats

Objective: 1)to investigate the potential of iron oxide nanoparticles to promote osteogenic differentiation of human periodontal stem cells(hPDLSCs)and to study the optimal osteogenic concentration,and transcriptome sequencing was used to analyze the possible molecular mechanism of transcriptome sequencing.2)explore the feasibility of iron oxide nanoparticles in repairing periodontal bone defects in rats.Methods: 1)(1)hPDLSCs were cloned by modified enzyme digestion and limited dilution technique,and the phenotype of the third-generation hPDLSCs was detected by flow cytometry.(2)The ability of osteogenesis,lipogenesis and chondrogenic differentiation of the third generation of hPDLSCs was observed after 21 days in vitro.(3)Peals blue staining and transmission electron microscopy(TEM)were used to observe the distribution of iron oxide nanoparticles in hPDLSCs.(4)Immunofluorescence assay was used to detect the phenotype of hPDLSCs treated with iron oxide nanoparticles.(5)The proliferation of hPDLSCs treated with different concentrations of 0 g/ mL,10 g/ml,25 g/ml,50 g/ml,100g/ml,300 g/ml,and 500 g/ml IONPs were detected by CCK-8 and immunofluorescence assay.(6)IONPs with different final concentrations of 0 g/ mL,10 g/ml,25 g/ml,50 g/ml,100 g/ml,300 g/ml,and 500 g/ml were used as experimental subjects,and the appropriate bone differentiation concentration was selected by using ALP quantitative detection method.(7)In order to detect their osteogenic differentiation ability.(OS group: osteogenic medium induction;50?g/ml iron oxide nanoparticles group: osteogenic medium induced group + iron oxide nanoparticles;100?g/ml iron oxide nanoparticles group: osteogenic medium induced group + iron oxide nanoparticles).The four groups were subjects:osteogenic related genes(COL1,ALP,RUNX2,BMP2)and the expression of corresponding proteins were analyzed by RT-PCR and Western blot.(8)The third-generation hPDLSCs were cultured with iron oxide nanoparticles for 7 days,and RNA transcription sequencing was used to detect the relevant mechanism at the molecular level.Theintervention was pretreated with the Wnt signaling pathway inhibitor Dickkopf1(DKK1).Three groups were divided into three groups: blank control group:ordinary medium,iron oxide nanoparticles group: ordinary medium + 50?g/ml iron oxide nanoparticles,DKK1 inhibition group: ordinary medium + 50?g/ml iron oxide nanoparticles +100ng/mlDKK1.In addition,the mechanism of Wnt pathway was preliminarily explored by detecting the expression of mRNA and related proteins of signaling molecules related to Wnt pathway.(9)Wnt signaling pathways and osteogenetic differentiation of the intimate relationship,with the blank control group: normal medium group,OS: osteogenesis induction media group,iron oxide nanoparticles group: ordinary medium + 50?g/ml iron oxide nanoparticles,DKK1 group: ordinary medium + 50?g/ml iron oxide nanoparticles + 100 ng/ml DKK1 four groups of subjects: test groups of osteogenesis related mRNA and osteogenesis related protein expression,further verify the iron oxide nanoparticles by Wnt signaling pathways to regulate human periodontal membrane stem cells differentiation.2)SD rats were used as experimental animals to make a defect model of 3mm × 2mm×1mm hole at the distal alveolar bone of the lower anterior teeth of each rat;The 30 rats(male or female)were equally divided into three groups : A:blank group;B: nanometer hydroxyapatite group;C:iron oxide nanoparticle composite nanometer hydroxyapatite group,n =10;Six weeks after the operation,the samples were collected,and the new tissues were observed.HE staining and Micro-CT were used to detect the defect repair.Results:1)(1)The primary hPDLSCs were obtained by modified enzyme digestion.Cells could be seen crawling out of the tissue blocks under the microscope for 4-5 days,forming colonies and growing with spindle cells and full cytoplasm.After purification by limited dilution,the cell morphology was uniform and the growth rate was fast.The results of flow cytometry showed that the mesenchymal stem cell phenotype of the third-generation hPDLSCs was highly expressed.(2)Identification of multidirectional differentiation ability of cells: the results of alizarin red staining in osteogenesis showed the formation of multiple calcification nodules.Lipid detection oil red O staining showed reddish lipid droplets in the cytoplasm.Detection of chondrogenesis by alcian blue staining showed bluish chondroid formation.(3)perls blue staining and transmission electron microscopy(TEM)were used to observe the distribution of iron oxide nanoparticles in hPDLSCs.peal blue staining showed that blue iron oxide nanoparticles were distributed in the cytoplasmof hPDLSCs.(4)hPDLSCs treated with iron oxide nanoparticles still showed high expression of mesenchymal stem cell phenotype: immunofluorescence test results showed positive expression of STRO-1,positive expression of vimentin and negative expression of keratin.(5)CCK-8 test results showed that the final IONPs concentration was within 100?g/ml,and the cell growth rate was not affected.The results of EdU immunofluorescence showed that the IONPs concentration reached 300?g/ml,and the number of new cells decreased significantly.(6)Results of quantitative determination of ALP: the ALP activity of IONPs group was significantly higher than that of control group than that of pure osteogenic medium group,and there was statistical significance between groups(P<0.05).According to the results,iron oxide nanoparticles have relatively strong osteogenic ability when the final concentration is 50?g/ml.(7)The results of RT-PCR and Western blot showed that compared with the control group,mRNA and its corresponding proteins in the osteogenic induction group and the iron oxide nanoparticle group were significantly increased,with statistical significance(P<0.05).The iron oxide nanoparticle group was higher than the osteogenic induction group,with statistical significance(P<0.05).(8)RNA transcriptome sequencing and the results show that: RT-PCR detection of iron oxide nanoparticles group high expression of Wnt pathway and factors related to main LRP5,LEF1,?-catenin,cyclind mRNA,Western blot confirmed the results,with statistical significance(P<0.05).(9)the result of RT-PCR showed that iron oxide nanoparticles of mRNA and protein expression of bone related results are higher than the control group,with statistical significance(P<0.05).However,the inhibition of bone-related mRNA and protein expression by adding DKK1 was lower than that of the iron oxide nanoparticle group,with statistical significance(P<0.05),suggesting that iron oxide nanoparticle may promote osteogenic differentiation of hPDLSCs through Wnt signaling pathway.2)The rats were sacrificed 6 weeks after the operation,and visual observation showed that the defects of the iron oxide nanoparticle composite nano-hydroxyapatite group were almost completely filled with new tissue,and the defect boundary was not clear.The defects of hydroxyapatite group also had more new tissue formation,and the defect boundary was visible.Histological observation of new tissue formation in the defect of blank control group showed that the iron oxide nanoparticles combined with nano-hydroxyapatite group had new bone tissue formation and bone regeneration significantly better than other groups.The results of Micro-CT were consistent with theresults of histological examination.Conclusion: 1)iron oxide nanoparticles can promote the osteogenic differentiation of human periodontal membrane stem cells,and in vitro IONPs final concentration of50?g/ml is the optimal osteogenic differentiation concentration,in addition,it is speculated that IONPs osteogenic differentiation of hPDLSCs may be regulated by the Wnt signaling pathway.2)the application of iron oxide nanoparticles combined with nano-hydroxyapatite to repair periodontal bone defects in rats can achieve a good repair effect.