Effects Of Inflammation On Osteogenesis And Autophagy Of Human Periodontal Ligament Stem Cells In Different Stages Of Osteogenic Differentiation

Objective: Human periodontal ligament stem cells from normal and inflammatory sources were identified by labeling,primary culture,MTT cell proliferation,osteogenesis and lipogenic differentiation.On the 3rd,7th and 14 th day of osteogenic differentiation of all periodontal ligament stem cells,the fluorescence expression of microtubule associated protein 1A / 1b light chain 3(lc3b)was detected by immunofluorescence,Real time PCR was used to detect Osteocalcin(OCN)and bone sialoprotein,BSP,Runx2,ALP and lc3b,Beclin1 and ATG5 autophagy related mRNA expression were compared.The expression of osteogenic and autophagic mRNA in inflammatory and normal periodontal ligament stem cells was different The expression of ALP and lc3b,Beclin1,ATG5 autophagy related proteins were detected by blot.The differences of osteogenic and autophagic protein expression between inflammatory and normal periodontal ligament stem cells were compared.The effects of inflammation on osteogenic ability and autophagy level of human periodontal ligament stem cells were studied.Methods:1.Sample collection and primary culture: the permanent teeth of healthy and periodontitis patients in oral and maxillofacial surgery of Stomatology Hospital of Hebei Medical University were collected.Normal tissue samples: three orthodontic or impacted healthy permanent teeth,marked with H1,H2 and H3,included in the standard: no gingivitis and periodontal pocket,no bleeding after probe.Inflammatory tissue samples:3 permanent teeth of periodontitis patients,marked P1,P2 and P3,inclusion criteria: PD > 4mm,gingivitis,probe bleeding,alveolar bone absorption,tooth loosening.After the teeth were obtained in the clinic,they were completely immersed in the solution containing 2% of penicillin streptomycin,PS)?-MEM,The primary periodontal ligament stem cells of H1,H2,H3,P1,P2 and P3 were obtained by tissue block method.The primary cells were changed every 2-3 days.After the cells crawled out of the tissue block and grew to local confluence,and the cell density was 70-80% of the total area,the primary periodontal ligament stem cells of H1,H2,H3,P1,P2 were obtained And P3 cells.The periodontal ligament stem cells were further isolated and purified by limited dilution method.H1,H2,H3,P1,P2 and P3 cells were subcultured,and the third to fifth generation cells were used for subsequent experiments.2.Identification of cell source: on the 1st,3rd,5th and 7th day after cell planing,the source of cultured periodontal ligament stem cells was identified by vimentin of H1,H2,H3,P1,P2 and P3 cells and STRO-1 immunofluorescence to verify whether the cultured periodontal ligament stem cells expressed mesenchymal stem cell markers,which were derived from mesenchymal stem cells.Whether the cultured periodontal ligament stem cells come from the ectoderm,whether there are epithelia l cell impurities and pollution.3.Validation of periodontal ligament stem cells: 1,3,5 and 7 day s after the laying of H1,H2,H3,P1,P2 and P3 cells,the proliferation activity of periodontal ligament stem cells was detected by MTT cell proliferation experiment to verify whether the cultured cells were periodontal ligament stem cells with strong self-renewal ability.After the third generation of H1,H2,H3,P1,P2 and P3 periodontal ligament stem cells were subcultured,they were inoculated into 6-well plate according to the density of 5 × 104 / well,replaced with osteogenic induction medium for 24 hours,fixed with 4% paraformaldehyde for 21 days,stained with alizarin red to observe the generation of mineralized nodules,and verified the osteogenic differentiation ability of periodontal ligament stem cells.After the third generation of H1,H2,H3,P1,P2 and P3 periodontal ligament stem cells were subcultured,they were inoculated into 6-well plates according to the density of 1 × 105 / well,and then replaced with the medium of induction of adipogenesis for 24 hours,fixed with 4% paraformaldehyde for 21 days,and stained with oil red O to observe the formation of lipid droplets and detect the ability of adipogenesis and differentiation of periodontal ligament stem cells.Osteogenic differentiation and lipogenic differentiation of cultured periodontal ligament stem cells.4.Osteoblast differentiation culture: the 4th generation of healthy source H1,H2 and H3 and inflammatory source P1,P2 and P3 periodontal ligament stem cells were subcultured,inoculated in a 10 cm dish with 1 × 106 / well,and then replaced with osteoblast culture medium for 24 hours.The culture medium was replaced every 48 hours,and the samples were collected at 3,7 and 14 days.The expression of OCN,BSP,Runx2 and ALP osteoblast related mRNA was detected by real-time PCR,and Western blot The expression level of ALP osteogenic related protein was detected by blot.Spss22.0 software package was used to analyze the data.5.Autophagy test: after the 4th generation of normal source H1,H2 and H3 and inflammatory source P1,P2 and P3 periodontal ligament stem cells were subcultured,inoculated in a 10 cm dish with 1 × 106 /well,the culture medium was changed into osteoblast culture medium for 24 hours,and the culture medium was changed every 48 hours,and the samples were collected on days 3,7 and 14,respectively.The cells were used for immunofluorescence staining,RT-PCR and Western blot detection.On the 3rd,7th and 14 th day of osteogenic differentiation of normal and inflammatory periodontal ligament stem cells,the expression of autophagy related protein lc3b was detected by immunofluorescence,lc3b,Beclin1,ATG5 autophagy related mRNA by real-time PCR,and lc3b,Beclin1,ATG5 autophagy related protein by Western blot.Spss22.0 software package was used to analyze the data.Results: 1.Immunofluorescence staining showed that vimentin was positive in H1,H2,H3,P1,P2 and P3 periodontal ligament stem cells,pan keratin was negative,STRO-1 was positive.2.The results of MTT assay showed that the proliferation of H1,H2,H3,P1,P2 and P3 cells was slow in the first five days,and accelerated in the fifth to seventh days.3.The cultured H1,H2,H3,P1,P2 and P3 cells were used for osteogenic differentiation and lipogenic differentiation.After 21 days of osteogenic differentiation,many dense,uniform and large-area mineralized stromal nodules could be seen in periodontal ligament stem cells.After 21 days of lipogenic differentiation,large and small lipid droplets could be seen in periodontal ligament stem cells.4.The expression levels of OCN,BSP,Runx2 and ALP mRNA of periodontal ligament stem cells from inflammatory sources were significantly lower than those of periodontal ligament stem cells from normal sources(P < 0.05),and the expression levels of ALP protein of periodontal ligament stem cells from inflammatory sources were significantly lo wer than those of periodontal ligament stem cells from normal sources(P < 0.05).5.Immunofluorescence staining showed that the fluorescence intensity of lc3b from inflammatory periodontal ligament stem cells was lower than that of normal periodontal ligament stem cells at 3,7 and 14 days after osteogenesis.6.RT-PCR showed that the mRNA expression levels of lc3b,Beclin 1 and ATG5 mRNA in inflammatory periodontal ligament stem cells were lower than those in healthy periodontal ligament stem cells,but there was no significant difference(P > 0.05).The expression levels of lc3b,Beclin 1 and ATG5 in inflammatory periodontal ligament stem cells were lower than those in healthy periodontal ligament stem cells on 7 and 14 days after osteogenesis The level of mRNA expression was significantly lower than that of healthy periodontal ligament stem cells(P < 0.05).7.Western blot showed that the expression levels of lc3b,Beclin1 and ATG5 in inflammatory periodontal ligament stem cells were lower than those in healthy periodontal ligament stem cells,but there was no significant difference(P > 0.05).The expression of lc3b,Beclin1 and ATG5 in inflammatory periodontal ligament stem cells was significantly lower than that in healthy periodontal ligament stem cells(P < 0.05).Conclusions:1.All the cultured cells expressed MSCs,and there was no epithelial impurity and pollution.It can be concluded that all the cells were derived from MSCs.2.All the cultured cells have strong ability of self-renewal,osteogenic differentiation and lipogenic differentiation.It can be concluded that all the cultured cells are human periodontal ligament stem cells.3.Inflammation reduces the ability of periodontal ligament stem cells to differentiate into osteoblasts.4.Inflammation had no significant effect on the autophagy level of periodontal ligament stem cells in the early stage of osteogenic differentiation,but decreased significantly in the middle stage of osteogenic differentiation.