| Backgroud and ObjectivesPeriodontitis is a chronic infectious and destructive disease that occurs in the periodontal tissues.It is the main cause of tooth loss in adults.Despite the improvement in living standards and oral hygiene awareness in recent years,the incidence of periodontitis remains high.Longterm periodontal inflammation ultimately leads to tooth loss and significantly affects normal oral functions such as chewing,pronunciation,and aesthetics,causing physiological,psychological,and economic losses to patients.In addition,periodontal disease,as a long-term chronic and persistent source of infection,has been recognized as a risk factor for systemic diseases such as diabetes,cardiovascular diseases,and osteoporosis.Therefore,the prevention and treatment of periodontitis are particularly important for maintaining oral and overall health.Periodontal epithelium includes gingival epithelium,sulcus epithelium,and junctional epithelium.The junctional epithelium,as the first line of defense in the periodontal tissues,acts as a physical barrier located at the base of the gingival sulcus and directly contacts the root surface,sealing off the soft and hard tissues.Additionally,the junctional epithelium has a fast metabolic turnover rate and possesses sensor functions,playing an important role in maintaining periodontal tissue homeostasis and defending against microbial invasion.However,the defense mechanisms of the junctional epithelium are not able to completely resist the occurrence and progression of periodontal disease.Under the influence of inflammation,the junctional epithelium proliferates towards the root and detaches from the tooth surface,undergoing a transition into pocket epithelium,which is considered a sign of periodontitis.Therefore,the ability of the junctional epithelium to resist detachment and maintain attachment to the tooth surface becomes an important factor in the prevention and progression of periodontitis.Odontogenic ameloblast-associated protein(ODAM)is a secretory protein expressed and secreted by ameloblasts in the late stage of enamel formation.It plays a role in the mineralization and maturation of tooth enamel.During development,as the tooth formation completes,the enamel organ gradually regresses to form the reduced enamel epithelium,which later transforms into the junctional epithelium after tooth eruption.Therefore,some studies generally believe that ameloblasts can serve as the precursor cells of junctional epithelium.In recent years,some studies have shown that the continuous and lifelong expression of ODAM in the junctional epithelium may have a stabilizing effect on the function of the junctional epithelium.Observations from Odam gene knockout mice have shown that the absence of the Odam gene does not affect the structure and morphology of tooth enamel.However,it does impact the healing of gingival tissue following gingivectomy surgery.Additionally,in elderly mice,the junctional epithelium exhibits reduced adhesion to tooth surfaces and a loss of sealing capability,though the exact mechanism remains unclear.Meanwhile,some clinical studies have indicated that ODAM levels in gingival crevicular fluid increase with probing depth in periodontitis patients,but expression is absent in immunohistochemical staining of periodontal tissues.There is currently no clear explanation for this phenomenon.Furthermore,recent research has shown that ODAM plays a significant role in the transition of mature enamel cells to junctional epithelium.Therefore,what changes and mechanisms are associated with ODAM in the development of normal junctional epithelium and periodontitis?Does supplementation with ODAM have a certain inhibitory effect on the occurrence and progression of periodontitis?Therefore,this study aims to explore the expression levels of ODAM in junctional epithelium under different periodontal conditions,starting from clinical samples of different periodontal states.Using transcriptome sequencing after knocking out the Odam gene in precursor cells that form junctional epithelial cells,to infer and validate the function and related mechanisms of ODAM in periodontal tissue epithelium.Additionally,the effects of exogenous recombinant ODAM protein on the occurrence and progression of periodontitis will be evaluated in an experimental periodontitis mouse model to determine whether it has a rescuing or inhibitory effect.To clarify the role and mechanism of ODAM in the development of periodontitis,and to provide new ideas and directions for the prevention and treatment of periodontitis.Methods1.The expression of ODAM in human periodontal tissueBased on the exclusion and inclusion criteria of experimental grouping,cases were collected,and the required sample size was calculated.In total,there were:12 cases in the periodontal health group(6 males and 6 females,age=36.5± 4.6 years),10 cases in the gingivitis group(6 males and 4 females,age=35.83 ± 6.5 years),and 12 cases in the periodontitis group(6 males and 6 females,age=39.6±3.5 years),with the periodontal health group serving as the control group.The periodontal tissue samples from the periodontal health group were obtained during the eruption of healthy impacted teeth or other teeth extraction procedures.The gingivitis group’s periodontal tissue samples were collected from erupted impacted teeth or other teeth during extraction procedures with evident signs of gingivitis.As for the periodontitis group.samples were acquired from surgically excised portions of diseased tissue during periodontal surgery.Each sample was divided into two parts:one part was stored in liquid nitrogen for realtime quantitative polymerase chain reaction(qRT-PCR)and Western Blot(WB)analysis to determine the mRNA and protein expression levels of ODAM in the tissues,while the other part was fixed in 4%paraformaldehyde,processed into tissue sections,and subjected to immunohistochemical staining for CK-19 and ODAM.CK-19 immunohistochemical staining was used to indicate the position and morphology of the junctional epithelium,and serve as a positive control for ODAM.2.Transcriptome sequencing analysis of function of OdamTake the ameloblasts cell line which is precursor cells of junctional epithelium for culture.Based on the establishment of the cell line and the characteristics to identify the cells as ameloblasts cells and have normal functions for subsequent experiments.Based on the expression pattern of ODAM in periodontitis,the Odam gene in ameloblasts was knocked out using CAS-9 lentiviral vectors.The efficiency of CAS-9 viral knockout was determined using mismatch repair assay.the transfection efficiency was assessed by qRT-PCR and WB.Ultimately,cells with a knockout efficiency of over 90%were selected for transcriptome sequencing.The samples were sent to Shanghai Meiji Corporation for quality inspection,library preparation,and sequencing.After obtaining the read counts of genes,differential gene expression analysis was conducted between samples.Based on the negative binomial distribution model,differential expression calculation was performed on the gene read count data,with the criteria for selection of significantly differentially expressed genes(DEGs)set as:FDR<0.05&|log2FC|≥ 1.When a gene satisfied both of these conditions,it was considered as a DEG.qRT-PCR was conducted to validate the reliability of the sequencing results.Using the Meiji platform,Gene Ontology(GO)database(http://www.geneontology.org/)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database(http://www.genome.jp/kegg/)were used for functional and pathway enrichment analysis of the DEGs.A gene set was established based on the functional analysis of the DEGs and uploaded to the STRING database for protein-protein interaction(PPI)analysis.Cytoscape 3.7.2 software was used for visualization and construction of the PPI network,and core genes were selected.3.Exploration of the function and mechanism of ODAM based on transcriptome sequencing analysis in vitro(1)Verification the functional of ODAM in vitroBased on the transcriptome sequencing analysis mentioned above,it was found that ODAM knockout affects cell adhesion,proliferation,and migration.Therefore,plasmid transfection was performed to overexpress ODAM in ameloblast cells.Cell adhesion ability of ODAM knockout and overexpressing cells was assessed using a cell adhesion assay at 4 hours,12 hours,and 24 hours.Cell proliferation ability of ODAM knockout and overexpressing cells was determined using CCK-8(Cell Counting Kit-8)and CFSE(Carboxyfluorescein succinimidyl ester)flow cytometry assays,while cell apoptosis was assessed using a cell apoptosis detection kit.The migration ability of ODAM knockout and overexpressing cells was evaluated using a Transwell assay,and changes in cell cytoskeleton were observed by rhodamine-phalloidin staining.(2)Preliminary exploration of the mechanism of ODAM affecting cell adhesionIn order to investigate the adhesive function mechanism of ODAM,the integrin α6β4(including ITGA6 and ITGB4)and the γ2 subunit(LAMC2)of laminin-α3β3γ2 were explored using qRT-PCR,Western blotting,immunohistochemistry,and Coimmunoprecipitation methods.(3)Preliminary exploration of the mechanism of ODAM affecting cell proliferation and migrationThe second part of the transcriptome sequencing results suggests that Odam gene knockout affects the regulation of cell proliferation and migration through the Src-MEK-ERK pathway.WB was used to detect the activation and inhibition of this pathway(p-Src,p-MEK,p-ERK)after ODAM knockout and overexpression.The Src kinase inhibitor PP2(10μmol/mL)was used to inhibit the activation of Src expression in ODAM knockout.After the addition of PP2 inhibitor for 24 hours,cell proliferation ability was assessed using CCK-8 and CFSE assays,while cell migration ability was determined using Transwell assay.Cell skeleton staining was performed using phalloidin after cell crawling assay.WB detection of p-MEK and p-ERK phosphorylation levels after Src inhibition.4.Expression of ODAM in experimental periodontitis mice and rescue experiment(1)Expression of ODAM in experimental periodontitis mice:Thirty adult male SPF C57 mice weighing 25-30 g were divided into three groups:normal group(n=10),7-day periodontitis group(n=10),and 28-day periodontitis group(n=10).For the periodontitis groups,1μL of Porphyromonas gingivalis lipopolysaccharide(LPS)solution(2μg/mL)was injected into the second maxillary molars on both sides every 2 days.After 7 days,the mice from the 7-day periodontitis group were euthanized with excess pentobarbital sodium,and the maxillary bones were collected.A portion of the bones was fixed with 4%paraformaldehyde for tissue section preparation,while another portion of the maxillary bones was dissected under a stereomicroscope to collect the periodontal soft tissues around the second molars of the mice,which were then stored in liquid nitrogen.The normal group and the 28day periodontitis group mice underwent the same procedures after 28 days of LPS injection.QRT-PCR and WB were performed to evaluate the mRNA and protein expression of ODAM in the three groups of mice.Immunohistochemical staining for CK-19 and ODAM was performed,and the expression and distribution of ODAM in the normal group,7-day periodontitis group,and 28-day periodontitis group were observed under a light microscope.(2)Rescue Experiment of ODAM Recombinant Protein in Experimental Periodontitis MiceThirty C57 mice were divided into three groups:normal group(n=10),periodontitis group(n=10),and ODAM rescue group(n=10).For the periodontitis and ODAM rescue groups,local injection of LPS was performed on the second maxillary molars on both sides to establish the periodontitis model.ODAM recombinant protein(5 μg/mL)was injected into the buccal and lingual sides of the second maxillary molars every 2 days in the ODAM rescue group.After 28 days,samples were collected from the three groups of mice.One side was fixed with 4%paraformaldehyde for tissue section preparation,and the other side was placed in an electron microscopy scanning solution.HE staining was performed to observe the inflammatory status of the periodontium in the three groups of mice.Electron microscopy scanning was performed to observe the attachment of the junctional epithelium.Immunohistochemical staining for ITGB4 was performed to evaluate its expression and localization in the three groups of mice.Immunohistochemical staining for p-Src,p-MEK,and p-ERK was performed to evaluate their expression and localization in the three groups of mice.Results1.The expression of ODAM in human periodontal tissueIn the histological samples of normal patients,full-layer expression of ODAM was observed in the junctional epithelium with consistent intensity,similar to the characteristic staining of CK-19.However,in gingivitis,the expression of ODAM was no longer limited to the junctional epithelium,but spilled over into the subepithelial tissue.Nevertheless,there were no significant differences in ODAM mRNA and protein expression levels at this stage(P>0.05).In periodontitis,the expression of ODAM was decreased,particularly in the basal region of the junctional epithelium,showing a significant decrease compared to the normal group both at the mRNA and protein levels(P<0.05).2.Transcriptome sequencing analysis of function of OdamAmeloblasts are columnar-shaped cells with polygonal outlines and exhibit dendritic processes that connect with each other,resembling a cobblestone pattern.Cells can express amelogenin and tuftelin,enamel proteins,when cultured in both SMEM limit medium and DMEM medium.However,only when cultured in DMEM medium,cells can be induced to express enamelin,an enamel matrix protein.The induction of enamelin expression allows the identification of these cells as ameloblasts and confirms their normal functionality.Therefore,we believe that these cells are ameloblasts and possess normal functionality,making them suitable for subsequent experiments.Through transcriptome sequencing of Odam gene knockout cells,we discovered a total of 2801 differentially expressed genes,including 1336 upregulated genes and 1465 downregulated genes.In the functional enrichment analysis of Gene Ontology(GO),we found that 2928 GO terms were significantly enriched.Among the top 20 enriched GO terms,they are related to cell proliferation,cell adhesion,cell-matrix adhesion,cell migration,cell apoptosis,and other cellular processes.324 enriched pathways were identified in the analysis of KEGG,,and the top 20 enriched pathways include the mitogen-activated protein kinase(MAPK)signaling,extracellular matrix receptor interaction,Wnt signaling,microRNAs in cancer,and cancer pathways.Two protein interaction networks were generated,with the main network containing Src,integrin,and laminin genes,and the subnetwork mainly containing laminin and integrin genes.QRT-PCR verified that the sequencing results were consistent with the expression trend of the selected genes,thus suggesting that the sequencing results were highly reliable.3.Exploration of the function and mechanism of ODAM based on transcriptome sequencing analysis in vitro(1)Verification the functional of ODAM in vitroAfter knocking out the Odam gene,the cell adhesion ability decreased by half within 4 hours compared to the control group.The cell adhesion ability remained lower than the control group even after 12 hours,but no significant difference in adhesion ability was observed after 24 hours.In contrast,overexpression of the ODAM increased cell adhesion ability compared to the control group and the empty vector group at 4 hours,but no significant difference was observed between groups at 12 hours and 24 hours.After 72 hours of culturing,the ODAM knockout group showed accelerated cell proliferation compared to the control group,as detected by CCK-8 and CFSE flow cytometry.However,there were no significant differences in cell proliferation observed between groups after overexpression of the ODAM.Moreover,no obvious changes in cell apoptosis were found after ODAM knockout or overexpression.The results of the Transwell experiment showed that the cell migration ability increased in the ODAM knockout group compared to the control group.Within 24 hours,there was an increased number of cells migrating to the lower layer of the Transwell chamber compared to the control group.However,there were no significant changes in cell migration ability observed after ODAM overexpression.During staining of the cell cytoskeleton with phalloidin,it was observed that in the ODAM knockout group,the cells in the control group exhibited a polygonal or star-shaped morphology,with more cell protrusions and filopodia.The cell cytoplasm exhibited pronounced filamentous structures and a well-defined cytoskeleton.However,in the ODAM knockout cells,the cell filaments were observed to aggregate mainly at the cell periphery,resulting in a change in the polarity of the cytoskeleton and less distinct cell protrusions.No significant differences in cytoskeletal morphology were observed in the ODAM overexpression group.(2)Preliminary exploration of the mechanism of ODAM affecting cell adhesionThe results of immunohistochemical staining,qRT-PCR,and WB detection in ODAM knockout and overexpression cells revealed that ODAM was positively correlated with the expression of ITGB4,which is one of the components of hemidesmosomes,the main adhesion structure that binds epithelial cells to the tooth surface.However,there were no significant changes in the expression of the other two components,ITGA6 and LAMC2.(3)Preliminary exploration of the mechanism of ODAM affecting cell proliferation and migrationAfter ODAM knockout,Western blot analysis showed that P-Src was activated and phosphorylation of p-MEK and p-ERK increased,indicating activation of the Src-MEK-ERK pathway.On the other hand,ODAM overexpression led to an increase in ODAM expression and a decrease in the expression levels of p-Src,p-MEK,and p-ERK.This suggests that ODAM overexpression can inhibit the activation of the ERK pathway.After knockout of the ODAM in cells and addition of Src inhibitor,the phosphorylation of downstream MEK and ERK was affected.CCK-8 and CFSE flow cytometry analysis showed a significant decrease in cell proliferation,which further declined with increasing time.Transwell migration assay revealed a decrease in cell migration after the addition of Src inhibitor.Immunostaining of the cell cytoskeleton with phalloidin showed that in the control group,the cytoskeleton was densely arranged around the cell periphery,while the cytoskeletal microfilaments in cell synapses were not prominent.However,after the addition of the inhibitor,the cytoskeletal microfilaments were uniformly distributed in the cytoplasm and cell synapses.4.Expression of ODAM in experimental periodontitis mice and rescue experiment(1)Expression of ODAM in experimental periodontitis miceImmunohistochemical staining of CK-19 in the gingival epithelium and junctional epithelium of normal mice,7-day periodontitis group mice,and 28-day periodontitis group mice showed obvious staining in the entire layer of the junctional epithelium and the basal part of the gingival epithelium.The expression sites and distribution were consistent among the three groups.ODAM expression in the 7-day group was similar to that in the normal group of mice,but the mRNA expression level was lower than that in the normal group.Western blot analysis showed no significant decrease in protein levels.In the 28-day periodontitis group mice,ODAM expression in the junctional epithelium was absent near the basal part,with only slight mRNA and protein expression levels in the near coronal part,which were decreased compared to the normal group.(2)Rescue experiment of recombinant ODAM protein in experimental periodontitis miceHE staining of normal mice,periodontitis mice,and ODAM rescue group mice revealed that the junctional epithelium of the normal group mice was intact,with no obvious inflammatory cells in the periodontal tissue,and clear subepithelial periodontal fibers.However,the junctional epithelium of the periodontitis group mice was incomplete,with continuous interruptions,tissue ulceration,prominent inflammatory cells in the subepithelium,discontinuity of subepithelial periodontal fibers,increased vascular proliferation,and significant gingival recession.In the ODAM rescue group mice,the gingival epithelium was thicker than that in the normal group mice,with no obvious discontinuity,and slight inflammatory cells were observed in the subepithelium,but significantly less than in the periodontitis group.The subepithelial fibrous tissue was edematous.Electron microscopy scanning showed that the periodontal tissue of the normal group mice was in close contact with the tooth surface,with visible gingival crevices,while the periodontal tissue of the periodontitis group mice was significantly separated from the tooth surface,with obvious ulceration.In contrast,the periodontal tissue of the ODAM protein rescue group mice showed less noticeable separation between the junctional epithelium and the tooth surface.After supplementation of recombinant ODAM protein,the expression of ITGB4 in the junctional epithelium was similar to the control group,but decreased in the periodontitis group.Compared to the normal group,the expression levels of p-Src,p-MEK,and p-ERK were increased in the periodontitis group.However,after supplementation with recombinant ODAM protein,there were no significant changes in the expression levels of p-Src,p-MEK,and p-ERK compared to the control group.Therefore,supplementation with recombinant ODAM protein did not fully activate the Src-MEK-ERK pathway.Conclusion1.ODAM can serve as a specific marker protein for normal junctional epithelium,with uniform expression throughout the entire layer of normal junctional epithelium.However,in the presence of gingivitis,ODAM expression is no longer limited to the junctional epithelium,but the expression level remains unchanged.As the junctional epithelium recedes below the cementoenamel junction,i.e.,after the formation of periodontitis,ODAM expression decreases in periodontal tissues and is significantly reduced in the junctional epithelium.2.By performing transcriptome sequencing on Odam knockout cells,we found a total of 2801 differentially expressed genes,including 1336 up-regulated genes and 1465 downregulated genes.Through functional and pathway enrichment analysis of these differentially expressed genes in GO and KEGG databases,it is speculated that ODAM may play a role in cell adhesion,proliferation,and migration,which may be related to core genes such as Src,integrins,and laminins in the differentially expressed genes.These factors may affect cell function through the integrin pathway,extracellular matrix-related pathways,and MAPK pathway.3.ODAM may interact with one of the components of the hemidesmosome,ITGB4,which is responsible for attaching the epithelium to the tooth surface.The expression of ODAM is positively correlated with cell adhesion ability,and it may influence early cell adhesion.However,after ODAM knockout,the cell proliferation and migration abilities are affected,and significant rearrangements occur in the cell cytoskeleton.This process may be caused by the downregulation of ODAM,leading to the activation of Src kinase and subsequent activation of the MEK-ERK pathway,resulting in functional changes.When ODAM is overexpressed,it can inhibit the activation of the Src-MEK-ERK pathway,but this does not affect cell proliferation and migration abilities compared to the control group.4.The expression of ODAM is downregulated in experimental periodontitis mice,similar to the expression pattern of ODAM in human periodontitis.Supplementing exogenous ODAM recombinant protein can improve the adhesion of the junctional epithelium to the tooth surface during periodontitis and has a certain inhibitory effect on the occurrence and development of experimental periodontitis.At the same time,supplementation of exogenous ODAM can increase the expression of ITGB4 and inhibit the activation of the Src-MEK-ERK pathway. |
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