Biological Effects Of Prolyl Hydroxylase Inhibitor DMOG On Periodontal Tissue Regeneration

Background and Objective:Chronic periodontitis,one of the most common bacterial oral diseases,is characterized by gingival inflammation,attachment loss,and alveolar bone resorption,and eventually leads to mobility,displacement and loss of teeth.The ultimate aim of periodontal therapy is the acquirement of periodontal tissue regeneration after eradicating local stimulating factors and controlling inflammation.In recent years,periodontal tissue engineering has obtained remarkable efficacies,and local application of growth factors is an effective method to promote periodontal tissue regeneration.However,these macromolecular proteins have short effective half-life,poor protein stability,high dose requirement and high cost,which limit their clinical application.In addition to superior biological effects,bioactive small molecule compounds have the advantages of good stability,low dose requirement and low cost.Therefore,studies based on small molecular compounds are of great significance for periodontal tissue regeneration.DMOG(dimethyloxalylglycine),a cell-permeable small molecule,is the competitive inhibitor of prolyl hydroxylases(PHDs).DMOG possesses various biological effects,and can play a protective role in many inflammatory diseases,such as inflammatory bowel disease(IBD).As a pharmacological inhibitor of PHDs,DMOG indirectly activates the hypoxia-inducible factor 1(HIF-1),and simulates the hypoxia environment,which promotes the formation of new blood vessels and ameliorates ischemia-reperfusion injury.The control of local inflammation and the realization of tissue regeneration are the key links in the treatment of periodontitis.However,the roles of DMOG in periodontal tissue regeneration have not been clarified.Therefore,this study was aimed to explore the regulatory effect of DMOG on lipopolysaccharide(LPS)-induced inflammatory model of human gingival fibroblasts(HGFs)in vitro and the potential mechanism,exploring the effect of DMOG on Fusobacterium nucleatum(F.nueleatum)-stimulated expression of inflammatory cytokines to further clarify the inflammatory regulation of DMOG,preparing DMOG-loaded electrospinning fibrous membrane and invetigating its immunoinflammatory regulation in the early stage of wound repair in vivo,preparing electrostatic spinning fiber membranes loaded with DMOG and bioactive two-dimensional nanomaterials nanosilicate(nSi)to improve the regenerative potential of DMOG,and studying its roles in in vitro osteogenic and angiogenic differentiation,in vivo stem cell recruitment,neovascularization,bone tissue remodeling and functional periodontal tissue regeneration,so as to establish a new treatment strategy for periodontitis.Materials and Methods:1.The regulatory effects of DMOG on LPS-induced inflammation in vitro and the underlying mechanismsFirstly,cell viability assay was used to screen out the concentrations of DMOG without cytotoxicity for subsequent experiments.Secondly,HGFs were pretreated with different concentrations of DMOG(10,50,100 μmol/L)for 24 h,and were respectively stimulated by LPS at 5 μg/mL for 2,6,12,24 h to establish in vitro inflammatory models.Quantitative real-time polymerase chain reaction(qRT-PCR)and enzyme linked immunosorbent assay(ELISA)were used to evaluate the expression of inflammatory cytokines at gene and protein levels.Western blot and immunofluorescence assay were used to detect the expression of relevant signal pathways.The different phenotypes of PHDs were knocked down by the small interference RNA(siRNA)technique,and then the expression of LPS-induced inflammatory cytokines was detected by qRT-PCR to determine the PHD phenotypic specificity for the regulatory effects of DMOG.2.The regulatory effects of DMOG on F.nucleatum-induced inflammation in vitroPolymerase chain reaction(PCR)was used to identify F.nucleatum ATCC 25586(from Shandong Key Laboratory of Oral Tissue Regeneration).F.nucleatum was used to infect HGFs with different multiplicities of infection(MOI=10,50,100)for 12 h,and the effect of bacterial infection on cell morphology and number was observed by crystal violet staining experiment.Then,HGFs were pretreated with different concentrations of DMOG(10,50,100 μmol/L)for 24 h,and F.nucleatum,with MOI of 100,was used to infect HGFs for 2 h,6 h,and 12 h,respectively,to establish an inflammation model in vitro.qRT-PCR and ELISA were used to detect the expression of inflammatory factors at gene and protein levels.3.Immune-inflammation regulatory roles of DMOG-loaded electrospinning fibers in the early stage of periodontal injury in vivoThe DMOG-loaded fibrous membrane was prepared by electrostatic spinning technique.PLGA(poly(lactic acid-co-glycolic acid))(LA:GA=75:25,MW=90,000)was dissolved in an organic solvent hexafluoroisopropanol(HFIP)to obtain the polymer solution with the concentration of 20%(weight/volume,w/v).Then,DMOG(1%w/w of PLGA)was dispersed in PLGA solution to prepare homogeneous solutions.The PLGA fibrous membrane(PLGA)and DMOG-loaded PLGA fibrous membrane(DMOG-PLGA)were further prepared by electrostatic spinning technology.Scanning electron microscope(SEM)and fourier transform infrared spectroscopy(FTIR)were used to characterize the prepared materials,and cytoskeleton staining and CCK-8(cell counting kit-8)assay were performed to assess the biocompatibility of the fibrous membranes.Subsequently,a mandibular periodontal defect model was created in Wistar rats,and the fibrous membrane materials were implanted into the periodontal defects.Hematoxylin and eosin(H&E)staining and immunohistochemical staining were used to evaluate its regulatory roles in the early inflammatory response after injury.4.Effect of DMOG/nSi-loaded electrospinning fibrous membranes on periodontal tissue regenerationThe composite fibrous membranes loaded with DMOG(1%w/w)and nSi(5%w/w)were prepared by electrospinning technique.The materials were characterized by SEM,FTIR and tensile experiment.In vitro releasing of DMOG and Si from the composite fibrous membranes was detected.Human periodontal ligament stem cells(PDLSCs)were seeded on the surface of the membranes,and cell adhesion and growth were evaluated by SEM.CCK-8 assay was performed to evaluate the cytotoxic effect of material leach liquor.Then,cells were inoculated on different fibrous membranes,and osteogenic differentiation potential of the fibrous membranes in vitro was evaluated by alkaline phosphatase activity(ALP)assay and qRT-PCR.Tubule formation assay and qRT-PCR were used to evaluate the angiogenic ability of the fibrous membrane in vitro.The membranes were implanted into the mandibular periodontal defect model of Wistar rats,and the recruitment of CD90+CD34-stem cells in vivo was detected by immunofluorescence double staining.The expression of angiogenic related markers was detected by immunohistochemical staining.H&E staining,tartrate-resistant acid phosphatase(TRAP)staining,immunohistochemical staining and micro-CT were used to evaluate the effect of fibrous membrane on bone remodeling.The angle of new periodontal ligament fibers was evaluated by H&E staining.Results:1.DMOG downregulated the expression of LPS-induced inflammatory cytokines through TLR4/MyD88-mediated Akt/NF-KB and MAPK signaling pathwaysHGFs were pre-treated with various concentrations of DMOG(10,50,100,250,500,1000 μmol/L)for 24 h and 48 h,respectively.CCK-8 assay showed that high concentrations of DMOG(250,500,1000 μmol/L)obviously suppressed cell proliferation,whereas low concentrations of 10,50,100 μmol/L had no significant cytotoxic effect.Therefore,10,50,100 μmol/L of DMOG were used for the subsequent experiments.Flow cytometry(FCM)also indicated that DMOG at 10,50,and 100 μmol/L did not lead to cell apoptosis.qRT-PCR and ELISA showed that DMOG inhibited the expression of LPS-induced inflammatory cytokines interleukin(IL)-6 and IL-8 at the gene and protein levels,and down-regulated the expression of inflammatory cytokines tumor necrosis factor(TNF)-α,IL-1β,toll-like receptor 4(TLR-4)on cell surface and downstream junction protein myeloiddifferentiationfactor-88(MyD88)at the gene level.Western blot and immunofluorescence staining confirmed that DMOG inhibited LPS-induced activation of Akt/NF-κB and MAPK signaling pathways.In addition,LPS-induced inflammatory cytokine expression was downregulated in PHD2 knockdown cells after PHD1 and PHD2 knockdown by siRNA technique.2.DMOG downregulated the expression of inflammatory cytokines in HGFs stimulated with F.nucleatumAgarose gel electrophoresis imaging showed that the DNA bands of the PCR amplification products of F.nucleatum were between 100 and 250 bp,and the fragment size was about 146 bp.The results of the crystal violet staining experiment showed that the morphology and number of HGFs did not change significantly with the infection of F.nucleatum(MOI=10,50,100)for 12 h.Therefore,F.nucleatum with MOI of 100 was selected for subsequent experiments.qRT-PCR results showed that DMOG inhibited the expression of inflammatory cytokines IL-6,IL-8,TNF-αand IL-1β stimulated with F.nucleatum at the gene level.Nevertheless,at the protein level,F.nucleatum did not stimulate the secretion of IL-1β,and DMOG inhibited the secretion of IL-6 and IL-8 at the early stage(2 h and 6 h),but had no significant effect on the expression of them at 12 h.3.DMOG-loaded electrospinning fibers ameliorated immuno-inflammatory responses in the early stage of periodontal injurySEM results indicated that the electrospinning membranes presented staggered fibrous structures,and the fibers were smooth without fracture,and evenly distributed in diameter.FTIR spectra showed that the functional groups on the surface of the fibers did not change significantly after DMOG incorporation.Cytoskeleton staining showed that PDLSCs adhered and grew normally on the surface of the material.CCK-8 assay demomstrated that fibrous membranes had no cytotoxic effect on cell proliferation,indicating that the material had good biocompatibility.At the early stage(1 and 2 w),H&E staining results showed that the inflammatory cell infiltration in the defect area was significantly reduced after implantation of the membrane,and bone volume was increased.Immunohistochemistry and immunofluorescence staining showed that the number of iNOS,CD11b and CD40L positive cells were significantly decreased,and CD206 positive cells were increased.4.DMOG and nSi-loaded osteogenic/angiogenic difunctional fibrous membranes promoted periodontal regenerationSEM results showed that the prepared fibrous membranes of each group had similar fibrous structure,well-distributed fiber diameter and smooth fibers.After DMOG and nSi incorporation,the surface functional groups of the materials did not change.The tensile test showed that the mechanical properties of the fiber membrane were enhanced after the incorporation of nSi.Moreover,the composite fibrous membrane released DMOG and Si slowly and continuously for about 30 days.SEM results showed that cells adhered to the material surface and grew after being inoculated on the material surface.CCK-8 assay proved that the material leach liquor had no obvious cytotoxic effect on cell proliferation.After induction,the osteogenic differentiation potential of cells inoculated on the surface of the composite fibrous membrane was significantly promoted,which was characterized by enhanced ALP activity and up-regulated expression of osteogenic markers.At the same time,the composite fibrous membrane significantly promoted the tubule formation and the expression of angiogenic markers in vitro.CD90+CD34-stem cell recruitment was significantly promoted after implantation of the material into periodontal bone defects.H&E staining,TRAP staining,immunohistochemical staining and micro-CT results showed that the fibrous membrane significantly faciliated bone remodeling,increased the expression of angiogenic related markers,and obtained the functional periodontal ligament regeneration.Conclusions:1.DMOG downregulated LPS-induced inflammation in vitro,and inhibited inflammatory cytokine expression via TLR4/MyD88-mediated Akt/NF-κB and MAPK pathways,which was dependent on PHD-2.2.The expression of inflammatory cytokines was significantly increased in HGFs infected with F.nucleatum,and DMOG could significantly inhibit the expression of inflammatory cytokines stimulated by F.nucleatum at the gene level.3.DMOG-loaded PLGA electrospinning fibrous membrane alleviated the early inflammatory response in periodontal defect,and regulated the expression of immune-inflammation markers.4.DMOG and nSi-loaded fibrous membranes enhanced osteogenic/angiogenic differentiation in vitro,and promoted stem cell recruitment,bone remodeling,neovascularization,and functional periodontal tissue regeneration in vivo.