Multifunctional Barrier Membranes Promote Periodontal Regeneration By Scavenging H₂O₂,Regulating Immune Response,and Generating O₂

Backround and ObjectiveGuided tissue regeneration(GTR)is a crucial strategy for periodontal regeneration and the barrier membrane is the key to success.Our team has fabricated a hydroxyapatite(HAp)/polylactic acid(PLA)barrier membrane(HP)for GTR,which has achieved efficient bone repairing by the barrier function at PLA side and osteogenesis promotion at HAp side.However,the efficiency of HP was limited as it lacked the regulation of other factors during bone regeneration,ROS,and immunity,for instance.The level of ROS,mainly hydrogen peroxide(H2O2),increases significantly during the process of tissue repairing.Specifically,excessive H2O2 not only increases intracellular oxidative stress and induces apoptosis,but also stimulates chronic inflammation and Ml polarization of macrophages.Therefore,eliminating excessive H2O2 is an important strategy to improve tissue regeneration and ROS-scavenging enzymes have attracted vast attention.Among them,manganese dioxide(MnO2)was admired as it can decompose H2O2 into O2 directly and degraded manganese ions can encourage osteogenesis.In this study,a novel multifunctional barrier membrane(HP@Mn)was prepared by loading our previous barrier membrane(HP)with MnO2.Subsequently,the effect of HP@Mn on periodontal regeneration was explored.Materials and Methods1.Preparation and characterization of materials.MnO2 nanosheets(NSs)were prepared by wet chemical method and HAp nanowires(NWs)were prepared by hydrothermal method.HAp,HAp@0.5%Mn,HAp@1%Mn,and HAp@2%Mn membranes were prepared by extraction and filtration method after mixing MnO2 NSs and HAp NWs.Subsequently,HP,HP@0.5%Mn,HP@1%Mn,and HP@2%Mn composite membranes were fabricated by the pervaporation of PLA solution.Transmission electron microscope(TEM)and scanning electron microscope(SEM)were applied to evaluate the morphologies and lattices.The ability to scavenge H2O2 of MnO2 NSs was evaluated by detecting the concentration of dissolved O2 in solution and 3,3’,5,5’-tetramethylbenzidine(TMB)test Energy dispersive X-ray spectroscopy(EDS)was adopted to observe elements.The hydrophilicity and mechanical property of HP@Mn membranes were assessed by water contact angle(WCA)test and universal testing machine.2.The evaluation on biocompatibility of HP@Mn membranes.The hemolysis rates of HP@Mn membranes were detected by the hemolysis test.Human periodontal ligament stem cells(PDLSCs)were cultured on tissue culture plate(TCP),HP,HP@0.5%Mn,HP@1%Mn,and HP@2%Mn membranes.LIVE/DEAD viability and CCK-8 were adopted to test the cell viability and proliferation.SEM and phalloidin staining were used to observe the morphologies.3.The evaluation of ROS scavenging,immunoregulation and osteogenesis promotion of HP@Mn membranes in vitro.(1)Assessment of oxidative stress elimination ability.α-MEM with 400 μM of H2O2 was treated with HP,HP@0.5%Mn,HP@1%Mn,and HP@2%Mn membranes for different time points respectively before stimulating PDLSCs.Cells treated with a-MEM without H2O2 were denoted as the control(Ctrl)group and fresh α-MEM with 400 μM of H2O2 was set as a positive control(PC)group.CCK-8 and LIVE/DEAD viability were used to test the cell viability and proliferation.ROS intracellular fluorescence and flow cytometry were to evaluate the level of intracellular ROS.(2)Evaluation of immunomodulation.The treatment procedure for PDLSCs was the same as the above mentioned.Enzyme-linked immunosorbent assay(ELISA)and quantitative realtime PCR(qRT-PCR)and were used to test the expression of inflammation-related factors.RAW 264.7 was adopted to observe the effect of composite membranes on the polarization of macrophages.The treatment procedure was the same as the above mentioned and the detection methods were qRT-PCR and immunofluorescence staining.(3)Assessment of osteogenic differentiation.PDLSCs were cultured on TCP,HP,and HP@2%Mn membranes.Alkaline phosphatase(ALP)staining,qRT-PCR ALP activity,and immunofluorescence(IF)were applied to detect the potential of osteogenesis.4.Evaluation of HP@2%Mn membrane on promoting periodontal tissue regeneration and regulating immunity in vivo.The rats were operated with a defect with a size of 4 × 4 × 1 mm.HP and HP@2%Mn groups were implanted with corresponding membranes,and the Ctrl group healed naturally.The condition of new bone formation was tested by H&E staining and Micro-CT.The periodontal regeneration was evaluated by H&E staining and the subsequent measurement of the angle of the periodontal ligament(PDL).Immunohistochemistry(IHC)was applied to assess the expression of myeloperoxidase(MPO)and immunofluorescence(IF)was utilized to evaluate the M1 polarization.Finally,the main visceral organs were excised for H&E staining to test the visceral toxicity.Results1.Homogeneous and pure MnO2 NSs and HAp NWs were prepared.MnO2 NSs could decompose H2O2 and produce O2 simultaneously.MnO2 NSs were successfully loaded on the HP@Mn membranes.The HAp side of HP,HP@0.5%Mn,HP@1%Mn,and HP@2%Mn membranes were highly hydrophilic,which indicated that the loading of MnO2 NSs did not affect this characteristic.Furthermore,the HP@2%Mn membrane possessed enough toughness to provide mechanical support during bone regeneration process.2.The HP@Mn membranes possessed good cytocompatibility and did not cause hemolysis of red blood cells.3.The in vitro results demonstrated that the level of intracellular ROS increased under the stimulation of H2O2,the reason for cell apoptosis,while the treatment of HP@2%Mn membrane relieved this oxidative damage,thereby maintaining cell activity.Besides,excessive H2O2 induced inflammation and promoted macrophage M1 polarization,while HP@2%Mn inhibited these tendencies.Moreover,HP@2%Mn membrane promoted osteogenic differentiation of PDLSCs both in the early and mineralization stages of osteogenesis.4.The in vivo results showed that there was more new bone in the HP@2%Mn group when compared with Ctrl and HP groups,and the newly formed bone in HP@2%Mn group was denser than other groups.Moreover,HP@2%Mn promoted cementum and PDL regeneration and guided PDL to attach to the root surface.HP@2%Mn also suppressed the recruitment and activation of neutrophils and inhibited M1 polarization.Finally,the HP@2%Mn membrane did not cause noticeable histological changes in organs.ConclusionIn summary,highly biocompatible HP@Mn membranes could remove the excessive H2O2 during bone repair and generate O2 meanwhile,protecting the cells from apoptosis caused by oxidative stress,eliminating inflammation,and regulating the immune microenvironment.In parallel,MnO2 NSs and nanostructured HAp synergistically promoted osteogenesis.Furthermore,HP@Mn membranes could efficiently promote functional periodontal tissue regeneration by guiding the alignment of the PDL and the formation of new cementum.Therefore,HP@Mn membranes with the ability of H2O2 scavenging possess a promising potential in GTR,as well as in other regenerative strategies.