Role And Mechanism Of Molybdenum-Containing Bioactive Glass Ceramics In Enhancing Periodontal Tissue Regeneration

BackgroudPeriodontitis is an infection-driven inflammatory disease characterized by the progressive destruction of the tooth-supporting tissues within the periodontium and untreated periodontitis is the leading cause of tooth loss in adults worldwide.Periodontal tissue regeneration faces two major challenges.First,periodontitis is an inflammation-driven disease characterized by irreversible bone loss due to the imbalance between osteogenesis and osteoclastogenesis.Positively regulating activity of bone-forming osteoblasts plus a negative feedback regulation of osteoclastic activity are keys in any regenerative strategies that aim to achieving a successful regenerative outcome.Most of current biomaterials mainly focus on how to improve the osteogenesis,while the role of osteoclasteogenesis in biomaterial design is largely ignored.This may be the reason why the present treatments cannot completely repair the damaged periodontal bone.Sencond,although the pathogenic progression of periodontitis remains elusive,macrophages have been found to be central players in the destructive and reparative phases of periodontitis.Macrophages exhibit a full spectrum of polarization phenotypes and polarize toward different functional phenotypes in response to a variety of biophysical/biochemical signals.Polarized macrophages are typically classified into M1 macrophages,which release proinflammatory factors to initiate and maintain inflammation,and M2 macrophages,which secrete anti-inflammatory mediators to modulate and control wound healing and regeneration.In the treatment of periodontitis,regulating phenotypic changes in macrophages is therefore central to restore the architecture and function of the damaged periodontium.The inclusion of bioactive molecules（e.g.,SDF-1α）or controlling the physiochemical properties of biomaterials is reliable strategies to enhance the osteogenic ability of osteoblasts,however,the bioactive effects of these modulatory factors is prone to be lost rapidly due to enzymatic degradation.In contrast to frequently used cytokines or drugs,bioactive ions released from transplants along with degradation are resistant to enzymolysis,which enables adequate concentrations of bioactive ions at the defect sites.More importantly,emerging evidence has indicated that the bioactive ions（e.g.,Sr,Mn and Zn ions）can exert multifunctional effects on tissue regeneration,which pave their ways for strategies co-modulating the cellular behavior.Previously,we found that the incorporation of molybdenum into 3D printed bioactive glass ceramics（BGC）can not only strength the BGC scaffolds,but also promote cartilage/bone regeneration.On the other hand,strategies targeting the mechanism of macrophage polarization may lead to advanced macrophage-regulating paradigms that enhance current periodontal regenerative outcomes.Molybdenum（Mo）,an essential trace element for nearly all organisms,has been identified as part of the active sites of more than fifty enzymes involved in various redox reactions and oxygen atom transfer.Although high doses of Mo may impair mitochondrial function,we previously found that the incorporation of Mo at a selected concentration into3-dimensional（3D）-printed bioactive glass ceramic（BGC）scaffolds effectively stimulated cell proliferation and differentiation in vitro.We want to certify that the Mo-BGC scaffold can promote the osteogenic differentiation potential of stem cells and inhibit the activity of osteoclasts.It also can change the phenotype of macrophages by regulating mitochondrial function for complex periodontal tissue regeneration.ObjectivesTo prepare BGC/Mo-BGC scaffolds;to clarify the osteogenesis,osteoclast and macrophage polarization regulation ability of BGC/Mo-BGC scaffolds in the extreme model of canine periodontal defect;to investigate the regulatory effect of BGC/Mo-BGC bioink extracts on the osteogenic differentiation potential and osteoclast activity of stem cells;to explore the regulatory effect of BGC/Mo-BGC bioink extracts on macrophage polarization type;to explore the effect of Mo ions on macrophage polarization by regulating mitochondria deep mechanisms of cell polarization types.Methods1.Preparation of BGC/Mo-BGC scaffolds and evaluation of periodontal tissue regeneration in response to BGC/Mo-BGC scaffolds transplantation:A 3D printed BGC/Mo-BGC scaffolds was prepared by the sol-gel method,and the scaffolds were characterized.The general morphology of the scaffolds was recorded by an ordinary optical camera,the microscopic morphology of the scaffolds surface was recorded by scanning electron microscopy,the ion distribution in the scaffolds was analyzed by energy dispersive spectrometer,the three-dimensional structure of the scaffolds was recorded by Micro-CT,and the degradation behavior of the scaffolds was separated by ICP-MS.Subsequently,mouse bone marrow-derived macrophages（BMDMs）were extracted and cultured,and the cells were seeded on the surface of the scaffolds to test the cytocompatibility of the scaffolds.The adhesion and morphology of cells on the surface of the scaffolds were observed by SEM,the cell viability was observed by live and dead cell staining,and cell apoptosis was detected by flow cytometry.Inspection and radiographs were used to detect material retention and wound healing in the defect area.Animals were sacrificed 1 week and 8 weeks after scaffolds implantation,respectively,and the formation of new bone in the periodontal defect area was analyzed by Micro-CT,methylene blue acid fuchsin staining and HE staining.After the scaffolds were implanted at 1 week,2weeks and 4 weeks,the canine periodontal defects were observed by alkaline phosphatase（ALP）staining and TRAP staining.After the scaffolds were implanted at 3 and 7 days,the dynamics of the polarization type state of macrophages in the canine periodontal defect area were observed by immunofluorescence staining.2.Evaluation of BGC/Mo-BGC on cellular osteogenic/osteoclastogenic activity:The BGC/Mo-BGC extracts with the dilution ratio of 1/4,1/32 or 1/128 was prepared as cell culture medium.The effect of BGC/Mo-BGC extracts on the osteogenic differentiation potential of mouse bone marrow mesenchymal stem cells（BMMSCs）were analyzed.The proliferation capacity of BMMSCs incubated in dilute solutions of BGC/Mo-BGC bioink extracts were analyzed using a Cell Counting Kit-8（CCK-8）assay Kit.The osteogenic differentiation of BMMSCs was analyzed with regard to the formation of calcified nodules（Alizarin Red S staining）and ALP activity.To assess the effects of Mo ions on osteogenic differentiation of BMMSCs,culture medium including Mo O₄^2-ion was prepared.Similarly,the osteogenic differentiation of BMMSCs following a 7-day induction was analyzed in terms of Alizarin Red S staining and ALP activity.In parallel,the osteogenesis-related proteins（Runx2 and BSP-1）and genes（Runx2,ALP and SP7）of BMMSCs were determined by Western blot and quantitative real-Time polymerase chain reaction.On the other hand,we investigated the effects of dilute solutions of BGC/Mo-BGC bioink extracts on osteoclastic differentiation of BMDMs.BMDMs were subjected to TRAP staining,immunofluorescent staining and q RT-PCR to observe the formation of F-actin ring,the specific protein markers（MMP9 and NFATc1）for osteoclastogenesis and osteoclastogenesis-related genes（MMP9,NFATc1 and RANKL）in BMDMs following a 7-day bioink extracts induction.Similar,the influence of the presence of Mo ions in culture medium on osteoclastic differentiation of BMDMs was investigated.The osteoclastic differentiation of BMDMs were analyzed in terms of TRAP staining,immunofluorescent staining and q RT-PCR.3.Evaluation of BGC/Mo-BGC on macrophage polarization:To prepare BGC/Mo-BGC extracts with a dilution ratio of 1/4,1/32 or 1/128 as cell culture medium,and observed the effects of BGC/Mo-BGC extracts on M0 macrophage polarization.M2polarization-related proteins（Arg/CD68 and CD206/CD68）and M1 polarization-related proteins（CCR7/CD68 and i NOS/CD68）were detected by immunofluorescence staining.Polarization-related genesexpression levels of M2（Arg,CD206 and IL）and M1（CCR7,IL-1βand TNF-α）were analyzed by q RT-PCR.Futher,M0 macrophages were induced to M1 macrophages,then observed the effect of Mo-BGC extracts on M1.The detection methods were as same as above.Finally,to prepare medium containing Mo ions to culture M0 macrophage and detect the M1 and M2 polarization-related genes and proteins.4.Mechanism underlying Mo-BGC-coaxed macrophage polarization:Mitochondrial morphology and function of macrophages following incubation with BGC/Mo-BGC powder extracts were detected.The mass of Mo,Si,Ca and P in the mitochondria of macrophages were determined by ICP-MS,the nanostructure of mitochondria was observed by transmission electron microscopy,the MMP value of macrophages was determined by JC-1,the ATP assay kit was used to determine the production of intracellular ATP in macrophages,and the levels of intracellular and mitochondrial ROS in macrophages were detected by flow cytometry.Then,mitochondrial metabolism of macrophages following incubation with BGC/Mo-BGC bioink extracts were detected.Mitochondrial metabolism was detected by OCR and ECAR methods,and the expression level of mitochondrial respiratory chain complexes in macrophages were determined by Western Blot.Further,macrophages were cultured with Mo O₄^2-ion-containing medium.The effects of Mo O₄^2-ions on mitochondrial metabolism of macrophages was detected by OCR and ECAR.Finally,by inhibiting the mitochondrial function of macrophages,the effects of BGC/Mo-BGC extracts on macrophages were observed.Immunofluorescence staining was used to observe the expression of M2 polarization-related proteins（Arg/CD68 and CD206/CD68）and M1 polarization-related proteins（CCR7/CD68 and i NOS/CD68）in macrophages.q RT-PCR was used to detect the expression of M2polarization related genes（Arg,CD206,and IL-10）and M1 polarization-related genes（CCR7,IL-1β,and TNF-α）in macrophages.Results1.Mo-BGC leads to robust periodontal tissue regenerationacross periodontal defects:BGC/Mo-BGC scaffolds were observed by optical camera,scanning electron microscope,energy dispersive spectrometer and Micro-CT.BGC/Mo-BGC scaffolds presented uniform properties and alveolar bone-like morphology,and Mo O₄^2-ions were uniformly doped in Mo-BGC scaffolds.Mo-BGC scaffolds released a significantly higher Mo O₄^2-ion concentration than BGC scaffolds.The weight loss of Mo-BGC scaffolds was significantly lower than that of BGC scaffolds.Subsequently,the cell morphology,cell viability and apoptosis rate of BMDMs on Mo-BGC scaffolds were same as BGC scaffolds.Finally,the canine periodontal defect model was successfully constructed.Eight weeks after BGC/Mo-BGC scaffolds transplantation,Micro CT,methylene blue acid fuchsin staining and H&E staining of the periodontal defect area showed that a relatively large amount of newly bone and new blood vessels can be observed around the root in Mo-BGC scaffolds.At 2 and 4 weeks post-surgery,the number of ALP-positive cells regulated by Mo-BGC scaffolds were significantly higher than that of BGC scaffolds.The quantitative analysis of TRAP staining showed a lager number of osteoclasts at the edge of the material-tissue interface in Mo-BGC scaffolds.Immunofluorescence showed more CD206+/CD68+cells（M2 macrophages）in Mo-BGC scaffolds at 7 days.2.Mo-BGC exerted an enhanced osteogenic plus an inhibited osteoclastogenic activity:Mo-BGC extracts with a dilution ratio of 1/4,1/32 or 1/128 were prepared as the cell culture medium to culture BMMSCs.CCK8 assays showed that Mo-BGC bioink extracts exhibited more significant proliferation-enhancing potential than BGC bioink extracts.BMMSCs incubated in each diluted solution of BGC/Mo-BGC bioink extracts exhibited robust potential to undergo osteogenic differentiation and to form Alizarin Red S-positive mineral nodules and ALP-positive cells.Further,analyzing the concentrations of Ca,P,Si and Mo ions in the Mo-BGC extracts with different dilution ratios,it was found that when the dilution ratio was less than 1/32,there was no significant difference in the concentration of Ca and P ions.Therefore,1/32 concentration of Mo-BGC extract was selected for the next study to exclude the biological effects of Ca and P ions on cells.The effects of Mo O₄^2-ions on the osteogenic differentiation potential of BMMSCs were analyzed by preparing a medium containing Mo O₄^2-ions.As demonstrated by Alizarin Red S staining and ALP staining,the presence of Mo ions in either complete medium or diluted solutions of BGC bioink extracts significantly increased the formation of calcium deposits and ALP positivity after a 7-day induction.The expression levels of osteogenesis-related proteins（Runx2 and BSP-1）and genes（SP7,Runx2,and ALP）were significantly increased in the presence of Mo ions in either complete medium or diluted solutions of BGC bioink extracts.On the other hand,the Mo-BGC extracts with a dilution ratio of 1/4,1/32 or 1/128 was prepared as cell culture medium,and the effects of the Mo-BGC extract on BMDMs activity was analyzed.TRAP staining showed that the number of osteoclasts in BGC/Mo-BGC extracts increased in a dose-dependent manner,and Mo-BGC extract with the same dilution ratio had a better effect on osteoclast formation than BGC extract.Immunofluorescence staining results showed that the Mo-BGC group had smaller F-actin rings and lower intensity of osteoclastogenesis-related proteins（MMP9 and NFATc1）,q RT-PCR assay showed that Mo-BGC extract significantly reduced the expression of osteoclastogenesis-related genes（MMP9,NFATc1and RANKL）.Further,mediums containing Mo ions were prepared to analyze the effect of Mo ions on the osteoclast differentiation of BMDMs.In all tests,culture medium or diluted solutions of BGC bioink extracts with Mo ions resulted in BMDM responses similar to those observed with Mo-BGC bioink extracts.3.Mo-BGC coaxes macrophage polarization toward an M2 phenotype:Mo-BGC extracts with a dilution ratio of 1/4,1/32 or 1/128 were prepared as the cell culture mediums,and the M0 macrophages were cultured.Immunofluorescence results showed that cells incubated in Mo-BGC extracts highly expressed M2 polarization markers（Arg and CD206）;q RT-PCR analysis showed that the expression levels of M2polarization-related genes（Arg and CD206）were significantly increased after incubation in Mo-BGC extracts.Again,prepare the Mo-BGC extracts with a dilution ratio of 1/4,1/32 or 1/128 as cell culture medium,induce M0 macrophages into M1 macrophages,and observe effects of Mo-BGC extracts played on M1 macrophage.Immunofluorescence staining revealed more M2 macrophages and fewer M1 macrophages in LPS-pretreated cells incubated in Mo-BGC extracts,q RT-PCR analysis showed that cells incubated in Mo-BGC extracts exhibited higher expression levels of M2 polarization-related genes（Arg,CD206,and IL-10）.Finally,M0 macrophages were cultured in a medium containing Mo ions.The effects of Mo ions on the polarization of M0 macrophages were observed.BGC bioink extracts with Mo ions also promoted the polarization of M0 macrophages to M2 macrophages consistent with Mo-BGC bioink extracts.4.Mo ions targeting immunometabolism and mitochondrial function for macrophage modulation:The macrophage was incubated in the Mo-BGC extracts to analyze the effect of the extracts on the mitochondria of macrophages.The mass of Mo in the mitochondria of macrophages increased significantly following incubation in Mo-BGC powder extract for 24 hours by ICP-MS.Mitochondrial swelling characterized by disappearance of mitochondrial cristae was frequently observed in cells incubated in BGC powder extracts.In immunofluorescent staining,obvious JC-1 aggregates were observed in the mitochondria of cells incubated in BGC/Mo-BGC extracts,while more monomeric JC-1was observed in the mitochondria of cells incubated in complete medium.The cells cultured in the Mo-BGC group had the highest ATP production.Flow cytometry showed that the Mo-BGC extracts significantly reduced the intracellular and mitochondrial ROS levels.Subsequently,macrophages were incubated in BGC/Mo-BGC extracts to analyze the effects of the extracts palyed on the mitochondrial metabolism of macrophages.OCR and ECAR results showed that the Mo-BGC extracts could enhance macrophage TCA metabolism and oxidative phosphorylation（associated with M2 polarization）,while reducing glycolytic metabolites（associated with M1 polarization）in macrophages.Western Blot assay showed that the expression levels of complex I subunit,complex II subunit and complex V subunit in the cells of the Mo-BGC group were significantly higher than those of the BGC group and the control group.Further,macrophages cultured with Mo ion-containing medium were detected by OCR and ECAR methods.The mitochondrial metabolism of BGC bioink extracts with Mo ions were consistent with the Mo-BGC extracts.Finally,the effects of Mo-BGC extracts on macrophage polarization were observed by inhibiting the mitochondrial function of macrophages.Immunofluorescence showed that inhibiting mitochondrial function of macrophages significantly reduced Arg+/CD68+and CD206+/CD68+cells（M2 macrophages）,while increasing the proportion of M1 polarization-related markers（i NOS and CCR7）.Further examination by q RT-PCR showed that all inhibitors significantly reduced M2polarization-related genes（Arg,CD206 and IL-10）and increased M1 polarization-related genes（CCR7 and IL-1β）.Conclusion1.In this study,a 3D printed BGC/Mo-BGC scaffolds with an alveolar bone-like morphology were successfully prepared.The scaffolds have good cytocompatibility and tissue regeneration regulation ability that promotes the formation of new bone and blood vessels in a canine periodontal defect model.Mo-BGC scaffolds has the ability to promote stem cell osteogenesis and inhibit osteoclastogenesis and also have the ability to promote the polarization of macrophages to M2 type in vivo.2.In vitro,Mo-BGC extracts have the ability to promote stem cell osteogenic differentiation potential and inhibit osteoclast activity.Mo ions palys an important role in regulating the osteogenic differentiation and osteoclast formation of BMMSC and BMDM by Mo-BGC extracts.3.In vitro,Mo-BGC extracts have the ability to promote the polarization of M0 and M1macrophages to M2 macrophages.Mo ions palys an important role in regulating macrophage polarization.4.Mo ions regulate the polarization of macrophages to M2 macrophages.Mo ions regulate the transition from cellular glycolytic metabolism to mitochondrial TCA and OXPHOS metabolism in macrophages.