| Diabetes mellitus(DM),as a chronic metabolic disease widely distributed worldwide,poses a major threat to human health.The repair of diabetic bone defects is faced with multiple adverse factors,including delayed activation of immune response,decreased vasodinnervation,and decreased osteogenic differentiation ability.These factors interact with each other,which together lead to the obstacle of bone defect repair in diabetes mellitus,and lead to the decrease of fracture healing speed and the increase of bone defect incidence in the whole skeletal system.In the oral and maxillofacial field,these challenges are manifested in hindered periodontal tissue regeneration,implant bone integration failure,and increased complexity in the repair of craniomaxillofacial bone defects.Despite the existence of a variety of bone repair biomaterials on the market,the application of these materials in patients with diabetes often fails to meet expectations,highlighting the urgent need to develop targeted bone repair materials and treatment options for patients with diabetes.Silicon(Si)is a key nutrient element in bone growth and development,and has been widely used in various bone defect repair materials.Silicon-based materials are capable of releasing bioactive silicic acid,which has a catalytic effect on a variety of cellular and biological processes.Under normal energy metabolic conditions,silicon-based materials have been shown to effectively regulate the process of bone defect repair,including promoting immune response activation and vascular nerve regeneration.However,there is still a lack of in-depth research and clear scientific evidence on the potential application of silicon-based materials in the repair of diabetic bone defects,as well as its specific impact and mechanism of action on the repair of diabetic bone defects.In this study,we systematically investigated the biological effects and mechanisms of silicon-based materials in the repair of diabetic bone defects.Firstly,the effects of silicon-based materials on immune activation,vaso-neural regeneration and bone deposition during bone defect repair in diabetic conditions were evaluated.Then,we explored the transfer of macrophage mitochondria to endothelial cells and nerve cells in diabetic conditions,and the role of silicic acid in the immune cascade of diabetic bone.In addition,the regulation mechanism of silicic acid on mitochondrial function homeostasis of macrophages in diabetic environment was studied.Finally,silicon-based materials were used to regulate macrophage mitochondrial dynamics to promote bone defect repair in diabetic conditions,and a combination treatment strategy for diabetic bone defect was constructed.1.Research ApproachIn the first part of this study,we explored the potential role of silicon-based materials for bone defect repair in diabetic conditions.Firstly,the levels of silicic acid in extracts of Collagen scaffold(CS)and Silicified collagen scaffold(SCS)were measured by silicomolybdic acid spectrophotometry.Atomic force microscope(AFM)was used to observe the microstructure and surface roughness characteristics of CS and SCS.Next,the effect of silicon-based materials on the repair of diabetic bone defects was evaluated by establishing critical size skull defects in a diabetic mouse model and implanting CS and SCS,respectively.Micro computed tomography(micro-CT)was used to analyze the restoration of the skull 1,2,4 and 8 weeks after operation.Hematoxylin and eosin(H&E)staining technology was used to observe the new bone tissue.Macrophage aggregation,vascular growth and innervation in new bone were evaluated by immunofluorescence staining.In addition,the primary Macrophage,Endothelial cell(ECs)and Dorsal root ganglion(DRG)cells were isolated and cultured.Untreated control conditions(Ctrl group)and simulated diabetes conditions(DM group)were set up.The effectiveness of the in vitro diabetes model was evaluated by measuring cell activity and intracellular reactive oxygen species levels.We compared the effects of blank conditions(DM group)and supplemental silicic acid(DM+Si group)on different cell types in a medium that mimics diabetic conditions.The Oxygen Consumption Rate(OCR)and Extracellular acidification rate of cells were measured by Seahorse XP96 assay.The oxidative phosphorylation and glycolysis of mitochondria were analyzed.Mitochondrial function was assessed by measuring the level of Adenosine triphosphate(ATP)and Mitochondrial membrane potential(MMP).The results of this part of the experiment showed that SCS can effectively promote the immune activation,vascular nerve regeneration and bone deposition during the repair of bone defect in diabetic mice.In addition,silicic acid significantly improved the energy metabolism and mitochondrial function of macrophages,but did not have significant effects on vascular endothelial cells or dorsal root ganglion cells.The above findings revealed that the biological effects of silicic acid on different cell types may be different in the condition of diabetes.However,whether silicic acid is involved in and regulates the interaction between macrophages and other cell types still needs further investigation.In the second part of this study,we explored how silicon influences the role of macrophages in the repair of diabetic bone defects.First,CS and SCS were implanted in a diabetic mouse model with macrophage depletion,and the effect of macrophage depletion on the early stage of angiogenesis was observed by immunofluorescence staining.Then,in a medium that mimics diabetic conditions,Culture Mouse mononuclear macrophages cell(RAW264.7)Endothelial progenitor cell,EPC)and peripheral nerve cells(Adrenal pheochromocytoma cell,PC-12).On this basis,we established an indirect co-culture system between macrophages and endothelial cells or macrophages and nerve cells.The experiment was divided into three groups:macrophage culture medium under simulated diabetic condition(DM group),macrophage culture medium supplemented with silicic acid under simulated diabetic condition(DM+Si group),and macrophage culture medium supplemented with silicic acid under simulated diabetic condition without extracellular vesicles(DM+Si+Fil group).Mitochondrial function of endothelial cells and nerve cells was evaluated by ATP and MMP levels.Endothelial and nerve cell differentiation was evaluated by angiogenesis and axon regeneration experiments.Further,extracellular vesicles in macrophage culture medium were isolated by gradient centrifugation and verified by particle size analysis,Transmission electron microscope(TEM)and Western Blot(WB).the expression of Translocase of the outer mitochondrial membrane-20(TOM20)and the activity of citrate synthase were detected by WB to analyze the mitochondrial content in extracellular vesicles.TEM imaging,ATP level,MMP level and oxidative phosphorylation complex enzyme activity were used to evaluate mitochondrial function in extracellular vesicles.In order to track the transfer of mitochondria between cells,mitochondrial specific fluorescent probe labeling technique was used in indirect co-culture model to label endogenous or exogenous mitochondria in endothelial cells or nerve cells respectively.Macrophages labeled with mitochondrial fluorescence probes were transplanted into healthy mice implanted with collagen scaffolds(Ctrl+CS group),diabetic mice implanted with collagen scaffolds(DM+CS group),and diabetic mice implanted with silicified collagen scaffolds(DM+SCS group),and the transfer of macrophage-derived mitochondria in the skull defect area was examined by laser confocal microscopy.The results of this part of the study show that the transfer of mitochondria from macrophages to vascular nerve cells is a common phenomenon in diabetic conditions,and the promotion of vascular nerve regeneration by silicon-based materials depends on the functional mitochondrial transfer of macrophages.However,the specific mechanisms of how silicon regulates mitochondrial transfer remain unclear and require further experimental studies to clarify.In the third part of this study,we explored the regulatory mechanism of silicic acid on mitochondrial function homeostasis in macrophages under diabetic conditions.Three different experimental conditions were set up to observe the motility and morphological changes of macrophage mitochondria:Control condition(Ctrl group),untreated simulated diabetes condition(DM group)and simulated diabetes condition supplemented with silicic acid(DM+Si group)were used to monitor the movement of mitochondria in cells in real time using live cell imaging system and immunofluorescence probe technology,and observe the position of mitochondria relative to the cell membrane.The morphology of intracellular mitochondria was observed and analyzed by immunofluorescence probe technique.The internal structure of macrophage mitochondria was observed by TEM.In addition,for macrophages in DM group and DM+Si group,real-time quantitative polymerase chain reaction(qPCR)and Western Blot were used to determine the changes in protein expression levels related to mitochondrial dynamics.The process of mitochondrial division of macrophages was recorded by live cell imaging system and immunofluorescence probe technology,and the division of central region(division point within 50%of the center)and peripheral region(division point less than 25%from the end)were quantitatively analyzed.In order to clarify the cascade effect caused by macrophage mitochondrial division,three different groups of indirect cell co-culture systems were constructed:Untreated macrophage conditioned medium in simulated diabetic condition(DM group),macrophage conditioned medium in simulated diabetic condition treated with mitochondrial division inhibitor Mdivi-1(DM+Mdivi-1 group),macrophage conditioned medium in simulated diabetic condition treated with mitochondrial division inhibitor Mdivi-1 and silicic acid(DM+Mdivi-1+Si group);The transfer of macrophage mitochondria to endothelial cells and nerve cells was detected by flow cytometry.Mitochondrial function of endothelial cells and nerve cells was evaluated by measuring ATP and MMP levels.Further,macrophages pretreated with the mitochondrial division inhibitor Mdivi-1 were transfused in a diabetic mouse model,and the effect of this treatment on vascular and nerve regeneration in mice was evaluated by immunofluorescence staining.The results of this part of the experiment revealed that the mitochondrial transfer process of macrophages in diabetic conditions depends on the mechanism of intracellular mitochondrial division mediated by Dynamin-related protein 1(DRP1).Silicate by promoting the expression of mitochondrial fission factor(Mitochondriafissionfactor MFF)promoting mitochondria physiological division,which increases the transfer function of mitochondria,and further promote the vascular remodeling of nerve cells.In the fourth part of this study,we discussed the application of silicon-based materials combined with therapeutic strategies in the repair of diabetic bone defects.In order to fully understand the effect of combination therapy on the fine biological function of macrophages,two experimental groups were set up:supplementation of silicic acid under simulated diabetic conditions(Si group),and supplementation of silicic acid and mitochondrial hyperdivision inhibitor P110 under simulated diabetic conditions(Si+P110 group).The oxidative stress level of macrophages was evaluated by detecting Superoxide Dismutase(SOD)activity,Catalase(CAT)activity and mitochondrial Reactive oxygen species(ROS)production.Furthermore,mitochondrial specific fluorescence probe technique was used to detect mitochondrial transfer level in indirect co-culture model.Mitochondrial function of endothelial cells and nerve cells was evaluated by measuring ATP and MMP levels.In addition,in the cranial defect model of diabetic mice,silicified collagen scaffolds were treated(SCS group),P110 injection therapy(P110 group)and silicified collagen scaffolds combined with P110 therapy(SCS+P110 group),respectively.The neurovascular regeneration in the bone defect area was analyzed by immunofluorescence staining.New bone formation was analyzed by Micro-CT to determine the effect of this combined treatment strategy in promoting the repair of diabetic bone defects.The results of this part of the experiment showed that the combined treatment of silicon-based materials and P110 could significantly enhance the mitochondrial activity of macrophages,and enhance the mitochondrial function of vascular nerve cells through mitochondrial transfer of macrophages.In the cranial defect model of diabetic mice,the combination of silicon-based materials and P110 effectively promoted the regeneration of blood vessels and nerves,and the process of bone deposition.2.Experimental ResultsPart 1:The Biological Effects of Silicon-Based Material on Diabetic Bone Microenvironment1.The results of silicomolybdic acid spectrophotometry showed that SCS could release silicic acid stably and continuously.AFM surface topography analysis showed that CS and SCS showed similar rhythmic microstructure and surface roughness of collagen.2.Micro-CT test results showed that from the 4th week after surgery,the bone volume fraction in the SCS group was significantly improved compared with the CS group.At 8 weeks after surgery,H&E staining showed that the SCS group had a larger area of bone-like tissue than the CS group.3.The results of immunofluorescence staining showed that the distribution area of CD31-positive blood vessels and TUbb3-positive nerves in the SCS group reached a peak at week 2 and remained until week 8.Blood vessel and innervation levels increased significantly at all observation time points and peaked earlier in the SCS group compared to the CS group.4.Immunofluorescence staining results showed that F4/80 positive macrophages in the SCS group reached their peak value in the first week,but F4/80 positive macrophages in the CS group reached their peak value in the second week.Subsequently,the expression of macrophages in both CS group and SCS group gradually decreased over time.5.The results of Seahorse XP96 experiment showed that under simulated diabetic conditions,only macrophages showed a significant increase in mitochondrial oxidative phosphorylation after silicic acid supplementation,while endothelial cells and nerve cells showed no significant changes.Under simulated diabetes conditions,the glycolysis capacity of the three types of cells did not change significantly,regardless of whether the silicic acid was supplemented.6.The detection of intracellular ATP and MMP levels showed that silicic acid significantly improved the mitochondrial function of macrophages under simulated diabetic conditions,but had no effect on the mitochondrial function of endothelial cells and dorsal root ganglion cells.Part 2:The Role of Silicic Acid in Regulating the Immunobiological Cascade of Diabetic Bone through Mitochondrial Transfer1.After consuming diabetic mouse macrophages with clophosphonate-containing liposomes,immunofluorescence staining results showed no significant difference in local neurovascularization between the SCS group and the CS group.2.Intracellular ATP and MMP levels showed that mitochondrial activity of endothelial cells and nerve cells in DM+Si group was significantly increased compared with that in DM group.However,the mitochondrial activity of DM+Si+Fil group was not significantly increased.3.Tubule formation experiment and axon elongation experiment showed that the differentiation ability of endothelial cells and nerve cells in DM+Si group was significantly improved compared with that in DM group.However,there was no significant improvement in cell differentiation in DM+Si+Fil group.4.Particle size analysis and TEM imaging results showed that there were extracellular vesicles in conditioned medium of DM group and DM+Si group.WB and citrate synthetase activity showed that mitochondria-related proteins were present in extracellular vesicles.5.The detection of ATP level,MMP level and respiratory chain complex activity showed that the extracellular mitochondrial activity in DM+Si group was higher than that in DM group.TEM images showed that compared with DM group,the extracellular mitochondria in DM+Si group showed more clear transverse ridge structure.6.Confocal microscopy showed that intercellular mitochondrial transfer was observed in both DM group and DM+Si group,but the degree of transfer was not significantly different.No donor mitochondria were observed in DM+Si+Fil group,which ruled out the influence of fluorescence probe spillover.7.Confocal microscopy showed that macrophage mitochondria transfer to vascular nerve cells was observed in both DM+CS group and DM+SCS group,and there was no significant difference in the transfer rate.No mitochondrial transfer was observed in the Ctrl group.Part 3:The Regulatory Mechanism of Silicic Acid on Mitochondrial Functional Homeostasis in Macrophages under Diabetic Condition1.Real-time cell imaging results showed that the mitochondria in macrophages of DM group and DM+Si group moved randomly compared with Ctrl group.In the DM group and the DM+Si group,the mitochondria were observed to be wrapped in cell membrane,similar to the vesicle release process.2.Confocal microscopy showed that the length of mitochondria in macrophages in DM and DM+Si groups was significantly shorter than that in Ctrl group.TEM images showed that mitochondria in macrophages maintained normal ridge structure in Ctrl group and DM+Si group,while mitochondrial ridge in DM group showed deformation and swelling.3.qPCR results showed that there were no significant differences in gene expression related to mitochondrial biogenesis and mitochondrial fusion between DM and DM+Si groups.The results of qPCR and WB experiments showed that only the expression level of MFF protein associated with mitochondrial division was significantly increased in DM+Si group.4.Real-time cell imaging results showed that mitochondria in DM group divided more in the peripheral region,while those in DM+Si group divided more frequently in the central region.5.Flow cytometry analysis showed that compared with DM group,DM+Mdivi-1 group significantly inhibited macrophages’ ability to transfer mitochondria,while DM+Mdivi-1+Si group could not restore mitochondrial transfer ability.6.The detection of intracellular ATP and MMP levels showed that,compared with the DM group,neither DM+Mdivi-1 group nor DM+Mdivi-1+Si group significantly improved the mitochondrial function of vascular nerve cells.7.Immunofluorescence staining results showed that in diabetic mouse models implanted with silicified collagen scaffolds,both mice without macrophage transplantation and mice implanted with Mdivi-1 pretreated macrophages showed reduced vascular and nerve regeneration ability compared with macrophage transplantation.Part 4:The Application of Silicon-Based Material Combined Treatment Strategy in Diabetic Bone Defect Repair1.The detection of SOD activity,CAT activity and mitochondrial ROS levels showed that the oxidative stress of macrophages was significantly reduced in Si+P110 group compared with Si group.2.Under the indirect co-culture condition,flow cytometry analysis showed that the mitochondrial transfer rate of macrophages in Si+P110 group was significantly increased compared with Si group.3.Under indirect co-culture conditions,ATP and MMP levels showed that mitochondrial activity of endothelial cells and nerve cells in Si+P110 group was improved compared with Si group.4.Immunofluorescence staining results showed that in the cranial defect experiment of diabetic mice,the expressions of CD31-positive blood vessels and TUbb3-positive nerves in SCS+P110 group were significantly higher than those in SCS+P110 group and P110 group.5.Micro-CT test results showed that in the cranial defect experiment of diabetic mice,the bone defect repair level of SCS+P110 group was significantly improved,and the effect was significantly better than that of SCS and P110 group.3.ConclusionSilicon-based materials can significantly accelerate the bone healing process in diabetic mice,mainly by promoting immune activation,vascular and nerve regeneration and bone deposition.Under simulated diabetic conditions,silicic acid significantly improved the energy metabolism and mitochondrial function of macrophages,but had no significant effects on endothelial cells and nerve cells.Silicic acid indirectly regulates the mitochondrial activity and differentiation ability of endothelial cells and nerve cells by promoting the transfer of functional mitochondria of macrophages,which is closely related to the process of mitochondrial division in macrophages.Specifically,in the diabetic environment,mitochondrial transfer of macrophages is mainly regulated by the mitochondrial division protein DRP1.Silicic acid effectively promotes the physiological division of mitochondria by enhancing the expression of DRP1 receptor MFF,thereby increasing the proportion of functional mitochondria in intercellular transfer.P110 can inhibit the interaction between DRP1 and its receptor FIS1.When P110 is combined with silicon-based materials,it can significantly inhibit the pathological division of mitochondria,further improve the transport efficiency of functional mitochondria,and thus enhance the energy metabolism and differentiation potential of endothelial cells and nerve cells.In a diabetic mouse skull defect model,silicon-based materials combined with P110 effectively promoted vascular and nerve regeneration,as well as bone formation,demonstrating its great potential in the repair of diabetic bone defects.In summary,the research results of this subject are of great significance for understanding and treating diabetic bone defects.On the one hand,the study reveals the mechanism of mitochondrial transfer of metabolic disorders in diabetic conditions,which has important theoretical implications for understanding how diabetes affects intercellular communication and tissue regeneration.On the other hand,the study proposed a new strategy for the combined treatment of silicon-based materials,which provides a new practical approach for the treatment of bone defects in diabetic patients. |
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