Metformin Carbon Dots For Promoting Periodontal Bone Regeneration Via Activation Of The ERK/AMPK Pathway

Periodontitis is one of the most common oral diseases.The clinical manifestations of periodontitis are bleeding gums,periodontal pocket formation,alveolar bone resorption,and even tooth loss.Periodontitis not only harms the oral function,but is also closely related to systemic diseases,such as cardiovascular disease,diabetes,lung disease,rheumatoid arthritis,cancer,and Alzheimer's disease.The ultimate goal of periodontitis treatment is to restore and rebuild the periodontal support tissue,and the regeneration of alveolar bone is crucial to the realization of the treatment goal.However,traditional treatment methods,such as ultrasonic scaling,subgingival scaling,root planning and flap surgery,can eliminate the stimulation of local pathogenic microorganisms and control the progression of the disease,but the regeneration of the absorbed alveolar bone is limited.In recent years,although regenerative surgery,development of biological agents,and stem cell tissue engineering technology have brought new opportunities for regeneration of the periodontal tissue,there are still limitations and deficiencies.As a new treatment concept,endogenous periodontal tissue regeneration is receiving extensive attention from researchers.Its core idea is to make full use of the body's inherent regenerative ability to induce the recruitment and homing of its own stem cells for realizing the regeneration of periodontal tissues.This treatment strategy avoids not only the high cost of biological agents and stem cell culturation,but also the complexity involved in surgery and tissue engineering technology,making regenerative treatment simple,safe and easy to accept,providing a broad development space for clinical applications.Therefore,how to make endogenous stem cells fully exert their regenerative potential is particularly important for endogenous periodontal tissue regeneration strategies.Bone marrow mesenchymal stem cells(BMSCs)are one of the main sources of stem cells for periodontal bone regeneration.Not only the BMSCs of alveolar bone have strong osteogenic ability,but the BMSCs in bone marrow can also migrate to periodontal defect site to repair the alveolar bone.However,continuous inflammation will seriously damage the osteogenic potential of BMSCs,which greatly reduces the osteogenic differentiation ability of BMSCs in the periodontitis microenvironment.Therefore,it is necessary to explore an effective strategy to rescue the osteogenic differentiation potential of BMSCs under inflammation.In recent years,nanomaterials have been widely used in the field of bone regeneration due to their small size,easily access to cells,abundant surface groups,and easy modification.As a new type of fluorescent nanomaterials,carbon dots have several distinct advantages,such as excellent optical properties,good biocompatibility,facile preparation,low cost,and extensive sources of raw materials.They have extensively contributed to various biomedical applications.More notably,carbon dots have shown great potential in the field of bone regeneration.For example,the citrate-based carbon dots,bioactive CDs from adenosine and aspirin can promote the osteogenic differentiation of BMSCs.Zinc gluconate carbon dots and ascorbic acid-polyethyleneimine carbon dots can promote the repair of bone defects.However,whether carbon dots can be used for bone regeneration under inflammatory conditions has not yet been reported.If carbon dots can be used to rescue the osteogenic differentiation potential of BMSCs under inflammatory conditions and promote periodontal bone regeneration,it will be significant for the treatment of periodontitis.In the second chapter,metformin carbon dots(MCDs)were synthesized from metformin hydrochloride and citric acid through the hydrothermal method.After purification,the morphology and structure of MCDs were characterized by transmission electron microscopy(TEM)and high-resolution TEM(HRTEM).It is found that MCDs are approximately spherical in shape and the average diameter is3.76 nm with a clear lattice structure.The lattice spacing is 0.22 nm.The X-ray powder diffraction(XRD)pattern of MCDs displays a broad peak at 26°,corresponding to the(002)crystal facet of graphite,which further supports the crystal facet observed in HRTEM.Fourier transform infrared spectroscopy confirms the existence of hydroxyl,amine and carboxyl groups in MCDs.MCDs manifest an excitation dependent feature according to fluorescence spectra.The UV-vis spectrum of MCDs indicates absorption peaks at 209 nm and 333 nm.The main fluorescent color of MCDs is blue under UV light.We also investigated the biological properties of MCDs.We tested the biocompatibility of MCDs with CCK-8 and flow cytometry.The results show that MCDs have no significant effect on the viability,cell cycle and apoptosis of rat bone marrow mesenchymal stem cells(r BMSCs).The MCDs can be rapidly cleared from the body and mainly have two metabolic pathways: kidney and liver system.After co-cultivating MCDs and r BMSCs cells,the fluorescence of MCDs in r BMSCs was observed using confocal laser scanning microscopy(CLSM).The blue fluorescence in r BMSCs indicates that MCDs can enter into cells.In this part,we synthesized MCDs and characterized the chemical and biological properties.It is proved that MCDs have good nano-sized structure,excellent optical properties and biocompatibility.Moreover,MCDs possess abundant hydroxyl,amine and carboxyl groups and can enter into cells to play a role.In the third chapter,we investigated the osteogenic effects promoted by MCDs under inflammatory and normal conditions.First,alkaline phosphatase(ALP)staining was performed to determine the effective concentration of MCDs on the osteogenic differentiation of r BMSCs under inflammatory and normal conditions.The results indicate that MCDs enhance the activity of ALP in r BMSCs in a dose-dependent manner under both inflammatory and normal conditions.In order to further compare the effects of MCDs and metformin on the osteogenic differentiation of r BMSCs,the real-time PCR was performed to detect the expression of early osteogenic differentiation genes(Runx2,Sp7,ALP and COL-1).It is found that MCDs can promote the gene expression of Runx2,Sp7,ALP and COL-1 in r BMSCs under both inflammatory and normal conditions.The effects are better than metformin.We also detected the expression of early osteogenic proteins(COL-1 and DMP1)by Western blot.It is found that MCDs promote the expression of COL-1 and DMP1 proteins of r BMSCs under both inflammatory and normal conditions,and the effects are better than metformin.In addition,the activity of ALP was detected by ALP staining.The results indicate that MCDs can enhance the activity of ALP in r BMSCs under inflammatory and normal conditions.The effect is also better than that of metformin.We further detected the formation of calcium nodules by ARS staining and evaluated the ability of r BMSCs osteogenic differentiation in later stage.The results show that MCDs can promote the formation of calcium nodules in r BMSCs under inflammatory and normal conditions,and the effect is better than metformin.Therefore,in this part,It is found that MCDs can promote the osteogenic differentiation of r BMSCs both under inflammatory and normal conditions,and the effects are better than metformin.In the fourth chapter,we explored the mechanism of MCDs promoting osteogenic differentiation of r BMSCs under both inflammatory and normal conditions.We detected the effect of MCDs on the expression of ERK and AMPK signaling pathway-related proteins in r BMSCs by Western blot.The results indicate that MCDs can activate ERK and AMPK signaling pathways in both inflammatory and normal conditions.In order to clarify the relationship between ERK and AMPK signaling pathways in the process of osteogenic differentiation induced by MCDs,the inhibitors were selected for related experiments.First,the AMPK inhibitor Compound C was used to block the AMPK signaling pathway in r BMSCs,and then the effect on the ERK signaling pathway was detected.The results show that the ERK signaling pathway is activated after the AMPK signaling pathway inhibited,suggesting that ERK signaling is not downstream of AMPK signaling pathway in the process of osteogenic differentiation induced by MCDs.Next,U0126(an inhibitor of ERK signaling)was used to block the ERK signaling pathway in r BMSCs,and then detected its effect on the AMPK signaling pathway.The results indicate that AMPK signal is inhibited when the ERK pathway is blocked in r BMSCs both in inflammatory and normal conditions.It is suggested that AMPK signaling is the downstream target of ERK signaling pathway during the process of osteogenic differentiation induced by MCDs.Therefore,MCDs can activate the ERK/AMPK signaling pathway in r BMSCs under both inflammatory and normal conditions.Moreover,the protein level of COL-1 and DMP1 is also decreased after pretreatment with U0126,indicating that the ability of MCDs-induced osteogenesis is weakened because the ERK/AMPK pathway is blocked both in inflammatory and normal conditions.In order to further verify the role of ERK/AMPK signaling pathway in MCDs-induced osteogenesis,U0126 was adopted to block the ERK/AMPK pathway,and then the ALP and ARS staining were evaluated.The results show that the ALP activity and calcium nodule formation of r BMSCs induced by MCDs are inhibited after the ERK/AMPK signaling pathway is blocked under both inflammatory and normal conditions.These findings support that the ERK/AMPK signaling pathway is involved in MCDs-induced osteogenesis of r BMSCs both inflammatory and normal conditions.Overall,in this part,it is proved that MCDs promote osteogenesis via activation of ERK/AMPK pathway both in inflammatory and normal conditions.In the fifth chapter,we evaluated the effects of MCDs on periodontal bone regeneration in vivo.The periodontitis model of Wistar rats was established by silk ligation,and given local injection of PBS,MCDs or metformin.After 4 weeks,all the rats were euthanized and maxillary samples were collected,and then the samples were scanned by micro-CT.The distance from cemento-enamel junction(CEJ)to the apex of alveolar bone crest(ABC)was measured as the index of alveolar bone loss.The relevant bone and bone trabecular parameters were also measured between the maxillary first and second molars.It is found that the alveolar bone resorption is severe in the periodontitis group.Moreover,the bone mass and bone mineralization density are also decreased.To our delight,the height of alveolar bone,bone mass,and bone mineralization density are almost restored to the normal level in the MCDs group.However,there is no significant difference between the metformin and periodontitis groups.In order to further evaluate the quality of periodontal bone regeneration,HE staining was performed.The results indicate that the apex of the ABC is smooth and there is no bone absorption in the control group.Nevertheless,in the periodontitis group,the alveolar bone has been absorbed close to the root apices.Most importantly,in the MCDs group,the absorbed alveolar bone is repaired obviously,suggesting new bone formation.The alveolar bone resorption is still obvious in the metformin group.Based on these results,it is reasonable to believe that MCDs can promote periodontal bone regeneration in vivo.In addition,the biosafety of MCDs in vivo was evaluated by HE staining based on vital organs,including hearts,livers,spleens,and kidneys.The results show that there is no significant difference in the cell morphology among control and MCDs groups.Therefore,this part of the experiment shows that MCDs are safe for applications in vivo and have great potentials in periodontal bone regeneration.In summary,we prepared MCDs using metformin hydrochloride and citric acid through a facile hydrothermal strategy.The MCDs exhibit excellent biocompatibility and have abundant groups,such as hydroxyl,amine and carboxyl groups.It is proved that the MCDs can promote the osteogenic differentiation of r BMSCs by activating the ERK/AMPK signaling pathway under both inflammatory and normal conditions.Importantly,MCDs can more effectively enhance the periodontal bone regeneration in rats with periodontitis than the raw material metformin in vivo.