| Polydopamine-templated hydroxyapatite(tHA)is a type of nanobiomaterial which biomimetic synthesized by using dopamine as template.Its crystal morphology and chemical composition are close to the microstructure of natural bone tissue,and it has an effect of promoting osteogenic differentiation of mesenchymal cells and repairing regeneration of bone tissue defects.The application of tHA as a scaffold material in periodontal bone tissue engineering may have great research value.However,High concentration of tHA causes the increase of reactive oxygen species(ROS),which leads to oxidative stress damage and apoptosis,inhibits cell activity and osteogenisis.Therefore,to improve the biocompatibility of tHA,prevent oxidative stress damage caused by ROS is an urgent problem to be solved.Metformin is currently the first-line drug treatment for type 2 diabetes,and it has been demonstrated to resist oxidative damage and preventing cell senescence and apoptosis,which may be related to the induction of autophagy by metformin-activated AMP-activated protein kinase(AMPK)signaling pathway.Autophagy is an important protective mechanism for cells to resist injury,inflammation and aging.It is reported that autophagy removes excess ROS production and organelles damaged by ROS,reduces cell damage and maintains normal physiological functions of cells.Furthermore,studies have reported that metformin has the potential to promote osteogenic differentiation,and has the role of assisted control of periodontitis.The present study was performed to investigate the potential application of tHA in combination with metformin in periodontal bone tissue engineering.In vitro and in vivo experiments were conducted to investigate whether metformin can promote autophagy to improve activity and osteogenic differentiation of human periodontal Ligament Stem Cells(hPDLSCs)on tHA materials.Firstly,tHA materials were synthesized and characterized.In vitro: hPDLSCs were cultured with tHA in combination with metformin.Cell viability and material biocompatibility were assessed by ROS generation,lactate dehydrogenase(LDH)leakage,apoptosis,CCK-8 proliferative assay,and cytoskeletal staining.Osteogenesis effect was detected via alkaline phosphatase(ALP)assay,calcium nodule mineralization,and gene expression of osteocalcin(OCN),osteopontin(OPN),and Runt-related transcription factor 2(RUNX2).Additionally,autophagy marker proteins Beclin-1 and LC3 and phosphorylation of the AMPK/mTOR signaling pathway were analyzed by using western-blot to clarify the mechanism of tHA combined with metformin in hPDLSCs.It was found that tHA in combination with metformin substantially reduced ROS generation and apoptosis,enhanced proliferation of hPDLSCs and cytoskeletal extension.tHA combined with metformin significantly increased ALP activity,mineralized nodule formation,and mRNA expression of OCN,OPN,and RUNX2.Enhanced protein levels of LC3Ⅱ and Beclin-1 were observed.The expression of phosphorylated AMPK was increased and phosphorylated mammalian target of rapamycin(mTOR)proteins was decreased after exposure to the combination.In vivo: The model of periodontal defect in SD rats was established.The combination of tHA and metformin were implanted in the bone defect.The sample was collected and reconstructed by Micro CT after 10 weeks.Calcein and alizarin double fluorochrome labeling of mineralization in different groups.The results showed that tHA combined with metformin significantly increased the formation of new bone,and improved the speed of new bone mineralization in periodontal bone tissue defects.Taken together,the results demonstrated that tHA combined with metformin increased the viability of hPDLSCs via the AMPK/mTOR signaling pathway by regulating autophagy and further improving the osteogenic effect.In addition,tHA combined with metformin had significant regeneration effects on rat periodontal bone defects.Therefore,tHA combined with metformin has great potential in the field of periodontal bone tissue regeneration. |
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