Effect Of Metformin Carbon Nanodots On Differentiation Of Dental Pulp Stem Cells Into Odontoblasts

Background:The classic treatment of endodontic and periapical diseases is root canal treatment,but it is difficult to solve the endodontic and periapical diseases of young permanent teeth with immature apex which is treated by pulpotomy and pulp revascularization clinically.In terms of mature permanent teeth,the problems such as tooth fracture and discoloration after root canal therapy are difficult to solve.More and more scholars focus on the ideal solution: dental pulp regeneration.However,human dental stem cells(hDPCs)are seed cells in dental pulp regeneration and have multi-lineage differentiation potential.Therefore,they may differentiate into unfavorable cell types during dental pulp regeneration.Nanomaterials can mimic the different microenvironment of stem cell differentiation by changing the material stiffness and surface micromorphology,and thus regulate the directed differentiation of stem cells.Recently,to explore new materials to regulate the differentiation of dental pulp stem cells into odontoblasts is research highlights.Carbon nanodots as a new kind of nanomaterials with the abilities such as biological compatibility,low cost,easy synthesis and so on,have attracted more and more scholars' attention since its appearance.Therefore,we have developed metformin carbon nanodots(MCD)to study the effects of MCD on hDPCs migration and odontogenic differentiation,providing scientific theoretical basis for the application of MCD in dental pulp regenerationMethods:1.Synthesis of MCD: Metformin carbon nanodots were synthesized by hydrothermal method using metformin and citric acid as raw materials.2.Characterization of MCD: The size and morphology of carbon dots were observed by transmission electron microscope(TEM),and the infrared spectrum(FT-IR)of the sample was obtained by Fourier transform,and the fluorescence spectrum of the sample was obtained by fluorescence spectrometer scanning.3.Biocompatibility test of MCD: hDPCs were co-cultured with MCD solution of gradient concentration(0,50,100,200,400,800 ?g/mL).Cell compatibility of MCD was detected by MTT assay and flow cytometry.4.Odontogenic differentiation of hDPCs after treatment of MCD: hDPCs and gradient concentration carbon dots(0,50,100,200 ?g/mL)medium were cultured.The mRNA expression of odontogenic genes was detected by qRT-PCR in 7 and 14 days and the expression of odontogenic proteins was detected by Western Bolt in 14 days.5.Promotion of the differentiation of hDPCs to odontoblast after treatment of MCD via autophagy: hDPCs after treatment of MCD(50 ?g/mL)were tested for autophagy through WB and transmission electron microscope at 0,6,12,24,48 and 72 h.After pretreatment with 3-MA,autophagy inhibitor,hDPCs and MCD(50 ?g/mL)medium were cultured for 3 days to detect the expression of autophagy protein and odontogenic mRNA.6.Promotion of the migration of hDPCs after treatment of MCD: hDPCs were inoculated into the upper chamber of Trans-well,and gradient concentration carbon nanodots(0,25,50,100 ?g/mL)medium solution were put into the lower chamber for co-culture for 48 h.The migration quantity of cells was determined by crystal violet staining and DAPI staining.Results:1.TEM results show that MCD with 5.9nm are successfully synthesized.FT-IR results show that there are hydroxyl,amino and carbonyl groups on carbon nanodots.The MCD is blue fluorescence.In the emission spectrum,the best excitation wavelength is 367 nm,and the best emission wavelength is 444 nm.2.MTT results show that MCD cells have no effect on cell viability,when the carbon concentration is below 200 ?g/mL.Flow cytometry shows that when MCD concentration is less than 200 ?g/mL,MCD do not cause apoptosis.3.The expression of odontogenic mRNA such as DSPP and DMP1 and so on in 7 and 14 days increases.The expression of odontogenic protein such as DSPP and DMP1 in 14 days also increases.4.MCD can promote the expression of autophagy-related proteins p62,LC3,Beclin1 inducing autophagy.Transmission electron microscopy is used to detect autophagosomes in MCD-treated dental pulp stem cells.At the same time,after inhibiting autophagy,the expression of odontogenic mRNA and autophagy proteins significantly reduce.5.MCD can promote hDPCs migration,and the migration effect is strongest when the concentration of MCD is 50 ?g/mL.Conclusion:1.MCD,with an average particle size of 5.9 nm,are spherical carbon nanoparticles with excitation wavelength dependence.2.MCD has good biocompatibility.3.MCD can promote differentiation of hDPCs into odontoblasts via autophagy.4.MCD can promote migration of hDPCs.