Low Level Diode Laser Therapy In Pulpotomy In Primary Teeth-Clinical Application Evaluation And Underlying Mechanism

BackgroundPulpotomy can preserve the radicular pulp to retain the tooth physiological function and the natural exfoliation of primary dentition.High-level lasers could result tissue thermal damage while low level laser irradiation(LLLI)ould produce bio-stimulation effect on target tissue,making it an appropriate candidate for clinical treatment.Although many researches have suggested that LLLI can be used for primary tooth pulpotomy,the most appropriate laser parameters and underlying mechanism still unclear.Hence,this study aims at exploring the security and efficiency of LLLI-assisted pulpotomy based on cell experiment and clinical trial to seek the most appropriate laser parameters and further investigate the pathway probably involved,laying a theoretical foundation on further clinical application.Materials and Methods1、Isolation and culture of stem cells from exfoliated deciduous teeth(SHED)by the method of enzymatic digestion,followed by the detection of stem cell surface markers by flow cytometry.The CCK-8 assay was performed to the viability of SHED.The experiment about the influence of LLLI on mineralization-related gene and protein expression was conducted as well.2、We explored the expression of BMP and Wnt pathways-related protein and further inhibit these pathways with the application of specific inhibitors(noggin and DKK1).Then,we tested the expression of BMP and Wnt pathways-related protein and mineralization-related protein,and mineralization staining.3、Total of 40 primary teeth with deep caries lesions which are close to pulp and reversible pulpitis were selected for the study and the pulpotomy teeth were randomly assigned into one of two groups.The treated teeth were assessed clinically and radiographically at 12-month follow-up.Results1、SHED has the the potential for trilineage differentiation.The result of CCK-8 assay showed that LLLI promoted the viability of SHED.According to the results of qRT-PCR and western blot,gene expression of mineralization markers ALP,RUNX2,BMP2,DMP1 and DSPP had a significant increase at 5 J/cm2 group and the protein expression markers BMP2、RUNX2 and DMP1 increased as well.We also conducted immunofluorescence(IF)staining analysis to confirm the expression of the RUNX2 protein with consistent with the result of western blot.From the results of ARS staining and ALP staining,LLLI could promote the odontogenic differentiation of SHED.2、The results of western blot indicated that LLLI caused the significant upregulation of p-Smad 1/5/9,so as well as β-catenin.Consistent with these findings,IF staining analysis confirmed β-catenin accumulation and increased expression of p-Smad 1/5/9.noggin and DKK1 were supplied to block BMP and Wnt pathways.The results showed that the expression of odontogenic makers(RUNX2,BMP2 and DMP1)and pathway-related protein(p-Smad 1/5/9 and βcatenin),indicating using noggin and DKK1 alone or together could significantly reduce the osteo/odontogenic differentiation:noggin plus DKK1>noggin>DKK1.From the results of ARS staining and ALP staining,the formation of mineralized nodules in the LLLI-induced group were obviously inhibited after inhibitor treatment.3、Total of 40 teeth were treated and 3 teeth lost follow-up.The clinical success rate for laser pulpotomy was 100%(at 12 months)and for iRoot-BP pulpotomy was 100%.There was no significant difference between these two groups.The radiographic success rate for laser was 94.4%(12 months)and 89.5%(at 12 months)for the control group.Again,no significant difference was found in radiographic success rate between these two groups.Conclusion1、The result showed that LLLI improved the odontogenic differentiation of SHED in vitro.The effect was most profound when the laser parament was 5 J/cm2.2、LLLI could activate BMP and Wnt signaling pathways.In addition,both signaling pathways and promoted odontogenesis were also blocked at the same time.However,this inhibition could be slightly reversed by the induction of LLLI.These findings demonstrated that LLLI plays a dominate role in the osteo/odontogenic of SHEDs through the cross-talk between the BMP and Wnt signaling pathways.3、Selected appropriate laser parament(5J/cm2)in the former study was used in laser-assisted pulpotomy.There is no significant difference between was found in clinical and radiographic success rate between these two groups.However,Larger sample sizes and long time to follow-up are needed for further investigation.