Effect Of Silver Diammine Fluoride On Micro-ecology Of Plaque From Extensive Caries Of Primary Teeth-a Preliminary In Vitro Study

ObjectiveSilver diammine fluoride（SDF）garnered great attention at home and abroad because its super ability in arresting and preventing caries.Previous studies on the working mechanism of SDF in the control of dental caries focused mainly on a few specific cariogenic bacteria,whereas according to the concept of ’ecological plaque hypothesis’,caries development is due to the loss of micro-ecology balance within the dental plaque.New evidence suggested that SDF had no specific selectivity in inhibiting bacteria.The effect of SDF on micro-ecology of dental plaque should be further investigated.In this study,metagenomic gene sequencing and analysis techniques were used to investigated micro-ecological changes in dental plaque on extensive caries of deciduous teeth after topical SDF treatment.MethodsSchool children aged 6-12 years were targeted in clinic,and the presence of at least one deciduous tooth with extensive caries（caries involved more than half of the teeth surface,with or without significant pulpitis）in the mouth,which needs to be extracted.The above decayed deciduous teeth were included in this study.Initial plaque sampling（plaque from carious cavities and plaque from the buccal of the other caries-free teeth in the mouth）and unstimulated saliva collection were done before tooth extraction.The above extracted decayed deciduous teeth were collected,38%SDF was topically treated onto dental surface within the extensive caries in vitro after tooth extraction.After treatment,these decayed deciduous teeth were stored respectively in a centrifuge tube containing a certain amount of artificial saliva and placed in a 3 7℃ conventional thermostatic incubator.Repeated plaque collections were done at 24 hours and 1 week post-intervention and saved respectively.Pyrosequencing on metagenomic DNA of each plaque or saliva sample collected during the study was conducted by using Illumina MiSeq high-throughput sequencing platform.According to the sequencing results,the micro-ecological characteristics of the samples before and after 38%SDF intervention,such as microbial diversity,microbial composition,inter-genera correlation and gene function were analyzed.ResultsIn this study,a total of 16 school-age children（aged 7-10 years,average age 8.06 years,56%boys）were recruited.Among these children,one had 3 qualified teeth in his mouth,two had 2 qualified teeth and each of the other children had 1 tooth in their mouths that met the inclusion criteria,therefore,a total of 20 decayed deciduous teeth were eligible for inclusion.At baseline,a total of 20 plaque samples from carious cavities（PLC）,16 plaque samples from the buccal of the caries-free teeth（PCF）and 16 saliva samples（S）were collected.According to the experience of previous studies,the significant effective antibacterial of SDF will lead to a large difference in plaque,5 children from the 16 children who met the above requirements were randomly selected,the caries teeth of the 5 children were applied with 38%SDF and subsequent sampling as well as analysis.Each of the 5 selected children had one caries tooth in their mouth.After intervention with 3 8%SDF in vitro,a total of 5 plaque samples from caries 24 hours after SDF intervention（PAI₂4h）,5 plaque samples from caries 1 week after SDF intervention（PAI_lw）were collected.All samples at baseline and after intervention passed qualification test for further DNA sequencing and completed the high-throughput gene sequencing.At baseline,the main micro-ecological characteristic results of each sample were briefly summarized and reported.The average microbial diversity of dental plaque from the extensive caries of the deciduous teeth in mixed dentition decreased.In terms of microbial composition,Pseudomonas,Olsenella,Prevotella₇,Bifidobacterium,Propionibacterium and Lactobacillus were noticeably increased in caries plaque（0.001<P ≤ 0.01）.Network analysis of microbial correlation showed that the lines among the genera in caries plaque were increased and the connection was strengthened,much more positive connections of synergism were observed than the negative ones of antagonism.Predictive analysis of microbial gene function confirmed that caries plaque had higher carbohydrate transport and metabolic functions with the parameter value was 0.073,while the parameters of PCF and S were 0.067 and 0.068,respectively.After treatment with 38%SDF,the diversity of microorganisms in caries plaque decreased,although not statistically significant（P>0.05）.In terms of microbial composition,Olsenella,Streptococcus,Bifidobacterium,Prevotella₇,Lactobacillus,Actinomyces,Leptotrichia,Selenomonas₃,and Veillonella from the caries plaque showed decreasing trend in different degrees compared with those before intervention,although not statistically significant（P>0.05）.Relative contents of the other three bacteria genus,including Pseudomonas,Fusobacterium and Pseudoramibacter were noticeably higher than before,although not statistically significant（P>0.05）.At the same time,this study found that after SDF intervention,the results of the Network analysis of microbial correlation showed that the inter-microbial associations became more complex,much more positive associations among survived bacteria were observed than negative ones.In addition,predictive analysis of microbial gene function showed carbohydrate transportation and metabolic functions in the plaque were significantly reduced at 24 hours and 1 week post-intervention,with the parameter value was 0.080,while the parameters of PAI₂4h and PAI_lw were 0.068 and 0.064,respectively.Meanwhile,the replication,recombination and repair function in the plaque were also significantly reduced at the same point,and the parameter values were 0.059,0.051 and 0.044,respectively.ConclusionsIn summary,the micro-ecological characteristics of plaque from extensive caries was noticeably different from that of dental plaque of sound tooth surface and salivary in mixed dentition.After treated with 38%SDF,the microbial diversity in caries plaque decreased,relative contents of the most microorganism were noticeably decreased,inter-microbial associations of the survived bacteria became more complex and closer,the synergistic effect was enhanced,the relative structure of microbial composition was changed noticeably,which helped to promote new balance of the plaque flora.Carbohydrate metabolism in dental caries plaque was significantly reduced.