| BackgroundThe impact of orthodontic appliance on periodontal tissue has been a concern for both orthodontists and periodontists.The fixed appliances could change the subgingival microbial environment by increasing the plaque accumulation,which could result in a risk of periodontal diseases.Compared with fixed appliances,clear aligners are well known for facilitating the oral hygiene maintenance due to its removable characteristic.Recently,most of clinical researches hold the view that clear aligners could decrease the periodontal diseases as compared to fixed appliances.However,this view is lack of microbial evidence to support.With the improvement of living standards,an increasing number of patients with periodontitis seek orthodontic treatment to improve their aesthetic and masticatory function.While,patients with periodontitis who receive orthodontic treatment are at a higher risk of periodontal diseases.At present,few studies have focused on the clinical and microbial changes that occur in patients with periodontitis during orthodontic treatment.With the development of 16S rRNA gene sequencing,the change in microbial community has been an indicator of periodontal health.The gingival swelling is one of the most common adverse effects during the orthodontic treatment.The mechanism of periodontal tissue inflammation is complicated.Recently,the autophagy has been reported to be involved in periodontitis.Autophagy could remove the periodontal pathogen infection and modulate the immune inflammatory response.With the development of sequencing and bioinformatics,long non-coding RNA(lncRNA)has been demonstrated to participate in the regulation of autophagy.At present,the role and function of lncRNA in periodontitis and whether lncRNA regulates autophagy in the inflammatory microenvironment remain unclear.ObjectivesThe first aim of this study was to explore the microbial and clinical changes of the female patients with fixed appliances,the female patients with clear aligners,and orthodontic patients with moderate to severe periodontitis in the early stage of orthodontic treatment,and to explore the periodontal changes with the microbial community changes.Besides,our study further explored the mechanism of periodontal tissue inflammation,which could help make early diagnosis and new treatment plan of periodontitis.Materials and Methods1.The microbial research during orthodontic treatment:Ten female patients with fixed appliances,ten female patients with clear aligners,and ten orthodontic patients with moderate to severe periodontitis,which were recruited from the orthodontic department of Peking University School and the Hospital of Stomatology,were selected in our study.For female patients with fixed appliances and clear aligners,the subgingival plaque sampling and periodontal examination(the Quigley-Hein plaque index and bleeding index)were performed at three time points:before orthodontic treatment(T0),one month after orthodontic treatment(T1)and three months after orthodontic treatment(T2).For orthodontic patients with moderate to severe periodontitis,the periodontal examination(the Quigley-Hein plaque index,bleeding index and probing pocket depth)were performed at four time points:before orthodontic treatment(T0),one month after orthodontic treatment(T1),three months after orthodontic treatment(T2)and six months after orthodontic treatment(T3);the saliva sampling was performed at three time points:before orthodontic treatment(T0),one month after orthodontic treatment(T1)and three months after orthodontic treatment(T2).DNA isolation,PCR amplification,16S rRNA sequencing and sequence processing were further performed for subgingival plaque and saliva samples.2.The mechanism research of periodontal tissue inflammation:The tumor necrosis factor ?(TNF?)and Porphyromonas gingivalis-derived lipopolysaccharide(LPS)were used to create an inflammatory microenvironment in periodontal ligament cells(PDLCs).The quantitative reverse-transcription polymerase chain reaction(qRT-PCR)was used to detect the level of lncRNA H19.Western blotting,flow cytometric analysis,and immunofluorescence staining were used to detect the autophagy flux.Overexpression and knockdown of H19 were used to confirm its function,and RNA sequencing was further performed to determine the differentially expressed genes.The periodontitis-affected human gingival tissue and ligature-induced periodontitis model in mice were used in vivo.The immunohistochemical staining was used to detect the autophagy level,and qRT-PCR was used to detect the level of lncRNA H19.Results1.The microbial research during orthodontic treatment:(1)For the female patients,the category and diversity of subgingival microbial community,and the relative abundance of core genera and periodontal pathogens,were stable during the first three-months fixed appliance treatment.The structure and composition of subgingival microbial community were changed in the third month of fixed appliance treatment.As for the clinical examination,the plaque index was transiently increased in the first month of fixed appliance treatment,and the bleeding index was significantly increased in the third month.(2)For the female patients,the category of subgingival microbial community,and the relative abundance of core genera and periodontal pathogens,were stable during the first three-months clear aligner treatment.The structure and composition of subgingival microbial community were changed in the first month of clear aligner treatment,and microbial diversity was slightly decreased without significant difference.As for the clinical examination,the plaque index and bleeding index were stable.(3)For the patients with moderate to severe periodontitis,the salivary microbial diversity was slightly increased in the first month of orthodontic treatment.The structure and composition of salivary microbial community were changed in the first month,and return to the pre-treatment level in the third month.The relative abundance of core genera and periodontal pathogens were stable during the three-months fixed appliance treatment.As for the clinical examination,the plaque index was increased in the first month,the bleeding index was slightly increased in the third month,and all returned to pre-treatment level in the sixth month.The probing pocket depth was stable during the first six-month fixed appliance treatment.2.The mechanism research of periodontal tissue inflammation:The autophagy level and the H19 expression were significantly increased in PDLCs after inflammatory stimulation as well as in a ligature-induced periodontitis model in mice and periodontitis-affected human gingival tissue.The levels of autophagy were significantly increased and decreased after overexpressing and knockdown H19 in PDLCs,separately.H19 mediates the activation of autophagy via PI3K/AKT pathway suppression in PDLCs.ConclusionsConsidering the microbial and clinical changes,the female patients might have a transient mild gingival inflammation in the early stage of fixed appliance treatment.The subgingival microbial community affected by clear aligner is considered to be less susceptible to periodontal diseases at the early stage of clear aligner treatment,the female patients have a healthy periodontal status.The salivary microbial community of patients with moderate to severe periodontitis during the first month of orthodontic treatment was slightly changed,which did not cause irreversible damage to periodontal tissue.The periodontal inflammation of patients with moderate to severe periodontitis during the six-months orthodontic treatment was under control.The autophagy regulated by lncRNA H19 plays an important role in periodontal inflammation.The level of autophagy in inflamed periodontal tissue was increased,lncRNA H19 mediates the activation of autophagy via PI3K/AKT pathway in periodontitis. |
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