牙周手术治疗对猴Ⅲ度根分叉病变模型龈下菌斑中牙周致病微生物的影响

Effect of periodontal surgery on the subgingival periodontal microbiota in artificial class Ⅲ furcation defects: an experimental study in monkeys

目的:纵向观察猴在自然状态下(Ⅲ度根分叉病变建立前),根分叉病变建立后牙周手术治疗前,以及手术治疗后6个月时,根分叉部位龈下菌斑中5种牙周可疑致病菌检出率的变化。方法:在猴下颌双侧第二前双尖牙、第一磨牙和第二磨牙制备慢性Ⅲ度根分叉病损后,行牙周手术治疗,3只猴共18个牙位。分别在自然状态下(Ⅲ度根分叉病变建立前),根分叉病变建立后牙周手术治疗前,以及手术治疗后6个月时,取根分叉部位(颊、舌侧)的龈下菌斑。每只猴每时间点有12个样本,3只猴36个菌斑样本,用16SrRNA为基础的PCR技术检测样本中5种牙周可疑致病菌:牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、福赛坦氏菌(Tannerella forsythensis,Tf)、齿垢密螺旋体(Treponema dinticola,Td)、伴放线放线杆菌(Actinobacillus actinomycetemcomitans,Aa)和具核梭杆菌(Fuso-bacterium nucleatum,Fn)。结果:根分叉病变模型建立后,局部炎症明显,牙周手术后6个月根分叉区牙周组织有一定程度的修复,但局部炎症依然存在,Pg、Tf、Td和Fn的检出率均逐渐显著增加,分别从58.3%、69.4%至88.9%(P<0.01),从47.2%、69.4%至83.3%(P<0.01),从13.9%、36.1%至61.1%(P<0.01),从69.4%、91.7%至91.7%(P<0.05)。Aa的检出率3次取样变化不大(从25.9%、13.9%至33.3%)。同时检出3种以上微生物的检出率从38.9%、61.1%至83.3%(P<0.01),以及红色复合体(Pg+Tf+Td)的同时检出(从8.3%、27.8%至44.4%,P<0.01)也逐渐明显增加。根据术后6个月组织学有无炎症细胞浸润,分成8个感染牙位和10个未感染牙位,术后6个月感染位点与其根分叉病变建立后牙周手术治疗前的红色复合体检出率(87.5%,62.5%)明显高于根分叉病变建立前(0.0%,P<0.01)。虽然均高于非感染部位(60.0%,40.0%),但差异无统计学意义(P均>0.05)。结论:红色复合体(Pg、Tf和Td)在牙周炎的发生和发展中有着重要的作用,Fn则可能是龈下菌斑中的常驻菌,而Aa可能不是慢性牙周炎的主要致病微生物。

Objective： This study was to longitudinally evaluate the change of prevalence of five periodontal putative pathogens in the subgingival plaque of artificial class Ⅲ furcation defects at the three time-points,including before the establishment of furcation defects,before and 6 months after periodontal surgery.Methods： Eighteen chronic infected class Ⅲ FI defects were created at the mandibular first molars,second molars and second premolars of three adult male Macaca fascicularis.The samples of subgingival plaque were obtained from the subgingival area of furcation defects in buccal and lingual sites before the establishment of furcation defects,before and 6 months after periodontal surgery.36 samples were obtained at one time-points.Five periodontal putative pathogens,including Porphyromonas gingivalis（Pg）,Tannerella forsythensis（Tf）,Treponema dinticola（Td）,Actinobacillus actinomycetemcomitans（Aa） and Fusobacterium nucleatum（Fn）,were detected with 16SrRNA based PCR.Results： 1.The prevalence of Pg,Tf,Td and Fn was gradually increased,from 58.3% to 69.4% to 88.9%,47,2% to 69.4% to 83.3%,13.9% to 36.1% to 61.1%（P0.01）,and 69.4% to 91.7% to 91.7%（P0.05）,respectively during the experimental period.The prevalence of Fn was higher than Pg,Tf and Td.The prevalence of Aa was the lowest and no obvious difference among the three samplings（from 25.9% to 13.9% to 33.3%）was detected.2.The prevalence of more than 3 species simultaneously detected was increased from 38.9% to 61.1% to 83.3%（P 0.01）.The red complex（Pg ＋ Tf ＋ Td） was detected from 8.3% to 27.8% to 44.4%（P0.01） at the different time point.3.The combined detection frequency of red complex in the inflammatory sites（87.5%）,which were histologically defined as inflammatory cells infiltrated in furcation area 6 months post-surgery,and the same sites pre-surgery（62.5%） was more than that in pre-creation of furcation defects（P0.01）.But there were no significant differences compared to that in non inflammatory area（60.0%,40.0%）,respectively.Conclusion： The prevalence of periodontal pathogenic bacteria correlated with the severity of local inflammation.The increase of coexistent rate of red complex at the second and third sampling times suggests that the red complex play important role in the pathogenesis of periodontitis.Fn may be a resident bacteria in the subgingival plaque,play a bridge role on the biofilm formation and maturation.Aa may not be a major cau-sative bacteria in the clinical periodontitis.