Analysis of bacterial biofilms as a virulence factor for peri-implantitis

我们用愈合基台研究了人类种植体沟中初始生物膜形成的形态学。在征得7例患者的知情同意后，于植入后第10、30、90和150天小心地取出20个愈合基牙，并进行扫描电镜观察。第10天，基牙表面部分形成单层生物膜。观察到所谓的早期殖民者：球菌和细丝。30天后，生物膜发育，在人类种植体沟中主要观察到长杆和短杆。接下来，免疫组织化学检查了人类种植体周围炎相关细菌。从4例自愿患者中提取8个种植体，制成半薄切片，采用改良革兰氏染色法和4种牙周病细菌抗体链霉亲和素法进行染色。革兰氏阳性菌不仅出现在种植体表面，而且出现在基牙钉的内部部位，牙龈卟啉单胞菌（Porphyromonas gingivalis）和直弯曲杆菌（Campylobacter rectus）均未在人种植体沟中发现特异性抗体标记。采用牙龈卟啉单胞菌（Porphyromonas gingivalis）生物膜体外模型，检测了1)生物活性（ATP）， 2)抗菌药物敏感性，3)生物膜发育必需基因（polyphosphate kinase, ppk）同源物。生物膜中ATP含量的增加速率较低，为浮游细胞的1/3 × 10^5。葡萄糖酸氯己定能有效降低牙龈卟啉菌生物膜的活力，而盐酸米诺环素和甲硝唑则没有变化效应。克隆的ppk基因与大肠杆菌ppk基因的同源性为35.8%，其中牙龈卟啉卟啉ppk基因高度保守

We investigated morphologically initial biofilm formation in the human implant sulcus using healing abutments. After obtaining informed consent from 7 patients, twenty healing abutments were removed carefully on day 10, 30, 90 and 150 after insertion and treated for a scanning electron microscopy. On day 10, mono-layer biofilm have partially formed on the abutment surfaces. So called early colonizers: cocci and filaments, were observed. After 30 days, biofilms developed, and long and short rods were predominantly observed in human implant sulcusNext, human periimplantitis associated bacteria have been examined immunohistochemically. Eight implants obtained from 4 voluntary patients were extracted and processed into semithin sections They were stained BrownBrenn modified Gram stain and streptavidinbiotin method using 4 kinds of antibodies with periodontopathic bactria. Gram positive bacteria were shown not only on the implant surface but also at the inner site of abutment screw, Porphyromonas gingivalis and Campylobacter rectus labeled with speciesspecific antibody were not found in the human implant sulcusBy use of the in vitro model with P. gingivalis biofilm, 1) the biological (ATP) activities, 2) the susceptibility to antimicrobial agents, and 3) the essential gene for development of biofilm (the homologue for polyphosphate kinase (ppk)) were examined. The rate of increase of ATP contents in the biofilm was quite low, and 1/3.0x10^5 compared with that of planktonic cells. Chlorhexidine gluconate was effective at reducing the viability of P. gingivalis biofilm, while minocycline hydrochloride and metronidazole did not have a variable effect. Identity of P. gingivalis ppk gene that was carried out cloning to Esherihia coli ppk gene was 35.8%, including \hat ppk from P. gingivalis was highly conserved