牙周菌斑生物膜的体外模型建立

目的观察血链球菌、牙龈卟啉单胞菌和具核梭杆菌在人工牙根面上随培养时间变化形成单菌种、双菌种和三菌种生物膜的能力,期望在体外建立龈下菌斑生物膜模型。方法培养血链球菌、牙龈卟啉单胞茵和具核梭杆菌试验菌株,制备胶原包被羟磷灰石的人工牙根面,在人工牙根面上培养形成试验菌株的单菌种、双菌种和多菌种生物膜,并用扫描电镜观察三种试验菌株分别在培养24、48和72h后形成单菌种、双菌种和三菌种生物膜的情况。结果单独培养24h的血链球菌在人工牙根面上形成的完整生物膜,具备三维立体结构;培养48和72h后,细菌密度逐渐增大,生物膜更加成熟。单独培养48h的牙龈卟啉单胞菌形成的完整生物膜,具备三维立体结构;培养72h后,细菌密度有所降低。牙龈卟啉单胞菌和具核梭杆菌培养24h形成的完整的双菌种生物膜,具备三维立体结构;培养48和72h后已看不出完整的生物膜结构,两菌的数量大幅度降低。血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时形成的较完整的三菌种生物膜,初步具备三维立体结构;三菌中,血链球菌、具核梭杆菌所占比列远大于牙龈卟啉单胞菌;培养48h后,三菌种生物膜更加成熟,牙龈卟啉单胞菌所占比例有所增加,此时的生物膜已具备三维立体结构;培养72h后,三菌种仍保持较完整的生物膜结构,但数量均有所下降。结论血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时间点已基本形成了完整的单菌种、双菌种和三菌种生物膜结构,故在今后建立龈下菌斑生物膜模型时可选取24h这一时间点。     

牙周菌斑生物膜的体外模型建立

目的 观察血链球菌、牙龈卟啉单胞菌和具核梭杆菌在人工牙根面上随培养时间变化形成单菌种、双菌种和三菌种生物膜的能力,期望在体外建立龈下菌斑生物膜模型.方法 培养血链球菌、牙龈卟啉单胞菌和具核梭杆菌试验菌株,制备胶原包被羟磷灰石的人工牙根面,在人工牙根面上培养形成试验菌株的单菌种、双菌种和多菌种生物膜,并用扫描电镜观察三种试验菌株分别在培养24、48和72 h后形成单菌种、双菌种和三菌种生物膜的情况.结果 单独培养24h的血链球菌在人工牙根面上形成的完整生物膜,具备三维立体结构;培养48和72 h后,细菌密度逐渐增大,生物膜更加成熟.单独培养48 h的牙龈卟啉单胞菌形成的完整生物膜,具备三维立体结构;培养72 h后,细菌密度有所降低.牙龈卟啉单胞菌和具核梭杆菌培养24h形成的完整的双菌种生物膜,具备三维立体结构;培养48和72h后已看不出完整的生物膜结构,两菌的数量大幅度降低.血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时形成的较完整的三菌种生物膜,初步具备三维立体结构;三菌中,血链球菌、具核梭杆菌所占比列远大于牙龈卟啉单胞菌;培养48 h后,三菌种生物膜更加成熟,牙龈卟啉单胞菌所占比例有所增加,此时的生物膜已具备三维立体结构;培养72 h后,三菌种仍保持较完整的生物膜结构,但数量均有所下降.结论 血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时间点已基本形成了完整的单菌种、双菌种和三菌种生物膜结构,故在今后建立龈下菌斑生物膜模型时可选取24h这一时间点.     

牙周致病菌和致龋菌间生长关系的体外动态观察

目的研究6种代表性牙周致病菌和致龋菌在菌斑生物膜和悬浮液中的消长关系。方法将双菌组（致龋菌＋牙周致病菌）接种于改良恒化器中,连续培养1h、24h、48h和96h,然后取在羟基磷灰石表面形成的生物膜和悬浮液再进行细菌培养。结果与血链球菌混合培养时,菌斑生物膜和悬浮液中牙周致病菌均增多,而血链球菌明显减少（P〈0.05）;具核梭杆菌与变形链球菌培养时,菌斑生物膜和悬浮液中具核梭杆菌明显增多,而变形链球菌减少（P〈0.05）。菌斑生物膜中致龋菌占优势,相应的悬浮液中牙周致病菌96h开始占优势。悬浮液中细菌量波动更明显。结论2类致病菌在菌斑生物膜与相应的悬浮液中的生长是不一致的。2类致病菌之间相互作用也许是决定菌斑生物膜内部生态环境发展方向的主要因素。     

菌斑生物膜原位干预模型的建立

目的 通过对原位菌斑生物膜发育过程及早期成熟菌斑生物膜的观察,探讨所建立的菌斑生物膜模型用于菌斑化学干预研究的可能性.方法 建立原位菌斑生物膜模型,运用激光共聚焦扫描显微镜结合荧光染色技术观察原位菌斑生物膜0～48 h的发育过程及48 h菌斑生物膜的活性和厚度.结果 可观察到0～48 h菌斑生物膜从无到形成到成熟,细菌排列趋于密集,菌斑厚度逐渐增加的过程.48 h的菌斑生物膜活性和厚度可检测.结论 建立的菌斑生物膜模型可观察原位菌斑的形成过程、形态及活性,适用于菌斑化学干预的研究.     

生物活性玻璃对3种细菌和龈下早期混合菌斑抗菌效果的研究

目的研究不同浓度生物活性玻璃(bioactive glass,BG)对3种浮游细菌及其形成的单菌种菌斑、三菌种早期龈下混合菌斑的抗菌效果。方法测定血链球菌(Streptococcus sanguinis,Ss)、具核梭杆菌(Fusobacterium nucleatum,Fn)、伴放线放线杆菌(Actinobacillus actinomycetemcomitans,Aa)的最小抑菌浓度(minimal inhibitory concentration,MIC)、最小杀菌浓度(minimal bactericidal concentration,MBC)、最小菌斑清除浓度(minimal biofilm eradication concentration,MBEC)。20、40、80 mg/m L的生物活性玻璃和惰性玻璃二氧化硅(Si O2)处理24 h早期三菌混合菌斑,于激光共聚焦显微镜(confocal laser scanning microscopy,CLSM)下摄片观察分析生物膜厚度和平均荧光强度。结果 BG对菌斑的MBEC明显大于对浮游菌的MIC和MBC,MBEC为MIC的2~16倍。随着生物玻璃浓度加大,混合菌斑生物膜厚度变薄,团块减小,菌斑活性比减低,与空白对照组和阳性对照Si O2组有显著统计学差异(P<0.05)。结论 BG可抑制和杀灭血链球菌、聚合梭杆菌、伴放线放线杆菌浮游菌及其单菌种菌斑生物膜,对浮游细菌效果优于菌斑生物膜。低浓度BG对龈下混合菌斑有一定抗菌效果。     

改良恒化器中牙周致病菌和致龋菌的激光共聚焦显微镜动态观察

目的　模拟口腔条件下研究牙周致病菌和致龋菌的动态关系。方法　选用牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌、中间普氏菌、变形链球菌、血链球菌、黏性放线菌和嗜酸乳杆菌8种牙周致病菌和致龋菌。按分组接种于模拟口腔的改良恒化器中 ,连续培养 1、2 4、4 8和 96h ,活菌革兰荧光染色与激光共聚焦显微镜结合测量羟基磷灰石圆片表面生物膜厚度 ,连续断层扫描及三维重建。结果　随时间变化各组生物膜厚度均显著增加 (P <0 0 0 1) ;同一时间点血链球菌生物膜明显厚于伴放线放线杆菌 ,8种菌明显比血链球菌和伴放线放线杆菌形成的生物膜厚。三维重建显示 ,G-牙周致病菌主要分布于G 致龋菌菌团中或膜表层。结论　人工菌斑生物膜中G 致龋菌首先定植 ,G -牙周致病菌的量和比例随时间增加 ;牙周致病菌和致龋菌的生态平衡与疾病之间的关系值得深入研究     

血链球菌细菌素对唾液生物膜中牙龈卟啉单胞菌和具核梭杆菌拮抗作用的实验研究

目的:提取血链球菌标准菌株(ATCC10556)细菌素,研究其对唾液生物膜中牙龈卟啉单胞菌和具核梭杆菌的拮抗作用.方法:通过超声破碎,高速离心,盐析等方法提取血链球菌细菌素;采用二倍稀释法,测定血链球菌细菌素对浮游状态下牙龈卟啉单胞菌和具核梭杆菌的最小抑制浓度(MIC);在体外建立牙龈卟啉单胞菌和具核梭杆菌唾液生物膜模...     

比较6种口腔材料对体外生物膜形成的影响

目的比较体外混合菌在不同充填材料表面形成早期生物膜的能力,探讨生物膜状态下不同充填材料细菌形成的差异。方法用6种不同修复材料按临床比例制成试件各3件,将各试件放入国际标准菌株牙龈卟啉单胞菌、具核梭杆菌、伴放线放线杆菌、粘性放线菌、血链球菌、变形链球菌混合培养24h,使之在试件表面形成生物膜。在激光共聚焦扫描显微镜(LCSM)下观察生物膜厚度和平均荧光强度,比较不同充填材料表面生物膜细菌形成的差异。结果形成的生物膜平均厚度依次为聚羧酸锌水门汀>磷酸锌水门汀>玻璃离子体水门汀>复合树脂>银汞合金>富士Ⅱ型玻璃离子水门汀。组间比较发现:四种充填材料生物膜厚度无统计学的差异,富士Ⅱ型玻璃离子水门汀、银汞合金的生物膜厚度与两种垫底材料之间都有显著性差异,复合树脂、玻璃离子体水门汀、磷酸锌水门汀的生物膜厚度与聚羧酸锌水门汀有显著性差异。结论材料表面形成的不同生物膜厚度提示:富士Ⅱ型玻璃离子水门汀、银汞合金具有较强的抑制生物膜形成的能力。     

具核梭杆菌在牙菌斑生物膜形成过程中的作用

牙周炎是一类由菌斑生物膜引起的慢性感染性疾病,研究菌斑生物膜的形成对于牙周炎的预防和治疗有重要意义.具核梭杆菌作为连接早晚期定植菌的桥梁菌,在牙菌斑生物膜的形成过程中具有重要的作用,本文就其在菌斑生物膜形成过程中的作用进行总结.     

三种漱口液对早期原位菌斑生物膜形成的影响

目的:应用口内牙菌斑生物膜模型评估替硝唑漱口液、西吡氯铵漱口液与氯己定漱口液对早期原位牙菌斑生物膜形成的影响.方法:在专业洁治后,志愿者佩戴一种上颌装置,6 个玻璃片置于装置内以形成口内菌斑生物膜.志愿者用指定漱口液每天漱口2 次,每次15 ml含漱1 min.48 h后样本从装置中移出,立即用2 种荧光染料染色,分别将死菌染成红色,活菌染成绿色,在激光共聚焦显微镜下观察,进行三维扫描,评估菌斑生物膜的厚度和活菌比率.在14 d洗脱期后,再应用另一种漱口液.结果:与清水相比, 3 种漱口液作用下的48 h菌斑生物膜平均厚度及平均活菌比率均有显著降低(P<0.001),且3 组之间有显著差异(P<0.05).结论:对于早期原位菌斑生物膜, 3 种漱口液均显示有抑制菌斑形成及抗菌的作用.     

Validation of an in vitro biofilm model of supragingival plaque

The study of biofilm structure and function mandates the use of model systems for which a host of environmental variables can be rigorously controlled. We describe a model of supragingival plaque containing Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, Streptococcus sobrinus, and Streptococcus oralis wherein cells are cultivated anaerobically in a saliva-based medium on hydroxyapatite discs coated with a salivary pellicle, with material and pieces of apparatus common to all microbiology laboratories. After 0.5 hr, 16.5 hrs, 40.5 hrs, and 64.5 hrs, the composition of adherent biofilms was analyzed by culture techniques, live/dead fluorescence staining, and confocal laser scanning microscopy. Repeated independent trials demonstrated the repeatability of biofilm formation after 40.5 hrs and 64.5 hrs. Brief exposures of biofilms to chlorhexidine or Triclosan produced losses in viability similar to those observed in vivo. This biofilm model should prove very useful for pre-clinical testing of prospective anti-plaque agents at clinically relevant concentrations.     

用激光共聚显微镜研究菌斑生物膜的结构

目的：研究菌斑生物膜的结构。方法：从牙齿表面获得完整的菌斑生物膜标本，运用激光共聚焦显微镜对其进行断层扫描和分析。结果：菌斑生物膜是由分布不均匀的细胞、基质和空隙构成的复杂结构。结论：应用激光共聚焦显微镜技术研究菌斑生物结构是一种可行的方法。     

MPC-polymer reduces adherence and biofilm formation by oral bacteria

Oral biofilms such as dental plaque cause dental caries and periodontitis, as well as aspiration pneumonia and infectious endocarditis by translocation. Hence, the suppression of oral biofilm formation is an issue of considerable importance. Mechanical removal, disinfectants, inhibition of polysaccharide formation, and artificial sugar have been used for the reduction of oral biofilm. From the viewpoint of the inhibition of bacterial adherence, we investigated whether aqueous biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer can reduce streptococcal colonization and biofilm formation. We examined the effects of MPC-polymer on streptococcal adherence to saliva-coated hydroxyapatite and oral epithelial cells, and the adherence of Fusobacterium nucleatum to streptococcal biofilm. MPC-polymer application markedly inhibited both the adherence and biofilm formation of Streptococcus mutans on saliva-coated hydroxyapatite and streptococcal adherence to oral epithelial cells, and reduced the adherence of F. nucleatum to streptococcal biofilms. A small-scale clinical trial revealed that mouthrinsing with MPC-polymer inhibited the increase of oral bacterial numbers, especially of S. mutans. These findings suggest that MPC-polymer is a potent inhibitor of bacterial adherence and biofilm development, and may be useful to prevent dental-plaque-related diseases. (UMIN Clinical Trial Registry UMIN000003471).     

Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro

The aim of this study was to examine the influence of glucosyltransferase-gene-negative (gtf-) Streptococcus mutans strains unable to synthesize water-insoluble or soluble glucan on the structure and macromolecular diffusion properties of in vitro grown mixed oral biofilms. Biofilms modeling supragingival plaque consisted of Actinomyces naeslundii OMZ 745, Candida albicans OMZ 110, Fusobacterium nucleatum KP-F2, Streptococcus oralis SK 248, Veillonella dispar ATCC 17748T and one of the S. mutans strains UA159, OMZ 966, OMZ 937 or OMZ 977. Biofilms were grown anaerobically on sintered hydroxyapatite disks for 64.5 h at 37 degrees C. To perform confocal laser scanning microscopy analyses, microorganisms were stained with Syto 13 and extracellular polysaccharides (EPS) with Calcofluor. Macromolecular diffusion properties were measured following timed biofilm exposure to Texas-Red-labeled 70-kDa dextran. Results showed that replacing wild-type S. mutans by a gtfC- mutant led to an increase in the volume fraction occupied by cells from 29 to 48% and a decrease of the EPS volume fraction from 51 to 33%. No such changes were observed when the S. mutans wild-type strain was replaced by a gtfB- or gtfD- mutant. The diffusion coefficient of 70-kDa dextran in biofilms containing the gtfC- S. mutans was 16-fold higher than in biofilms with the wild-type strain indicating a strong macromolecular sieving effect of GTF C-generated glucans. Our data demonstrate the influence of EPS on the structure and macromolecular diffusion properties of an oral biofilm model and uncover our still limited knowledge of the function of EPS in biofilms and plaque.     

Sensitization of oral bacteria in biofilms to killing by light from a low-power laser.

Biofilms of Streptococcus sanguis, Porphyromonas gingivalis, Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans were prepared on the surfaces of agar plates and a number of compounds were screened for their ability to sensitize bacteria in these biofilms to killing by light from a 7.3 mW Helium/Neon (He/Ne) laser. Toluidine blue O and methylene blue enabled detectable killing of all four target organisms after exposure to He/Ne light for 30 s. Aluminium disulphonated phthalocyanine, haematoporphyrin HCl and haematoporphyrin ester were effective photosensitizers of only some of the target organisms. These findings suggest that lethal photosensitization may be an effective means of eliminating periodontopathogenic bacteria from dental plaque.     

Effects of Er:YAG laser irradiation on biofilm-forming bacteria associated with endodontic pathogens in vitro

With the development of dental laser delivery systems that can enter into the root canals, it is possible to use Er:YAG lasers to remove the residual biofilm associated with infected root canals. We examined their effects against biofilms made of Actinomyces naeslundii, Enterococcus faecalis, Lactobacillus casei, Propionibacterium acnes, Fusobacterium nucleatum, Porphyromonas gingivalis, or Prevotella nigrescens in vitro. After Er:YAG laser irradiation with energy densities ranging between 0.38-0.98 J/cm(2), the biofilm samples on hydroxyapatite disks were quantitatively and morphologically evaluated. The Er:YAG laser was effective against biofilms of 6 of the bacterial species examined, with the exception of those formed by L. casei. After irradiation, the numbers of viable cells in the biofilms were significantly decreased, whereas atrophic changes in bacterial cells and reductions in biofilm cell density were seen morphologically. Er: YAG lasers might be suitable for clinical application as a suppressive and removal device of biofilms in endodontic treatments.     

Synergy between Tannerella forsythia and Fusobacterium nucleatum in biofilm formation

During dental plaque formation, the interaction of different organisms is important in the development of complex communities. Fusobacterium nucleatum is considered a 'bridge-organism' that facilitates colonization of other bacteria by coaggregation-mediated mechanisms and possibly by making the environment conducive for oxygen intolerant anaerobes. These studies were carried out to determine whether coaggregation between F. nucleatum and Tannerella forsythia is important in the formation of mixed species biofilms. Further, the role of BspA protein, a surface adhesin of T. forsythia, in coaggregation and biofilm formation was investigated. The results showed the development of synergistic mixed biofilms of F. nucleatum and T. forsythia when these bacteria were cocultured. The BspA protein was not involved in biofilm formation. Though BspA plays a role in coaggregation with F. nucleatum, presumably other adhesins are also involved. The synergistic biofilm formation between the two species was dependent on cell-cell contact and soluble components of the bacteria were not required. This study demonstrates that there is a positive synergy between F. nucleatum and T. forsythia in the development of mixed biofilms and that the cell-cell interaction is essential for this phenomenon.