HMME—PDT对致龋模型中变形链球菌的杀伤作用及组织病理研究

目的：探讨光动力疗法（PDT）对大鼠口腔变形链球菌的杀伤作用。方法：以Wistar大鼠构建致龋模型,随机分成7组：照射60、90、120 s的PDT组、单纯光敏剂、单纯激光、0.2%NaF阳性组、0.9%生理盐水阴性组。选择半导体激光为光源,血卟啉单甲醚（HMME）为光敏剂。通过平板菌落计数法观察各组对变形链球菌的杀伤作用,组织病理观察大鼠口腔软组织变化。结果：平板菌落计数显示,与对照组比较,PDT防龋处理可使大鼠口腔中变形链球菌数量显著减少（P〈0.05）,光照时间为90s时对大鼠口腔软组织的损伤较小。结论：HMME-PDT可有效杀灭大鼠口腔中的变形链球菌,选择合适照射时间,不仅可以提高灭菌率还可减少对口腔其他组织的损伤。     

光动力疗法联合EDTA进行根管消毒的体外研究

目的：探究光动力疗法（photodynamictherapy,PDT）与EDTA联合应用进行根管消毒的效果。方法：选取75颗人单根管离体前磨牙建立感染根管模型,随机均分为5组,A组：PDT处理组;B组：EDTA处理组;C组：PDT联合EDTA处理组;D组：NaCIO阳性对照组;E组：生理盐水阴性对照组。各组处理前、处理后分别用纸尖在根管内取样,进行平板菌落计数。回复实验中在根管内注满无菌液体培养基,培养7d后用纸尖取样,统计检出细菌的根管个数。结果：PDT联合EDTA组杀菌率达100％,明显高于其余4组（P〈0．05）。回复实验中PDT联合EDTA组检出细菌根管数为4个,明显少于其余4组（P〈O．05）。结论：PDT联合EDTA可有效进行根管消毒,有望为临床提高根管消毒效率提供一条新途径。     

光动力疗法对牙菌斑生物膜内致龋菌的影响及作用机制的研究

目的：探讨以血卟啉单甲醚（hematoporphyrin monomethylether,HMME）为光敏剂的光动力疗法（photo-dynamictherapy,PDT）对牙菌斑生物膜内致龋菌的杀灭作用及机制。方法：以变形链球菌、血链球菌、嗜酸性乳杆菌和粘性放线菌为实验菌株,建立牙菌斑生物膜模型。实验分为4组：HMME-PDT组、HMME-PDT加叠氮钠组、西吡氯铵处理组、生理盐水处理组。平板菌落计数观察牙菌斑内致龋菌的活力,并测定不同处理前后pH的变化值（么pH）观察其对致龋菌产酸能力的影响。结果：与生理盐水组相比,HMME-PDT组和西吡氯铵组牙菌斑内致龋菌的数量（CFU／mL）显著下降（P〈0．05）,其杀菌率分别为94．92％和51．86％;而HMME-PDT加叠氮钠组牙菌斑内致龋菌数量无显著性差异,杀菌率仅为9．47％。么pH值测定结果显示,在一定时间范围内（O～24h）,与生理盐水组相比,HMME-PDT组牙菌斑内致龋菌的产酸能力显著下降。结论：PDT可有效杀灭菌斑生物膜内的致龋菌,并抑制其产酸,同时单线态氧在PDT杀菌过程中起着极其重要的作用。     

The susceptibility of Streptococcus mutans to antibacterial photodynamic therapy: a comparison of two different photosensitizers and light sources

Streptococcus mutans is the main etiological agent for dental caries. Recently, photodynamic therapy (PDT) has been introduced as a new modality in bacterial decontamination.

Objective

This in vitro study was carried out to evaluate the susceptibility of Streptococcus mutans to antibacterial PDT using two different photosensitizers and light sources.

Material and Methods

Standard suspensions of S. mutans were exposed to laser light at 662 nm and Radachlorin^® or LED 630 nm in combination with Toluidine blue O (TBO). Radiation-only groups, photosensitizer alone, and groups with no treatment were used as controls. Bacterial suspension from each treatment was subcultured onto the surface of Mueller-Hinton agar plates and bacterial growth was assessed. The results were analyzed by analysis of variance and Tukey test (p<0.05).

Results

PDT with TBO and Radachlorin^® significantly reduced S. mutans viability, whereas no difference was observed between two groups of PDT. In the groups treated just with the photosensitizer or irradiated alone, no significant reduction of S. mutans colonies was observed.

Conclusion

S. mutans colonies were susceptible to either 662 nm laser or LED light in the presence of Radachlorin^® and TBO respectively with no priority.     

Use of hand held photopolymerizer to photoinactivate Streptococcus mutans

OBJECTIVES: The main focus of this research was to investigate the photodynamic therapy (PDT), in vitro, acting on Streptococcus mutans and fibroblasts. A hand held photopolymerizer (HHP) and a classical photosensitizer (Rose Bengal) were used to induce photodynamic response. METHODS: S. mutans and fibroblast were treated with different concentrations of Rose Bengal (0-50 microM) irradiated with light (400-500 nm) for different time periods (0-40s) and then cell viability was evaluated. RESULTS: It was observed that the light (per se) is not toxic and in the dark Rose Bengal is toxic to the cells tested only at concentrations above 2.5 microM. Under light exposure concentrations of Rose Bengal above 0.5 microM all S. mutans were killed with no cytotoxic effects to fibroblasts. CONCLUSIONS: For the purpose of this work, the photoactivation of Rose Bengal, using the HHP, inactivated the bacteria without affecting the fibroblast viability.     

Effect of reduced exposure times on the cytotoxicity of resin luting cements cured by high-power led

Objective

Applications of resin luting agents and high-power light-emitting diodes (LED) light-curing units (LCUs) have increased considerably over the last few years. However, it is not clear whether the effect of reduced exposure time on cytotoxicity of such products have adequate biocompatibility to meet clinical success. This study aimed at assessing the effect of reduced curing time of five resin luting cements (RLCs) polymerized by high-power LED curing unit on the viability of a cell of L-929 fibroblast cells.

Material and Methods

Disc-shaped samples were prepared in polytetrafluoroethylene moulds with cylindrical cavities. The samples were irradiated from the top through the ceramic discs and acetate strips using LED LCU for 20 s (50% of the manufacturer''s recommended exposure time) and 40 s (100% exposure time). After curing, the samples were transferred into a culture medium for 24 h. The eluates were obtained and pipetted onto L-929 fibroblast cultures (3x10⁴ per well) and incubated for evaluating after 24 h. Measurements were performed by dimethylthiazol diphenyltetrazolium assay. Statistical significance was determined by two-way ANOVA and two independent samples were compared by t-test.

Results

Results showed that eluates of most of the materials polymerized for 20 s (except Rely X Unicem and Illusion) reduced to a higher extent cell viability compared to samples of the same materials polymerized for 40 s. Illusion exhibited the least cytotoxicity for 20 s exposure time compared to the control (culture without samples) followed by Rely X Unicem and Rely X ARC (90.81%, 88.90%, and 83.11%, respectively). For Rely X ARC, Duolink and Lute-It 40 s exposure time was better (t=-1.262 p=0,276; t=-9.399 p=0.001; and t=-20.418 p<0.001, respectively).

Conclusion

The results of this study suggest that reduction of curing time significantly enhances the cytotoxicity of the studied resin cement materials, therefore compromising their clinical performance.     

不同人群唾液中变形链球菌的定量检测及其意义

目的 建立定量检测唾液样本中变形链球菌和细菌总数的方法 ,比较变形链球菌和细胞总数的存在与不同人群中龋齿患病率的关系.方法 针对染色体DNA印迹法检测变形链球菌中出现的14 kb的HaeⅢ酶切片段,合成特异性引物(Sm~*),运用实时荧光定量聚合酶链反应(PCR)技术定量检测变形链球菌;对单纯随机抽样方法 抽取的99份唾液标本按照龋齿牙数是否为0分为龋齿组和无龋组,龋齿组72人,无龋组27人(包括从未患过龋齿者和曾患龋齿但已经过治疗和填充的无新鲜龋齿者),分别进行变形链球菌和细菌总量的检测及统计学分析.结果引物Sm~*仅对变形链球菌有特异性,定量PCR可检测的下限为0.1 μg/L;细菌总数在龋齿组和无龋组分别为51.4×10~8个/L和221.6×10~8个/L,变形链球菌占细菌总数比值分别为0.0193和0.0059,细菌总数和变形链球菌所占比值在两组间差异有统计学意义(P=0.022).结论 引物Sm~*可用于变形链球菌的定量检测;唾液中变形链球菌与总细菌数的比值与龋齿患病率相关.     

Hydroalcoholic extracts of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves on Streptococcus mutans biofilm and tooth demineralization

ObjectivesThis study evaluated the effect of the hydroalcoholic extracts of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves (alone or combined) on the viability of Streptococcus mutans biofilm and on the prevention of enamel demineralization.MethodsStrain of S. mutans (ATCC 21175) was reactivated in BHI broth. Minimum inhibitory concentration, minimum bactericidal concentration, minimum inhibition biofilm concentration and minimum eradication biofilm concentration were determined in order to choose the concentrations to be tested under biofilm model. S. mutans biofilm (5 × 10⁵ CFU/ml) was produced on bovine enamel, using McBain saliva under 0.2% sucrose exposure, for 3 days. The biofilm was daily treated with the extracts for 1 min. The biofilm viability was tested by fluorescence and the enamel demineralization was measured using TMR.ResultsMyracrodruon urundeuva All. (Isolated or combined) at the concentrationsc ≥0.625 mg/ml was able to reduce bacteria viability, while Qualea Grandflora Mart. alone had antimicrobial effect at 5 mg/ml only (p < 0.05). On the other hand, none of the extracts were able to reduce enamel demineralization.ConclusionsThe hydroalcoholic extracts of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves (isolated or combined) have antimicrobial action; however, they do not prevent enamel caries under S. mutans biofilm model.     

变链菌细胞外多糖合成与乳牙高龋的关系研究

目的:比较高龋和无龋幼儿变链菌临床分离株在体外利用蔗糖合成水溶性、水不溶性葡聚糖的能力。方法:蒽酮法定量测定从高龋(dmft≥5)和无龋幼儿(dmft=0)牙菌斑分离的变形链球菌临床分离株水溶性和水不溶性葡聚糖产量。结果:高龋组菌株合成水溶性与水不溶性葡聚糖量平均分别为0.0332mg/ml和0.0357mg/ml,高于无龋组菌株的0.0215mg/ml和0.0192mg/ml;高龋幼儿定植的合成水溶性及水不溶性葡聚糖能力强的菌株所占比例也高于无龋幼儿;变链菌临床菌株水不溶性葡聚糖产量平均为0.0301mg/ml,高于水溶性葡聚糖产量(0.0267mg/ml);差异有统计学意义。结论:高龋幼儿的龋活跃性与其口腔中变形链球菌临床菌株合成细胞外多糖能力强有关。     

Efficacy of red propolis hydro-alcoholic extract in controlling Streptococcus mutans biofilm build-up and dental enamel demineralization

ObjectiveThe efficacy of a red propolis hydro-alcoholic extract (RP) in controlling Streptococcus mutans biofilm colonization was evaluated. The effect of RP on dental demineralization was also investigated.MethodsChemical composition was determined by High Performance Liquid Chromatography (HPLC). Minimum Inhibitory and Bactericidal Concentration (MIC and MBC, respectively) were investigated against Streptococcus mutans (ATCC 25175). The cytotoxic potential of 3% RP in oral fibroblasts was observed after 1 and 3 min. Bovine dental enamel blocks (N = 24) were used for S. mutans biofilm formation (48 h), simulating ‘feast or famine’ episodes. Blocks/biofilms were exposed 2×/day, for 3 days, to a cariogenic challenge with sucrose 10% (5 min) and treated (1 min) with: 0.85% saline solution (negative control), 0.12% Chlorhexidine (CHX, positive control for biofilm colonization), 0.05% Sodium Fluoride (NaF, positive control to avoid demineralization) and 3% RP. Biofilms were assessed for viability (CFU/mL), and to observe the concentration of soluble and insoluble extracellular polysaccharides (SEPS and IEPS). Dental demineralization was assessed by the percentage of surface hardness loss (%SHL) and through polarized light microscopy (PLM).ResultsThe RP presented 4.0 pH and ºBrix = 4.8. The p-coumaric acid (17.2 μg/mL) and luteolin (15.23 μg/mL) were the largest contents of phenolic acids and flavonoids, respectively. MIC and MBC of RP were 293 μg/mL and 1172 μg/mL, respectively. The 3% RP showed 43% of viably cells after 1 min. Lower number (p < 0.05) of viable bacteria (CFU/mL) was observed after CHX (1.8 × 10⁵) followed by RP (1.8 × 10⁷) treatments. The lowest concentration (μg/CFU) of SEPS (12.6) and IEPS (25.9) was observed in CHX (p < 0.05) followed by RP (17.1 and 54.3), and both differed from the negative control (34.4 and 63.9) (p < 0.05). Considering the %SHL, all groups differed statistically (p < 0.05) from the negative control (46.6%); but NaF (13.9%), CHX (20.1%) and RP (20.7%) did not differ among them (p > 0.05). After all treatments, suggestive areas of caries lesions were observed by PLM, which were lower for CHX and NaF.ConclusionThe 3% RP reduced S. mutans colonization, decreased concentration of extracellular polysaccharides and reduced dental enamel demineralization.     

变形链球菌(血清型C)临床分离株AP-PCR基因分型

目的 探讨变形链球菌（血清型C）菌珠基因型与龋发生的关系。方法 用Chelex法撮细菌染色体DNA,经多次实验自行确立AP－PCR最佳反应条件,并对278株来自不同龋敏感个体的临床分离株进行AP－PCR基因型分析。结果 278株变链菌临床分离株共区分出105种基因型,无龋个体84．21％携带1种基因型,而高龋患者95％携带2种或2种以上基因型。结论 变链菌菌株间存在明显的遗传多态性,个体携带变链球菌基因型的种数与其致龋性密切相关。     

Genotyping of Streptococcus mutans by using arbitrarily primed polymerase chain reaction in children with Down Syndrome

OBJECTIVE: The aim of this study was to compare the caries prevalence between Down Syndrome (DS) and non-DS children and to investigate the difference between the genotypes of Streptococcus mutans (S. mutans) colonized in both DS and non-DS groups. DESIGN: Sixty children with DS and 64 non-DS children aged between 7 and 12 years old were included to this study. All erupted teeth were evaluated according to the criteria recommended by the World Health Organization. Unstimulated saliva samples were carried out from the children and cultivated on S. mutans selective Tryptone-yeast-cystine (TYC) agar with 0.2 U/ml bacitracin and 15% sucrose. Molecular typing of S. mutans strains was performed by using arbitrarily primed polymerase chain reaction (AP-PCR) with OPA-05 primer. All data were analysed by using SPSS (SPSS Inc., Chicago, IL, USA) 11.0 software program for windows. RESULTS: The caries index scores were found significantly lower in DS individuals than the non-DS group (p < 0.05). The salivary S. mutans levels between DS and non-DS groups did not show significant difference (p > 0.05). The difference between dental caries and salivary S. mutans levels also was not statistically significant (p > 0.05). According to the results of the AP-PCR typing, all profiles of S. mutans which colonized in DS group were different from the control group. The relationship between these different profiles and dental caries prevalence was statistically significant (p < 0.05). CONCLUSION: The profiles of S. mutans colonized in DS group might be a reason of low caries prevalence.     

Description of Streptococcus mutans,Streptococcus sanguinis,and Candida albicans biofilms after exposure to propolis dentifrice by using OpenCFU method

ContextDental caries is a major and chronic dental public health problem, which can usually be prevented by regular oral hygiene. The most common oral hygiene practice is brushing teeth with a dentifrice. Propolis has emerged as a promising anti-cariogenic agent, which is considered to be a good oral antiseptic for prevention of caries. Several studies have shown that the use of C has an influence in the growth of oral biofilms. There are several standard methods used to count bacterial colonies, such as crystal violet and CFU Count assays. OpenCFU method is a technique that can be used to calculate biofilm colonies more faster, precisely, and accurately.AimTo compare several methods for evaluating the number of biofilm colonies formed with exposure to a standard dentifrice and propolis.Methods and materialsBiofilm assays were carried out on 96-well microplates. Reference strains of oral Streptococcus species (S. mutans ATCC 25175^T and S. sanguinis ATCC 10566^T) and yeast (Candida albicans ATCC 10231^T) were inoculated into wells, and 200 µL of standard and propolis dentifrice solution were added to each well and incubated for 18 h at 37 °C. Bacteria and yeast were then sub-cultured on respective media and the colony-forming units (CFU) were counted manually. The other wells were stained by crystal violet and incubated for 15 min, followed by observation using an inverted microscope and evaluated using crystal violet analysis and the OpenCFU automated method.ResultsThe numbers of CFUs determined for all strains were similar in the standard-dentifrice group and propolis-dentifrice group, and were similar among the three methods: crystal violet staining, manual CFU count, and OpenCFU analysis.ConclusionOpenCFU analysis can be reliably used as a rapid and a more practical method to analyse the growth of oral microorganism biofilms. However, high digital image quality is required to provide an accurate analysis for colony counting.     

高发龋和无龋儿童菌斑中变形链球菌及远缘链球菌的任意引物PCR检测

目的采用任意引物聚合酶链反应（arbitrarily primed-polymerase chain reaction，AP-PCR）法探讨高发龋和无龋儿童牙菌斑致龋菌的检出情况，分析变形链球菌及远缘链球菌与乳牙龋齿发生的关系。方法从20例高发龋患儿和20名无龋儿童的牙菌斑中分离、鉴定变形链球菌和远缘链球菌，以Shiroza的DNA提取方法提取细菌基因组DNA，以形态学、生化和AP-PCR的方法鉴定变形链球菌和远缘链球菌。结果AP-PCR法检测发现高发龋患儿变形链球菌和远缘链球菌的检出率分别为100％和40％，而无龋儿童两种细菌的检出率分别为75％和5％，差异有统计学意义。结论用AP-PCR法检测发现高发龋患儿变形链球菌和远缘链球菌的检出率均明显高于无龋儿童，口腔中定植的变形链球菌和远缘链球菌是乳牙龋高发的危险因素。     

Genotypes of Streptococcus mutans in saliva versus dental plaque

OBJECTIVE: The aim of this study was to investigate whether genotypes of Streptococcus mutans strain can be detected as effectively in saliva samples as in plaque samples from buccal surfaces and occlusal surface fissures of permanent first molars. DESIGN: The study included 20 school children aged 6-7 years who were positive for mutans streptococci. Samples of stimulated saliva and of dental plaque on buccal surfaces and occlusal surface fissures of permanent first molars were collected. Samples were cultivated in MSB agar. Up to nine isolates compatible with mutans streptococci were obtained and identified by means of biochemical tests. All isolates identified as S. mutans were genotyped by arbitrarily primed polymerase chain reaction. RESULTS: A total of 28 genotypes of S. mutans were isolated: 23 in saliva samples, 23 in buccal surface plaque samples, and 16 in plaque samples from occlusal surface fissures. CONCLUSIONS: Although, saliva sampling did not reveal all genotypes isolated, it was equally as effective as plaque sampling from the buccal surfaces of permanent first molars, and more effective than plaque sampling from fissures on their occlusal surfaces.     

甲苯胺蓝为光敏剂的光动力疗法对龈下菌斑灭菌效果的研究

目的：研究甲苯胺蓝为光敏剂的光动力疗法对牙周龈下致病菌的灭菌效果。方法：从门诊25例牙周病病人随机提取龈下菌斑各1份,放人装有1mL预还原转送液的离心管,振荡,稀释。2h内送到微生物室,接种于牛心脑浸液平皿,在厌氧环境（含CO2、H1、N2的体积分数分别为10％、10％、80％）培养。培养出黑色菌落并与牙龈卟啉单胞菌标准菌株相鉴别。将菌样随机分为4组,A甲苯胺蓝组,甲苯胺蓝浓度分别为0．1、0．5、1．0、2．5g／L。B组光剂量组,光剂量为53、106、159、212mW／cm^2。C组光强度组,光强度为6、12、24、48J／cm^2。D组空白组。观察比较各组的灭菌效果,并确定最佳治疗组合。结果：光动力疗法的灭菌效果最佳组合为甲苯胺蓝1．0g／L＋光强度48J／cm^2＋光剂量212mW／cm^2,其灭菌效果平均23．45％。结论：本研究发现以甲苯胺蓝为光敏剂的光动力疗法对龈下菌斑有灭菌效果,最佳光敏剂浓度仍为1．0g／L,而龈下组所需光剂量和光强度均比前期研究的龈上组剂量大。不同病人的取菌条件不同,其灭菌效果也有差异。     

Photodynamic therapy in the management of lesions of the head and neck

Photodynamic therapy (PDT) is a promising and effective treatment for lesions of the head and neck. It uses illumination with light of a specific wavelength, which activates a photosensitising drug in the presence of oxygen. It can be used in combination with other treatments or on its own, and results in the cellular destruction of the lesion through a free-radical process. Photosensitisers can be applied topically or given systemically depending on the lesion being treated. Results indicate that PDT is an effective adjunct to standard conventional treatments. We review its use.     

变形链球菌GbpA的GBD免疫防龋实验研究

目的：观察变形链球菌GbpA的GBD融合蛋白免疫SD大鼠的防龋效果。方法：用纯化的变形链球菌GbpA的GBD融合蛋白皮下免疫SD大鼠,喂Keyes改良的高糖Diet 2000,第1次免疫20d时,连续3d大鼠口腔中接种S．mutans Ingbritt,在接种S．mutans后第77天处死大鼠,收集大鼠颌骨标本,用于龋齿记分分析,用t检验进行统计学分析。结果：GBD融合蛋白免疫大鼠,实验组龋损范围及龋坏程度均显著低于对照组,P＜0．01。结论：变形链球菌GbpA的GBD融合蛋白疫苗可有效降低龋齿的发生。     

光动力疗法对II型糖尿病患者合并慢性牙周炎的治疗效果

目的:探究光动力疗法对II型糖尿病患者合并慢性牙周炎的治疗效果及对炎症因子的影响。方法::48例患有慢性牙周炎的II型糖尿病患者随机分为PDT组(n=24)和对照组(n=24),牙齿洁治后1周行龈下刮治和根面平整(SRP)治疗,PDT组在此基础上于洁治后1周和SRP后6周行光动力疗法(PDT)治疗,对比患者疗效,空腹血糖(FGP)、糖化血红蛋白(HbAle)含量,牙周检查结果及基质金属蛋白酶-8(MMP-8)和白细胞介素-1β(IL-1β)水平的变化。结果:PDT组治疗总有效率95.83%,明显高于对照组(P<0.05);SRP治疗后3个月PDT组HbAle水平明显低于牙齿洁治后1周及对照组(P<0.05),SRP治疗后6周和治疗后3个月两组PD、AL、BI、PLI均显著下降,但与对照组相比,PDT组在下降更明显(P<0.05);治疗后两组MMP-8和IL-1β均明显下降(P<0.05),但PDT组下降更明显(P<0.05)。结论:光动力疗法能有效改善II型糖尿病患者牙周炎症状,降低患者炎症水平,效果显著,值得推广使用。