五倍子在体外生物膜模型中对口腔常驻菌形成的致龋生物膜的影响

目的　研究五倍子对口腔常驻菌形成的人工致龋生物膜的影响,探讨其防龋应用前景。方法　在体外模型中的羟基磷灰石片上定植4种口腔常驻菌,以形成实验性生物膜。实验组定时给予质量浓度4 g/L的五倍子提取物的蔗糖溶液处理生物膜,观察其pH的动态变化,生物膜表面微结构与微生物组成的改变。以同质量浓度的蔗糖溶液与蒸馏水分别作为阳性与阴性对照。结果　①实验组培养室内pH值呈先下降,再逐步恢复正常的波浪形变化;②五倍子的加入能抑制变形链球菌对羟基磷灰石片的粘附;③五倍子的加入可使生物膜表面胞外基质的量减少。结论　五倍子作为一种潜在的防龋药物可能有着良好的应用前景。     

柠檬提取物对变形链球菌乳酸脱氢酶和蔗糖酶活性的影响

目的 观察柠檬提取物对变形链球菌(Streptococcus mutans,Sm)乳酸脱氢酶、蔗糖酶活性的影响,探讨柠檬酸提取物抑制Sm致龋活力的相关机制.方法 采用二倍稀释法,用含2%蔗糖的胰蛋白胨大豆肉汤将柠檬提取物的抑菌浓度稀释为0.64、0.32、0.16、0.08及0.04 g/L共5个质量浓度的溶液(5个实验组),以胰蛋白胨大豆肉汤液体培养基作为空白对照组.加入Sm菌液,厌氧培养6、18、24及48 h,采用还原性辅酶Ⅰ氧化法测定乳酸脱氢酶活性、用pH计测定培养液的pH变化值(△pH),同时采用3,5-二硝基水杨酸显色法测定蔗糖酶的活性.结果 随着柠檬提取物浓度的升高(0.04～0.64 g/L),乳酸脱氢酶、蔗糖酶活性和△pH均逐渐降低(P＜0.01):加入Sm厌氧培养24 h后,Sm乳酸脱氢酶活性从(0.8025±0.0913)×103 U/L降至(0.2099±0.0283)×103 U/L,Sm蔗糖酶活性从(-0.0107±0.0003)×103U/L降至(-0.0078±0.0002)×103 U/L,Sm △pH从2.8067±0.0404降至2.5033±0.0416(24 h).各实验组之间及与空白对照组之间差异有统计学意义(P＜0.01);柠檬提取物对Sm产酸的抑制作用与对乳酸脱氢酶活性的抑制作用之间呈正相关(r=0.8120～0.9918,P＜0.01).结论 低于抑菌浓度的柠檬提取物对Sm乳酸脱氢酶活性和蔗糖酶活性及产酸能力都具有显著抑制作用,作用强度具有浓度依赖性,对对数期细菌抑制作用强于其他生长周期,具有防龋药物的潜能.     

银杏叶提取物对口腔致龋变形链球菌的作用研究

目的 :为探讨天然植物银杏叶的提取物对临床分离的致龋变形链球菌的体外抗菌实验。方法 :随机选取DMFT≥ 3的成人 5 4名 ,取牙菌斑接种于培养基中 37℃厌氧培养筛选传代 3次 ,每株均在 6个不同的药物浓度下进行抑菌实验 ,观察最小抑菌浓度 (MIC)。结果 :银杏叶的提取物对临床筛选出的 33例致龋变形链球菌株 ,最小抑菌浓度MIC5 0和MIC90分别为6 2 5g/L和 12 5g/L。结论 :在天然植物的提取物中 ,银杏叶提取物对临床致龋变形链球菌有较好的抑制作用     

中药蜂房对口腔常驻菌形成人工致龋生物膜影响的实验研究

目的　研究蜂房对口腔常驻菌形成人工致龋生物膜的影响,探讨其防龋应用前景。方法　在体外模型中的羟磷灰石片上定植4种口腔常驻菌,以形成实验性生物膜。实验组定时给予含4 g/L蜂房提取物的蔗糖溶液处理生物膜,观察其pH的动态变化,以及生物膜表面微结构与微生物组成的改变。以同浓度的蔗糖溶液与蒸馏水分别作为阳性与阴性对照。结果　①实验组培养室内pH值呈先下降,再逐步恢复正常的波浪形变化。②蜂房的加入能抑制变链对羟磷灰石片的黏附。③蜂房的加入可使生物膜表面胞外基质的量减少。结论　蜂房作为一种潜在的防龋药物可能有着良好的应用前景。     

菌斑固相、变形链球菌及葡聚糖对酸的缓冲作用

目的研究糖代谢后菌斑固相缓冲力的变化及影响因素。方法采集40名18~21岁的大学生的饥饿牙菌斑,体外10%蔗糖孵育1h。体外制备无糖培养和2%蔗糖培养的变链菌团。以25mmol/L KCl制备菌斑固相、变链菌团和不溶性葡聚糖混悬液及可溶性葡聚糖溶液,用1mmol/LHCl滴定并计数细菌密度,统计学分析。结果变链菌团代谢蔗糖后的缓冲容量为(0·099±0·047)mmol/L,比无蔗糖培养的变链菌团的缓冲容量(0·609±0·202)mmol/L低,且缓冲力随细菌密度降低直线下降,葡聚糖几乎没有缓冲作用[(0·028~0·032)mmol/L]。人牙菌斑固相缓冲力的变化规律与体外纯菌培养研究结果一致。结论菌斑固相缓冲力与所含细菌密度密切相关。     

内氏放线菌尿素酶对牙菌斑生物膜酸碱平衡调节作用的初步研究

目的：初步探讨在牙菌斑生物膜天然环境中,内氏放线菌尿素酶能否发挥高效尿素水解反应,以及尿素水解对口腔环境中pH值的调节作用。方法：通过酶促反应动力学实验寻找内氏放线菌尿素水解的最适条件．监测尿素水解调节细菌产酸后的pH值变化。采用SPSS软件包,对酶促动力学实验数据进行线性回归与相关分析。结果：内氏放线菌尿素酶米氏常数Km=7．5mmol／L,在口腔中正常尿素浓度3-10mmol／L范围内,内氏放线菌尿素酶催化活性大约保持在最大活性的20％-63％;内氏放线菌尿素酶最适pH值=6．5,但是在牙菌斑临界pH5．0,尿素酶活性仍保持40％的最大活性;在口腔正常尿素浓度范围内,内氏放线菌尿素水解产物中和细菌产酸后,pH值不会下降到牙菌斑临界pH5．0以下。结论：在牙菌斑生物膜中,内氏放线菌尿素酶可以发挥高效尿素水解反应．尿素水解对口腔环境pH值具有明显的调节作用。     

伊犁黑蜂蜂胶对口腔主要致龋细菌生物膜作用的实验研究

目的 研究伊犁黑蜂蜂胶对口腔常见致龋细菌生物膜的影响,探讨其防龋效果及可能的防龋机制。方法 通过结晶紫染色法测定黑蜂蜂胶对口腔常见致龋菌（变异链球菌、表兄链球菌、血链球菌、黏性放线菌、内氏放线菌）的最小生物膜清除浓度（MBEC）;培养测试细菌24 h单菌生物膜,加入MBEC及以下的3个浓度配置成初始pH值为7.0的含药培养基,厌氧培养24 h后测pH值,并计算pH变化值以检测不同浓度黑蜂蜂胶对测试菌单菌生物膜产酸能力的影响。蒽酮法测定MBEC及以下的3个浓度的黑蜂蜂胶对变异链球菌24 h单菌生物膜产生水不溶性胞外多糖的影响。结果 黑蜂蜂胶对变异链球菌、表兄链球菌、血链球菌、黏性放线菌、内氏放线菌的MBEC分别是6.25、1.56、3.13、0.78、0.78 mg•mL-1;黑蜂蜂胶可使各测试菌单菌生物膜ΔpH降低,蜂胶各组与对照组相比差异均有统计学意义（P<0.05）;在MBEC浓度时,蜂胶可使变异链球菌合成水不溶性胞外多糖的能力降低。结论 伊犁黑蜂蜂胶具有一定的防龋效果,其可能的防龋机制是通过有效清除口腔主要致龋细菌单菌生物膜,抑制测试菌株产酸、合成水不溶性胞外多糖的能力起作用的。     

多孔HAPw/n-ZnO骨修复材料的抗菌机理及抗生物膜研究

目的:评估多孔羟基磷灰石晶须/纳米氧化锌(HAPw/n-ZnO)骨修复材料在抑制细菌生物膜中的作用,并探讨其抗菌机理。方法:分别建立金黄色葡萄球菌、变形链球菌、牙龈卟啉单胞菌生物膜体外模型。无光条件在相同的多孔HAPw/n-ZnO浓度梯度(0~2 g/L)下分别进行生物膜抑制试验与细胞内活性氧(ROS)检测,并利用扫描电子显微镜分析材料性能,未加入材料组作为对照。结果:无光条件下随着材料浓度递增,各浓度多孔HAPw/n-ZnO组(1.00、1.25、1.50、1.75、2.00 g/L)相对于对照组(0 g/L),3种细菌的生物膜形成量(A值)逐渐减少以及细胞内活性氧(ROS)水平(荧光强度)逐渐升高,差异有统计学意义(P＜0.05)。扫描电镜图片显示,复合材料组细菌黏附减少,生物膜形成受到抑制;细胞形态异常,细胞膜受损导致内容物泄露。结论:无光条件下多孔HAPw/n-ZnO材料对金黄色葡萄球菌、变形链球菌、牙龈卟啉单胞菌生物膜均有一定程度的抑制作用,其抗菌机理之一是活性氧(ROS)的形成。     

木糖醇对黏性放线菌生长及产酸影响的体外研究

目的对比不同质量浓度下木糖醇对黏性放线菌生长及产酸的影响。方法分别用含不同质量浓度（128、64、32、16、8、4 g·L^-1）木糖醇的脑心浸液（BHI）液体培养基在厌氧条件下培养黏性放线菌,测定其最小抑菌质量浓度（MIC）;然后测量对照组以及1/2、1/4、1/8 MIC和MIC质量浓度时培养1.5、3、6、12、24、48 h液体培养基的pH值,计算ΔpH值,同时测量2、4、6、8、10、12 h液体培养基的光密度（OD550）值;最后测定抑制50%及 90%黏性放线菌生物膜形成的最低木糖醇质量浓度（即MBIC50和MBIC90）。运用SPSS 19.0进行数据分析。结果木糖醇能抑制黏性放线菌的生长,MIC为64 g·L^-1。培养12 h后,各组之间的ΔpH值差异均有统计学意义（P<0.05）,且ΔpH值随MIC质量浓度的增大而减小;此时,除1/2 MIC以及MIC组之外,其余各组的OD550差异均无统计学意义（P>0.05）,OD550值随MIC质量浓度的增加而减小。提示黏性放线菌在含1/2 MIC及MIC的木糖醇培养基里生长及产酸能力随木糖醇质量浓度的升高而下降。木糖醇的MIBC50为64 g·L^-1,MIBC90为128 g·L^-1,说明木糖醇培养基能抑制黏性放线菌生物膜的形成。结论木糖醇能有效地抑制黏性放线菌的生长、黏附以及产酸,有一定的抑制致龋菌和防治龋病的效果。     

不同pH对变异链球菌体外生物膜形成的影响

目的：了解变异链球菌临床株体外生物膜形成规律以及不同pH对生物膜形成的影响。方法：采用微孔板培养,染色、分光光度测定法（A630）绘制体外不同pH条件下（pH=7.0～5.0）593号、18号菌株以及变异链球菌标准株（ATCC25175）的生物膜生长曲线。结果：体外变异链球菌各株在pH=5.0时均不能形成生物膜;pH=7.0时细菌生物膜形成表现为缓慢的非线性生长,12～24h生物膜开始成熟,24～36h出现一相对的生长停滞期;pH=5.0时对已形成12h的变链菌生物膜生长有明显的抑制作用,但经历12h的酸休克后各菌株的生物膜均能恢复生长。结论：变异链球菌在体外pH=7.0时于12～24h形成稳定的生物膜,该生物膜能抵抗一定程度的酸（pH=5.0）攻击,而浮游状态的细菌则不能。     

Inhibition of acid production in dental plaque bacteria by green tea catechins

The inhibition of acid production from dental plaque and mutans streptococci by epigallocatechin gallate (EGCg), one of the green tea catechins, was examined. The effect of EGCg solution on dental plaque pH was investigated.Subjects rinsed their mouths with 2 mg/ml EGCg solution and then, after 30-min interval, rinsed their mouths with 10% sucrose. Plaque samples were collected at appropriate times and the pH was measured. The pH values of plaque samples from 15 volunteers were significantly higher after treatment with catechin than after treatment with water. EGCg inhibited pH fall when cariogenic bacteria grown in medium with or without sucrose were incubated with sugar. In medium without sucrose, cultured cells were killed time-dependently by EGCg treatment. However, EGCg did not kill cells cultured in medium containing sucrose. Also, EGCg did not kill oral streptococci adhering to a saliva-coated hydroxyapatite disk. EGCg and epicatechin gallate inhibited lactate dehydrogenase activity much more efficiently than epigallocatechin, epicatechin, catechin or gallocatechin. These results suggest that EGCg is effective in reducing acid production in dental plaque and mutans streptococci.     

The effects of different concentrations of sucrose, fructose, and glucose on pH changes by Streptococcus mitior in an artificial mouth

The aim of this study was to determine the effects of the initial sucrose (S) concentration, as well as those of fructose (F), glucose (G), and invert sugar (F/G), on the pH developed by a layer of S. mitior, to represent dental plaque, in an artificial mouth which simulates the process of oral sugar clearance. At S, F. G, and F/G concentrations of 2% and with normal oral sugar clearance rates, the bacteria produced a smaller pH fall from S than from the other sugars; at initial concentrations of 20%, however, the differences were not significant. With constant S concentrations of 0.5-35%, the minimum pH reached was 4.03 +/- 0.14 (S.D.); with S concentrations of 50% and above, slightly but significantly higher values (4.41 +/- 0.34) occurred. However, with normal sugar clearance, the pH fall was much less than with a constant concentration and was dependent on the S concentration over the range of 0-10%, but was independent at higher concentrations. Exposure of the bacteria to S for as short a period as two min during normal sugar clearance gave a nearly maximum pH fall. This suggests that rinsing the mouth with water more than two min after consumption of sucrose in liquid form will have very little effect in reducing the pH fall in dental plaque. A more appropriate method for reducing acid formation by dental plaque would be consumption of a salivary stimulant which would increase the flow rate and buffer capacity of the saliva.     

菌斑pH值、游离钙和总蛋白水平与根龋易感性的关系

目的　分析菌斑液成分的动态变化与根龋易感性的关系。方法　比较无根龋组和有根龋组根面牙菌斑液在糖溶液漱口前后 ,所测定pH值、游离钙 (微电极法 )和总蛋白水平 (分光光度法 )的差异。结果　静止状态下 ,无根龋组和有根龋组的pH值 (无根龋组 6 2 4± 0 74,有根龋组 5 96±0 70 )和游离钙 [无根龋组 (0 6 0± 0 46 )mmol/L ,有根龋组 (0 89± 0 5 4)mmol/L]两项指标差异无统计学意义 ;糖漱口后 ,有根龋组的pH(5 14± 0 19)和游离钙 [(1 73± 0 74)mmol/L]水平均低于无根龋组[pH 5 2 8± 0 16 ,游离钙 (2 73± 1 2 5 )mmol/L],差异有统计学意义。总蛋白水平与游离钙含量之间没有相关性。同一个体牙根面与冠面之间菌斑液的三项指标均有显著的相关关系。结论　有根龋组的菌斑液有较强的致龋倾向 ,在细菌酵解糖产酸方面 ,牙根面 (牙骨质 )菌斑与牙冠面 (牙釉质 )菌斑无根本差别     

Effect of dental plaque age and bacterial composition on the pH of artificial fissures in human volunteers

Changes in sucrose-induced plaque pH profiles and the microbial composition of occlusal tooth surface fissures were analyzed using wire telemetry and bacterial culturing techniques. Four human volunteers wore appliances containing artificial fissures constructed with ion-sensitive field-effect transistor (ISFET) electrodes for 1, 2 and 4 days; 1 subject kept the electrode for 3 weeks. After monitoring the plaque pH response at the base of the fissure to a 10% (w/v) sucrose rinse the plaque was removed and analyzed for total viable bacteria, total and specific streptococci, lactobacilli and Actinomyces spp. One-day-old plaque showed a rapid drop in plaque pH to a minimum of 4.8 +/- 0.2, with 2-day-old plaque showing the most acidogenic pH profile (minimum pH 4.6 +/- 0.2). The 4-day-old plaque response was less acidogenic (minimum pH 5.0 +/- 0.3) than the results from days 1 and 2. Responses from 13- and 21-day-old fissure plaques showed greatly decreased acidogenic responses (day 21 minimum pH 5.7). Viable bacteria recovered from the fissure increased from approximately 4 x 10(6) colony-forming units on day 1 to 1.2 x 10(7) on days 2 and 4 and 1.7 x 10(7) on day 21. Streptococci (greater than 50%) and Actinomyces (greater than 10%) dominated in the fissure plaques and their levels were related to minimum pH. Since fissure plaque of all ages tested contained high concentrations of acidogenic bacteria, the decreased acidogenic response at the base of fissures with increasing plaque age suggests that maturing fissure plaques provide an increasingly greater diffusion barrier to fermentable carbohydrates.     

Human dental plaque microcosm biofilms: effect of nutrient variation on calcium phosphate deposition and growth

Plaque mineralisation is a multi-factorial process involving plaque pH, nucleation, inhibitors and promotors. It is poorly understood because of its complexity. OBJECTIVE: To establish the effects of amino acids and peptones in the simulated oral fluid BMM, a saliva analogue DMM and modifications of these on mineral deposition into dental plaque biofilm microcosms. METHODS: Microcosms were cultured for up to 35 days in an Artificial Mouth pulsed with sucrose, followed by 10 days periodic treatment with a pH 5.0 calcium-phosphate-monofluorophosphate-urea solution (CPMU). RESULTS: Initial biofilm doubling times were 3-7h, which then slowed and varied under the different nutrient conditions although their pH behaviour was similar. In BMM, mineral deposition was 20% that of DMM, but removal of BMM peptones increased deposition 12-fold. Substitution of the amino acids in DMM by casein did not affect deposition levels, but their removal leaving mucin the sole macronutrient, increased mineral deposition three-fold, reaching 40 mmol Ca/g protein. CONCLUSIONS: These substantial increases in mineral deposition when the macronutrient concentration is reduced indicates probable changes in the nucleating, inhibitory and Ca-binding properties of the simulated oral fluids themselves and/or changes in the plaque microbiota and their crystal nucleators and inhibitors.     

A comparison of human dental plaque microcosm biofilms grown in an undefined medium and a chemically defined artificial saliva

The growth and pathogenic properties of dental plaque result from interactions between the microbiota and the oral environment and have been studied in laboratory experimental systems ranging from single or a few species (such as in chemostats) to dental plaque microcosms. Microcosm plaque is an in vitro version of natural plaque and has been explored as a microflora model because it is sited a more manipulable and controllable environment. It is obtained as microcosm biofilms in an 'artificial mouth' plaque culture system by culturing the bacteria in natural plaque-enriched saliva (i.e. salivary bacteria where a whole-saliva donor has abstained from oral hygiene for 24 h to increase the plaque bacteria in the saliva). The aim here was to examine whether a new, chemically defined analogue of saliva (defined medium mucin, DMM) could substitute for a previously used, chemically undefined medium (basal medium mucin, BMM) as an analogue of saliva for large-scale biofilm culturing. DMM contains various ions, mucin, amino acids, vitamins and growth factors at concentrations generally similar to those in saliva, whereas BMM contains yeast extract, peptones and mucin. To model the nutrient functions of salivary proteins, amino acids equivalent to 5 g/l casein were also included in DMM. In earlier studies, BMM-grown plaques were similar to natural plaques in structure, composition, growth rate and pH response to substrates. Their doubling-time patterns over a 20-day period were similar, except that the DMM-grown plaques showed biphasic growth patterns that were more pronounced than with BMM. Variation in enzyme profiles between BMM- and DMM-grown plaque, measured using the API-ZYM technique, provided evidence of nutritional effects on plaque composition. It was concluded that realistic growth rates and patterns are generated in microcosm plaque biofilms by supplying both DMM and BMM. However, the use of DMM enables specific modifications to be made to nutrient conditions during large-scale culture in our 'artificial mouth' biofilm system.     

Composition of dental plaque formed in the presence of sucrose and after its interruption

Since dental plaque reservoirs of fluoride (F), calcium (Ca) and inorganic phosphorus (Pi) are susceptible to decreases in pH, this in situ crossover study was conducted to test the hypothesis that the low concentration of these ions in plaque, formed in the presence of sucrose, could be attributed merely to the fermentation of this sugar. Eleven volunteers wore palatal appliances containing 6 human enamel blocks during two stages. In each stage, the treatments were either 20% sucrose solution or distilled deionized water, which were dripped onto the blocks 8 times a day. After 28 days, in each stage, the dental plaque formed on two blocks was collected, the treatment was inverted and after a further 24 and 48 h, the biofilm formed was collected from the other blocks. The concentration of acid-soluble F, Ca and Pi, and the concentration of insoluble polysaccharide (IP) were determined in the dental plaque. Statistically lower concentrations of F, Ca and Pi, and a higher concentration of IP were found in the 28-day biofilm formed in the presence of sucrose than in its absence; after the treatment inversion the change in F, Ca and Pi was not statistically significant, but the IP concentration changed significantly. The hypothesis was rejected because change in concentration of F, Ca and Pi is not due to fermentation of the sucrose.     

Method for determination of invertase activity in homogenates of human dental plaque

Supragingival human dental plaque was collected from patients with evidence of caries. The plaque was frozen and stored at -20 degrees C. Pooled plaque was homogenized in acetate buffer pH 5.0 in an ice-water bath. By incubating the homogenate at pH 5.0 with [U-14C]-sucrose the formation of glucose and fructose was followed. Incubation in acetate buffer at pH 5.0 eliminated the glycosyltransferase activities and the glycolytic pathway. Normal Michaelis-Menten kinetics were observed until about 40 mM sucrose. At higher concentration of sucrose, excess substrate inhibition occurred. Storage of the homogenate at -20 degrees C resulted in decrease of the invertase activity with time.     

Method for determination of invertase acitivity in homogentes of human dental plaque

Abstract – Supragingival human dental plaque was ecllected from patients with evidence of caries. The plaque was frozen and stored at-20°C. Pooled plaque was homogenized in acelate buffer pH 5.0 in an ice-water bath. By incubating the homogenate at pH 5.0 with [U-°C]-sucrose the formation fo glucose and fructose was followed. Incubation in acetate buffer at pH 5.0 eliminated the glycosyltransferase activities adn teh glycolytic pathway. Normal Michaclis-Menten kinetics were observed until about 40 mM sucrose. At highert concentration of sucrose, excess substrate inhibition occurred. Storage of the homogenate at −20° C resulted in decrease of the invertase activity with time.