Effect of bovine lactoferrin on enamel demineralization and acid fermentation by Streptococcus mutans

The purpose of this study was to evaluate the effects of bovine lactoferrin on acid fermentation and enamel demineralization using Streptococcus mutans in a culture system and an artificial mouth model system. The antibacterial activity of bovine lactoferrin (bLF) against S. mutans was analyzed by a radial diffusion assay. In the culture system, the effect of bLF on the synthesis and adherence of water insoluble glucan (WIG) and the adherence of S. mutans to a glass surface was examined by a batch culture. In the artificial mouth model system, cell suspension of S. mutans, heart infusion broth supplemented with sucrose, and PBS or lactoferrin solution were supplied separately and constantly for 21 hours. The following parameters were determined for evaluation: the amount of artificial biofilm, the changes in pH underneath the biofilm; and the changes in enamel microhardness measured by a Vicker's hardness tester. The antibacterial activity of bLF against S. mutans was observed. The amounts of bacterial cells in the total adherent fractions were inhibited by bLF in a dose dependent manner. The amounts of WIG in a firm-adherent fraction were significantly inhibited by 0.1–1.0% bLF. The changes in microhardness on enamel slabs in the bLF group (2.4 ± 0.8) showed significantly less hardness reduction than those in the control group (22.3 ± 2.5) (P < 0.001). The artificial biofilm accumulation was not reduced by bLF. The results of this study suggest that bLF might have inhibitory effects against acid fermentation and demineralization of enamel by S. mutans.     

Bovine enamel hardness and its Ca-, P-, Mg- and F-contents modified by the bacterium Streptococcus mutans, artificial dental plaque and fluoride

A layer of cells from buffered Strep. mutans suspension (pH 5.8), with or without sucrose, was centrifuged on bovine enamel surfaces. Fluoride was added to a part of the samples (final F 25 parts/10(6]. Control and test samples were incubated at 37 degrees C for 18 h. The pH of the fermenting plaque dropped to 4.15. When F was present, the final pH was 4.45. Microhardness of the enamel surface was reduced by the presence of sucrose but less in the presence of F. Enamel Ca was liberated during fermentation, but only into the fluid and less in the presence of F. The weight ratio of Ca and P released by sugar fermentation was 3.14 and 1.91 when F was present. The F content of enamel surface was only slightly increased (130 parts/10(6]by the F in distilled water alone. Subsurface enamel gained more F (280 parts/10(6]. When artificial plaque was present, addition of F increased the F content of enamel surface by 450 parts/10(6) and F of subsurface by 210 parts/10(6). The addition of F increased the enamel F content to the greatest extent under the fermenting plaque, 680 parts/10(6) in surface and 400 parts/10(6) in subsurface enamel, compared to the values of the enamel under non-fermenting plaque.     

Protection of sound enamel and artificial enamel lesions against demineralisation: Caries infiltrant versus adhesive

Objective

To compare the protective potential of a conventional adhesive, a caries infiltrant and a combination of both against acidic challenge in vitro.

Methods

One-hundred-and-fifty discs from bovine lower central incisors were fabricated. Seventy-five samples remained untreated, whereas the other half was subjected to a demineralisation process (14 days, acidic buffer, and pH 5) to create artificial enamel lesions. Specimens were then radioactively irradiated, and each 15 sound and demineralised specimens were treated with a caries infiltrant (Icon, DMG), an unfilled adhesive (Heliobond, IvoclarVivadent) or a combination of infiltrant and adhesive. Specimens treated with the adhesive followed by a flowable composite (TetricEvoFlow, IvoclarVivadent) served as positive control, while untreated specimens served as negative control. All samples were then subjected to lactic acid for 3 weeks at pH 4. Loss of apatite was determined using the radiochemical method of liquid scintillation. Data were statistically analysed by Kruskal–Wallis-test, one-way ANOVA and Scheffe's post hoc tests (p ≤ .05).

Results

In both sound enamel and artificial caries lesions, untreated specimens showed the highest rate of apatite loss, whereas enamel treated with the adhesive and the flowable composite showed almost complete protection surface against dissolution. The caries infiltrant, the adhesive and the combination of both were able to decrease enamel dissolution, but the adhesive and the combination of adhesive and infiltrant were more effective than the infiltrant alone.

Conclusion

Within the limitations of this in vitro study, the application of an adhesive (alone or in combination with the caries infiltrant) is more effective to protect enamel dissolution than the infiltrant alone.     

Detection of Streptococcus mutans and Streptococcus sobrinus in dental plaque samples from Brazilian preschool children by polymerase chain reaction

The purposes of this study were to detect S. mutans and S. sobrinus by polymerase chain reaction (PCR) amplification, and to relate their presence to the incidence of dental caries in 42 Brazilian preschool children. Dental plaque samples were collected from the cervical margin of all erupted teeth of 5-6 years old children with primary dentition, using a sterile explorer. Examination of the dmft (decayed, missing, filled teeth) index, performed following the World Health Organization (WHO) caries diagnostic criteria, showed a 2.71 score. Prevalence of S. mutans and S. sobrinus was respectively, of 85.7% and 14.3%; no dental plaque sample was either positive or negative for both bacterial species. Children harboring either S. mutans or S. sobrinus presented the same caries prevalence. PCR showed good discriminative ability for differentiation between these species, and suggested that it is a technique suitable for epidemiological studies on mutans streptococci.     

人工牙菌斑致釉质表面钙,磷变化的化学分析

目的：采用多菌系人工龋菌斑模型系统对菌斑－牙釉质界面钙磷运动情况进行化学分析。方法：用微量化学分析法测定钙、磷含量。结果：菌斑一经接触糖后，ｐＨ开始下降，菌斑液中钙磷浓度随之增加，并且同ｐＨ下降程度成正相关。结论：认为早期龋形成中钙磷离子运动直接影响早期釉质龋表层下损害的形成。     

Antibacterial synergistic effect of chlorhexidine and hydrogen peroxide against Streptococcus sobrinus, Streptococcus faecalis and Staphylococcus aureus

Chlorhexidine (CHX) and Hydrogen peroxide (HP) are potent antibacterial agents that are used in controlling dental plaque. However, both agents bear undesired side-effects. We have tested the hypothesis that an antibacterial synergistic effect can occur between the two agents against Streptococcus sobrinus, Streptococcus faecalis and Staphylococcus aureus. We have found that at several combinations of HP and CHX an antibacterial synergistic effect does occur, while at other combinations a on-significant synergism was noticed. No antagonism between the two agents was found in our experimental system. It can be postulated that the mechanism of this synergistic effect is via alteration of the bacterial cell-surface by CHX thereby allowing for an increased amount of HP to penetrate and to react with the intercellular organelles of the bacteria. These results suggest that CHX and HP can be of use in controlling the dental plaque in the oral cavity.     

远缘链球菌黏附与游离状态下合成胞外多糖的比较

目的：比较远缘链球菌在游离和黏附状态下合成水溶性、水不溶性多糖的能力并探讨其原因。方法：在不同培养时间和多种蔗糖浓度条件下,对远缘链球菌进行摇动培养和静止培养,以模拟该菌在游离和黏附的状态下生长。采用蒽酮法测定细菌合成的水溶性和水不溶性多糖的含量,实验数据采用SPSS10.0软件包分析,选用单因素方差分析的Dunnett双侧检验比较组间的差异有无统计学意义。结果：生物膜形成发展期,游离菌合成的水溶性多糖随培养时间的延长而增多,黏附菌合成的水溶性多糖则随时间呈下降趋势;在生物膜成熟期,黏附菌合成的水不溶性多糖多于游离菌（P〈0．05）,而两者合成的水溶性多糖量的差异无显著性（P〉0．05）。细菌合成的水溶性、水不溶性多糖随蔗糖浓度的增加而增加;在各个蔗糖浓度条件下,黏附菌合成的水不溶性胞外多糖量显著多于游离菌（P〈0.05）,而水溶性胞外多糖的量差异不大（P〉0．05）。结论：培养时间和蔗糖浓度增加,导致远缘链球菌合成的水不溶性胞外多糖较游离状态明显增加;生物膜特殊的生长环境,是造成这种差异的可能原因。     

Stability and activity of specific antibodies against Streptococcus mutans and Streptococcus sobrinus in bovine milk fermented with Lactobacillus rhamnosus strain GG or treated at ultra-high temperature

Passive local immunization against dental caries is a promising approach to its prevention, as clinical evidence of active oral or nasal immunization is still limited and controversial. By means of systemic immunization of pregnant cows with a multivalent vaccine, high titres of IgG antibodies against human cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus, were produced in bovine colostrum. The purified immune product (IP) of this preparation has a number of anticariogenic properties, such as inhibition of streptococcal adherence to saliva-coated hydroxyapatite and inhibition of glucosyltransferase enzymes. This study investigated whether IP antibodies remained active and functional when added to ultra-high temperature (UHT)-treated milk or to Lactobacillus rhamnosus GG (LGG)-fermented milk stored for an extended time. LGG was chosen because of its widely known health benefits in humans and animals. A commercial UHT toddler's milk was supplemented with IP and stored for 2 months at 5, 21 and 30 degrees C. The antistreptococcal titres in UHT milk did not decline at any temperature during storage, and UHT-IP inhibited the adherence of S. mutans for up to 2 months. This was not the case with UHT toddler's milk without IgG antibodies. Milk was fermented with live LGG cells in the presence or absence of 5% IP. The antistreptococcal titres declined to about 30% of the original titres after storage. Fresh milk alone slightly enhanced streptococcal adhesion but fresh milk with IP inhibited the adherence of S. mutans by over 50%. LGG-positive fermented milk without antibodies also inhibited (P < 0.05) the adhesion by about 40%. In both LGG-fermented and UHT immune milk, the activity of antibodies against cariogenic streptococci was maintained during the expected shelf-life of these products. From the anticariogenic point of view it may be beneficial to add bovine-specific antibodies against mutans streptococci to probiotic LGG-containing milk products.     

Ultrastructural changes in Streptococcus sobrinus induced by xylitol, NaF and ZnCl2

The effect of xylitol, NaF, or ZnCl2 on the ultrastructure of Streptococcus sobrinus OMZ 176 was studied. The bacteria were grown statically in air for 8 h in brain-heart infusion broth supplemented with 33 mM Xylitol, 2.5 mM NaF, 0.5 mM ZnCl2, separately or in combination. The bacteria were washed briefly and prepared for scanning and transmission electron microscopy. All three agents and their combinations induced distinct ultrastructural alterations. The alterations were related to various aspects of cell morphology and varied in nature and extent among the agents. The most pronounced ultrastructural alterations were seen in bacteria grown in the presence of zinc ions. At the concentrations used, fluoride appeared to induce the least effect.     

Effect of desalivation and age on susceptibility to infection by Streptococcus sobrinus

The influence of desalivation and age on susceptibility to infection by Streptococcus sobrinus was studied using 80 Sprague-Dawley female rats weaned at age 21 days. Forty animals (donors) were infected with S. sobrinus 6715 (ATCC 27352), formed into ten groups of 4, fed diet 2000, and offered 10% sucrose water ad libitum. The remaining 40 animals were fed laboratory chow and sterile distilled water until introduced into the experiment. When the animals were aged 25 days, 10 noninfected animals were anesthetized, desalivated, and caged with 10 infected (donor) animals (group I). On day 36 these procedures were repeated on the remaining animals (group II). Infection was checked daily. Animals were regarded as infected when S. sobrinus was detected on 2 consecutive days and confirmed 1 week later. Animals were killed, jaws dissected, and populations of S. sobrinus and total bacterial viable count of each jaw estimated. The average number of days from pairing to detecting infection (2 consecutive days) was as follows: group I (age 25 days): desalivated 5.4 (SD 2.5), intact 8.8 (SD 4.5); group II (age 36 days): desalivated 7.8 (SD 2.9). Only 3 intact animals became infected in group II after a total elapsed time of 21 days. The mean viable counts of S. sobrinus per jaw in group 1 were as follows: desalivated 3.6 x 10(6) (SD 4.1); donor cagemate 13.0 x 10(6) (SD 9.7); intact 0.26 x 10(6) (SD 0.39); donor cagemate 4.5 x 10(6) (SD 4.3). Desalivated animals tended to be more heavily infected than intact animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of antibodies on chemiluminescence and on killing of Streptococcus sobrinus by polymorphonuclear leukocytes

The effect of a polyclonal antiserum and OMVU10, a monoclonal antibody reactive with Antigen B of Streptococcus sobrinus , on the interaction of polymorphonuclear leukocytes with S. sobrinus was studied, using chemiluminescence and bacterial killing assays. Increased stimulation of neutrophils as measured in the chemiluminescence assays was established when S. sobrinus was preincubated with polyclonal antiserum or when polyclonal antiserum was added to the reaction mixture. Higher counts were measured in comparison to preimmune serum. After 90 min, 52% of S. sobrinus preincubated with polyclonal antiserum was killed. Killing was also increased when polyclonal antiserum was added to the reaction mixture in comparison to the controls. No killing was found when bacteria were preincubated with OMVU10 or when OMVU10 was added to the reaction mixture in comparison to Clone 24, a control antibody.     

Stability and activity of specific antibodies against Streptococcus mutans and Streptococcus sobrinus in bovine milk fermented with Lactobacillus rhamnosus strain GG or treated at ultra‐high temperature

Passive local immunization against dental caries is a promising approach to its prevention, as clinical evidence of active oral or nasal immunization is still limited and controversial. By means of systemic immunization of pregnant cows with a multivalent vaccine, high titres of IgG antibodies against human cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus, were produced in bovine colostrum. The purified immune product (IP) of this preparation has a number of anticariogenic properties, such as inhibition of streptococcal adherence to saliva‐coated hydroxyapatite and inhibition of glucosyltransferase enzymes. This study investigated whether IP antibodies remained active and functional when added to ultra‐high temperature (UHT)‐treated milk or to Lactobacillus rhamnosus GG (LGG)‐fermented milk stored for an extended time. LGG was chosen because of its widely known health benefits in humans and animals. A commercial UHT toddler's milk was supplemented with IP and stored for 2 months at 5, 21 and 30°C. The antistreptococcal titres in UHT milk did not decline at any temperature during storage, and UHT‐IP inhibited the adherence of S. mutans for up to 2 months. This was not the case with UHT toddler's milk without IgG antibodies. Milk was fermented with live LGG cells in the presence or absence of 5% IP. The antistrept?ococcal titres declined to about 30% of the original titres after storage. Fresh milk alone slightly enhanced streptococcal adhesion but fresh milk with IP inhibited the adherence of S. mutans by over 50%. LGG‐positive fermented milk without antibodies also inhibited (P < 0.05) the adhesion by about 40%. In both LGG‐fermented and UHT immune milk, the activity of antibodies against cariogenic streptococci was maintained during the expected shelf‐life of these products. From the anticariogenic point of view it may be beneficial to add bovine‐specific antibodies against mutans streptococci to probiotic LGG‐containing milk products.     

远缘链球菌g型对铸钛、钴铬合金、镍铬合金修复体腐蚀失泽的研究

目的:研究远缘链球菌g 型对铸钛、Co-Cr 合金、Ni-Cr 合金修复体失泽腐蚀的影响。方法:将Co-Cr 合金、Ni-Cr合金、纯钛分别铸造制备成10 mm×10 mm×1 mm板片,每组30 片。将上述3 组每组随机分空白对照组,培养基对照组、实验组各10 片。需氧环境中,将g 型远缘链球菌接种到蔗糖琼脂培养基后,将试件贴附其表面,每周转换传递一次,共10 周。0105 %戊二醛消灭细菌,清洗,2 周后用MINOLTA CR-100 型色度计观察。结果:经统计学分析可知铸钛、Co-Cr 合金、Ni-Cr 合金修复体的空白对照组与培养基对照组的L*、a*、b*值无显著差异( P > 0105) 。铸钛、Co-Cr 合金、Ni-Cr 合金修复体实验组与空白对照组、培养基对照组的L*、a*、b*值有明显差异( P < 0105) 。结论:按CIE1976 标准检测得出远缘链球菌g 型对Co-Cr 合金、Ni-Cr 合金、铸钛修复体色泽有影响,但未超出黄绿区域。     

Inhibition of the growth of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei by oral peroxidase systems in human saliva

Streptococcus mutans, Strep. sobrinus and Lactobacillus casei were grown in glucose-supplemented, sterilized, human whole saliva, adjusted to pH 5, 6 or 7. Components of the antibacterial peroxidase system--hypothiocyanous acid (HOSCN) and hypothiocyanite ions (OSCN-)--were generated by adding exogenous H2O2 to sterilized saliva containing endogenous peroxidases and thiocyanate (SCN-) ions. HOSCN/OSCN- generation was proportional to the amount of H2O2 added, and more HOSCN/OSCN- was detected in saliva at pH 7 than at pH 5. However, the growth of mutans streptococci and L. casei was inhibited at pH 5 by HOSCN/OSCN-, whereas no inhibition was found at pH 7. The findings show that (a) sufficient amount of HOSCN/OSCN- will inhibit the growth of cariogenic bacteria in human saliva at pH 5; (b) this amount of HOSCN/OSCN- can be generated in saliva by exogenously added H2O2; and (c) peroxidase systems have stronger antistreptococcal effects in human whole saliva than in phosphate buffer.     

The effect of artificially produced pellicle and plaque on the uptake of 18F by human enamel in vitro.

The presence of artificial pellicle and plaque alters the mechanism of uptake of ¹⁸F by enamel from an acidulated phosphate fluoride solution (APF) labelled with ¹⁸F but has no apparent effect on the mechanism of uptake of the isotope from a labelled solution of NaF with a similar fluoride content (1.2 per cent). The presence of artificial plaque has no significant effect on the quantitative uptake of ¹⁸F by enamel from labelled solutions of either APF or NaF in 4 min. Results from a similar experiment using plaque formed in vivo support these findings.     

Suitability of human, bovine, equine, and ovine tooth enamel for studies of artificial bacterial carious lesions

The progress of artificial caries-like lesions created in human, bovine, equine, and ovine enamel has been studied. Lesions were produced by exposure to an acid gel system or by 5-day exposure to a sequential batch culture technique using Streptococcus mutans NCTC 10832. Longitudinal ground sections were prepared. The lesions were of similar appearance in all species when examined in polarized light. The depth in human enamel was approximately half that in the animal species. Microradiography confirmed subsurface demineralization in all four species. Similar depth ratios were seen in the scanning electron microscope, but there were structural differences between lesions in human and animal teeth. Lesions in bovine teeth were more like those in human, but lesions in equine and ovine teeth were markedly different. Substitution of these animal enamels for human enamel in caries experiments demands that these differences be taken into account. Scanning electron microscopy is capable of resolving features in artificial caries lesions which cannot be differentiated by polarized light techniques. The latter will demonstrate generalized mineral loss, but scanning electron microscopy is required to characterize the sites of mineral loss.     

The Ca, Pi, F, and proline content of developing bovine enamel under GBHA-stained and unstained bands

The surface enamel of fetal bovine teeth was stained with GBHA to indicate the position of bands of smooth-ended and ruffle-ended ameloblasts relative to the developing enamel. The boundaries of the bands were scored, under a dissecting microscope, and the bulk enamel under each band was collected. The enamel samples were assayed for Ca, Pi, F, and proline. The amount of Ca and Pi in the enamel increased in successive bands and seemed unrelated to the overlying ameloblast cell type. The loss of proline seemed unrelated to cell type. The fluoride content of enamel increased by approximately 50% in the first stained band immediately adjacent to the secretory zone. The F level returned to secretory values in the succeeding unstained band. Thus, only changes in the F level of developing enamel appeared to be related to GBHA staining patterns.