Epithelial cells associated with the development of dental plaque.

Transmission and scanning electron microscopy were used to study desquamated epithelial cells in the developing and mature dental plaque. Epithelial cells were found attached to enamel as early as one hour after clean enamel cylinders were placed in the mouth, and they were seen in great numbers after two days. These cells generally had bacteria attached to their surfaces. Epithelial cells adsorbed to the surface of the enamel appeared to alter their morphology, and mineralization of these structures was also observed. Desquamated epithelial cells could play a role in the initial bacterial colonization of tooth enamel since it is known that bacteria selectively adsorb onto their surfaces. Hydroxyapatite may affect the morphologic alteration of epithelial cells adsorbed to tooth enamel. From the observation of developing and mature plaque samples, it seems that certain suprasurface pelliclelike structures might be epithelial cells.     

十二烷基硫酸钠抗牙菌斑作用的实验研究

十二烷基硫酸钠（ｓｏｄｉｕｍｄｏｅｃｙｌｓｕｌｆａｔｅ，ＳＤＳ）常作为去垢剂广泛地应用于牙膏之中。已证实ＳＤＳ与羟基磷灰石（ＨＡ）有高度亲合力，通过ＳＤＳ与ＨＡ的结合，干扰蛋白质对ＨＡ的吸附，从而影响牙面获得性膜的形成。为进一步了解ＳＤＳ的抑菌和抗菌斑作用，本研究利用口腔恒化器和扫描电镜及同位素标记液闪检测技术，对ＳＤＳ的抗菌斑作用进行了观察。结果显示，经ＳＤＳ处理后的牙釉质表面没有成熟的牙菌斑形成，仅有少数细菌散在附着，其放射性明显少于对照组。提示ＳＤＳ具有抗菌斑作用，是牙膏中的天然抗菌斑成分之一。     

The "plaque-free zone" in health and disease: a scanning electron microscope study.

A "plaque-free" zone has been described on the enamel surface of healthy extracted teeth. This study examined this zone on chronic adult periodontitis-affected teeth (CAPT). Ten healthy controls and 16 CAPT were collected immediately after extraction, fixed, dehydrated, critical point dried, coated, and viewed by scanning electron microscopy (SEM). The "plaque-free" zone was observed in both groups as an area with few bacteria between the apical plaque border and the coronal limit of an epithelial layer on the root surface, extending to the residual periodontal ligament. On the healthy specimens, the apical plaque border consisted mainly of cocci and short rods, while on the CAPT specimens spirochetes predominated. Isolated or small groups of microorganisms were always present in the "plaque-free" zone and at its apical limit, close to or in contact with junctional epithelial cells. This zone is therefore not completely free of plaque, as suggested. It was concluded that a tissue complex, analogous to that in health on enamel, persists on the root surfaces of CAPT throughout the disease process. It comprises a discrete plaque border, a dental cuticle with sparse organisms, and an epithelium analogous to junctional epithelium. Its main function would appear to be to prevent bulk access of plaque to the surrounding tissues, including direct contact of bacteria with underlying ligament.     

建立口内菌斑模型研究菌斑超微结构

目的:系统了解菌斑形成的超微结构。方法:建立一种口内的菌斑模型,并将1、5、9d形成的菌斑标本制成电镜切片后用透射电镜观察。结果:显示初期菌斑的细菌较少,主要为球状菌。随着时间的延长,细菌的数量和种类开始增加,出现丝状菌和杆状菌。成熟菌斑结构是两侧为丝状菌中间为球状菌的栅栏状结构,其中部分细菌会出现坏死的现象。结论:所建立的菌斑模型较接近天然菌斑结构,具有一定的参考价值。     

Studies on artificial dental plaque. Preliminary observation on the morphology of developing artificial dental plaque

This paper introduced a new method which was used to study the formation of dental plaque in vitro. From this investigation, it can be regarded that artificial dental plaque is similar to natural dental plaque in structure. In the 1st day, artificial dental plaque was mainly coccal shaped bacteria adherent on the tooth enamel. At the third day, the number of filaments and rods increased obviously, and in 5th and 7th day plaques, main bacteria were filaments, with a few rods and cocci, and the special palisade and corn cob structures were occurred. The morphology of 2-week, 3-week and 4-week plaque were almost the same, but denser in structure.     

Enamel decalcification by natural dental plaque in sugar solutions

The edge-decalcification technique has been employed to observe the decalcification of enamel in vitro by dental plaque obtained in vivo. Four different factors were studied for their relative importance on penetration rate of enamel under attack by small quantities of natural dental plaque, treated with sugar solutions. The factors were the kind of sugar and its concentration, the concentration of buffer and the time of exposure of the plaque to a sugar solution. Concentration of buffer and the kind of sugar, whether sucrose or glucose, appeared to be unimportant under the conditions selected. The concentration of sugar was unimportant over the range of 1 molar to 0.1 molar and only a 20 per cent decrease in depth of penetration was found where the concentration of glucose was reduced to 10 millimolar.

Plaque treated 30 min per day with sugar caused changes in the underlying enamel which was only slightly less than plaque treated for 5 hr per day, and only 45 per cent less than those treated 24 hr per day. The surprising finding was the relatively high values of enamel penetration that occurred when plaque had access to sugar only 2 per cent of the time.     

Early formation of dental plaque on plastic films

This study was designed to gain further information on early plaque formation using a previously described method employing plastic films for the collection of the initial deposits.
To prevent the loss of loosely adhering material during processing of dental plaque for microscopic observation, the method was further developed by introducing an agar protection of the deposits formed on plastic films prior to fixation, dehydration and embedding procedures.
Four human subjects with healthy gingiva developed microbial plaque during periods of 4 and 8 hours on plastic films applied on the buccal surfaces of premolars and cuspids. After 4 hours the plastic films were covered by a surface coating of an acellular material in or on which bacteria, epithelial cells and leukocytes were observed. The microorganisms were almost exclusively identified as Gram-positive cocci. The deposit in the 8 hour samples exhibited the same general morphology, but with more cellular components. Bacterial aggregation to each other or to epithelial cells was frequently mediated by filamentous appendages on the bacterial cell walls. The frequent observation of a close association between bacteria and epithelial cells suggests that the latter may play an important role as a vehicle for transporting microorganisms to the solid surfaces in the mouth in the initial stage of plaque formation.     

The influence of surface free energy and surface roughness on early plaque formation

Previous in vivo studies suggested that a high substratum surface free energy (s.f.e.) and an increased surface roughness facilitate the supragingival plaque accumulation. It is the aim of this clinical trial to explore the "relative" effect of a combination of these surface characteristics on plaque growth. 2 strips, one made of fluorethylenepropylene (FEP) and the other made of cellulose acetate (CA) (polymers with surface free energies of 20 and 58 erg/cm2, respectively) were stuck to the labial surface of the central incisors of 16 volunteers. Half the surface of each strip was smooth (Ra +/- 0.1 microns) and the other half was rough (Ra +/- 2.2 microns). The undisturbed plaque formation on these strips was followed over a period of 6 days. The plaque extension at day 3 and 6 was scored planimetrically from color slides. Finally, of 6 subjects samples were taken from the strips as well as from a neighbouring smooth tooth surface (s.f.e. 88 erg/cm2; Ra +/- 0.14 microns). These samples were analysed with a light microscope to score the proportion of coccoid cells, and small, medium, and large rods or fusiform bacteria. At day 3, a significant difference in plaque accumulation was only obtained when a rough surface was compared with a smooth surface. However, at day 6, significantly less plaque was recorded on FEP smooth (19.4%) when compared with CA smooth (39.5%). Between FEP rough (96.8%) and CA rough (98.2%), no significant difference appeared.(ABSTRACT TRUNCATED AT 250 WORDS)     

The acid-tolerant microbiota associated with plaque from initial caries and healthy tooth surfaces

The intent of this study was to compare the inherent acid tolerance of bacteria in samples of dental plaque from tooth sites in subjects with and without initial caries. Plaque was collected from approximal surfaces showing early enamel caries and from healthy tooth surfaces in the same subjects, as well as from enamel surfaces of caries-free individuals. In addition to plating on blood agar, the plaque samples were plated directly on non-selective solid agar medium buffered to pH 7.0, 6.0, 5.5, 5.0, 4.5 and 4.0 to avoid any loss of adaptation to acid during primary isolation of plaque bacteria. The results showed that approximately 50% of the total cultivable plaque microbiota from caries, as well as healthy tooth sites, was able to grow at pH 5.5 and 1% at pH 5.0, pH values regarded as critical for the demineralization of tooth enamel. At pH 5.0, members of the genus Streptococcus were the dominant group, but mutans streptococci accounted for less than half of the streptococcal viable count. The other acid-tolerant streptococcal isolates included Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordinii, Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius and SStreptococcus sanguis. Analysis of the results indicated that the mutans streptococci in dental plaque were highly variable with respect to acid tolerance, and that both caries and healthy sites harboured significant numbers of mutans streptococci that were not acid-tolerant.     

Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose

The composition of dental plaque formed in the presence of sucrose or glucose and fructose and its relation to cariogenicity was evaluated. Twelve adult volunteers took part in this crossover study done in three phases of 28 days each. For each phase, an acrylic resin appliance containing four human dental enamel blocks was constructed for the volunteers. Solutions containing 20% sucrose or 10% glucose + 10% fructose were dripped onto the enamel blocks 8 times a day, while in the control group no solution was used. Enamel surface and cross-sectional microhardness results showed that dental plaque formed in the presence of sucrose was more cariogenic than that formed in the presence of glucose + fructose (p<0.05). The concentration of alkali-soluble carbohydrates in dental plaque was higher in the sucrose group than in the control and glucose + fructose groups (p<0.05). Although concentrations of Ca, P and F were lower in plaque formed in the presence of sucrose than glucose + fructose and the control, significant differences (p<0.05) were only observed in relation to control. The electrophoretic profile of the matrix proteins of dental plaque showed distinct patterns when it was formed in the absence or presence of the different carbohydrates. Although the results suggest that the high cariogenicity of dental plaque formed in the presence of sucrose can be mainly explained by the high concentration of insoluble glucans of its matrix, the low inorganic concentration and its protein composition may have some contribution.     

Enamel microhardness and fluoride uptake underneath fermenting and non-fermenting artificial plaque.

Washed cells of Streptococcus sanguis were used to form artificial plaque on the surface of bovine enamel and incubated underneath buffer solutions, initial pH 6, for 36 h at 37 degrees C. The decrease in the microhardness of the enamel surface under fermenting "plaque" could be prevented with fluoride. Enamel under a fermenting "plaque" took up significantly more (P less than 0.0u) fluoride than enamel under a non-fermenting "plaque" (initial F- in buffer: 10 parts/10(6)). The artificial plaque did not accumulate fluoride. Within fermenting "plaques/, the pH decreased significantly more without flouride (P less than 0.01) than with fluoride. Fluoride combined with sucrose more than negated the softening of the enamel caused by sucrose fermentation, i.e. it increased the hardness above the original values. The diffusion of fluoride through the fermenting artificial plaque was more rapid than through a non-fermenting plaque. These findings suggest that caries-conducive circumstances may promote fluoride uptake by enamel compared with non-caries-conducive circumstances.     

Microbial vitality of supragingival dental plaque during initial stages of experimental gingivitis in humans

Although vital plaque micro-organisms are part of the natural ecosystem in the oral cavity they are also the key factor in the development of diseases induced by the human dental plaque. In a previous study (9) the portion of vital bacteria related to the total number of plaque micro-organisms (i.e. the microbial vitality) appeared low in small plaque samples. The objective of this investigation was to determine the exact relationship of microbial vitality and age of supragingival plaque during the early phases of human dental plaque formation. Between intervals of optimal oral hygiene, thirteen participants refrained from all oral hygiene measures for periods of 1, 2, 4, 8, 24 and 72 h. Plaque was completely sampled from a defined area situated on the vestibular surface of the teeth 13, 14, 15, 23, 24 and 25. The pooled plaque from these areas was immediately processed. Total bacterial counts (BC) as enumerated by darkfield microscopy, and colony-forming units (CFU) were recorded. The microbial vitality was calculated indirectly as plating efficiency (PE=CFU per BC) and directly assessed using a vital fluorescence (VF) technique. In the 1 h old plaque samples the median values of PE and VF were 29% and 18%, respectively. Thereafter, the microbial vitality increased significantly with plaque age. The 24 h old plaque samples yielded values of 77% (PE) and 62% (VF). It was concluded that the microbial vitality of the early dental plaque investigated was considerably lower compared to that of a more mature plaque.     

Bacterial composition and red fluorescence of plaque in relation to primary and secondary caries next to composite: an in situ study

BACKGROUND/HYPOTHESIS: Secondary caries has been suggested as the main reason for restoration replacement. We hypothesized that more caries-associated bacteria are found on composite resin restoration material, compared to sound tooth tissue. METHODS: Both restored and unrestored dentin and enamel samples were placed in a full denture of eight subjects for 20 weeks. The microbiological composition of approximal plaque and the association between caries-associated bacteria and red autofluorescence of dental plaque was studied. Every 4 weeks the specimens were microradiographed using transversal wavelength independent microradiography (T-WIM). After 1 and 20 weeks red fluorescence pictures and plaque samples were taken. Samples were cultured for total anaerobic counts, mutans streptococci, lactobacilli, candida and Actinomyces odontolyticus. RESULTS: Lesion depth in the dentin and enamel was positively associated with lactobacilli, and lesion depth in dentin was positively associated with A. odontolyticus, whereas no association was found between mutans streptococci and lesion depth. The red-fluorescent bacteria A. odontolyticus and lactobacilli did not correlate with red-fluorescent plaque, indicating that red fluorescence is probably not caused by a single species of these bacteria. After 20 weeks, a higher proportion of combined mutans streptococci and lactobacilli was found on restored tissue compared to non-restored tissue (P = 0.04). CONCLUSION: The higher proportion of caries-associated bacteria on restored tissue indicates that the ecology on the surface of primary lesions differs from that on lesions next to composite, and that secondary caries next to composite may differ from the primary caries process.     

Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development

This study employed in situ hybridisation using a probe recognising all isoforms of the molecule. Expression of the molecule in tooth germs started at embryonic day 13, when they were at the bud stage. Both inner cells of the epithelial bud and peripheral cells of the dental mesenchyme were positive. At the cap stage, positive cells were found in the inner part of the enamel organ but only in a limited area near the outer enamel epithelium. In the mesenchyme at the cap stage, expression was weak in the dental papilla and strong in the follicle. From the bell stage onward, epithelial cells in the enamel organ were negative except for the cells of the stratum intermedium, which were transiently positive at early and late bell stages. In the dental papilla, expression had mostly ceased during and after the bell stage, although transient expression was found in cuspal areas at the early bell stage. The dental follicle strongly expressed neural cell-adhesion molecule (NCAM) to the end of the experimental period, at post-natal day 4. In contrast to the first molar at its earliest stage of appearance, in which both the thickened epithelium and surrounding mesenchyme were negative for the expression of the molecule, the second molar appeared as a combination of extending epithelial thickenings and mesenchymal cells strongly positive for its expression. This study newly identifies the dental papilla and the stratum intermedium as NCAM-expressing sites.     

Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms

The importance of Streptococcus mutans in the etiology and pathogenesis of dental caries is certainly controversial, in part because excessive attention is paid to the numbers of S. mutans and acid production while the matrix within dental plaque has been neglected. S. mutans does not always dominate within plaque; many organisms are equally acidogenic and aciduric. It is also recognized that glucosyltransferases from S. mutans (Gtfs) play critical roles in the development of virulent dental plaque. Gtfs adsorb to enamel synthesizing glucans in situ, providing sites for avid colonization by microorganisms and an insoluble matrix for plaque. Gtfs also adsorb to surfaces of other oral microorganisms converting them to glucan producers. S. mutans expresses 3 genetically distinct Gtfs; each appears to play a different but overlapping role in the formation of virulent plaque. GtfC is adsorbed to enamel within pellicle whereas GtfB binds avidly to bacteria promoting tight cell clustering, and enhancing cohesion of plaque. GtfD forms a soluble, readily metabolizable polysaccharide and acts as a primer for GtfB. The behavior of soluble Gtfs does not mirror that observed with surface-adsorbed enzymes. Furthermore, the structure of polysaccharide matrix changes over time as a result of the action of mutanases and dextranases within plaque. Gtfs at distinct loci offer chemotherapeutic targets to prevent caries. Nevertheless, agents that inhibit Gtfs in solution frequently have a reduced or no effect on adsorbed enzymes. Clearly, conformational changes and reactions of Gtfs on surfaces are complex and modulate the pathogenesis of dental caries in situ, deserving further investigation.     

Enamel microhardness and fluoride uptake underneath fermenting and non-fermenting artificial plaque

abstract – Washed cells of Streptococcus sanguis were used to form artificial plaque on the surface of bovine enamel and incubated underneath buffer solutions, initial pH 6, for 36 h at 37°C. The decrease in the microhardness of the enamel surface under fermenting "plaque" could be prevented with fluoride. Enamel under a fermenting "plaque" took up significantly more (P < 0.01) fluoride than enamel under a non-fermenting "plaque" (initial F⁻ in buffer: 10 parts/10⁶). The) artificial plaque did not accumulate fluoride. Within fermenting "plaques", the pH decreased significantly more without fluoride ( P <0.01) than with fluoride. Fluoride combined with sucrose more than negated the softening of the enamel caused by sucrose fermentation, i.e. it increased the hardness above the original values. The diffusion of fluoride through the fermenting artificial plaque was more rapid than through a non-fermenting plaque. These findings suggest that caries-conducive circumstances may promote fluoride uptake by enamel compared with non-caries-conducive circumstances.     

Ultrastructural observations on the human pre-eruptive enamel cuticle

The enamel cuticle in the human gingival sulcus was electron-dense and 30 nmdash5 μm thick. Accretions to this cuticle were amorphous, granular or striated, sometimes with linear splits, and occasionally enclosed organisms. The cuticle where colonized by bacteria was often scalloped. Neutrophils and other host cells as well as bacteria adhered to the cuticle. The thick form of cuticle contained no polysaccharide or mucopolysaccharide, although it possessed an adsorbed surface layer of such matter. Both forms of cuticle seem to be composed largely of protein and possibly also proteoglycan. Use of contrasting techniques both in the block and on the grid showed that lack of polysaccharide content was not due to failure of reagents to penetrate the cuticle. Thus enamel cuticle pre-eruptively formed is derived initially from the enamel organ, its main macromolecular constituent is protein and additions to its substance in the region of the oral sulcular epithelium may derive from contiguous epithelial cells or gingival fluid.     

Ultrastructural study of early dental plaque formation

Initial Colonization and early plaque formation were studied using hydroxyapatite splint segments attached to buccal surfaces of maxillary molar and premolar teeth in six young adults given a low-sucrose diet. Segments were removed at intervals of 2, 4, 6, 12, 24 and 48 hr. Bacteria were first regularly seen in 4 or 6 hr specimens where they occurred as individual cells or as small groups of cells. No structures resembling preformed aggregates or hemispherical bacterial "globules" could be demonstrated. The bacteria most frequently attached to the pellicle surface diectly by their cell wall and gram-positive cocci were most abundant. Another mode of bacterial attachment to pellicle was by means of fine bifbrils or coarser thread-like structures. Occasionally organisms were seen attaching to apatite surfaces without interjacent pellicle material and sometimes they appeared to be completely embedded in the pellicle. Bacteria colonizing on epithelial cells regularly displayed a "fuzzy coat". Extensions of organic material from the pellicle surface sometimes made it difficult to distinguish between pellicle and plaque matrix. No clear indication of bacteria metabolizing the pellicle was seen. Trilaminar vesicles probably originating from the surface of degenerating cells were especially abundant in areas with Gramnegative bacteria. In areas with Gram-positive cells the amount of plaque matrix was greater and a number of cells displayed surface threads. The outer surface of the plaque ordinarily did not show a layer of extracellular organic material although a granular laer could be seen in local areas. These findings lend support to a concept of plaque formation as a sequential build-up resulting from selective attachment and growth of individual organisms rather than resulting from attachment of aggregates of bacteria or a passive entrapment of organisms in a matrix.     

Invasion of bacteria in enamel carious lesions

A review of recent findings concerning enamel carious lesions is presented. This lesion represents the initial phase of dental caries and is characterized by a demineralization of the subsurface enamel caused by acids of the plaque bacteria. Streptococcus mutans has been described as the etiologic agent of the dental caries and the most acidogenic plaque bacteria. Morphological studies have shown an invasion of microorganisms inside the enamel carious lesion. Unfortunately, several technical problems are associated with such studies. The identification of the invading bacteria has not yet been achieved. The future identification of bacteria inside the subsurface enamel lesions will represent an important step in the prevention of the carious progression.     

A light and scanning electron microscopic study of the development of enamel-free areas on the molar teeth of the rat

At the late bell of enamel organ development (about 20 days in utero) segments of the internal dental epithelium (IDE) at the future cusp tips of first mandibular molars appeared to make contact with the external dental epithelium (EDE); this relationship was maintained until about 3 to 4 days after birth. Dentinogenesis began at about birth; enamel formation began about the third day on the cusp slopes. However, those cells of the IDE at the enamel-free areas (EFAs) on the cusp tips which were in contact with the EDE remained low columnar and many of their nuclei failed to polarize; enamel was deposited only in sporadic fashion on the EFAs. Later, the EFA epithelial cells became shorter and less regularly arranged. The patches of enamel on EFAs, and even the dentine surface, showed resorption and, just before tooth emergence, these areas were virtually free of enamel.The enamel forming capacity of the EFA epithelium appears to be inhibited, in most cases totally but in some cases partially. The hypothesis is proposed that the EDE layer inhibits the enamel-forming capacity of those IDE cells which come into contact with it in early crown morphogenesis; EFA cells later resorb any enamel they deposited and some of the dentine beneath.