Morphology and microflora of intercusp fissures in teeth

Fissures on masticatory surfaces of lateral teeth are created during development of cusps in odontogenesis, which begins in the first weeks of fetal life with formation of deciduous tooth buds, and terminates about the age of 12 years, or 15-18 years with wisdom teeth. If some disturbances occur in the process of odontogenesis, they cause anomalies within forming dentine or enamel. During early dentinogenesis, insufficient supply of whole protein, amino acids and vitamins or introduction of chemical compounds such as multifunction phosphonic acids, diuretics, cytostatics results in impairment of odontoblast function, thus inadequate base substance for developing collagen matrix is produced.

During later dentinogenesis and biosynthesis of collagen, the above mentioned factors can cause a decrease of hydrolyzing enzyme's activity, thus lowering the amount of hydroxyproline in procollagen, which in turn results in production of defective organic dentine matrix. The created dentine has reduced number of dentinal tubules, which are sinuous along their length. If the harmful factors are active during mineralization of collagen matrix, predentine is formed. Odontoblast cells produce centers of intermediate tissue between dentine and bone; so-called osteodentin is formed.

Experimental studies proved that dentine mineralization emits an inductive signal for creation of soft protein enamel matrix. If dentine mineralization is disturbed, formation of primary protein enamel matrix is delayed. Stimulation by ameloblasts results in genetically conditioned activity of odontoblasts. If ameloblasts do not develop normally, development of odontoblast cells is inhibited and their function is impaired. Due to odontoblasts' impairment, enamel is subjected to underdevelopment, such as: hypoplasias and hypomineralization. During transition from amorphous to crystal phase, inadequate supply of elements such as calcium, phosphorus, fluorine and other microelements, or introduction of substances regarded as cytotoxic, disturb enamel mineralization. Disturbance of enamel mineralization proved to be a very complex process.

In properly structured and mineralized enamel, geometrical shapes of fissures are significantly diversified. Sizes and geometrical shapes of fissures also vary considerably in each individual. Shallow and deep irregularly shaped fissures are observed, narrow at the entrance and spreading near the base. The depth of fissures varies from a few to between 10 and 20 micrometers, depending on tooth's anatomical group. It happens that the central fissure reaches enamel-dentine junction, and sometimes even ends in dentine. However its bottom is usually covered by a thin layer of enamel (27). A fissure in an erupted tooth is filled with dental plaque with microflora changing with age, and residual food particles.

Longitudinal stripping of molars allowed assessment of mean sizes of fissures in those teeth. It was observed that the central fissure in molars has an average depth of 1.1mm, and its width at fissure entrance ranges from 0.2 to 0.5mm, and the width at fissure base has a mean value of 0.1 mm. Premolars indicate a larger diversity. According to Taylor and Gwinett (81) most commonly they are funnel-shaped with a narrowing at the middle of fissure's depth; inferior premolars have more varied shapes than superior ones. Generally in inferior premolars predominant fissures are narrow at the entrance and expanding at 1/3 of length towards the base. This creates conducive conditions for development of bacterial plaque and deposition of food remains, which leads to beginning and development of carious lesions. It was also observed that enamel covering fissures, especially on first permanent molars, demonstrates a smaller fluorine content than enamel on smooth surfaces of those teeth (16,72).

A lot of interesting information has been collected on the structure and composition of dental plaque residing in hollows of masticatory surfaces of teeth. The information comes from experimental studies carried out on gnotobiotic animals and from studies on people with the use of fissure models made of Mylar foil, placed in large amalgam fillings on molars' masticatory surfaces with the use of dentures as carriers for fragments of natural teeth (third molars). Dental plaque accumulating on smooth surfaces is different from plaque deposits in fissures. Plaque in fissures on masticatory surfaces is made of Gram positive cocci bacteria, which constitute 77-89% of total bacterial flora, short rods, a small amount of filiform microorganisms and blastomyces that are an almost regular ingredient of most plaque in intercusp fissures. Cocci form microcolonies, and bacterial plaque's matrix varies depending on the amount of culturable bacterial flora, plaque's thickness and its position in the fissure. Streptococcus mutans does not play any important role in early bacterial colonization of intercusp fissures. In one-day-old plaque this species can hardly be found. With time the number of these bacteria increases, but they constitute less than 10% of total streptococcus flora. Cocci are first to settle in fissures. As plaque ages, their content decreases from 62% on the first day to 46% after three days and down to 28% after eight days. After initial homing in fissures, probability of further addition of new microorganisms seems low. The structure of bacterial plaque in fissures differs from that of plaque accumulating on smooth surfaces. At fissure entrance, cocci are positioned in palisade arrangement, perpendicular to fissure's bottom, and they occur together with fusobacteria, which are considerably less numerous than in plaque on smooth surfaces. During colonization of smooth surfaces, a selection of homing microorganisms takes place. Only those microorganisms can settle which are capable of producing polysaccharides of increased viscosity. In case of fissure plaque, practically any microorganism can become its part. During early stage, the development of plaque on smooth surfaces results from bacterial cell divisions and by addition of microorganisms from saliva. Streptococci constitute almost 90% of bacteria in early plaque.

With development of methods for intercusp fissure sealing, there has been an increased interest in microflora status in carious cavities covered with sealant (75). Because of anatomical characteristics of masticatory surfaces of teeth, sealing material is introduced on bacterial plaque accumulated on the fissure's bottom. Therefore it is critical to obtain information about sealant's impact on microflora in the fissure. The sealant's impact on microflora of fissures not affected by caries has been the subject of numerous studies. Those studies were performed on implanted fragments of masticatory surfaces of teeth. Data indicate that after sealing, microflora in carious lesions becomes suppressed. However, under the sealant a presence of microorganisms capable of growing was observed, which are believed to be etiologically connected with carious processes. It was also demonstrated that sealing materials do not constitute a fully tight barrier separating fissures from oral environment, which occurs very soon after treatment.

Microbiological studies conducted by Jodkowska (32) showed that sealing of intercusp fissures not affected by caries led to a considerable reduction in the number of bacteria. The impact of this treatment on intercusp microflora varied depending on time elapsed after sealing and on the type of applied sealant. In a short time, which means 30 minutes after sealing with Nuva-Seal material, no bacteria growth was observed in 89% of examined samples. In further observation periods, after application of this material, a slow decrease of percentage of aseptic samples was observed, which reached the value of 17% after 18 months. After sealing with Concise BWSS (Brand White Sealant System) a reverse tendency was observed. Nuva-Seal material was more effective in fissure sealing than Concise (BWSS) in a short period immediately after treatment. The efficacy of sealing with this material decreased with time. On the other hand Concise (BWSS), which initially inhibited bacteria growth to a lesser degree than Nuva-Seal, proved more effective in later period of observation. After 18 months the activity inhibiting bacteria growth of both evaluated materials was similar. The most frequently isolated bacterial stains were Streptococcus mutans (25%), however more rarely strains classified as Staphylococcus aureus were obtained

(3,4%). In 71% of cases growth of only one strain was obtained, whereas in remaining cases various bacterial strains were isolated. Mixed bacterial flora was observed more frequently in material coming from teeth sealed with Nuva-Seal than from teeth sealed with Concise (BWSS).

The author also conducted microbiological studies on 30 first and second permanent molars in children whose condition of hard tissues in initial examination (visual - mirror and probe) in 16 cases suggested a suspicion of an early stage of initial caries and in 14 cases evident initial caries was diagnosed. Samples of material collected for bacteriological examination were collected after 30 minutes, two weeks, and also after two, eight, twelve and eighteen months from sealing. Samples were collected from occlusal surfaces of carious teeth covered with light-cured Nuva-Seal material and with chemically-cured Concise (BWSS). The research demonstrated that the highest reduction of culturable microorganisms in samples collected from fissures sealed with Nuva-Seal occurred 30 minutes after treatment. Then the reduction increased within the period from 2 weeks to 8 months and reached the level which was 300-fold lower compared to samples collected from control teeth. After 12 months of observation 70-fold less bacteria were cultured from sealed teeth, and after 18 months only 6.5-fold less than in samples collected from control teeth. On the other hand Concise (BWSS) material which initially inhibited bacteria growth to a lesser degree than Nuva-Seal, within the period from 2 to 8 months, indicated a 500-fold reduction of microflora compared to samples collected from control teeth. After 12 months of observation the number of culturable bacteria indicated a 100-fold reduction, and after 18 months the activity inhibiting bacteria growth of both assessed sealing materials was similar.

An analysis of isolation of individual bacteria strains and the number of isolated microorganisms in various periods of observation after sealing with both assessed materials indicated that Concise (BWSS) material had a more favorable effect on microflora of carious lesions than Nuva-Seal material. After application of Concise (BWSS) a less frequent development of bacteria etiologically associated with caries, belonging to Streptococcus mutans (S. mutans) species was observed. Microorganisms belonging to this species occurred solely in material collected from fissures sealed only during certain periods of observation. Numbers of S. mutans in material collected after sealing with Concise (BWSS) material were - in various periods of observation - generally much lower than numbers observed after application of Nuva-Seal material. After use of Nuva-Seal material, a less frequent development of mixed bacterial flora was observed. Clinical observations of carious teeth sealed with both assessed materials, which were performed during 18-month period, indicated that only in three cases, where sealant was damaged, slightly deepened carious cavities were observed under sealant. Studies by other researchers confirm the results obtained by the author and conform with the above results, while deepening of carious cavities, if it occurs, seems small (39). This is due mainly to the presence of a mechanical barrier which blocks an inflow of new microorganisms and of substrate for bacterial transformations. Moreover, acid etching of sealed teeth and regenerative capability of dentine may to some degree inhibit further progression of existing carious lesions in fissures (1). Study by Heller et al., who assessed occurrence of caries in previously healthy teeth and in teeth with initial caries, where fissures were sealed in some patients of each group, observed after five years that among previously healthy teeth caries developed in 13% of not sealed teeth, whereas in case of sealed teeth the percentage was smaller and equaled 8% (28). After five years, in the group with previously diagnosed caries the condition of 52% of not sealed teeth and of 11% of sealed teeth deteriorated. According to the authors, sealing of teeth previously affected by caries was fully justified because through application of sealants - instead of conventional preparation and filling of cavities - one of the prophylactic methods against development of carious centers can be applied. Further research may bring more data on this issue.

Healthy enamel, especially its surface layer, however well mineralized during tooth eruption, undergoes continual chemical and physical changes throughout organism's life. Those changes may be favorable, yielding better mineralized, more mature enamel, or they may be unfavorable, especially when enamel is covered by bacterial plaque. Fermentable carbohydrates diffuse into dental plaque where they are metabolized and transformed into organic acids with low pH (lactic, formic, pyruvic acids) and with high pH (butyric, propionic, acetic acids). These acids may be buffered by various systems or they can diffuse into dental plaque or into oral fluid. However, they can also penetrate through the plaque-enamel surface and partly demineralize crystals on and immediately under the surface of enamel. The external surface of enamel is in the state of dynamic balance between demineralization and remineralization, which periodically follow each other.

Fissures in lateral teeth are particularly predisposed for caries development. Most typically caries starts in enamel of fissures. We know that it develops due to disturbances between demineralization and remineralization phases of enamel covered with bacterial plaque. Organic acids produced by bacteria penetrate through the plaque-enamel contact surface in a dissociated or non-dissociated form and diffuse in the liquid phase among enamel crystals or inside crystals. Continual elution of calcium and phosphorus ions, decrease of pH value, ion concentration and other factors have impact on diffusion, on amount of acids penetrating into enamel and on loss of majority of minerals from hydroxyapatites. Progression of carious processes in enamel of fissures depends on consecutive attacks of acids and further destruction of enamel's crystal structure. Early carious lesions are characterized by varied advancement of demineralization in the surface layer and inside the tissue, where the highest loss of non-organic compounds and degradation of prismatic structure is observed in the so-called subsurface layer. With a growing loss of non-organic components of enamel, its porosity increases. Demineralization of enamel in fissures starts at fissure entrance. The main demineralization center spreads sideways from isthmus of fissure along central fissure. Outside layer of enamel with early carious lesions observed on microphotographs indicated a considerably higher degree of mineralization compared to underlying areas of enamel and dentine. Conducted research confirms that in the beginning stage of carious lesion's development the degree of demineralization of fissures' enamel depends on topography and depth of fissures. The deeper and more diversified in shape the fissure, the higher the observable degree of demineralization. The degree of demineralization decreases towards fissure base. In wide fissures the degree of demineralization was smaller. The period of the first three years after tooth eruption is regarded to be of the highest susceptibility for caries development on masticatory surfaces. In dentine, demineralization period depends on the depth and width of fissures. At the entrance of all fissures, both narrow and wide, demineralization area was the largest. At fissure base the area of dentine was preserved and did not indicate symptoms of demineralization, whereas in deep fissures demineralization at fissure base was less pronounced. Narrow fissures indicated a smaller loss of non-organic compounds. In the narrowest fissures demineralization was deepest but in majority of fissures it was limited to the upper part, not reaching the fissure base. The described associations between dentine fissure morphology and range of tissue demineralization in conditions of experimental exposure to caries-inducing factor continued for eight weeks. However, the loss of minerals and dentine tissues was higher on the seventh day of the experiment compared to the loss observed eight weeks after the experiment (45).

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