Pellicle Surface Research Articles

Application and assessment of laser Doppler velocimetry for underwater acoustic measurements

The majority of traditional methods for making underwater acoustic pressure measurements involve placing all or part of a measurement transducer in the acoustic field. A variety of optical metrology techniques have been developed in an attempt to reduce or remove any perturbing effects. An example of this is the use of laser interferometry which has been developed as the primary method of calibrating hydrophones in the frequency range 500 kHz – 20 MHz at the National Physical Laboratory (NPL). This technique involves suspending a thin Mylar pellicle in the acoustic field and recording the displacement of the pellicle surface using a Michelson Interferometer. This study details a comparison of a Laser Doppler Velocimeter (LDV) with the NPL Laser Interferometer, which gives a good correlation where agreement is within approximately 4% and 7% for two different power levels from a 500 kHz plane piston transducer and within 2.5% and 1% for the same power levels from a 1 MHz plane piston transducer. A novel, non-perturbing method of recording temporally resolved acoustic pressure distributions in water using an LDV is also described. The technique is shown to benefit from the consistent frequency response of the LDV detection system, such that the measured output resembles the drive voltage input to the transducer more closely than a similar hydrophone measurement. For the experimental arrangement described, the LDV system is shown to be sensitive to minimum pressure amplitudes of nominally 18.9 mPa/√ Hz .

Atomic force microscopy study of salivary pellicles formed on enamel and glass in vivo

This study was performed to evaluate the use of atomic force microscopy (AFM) in examining the surface of the adsorbed layer of salivary proteins (salivary pellicle) formed in vivo on dental enamel and glass surfaces. Enamel and glass test pieces were attached to the buccal surfaces of the upper first molar teeth in two adults using removable intraoral splints. The splints were carried intraorally over periods ranging from 10 min to1 h. Using the contact mode of AFM, pellicle structures could be recognised on intraorally exposed specimens compared to nonexposed enamel and glass surfaces. The surface of the adsorbed salivary pellicle was characterised by a dense globular appearance. The diameter of the globulelike protein aggregates adsorbed onto enamel and glass varied between 80 and 200 nm and 80 and 150 nm, respectively. The structure of the adsorbed protein layer was clearly visible on glass surfaces, even though minor differences in the protein layer between glass and enamel specimens were observed. This study indicates that AFM is a powerful tool for high-resolution examination of the salivary pellicle surface structure in its native (hydrated) state. AFM avoids artefacts due to fixing, dehydration and sputter-coating which occur with scanning electron microscopic analyses.

Mechanisms of Stain Formation on Teeth, in Particular Associated with Metal Ions and Antiseptics

Much has been written on the subject of extrinsic tooth discoloration, but, except when the pigment is intentionally applied, the etiologies and mechanisms are poorly understood. Extrinsic stains have been classified as non-metallic or metallic. The pigment usually lies not on or in the dental tissues, but in surface deposits, particularly the acquired pellicle layer and at sites receiving limited cleaning. Whether pigments absorb, adsorb, or chemically interact with dental surfaces is unclear. Some stains merely seem to reflect the color of the apparent source, whereas others have been ascribed to a secondary chemical alteration of a substance at the tooth or pellicle surface. Theories of chromogenic bacteria and formation of metal sulfides are frequently propounded but without clear supportive evidence. Staining by cationic antiseptics and, to a lesser extent, metal salts has attracted research interest. Chlorhexidine and other cationic antiseptics, it is hypothesized, may catalyze browning reactions or facilitate metal sulfide formation in pellicle. Controlled clinical studies have repeatedly shown that dental and mucosal staining associated with the use of chlorhexidine and some metal salts is dependent upon volunteers' imbibing reasonable quantities of chromogenic beverages, such as tea. However, it must be appreciated that cationic antiseptics and polyvalent metals can precipitate chromogenic material from a large range of dietary compounds. The control of dental staining, at least that associated with chlorhexidine, can be achieved both in vitro and in vivo by the use of oxidizing agents which appear to remove the stain physically from the surfaces.

Studies on Dental Stains Induced by Antibacterial Agents and Rational Approaches for Bleaching Dental Stains

Extrinsic stain resides in the dental pellicle and can be caused by introduction of chromogenic materials or therapeutic agents into the oral cavity. In contrast, intrinsic tooth stain is found within the tooth structure and can be caused by a variety of agents, including hematological and developmental abnormalities and drugs such as tetracycline. The mechanisms of extrinsic stain formation differ with respect to the causative agent. For example, stain induced by chlorhexidine (CH) can be explained by an increased rate in the non-enzymatic browning reactions occurring at the pellicle surface, while food stains are retained on the surface via ion exchange mechanisms. Although most extrinsic dental stain can be removed by abrasive and/or surface-active materials, removal of certain types of surface stain, e.g., staining due to cationic antimicrobial agents, requires specific agents such as aminoguanidine to reduce the stain.A broad-spectrum approach to reduce both intrinsic and extrinsic dental stains clinically requires oxygenating agents. To evaluate this approach and understand the mechanisms of stain removal, we developed a spectroscopic method for measuring stain in vivo. A series of clinical studies was performed to evaluate stain removal by the agents. The results showed that carbamide peroxide in combination with surfactants and anti-redeposition agents, e.g., sodium pyrophosphate, was more effective in bleaching dental stain compared with carbamide peroxide alone. A detailed examination of the tooth structure by microhardness measurements, x-ray photoelectron spectroscopy, and scanning electron microscopy showed that stain decolorization with this system did not have any adverse effects.

Characterization of GlucosyltransferaseB, GtfC, and GtfD in Solution and on the Surface of Hydroxyapatite

GlucosyltransferaseB, GtfC, and GtfD were purified by hydroxyapatite column chromatography, followed by ultrafiltration from the culture supernatant fluids of three Streptococcus milleri constructs (gift from Dr. H.K. Kuramitsu) which harbored individual gtf genes of Streptococcus mutans GS5. GtfB, GtfC, and GtfD were enzymatically active both in solution and in an experimental pellicle (HA-CWS-Gtf) formed by adsorbing Gtf onto the surface of clarified human whole saliva (CWS)-coated hydroxyapatite (HA). The Km values for sucrose for all three enzymes were lower when the enzyme was adsorbed to a surface, compared with when it was in solution. In solution phase assays, and in the absence of primer dextran, glucan production was enhanced 75% when both GtfB and GtfD were present in the reaction mixture, compared with the sum of the individual enzyme activities (p < 0.005). This enhancement did not occur when GtfC was additionally present, or when the GtfB+GtfD enzyme pair was adsorbed onto HA-CWS. In additional experiments, glucan formed by GtfB or GtfC, but not by GtfD, on a HA-CWS-Gtf surface increased adherence of Streptococcus mutans GS5 and Streptococcus sobrinus 6715 by seven- to nine-fold compared with adherence when no glucan was present on the pellicle surface (p < 0.001). Further, treatment of the HA-CWS-GtfB-glucan or HA-CWS-GtfC-glucan pellicle with alpha-1,6 dextranase significantly reduced adherence of both streptococcal strains (p < 0.001). These results show that GtfB, GtfC, and GtfD are enzymatically active in an adsorbed state and that the nature of their product glucan can influence the adherence of cariogenic oral streptococci to an experimental pellicle.

The effect of milk and kappa casein on streptococcal glucosyltransferase.

Many dietary components such as carbohydrates, lipids and proteins may be incorporated into the salivary pellicle and thus may affect glucosyltransferase (GTF) activity on pellicle surfaces. The effect of milk on streptococcal GTF activity was determined. Milk, when coated onto buffer-coated hydroxyapatite or saliva-coated hydroxyapatite (sHA), reduced the subsequent adsorption of GTF onto the surfaces. Milk also reduced the expression of enzymatic activity of GTF adsorbed onto sHA. kappa-Casein, when present on the surface of sHA, reduced the adsorption of GTF activity onto sHA, resulting in reduced glucan formation. alpha-Casein had no effect on the adsorption of GTF onto sHA or on subsequent glucan formation. Both milk and kappa-casein reduced activity of the enzyme in solution. The presence of milk and kappa-casein fractions on the surface of sHA and in solution with GTF can clearly modulate glucan formation in vitro.

A transmission electron microscopy study of the adsorption patterns of early developing artificial pellicles on human enamel

Adsorption patterns of pellicles formed in a flow cell system under a moderate shear rate of 21 s −1, were studied with a replica technique. Characteristic features of the bare enamel surface disappeared within seconds after exposure to reconstituted saliva, indicating the adsorption of a homogeneous film. After that, three to four distinct homogeneous films were adsorbed on top of each other within 10 s and an uneven, knotted structure developed with stalky projections on the outer surface. This heterogeneous adsorption pattern determined the structure of the pellicle surface for at least 2 h.

External radiolabelling of components of pellicle on human enamel and cementum

Enamel and cementum pellicles form by different adsorption of salivary and serum components to the tooth surface. The authors compared the constituents of surface pellicle formed on human enamel and cementum under three conditions: (1) natural pellicle, present on extracted teeth, which was formed by prolonged exposure to human salivary and serum components in vivo; (2) short-term in-vivo pellicle, formed by exposing enamel and cementum slabs to the oral environment for 0-60 min; (3) in-vivo pellicle, formed by incubating enamel and cementum slabs in a 1:1 mixture of parotid and submandibular/sublingual saliva for 0-60 min. Pellicle composition was characterized by external radiolabelling techniques specific for exposed carbohydrate (sialic acid and galactose) and amino-acid (tyrosine) residues. There were differences between cementum and enamel in the electrophoretic profiles of natural-pellicle components; notably, a major 180 kda 3H-labelled sialoglycoprotein, unique to the cementum pellicle, had the same electrophoretic mobility as the low-molecular-weight mucin from human submandibular/sublingual saliva. After alkaline-borohydride treatment, 3H-labelled natural-pellicle oligosaccharides chromatographed in the di- to tetrasaccharide region of a Bio-Gel P-2 column. The most prominently labelled components of short-term enamel and cementum pellicles in vivo and in vitro had the same electrophoretic mobility as the low-molecular-weight salivary mucin. The pellicle components formed in vitro, unlike those formed for the same period of time in vivo, were rapidly desorbed from the cementum, but not from the enamel surface. We conclude that: (1) external labelling techniques are useful for obtaining a profile of pellicle components; (2) submandibular/salivary mucins are major constituents of salivary pellicles on tooth surfaces; (3) glycoproteins that carry low-molecular-weight, sialic-acid-containing saccharides are important determinants of pellicle surface properties [corrected].

Ac conductivity of anhydrous pellicular zirconium phosphate in hydrogen form

Aqueous suspensions of delaminated zirconium phosphate have been recently obtained in our laboratory by a suitable intercalation-deintercalation of alkylamines. By filtering these suspensions thin and flexible sheets or pellicles of lamellar α-zirconium phosphate, with the most part of the layers oriented parallel to the pellicle surface, can be easily prepared. ac conductivity measurements have been carried out, in the temperature range 150–300°C, on pellets made of α-Zr(HPO 4) 2 pressed pellicles, oriented both parallel and perpendicular to the electric field. Independently of the pellet orientation, a slope variation was observed in the Arrhenius plot at about 220°C. As already found for the α-Zr(HPO 4) 2 powder, this is associated with a phase transition at this temperature, which causes a discontinuous change of the interlayer distance from 7.4 Å to 6.8 Å. The conductivity of samples oriented parallel to the electric field ( ∼10 −4 Ω −1 cm −1 at 300° C ) is about two orders higher than the conductivity of the anhydrous microcrystalline powder, which, in turn, is higher than that of perpendicularly oriented samples ( ∼3 × 10 −7 Ω −1 cm −1 at 300° C ).

Bacterial adherence and dental plaque formation.

Bacterial masses on the crowns of the teeth, termed dental plaques, are generally separated from the tooth enamel by a pellicle of salivary origin. The initiation of plaque formation entails the firm adherence of bacteria to the pellicle andin situ growth of attached bacteria. Plaque accumulation, after saturation of the pellicle surface, involves adhesive interactions between growing attached bacteria via the interbacterial matrix (host- and bacteria-derived products) and further attachment. Bacterial adherence to the pellicle is a prerequisite for plaque initiation. The attachment of different bacterial species exhibits a great deal of specificity and appears to involve specific receptors on the bacterial and pellicle surfaces. Different salivary glycoproteins, e. g. high M. W. polymers or immunoglubulins in the pellicle, as well as bacterial cell surface appendages (fimbriae) have been implicated, and some evidence suggests involvement of lectin-like interactions. Plaque accumulation may be mediated by bacterial extracellular polysaccharides, salivary components as well as direct cell-to-cell binding. These interactions also exhibit great selectivity, and evidence for a lectinlike nature of some of them has been obtained. Extracellular glucans synthesized from sucrose appear to favor specifically the accumulation ofStreptococcus mutans cells. The role of other homo- and heteropoly-saccharides is much less clear. Salivary components play a dual role in plaque formation. They can mediate bacterial attachment to the pellicle or plaque periphery, but by binding to unattached bacteria they can also diminish the attachment of such bacteria. The bacterial composition of plaque in its early phase of formation appears to be dictated by the relative adherence ability of participating organisms. Shifts in the bacterial composition of plaque in later development stages can also be caused by changes in bacterial growth conditions. In contrast to plaque composition, total plaque mass is governed mainly by growth of attached organisms rather than by continuous new adherence of bacteria to the plaque periphery. Successful practical methods for inhibiting or preventing plaque formation by interfering with bacterial adhesive processes have not yet been developed.

Distribution of the receptors for concanavalin A on the surface of Euglena gracilis as revealed by fluorescence microscopy

The binding of concanavalin A (Con A) to the cell surface of Euglena gracilis was explored in various conditions by fluorescence microscopy. In both living and glutaraldehyde-fixed cells, the membrane of the emergent flagellum and of the reservoir are intensely and uniformly labelled by the fluorescein isothiocyanate-conjugated lectin. In contrast, the pellicular surface shows an alternation of reactive and unreactive strips. The streaks of binding, weak in brightness, pass helically around the cell body, probably at the level of the grooves and/or of the posterior margin of each ridge. Neither temperature variations nor exposure to the fluoresceinated ligand over a 30-min period consistently modify the binding pattern which in all experimental conditions occurs almost immediately at the flagellum level and subsequently at the reservoir and pellicle surface. Two main indications arise from the results: (i) concanavalin A receptors are especially localized in membrane areas possessing an unstable structure and numerous external glycosylated components; (ii) the distribution pattern of the lectin along the pellicle is similar to that known for the antipellicular antibodies.

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.

Dental plaque morphology as revealed by direct observation and by replicating techniques

The ability of four elastomer impression materials to reproduce details of bacterial plaque structures was studied by comparing areas of the replica models with corresponding areas of the replicated plaque. Plaque was grown on hydroxyapatite splint segments for 48 hours and replica models were made from three different resins. One of the resins, a methacrylate material, was not suited for this purpose due to its content of spherical particles. The polysulfide and polyether impression materials reacted with the heat curing epoxy resin, and less satisfactory results were obtained with cold curing epoxy resin. Best results were obtained by a combined use of low viscosity silicone impression materials and cold or heat curing epoxy resins as model materials. However, a considerable loss of detail occurred in the replica models compared with observations of plaque directly on splint surfaces. Outlines of individual bacteria could sometimes be seen in the models, but generally several cells seemed to fuse, and appeared like small globular structures where cells and pellicle were difficult to distinguish. The bacterial colonization started near the gingival border of the surface, initiated by attachment of individual bacteria to the pellicle surface.

Early dental plaque morphogenesis. A scanning electron microscope study using the hydroxyapatite splint model and a low-sucrose diet.

Bacterial colonization of hydroxyapatite splint segments attached to buccal surfaces of maxillary molar and premolar teeth, were studied at intervals of 2, 4, 6, 12, 24 and 48 hr in six subjects given a low‐sucrose diet. Bacteria were first encountered in 4 or 6 hr specimens and considerable variation existed between subjects regarding time of appearance, rate, and extent of microbial growth. Individual organisms were seen attaching to a pellicle surface with or without intervening thread‐like or coat‐like structures. Similar thread‐like structures were sometimes seen connecting individual organisms. The colonization started frequently in grooves or pits in the surface and the bacteria spread as monolayered cells from these starting points. Bacteria were also seen colonizing on epithelial cells. Cocci dominated in numbers in all phases of this study although rods and filaments appeared in increasing amounts in 24 and 48 hr specimens. Structures resembling corncobs were seen in 48 hr specimens. The gross morphology of plaque development was characterized by patches or strands of bacterial growth and a heterogeneous pattern of apparently freshly colonized areas interspersed between areas of more extensive colonization. Except for a few bacterial clumps, no aggregates or hemispherical globules appeared on the splint surface. These findings warrant a revision of how the salivary aggregating factors are functioning in vivo: Their importance may be to mediate attachment between individual cells in the early monolayer as well as between cells and pellicle, and later contribute to the cohesion of the plaque, rather than by forming bacterial aggregates that settle down on the tooth surface.