Journal of Dentistry,
7, No. 3, 1979,
pp. 235-245.
Printed in Great Britain
The host-organism interface human dental plaque H. N. Newman, Department
in natural
BDent SC, MA, MDS, PhD
of Periodontology,
lnstitu te of Dental
Surgery, Eastman Dental Hospital,
University of London
ABSTRACT
This study concerns the adhesion of natural apical border plaque to approximal surfaces of children’s posterior teeth. The predominant intact cells were polysaccharide-containing cocci linked to the cuticle by means of mainly fibrillar or globular polysaccharide. Correlation of the features observed with those from studies of plaque formation and microbial adhesion in other habitats suggests that approximation of bacteria to enamel cuticle (and salivary pellicle) may be due to cross-linking and salt bridging of polysaccharide until mutually repulsive net negative surface charges are overcome. Low radius of curvature of bacterial processes and low pH, the latter to be expected at the subcontact area apical border in children, would favour reduction in bacterial surface potential. The final stages in bacterial adhesion in plaque seem to involve strong forces producing flattening of the cell wall at the site of the closest contact.
INTRODUCTION Concomitant with the study of individual bacterial and host binding factors is the need to establish the mechanisms mediating adhesion of natural dental plaque to teeth in viva. It is also necessary to correlate observations from studies of short term plaque formation with the same features in natural plaque. The present study is part of an attempt at such a correlation, being based on the carbohydrate electronhistochemistry of natural approximal plaque from children’s teeth. The importance of studying the cuticle-plaque (and acquired pellicle-plaque) interface needs to be emphasized in view of the observation that components located more than 10 nm from the surface will have no significant influence on surface characteristics (Glantz, 1977). Even after a few seconds, it is possible to detect such an organic film on clean solid surfaces (Glantz, 1977). Hydroxyapatite properties are relevant mainly to the binding of cuticle or salivary glycoprotein to enamel.
MATERIALS Premolar
AND METHODS
and molar
teeth
removed
from
children
for orthodontic
reasons
were
rinsed
to
remove loose debris and blood, and then placed immediately in a primary fixative solution (3 per cent glutaraldehyde in cacodylate buffer, pH 7.4) and fixed for 3 h at 4 “C. They were then washed overnight in sodium cacodylate buffer, pH 7.4, containing 5 per cent sucrose. As teeth were removed in the morning under general anaesthesia, all the patients had been fasting for at least 10 h. None of the specimens had clinically apparent gingivitis or approximal caries. Gingival crevices were not probed, to avoid disturbance of the apical border of the plaque. Specimens were processed by the following methods with controls as appropriate. Methods 3 and 4 also served as controls for each other, as did comparison of the features present on specifically and non-specifically stained specimens. 1. Post-fixation for 2 h in 2 per cent 0~0~ in veronal acetate buffer (Zetterqvist, 1956). 2. Periodic acid-thiosemicarbazide-silver proteinate technique (PA-TSC-AgP) (grid stain) (Thiery, 1967).
236
Fig. I. Routine fuation. Average enamel cuticle. E, enamel. Bar = 0.i pm. (X 18 885.) Fig. 2. Ruthenium red. Amorphous thick enamel cuticle. Note external surface coating with amorphous and globular ruthenium red-positive material. Lines and holes in cuticle are section artefacts. E, enamel. Bar = 0.5 pm. (X 6110.) Fig 3. Safranin O-positive enamel cuticle. The most coronal plaque is at the bottom of the micrograph. Note that initial scalloping of the cuticle is followed by a diminution in thickness until only a thin primordial layer remains. Electron-lucent vesicles in most organisms are polysaccharide granules. E, enamel. Bar = 0.5 pm. (X 11 110.)
3. Ruthenium red (Brooker and Fuller, 1975). 4. Colloidal iron (Mowry, 1963). 5. Alcian blue/lanthanum nitrate (Shea, 1971). 6. Safranin 0 (Shepard and Mitchell, 1976). 7. Sodium chlorite (grid stain) (DiPersio and Deal, 1974; DiPersio et al., 1974). The approximal plaque-bearing surfaces were removed from processed teeth with a diamond disc and cooled and rinsed with an appropriate buffer. All specimens were dehydrated in graded ethanol series and ethoxypropane, and embedded in Araldite (CY2 12, EMScope, London, England). From the embedded surfaces specimens incorporating the following sites were removed: (a) subcontact area or mid-approximal apical border; @) corresponding embrasure specimen. These were demineralized from the enamel side in EDTA buffered to pH 7.4 with NaOH. After removal from enamel, the specimens were dehydrated as before and reembedded in Araldite. Sections were cut to give an interference colour of silver to light gold (80-I 20 nm).
Newman:
Host-organism
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in dental plaque
237
F&Y 4. PA-TSC-AgP. Predominant intact forms in apical border plaque subjacent to the contact area are Gram-negative and positive polysaccharide-containing cocci. Gram-negative forms include many large cells. E, enamel. Bar = 0.8 pm. (x 4800.) Fig. 5. PA-TSC-AgP. Apical organisms are either lysed or polysaccharidecontaining. Note poorly contrasted cuticle. E, enamel. Bar = 0.8 pm. (X 22 400.)
Sections from specimens fixed routinely with glutaraldehyde and osmium tetroxide were stained further with 0.4 per cent ethanolic uranyl acetate for 4 mm, followed by O-4 per cent lead nitrate in aqueous N/l0 NaOH for 4 min.
RESULTS The enamel cuticle This organic structure formed on the enamel surface before eruption, consisted of an electron-dense layer as observed by conventional and proteoglycan contrasting (Figs. I-S), but was more electron-lucent when contrasted with PA-TSC-AgP (Fig. 5). The cuticle was continuous with prolongations of organic matter between the spaces occupied before demineralization by the enamel crystals. Subsurface organic matter was variable in amount,
238
Journal of Dentistry, Vol. ~/NO. 3
Fig. 6. PA-TSC-AgP. Bacteria are separated from enamel by thin cuticle and glycocalyx. Note flattening of cell walls at regions of close contact. E, enamel. Bar = 0.7 brn. (X 34 300.) Fig. 7. Ruthenium red. Intact cell has irregular amorphous mucopolysaccharide glycocalyx. E, enamel. Bar = 0.7 Mm. (X 23 800.)
being most abundant in relation to irregularities such as lamellae and striae of Retzms. In many sections, subsurface organic matter was sparse. The cuticle was usually between 30 and 200 nm thick, but occasionally exceeded 5 pm (Fig. 2) especially in the region under examination, i.e. between junctional epithelium and the apical border of the plaque. Occasionally it extended beneath the plaque, in which case it was scalloped by organisms and reduced in thickness until only a thin layer similar to the usual thin cuticle remained (Fig. 3). This layer persisted between the plaque and the enamel. Homogeneous, ‘fuzzy’, vacuolated and striated varieties of thick cuticle were observed. Thick cuticles were more common in embrasures than in subcontact area loci, and were sometimes more continuous with proteoglycan-like matter at the suface of the apical border of the plaque in the former location. The apical border of plaque The predominant organisms at the border were Gram-positive and those less abundant were Gram-negative cocci (Fig. 4). The border was usually discrete in the embrasure and tapering apical to the contact area. A more varied flora was found in embrasures.,The most apical organisms of the tapering border were either lysed cells comprising cell walls with or without vestiges of cytoplasm, or IPScontaining, apparently intact cells (Figs. 3-5). Apparently intact organisms were often present in microcolonies. In the outer plaque, even in thin layers, organisms were more randomly arranged (not palisaded) and the proportion of matrix to bacteria was higher.
Newman:
Host-organism
interface
in dental plaque
Fig 8. Colloidal iron. Partially demineralized. Note coating of both cells by acidic mucopolysaccharide or proteoglycan and thinning of this layer in relation to portion of left-hand cell in contact with cuticle. Bar = 1 pm. (X 34 000.)
The plaque-host
Fig. 9. Ruthenium red. This cell shows tine fibrillar elements on the aspects away from the cuticle and globules of mucopolysaccharide between cell wall and cuticle. Black intracellular globule is a common ruthenium artefact. E, enamel. Bar = 1 pm. (X 34 000.)
interface
were not found with their cell walls in direct contact with crystal spaces of the surface enamel, but were separated from them by enamel cuticle and bacterial glycocalyx (Fig. 6). Staining with ruthenium red, colloidal iron or Alcian blue/lanthanum nitrate invariably disclosed a glycocalyx which appeared to mediate host-bacterial (and interbacterial) attachment and which was absent on routinely fixed, nonspecifically contrasted specimens (Figs. 7-13). This irregular, fuzzy coat external to the cell walls varied considerably in thickness. The more abundant glycocalyx contained amorphous material (Figs. 7, 8) and often possessed globular (Figs. 9-1 Z) and long and short fibrillar elements and networks (Figs. 9, II, 12). Some conventionally fixed and contrasted cells possessed radially-arranged or polar fimbriae. Organisms in the outer plaque were often separated from one another by areas of mostly non-contrasting matrix, with occasional fine strands between organisms. Similar fibrils often connected bacteria and cuticle. Initial attachment of bacteria to cuticle and bacteria to each other when surfaces were furthest apart was by fine polysaccharide tibrils or globules (Figs. 9, ZO), amorphous or fibrillar networks (Fig. 11) or nonpolysaccharide or proteoglycan fimbriae (i.e. fixed routinely and contrasted with lead was associated with similar, citrate and uranyl acetate) (Fig. IS). Closer approximation denser, less discrete links, as well as interspersed globular or vesicular elements (Fig. 12). The next stage seemed to be a closer approximation of cells to each other and the cuticle, and the loss in discrete morphology of the matrix components (Figs. 6, 8). Acidic mucopolysaccharide was often found on the external surface of the cuticle in the absence of adjacent microorganisms (Fig. 2). Closer approximation to cuticle resulted in flattening of that portion of bacterial cell wall contiguous to the cuticle (Fig. 6). Infrequently bacteria were attached to the cuticle through epithelial cells. Organisms
240
Journal of Dentistry, Vol. ~/NO. 3
Fig. IO. Ruthenium
red. Outermost cell has round mucopolysaccharide elements .between it and cuticle. These seem to either fuse or become less discrete closer to the cuticle surface. Bar = 1 om. (X 22000.)
Fig. Il.
Fig. 12. Ruthenium
Fig. 13. Routine fixation. This organism is attached
red. A fibrillar network seems to have extended from the bacteria to coat the cuticle. The closer the organisms approach to the cuticle, the less discrete become the separating polysaccharide structures. Bar = 1 pm. (X 34 000).
Ruthenium red. This cell has an abundant fibrillar glycocalyx with interspersed globular elements. Note long extensions to cuticle. Bar = 1 pm. (X 34 000).
to a striated cuticle by a fine fimbrial network. E, enamel. Bar = 1 pm. (X 63 000.)
Newman:
Host-organism
interface
in dental plaque
241
DISCUSSION The observations may be correlated in many respects with known data on bacterial adhesion at interfaces and colonization of teeth as studied in defined systems. The histochemical techniques employed in this study are established procedures for the ultrastructural demonstration of carbohydrate-containing macromolecules. PA-TSC-AgP is considered specific for aldehyde groups formed by periodic acid oxidation in polysaccharides other than proteoglycan/glycoprotein/mucopolysaccharide, although prolonged incubation can label some molecules (Thiery, 1967). Ruthenium red and colloidal iron at low pH bind to polyanionic proteoglycans/mucopolysaccharides but not to proteins, polypeptides or neutral polysaccharides (Mowry, 1963; Brooker and Fuller, 1975). Polysaccharides and polysaccharideeprotein complexes are stained by Alcian blue at neutral pH. Their contrast is further improved by the incorporation of lanthanum in the reagent mixture (Shea, 1971). Safranin 0 binds ruthenium red and colloidal iron to polyanionic polysaccharides, but penetrates tissues more efficiently because of its smaller molecular weight, its contrasting effect being proportional to the tissue glycosaminoglycan content (Shepard and Mitchell, 1976). Irregularities in dimensions, morphology and texture of the enamel cuticle in the region of the gingival crevice have been observed previously (Newman, 1975a). The present study suggests that glycoprotein or proteoglycan may contribute to its content, while a more polyanionic acidic mucopolysaccharide is localized to its external aspect. The polysaccharide may derive from epithelial attachment substance as well as bacteria. Schroeder and Listgarten (197 1) state that the cuticle is composed mainly of protein resistant to peptic and tryptic digestion, and contains some mucopolysaccharide and protein-bound lipids. The continuity of the cuticle with organic matrix around enamel crystals suggests that the subsurface and thin surface cuticles comprise one organic structure. It is possible that the external layers of thick cuticles may derive from accretions from gingival fluid or contiguous crevicular epithelial cells. It still has to be ascertained whether the bacterial glycocalyx in situ in natural plaque derives from the producer organism, or contains elements from other bacteria, crevicular fluid, epithelium or saliva. Regarding bacterial adhesion to and subsequent scalloping of the enamel cuticle, Corpe and Winters (1972) have noted the ability of marine periphytic bacteria to utilize protein and polysaccharide components of their host surface. The presence of an organic cuticle between host and bacteria is common to a variety of habitats (Newman, 1974). Diminishing radius of curvature reduces the potential energy barrier to contact (Weiss, 1970) and helps to explain preferential adhesion of coccal cells with surface fine filamentous or globular polysaccharide extensions. Cross-linked polysaccharide meshes have been observed to mediate bacterial attachment in a marine habitat (Corpe, 1970). Irregularities in glycocalyx structure and contact with cuticle support suggestions of specific binding sites on bacterial surfaces (Sutherland, 1977). The most likely mechanism of closer approximation of bacteria to cuticle may be hydrogen bonding or salt bridge formation between polysaccharide chains (Sutherland, 1977). Rolla (1976, 1977) has shown that divalent cations may mediate attachment by polysaccharides. The sequence of colonization observed resembles that in developing plaque (Hardie and Bowden, 1974; Newman and Poole, 1974) and in plaque on healthy gingiva and on gingiva affected by early chronic gingivitis (Listgarten, 1976). The structural features of the plaque-~ cuticle interface resemble those in microbial marine habitats. It is possible that the
Journal of Dentistry, Vol. ~/NO. 3
242
sequence of events in colonization is similar, i.e. sorption of polymers to the surface, the attraction of bacteria, (? reversible sorption), irreversible sorption and development of secondary microflora (Mitchell, 1976). The main disparity with the marine habitat seems to be that non-motile bacteria are the first to colonize the tooth. Unlike lysed organisms the intact cells at the subcontact area apical border of plaque contained IPS (Newman, 1975b) and possessed glycocalyx. The resistance of the former to lysis is due probably to the hydrophilic nature of the glycocalyx, which confers on a producer organism a contact angle with saline less than that of human neutrophils, which are abundant in cievicular fluid. Bacteria will neither adhere to nor be phagocytosed by polymorphs unless their surface hydrophobicity is sufficient for the contact area they make with saline to exceed that made by polymorphs, allowing the latter to spread around and engulf the organisms. Specific antibodies and complement may enhance both contact area angle and phagocytosis. Polysaccharide glycocalyx and possibly also polypeptide fimbriae may serve as antiphagocytic shields by being highly hydrophilic and thereby not readily contacted by the more hydrophobic phagocytes (Dudman, 1977). However, it is possible that lysed organisms did possess a glycocalyx prior to lysis. Carbohydrate-containing macromolecules (glycomacromolecules) in one form or another constitute the outermost surface layers of cells of all taxa, although they do not appear to be essential to viability (Dudman, 1977). Their formation is particularly abundant where growth nutrients are limited, carbohydrate such as sucrose plentiful, and pH low (Newman, 1974), as in the subcontact area locus on children’s teeth (Newman, 1975b). Their synthesis may be necessary for bacterial adhesion (Koga and Inoue, 1978), while other organisms may depend on them if they are not capable themselves of adhering directly to the cuticle. Polysaccharide-containing substances are highly hydrated and shrink extensively on dehydration (Bayer and Thurow, 1977; Roth, 1977). The fine fibrils observed in this study are unlikely to be shrinkage artefacts since they are also present in relation to freeze-etched bacteria in pure culture and in plaque (Newman, 1972; Newman and Britton, 1973,1974). Some fimbriae of plaque organisms are likely to be protein on the basis of their contrasting reaction on routine fixation and staining with lead citrate and uranyl acetate, which would conform to their main composition in other forms (Ottow, 1975). Fimbriae of strains of Bacteroides melaninogenicus have been suggested as the mechanism of adherence of this organism in the gingival crevice (Okuda and Takazoe, 1974). Vesicles as observed in relation to bacteria in this study are produced by many plaque bacteria, and may have a role in attachment in other aqueous habitats (Ridgway and Lewin, 1973; Kerebel et al., 1975; Newman et al., 1976; Theilade et al., 1976). They may contain lipoteichoic acids (LTA), which have been found external to several Gram-positive species and shown to take part in bacterial adhesion. The lipids of LTA anchor cell wall teichoic acids to cytoplasmic membrane, and may function similarly in bacterial adhesion (Jones, 1977). Rolla (1977) suggests that extracellular LTA is trapped in the EPS around sucrosegrown Streptococcus mutans. It is possible that the vesicles enclosed in the glycocalyces of plaque organisms are similarly enclosed LTA. However, the adhesive properties of LTA, if present, must be explained on the basis of adhesion, not to the rarely exposed hydroxyapatite (Ciardi et al., 1977) but to the organic cuticle.
CONCLUSlON Correlating
the findings
of this and previous studies,
a likely sequence of events in initial
Newman:
Host-organism
interface
in dental plaque
243
bacterial colonization at the apical plaque border on children’s teeth would be as follows: 1. Selective survival of predominantly coccal IPScontaining forms. 2. Adhesion of bacterial glycocalyx to cuticle by means of mainly fibrillar and globular polysaccharides. On the basis of the semi-specific nature of the histochemical reagents used, these could include mucopolysaccharide/glycoprotein and heteroglycans. 3. Approximation of bacteria and cuticle, by cross-linking and salt bridging of polysaccharide, possibly until mutually repulsive negative surface charges are overcome. Low radius of curvature of polysaccharide processes and low pH would favour reduction in bacterial surface potential. 4. Flattening of bacterial cell walls at the sites of contact or close approximation, due presumably to short range attractive forces.
Acknowledgements I would like to thank Mrs J. Downton and the staff of the Oral Surgery Department, Eastman Dental Hospital, who obtained the dental specimens used in this study, and Miss C. Rundle and Mrs P. Quirke for their assistance. I acknowledge gratefully support of this work by a grant from the Medical Research Council. REFERENCES Bayer
M. E. and Thurow H. (1977) Polysaccharide capsule of Escherichia cob: microscope study of its size, structure and sites of synthesis. J. Bacterial. 130,9 1 l-936. Brooker B. E. and Fuller R. (1975) Adhesion of lactobacilli to the chicken crop epithelium. J. Ultrastruct. Res. 52, 21-31.
Ciardi J. E., Rolla G., Bowen W. H. et al. (1977) Adsorption of Streptococcus mutans lipoteichoic acid to hydroxyapatite. Stand. J. Dent. Res. 85, 387-391. Corpe W. A. (1970) Attachment of marine bacteria to solid surfaces. In: Manly R. S. (ed.) Adhesion in BioZogicaZSystems. New York, London, Academic Press, pp. 73-87. Corpe W. A. and Winters H. (1972) Hydrolytic enzymes of some periphytic marine bacteria. Can. J. Microbial.
18, 1483-1490.
DiPersio J. R. and Deal S. J. (1974) Identification of intracellular polysaccharide granules in thin sections of Nocardia asteroides. J. Gen. Microbial. 83, 349-358. DiPersio J. R., Mattingly S. J., Higgins M. L. et al. (1974) Measurement of intracellular iodophilic polysaccharide in two cariogenic strains of Streptococcus mutans by cytochemical and chemical methods. Infect. Immunol. 10, 597-604. Dudman W. F. (1977) The role of surface polysaccharides in natural environments. In: Sutherland I. (ed.) Surface Carbohydrates of the Procaryotic Cell. London, New York, Academic Press, pp. 357-414. Glantz P.-O. (1977) Adhesion to teeth. Znt. Dent. J. 27, 324-332. Hardie J. M. and Bowden G. H. (1974) The normal microbial flora of the mouth. In: Skinner F. A. and Carr J. G. (ed.) The Normal Microbial Flora of Man. London, New York, Academic Press, pp. 47-83. Jones G. W. (1977) The attachment of bacteria to the surfaces of animal cells. In: Reissig J. J. (ed.) Receptors and Recognition. Series B. Volume 3. Microbial Interactions. London, Chapman and Hall, pp. 1399176. Kerebel B., Clergeau-Gudrithault S. and Forlot P. (1975) Etude ultrastructurale de plaques microbiennes dentaires chez des sujets indemnes de carie. Ann. Microbial. (Inst. Pasteur) 126A, 203-229.
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of Dentistry,
Vol. ~/NO.
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Koga T. and Inoue M. (1978) Cellular adherence, glucosyltransferase adsorption and glucan synthesis of Streptococcus mutans AHT mutants. Infect. Immunol. 19,402-410. Listgarten M. A. (1976) Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic study. J. Periodont. 47, l-l 8. Mitchell R. (1976) Mechanism of attachment of microorganisms to surfaces. In: Stiles H. M., Loesche W. .I. and O’Brien T. C. (ed.) Microbial Aspects of Dental Caries. Washington D.C., Information Retrieval Inc., pp. 47-54. Mowry R. W. (1963) The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mucins. With revised directions for the colloidal iron stain, the use of Alcian blue G8X and their combinations with the periodic acid - Schiff reaction. Ann. NY Acad. Sci. 106,402-423. Newman H. N. (1972) Freeze-etching and dental research. J. Periodont. Res. 7, 9 1 - 101. Newman H. N. (1974) Microbial films in nature. Microbios 9, 247-257. Newman H. N. (1975a) The pre-eruptive portion of the human enamel integument. J. Dent. 3,110-l 20. Newman H. N. (1975b) The gingival border of plaque. Morphological studies in 8 to 15 year old children. Br. Dent. J. 138, 335-345. Newman H. N. and Britton A. B. (1973) Ultrastructure of selected bacteria isolated from dental plaque as revealed by freeze-etching. J. Dent. Res. 52, 1194- 120 1. Newman H. N. and Britton A. B. (1974) Dental plaque ultrastructure as revealed by freezeetching. J. Periodont. 45,478-488. Newman H. N., Donoghue H. D. and Britton A. B. (1976) Effect of glucose and sucrose on the survival in batch culture of Streptococcus mutans C67-1 and a non-cariogenic mutant C67-25. Morphological studies. Microbios 15, 113-125. Newman H. N. and Poole D. F. G. (1974) Structural and ecological aspects of dental plaque. In: Skinner F. A. and Carr J. G. (ed.) The Normal Microbial Flora of Man. London, Academic Press, pp. 11 l-l 34. Okuda K. and Takazoe I. (1974) Haemagglutinating activity of Bacteroides melaninogenicus. Arch. OralBiol.
19,415-416.
Ottow J. C. G. (1975)
Ecology,
physiology
and genetics
of fimbriae
and pili. Ann. Rev.
Microbial. 29, 79-108.
Ridgway H. F. and Lewin R. A. (1973) Goblet-shaped sub-units from the wall of a marine gliding microbe. J. Gen. Microbial. 79, 119-128. Rolla G. (1976) Inhibition of adsorption - general considerations. In: Stiles H. M., Loesche W. J. and O’Brien T. C. (ed.) Microbial Aspects of Dental Curies. Washington D.C., Information Retrieval Inc., pp. 309-324. Rolla G. (1977) Formation of dental integuments - some basic chemical considerations. Swed. Dent. J. 1,241-251. Roth I. L. (1977) Physical structure of surface carbohydrates. In: Sutherland 1. (ed.) Surface Carbohydrates of the Procaryotic Cell. London, Academic Press, pp. 5-26. Schroeder H. E. and Listgarten M. A. (1971) In: Wolsky A. (ed.) Fine Structure of the Developing Epithelial A ttachment of Human Teeth. Basel, Karger. Shea S. M. (197 1) Lanthanum staining of the surface coat of cells. J. Cell Biol. 51,6 11-620. Shepard N. and Mitchell N. (1976) The localization of proteoglycan by light and electron microscopy using safranin 0. J. Ultrastruct. Res. 54,451-460. Sutherland 1. W. (1977) Bacterial exopolysaccharides - their nature and production. In: Sutherland I. (ed.) Surface Carbohydrates of the Procaryotic Cell. London, Academic Press, pp. 27-96. Theilade J., Fejerskov 0. and Hbrsted M. (1976) A transmission electron microscopic study of 7-day old bacterial plaque in human tooth fissures. Arch. Oral Biol. 21, 587-598.
Newman:
Host-organism
interface
in dental plaque
245
Thiery J.-P. (1967) Mise en evidence des polysaccharides sur coupes fines en microscopic electronique. J. Microscopic 6, 987-l 018. Weiss L. (1970) A biophysical consideration of cell contact phenomena. In: Manly R. S. (ed.) Adhesion in Biological Systems. New York, London, Academic Press, pp. l-14. Zetterqvist H. (1956) Thesis, Stockholm. Quoted in: Glauert A. M. The fixation and embedding of biological specimens. In: Kay D. (ed.) Techniques for Electron Microscopy. Oxford, Blackwell Scientific Publications, pp. 16662 12.
7, No. 3, 1979,
pp. 235-245.
Printed in Great Britain
The host-organism interface human dental plaque H. N. Newman, Department
in natural
BDent SC, MA, MDS, PhD
of Periodontology,
lnstitu te of Dental
Surgery, Eastman Dental Hospital,
University of London
ABSTRACT
This study concerns the adhesion of natural apical border plaque to approximal surfaces of children’s posterior teeth. The predominant intact cells were polysaccharide-containing cocci linked to the cuticle by means of mainly fibrillar or globular polysaccharide. Correlation of the features observed with those from studies of plaque formation and microbial adhesion in other habitats suggests that approximation of bacteria to enamel cuticle (and salivary pellicle) may be due to cross-linking and salt bridging of polysaccharide until mutually repulsive net negative surface charges are overcome. Low radius of curvature of bacterial processes and low pH, the latter to be expected at the subcontact area apical border in children, would favour reduction in bacterial surface potential. The final stages in bacterial adhesion in plaque seem to involve strong forces producing flattening of the cell wall at the site of the closest contact.
INTRODUCTION Concomitant with the study of individual bacterial and host binding factors is the need to establish the mechanisms mediating adhesion of natural dental plaque to teeth in viva. It is also necessary to correlate observations from studies of short term plaque formation with the same features in natural plaque. The present study is part of an attempt at such a correlation, being based on the carbohydrate electronhistochemistry of natural approximal plaque from children’s teeth. The importance of studying the cuticle-plaque (and acquired pellicle-plaque) interface needs to be emphasized in view of the observation that components located more than 10 nm from the surface will have no significant influence on surface characteristics (Glantz, 1977). Even after a few seconds, it is possible to detect such an organic film on clean solid surfaces (Glantz, 1977). Hydroxyapatite properties are relevant mainly to the binding of cuticle or salivary glycoprotein to enamel.
MATERIALS Premolar
AND METHODS
and molar
teeth
removed
from
children
for orthodontic
reasons
were
rinsed
to
remove loose debris and blood, and then placed immediately in a primary fixative solution (3 per cent glutaraldehyde in cacodylate buffer, pH 7.4) and fixed for 3 h at 4 “C. They were then washed overnight in sodium cacodylate buffer, pH 7.4, containing 5 per cent sucrose. As teeth were removed in the morning under general anaesthesia, all the patients had been fasting for at least 10 h. None of the specimens had clinically apparent gingivitis or approximal caries. Gingival crevices were not probed, to avoid disturbance of the apical border of the plaque. Specimens were processed by the following methods with controls as appropriate. Methods 3 and 4 also served as controls for each other, as did comparison of the features present on specifically and non-specifically stained specimens. 1. Post-fixation for 2 h in 2 per cent 0~0~ in veronal acetate buffer (Zetterqvist, 1956). 2. Periodic acid-thiosemicarbazide-silver proteinate technique (PA-TSC-AgP) (grid stain) (Thiery, 1967).
236
Fig. I. Routine fuation. Average enamel cuticle. E, enamel. Bar = 0.i pm. (X 18 885.) Fig. 2. Ruthenium red. Amorphous thick enamel cuticle. Note external surface coating with amorphous and globular ruthenium red-positive material. Lines and holes in cuticle are section artefacts. E, enamel. Bar = 0.5 pm. (X 6110.) Fig 3. Safranin O-positive enamel cuticle. The most coronal plaque is at the bottom of the micrograph. Note that initial scalloping of the cuticle is followed by a diminution in thickness until only a thin primordial layer remains. Electron-lucent vesicles in most organisms are polysaccharide granules. E, enamel. Bar = 0.5 pm. (X 11 110.)
3. Ruthenium red (Brooker and Fuller, 1975). 4. Colloidal iron (Mowry, 1963). 5. Alcian blue/lanthanum nitrate (Shea, 1971). 6. Safranin 0 (Shepard and Mitchell, 1976). 7. Sodium chlorite (grid stain) (DiPersio and Deal, 1974; DiPersio et al., 1974). The approximal plaque-bearing surfaces were removed from processed teeth with a diamond disc and cooled and rinsed with an appropriate buffer. All specimens were dehydrated in graded ethanol series and ethoxypropane, and embedded in Araldite (CY2 12, EMScope, London, England). From the embedded surfaces specimens incorporating the following sites were removed: (a) subcontact area or mid-approximal apical border; @) corresponding embrasure specimen. These were demineralized from the enamel side in EDTA buffered to pH 7.4 with NaOH. After removal from enamel, the specimens were dehydrated as before and reembedded in Araldite. Sections were cut to give an interference colour of silver to light gold (80-I 20 nm).
Newman:
Host-organism
interface
in dental plaque
237
F&Y 4. PA-TSC-AgP. Predominant intact forms in apical border plaque subjacent to the contact area are Gram-negative and positive polysaccharide-containing cocci. Gram-negative forms include many large cells. E, enamel. Bar = 0.8 pm. (x 4800.) Fig. 5. PA-TSC-AgP. Apical organisms are either lysed or polysaccharidecontaining. Note poorly contrasted cuticle. E, enamel. Bar = 0.8 pm. (X 22 400.)
Sections from specimens fixed routinely with glutaraldehyde and osmium tetroxide were stained further with 0.4 per cent ethanolic uranyl acetate for 4 mm, followed by O-4 per cent lead nitrate in aqueous N/l0 NaOH for 4 min.
RESULTS The enamel cuticle This organic structure formed on the enamel surface before eruption, consisted of an electron-dense layer as observed by conventional and proteoglycan contrasting (Figs. I-S), but was more electron-lucent when contrasted with PA-TSC-AgP (Fig. 5). The cuticle was continuous with prolongations of organic matter between the spaces occupied before demineralization by the enamel crystals. Subsurface organic matter was variable in amount,
238
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Fig. 6. PA-TSC-AgP. Bacteria are separated from enamel by thin cuticle and glycocalyx. Note flattening of cell walls at regions of close contact. E, enamel. Bar = 0.7 brn. (X 34 300.) Fig. 7. Ruthenium red. Intact cell has irregular amorphous mucopolysaccharide glycocalyx. E, enamel. Bar = 0.7 Mm. (X 23 800.)
being most abundant in relation to irregularities such as lamellae and striae of Retzms. In many sections, subsurface organic matter was sparse. The cuticle was usually between 30 and 200 nm thick, but occasionally exceeded 5 pm (Fig. 2) especially in the region under examination, i.e. between junctional epithelium and the apical border of the plaque. Occasionally it extended beneath the plaque, in which case it was scalloped by organisms and reduced in thickness until only a thin layer similar to the usual thin cuticle remained (Fig. 3). This layer persisted between the plaque and the enamel. Homogeneous, ‘fuzzy’, vacuolated and striated varieties of thick cuticle were observed. Thick cuticles were more common in embrasures than in subcontact area loci, and were sometimes more continuous with proteoglycan-like matter at the suface of the apical border of the plaque in the former location. The apical border of plaque The predominant organisms at the border were Gram-positive and those less abundant were Gram-negative cocci (Fig. 4). The border was usually discrete in the embrasure and tapering apical to the contact area. A more varied flora was found in embrasures.,The most apical organisms of the tapering border were either lysed cells comprising cell walls with or without vestiges of cytoplasm, or IPScontaining, apparently intact cells (Figs. 3-5). Apparently intact organisms were often present in microcolonies. In the outer plaque, even in thin layers, organisms were more randomly arranged (not palisaded) and the proportion of matrix to bacteria was higher.
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Fig 8. Colloidal iron. Partially demineralized. Note coating of both cells by acidic mucopolysaccharide or proteoglycan and thinning of this layer in relation to portion of left-hand cell in contact with cuticle. Bar = 1 pm. (X 34 000.)
The plaque-host
Fig. 9. Ruthenium red. This cell shows tine fibrillar elements on the aspects away from the cuticle and globules of mucopolysaccharide between cell wall and cuticle. Black intracellular globule is a common ruthenium artefact. E, enamel. Bar = 1 pm. (X 34 000.)
interface
were not found with their cell walls in direct contact with crystal spaces of the surface enamel, but were separated from them by enamel cuticle and bacterial glycocalyx (Fig. 6). Staining with ruthenium red, colloidal iron or Alcian blue/lanthanum nitrate invariably disclosed a glycocalyx which appeared to mediate host-bacterial (and interbacterial) attachment and which was absent on routinely fixed, nonspecifically contrasted specimens (Figs. 7-13). This irregular, fuzzy coat external to the cell walls varied considerably in thickness. The more abundant glycocalyx contained amorphous material (Figs. 7, 8) and often possessed globular (Figs. 9-1 Z) and long and short fibrillar elements and networks (Figs. 9, II, 12). Some conventionally fixed and contrasted cells possessed radially-arranged or polar fimbriae. Organisms in the outer plaque were often separated from one another by areas of mostly non-contrasting matrix, with occasional fine strands between organisms. Similar fibrils often connected bacteria and cuticle. Initial attachment of bacteria to cuticle and bacteria to each other when surfaces were furthest apart was by fine polysaccharide tibrils or globules (Figs. 9, ZO), amorphous or fibrillar networks (Fig. 11) or nonpolysaccharide or proteoglycan fimbriae (i.e. fixed routinely and contrasted with lead was associated with similar, citrate and uranyl acetate) (Fig. IS). Closer approximation denser, less discrete links, as well as interspersed globular or vesicular elements (Fig. 12). The next stage seemed to be a closer approximation of cells to each other and the cuticle, and the loss in discrete morphology of the matrix components (Figs. 6, 8). Acidic mucopolysaccharide was often found on the external surface of the cuticle in the absence of adjacent microorganisms (Fig. 2). Closer approximation to cuticle resulted in flattening of that portion of bacterial cell wall contiguous to the cuticle (Fig. 6). Infrequently bacteria were attached to the cuticle through epithelial cells. Organisms
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Fig. IO. Ruthenium
red. Outermost cell has round mucopolysaccharide elements .between it and cuticle. These seem to either fuse or become less discrete closer to the cuticle surface. Bar = 1 om. (X 22000.)
Fig. Il.
Fig. 12. Ruthenium
Fig. 13. Routine fixation. This organism is attached
red. A fibrillar network seems to have extended from the bacteria to coat the cuticle. The closer the organisms approach to the cuticle, the less discrete become the separating polysaccharide structures. Bar = 1 pm. (X 34 000).
Ruthenium red. This cell has an abundant fibrillar glycocalyx with interspersed globular elements. Note long extensions to cuticle. Bar = 1 pm. (X 34 000).
to a striated cuticle by a fine fimbrial network. E, enamel. Bar = 1 pm. (X 63 000.)
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DISCUSSION The observations may be correlated in many respects with known data on bacterial adhesion at interfaces and colonization of teeth as studied in defined systems. The histochemical techniques employed in this study are established procedures for the ultrastructural demonstration of carbohydrate-containing macromolecules. PA-TSC-AgP is considered specific for aldehyde groups formed by periodic acid oxidation in polysaccharides other than proteoglycan/glycoprotein/mucopolysaccharide, although prolonged incubation can label some molecules (Thiery, 1967). Ruthenium red and colloidal iron at low pH bind to polyanionic proteoglycans/mucopolysaccharides but not to proteins, polypeptides or neutral polysaccharides (Mowry, 1963; Brooker and Fuller, 1975). Polysaccharides and polysaccharideeprotein complexes are stained by Alcian blue at neutral pH. Their contrast is further improved by the incorporation of lanthanum in the reagent mixture (Shea, 1971). Safranin 0 binds ruthenium red and colloidal iron to polyanionic polysaccharides, but penetrates tissues more efficiently because of its smaller molecular weight, its contrasting effect being proportional to the tissue glycosaminoglycan content (Shepard and Mitchell, 1976). Irregularities in dimensions, morphology and texture of the enamel cuticle in the region of the gingival crevice have been observed previously (Newman, 1975a). The present study suggests that glycoprotein or proteoglycan may contribute to its content, while a more polyanionic acidic mucopolysaccharide is localized to its external aspect. The polysaccharide may derive from epithelial attachment substance as well as bacteria. Schroeder and Listgarten (197 1) state that the cuticle is composed mainly of protein resistant to peptic and tryptic digestion, and contains some mucopolysaccharide and protein-bound lipids. The continuity of the cuticle with organic matrix around enamel crystals suggests that the subsurface and thin surface cuticles comprise one organic structure. It is possible that the external layers of thick cuticles may derive from accretions from gingival fluid or contiguous crevicular epithelial cells. It still has to be ascertained whether the bacterial glycocalyx in situ in natural plaque derives from the producer organism, or contains elements from other bacteria, crevicular fluid, epithelium or saliva. Regarding bacterial adhesion to and subsequent scalloping of the enamel cuticle, Corpe and Winters (1972) have noted the ability of marine periphytic bacteria to utilize protein and polysaccharide components of their host surface. The presence of an organic cuticle between host and bacteria is common to a variety of habitats (Newman, 1974). Diminishing radius of curvature reduces the potential energy barrier to contact (Weiss, 1970) and helps to explain preferential adhesion of coccal cells with surface fine filamentous or globular polysaccharide extensions. Cross-linked polysaccharide meshes have been observed to mediate bacterial attachment in a marine habitat (Corpe, 1970). Irregularities in glycocalyx structure and contact with cuticle support suggestions of specific binding sites on bacterial surfaces (Sutherland, 1977). The most likely mechanism of closer approximation of bacteria to cuticle may be hydrogen bonding or salt bridge formation between polysaccharide chains (Sutherland, 1977). Rolla (1976, 1977) has shown that divalent cations may mediate attachment by polysaccharides. The sequence of colonization observed resembles that in developing plaque (Hardie and Bowden, 1974; Newman and Poole, 1974) and in plaque on healthy gingiva and on gingiva affected by early chronic gingivitis (Listgarten, 1976). The structural features of the plaque-~ cuticle interface resemble those in microbial marine habitats. It is possible that the
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sequence of events in colonization is similar, i.e. sorption of polymers to the surface, the attraction of bacteria, (? reversible sorption), irreversible sorption and development of secondary microflora (Mitchell, 1976). The main disparity with the marine habitat seems to be that non-motile bacteria are the first to colonize the tooth. Unlike lysed organisms the intact cells at the subcontact area apical border of plaque contained IPS (Newman, 1975b) and possessed glycocalyx. The resistance of the former to lysis is due probably to the hydrophilic nature of the glycocalyx, which confers on a producer organism a contact angle with saline less than that of human neutrophils, which are abundant in cievicular fluid. Bacteria will neither adhere to nor be phagocytosed by polymorphs unless their surface hydrophobicity is sufficient for the contact area they make with saline to exceed that made by polymorphs, allowing the latter to spread around and engulf the organisms. Specific antibodies and complement may enhance both contact area angle and phagocytosis. Polysaccharide glycocalyx and possibly also polypeptide fimbriae may serve as antiphagocytic shields by being highly hydrophilic and thereby not readily contacted by the more hydrophobic phagocytes (Dudman, 1977). However, it is possible that lysed organisms did possess a glycocalyx prior to lysis. Carbohydrate-containing macromolecules (glycomacromolecules) in one form or another constitute the outermost surface layers of cells of all taxa, although they do not appear to be essential to viability (Dudman, 1977). Their formation is particularly abundant where growth nutrients are limited, carbohydrate such as sucrose plentiful, and pH low (Newman, 1974), as in the subcontact area locus on children’s teeth (Newman, 1975b). Their synthesis may be necessary for bacterial adhesion (Koga and Inoue, 1978), while other organisms may depend on them if they are not capable themselves of adhering directly to the cuticle. Polysaccharide-containing substances are highly hydrated and shrink extensively on dehydration (Bayer and Thurow, 1977; Roth, 1977). The fine fibrils observed in this study are unlikely to be shrinkage artefacts since they are also present in relation to freeze-etched bacteria in pure culture and in plaque (Newman, 1972; Newman and Britton, 1973,1974). Some fimbriae of plaque organisms are likely to be protein on the basis of their contrasting reaction on routine fixation and staining with lead citrate and uranyl acetate, which would conform to their main composition in other forms (Ottow, 1975). Fimbriae of strains of Bacteroides melaninogenicus have been suggested as the mechanism of adherence of this organism in the gingival crevice (Okuda and Takazoe, 1974). Vesicles as observed in relation to bacteria in this study are produced by many plaque bacteria, and may have a role in attachment in other aqueous habitats (Ridgway and Lewin, 1973; Kerebel et al., 1975; Newman et al., 1976; Theilade et al., 1976). They may contain lipoteichoic acids (LTA), which have been found external to several Gram-positive species and shown to take part in bacterial adhesion. The lipids of LTA anchor cell wall teichoic acids to cytoplasmic membrane, and may function similarly in bacterial adhesion (Jones, 1977). Rolla (1977) suggests that extracellular LTA is trapped in the EPS around sucrosegrown Streptococcus mutans. It is possible that the vesicles enclosed in the glycocalyces of plaque organisms are similarly enclosed LTA. However, the adhesive properties of LTA, if present, must be explained on the basis of adhesion, not to the rarely exposed hydroxyapatite (Ciardi et al., 1977) but to the organic cuticle.
CONCLUSlON Correlating
the findings
of this and previous studies,
a likely sequence of events in initial
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bacterial colonization at the apical plaque border on children’s teeth would be as follows: 1. Selective survival of predominantly coccal IPScontaining forms. 2. Adhesion of bacterial glycocalyx to cuticle by means of mainly fibrillar and globular polysaccharides. On the basis of the semi-specific nature of the histochemical reagents used, these could include mucopolysaccharide/glycoprotein and heteroglycans. 3. Approximation of bacteria and cuticle, by cross-linking and salt bridging of polysaccharide, possibly until mutually repulsive negative surface charges are overcome. Low radius of curvature of polysaccharide processes and low pH would favour reduction in bacterial surface potential. 4. Flattening of bacterial cell walls at the sites of contact or close approximation, due presumably to short range attractive forces.
Acknowledgements I would like to thank Mrs J. Downton and the staff of the Oral Surgery Department, Eastman Dental Hospital, who obtained the dental specimens used in this study, and Miss C. Rundle and Mrs P. Quirke for their assistance. I acknowledge gratefully support of this work by a grant from the Medical Research Council. REFERENCES Bayer
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