Calcif. Tiss. Res. 21, 47--55 (1976) 9 by Springer-Verlag 1976

Scanning Electron Microscopy of Dental Calculus J. L u s t m a n n , J. L e w i n - E p s t e i n a n d A. Shteyer Department of Oral Surgery, The Hebrew University, I-Iadassah School of Dental Medicine, Jerusalem Received January 13, accepted April 7, 1976 The morphologic structure of anorganic dental calculus was studied by means of the scanning electron microscope. From surface observations, calculus is apparently composed of two components with distinguishable patters of calcification. One component is formed by the precipitation of minute calcific crystals on microorganisms and intermicrobial substances (plaque matrix). Such calcified masses, often spherical in shape, have a sponge-like appearance with empty spaces representing the former sites of entombed and degenerated organisms. Thus, intracellular calcification is not evident at this stage of calculus development. The other component, although having at least one common calcification front with the former, does not appear to be directly associated with microbial calcification. It exhibits a configuration of generally larger crystal growths of varying shapes and sizes. These two calcification patterns are comparable, both in distribution and size, to what has been observed by means of the transmission electron microscope, and what Schroeder has designated as "types A& B centers of mineralization," respectively. The calcific precipitation in type A centers have been identified by X-ray diffraction as hydroxyapatite. It is, therefore, speculated that the crystal patters in type B centers might represent other known forms of calcium phosphates present in calculus, such as octacalcium phosphate, whitlockite and brushite. Key words: Dental calulus - - Scanning Electron microscopy - - Calcification- - Microorganism.

Introduction D e n t a l calculus, a p r o d u c t of calcification of the d e n t a l plaque, is the most c o m m o n ectopic calcific deposit i n m a n a n d can be f o u n d a r o u n d t e e t h of most h u m a n s . Macroscopically, the surface of d e n t a l calculus is observed to be irregular, coarse, a n d covered b y a t h i n layer of plaque. X - r a y diffraction studies have shown the d e n t a l calculus to be composed of four principle minerals: h y d r o x y a p a t i t e , octacalcium phosphate, brushite, a n d whitlockite. The ratio of these c o m p o n e n t s v a r y a n d is m a i n l y d e p e n d e n t on the age a n d site of the calculus [16]. F u r t h e r m o r e , Schroeder [16], i n his investigations using histochemistry, polarized light, a n d t r a n s m i s s i o n electron microscopy, has concluded t h a t two d i s t i n c t types of m i n e r a l i z a t i o n centers can be distinguished, which a p p a r e n t l y c o n s t i t u t e different m e c h a n i s m s of formation. He called these centers of m i n e r a l i z a t i o n " t y p e s A a n d B ". Mineralization centers of type A are i n i t i a t e d a n d formed only i n the presence of, a n d i n association with, microorganisms, whereas B-centers, which i n v a r i a b l y have a t least one c o m m o n border with A-centers, are a p p a r e n t l y n o t directly related to microorganisms in their formation.

For reprints: Dr. J. Lnstmann, Department of Oral Surgery, Hebrew University-Hadassah School of Dental Medicine, P.O.B. 499, Jerusalem, Israel.

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Fig. 1 A and B. Cross section of fractured surfaces of dental calculi. (A) I-Iomogeneous surface in thin, dark calculus of lower anterior tooth ( • 162). (B) Laminated surface from large white chalky calculus removed from upper molar ( • 57)

R e c e n t studies h a v e d e m o n s t r a t e d t h e m o r p h o l o g y of d e n t a l eMeulus b y m e a n s of t h e scanning electron microscope (SEM) [1, 7-10, 18]. The p u r p o s e of this s t u d y is to p r e s e n t f u r t h e r SEM o b s e r v a t i o n s on t h e m o r p h o l o g y of d e n t a l calculus, especially as t h e y relate to r e p o r t e d o b s e r v a t i o n s using t r a n s m i s s i o n electron microscopy. Materials and Methods Extracted teeth with attached and undamaged calculus were fixed in 10% buffered formaldehyde. All specimens were treated with sodium hypochlorite (NaOCI) for 20 minin order to remove plaque and other organic debris. This was followed by rinsing in running water for 15 min. In many specimens parts of the attached calculus were purposely split. from the main body to reveal the fractured surface. Following the rinsing, all specimens were dried, glued to aluminium stubs, coated with about 300 A of gold in a vacuum evaporator (Polaron Equipment Ltd., SEM Coating Unit E 5000) and examined with an $4-10 Stereosean, (Cambridge Scientific Instruments Co. Cambridge, England) operated at 30 KV.

Results W i t h low m a g n i f i c a t i o n t h e superficial surface of d e n t a l calculus a p p e a r e d irregular a n d coarse. The cross section of f r a c t u r e d surfaces in some specimens was h o m o g e n e o u s a n d c o m p a c t (Fig. 1 A), while in others it was l a m i n a t e d a n d exh i b i t e d a high degree of p o r o s i t y (Fig. 1 B). W i t h higher m a g n i f i c a t i o n t h e essential configuration was t h a t of calcified plaque, a n d we could distinguish b e t w e e n t h e older a n d t h e m o r e r e c e n t l y calcified structures. A t t h e p e r i p h e r y of t h e a t t a c h e d calculus we o b s e r v e d a m o r p h i c calcifie deposits on t h e n o r m a l g l o b u l a r surface of t h e c e m e n t u m ; t h e y a p p e a r e d as isolated d r o p l e t s in some areas a n d as larger a g g r e g a t i o n s in others. This is c o m p a t i b l e w i t h t h e e a r l y stages of calculus f o r m a t i o n , which c o m p l e t e l y covers

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Fig. 2. Early calculus formation on surface of cementum. Note normal globular anatomy (G), calcific deposits on cemental surface (/~), and more advanced calcification (C) encompassing microorganisms (arrows), ( • 5,000)

the cementum by successive calcific accretions also encompassing microorganisms, and essentially represents the morphology of early dental calculus (Fig. 2). Clusters of calcified spherical masses measuring 5--20 ~ in diameter were observed. They contained variegated empty spaces which gave them a sponge-like appearance. These spherical masses were clustered on or near larger and less porous calcified masses. They were probably more recently calcified accretions of microcolonies in plaque which had accumulated on previously formed calculus (Fig. 3). I n some fields, calcified rod-like or filamentous microorganisms could be identified projecting almost perpendicularly from these calcified masses (Fig. 4A and B). While some of the projections appeared to be solid mineral, others exhibited hollow tips. The diameters of the ringed spaces in the spherical masses were the same as those of the calcified microorganisms (Fig. 5). Occasionally, we noted minute crystals 5004000 A long deposited upon these mineralized microorganisms, apparently representing extracellular plaque matrix calcification and leading to intercellular coalescence (Fig. 6A and B). A different configuration of crystals was observed in juxtaposition to the sponge-like spherical masses, but apparently was not related to microbial calcification (Fig. 7). These crystals were varied in shape, sometimes applaring elongated or needle-like and measuring about 2,000 A in width (Fig. 8A and B), but more often as platelets of different shapes and sizes, 1,000-2,000 A thick. (Fig. 8C, D, and E). I t was impossible to estimate their true lengths, although we observed

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Fig. 3. Clusters of calcified sponge-like spherical masses with demonstrable calcified rod-like and filamentous microorganisms. Note relation of these spherical musses to a larger calcified mass (C), ( x 2,000)

Fig. 4A and B. Calcified filamentous microorganisms projecting almost perpendicularly from calculus surface. (A x 1,100, B x 5,500)

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Fig. 5. Some calcified microorganisms appear as solid mineral (M) while others appear hollow (H). Note many empty ringed spaces probably representing fractured bacterial molds ( • 10,000)

Fig. 6. (A) Demonstration of calcified microorganisms (M), with fine intermicrobial crystal growth (arrows) (• (B) Higher magnification of intermicrobial crystal deposits. ( • 20,000)

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Fig. 7. Configuration of platelet or needle-like crystal growths (CR) bordering on cluster of spherical masses (S), ( • 500)

that some measured more than 8 ~. Less frequently, tiny euboidal crystal aggregates measuring up to 1 ~z in diameter could also be observed (Fig. 8F).

Discussion I n vitro investigations of microorganisms isolated from dental plaque have demonstrated two morphologie patterns of microbial eMeifieation: one intraeellular and the other extraeellular [2-5, 11, 12, 14, 15, 17]. I n both instances the resultant precipitation was that of calcium phosphate identified by X-ray diffraction, as eMeium hydroxyapatite. Intracellular calcification apparently results from the ability of the microorganisms to convert intraeellular calcium salts to an insoluble form [2]. I t is not clear whether microorganisms, either in their viable or nonviable state, can initiate or catalyze extraeellular and intermicrobiM (matrix) ealeifiability, or if they are passively engulfed in such erystMlization. Appropriate environmental conditions are evidently prerequisite for both intraeellular and extraeellular calcification. I t is possible that some of the intact, eMeified, filamentous microorganisms, closely associated with the spherieM sponge-like masses, represent solid mineral as a result of both intracellular and extraeellular calcification. Gonzales and Sognnaes [6] and Zander et al. [19] observed in electron mierographs of dental calculi, that in densley calcified areas, crystal deposits were found both within

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Fig. 8 A--F. Various forms of crystal growths: (A) and (B) needle-like or elongated shapes; (C), (D) and (E) different shapes and sizes of platelet forms, and (F) euboidal crystals. (A, B, C, D and E • 5000, F • t0,000)

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and between microorganisms. However, where calcification was less advanced they noted crystal deposition only on the surface and between microorganisms. Our findings substantiate these latter observations. The spherical and larger coalesced calcified masses were noted in all specimes and were found to be near the plaque-calculus interface, that is, on the surface after removal of the organic material and therefore of more recent formation. These configurations suggest microbial surface and intermicrobial calcifications, with little if any apparent intracellular precipitation, at least not at this early stage. Thus, when damaged in situ or during preparation of the specimen, the calcified bacterial molds fracture revealing variegated empty spaces representing cellular outlines longitudinaly, tangentialy, or in cross section. Such a space in cross section has a diameter about 1 [z which is compatible with the size of the microorganism. With time, further calcific accretions may fill these spaces, presenting a more solid crystallized mass as seen in the fractured surface of older dental calculi. These crystal growths are identical with structures that Schroeder [16] terms " t y p e A centers of mineralization". According to Schroeder's observations, based on electron micrographic measurements, the crystals im A-centers fluctuate in size, from 175 A to 3,100 A in length and from 25 A to 300 A in thickness. He presumed that this fluctuation depended on the duration of crystal formation or the age of the centers. Our crystals measured 5004000 A. However, after subtracting the thickness of the gold coating, which added approximately 300 A to the radius, the dimensions were essentially similar to those reported by Schroeder. The cuboidal crystals, also observed in calculus by Kerebel [10] and Yamaoka et al [18], are dissimilar to the other reported crystal configurations. They could be artifacts such as sodium chloride crystals formed during processing as demonstrated by Meyer et al. [13]. Curiously, we have never observed cuboidal crystals in hundreds of specimens, such as enamel, dentine, cementum, bone, and kidney and salivary calculi, similarly processed. The other crystal growths that were apparently not directly associated with microbial calcification, correspond to the " t y p e - B centers of mineralization" described by Schroeder [16], both in their location and their size. Possibly the diversity in form and size represents different calcium phosphate crystal types, such as brushite, whitlockite, octacalcium phosphate, or hydroxyapatite. Acknowledgement: We wish to thank Mr. Avi Ben-Hamo and Mr. Moshe Rosenberg for their excellent technical and photographic assistance. Relerenees 1. Baumhammers, A., Conway, J.C., Saltzberg, D., Matta, R.K.: Scanning electron microscopy of supragingival calculus. J. Periodont. 44, 92-95 (1973) 2. Ennever, J. : Intracellular calcification by oral filamentous microorganisms. J. Periodont. 31, 304 307 (1960) 3. Ennever, J.: Microbiologic calcification. Ann. N. Y. Acad Sci 169, 4-13 (1963) 4. Ennever, J., Creamer, H. : Microbiologic calcification: Bone mineral and bacteria. Calcif. Tiss. Res. 1, 87-93 (1967) 5. Ennever, J., Streckfuss, J. L., Takazoa, I.: Calcification of bacillary and streptococcal variants of Bacterionema matruchotii. J. dent. Res. 527 305-308 (1973) 156-158 (1960)

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6. Gonzales, F., Sognnaes, R . F . : Electronmicroscopy of dental calculus. Science 131, 156-158 (1960) 7. Jones, S. J.: Calculus on human teeth. Apex 6, 55-59 (1972) 8. Jones, S. J.: Morphology of calculus formation on the human tooth surface. Proc. roy. Soc. Med. 65, 29-31 (1972) 9. Jon~s,S. J.: The tooth surface in periodontal disease. Dent. Practit. dent. Rec. 22, 462473 (1972) 10. Kerebel, B. : Apports du microscope electronique a balayage a l'histologie et a la pathologie dentaires. Actualitds odonto-stomat. 96, 449-472 (1971) 11. Lie, T., Selvig, K . A . : Calcification of oral bacteria: an ultrastructural study of two strains of Bacterionema matruchotii. Scand J. dent. Res. 82, 8-18 (1974) 12. Lie, T., Selvig, K. A. : Effect of salivary proteins on calcification of oral bacteria. Scand. J. dent. Res. 82, 135-143 (1974) 13. Meyer, J. L., Eick, J. D., Nancollas, G. H., Johnson, L. N.: A scanning electron microscopic study of the growth of hydroxyapatite crystals. Calcif. Tiss. Res. 10, 91-102 (1972) 14. Rizzo, A. A., Martin, G. R., Scott, D. B., Mergenhagen, S. E. : Mineralization of bacteria. Science 135, 439-441 (1962) 15. Rizzo, A. A., Scott, D. B., Bladen, H. A. : Calcification of oral bacteria. Ann N. Y. Acad. Sci. 109, 14 22 (1963) 16. Schroeder, H . E . : Formation and inhibition of dental calculus, pp. 94-122, Berne: Hans Huber 1969 17. Takazoe, I., Kurahashi, Y., Takuma, S. : Electron microscopy of intracellular mineralization of oral filamentous microorganisms in vitro J. dent. Res. 42, 681 685 (1963) 18. Yamaoka, A., Nishimura, S., Tajime, Y., Yokoyama, K., Sagawa, It., Mashimo, It. : Scan.uing electronmicroscopic observations of dental calculus and the root surface. J. Osaka Odont. Soc. 84, 341-350 (1971) 19. Zander, H. A., Hazen, S.P., Scott, D. B.: Mineralization of dental calculus. Proc. Soc. exp. Biol. (N.Y.) 103, 257-260 (1960)

Scanning electron microscopy of dental calculus.

Calcif. Tiss. Res. 21, 47--55 (1976) 9 by Springer-Verlag 1976 Scanning Electron Microscopy of Dental Calculus J. L u s t m a n n , J. L e w i n - E...
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