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Tan B, Gillam DG, Mordan NJ, Galgut PN. A preliminary investigation into the
ultrastructure of dental calculus and associated bacteria. J Clin Periodontol 2004; 31:
364369 doi: 10.1111/j.1600-051X.2004.00484.x. r Blackwell Munksgaard, 2004.
Abstract
Introduction: Though dental calculus is generally recognised as comprising
mineralised bacteria, areas of non-mineralised bacteria may be present.
Aim: To investigate the ultrastructure of non-decalcified young and mature
supragingival calculus and subgingival calculus, and the possible presence of internal
viable bacteria.
Materials and methods: Supragingival calculus was harvested from five patients,
910 weeks after scaling and root debridement. Five samples of mature supragingival
and subgingival calculus were taken from patients presenting with adult periodontitis.
Specimens were fixed and embedded for transmission electron microscopy.
Results: The ultrastructure of young and mature supragingival calculus was similar
with various large and small crystal types. Non-mineralised channels were observed
extending into the calculus, often joining extensive lacunae, both containing intact
non-mineralised coccoid and rod-shaped microorganisms. Subgingival calculus
possessed more uniform mineralisation without non-mineralised channels and lacunae.
Conclusion: Supragingival calculus contains non-mineralised areas which contain
bacteria and other debris. The viability of the bacteria, and their identification could
Key words: bacteria; calculus; plaque;
not be determined in this preliminary investigation. As viable bacteria within these subgingival; supragingival.
lacunae may provide a source of re-infection, further work needs to be done to identify
the bacteria in the lacunae, and to determine their viability. Accepted for publication 19 June 2003
Dental calculus is defined as miner- While traditionally regarded as an nature of periodontal disease is site
alised dental plaque that is permeated aetiologic agent of periodontal disease specific (Schroeder 1969). Furthermore,
with crystals of various calcium phos- (Weinberger 1948), the importance the studies did not include plaque
phates (Schroeder 1965, 1969). X-ray placed on calculus changed with the adherent to dental calculus in the
diffraction studies have revealed the advent of studies on dental plaque. evaluation (Schroeder 1969). The cur-
presence of four main crystal structures; However, the evidence for the role of rent view is that dental calculus is not in
hydroxyapatite (HA), whitlockite (WH), calculus in the initiation and progres- itself harmful and that the major reason
octacalcium phosphate (OCP) and di- sion of periodontal diseases is incon- for preventing its formation or removing
calcium phosphate dihydrate (DCPD). clusive. Earlier epidemiologic studies it once it has formed is because it is
Crystallographic aspects of dental cal- (Ramjford 1961, Lilienthal et al. 1965) always covered by a layer of unminer-
culus were described on a systematic showed a stronger correlation between alised, viable and metabolically active
basis by Jensen & Dan (1954), Jensen calculus and disease than plaque and bacteria (Newman 1994).
& Rowles (1957), Grn et al. (1967) and disease. These studies unfortunately, Supragingival calculus has been
more recently by Sundberg & Friskopp could not provide significant informa- shown to contain non-mineralised areas
(1985). HA and OCP were found to be tion of causality because they, as well as and, by nature of its porosity (Friskopp
most abundant in supragingival calcu- their antecedents, employed indices & Hammarstrom 1980, Friskopp 1983),
lus, and WH to be most abundant in attempting to correlate mean values for it has been proposed that it may act as a
subgingival calculus by these workers. deposits and disease, while in fact the reservoir for irritating substances such
Ultrastructure of calculus 365
as endotoxins, which can affect the fixed calculus specimens were post- with the external bacterial plaque, and
chronicity and progression of period- fixed in 1% osmium tetroxide for 2 h extended to the calculus/tooth interface
ontal disease (Mandel & Gaffar 1986). at 41C. After the fixation procedures all (Fig. 1a, arrows).
Furthermore, the mineralisation of cal- specimens were dehydrated in a graded
culus has been shown to be highly series of ethanols (20%, 50%, 70%,
Subgingival calculus
variable, containing a variety of differ- 90% and 3 100%) at room tempera-
ent crystalline forms, which seem to ture, and then infiltrated with 100% Semi-thin sections of subgingival calcu-
predominate depending on the age of propylene oxide. All the specimens lus (Fig. 1c) demonstrated that the
the calculus. Although some mineralisa- were embedded in Araldite CY212 resin mineralised component was uniform.
tion may occur within a few days of (Agar Scientific Ltd, Stansted, UK). Unlike supragingival calculus, lacunae
professional prophylaxis (Theilade Semi-thin sections of 0.51 mm were of stained organic material were not
1964), and in some individuals may be cut on an ultramicrotome (Reichert, seen within the body of subgingival
clinically evident as soon as 2 weeks Leica, UK) with a glass knife and then calculus. The calculus surface pre-
(Galgut 1996), it appears clinically to be stained with toluidine blue for light viously in contact with the tooth was
relatively chalk-like. With time it be- microscopic examination. The speci- usually flat and mineralised whilst the
comes more condensed and crystalline mens were viewed under a standard external/oral surface was fairly regular
(Schroeder & Baumbauer 1966). Var- light microscope (Carl Zeiss, Oberko- and covered by a non-mineralised
ious terms for the development of chen, Germany) and the images cap- plaque layer of variable thickness.
calculus with time are found in the tured with a colour video camera (JVC
literature. Terms such as young, mature, TK-870E, Victor Company, Tokyo,
crystalline or old calculus are found in Japan) and digitised (Image Pro Plus v Transmission Electron Microscopy
the literature, but are not defined. In this 3.01, DataCell, Wokingham, UK). Young and mature supragingival calculus
paper, young calculus is defined as For transmission electron microscopy
calculus that had reformed within 12 (TEM), ultra-thin sections of 90100 nm The ultrastructure of young and mature
weeks after thorough professional pro- were cut on an ultramicrotome (Reichert, supragingival calculus was similar. The
phylaxis, as opposed to mature calculus, Leica, UK) with a diamond knife (Dia- mineralised intermicrobial areas of the
which is defined as calculus harvested tome, Bienne, Switzerland). These sec- body of the calculus contained predo-
from subjects requiring periodontal tions were mounted onto carbon-formvar minantly small, randomly orientated
treatment who had not received profes- coated 200 mesh copper grids, stained and needle-shaped/platelet-shaped crystals
sional prophylaxis for at least 6 months viewed with a JEOL 100CXII transmis- (Fig. 2a). Areas containing crystals of
prior to harvesting the samples. sion electron microscope (JEOL, Oberko- larger columnar and roof-tile shapes
The aim of the present study is to chen, Germany) operating at 80 kV. were also observed (Fig. 2b). Individual
investigate the ultrastructure of non- microorganisms present within the
decalcified young and mature supra- mineralised matrix showed varying
gingival calculus and subgingival Results degrees of calcification, in particular,
calculus and the possible presence of individual non-mineralised coccoid and
Light microscopy
viable bacteria within them. rod/filamentous bacteria were observed
Young and mature supragingival within fully mineralised matrix (Fig.
calculus 2c). Some of these bacteria showed
vacuolation but appeared otherwise
Materials and Methods In toluidine blue stained sections, the normal and possessed intact cell walls,
Recently formed supragingival calculus organic material was stained blue, with good ultrastructural preservation
was harvested from five patients, 910 mineralised areas remained unstained evident in the cell walls between two
weeks after thorough scaling and root and appeared grey and white areas were filamentous bacteria (Fig. 2d).
debridement. Mature supragingival cal- artefacts due to the loss of mineralised Large thin crystals, not associated
culus was taken from five patients material during sectioning. with the microorganisms, appeared to be
presenting with moderate to advanced The light microscope pictures pre- growing from the previously formed
adult periodontitis as part of their sented of young supragingival calculus calculus (Fig. 2e). These crystals were
periodontal treatment. Subgingival cal- (Fig. 1a) and mature supragingival found both at the calculus/plaque inter-
culus was harvested from three patients calculus (Fig. 1b) reflect the variation face where there was no overlying
undergoing surgical therapy for moder- observed in these specimens, though established plaque, and in apparent splits
ate to advanced adult periodontitis and generally the mature supragingival cal- in the mineralised areas. Such splits
two patients who had their teeth ex- culus specimens were larger. The inter- differed from the non-mineralised chan-
tracted due to advanced adult perio- face with the tooth surface was fairly nels in that they contained few bacteria.
dontitis. Care was taken to obtain large, smooth and slightly curved following the Channels of organic matrix containing
single pieces of calculus and to maintain shape of the tooth whereas the external non-mineralised bacteria were often ob-
the cross-sectional integrity of the mineralised surface was generally irre- served extending into the calculus from
structure from the tooth surface through gular and covered by a non-mineralised the calculus/plaque interface (Fig. 3a).
to the external surface. plaque layer of variable thickness. Speci- The width of these channels ranged from
The harvested calculus was immedi- mens ranged from containing many (Fig. a single bacterial cell to many cells
ately placed in a fixative solution of 3% 1a) to fewer (Fig. 1b) non-mineralised although not all channels contained
glutaraldehyde in 0.1 M cacodylate buf- lacunae and, in some sections, the bacteria. Channels were observed to join
fer, for a minimum of 3 h at 41C. The lacunae formed a continuous connection extensive non-mineralised lacunae within
366 Tan et al.
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