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Light and scanning electron microscopic observations of the canalicular system in human cellular cementum.

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THE ANATOMICAL RECORD 222:121-127 (1988)
Light and Scanning Electron Microscopic
Observations of the Canalicular System in Human
Cellular Cementum
Histology, University of Illinois, College of Dentistry, 801 South Paulina,
Chicago, Illinois 60612
A scanning electron microscopic cast technique was used to determine the nature of the canalicular system in human cellular cementum. Prior light
microscopicobservations suggested the presence of two distinct types of lacunae: bonelike and a large irregular type generally confined to the interradicular region. Only
the bone-like lacunae were visualized in the SEM cast preparations. The canalicular
system associated with the bone-like lacunae was usually continuous from the surface
of the dentin to the surface of the cementum in newly-erupted teeth. Casts having a
sponge-like configuration were observed near the cementodentinal junction in some
of the specimens from the interradicular region. The presence of these casts could
not be predicted from prior light microscopic observations and it was concluded that
they may represent infiltration of hypomineralized matrix rather than lacunae.
Previous transmission electron microscopic (TEM)
studies have shown that the more deeply-positioned
cells in cellular cementum in man (e.g., Furseth, 1967,
1969)and other species (e.g., Jande and Belanger, 1970;
Furseth, 1970, 1977) appear to be dead or moribund.
One explanation for this apparent morbidity is the canalicular mechanism’s lack of enough efficiency to maintain the vitality of cells when, as a result of continued
appositional growth of the tissue, they come to lie at
progressively greater distances from the periodontal
vasculature. Other reasons for the observed cellular
morbidity must also be considered.
Cellular cementum is a difficult tissue to fix. It is
essentially avascular and the cells are accessible to the
fixative only via minute canals (the canaliculi) having
a diameter of a micrometer or less. The rate of diffusion
of the fixative through these channels may not be rapid
enough to adequately fur the more deeply-positioned
cells. This apparent morbidity may therefore be the
result of poor fixation rather than cell death. The nature
of the canalicular network must also be considered. Is
there a continuous canalicular network interconnecting
the lacunae throughout the full thickness of cellular
Classical light microscopic and TEM methods cannot
unequivocally answer this question. Light microscopic
observations show that the canaliculi in cellular cementum tend to radiate toward the source of nutrition,
the periodontal ligament, and often present the illusion
that the lacunae and canaliculi are isolated units. Vital
cells, of course, could not be maintained in isolated lacunar units. The purpose of this present investigation
was to determine to what extent, if any, the lacunae in
cellular cementum are interconnected, both with each
other and ultimately with the surface of the cementum.
A scanning electron microscopic (SEM) cast technique
(Weber, 1983; Curtis et al., 1985) which permits the
1988 ALAN R. LISS, mc.
three-dimensional visualization of internal spaces in
hard tissues was used to examine the canalicular system in human cellular cementum.
Fifty non-carious, newly-erupted human third molars
obtained from the University of Illinois Oral Surgery
Clinic were used in the present investigation. A saw
microtome employing an annular water-cooled diamond blade was used to obtain 2 to 3 300pm mesiodistal longitudinal sections from the central region of
each tooth. These sections were examined light microscopically to determine the location and the amount
of cellular cementum present. Although the amount of
cellular cementum present varied considerably, all of
the teeth had at least some cellular cementum present
in the interradicular regions. A heavy gauge razor blade
was used to cut small (approx. lmm x 1.5mm) segments from selected areas of the root. These segments
contained the full thickness of the cementum along with
a thin layer of the underlying dentin. The interradicular
cellular cementum often contained many large irregular lacunae, whereas the lacunae seen in other regions
were more similar in size and configuration to those
seen in bone. Therefore, segments taken from the interradicular regions were separated from those taken
elsewhere. The segments were infiltrated with methyl
methacrylate monomer by either vacuum or replacement infiltration as previously described (Weber, 1983).
After polymerization under UV light in 16mm flatbottom glass vials, the segments were reduced to 3080pm on plastic microscope slides using an integrated
Received September 29, 1987; accepted January 28, 1988.
Address reprint requests to Dr. Dennis F. Weber, Department of
Histology, University of Illinois, College of Dentistry, 801 South Paulina, Chicago, IL 60612.
mounting and reduction technique (Weber et al., 1982).
Photomicrographs of selected areas were taken with
ordinary transmitted light. The areas photographed were
scored with a razor blade so that light and SEM image$
could be compared. For SEM processing, a lOmm x
lOmm segment containing the specimen was cut out of
the plastic microscope slide with a revolving diamond
disk. Complete matrix removal cast preparations were
processed for SEM observations. These were produced
as previously described in our studies of the canalicular
system in human cortical bone (Curtis et al., 1985).
The light microscopic image of the initial saw microtome-cut sections differed from that of the same section
after methacrylate embedment and further reduction
in thickness. The lacunae and canaliculi in the uninfiltrated thicker sections were readily visible as darkened structures, whereas in the thinner embedded
sections, many (but not all) of the lacunae were translucent and the canaliculi of these lacunae were not visible at all. A variable number of lacunae, however, often
retained a darkened appearance, suggesting that these
lacunae represent isolated units which do not become
infiltrated with the resin because their canaliculi are
not part of a continuous system.
A different series of light microscopic observations
were made to determine if this apparent absence of
infiltration was a peculiarity of the resin embedding
and reduction technique. Additional saw-microtome
sections were cut at 200pm as previously described and
dried overnight in a vacuum desiccator. Selected areas
containing a high density of lacunae were photographed
without using mounting medium or cover slips. These
sections were then slowly infiltrated with mounting medium by first placing the specimens in xylol and gradually adding increasing amounts of a synthetic mounting
media over a twenty-four hour period. A cover slip was
then placed over the specimen and the same area rephotographed. Some of these specimens were then
embedded in methyl methacrylate to produce SEM cast
opaque in light microscopic observations (Fig. 51, no
lacunar casts were observed in the SEM preparations
even though the underlying dentinal tubules were obviously well-infiltrated with the resin (Fig. 6). In sections in which most of the lacunae became translucent
after resin embedding, an extensive canalicular cast
system was observed. Lacunae-to-lacunae canalicular
connections appeared to form a continuous network
throughout the full thickness of the cellular cementum
(Figs. 7-10). The lacunae, particularly in the thicker
sections (e.g., Fig. 71, were often difficult to visualize
because of the surrounding superimposed dense network formed by the canalicular system. Furthermore,
the canalicular network did not appear t o be formed
from a one-to-one direct connection between canaliculi
of adjacent lacunae, as seen in lamellar bone. Instead,
the canalicular system appeared to have a complicated
branching system which had a reticular configuration
(Fig. 9).
Casts having a sponge-like configuration were occasionally observed in the interradicular regions near the
cemento-dentinal junction (Fig. 10). In low magnification scanning electron micrographs, these casts appeared to be individual, interconnected units which were
much larger than the bone-like lacunar casts (Fig. 10).
Prior light microscopic observations of the sections containing these casts did not suggest their presence.
Fig. 2. Ordinary light photomicrograph of the area shown in Fig. 1
after infiltration of the specimen with mounting media. The bonelike
lacunae have become translucent (arrows) and are difficult to visualize.
Canaliculi are no longer visible; x 142.
As observed with the light microscope, the lacunae
and canaliculi in cellular cementum were readily visible
as opaque structures in unmounted dried sections (Fig.
1).They appeared to be similar in size to those observed
in bone. In most of the specimens, all of the lacunae
became translucent and the canaliculi were no longer
visible after thorough infiltration with mounting medium (Fig. 2). In some specimens, however, varying
numbers of lacunae remained as opaque structures despite repeated efforts to infiltrate the specimen with
mounting media (Fig. 3). Larger irregularly-shaped lacunae were often observed in the interradicular dentin.
These lacunae were usually elongate in configuration,
with their long axes oriented generally perpendicular
to the surface of the cementum (Fig. 4). As with the
bone-like lacunae, these lacunae often became translucent (not shown) after infiltration with mounting media. No casts corresponding in size and configuration
to these lacunae, if they are indeed lacunae, were seen
in subsequent SEM observations.
As with mounting media infiltration, the light microscopic image of the lacunae varied in the methacrylate-embeddedspecimens. When the lacunae appeared
The light microscopic and SEM observations in the
present investigation show that the lacunar-canalicular
system in human cementum is quite heterogeneous in
Fig. 1. Ordinary light photomicrograph of a thick (100-um) air-dned
ground section of cellular cementum from the interrahcular region of a
human molar tooth. Outer surface of the cementum can be seen in the
left-hand portion of the field. Arrows indicate cemento-dentinal junction.
Many dark, bonelike lacunae can be seen distributed throughout the
thickness of the cellular cementum; x 142.
Fig. 3. Ordinary light photomicrograph of a ground section of cellular
cementum that was infiitrated with mounting media. Many bonelike
lacunae have become translucent (small arrow); others remain dark and
retain the appearance seen in uninfidtrated specimens. Canaliculi can
be observed in association with the dark lacunae; x 142.
Fig. 4. Ordinary light photomicrograph of an Aral&te-infiltratedground
section of cellular cementum taken from the interradicular region. A
number of large, dark, elongated lacunae can be seen. When this specimen was prepared for scanning electron microscopy, no casts corresponding to these lacunae could be observed; ~ 6 8 .
Fig. 5. Ordinary light photomicrograph of a methyl methacrylateinfiltrated ground section of cellular cementum ( C ) and a portion of the
underlying dentin (I)). Many dark, bonelike lacunae and canaliculi can
be seen distributed throughout the full thickness of the cementum. Open
arrow indicates the cemento-dentinaljunction; X 72.
Fig. 6. Scanning electron micrograph of the preparation shown in Fig.
5. With the exception of one small area in the upper left portion of the
field, no casts of lacunae or canaliculi can be seen in the region previously
occupied by the cellular cementum. Dentinal tubules (T) have been infiltrated. They do not, however, extend to the cemento-dentinal junction
(open arrow), probably because the outermost portion of the radicular
dentine had undergone sclerosis. x 72.
a number of respects. The size and shape of the lacunae
vary and the number of lacunae per unit volume also
vary. The canalicular system was found to vary, from
a continuous network (extending from the surface of
the dentin to the surface of the cementum) to totally
isolated lacunar-canalicular units. Vital cells cannot be
maintained in the latter arrangement, but the potential
for maintaining vital cells in a continuous system clearly
exists. Since the most frequently observed lacunarcanalicular system had bone-like lacunae interconnected by a continuous canalicular system, we suggest
that the ultrastructure of the more deeply-positioned
cells in cellular cementum should be re-evaluated.
As was mentioned in the Introduction, most authors
have suggested that these deeper cells are moribund.
As has been suggested for bone (Matthews, 1980), it
may be that the degenerative appearance of these cells
is the result of poor fixation rather than cell morbidity.
In order for a fixative to reach the cells in a mineralized
tissue such as celluar cementum or bone, the fixative
must diffuse through a series of minute channels over
a distance which can be quite substantial when considering the distance that the deepest cells lie from the
vascular elements. In previous studies of human cellular cementum, the intact tissue was fixed by immersion and therefore the only surface of the tissue exposed
to the fixative was the outer surface of the cementum.
Perfusion fixation has been used in studies of non-hu-
Fig. 7. Split screen scanning electron microgrpah of the specimens
shown in Figs. 1and 2. After viewing the specimen with the light microscope as previously described, the specimen was reembedded in methyl
methacrylate and prepared as a cast preparation. The low-power view
on the lee includes the entire thickness of the cellular cementum. An
extensive and apparently continuous canalicular cast system extends
throughout the full thickness of the cementum in this relatively thick
preparation; x 206. Higher magmilcation of the framed area seen in the
right hand portion of the field illustrates even more dramatically the
extensive nature of the canalicular system, which tends to obscure most
of the lacunae (arrows).Background is cast system of underlying lacunae
and canaliculi; x 1,069.
Fig. 8. Split screen scanning electron micrograph of a cast preparation.
The low-power view on the left shows a segment of cellular cementum
taken from the apical region of a human molar. A portion of another
segment can be seen in the upper righthand portion of the field. An
extensive cast system can be seen throughout the full thickness of apical
segment; x 35. A higher magmtkation of the framed area is shown on
the right. Lacunae (arrows) are more readily visible in this thin preparation. Dark background represents surface of the plastic microscope
slide on which the specimen was prepared, x 357.
Fig. 9. Split-frame scanning electron micrograph taken from a different
area of the specimen shown in Fig. 8. The lower-power view on the top
is taken from the central one-third of the cellular cementum. The canalicular system appears to be continuous throughout the field; x 189.
The lower portion of the field is a higher magnification of the framed
area: one lacuna (L) is visible in the lower portion of the field, and an
extensive branching canalicular network can be seen throughout the
field x2197.
Fig. 10. Split-screen scanning electron micrograph; the upper one-half
of the field is a lower-power view of a portion of a segment taken from
the interradicular region of a human molar. An extensive cast system
can be seen throughout the full thickness of the cellular cementum, and
casts of the tubules can be seen in the underlying dentin (D).The casts
in the framed area near the cernento-dentinal junction appear to be
larger than those seen elsewhere in the cementum; x 45. The lower onehalf of the field is a higher magnification view of the framed area. An
extensive cast system with a spongelike appearance can be seen within
the field; x 198.
man cellular cementum. Although this method is generally superior t o immersion fixation, it would appear
to be only minimally superior for the fixation of hard
tissues because the fmative must still diffuse through
the canalicular system to reach deeply-positioned cells.
It is suggested, therefore, that optimal fixation of human cellular cementum would be enhanced by rapidly
sectioning the tissue immediately prior to initial exposure to the fixative. Such a technique has been successfully employed to fix the contents of the dentinal
tubules in human coronal dentine (Gaynor et al., 1984).
Sections of cementum in the range of 50-150 um can
be easily cut with a saw microtome using a water-cooled
diamond-edged blade. Although some of the cells along
the cut surface may be destroyed and heat damage may
occur along this surface, many cells within the bulk of
the section should remain undamaged. When such a
specimen is placed in the fixative, the surface area of
the cellular cementum exposed to the fixative is greatly
increased. The fixative now has a very short distance
to diffuse to reach the cells via lacunae and canaliculi
which have been exposed on both cut surfaces of the
section. The fixative will now reach cells located at all
points between the surface of the cementum and the
surface of the dentine at the same time.
Many of the large irregular lacunae appeared dark
both before and after mounting media infiltration and
also appeared dark after embedment in methyl methacrylate. Other large irregular lacunae became infiltrated
with the mounting medium or methly methacrylate
and appeared translucent after infiltration. No casts
corresponding in size or configuration to these lacunae
were seen in the SEM preparations. These large irregular lacunae were probably infiltrated because the lacunae and canaliculi were made accessible to the
mounting media or methyl methacrylate by exposure
through sectioning rather than because they had a continuous interconnecting canalicular system which ultimately opened onto the surface of the cementum.
Although these lacunae had associated canaliculi, the
canaliculi differed from those associated with the bonelike lacunae in that they presented a tree-like branching
configuration. Such a configuration in itself, however,
would not mitigate against a continuous interlacunar
canalicular system. The nature of the large irregular
lacunae and their contents must remain speculative.
Cementoblasts and epithelial rest cells (Lester, 1969;
Jande and Belanger, 1970) are known to become entrapped in cellular cementum. Of these two cellular
elements, only cementoblasts are known to produce cellular processes which could be surrounded by matrix
to produce canaliculi. Therefore, if these lacunae contain or contained cells, it would appear most likely that
the cells were large cementocytes or that more than one
cementocyte resides in these lacunae. In either case,
the processes of these cells, for some reason, never established contact with neighboring cells. Although it
seems unlikely, the possibility that these darkened regions represent isolated modifications of the extracellular matrix rather than actual lacunae cannot be totally
The observation of the sponge-like casts near the surface of the dentine was surprising because light microscopicobservations of the sections prior to matrix removal
did not suggest their presence. The size and configu-
ration of these casts suggest that they do not represent
an aberrant form of lacunae. They are much larger than
both the bone-like and the large irregular lacunae, and
the reticular configuration of the casts does not correspond to the canalicular network observed in cellular
cementum in the present study or the canalicular network previously observed in bone (Curtis et al., 1985).
Since unmineralized areas of varying size have been
previously observed near the surface of the dentine
(Furseth, 19691, it is possible that these casts represent
an infiltration of the matrix rather than an infiltration
of spaces housing cells and their processes. Re-embedding
the casts and sectioning them for transmission electron
microscopy would answer this question. If extracellular
matrix is seen in TEM preparations, then the casts
would represent an infiltration of the matrix. If nothing
is observed in the casts, this would suggest that the
casts represent empty spaces which were previously
occupied by cells. The presence of cellular elements
would, certainly, confirm the latter suggestion.
Belanger, L.F. 1968 Resorption of cementum by cementwyte activity
("cememtolysis").CalcS. Tissue Res. 2229-236.
Curtis, T., S. Ashrafi, and D.F. Weber 1985 Canalicular communication
in the cortices of human long bones. Anat. Rec. 212:336-344.
Furseth, R. 1967 A microradiographic and electron microscopic study of
the cementum of human deciduous teeth. Ada Odontol. Scand.
Furseth, R. 1969 The fine structure of the cellular cementum of young
human teeth. Arch. Oral Biol. 14:1147-1158.
Furseth, R. 1970 A microradiographic, light microscopic and electron
microscopic study of the cementum from deciduous teeth of pigs.
Acta Odontol. Scand. 28~811-831.
Furseth, R. 1977 Further observations on the fine structure of cellular
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Gaynor, M.K., D.F. Weber, and J . Everingham 1984 A M e r e n t approach
to immersion fmtion of human dental pulp and odontoblast processes. Arch Oral Biol. 29:243-248.
Jande, S.S., and L.F. Belanger 1970 Fine structural study of rat molar
cementum. Anat. Rec. 167:439-464.
Lester, K.S. 1969 The incorporation of epithelial cells by cementum. J .
Ultrastruct. Res. 27~63-87.
Mathews, J.L. 1980 Bone structure and ultrastructure. In: Fundamental
and Clinical Bone Physiology. M.R. Urist, ed. J.P. Lippincott, Philadelphia, p. 31.
Weber, D.F., D.R. Eisenmann, and A.E. Zaki 1982 A grinding method
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cementum, canaliculus, observations, microscopy, light, scanning, electro, system, cellular, human
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