Journal of Periodontal Research 18: 353-36!

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Cell turnover in the periodontium in

health and periodontal disease
T R. L. GOI;LD, D . M . BRUNETTB AND J. DOREY
Department of Orai Medicine, Faculty of Dentistry, Vancouver, B.C., Canada

The use of tritiated thymidine labelling given by continuotis delivery over long periods by means of lan
implanted osmotic minipump is a convenient method of accurately determining the rate of cellular
proliferation in various tissues, but is especially advantageous for the periodontal ligament (PDL), the
fibroblasts of which tum over at an extremely low rate. This technique was used to examine the
hypothesis that differences exist between cell turnover in normal hamsters (N.D.) and those which had
been placed on a periodontal disease inducing diet (S.S.D.). Tumover rates were then calculated for PDL
and epithelial attachment (EA) for N.D. and S.S.D. animals before and after disease had been induced.
No efiect of disease was noted for either PDL or EA. However, the technique was sufficiently sensitive to
demonstrate a significant effect of age on Ihe turnover of celts in the epithelial attachment.

(Accepted for publication March 15, 1983) :

(Rodent Laboratory Chow #5001, Ralston

Introduction
Purina Ltd.). The other {S.S.D.. - 14 animals)
It appears likely that the fibroblasts of the were fed on a soft sweet diet known to cause
periodontal ligament (PDL) constittite a cell periodontal disease (King 1951). Animals were
renewal system, albeit one with a relatively slow maintained in cages with large-mesh wire floors
tumover time of about 90 days in the hamster to eliminate the effects of intraorai impaction of
(Gould, Brunette & Dorey 1982), but little is bedding material. .. "'
known about the effects of periodontal disease
on cell kinetics within the periodontium. The Start of diet period .; .
purpose of this study was to compare cell Between 9 a.m. and 11 a.m., 8 animals from the
tumover in hamster periodontium both in N.D. group and 6 from the S.S.D. group were
normal animals and those which had been anesthetized with sodium pentobarbital and an
placed on a diet known to produce periodontal osmotic minipump (Alza Corp., Palo Alto,
disease (King 1951). Califomia) which had been previously filled
with 200 fiO ff-TdR (New England Nuclear
Co., s,pec act. 73.5 ci/mM) implanted intra-
Materials and Methods
peritoneally. Tissues were sutured in layers
Thirty male Goldeti Syrian Hamsters 16 weeks using 5.0 silk sutures. Two animals from each
old weighing approximately 100 grams were group were killed by ether at each of 2, 4, and 7
allowed to acclimatize in their cages for 1 week. days later. Also, two animals from the N.D.
Food and water were provided ad libitum. group were killed at 4 hours.
Animals were then divided into two groups.
Those in the first group (N.D. - 16. animals) End of diet period
remained on a standard laboratory rodent diet Flash labelling. At the end of a three month
354. G'OULD, BRUNETTE AND DOREY

period (84 days), two animals from each group containing four sections. Slides were dipped in
were injected with 1 /iCi/gm H^-TdR and killed Kodak NTB2 emulsion and exposed for twelve
one hour later. weeks, developed iti Dektol diluted 1:1 with
water,, fixed in Kodak rapid fixer, and stained
Continuous labellmg. Six animals from each with hematoxylin and eosin.
group had osmotic minipumps implanted as In each specimen a minimum of three serial
described previously. Two animals were killed radioautographs separated by at least 15 p.ra
2, 4, and 7 days later. was studied. The total number of labelled
The right and left halves ofthe mandible were fibroblasts and interphase cells in the PDL were
immediately excised and placed in Bouins counted and the labelling index calculated as:
fixative for two days. Specimens were decalci- LABELLING INDEX= .- \
fied in 4.13% ethylenediaminetetra-acetic acid Number of labelled cells ^.
for four Veeks with constant stirring and total number of cells . \
frequent solution change. Specimens were then
trimmed with a razor blade to leave only the for each section.
lower first molar, embedded in plastic (plastic LI data from different sections were com-
embedding media, Polysciences Inc., Warring- bined to give a LI-for an individual animal.
ton, Pennsylvania) and oriented in a coronal Mean LI and standard error of the mean were
plane along the long axis ofthe root. Sections of calculated for each time period of continuous
2 foa thickness were prepared on a Sorval JB4 labelling (Table 1). Similarly, labelling indices
microtome. Sections were cut, stained with were also prepared for the epithelial attach-
toluidene blue, and examined until the mid ment. Graphs were plotted of these labelling
portion of the root was reached. Four slides indices and the least squares fit calculated.
were then prepared from each specimen, each Regression lines given by this calculation were

Table 1
. . , . .

Normal dset Soft sweet diet

100% labelling (days) 100% labellii'ng (days) ' - ' /\

0-7 days 84-91 days 0-7 days 84-91 days

Fibroblasts 4S1 51+3 43 + 2 51 2

Epithelial attachment . 1.12 20+3 133 203 . ;

Turnover times calculated by extrapolation ol least sq'yares fit to a siraight line. The error estimates were calculated by
inverse prediction, using the equation:
. Y i - a +S,E.- ; ' - ,. . . . / ; : ' . .., . ,: .; , .,.;;;
A '' ' ' ".. - . ". - . "

Tabie 2
Normai diet Soft sweet diet

PDt. width PDL area Totai number PDL width PDL area Totai number .
Time (mm=) of Ilbrobtasts {pm) (mm) ol tibroblasts
(days) (mean) (mean) (mean) (mean) (mean) (mean)

0-7 112 0.6 2018 111 0.4 1510

84-91 111 0.5 2173 101 0.3 2126 . ^
 CELL TURNOVER IN THE PERIODONTIUM 355

R
D

Fifl. 1. Epitheiial allachment in animais on (a) Normai Diet, (b) Soft Sweet Diet E, Epitheliai attaohmieniarea. R, Root. 0 ,
Debris and piaque. x163.
 GCU-D DOREY

Fifl. 2. Labellmg in epithelial attachment after (a) four hours and (b) 7 days exposure to H'-TdR. R, Root L, LabBlleC
epithelial cells x 1152.
 CELL TURNOVER
then comp,ared using a modified Student's r test
to detect any significant differences (Zar 1974).
The area ofthe PDL was calculated using the
graphics tablet of a microcomputer (Apple,
Cupertino, Califomia). A light emitting diode
was attached to the stylus of the tablet, the
position of the stylus was observed, and the
outline ofthe PDL was traced onto the tablet by
means of a camera lucida attached to the
microscope. By this means, it was possible to
determine values of PDL width and area and
Defieshed specimens at end of three months diet (a) Normal Diet, (b) Soft Sweet Diet x 2 0
IN THE PERIODONTIUM 357
hence calculate cell density (Table 2).
Differences were compared using unpaired f
tests. .. v> . : . ' . ; ^ ,;
Results
The histological appearance ofthe radioautog-
raphs was similar to that described previously
(Gould et al. 1982). Coronal and radicuiar
dentin enclosed normal pulpal tissue. Epithelial
tissue overlay the alveolar bone and the sulcuiar
epithelium was keratinized. In some sections
,358 GOULD. BRUNETTE AND DOREY

food debris was evident in the gingival sulcus. revealed greater aiveolar bone loss, inthe S.S:D.
In the S.S.D. group the epithelial attachment group (Fig. 3).
had migrated onto the root stirface, the gingival The subjective observations of a decrease in
sulcus 'twas deepened, and .large amounts of ligament width and area were confirmed using
debris and plaque were evident (Fig. 1). In the morphometdcal techniques. After three
severe cases of plaque accutnulation, disruption months, the animals on the S.S.D. had a
of the epithelial cells was evident. The PDL significant reduction in the PDL area (Table 2).
appeared to be narrower in the S.S.D. group, However, there were no significant differences
although the alveolar bone appeared normal in in the ftbroblast population density, defined as
both groups with nntnerons lacunae housing cells/cm^., in the pedodontal ligament of the two
osteocytes witb tbe occasional resting or groups (3648 + 879 cells./mm^ for N.D. and
reversal line evident. Osteoclasts were evident in 3708 + 806 cells/mm^ for S.S.D.). There was
both groups adjacent to the alveolar bone. also no significant change in this parameter
Within the periodontal ligament, nutnerous, over the time period studied for either of the
plump, irregularly-shaped fibroblasts with dark groups.
basopbilic nuclei and lighter staining cytoplasm In the study on cell proliferation, animals in
could be seen. Radioactive label was located each diet group were continuously infused with
over the actively dividing fibroblasts although tritiated thymidine for periods of 2, 4, and 7
non-labeledfibrocyteswere also evident within days after the start of the experiment. This was
the collagenous. matrix. Numerous capillaries done to see if there was any imtnediate effect of
containing red blood cells and neutrophils were the diet on the rate of proliferation. At day 84 of
visible both in longitudinal and cross-section, the experiment the animals in e.ach group were
and appeared to be mainly on the bone side of continuously infused with H'-TdR for 2,4, and
the PDL. The capillaries were surrounded by a 7 days. The intent of this comparison was to
single layer of flattened endothelial cells which determine if there was a difference in tbe
were often labelled with H^-TdR. Within the proliferation pattern when tbe periodontal
epithelium and epithelial attachment numerous disease process was well established. The data
labeiied epithelial cells were visible (Fig. 2). are shown in Figs. 4-7. As the label is avaiiabie
Gross examination of deOeshed specimens continuously, tbe number of labelled cells

.NO
. SSD

Fig. 4. Labelling iridex wrtn time for PQL

frbroblast including standard error o-
estimate (0-7 days).
 CELL TURNOVER IN THE PERIODONTIUP.;] 359

Fig. 5 Labeiling incex wt^' lime Tor PDL

fib'"OD,asts including standard error of
esiimate ,'d4 3"* daysi

Fifl. 6. Labellmg index with time for

epithelial attachmenl irtciuding standard
error olf estimate (0-7 days),

-NO
:SSD

iFig. 7. Labeilling index with time for

epitheijal attachment imduding standard
error of estimate (84-31 days).
360 GOULD, BRUNETTE AND DOREY
shotild increase continuously with time. The reduce the forces of mastication transmitted to
rate of proliferation is given by the slope of the the ligament, and this lack of function may
straight line obtained using a least squares fit. account for our observation that the pedodon-
The error bars indicate the standard error of tal space decreased over the 91 day period of the
estimate (Zar 1974). As expected there was a experiment.
continuous rise in the number of labeiied cells The experiment was designed in such a way
with increasing exposure to H'-TdR for both that it was possible to distiaguisb between the
the epithelium and tbe fibroblasts. There were effect of aging of the animals and the effect of
BO significant differences between the N.D. and diet. Thus, for both fibroblasts and epithelium,
the S.S.D. in the proliferative rate of either the rate of proliferation after 91 days could be
fibroblasts of tbe PDL or the epithelium. compared between diets and also with the value
However, a significant difference was seen for obtained in thefirstweek of the experiment. NO'
the epithelium within the same diet group when differences in tbe rate of proliferation was
the early (days 0-7) and late (days 84-91) time found between the two diets, but the technique
periods were compared. ....;. - . , .. used was sufficiently sensitive to detect a
statistically significant difference in the pro-
liferative rate of the attachment epithelium
Discussion
betweentheA month and7-month-old hamsters.
The use of osmotic minipumps has several This difference could be demonstrated by
advanta.ges over other methods for the continu- statistical comparison of the slopes of tbe
ous delivery of H'-TdR over relatively long time labelling index with time ctirves or analysis of
periods (Gould et al. 1982). These include variance. We chose the slope of the labelling.
consistent and predictable maintenance of index curve tvith time as the parameter of
serum levels of the isotope and a reduction of interest in these comparisons because this is tbe
stress to the experimentai animals (such as value one would use to extrapolate to a value of
would be experienced if multiple injections were 100% labelling of the population which, under
used). Since the animals were handled and the certain conditions, corresponds to the turnover
minipumps implanted only at the same time time. Using this approach we estimate a
each day, problems with diumal variation turnover time of 11+2 days for the epithelial
shotild also have been reduced (Roberts 1975). attachment of young hamsters and 20 + 3 days
A number of bistologica! stuides have docum- for the older animals. These values are similar
ented the progression of periodonta] disease in to those found by other workers using the
tbe hamster (King 1951, Costich 1955, Gupta & transit time approach to measure cell turnover
Shaw 1960, Gotcher & Jee 1981). With the in mice (Beagrie & Skougard 1961). A decrease
exception that we did not see any widening of in labelling index of periodontal tissues witb
the periodontal ligament, a similar pattern was time has also previously been observed in mice
found in this study. An explanation for this (Weiss, Tonna & Stabl 1968). Another ad-
difference may reside in the diet employed to vantage of using the slope in these comparisons
produce the diseased condition. Although is that it is based on al! the data as opposed to
pedodontal disease increases with advancing comparison based on the mean LI at a given
age in tbe Syrian hamster (Saffar 1982), a time point which are less representative. In
special soft high carbohydrate diet has been considering continuous labelling experiments in
found to enhance the tissue destruction prob- which the thymidine was administered by
ably by promoting the accumulation of basop- multiple injections, Potten and Major (1980)
hilic, plaque-like materiai in the gingiva] crevice pointed out that it is impossible to make any
(King 1951). This soft diet would be expected to firm statements about the proliferative model in
 G^ELL T U R N O V E R IN T H E
terms of the relative number of stem cells in the
tissue. Moreover in situations were there is a
loog cell cycle time such as would appear to be
the case of the fibroblasts of the PDL of hamster
(Gould et al. 1982), it may be difficult to
accurately measure the growth fraction, be-
cause Mils may leave the cycie after mitosis and
enter the non-proliferating subpopulation
(Steel 1977). Thus, the absolute values of the
turnover times calculated above may not be
accurate. However, the intent .of this ex.peri-
ment was not to obtain absolute values btit to
assess the effect on proliferative activity of a diet
which induces periodontal disease. It appears to
be reasonable to state that the effect, if any, of
the diet and resultant diseased condition on cell
production of either fibroblasts or epithelium
would appear to be small. Although a marked
effect in the rate of cell production between the
diets was not found, it is still possible that there
may be a difference in the rate of cell death or
the average lifetime of a fibroblast in the
periodontal ligament. In this regard the pre-
sence of bacterial toxins and other sequelae of
plaque accumulation would provide a likely
explanation, but a direct test of this hypothesis
is required.
References
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PERIODONTIUM 361
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