Journal of
Dental Research
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In vitro Root Caries Progression Measured by 125I Absorptiometry: Comparison with Chemical
Analysis
H. Almqvist, J.S. Wefel, F. Lagerlof, J. Ekstrand and C.O. Henrikson
J DENT RES 1988 67: 1217
DOI: 10.1177/00220345880670091301
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In vitro Root Caries Progression Measured by
1251 Absorptiometry: Comparison with Chemical Analysis
H. ALMQVIST, J.S. WEFEL1, F. LAGERLOF, J. EKSTRAND', and C.O. HENRIKSON2
Departments of Cariology and 2Oral Radiology, Karolinska Institutet, School ofDentistry, Box 4064, S-141 04 Huddinge, Sweden; and 'Specialized
Caries Research Center, College of Dentistry, The University of Iowa, Iowa City, Iowa 52242
Radiation from a 125I source and a non-image-forming detector was
used for non-destructive measurements of root caries progression.
Blocks were cut parallel to the cementum surface of unexposed human
roots. These blocks were then individually demineralized in undersaturated calcium phosphate solutions over an 84-hour period. In
order for the in vitro root surface demineralization to be followed,
the changes in transmission (AT) through the blocks were measured,
by 125I absorptiometry, eight times during the course of the experiment. Chemical analyses of the calcium output (ACa) from the blocks
into the demineralizing solutions were also performed, and the rate
of demineralization (Vdem) was calculated from these values. The precision of 125I absorptiometry was calculated from 176 duplicate transmission measurements, and the coefficient of variation was found to
be 0. 20%. The correlation coefficient between AT and total ACa for
each of 22 cementum/dentin blocks ranged between r = 0.934 and r
= 0.998. The progression of root hard-tissue lesions observed by
these two methods and by the calculated Vdem was found to be proportional to the square and cubic roots of time. The study shows that
125I absorptiometry can be used for continuous non-destructive measurements of root hard-tissue demineralization in vitro.
J Dent Res 67(9):1217-1220, September, 1988
Introduction.
Root caries may be an increasing problem in the future as
proportionately more elderly people retain their natural teeth,
and gingival recession will result in exposure of root surfaces
and increased caries risk. It therefore seems important for studies on this incompletely understood disease to be conducted.
The traditional methods for evaluation of the change in mineral content in the tooth hard tissues are: hardness test (Koulourides et al., 1974), polarized light microscopy (e.g.,
Carlstrom, 1964), and microradiography (e.g., Angmar et al.,
1963). These methods are destructive, and repetitive measurements of the same area are not possible. A method for documenting root caries progression, during a time period, would
improve our knowledge of the disease process and allow for
studies on the effect of different root caries preventive measures.
Only a few methods are available for continuous measurements of mineral changes in teeth. One method is the single-
section technique, developed by Featherstone and Silverstone
(1982). With this technique, the single-section slice is very
sensitive to mechanical trauma and dehydration, and the determinations of the specimens are usually restricted to one measurement before and one after the experimental procedure. Other
methods used are chemical analyses of the demineralizing solution (e.g., ten Cate and Duijsters, 1983), longitudinal microradiography (de Josselin de Jong et al., 1987), and scanning
optical monitoring (ten Bosch et al., 1984). Up to now the
two latter methods have not been adapted to root hard tissues.
Photon absorptiometry with a radioactive source (1251) and
a non-image-forming detector has previously been used for
longitudinal measurements of the mineral content of enamel
undergoing artificial demineralization (Henrikson and Linden,
1974). The method was further developed by Julin (1975).
During mineral loss the transmission of the radiation (through
a mineralized sample) increases, and this increase may be recorded with high precision. The aim of this study was to use
125I absorptiometry for continuous non-destructive measurements of root hard-tissue demineralization in vitro and to compare these measurements with the results of chemical analyses.
Materials and methods.
Root hard-tissue specimens.-The specimens consisted of
root hard tissue from eight impacted human molars. After surgical removal, the teeth were placed in distilled water with the
addition of a few thymol crystals. The roots were gently cleaned
of organic material with a wooden spatula, and efforts were
made to avoid dehydration. The roots were examined with a
dissecting microscope, and only those roots without surface
defects were selected. Cross-sectional cuts were made just below the cemento-enamel junction and at 4 mm below by use
of a diamond disc under continuous cooling with distilled water
(Fig. 1). This slice was divided into from two to four blocks
(total, 22 blocks) which were ground with a water-cooled diamond disc, at the pulpal surface, until the thickness was approximately 1 mm. The outer surface (the cementum surface)
was left intact. Each block was covered with nail varnish except on the pulpal surface and a window on the cementum
surface. The area of the exposed cementum surface was determined from an enlarged digitized image and was found to
be 3.8 ± 0.3 mm2 (mean ± S.D.). Sticky wax was used to
mount the blocks in plastic holders. The thickness of the plastic
beneath each block was approximately 0.5 mm, and no wax
was placed on the pulpal and cementum surfaces. The blocks
were mounted and centered in the plastic holder, so that the
beam from the radioactive source always struck perpendicularly to the exposed cementum surface.
Demineralizing procedure. -All 22 blocks were treated with
A
B
C
D
E
Fig. 1 Schematic drawing of the preparation of the cementum/dentin
Received for publication December 7, 1987
blocks. Previously impacted third molars (A) were cut just below the
Accepted for publication April 20, 1988
cemento-enamel junction and 4 mm below (B). The obtained slice (C)
This study was supported by grants from the Swedish Medical Rewas divided into blocks (D), which were ground at the pulpal surface,
search Council, Projects No. 6002 and 7203, Swedish Patent Revenue
painted with nail varnish (except on a window on the cementum surface),
Research Fund, and NIH-NIDR Grant P50 DE07010.
and mounted into plastic holders (E).
1217
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-
ALMQVIST et al.
1218
a demineralizing solution, which was made from analyticalgrade chemicals and contained 2.2 mmol/L calcium (as CaCI2),
2.2 mmol/L phosphate (as NaH2PO4), and 50 mmol/L acetic
acid. The pH was adjusted with HCl to pH 4.5. The negative
logarithm of the ionic-activity product with respect to hydroxyapatite (PIOHA) was approximately 127.8, calculated according
to Lagerlkf (1983). Each block was individually placed in a
small plastic beaker with 5 mL demineralizing solution. In
order to produce circulation of the solutions, we placed ail
beakers on a custom-made shaking table. The treatment continued, at room temperature, for 84 hours. After six, 12, 24,
36, 48, and 72 hours, the solutions were replaced.
Chemical analysis.-In order for the calcium loss from each
block to be measured, the calcium content in the solution, from
each beaker, was analyzed by atomic absorption spectrophotometry. All analyses were carried out in duplicate. The calcium loss, in pug per square mm area (ACa), of each block
was calculated as the difference between analyzed calcium
content and initial calcium content of the demineralizing solution for each beaker, divided by the area of the exposed
cementum surface. The total calcium output (ACa505) value for
each block was calculated by summation of the seven ACa
during the experiment. The rate of demineralization (Vdem), in
mole hydroxyapatite/cm2 sec, was calculated from the changes
in calcium concentration in the buffer solutions by use of the
formula of Theuns et al. (1985).
Photon absorptiometry.-In Fig. 2 the apparatus used for
photon absorptiometry is shown. Radiation from a 1251 source
(Studsvik Energiteknik AB, Sweden), which had an activity
of approximately 20 mCi, was used. A tin filter of 0.12-mm
thickness was placed in front of the source to reduce energies
above 29 keV, and the resultant radiation was found to be
nearly monochromatic (96%), with an energy of 27.4 keV.
The beam was collimated to a cross-sectional area of 3.14
mm2. The radiation source was fixed to a base, and a brass
holder was constructed to maintain the plastic holder with the
block in a reproducible position during measurement. The brass
holder was mounted in the beam and fixed in relation to the
source and detector, resulting in distances between source and
specimen and between source and detector of 30 and 90 mm,
respectively. The radiation was detected with a thallium-activated sodium iodide crystal of 25-mm diameter and 2.5-mm
thickness. This scintillation crystal gave almost total absorption of radiation from the source. The crystal was mounted on
a photomultiplier tube, and the pulses were amplified and connected with a single-channel pulse-height analyzer, which
showed the number of photons registered.
In order to measure the transmission (T) through a block,
we immediately recorded the incident radiation before measuring a block. The block was then placed in the beam (fixed
to the brass holder), and the transmitted radiation was recorded
twice. After the block had been removed, a new recording of
the incident radiation was made. The same procedure was repeated, and T, in percent (the ratio of the transmitted and
J Dent Res September 1988
incident radiation x 100), was calculated from this duplicate
measurement. Measurement of T was carried out on each block
after zero (initial T value), six, 12, 24, 36, 48, 72, and 84
hours of demineralization. For each block, the total resultant
exposure time for radiation was 16 minutes. The change in
transmission (AT) for each block was calculated as the difference between the obtained T value at each time point and the
initial value.
Precision of analysis. -In order to estimate the precision of
the methods, we performed 176 and 154 duplicate measurements of T and of ACa, respectively. The standard deviations
of the duplicate measurements (S.D.,), at different time points,
were calculated by the following formula: S.D., = S.D.d/
\r2, where S.D.d is the standard deviation of the paired differences between the duplicates. A coefficient of variation, in
percent, was calculated for each time period. The mean coefficient of variation for each method was then calculated from
the different values.
Results.
The average coefficient of variation was found to be 0.20%
(range = 0.17-0.23%) for the T measurements and 0.75%
(range 0.65-0.99%) for the chemical analyses.
A typical example of a plot of AT as a function of time is
shown in Fig. 3a. It appears that AT increases faster initially
a
-100
E
E
2:
z
0
Un
1251
Absorptiometry
U)
I
U
analysis
z
4
-SD
2:
<D
2L
-25
w
CD
4
z
I
012
48
36
TIME (HOUR)
24
84
60
b
E
E 1400
'
120-
100_
\
'I 80*10
2:z
2 60-
~--0---
0
radioactive
1-5
-275 00)
2:
o 40-.
sourceI
w
t4
12
24
36
48
TIME (HOUR)
772
884
Fig. 3 - (a) The change in transmission and the change in output of
total calcium for a representative cementum/dentin block immersed for 84
hours in frequently changed acetic acid buffer, pH 4.5, containing 2.2
mmol/L of calcium and inorganic phosphate. (b) The rate of demineralization (pmol/cm2-sec) of the same block calculated from the change in
calcium concentration in the buffer solutions.
Fig. 2 Schematic drawing of the equipment required for transmission
measurements using 1251 absorptiometry.
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-
60o6
IN VITRO ROOT CARIES PROGRESSION
Vol. 6 7No. 9
as compared with the later time periods. In the Fig. the corresponding data for ACatt are plotted after the scale of the right
y-axis is adapted by means of a least-squares technique. The
AT and ACa,0, correlate to a high degree (r = 0.983), which
was also true for all 22 individual blocks (r = 0.934 - 0.998;
mean = 0.980). The rate of demineralization, Vdem, for the
same block, calculated from the changes in calcium concentration in the buffer solutions, decreased with time (Fig. 3b).
Fig. 4 shows the relationship between ACaO, and AT for
the 22 cementum/dentin blocks. The correlation coefficient for
these 154 individual measurements was 0.825.
In the Table the average values of AT, ACa,0t, and Vdem
are presented. After six hours, the transmission through the
cementum/dentin blocks had significantly (p<0.001, paired t
test) increased, with an average value of 0.78%. The Vdem was
approximately twice as high during the first six hours com-
pared with the last 12 hours. The data from the Table were
plotted versus the square root of time (t), and by linear regression the relationship was found to be
AT = 0.37 Vi - 0.23 (r = 0.997)
ACatt = 12.0 Vt - 22.4 (r = 0.996)
Vdem
8.7
=
Vi
+
133.2
(r
=
-0.955)
AT, ACatt, and Vdem were also found to be linear versus the
5-
i?- 4z
cn
U)
U)
0
z
3
z
w
2
z
7:
I
I. .. .
1-
s~~~~~
25
50
75
100
CALCIUM OUTPUT(1ug/mm2)
125
Fig. 4 The relationship between the change in output of total calcium
measured by atomic absorption spectrophotometry and the change in transmission measured by 1251 absorptiometry for 22 cementum/dentin blocks
-
(154 determinations,
r
=
0.825).
1219
cubed root of time (r = 0.993, r = 0.989, and r = -0.969,
respectively).
All r values given in the text are statistically significant
(p <0.001).
Discussion.
1251 absorptiometry, as well as microradiography (Angmar
et at., 1963), can be used for quantification of the mineral
content in dental hard tissue. The two methods have been used
for comparative measurements of enamel slices, and the accuracy has been found to be high (Henrikson and Linden,
1974; Julin, 1975). The advantage of '2-I absorptiometry compared with microradiography is that non-destructive measurements can be performed.
In the present study, we showed that for an individual root
hard-tissue specimen, the change in transmission, determined
by 1251 absorptiometry, followed closely the calcium output
into the demineralizing solution, as indicated by the good correlation between these parameters. Furthermore, it was found
that the absorptiometric method had a high precision and thus
can be used for detection of small changes in the mineral content. After only six hours, the transmission through the cementum/dentin blocks had significantly increased.
During the first 84 hours of root hard-tissue demineralization, the changes in transmission, in calcium output, and in
rate of demineralization were found to be proportional to the
square root of time. A linear relationship with a similar correlation was also found with the cubic root of time. Three
possible reasons for this phenomenon can be suggested. First,
this could be due to an immediate etching or surface softening
of the cementum, since the pIoHA (the negative logarithm of
the ion-activity product of the solutions with respect to hydroxyapatite) of the buffer solutions was initially about 127.8,
i.e., highly undersaturated with respect to hydroxyapatite, which
has an approximate solubility product of 117.2 (Theuns et al.,
1985). This degree of undersaturation remained approximately
constant during the experiment, since the buffer solutions were
frequently changed, and the calcium concentration did not increase more than 0.5 mmol/L, giving a PIoHA of 126.6 in the
demineralizing solution. It has previously been shown for enamel
(Theuns et al., 1985) that the combination of high PIOHA and
low pH resulted in a higher rate of initial demineralization.
When the higher vulnerability and solubility of root hard tissue
are considered compared with those of enamel (Phankosol et
al., 1985; Hoppenbrouwers et al., 1986), it is logical to believe that this combination of physico-chemical factors is disadvantageous for root hard tissue as well. In a recent publication,
Hoppenbrouwers et al. (1986) calculated the rate of demineralization of previously unexposed human roots, using microradiography. It was shown that at pH 5.0 the rate of
demineralization, measured at days 3.5, seven, and 14, was
TABLE
THE CHANGE IN TRANSMISSION (AT), THE TOTAL CALCIUM OUTPUT (ACa,.,), AND THE RATE OF DEMINERALIZATION (Vdem)
DURING 84 HOURS OF DEMINERALIZATION OF CEMENTUM/DENTIN BLOCKS
AT
Time
Vdem
ACa0tt
(%)
(hours)
(ptg/mm2)
(pmol/cm2 sec)
6
0.78 (± 0.48)*
10.7 (± 2.3)*
123.6 (± 26.5)*
12
0.97 (+ 0.48)
19.1 (± 4.3)
97.1 (± 25.2)
24
1.50 (± 0.58)
33.8 (± 6.6)
85.1 (± 15.7)
2.06 (± 0.60)
36
47.3 (± 8.7)
77.6 (+ 13.1)
48
2.39 (± 0.61)
59.4 (± 10.9)
69.8 (± 14.7)
72
2.94 (± 0.66)
80.4
60.8 (+ 8.6)
13.6)
84
3.17 (+ 0.67)
90.9 (± 15.6)
60.2 (± 12.7)
= 22).
*The average + the standard
deviation
(n
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1220
ALMQVIST et al.
constant, but that the experimental conditions led to a gradually
increasing surface softening. When our results of the rate of
demineralization are compared with those of Hoppenbrouwers
et al. (1986), there is a marked difference. Our rate of demineralization was much higher and could be explained by the
lower pH used in our study. Hoppenbrouwers et al. (1986)
found a ratio between the rate of demineralization for roots
and for enamel of about 6:1. Comparing our results for the
rate of demineralization for roots with those of Theuns et al.
(1985), for enamel, results in the same ratio, i.e., about 6:1.
Second, the phenomenon with initially higher changes in the
transmission, in the calcium output, and in the calculated rate
of demineralization could be explained by a diffusion-controlled dissolution process, which has been proposed previously. The rate of demineralization of enamel (Featherstone et
al., 1979; Poole et al., 1981) and of root hard tissue (Arends
et al., 1987; Featherstone et aL, 1987) as measured by lesion
depth was found to be proportional to the square root of time.
On the other hand, Christoffersen and Arends (1982) showed
this relationship to be cubic for enamel. Both views are supported by our results. However, no exactly comparable data
are available, since this study differs from the previous ones
in the short duration of the experiment. Third, the gradually
decreasing rate of demineralization may be due to a combination of surface softening and a diffusion-controlled dissolution process. It seems likely that the high rate of change in
transmission and in calcium output initially is due to an etching
of the surface, while diffusion processes control these parameters in the later stages of the demineralization process.
From the present research, we conclude that 125J absorptiometry can be used to follow the initial demineralization of
cementum/dentin blocks. This method should be regarded as
a complement to the other methods for studies of caries development. 1251 absorptiometry makes it possible to study in
situ the disease process and the effects of different root-cariespreventive measures. Future studies will deal with these questions.
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