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DEDICATION
To my mother and father who have

helped make my education possible.

THE INCIDENCE OF DENTAL CARIES IN CARIES SUSCEPTIBLE AND
CARIES RESISTANT ALBINO RATS, (RATTUS NORVEGICUS),
WHEN FED DIETS CONTAINING GRANUIATED AND
POWDERED SUCROSE

By
WALTER HENRY 3333mm, JR.

A THESIS ‘
Submitted to the Graduate School of Michigan State College
of Agriculture and Applied Science in partial
fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Zoology
1952

“”195

ACKNOWLEDGMENTS

I wish to thank Dr. Hunt and Dr. Hoppert for their
many opinions, criticisms and suggestions. Dr. Hunt guided
my experimental progress with many personal consultations.
Dr. Hoppert furnished me with data on my diets and gave
advice as to experimental procedure in mixing and formulat-
ing the diets.

Mn.Earl Harrison was helpful in feeding and caring for
the animals, when I could not be present. He also assisted
me in marking and examining the animals regularly.

Mr. Klever of the chemistry rodent laboratory and Mrs.
Bernadette Henderson (Miss Mac), of the Zoology Department
helped me many times to acquire the needed materials for my
experiment.

To these people I wish to extend a feeling of genuine
appreciation, for without their cooperation this paper could

not have been written.

‘V Q old, 2 'I a - g Q ‘P.
' 4' L 5.. £
f‘l.’ b :g‘ .: ; _‘ ‘5‘ g 3

TABLE OF CONTENTS

INTRODUCTION .....................................
HISTORY AND REVIEW OF LITERATURE .................
THE HUNT AND HOPPERT EXPERIMENT ..................
THE PROBLEM .......................ooo............
PROCEDURE ........................................
THE GRANULATED SUCROSE DIET ..................o...
DATA AND OBSERVATIONS ........o......o............
FIGURE 1 ........................................o
TABLE I ..................oo....o.................
TABLE II .......o.................................
FIGURE 2 ......oooo....oo.........................
FIGURE 3 ....................................o....
TABLE III o.......................................
DISCUSSION .......................................
FIGURE 4 o......o.o...............................
FIGURE 5 .......................................o.
FIGURE 6 ................a........................
FIGURE 7 .....................c....o..............
CONCLUSIONS .......oo.o.o........................o

BIBLIOGRAPHY .....OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

PAGE

12
12
11+
18
2o
22
24
25
28
29
33
34
35
36
37
40
41

1.

INTRODUCTION

Hunt and Hoppert have established the fact that heredity
is an important factor in the development of dental caries in
albino rats. The diet used in this experiment consisted of
66% coarsely ground hulled rice, 30% powdered whole milk, 3%
alfalfa meal, and 1% sodium chloride. The question arises as
whether the hereditary differences discovered are specific for
the above diet, or whether these differences persist when
various other diets are used.‘ This is the problem attacked

in the present investigation.
History and Review of the Literature

Many ideas have been presented as to the cause of tooth
decay or dental caries. Four general theories have been
prominent at various times. Hippocrates in #50 B. C.believed
in the inflamation theory, and thought the carious process was
due to the stagnation of depraved Juices within the tooth. 2
More recent investigators supporting this theory believed the
dentine contained capillaries and a circulatory system.
Histological work has shown the dentine to be without these
tissue constituents.11,4

As late as 1824 the worm theory was still accepted as a

 

cause of dental caries. Worms were believed literally to
subsist on the enamel.-2 In 1861 Bridgeman presented the
electrical theory. He thought of the tooth as a battery and
believed differences in electrical potential between the root
and the crown caused deterioration of the enamel.

A fourth theory of tooth decay is the acid theory. In

1530 a German investigator, Chr. Egenolff, made the statement

2
that caries was due to the decomposition of food particles.

Robertson in 1835 concurred in Egenolff's views and stated

that acid from the decomposition of food caused caries.2

The first men to introduce the theory that bacteria may be

the cause of caries, were Professors Erdl and.Ficinus, Dresden
physicians.(1843) In 1841 Miles and Underwood working with
Kock demonstrated that carious lesions invariably contain micro-
organisms.2 About this time the discovery of aniline dyes made
accurate work on the indentification of bacteria possible.
Underwood and Miles believed the carious lesions were due to:
(l) the action of the acids, (2) the action of germs.6'2
Although these investigators demonstrated the presence of
bacteria in dental caries, they were unable to prove that the
bacteria were the causitive agents.

It was left for W. D. Miller to establish the fact that
bacteria are active factors in the carious process. In 1891
Miller showed that when calcified tooth substance is incubated
in saliva-food mixtures, the enamel is attacked only when the
carbohydrate foods are used.2’9 When the saliva was sterilized
with heat, and carbohydrates were added to the mixture the
calcified tooth substance remained intact. Miller showed that
an acid fermentation takes place which may cause the primary
carious lesion in the enamel. He also believed that proteolytic
bacteriagmay be active in the secondary stage of the carious

process.

Dietz, who devised.an artificial mouth with a tooth in it
which could be observed with a microsc0pe noted the following

facts:

l. Caries begins on the surface of a tooth.

2. A bacterial plaque forms, sometimes producing acid.

3. Microorganisms in this plaque live on the same food
as the animal lives on.

4. The bacterial plaque produces acid and the caries
begins where the carbohydrates rest against the
enamel.

5. The bacteria are producing acid on a surface which
can be dissolved by acid.

The classical Millerian acid hypothesis remains in its present
form today.

In 1915 Kligler made the observation that certain types of
microorganisms grow frequently on the teeth of individuals with
active caries.37 The most important of these organisms were
Lacobacillus acidophilus. Subsequent research work of Dr. Philip
Jay and Dr. R. W. Bunting support the findings of Kligler.35,36’19

Dental caries is a pathological process.6 Bunting describes
caries as a disintegration of the teeth characterized by the
formation of cpen lesions in the enamel, dentine and cementum.2

Bernhard Gottlieb6, a recent investigator, does not agree
with the classical Millerian hypothesis of tooth decay. Gottlieb
believes the carious process begins in the organic components of
the enamel.6 By staining teeth with silver nitrate, he has shown
lamellae extending from the exterior surface of the enamel to the
dentine. The lamellae are thought to be organic constituents
between the calcium prisms of the enamel. Many investigators

disagree with this hypothesis. However, it may be stated that

the formation of dental enamel is among the most controversial

1,6,13,14
subjects of dental histology and embryology.

Gottlieb points out that naturally caries immune individuals
may consume sugar as much as they like and no caries will develOp.
This would point to another explanation of tooth decay than B.

6 I.

acidophilus in the saliva. Soluble sugar may penetrate the

 

lamellae according to Gottlieb. Fones and Boyle have shown
4 l
bacteria in carious dentine tubules. , The enamel lamellae

might thus be a possible path along which caries develops. Thus
it is evident that the causes of tooth decay have been by no
means completely identified, and numerous investigations concern-
ing the histological, embryological, bacteriological, nutritional,
chemical and genetical aspects of the decay process need to be
carried on. Two main types of research have been followed:

(1) Studies of the resistance of the tooth, (2) The natures of
the attacking forces.

Although caries has occurred in all stages of man's history,
the frequency has increased.with the degree of civilization.1,2
Bunting and others estimate that modern man has a 90-95% incidence
of tooth decay.2 Why is this true? In some populations in which
the incidence of caries is low, there is a noticeaglg lack of

.

refined carbohydrates even on a sub-standard diet. Dental
1

caries as we know it is a disease of civilization. An.English

investigator, Bresse, showed that during the war when sweets were 18

eliminated from the diet, caries was markedly reduced in children.

Bibby has also shown by numerous population studies that carbo-
hydrates influence caries susceptibility. Waugh showed that

refined sugars increased the B. acidophilus count and caries
42
incidence in Eskimos.

Price made extensive studies of caries in Africa and found

an increase in the incidence of caries whenever the tribes were

39
in contact with civilization. Observations of teeth in an

isolated mountain village in Switzerland, where sugar and refined

l
carbohydrates were unknown, show a negative incidence of caries.

These facts suggest that carbohydrates are an active factor

in tooth decay. However, all carbohydrates do not produce the

19
same effect. Inhabitants of the Pacific Island, Tristan da

Cunha, consume potatoes as a staple in their diet, yet they are
19
free from caries. Sugar appears to b: a more important factor
2

in producing caries than starchy foods. Also, to be noted is
the important fact that some persons teeth exhibit remarkable
resistance to caries even with high sugar consumption.

Mellanby, Rumsey and Rosebury believe that the diet is the
controlling factor in the incidence of dental caries.21 The diet
may alter the saliva or the teeth themselves. Bunting noted that

19
g. acidophilus counts are changed by regulating the diet.

 

Fosdick has shown a difference in Ph indiceg in the salivas of
2

caries immune and caries resistant people.

Fosdick states that acids in the mouth come from carbo-
hydrate materials: however, most oral acids under normal
conditions are derived from fermentable sugars. 3 Common
sugar or sucrose is a double sugar, each molecule consisting
of one glucose molecule and one fructose molecule linked together
rather loosely. When sucrose is exposed to acids or certain
digestive fluids in the presence of water, suzrose is quickly
split into a mixture of glucose and fructose. 3’5 These latter

43.5
sugars are known as inverted sugars. The biochemical equation

6.

43,5
for the process is as follows:
C H O -+~ H O or invertase or acids(HCl)
12 22 ll 2
(digestive enzymes)
Sucrose
(disaccharide)

(beet sugar)

CHO + CHO
6 l2 6 6 l2 6
d- Glucose d- Fructose
(dextrose) (levulose)
(monosaccharides)

The degradation of carbohydrates to lactic acid is thought
2 22 21
to be an etiological factor in the process of dental caries. ’

The formula for the formation of lactic acid from the glucose
molecule is as follows:
C H O ‘4‘ enzyme(Bacillus acidophilus)
6 12 6
Glucose
2 C H O
3 6 3
lactic acid
113
Hockett, in his article on natural and refined sugars
states that human saliva is able to convert starch to glucose,
but the saliva of rats and dogs does not affect starch. This
19
would support Bunting's statement that different carbohydrates
play different roles in the production of dental caries.
Stegerradas study of the Navajo and Mays Indians showed a

A" on».
marked difference in the amount of tooth decay te—the decay in

7.

41
our present civilization. His study suggested that there

may be racial and hereditary factors in tooth decay. Bunting
also makes the statement that heredity or individual
characteristics may determine caries susceptibility rather
than dietary considerations alone.19
Thus the four current types of theories concerning tooth
10,29,30
decay are:
l) Chemico-parasitig
a. Acid production by bacteria.
2) Specific-bacterial
a. Bacterial degradation of the tooth structure.
3) Metabolic
a. Dietary and vitamin causes.
4) Heredity

a. Influence of genes on tooth structure or

physiological processes.

It would appear that acid production and an oral environment
favorable to acid producing bacteria are important in the
causation of dental caries if acid is an etiological factor
in that process. We now return to the problem of what causes
some teeth to be resistant to sugar diets and others to be
highly susceptible?. What factors are involved 12.222
susceptibility and resistance 32 tggth.ggg§y?

The Hunt and Hoppert Experiment

Hoppert, Webber and.Canniff in 1932 found that they could

8.

induce4caries in laboratary rats which were fed an adequate
diet.2 ’25 They showed that vitamins, calcium and phosphorus
did not appreciably retard the decay of the teeth when added
to the caries producing ration. This diet contained cornmeal
as the major constituent. When oatmeal was added in place of
the cornmeal no caries appeared. The finer the cornmeal was
ground the lesser the incidence of caries. In this paper,
retention of food by the teeth was thought to be a contributing
factor to caries.25

Hunt and Hoppert conducted their eXperiment to determine
whether there is an inheritance factor in tooth decay, and if
so)how many genes are involved and how do they produce their
effects? The diet devised by Hoppert, Webber and Canniff
produced caries in the rats and maintained their health, growth

and fertility. The composition of the diet was the following:

Coarsely ground hulled rice:----------- 66 %

Whole milk powder--- -------- --- ------ ~--- 30 %

Alfalfa, meal-D-- ...... -------------------- 3%

SOdillm Chloride-----“u- ----------- -..-co 1
“—1100 x

* Seventy percent of the rice was retained on
a 20 mesh screen when tested for fineness.30
Hunt and Hoppert with this diet in 1937 started their
inheritance experiment. Phenotypic selection, progeny testing
and brother and sister inbreeding have been used to build a
caries resistant and caries susceptible strain. Seventeen
generations of resistants and twenty-four generations of the

susceptibles have been bred and examined.

 

‘A'II qiji Illa

9.

With this type of mating system the proportion of
heterozygosity in the stock steadily decreases. Li has shown
that the proportion of heterozygosity in animals practicing
brother and sister matings after n generations to be reduced
systematically.7 The generations of animals which I worked

with would have the following percentagesof heterozygosity:

(Res.s+.n+s .Suscep+nb\cs
H17 ; 1.961% H21 3 .824%

This shows a very small amount of heterozygosity left in
the stock at the end of 17 and 21 generations of brother and
sister matings.

During the inbreeding experiment the caries time has

fluctuated for resistants and susceptibles, but a significant

 

 

 

difference is apparent between the two strains.30 The average
caries times for the susceptibles and resistants have been
as followszeg,30 (16th and 22nd generations-anublished data)
Gen. Susceptible Gen. Resistant
Ave. C. T. Avg. 0. T.
2 57 days 2 116 days
15 13 " 15 470 “
16* 43 " 16* 379 "
22 42 "

*Fine rice added to the diet.

A caries susceptible and caries resistant strain of rats

have been produced by selection, progeny testing and inbreeding.
30

The coarseness of the rice was found to be a detegmining
25 1 .17
factor in producing dental caries. Braunschneider,

 

lO.

discovered that if'the rice component of the ration is in the
form of flour, caries in the rats of the susceptible strain is
rare until an age of 100 to 150 days.

Braunschneider studied the influence of age on the incidence
of dental caries.l6’17 Caries could be delayed in susceptibles
until 150 days of age by using the diet which contained rice
flour.17 The composition of the diet was the same as that
described on page 8, except the rice flour was substituted for
coarse rice. Two groups of animals were raised to 100 and 150
days of age caries free. These animals were then put on a
caries producing diet which contained the coarse rice particles.
These two groups showed more resistance to caries than the
animals at the 35 day growth stage.17

Hunt and HOppert found that sex is not important in
influencing dental caries in rats.32 These investigators
studied the distribution of 10,048 gross carious cavities in

V 34
‘the lower molars. Some areas of the occlusal surfaces of the

rat's lower molar teeth are more susceptible to caries than
others?‘ .The right lower molars develop a higher percentage
of cavities than the left in susceptible and resistant animals.34
Hunt and Hoppert proved that use is an important factor in
developing cavities. It was found that when the opposing

upper molar was removed from the mouth it lengthened greatly the

caries time in the corresponding lower molar in susceptible

33
rats.

Nakfoor, Hunt and Hoppert studied the resistance to
fracturing in resistant and susceptible teeth. The lower

molars of resistant rats were found to be more resistant to

11.

fracturing at 60 days of age than susceptible lower molars

at the same age. 8 This may have been due to a slight weak-
ening of the structure of the susceptible teeth by the carious
processes.38 About one-half of Nakfoons susceptible rats
unexpectedly produced caries on the rice flour diet. If
natural fracturing is a prominent cause of dental caries, then
the teeth of these rate should have a considerable amount of
the fracturing. But, they did not. Hence natural breakage

is probably not important in the carious process.

Keller has severed the parotid gland duct in susceptible
and resistant animals and found that the parotid secretion is
not of great importance in the carious process of the animals.
(Unpublished data) He also found that the resistant strain
is practically immune to caries in the upper molars, while
there is a 42 percent incidence of caries in the upper molars
of susceptibles.(Unpublished data)

R. L. 61186 has studied the growth curves of resistant and
susceptible rats and found that curves for the two lines are
substantially the same until 147 days of age.(Unpublished data)
Beyond the 147 days the susceptibles show a slower growth
rate than the resistants. He also found that the susceptibles
have significantly less hair than the resistants.(Unpublished
data)

A. E. Epstein has made a study on the effect of sugar-acid
drinks on the erosion of susceptible and resistant teeth.
Susceptible animals appear to show more erosion than resistants
on this drink.(Unpublished data) He has also delayed caries
in susceptible animals until 170-210 days by using a powdered

milk diet of the following composition:

12.

Powdered milk--------80 % '
Alfalfa meal---------19 %

NaCl--- ------------- - l
105%

THE PROBLEM

Hunt and Hoppert's caries resistant and caries susceptible
strainfof rats ate a ground rice, powdered milk and alfalfa
meal diet. Would.this hereditary difference be apparent if
gthgg diets are consumed?

Sognnaes has found that a highly concentrated sugar diet
would przduce a high degree of caries if fed before the teeth
erupted. 3,40 With this information it was decided to test
the susceptible and resistant strainsof rats on a sugar diet
similar to the one Sognnaes used..40'43 Would the resistant
animals still be resistant to caries with a high concentration
of sucrose, and different from the susceptible rats? In

other words, are the hereditazy differences giscovered'gy

Hunt and Hogpert specific for one diet only?

Procedure

Breeder animals were obtained from Dr. Hunt's 17th.and
21st generation resistant and susceptible r;:::I;N0ctober, 1950.
Breeders were selected for their general health,and the matings
were between animals of as nearly the same caries time as possible.
The same environmental controls which have been maintained for

13 vears during the Hunt and Hoppert experiment were used.

13.

The temperature of the animal laboratory was held
between 77° and 80° Fahrenheit. The water was furnished
from the college deep well system. Water was supplied to
the experimental animals by drip bottles and these were
kept clean by periodic cleaning. Metal cages were used which
measured 2 1/2 x 1 1/2 x 1 feet. Five animals or less were
kept in a cage. Pine shavings provided litter for the
animals. The cages were cleaned about every 10 days. Kerosene
was used as an insecticide under the shavings. DDT was
added in small amounts to the kerosene.

Breeder females were isolated when they appeared pregnant.
The females were observed each day in order that the exact
date of birth of the litter could be recorded. When the
litters were 25 days old the females were removed and the
young animals were put on the experimental diet. The breeder
females were put in a rest cage for seven days and then
mated again. All males and females in the litters were sep-
arated before they matured, usually about 40 days. The
animals were all numbered by the regular toe and ear marking
system used by Hunt on all of his animals. A description

of the experimental diets follows:

The Beet_Sucrose Diet

In order to determine whether the difference in heredity
in the Hunt-Hoppert lines is specific for only one diet the

following ration was used:

14.

Granulated beet sugar(Michigan beet)------- 57 %

"Fine" rice ------------ - ........... -------- 10
Casein ------- - ............................. 18
/ Corn oi1(Mazola) ------- - ----------------- -- 2
Brewers yeast --------- --- --------- - ..... --- 5
Alfalfa meal--- ----------------------- ---- 5
Salt mixture ---------- - ------------------- .;_2
100 %
26

Description 9; the Salt Mixture

 

Ca003 --------------------- ---- 544.08 gm.
M3003 ------------------------ - 25.00
MgSO4 -- -------- - ----------- --- 16.00
NaCl ------- ------- ----------- 69.00
KCl - ----- ------------------- 112.00
KH2P04 ------- ----- - ------------ 212.00
FePO4'4H20 -------- ----------- 20.50

MhSO4 ------------------------- .35

A12(SO4)3‘KQSQ4 --------------- .17

CuSO4 - ----------------------- - .20

1000.00 gm.
Granulated beet sugar is a cuboidal crystal, which

dissolves easily and passes through a screen with 20 meshes
to the inch. The fine rice in this diet has a 2 percent
retention on a 20 mesh screen. Examinations of this diet
on a 20 mesh screen showed only oil adhesions and brewers

yeast as the major portion of the residue. Some fine rice

15.

and KCl particles from the salt mixture were retained on this
screen.

The rats on this sucrose diet liked the food and grew
normally in all visible respects. A good coat of fur
developed in both the resistant and susceptible animals on
the sucrose containing diet. There was, however, a susceptibility
to respiratory discharges in the older resistants. This type
of discharge was not noticed in the susceptibles as they were
usually killed when comparatively young. The blood was of
a dark purple color and anemia was improbable. The internal
organs appeared healtly on autopsy in both strains.

The control diet used was the same as on page 8 except
fine rice is substituted for coarse rice. This diet was

{FFercnh'ej'e

known tod a caries resistantfggapcaries susceptible
rat.
During the experiment it was found that the beet sugar
containing diet seemed to lengthen the caries time as compared
with the effects of the fine rice ration in the susceptible
strain. The question then arose as to whether this might be
due to a decrease in the percentage of fine rice particles or
the presence of sugar crystals. In order to test the effect

of a diet having no fine rice particles or sugar crystals
the following ration was fed to the susceptible animals:

The Powdered Sugar Diet

Powdered sugar(commercial)------- ------ 67 fl

0 a 8 Gin---“--------------------------- .- 18

Com Oil----- -------------- ----- ----- --- 2

l6.

Brewers yeast ----- ------- ------- ------- 5 %
Alfalfa meal--------------------------- 5
Salt mixture---------------------------1_;2_
100 f

The salt mixture in this is the same as the mixture used
in the beet sugar diet. The powdered sugar contains about
3 per cent starch in order to keep the sugar in a powdery
consistency.

This diet was extremely fine. Examination of the coarse
particles in this diet based on 75 gram samples showed a
15 percent retention on a 20 mesh screen. The residue was
found to be mostly oil adhesions and flakes of brewers yeast.
However, there were some hard particles of K01 from the salt
mixture. The K01 was ground in a mortar before it was added
to the salt mixture; therefore these particles would constitute
only a small percentage of the diet. The powdered sugar diet
was fed only to the susceptible rats. They grew slower at
first than the susceptibles on the beet sugar diet. No
comparative weighings were made.

Another observation was that the young susceptible rats
did not grow hair in the normal pattern on the powdered
sucrose diet. The hair appeared blotchy and thin toward the
tail. After this condition cleared the animals grew and had

a good coat of fur and appeared normal.
Method of Mixing the Sugar Diets

After measuring the ingredients on an appropriate

balance, the diet was thoroughly rubbed together and mixed

17.

in a galvanized iron can. The oil adhesions were thoroughly
rubbed into the mixture. The diets were dry and had no
tendency to cake unless water came into contact with the

mixtures.
Method of Determining Caries

Dr. Hoppert made periodic 14 day inspections of the
lower molars of all the susceptible rats. In a few cases the
14 day period was extended. Samples of the resistants were
observed at the same intervals until decay was observed, then
the resistants were all inspected from that date at fortnightly
periods. A nasal speculum was used to pry open the mouths
and observe the teeth. The light was furnished'by a 100 watt
bulb about three feet away. Since Dr. Hoppert observes the
teeth for the Hunt-and Hoppert experiment,his observations
compare with those he made on the Hunt-Hoppert rats. The
main point is that one man observes the teeth, and the
determination of cavities is constant.

Each animal had a page in a record book. A rubber stamped
picture of the lower molars was entered in the book.
Observations of the lower molars were entered as no caries, or
if caries was present a (/) sign with the date of occurance
was entered at the spot on the tooth where the caries was
seen. If there was an impaction so that we could not make a
positive identification, or if the cavity was at all doubtful
a (7) was entered by that tooth with the date and the
observations were continued. When a (f) was entered the

animal was killed and the head put into 95% alcohol.

18.

The heads were dissected.with care so as not to injure
the upper and lower molar teeth. These teeth were then
observed with a binocular microscope of 20 power magnification
and the observations were entered in a table. The caries
timgwin this experiment was calculated.§g the time the animal
(£Q_observation.

One or two controls from most litters were put on the
fine rice ration used by Hunt and.Hoppert. However, Hunt
and Hoppert; averagefor the same generation could have
been used for comparison.

A few animals were not treated in accordance with the
above experimental procedure. Two resistant litters were
put on the experimental sucrose diet at 36 and 32 days of
age instead of the prescribed 25 day limit. Magnesium
sulfate was substituted for magnesium carbonate in one

mixture of 16 kilograms of the beet sugar feed.
DATA AND OBSERVATIONS

Five groups of animals are present in this experiment.

They will be designated by the following subscripts:

Rr-oo Resistant animals on the fine-rice containing diet.
Rs"' Resistant animals on the beet sugar containing diet.
Sr“‘ Susceptible animals on the fine-rice diet.

Ss-- Susceptible animals on the beet sugar diet.

3x7" Susceptible animals on the powdered sugar
containing diet.

19.

The graph(figure l) of the resistant and susceptible
animals on the best sucrose diet shows the distribution of the
two strains on the same diet. The mode for the susceptibles
is at the 71-110 day caries time interval, while the mode
for the resistants is at the 311-350 day interval. The
difference between the two strains on the fine rice ration
is also apparent. It is clear that the hereditary difference
revealed by the rice containing ration is likewise revealed by
the sucrose containg ration. 2222 £22 contrast between

susceptibles and resistants is not specific for the rice

 

containing,ration.

The data in this graph actually underestimate the difference
between the resistant and susceptible strains of rats. It
was decided to tabulate these data before all the resistant
animals showed caries or died because I had to conform to a
terminal date for submitting the thesis. Some of the resistant
rats in Hunt's data have reached the age of 784 days without
showing caries.30 Moreover, resistants have died without
caries. The high mode of resistants at the 471-510 day interval
is occasioned by the animals we completed observations on
before they developed caries. Had these animals lived they
would have increased the variability of the resistant strain.
None of the 8 control resistants on the fine rice diet showed
any caries. These 8 rats represented 6 sibships and were a
random sample from the resistant rats put on the best sugar
diet.

The average age in caries time of the 16 resistant animals

which did not show caries and were included in these data

FIGURE 1, Comparison of Susceptible and.Hesistant

Rats on the Sucrose and Fine Rice Diets

Caries Frequency
Time

2 1 § 8’ 1:01:21141361182'92224

20.

 

l l '

31-70

71-110

111-150

151-190

191-230

231-270

271-310

311-350

351-390

391-430

431-470

 

471-510

 

21.

was 453.8 days. If the observation of these animals were
continued they would certainly have raised the mean caries
time of the resistant strain on the sucrose diet.

All the susceptible animals were observed to have caries.
Seventeen susceptibles were randomly picked from each litter
and put on the fine rice containing diet. There were 13
sibships represented in the susceptible control group(8r). The
graph, figure 1, seems to indicate that the sucrose diet
increased the variability of both the susceptible and
resistant rats.

Table 1 shows the means, variances, and standard deviations
of the five groups of animals. The first statistical question
that presents itself is whether or not the data and mean
differences between the five groups; He, Rr, 58’ Sr and SK
are significant or are they explained in terms of chance.

An analysis of variance test described by Snedecor gives a

12
very good description of this test and its applications.

 

 

 

 

Source of Variation D. F. Sum of Squares Mean Square
Total 129 3123636.63

Between Groups 4 2684440.30 671,110.07
Within Groups 125 #39,196-33 3513-57

F4’125 ; 671,110.07/ 3513.57 3 191 *s

This value of F lies far from the 1 percent probability
level given in the tables of F in Snedecor.12 F needed only
to exceed 3.45 to be significant at the 1 percent level.
Hence the group means are not different due to chance alone.

The hereditary difference is very evident between the resistants

 

 

 

 

 

Sum of X

N!

Sum of x2

C. T.”

Variance
Standard Dev.
Sum of (X - X)?
No. of Litters

Avg. Size of
Litter

*12

SS (1)

 

4059

44

92.25

417405

374442.75

999.12

31.60

42962.25

18

3.8

 

991

17

58.29

60769

57769.47

187.47

13.69

2999.53

13
(sampled)

Table I

Groups

Sx (3)

 

723

11

65.72

50435

47520.81

291.41

17.07

2914.19

2.8

Rr (5)

 

 

18387

50

367~75

7139199

6761635-58

7705.37

87.77

377565.42

11

5-5

2977

372-13

1120573

1107816.13

1822.41

42.60

12756.87

6
(sampled)

 

 

23.

and the susceptibles.

 

The next calculation is to determine whether there are
significant differences between data for pairs of groups.
Table 11 contains F and T tests between all the pairs of groups.
F tests the significance of the variances, while T tests the
signifiance of the means. Lindquist gives a good description
of these tests.8

We are mainly interested in the differences between groups
1:4 and 2:5. F and T are highly significant in comparison of
the susceptibles and resistants on the sugar containing diet.
Groups 2-5 are also significantly different, but we knew this
before. Again it_i§ evident that the hereditary differences
between the two lines are revealed by the sugar containing diet.

The numbers are small in the other groups which we compared.
The meaning of the significance of these group comparisons can
only be speculated on. However, one interesting fact emerges.
This is the significant F and T test between groups 1-2. Two
different diets were fed to a highly inbred strain of susceptible
rats. The sugar containing diet increased the variability and
mean caries time of these susceptible animals. The comparison
between groups 1-2 show that the 57 percent sucrose diet in
some way delayed the development of caries as compared with the
fine rice containing ration. Both F and T tests confirm the
significance of this difference.

Figure 2 shows the distributions of the susceptible animals

on the sucrose, fine rice and powdered sucrose diets. The diff-

erence between groups 88 and Sparerclearly shown on this graph

Groups

 

Values of F and T Tests Between All

Combinations of the Five Groups

Group with larger variance

Table II

F- test

 

5.32s»
3.42*
7.71**
1.82
1055

41.10**

9.72**

26.44**

6.25*-
4.22-

*significant between 1 and 5%

*(T)

** beyond 1% significance

beyond 1% significance

Group with smaller variance

 

T-test

 

4.27-
2.68*
19.89*
6.91*
1.29
14.48“
28.04*
11.35*
21.74*
.137

 

 

d'2 3 Sum of (x - x)2
X1 - X2
2 2
n1 n2 - 2 n1 n2

24.

FIGURE 2,

Caries
Time

30-44

45-59

50-74

75-89

90-104

105-119 ‘

120-134 ‘

135-149"

150-164"

165-179"

180-194 ‘

25.

Distribution of Susceptible Rats on
Three Diets

Frequency

 

5?

1?

10 11 12 1; 14
l I lfl I '.

// Group SK
1

Group SB

 

195-209 J

26.

with 14 day intervals. It would appear that the modal caries
time is lengthened in the susceptible strain on the sucrose
containing diet.

The two diets differed in content; both mineral, fine
rice particles, sugar particles and carbohydrates. Which
one of these constituents changed the carious effect in the
susceptible animals? Did the bacterial or saliva threshold
change? 0f the 61 susceptibles raised, 17 were randomly
picked from the litters and put on the fine rice ration.

The two groups were similar in all respects except diet.
Comments on the Powdered Sucrose Containing Diet

'The powdered sucrose diet was fed to the susceptibles
in order to observe the effect of a diet with no rice
particles or sugar crystals. The susceptible animals on
the powdered sugar diet behaveflguch the same as the animals
on the fine rice ration.(figure 2) However, one susceptible
rat has gone 164 days, without caries on the powdered sugar
diet. The number of animals on the powdered sugar diet is
small, and more research is indicated with this type of ration.
However, the susceptibles stlll developed caries gg'§_
dlgt consisting'gg powdered s ar, casein, corn oil, brewers
ygagt, alfalfa meal, and salt mixture. The number of coarse
particles in this diet were less than .3 percent of the
total weight.(no fine rice particles at all) These observations
emphasize the point that dental caries can develop in the
almost complete absence of coarse food particles of any kind.

Possibly the bacterial content of the mouths was different for

27.

the different diets; this subject deserves further investigation.
OBSERVATIONS OF THE TEETH

A study was made of the distribution of the cavities in
the susceptible and resistant rats. The rats were usually
killed when the first (/) cavity was seen or on the next
observation two weeks later. Figure 3 shows how the cavities were
scored when decay was observed in each molar. Because of the
size of the cavities in the susceptible teeth, only the tooth
on which a cavity occurred was recorded. No attention was
given to decay in the crevices. The tooth was considered as
one area.
The following groups were investigated and the number of

heads observed under the microscope are as follows:

 

figgup No. Observed
as 30
S, 44
Sr 17
8x 9

According to figure 3 the teeth were numbered individually
as lst, 2nd and 3rd molars. The teeth were classified in
groups; right and left molars, upper and lower molars. When
a carious cavity was noted in a tooth it was tabulated as a
(/). These (/)'8 were added together and the results compiled
in the form shown in table III. The percentage of carious cavities
in each location was calculated by dividing the number recorded
for that location by the total number of cavities in that group.

Carious cavities usually have rounded centers and generally

FIGURE 3, Classification of Upper and Lower Molars

3 3

Right Left
UPPER MOLARS

 

 

 

 

 

 

 

Q: 3
2
1

 

Left R 15m.
LOWER MOLARS

 

Table III

Distribution of Caries in the Upper and Lower

Molars of the Susceptible and.Hesistant Groups

Right
1 2

44 44
15.4% 15.4%

14

17
5.96% 4.91%

(3,.)

17 17
14.5% 14.5%
5
4.27%
9
a 11.6%
7
9.09%

21
23.3%

25
27.7%

Lower

4.56%
Upper

14
4.91%

Lower

15
12.8%

Upper

3
2.56%

Lower

Molars

22m.

1.

43
15.0%

Molars

11
3.85%

Molars

17
14.5%

Molars

3
2.56%

Molars

9
11.6%
Molars

1
1.2%

Molars

19
21.1%

Molars

2

44
15.4%

14
4.91%

17
14.5%
5.12%
9
11.6%
5.19%

24
26.6%

2

12
4.21%

15
5.26%

14
11.9%

7-7%

10.38%

Grand Total

29.

Total

285

117

77

90

569

30.

extend into the dentine and even the pulp. The percentage of
cavities in the upper molars of the susceptibles are somewhat
misleading because table III does not take into consideration
the extent of the carious process in each tooth. In some cases
the whole lower molar was gone, or only an enamel shell was
left. Comparatively speaking, the lower molars had larger
cavities than the upper ones. Figures 4,5,6 and 7 are photo-
graphs showing cavities in the upper and lower molars of the
four groups of rats on the experimental diets.

Sucrose appeared to lengthen the caries time in the sus-
ceptible rats; however, this diet produced rampant caries after
the initial lesion began. The dentine seemed to disintegrate
completely in some cases. I would say that the sucrose contain-
ing diet produced a more rampant caries than the fine rice ration.

The data presented in table III suggests that the 3rd
lower molars in the susceptibles on sugar (88) is more
resistant to caries (8-77%) than the 3rd lower molars on the
rice ration (Sr), 24.7%. Also, the sucrose diet tends to
increase the susceptibility of the upper molars to caries
when compared with caries in the uppers of susceptible rats
on the fine rice diet.

One unusual finding is that the upper molars of the
resistant rats did not show any carious cavities at all on the
sucrose diet. However, the susceptible upper molars on the
same diet had an incidence of approximately 30% of the total
carious sites: The upper molars hadiimpacted food material
in their crevices which was very solid. This impaction

affords a good place for the bacterial decomposition

31.

of food with resultant acid production. What can be said
from the observations tabulated here is that the reistant
uppers are more resistant than the susceptible uppers to
caries on a high concentrated sucrose diet.

Nine observations were made on the susceptible molars
on the powdered_sucrose containing diet. These animals were
not killed on the (f) observation. We thought we might need
breeders and they were kept longer on the powdered sugar diet.
The high incidence of caries in the upper molars was probably
due to the extended time the rats were on the diet. The
susceptible teeth on the powdered sucrose diet showed more
extensive decay in the lower molars thafiifihe other diets. In
some cases only the outermost shell of the enamel was left.
Figure 7 shows the decay in the susceptible teeth on the
powdered sugar diet.

The 1st and 2nd lower molars still show the highest
incidence of caries. This is similar to the findings of
Hunt and HOppert.34 Inspection of the percentages in table
III shows this trend. The number of cavities on the right

side appear to be greater than on the left side.

 

Q3232 Right Molars Left Molars
8, 35-36% 34.61%
5,. 41.8% 40.9%

RII 52-1% 47.7%

Due to the small numbers of observations too much
significance can not be attached to these differences. However,
the excess of cavities on the right side does persist.

The caries I observed was usually in the advanced stage

and the exact nature of the initial carious lesion is still

unknown. my observations of the heads did not support the
fracture hypothesis of caries origin. The smaller cavities
were seen usually at the bottom of the sulci in the lower
and upper molars. The large carious cavities were all
centered around the deep sulci. The early cavities would
not originate at the bottom of the crevices if the caries
were initiated by fracturing.

Observations of the susceptible teeth showed.that there
was more impaction in the lower molars on the rice ration,
than on the sugar diet. This may have been due to the ability
of the rice to pack into the crevices easier than the gran-
ulated sucrose, which dissolves quickly.

Uppers may be less susceptible to caries because the
crevices are not as wide as those of the lower molars. In
picking food particles from the crevices of both lowers and
uppers this was a general observation.

Some teeth appeared to be resistant to caries, next to
teeth with rampant caries. Regardless of whether the rat
was a susceptibha or a resistant; pinkish teeth seemed to be
associated with resistance. If a tooth was white or cream
colored,caries was usually present. A white radiating color
change was noted in some small cavities on the enamel of the
tooth around the lesion. Gottlieb, in his book on dental
caries, goes into detail dn color changes.6

There was a brownish deposit on the molars. This was
especially noticeable in resistant uppers. It flaked off like
a dried membranne when the teeth were dry. Further research

is indicated in determining what effect color changes and

deposits on the teeth have on dental caries.

33.

Some teeth were observed to have erosion under the gum,
but no caries was observed. A decay process of the lower
dentary bone around the tooth structure was observed in some
heads. Figure 4 shows this type of carious process of the
bone by a resistant rat's 3rd molar. (Figure 4- resistant
male-l4). The relationship of caries with this type of
periodontoclasia is unknown. Further research should be
cognizant of this type of pathological process.

On the following pages, figures 4,5,6 and 7 show the
carious cavities in the differfiet groups of rats on the ex-
permental diets. The caries time is given for each set of

lower molars under the photograph.

DISCUSSION

Charts and graphs show the distribution in caries time
of the susceptible and resistant rats on the granulated sucrose
diet. Caries resistant and caries susceptible animals still
differ on a diet containing 57 percent sucrose. This fact is
shown by a graph (figure 1), analysis of variance test and
significant F and T tests. The hereditary difference of the
Hunt-Hogpert strain of rats is not specific for only the fine
rice diet. A 51 percent sucrose diet still reveals the
hereditary difference between the two strains.

A difference in the caries time has been observed in
the susceptible animals on the sucrose and fine rice diets.
The variances and means of groups 88 and Sr are different
in terms of days to the first (/) observation of caries.

The number of rats are small for any final conclusion.

34.

FIGURE 4, Resistant Rats Upper and Lower Molars on

the Sucrose Diet (Group RB)

 

Top Row:

F- 85 F- 82 Me 14

(bone caries)

CariesTime 213 177 428
Bottom Howl

Me 58 F- 5 F- 21 Me 27
Caries Time 308 401 313 363

* M- male
F- female

A magnifying glass can be used to
advantage in examining these molars.

35-

FIGURE 5, Susceptible Rats Upper and Lower Molars on

the Sucrose Diet (Group as)

 

 

 

19232.2:

M- 96 Me 98 F- 71
Caries Time 69 55 49
Bottom Row:

F- 114 M— 105 F- 111

Caries Time 73 203 80

30-

FIGURE 6, Susceptible Rats Upper and Lower Molars on

the Fine Rice Diet (Group Sr)

 

122.1321:
M- 108
(upper molars
reversed)

Caries Time 66

Bottom Row:
F- 42

Caries Time 64

Me 103 I? 119

41 37

M- 60 M- 79

63 84

37-

FIGURE 7, Susceptible Rats Upper and Lower Molars

on the Powdered Sucrose Diet (Group S

x)

 

Caries Time 43

Bottom Row: F- 122

Caries Tim}; 78
(139 days
on diet)

F- 123

87

M5121

92

F- 124

37
(57 days on
diet)

M? 131
85

(85 days
on diet)

38.

However, what factors caused this fluctuation in a highly
inbred strain of caries susceptible rats?

This difference may have been due to the impactibility
of food, number of coarse particles or some quality of the
carbohydrate degradation in the mouths of the susceptible rats.
Examinations of the teeth in the susceptible rats on the n.ne
rice and sucrose diets showed the following facts, (Groups
' s8 and Sr):

1. More food impaction in the lower molars on the
rice diet.

2. The 3rd lower molar may be more resistant to caries
on the sugar diet thanfithe fine rice diet.

3. The sucrose diet increased the susceptibility of
the upper molars to caries in the susceptible rats.

A difference in the effect of these diets has been noted.
Further research is indicated in analyzing this effect.

Analysis of the granulated sucrose diet and changing the
constituents in it would be valuable in determining what sub-
stance is important in the causation of tooth decay. Would an
increase in the sucrose level over 57% increase the susceptibil-
ity to caries? Would a decrease lower the incidence?

Resistant rats have reached over 500 days of age without
tooth decay and may die without showing any carious effect on
the granulated sucrose diet. The granulated sucrose produced
'initially slower caries in the susceptible animals, but pro-
duced rampant caries after the initial lesion. All the sus-
ceptibles showed caries at an average of 92.25 days on the

granulated sucrose diet.

39.

Again the hereditary difference is apparent between the

 

 

caries resistant and caries susceptible strain 9: rats pp the

granulated sucrose diet.

 

A very fine powdered sucrose diet has produced caries in
susceptible rats at an early age. Fracturing by coarse par-
ticles does not appear to be an initiator of caries. Exami-
nation of small cavities deep in the sulci supports this
statement. No resistant rats were fed the powdered sucrose
diet, research is needed in testing its effect on these animals.

The variabilities of the different groups of rats on the
different diets shows that the caries process is not a simple
cause and effect relationship. There are many interacting
factors present in the process. Experimental error may also
be a cause of variability, however, the animals were controlled
in all experimental procedures.

Upper molars have been shown to be more resistant to decay
than the lowers. No decay was observed in resistant upper molars
on the granulated sucrose diet. Caries was observed in the sus-
ceptible uppers on this same diet. Uppers in the resistant rats
are more resistant to caries than uppers in susceptible rats.

Pinkish teeth seem to show more resistance to caries than
white ones. The cause of the initial carious lesion can only
be speculated on.Is the decay process due to the basic nature
of the tooth as shown by the color difference or is the action
of bacteria on the enamel through the decomposition of the food
the primary cause? More experimentation is suggested to deter-
mine this cause and effect relationship.

A finer date for the determination of the initial carious

40.

lesion would be productive when working with diets producing

rampant caries. Only gross carious lesions are seen. An

early investigation in-vivo of the carious process in the

lower molars may show some pertinent facts concerning the

initial carious lesion. However, the observations a3

carried out in this experiment showed the hereditary difference

 

ip the caries resistant and caries susceptible rats very

well pp the granulated sucrose diet.

1.

2.

4.

CONCLUSIONS

The hereditary difference is still apparent for the
caries resistant and caries susceptible strain of

rats when fed a diet containing a high percentage of
granulated sucrose. The resistance and susceptibility

is not specific for only the fine rice containing

 

diet.

 

The granulated sucrose diet increased the variability
of the incidence of caries in the susceptible

animals, when compared to the control, fine rice diet.
A very fine powdered sucrose diet produces early caries
in the susceptible animals. This conflicts with

other data on the effect of fine diets on the

incidence of caries.

Some areas of the resistant and susceptible lower and
upper molars are more resistant to caries than other
areas on the sucrose diet. Namely, these are the

3rd lower molars, and the upper molars generally.

Resistant upper molars are more resistant to dental

caries than susceptible uppers on the granulated

sucrose diet.

Sucrose diets produce rampant caries.

41.

1.

10.

11.

12.

13.

42.

BIBLIOGRAPHY

Books

Boyle, Rudolf, Kronfeld's HistOpathology pf the Teeth
and Their Surrounding Structures, Lea
and Febiger, Philadelphia, 1949.

Bunting, Russell W., Oral Pathology, Lea and Febiger,
Philadelphia, 1929.

Easlick, C. V., Dental Caries, Mechanism and Present
Control Techniques, C. V. Mosby Co.,
St e 1101.11 5, 19480

Fones, A. C., Mouth Hygiene, Lea and Febiger,
Philadelphia, 1934.

Garard, I. D., Introduction to Organic Chemistpy,
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1932.

Gottlieb, B., Dental Caries, Lea and Febiger,
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Li, Ching Chun, Population Geneticp, National Peking
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Lindquist, E. F., Statistical Analysis in Educational
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M8380 , 19400

Miller, W. D., The Microorganisms of the Human Mouth,
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Miner, Leroy 8., Why the Teeth, Funk and Wagnalls Co.,
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Orban, B., Oral Histology and Embryology, C. V. Mosby
U00, Ste 1101113, 19490

 

 

Snedecor, G. W., Statistical Methods, The Iowa State
College Press, Ames, Iowa, 1946.

Research Conference pp the Cause and Prevention 2;
Dental Caries, Chicago, Illinois, Good Teeth Council
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14.

15.

16.

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24._

43.

Periodicals and Theses

Beust, T. B., "Enamel and Dentine Metabolism Illustrated
by Reaction to Caries," Journal 2; the
American Dental Association, 23, p. 827,
1936.

Bibby, B. G., "Neglected Factors in the Study of Dental
Caries," Journal 2; the American Dental
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Braunschneider, G. E., |‘Rice Flour in the Diet, and.Age
as Factors in the Incidence of Dental
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Rats (Rattus norvegicus)," Theses for the
Me Se degree, Me Se Ce ’ 1946e
Braunschneider, G. E., H. R. Hunt and C. A. Hoppert,
'The Influence of Age in the DevelOpment
of Dental Caries in the Rat (Rattus
norvegicus), " The Journal p§_Dental
Research, Vol. 27, p. 1547 194 .

Bresse, F., " Diet and Teeth," British Dental Journal,56,
120, (Feb. 1) 1934—. ""'—" —"—

Bunting, R. W., ”Diet and.Dental Caries," Journal 23 the
American Dental Association, 22, p. 114,

1935-

Erwin, W. G., "A Genetic Study of Dental Caries in the
Albino Rat," Thesis for the Ph.D. degree,
Michigan State College, 1940.

Fosdick, L. S. and H. L. Hansen, ” Theoretical Considerations
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to Dental Caries," Journal 2; phg
American Dental Association, 23, p. 401,
1936.

Fosdick, L. S. and A. C. Stark, "Carbohydrate Degeneration
- by Mouth Organisms," Journal of the
American Dental Association, 28, p. 234,
1941.

Fosdick, Hansen and Epple, "Enamel Decalcification by
Mouth Organisms and Dental Caries,"
Journal of the American Dental Association,

'24", "_"p . 12757—1937 -

Hoppert, C. A., P. A. Webber and T. L. Canniff, "The
Production of Dental Caries in Rats Fed on
an Adequate Diet," Journal of Dental
ResearCh, VD].- 12, 130 161, 1932e

25.

26.

27.

28.

29.

30.

31-

32.

33-

34.

35-

36-

HOppert, C. A., P. A. Webber, and T. L. Canniff, "The
Production of Dental Caries in Rats Fed
on an Adequate Diet," Science, Vol. 74,
p. 77, 19310

Hubbell, R. B., In B. Mendel and A. J. Wakeman, "A New
Salt Mixture for Use in Experimental
Diets," Journal hT_Nutrition, Vol.23,

p. 273. 1937-

Hunt, H0 R-. and C- A- Hoppert, "Inheritance in Rat Caries,"

Journal QT the American College 9T
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Hunt, H. R., and C. A. H0ppert, "Inheritance of .
Susceptibility and Resistance to Caries in
Albino Rats," Journal ET the Amggican
College 9T Dentists, Vol.11, p. 33, 1944.

Hunt, H. R., C. A. Hoppert, and W. G. Erwin, "Inheritance
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Hunt, H. R., C. A. Hoppert, and.G. E. Braunschneider,
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