2

( I
L
|

AIM

Ml

lNl‘leIHM

NW

»

 

'é‘é’é "NW“

A STUDY OF sewazuw GF T'FE SENTM.
ENM'AEE. OF CAQZES-RESGSTANT
AN? CARiES-SUSCE "féBiE ALBENQ RA“?

Thesis b: the Began at“ #3.. S.
MQC‘diGAN STATE CORLEGE
Garda Macks-e
W54:

Or

,1: i-‘t “*1 I. -’ www ”ﬂ ' 3... ’ “‘ ‘ . I W.‘ ‘ N" ’ ' ‘ 'i - “-’
LI 5! U! -,.1Luiua.$31 'J.‘ 43“.; LLJJ-d-‘u. outrun“;- 91' L.‘.:.A¢ 5"?3‘11!‘ It“!

- n
3 ' r v

k'?"{ 0 -_ was '1: -‘ -" _u‘1_“'t F4 ' :‘ r‘f .Z.‘ ”L
(-1.15: {4&154 LL1.“'8 V's/v ('4. 1&Mb" ‘LLdrusai‘J tblf-ib

ﬂy

Gerda mootuo

{unalttgd a; has: whoa]. cl Gtéaéﬁbﬂ Satanic-z: vi“ relics-21;; u

itata Cullegu u! AdfiCALtirﬁ an; Aggliﬁﬁ

clause

rl-N

in raftial fultillmunt u; ta: re14lrugautb
for La ware-s: of

'ﬁ ‘5‘? -. 3“ . «it i.:', f i' :4." 4'
15‘s...» an uv‘unou

Euguruscut us; (imaginary

19:4

During tun d8C¢d€E whicn {$110388 tsa ru¢h&;tiaa cf ﬁllier,
tnﬁt deuaal Cnrlcﬁ 13 Cdﬁ Lu relation of the inwr¢.nic yOPLiUu of
ﬁnmuui and dugbia la ﬁciae‘. traum- _..:‘O._,I‘t3:.h has barn mum is rev-'3 ling
the facturs involveg. Iv gr&zunn a cumgicta ,ictzra at tae cavelag-
want 01 caries, tug Ltrxct4rm ﬁn; CGQﬁU£1§iUu of tag tavth mutt be
inciaqed.

Evidmnca of tau Varying rcpiecvnce-uf tuetn t9 dantgl cnriaa
has been greuoutod by HJnt and ﬁﬁggart “no bred a carieauraciatunt uni
carisa—auacartlble strain or aLhinJ rat». Tna LVlenbiiibj o: theae
attains made it gdikiblﬁ to Aztumgt to find Out whether dental cariaa
wan carrelutad to tug avg,0ainicn of aunt l “ﬂaﬁcla Csu:e_saubly a
study of the eoiubiiitj o; yuwuarc¢ enuaei frsu 6&riea-ra;ibt at and
Chrimﬁ-auaceytible aiblna Put; in ha.rer¢a (elation; of u ya 4~8 was
undertﬁkan. The dotarainatioa 9f calcium and ghannurue 13 wall
aduytud to tun Etuuy of noiubilitj of tau teutn enamel tines anagel
cogpriaea about éﬁ Par cent Ca and 17 par cent ?. Calcium wha ﬁre-
Ciyittted £5 tun oxainta And titrﬁteo with C.Q1N xmuaé. fhgcgnyruﬁ
was detarmlued colorimetrlcally bg'tﬂﬂ Brigg; mueifichtian of £30
3611*EOiiy metuud u:iug a ghotoeiectric Qulurimazur.

50 airfareuce wan foamy in zn¢ LGLUUiLiL} u: tha toutn enamel
of tha two atrgiuu; The enamel 0f the invigor: of both atraiua summed
tam what lowﬁr auiauilizy tuna tau Ednmﬁl or £43 mulwr taﬁtn.

Unleaﬁ it Can be shown taut Lac {oiubiilty 0f tug neutai ﬁﬂ.£ﬁl
01' Law intact $9211.51 0;; Lita-‘3 Pram: 3.1.530. in thin titan”; his} :1“:-“.- Iran; $14.4, of
tha annual #uwdsr it ma;t he Concluuvu taut otuer faster: account {or

tha diffarﬁub Cariea rutaa.

344081

A bTULY 0F SGLUEILITI UP TILE TLJTiL Ella-ml: 0F CnRIES-I'iELSISTEﬁT

AND CAkLIEb~£ 'SCL’TIBLE MBIJO Ré'I’S

BY

GEEK}; MOO TEE

A THESIS
Submitted to the School of Graduate Studies of Michigan State
College of Agriculture and Agplied Science in partial
fulfillment of the requirements

for the degree of

MASTER OF SCI 121 CE

Department of Chemistry

1954

ACKNOKLEDGEUENT

The author is deeply indebted to Dr. Carl A. Hoppert
for suggesting the problem und for his constant advice and encourage-
ment without which this study would not hive been possible.

Sincere thanks are also due Dr. E. Leininger for providing
some equigment uoed in this investigation and Dr. H. A. Lillevik for

high vacuum distilled bromoform used in the separation procedures.

INTRODUCTION
HISTORICAL .
EXPERIMENTAL
RESULTS . .
Table I .
Table II .
Table III.
table IV .
DISCUESION .
SUMMARI o o

BIBLIOGRAPHX

TLELE

OF CCRTEﬁTS

Page

12
16
17
18

19

9-3 £3

33

INTRODUCTION

INTRODUCTION

In 1944 Hunt, Hoppert and Brain (1) published dets thnt
with environmental factors held fsirly constant, most of the dif-
ference between the caries-susceptible and caries-resistant lines
of albino rats was hereditary.

These two strains of rats were developed using pheno-
typic selection, brother and sister inbreeding and progency testing
of breeders.

A full understanding of the structure and behsvior of the
tooth can be obtained, however, only by applying all available methods
of study, physical, chemical and anatomical, and by interpreting
these in terms of the general biochemical and physiological principles
which govern living organisms.

In this investigation a comoerison of the enamel solubility
of the resistant and suscegtible strains of albino rats was made
with regard to (u) the position of the tooth in the dental arch (b)
the pH of the solvent.

Ever since the acid theory of caries was formulated by
W. D. Miller, great interest has been shown in the solubility of teeth.
Since the primary attack in caries is on enamel, almost all the
solubility studies heve been made on this tissue. Being composed
of only around one per cent organic material, chiefly a modified
protein,'the enamel should be subject to total disintegration by

exposure to acid.

Lullemegne and Melon (2) indicated thnt the enamel of
the tooth is comyosed of about 60 per cent csrbonete-apatite, 50 per
cent -tricelcium phosphate, and l per cent calcium carbonate, the
residue being minor minerals and organic matter. Since apatite
is denser and less readily attacked by acids than is tricelcium
phosphste, the chief constituent of tooth dentin and cementum, such
of the strength of enamel may be due to its content of carbonate-
apatite.

Miller's (5) main conclusion following his two year study
of an estimated 8000 teeth was: ' Caries of the enamel is purely
chemical, the decnlcificetion resulting at once in the complete
dissolution of the tissue." But Miller did not associate any enamel
structures with either freedom from or susceptibility to decay.

The present study was carried out to determine whether
caries susceptibility might be associated with the relative solubility

of the dental enamel.

HISTORICAL

HISTORICAL

Miller's theory has been until recently the foundation
upon which almost all theoretical considerations of caries have been
built. The solubility of tooth enamel has been studied by various
investigators. The earlier work was ineccurete, because the effect
of dissolved material on the acidity of the solvent was disregarded.

Rodriguez (4) end McIntosh et el (5) immersed whole teeth
in acidic solutions and did not find any decelcification of the enemel.
Hartsell et a1 (6) observed only slight opacity. hummery (7) exposed
the crowns of various teeth to 0.075 per cent lactic acid and tested
the surfaces daily by scratching. Es observed decslcificetion of
cusps and incisal edges first. Bunting et'el (8) and Friesell et
al (9) exposed small areas of teeth to acidic solutions and examined
the exposed area microscopically. Thurlow and Bunzell (10) investigated
the activity of various organic acids in decelcification of enamel
and noticed that the solubility appeared to depend upon factors
other than 9H alone. Whereas oxalic acid actually tends to prevent
solution of enamel, citric acid dissolves enamel strongly. McClellend
(11) used buffer solutions of various pH leVels in which to suspend
pieces of ennmel and determined the rate of dissolution by weighing
before and after immersion. Enright et el (12) maintained a constant
hydrogen ion concentration by allowing a continuous stream of acid
to flow over the surface of enamel, so that the dissolved products
did not retard the progress of the reaction. They found that all the

lactate and citrate buffer solutions from pH 4 to pH 8 decalcified

the enamel. When these buffers had been saturated with tricelcium

phosphate, only tAOEe buffers with a pH more acid than 5.0 etched
the enamel.

These quelitntive results can not safely be used for
comynrison. The size of surface of the whole teeth as well as of
pieces of enamel varies and often one or only few teeth were used.
This procedure is not satisfactory, since it is now known that in-
dividual intact teeth vary greatly in their resistance to acid decalci-
fication.

The factor of individual variation in composition of the
teeth ccn be overcome by using a statistically significant number of
teeth. The variation of enamel surface exgosed to the acids action
was overcome by using a relatively homogeneous enamel powder. Pro—
cedures commonly employed for the pregnretion of enemel mo;t of
which utilized mechanical forces-(a) splitting off fishes of enamel
from the dentin (b) grinding out the dentin from the pulpel side
or (t) grinding off the enamel fr0m outside-usually gave low yields
and impure products.

An outstanding contribution was made by Erekhus and Arm~
strong (15) in 1955 by the introduction of e flotation method for
separating enamel, dentin and cementum. The method takes advantage
of the differences in the specific gravity of the various dental
tissues.

The method was greatly improved by Manly and Hodge (14)
who spylied a centrifugal technique to the flotation method. By

this method the degree of purity of the fractions obtained can be

followed by determining the indices of refraction of the dentin and

enamel particles.

Benedict and Kenthnk (15) determined the rate of solution
of powdered human enamel in various buffered solutions and found the
rate to be proportional to the acidity of the solvent. The rate was
rapid during the first 15 minutes and then reached an even, and slower
rate during the next hour. Volker (16) exoleined the rate difference
due to the greater regicity of solution of smaller oerticles of enamel.

Benedict et a1 were the first to observe a difference
in solubility related to enamel structure. Studying the decelcified
enamel under the reflecting microscope, they noted that the rods were
more easily decalcified then the interprismetic material.

Dallemagne et Melon (I?) explained that the presence in
and eround the interorismetic materiel oforgunic matter slowed down
the rate of solution, and so gave the effect of a greater solubility
of the rods. When the organic matter was removed, the greater
solubility of a ~tricalcium phosphate over the carbonate apatite
of the rods became apparent.

Karshen end Rosebury (18) determined the pH relationshios
at equilibrium to dissolved phosphorus and calcium. The results
indicated that in lactate buffers, pH change at each time level
was proportional to dissolved yhosphorus at thnt level. The calcium
values were about twice the ohosghorus Concentration.

Forbes (19) showed, thnt the solubility of sound enamel
from curious and noncarious teeth was the same.

Volker (20) used a standard technique in his quantitative
measurements on solubility of tooth tissues. He noted that if the

powder use air dried, the solubility did not change, but if it was

dried in e vacuum, the eolubility decreased as much as 25 per cent.
He studied the effect of surface area on the amount of enamel dissolved.
He also observed the greater solubility of deciduous enamel and ex.
plained it on the basis (a) of the greater content of Mg salts or
(b) lower content of fluoride. Volker did not find a significant
difference between the solubility of enamel from carious and non-
carioue teeth and concluded, that the resistance or susceptibility of
a tooth to caries is not degendent on the relative decclcificetion
of the enamel in acid, but tnet all teeth would be discolved by
acid solutions at approximately the same rate. He noticed a slightly
lowered solubility of Curious enamel. The first observation on
dentin was recorded by Velker (20) who found that it was alwcys more
soluble than enamel.

A number of substances with resyect to their power to reduce
the solubility of powdered enamel was studied by Buonocore et el (21).
In general, they used lone Which could be exoected to enter the apatite
lattice and alter its solubility. Laud fluoride proved to decrease
the solubility of powdered enamel most.

Seve‘nl of the substances which can reduce enamel solubility
in vitro heve been tested for their action in vivo. Manly et a1 (22)
attributed the difference bcteeen the clinical and the in vitro tests
to the _reeiatance of the protective layer on the intact enumel surface.
Enough of tne results were ne~etive to inVclidnte a generclizetion that

acid-solubility reduction invariably brings about caries reduction.

Although the destructive effects of acids were confirmed
by many investigators, the question arose, as to whether any specific
organic ion might not have been gsrticulerly active in destroying
the tooth surfaces.

McClure et sl (£5) exbosed the enamel of rats' molar teeth
to the action of diluted solutions of Ne—citrste at pH 5.5 - 7.2
such as prevail within the oral cavity. Although the solubilizing
action of the citrate ion on the enamel bears no gross similarity to
ordinary dental caries, it is suggestive of the erosion observed in
human teeth. The action of the ion was eiglsined as due to binding
of the Ca of the enumel to form a soluble, slightly ionized Ce—citrete
complex. The authors suggest thet in considering the etiology
of dental caries less emyhusis should be placed on acid deculcificetion
and that possibilities of a nonnacid decelcificetion, brought about
by bacterial metubolites, be given more attention in dental caries
research.

One may question the imgortcnce of tooth resistence to
decelcificstion and the applications of solutions of the teeth to
decrease it when there normally exists a wide variation in resistance
to decelcificetion among non—carious teeth from the same individual
as well as among different individuals.

Brudevold (24) has develoged a method for qusntitutively
testing the surface solubility of enamel in acid solutions. The tooth
is covered with wax except for e standard—sized window, and after ex?
posure to acid the amount of dissolved P is analysed. In a study of

forty permanent teeth, four consecutive exposures of an area with a

diameter of 4 mm to 5 cc of acetate buffer of 9H 4 for 10 minute
periods showed wide variations. The first exgosire showed lover P
values in 85 per cent of teeth, indicating that the aurfuce is more
acid resistant than deeper layers. In contrast ground surfaces

were more soluble than intact enamel surfaces. The first exgosnre
of a ground surface showed a higher average solubility than the
second. Solubility of different areas on one tooth may very as much
as 50 per cent. In thirty areas on twelve erugted t.eth svereg: de-
viation was 19.5 per cent. In six erens on three teeth the avarige
deviation in solubility of each tooth was 17 yer cent. ~31§£V0li
showed also that brown pigmentation of enamel Wes frequently associated
with low solubility. Repeated acid extracts from intact end ground
enamel surfaces frequently showed variations in the Cn/P ratio. He
suggested that there may be differences in the inorganic comgos‘tion
of the enemel from the acne tooth.

Isrdeni (25) exposed whole teeth and powdered enamel (for
comparison) to buffers at varying pH levels and measured the weight
loss. The rate of dissolution of teeth was essentially a linear
function of hydrogen ion concentration. The loss of weight did not
appear to increase linearly with the time of ex; sure, 1.6., the
decomposition did not proceed at a constant rate. Powdered enamel
showed a linear course probably because of its greeter~homogeneity.
The attack on intact teeth was at first slow, e lag per'od being
present. He explained it as a threshold necessary to overcome some
kind of a biological barrier ﬁguiﬂ5t acid diriusion, grobebly the

continuous organic matrix evenly distributed throughout tne enamel.

10

The slight dissolution near the neutral region he interyreted as

due to the carbonate fraction of enamel. With decreasing pH the
dissolution steadily increased ug to a certain critical point.

Further loss was very slight either because of a decrease of rate

of attack with the loss of minerals by the tooth structure or because
of a change of electrical chsrge at the isoelectric point of the tooth
at a certain.low pH value.

The diminishing rate of dissolution is in agreement with the
clinical observation that carious enamel is more reeietent to acids.
He postulated thnt in vivo the decelcificntion degends on the quelity
of the structure of the tooth, the size of the apatite crystallites,
and the width of tne organic channel: open for diffusion.

In 1952 Inrdeni (26) publiehed_datn on solubilities of
powdered ennmel and dentin. He criticized the postulation that the
more mineralized enamel is more susceptible to decalcificetion than
dentin. According to Armstrong (27) the apatite crystallites are
smaller in dentin then those in Inamel, and hence present a greater
surface for absorption. Yerdeni says:

”The fact is, that enamel contains larger apatite crystellitee,
is more compact, and therefore, 198p permeable to the acid
solution. The qunlity of the minernl is the same in enamel
as in dentin. Po;cibly, it is the anions which determine the
different rate of solubility; first the carbonates readily
go into solution, then the ghosphntee, and finally the more
resistant fluorides. But there may be else n reaction of the

enamel protein with the comgonente of the buffer solution.‘'

Swsrtz and Phillips (28) determined the hardness of the
tooth enamel and tried to correlate it with solubility. Enamel
varied widely in hardness and solubility from tooth to tooth es
well as from area to area on the same tooth. The maximum area of
approximately uniform hardness wns found to be £ mm in diameter.
No correlation between hardness and solubility was detected. The
decalcified areas of intact teeth were enemined microscopically.
When the depth of penetration was measured, the floor of the decelci-
fied area wee uneven, indicating that the acid hsd penetrated to
greater depths in some areas then others. The Ca/P ratio of the
enamel dissolved during the solubility tests Was slightly higher

than the 2:1 known to exist in enamel.

EXP ERIM LN TAL

EXPERIMENTAL
AﬂIﬁsLS

Twenty-fifth generation albino rats from Hunt and Heppert'e
caries-susceptible and twentieth generation caries-resistant strains
were used for the dental tissue studies. The animals were killed

with ether.
TREnTs-im '1' OF TEETH

The teeth of sacrificed animals were dissected out, stripped
of adhering tissues and separated into collective groups of upper end
lower incisors and upper end lower molars of caries-susceptible and
caries-resistant strains. The incisors were Split into fragments
to facilitate the removal of dentsl pulp. Subsequently the teeth
were extracted with absolute ethanol for tselve hours followed by a
similar extraction with ethyl ether. Extraction appeared to decrease
the tendency of particles to adhere during subsequent pulverizetion.
Samples were dried at 60° and pulverized in en agate mortar with
repeated sifting until the whole qusntity passed a sixty mesh screen.
The powder was egnin extrscted with an ethanol-ether mixture and
dried.

The powdered teeth were fractionated into enamel and dentin
and cementum components by the flotation method of Manly et Hodge (14)
who used a bromoiorm.scetone mixture of 59. gr. 2.70 for separating
human enamel. It was found thst the corresponding rat tissues

appeared to be slightly less dense end in a liquid of sp. gr. 2.70

14

practically no enamel Segnreted. Consequently a mixture of bromoiorm
and acetone of 59. gr. 2.65 was used and centrifugetion carried out

at 1500 RPM in a swell clinical centrifuge. The percentage composition
of the fraction of enamel was determined by direct count of one hundred
particles under the microscope, using the Becke line hosed on the
different refractive indices of dentin and enamel as a means of dif-
ferentiating. (14) The enamel fraction was considerably contaminated,
the less dense material carried along into the outer tube. Purity

rose as teeth were ground finer to pass the two hundred mesh sieve.

To insure uniform particle size for solubility studies only that
portion thet yeSeed the two hundred mesh sieve and failed to pass a

525 mesh screen was used. The seporntion of the two fractions appeared
to be connlete after the fourth refractionution. The enamel eemgles
were air dried, stored in a vacuum desiccator over anhydrous Ca012 to

constant weight and weighed.
ANALYTICnL PROCEDURE

The phthnlete buffers were pregered from pH 4 - pH 6
and checked by means of the pd meter. Ten milligram eemoles of the
powdered enamel were added to 10 ml of the buffered solutions, shaken
for fifteen minutes and filtered.(29) The analytical figures refer

to the acid soluble Ca and P.

15

. r‘m‘_ :" Y.-.'HT”.'.Y
Ch LR¢LJ ullu‘ol' 65““!{1

Suitable Eemples of the filtered enamel solutions were
analyzed by precipitation of the Ca es oxalate in 15 ml centrifuge
ltnbes. Further ogeretione of centrifuging, washing and titration
with 0.01N K3n04 were all carried out in the centrifuge tube by

the Clerk-001119 modification of the Kramer-Tisdell method. (50)
P DETiuMlﬂnlluﬂ

Suitably diluted portione of the filtered enamel solutions
were analyzed for P by the nethod of Fieke-Subberow modified by Briggs
using a photoelectric calorimeter. (31) Both methods gave reproducible
results.

.“

RESULTS

Th5 Eliimi SuinILITI AT pH 6

(expressed as use. Cu/lOOcc)

 

W .:—

i

17

 

 

TOOTH I II III Average
Upper resistant molars 2.62 2.59 2.6 2.6
Lower resistant molars 2.55 2.57 2.6 2.57
Upper susceptible molars 2.59 2.6 2.57 2.59
Upper resistant incisors 2.43 2.4 2.42 2.42
Lever resistant incisors 2.4 2.42 2.42 2.41
Upper susceptible incisors 2.4 2.42 2.42 2.41
Lower susceptible incisors 2.58 2.4 2.4 2.4

 

(expressed as mgs. 2/100 cc)

 

 

 

TOOTH I II III Average
Upper resistant molars 1.22 1.24 1.25 1.24
Lower resistant molars 1.2 1.24 1.25 1.22
Upper susceptible molars 1.25 1.2 1.25 1.22
Upper resistant incisors 1.14 1.12 1.15 1.14
Lower resistant incisors 1.12 1.15 1.15 1.13
Upper susceptible incisors 1.15 1.15 1.08 1.12
Lower susceptible incisors 1.15 1.14 1.13 1.14

 

 

TUE EHAHLL SOLUBILITI AT pH 5.5

TABLE II

(expressed as mgs. 04/100 cc)

18

 

 

 

 

TOOTH I II III Average
Upper resistant molars 4.6 4.58 4.59 4.59
Lower resistant molars 4.62 4.6 4.65 4.62
Upper susceptible molars 4.61 4.65 4.58 4.61
Upper resistant incisors 4.18 4.2 4.17 4.18
Lower resistant incisors 4.19 4.21 4.2 4.2
Upper susceptible incisors 4.2 4.19 4.18 4.19
Lower susceptible incisors 4.2 4.25 4.2 4.21

(expressed as mgs. P/lOO cc)

 

 

 

TOOTH I II III Average
Upper resistant molars 2.2 2.19 2.2 2.2
Lower resistant molars 2.18 2.19 2.2 2.19
Upper susceptible molars 2.6 2.2 2.2 2.2
Upper resistant incisors 1.97 1.96 1.92 1.95
Lower resistant incisors 1.92 1.99 1.95 1.95
Upper susceptible incisors 1.97 1.99 1.95 1.97
Lower susceptible incisors 1.94 1.97 1.98 1.96

W

TABLE III

THE ENAUEL SOIUBIIITY AT pH 5

(expressed as mgs. Cs/lOOcc)

19

W

 

TOOTH I II III Average
Upper resistant molars 8.4 8.54 8.53 8.49
Lower resistant molars 8.57 8.6 8.55 8.57
Upper susceptible molars 8.6 8.58 8.57 8.58
Upper resistant incisors 6.7 6.9 6.78 6.79
Lower resistant incisors 6.9 6.8 6.82 6.84
Upper susceptible incisors 6.84 6.9 6.86 6.87
Lower susceptible incisors 6.85 7.0 6.85 6.89

(expressed as mgs. P/lOOcc)

 

 

 

 

TOOTH I II III Average
Upper resistant molars 4.1 4.07 4.08 4.08
Lower resistant molars 4.13 4.09 4.12 4.11
Upper susceptible molars 4.1 4.12 4.09 4.1
Upper resistant incisors 5.27 3.29 5.27 5.28.
Lower resistant incisors 5.27 3.29 3.29 5.29
Upper susceptible incisors 6.5 6.28 5.27 5.28
Lower susceptible incisors 5.28 5.5 6.29 5.29

 

 

t“

RABIES I V

' iii-I. LULJJILI‘I‘I .11" pH 4

'1": 13'. 5
‘91:. H‘OLH

(expressed as mas. CsflGO cc)

 

TOOTH I II III Average
Upper resistant molars £2.98 25.55 25.05 25.02
Lower resistant molars 21.98 15.05 25.05 25.02
Upper susceptible molars 25.1 25.2 25.0; 25.12
Lower resistant incisors 18.27 18.5 18.L. 18.29
Laser susceptible incisors 18.54 13.4 13.5 18.55

W

(expressed as mas. 2/100 cc)

 

10018 I II III Average
Upper resistant molars 11.04 11.1 11.0 11.05
Lower resistsnt molars 11.03 11.06 11.0 11.05
Upper susceptible molars 11.16 11.04 11.18 11.15
Lower resistant incisors 8.59 8.75 8.68 8.64
Lower susceptible incisors 8.68 8.6 8.7 8.66
a“:— “1‘“? ‘4 m

 

 

 

DISCUSSION

DISCUSSION

The pepularly accepted concept of dental caries, namely,
that acid formation on the surface of the tooth is the complete cause
of its destruction, has been followed for more than hslf a century.

It has not led to a complete comprehension of the dental caries
mechsnisn as evidenced by clinical observation.

The experimental biological approach to the problem of dental
caries is comparatively recent. The hypothesis of this thought has
been set up thst the factors causing dental caries are twofold:

1. The presence of exciting (exogenous) factors, (food
retention, bacteria) has been demonstrated.

2. The predisposing (endogenous) factors, nhicn could
explain the immunity to dental caries of certain individuals, still
are Somewhat obscure.

It has been proven that susceptibility and resistance to
caries in rats are in part due to heredity. (1) Genes produce their
effects by initiating events which lead to the develoyment of structures,
and to chemical processes. Both the susceptibles end the resistants
have consumed the same kind of food, drunk water from the some
source, lived in the some kind of cage, and been handled by the same
caretaker in the same building.

The process of caries is very complex, involving the interplay
and interaction of a great many different factors, no one of which

alone can be considered the cause of caries, yet any one of which may

25

be decisive in the progress or prevention of the condition. Variable
resistance of teeth to attack concurs to be linked more with endogenous
factors. These factors may relate to abnormal structural variations,
such as permeable enamel lemellae, varying chemical comyosition,
chemical differences in tissue fluid within the tooth, which in turn
is dependent on blood conditions.

The present exocriment considers the possibility that the
genes for ceries susceptibility not through producing an effect on the
composition of the enamel.

In solubility studies we hsve to consider the chemical
conyosition as well as the anatomicsl structure of the teeth. That
they both influence the solubility has been confirmed by various
investigators. Dellemegne et el (17) showed the difference in solubility
to be due to the difference of the organic matter in enamel. Enamel
protein has caused much controversy which.mey be due in part to the
rather wide variation in amount. Rosebury (52) decslcified the enamel
with acetic acid and obtained an insoluble protein, very resistant to
hydrolysis with hydrochloric acid or potassium hydroxide. According
to Volker (16) the difference in moisture content of the enemel
properttion.mny effect the solubility as much as 25 per cent. Yerdeni (26)
postulates the solubility to be dependent on the structure of the tooth
as well as on the chemiCul comgosition.

There is abundant evidence that intact teeth are highly
variable in their resistance to acid decelcificstion in vitro and there
is indication that the resistance is associated with the nature of

the surface.

The only plausible explanation of failure of some teeth or
erees of teeth to be attncked in en in vitro experiment is a difference
in surface structure between different teeth end different parts of the
same tooth. However, if there ere indeed reel structures of dentel
enamel surfaces tnnt confer resistance to caries, the origin is in
vivo (55). By inspection of enamel with the ﬁoetgenspectroscope,
differences in crystalline structure between outer and inner layers
can be demonstrated.

Our data comyeres only the mean inorganic phase of the tooth
enamel of theee two strains of rats. On the basis of these results one
may conclude thet there are no hereditary differences as to the in-
organic cameosition of the enamel in the two strains. In our exyeriments
all the nannies were treated similarily and conditions were keyt constant.
(In order to avoid the differences in the moisture content or of the or-
ganic matter.) By refrectionetion the samples got very “herd" treatment.
The enamel tended to carry less dense materiel along. In order to avoid
such contamination, the powder was added stepwise in four portions with
a short centrifugetion immediately after eech addition. Although the
bromoforn was redistilled freshly under high Vacuum etc, the centrifuge
used did not permit control of the temperature.

Gilda (34) studied the hanly-Hodge (l4) separation method as
applied to dental tissues of rodents and explained the difficulties
in eeperetion due to a higher percentage of junction particles (a
smaller volume of enemel per unit area). According to Gilda (55) the

enamel of albino rats closely resembles hydroxyepatite in X—Ray

25

diffraction pattern. The lower density (2.720 - 2.930 Gm per cc) com-
pared to humen tissue he attributes to a relatively greater smount of
organic matter in rat enamel. That would account for greater differences
in solubility of e ret's intact tooth end of cnnmol powder grepcred from
rats' teeth. .33 the literature there is much confusion resulting from
lattempts to analyze and assign fonmulss to definite consounos which were
thought to hsve been isolated from the tooth.

The stable entity in the tooth is the nputite lattice itself,
the arrangement of stems in hyace being relatchly constunt. The out—
standing socilinr ty of this lattice is that it ccn tOIGTLtB e large hump
ber of substitucnts for the usual Ce, P, 02 And “2 atoms anion it contains.
If the substituting elements are about the same atomic radius as the
elements which they reelece, the unit cell of the eyetite lattice is
not greatly altered, and the diffraction pattern whicn it gives remains
essentially the some. That the composition of nny anetite will depend
noon the conditions union greVall at the time it is formed has been
shown by Thewlis et el (56). In the case of teeth this will obviously
reflect the comoosition of the blooo serum from which it originated.

Sobel et Hench (57) confirm thet there is e relationshit
between the inorganic congosition of the upper incisor of the albino
rat and blood serum, which in turn is regulated by the diet. They do
not exgress an sheet constitutive relationsnig beseuse the activities
of the tooth forming ions at the site of edepo;ition and exect relation-
ship between congenition of tne tooth and the activities of the ions

that form the tooth are not known.

In vise of the findings, thbt the P043003 ratios in rat
enanel are in ell cases lower than in dentin (58) (In human teeth
Armstrong and Brekhus (59) regorted higher PO4:CO5 ratios in the enamel
than in the dentin) they pointed out thnt the dlfiershce in congenition
between enamel and dentin of human and rate may be due to the particular
diet given rather than to species differentiation (40).

The investigations by Deenins (41) indicate the inorganic
concosition oi encnei to be count at. In developing pig enamel the Ca,
P and 002 contents were found to increase linearly end in constant
ratio to each other throughout the range of calcifiCation. It was
concluded that these elements are degocited as a conglex compound
having a filSQiCOmROSitiOﬂ. The calcificstion gradients in the sets
tooth they attributed to the start of infiltration of the mineral
phase into the substance of a yreformed organic matrix at the cusp
tips and to the granunl spread over the whole crown of the tooth.

We do not know whether the limitations of the analytical
methods for detennining the fine structure of tooth substance led to
varying results and conclusions.

It is peerible to sum no the foregoing discussion by the
statement that the nature of the agatite lattice possesses greet
signifigance fr0m the standpoint of the structure and behavior of
the tooth. It explains the constancy of the chief properties of the
tooth in shite of variations in conuosition. It enslains also the
extreme sensitivity of the deveIOying tooth to any metabolic changes
as well as same of the Changes which the fully formed tooth can

undergo.

27

There is, however, evidence that teeth are tissues in equili-
brium with body fluids. Greenberg (42) Armstrong et el (45) fed Ca45 and
P52 to albino rats and measured the amounts found in the teeth. They
found that the turnover of the isotooes in the incisor enamel was about
one-third of that in the femur. In the case of molar enamel the turn-
over rate was only from 1.5 to 5 per cent of that of the femur.

As permanent teeth become more comyletely calcified, euuili-
briun with body fluids slows up to such an extent thst only minor changes
can be observed. According to Gilda (55) comparatively small changes
in the comyosition of the tooth may affect tooth solubility.

Fran this we can conclude that the caries—resistant and caries-
susceptible strains of albino rats have fairly constant inorganic com~
position of tooth enamel with no correlation with suscegtibility or
resistance to decay of the teeth. This is in agreement with the results
of Armstrong et al (33). The same 05:? ratio indicates a homogeneous
inorganic phase.

The lower solubility, of the incisor enamel of albino rats is
hard to explain. It may merely be incidental to the need for structural
materiel thst is best suited for the special function of the incisors.
Perhaps pigmentation may be of some significance for it is well known
that the incisors of rats receiving an excessive amount of fluoride
show depigmentution and are easily broken. If solution in acid is a
prime factor in the initiation of caries, it will be easy to see the
basis of the statement of Hunt and Hoppert (44):

"In more than 6,400 rats we have never observed a curious lesion

in the incisors.‘

The euaael gouder of ugper and lower teeth teams to hgvu tne tame
comgosition. This is not in aéreement with the work of watsuda (45) who
investigated the congLition of whole incisors of rats and found a difforence
in comyuaitioa of quor cud lo a: tooth. The fact tumt cugmel and dentin.
were not segcrcted make; thuae result: less significant.

The increased rolability with decreasing pH may to eh lained on
the basis of a diijeront 'cte of rolnbility of aifferont anions. Yardeni
(£8). Thu fict, that rat enuhel and dentin Show about the some solubility
as the corrcagonding human tissue gregarations of Volker (46) gives more
significance to the greaeut work. Thus the solubility of enaael yowder
was relatively alight above pH 6.0 but increased with decreasing pH.

, to aboald leu combiner tau goasiolc role of genes influencing
the tooth form, which could bu correlmtuc to the auoceytibility to caries.

It has long been recognized that tooth form and thm shage of con-
tacts between teeth may influence the onset of Gental caries. In 1952 Nak—
foor, Hunt, Hoppert (47) measuring the reoistnnce of lower molar teeth to
mechanical fracturing, obaerved greatar mGChudiCul weakness in the susceptible
rats. They also observed (beta wita toe unaided eye and with a binocular
microscoge) taut tne crevices of tae molars of the suscegtible strain were
wider than taste of the resistlnt rats. tide crevices would permit im-
paction of food more readily waicu in turn would allow formation by bacteria,
and finally caries. As a matter of fact, 69.4 per cent of the caries in
susceytiblec has occurred ot the major crevices of the fir;t and second
lower molara, whereas in tho reaibtants only 15.7 yer cent of the cavities

appeared at thoae locations.

29

Nevin and Walsh (48) investigated the possible effects of certain
physico-chemicel factors in relationshiy to the cause of caries. They
obtained results which Can exglnin the differences in interproximal eur-
fecee of the same tooth and between interproximel surfaces in different
parts of the mouth. The indications are that variations in degree of
separation of teeth and the ennpe and width of contacts are more im-
portant than the buffering Capacity of saliva.

Cox (49) showed that rat molars exhibited Variations in

(a) subdivision of major cusge, (b) fusion of minor to mejor cusps,
(c) absence of minor cusps, (d) presence of extra cuepe, and (e) bi-
furcation of minor cusps. Some of these Variations are related, in
frequency, to the diet of the mother during gregnency and lactation;
others may be related to familial, and hereditary factors.

From the present study it beconee obvious thet further work
will be neceeeery to gain a full understending of the reletionehig of
tooth structure to sueceytibility to caries.

The exact path of every individual lesion may be different
and will depend on tho mhjor factors. One, the external, is determined
by the types of bacteria, acidogenic end yroteolytic, together with
the suggly of suitable substrates upon which they can act, and the con-
ditions of pltque, pit or fissure which bring them into contact with
tne enamel surface.

The second major factor is the moryhologicel and histo-
logical etete of the tooth itself, the condition of the surface and the

amount and distribution of orgnnic and inorgenic matter available for

attack at the surface and within the enamel itself.

In the background of the production of acid is the still
greater problen of clinical caries with systemic factors not yet
determined and bacteriul entugcnisms and synergisms undoubtedly playing
a major role in the comelex gicture. Yerdeni (25)

Although a genetic factor related to caries registence has been
clearly established in laboratory animals, only familial relations have

been demonstrated for men.

52

L "J silt}. 111

l. A study of the Loinbiiitj oi gendered tooth encnei
fFOm tne tecntyafifth generntion of Cariﬁﬂ-tUiCﬁytlblB end the
treatieth generation 01 corieeereoietunt Ltrein; of albino rets in
buffered soiutione of a pH of 4, 5, 8.5 and 6 Cid not show signi-
ficant differences between tde tee etrnins.

2. No difference in solnhility Was found between the
enhmel of the anger and lower molnre of the recistrnt strain.

3. The incisor BRLmCl yonder «he mcrhealy lees soluble
than the i“endeared eunhel of holers.

4. The soluble Cat? rntio was in all Cases eeeentielly
tho 3133830 0

5. The solubility incrceeed with decrcheing pH. The
increcee nus not pregortionhl to the H ion concentration, but was

much higher at the lower ph level.

BIBLIOGRAPHI

1.

5.

t.

5.

6.

7.

8.

9.

10.

11.

BIBLIOGRAPHY

Hunt. HOBO. HOppert, COAO, and EMn, W000, Inheritance 0f
susceptibility to caries in albino rats.,
J. Dental Research 253585 (1944)

Dullomagne, H.J., and Nolan, J., Density and refrective index
of bone end of tootn enamel, dentine and cement.
First results on the determination of tee chemical constituents
of the minerel portion of the teeth.
Enll. Soc. Chim. Biol. 27:85, 597 (1945)
State of combination of the carbonate of tooth enamel, dentine
and cement.
Compt. rend. Soc. biol. 1393804 (1945)

Miller, W.D., Caries of the human teeth.
Independent Fractioner 4:629 (1885)

Rodriguez, F.E., Studies in the specific bacteriology of dental
caries.
Military Dental J. 5:199 (1922)

McIntosh» J., and Lazaruaaﬁerlow, an investigetion into the etiology
of dental caries. I The nature of the destructive agent and the
production of artificial caries.

Brit. J. Exp. Path. 5:138 (1922)

Hartzell, T.B. and Larson, W.P., Preliminary retort of some experi--
ments on tne destruction of human enamel.
J. Am. Dental Aeeoc. 118649 (1924)

Mammary, S.P., Some experiments on the reletive suaceptibility of
different teeth to dental Caries.
Proc. Roy. Soc. ﬂed (Sec. Odont.) 3:71 (1910)

Bunting, H.W., Palmerlee, F., The role of the Becillne acidophilus
in dental caries.
J. Am. Dental Assoc. 121581 (1925)

Friesell, H.E., and Vogt, 0.0., A report concerning the etiology of
dental caries.
J. Am. Dental Assoc. 153748 (1926)

rhurlov 5., and Bunzell, H.H., Tooth decey studies. I On tne effect
of acids on tootn tissue.
Dental Cosmos 69:29 (1927)

McClellend, J.R., Decelcificetion of human tooth enamel.
Dentel Cosmos 683127 (1926)

12.

13.

14.

15.

16.

17.

18.

19.

20.

25.

24.

25.

55

Enright, J.J., Friesell, H.E., end Treechcr, M.U., Etunies of the
cause and nature of dental caries.
J. Dental Research 12:759 (1932)

Brekhus, P.J., and Armstrong, F.D., A method for tne e Heration of
enamel, dentin and cementum.
J. Dental Research 15:23 (1955)

Manly, R.S., and Hodge, H.C., Density and refractive index studies
of dental hard tissues.
J. Dental Research 18:153 (1959)

Benedict H.D., and Kanthnk, F.F., The solubility of dental enamel
in various buffered aolutions.
J. Dental Research 123277 (1952)

Volker, J.F., Studies on the acid solubility vf human enamel.
J. Dental Research 19:55 (1940)

Dallemagne, H.J., and Melon, J.
”Ch. biOlo Paris 57879 (1946)

Kerehen, ﬂ., and Rosebury, T., Relationship of pH, Calcium and
Phosphorus to solubility of enamel.
J. Dental Research 143220 (1954)

Forbes, J.C., Solubility of enamel.
J. Dental Research 15:549 (1953)

Volker, J.F., Effect of fluorine on solubility of enemel and
dentin.
Proc. SOC. EXP. B1010 Med 428725 (1939)

Buonocore, ﬁ.G., and Bibby, 8.0., The effects of various ions
on enamel solubility.
J. Dental Research 24:105 (1945)

Manly, 3.6., and Bibby, 9.6., Substances capable of decreasing
the acid solubility at tootn enamel.
J. Dental Research 28:160 (1949)

MCClure, FoJ., can Ruzicka, S.J., Destructive effect of citrate
versus lactate ions on rat molar tootn surfaces, in vivo.
J. Dental Research 25:1 (1946)

Brudevold, F., A study of the yhoeyhete solubility of the humen
enamel surface.
J. Dental Research 27:520 (1948)

Iardeni, J., The weight loeees of intact teeth in oifrerent buffers
at varying pH values.
J. Dental Research 293278 (1950)

26.

27.

28.

50.

51.

52.

55.

55.

56.

57.

58.

59.

56

Iardeni, J., Further experiments with intact teetn placed in various
buffer solutions.
J. Dental Research 51:595 (1952)

Armstrong, E.B., Proc. Soc. Exp. Biol. ﬂed 44:28 (1940)

Swertz, M.L. and Philliys, B.W., Solubility of enamel on areas
of known hardness.
J. Dental Reeeerch 513295 (1952)

Suess, P.H., and Fosdick, L.S., The solubility of normal and
fluoridized ensmel.
J. Dental Research 50:177 (1951)

Clark, E.P., and Collip, J.B., A study of the Tisdnll method for
the determination of blood serum On with a suggested monification.
J. 8101. Chen. 653461 (1952)

Briggs, A.P., A modification of the Bell—Doisy yhosohste method.
J. 3101. Chem. 55315 (1922)

Rosehury, T., Biochemical study of tee protein in dental enamel.
J. Dental Research 103187 (1950)

Committee on Dental Health. A survey of the literature of dental
caries. (1952)

Gilda, J.E., Studies on gnysicel progerties of radiant enamel,
I The Manly-Hodge separation method as applied to the teeth of
the rat and hamster.
J. Dental Research 50:445 (1951)

Gilda, J.E., Studies on physicsl proserties of rodent enamel,
II Density distribution and X-Ray diffraction pattern.
J. Dental Research 50:828 (1951)

Thewlis, J., Clock, G.E., and Murray, m.n., Trans Faraday Soc.
35:358 (1959)

Sobe1, A.E., and Hanok, A., Calcification of teeth. I C0mgoaition
in relation to blood and diet.
J. Biol. Chem 176:1105 (1948)

SObel, AOEO’ CQlCifiChtion of teeth.
J. Dental Research 28:81 (1949)

Armstrong, W.D., Brekhus, P.J., Chemical constitution of enamel and
dentin. I Principal components.
J. Biol. Chem. 120x677 (1957)

40.

41.

43.

44.

45.

46.

47.

48.

49.

57

Korehner, H., Sobel, A.E., Henok, A., I Division or 5101051051
Chemnniry, American Cnenicel bociety, 114th meeting, teshington,
Aug. 50 - sept. 5 (1948)

Deckins, M. and Burt, R., The OGgOtithﬂ of Ca, P and Carbon dioxide
in calcifying dental enamel.
J. 3101. Chem. 56:77 (1944)

Greenberﬁ, D.M., Studies in mincrul metabolism with the aid of
artificial rnaiorctive isotoges. vxxl Tracer experiment: with
radioactive Ca and Er on tne mecnnniem of vitamin D action in
rachitic rats.

J. Biol. Chem. 157.99 (1945)

Armstrong, K.D., and Barnum, C.P., Concurrent use of radioactive
isotopes of Ca and P in the stuny of the metcboliem of calcified
tissues. .

J. B1010 Chem. 1723199 (1948)

Hunt, H.R., Hoypert, C.A., The distribution of carious cavities in
the lower molars of ceriee~susceptible and caries-resistant albino
ratBo
J. Dental Research 293153 (1950)

Metcudu, I.A., Biothemicel study of tooth growth.
J. Biol. Chem. 71:45? (1927(

Voleer, J.F., Proc. Soc. E13. Biol. and ﬁed. 45:645 (1940)

Nekfoor, E.C., hunt, H.R., Hoppert, C.A., Fracturing of the molar
teeth in caries-succegtible and ceries—reeintent albino rats.
J. Dental Research 51:145 (1952)

NeVin, BhB., Welsh, J.P., Some ghyeico-chenieel factors in relation
to the causation of intergroximel caries.
J. Dental research 50x255 (1955)

Cox, G.J., Methane, J., Wirth, L.J., A method of study of molar
form in the rat.
J. Dental Research 313466 (1952

Michigan State College
East Lancing, Michigan