Loyola University ChicagoLoyola eCommons

Master's Theses Theses and Dissertations

1969

The Effect of Cortical Steroids on the Rate ofEruption of the First Maxillary Molar in the AlbinoRatStephanie Jean ZayachekLoyola University Chicago

This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion inMaster's Theses by an authorized administrator of Loyola eCommons. For more information, please contact ecommons@luc.edu.Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.Copyright © 1969 Stephanie Jean Zayachek

Recommended CitationZayachek, Stephanie Jean, "The Effect of Cortical Steroids on the Rate of Eruption of the First Maxillary Molar in the Albino Rat"(1969). Master's Theses. Paper 2352.http://ecommons.luc.edu/luc_theses/2352

THE EFFECT OF CORTICAL STEROIDS ON THE RATE

OF ERUPTION OF THE FIRST MAXILLARY MOLAR

IN THE ALBINO RAT

by

Stephanie Jeazi Zayachek

A Thesis Submitted to the Faculty of the Graduate School

of Loyola University in Partial Fulfillment of

the Requirements for the Degree of

Master of Science

JWle

1969

Pbflf')' ··Loyola Unlvorsity Medical Center

LIFE

Stephanie Jean Zayachek was born in Granville, New York, on

January 23, 1942.

After graduating from Granville Central High School in

June, 1959, she attended the State University College of Educa­

tion at Albany, New York, and The Catholic University of America

in Washington, n.c., from which institution she received the

Bachelor of Arts degree 1n absentia in October, 1963. In Sep­

tember, 1963, she began graduate work at the University of

Chicago, Chicago, Illinois, as a United States Public Health

Service Fellow in Anatomy. In September, 1965, she transferred

to Loyola University Stritch School of Medicine, Chica.go, where

she continued her graduate studies in Anatomy.

During the years 1965-1968, she held a Loyola University

Graduate Student Teaching Assistantship and during the year

1968-1969, a Research Assistantship in the Department of

Anatomy.

ii

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to my advisor,

Dr. Lincoln v. Domm, Professor of Anatomy, Emeritus, Department

of Anatomy, for his suggestion of the problem, constructive

cotmsel and criticism, and enthusiastic support throughout this

investigation~

I am also grateful to Dr. Leslie A. Emmert, Assistant

Professor of Anatomy, for his tmfailing help, particularly with

respect to some of the histological phases of the investigation.

I am especially grateful to Dr. Joseph Gowgeil of the •

Loyola University School of Dentis~ry for his invaluable and

helpful criticism, assistance, and encouragement.

Sincere appreciation is extended to Mrs. Lucia Smelte,

Senior Technician in the Department of Anatomy, for her assis­

tance with the histological aspects of the project.

Appreciation is also extended to Merck, Sharp and Dohme,

Division of Merck and Company, Inc., ·Philadelphia, Pa., for the

cortisone and hydrocortisone generously supplied to my advisor,

Dr. Domm, for use in this investigation.

The study was supported in part by USPHS Research Grant

DE-00694, and a USPHS, NIH, General Research Support Grant.

Grants administered by Professor Lincoln v. Domm.

iii

TABLE OF CONTENTS

Page

·- I INTRODUCTION •••••• •• •••• .- ••• •.•• •••• ••• o ••••• • •••• l

II REVIEW OF LITERATURE

A. Theories of Eruption•••••••••••••••o••••o••3

B. Hormones and Eruption•••••o••••••••••o•••••9

III MATERIALS AND METHODS••••••••••••••••o•••••••••••l3

IV EXPERIMENTAL RESULTS

A. Gross Observations •••••••••••••••••••••••• 17 • B. Radiological Observations ••••.••• o ••••••••• 18

c. Histological Observations.•0••••••••••••••20

V DISCUSSION

A. Cortisone and Molar Eruption •••••••••••••• 26

B. Cortisone and the Cellular Proliferation

Theocy ••••••••••••••• ,• •••••••••••••••••••• 29

C. Cortisone and the Vascular Theory ••••••••• 31

D. Cortisone and the Connective Tissue

Theory ..................................... 31

E. Cortisone and Other Hormones •••••••••••••• 32

F. Cortisone and Body Weight ••••••••••••••••• 38

a. X-ray as an Experimental Procedure •••••••• 43

iv

'

VI

VII

VIII

IX

Page

H. True or Apparent Precocious Eruption ••••••• ~3

I. Cortisone and Gingival Keratinization •••••• 44

J. Cortisone and Hair Growth •••••••••••••••••• 45

K. Cortisone and Bone·Growth •••••••••••••••••• 46

L. Histological ·observations •••••••••••••••••• 48,

SUMMARY AND CONCLUSIONS ••••••••••••••••••••••••••• 52

LITERATURE CITED.••• ••••• ••.•.•••·• o • • • •••• • ••••••• 54

TABLES••••••••••••••••••••••••••••••••••••••••••••65

FIGURES AND PLATES .••• • •• • ••••• • •••••••••••• o •••••• 71

v

Table

1.

2.

4.

6.

LIST OF TABLES

Page

BODY WEIGHT CHANGES IN RATS INJECTED BETWEEN DAYS 11AND16 ••••••• 00•••••0••••••••••••••••••••••0•065

ERUPTION RATES OF THE FIRST MAXILLARY MOLAR IN RATS INJECTED BETWEEN DAYS 11AND16••••0••••••••66

ERUPTION RATES OF THE FIRST MANDIBULAR MOLAR IN RATS INJECTED BETWEEN DAYS 11AND16•••••000•••••67

HISTOLOGICAL MEASUREMENTS OF THE FIRST MAXILLARY MOLAR REG ION ••••••••••••••••••••••••••••••••••••• 68

MEASUREMENTS SHOWING GROWTH OF ALVEOLAR BONE IN RATS INJECTED BETWEEN DAYS 11AND16••••••••0••••69

X-RAY AS AN EXPERIMENTAL PRoCEDURE, A STATISTICAL ANALYSIS • •••••••••••••• : ••••••••••••••••••••••••• 70

vi

LIST OF FIGURES

Figure Page

1. A PHOTOGRAPH SHOWING THE CEPHALOSTAT AND INDUSTRIAL X-RAY UNIT USED IN THE EXPERIMENTS ON THE FIRST MAXILLARY MOLAR.•••••••••••••••••••••••••••••••••••71

2. A PHOTOGRAPH SHOWING PRECOCIOUS ERUPTION OF THE FIRST MAXILLARY MOLAR IN A 0.5 MG CORTISONE­TREATED RAT AND NORMAL ERUPTION IN A CONTROL RAT (AGE: 16 DAYS)•••••••••••••••••••••••••••••••••72

3. A PHOTOMICROGRAPH OF THE FIRST MAXILLARY MOLAR OF A CONTROL RAT SHOWING PERIAPICAL AND DENTAL TISSUES x l00 •••••••••••••••••••••••••••••••••••••••••••••• 73

4. A PHOTOMICROGRAPH OF THE FIRST MAXILLARY MOLAR OF A CONTROL RAT SHOWING PERIAPICAL AND DENTAL TISSUES x 100 ••••••••••••••••••••••••••••••••••• 0 ••••••• 0 •• 73

5. A GRAPH SHOWING THE EFF;;CT OF DIFFERENT DOSAGES OF CORTISONE AND HYDROCORTISONE ON THE TOTAL ERUPTION DURING A FIVE DAY PERIOD OF THE FIRST MAXILLARY MOLAR (AGE: 16 DAYS)••••••••••••••••••••••••••••••74

6. A GRAPH SHOWING THE EFFECT OF DIFFERENT DOSAGES OF CORTISONE AND HYDROCORTISONE ON BODY WEIGHT GAIN DURING A FIVE DAY PERIOD (AGE: 16 DAYS) •••••••••••• 75

7. A ROEN'J;'GENOGRAM OF A RAT HEAD SHOWING THE LINES SUPERIMPOSED TO ILLUSTRATE THE METHOD OF BONE GROWTH AND MOLAR ERUPTION RATE MEASUREMENTS X 4 •••• 76

8. A PHOTOMICROGRAPH OF THE FIRST MAXILLARY MOLAR OF A CONTROL RAT SHOWING PERIAPICAL AND DENTAL TISSUES x 100 •••••••••••••••••••••••••••••• 0 ••••••••• 0 ••••• 77

9. A PHOTOMICROGRAPH OF THE FIRST MAXILLARY MOLAR OF A 1.0 MG CORTISONE-TREATED RAT SHOWING PERIAPICAL AND DENTAL TISSUES X 100•••••••••••••••••••••••••••••••77

10. A PHOTOMICROGRAPH OF THE FIRST MAXILLARY MOLAR OF A CONTROL RAT SHOWING NORMAL ERUPTION (AGE: 16 DAYS) X 100•o•••••••••••••••••••••••••••a••••••••••••••••78

11. A PHOTOMICROGRAPH OF THE FIRST MAXILLARY MOLAR OF AN EXPERIMENTAL RAT (l.O MG CORTISONE INJECTED) SHOWING APPARE~""T PRECOCIOUS ERUPTION (AGE: 16 DAYS) x 100 ••••••••••• 0 ••••• ••••••••• 0 0. 0 ••••••• ••••••••• 78

vii

ABSTRACT

This study is concerned with the effect of adrenal cortical

steroids on the eruption of the first maxillary molar of the

albino rat. The rate of eruption was determined by means of

measurements on roentgenogram exposures, and histological

observations were made on hematoxylin and eosin stained sec­

tions or the molar area.

Tbe treated rats received varying dosages (O.l to 2.0 mg)

or either cortisone {cortone acetate) or hydrocortisone (hydro­

cortone acetate). Litter mate controls received eqUivalent

volumes or saline. Five consecutive daily injections were

given beginning on day 11. The rats were exsangUinated by

d~cap1tat1on on day 16, and the heads immediately placed in

cold 80% alcohol for subsequent gross and histological ex~~­

ination. Radiological measurements were made from exposures

taken on days 11 and 16 in order to measure the rate of eruption

and bone growth.

The cortical steroids administered elicited a gradient

decrease in weight gain proportional to dosage and brought .

about a decrease in growth of body hair. Upon gross examin­

ation, precocious eruption of the first maxillary molar ap­

peared to have occurred in all steroid treated animals.

Histological exa~ination of hematoxylin and eosin stained

sections of molar regions of high (l.Omg) and low (O.lmg) viii

L

ix dosage animals showed no effect on dental tissues. Bone

resorption was extensive at the 1.0 mg dosage. Depletion of

loose connective tissue was seen in all the steroid treated

animals, as well as marked hypoplasia of bone marrow cells

with evidence of tat storage in the marrow and adjacent to

muscle.

Radiological measurements revealed no significant bone

growth in the 0.1 mg cortisone treated rats while in those

receiving 1.0 mg there was significant resorption at the molar

alveolar crest.

In contrast to the initial gross observations, radio­

logical measurements showed that the eruption rate of the

first maxillary molar was accelerated only at the lower dos­

ages of cortisone and hydrocortisone. The effect was in­

versely proportional to dosage; accelerated 39 and 32% res­

pectively at 0.1 mg; 25 and 20% at 0.5 mg; and retarded at the

l.O, 1.5 and 2.0 mg dosages.

I INTRODUCTION

Tomes ( 114), in discussing the evolution of teeth, states

that the various types of teeth seen in vertebrates were prob­

ably derived from a common form, a mono-cusp, mono-rooted, con­

ical form which, in the course of time, became modified through

natural selection. A living example of this type of tooth is

seen in the ungUlate, Anoplotherium. According to Tomes,

Gervais (1854) postUlated that molars, as we know them today,

evolved from conical teeth which fused as the jaw shortened in

length. Tomes further cites Virchqw as having observed a molar

made up of separate conical denticles each with a root of its

o\'m. Virchow viewed this as a case of atavism. Ness ( 1 56)

concluded that eruption of the different types of vertebrate

teeth may be considered to be fundamentally the same.

This paper reports the resUlts of an investigation on the

effects of cortical steroids on the first maxillary molar of

the rat. It has been shown by a number of investigators that

cortisone accelerates the rate of eruption of the continuously

erupting rat incisor (Parmer et al., 1 51; Domm and Marzano, 1 54;

Goldsmith and Ross, 1 54; Domm and Leroy, 1 55; Leroy and Domm,

'55; Domm and Wellband, 1 60; Garren and Greep, 160). Domrn

and Wellband ( 1 60, 1 61) reported that cortisone elicits this

reaction not only in normal rats but also in adrenalectomized

1

f

2

and thyroidectomized animals. However, these studies did not

explain the actual mechanism of incisor eruption or how cor­

tisone acts to produce accelerated eruption.

Eruption of the rat molar is believed to be continuous

throughout the life of the rat, but is so slow as to be Wlde­

cernable (Schour and Massler, 1 49). We were interested in

determining, if possible, whether the mechanism operative in

the limited eruption of the molars is similar to that in the

continuously erupting incisors. In order to shed light on this

problem, we studied the effects of cortical steroids on the

first maxillary molar of the rat, Specifically, we were inter­

ested in detennining any change in rate of eruption or histo­

logically apparent modification of growth following injection

of these steroids •. If the molars, under the influence of

cortical steroids also reveal accelerated eruption, it would

indicate that this effect is common to the teeth of the rat and

would seem to lend support to the theory that both types had a

common origin. Conversely, if cortisone does not accelerate

eruption, it can be assumed either, that the mechanism of

eruption involved in the two types of teeth is different, or

that cortical steroids are not effective on molars at the

dosages administered.

II REVIEW OF LITERATURE

A. Theories· of Eruption

Constant (1900) theorized that tooth eruption is caused by

blood pressure "pushing" the tooth into the oral cavity. A

parallel to this theory was proposed by Addison and Appleton

('15) who attributed tooth movement to the pressure exerted by

dividing cells at the basal or proliferative zone of the tooth.

The blood pressure theory was supported by Schour and Van

Dyke ( 1 32a} who hypophysectomized rats and noted that the rate

of eruption was more seriously affected than the growth of

dental tissues. These investigators suggested that the vas­

cular disturbances involved in hypophysectomy may be a primary

factor in the observed decrease in eruption rate. Massler and

Schour ( 1 41), in an evaluation of eruption theories, supported

this proposal and suggested that other factors may also be

involved. Bryer ( 1 57) concluded that the tooth movement,

inherent in eruption, is the direct result of hydrostatic

tissue pressure in the pUlp and periodontal membrane.

Sieber ( 1 42a, 1 42b) described a "cushioned hammock lig­

ament" slung beneath the root of the tooth against which the

proliferating cells push, thus forcing the tooth occlusally.

Scott ( 153) also observed this structure and attributed to it

the role proposed by Sicher. Baume et al. ( 1 54a, '54b, 1 54c)

3

4

reported a decrease in vascuJ.arity correlated with a retar­

dation in rate of eruption of incisors in rats following hypo­

physectomy and thyroidectomy. These investigators concluded,

however, that eruption is the direct result of dental and

periodontal tissue growth. Domm and Kiely ( 1 68) correlated

increase in the number of colchicine-arrested cells in the

basal proliferative loop of the rat incisor with acceleration

in eruption rate following the administration of cortisone.

Taylor and Butcher (.• 51) cut the inferior dental nerve

in a sympathectomized rat and showed that the incisor eruption

rate is not sensitive to even large changes in blood flow.

Miller ( 1 57) changed blood flow in ·the dental area of the rat

and rabbit by carotid ligation or sympathectomy and observed

no correlation between blood flow and eruption rate. Stunnan

('57) injected the vasodilator drug, Priscoline, and the

vasoconstrictor drug, Levophed, and observed fluctuations in the

eruption rate of the rat incisor. However, he questioned

this effect and described an "excitability factor", needle

injections, which produce·d similar resuJ.ts. The vasoconstrictor

maintained eruption at the normal rate for one week and then

accelerated it. From this, Stunnan concluded that the actual

eruptive force, though locally affected by blood pressure, is

the manifestation of mitotic activity in the odontogenic epi­

thelium and puJ.p cells. Main ( 1 61) and Main and Adams ( 1 62,

166) produced a hypotensive state in rats with hydralazine and

guanethidine and noted no modification in incisor eruption

rate.

5

The persistence of eruption following the removal of both

the pulp and Hertwig's sheath {odontogenic epithelium) demon­

strated that cell proliferation at these loci is a process

separate from that of the eruptive movement {Herzberg and

Schour, 1 41; Bryer, 1 57; Kostlan et al., 160). Main {165)

disavowed the existence of a functional hammock-like ligament

as the result of an extensive histological examination of

actively-erupting teeth in rats, dogs, cats, sheep, pigs,

calves, guinea pigs, rabbits and mice. Main and Adams {166)

repeated their 1962 experiments with hypotensive drugs and

did a parallel experiment with the nucleotoxic drugs, deme­

colcine and triethylene melamine {TEM). They measured the

effect of these drugs on the growth of incisors cut out of

occlusion, thereby removing the variable of attrition. They

found no fluctuation ~n the eruption rate of the treated

when compared with that of untreated rats. Hence, they con­

cluded that, neither blood pressure nor cellular proliferation

is the source of the eruptive force.

The only proposed concept left to explore is that of the

collagen fibers of the periodontal membrane, which by their

contraction are believed by some investigators to pull the

tooth into the oral cavity. The first to suggest that eruption

may be due to factors inherent in the periapical tissues was

6

Underwood cited by Tomes ( 1 14). Verzar ( 1 63) noted that nor­

mal collagen under tension will contract as it ages. The

nature of this contraction is due to the accumulation of inter­

molecular cross-linkages. These cross-linkages are believed

to consist of hydrogen bonding, covalent bonding, and (pos­

sibly) ester linkages. It is thought that the hexose molecules

gained from the mucopolysaccharide ground substance are the

source of this bonding. Levene and Gross ( 1 59) and Van den

Hooff et al. ( 1 59) noted that the drug, alpha-aminopropio­

nitrile, interfered with the nonnal {cross-linking) maturation

process or collagen. Martin et p.i. ( 1 62) produced lathyritic

rats (a condition in which the teeth do not erupt) by injecting

this drug. From the results or this study on collagen ext­

raction they came to the conclusion that these rats showed

a defect in the cross-linking or collagen, specifically a

failure in chain-pair linking. They therefore concluded that

the collagen or the periodontal memb~ane nonnally contracts

and, in this manner, produces occlusal tooth movement. Thomas

( 164, 165, 167) demonstrated this phenomenon more completely.

In the 1965 experiment he showed that the collagen cross­

linkage breaking drug, beta-aminoaceton1tr1le, retarded molar

eruption and slowed the eruption rate or' the con-tinuously

erupting incisor. He showed that while dentin, bone, and

vasculari ty or the pulp and periodontium were nonnal, the

collagen fibers of the periodontal membrane were palisaded

p

7

rather than obliquely oriented. Eruptio~ in normal rats,

said Thomas, is initiated by growth of the tooth genn away

from the oral cavity. Fibroblasts orient themselves along

the lines or stress {Weiss, 1 59), set up by this growth. The

collagen fibers are consequently laid down in a functional

manner between the cementum and alveolar bone. Thomas main­

tained that as the root is established, its growth away from

the oral cavity causes a stretching of the collagen fibers

leading to an increase in cross-linkages, subsequent con­

traction of the fibers, and consequent tooth movement. For

more information on the nature ~ collagen see Verzar {1 63),

Piez et al. ( 1 61), and Bear ( 1 44).

' For the above phenomenon to occur, the connective tissue

fibers would have to connect the alveolar bone to the tooth

and the points of tooth connection would have to move with the

erupting tooth. This, in fact, they do. Melcher {1 67) made

a lesion with a slow-running burr {size 6) in the mandibular

incisor or the adult rat. This lesion included the alveolar

bone, periodontal membrarie, cementum, and dentin. After four

days or healing, histological examination showed that the

intact periodontal fibers and the adjacent cementum moved into

the area of the lesion marked by the absence or intact bone.

This would not have been possible had the fibers not been

carried there by the erupting tooth. Ramos and Hunt ( 1 67),

using tritiated proline, demonstrated a similar movement ,.

8 •

of the periodontal fibers in the continuously erupting guinea

pig molar.

It is of interest here to note.the relevance of the

discussion on eruption theories by Massler and Schour ( 1 41).

They noted a consistent correlation between increase in vas­

culari ty and positive tooth eruption. For instance, they

observed that when a denture irritates a submerged tooth, or if

the tooth is rubbed, it will erupt; they observed increased

hyperemia in such conditions and correlated this with the

increase in rate of eruption. This data could be applied in

the light of the infonnation on.connective tissue contraction.

Such downward pressure from a denture could stimulate the

fibroblastic and collagen fiber orientation and place the

fibers under tension. With subsequent maturation (increase in

cross-linkages), the teeth would erupt. With this increased

movement, chain reaction events could occur, such as dental

tissue growth, increased vascularity of the physiologically

active tissue and fibroblastic replacement of cells and per­

iodontal fibers. Biting ·forces in increased attrition would

stimulate the process. In incisors cut out of occlusion, the

.incisor eruption rate is increased above nonnal and the enamel

and dentin are decreased in amount (Noe, 1 02; Addison and

Appleton, '15; Dalldorf and Zall, 1 30; Hunter and Sawin, 1 42;

Schour and Medak, 1 51; Taylor and Butcher, '51; Ness, 1 56;

Main, 1 65; Ness, 1 67). Under similar conditions the molar

....

9

eruption rate is also increased (Sognnaes, 1 41). In such a

tooth the accelerated rate of eruption would reflect the rate

of collagen maturation and cell proliferation, and the

decrease in amounts of enamel and dentin (hard substance

apposition) would indicate that fonnation of these substances

would have to keep pace with the accelerated eruption.

B. Honnones ~ Eruption

Hoskins ( 1 27) reported that as little as 0.1 mg of

acetyl thyroxine stimulated precocious eruption of incisors in

newborn rats. Growth of the nasal bone, otherwise normal,

was accelerated. No observable effect was apparent in the

molars. A decrease in the eruption rate of the incisors in

hypophysectomized rats was reported by Schour and Van Dyke

( 1 31). The incisors, two-thirds the size of controls, showed

degeneration of Hertw1g 1 s epithelial sheath (odontogenic

epitheliwn), the periodontal membrane and the labial alveolar

periosteum. Examination of the dentin and enamel revealed

foldings at the basal end of the tooth and a thickening of

cementum. A decrease in calcification of the dentin was

evident. No changes were seen in the molars which had Just

come into occlusal contact at the start of the experiment. In

1932, Schour and Van Dyke repeated this experiment, confinning

their earlier findings, and attempted replacement therapy with

hypophyseal extract. They reported an acceleration in rate of

eruption and a restoration of a more nonnal histological picture.

They noted that hypophysectomy had a

10

more pronoW1ced effect on rate of eruption than on growth rate

of the dental tissues and attributed this to vascular distur­

bances (Schour and Van Dyke, 1 32a). That same year they also

reported the effect of growth honnone on the dentition of

hypophysectomized rats. This hormone, in addition to re­

placing lost body weight, also accelerated the depressed

eruption rate {Schour and Van Dyke, 1 32b). Their work was

confinned by Collins et: al. ( 1 49). Schour and Rogoff ( 1 36)

reported the additional observation that growth honnone did

not accelerate the eruption of incisors in nonnal rats. They,

in turn, adrenalectomized rats and observed, in addition to

disturbed dentin calcification and degeneration of cells, a

decrease in incisor eruption rate. They concluded that the

causative factor was the effect of the adrenal on calcification.

Pnnner ( 1 47~ hot~d that when DOCA (desoxycorticosterone)

was administered to thiouracil-treated (thyroxin-inhibited)

animals, the impaired incisor eruption rate and delayed eye­

opening returned to nonnal. However, this honnone did not

restore the thyroid to normal, suggesting that the process of

eruption is not necessarily mediated through this gland.

Cortisone, found to be the most potent growth inhibitor and

the most potent stimulus for eruption of incisors, eye~opening

and gingival development, was compared in its effect on new­

born rats with ACTH, aqueous adrenal extract, corticosterone,

desoxycorticosterone, and pregnenolone (Panner et al., 1 51).

PJ': _______________ ___, 11

These investigators concluded that the mode or activity of

cortisone was probably quite specific since pregnenolone, which

1s of a similar configuration, had no effect. Domm and Marzano

(•54) observed that the incisors of newborn rats treated with

cortisone erupted three days earlier than those or the controls.

Growth honnone had little or no effect on the eruption rate of

the incisors in nonnal rats. Administered in hypophysectomized

rats, cortisone brought about a complete recovery of the incisor

eruption rate. Garren ( 1 55), in studies on rats, confinned the

work of Domm and Marzano. Goldsmith and Ross ( 1 54), also

observed a precocity in the rat~ of incisor eruption in cor­

tisone-treated nonnal rats. They reported a stimulation of

· gingival keratinization, osteodentin fonnation, amelogenesis

af:ld odontogenesis. However, growth of' the periodontal con­

nective tissue appeared to be depressed. Domm and Leroy ('55)

and Leroy and Domm ('55) administered cortisone to pregnant

rats and to rat fetuses in utero. In both instances they ' ----

observed a precocious eruption or the incisors in the neonatal

rats. Goldsmith and Ross ( 156) administered different dosages

of cortisone to pregnant rats and observed accelerated incisor

development in the 18 and 20 day old fetuses. Cortisone treat­

ment in newborn rats elicited a 2.9 day precocity in incisor

eruption. In addition to an increase in alkaline phosphatase

and RNA content or the tooth bud cells and odontoblasts, a dis­

organization of the periodontal membrane and alveolar bone was

~-· -------------------------,

L

12

seen. These effects of cortisone administration were believed

to be responsible for the early eruption observed. Domm and

Wellband ( 1 60) observed a decrease in the incisor eruption

rate in adrenalectomized rats. The administration of cortisone

brought about an acceleration in the rate or eruption of 21%

above nonnal. In 1961, these investigators, in their studies

on adrenalectomized, thyroidectomized and thyro-adrenalectomized

rats, reported that the thyoid gland was also involved in

stimulating eruption. Garren and Greep ( 1 60) reported that

cortisone had the effect of nonnalizing the rate of incisor

eruption in adrenalectomized as well as in hypophysectomized •

rats. Wellband and Domm ( 1 64) reported an experiment showing

that cortisone accelerates the rate of eruption in incisors

cut out of occlusion. Domm and Kiely ( 1 68), by application

of the colchicine metaphase arrest technique, reported an inc­

rease in the number of mitoses in the basal or proliferative

zone of the rat incisor following cortisone administration. On

the basis of their observations, they concluded that mitotic

activity was probably one of the factors responsible for the

acceleration in the rate of eruption observed in cortisone- .

treated rats.

~-----------------~ III MATERIALS AND METHODS

Pregnant, multiparous, albino rats of the Sprague-Dawleyl

strain, in about the fifteenth day of pregnancy, were purchased

commercially. They were housed in separate cages and main­

tained on Rockland Chow pellets and tap water ad libitum. On

the s1xteenth day or gestation, the wire mesh floors of the

cages were removed and the rats placed into the attached pans

and provided with appropriate nesting materials. This measure

was taken so that the newborn would not be subject to injury

from contact with the coarse wtre mesh floors. Except for

removal of dead newborn, all young remained untouched for four

days after delivery. This precaution insured maximum survival

of the young since it minimized the danger of cannibalism.

The newborn were weighed daily, beginning on day four, at

which time each animal was marked with picric acid for ident­

fication. Their' condition, date of incisor appearance and date

of eye opening were recorded.

Injections were begun on day 11 and continued to the day

of sacrifice, day 16. All animals were injected intraperiton­

eally with either 0.1, 0.5, 0.75, 1.0, 1.5 or 2.0 mg of cor­

tisone or 0.1, 0.5, 0.75, 1.0, 1.5 or 2.0 mg of hydrocortisone.2 !Purchased from the Honnone Assay Laboratories, Inc., Chicago,

Illinois. 2The cortisone (cortone acetate) and bydrocortisone~(hydro­cortone acetate) were generously supplied to Dr. L.v. Dornm by Sharpe and Dohrne, Division of the Merck and Company, Philadel-phia, Pa. 13

~ -------------~--------------------------------------------------.

L

Litter mates served as controls and were administered equiv­

a.J..ent volumes of saline to simulate the trauma or injection.

14

A second series or controls withou~ injec~ion or X-ray was also

maintained.

The degree of molar eruption was detennined by means or

measurements on roentgenogram exposures. At the completion or

experiments, the animals were exsanguinated by decapitation. -

The heads were immediately placed in cold 80% alcohol for not

less than 24 hours. Subsequently, gross observations on the

molar areas were recorded.

All animals were anaesthetized before each or two X-ray

exposures with 32.5 mg/kg of nembutal, injected intraperit­

oneally. A cephalostat was designed and constructed to hold

·the head in a fixed position during exposure. It was found

that placing the ear posts of this instrument into the ears

was unsatisfactory since the ears of the newborn rat are very

flexible and movable over the bony skull. A more suitable

method for head stabilization was to place the ear posts in

the temporal rossaeo

Kodak ultra-speed, fine emulsion, dental occlusal film

was placed in:a fixed cassette in the cephalostat beneath the

animal's head. Lead foil letters were placed on the cassette

for identification of exposed film. Roentgenograrns were taken

in a Universal V-U X-ray machine at 3 M.A., 60 K.V., for an

exposure time of one second at a distance of 16 inches. The

,.,-:~·'------------~~

L

15

films were placed in standard X-ray developer (D-19) for 3k minutes, rinsed in clean water for seven seconds, then placed

in standard fixer for 10 minutes and washed in clean running

water for another 20 minutes. The films were processed at

73° F. during each step.

The measurements were made on roentgenograms that had been

enlarged about 4 x. The distance between the most mesial

(rostral) cusp of the first maxillary molar and its alveolar

crest was measured. The difference between the day 11 meas­

urement and that of day 16 was calculated as the distance the

tooth had erupted into the oral cavity. By this method, the •

effect of the steroid on eruption can be compared with that of

the control.

To insure that the data obtained from measurements repre­

sent actual eruption and not merely bone resorption, the fol­

lowing additional measurements were made on the roentgenograms

(fig. 7). A line was drawn connecting the lingual alveolar

crest of the maxillary incisor {I) with the molar alveolar

crest {M). A line perpendicular to this line was constructed

connecting the midpoint of the intercrestal line to the sup­

erior aspect of the nasal bone {N). The perpendicular line,

representative of the extent of bone growth, was then measured.

Values on experimental and control animals were compared. To

determine the possible physiological effect of X-ray as a

variable, the weights of the X-rayed and non-X-rayed animals

~~/---------------,

L

16

-were compared and the standard error of difference (s.eod.)

test or significance applied. These weights were then tested

against those of a second control group.

In preparation for histological examination, specimens of

the entire molar region were cut and placed in a sodium citrate­

formic acid decalcifying agent. Complete decalcification was

detennined by the negative oxalate test (Humason, 1 62). The

specimens were then dehydra~ed and double-imbedded in celloi­

din benzoate and paraffin. The blocks were sectioned at 8µ,

stained with Harris hematoxylin and eosin Y. In order to

compare growth effects, a series of measurements were made on •

histological sections by means of an ocular micrometero The

measurements included connective. tissue thickness, the distance ,

between the most superior portion of the alveolar bone and the

dentino-enamel junction, trabecular thickness, height of the

Haversian space and diameter of the major pulpal vessels. The

measurements were expressed in high and low power units.

~-----) ---------. "IV EXPERIMENTAL RESULTS

True precocious eruption in this study is defined as an

earlier than normal appearance in the oral cavity of any

part or the molar crown before that of' a control animal.

our initial gross observations on the effects of cortical

steroids in the molar area led us to the conclusion that

eruption of the first maxillary molar had been accelerated

in the l.O, 1.5 and 2.0 mg injected animalso However, radio­

logical measurements showed that its eruption was, in fact,

retarded. The gross appearance of the molar area was •

probably due to shrinkage or a thinning of the gingivae so

that the molar cusps appeared precociously (figs. 10, 11).

A. Gross Observations

In our study on the effects of cortical steroids on the

molar eruption rate, we observed precocious eruption of' the

molars (fig. 2) early eye-opening, a decrease in hair growth

and a decrease in weight gain and body size.

These effects, with the exception of hair growth, varied

directly with increase in dosage administered. After five

days of cortisone treatment, the 16 day-old rats appeared

to have somewhat less than a normal growth or hair when

compared with their litter mate controls. The decrease in

body weight tended to increase with an increase in dosage

17

~-/ ______ ' ___,

L

i8 (fig. 6, table 1). This decrease in weight, following five

days or cortisone injection, went from 4% at the 0.1 mg dos­

age to 66% at 1.0 mg and 83% at 2.0 mg. Hydrocortisone was

round to have a more severe effect on body weight. Following

five days of injection with this steroid, total weight loss

went from 31% at O.l mg to 93% at 1.0 mg and 88% at 2.0 mg

when compared with the weight or controls.

B. Radiological Observations

The validity or the use of X-ray as an experimental

tool was determined by a comparison of body weight gain of

the rats during this experimen~. By the absence of stat­

istical significance in the groups compared, it was demon­

strated that X-ray did not introduce a variable into the

experiments (table 6).

The mean measurement of all control molar eruption

rates was calcUlated to be 0~201 mm per day in the period be­

tween age 11 and 16 days. Corrected for magnification in­

trinsic to the X-ray procedure (l.038X), the normal rate of

eruption of the first maxillary molar during this five

day period is 0.194 mm per day. Cortisone accelerated this

rate to 0.244.mm per day.

Daily administration for five consecutive days of 0.1 mg

of cortisone and 0.1 mg of hydrocortisone produced an

increase in the rate of eruption of the first maxillary molar

of 39 and 32% respectively while the rate was accelerated 25

r:~·· ---------------~ ·19

and 20% at the 0.5 mg dosage. This effect tended to decrease

as the dosage was increased (fig. 5, table 2). Cortisone

accelerated the molar eruption rate 26% at the 0.75 mg

dosage whereas the rate was retarded 2% at the l.O mg and

25% at the 1.5 mg dosages. I~ should also be noted that

the eruption rate of the first mandibular molar measured

at the 0.1 mg dosage of cortisone and bydrocortisone also

exhibited acceleration in eruption.(table 3).

In the 0.1, 0.5 and 0.75 mg injected groups, the first

maxillary molars or the hydrocortisone treated animals

appeared to have erupted somewhat earlier than those of the •

cortisone treated animals. This ·observation is based on

gross examination of the molar cusps and the gingivae

immediately following exsanguination. However, X-ray meas­

urements showed that the molars of the cortisone-treated

animals had in fact erupted before those of rats treated

with an equal concentration of hydrocortisone. This data

would seem to show that a given concentration of hydro­

cortisone has a more sever_e wasting or dehydrating effect

on the gingivae than does an equal concentration of cortisone.

The extent of eruption of the first maxillary molar was

determined from measurements between the most rostral molar

cusp (C) and its adjacent alveolar crest (fig. 7, (M)).

Neither cortisone nor hydrocortisone produced a change in

bone growth in the 0.1 mg injected animals as shown by X-ray

L

20

measurements. Therefore, decreased bone growth can not be

regarded as a factor in the measured precocious (accelerated)

molar eruption in these animals. The absence or bone appos­

ition in the 1.0 mg injected rats when compared with control

measurements, indicated that the observed retardation in

eruption is not a manifestation of alveolar bone apposition.

In fact, a decrease was observed in molar alveolar bone growth

in measurements on experimental rats when compared with litter

mate controlso These findings were detennined from radio­

logical measurements (fig. 7,(line N to IM), table 5).

c. Histological Observations ..

Specimens were prepared from all animals for histo­

logical studyo Sagittal sections, taking every 12-15th section,

or the entire maxillary molar region were made at Bp. The

customary exposure to the decalcifying agent, sodium citrate­

fonnic acid, accounts for the absence of enamel and removal or

the dentinal matrix.

In order to determine histological growth effects on

dental structures, a seri'es of measurements was taken on O. l

and 1.0 mg cortisone treated rats, by means of an ocular micro­

meter. The measurements included: connective tissue thick-

ness, the distance between the most superior portion or the

alveolar bone and the dentino-enamel Junction, trabecular

thickness, height or the Haversian space and diameter of major

pUlpal vessels (table 4).

r:--J ____ ______,

l

21

The treated rats showed a histological picture very

similar to that or their litter mate controls (figs. 3.4.8,9).

The basal loop (Hertwig 1 s sheath or odontogenic epithelium)

consisted or two rows or cells forming a loop at the base or

the pulpal chamber. The inner row of cells gives rise to the

dentin producing odontoblasts, and the outer may give rise to

the enamel forming ameloblasts (Sieber, 166). The odontoblasts

or the steroid treated animals appear to be normal as judged

by the morphology or the cells, regularity of arrangement and

thickness of odontoblastic row. These cells have a columnar

~ody with an oval nucleus. Some of the bodies are long, others

short. Their irregular arrangement higher up in the pulpal

cavity is normal.

In the hematoxylin and eosin stained sections, dentin

fonns the heavily eosin-stained region surrounding the pUlpal

chambero The dentinal tubules run outward and curve occlu­

sally from the odontoblastic row of cells. In the living

animal, the dentin consists of an organic and an inorganic

part. The inorganic component (hydroxyapetite) has been

removed in these sections by decalc1f1cat1on. The dentin was

measured for thickness to determine whether or not the rate

of dentinal apposition was affected by cortisone treatmento

This measurement was made at the dentino-enamel junction at a

plane perpendicular to the plane of growth. Its apposition

appears to be unaffected by the administration of either

~-/ _____ _,

22

dosage or cortisone.

VascUlarity or the pUlpal region was closely examined

and no consistent variation was seen in either the low or high

dosage treated animals {O~l-1.0 mg). At times, hypertrophied

vessels were seen as was an increase in the number or vessels.

However, this condition was also seen in control animals.

Hence, the variations in vascUlar caliber and number were

nonnal. Our lack or positive findings in the dental tissues

{odontoblasts and ameloblasts) or the steroid treated animals

concurs with the work of others {Goldsmith and Ross, 1 54;

Garren, '55; Wellband, 1 61; and Kiely, 1 67).

Active resorption and appostition is the normal conditio

of the molar alveolar bone. These processes are only con­

sidered pathological when one process for outweighs the other

resulting in an abnormal loss or gain in bone. In the steroid

treated animals, resorption was characterized by a greater

than normal number or osteoclasts on all surfaces or the

trabeculae. This finding indicates pathology which agrees with

observations or Frost ( 166). Measurements of the trabeculae

showed a marked decrease in thickness most evident in the

alveolar bone of rats treated with the higher dosage {l.O mg) 0

The heigh~ or the alveolar bone when measured from the

dentine-enamel junction was decreased in the steroid treated

animals. In order to detennine whether this was due to an

increase in eruption or a decrease in bone growth, we examined

~~i' -----------------~ , . 23

our X-ray data. Roentgenographic bone measurements revealed

an accelerated eruption at this dosage. Hence, we concluded

that the decreased height of the alveolar bone, as measured

from the dentino-enamel junction, is a manifestation of an

acceleration in rate of erup~ion in this group. Roentgeno­

graphic bone measurements in the 1.0 mg cortisone injected

animals revealed a decrease in bone growth at the alveolar

crest, while measurements on eruption revealed a retardation

in rate. We therefore concluded that the decreased height

of the alveolar bone in this group is a manifestation of bone

resorption. •

Osteoporosis is defined as a condition characterized

by a decrease in the normal amount of bone, i.e., negative

skeletal balance (Frost, 1 66). The presence of this condition

was detennined by measurements on the size of the Haversian

space {bone marrow space) and by the thickness of its trab­

ecular envelope. The Haversian space was found to be larger

and the trabecular wall markedly thinner than that of controls

in both 0.1 and 1.0 mg cortisone treated rats (table 4). This

represents an enlargement of the Haversian space at the

expense of the surrounding matrix and in the case of the loO mg

treated rats, in a net loss of alveolar bone.

The number of hematopoietic cells in the marrow was

!reduced in all steroid tl.'.59ated (cortisone and hydrocortisone)

rats when compared with controls (figs. 3,4,8,9).

~---) ____ ____,

L

24

The most remarkable effect noticed in the histological

sections or all our steroid treated rats was the striking

accumUlation or rat in the bone marrow, filling the spaces

vacated by the blood cell precursors. · Normally, (note control)

only a few scattered fat cells are found in the area. Furthex­

more, in the muscle seen adjacent to the alveolar process, we

also noted an increase in the accumulation or rat cells

surrounding the muscle belly (fig.4). These conditions

were seen in animals treated with either hydrocortisone or

cortisone regardless or the dosage administered.

These observations on th& presence of osteoporosis,

hypoplasia of blood cell precursors, and replacement or these

cells by rat cells confirm the results or Baker and Ingle (•48)

who injected adult rats with 1.0 to 3.0 mg of ACTH for a

period of 21 days. The occurrence or osteoporosis correlates

with the findings of Becks et al._ {1 44) who observed a de­

crease in the amount of bone in cortisone treated rats, and

Ragan et al. ( 1 49) who observed this condition in ACTH treated

normal rats.

The connective tissue of the oral epithelium had

decreased in thi~kness in the O.l and 1.0 mg cortisone treated

animals: the decrease in thickness was determined by direct

histological measurements (table 4). This finding supports our

conclusion that the "apparent" precocious eruption observed

in our high dosage treated rats is not due to an accelerated

25 movement of the tooth. Rather it may be due to a thinning of

the overlying gingivae, thus leading to its rupture over the

molar cusps (figs 10, 11).

•

rr--) --------, !·

l

V DISCUSSION

A. Cortisone and Molar Eruption

The effects of cortical steroid administration on molar

eruption in the albino rat show that five consecutive daily

injections begun on eleven day old rats will accelerate the

eruption rate. The effect was inversely proportional to the

dosage administered showing that this steroid bas an optimal

dosage preference. An acceleration in rate of eruption of the

first maxillary molar was observed at 0.1 and 0.5 mg and a

retardation at the l.O, 1-.5 and ~.o mg dosages.

An acceleration in the rate or· eruption of incisors has

been observed by other investigators following the administratio

of cortisone in neonatal rats (Panner et al., 151; Leroy and

Domm, 151; Goldsmith and Ross, 154; Donm1 and Leroy, 1·55;

Goldsmith and Ross, 1 56) and in adult rats {Domm and Marzano,

1 54; Garren, 1 55; Domm and Wellband, 1 60; Garren and Greep,

•60).

To our knowledge there have been no studies on the effects

of cortical steroids on molar eruption in the rat. Indirect

evidence was furnished by the work of Schour and Van Dyke ( 1 31)

who noted that hypophysectorny, which induces a decrease in

adrenal function in rats, caused no observable gross change in

the molar. However, their study was begun on day 34 when the

26

~---' -------,

L

27

molars had already achieved functional occlusion and without a

reliable method of measurement, they could not have detected

fluctuations in the molar eruption rate.

Bundgard-Jorgensen et al. ( 1 58) administered 1.0 mg of

cortisone daily for the first two weeks of life and 2.0 mg

thereafter until day 49. They observed inflammation and ulcer­

ation in the molar areas which they attributed to tra~~a from

biting on foreign bodies found in the lesions of this area.

In the light of our experiments, eruption of the molars would

have been retarded with the alli~inistration of such a dosage,

and the anti-repair effects (inhioition of protein synthesis)

of cortisone would have been severe enough to explain their

observations on inflammation and ulceration. Johannessen ( 164)

administered 1.0 to 2.0 mg of cortisone daily via food intake

for 41 days. The first mandibular molars were removed and the

growth of dentin measured. He found that dentinogenesis had

been inhibited and was not restored in animals kept intact for

later examination. Furthermore, in experiments on semi-star­

vation of normal rats, he noted that somatic growth was more

severely affected than dentinogenesis.

Sobko\'i~ki and Srni t~all ( ' 41), Garren ( 1 55), and Garren

and Greep ( 1 60) observed that even at high dosages (8.0-10.0mg)

cortisone accelerated the eruption rate of incisors in adult

rats. However, in our experiments, a comparably high dose per

r; _______ _____, 28

kilogram of body weight {l.O and 1.5 mg in newborn rats)

induced a retardation of molar eruption. We attributed this ~

to a lack of tolerance in the younger rat to the toxicity

induced by the cortisone. · If weight gain is taken as a

parameter of intensity of the cortisone effect, our results

show that the weight loss at higher dosages is greater than

at lower dosages in animals of the same age. Smith ( 1 64)

reported, using body weight change and skin collagen per total

skin area as parameters or toxicity, that weanling rats are

less tolerant to corj;isone treatment than young adult or adult

rats. He used a 10 mg/kg dosage,. the same dosage employed by

us. Therefore, from bis data, it can be deduced that the

retardation of molar eruption at 1.0 and 1.5 mg is the manifes­

tation of a toxic effect of cortisone and hydrocortisone in

the young animal.

It occurred to us that cortisone may be accelerating the

rate of molar eruption in an indirect manner, that is, it may

be exerting its effect on the overlying connective tissue

rather than directly on the eruptive mechanism of the molar.

Massler and Schour ( 1 41) in their review on theories of

eruption pointed out that surgical "release" {incision of the

overlying tissues) of an unerupted tooth often precipitates

1 ts eruption. Cortisone may well have an ~"'1alogous effect on

the overlying tissues through which the unerupted molar must

pass before attaining its future occlusal position. It has

,-: __ - ! --------.

L

29 been reported that cortisone exerts a protein catabolic effect

and therefore an anti-repair effect on connective tissues.

{Clark., '53). In an environment or trauma from nursing and

other masticatory activity and even trauma from the force of

the erupting tooth, cortisone, because of its anti-repair

effect, could release the erupting tooth from restraint. If

this were the case., the molar could erupt at a faster rate

similar to a molar released from occlusal contact {Soggnaes,

• 41).

B. Cortisone ~ the Cellular Proliferation Theory

The observed acceleration in. the rate of eruption of the

rat molar tUlder the influence or cortisone could be due to an

acceleration or cellular activity (differentiation and/or

mitosis) in the basal or proliferative zone of the tooth. This

view gains support because or earlier observations on the

proliferative area in incisors under the influence of cortisone.

Goldsmith and Ross ( 1 53) administered 15 and 20 mg of cortisone

daily to. pregnant rats and observed that the fetuses at twenty

days showed accelerated ameloblastic and odontoblastic dif­

ferentiation. Goldsmith and Ross ( 1 54) reported histologic

precocity in three day old rats given 0.1 mg of cortisone in

two daily injections beginning at 24 hours of age. The amel­

oblasts and odontoblasts, though they appeared normal, were

producing above normal amounts of enamel and dentin. These

investigators, in conjunction with another experiment~ confirmed

~----------------------~ .30

their earlier observation on acceleration of arneloblastic and

odontoblastic development with a O.l mg daily dosage (Goldsmith -

and Ross 1 56). Anneroth and Bloom ( 1 66) noted that 50 mg of

cortisone administered daily for eight days in adult rats

caused Hertwig 1 s epithelial sheath (odontogenic epithelium)

to be abnormally proliferated. They also noted an increase in

the number of pulpal cells. Domm and Kiely ( 168) correlated

an increase in colchicine-arrested (dividing) cells in the

basal or proliferative ioop with accelerated eruption of

incisors in young adult rats following daily administration of

0.5 and 1.0 mg of cortisone.

Although cortisone gener8.J.l~ depresses mitotic activity,

(Baker et al., 1 51; Thiersch et ,.al., 1 52; Wellington and

Moon, 160; Bass and Snell, 1 61) and growth of cells (Kaufman

et al., '53; Wellington and Moon, 1 60) stimulatory effects on

mitosis following cortisone administration have been reported.

For example, cortisone accelerated mitotic activity of bone

marrow erythroblasts (Fruman, 155). In an .!!l vitro study,

Aujard and Chany ( 1 63) found that although cortisone usually

depressed mitotic activity, low dosages sometimes stimulated

an increased yield in tissue culture. Such seemingly contra­

dictory results may perhaps be explained in terms of specific

actions confined to specific dosages. It should be considered

that cells may have different types or mitotic triggers,

some of which may be sensitive to cortisone only at

~--------------. 31

certain dosages (Mazia, : '60).

c. Cortisone ~ the Vascular Theory

In view or the reported effects or cortisone on blood

vessels and blood pressure there is some support for the

vascular theory of eruption, if in fact increased vascularity

or blood pressure is the source of the eruptive force.

Anneroth and Bloom ( 166) recorded an increase in pulpal vas­

culari ty with cortisone administration. Wellband ( 1 61),

working under the supervision or Dr. L. v. Domm in this lab­

oratory, reported an increase in vascularization of the pulp

correlated with injections of 1.5 mg or cort:tson&. in MW.t

hypophysectomized, thyro-adrenalectomized, thiouracil-fed,

· and nonnal rats.

Also or interest is the observation that some gluco­

corticoids cause sodiurn retention, that is, they induce an

exchange of extracellular sodium for intracellular potassium.

The potassium is excreted by the kidney and the sodium is

mobilized from connective tissue into the extracellular com­

partment. There is a concomitant increase in extracellular

water, inducing a rise in·blood pressure and a diuresis

(White et al., •64). These factors may effect an increase in

molar eruption if vascularity and blood pressure are causative

agents in this phenomenon.

:r>.' Cortisone ~the Connective Tissue Theorz

In any discussion on the collagen contraction theory of

32

tooth eruption, the role of cortisone must be considered.

Thomas ( 167), a proponent of this theory, suggested that cor­

tisone may specifically affect the connective tissue of the

periodontal membrane, i.e., induce catabolism of the collagen

and thereby speed up eruption. Collagen shortening has been

shown to be a manifestation of increased hydrogen, covalent, and

(possibly) ester bonding {cross-linking) of the molecule {Verzar,

•63). We assume that Thomas is implying that cortisone inc­

reases the maturation (cross-linking) of collagen fibers, thus

inducing their contraction and thereby accelerating eruption.

This has not as yet been shown. •

Thomas ( 167) has also suggested that cortisone may elicit

an accelerated eruption by its dehydration effect on collagen

fibers. This is supported by the work of Bear ( 1 44) who by

means of X-ray diffraction studies reported collagen contraction

as a result of dehydration.

E. Cortisone and Other Hormones

Schour and Rogoff ( 1 36) noted that adrenalectomy in the

rat caused a decrease but not a complete cessation in incisor

eruption rate. Their finding suggests that there may be factors

other than adrenal influences that regulate the eruption rate.

Schour and Van Dyke ( 1 32b) reported that hypophysectomy com­

pletely stopped eruption of the incisors in the adult rat. At

the same time, they noted that growth hormone (GH) replacement

therapy brought about a partial recovery of the eruption rate.

~------------~--------, 33

or interest is the fact that in their experiment, growth hormone

bad no effect on the eruption rate in normal animals. Collins

et al. ( 1 49) also administered growth hormone to hypophysectom­

ized rats and observed that the eruption rate returned to one­

half that of normal. At the same time they also reported that

the incisors were larger than those or the hypophysectomized

controls. Enamel formation had ceased altogether in the apical

one-third leaving the dentin in direct contact with the peri­

odontal membrane. They also observed oral epithelial degen­

eration, scattered dentin deposits in the pulp and a general

increase in bone accretion in the.jaw and calvarium. Domm and

Marzano ('54) while reporting cortisone stimulation of incisor

eruption rate in newborn rats, confirmed the findings of Schour

and Van Dyke ( 1 32b) noting that growth hormone had no effect on

the eruption rate in normal rats.

Paralleling the above research, were studies being per­

formed concerning the effect of thyroid hormone on eruption.

By 19271 it had been shown that acetyl thyroxin could stimulate

incisor eruption in the newborn rat (Hoskins, 1 27), and, by

1947, that thiouracil, a thyroid gland inhibitor, caused an

inhibition in the rate of eruption of the incisor in the rat.

(Parmer, 1 47)0

Baume et al. ('S4a, 1 54b, 154c) in a series or three

consecutive papers reported initially that in a group of 60

thyroidectomized rats, the incisor eruption rate was decreased

34 45%; growth hormone stimulated this rate to 10% over that of

control thyroidectom1zed rats; thyroxin to 27%; and a combin­

ation of growth hormone and thyroxin stimUlated it slightly

more than 27%. The histology of the incisors in the thyroi­

dectomized, thyroxin-treated animals did not return to normal

whereas that of the growth hormone thyroxin-treated ones had

returned to normal.

In their second paper ( 1 54b) they compared incisor eruption

rates of normal and hypophysectomized rats and found that the

normal rate was 2.53 mm per week, while the hypophysectomized

rate had decreased 76% to 0.55 mm.per week. The fact that

bypophysectomy decreases the eruption rate more than thyroidec­

tomy may indicate that another pituitary factor(s) is involved.

In their third paper (' 54c) Baume 1 s group reported the

effects of growth hormone and thyroxin in hypophysectomized rats.

Growth hormone, while increasing the size of the incisor, had no

effect on the eruption rate whereas thyroxin administration

stimulated the rate by as much as 46% over that of hypophysec­

tomized controls. The simultaneous administration of growth

hormone and thyroxin restored tne eruption rate to normal. In

evaluating their data these investigators postulated that growth

hormone and thyroxin synergistically regulate the eruption pro­

cess,; 1.(~., growth hormone affects the basic growth process,

while tbyroxin controls maturation and differentiation.

Goldsmith and Ross ( 156) injected 0.1 mg of cortisone. into

rr---------------~ ' 35

L

newborn rats and observed an acceleration in the rate of eruption

of incisors. They also noted from the work of Schour and Rogoff

(•36) that adrenalectomy only blocks eruption partially. Extra­

polating from the data in the literature, they concluded that

growth hormone and thyroxin directly contro,l eruption and, on

the basis of their own study, they concluded that cortisone is

vital in normal eruption because of its effects on RNA and

alkaline phosphatase levels. '

Domm and Wellband ( 1 61) measured eruption rates in thyroi­

dectomized, adrenalectomized, thyro-adrenalectomized, and normal

adult female rats. Each of these.groups were then divided;

one was treated with cortisone, while tlie other was kept as an

operated control. Thyroidectomy decreased the eruption rate

of.incisors 19%; adrenalectomy 33%; and thyro-adrenalectomy 53%.

The administration of 1.5 mg of cortisone restored the eruption

rate to normal in each of these groups. They concluded on the

basis of percentage results in operated animals that normal

eruption is synergistically controlled by the thyroid and the

adrenal glands.

For purposes of clarification of the many facts presented

on hormones and tooth eruption, we have constructed the table

shown below. On the·basis of this data, we believe we can make

certain tentative conclusions and predictions.

In view of the wide application of cortisone as replacement

therapy in many honnonally-deficient states (row three) we could

36 The Effect of Honnones on Eruption Rate in the Rat

Nor- Hypophy- Thyroi- Adrenal- Thyro-mals sectomy dectomy ectomy Adrenal-

or ectomy Thiou-racil

No GH,'l',C* .No 'J!; nonna1 .NO C; nonna.J. No ·r, c . GH & C GH & T nonnal GB

Growth --- for --- 1' ? ? Honn one 2,4 2,3_,9 7 Predict-or~ Predict-o/l

5 1' t 1 ? ? t Thyroxin 9 7 Predict t Predict:M1

11,l.2 WNL WNL WNL WNL Cortisone 4,10, 12 6,11,12 11,12 11,12

GH & Thy- ? WNL t ? ?

roxin P~d1 · 9 7 Predict t PredictWN

No ther- WNL t 01- STOP ~

"' ~

apy 2,8,12 6,7,11 1,11,12 11,12 .. ··- . ~

* GH Growth Honnone; T Thyroxin or acetyl thyroxin; c Cor-tisone.

WNL Within Nonnal Limitso

1. Schour and Rogoff ( 1 36) 1. Baume et al, {'54n} 2. Schour and Van Dyke ( 1 32) a. 1 54b 3. Collins et al. ( 1 49) 9. '54c 4. Domm and Marzano ( 1 54) 10. Goldsmith and Ross ( 1 56) 5. Hoskins (' 2}) · 11. Domm and Wellband ( 161) 6. Panner ( 1 47 12. Wellband ( 161)

postUlate that the use of phannacological doses is sUfficient .;, ·- ' ; . ~

. ~ ~ .. ·-· . - ·-for the maintenance of nonnal tooth eruption in rats whether

they are hypophysectomized, thyroidectomized, chemically-thy-

roidectomized, adrenalectomized, thyro-adrenalectomized, or

nonnal. Noting that growth bonnone and thyroxin combined

(row four} can restore the eruption rate to within nonnal

~----------, 38

limits, we might question whether cortisone is necessary at

all. In the normal animal in which only physiological doses

of each of these hormones are acting, we may tentatively

suggest that these three hormones, growth hormone, thyroxin,

and cortisone, all act synergistically to control tooth

eruption; or, that cortisone potentiates the other two hor­

mones. The studies performed on animals, not receiving

replacement therapy (row five), that were hypophysectomized

(eruption stops), thyroidectomized (eruption rate slows),

and adrenalectomized (eruption rate slows) would seem to

support this assumption. •

The question marks in the table indicate research which

to our knowledge has not yet been reported. Our predictions

are based on the publications cited. We believe that these

missing links are the factors that, once reported, may

explain the hormonal mechanisms acting in normal tooth

eruption.

F. Cortisone and Body Weight

The degree of severity of the cortical steroid effect

was obvious. Our gross observations included weight loss,

diarrhea, loose yellow stools, and decreased hair growth.

These observations confirm the report of Parmer et al. ( 1 51)

who administered daily, for five consecutive days, 0.1 mg

injections or cortisone to one day-old rats. In our exp­

eriments we began O.l mg injections on day llo Our gross

rr-------------------3-9____, obser-vations did not include effects so severe as those

reported by Panner's group, but in a previous experiment

using the same dosage (0.1 mg) with injections beginning

on day 4, our observat1·ons correlated very well.

In this discussion, weight gain refers to the total

gain from the onset of honnone injection to the day of sac­

rifice, a five day period. The immediate effect of the cor­

tical steroids was reflected in all treated animals as a

weight loss at the end of the first day and a gradual re­

covery on subsequent days. As shown in table 1, weight

gain decreased as the dosage increased. Hydrocortisone had

a slightly greater effect in this regard than did cortisone.

A decrease in weight gain has been reported by several

investigators in work on newborn rats (Leroy and Domm, 1 51;

Parmer et al., 1 51) and adult rats (Garren, 1 54; Bundgard­

Jorgensen et al., 1 58; Johannessen 1 64; Anneroth and Bloom

'99); and in newborn mice (Stock et al., 151).

Parmer et al. ( 1 51) explained the weight loss in tel,ns

of dehydration due possibly to decreased nursing by the

newborn rats under the influence of cortisone. This exp­

lanation is not confinned by the data of Johannessen ( 1 64)

who administered 1.0 mg of cortisone per day to adUlt rats

and also observed weight loss with decreased food intake.

However, upon the discontinuance of treatment, the increase

in food intake was not sufficient to explain the observed

rr----------, ! ~

gain in weight. Russell and Wilhelmi ( 1 54) note that.in

humans as well as in animals there tends to be an increase

in appetite with the administration of cortical steroids.

These varying reports sUggest that the weight loss observed

in cortisone-treated rats is not due to a decrease in food

intake but rather to a decrease in the utilization of metab­

olites at the celluJ.ar level, or to a change in weight due

to some other factor such as water balance.

Cortisone does affect water balance. This honnone may

directly affect kidney function (Harrison and Darrow, 1 38),

however, extrarenal effects were also observed. Cortical

steroids may alter water balance.by decreasing the serum

potassium in adrenalectomized and nephrectomized rats {Ingle

et al., '37). In this way, these steroids may affect elec­

trolyte balance. Conn et al. ( 1 51) reported that hydro­

cortisone induces sodium retention (and, therefore, water

retention} in normal people. In other investigat~ons, . cortisone induced sodium excretion and diuresis {Thorn et al.

1 4lj 'Dorfman, 1 49). This difference may be due to the

dosages used. Dehydration due to transfer and/or excretion

or water could be a factor to consider in the weight loss in

cortisone-treated rats.

, Weight loss in the cortisone-treated rat may be ex- .

plained by the effect of cortisone on carbohydrate, lipid,

and/or protein metabolism. Cortisone administration in rats

41 increases the carbohydrate content or blood and tissues, much

or this increase being stored as liver glycogen. The effect

of cortisone in 11 diabetogen1c 11, i.e., there is an increase

in blood glucose and urinary nitrogen (Long et al., 1 40).

The increase in urinary nitrogen excretion is a reflection of

protein catabolism and is referred to as a negative nitrogen

balance. Cortisone administration also leads to a break­

down or a blockage in the synthesis or proteins in rats and

man which has been reported to be the source or the excess

urinary nitrogen (Clark, 1 53). This protein is altered

metabolically and released as.carbohycrate into the blood

and tissues. The liver, under the effect of cortisone,

stores the blood carbohydrate as hepatic glycogen {Greengard

et al., •63a, 1 63b) by st~mulating the enzymes necessary for

storage. In the second paper (•63b), they reported on

injections of cortisone into starved adult Sprague-Dawley

rats. Puromycin· (an .inhibitor or cortisone-induced hepatic

enzymes, i.g., tyrosine transaminase and tryptophane Pyr­

rolase) were injected in these rats to block hepatic

glycogen storage. These antimetabolites did inhibit the

cortisone-induced storage of glycogen. From this, they

concluded that cortisone stores liver glycogen by stim-­

ulating an increase of the necessary hepatic enzymes.

In balance experiments in which adrenal corticoids were

administered, the increase in excretion or glucose far

~--------____, 42

exceeds the amoWlt of nitrogen excreted. This implies that the

source of this glucose is not entirely protein cataboli·BITh: b.Ut

that the normal utilization of carbohydrate may be blocked

as well (Ingle and Thorn, 1 41; Russell and Wilhelmi, 154).

Weight loss in rats on cortical steroid treatment could there­

fore be explained in terms of a tissue breakdown and rep~ir

inhibition which, specifically, is a manifestation of protein

catabolism and blockage of carbohydrate utilization at the

cellular level.

Since, Wlder the effects of corticoids, the body is

suffering a protein catabolism atld a blocked utilization of

blood carbohydrates, it seems logical to assume that the body

would tap fat stores for required energy. Cahn and Houget

( 164) noted that in the cortisone-treated doe-rabbit, the fat

stores are mobilized to compensate for the decreased com­

bustion of glucides (carbohydrates and glycosides). Cortisone

enhances the storage of hepatic fat and muscle fat and burns

the blood lipids for energy. According to Jeanrenaud and

Renold ( 1 66), the glucoco·rticoid action on rat adipose tissue

in vitro is that of increasing the output of free fatty acids.

But they do not act by breaking down (lipolysis) fat stores

as such, but by decreasing the rate of re-esterification of free

fatty acids. The most likely explanation is that the decreased

glucose metabolism and the inhibition of re-esterification are

somehow directly linked, possibly through a decreased avail-

rr-------------1 ~

ability of glycerophosphate. Whether this glucose metabolism

is at the cell membrane or in the Kreb's cycle itself is not

known.

G. X-ray~~ Experimental Procedure

The use of the X-ray as an experimental procedure was

evaluated by application of the standard error of difference

(s.e.d.) test of significance to comparisons of body weights

(significant deviation in body weight was chosen as an indic­

ator of procedural variation (Smith, 164)).-Compared were the

groups outlined on table 6. Statistical significance is

present if D (the difference between the means of the compared

groups) exceeds 3 s.e.d. There was no statistical signi­

ficance in any of the groups compared. Therefore, use of the

X-ray procedure did not introduce a variable into these exp-

eriments.

H. True ~ Apparent Precocious Eruption

Our gross observations on the gingivae overlying the

first maxillary molar, initially led us to conclude that we

were observing true precocious eruption of this molar in rats

injected with 1.0 and 1.5 mg of cortisone. However, meas-

urements on roentgenograms showed that molar eruption was in __ _

fact retarded. What we had viewed grossly was the molar cusps

through a dehydrated or thinner-than-nonnal gingivae.

We have already discussed cortisone-induced dehydration.

In this case, we may simply have been observing a dehydrated

r:---------i 44

g1ngivae that weakened and separated over the advancing molar

cusps. We also discussed the data on cortisone-induced protein

catabolism. With this in mind, the gingival condition may be

due to a tbinning of the subjacent connective tissue due to

loss or protein.

I. Cortisone and Gingival Keratinization

We did consider the effect or cortisone on keratinization

or the molar gingivae. Goldsmith and Ross ( 1 54) noted that the

oral epithelium in the incisor area became completely kerat­

inized following the administration of 0.1 mg of cortisone

in newborn rats. Panner et al, ( 1 51) found that in addition

to accelerated eruption and early eye-opening, cortisone in­

duced an accelerated gingival development. Mangine ( 1 65),

wprking under the supervision or Professor L. V. Domm of this

department, did a careful study on the effects or cortisone

on the gingivae and oral mucosa or the hard palate. She noted

that 0.5 and 1.5 mg or cortisone stimulated keratinization in

these areas in the neonatal rat. Accompanying this increase

in keratinization there was a decrease in the thickness of the

non-keratinized epithelial layer. She concluded that, in

consequence, an erupting tooth has a thinner layer or stratum

corneum in its path· to occlusion which may, in part, account

for the precocious eruption observed in cortisone-treated neo­

natal rats. Our observations of "apparent" precocious eruption

or the rat molar under the effect of cortisone may be explained

rr--------------------45---. in tenns or the above data. Cortisone may have stimulated the

keratinization of the molar gingivae and reduced the thickness

or the underlying non-keratinized layers in our experiments.

The thinned gingivae may then have been pulled down over the

molar cusps, rupturing the gingivae, thus giving the appearance

of accelerated growth or the molar and precocious eruption.

J. Cortisone and Hair Growth

In our experiments we observed a decrease in hair growth

as a result of the administration of cortisone and or hydro­

cortisone. This failure or growth was consistent and tende.d

to be or greater severity when toe nonnone treatment was

begun on younger rats. Other investigators have reported

decreased hair fonnation following cortisone administration

(Baker et al., •48; Baker and Whitaker, 1 48; Castor and Baker,

1 50) but the mechanism or action is unkno~m. Capillary

fragility and permeability increase with adrenalectomy and

are correlated with precocious hair growth, (Kozam, 1 52).

Therefore, it would seem that cortisone administration would

decrease this fragility and penneability. This could deprive

the hair follicle cells of needed nutrients, of metabolite

exchange, or of other growth factors (Chase, '55). Cortisone

is known to deprive· body cells of needed carbohydrates, lipids,

and proteins that may be necessary for hair growth. It has

been reported by many authors that cortisone, minerals, vita-e

mins, and sulfhydryls affect hair growth (Ralli and Graef, 1 43,

rr------------------46~ ~

L

•45; Anderson et al., 1 51; Hundley and Ing, 1 51; Meites, 1 51

and others).

In our results, decreased hair growth tended to be more

severe in the younger rats treated with cortical steroids.

Mohn ( 1 58) .round in his work on rats that only follicle

growth is af'fected by cortisone. This would explain our

observations of a more severe decrease in hair growth in the

younger rats treated with either cortisone or hydrocortisone.

K. Cortisone and Bone Growth

The degree of eruption was measured as the distance

between the molar alveolar crest. and the most mesial (rostral)

molar cusp (fig. 7). Radiographic8.J. bone measurements were

made on the roentgenograms of 0.1 and l.O mg cortisone-

treated and control animals to determine the validity of the

radiological measurements which indicated acceleration and

retardation of molar eruption in these groups. In this way,

the effect of cortisone on bone growth could be measured and

compared with control values.

We found no significant difference in the amount of bone

growth between the 0.1 mg cortisone-treated and control

animals. Therefore, since there was no manifestation of bone

resorption at the molar alveolar crest we concluded that

accelerated molar eruption had actually occurred. We observed

a retardation in eruption of the 1.0 mg cortisone-treated rats.

This finding is not a manifestation or bone apposition;at the

rr----------------------4-7__, t

L

molar alveolar crest. We did in fact observe a statistically

significant decrease in bone growth at the alveolar crest.

Schour and Van Dyke {1 32a) noted that hypophysectomy

~inducing an adrenal insufficiency} led to a decrease in

alveolar bone growth in adult rats. Specifically, they noted

a thinning and degeneration of the periosteumo The effect of

cortisone in such rats was not known is 1932. Cortisone when

administered to growing rats in various dosages from 0.25 to

100 mg/kg (1 to lO_mg/kg in our experiments) induces the

fonnation of a dense zone of calcified cartilagenous spicules

in long bones (Urist et al., 1 46; Ragan et al., 1 49). Leroy

and Domm ( 1 51) injected 5 mg of cortisone into nonnal pregnant

rats and noted that X-ray examination of the skull in the ten

~ay-old rat showed a decrease in calcification. Goldsmith and

Stahl ( 1 53) injected 3.0 mg of cortisone in adult rats and

reported an increase in growth or alveolar bone. Goldsmith

and Ross ( 1 56) adm'inistered 0.1 mg of cortisone in newborn

rats and observed a marked overgrowth of alveolar bone and a

precocity in the rate of incisor eruption of 2.9 days. These

studies reporting an increase in bone growth lead us to conclude

that cortisone may possess a specificity for accelerating b9ne

growth at certain dosages.

The observations or Garren and Greep ( 1 60) are in partial

agreement with our results. Tney administered 1 to 10 mg/kg

in adult rats (the dosage employed by us) and reported no

rr--------------~~ change in alveolar bone. In our studies, newborn rats re­

ceiving the lower dosage (0.1 mg) revealed no significant

change in alveolar bone growth. At the higher dosage, how­

ever, we observed a resorption of bone (determined by radio­

logic measurements).

It has been reported that the alveolar crest is more

stable.in an environment of physiological change than the

gingivae for the purpose of measuring rate of eruption

(Ness, 1 54; Miller, '57). The choice of the alveolar crest

as a reference point in the measurement of molar eruption is

reinforced by the consistency o~ our results (table 2) in each

dosage grouping.

L. Histological Observations

The molars of rats treated with cortisone and hydro­

cortisone showed no morphologic modifications with respect

to odontoblasts, arneloblasts, dentin fonnation or pulpal

cellularity and vascularity. It was not possible to make

observaions on enamel on the decalcified sections. Since

arnelogenesis is complete at 11 days (Schour and Massler, 1 49)

we did not expect any change in enamel fonnation as a result

of the experimental procedures which began on day 11.

Our findings confirm those of Kiely ( 1 67) who found no

significant histological modifications in his control rats,

0.25, 0.5 and 1.0 mg cortisone-treated rats, or normal un­

treated controls. Wellband ( 161) also noted no histological

49 differences between his 0.25, 0.5 and 1.5 mg cortisone-

treated and control rats.

or significance in our study was the increase in alveolar

bone resorption in the 1.0 mg treated rats. This was first

observed as an increase in the number of multinucleated osteo­

blasts on the walls of all trabeculae. _Actual measurements

on the thi"Qkness or the tr.abeculae supported this observation

by indicating a marked decrease in thickness in the treated

rats. Measurements on the height of the alveolar crest from

the dentine-enamel junction also gave an indication that a

resorption process was taking place. These measurements,

(table 4) combined with the radiological observations on bone

growth (table 5) and eruption {table 2) indicated that bone

r.esorption increased noticably in the 1.0 mg cortisone­

treated rats when compared with those receiving 0.1 mg. It

has been shown by Follis ( 1 51) and StanisavlJevic et al., ( 1 62)

that cortisone suppresses bone formation more than bone

resorption in the rat. Hence, resorption quickly overcomes

formation, resulting in a net bone loss.

This resorption in the alveolus of the rat may be a

response by bone to the cortisone-induced alteration in calcium

and phosphorus metabolism (Storey, 1 60). Because of this

alteration, the bone matrix cannot be mineralized and it is

subsequently broken down (resorbed).

~---------T-h_i_s __ r_e_s_o_rp __ t_i_o_n_b __ r_i_ng_~_s __ ab--ou __ t_'_'_o_st_e_o_p_o_r_o __ si_s_'_'_d_e_r_i_n_e_d __ b_y ____ _,

50 Frost ( 1 66) as the condition resulting in the net loss of bone.

A more popUlar theory accounting for this extensive bone re­

sorption holds that cortisone brings about a "negative skel­

etal balance" caused by the immediate increase_in number of

osteoclasts (Frost, 1 66). However, the mechanism of action is

unknown. Robbins ( 164) suggests that in byperadrenalism

(Cush1ng 1 s syndrome) there is an increase in protein catabolism

leading to a breakdown of the organic matrix of bone. Since

there is then no place where calcium can be deposited, the

resorption-f'onnation balance is upset--resUlting in a net

loss of' bone.

The marrow space of' the molar alveolar bone was marked

by a hypoplasia or the blood cell precursors and an increase

in fat cells. Baker and Ingle ( 1 48) injected l. 0 to 3. O:-mg

of' ACTH in adult rats and observed red marrow atrophy and

replacement of' these cells with fat cells.

The presence or histologically detectable f'at storage

was a noticable difference between experimentals and controls·

in all our cortical steroid treated rats. This accumulation

of' fat was seen in the marrow spaces or the alveolar bone

and surrounding the muscle in these section. As mentioned

above, rat stores are usually mobilized to compensate for

decreased combustion of carbohydrates; moreover, cortisone is

known to enhance the storage or hepatic and muscle rat

(Cahn and Houget, '64) •

51 A deficiency in the loose connective tissue of the molar

gingivae was observed in all cortisone-treated rats. This

would seem to lend support to our conclusion that gingival

rupture over the molar cusps brings about an apparent

precocious eruption rather than a true eruption. We believe

that the connective tissue deficiency is in some way related

to protein catabolism. Ragan et al. ( 1 49) also observed an

inhibition in the fonnation of connective tissue in rabbits

treated with 12.5 mg of cortisone twice daily for three days,

and Castor and Baker ( 1 50) reported a decrease in the col­

lagenous part of connective tissue in rats treated with cor­

tisone.

r ______________ ~ VI SUMMARY AND CONCLUSIONS

1. The administration or cortisone and hydrocortisone in five

consecutive daily doses brought about precocious eruption

of the molars (gross observation). The injection of l.O mg

of either or those steroids did not result in an increase

in the eruption rate a.S shown by neasurements on roentgeno­

grams. It is concluded that shrinkage or thinning of the

gingivae over tbe erupting cusps rather than acceleration in

eruption facilitated this appearance in the molar region.

Hydrocortisone at comparable dosages was somewhat more

effective than cortisone in this response.

2. A reduced gain in body weight and a decrease in the density

of hair were observed regardless of the steroid employed or

the dosage administered.

3. The molar eruption rate was accelerated by both cortisone

and hydrocortisone at dosages of 0.1 and 0.5 mg while it was

actually retarded at the l.O, 1.5 and 2.0 mg dosages. Cor­

tisone accelerated eruption slightly more than hydrocor­

tisone {roentgenographic observation).

4. ' The measurements on rate or eruption which were obtained

radiographically are considered to be valid and are not

manifestations of increased or decreased alveolar bone

growth.

5. The radiographical measurements on bone growth showed that

there was little fluctuation in growth at a dosage or 0.1 mg t52

r----------------53

or cortisone whereas at the -1.0 mg dosage, the molar

alveolar bone revealed a resorption when compared with

controls.

6. Neither cortisone nor hydrocortisone had any histologically

observable influence on basal loop proliferation, odonto­

blastic and ameloblastic morphology, dentin fonnation, or

pulpal vascularity. However, connective tissues were de­

pleted. Extensive decrease in growth or the molar alveolar

crest was observed with high (l.O mg) dosages of cort­

isone while on the contrary it was minimal at the lower

(O.l mg) dosage.

Cortisone caused a hypoplasia or blood cell pre­

cursors in the bone marrow. Accompanying this there was

extensive storage of fat in the bone marrow.

7. The X-ray procedure, as employed in this study, is a

valid experimental toolo Using body weight as a measure

of variation, it was shown that the use of X-ray did not

introduce a variable into the experiments.

8. Both cortisone and hydrocortisone accelerated the rate of

eruption or molars. These observations support the hypo­

thesis that similar mechanisms operate in the eruptive

process or both the continuously erupting incisors and

the limited erupting molars or the rat.

rr--------------~ VII LITERATURE CITED

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Anderson, G.E., L.L. Wiesel, R.W. Hillman, and W.M. Stumpe 1951 sulfhydryl inhibition as a mechanism in the effects of ACT and cortisone. Proc. Soc. Expo Biol. Med., 76: 825-827.

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54

rr-----------------5-5~ Baume, L.J., H. Becks, J.C. Ray, and H.M. Evans 1954b Honnonal

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Castor, c.w., and B.L. Baker 1950 The local action of adreno­cortical steroids on epidermis and connective tissue of the skin. Endocrinology, 47: 234-241.

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~------------~-----5-6~ conn, J.W., L.H. Louis, and s.s. Fajans 1951 The probability

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constant, T.E. 1900 The eruption of teeth. Third Congress Dentaire Intern. Paris, 2: 180-194.

Dalldorf, G., and c. Zall 1930 Tooth growth in experimental scurvy. J. Exp. Med., 52: 57-63.

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57

Garren, L.D. 1954 The effect of hormones on the eruption rate of the rat incisor. Harvard Dent. Alumni Bull., 14: 3-6.

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(Abstract). . ·

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~---------r r 58

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rr--------------59 f

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60

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61

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62

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. 63

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64

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# or Rats 7

11 7

13

~ 6 7 5

17 13 11

7

l 6 5 5

4

TABLE 1

Body Weight Changes in Rats

'Injected Between Days 11 and 16

Treatment Weight (gm)

Day 11 Day 16 Control* 0.1 mg E* O.l mg F*

20.7 31.5 24.4 34.8 22.4 29.8

Control 0.5 mg E 0.5 mg F

Control 0.75 mg E 0.75 mg F

Control l.O mg E 1.0 mg F

22.7 23.3 21.8

23.4 21.3 22.2

18.4 18.8 19.3

Control 20.8 1.5 mg E 19.9 1.5 mg F 20.8

Control 21.3 2.0 mg E 19o3 2.0 mg F 2.01

.control II** 21.2

33.7 30.1 22.9

34.3 • 25.7

23.9

28.2 22.1 20.0

31.7 22.8 21.7

33.6 21.3 21.6

3206

gm Gain 10.8 10.4 7.4

11.0 6.8 1.1

10.9 4.4 1.7

9.8 3.3 0.1

10.9 2.9 0.9

12.3 2.1 1.5

11.4

65

:t. .. ... s.n. s:-E. s.E.n. 3.72 1.52 1.72 0.81 1.72 2.49 1.02 1.83

1.90 0.53 ----3.21 1.07 1.19# 2.24 0.85 1.00#

4.18 2.30 ----3.68 1.84 2.95 1.34 0.95 2.19#

1.48 0.38 ----1. 70 0.54 o.66# 1.17 0.35 0.52#

1.62 0.72 ----1.20 o.69 l.OO# 1.85 Oo92 1.17#

1.98 0.10 ----1.60 0.54 0.92# 2.12 Oo75 1.03#

1.66 0.83 ----

* Control: All control animals received o.85% saline. E: Compound E acetate (cortisone). F: Compound F acetate (hydrocortisone).

• S.D.: Standard deviation - S.E.: Standard error

S.E.D.: Standard error of difference between experimental and control means.

# Significance equal to +/- 3 S.E.D. **Control II animals received neither X-ray nor injection.

TABLE 2

Eruption Rates of the First Maxillary Molar

in Rats Injected Between Days 11 and 16

# of Eruption (mmt mm mm/ % of + + Rats Treatment Day 11 Day 1- Total Day Cont. s:-D. s:-E. 7 Control* ·~99 l.io9 1.015 .182 100 .133 -~03 g O.l mg E* • 22 1. 89 1.267 .2~3 139 .193 • 43

O.l mg F* .706 1.911 1.204 .2 1 132 .073 .0299

6 Control .581 1.598 1.017 .203 100 0133 .0504 6 0.5 mg E .564 1.828 1.264 .253 125 .080 .0328 6 0.5 mg F .524 1.743 1.219 .244 120 .o64 .0260

5 Control .632 1.673 1.041 .208 100 .132 .0590 3 0.75 mg E .522 1.830 1.308 .262 126 .134 .1774 2 0.75 mg F .550 1.481 0.921 .184 88 .005 .0035

6 Control .599 1.580 o.98i .196 100 .095 .0361 6 loO mg E .671 1.635 0.964 .193 98 .179 .0729 6 l.O mg F .464 1.492 1.028 .206 105 .093 .0378

4 Control .569 1.538 0.969 .194 100 .095 .0361 ·2 1.5 mg E .831 1.558 0.727 .145 75 .025 .0175

3 1.5 mg F .519 1.597 1.078 .216 113 .052 .0297

6 Control .572 1.598 1.026 .205 100 .072 .0293 7 2.0 mg E ·~3 1.492 1.989 .198 97 .1o6 .04o1 7 2.0 mg F • 6 1.347 0.881 .176 86 .235 .0890

* Control: All controls received o.85% saline. E: Compound E acetate ~cortisone). F: Compound F acetate hydrocortisone).

+ S.D.: Standard deviation - s. E. : Standard error

s.E.D.: Standard error of difference between experimental and control means.

# Indicates significance equal to +/- 3 S.E.D.

66

... S.E.D. -------.081'6# .0586#

----.0743# .0715#

--.. --\. .0973 .0591

----.0813 .0523

-----.0637# .o681

----.0496 .0937

No. Rats

9

9

6

* #

TABLE 3

Eruption Rates of the First Mandibular Molar

in Rats· InJect.ed Between Days 11 and 16

b;rupt1on {~l mm mm/ ~ or + .t Treatment Day 11 Day 1 Total Day Cont. s:-D. S.E. Control* .$20 1.269 .749 .150 100 .135 .045

0.1 mg E* .491 1.354 .863 .171 114 .174 .058

O.l mg F* .520 1.478 .958 .195 130 .164 .067

Same footnotes as TABLE 2. •

Indicates significance equal to +/- 3 S.E.D.

67

+ S.E.D. _ .. __

.022#

.024#

68

TABLE 4

Histological Measurements

of the First Maxillary Molar Region*

No. • Me as urements • • . . Obs. Treatment • H.S. T. A.h. D. C.To b.vo: •

• . • •

5 Control 34.9 9.9 38.6 9.9 11.7 25.8

7 O.l mg E** 46.9 5.9 31.4 9.4 9o3 22.9

8 Control 47.8 10.2 48.8 9.5 16.2 27.9

5 1.0 mg E 50.3 6:5 4o.6 11.9 11.9 32.5

* All measurements were made with an ocular micrometer and are expressed in low power units with the exception of blood vessel measurements which are in high power units.

H.S.: Haversian space or alveolar bone. T.: Trabecular envelope surrounding H.s. A.h.: Height or alveolar bone, ie., Superior aspect

of alveolar bone to the dentine-enamel junction. D.: Thickness of dentin at the dentino-ena~el junc­

tion. C.T.: Thickness of connective tissue of the gingivae. b.v.: Diameter of major pulpal vesselso

** E refers to Kendall 1 s compound E or cortone acetateo Treated rats were injected daily from 11 and 16 dayso

TABLE 5

Measurements Showing Growth of Alveolar Bone*

in Rats Injected Between Days 11 and 16

O.l mg Cortisone (mm) 1.o mg Cortisone

mean s.n. S.E. S.E.D.

6.881 6.114 7.133 6.709

+ .644** -; .372** ± .394**

mean S.D. S.E. S.E.D.

• 60605 6.359 6.369 6.234 6,535 6.443

+ .120 .. .049 + .123

69

(mm)

0.1 mg HYdrocortisone (mm) • 1.0 mg HYdrocortisone(mm)

mean S.D. S.E. S,E.D.

6.788 6.7(){ 6.853 6.602 6.738

+ .109 + .055 :t .14o

Control {mm)

6.797 6.694 7.o61 6.441

mean 6.748 S.D. + .257 S.E. _. .129

mean S.D. S.E. S.E.D.

mean S.D. S.Eo

• 6.013 5.971 6,353 6.267

~ .1 + .151 + .062 • .129

Control (mm}

.932 6.977 70469 6.811 6.805 6.683 6.946

+ .276 ± .113

*Measurements according to method outlined on p.rs. **S.D. Standard deviation; S.E. Standard error; S.E.D. Stan­

dard error of difference between experimental & control means.

TABLE 6

X-ray as an Experimental Procedure

Test D* S.E.D.* Significance*

1:2 0.30 gm . 0.924 gm No

1:3 0.94 gm 1.012 gm No

2:3 0.54 gm 0.706 gm No

4:5 0.38 gm 2.797 gm No

6:7 0.06 gm 0.995 gm No

1: Non-injected, non-X-rayed control: N*= 4 mean= 11.4 gm s.D.*.:: i 1.66 gm s.E.*= ± o.83 gm

Saline-injected, X-rayed eontrol: N 36 mean·=ll.O gm S.D.= i 2.43 gm S.E.= i 0.41 gm

2:

Saline-injected, non-X-rayed control; N= 17 mean= 10. 46 gm

3:

S.D.= i 2.39. S.E.=+ O. 58 gm

0.5 mg cortisone-injected, X-rayed experimental: N= 6 . mean= 6.95 gm S.D.= i 2.98 S.E~ 1.21 gm

4:

5: 0.5 mg cortisone-injected, non-X-rayed experimental: N;: 3 mean= 6. 57 gm S.D.::± 4.37 gm S.E.=f. 2.52 gm

6: 1.0 mg cortisone-injected, X-rayed experimental: N = 6 mean= 3. 32 gm s.D.= ± 2.17 gm s.E.=± 0.89 gm

7: 1.0 mg cortisone-injected, non-X-rayed experimental: N ;.4 mean~3. 38 gm s.D.= + 0.90 gm s.E.= ± o.45 gm

* D: Dii'f'erence between means oi' compared groups. S.E.D.: Standard error or difference between com-

pared means. N: Number of' animals in group. Mean: Average, or mean weight gain.

70

S.D.: Standard deviation~ S.E.: Standard error of mean.

'•

PLATE 1

EXPLA..~ATION OF FIGURE

1 A photograph showing the cephalostat and industrial X- ray unit used in the experiments on the first maxillary molar of the rat.

71

FIGURE 1

PLATE 2

EXPLANATION OF FIGURE

2 A photograph showing precocious eruption in a 0.5 mg cortisone-treated rat and norwal eruption in a control (ages 16 days). Eruption of experimental rat: 1.323 mm; control rat: 0.760 mm. Arrow points to first maxillary molar.

Experimental

72

control

FIGURE 2

3

"

PLATE 3

EXPLANATION OF FIGURES

A photomicrograph of the first maxillary molar of a control rat showing periapical and dental tissues X 100.

4 A photomicrograph of the first maxillary molar of a O.l mg cortisone-treated rat showing pericpical and dental tis~ sues X 100. A: fuueloblasts ; CT: connective tissue; D: den­tin; ES: enamel space; HS Haversian space; O: odonto­blasts; P: pulp cavity; T: trabecular wall of alveolar bone.

73

FIGURE 3

FIGURE 4

5

..

PLATE 4

EXPLANATION OF FIGURE

A graph showing the effect of different dosages of cortisone on the total eruption during a five day period of the first maxillary molar of newborn rats. The bra­ckets indicate the standard error. E: cortone acetate; F : hydrocortone acetate; C: control.

0 < "' "' ... < "' c ~ .,, "' "' 0 c ~

3 !

1.35

1.'25

1.15

1.05

095

0 .85

0.75

0.70

-----t I \ L \

\ \ \ \ : \ - \ - \ . \ .

. " .. ·4.; : J.. .. ···· \ : · .... bt ................... ~. \ ~ ·····1. : . \ : /• ~ \ i.. I ~ . \ I

74

r _,-\

.;-' I\ _,-' L. \ .,..,, \

r ' \ .. . . .. ·'i . ... \

········ ········ .l···· ....... ~· \ : \ ... \ I

- \ I . \ I

~

0.1 0.5 0.75 1.0 1.5

DOSAGE OF ADRENAL STEROID (mg>

' . I ' I \ I

~F I I I I I L

2.0

6

"

PLATE 5

EXPLANATION OF FIGURE A graph showing the effect of different dosages of cor­tisone on body weight gain in newborn rats during a five day period. The brackets indicate the standard error. E: cortone acetate; F: hydrocortone acetate; C: control.

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DOSAGE OF ADRENAL STEROID Cmg>

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PLATE 6

EXPLANATION OF FIGURE

7 A roentgenogram of a {16 day old) rat head showing the lines superimposed to illustrate the method of bone growt b and molar eruption rate me asurement X 4. C: most rostral cusp of first maxillary molar; M: molar alveolar crest; I: incisor lingual alveolar crest; N: superior aspect of nasal bone; Line N-IM is indicative of bone growth.

76

FIGURE 7

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PLATE 7

EXPLANATION OF FIGURES

A photomicrograph of the first maxillary molar .trol rat showing periapical and dental tissues

of a con­X 100.

9 A photomicrograph of the first maxillary molar of a loO mg cortisone-treated rat showing periapical and dental tis~ sues X 100. A: ameloblasts; CT: connective tissue; D: dentin; ES enamel space; HS: Haversian space; O: odonto­blasts; P: pulp cavity; T: trabecular wall of alveolar bone.

77

FIGURE 8

FIGURE 9

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PLATE 8

EXPLANATION OF FIGURES

A photomicrograph of the first maxillary trol rat showing normal eruption (age 16

molar days)

of a con­X 100.

A photomicrograph of cortisone-treated rat tion. (age 16 days) X

the first showing 100.

maxillary molar of apparent precocious

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FIGURE 10

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APPROVAL SHEET

The thesis submitted by Stephanie Jean Zayachek bas '

been read and approved by three members of the tacul ty of

the Graduate School.

The final copies have been examined by the director

ot the thesis, and the signature wb1cb appears below verities

the tact that any necessary changes have been incorporated. ! • .. .

and that the thesis is now given final approval with ref-

erence to content, to.nn, and mechanical accuracy.

The thesis is therefore accepted in pa~ial fulfillment

ot the requirements to~ the degree or Master ot Science.