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Experimental anaylsis of the growth pattern and rates of appositional and longitudinal growth in the rat femur

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NAME AND ADDRESS
DATE
NORTHWESTERN UNIVERSITY
EXPERIMENTAL ANALYSIS OP THE GROWTH
PATTERN AND RATES OP APPOSITIONAL AND LONGI­
TUDINAL GROWTH IN THE RAT FEMUR
A DISSERTATION
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
for tiie degree
DOCTOR OF PHILOSOPHY
DEPARTMENT OF SURGERY
BY
LEON J. ARIES
EVANSTON, ILLINOIS
APRIL, 1940
P ro Q u e s t N u m b e r: 10060838
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uest
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EXPERIMENTAL ANALYSIS OP THE
GROWTH PATTERN AND RATES OP APPOSITIONAL AND
LONGITUDINAL GROWTH IN THE RAT FEMUR
Introduc tion
Purpose of study
Review of Literature
History of study of "bone growth
Embryology and growth of bone
Madder
Alizarine
Statement of the Problem and Approach to the Problem
Method and Materials
Bone sections
Model and preparation
Results of Experiments and Observations
Gross observations
Microscopic observations
Periosteal growth
Haversian bone
Quantitative Assessments
Longitudinal growth - Gross and microscopic
Rates
Gradients
Subperiosteal growth
Discussion of Inference Drawn from Results
Pattern of growth
Mechanics of growth pattern
Model - Method of Preparation
Lines of arrested growth
Outlook
Summary and Conclusions
Tables» Graphs. Diagrams. Translites, and Model
Bibliography
Introduc tion
The problem of bone growth has been studied for cen­
turies and to the present day is not completely understood.
Prom the earliest records, information has been gathered
from purely morphologic studies.
The chief advances in
the knowledge of bone growth have been made using the
physiologic method of vital staining.
The purpose of this
study was to experimentally investigate the growth pattern
and quantitatively assess the rates of growth in long bones
by the method of vital staining.
Review of Literature
The foundation of our knowledge of the mode of bone
33
growth began in 1736 when John Belchier,
an English sur­
geon, was entertained at a dinner by a calico printer.
The entree was roast pig and a joint of this pig was served
to Belchier.
The pink, ruddy color of the bones of this
pork aroused the scientists curiosity and he asked his
host about the origin of the pig.
The calico printer,
being an economical man, used the madder soaked bran from
his dye vats to feed his pigs.
feed to his friends.
The pigs he then used to
Subsequently, Belchier fed madder
root to a cock forcibly and it died in sixteen days.
This
single result of the pink color of the fowl!s bones, he
reported to the Hoyal Society^ and that finished his re­
search.
He was satisfied with his teacher Cheselden1s
2
ry
dicta
that hones grew by addition of ossifying matter
and then, because their hardness resisted further growth,
they stopped growing*
Henri Louis Duhamel, a Frenchman, had spent his early
life in Paris as a student of law and the work of Belchier
was brought to his attention by a physician friend.
In
attempting to verify Belchierfs observations, Duhamel fed
madder to fowls, turkeys, pigeons, and pigs and found
their bones and only their bones were stained.
mal bones stained more deeply than the old.
Young ani­
He submitted
these observations to the Academy of Science. 12
This
paper added little new material to BelchierTs report, but
in 1741-2-3, Duhamel reported work
12
on fractures of bones
and found that the periosteum became swollen about a frac­
ture and stained red when the animal was fed madder.
Hav­
ing given a course in madder feeding and then withdrawing
the dye, he observed that upon later sacrificing the animal,
nearly all the redness was gone from the bone.
He was
then led to believe that the stain was only temporary.
When these bones were sectioned, he observed that the mad­
der colored layer was only covered and hidden by an un­
stained surface layer of bone laid down after the madder
diet had been suspended.
Duhamel then found by alternating
madder diet with periods without madder he obtained red and
white layers on the circumference of the bone.
The last
3
formed layer or plate was on the surface of the bone im­
mediately under the periosteum.
He concluded that bone
grew as wood grew with superposition of layer upon layer.
He also concluded that it was the periosteum that possessed
the osteogenic function.
It was impossible for Duhamel to
explain the enlargement of the medullary cavity which aLso
increased in diameter as the bone grew.
He explained this
on the basis of an expansion of the cortex or shaft as a
whole, while the superficial layers were being deposited.
To prove this he encircled the shaft of growing bones with
rings of silver wire and, over a period of time, found
they had t?cutM their way into the medullary cavity. Duhamel
was thus convinced that the result could be obtained only
by expansion of the shaft.
He then used the experiment
of measuring the longitudinal growth of bone by making
!,benchn marks In the shafts of the growing bone just as
"bench*1 marks are made In the trunks of trees and do not
alter, but remain at a constant level year after year.
Duhamel drilled holes at measured intervals in the shafts
of long bones and placed silver stylets in them to keep
the holes open.
He then measured the distance between
these holes at a later date and found that in the mid­
shaft the distances remained the same even though the total
length of the bone was greater.
Thus he concluded that
growth must take place at the ends of the bones.
He also
observed the epiphyseal lines but left them without fur-
4
tlier study*
Haller,
24
1748, was of the opinion that it was the
arteries which, deposited bone anywhere within the limits
of periosteum since he noticed that ossification always
started at the terminal meshwork of an ingrowing group
of blood vessels.
He believed periosteum was merely a
vascular covering to serve for the nourishment of bone*
This initiated the two schools of thought:
(1) Duhamel1s
osteogenic function of periosteum and (2) Haller’s and
Hunter’s which regarded periosteum as devoid of any bone
forming power*
It was Hunter 41 who recognized the power of living
tissue to resorb and absorb parts of tissues*
In 1754,
Hunter noticed that the three permanent molar teeth had
to find room In the ascending ramus of the mandible which
had to be remodelled to accommodate them.
Thus, he con­
cluded that the bone growth entailed two distinct pro­
cesses, one of deposition and one of absorption*
In 1764
he resorted to Duhamel’s method to test the proof of his
theory of bone growth.
month.
He fed madder to two pigs for one
At the end of this time, he sacrificed one*
The
other he kept for an additional month on ordinary food
before killing it.
The upper surface of the bone was seen
to be surmounted by new bone and a clear Indication of ab­
sorption was present within the bone.
Hunter regarded
5
absorption or bone to take place via the lymphatics.
His
conclusion in the main agreed with those of Duhamel, but
the two investigators did not agree on the property of
periosteum.
Hunter^-*- repeated the experiments of placing
pellets in the tibia of a pig at measured intervals and
later observed that the pellets were lying in the medul­
lary cavity exactly the same distance apart.
The longitudinal growth of bone was studied by
Stephen Hales,
a clergyman and scientist who recorded
an experiment performed in 1747 when he drilled two holes
in a shaft of the tibia of a chicken.
After two months
he examined the shaft and found the distance between the
holes remained the same although the shaft had increased
over an inch in length.
The Bisgards
3
have offered a detailed study of the
accurate direct measurements of the longitudinal length
of bones.
These authors used newborn goats and placed
steel shot in drill holes made in the shaft.
Roentgeno­
grams were taken of the bones at the beginning and end
of the experiment and at intervals during the course of
the investigations over a 12-18 month period.
Final cal­
culations were made from actual post mortem measurements
between shots and the epiphysis.
They concluded that
the lengthening of long bones occurs at the ends and takes
6
place by deposition or layers or new bone between the end
or the diaphysls and the epiphyseal cartilage.
The rate
or growth remains constant during the rirst two months
and then progresses at hair its rormer rate*
Gatewood^ had perrormed this work in 1927 using
shot at varied intervals on the shart or long bones and
comparing the increase in length or the bone. Neither
Gatewood 19 nor the Bisgards *5 were able to determine both
the longitudinal and appositional growth at the same time
in the same animals.
Bone Growth
A brier review or the embryologic development or long
bone is necessary as a basis or study.
Connective tissue,
cartilage, and bone arise from mesenchyme, an unspecial­
ized pack or cells between an internal and external epi­
thelium.
Approximately at the seventh week, the rorma-
tion or enchondral membrane or cartilage bone appears.
Membranous bone appears as a sheet of cells with islands
coalescing and rorming a membrane.
Cells become trapped
in clusters and a bone matrix is dirrerentiated within
this periosteal membrane rorming lamellae or compact bone.
The matrix or ground substance or this bone is either a
syncytial ectoplasm rrom mesenchyme and is Intracellular
or it is a product or secretion or the cells and is Inter-
7
cellular.
With, the appearance of matrix, the cells are
now called osteoblasts and arrange themselves on this
bony plate in a single layer.
As the bone grows, the
osteoblasts become engulfed or trapped in the matrix and
are termed bone cells*
In contradistinction to membranous bone formation,
the bones represented in the skeleton of cartilaginous
fishes pass, in higher vertebrates, through a cartilagin­
ous stage.
These bones are known as cartilage bones*
Additional bones in the skull, made necessary by the
greater development of the brain, develop as membrane
bone, however?^
Cartilage bone may be of two types:
1*
Enchondral and perichondral.
Long bones are pre­
formed in hyaline cartilage which is formed from mesen­
chyme by a multiplication and rounding up of mesenchymal
cells, a decrease in blood supplyaand an increase in inter
cellular material.
This cartilage is suspended in an
elastic hyaline matrix, the outer portion of which is a
fibrous condensation of closely packed cells, and the
inner part, cartilage cells with few or no blood vessels*
The shape of the bone is determined by its cartilage
model.
There are centers of ossification appearing in
each long bone, usually one in the diaphysis and on^in
8
each, epiphysis.
This is a condensation of mesenchymal
cells with no particular arrangement and which is followed
"by an enlargement of the cartilage cells.
These cells ar­
range themselves in rows at right angles to the long axis
of the shaft.
Prom this central ossification center cellu­
lar spaces appear.
The cartilage cells which are mature
and no longer divide, tend to disappear leaving a prim­
ordial marrow cavity.
In this area, calcification begins
to take place on the spicules of remaining cartilage,
forming spicules of bone lined by a layer of osteoblasts.
This forms spongy bone, a phase of chondrogenic osteo­
genesis •
While this is happening, the perichondrium with its
oblong cells covers the whole shaft except the articular
surfaces.
The periosteal buds of osteogenic tissue push
their way into the degenerating cartilage cells and pro­
ceed to proliferate and differentiate.
The cartilage cells
have begun to swell up and the intercellular material to
rarefy.
Blood vessels invading with the periosteal buds,
accompanied by osteoblasts actively Invade the cartilage
in several places along the shaft, making up the primary
center of enchondral bone formation.
Is now known as periosteum.
The perichondrium
The perichondrium in the
interim has laid down a layer of bone about the periphery.
We now have a tube of bone surrounding cartilage with
9
periosteal buds forming new bony trabeculae within the
shaft •
Continued resorption and erosion of cartilage cells
about the entrance of the large blood vessels in the
shaft forms the medullary cavity.
New bone continues to
be deposited on the periphery and in the long axis of
the bone in the form of axial channels forming the
Haversian canals.
The longitudinal growth of bone also occurs by car­
tilage proliferation with invasion of cartilage cells from
the diaphyseal ossification centers.
The youngest cells
are near the epiphyseal line while the more mature are
toward the diaphysis where they are degenerating and being
invaded by the osteogenic bud.
Thus the cartilaginous
epiphyseal plate is a series of rows of cartilage cells in
long columns undergoing cellular division near the epi­
physeal line.
The eytomorphosis is as follows: As the
cartilage cells recede from the epiphysis they become lar­
ger and vacuolize.
appear.
fies.
The nuclei shrink and mitochondria dis­
The anpty lacunae coalesce and the matrix calci­
These lacunae become invaded by osteogenic cells
from the diaphysis and calcify these cartilaginous septa
converting them into bone trabeculae.
Secondary centers
of enchondral ossification take place at the ends of the
10
bones In the same manner.
They are termed epiphyses.
In the femur there is one appearing in the first year
being the head of the femur, and the seond in the car­
tilaginous great trochanter in the fifth year.
The enchondral bone spreads in the shaft to occupy
the whole shaft down to the epiphyseal plate and in each
epiphysis from the cartilaginous surface of the joint
space toward the epiphyseal plate bordering on the dia­
physeal end of the bone.
There is a continually progres­
sive replacement of cartilage by enchondral bone.
En­
chondral bone later replaces the cartilage in the tro­
chanters, extends upward toward the neck of the femur and
replaces all the cartilage except the epiphyseal plates.
It is at these plates that extension of enchondral bone
of the diaphysis is said to take place.
It is also stated
that periosteal bone formation Is more active on the proxi­
mal or upper side of the neck than on the lower side.
It has been demonstrated that pegs inserted in the
shaft of a long bone a measured distance apart remain the
same distance apart even when there is an elongation In
the total length of the bone.
This is not true of the
diaphyseal end of the shaft near the epiphyseal cartilagin­
ous plate
When one of the pegs is placed in the lower
shaft above the cartilage plate, and the other peg in the
epiphysis, they separate widely as the bone increases in
11
length..
This was said to prove that the growth takes
place in the epiphyseal cartilage plates which inter­
vene "between the secondary ossification centers (epiphy­
sis) and the primary center (diaphysis).
This type of
"bone deposition and ossification is classified as appositional growth where cartilage cells are changed to
"bone and are added to the ends of the bone.
This differs
from interstitial growth or internal growth by which ex­
pansion of the original cartilage model takes place.
When a metal band is placed about the shaft of a
young bone it will not be visible on the external surface
of the shaft after several weeks, but layer may be found
lying free in the marrow cavity.
This is brought about
by deposition of new compact bone on the outer surface
of the shaft of the bone with a disappearance or resorp­
tion of bone on the inside enlarging the marrow cavity.
Madder
The method of vital staining with madder was not new
although it was noticed by Belchier in 1736. It was
known to the Greeks. 27 The Egyptians brought madder
from India where they learned the trick of combining it
with calcium, aluminum salts, and polymerized fatty acids
as mordants. 42
Madder is a powdered root of Rubia Cordifolia in
12
India and Central Asia,
In Europe, it Is the Hub la
Peregrina (native wild plant) of England, Wales, and
Ireland.
The U. S. Dispensatory refers to it as Rubia
Tinctorium*^
The active principle of the madder root
dye Is alizarine and purpurin and its name was received
from the trade name Levantine madder root.
The dye
occurs as a glueoside (ruberythric acid) in the roots
in 2-4 per cent proportions and is obtained by fermenta­
tion or boiling with dilute acids.
It is a reddish yel­
low powder insoluble in cold water and slightly soluble
in boiling water.
However, it dissolves in hot alcohol,
ether, benzine, glycerine, or glacial acetic acid.
Alizarine
Alizarine was isolated from madder by Robiquet and
p
Cobin In 1827 •
G-raebe and Liebermann (1870)
22
showed
it was a glucoside of the anthraquinone type, 1-2 dihydroxyanthraquinone •
Ott
- OH
it
0
15
Perkin (1870)^6 found the sodium sulphonate salt, ali­
zarine red S which was more soluble and more stable than
alizarine*
^
ii
-OH
u
O
Alizarine has a varied tinctorial gradient depend­
ing on the pH of the medium*
At pH » 5, it is yellow;
at pH b 5 to 6*8, red; and at pH greater than 6*8, it is
purple*6
Alizarine, when injected into fish and amphibia, is
toxic*
Tadpoles and frongs can take small doses*
not as toxic for reptiles, snakes, and lizards.
It is
In ver-
0
tebrates, it is toxic in large doses*
Following the intraperitoneal injection of 1 cc. of
a 2 per cent solution of alizarine red S, there is a
staining within thirty minutes of the intercellular
deposits of calcium.
The liver and kidneys are stained but
this coloring is quickly removed.
The dye continues to be
excreted by the kidney and gastrointestinal tract for five
to six days.
Some dye remains in the bones and teeth*
14
The younger the age of the animal injected, the deeper Is
the color of the stain in the bones and teeth while old
animals take the stain poorly or not at all*6
Thus,
there is a selective staining of bone and teeth which
are growing and calcifying at the time of injection*
The actual staining of bones was first recorded by
Lemnius (1576)^ and Mizaldus (1592)^ and then by
Belchier in 17 36 * ^
Gibson, ^
in 1805, noticed the
staining powers of young and old animals with alizarine#
Wolf, 1868, worked with pigeons*63
Mass®® (1872) worked
with hens, Strelzoff5^ (1873) with doves, Kastschenko3^
(1882) with frogs, Schreiber5^ (1904) with rabbits and
frogs, Reimers and B o g e ^ (1905) with dogs, Brash^ (1924)
with pigs, Broell 43 (1926) with hens, dogs, and cats, and
Handelsman and Gordon^3 (1930) with rats*
Strelzoff,
52
in 1873, tried to remove the organic
constituent of alizarine-stained bone with boiling caustic
soda, and found the color persisted*
Gottlieb
21
,
(1914)
concluded that the alizarine stain deposited in those
tissues where calcium was being deposited at the time of
administration.
Cameron
concluded that alizarine could
form dye lakes with calcium*
When given to vertebrates,
there was a selective staining for calcifying bones*
The staining was independent of all soluble constituents
15
of bone, of the blood vessels of bone and of the living
bone cell.
It appeared to be dependent on the earthy
salt content, especially of recent deposits of calcium.
Hoffman^® (1939) has demonstrated that the staining
effect of alizarine red S in bones is dependent upon the
pH value of the colloidal matrix at the moment the cal­
cium precipitates to its earthen state.
The Problem
The purpose of these experiments was to determine an
adequate method of investigating the growth pattern of
bone and quantitatively assess the longitudinal and appositional growth of long bone by the method of vital
staining.
Review of Approach to the
Problem
Various fractures occurring in the bones of the body
do not heal adequately or completely fail to unite.
Frac­
tures through the neck of the femur are notorious for
their high incidence of non-union and resorption of the
capital fragment.
The numerous types of treatment in use
is evidence of the inadequacy of our present therapy.
Could one stimulate the rate of growth of bone by some
chemical means, it would promote healing of the fragments
before resorption of the bone could take place.
The literature abounds with studies on the value of
16
. .
35,16
-i ■ _ 13 -i
certain
procedures,
*
chemicals,
hormones, 2,14,15,17
• * *
diets, 40 or enzymes 54 Tor the increase or decrease in the
rate or hone deposition.
Wo one of the investigators has
an adequate method by which he can accurately measure
the increase in deposition of hone hut has used crude
methods as x-ray comparisons or morphologic measurements
of the gross specimen.
Our original experiments were carried out in the same
manner hy producing artificial fractures through the neck
of the femur in dogs and approximating and immobilizing
the fractured ends so that healing could take place.
The
only available methods of studying the rate of hone growth
are those of repeated x-ray studies and Judging the rela­
tive amount of callus deposited about the fracture.
method of comparison is of no scientific value since only
a vague impression is gathered of the amount of calcium
deposited about the fracture site as viewed on the x-ray
film.
Fractures of the femur neck do not heal with callus
formation.
Hesuits of examination of our dogs under thera­
py, with glutathione^® and "sulphydryl containing sub30
stances" varied greatly within each experimental group.
A controlled series of normal animals with fractures also
varied so greatly that no conclusions could he drawn from
the experimental work.
This resulted because of the fact
that, to date, there has been no adequate method for the
17
measurement of appositional and longitudinal growth.*
The method of total hone enlargement hy direct measure­
ment was used, hut since there is a constant remodelling
of hone there is no fixed point or plane from which to
start*
The amount of bone deposited in any given inter­
val cannot he ascertained even after sacrificing the
animal and measuring directly*
Direct measurements and
comparative x-ray studies were abandoned since they were
confusing and inconclusive*
The vital staining of animal hone would place a
definite plane or landmark in the hone from wiich measure­
ments could he made.
The following experiments with
madder root dye were attempted.
Dogs, rabbits, and rats
were fed synthetic alizarine hy placing 2 per cent
aqueous solution of alizarine sulphonate over their food
for varying intervals of time.
Animals will not ingest
natural madder root dye hut do not object to the food
covered with synthetic alizarine sulphonate.
The dye
deposits in the bone ossifying only at the time of inges­
tion.
That hone already present does not take the stain.
The dye was given for one, two, or three weeks and then
withdrawn from the diet.
This resulted in a diffuse pink
stain about the sites of the fractures but there was
practically no staining of the periosteal hone deposit.
The animals were too old and not growing sufficiently
18
rapidly.
Feeding the dye to younger animals resulted in
wide zones of diffuse pink stain along the periosteal
layers and a more intensely diffuse pink stain at the ends
of bones.
This wide pink zone in the bone was not sharp­
ly demarcated from the surrounding previously deposited
bone.
This was in reality a repetition of the work of
Hunter and did not outline the pattern of growth.
Thus
feeding of alizarine did not offer a method of measurement
of the amount of bone deposited over any given period as
the staining was too diffuse.
Schour and H o ff m a n ^ >
^ ,50,29 measured the apposi-
tional growth of the incisor and molar teeth in the rat,
as demonstrated by alizarine red S.
These workers in­
jected the rat with alizarine red S at varying intervals
and measured the amount of appositional growth over that
given period.
Their work opened a new field of research
on growth of teeth and bones and directly suggested a simi­
lar study on the rat femur.
Method and Materials
This study is based on the examination of 180 albino
rats ranging from 7 to 304 days of age.
Intraperitoneal
injections of a 2 per cent isotonic alizarine red S solu­
tion (Coleman and Bell Co.), Color index #1034, were
given at intervals of 7 to 101 days.
A two per cent solu-
19
tlon of tlie alizarine dye was made by dissolving finely
powdered alizarine in 0*4 per cent warm saline solution.
The 0.4 per cent saline is used to make the solution
equivalent to the osmotic pressure of mammalian serum. ^
An early series of young rats was injected with
various percentage strengths of alizarine solution.
The
animals either died of the large dosage injected or the
bones were not stained when the smaller dosage was ad­
ministered.
Intravenous injection caused death of the
animals in a few minutes in respiratory and cardiac fail­
ure.
There were no convulsions.
The smaller dosage of
alizarine Intravenously did not produce a definite stain­
ing of the bone although the teeth were stained.
Spilling
of alizarine solution into the subcutaneous tissue caused
a hard indurated area about the Injection site followed
by a slough.
The intraperitoneal route was most satisfac­
tory as the animals could tolerate a sufficiently large
dose for staining of the bone.
The pharmacological dosage
was experimentally found to be approximately 100 mg./kg.
The macro- and microscopic measurements were based on 52
animals (Tables 1, 2, 3, and 4).
Number of Injections:
On the basis of thirty pre­
liminary trial animals, injections were given to three
groups of ten animals each on the seventh, fourteenth, and
20
twenty-first days respectively#
The animals injected
weekly and bi-monthly had lines in their bones that were
overlapping or too close together to be of value, espe­
cially when the animal was over 100 days of age.
The
younger animals injected at 21-day Intervals presented
lines that were far enough apart to be accurately measured.
In older animals it was necessary to allow thirty or more
days to elapse between Injections.
The trial animals
were given as many as ten Injections and when sacrificed
presented alizarine patterns so complex it was impossible
to follow any one injection throughout the bone.
It was found advisable to limit the number of injec­
tions to one or two for each animal since more than two
lines caused confusion as to which injection produced
which line.
For convenience, the animals were allocated
into Incremental age groups depending upon the days on which
they were injected (Table l).
Dissection and the Preparation of Microscopic Sections:
The usual method of making microscopic sections
of bone for histologic study by decalcification in acid,
could not be employed in this experiment since the red to
purplish effect of the alizarine red S in the bone is de­
colorized at a pH of 5.
G-ross specimens were preserved
in 10 per cent formalin to which magnesium carbonate had
21
been added.
It Is important to keep the formalin alka­
line as numerous early specimens were decolorized in
formalin labelled neutral, but actually was slightly
acid.
The presence of a precipitate of magnesium car­
bonate In the formalin assured alkalinity.
After clean­
ing the bone of all muscular and tendinous attachments,
cut sections were prepared at the following levels
(Pig. 1 and 2):
(A) through the neck and head, (B) trans­
versely through the shaft, distal to the lesser trochan­
ter, (C) transversely through the diaphysis, and (D)
longitudinally, mid-sagittal medio-lateral sections*
The original grinding of sections of bone was done
by hand on an oil stone.
The bone was ground to a thin­
ness sufficient to allow for microscopic examination.
The
sections were thick and filled with debris of oil in the
Volkman’s canals obscuring the detail.
the bone fragmented and was lost.
When ground thin
Long sections could not
be made by this method as the bone would crack and break
before It was ground sufficiently to be transparent.
The following method was evolved.
The bone was cut with a 0.5 mm.
1ewelerTs saw blade
which was mounted in an appropriate frame.
One surface
of the cut section was ground smooth on a circular fine
grade carborundum stone attached to a laboratory lathe
22
(Pig* 3).
This section was dehydrated in alcohol and
treated with acetone.
The smooth surface was fixed in
cellulose cement to the flat ground facing of a cork#
The cement served as an embedding medium and to attach
the section to the <york.
When the cement was completely
set (4 hours) the exposed surface was ground until a
thickness of 20 to 50 microns was obtained.
While grind­
ing, the bone was continuously cooled and washed free
of debris by a stream of water passing through a brush
placed against the stone (Pig# 5)#
Pinal polishing of
the section was completed on a hard Arkansas stone (Fig.3)#
The bone was removed from the
acetone.
cork by immersion in
Several changes in fresh acetone were neces­
sary to free the sections of excess cellulose cement#
The sections were then dried and cleared for 24 hours in
xylol and mounted on the slide in gum damar#
It was
found that the red stain faded and disappeared in several
weeks from sections which were mounted in balsam.
One
half of the longitudinal section of the femur was ground
and mounted as above while the opposite half was immersed
in xylol for clearing.
The cleared bones were examined
with the aid of a binocular dissecting microscope while
suspended in the clearing solution.
The red injection
effects could be studied from a three dimensional aspect.
23
Model:
To clarify in our minds the three dimension­
al effects of alizarine red S in the bone it was neces­
sary to construct a model of the femur.
A model approxi­
mately six times the size of that of a 150 day old rat
femur was first carved of a block of dental modelling
wax.
The positive produced was cast in a hydrocolloid to
make a negative.
A positive plaster cast of the femur
was made but since it was not transparent did not give
the three demensional superimposition of alizarine effects.
A transparent model was necessary.
G-lass was considered
first but the necessity of a metal negative flask made
this impractical because of the expense Involved In making
a few models.
Recent advances in the fields of plastics have produced transparent non-breakable objects of acrylic resins 18
(resinous products are obtained by polymerization of mono­
meric derivatives of acrylic acid).
and methacrylic acids and esters.
These include acrylic
The formula of acrylic
acid being CHr>=CH-C0QH, the alpha methyl derivative is
CH2=C(CHg)-C00H.
The esters of methacrylic and acrylic
acid polymerize under heat and give sodium peroxide, per­
oxide and benzyl peroxide.
The acrylic resins vary from
a soft sticky semi-solid liquid to a hard, tough, thermo­
plastic solid.
Polymethylmethacrylate Is used in cast
sheets and is light and transparent.
The monomers are
24
are mobile liquids which polymerize on heating in the
presence of a catylist.
The sheets and bars may be
moulded at temperatures of 180 to 250 degrees Fahrenheit.
The combination of the monomer and polymer of methylmethacrylate were used.
It is necessary to compress the
methacrylate mixture under pressures of 2000 to 3000
pounds.
This necessitates a negative mould strong enough
to withstand this pressure.
A steel flask was constructed
in two halves and impression stone was poured into this
form about the original wax model.
When the stone was set,
the wax model was melted out of the stone negative.
Each
half of the model was made separately to form a medullary
cavity.
The two half stone negatives encased in steel
flasks were covered with tin foil cemented to the stone
with rubber cement to make a smooth surface.
The powdered polymer and liquid monomer were mixed
in the proportion of 3 to 1 respectively and placed in
the foiled negative after the flask was heated to 80 de­
grees in a water bath.
A test packing was made using
cellophane to prevent sticking.
The negative was filled
repeatedly and packed until the excess ran over the sides
as a sticky, soft resin and was trimmed away.
The flask
was now closed and compressed by threaded bolts and nuts,
made for the purpose.
The whole flask was heated at
25
256 degrees Fahrenheit for three hours in an autoclave#
When allowed to cool the model was removed and except
for a few bubbles which are difficult to exclude, using
the crude type of flask and compression, the model was
transparent•
The model was made in two half sections so that the
pattern of growth could be represented within the medul­
lary cavity.
With a dental burr and disc, the pattern
of growth was carved into the transparent model and the
grooved lines thus produced were filled with colored inks.
This was covered with transparent shellac to prevent its
wearing away.
The result is a three dimensional model
to elucidate the findings described in this paper.
Transparent model, Figure 4.
Method of Examination:
Gross specimens were measured
for length and diameters over-all with a Boly gauge.
Readings were made correctly to l/lOO of a millimeter.
Microscopic studies of the ground sections were made
under the 52 and 16 mm. objectives.
A vernier filar
micrometer eyepiece which was calibrated to a standard
micrometer stage for the microscope and lenses was utilized
26
to make the measurements between the injection effects
of alizarine red S*
The measurements were made in mi­
crons and were calculated correctly to within the first
decimal place.
Measurements were made at the levels shown in
Figures 1 and 2.
The mean values, standard deviations, and error of
the mean v/ere calculated for the specific levels of
animals within specific age groups (Table 1).
The measurements of the means for each animal were
plotted again^ttime (Figures 7, 8, 9, 10), both arith­
metically and logarithmetically.
Results of Experiments and Observations
Gross Observations:
Gross examination revealed a
diffuse, purplish-red stain of the femur in animals in­
jected with alizarine red S from birth to 15 days of age.
Animals injected at 15 days of age and sacrificed at 50
to 60 days of age presented a diffuse, purplish-red stain
in the metaphyseal portions of the bone and a colorless
diaphyseal bone s.s well as a colorless subcapital seg­
ment.
The junction of the colorless diaphysis with the
shaft showed an intensely stained band which encircled
the bone at that level.
The longitudinally cut section
27
(Fig. 2) of the bone presented a diffuse red stain in the
medullary cavity, but the sharp differentiation between
colorless diaphysis and stained shaft was not seen as a
sharp line and faded gradually toward the distal end.
The circumferential colored lines on the surface of
the cortex in the shaft of the bone were parallel to the
epiphyseal plate and varied in their distance from the
epiphyseal plate in approximately direct relation to the
number of days intervening between injections.
The ani­
mals which were given injections at varied intervals of
time presented parallel colored rings about the shaft, the
distance between which was the amount of longitudinal
growth at the diaphyseal ends of the bone for that interval.
The longitudinal mid-sagittal sections (Fig. 5) pre­
sented oblique, red lines extending from the medulla of
the shaft outward and toward the epiphysis to end tangentially at the surface of the bone with the parallel
lines mentioned above.
This would be at the epiphysis if
the animal were injected and sacrificed within a few days.
When 10 to 30 days elapsed between an injection and the
time the animal was sacrificed, these oblique red lines
were found a distance away from the epiphyseal plate and
ended at the surface or periosteal red line.
This arrange­
ment of a circular base with oblique lines beginning at the
28
cortical margin and extending away from the epiphysis and
medially formed a red truncated cone.
The cleared femurs
of animals which had been given one injection presented
this picture in three dimensions when examined with a bi­
nocular dissecting microscope.
This is readily seen in
the transparent model (Pig. 4) and corresponds to the blue
line in Figure 5.
When a second injection was given SO
days later the oblique red lines were found epiphysially
to the first line and were parallel to them.
These oblique
lines ended at the periosteal surface of the cortex where
the red circular lines were found (Fig. 4).
The perimeter
of the base of this cone formed the circumferential lines
seen on the surface of the cortex (Fig. 6).
Each succeed­
ing injection line was found epiphyseally to the previous
one in the form of one cone within another.
The base was
the epiphyseal plate but as more bone was deposited, the
legs and the perimeter of the cone (Fig. 5) remained in the
shaft as fixed planes.
The base remained as the epiphyseal
plate and did not take the alizarine stain.
Subsequent injections formed new cones which were seen
to dove-tail within the cones formed by the previous
alizarine red S effects.
The epiphyseal plate was now
found to have receded from the levels of the initial ali­
zarine red S rings (on the surface of the bone) and was
approximately parallel to them.
29
The Neck:
Longitudinal sections through the neck
(Fig# la) included an oblique section through the shaft
and lesser trochanter.
The oblique endosteal red lines
passed from the marrow cavity of the neck upward and out­
ward toward the capital epiphysis.
The periosteal de­
posit was found about the neck and was continuous with
the red line in the trochanter and shaft (Figs. la and
5).
A morphologic arrangement of the alizarine red S
lines, similar to that of the distal end of the femur,
existed in the neck.
Similar truncated cones appeared
superposed one within the other excepting that the base
was proximal while in the condylar end of the femur the
base of the cone was distal.
Periosteal Growth:
Concomitant with the longitudinal
growth of bone there was seen a subperiosteal deposition
of bone about the entire shaft.
This subperiosteal de­
position of bone showed the alizarine red S effects.
When
viewed in cross section, circular red rings were seen to
parallel the periphery of the bone.
Cross sections through
the shaft at the subtrochanteric level (Fig. lb) showed
circular red lines in the cortex.
The number of concen­
tric lines at this level was directly representative of
the number of injections administered.
Transverse sections through the diaphysis presented
30
three different findings dependent upon (l) the level of
the diaphysis through which the section was made, (2) the
number of injections, and (3) the interval of time be­
tween injections.
When one injection was given one or two circular
lines were found.
When two injections were given, two,
three, or four concentric red rings were seen (Fig. 5a,
b, c).
The distance between the lines was not regularly
equal as those found in the subtrochanteric region of
the shaft.
To clarify the discrepancy in the number of
circular red lines found in the diaphysis as compared
with the number of injections given, it was necessary to
restudy
the cleared
sections.The junction of the red
line of
the cone on
the innersurface of the shaft with
the periosteal deposit showed two lines on cross section
(Fig. 5c).
These junctions are demonstrated in the di­
aphysis of the transparent model when opened and viewed
from within
size of
(Figs. 4 and 5). The blue line indicates the
the bone at
the time of the first injection.
The
red line indicates the size of the bone at the time of
the second injection.
Harersian Bone:
in the
The walls of the vascular channels
Haversian systems showed the alizarine red S ef­
fects in the arrangement of concentric rings.
The con­
centric lamellae of the bone cells were parallel to these
31
red rings*
Bone growth within the Haversian systems oc­
curred as a result of deposition of new hone on the wall
of the vascular channel and caused a diminution in the
diameter of the channel*
Quantitative Assessments
Longitudinal Growth - Gross:
The lengths over-all
of the femurs on the hasis of 10 day intervals from 10
to 300 days showed that the daily rates of growth de­
celerated with age increase (Table 3, Pig* 8)*
These
measurements closely coincided with similar data on longi­
tudinal growth of the femur as shown by Donaldson*^
Microscopic:
Measurements which were made between
the last two alizarine red S effects for specific age
groups (Table 1) showed that the increase at either the
distal or the proximal ends alone was not equivalent to
the total increase over-all for the femur at any given
period*
The sum, however, of the vertical increases at the
distal and proximal ends closely coincided with the total
increase over-all (Table 2, Pig* 7)*
Rates:
The daily rates of growth, both experimental
and calculated, as shown by the alizarine red S effects
decelerated with age increase*
The latter assessments of
daily rates (sum of distal plus proximal) closely coin­
cided with the calculated rates which were obtained from
32
the curve on measurements of length over-all (Table 2,
Pig. 7).
Gradients;
Prom the plotting of the decelerating
rates or semi-logarithmic graphy paper (Pig* 8), it is
evident that a gradient of growth exists in the longi­
tudinal growth of the rat femur*
Logarithmic plotting of the total increments (Fig* 9)
resulted in a straight line graph and also makes it evident that a gradient of growth exists (Huxley31 ).
Subperiosteal Growth:
Measurements between injec­
tion effects in cross sections below the lesser trochan­
ter showed that the distance between any two red lines
was the amount of bone deposited for the given period at
that level*
The rates of growth were seen to be greater on the
anterior than on the posterior surface (Table 4, Pig* 10)*
The growth of bone beneath the periosteum also de­
celerated with age increase for anterior and posterior
surfaces (Table 4, Pig* 10)*
Statistical Evaluation on the basis of 1034 trial
measurements:
£
The standard deviation was found to be
0*990 with a mean error of t
0.249*
33
£ fd»
M
M
=
'
28.0
7
x 0.2
M
mean
G
assumed mean
a1
deviation from
assumed mean
f
frequency
C
interval (0.2)
arbi trary
N
M
=
Standard Deviation
C.
=.
_( ^
0 2^23
19
= 0.2 W 31.85 - 7.344
- 0.2 W 24.506
• 0.2 x 4.95
O
= ±
0.990
Probable Standard Error
ME
-
total measurements
standard deviation
28.542
6
=
O'
x 100
=
eg
\fiP
0.990 x 100 = 37 x 100
2.64
Probable error of the mean
« 0.6745
Q
x
■ +0.249
vTT
34
Discussion
Pattern of Growth:
■zp Q
bryology
Textbooks of histology and em-
-1
* 9
have, to the present time, described the
longitudinal growth of bone to take place by apposition
at the diaphyseal side of the epiphyseal plate in the
form of lines parallel to the epiphyseal plate.
Our
findings show the longitudinal apposition to be on the
endosteal surface in the form of a cone whose base is
the epiphyseal plate.
Multiple injections of alizarine
red S have demonstrated that the appositional process re­
sults in a series of cones, each
succeeding injection
effect dovetailed within the previous.
From a three di­
mensional aspect (Fig. 4), it can be demonstrated that
each successive endosteal deposit at the base (epiphyseal
plate) possesses a greater perimeter than the previous one,
and consequently produces a flare at the piphyseal line.
The latter accounts for the increasing flare at the epi­
physeal line.
The latter accounts for the increasing
flare at the epiphyseal end of the bone with advance in
age•
As a result, there is a tubularization pattern in
the growth of the long bones.
The growth of the periosteal bone would, off-hand,
appear to limit the pattern of bone to a straight cylinder
with parallel walls.
However, the periosteal growth follows
35
the pattern of the internal cone and serves to increase
the width of the cortex with increase in age.
Preliminary
assessments of periosteal growth on the same material
have shown that the rate of growth is greater in the region
of the shaft near the subtrochanteric area than at the
diaphysis.
Hence, approximately the total diameter of the
bone at the epiphyseal line is due to the flare of the legs
of the cone within the diaphysis (Fig. 5).
This morpho­
logic pattern which is found at the distal end of the
femur is also seen in the neck, though quantitatively to
a lesser degree in the latter.
The endosteal arrange­
ment in cones as found in the femur was also found in the
other long bones.
Mechanics of Growth Pattern:
The structural growing
pattern of the femur consists of two series of truncated
cones with bases facing away from the shaft at both ends.
The tibia at its proximal end presents a pattern which is
similar to, and approximately a mirror image of, the
growth pattern of the distal end of the femur.
Hence, on either side of the knee joint there is a
series of truncated cones whose bases are only separated
by the epiphysis of the femur and tibia respectively.
Weight is transmitted from the shaft of the femur to its
wide distal base and received by the corresponding cap
36
at the proximal end of the tibia.
Such an arrangement is one of the strongest geo­
metric configurations known to engineering for weight
support and serves for balanced equilibrium in force
distribution.
Columns with a cap and base are commonly
employed by architects to support buildings.
Model;
It was necessary to construct a trans­
parent model of the femur to clarify in our minds the
three dimensional effects of alizarine red S.
The model
may be illuminated by placing a light at one end*
This
plastic transmits light to its opposite end and demon­
strates the alizarine effects, carved into the surface.
Lines of Arrested Growth;
The peripheral base of
each cone explains the so-called Harris1 f,lines of arrested growth.*'
on x-ray films.
26
Harris has described these lines seen
Brash
has superposed serial x-rays of
Harris’ and has attempted to explain tubularIzation of
bone on this basis.
At present we are utilizing the
alizarine red S method of vital injections to experimental
ly elucidate the significance of these "lines of arrested
growth.11
Outlook:
The method for study of normal bone growth
as described in this investigation may be readily em—
37
ployed in experiments dealing with studies (1) involving
growth stimulating or growth arresting substances, (2)
healing of fractures, (3) bone grafting, and (4) the fate
of grafts, and other problems related to normal and patho­
logic conditions in bone.
Summary and Gone 1us ions
1.
This study was based on the gross examination
and microscopic study of the ground longitudinal and cross
sections of the femurs of 52 albino rats, 10 to 305 days
of age, which were given multiple intrap er it one al injec­
tions of a 2 per cent solution of alizarine red S (100 mg./ljg.)
at specific intervals.
Quantitative assessments were made of the rates of
longitudinal and subperiosteal growth.
The pattern of
morphologic growth was analyzed as a result of the alizarine
red S effects.
2.
Findings show:
(a)
The longitudinal growth of long bones is
a result of superposition of serial cones within the
diaphyseal portion of the shaft, and the bases of which
cones are the epiphyseal plate at any given period.
(b)
The periosteum deposits bone on the circum­
ferential surface of the shaft only, and does not con-
38
tribute toward the longitudinal growth of the femur*
(c)
The distal end of the femur grows faster
than the proximal end (neck)*
The sum of the growth at
both ends accounts for the total increase in length*
(d)
The longitudinal growth and the growth
in width follows a growth gradient--the rates decelerating
with Increase In age*
(e)
Haversian system growth as shown by the
alizarine red S effects occurs on the surfaces of the
vascular canals*
The red rings were seen to parallel the
Haversian lamellae*
The diameter of the canals during
appositional growth decreases with age.
3.
Colored photomicrographs, diagrams, tables of
measurements, graphs, and a three dimensional model are
presented for further elucidation of the findings.
4.
The morphologic pattern of the tubular!zation of
bone is compared to mechanical supports as utilized In
engineering and bears out the application of this mechani­
cal arrangement to the knee joint.
5.
Suggestions are offered for further investigation
on normal and pathologic studies on bone, utilizing the
vital injections of alizarine red S.
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51.
Schreiber: Arbeiten aus d. path. inst. Tubingen,
4:257, 1904
52.
Strelzoff, S. J.: Zur Lehre von der Knochenentwi eke lung,
Gentralbl f. d. med. Wissensch. 11: 1873
53.
Wolff, J.: Ueber Hnochenwachsthum vorlaufige Mittheilungen, Berliner Klin. Wchnschr. 110:1868
54.
Zaivisch-Ossenitz, Carla: Die Beeinflussung des
Knochenwachstums durch Fermentwirkung, Wien. klin.
Wchnschr. 45:321, 1932
Table 1: Experimental History or 52 Albino Rats given
Injections of* Alizarine Red S from 11 to 299
Days of Age*
Group
No.
Arbitrary
Age Range
of Animals
in Days
1
10-30
2
30-60
Number of
Animals Al­
located to
Each. Group
10
Age in
Interval Age
Days when Between
when
Injected Injection Sacrificed
Days
11-14-18
26
7-14-25
30-33
32-33-48
63-64-71
31-32-36
38-57
18-24-28
30-33-37
111-120
64-68-102
123
3
60-100
14
62-72-77
78-85-87
93-99
14-21-30
41-61-62
99-113-121
133-140
141-154
4
100-150
13
102-103106-107
108-123
142-143
9-14-30
31-33-40
41-60
113-123
129-133
140-141
154-171
173-192
2Q5
5
150-200
162-163
172
9-29-30
31-33-61
171-192
205
200-305
205-233
299
5-28-30
33-94
245-256
304-292
101
Table 2: Showing Total Longitudinal Increments of Growth
In the Femurs of 52 Albino Rats from 11 to 304
Days of Age.
No.
of
Rat
Age
In
Days
151 32
132 40
150 48
148 48
105 63
127 63
106 64
106 64
135 68
104 71
105a 71
138 99
139 99
108 102
108 102
110 102
111 111
140 113
140 113
100 120
142 121
109 123
109 123
160 129
113 133
101 140
103 141
103 141
112 154
114 154
116 171
115 173
117 173
118 192
119 205
119 205
120 205
126 256
79 292
Growth
at Dis­
tal End
in mm.
4.2
2.1
4.32
3.90
3.75
3.00
3.84
6.72
4 .60
3.91
13.00
3.08
2.80
3.80
5.0
3.10
3.60
1.25
3.78
2.10
2.15
2.33
5.22
2.35
2.97
1.80
2.04
4.62
1.89
2.25
2.60
3.40
2.01
1.05
2.17
0.88
0.75
1.78
0.245
Growth
at
Neck
In mm.
Length
of
Femur
1.04
0.40
0.80
0.80
0.53
0.54
0.53
0.78
0.80
0.69
2.86
0.50
0.39
1.14
1.10
0.60
0.80
0.35
1.05
0.48
0.22
0.31
0.67
0.31
0.48
0.03
0.38
0.75
0.27
0.125
0.40
0.60
0.66
0.31
0.50
0.20
0.58
0.86
0.01
13.4
17.6
19.2
19.6
24.3
24.3
24.3
19.2
24.3
24.3
13.6
29.17
29.17
28.0
28.0
29.17
29.10
32.1
29.0
31.2
31.2
31.2
29.0
31.5
32.2
32.5
32.4
31.4
32.5
33
34.2
33.5
33.5
35.6
35.8
36 •6
36.6
37.0
37.0
at
Age
(In
days)
Total
Length
in mm.
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
at
18
26
32
33
48
48
48
32
48
48
14
71
71
63
63
71
71
99
71
99
99
99
71
99
113d
120
120
99
120
129
154
140
140
171
173
192
192
205
205
18.6
19.2
24.3
24.3
28.58
27.84
28.67
26.68
29.70
28.89
29.46
32.68
32.36
34.10
34.10
33.80
33.5©
33.70
33.78
33.78
33.57
33.80
34.80
34.60
34.75
34.33
34.81
36.77
34.66
35.37
37.20
37.40
36.70
36.96
38.40
36.79
37.69
39.6
37.3
Table 3:
Calculated Dally Rates of Longitudinal Growth.
of Femurs for Ten Day Increments
Distance for period
Rate for any period « Time of that period
Day-
Mm./Day
Days
Mm. /Day
10
1.06
160
0.06
20
0.56
170
0.02
30
0.28
180
0.03
40
0.16
190
0.02
50
0.29
200
0.03
60
0.27
210
0.04
70
0.23
220
0.03
80
0.13
230
0.00
90
0.08
240
0.00
100
0.09
250
0.02
110
0.09
260
0.00
120
0.09
270
0.00
130
0.02
280
0.01
140
0.08
290
0.00
150
0.06
300
0.00
Table 4:
Calculated Daily Hates of Periosteal Grovirth
at the Anterior and Posterior Surface of the
Demur Just Below the Lesser Trochanter in
52 Albino Rats*
Age Interval
Anterior
Amount of Increase
Growth in per Day
Microns
in Microns
10- 20
20- 30
30- 40
40- 50
50- 60
60- 70
70- 80
80- 90
90-100
100-110
110-120
120-130
130-140
140-150
150-160
160-170
170-180
180-190
190-200
200-210
210-220
220-230
230-240
240-250
250-260
260-270
270-280
280-290
290-300
130.0
120.0
100.0
90.0
60.0
40.0
30.0
29.9
29.5
29.0
28.0
24.0
22.0
20.0
19.9
16.0
15.8
14.3
12.0
10.0
9.0
8.5
7.0
7.2
6.8
6.0
5.5
5.0
5.0
13.0
12.0
10.0
9.0
6.0
3.0
2.99
2.99
2.90
2.80
2.40
2.20
2.20
1.99
1.60
1.58
1.51
1.43
1.20
1.00
.90
.85
.70
.72
.68
.60
.55
.50
.50
Posterior
Amount of Increase
Growth in per Day
Microns
in Microns
110.0
80.0
70.0
60.0
55.0
65.0
50.0
35.0
35.0
40.0
30.0
25.0
20.0
25.0
20.0
15.0
15.0
15.0
15.0
10 .0
7.0
8.0
8.0
7.0
7.0
6.0
5.0
5.0
5.0
11.0
8.0
7.0
6.0
5.5
6.5
5.0
3.5
3.5
4.0
3.0
2.5
2.0
2.5
2.0
1.5
1.5
1.5
1.5
1.0
0.7
0.8
0.8
0.7
0.7
0.6
0.5
0.5
0.5
Photomicrographs of transverse ground sections and levels
at which they were made through the femurs of alizarinated rats.
(A) Longitudinal through head and neck and obliquely
through the shaft distal to the greater trochanter.
(B) Shaft, just distal to the lesser trochanter.
(C) Diaphyseal end of shaft.
3>
Figure 2
Longitudinal raid-sagittal section through the shaft of
the femurs of alizarinated rats« (E) Medio-lateral*
‘(F) Antero-posterior.
Courtesy of M. Hoffman
Figure 3.
1.
2.
3*
4.
5.
6.
7.
8.
9.
10.
Photograph of Apparatus Used for Grinding
Sections*
Laboratory Lathe
Dental Splash Pan
Aluminum Shield
Cork Washer
Fine Grade Carborundum
Glass Shield to Prevent Splashing of Water
Bristle End of Tooth Brush
Water Feed to Brush
Copper Drain Pipe
Hard Arkansas Stone for Polishing Sections
Figure 4
TRANSPARENT METHYL METHACRYLATE MODEL
of Femur of a 150 Day Old Rat
The hlack lines represent the
epiphyseal lines.
The blue lines represent the peri­
osteal and cone-like deposit of alizar­
ine red S effect in the femur of injec­
tion given at 60 days of age.
The red line represents the alizar­
ine red S effect at 100 days.
Animal was sacrificed at 150 days.
Magnified six times.
To be viewed open and closed.
~~Le<^
of
Second
First
B
C
Txr-si
Cone
mi e c t x o ' n .
itviection
m iectiorv
Second
tni&cUou
Figure 5
Longitudinal section through femur showing two alizarine
red S effects in a 150 day old animal* Blue represents
the first injection at 60 days of age. Red represents
the second injection at 100 days of age. This is a
diagrammatic representation of the model (Figure 4)*
B
A
Figure 6
Colored photograph of the femur of a 178 day old rat
shoving the effect of one Injection of alizarine red
3 given at 87 days of age*
(A) Mid-sagittal longitudinal section of left
femur shoving alizarine red 3 effect in cortex.
(B) Outer surface of the cortex of the right
femur shoving the alizarine red S effect at the
proximal and distal end.
40 -
30-
20 -
10-
A= LENGTH ON BASIS OF INCREMENTS AS SHOWN BY ALIZARINE RED S EFFECTS
• -TOTAL LENGTH
10 20 30 40 50 60 70 6 0 90 100 210 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 260290 300 310 320 330 340 350 360
1 Figure 7#
Graph, showing total lengths in millimeters
and total longitudinal increments as shown
by alizarine red S effects in the femurs of
fifty-two albino rats from 11 to 364 days
of age* (From Table 2)
2.0
1.0
tul
o
o
0.1
0.9
Q8
0.7
0.6
0.5
0.4
Q3
0.2
10 20 30 4 0 5 0 6 0 7 0 8 0 9 0 100110 120130 1401501601 7 0 1 8 0 1 9 0 2 0 0 2 1 0 220230 2 4 0 2 5 0 260270 260 290300
DAYS (ARITHMETIC)
Figure 8*
Graph, showing semi-logarithmic representation
of the daily rates of longitudinal growth in
the rat femur from 10 to 304 days of age.
LOG OF 5
LENGTHS
I N M.M. '
30-
20
30 405060708090100
200
300
LOG OF DAYS
Figure 9.
Graph showing the logarithmic plotting of the
increments of longitudinal gorwth in the
femur of 52 albino rats from 11 to 304 days
of age.
1400
1300
1200
1100
1000
900
800
700
600
500
400
• SUM OF INCREMENTS ON ANTERIOR SURFACE OF FEMUR.
A S U M OF INCREMENTS ON POSTERIOR SURFACE OF FEMUR.
300
200100-
0
9
DAYS
0 10 20 30 4 0 50 60 70 80 9 0 100110120 130140150160170160190200210220230240 250250270280290 300
Figure 10.
Graph, showing calculated daily rates of peri
osteal growth at the anterior and posterior
surface of the femur just below the lesser
trochanter in 52 albino rats*
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