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The relative number of osteoclasts in normal and rachitogenic guinea pig mandibular condyles.

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THE RELATIVE NUMBER O F OSTEOCLASTS I N
NORMAL AND RACHITOGENIC GUINEA
PIG MANDIBULAR CONDYLES
HUGH 1. MYERS, J. M. WATERMAN, RICHARD BLACK
VIOLA FLANAGAN
School of Dentistry, University of Eansas City,
Eansas City, Missouri
AND
SEVEN FIGURES
This investigation was concerned with 4 somewhat related
fields of study. The first of these involved the growth mechanism of the mandibular condyle. According to Rushton ( ' M ) ,
Sicher ( '45), Collins, Becks, Simpson and Evans ( '46), and
Engel and Brodie ('48)' this mechanism consists of the proliferation of the outer fibrous layer, its conversion to hyaline
cartilage which becomes calcified, destroyed and replaced by
bone. This is a continuous process as long as growth continues.
The second field of stu'dy involved modeling resorption.
Weinmann and Sicher ( '55), illustrate the logic for this process in the tibia. In the chapter written by these same two authors in Sarnat's book ('51), it is indicated such a process
must occur in the mandibular condyle. Brash ('34) earlier
came to the same conclusion. Since the condylar head is larger
than the ramus, especially in its medial-lateral dimension,
this is a logical assumption. Since osteoclasts are usually
present where bone is being destroyed, it is also logical they
would be seen here. Myers ('52) showed them to be present
with great regularity on the condylar neck surfaces and internally next to the calcified cartilage. Since the shape of the
condylar head remains constant during normal growth, the
* This investigation was supported by research grant D-184 from the National
Institute of Dental Research, Public Health Service.
487
488
M Y E R S A N D OTHERS
modeling resorption rate has to equal the growth rate. It
would also follow that the osteoclast nuniber perhaps would
be different in clifferent age animals. A pre1iminar;v study by
Myers, Reeve and Flanagnn ( '56) involving only a few animals, demonstrated the probability of this.
The third field of stadp conccrned the role of vitamin D in
rickets.
recent book edited liy Sebrell and Harris ('54) contains most of the currenl information in this field. TVeinmann
('46) demonstrated that the typical increase in width of the
growth cartilage and excessive osteoid, usually studied i n the
epiplipeal plates of the tibia and costochondral junction in
the rib, also could be found in the niandibiilar condyle in vitamin Id deficiency i n the rat. Probably as a result of its propensity f o r developing scurvy and fastidiousness in eatin?,
the guinea pip generally has not been used in vitamin D stiidies. Kodiecek and hfurray ('43) were unable to produce rickets in these animals on a diet devoid of vitamin D over a 90
day period. The ratio of calcium to phosphorous in their
diet was within a normal range. Enieriqiie ('37) reported
rickets using a high calcium-low phosphorous diet, mhereas
Howe, Wesson, Boyle and Holbach ('40) reported rickets in
guinea pigs using 10~17 calcium-high phosphorous diet. This
diet produced a 50% mortality in 60 days.
Lastly, the study to be reported here involved the osteoclasts as cellular entities and their possible role in hone resorplion. Tt is well knomii that these giant cells arc. usually
present where bone is being resorbed, but relatively little is
known concerning their physiology and possible role in hone
resorption. Most of the histochemical and related observations
in this connection have been r e r i e m d by €€ancox in Bourne's
book ( '56) on bone.
J t was felt that, if a larger series of animals would demonstrate that a growth curve could be based on the relative m n i ber of osteoelasts in the mandibular condyle and that if this
nurnbcr coiild be shown to bc clianyed by disturbin? the normal
growth pattern through the production of some con& tion such
as rickets, close association of osteoclasts with bone resorption
MASDIBULBB COXUYLE OSTEOCLASTS
489
would be indicated and a background T V O U be
I ~ provided for
possible future liistocliemical stu'dies.
~ U L T E R I A L SAND hIETHODS
Healthy male guinea pigs of known ages, maintained in a n
air-conditioned animal house, were used. The first study, dcsignated the normal control, consisted of securing mandibular
condyles to determine the relative number of osteoclasts in
normal aninials of different ages. This ~ v a sfollowed by experiments designed t o determine the effect vitamin D would
have on this number of osteoclasts.
The animals for the normal controls were fed a daily diet of
P u r i m pellets, oats, alfalfa hay and green vegetables. The
tissue speciniens consisted of the following : 19 condyles secured from 10 animals two wceks old, 14 condyles secured
from 7 animals 6 weeks old, 17 condyles secured from 9 animals 8 weeks old, 18 condyles secured from 9 animals 1 2 weelis
old, 11 condyles secured from 6 animals 24 weeks old, and 14
condyles secured from 7 animals 48 weeks old.
For the observations involving vitamin D, designated experimental studies, the animals were maintained in a sunlightfree part of the animal quarters. Their basic diet consisted of
a semisynthetic formula devised by Mr. House of the Midwest
Research Institute (table 1). It was designed to provide all
the necessary salts and vitamins, except vitamin D. Twentyfive grams were fed daily, the residual portion being weighed
before the next feeding. A minimum of 3 mg ascorbic acid
was given separately daily. This was mixed with lactose and
placed in gelatin capsules. After crushing the capsules in the
animals' mouths for a few days, they usually learned t o take
the capsules voluntarily. The animals were fed the semisynthetic diet beginning a t two weelis of age in all of the experiments. Each animal was weighed daily.
The first experimental study, designated the experimental
control, consisted of feeding the animals the semisynthetic
diet supplemented with 2 t o 5 units of vitamin D, daily
490
MYERS AND OTHERS
f o r 10 weeks. Six condyles were secured from three animals
(age 12 weeks).
I n the preliminary vitamin D deprivation experiment, designated the experimental rachitogenic, 6 animals were sacrificed
and the mandibular condyles secured after being maintained
TABLE 1
Sernisynthrtic diet f o r guinea p i g s
%
Casein
Sucrose
Gum arabic
Potassium acetate
Magnesium oxide
Calcium carbonate
Salt mix
Vitamin mix A E K no. 12 *
B vitamin mix no. 1*
Choline chloride solution (24 g m % )
(approx.)
30.0
40.2
15.0
2.6
0.4
1.0
4.0
2.4
0.45
Hubbell, Mendel and Wakeman ; from Nutritional Biochemicals Gorp.
Base: Lard, Solvent; Ether (diet)
Vitamin A Acetate
0.5 gm
50 mg
Menadione
3 gm
a-Tocopherol
'Base: Powdered sugar 98%
Riboflavin
112 mg
Pyridoxine
80 mg
p-Amino benzoic acid
800 mg
Ca-pantothenate
240 mg
Nicotinic acid
800 m g
Thiamin
80 mg
Inositol
1.6 gm
Biotin
3.2 mg
F o l k acid
48 m g
on the diet for 6 weeks (age 8 weeks). The condyles from
another 6 animals were secured after they had been fed the
diet 9 weeks (age 11weeks).
The above preliminary experiment was followed by an experimental rachitogenic study with a full complement of animals. Twenty-four condyles were secured from 1 2 animals
after having been fed the semisynthetic diet f o r 10 weeks
(age 1 2 weeks).
M A N D I B U L A R CONDYLE OSTEOCLASTS
491
Next, an experiment was designed to produce rickets and
then to cause a recovery. This was designated the experimental rachitogenic recovery. The animals were fed the semisynthetic diet for 10 weeks (age 12 weeks). Then they were
given 2 to 5 units of vitamin D, daily f o r an additional
10 days (age 13 weeks, three days). This was given by
adding it to the gelatin capsules containing the vitamin C and
lactose. Both condyles from 8 animals that had received this
treatment were secured.
The condyle tissue from all of the experiments was fixed in
formalin, electrolytically decalcified, imbedded in paraffin and
sections cut at 10 p . Every tenth section was mounted and
stained with hematoxylin and eosin. A consi'derable number of
the condyles from the normal animals were cut in cross section (fig.2) or in the saggital plane (fig. 3). The rest of the
condyles from the normal animals and all of the semisynthetic
diet animals were sectioned in the frontal plane (fig. 4).
Counts of the osteoclasts were made on the slides (i.e.,
every tenth section) at 430 magnification. I n the cross sections, this was done by examining in order all of the slides
from the upper cartilage tip to well below the condylar neck.
I n the sagittal sections, the osteoclasts were counted as follows: the highest point of the cartilage was found and the
slide was moved across the cartilage and osteoclasts counted.
The section was progressively moved one microscopic field
lower after having moved across the section. This was repeated until no osteoclasts were seen internally adjacent to
the marrow spaces. The section then was moved to the junction of the periosteum and bone on the posterior margin beneath the cartilage cap. It then was progressively moved
away from the cartilage until no additional osteoclasts were
seen. This procedure was repeated on the anterior margin.
Essentially the same procedure was repeated for the frontal
sections except, of course, the periosteal-bone junctions represented the medial and lateral sides.
The data obtained from these counts were recorded as the
total number of osteoclasts counted in each condyle. The
492
M Y E R S A N D OTHERS
means, standard deviations and standard errors \yere derived
in each case by the statistical forrnulas f r o m Chilton ('53).
DATA
A 1though the seniisynthetic diet was sufficiently adequate to
secure the data needed for this study and the animals gained
30-15 gm per week, it was far from ideal. I n the latter part
of tlic experiments, a considerable number of tlie aninids were
lost. They had enlarged abdomens, prolapsed rectums, and
roughened hair. Death was dne usually to respira-ior? coinplications.
The form of the osteoclasts seen in this study was the same
as is generally found in osteoclasts (fig. 5). The variability
in number of nuclei and cell size m7as commonplace. It was
felt that the vesicular nuclei and intense eosin staining cytoplasm was more frequently seen in the normal animals, \&ereas pyknotic nuclei and basophilic cytoplasm were more frequently seen in tlie animals maintained without vitamin D.
Osteoclasts were not found to be uniformly distributed
throughout the condyle arid ramus. Areas of concentration occurred beneath the periosteum on the niedial, lateral and anterior surfaces. They were also found internally in the marrow
adjawnt to the lower edge of the calcified cartilage.
I n the normal control experiment, the most pronounced morphological change in the condyles was the progressive diminution in the width of the cartilage cap with increase in age. At
2 1 weeks of age it was very narrow, and in animals 48 weeks
old a horizontal layer of bone had become deposited on the
cartilage separating it from the marrow. The osteoclast counts
showed a rather rapid relative decrease in number from two
weeks to 24 weeks of age (fig. 1). From 24 t o 48 weeks of age
there was only a slight additional decrease.
I n the experimental control animals, the morphology of the
condyle was essentially the same as in the normal control
(figs. 2-4). The osteoclast count at 1 2 weeks of age was only
slightly below that for the normal control (fig. 1).
MANDIBULAR CONDYLE OSTEOCLASTS
493
The preliminary experimental rachitogenic group showed
some variability of results. Those maintained on the diet f o r
4 weeks (age 6 weeks) and two of the 6 fed the diet f o r 7
weeks (age 9 weeks) appeared normal morphologically. No osteoclast counts were made on these. The other four 8-~veeli-old
animals showed both a morphological change in ilie condyle
and a deviation in the osteoclast count. The cartilage layer
was considerably thickened - the characteristic picture f o r
rickets (fig. 6). Unlike typical rickets, however, no distinct
layer of uncalcified bone matrix v a s discernible below t h e
cartilage. The osteoclast count was approximately 50% below
the normal control (fig. 1).
L
14
F
A G E I N WEEKS
Fig. 1 Comparison of number of osteoclasts per mandibular condyle in normal
and rachitogenic guinea pigs. Solid line indicates normal control animals; A,
experimental control animals; €3, preliminary experimental rnehitogenic aniinals;
C , experimental rachitogenic animals ; D, experimental rachitogenic recovery mimals.
494
MYERS AND OTHERS
I n the definitive experimental rachitogenic experiment in
which the animals were maintained on the non-vitamin D
semisynthetic diet for 10 weeks (age 12 weeks), all of the
condyles showed the thickened cartilage layer (fig. 6). The
osteoclast count also was approximately 50% below the normal
control (fig. 1).
The condyles from the experimental rachitogenic recovery
experiment in which the animals were fed the semisynthetic
diet without vitamin D for 10 weeks followed by the 10 day
addition of vitamin D, showed a cartilage width comparable
to the normal control with features, such as hyperemia in the
marrow, indicative of rapid cartilage dissolution (fig. 7). The
osteoclast count was felt to be comparable to the normal control animals of comparable age. The experimental count actually was slightly higher than the normal, but the difference
probably was not of sufficient magnitude to be significant
(fig. 1)DISCUSSION
It was indicated in the results the semisynthetic diet was
not entirely adequate, but the literature also showed a similar
difficulty in working with guinea pigs for rachitogenic studies. Those animals which survived, however, did develop rickets and thus provided the necessary material f o r this study.
We definitely did gain the feeling that this diet could become
very useful potentially. Apparently the fundamental difficulty
was inherent in the diet and not simply due to the lack of
vitamin D, since the mortality was as great in the experimental
control group as it was in the experimental rachitogenic
group. Perhaps the addition of some inert bulk to the diet
would have been helpful. Additional experiments on the nutrition of the guinea pig using modifications of this diet could
provide an interesting study.
As it is understood a t the present time, Sebrell and Harris
('54),vitamin D facilitates the absorption of calcium from the
intestinal tract, it controls in part the clearance of inorganic
phosphorous from the kidney, and in some way it is necessary
MANT)IBULAR C O S D Y LE OS‘rEOCLhSTS
193
f o r the deposition of calcium a s phosphate and carbonate in
osteoid ant1 cartilage matrix. In the rachitic state in the yomig
individual, one usually sees a thickened cpipliyseal cartilage
layer. The cartilage coiitiimcs to prolifei-ate hut it cannot hecome calcified, ~vliicliin turn seems to inhiliit its destruction.
Since hone matrix likewise eoiitinucs to he formed without
calcification, a laycr of osteoid usually is seen adjacent to tlie
cartilage. 111 these experiments the ostcoid layer was absent.
One could assume then that cartilagc matrix conld contiiiuc
to hc formed wlien ostcoid protluction is sapprcsse(1, that calcification conld hcl suppressed in tlic cartilage matrix, and
not in bone matrix, or that the cartilage could he calcified aiitl
tlic liqucfactivc proccss sorric~liowsupprt cd. Tlic purpose of
this study was basically conccrnecl \\-it11 the ostcoclasts, hence,
tliese vai*ioiispossi1,ilities n-crc not csplorccl. Since the fundaiiiciital action of vitaniin TI in clcpositioii of calciniii in cartilag^ a~iclboiic matrix is not liiion’ii, liistocliciiiical stutlies of
this material coizltl lie frnitful.
The pi.ogressivc rliiiiinutioii in width of tlic. cartilag:.c c a p
with iiici-caw in agcb in iiornial niiininls lias 1)ccii iiotcd ill other
\I)ccics, inclucliiig tlic Iiiminii. (’olliiis
a l . ( ’46) iiottd the
l i o i h n t a l layer of lmiic hcpiiratiiig thc c a r t i l a p froin the
itiai.i*ow in oltl rats. Tlicv fcllt this iiitlicatc~cl ccssatioii of
yi.0 n-t 11. Tliey a1so h\ve(l t li a t , 11I I I ilw t 1I t> hunia 11, t 11c ca 1-ti 1agc
iicver coiiiplctely t1isap~)eaiwl.,4l1pai*c~iitly
t l i c quiiica 1)iq ant1
r a t a r c alike. iii this rcsp,ccat.
Tlitl tlistrihntion of tlie oitcoclasts is loqical. Those fouiitl
mitler the p r i o > t c u n i 011 thc a n t erioi., mcdiul and lateral surfaces woiild indicate niodcliiig rcsorptioii of thc~iioiic. If oiic
I-tlalizcs the niaiidihlc gro\\-.; iipwai~lam1 l ) a c k w a i ~ lhp endoclioiitl~alforniatioii ill the coiicl~la1*
lieacl ant1 al)positioiial 1)oiie
f o mint i on 011 the 11o st c I- i or i’arriii1 1x11 ~c11’ wit 11 c oi I corn i t a i i t 1-esorption on tlie anterior, mcdial and lateral lmrtle~s,the presence of osteochsts in these lattcr areas ~ v o u l dhe q x c t e i l .
Since thc calcified cartilage is progressively tlcstiwpeil at its
lower cdgc, osteoclasts 1icli.c a r c logicd also.
clt
I t is rcxogiiizecl the tabulations of the nuinl)er of osteoclasts
coulcl be dorie iii diff erent ways. Indeccl, prelimiiia.rp rcsdts
w\.cirecomputcd as the average iiunihr of ostctocla.sts see11 per
slidc, instcacl of the total number countcd. For tlic purposes
of this stucly the t.otal nuiiiber of t.1iese cells in a coidylc WIS
iri-c+waut, beca.use relative differences in iiumbers wcrci
songlit. If one were to detcrrniiie the total number of ostooclasts found in a. coiirlyle the size of t.hese cclls i ~ o u l dIitwc to
be cl~t~riuiiicd
and then thc nunilner oalculatcd (Sheinin, '30).
It is nlso recognized the decrease in the ostcoclast counts in
older age groups and in. the rachitogenic animals could liare
bccm ail actual disappearance of some of these cells, hut it
also could have been simply a. decrease in the size of tlie cells.
The latter would not he too surprising if osteoclasts have a
very actirc role in bone rcsorption and, if it had ccasecl, especiully in the racliitogenic animals. If the ostcoc1ast.swere actnally destroyed, then new ones must have been formed in tlic
rachitogenic recorcry animals. No determination was reached
as to wliethcr the absence of transition cell forms from some
ot.lier cell type was due to thc time the animals were sacrificed
during the recovery period, or whether no new cclls were actually formed. Therefore, the actual ccll lincage origin for
osteoclasts still is as confused as it has been in the past.
It is fclt the removal of our knowledge of osteoclast fmictioil from the realm of speculation probably must await many
additioiid future histochemical observations. However, the
demonstration in this study that alteration in the growth of
the maiidibular condyle and its consequent modeling resorption of bone are correlated with changes in relative osteoclast
niinhrs, does provide presumptive evidence there is a causal
relationship with their function and bone rcsorption.
SUMMARY
1. The relative number of osteoclasts in the mandihular
condyle decreases with increasing age in normal guinea pigs.
2. The number of osteoclasts in the mandibular condyle is
sharply reduced when rickets is produced.
1 ~ 1 1 ~ ~ 1 ~ 1C
~ 1r r~ m
111~
B. IT. 1936 The Biochcinistiy aiitl Plij siologj of Roiie. Alc.rdeniic
P ~ e s ? ,Iiic.. K t w York.
B K ISH, J. C. 1934 Soilre p i olllcni\ oil the grorr tli n i i t l der c310]mic~iit,ili i i e c l i , i i ~ i c ~
of hone. E c h . 3Lrtl. Jour., 41: K. 8. IT, 303-319, 363-3ST.
111 tlentnl icseaicli. Office of Techiiic.,il Seiric c.5,
i c c , W\Ta\liingtoii, n. C.
S I M P h O K XND 11. 11. E V
1946 G i o n t h aiid
trnnsforiiiation of the nimdlhiilar joint in tlie rat. T. S o i i n a l fein:tle
i a t s . 4111. J. Ortli. Oral Suig.,
NVEKIQUE,I,. 1937 T x I l a c l i i t i ~ i n eexpeiiinc~ntaltlrrz lc cobaye. C’. 11. Ac:irl. Ssc.,
20;: 879-882.
I ~ G E L , M., LKD A . G . GKODIE 1948 C o d >liir gron th a n d m:indibnl,i~ tlefoi niitics.
Oral Ruig., Oinl JIcd., O r d l’;itli., 1: 790-8OG.
HOWE,P. R., L. G. TY’TISON, P. E. BOlLE 4 S D S. R. HOIALCH19-10 T,olV
enlciuni iicket\ in the guinca pig. Proc. Soc. Exp. Biol. Jlcd., f 5 :
298-301.
K O D I E C r K , E., AKD P. D. F. X U R R ~ 1943
Y
Influence of a proloiigcil partial rlefieiency of rit:imin C on the recovery of guinea pigs from injnrr t o
bone aiid ninscles. S a t n r e , 151 : 393-396.
LIYE RS, H. I. 1932 The di\tlibutioii of ostcoela5ts i n thc ninnclibular j o i n t of
the gniriea p i g . J. 1)crit. Res., 31: 46.5.
N I E R S , H. I., TV. L. RFELE A N D V. F L I N I G ~1956
N
A denioiistr:ition of the
osteoclasts asiocixted witli n1odeling resoi ption in the mandibular c 011dyle of the guinea pig. Ibid., 3 ; : 137-146.
RUSHTOS, M. A. 1944 Gronth at the m:indibular corid-yle in relation t o snnie
deformities. Brit. Dcnt. J., 76: 37-68.
SLXNAT,
€3. G. 1951 Tlir Terriporoniniidibiilnr Joint. Charles C” Tlioni:i.;, Spring
ficld, 111.
SEBRELL,
TV, II., AND R. S. I1 IRRIS 19.54 The Vitamins, 11. iicadernic Press, In?.,
Nem Yolk.
SITEININ, J. J. 1930 Typing of thr cells of the nieseiicephalic nucleus of the
trigeiniiial nerve i n the dog, 11:rsed 0x1 Sissl-granule nrr:ingemcnt. J.
Conip. S r n r . , t50: 109-132.
SICI-IER,
H. 194.5 Giomtli of t h c niniidihle. J. Periodont., 16: 87-93.
WEINNBNN,J. P. 1946 Racliitic elianges i n inaridibular cond!lc of the rat. J .
Dent. Req., 22: 309-512.
WEINMANN,J. P., A N D H. SICHER 1955 Bone and Eones. St. Louis, The C. V.
Mosby Co.. p. 80.
J~OUXSE,
PLATTG 1
2
('ross rwtioti of iiirtitdiliii1;tr coiiilyk of 11 giiiiwrt. pig X \welts oltl. Tlic outcsr riin of tisstic
is tlic. fil)rotin tlisc. liiwitlc this is tlic. xpovirtl erivity. Tlic contlylc ~ W O ~ J Wiiisiclc tlw synovial cavity uho~w:ti1 outer fihrociu coniicctivc tissue layer. Jiiiniediately adjacent is tlic cartil:tgc. Tltc. rettiaining ceiitrril portion i a eoiuposetl of Iio1tt1 spicules auil red I)OIIC 1it:trrow.
S:igitt:tl wctioii of a. ntantlibulrir condyle of i i guincri pig 8 acvks old. The fibrouri tlisc is
sitpwior, follomed i i i order Lj- syuovial cavity, filmxis conncctive tissuc, cartilngv, ri111'1 bone
spicii1c.w m d Inarrow.
1 Froatrtl sectioii of xi mtindihulnr conayle from a gilinea pig 8 weeks Old.
3 Typical ostcoclasts seen in the periostcuni in the region of thc 1iec.k of tl,e co~dylc.
3
198
PLATE 2
#ANDIBUJAK CONDYLE 08TEOCLASTS
PYIDUB AND OTHERS
6
Frontal section of n ninndibulnr eoudyle from a 12-?;eck-old guiiien pig fed the raeliitogenic
diet for 10 weeks. The thickened cartilage is proncuueed.
5
Frontal section of a ninndibular condyle from a lhveek, tliree-cla.y-old guinea. pig. The
animal was maintained on the racliitogcnic diet for 10 weeks, followecl by a 10-day recovery
period in which vitamin 1) wzis added to the diet.
499
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