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Mitochondrial changes associated with aging of periosteal osteoblasts.

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MITOCHONDRIAL CHANGES ASSOCIATED WITH
AGING O F PERIOSTEAL OSTEOBLASTS
EDGAR A. TONK'ilZ AND NAN PILLSRURP
T h e Hospital f o r Special Surgery, Philip D. Wilson Research Foundation
afiliated with Cornell University Medical College, N e w Pork
FOURTEEN FIGURES
INTRODUCTION
As far back as 1914, mitochondria have been noted to increase in number with increasing osteoblastic activity during
bone formation (Dieneka, '14). Pritchard ( '52) made similar
observations. Resting osteoblasts contained fewer, thicker,
shorter and more dispersed mitochondria (Hill, '36). Morphological descriptions of the mitochondria obtained from
osteoblasts differed. I n pig (Dieneka, '14), and tissue culture
(Hyslop, '52), mitochondria were described as being primarily short, thick rods, however, granular filamentous and
beaded forms were also noted. I n the chick (Fell, '25), rat
(Pritchard, '52) and tissue culture (Hill, '36), mitochondria
appeared predominantly as filamentous forms usually parallel to the long axis of the osteoblast. Fischer ('45) reported
the presence of long, smooth rods in chick tissue cultured cells.
Perhaps the morphological differences reported were due to
the extremely labile nature of mitochondria to a variety of
external and internal circumstances. Mitochondria behave as
perfect osmometers (Harman and Kitiyakara, '55 ; Tedeschi
and Harris, '55) and are therefore subject to morphological
variations with changes in the fluid environment. An injury
incurred during preparation was shown to cause swelling and
rounding up of mitochondria (Haguenau and Rernhard, '56).
' Aspects of this work were presented in Philadelphia to the Gerontological
Society in November, 1958.
Present address: M d i e a l Research Center Div. Experimental Pathology,
Rrookhaven National Laboratory, Upton, I,. I., IT. Y.
739
740
EDGAR A. TONNA AND NAN PILLSBURY
Variations may have been due also to the use of different
experimental subjects.
Tonna ('%a, b, '59a) reported that rat periosteal osteoblasts exhibited reduced respiratory enzyme activity (succinic dehydrogenase and cytochrome oxidase) with increasing
age. Since succinoxidase activity has been shown to be associated with mitochondria, (Hogeboom et al., '46 ;Holter, '54;
Chance et al.,
b), enzyme variations noted during aging
of periosteal osteoblast (Tonna, '%a, b, '59a) are believed to
be reflected by coincidental changes in oxidative-phosphorylation and perhaps mitochondrial morphology. It was the
aim of the present investigation to: (1) study such population and morphological changes which may occur in periosteal osteoblasts and their mitochondria, and (2) to compare
these changes with previously reported variations in respiratory enzymes for a more comprehensive cytochemical understanding of normal skeletal growth, development and aging.
XMATERIATA AND METHODS
Cytological studies were performed on 60 Long-Evans
strain rats which were divided into 6 age groups, namely; 1,
5, 8, 26, 52 and 104 weeks of age. Each group was subdivided
into males and females. Immediately after the animals were
sacrificed with ether, samples of periosteum were removed
from the mid-diaphyseal region of each femur, by shaving
with a scalpel. The shaving was deep enough to include
slivers of bone, thereby insuring obtaining undisturbed, intact periosteum with bone surfaces. The bones of one and
5-week-old rats were not shaved since they could be sectioned
routinely without difficulty. Spleen or liver sections were included as controls against defective fixation, mordanting,
staining, etc., since the normal morphology of these mitochondria is well known. ,
4 comparison between mitochondria1
morphology of liver cells and osteoblasts is seen in figures 7
and 8. Fixation was carried out wtthout prior decalcification
in Regaud's solution. Paraffjn blocks were prepared routinely
AQINQ MITOCHONDRIA OF OSTEOBLASTS
741
and sectioned at 5 p. Tissue sections were stained with
Regaud's iron hematoxylin3 (Gurr, '56). Additional pieces
of fresh tissue were frozen-sectioned and stained supravitally
with Janus green B, for comparison with fixed samples.
All the studies were made on periosteal osteoblasts which
were in juxtaposition to the preosseous zone. These cells are
intimately involved with formation of osteoid. The number
of osteoblasts per unit area (To)were counted in all age
groups and comprised the population studies. The following
cytological measurements of mitochondria were made under
oil immersion using a filar ocular micrometer and Kohler
illumination.
1. Average number of mitochondria per osteoblast (Rm)*
2. Average number of mitochondria per unit distance per
group
3. Average mitochondrial length and width (fix2), (iiw8).
4. Average mitochondrial surf ace and volume (iim2),(fim8).
5. Average mitochondrial surface and volume per osteoblast (Go2), (iiO3).
6. Average mitochondrial surf ace and volume available
per unit distance of periosteum per age group (iig2) or (fi,").
The mitochondrial surface [2 (nr2) 2nrh =u2], and volume (rrr2h= u3), were estimated on the basis of a cylinder.
The average number of mitochondria per unit distance of
periosteum per group (3,) is the result of the product of the
average number of mitochondria per osteoblast (RJ, and the
average number of osteoblasts per unit area per group (Ro).
Average mitochondrial surface (iio2) and volume (Go3) per
osteoblast, were obtained from the product of the average
number of mitochondria per osteoblast (Em),calculated for
each age group, and the average mitochondrial surface (fim2)
or volume (iim'), respectively. The average mitochondrial sur-
(X)'
+
a The following stains have been used to demonstrate mitochondria, namely :
Regaud 's aniline fuchsin-iodine green, Altmann 7s aniline f uchsin-picric acid,
Champy-Kull's aniline fuchsin-toluidine blue and aurantia, Bensley 's copper
chrome hematoxylin, Pritchard 7s silver stain and Regaud's iron hematoxylin.
The last proved most successful in our hands and gave consistently good results.
74%
EDGAR A. T O N N A A N D N A N PILLSBUBY
face and volume available to an animal of a particular age
group, per unit distance of periosteum is represented by the
product of the average mitochondrial surface (iio2)or volume
(Go3) per osteoblast, and the average number of osteoblasts
(Ro)per unit distance of periosteum. The numerical data
was evaluated f o r significance by statistical methods using
Fisher's t, having a critical ratio of 2.797 and a probability of
P < 0.001.
In attempting to obtain the average measurements, certain
pitfalls due to cellular and mitochondrial disposition were
encountered. Cells which exhibited any of the conditions represented diagrammatically in figure 1 were discarded from
the analysis. These dispositions, however, did not interfere
with accurate population counting.
Pitfalls in Mitochondria1 Counting'
1
2
3
4
Fig. 1 The diagram represents various dispositions of periosteal osteoblasts
and their mitochondria adjacent to the preosseous zone. 1, Cells show overlapping,
rendering almost impossible an accurate evaluation of the number of mitochondria per osteoblast. 2, The mitochondrial population is too large and too compact to allow suitable counting. 3, Overlapping of a cell upon itself a t times
makes certain regions of that cell impossible to count. 4, Only a portion of tho
cell appears in the section. Any of these conditions make the cell unsuitable f o r
mitochondrial analysis.
A G I N G M I T O C H O N D R I A OF OSTEOBLABTS
743
REBULTS
The mitochondria1 complement of periosteal osteoblasts
varied considerably in size, ranging from long, filamentous
rods to structures whose length was equal to the width. I n
older age groups, mitochondria or their fragments were found
which measured close to the resolution of the microscope.
Within a given age group, the number of mitochondria also
varied. This variation appeared to depend upon the activity
of the various osteoblasts taking part in bone formation. The
number of large mitochondria present within osteoblasts was
found to vary from cell to cell. Such differences, however,
generally occurred within a certain limit, so that significant
variations were found when one age was compared with
another age group. The frequency with which larger mitochondria were found diminished with increasing age, until
neither filamentous nor large rods were observed. Large mitochondria were oriented, more or less, parallel with the long
axis of the cell. Short rods were more often non-oriented.
With increasing age, orientation became less apparent as the
number of large mitochondria diminished. Mitochondria were
found t o be present in all parts of the cytoplasm, and in
many instances over the juxta-nuclear structure.
From birth to 8 weeks of age, numerous active osteoblasts
were found adjacent to the preosseous zone. Their number
increased only slightly within this period. Examination of
the sections taken from the 26-week-old rats revealed less
than one-third of the number of osteoblasts present in one to
8 weeks of age. The majority of the cells which were once
active osteoblasts, at 26 weeks of age were morphologically
different. Generally, they appeared spindle-shaped, having
elongated, more densely staining nuclei and less cytoplasm.
From 26 to 104 weeks, somewhat fewer cells were found at
the preosseous zone (table 1). A plotting of the numerical
data shows two distinct levels. One level occurring after birth
(one to 8 weeks of age), and is coincidental with the period of
active bone formation. The other level, 26 to 104 weeks of
744
EDQAR A. TONNA AND N A N PILLSBURY
AGING MITOCHONDRIA OF OSTEOBLASTS
745
age, is comparable to the period at which time the femoral
growth rate has reached a plateau (fig. 2).
Counts were made at each age of the number of mitochondria per osteoblast (table 2). Occasionally large active osteoblasts were noted containing large quantities of mitochondria
(fig. 9 ) , somewhat similar to what was seen in osteoclasts
(fig. 10). I n such cases, accurate counts could not be made,
'2
I
I
3
& 12-
Age (weeks)
Fig. 2 A plot representing the available number of osteoblasts present and
in contact with the preosseous zone of the femur of rats a t different ages. Two
distinct levels are shown; one ending at 8 weeks of age, the other beginning at
26 weeks. From 8 to 26 weeks of age, the number of osteoblasts diminish rapidly
from the preosseous zone of the periosteum.
and were therefore left out of the calculations. Had they
been included, the mean number of mitochondria of groups I
and I1 animals would have been somewhat larger. These exclusions, however, were not sufficient to produce a different
picture of mitochondria1 changes associated with aging.
The tabulated data showed a significant rise in the number
of mitochondria from birth up to 5 weeks of age. This increase was followed by a considerable decrease in mitochondria to less than half the number observed at 8 weeks of age.
746
EDGAR A. T O N N A A N D NAN PILLSBURY
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AGING M I T O C H O N D R I A O F OSTEOBLASTS
747
A progressive fall in the mitochondrial population followed
with increasing age (fig. 3 ) . Largely, non-significant differences were found between males and females.
Cytological analysis of periosteal osteoblasts revealed profound changes in mitochondrial morphology. The numerical
data is presented in table 3. With increasing age, mitochondria, on the average, became progressively shorter.
ti
P
040
1
-
c
x
Fig. 3 A plot of the mean number of mitochondria per osteoblast at each age
group shows an increase in the number of mitochondria t o 5 weeks of age, a
period at which time the rate of bone growth is about maximum. This period is
followed by a rapid reduction within three weeks of the mitochondria1 population
then a progressive but continued reduction.
Shortening to approximately 2870, W R S most pronounced between the ages of one and 8 weeks. Slopes were considerably
smaller among older animals. The average mitochondria1
width actually changes very little from one age to another,
except for a small increase noted between one and 5 weeks of
age, and a similarly small decrease found between the ages
of 5 and 8 weeks (fig. 4). Curves for the average surface and
volume per mitochondrion, although similar in shape (fig. 5),
were increased between one and 5 weeks of age. The surface
&=
0.49 rt 0.03
0.51 & 0.02
0.35 2 0.02
0.40 & 0.01
0.06 f 0.01
0.07 & 0.00
0.09 f 0.00
0.20 f 0.01
0.52 rtr 0.01
0.42 k 0.01
0.35 2 0.01
0.65 1 0 . 0 1
'Sixty mitochondria were counted in each case.
(104)
Group VI
(52)
Group V
(26)
Group IV
0.62 f 0.01
0.82 t 0.02
Group I11
(8)
1.00 rt 0.02
0.27 f 0.02
fi
0.54 t 0.01
L
1.13 f 0.06
Group I1
(5)
(1)
Group I
AVERAGE
VOLUME
2 SE
AVERAGE
WIDTH
2 BE
AVERAGE
LENGTH
2 SE
0.21 f 0.00
0.69 f 0.00
1.90 t 0.06
3.23 f 0.01
12.82 +- 0.03
7.99 t 0.03
P
AVERAGE
MITOCHONDRIAL
VOL/CELL
2 BE
SB
-+ 0.06
0.74 rt 0.06
0.92
1.09 t 0.05
2.61 & 0.08
3.90 f.0.15
3.47 2 0.14
P
2
AVERAGE
SURFACE
2.67 f0.03
8.58 f 0.03
24.31 f 0.06
43.20 & 0.08
144.96 2 0.26
100.77 f0.20
fi=
AVERAGE
MITOOHONDRIAL
SUR~ACE/CELL
2 BE
Morphological analysis of the mitO0hOndTial complement of periosteal osteoblasts of the femora o f male rats at direrent ages
TABLE 3
749
AGING MITOCHONDRIA OF OSTEOBLASTS
was approximately 10% larger and the volume 20%. This
increase was followed by a sharp decrease of up to 72% by
26 weeks of age. Little change was noted in mitochondrial
surface or volume in older animals. Plots of the data obtained for the average mitochondrial surface and volume per
osteoblast (fig. 6), show an even larger variation with increasing age, i.e., between one and 5 weeks of age the surface
increases by approximately 44% while the volume by 60%.
This increase is due mainly to an increased mitochondrial
population. From 5 to 26 weeks of age the surface decreases
by approximately 83% and the volume by S5%, as a result of
0
I
I
I
I
I
1
I
70
I
I
I
110
Age (weeks)
Fig. 4 The shortening of mitochondria is most pronounced up t o 8 weeks of
age. The average mitochondrial width, however, is little effected.
continued population loss and shortening of mitochondria.
Curves for the average measurements of surface and volume
per mitochondrion were similar to those representing the
average number of mitochondria available to an animal per
unit distance of periosteum, and the average mitochondrial
surface and volume available per unit distance per group.
The magnitude of the different units varies, but the shape of
the curves remains the same.
Initially, (one to 5 weeks of age) a sharp increase in
mitochondrial surface and volume appears available to the
osteoblast. This period is comparable to the time at which
the maximum rate of bone formation occurs in the rat.
'750
EDGAR A. T O N N A A N D N A N PILLSBURY
_
~
-
Age (weeks)
Fig. 5 Plots of the average surface and volume per mitochondrion indicate a
loss of approximately 72% of the surface and volume bp 26 weeks of :igcl. This
value is the result of the reduction in mitochondria1 length.
Average Mltochondrial Surface
0- - - -0 Average M i b c h h i a l Voium
L
I I
Y
A ;
$
v)
-
I
I
-3T
-
158
26
52
Age
$
104
(weeks)
Fig. 6 Plots of the average mitochondria1 surface and volume available t o
periosteal osteoblasts a t various ages indicate a loss of approximately 83% of
the surface and 85% of the volume by 26 weeks of age. The values are a result
of the reduction in mitochondria1 length and mitochondrial population.
AGING MITOCHONDRIA O F OSTEOBLASTS
751
Between 5 and 8 weeks there occurs an acute reduction in
volume and surface because of changes in mitochondrial size
and number. Average mitochondrial surface and volume per
osteoblast continue to decrease up to 104 weeks of age, however, with progressively diminishing slopes.
The microscopic changes seen in periosteal osteoblasts
and their mitochondrial complement are shown in figures 12,
13 and 14.
DISCUSSIOK
Dempsey ('56) pointed out that aging moribund cells, as
exemplified by the zona reticularis of the adrenal gland and
the yolk sac, contained fewer and degenerating mitochondria.
This is also true of the periosteum especially the periosteal
osteoblasts. It appears that the morphological picture of
mitochondria cannot be considered as a static one since considerable changes are found from one age to another and
even from cell t o cell. VC7hen mitochondria change shape as a
result of aging, oxidative phosphorylation, an essential energy
producing mechanism in animal cells, becomes diminished
(Harman and Fiegelson, '51). The ability for mitochondria
to carry out oxidative phosphorylation is also reduced as a
result of iw vitro "aging" (Siekevitz and Potter, '55; Ernster,
'56 and Hunter et al., '56). Active oxidative phosphorylation,
however, is not necessarily a prerequisite for the maintenance
of the morphological stability of mitochondria (Ernster and
Lindberg, '58). The increased mitochondrial population of
periosteal osteoblasts up to 5 .~r-eelssof age and the subsequent
fall in number is coincidental with succinic dehydrogenase
and cytochrome oxidase activity previously reported (Tonna,
'58a). The shape of both curves, representing the mitochondrial population and the respiratory enzyme activity, are
similar from birth to old age, indicating morphological as
well as biochemical changes. It is interesting to point out,
that although, significant falls have been observed in respirenzyme activity and mitochondrial number in rats between the
ages of 5 and 8 weeks, the population of osteoblasts and other
752
EDGAR A. TONNA AND N A N PILLSBURY
morphological characteristics appear not to have been altered.
By 26 weeks of age, however, the osteoblastic population was
considerably reduced and changes were noted in size, shape
and staining characteristics, rendering them frequently indistinguishable from connective tissue fibroblasts (Tonna,
'59b, c). Alterations in respiratory enzyme activity and mitochondrial morphology preceded the changes seen in osteoblasts.
Periosteal osteoblasts, unlike many other cells of the body,
exhibit a critical period early in the life of the animal, in
which drastic changes occur in respiratory enzyme activity.
Coincidental changes have also been noted in the mitochondrial population and mitochondrial morphology. It is not
known whether the variations in mitochondria, are the result
of the biochemical changes observed or vice versa. We do
know, however, that respiratory enzymes are intimately associated with mitochondria, and mitochondria are the generators of cellular energy. It has been shown (Ernster and
Lindberg, '58) that active oxidative phosphorylation is not
necessarily a prerequisite for the maintenance of the morphological stability of mitochondria. On the other hand, it
appears from the work of Harman and Fiegelson ( X ) ,that
the level of oxidative phosphorylation is dependent upon
mitochondrial morphology.
I n any event the fall-off in the rate of bone formation
with increasing age is believed to be a consequence of the
rapid degenerative changes observed in both the respiratory
enzyme activity of osteoblasts and their mitochondrial complement. These cells are probably deprived of the extra energy
required for bone matrix production, and maintenance ener,v,
resulting in the diminished population and altered morphology associated with aging.
It is important to point out that the findings described
in this paper do not refer to endosteal osteoblasts, which are
generally found lining trabecular bone.
Although much of the biochemical detail is obscure, a particular situation exists, which is believed to be a factor re-
A G I N G MITOCHONDRIA O F OSTEOBLASTS
753
sponsible for the degenerative changes observed. Immediately before these events take place, osteoblasts are known to
be actively engaged in bone formation. The task is so demanding at this time that trauma to the bone will fail to stimulate
the respiratory enzyme activity. Nevertheless, traumatization at any other age was shown to stimulate the respiratory
activity of periosteal osteoblasts (Tonna, '58b, '59a). These
findings indicated that the cells were already respiring at a
maximum rate. Periosteal osteoblasts appear to be driven
to exhaustion early in life by the great demand placed on them
at about 5 weeks of age in the rat. The period of activity
for a periosteal osteoblast is short, and so is its life span.
By 26 weeks of age numerous osteoblasts have already disappeared from the periosteum, and those that persist beyond
this age are considerably altered.
SUMMaRY AND CONCLTXXONS
1. The population and morphological changes occurring in
periosteal osteoblasts and their mitochondrial complement
have been studied in femora of rats from birth to old age.
2. Throughout the life of the rat, the osteoblastic population was represented by two levels ; one high from birth to 8
weeks of age, and the other low from 26 weeks on. A critical
period was seen between 8 and 26 weeks, during which time
the population diminished considerably.
3. After birth the mitochondrial numbers of each osteoblast increased up to the period of the maximum rate of bone
formation. Mitochondria1 population fell sharply so that only
half the number of mitochondria were found by 8 weeks of
age, and progressively less were found with increasing age.
4. Drastic changes in mitochondrial numbers occurred
prior to population changes in osteoblasts.
5. Shortening of mitochondria was observed from 5 weeks
to old age, however, it was most prominent between the ages
of one and 8 weeks.
754
EDGAR 4. TONNA AND N A N PILLSBURY
LITERATURE CITED
CHANCE, B., AND G . R. WILLIAMS 19353, Respiratory cnzymcs i n oxidative
phosphorylation. I. Kinetics of oxygen utilization. 11. Difference
spectra. Ill. The steady state. IV. The respiratory chain. J. Biol.
Cheni., d f 7 : 383-438.
CHANCE,B., G. R. WILLIAMS,
W. F. HOLMESAND J. HIGGINS 1955b Respiratory eiizyincs in oxidative phosphorylation. V. A mechanism f o r
oxidative phosphorylation. Ibid., 2 2 7 : 439-451.
DEINEKA,
D. 1914 Boebachtungen uber die entwickclung knockengewebes mittels der versilberungsmethode. I. Die entwickeluiig der knockenzellen
im perichondralen prozesse. Anat. Anz., 4 6 : 97-126.
DEMPSEY,
E. W. 1956 Mitochondria1 changes i n different physiological states.
I n : Ageing in Transicnt Tissues, 1-01. 2. Ciba Foundation Colloquia
on Ageing. G. E. W. Wolstenholme and E. C. P. Millar J. and A.
Chnrchill Ltd. London, pp. 100-104.
ERNSTER,L. 1956 Organization of mitochondria1 DPN-linked systems. 1.
Reversible uncoupling of oxidative phosphorylation. Exp. Cell Res.,
10: 704-720.
ERNSTER,
L., AND 0. LINDBERG1958 Animal mitochondria. Ann. Rev. Phpsiol.,
20: 13-42.
FELL,H. B. 1925 Histogcnesis of cartilage and bone in the long bones of the
fowl. J . Morph. Physiol., 40: 417-459.
FISCHER,A. 1948 Morphological aspects of animal tissuc cells in synthetic
media. Acta Anat., 5: 57-71.
GYRR,E. 1956 A practical manual of medical and biological staining tech
niques, 2nd edition. Interscience Publishers, N. Y., pp. 302-303.
1956 1,’apparcil de golgi clans les eelliilcs
I I ~ G U E N AF.,
U , A N D W. RERNHARD
normale rt cancereuses des vert8bri.s. Arch. h r i n t . niicroscop. et
morphol. comparke, 4 4 : 27-51.
HARMAN,
J. W., AND M. FIEGELSON
1951 Studies on mitochondria. V. Thc
relationship of structure and oxidative phosphorylation i n mitochondria of heart muscle. Exp. Cell Res., 3: 509-525.
HARMAN,
J. W.,A N D A. KIT1YAKARA4 1955 Studies on mitochondria. VI. The
relationship between the structure, osmotic reactivity and ATPase
activity of mitochondria from pigeon skeletal muscle. Ibid., 8:
411-435.
XIII~L,J. C. 1936 The cytology niid histochriniitry of osteohlnsts grown in vitro.
Arch. Exptl. Zcllforsch. Gewebez., IS: 496-511.
HOGEBOOM,
G . H., A. CL4UDE AND R. D. HOTCHKISS1946 The distribution of
cytochrome oxidase and suceinoxidase in the cytoplasm of the mammalian liver cell. J. Biol. Chrm., 166: 615-629.
HOLTER,
H. 3954 Distribution of some enzymes in t h e cytoplasm of amoebae.
Proe. Roy. SOC., 142B: 140-146.
HUNTER,F. E., J. DAVIS AND 1,. CARLAT 1956 The stability of oxidative and
phosphorylative systems i n mitochondria under anaerobic conditionq.
Biochem. e t Biophps. Acta, 90.- 237-242.
AGING MITOCHONDRIA OF OSTEOBLASTS
HYSLOP,D. B.
755
1952 The effect of supravital dyes on osteoclaslts in tissue eulture. M.Sc. thesis, University of Liverpool.
PBITCHAFD,
J. J. 1952 A cytological and histochemical study of bone and
cartilage formation in the rat. J. Anat., 86: 259-277.
SIEKEVITZ,
P., AND V. B. POTTER
1955 Biochemical structure of mitochondria.
I. Intramitochondrial components and oxidative phosphorylation. J.
Biol. Chem., 625: 221-235.
TEDESCHI,
H., AND D. C. HAF~RIS1955 The osmotic behavior and permeability
of mitochondria t o non-electrolytes. Arch. Biochem. Biophys., 58:
52-67.
TONNA,E. A. 19583, Histologic and histochemical studies on the periosteum
of male and female rats a t different ages. J. Gerontol., 23: 14-19.
195813 Enzyme changes in the aging periosteum. Nature, 281: 486.
1959a Post-traumatic variations in phosphatase and respiratory
enzyme activities of the periosteum of aging rats. J. Gerontol., 24(6) :
159-163.
1959b The effects of aging on the mucopolysaccharide content of
the periosteum as revealed by histochemistry and autoradiography. J.
Bone & Joint Surg., 4 2 8 ( 4 ) : 770.
TONNA,E. A., AND E. P. CRONKITE 1959e Histochemical and autoradiographie
studies on the effects of aging on the mucopolysaccharjdes of the
periosteum. J. Biophysic. & Biochem. Cytol., 6(6) : 171-1 78.
PLATE 1
EXPLANATION OF FIGURES
7
The mitochondrial complement of the liver cells of a r a t one week of age
is seen throughout the cytoplasm. The nuclei stand out clearly. X 1000.
8
The mitochondrial complement of osteoblasts involved in the formation of
new trabeeulae, taken from the feniur of a one-week-old rat. The nucleoli
can be seen as large dark spots within the clear nucleus. X 1000.
9
An osteoblast from a one-meek-old r a t cxhibiting a n enormous mitochondrial
population. The mitochondrial-free nucleus reveals a large nucleolus. x 1000.
10 The photomicrograph reveals two osteoclasts from a one-week-old rat ex-
hibiting a very large population of mitochondria. The large multinuclei can
be seen with their respective nucleoli. x 1000.
756
PLATE 1
LGIPU’G MITOCHONDRL4 O F OSTEOBLASTS
l D G A R A . TOXNA A N D N A N PILLSRURY
757
PLATE 2
EXPLANATIOK
OF
FIGURES
1 1 Several ohteoblasts of a one-week-old r a t , cxliillitiiig distinct mitochoiiclris
are seen actively involved in mntriv formation. X 1000.
12
Osteoblasts of a 5-week-old r a t showing s n abundant mitochondria1 coinplement. The cells are lining the preosseous zone found a t the left of the
photomicrograph. x 1600.
13 Periosteal osteoblasts of a n 8-weck-old r a t exhibiting considerably less niitoclioiidria than the cells seen in t h e previons figure. X 1000.
14 A further reduction in mitochondria1 population is seen with accompanying
niorphological changes in periosteal ostroblasts of a 26-\veek-old rat. x 1000.
758
A G I N G MITOCHONDRIA O F OSTKOULASTS
IIUGAR A . T O N N A A S D N.\K
PLATE 2
I’II,1.SL1U&Y
759
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