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Protein and ribonucleic acid synthesis in articular cartilage of osteoarthritic dogs.

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Protein and Ribonucleic Acid Synthesis in Articular
Cartilage of Osteoarthritic Dogs
By HENRY
J. MANKINAND PATRICK
G. LAING
T
IIE HISTOLOGICAL alterations in articuproach from those previously reported, and
lar cartilage from osteoarthritic joints measures the rates of synthesis of protein
and ribonucleic acid (RNA) as metabolic
have been well known for many year~,lJ-.",~
h i t the biochemical abnormalities are less indicators. Since the protein core of the
well defined, and most of the knowledge protein-polysaccharide molecule is syntheof the metabolism is speculative. Early ob- sized at the ribosome, and presumably unservations of changes in the metachromatic der control of genetic information transstaining3g4of the matrix were corroborated mitted by messenger RNA, assay of this
by studies demonstrating a decrease in would seem a more direct method of dechondroitin sulfate content with little al- fining the metabolic state of the cell. In a
teration in the ~ o l l a g e n . ~ , Collins
~.'
and prior study from this laboratory, assays of
his co-workers studied S'500,z incorpora- incorporated glycine-H3 and cytidine-H3
tion into osteoarthritic human cartilage, into cartilage were used to measure protein
and showed an increase in rate approxi- and RNA synthesis and were shown to be
mately proportional to the severity of the an excellent indicator of cellular injury."'
process.8 These 2 sets of experimental data
Over the past 5 years, the authors have
led to the theory that there was an in- studied 9 German Shepherd dogs which
creased turnover of protein-polysaccharide developed osteoarthritic changes in both
in osteoarthritic cartilage, and recent re- femoral heads as a result of dysplasia of the
ports regarding the presence of catheptic hips. Recently this study was terminated,
enzymes in cartilage have added consider- and one phase of the final examination of
ably to the credibility of this concept.9
the mechanical and biochemical abnormalThe radiosulfate technic is an excellent ities associated with the degenerative procmethod for the quantitative study of ess was the determination
of the in vitro
the metabolism of protein-polysaccharates of incorporation of glycine-H7 and
ride,1°~11~12J"
but it is not a direct indicytidine-H? into the cartilage from several
cator, since the sulfation of the macromolecule is known to occur as a separate step areas of the damaged joint surfaces.
The data obtained demonstrated a sigand is probably not directly related to
nificant
decrease in the rates of synthesis
either messenger RNA or protein synthesis
at the r i b o ~ o m e . ~Recent
~ . * ~ studies have of RNA and protein in the weight bearing
indicated that there may be a temporal area of the femoral heads of these osteolag between synthesis of the complex and arthritic dogs. In the non-weight bearing
its ~ u l f a t i o n . ' ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ areas, which were less severely involved,
This study of the metabolic activity of the rates of synthetic activity did not differ
osteoarthritic cartilage uses a different ap- significantly from those of normal cartilage.
From the Department of Orthopaedic Surgery,
Uninersity of Pittsburgh School of Medicine,
Pittsburgh, Pennsyli,ania 1521 3.
Supported in part by N.I.H. Service Grant
AM 01863-03.
HENRYJ. MANKIN,M.D.: Director of Ortho-
paedics, The Hoepita1 for Joint Diseases; Professor
of Orthopaedics, The Mount Sinai Medical School.
Current address: The Hospital for Joint Diseases,
New York, N.Y. PATRICKG. LAING,M.D.: Associate Professor of Orthopaedic Surgery, University
of Pittshurglz School of A4edicine.
444
ARTHR:-rIS AND
RHEUMATISM,
V O L . 10, NO. 5 (October 1967)
PROTEIN AND RIBONUCLEIC SYNTHESIS
Fig. 1.-Photographs
of the femoral head
from a German Shepherd dog with osteoarthritis. The “weight bearing area” is indicated. Note the erosive changes and
irregdarity of surface. Osteophytes a r e present
on t h e inferior posterior margin.
MATERIALSAND METHODS
The 9 German Shepherd dogs used for this
study were donated by a number of dog owners
in western Pennsylvania who found the animals
nnsatisfactory for breeding purposes because of
“congenital” dysplasia of the hips. In every case
the condition had progressed to frank osteoarthritis by the time they were first seen at the Orthop e d i c Laboratories of the University of Pittsburgh.
‘Three additional large ( b u t not thoroughbred) animals were obtained to serve as controls. None of
these hacl either gross or microscopic evidence of
osteoarthritis. All the osteoarthritic animals were
caged in standard dog pens over a 5-year period
and fed ad libitum with Purina Dog Chow.
Basal Eagle’s medium was obtained from the
Baltimore Biological Corporation, Baltimore, Maryland and glycine-H3 (Sp. Ac.200 mc./mM.) and
cytidine-HS (Sp. Ac.7 mc./mM. ) were obtained
from the New England Nuclear Corporation, Boston, Mass.
The assay of the in vitro rates of incorporation
of glycine-H3 and cytidine-H3 into the articular
cartilage of the femoral heads was performed at
the time of sacrifice. Since the animals varied in
age, size and possibly purity of breed, it was
necessary to have an “internal” control. This was
achieved by measuring the rate of isotope incor-
445
poration into the cartilage from the knees of the
osteoarthritic animals and from both hips and
knees of the control animals, thus providing a
ratio of rates of incorporation for hips and knees
from both groups. Since cell population varied
considerably depending on the area of study,
DNA content was also determined, so that these
ratios could be expressed on the basis of rates of
incorporation per pgm of DNA.
When the animal was killed, the hip and knee
joints were rapidly exposed, examined grossly and
photographed. It was apparent that the degree of
osteoarthritic change varied considerably in different areas of the femoral head, so the cartilage
surface was arbitrarily divided into 2 zones: the
weight bearing area, which lies in the anterior
superior quadrant in the dog; and the non-weight
bearing area, consisting of the remainder of the
femoral head (Fig. 1 ) . The cartilage covering the
distal femur showed no abnormality in any of the
animals. From these 3 areas-weight-bearing area,
non-weight bearing area, and distal femoraslices of cartilage were resected (taking care to
exclude the underlying bone) and the following
studies performed.
In vitro incorporation rates for glycine-H.’ and
cytidine-H”. Ten cartilage slices from each area
were separately incubated at 37 C in Eagle’s
medium containing glycine-H3 or cytidine-H?
(125 pc/ml.). After 1 hour the slices were harvested, fixed in formalin and dried and defatted
in absolute alcohol. The dried samples were
weighed to the nearest ,001 mgm. on a Mettler
Micro Gramatic Balance, and the trichloracetic
acid-insoluble fraction was assayed for incorporation radioactivity by a method previously described.“ Values obtained were expressed as
counts per mgm. (dry weight) per minute.
DNA determination and histological controls.
Another 10 samples of the cartilage from each
site were separately dried, weighed and assayed
for DNA content by the indole microcolorimetric
method described by Bonting and Jones.= Values
obtained were expressed as micrograms of DNA
per mgm. of dry weight. Standard hematoxylin
and eosin sections were made of representative
tissue from the same area (Fig. 2 ) .
I t was thus possible to obtain expressions for
the incorporation rates of glycine-H3 and cytidineH* per microgram of DNA for the 2 zones of the
femoral head and the knees of both osteoarthritic
and control animals. The incorporation rates for
the hips were then expressed as multiples of those
for the knees of the same animal for both osteoarthritic and control dogs, and thus allowed
critical comparison.
446
MANKIN AND LAING
Fig. 2.-Photomicrographs of the articular surfaces from the femoral head of a
dog with osteoarthritis. A: weight bearing area. This shows severe changes with
marked fibrillization of the surfaces. Cells are relatively well preserved ( x 100).
B: non weight bearing area. There is mild hypercellularity noted but the chief finding
is vascular buds crossing the "tideinark"-the
basophilic line separating the zone
of calcified cartilage (x 100).
the rate, as measured in counts per kgm
Figs. 3 and 4 are graphic representa- DNA per minute, appears to be approxtions of the data obtained in this study. Fig. imately the same for hip and knee. The
3 shows the rates of incorporation of gly- rate of incorporation is moderately recine-H3 and Fig. 4 the rates of cytidine-H3 duced in the cartilage of the weight bearinto articular cartilage of normal hips ing area of the arthritic hips, and essen(from control animals), and the weight tially unchanged or possibly slightly inbearing area and the non-weight bearing creased in the non-weight bearing area.
areas of the osteoarthritic hips. Each value
DISCUSSION
is expressed as a multiple of the values
From the date described above it is apfor the rates of isotope incorporation per
microgram of DNA for the knee of the parent that the in vitro incorporation rates
same animal, and each point on the graph of glycine-H3 and cytidine-H3 are considrepresents the mean of 10 such separate erably reduced in the cartilage from the
determinations.
weight bearing areas of osteoarthritic hips
Upon analysis of Fig. 3, it is evident that of dogs and, if not reduced, at Ieast are not
glycine-H3 incorporation is normally ap- increased in the non-weight bearing areas.
proximately 1.4 times as rapid in the hip One could assume from these data that
as in the knee. Also, in the weight bearing RNA and protein synthesis are significantly
area of osteoarthritic hips, it is sharply re- decreased in these tissues, and that the
duced to less than 60 per cent of the nor- degree of depression is proportional to the
mal mean value. In the non-weight bearing severity of the disease. Before accepting
area, there is a much wider scatter but these observations, it is necessary to be certhere appears to be little or no decline in tain that the results expressed above are
rate.
valid and that they indeed reflect the metFig. 4 demonstrates the results of cyti- abolic activity of the tissue.
dine-H3 incorporation. In normal animals
There is ample evidence that glycine-H3,
RESULTS
447
PROTEIN AND RIBONUCLEIC SYNTHESIS
-I
in
4
2.00
5
1.80
ZP
f
\
J
4
I
WT. BEARINO
2.00
1.60
z a
v,
1.40
1.20
1.00
1.00
0.80
0.60
0.80
o o
0
0.40
0.60
0.20
0.40
Fig. 3.-Glycine-H3 incorporation into the
cartilage of femoral heads of normal and
osteoarthritic dogs. Data is expressed as
multiples of values for the knee of the same
animal (recorded as counts/pgm. DNA/min.).
It is apparent that the cartilage from normal
hips incorporates glycine-H3 about 1.4 times
as rapidly as the knees. The cartilage from the
weight bearing areas of arthritic hips shows
marked reduction in glycine-H3 incorporation,
and that from the non-weight bearing areas a
slight reduction (or at least no increase.)
incorporated into the cartilage cell, is utilized in protein-polysaccharide synthesis.
Prior studies in this laboratory have indicated the shunt into nucleic acid metabolism23 is insignificant in articular cartilage,20*24 and that the majority of the
labeled substrate is entering the proteinp o l y ~ a c c h a r i d e . 2Degradation
~~~~
rates for
the protein-polysaccharides are much
longer than the experimental time in this
~ t u d y , 2 ~negating
~~7
the possibility that the
counts obtained are indicative of decay
rates as well as the rate of incorporation.
Cytidine, although a precursor of both
RNA and DNA,28,2gmay be assumed to be
entering RNA synthesis in a tissue where
DNA synthesis is minimal.29 Although
there is some increase in DNA synthesis in
osteoarthritic ~artilage,2*~O
the amount is so
small as to be insignificant, and it is therefore assumed that the rate of cytidine-H3
incorporation is indicative of RNA synthesis.
0.20
If:.
AV = 0.38
AV
= 0.76
I
AV = 1.051
Fig. 4.-Cytidine-H3
incorporation into the
cartilage of femoral heads of normal and
osteoarthritic dogs. Data is expressed as
multiples of values for the knee of the same
animal (see text). Cartilage from normal hips
incorporates cytidine-H3 at approximately the
same rate as the knees. There is a reduction
in the rate in the weight bearing area of the
arthritic hips and a wide scatter but probably
no change in the non-weight bearing area.
(Each point on the graph represents the mean
of 10 determinations on cartilage from only
the femoral head.)
Before accepting the validity of these
counts, it is important to point out that
there are 2 sources of potential error which
cannot be controlled. The quantity of
labeled substrate which is present in a tissue at the time of counting will depend
not only on the synthesis of t h e product
and the rate of degradation but also on the
pool of unlabeled substrate present in and
around the ce11.3I It is therefore possible
that the intracellular pools for glycine or
cytidine (or even sulfate) vary with the
degree of osteoarthritis and possibly independently of each other, thus affecting the
rates of incorporation of the labeled substrates to a lesser or greater extent. A second problem exists in attempting to define
the metabolism of a damaged cell by comparing it with a "healthy" one. It is possible
that glycine or cytidine utilization by the
448
MANKIN AND LAINC
Table 1.-Average
DNA Concentrations in Normal and Osterarthritir
of DNA per mgm. ( d r y weight).
Cartilag-mgm.
Krires ( 3 0 )
17.4 & 5.0
Normal Hips (6)
Osteoarthritic Hills (9)
U'eiglit
Ilearing
Area
Non-weight
Bearing
Area
Weight
Ilearing
Area
Nori-weight
Bearing
Area
16.7 r+. 6.2
(96%)
15.9 ? 4.5
(91%)
21.2 ? 6.1
(117%)
13.8 _t 5.4
(80%)
osteoarthritic cartilage cell is abnormal in
terms of either product or pathway, thus
to some extent invalidating these results.
If, however, one assumes that there is
little variation in the pools or utilization,
it is possible to conclude that the rates of
incorporation of glycine-HI and cytidineH I are indicative of the rates of protein
and RNA synthesis respectively in both
normal and abnormal cartilage.
The DNA determinations, as performed
by the method of Honting and Jones,22 are
indicators of the number of cells present
per milligram of tissue and therefore reflect, not only cell replication, but any
change in ratio of cells to quantity of
matrix. Table 1 illustrates the variations in
quantity of DNA in the weight bearing
and non-weight bearing areas of the osteoarthritic hips as cornpared with the normal
hips and knees. It is quite evident that
there is an increase in the amount of DNA
per milligram of tissue in the more severly
damaged (weight bearing) areas of the
femoral heads, probably indicating a decrease in matrix rather than an absolute
increase in the number of cells. Such an
observation is certainly consistent with the
histological appearance (Fig. 2 ) , in which
there is obvious degradation of matrix and
fibrillization of the articular cartilage with
relatively good preservation of the cells.
If the data are combined, it is possible to
define a rate of synthesis of RNA and pro-
tein per microgram of DNA, and thus establish an index of cellular metabolism for
the osteoarthritic cartilage as compared to
the hips of the normal animals. It is quite
evident that, barring the possible sources
of error described above, there is a decrease in metabolic activity in the severely
affected areas in the weight bearing portion of the femoral head.
At first glance, these results vary considerably from those reported by Collins and
his co-workers who have shown, by both
qualitative autoradiography and quantitative assay, an increased S'"O,= fixation in
articular cartilage from osteoarthritic human jointsHIn critical analysis, however, it
is apparent that species variation or technical difference in the procedures, or variation in the pathology, make a positive
statement of this sort impossible. If one
wishes to speculate that such a dichotomy
exists, the findings may imply that there is
some variation in the synthesis or degradation of the protein-polysaccharides in osteoarthritic cartilage which is associated with
a decline in RNA and protein synthesis
and an increase in sulfate incorporation.
In conclusion, the authors have found
that in cartilage from the femora of osteoarthritic hips of dogs, there is a substantial
decrease in protein and RNA synthesis as
compared with normals, and this decrease
seems proportional to the severity of the
process.
SUMMARY
The in vitro rates of incorporation in glycine-H:<and cytidine-H3 were determined
for the articular cartilage from the femoral heads of 9 German Shepherd dogs with
osteoarthritis secondary to congenital dysplasia, and 3 normal animals of approximately
449
PROTEIN AND RIBONUCLEIC SYNTHESIS
the same size and weight. These data were expressed per pgm of DNA and compared,
using the cartilage from the knees as internal control. On the basis of prior studies,
these rate values can be equated to the rates of synthesis of protein and RNA respectively. From the data obtained, it is apparent that protein and RNA synthesis are
markedly decreased in the most severely involved areas and, if not decreased, at least
riot increased in the less damaged areas.
SUhlMAFUO I N INTERLINGUA
Le rapiditate del incorporation de tritiate glycina e de tritiate cytidina esseva determinate in vitro in specimens de cartilagine articular ab le capites femoral de 9 canes de
pastor german con osteoarthritis secundari a congenite dysplasia e de 3 tal canes normal
de approximativemente le mesme grandor e peso. Le valores esseva exprimite como
fig de acido desoxyribonucleic e comparate con le us0 de cartilagine ab le genu como
control0 interne. A base de studios effectuate in le passato, il pare justificate equar le
valores obtenite con le rapiditates de synthese de proteina e de acido ribonucleic,
respectivemente. Ab le datos obtenite le conclusion pote esser derivate que le synthese
de proteina e de acido ribonucleic es marcatemente relentate in le areas le plus
severmente afficite. In areas minus servmente afficite, ille synthese-si non relentatees certo non accelerate.
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