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Experimentally induced arthritis using intra-articular papain.

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Experimentally Induced Arthritis Using
Intra-articular Papain
By DAVIDG. MURRAY
Daily injection of a crude papain solution into the knee joint of young rabbitst
continued over a six day period resulted
in a degeneration of the articular cartilage within two weeks. The initial findings were elevation and loosening of the
cartilage in the weight bearing area followed by thinning and fibrillation of the
remaining surface. Microscopic changes
consisted of disruption of the cartilage
adjacent to the subchondral bone and
loss of the superficial layers. Changes in
the articular cartilage were similar to
changes seen in clinical forms of degenerative arthritis.
Le injection diurne de un solution de
papaina crude ad in le articulation genicular de juvene conilios, quando continuate durante sex dies, resultava intra duo
septimanas in un degeneration del cartilagine articular. Le constatationes initial
esseva elevation e distachamento del
cartilagine in le in areas sub pression
de peso, sequite de tenuifi cation e fibrillation del remanente superficie. Le alterationes microscopic que esseva notate
consisteva de disruption del cartilagine
adjacente a1 osso subchondral con perdita del stratos superficial. Alterationes
in le cartilagine articular esseva simile
a alterationes vidite in formas clinic de
arthritis degenerative.
M
ANY ATTEMPTS have been made to duplicate in the experimental animal varieties of joint disease found in man. A recent survey by Gardnerl
on the experimental production of arthritis produced an extensive list of techniques and agents which have been used with varying degrees of success.
Some of these efforts have produced forms of arthritis with features analogous
to clinical
but many variables have made it difficult to draw significant conclusions. Continued work is being done to find methods for inducing
pathologic joint changes which will shed light on the etiology of chronic articular disease.
Intra-articular papain was selected in the present investigation to alter the
composition of articular cartilage. Papain, a proteolytic enzyme, has a profound effect on cartilage in young animals when administered intravenou~ly.~
This effect has been related to the release of mucopolysaccharide from cartilage matrix without apparent alteration of the collagena6Because of this
specific activity it was felt that local injection might provide information as
to the relative roles of mucopolysaccharide, collagen and cellular elements in
maintaining joint cartilage integrity.
METHOD
New Zealand white rabbits weighing approximately 1 kilogram were used as experimental animals because of their known susceptability to the effects of papain. Thirty
animals were divided into groups of 15 and the experiment was conducted as two separate
From the Department of Surgery, Division of Orthopedics, State Unioersity of New
York, Upstate Medical Center, Syracuse, N e w York.
211
ARTHRITISAND
RHEUMATISM, VOL.
7 , No. 3
(JUNE),
188.1
212
DAVID G . MURRAY
series. They were kept in standard cages on a routine maintenance diet and no attempt
was made to encourage or discourage other than normal exercisr.
A solution of crude papain was prepared by dissolving 1 gram of powder in 100 ml. ot
isotonic saline and filtering. The prepared solution was kept refrigerated and renewed
every 48 hours. A total of 2 ml. of crude papain solution was administered locally in
one knee joint of the rabbit in six divided daily doses. The initial dose was 0.5 ml. of solution followed by 0.5 ml. on the second day and 0.25 ml. on each of the following four days.
Isotonic saline was injected into the opposite knee in identical volumes as a control. The
pH of the solutions varied between 5.6 and 6.0 for the papain and 6.6 and 7.0 of the
saline. The solutions were accepted as slightly acid to avoid possible effect on papain
activity by adjusting to neutral. Previous work by Key2 has indicated that the p H of the
injected solution was not a primary factor in the experimental production of arthritis.
The technique for injection consisted of inserting a 25 gauge needle into the knee
joint of the unanesthetized rabbit, starting at the lateral inferior aspect of the patella
and directing the needle toward the suprapatellar pouch. In this manner inadvertent
damage to the cartilage of the tibia1 plateau was avoided.
An animal was sacrificed daily for the first 10 days and then at weekly intervals until
no further changes were noted. Tibia1 plateaus were taken for examination, split and fixed
in 10 per cent formalin prior to decalcification and sectioning. Sections were stained
with hemotoxylin and eosin for routine microscopic examination.
RESULTS
All animals showed some general effect of the papain following the initidl
intra-articular injection. This effect, which consisted of drooping of the tips
of the ears, resolved after approximately four days in spite of the repeated
injections. There were no other outward manifestations of papain effect. No
significant clinical evidence of discomfort appeared in the treated knee joints,
either at the time of injection or afterward. The animals were normally active
in their cages and did not limp or otherwise favor either leg during the period
of the experiment.
An increased amount of joint fluid which was faintly straw colored and
slightly cloudy was demonstrable by the third day of the experiment in the
joint receiving papain. Joint effusion began to diminish two weeks after
the last injection. An enlargement of the entire knee was noted during this
time which was still present at the time of sacrifice of the last animal. The
only other clinical finding manifest during the experiment was a mild flexion
contracture of the treated knee noted after four weeks.
Gross changes present on the articular surface of the experimental knee
at the time the animals were sacrificed were directly related to the duration
of the experiment. These changes were consistent depending upon the time
interval from the first injection of papain.
After 24 hours the articular cartilage was slightly opalescent and whiter
than the control side but remained smooth and glistening. The menisci were
normal in appearance, and except for a barely detectable increase in vascularity
the synovial membrane was unchanged.
The first localized change in the articular cartilage became apparent on
the 4th day after the initial injection. On the lateral condyle of the tibia a
circular patch of slightly wrinkled cartilage appeared in the area not protected by the lateral meniscus. The menisci appeared unchanged and the
synovium exhibited mild edema with minimal vascular injection.
INDUCED ARTHRITIS USING INTRA-ARTICULAR PAPAIN
213
Within the next two days the area of roughened cartilage on the lateral
condyle became elevated and rolled on one margin as if loosened from its attachment to the underlying bone. This area was distinctly whiter than the
surrounding cartilage and in some cases was separated from adjacent articular
cartilage (fig. 1 ) .
Two weeks from the beginning of treatment the central areas on both the
lateral and medial plateaus were slightly depressed, dull and roughened,
giving a coarse granular sensation when scraped with a smooth metal instrument. The synovial membrane was thickened with mild vascular injection and
the intra-articular fluid was increased in amount to a maximum of about 0.5
ml.
At 6 weeks the entire articular surface of the tibia was roughened and
dull with the most pronounced changes in the central areas (fig. 2 ) . Menisci
were thickened and frayed at the inner margins which were rounded in
contrast to the normal sharp semilunar border. The amount of intra-articular
fluid was less than noted at 2 weeks. The synovial tissue was thickened and
mildly inflamed with numerous fine intra-articular adhesions present, predominantly in the region of the cruciate ligaments.
On microscopic examination the earliest changes appeared on the first day
with swelling of cartilage cells and a decrease in intercellular material. By the
second and third day a zone of cell necrosis as compared to normal appeared
adjacent to the subchondral plate in the area of contact with the femoral condyle (figs. 3 and 4 ) . This area was devoid of viable cells and took on a fibrous
appearance with the orientation being parallel to the joint surface. By the fourth
day definite horizontal clefts appeared in this area (fig. 5) and by the sixth
day the clefts coalesced to a major rent which separated the superficial
viable layer of cartilage from the underlying subchondral plate leaving a thin
layer of collagenous tissue over the bone (fig. 6 ) . At later stages the superficial layer disappeared leaving a frayed fibrillated fibrous layer covering the
bone with attempts at repair reproducing clumps and clusters of cartilage
cells (figures 7, 8, 9). Several specimens showed an increased density of the
subchondral bone particularly in the area subjected to weight bearing stresses.
The synovial tissue throughout the experiment showed slightly increased
vascularity with a diffuse infiltration of inflammatory cells. A significant increase in the number of eosinophiles appeared noteworthy. In the later stages
there was a tendency for synovial tissue to blend with and replace articular
cartilage at the margins of the joint.
DISCUSSION
Parenteral administration of papain in young rabbits affects cartilage
generally by liberating chondroitin sulfate from the matrix. This is accomplished by breaking down the protein bonds between the mucopolysaccharide
and collagen and dowing the mucopolysaccharide to enter the blood ~ t r e a m . ~
The collagen remains unaffected by the enzyme. Unless repeated doses are
given chondroitin sulfate is reproduced and the normal constituency of the
cartilage is restored within 4 6 days. Permanent damage in sensitive areas
such as epiphyseal cartilage may result from repeated doses.6 There are no
214
DAVID G. MURRAY
Fig. 1,-Control and treated knee joints from a rabbit 48 hours after completing
six daily intra-articular injections of crude papain solution. The tibia on the right
shows loosening and marginal heaping of articular cartilage in an oval area where
it is in contact with the femoral condyle. For purposes of comparison the right and
left tibias have been transposed so that the lateral plateaus are adjacent. x4
Fig. 2.-Knee
joints from a rabbit sacrificed five weeks after completion of
the six day course of intra-articular papain. The cartilage on the treated side is now
generally dull and fibrillated with edema of the synovial tissue and marginal encroachment of synovial tissue on the degenerating joint surface. x4
INDUCED ARTHRITIS USING INTRA-ARTICULAR PAPAIN
215
Fig. 3.-Section through the weight bearing area of the lateral tibia1 plateau in
an untreated rabbit. The joint surface (S) is relativelv smooth with fairly uniform
distribution of cells throughout the articular cartilage ( C ). The subchondral bone
( B ) shows normal trabeculation. H & E x150
Fig. 4.--Section through an area comparable to Fig. 3. from an animal sacrificed
after two successive daily injections of crude papain into the knee joint. Note the
disappearance of viable cells in the area of cartilage ( D ) adjacent to the subchondral plate with irregularity and slight roughening of the articular surface.
H & E x150
216
DAVID G. MURRAY
Fig. 5.--Section of the tibia1 plateau taken 6 days from onset of treatment.
A this time clefts ( K ) have begun to appear in the deeper layers of the articuIar
C rtilage with a general orientation parallel to the joint surface. Friction of the
opposing femoral condyle has started to displace the superficial layer of cartilage
causing wrinkling ( W ) to occur at the margin of the contact area. H & E x150
Fig. 6.--Section taken 8 days from the onset of treatment corresponding to the
tibia shown in Fig. 1. The superificial layer of cartilage is now further separated
from the underlying fibrocartilagenous layer and displacement of the cartilage. has
continued. H & E x150
INDUCED ARTHRITIS USING INTRA-ARTICULAR PAPAIN
217
Fig. 7.-Section taken 10 days from onset of treatment and four days after the
final injection. The separated layer of cartilage ( C ) has begun to fragment and
disappear leaving a thin layer ( L ) of fibrocartilage over the epiphyseal bone. H & E
x70
Fig. &--Section taken 6 weeks from the onset of treatment and 5 weeks after
the final injection corresponding to the tibia shown in Fig. 2. The weight bearing
area is characterized by thin, fibrillated fibrocartilage (F) with attempts at repair
( R ) . At this point sclerosis (SC) of the subchondral plate is particularly marked in
the area of degeneration. H & E x150
218
DAVID G. MURRAY
Fig. 9.-Section from corresponding area of control tibia shown in Fig. 2.
Cartilage cells are viable with inainteiiaiice of relatively normal thickness of the
joint cartilage. There is no significant change in the subchondral bone from the
normal joint (Fig. 3 . ) . H & E x150
reports of arthritis caused by parenteral papain administration despite this
profound general effect on cartilage.
The changes prcduced in this experiment probably reflect the high concentration of enzyme in direct contact with the knee cartilage as well as to
the frequency of injections. That a certain amount was quickly absorbed
into the circulation was obvious from the slight drooping of the ears which
appeared as promptly as following intravenous administration but to a lesser
degree. The opposite knee cartilage on all animals showed some change during
the six days injections were given but reverted to normal within a week after
the last dose. Destructive changes might be produced in all joints if high
enough intravenous doses could be tolerated by the animal.
Two findings indicate that the changes in the cartilage were not due to a
local necrotizing effect of the enzyme. In the first place, though the material
was instilled into the suprapatellar pouch and should have diffused evenly
down to the region of the tibial plateaus, only a localized area was involved
in the first phase of degeneration. This was consistently in the central region
of the tibial plateau. In the second place, microscopic examination during
the’early stages showed the superficial cartilage cells to be viable while degeneration with loss of cells and cleft formation took place in the deeper
layer adjacent to the subchondral plate. In addition the superficial synovial
tissue showed no evidence of necrosis and actually a limited degree of inflammation.
Trauma to the extent associated with normal activity appeared to initiate
the actual destruction of the joint surface. The area in which the first changes
INDUCED ARTHRITIS USING INTRA-ARTICULAR PAPAIN
219
were noted was that area, usually on the lateral plateau, exposed directly to
contact with the femoral condyles and delineated by the margins of the
menisci. The opposing surfaces of the femur which during the course of
flexion and extension presented a relatively much greater surface of contact
with the localized tibial area showed no such isolated destruction, but ultimately became generally dull and frayed to gross inspection.
If one accepts the hypothesis that papain affects only the mucopolysaccharide component of the articular cartilage then the importance of this element in maintaining the integrity of the joint surface under the stress of
normal activity becomes apparent. It would seem from this experiment and
from some previously reported
that the enzyme also has a detrimental effect on the chondrocytes themselves and is a factor to consider in
the interpretation of results.
CONCLUSIONS
The combination of local enzymatic activity and unrestrained joint motion
produced a primary cartilage degeneration in the rabbit knee joint. Altering
the mucopolysaccharide content of the articular cartilage by repeated intraarticular papain injection appeared to weaken the attachment of the articular
cartilage to the subchondral plate. Separation of the superficial layer of cartilage was initiated by the trauma associated with friction of opposing surfaces
in a weight bearing area and attempts at repair were inhibited by a loss of
chondrocytes in this zone. The resultant picture of frayed fibrillated fibrocartilage in the weight bearing area of the tibial condyle with clumping of
chondrocytes resembled later stage? of clinical forms of degenerative arthritis.
REFERENCES
1. Gardner, U. L.: The experimental Pro-
2.
3.
4.
5.
duction of arthritis. Ann. Rheum.
Dis. 19:297, 1960.
Key, J. A.: The production of chronic
arthritis by the injection of weak acids,
alkalies, distilled water and salt d u tion into joints. J. Bone and Joint
Surg. 15:67, 1933.
Barnett, C. H.: Wear and tear in joints.
J. Bone and Joint Surg. 38-B:567,
1956.
Gardner, D. L.: Production of arthritis
in the rabbit by the local injection of
the mucopolysaccharide caragheenin.
Ann. Rheum. Dis. 19:369, 1980.
Thomas, L.: Reversible collapse of rabbit ears by intravenous papain and
6.
7.
8.
9.
prevention of recovery by cortisone.
J. Exp. Med. 104:246, 1956.
Bryant, J. H., Leder, I. G., and Stetten,
11.: Release of chondroitin sulfate from
rabbit cartilage after injection with papain. Arch. Biochem. and Biophys. 76:
122, 1958.
Tsaltas, T. T.: Papain induced changes
in rabbit cartilage. J. Exp. Med. 108:
507, 1958.
Hulth, A,: Growth inhibiting effect by
papain on young rabbits. Acta Or&.
Scand. 27:Fasc. 3, 1958.
Spicer, S. S.: Cartilage changes in papain
treated rabbits. Am. J. Path. 33:1237,
1957.
David G . Murray, M . D., Assistant Professor of Orthopedic
Surgery, State University of New York, Upstate Medical
Center, Syracuse, New York,
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