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Lubrication of animal joints. iii. the effect of certain chemical alterations of the cartilage and lubricant

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Lubrication of Animal Joints.
111. The Effect of Certain Chemical Alterations
of the Cartilage and Lubricant
By FRANK
C. LI"
The coefficient of friction of dog ankle
joints measured in oitro was found to be
greatly affected by certain enzymatic
treatments of bovine synovial mucin. Testicular hyaluronidase had no effect on
its lubricating action even though it
abolished the viscosity. By contrast, tryptic digestion destroyed the ability of the
mucin to lubricate without reducing the
viscosity. These observations indicate
that a protein moiety is an intrinsic component of the mucin molecule; and that,
together with an only partly polymerized
hyaluronate, it is responsible for the mucin's function as a lubricant. The protein
may serve as a prosthetic group for adsorption of the mucin onto the surface
of the cartilage. Sodium heparin inhib-
U
AND
ERICL. RADIN
ited lubrication by the m u c h In mucinfree buffers, the pH and molarity of the
lubricant bath altered both the friction
and deformability of the cartilage. The
change in friction was not consistently
related to the change in deformability.
The amphoteric effect of pH on the
friction raises the possibility that electrostatic surface effects contribute to adhesive friction. Formalin made the cartilage rigid and increased the coefficient of
friction. It did not, however, have much
effect on the lubricating action of the
mucin. A small number of pathological
human synovial fluids were studied and
found to lubricate as well or better than
healthy bovine material.
the lubrication of animal joints was believed to be hydrodynamic and dependent on the viscosity of
synovial fluid. It has now been established
that a combination of hydrostatic and boundary mechanisms is mainly responsible for
the low friction.1-5Joints are slippery even
when lubricated with saline solutions, but
the addition of synovial much lowers the
coefficient of friction by about 45 per cent.5
McCutchen4 and L i n d have shown that
when the viscosity of synovial fluid is reduced to that of water by digestion with
hyaluronidase, it loses little or none of this
lubricating advantage.
This paper deals with certain chemical
From the Section on Rheumatic Diseases, Laboratory of Experimental Pathology, National Institute of Arthritis and Metabolic Diseases.
Requests for reprints should be addressed to
Section on Rheumatic Diseases, 10:3N-114,
N.I.A.M.D., Bethesda, Md.20014.
FRANK
C. LINN,B.A. IN M.E.: Research Engineer (retired}, Section on Rheumatic Diseases, Laboratory of Experimental Pathology, National I n stitute of Arthritis and Metabolic Diseases,
National Znstitutes of Health, Bethesda, Maryland
20014. ERIC L. RADIN, M.D.: Formerly Captain
674
NTIL RECENTLY
U.S.A.F., M.C., Malcolm Grow Clinical Center,
Andrew Air Force Base, Maryland; Guest Inoestigator, Section on Rheumatic Diseases, Laboratory
of Experimental Pathology, National Institute of
Arthritis and Metabolic Diseases, National Znstitutes of Health, Bethesda, Maryland. Presently
Instructor in Orthopedic Surgery, Haruard Medical
School. Address: Massachusetts General Ilo.cpita1,
Boston, Mass. 02114.
ARTHRITISAND RHEUMATISM,
VOL.11, No. 5 (OCTOBER
1968)
675
LUBRICATION OF ANIMAL JOINTS. 111
Table 1.-E#eet
Lxge8tion:
Ezperiment no.:
Lubricant
Buffer
Control mucin
Buffer
Digested mucin
Buffer
of Terticular Hyaluronidaae Digation on Lubrication by Synovial M u c h
Mild (3 hours, 23"C, p H 7.2)
Exhaustive (24 hours, 37"C, p H 5.5)
1
2
3
4
Coef. of Relative
friction viscosity
Coef. of Relative
friction viscosity
Coef. of Relative
friction viscosity
Coef. of Relative
friction viscosity
.0077*
.0052
.0076
.0056
.0076
.0083
2.38
.o040
1.00
.0082
,0041
.0086
Uronic acid
(micmgrams/ml. )
Acetylglucostlmine
(micrograms/ml.)
.0061
3.27
.oO40
1.15
.0062
.0057
,0059
.0063
3.50
.0035
3.31
1.05
.0061
.0043
.0065
1.07
240
245
240
265
*A different ankle was used in each experiment.
alterations of articular cartilage and mucin
which further elucidate their particular contributions to joint lubrication.
MATERIALS
AND METHODS
Lubrication was tested by measuring in uitro
the coefficient of friction of dog ankles during
continuous oscillation under constant load. The
apparatus, techniques for conducting the tests and
analyzing the results have been established and a
high degree of reproducibility has been obtained.0
Thc friction tests were conducted at 23 C with a
load of 40 pounds at 40 cycles per minute. Deformation of the joint cartilage also was measured by
previously described techniques.0
Bovine synovial fluid was obtained from the
hocks of freshly slaughtered calves. Cells were removed by centrifugation in the cold at 6000 rpm
for 20 minutes. The fluid was then stored at 4 C
until just before testing. A mucin fraction was
prepared by filtering the centrifuged fluid through
a 220 mp filter (Millipore Filter Corp., Bedford,
Mass.) and redissolving it in cold buffer. Because
phosphate buffer is known to cause slow degradation of synovial mucin,' 0.155 M veronate buffer
(.088M NaCl, .00408M Na 5-5 diethylbarbiturate,
.0625M HCI, pH 7.2) was employed.
Friction measurements were carried out with
veronate buffer before and after each test to check
the reliability of the cartilage and measuring devices. After each test, the surfaces were washed
with buffer and wiped clean with tissue paper.
The viscosity of the lubricants relative to water
was measured by a capillary flow method ancl pFI
with a glass electrode.
The effects of p H and ionic strength were determined by using a series of buffers as lubricants
without synovial mucin. The tissues were equilibrated with each buffer by oscillating them under
load for 7 minutes and then replacing the buffer
with a second aliquot of the same fluid.
Synovial fluid was subjected to mild and to exhaustive digestion with testicular hyaluronidase
(Worthington Biochemical Corp., Freehold, N.J.,
HSE 7BA) in concentrations of 75 USP units per
ml. The mild conditions were a digestion time of
3 hours as 23 C, p H 7.2; the exhaustive conditions
were 24 hours at 37 C, pH 5.5. Completeness of
the digestion was determined by measuring the
uronic acids and acetylglucosamineg before and
after treatment with the enzyme. At the suggestion
of Dr. David Hamerman, the mucin was prevented
from clotting at pH 5.5 by first dialyzing it
against 0.5 M NaCl solution. Before testing, the
pH was restored to 7.2 and the molarity to .155
by dialyzing against veronate buffer. Streptococcal
hyaluronidase (Wyeth Corp., Philadelphia, Pa.)
was found unsuitable for these experiments because it acted on the cartilage and itself alterecl
the friction characteristics.
Tryptic digestion of the synovial mucin was
carried out with bovine pancreatic trypsin (Worthington Biochemical Corp., Freehold, N.J., TRL
7AA) in a concentration of 60 turbidity reducing
units per ml. for 72 hours at 4 C, pH 7.2. The
cartilage was protected from tryptic digestion by
addition of an excess of soybean -sin
inhibitor
(Worthington Biochemical Corp., SI 61B) to the
synovial digestate.
Sodium heparin, without added preservatives,
was obtained from two independent sources (Up-
676
LI" AND W I N
Table t.-Efecr
of Tryptic Digestion on Lubrication b y Synovial Mucin
Experiment m.:
Lubricant
2
1
Coef. of
friction
Relative
viscosity
Coef. of
friction
3
Relative
viscosity
Coef. of
friction
Relative
viscosity
2.37
.0056
.0031
.0052
2.49
~~
Buffer
Mucin and inhibitor
Buffer
Digested much and inhibitor
Buffer
.0103*
.0059
.0098
.0089
.0100
3.18
3.19
.0053
.0028
.0055
.0054
.0053
2.38
.0048
.0054
2.49
*A different ankle w&s used in each experiment.
range of 3 to 9.5 in .155 M acetate and
veronate buffers. When acetate solutions
were employed, the value was quite constant between pH 6.2 and 8 (Fig. 1).Thc
RESULTS
coefficient of friction was highest at about
Enzymatic digestion of mucin. Partial di- pH 5 and dropped perceptibly above and
gestion of whole synovial fluid or its mucin below this hydrogen ion level. There was
fraction by testicular hyaluronidase, suffi- a slight but progressive decrease in the
cient to reduce the viscosity to that of the friction obtained with veronate buffers bebuffer sohtion, had no effect on their ability tween pH 6.5 and 9.5. Differing dissociation
to lubricate the cartilage (Table 1). Com- constants of the two buffers may account
plete digestion of synovial mucin, though for slight variation in the friction values
not of whole synovial fluid, increased the of comparable pH.
Deformation of the cartilage, as it was
coefficient of friction appreciably but it still
compressed,
increased when pH fell below
was considerably below values obtained
5.5.
Above
this
value, it was constant (Fig.
with the control buffer solution. Protein was
1).
This
relationship
held both in tests constill precipitable with 10 per cent trichlorducted
after
the
joint
was loaded with 40
acetic acid in this exhaustively digested
pounds
for
7
minutes
or with only a tare
mucin.
load.
Tryptic digestion of synovial mucin did
When the pH was kept constant at 7.2
not alter its viscosity but consistently caused
varying
the molarity of acetate buffer over
the coefficient of friction to rise to values
a
range
of .002 to 1.0 also affected the
close to those obtained with the buffer solutions (Table 2). This effect was not ob- coefficient of friction. It was lowest at conserved when whole synovial fluid rather centrations of .4 to .6 moles per liter. Dethan mucin was subjected to digestion. formation of the cartilage increased with
Soybean inhibitor, by itself or added to the molarity (Fig. 2).
The changes in friction and deformability
synovial fluid or mucin, had no effect on
of
the cartilage produced by varying the
the friction.
pH
and molarity were reversible.
Fresh frozen citrated plasma and fresh
Znhibitory efect of heparin. Heparin, in
serum had no lubricating advantage over
concentration of at least 18 USP units per
buffer solution.
Effect of p H and molarity. The coeffi- ml., decreased the lubricating effectiveness
cient of friction varied with pH over n of whole synovial fluid or its mucin. This
john Co., Kalamazoo, Mich., and Mann Research
Laboratories, New York, N.Y.). Both preparations
behaved identically and no distinction is made
between them in the results.
677
LUBRICATION OF ANIMAL JOINTS. 111
0 HCf
- Veronof No Buffer
Aceioie Buffer
E
.OlO
0
rn
n
.008
2
,006
on
0
rn
z
-I
1 ;
ob: b b
i
.05
I
I
:
1,
f
d
,bl 0
VH
Fig. 1.-Effect of pH on coefficient of frictionand deformation of cartilage.
inhibitory effect was incomplete but related novial fluid as lubricants (Fig. 4). Synovial
to the amount of heparin (Fig. 3). Even at fluid still retained, however, considerable
a level of 290 units per ml., the average lubricating advantage under these condimaximal reduction was never greater than tions.
Lubricating ability of human synovial
20 per cent of the lubricating advantage.
Effect of formalin fixation of cartilage. fluids. Synovial fluid from the knees of 2
Fixation of the cartilage in 10 per cent patients with rheumatoid arthritis and 2
neutral formalin for 7 days greatly reduced with traumatic effusions secondary to a torn
the deformation of the cartilage. It also re- medical meniscus lubricated with as low or
sulted in a several-fold increase in the co- lower coe5cients of friction than did the
efficient of friction, both with buffer or sy- control bovine synovial fluid (Table 3).
-.020
.020
- . o.ole
le
;
n
2
g
z
-I
- ,016
,016 2
a
n
LL
0
I)
2 .05
-.014
,014 zi
4
-I
I
I
a
I
I
I
0.3
0.4
0.5
a
8
4
015 0.6
d6 0.7
017
o(8
CONCENTRATION, Moles per liter
ACETATE BUFFER
8
- .01
.012
2
I
1
0.9
1.0
It0
Fig. 2.-Effect of molarity on coefficient of friction and deformation of cartilage.
LI" AND W I N
.010
Saline t Heparin
Synovia t Heparin
w
0
8
'i,,,
0
100
50
150
250
200
300
HEPARIN, U. S. I? UNITS / M L
Fig. 3.-Inhibition of synovial fluid lubrication by heparin. The values obtained
for undiluted synovia remained constant throughout the test.
The viscosity of the human fluids was higher than that of the bovine.
Efect of diluting synovial fluid. Bovine
synovial fluid retained its lubricating effect
until it was diluted with more than 3 parts
of veronate buffer despite the fact that the
relative viscosity decreased ( Fig. 5 ) .
DISCUSSION
The present experiments show that the
friction of joints is greatly affected by certain enzymatic treatments or addition of
heparin to synovial mucin; by the pH and
molarity of the lubricating bath; and by
formalin fixation of the articular cartilage.
The lubricating advantage of synovial
Table 3.-Lubricating
Patient:
Diagnosis:
Lubricant
Buffer
Patient's synovial fluid
Buffer
Bovine synovial fluid
Buffer
32 yr. male
Torn medial
meniscus
mucin is not related to its viscosity. Digestion of the mucin by testicular hyaluronidase abolished the high viscosity but did
not reduce its lubricating ability. By contrast, tryptic digestion, although not affecting the viscosity, completely destroyed the
lubricant action. This effect of trypsin was
observed on isolated synovial mucin although not on whole synovial fluid. The
explanation undoubtedly has to do with
the anti-tryptic activity of the synovial
fluid.1°
Opinion is divided as to whether protein
is an integral part of the mucin molecule
or a contaminant.ll Trypsin together with
chymotrypsin removes 65 per cent of pro-
Action of Human Synodal Fluid8
Coef. of Relative Coef. of Relative
friction viscosity friction viscosity
,0077*
,0026
.0077
,0043
,0078
61.40
5.11
46 yr. female
Rheumatoid
arthritis
51 yr. male
Rheumatoid
arthritis
Coef. of Relative
friction viscosity
Coef. of Relative
friction viscosity
21 yr. male
Torn medial
meniscus
,0073
.0035
,0077
,0047
,0075
40.05
5.20
* A different dog ankle was used to test the fluid of each patient.
,0098
,0049
.0096
,0062
,0095
27.55
5.17
.0083
,0043
.0078
,0049
,0083
20.30
6.30
679
LUBRICATION OF ANIMAL JOMTS. I11
.OL
SALl NE
0:
P
'
I-
SY NOW A
ua
LL
LL
0
k
-
.02
uLL
LL
W
0
u
.01
C
Unfixed
1
Unfixed
Fig. 4.-Effect of formalin fixation on
Lubrication.
tein from hyaluronate-protein complex of
synovial fluid.12 The fact that trypsin abolishes the lubricating action of the mucin
siiggests that a protein moiety is a necessary component of the mucin. One possibility is that protein may serve to attach the
hyaluronate to the cartilage. Adsorption of
the mucin is an essential prerequisite for
boundary lubrication. In experiments concurrent with these, Wilkins has observed
similar effects of enzymatic digestion on the
ability of synovial mucin to lubricate a rubber-glass friction system.13
Since viscosity is related to the degree
of polymerization of the hyalur~nate,'~
other things being equal, the data obtained
with hyaluronidase suggest that highly
polymerized hyaluronate is not necessary
for synovial mucin to lubricate. Exhaustive
digestion with testicular hyaluronidase did
cause a rise in the coefficient of friction.
The completeness of the digestion by hyaluronidase was attested to by the acetylglucosamine determination (Table 1).It is
likely that some partly polymerized hyaluronate, in conjunction with protein, is responsible for the lubricating action of
synovial mucin. Our tests show that blood
proteins are not lubricants. That ovalbumin
or purified hyaluronate solutions alone have
no lubricating ability has been demonstrated previously.5
Heparin is a sulfated mucopolysaccharide. It may conceivably act as an inhibitor
in this situation by blocking the attachment
sites on the cartilage for the boundary lubricant, or it may in some manner react
with the lubricant. It has been reported
that heparin competes with hyaluronate of
streptococcal origin in certain other systems.];'
25
50
75
PERCENT SYNOVIA IN THE SOLUTION
100
Fig. 5.-Lubricating ability and viscosity of synovial fluid at various dilutions.
680
The variation in the friction with pH
and molarity indicates that these factors act
on the articular cartilage itself. The elastic
properties of this tissue and its lubrication
depend largely on the fixed anionic charges
of its ground substance."' McCutchen has
proposed that cartilage acts as a self-pressurized sponge. It has been established that
fluid flows from cartilage under pressure;
that this flow can be enhanced by increasing the molarity of the interstitial fluidlT;
that polyvalent cations increase deformability of the cartilage and trivalent ones depress elastic recovery.18
The deformability of articular cartilage
under these conditions was studied because
it is relevant to several known aspects of
the lubrication of joints. Interstitial fluid is
held osmotically and its displacement during loading is necessary for the boundary
mechanism, inasmuch as synovial fluid
lubricates deformable but not rigid beari n g ~ . ~"Plowing"
-~
friction" is associated
with compressive deformation of the cartilage and there is a small shear component.G
The slow rise in deformation of the cartilage as the ionic strength of the immersion
bath increased is in agreement with previous report^.^^'^^^^ Although swelling of articular cartilage in the presence of acid was
not as pronounced as in other collagenous
tissues, a distinct softening, i.e., an increase
in deformation per unit load, was observed
below pH 5. Under most conditions, softening was associated with a reduction in friction. Nevertheless the relationship between
deformability and friction was not consistent: softening induced by molar concentrations greater than 0.5 were associated with
some increase of friction. This suggests that
'Plowing involves the expenditure of energy to
deform the cartilage compressively as the joint
oscillates, i s . , the force necessary to move the
depressions which the depressible bearing siirfnces
make in each other.
LI" AND W I N
factors other than deformability of the cartilage may also be involved.
One possiblity is that there is a surface
phenomenon related to the electrostatic
charges on the cartilages. This is suggested
by the amphoteric character of the relationship between the coefficient of friction and
pH. Maximum friction was observed at
pH 5; this decreased at higher and lower
hydrogen ion concentrations. It may be that
the surface was isoelectric at pH 5 and
positively or negatively charged at other
points on the curve. The rubbing surfaces
might now be separated by their mutual
electrostatic repulsion. Since these experiments were carried out in the absence of
mucin, no conclusions can be drawn about
this phenomenon under conditions of sy
novial fluid lubrication.
The nature of the sites on the cartilage
surface for attaching synovial much is not
known. Although formalin greatly stiffened
cartilage and increased the coefficient of
friction by a factor of at least three, it did
little to impair boundary lubrication by the
mucin. One may conclude then that the
attachment sites are not susceptible to
blocking by formaldehyde.
The human synovial fluids lubricated better than the bovine. Even though they
were pathological, their viscosity was higher than that of the calf material-a well
recognized species difference. Considerable
dilution of the bovine fluid could be made
before it failed to lubricate. Although viscosity itself is not a determinant of lubrication by synovial fluid, these observations
raise the possibility that the amount of
synovial mucin in joints may greatly exceed
the requirements for reducing friction ,and
thus be a safety factor. It is conceivablc
that long-term clinical effects of small
changes in lubrication are not adequately
represented by these acute experiments,
but we have found bovine synovial fluid
681
LUBRICATION OF ANIMAL JOINTS. 111
worked well in this test system over a
period of 36 hours of continuousoperation,
ACKNOWLEDGMENTS
We thank Drs. Leon Sokoloff and David Hamerman for valuable contributions to this paper. Dr.
Charles W. McCutchen gave helpful criticisms.
Kenneth T. Cullen provided capable technical
assistance.
Dr. Roger Winand made the chemical analyses.
Streptococcal hyaluronidase was contributed by
Dr. George H. Warren, Wyeth Laboratories, and
heparin by the Upjohn Company.
SUMMARIOIN INTERLINGUA
Esseva trovate que le coefficiente de friction in le articulationes talocrural del
can mesurate in vitro es afficite marcatemente per certe tractamentos enzymatic
a base de bovin mucina synovial. Hyaluronidase testicular habeba nulle effecto super
le efficacitate del agente lubricatori ben q u e ill0 aboleva su viscositate. Per contrasto con isto, digestion tryptic destrueva le capacitate lubricatori del mucina sin
reducer su viscositate. Iste observationes indica que un ingrediente proteinic es un
componente intrinsec del molecula de mucina e que, insimul con un solo partialmente polymerisate hyaluronato, ille ingrediente es responsabile pro le function lubricatori del mucina. I1 es possibile q u e le proteina servi como gruppo prosthetic
pro le adsorption del mucina a1 superficie del cartilagine. Heparina a natrium inhibiva le effecto lubricatori del mucina. In tampones libere de mucina, tanto le
pH como etiam le molaritate del banio lubricatori alterava non solmente le friction
sed etiam le deformabilitate del cartilagine. Le alteration in friction non esseva systematicamente relationate con le alteration in deformabilitate. Le effecto amphoteric
del pH super le friction suggestiona le possibilitate que effectos electrostatic superficial contribue a1 friction adhesive. Formalina rendeva le cartilagine rigide e augmentava le coefficiente de friction. Tamen, ill0 non exerceva un marcate effecto
super le action lubricatori del mucina. Un micre numero de specimens pathologic
de human fluido synovial esseva studiate e il esseva trovate que illos lubricava tanto
ben como o melio q u e normal material bovin.
REFERENCES
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9. Ressing, J. L., Strominger, J. L. and Leloir,
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1959, pp. 12-22.
2. McCutchen, C. W.: Mechanism of animal
joints. Sponge-hydrostatic and weeping bearings.
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3. McCutchen, C. W.: The frictional propertie.;
of animal joints. Wear 5:1, 1963.
4. McCutchen, C. W.: Boundary lubrication by
synovial fluid: demonstration and possible osmotic
cxplauation. Fed. Proc. 25: 1061, 1966.
5. Linn, F. C.: Lubrication of animal joints. 11.
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I,. F.: A modified colorimetric method for the es-
1079,1967.
7. Pigman, W. and Rizvi, S.: Hyaluronic acid
and the ORD reaction. Biochem. Biophys. Res.
Commun. 1:39, 1959.
8. Dische, Z.: A new specific color reaction of
hexuronic acids. J. Biochem. (Engl.) 167:189,
1947.
timation of N-acetylamino sugars. J. Biochem.
(Engl.) 217:959, 1955.
10. Holmes, W. J., Jr., Keefer, C. S. and Myers,
W. K.: Antitryptic activity of synovial fluid in patients with various types of arthritis. J. Clin. Invest.
14:124, 1935.
11. Hamerman, D., Rojkind, M. and Sandson, J.:
Protein bound to hyaluronate: chemical and immunological studies. Fed. Proc. 25:1040, 1966.
12. Ogston, A. G . and Sherman, T. F.: Degradation of hyaluronic acid complex of synovial fluid
by proteolytic enzymes and by EDTA. J. Biochem.
(Engl.) 72:301, 1959.
13. Wilkins, J. F.: Destruction of synovial 11111cin boundary lubrication by proteolytic enzymes.
(In preparation)
14. Ogston, A. G. and Stanier, J. C.: The dimensions of the particle of the hyaluronic acid complex
in synovial fluid. Biochem. J. 49:585, 19 1
15. McClean, D.: The in uioo clecapsulation of
682
streptococci by hyaluronidase. J. Path. Bact. 54:
284,1942.
16. Schubert, M.: Structure of connective tissue. A chemical point of view. Fed. Proc. 25:1047.
1966.
17. Linn, I;. C. and Sokoloff, L.: Movement ant1
composition of interstitial flriid ol cartilage. Arthritis Rheum. 8:481, 1965.
LINN AND W I N
18. Sokoloff, L.: Elasticity of articular cartilage:
effect of ions and viscous solutions. Science 141:
1055, 1963.
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