ARTHRITIS & RHEUMATISM

Vol. 64, No. 12, December 2012, pp 39633971

DOI 10.1002/art.34674
2012, American College of Rheumatology

Diminished Cartilage-Lubricating Ability of Human

Osteoarthritic Synovial Fluid Deficient in Proteoglycan 4
Restoration Through Proteoglycan 4 Supplementation
Taryn E. Ludwig, Jenelle R. McAllister, Victor Lun, J. Preston Wiley, and Tannin A. Schmidt
in normal SF was 287.1 31.8 g/ml. OA SF samples
deficient in PRG4 (146.5 28.2 g/ml) as compared to
normal were identified and selected for lubrication
testing. The HA concentration in PRG4-deficient OA SF
(mean SEM 0.73 0.08 mg/ml) was not significantly
different from that in normal SF (0.54 0.09 mg/ml).
In PRG4-deficient OA SF, the molecular weight distribution of HA was shifted toward the lower range. The
cartilage boundarylubricating ability of PRG4deficient OA SF was significantly diminished as compared to normal (mean SEM <kinetic, Neq>
0.043 0.008 versus 0.025 0.002; P < 0.05) and was
restored when supplemented with PRG4 (<kinetic, Neq>
0.023 0.003; P < 0.05).
Conclusion. These results indicate that some OA
SF may have decreased PRG4 levels and diminished
cartilage boundarylubricating ability as compared to
normal SF and that PRG4 supplementation can restore
normal cartilage boundary lubrication function to these
OA SF.

Objective. The purposes of this study were 1) to

quantify the proteoglycan 4 (PRG4) and hyaluronan
(HA) content in synovial fluid (SF) from normal donors
and from patients with chronic osteoarthritis (OA) and
2) to assess the cartilage boundarylubricating ability
of PRG4-deficient OA SF as compared to that of normal SF, with and without supplementation with PRG4
and/or HA.
Methods. OA SF was aspirated from the knee
joints of patients with symptomatic chronic knee OA
prior to therapeutic injection. PRG4 concentrations
were measured using a custom sandwich enzyme-linked
immunosorbent assay (ELISA), and HA concentrations
were measured using a commercially available ELISA.
The molecular weight distribution of HA was measured
by agarose gel electrophoresis. The cartilage boundary
lubricating ability of PRG4-deficient OA SF, PRG4deficient OA SF supplemented with PRG4 and/or HA,
and normal SF was assessed using a cartilage-oncartilage friction test. Two friction coefficients () were
calculated: static (static, Neq) and kinetic (<kinetic, Neq>)
(where Neq represents equilibrium axial load and angle
brackets indicate that the value is an average).
Results. The mean SEM PRG4 concentration

The proteoglycan 4 (PRG4) gene (1) encodes for

mucin-like O-linked glycosylated proteins, including lubricin (2) and superficial zone protein (3). PRG4 proteins, collectively referred to as PRG4, are synthesized
and secreted by cells within articular joints, including
superficial zone articular chondrocytes (3) and synoviocytes (4). PRG4 is present in synovial fluid (SF) (5) and
at the articular cartilage surface (6). PRG4 acts as a
boundary lubricant; it mediates friction during cartilageon-cartilage contact between the articular surfaces,
where lubrication is provided by molecular interactions
at the surface (7). While PRG4 alone is an effective
boundary lubricant, it also acts synergistically with hyaluronan (HA) to further reduce friction to levels approaching that of whole SF (8). HA, a linear polymer of

Supported by the National Science and Engineering Research

Council of Canada, the Canadian Arthritis Network, Alberta Innovates Technology Futures, Alberta Innovates Health Solutions (OA
Team Grant), and the University of Calgary (funding from the Faculty
of Kinesiology and from the Center for Bioengineering Research and
Education, Schulich School of Engineering).
Taryn E. Ludwig, BSc, Jenelle R. McAllister, MSc, Victor
Lun, MSc, MD, J. Preston Wiley, MPE, MD, Tannin A. Schmidt, PhD:
University of Calgary, Calgary, Alberta, Canada.
Address correspondence to Tannin A. Schmidt, PhD, Faculty
of Kinesiology, University of Calgary, 2500 University Drive NW,
KNB 426, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
E-mail: tschmidt@ucalgary.ca.
Submitted for publication March 23, 2012; accepted in revised
form August 9, 2012.
3963

3964

repeating disaccharides composed of D-glucuronic acid

and D-N-acetylglucosamine (9), is another boundary
lubricant that is present in SF (8). It appears that both
PRG4 and HA are critical to the boundary-lubricating
function of human SF.
Changes in the PRG4 composition of human SF
after acute injury and in osteoarthritis (OA) have been
observed. Average concentrations of PRG4 in normal
SF between 35 and 250 g/ml (1015) have been reported. PRG4 concentrations have been observed to
decrease significantly after anterior cruciate ligament
injury, returning to normal within 1 year (12). Concentrations have been observed to increase after intraarticular fracture (11), remain normal after internal
derangement (13), and be elevated in late-stage OA
(10,14). However, animal models have suggested that
the PRG4 concentration in SF and its presence in the
superficial zone can decrease in secondary OA (1618).
Along with an altered lubricant composition, compromised boundary-lubricating ability was observed after
intraarticular fracture (11). However, no difference between the steady-state boundary-lubricating ability of
OA and normal SF has been observed (14,19). Mutations in the PRG4 gene in humans cause an autosomalrecessive disorder known as camptodactyly-arthropathy
coxa varapericarditis (CACP) syndrome (20). SF from
these patients is void of PRG4 and fails to lubricate (21).
Collectively, these findings in normal, injured, and diseased human SF suggest that SF deficient in PRG4 lacks
normal boundary-lubricating ability.
The HA composition of human SF has also been
observed to change with injury and disease. Average
normal concentrations of HA in human SF samples
range between 1.8 and 3.33 mg/ml (11,13,14,19,21,22).
The HA concentration in human SF has been observed
to remain normal in internal derangement injuries (13),
to significantly decrease with intraarticular fracture (11),
effusive joint injury, and arthritic disease (2224), and to
remain normal during OA (14,19,25) and CACP syndrome (21). The HA concentration has also been observed to be correlated with the age of the patient (25).
The molecular weight distribution (MWD) of HA has
been shown to range continuously between 27 kd and 10
Md in normal SF, peaking between 6 and 7 Md (2528).
The MWD of HA has been observed to shift to the lower
range during injury (13) and OA (14), but has also been
observed to remain constant between normal SF and
OA SF (25). The HA MWD in SF is of interest for the
potential difference in lubricating ability and interaction
with PRG4 of different MW species of HA (29). It has
been observed that HA supplementation of HA-

LUDWIG ET AL

deficient equine SF after acute injury was able to restore

compromised boundary-lubricating ability (30).
Intraarticular injection of HA is currently used to
treat OA. Commercially available formulations of intraarticular HA range from 0.5 to 6 Md and from 8 to
15 mg/ml (31,32). It has been demonstrated in injury
models of OA in rats that intraarticular injection of
PRG4 protects against cartilage degeneration (3335).
The potential application of PRG4 as a new and improved therapy for postinjury and OA knee joints, as
well as for maintenance of healthy joints, is promising.
However, it is unclear if PRG4 concentrations remain
normal in OA SF, and the biomechanical effects of
supplemental PRG4 on the boundary-lubricating ability
of SF, especially SF deficient in PRG4, in normal human
cartilage are unknown.
The objectives of this study were therefore to
quantify the PRG4 and HA content in SF samples from
normal donors and patients with chronic OA and to
assess the human cartilage boundarylubricating ability
of PRG4-deficient OA SF as compared to that of normal
SF, with and without supplementation with PRG4
and/or HA.
MATERIALS AND METHODS
Materials. Materials for the PRG4 enzyme-linked
immunosorbent assay (ELISA) (36) and PRG4 preparation
and lubrication testing (8) were obtained as described previously. In addition, disodium EDTA, benzamidine HCl, Nethylmaleimide, and a bicinchoninic acid (BCA) protein assay
kit were obtained from Thermo Fisher Scientific. Phenylmethylsulfonyl fluoride was from Bio Basic. Costar EIA/RIA high
binding plates were from Corning. Horseradish peroxidase
(HRP)conjugated peanut agglutinin (PNA), 3,3,5,5tetramethylbenzidine (TMB) tablets, DMSO, hydrogen peroxide (30%), dibasic sodium phosphate, citric acid, H2SO4
(95.098.0%), and Stains-All were obtained from SigmaAldrich. A hyaluronan DuoSet ELISA development kit was
obtained from R&D Systems, proteinase K was from Roche
Applied Science, and MegaLadder and HiLadder HA molecular weight markers were from Hyalose. Sodium hyaluronate
(1.5 Md) was from Lifecore Biomedical. Materials and equipment for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and protein staining
were obtained from Invitrogen.
Samples. Collection of all human tissues and fluids was
approved by the University of Calgary Conjoint Health Research Ethics Board. OA SF was aspirated from patients with
symptomatic chronic knee OA requiring aspiration (performed
prior to therapeutic injection). Patients were diagnosed as
having knee OA by 2 sports medicine physicians (VL and
JPW) following a review of the patients symptoms, a physical
examination, and plain-film radiography. OA SF was aspirated
using standard sterile knee aspiration technique. As much fluid
as possible was aspirated with each attempt.

RESTORATION OF CARTILAGE-LUBRICATING ABILITY WITH PRG4 SUPPLEMENTATION

Normal SF samples and the distal portion of normal

femurs were obtained through the Joint Transplantation Program at the University of Calgary and had been harvested
within 4 hours of the death of the donors. Femurs were stored
at 80C until used. Articular cartilage was macroscopically
normal (International Cartilage Repair Society grade 12), as
assessed at time of use.
Samples of normal and OA SF were clarified by
centrifugation (3,000g for 30 minutes at 4C [11,12,19]) prior
to storage at 80C with protease inhibitors (PIs) and when
sufficient volume was available, without PIs for HA MW
analysis. Sixteen OA SF samples were screened for PRG4
concentrations. Samples with low levels of PRG4 (defined as
an average PRG4 concentration below the average in normal
SF) were selected for lubrication testing and were assessed as
a distinct group. Patients had no history of therapeutic injection or injury within 4 months of aspiration.
Biochemical characterization of human SF. Biochemical characterization was performed on 16 OA and 13
normal SF samples. Since this is an ongoing study, PRG4deficient samples were selected for lubrication testing as they
were identified. PRG4-deficient samples were selected if patients had no recent history of injury or prior therapeutic
injection, sufficient volume for lubrication testing, and no
visible contamination with blood after clarification. The number of PRG4-deficient samples selected is not intended
to be an indicator of the actual proportion of the OA population that has low levels of PRG4. The total protein concentration in SF samples was measured in duplicate by BCA assay in
samples diluted 30 and 60 in deionized H2O.
Measurement of PRG4 concentrations. PRG4 concentrations in human SF samples were measured in triplicate by a
custom sandwich ELISA. An antipeptide capture antibody
(LPN) recognizing amino acids 13561373 at the C-terminal of
the full-length PRG4 molecule (36) was used, followed by
detection with HRPPNA (37). SF was digested with Streptomyces hyaluronidase (1 unit/ml for 3 hours at 37C) and subsequently with Sialidase A-66 (overnight at 37C) prior to
quantification. Purified PRG4 controls (described below) were
also treated with Sialidase A-66.
Purified control PRG4 for the ELISA was prepared
from culture medium conditioned with bovine cartilage explants, as described previously (8). PRG4 standards used to
determine SF PRG4 concentrations were purified by DEAESepharose anion exchange chromatography and Superose 6
size-exclusion chromatography, verified for purity by Western
blot analysis, and quantified by BCA assay. An appropriate
diluent was used so that the slopes of the control and sample
absorbance curves were equivalent in the linear range of the
sigmoidal curve.
High-binding ELISA plates were coated overnight at
4C with capture antibody (50 l of LPN at 2 g/ml). Plates
were then washed and blocked for 1 hour at 37C with 5% milk
in phosphate buffered saline (PBS). After the block was
removed, SF samples diluted to 4 and PRG4 controls at
320 g/ml were loaded in triplicate, serially diluted (2), and
incubated for 1 hour at 37C with nutation. The plates were
then washed and incubated for 1 hour at 37C with detection
by HRPPNA (50 l at 5 g/ml). Plates were washed, developed with TMB, and the development was stopped with 2M
H2SO4. Plates were read at 450 nm and 540 nm; readings at

3965

540 nm were subtracted from those at 450 nm to correct for

optical properties of the plastic, according to the manufacturers recommendation.
The assay was able to detect PRG4 to 10 g/ml in 90 l
of SF diluted to 4. The coefficient of variation for triplicates
averaged 12 9% (mean SD). Variation between plates
averaged 17 9% (mean SD). ELISA specificity for high
MW PRG4 that was immunoreactive to both LPN and HRP
PNA was confirmed by Western blotting on purified PRG4
and SF following 38% Trisacetate SDS-PAGE and transfer
to PVDF membrane (Figure 1).
Measurement of the HA concentration. The HA concentration in human SF was measured in triplicate using a
commercially available sandwich ELISA, which provided recombinant human aggrecan as a capture reagent and biotinylated recombinant human aggrecan for detection. SF samples
were diluted 1:40,000 in 5% Tween 20 in PBS. Intraassay
variation averaged 18 10% (mean SD) and interassay
variation was 13 12% (mean SD).
Determination of the MW distribution of HA. The MWD
of HA in SF samples stored without PIs and treated with
proteinase K was measured in duplicate by 1% agarose gel
electrophoresis, as described previously (38). The HA MWD
was measured in 8 normal SF samples and the 5 PRG4deficient OA SF samples. Briefly, HiLadder (0.51.5 Md) and
MegaLadder (1.56.1 Md) MW markers were used as HA
controls. One blank lane was left between samples for background measurement. After electrophoresis for 3 hours at

Figure 1. Characterization of the proteoglycan 4 (PRG4) enzymelinked immunosorbent assay control by protein staining (A) and
characterization of high molecular weight PRG4 immunoreactivity in
PRG4 control, normal (NL) human synovial fluid (hSF), and osteoarthritic (OA) SF samples by Western blotting using antipeptide
antibody LPN (capture) (B) and horseradish peroxidase (HRP)
conjugated peanut agglutinin (PNA) (detection) (C). Samples were
subjected to 38% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, followed by protein staining or Western blotting as described
in Materials and Methods. PRG4 controls treated with neuraminidase
and SF treated with hyaluronidase and neuraminidase were probed
with LPN and with HRPconjugated PNA.

3966

LUDWIG ET AL

Table 1. Characteristics of SF from normal subjects and from OA patients whose SF samples were
identified as PRG4-deficient and selected for lubrication testing*
Study group
OA patients
PRG4-deficient SF
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Mean SEM or total
Normal subjects
Mean SEM or total

Age,
years

Sex

Aspirate volume,
ml

Total protein,
mg/ml

56
79
54
62
66
63 4

Male
Male
Male
Male
Female
4 male, 1 female

9
12
42
10
13
17.2 6.2

22.2
33.2
30.8
31.4
26.6
28.8 2.0

58 3

10 male, 3 female

4.5 1.3

15.6 1.3

* SF synovial fluid; OA osteoarthritis; PRG4 proteoglycan 4.

P 0.05 versus normal subjects.

50V, the gels were stained with 0.005% Stains-All in 50%

ethanol and destained in 10% ethanol. The migration of HA
was assessed by densitometric analysis with ImageJ software
(National Institutes of Health).
Assessment of cartilage-lubricating ability. The cartilage
boundarylubricating ability of the SF samples was evaluated
in a cartilage-on-cartilage friction test in the boundary lubrication regimen using normal human osteochondral cores, as
described previously (39). Briefly, annulus and core-shaped
osteochondral samples were harvested from macroscopically
normal areas of the patellofemoral groove of the distal femur
samples (3 donors, mean SD age 64 4 years). Samples
were shaken vigorously overnight at 4C in 40 ml of PBS to
rinse residual SF from the articular surface (previously confirmed by lubrication testing [8,39]). Samples were bathed
overnight at 4C in the subsequent test lubricant prior to
lubrication testing; the cartilage surface was completely immersed in 0.1 ml (annulus) and 0.2 ml (core).
Cartilage boundary lubrication tests were performed
on an ELF 3200 instrument (Bose-EnduraTEC) as described
previously (39). Samples were first compressed at 0.002 mm/
second to 18% of the total cartilage thickness, followed by a
40-minute stress relaxation period to allow for an interstitial
fluid depressurization period. Using an exponential decay
curve fit for load during stress relaxation confirmed that
63.2% of the equilibrium load was reached after an average
time constant of 6.7 minutes and 98.1% was reached at 27
minutes. Furthermore, predicted values of load at 40 minutes
and 60 minutes were within 0.002N of one another. This
indicates that fluid depressurization was achieved at 40 minutes, nearly 6 times the time constant. Without removing
compression, samples were rotated 2 revolutions and 2
revolutions at 0.3 mm/second with presliding durations (Tps;
the duration of time the samples are stationary prior to
rotation) of 120, 12, and 1.2 seconds. The test sequence was
then repeated in the opposite direction of rotation. This friction
test has been shown to maintain boundary lubrication at a
depressurized cartilagecartilage interface (39).
In all experiments, each osteochondral pair (annulus
and core from the same donor but not necessarily the same
joint) was tested sequentially in each of the 5 test lubricants.
Each OA SF sample found to be deficient in PRG4 (n 5) was

tested in triplicate in the following sequence: 1) PBS (negative

control lubricant), 2) PRG4-deficient OA SF alone, 3) PRG4deficient OA SF plus PRG4, 4) PRG4-deficient OA SF plus
PRG4 plus HA, and 5) normal SF (positive control lubricant).
Normal SF from 1 donor (left and right knee, mean SEM
PRG4 concentration 254.7 118.5 g/ml, mean SEM HA
concentration 0.23 0.12 mg/ml, age 59 years) was used for all
experiments. PRG4-deficient OA SF was supplemented with
PRG4 and HA at concentrations based on preliminary ELISA
measurements in normal SF. Purified PRG4 at 450 g/ml
(obtained as described above) and 1.5-Md HA at 1 mg/ml were
dried and resuspended in PRG4-deficient OA SF. Two friction
coefficients () were calculated (39): static (static, Neq), representing resistance to the onset of motion, and kinetic
(kinetic, Neq; Neq represents equilibrium axial load and
angle brackets indicate that the value is an average), representing resistance to steady motion.
Statistical analysis. Data are presented as the mean
SEM except where indicated otherwise. Repeated-measures
analysis of variance (ANOVA) was used to assess the effects of
lubricant solution and Tps (as repeated factors) on static, Neq
and kinetic, Neq. The effect of test lubricant on kinetic, Neq
at Tps 1.2 seconds was assessed by ANOVA with Tukey
post hoc testing. ANOVA was used to assess differences in
PRG4 and HA composition. Arcsine square root transformation was used to improve uniformity of the variance for the
proportional (%) distribution of the MW of HA (40). Statistical analysis was performed with Systat 12 software.

RESULTS
Biochemical characteristics of SF. OA SF samples identified as PRG4-deficient and selected for friction testing were similar to normal samples in terms of
the characteristics of the donors (Table 1). There was no
significant difference between the ages of the OA patients with PRG4-deficient SF and the normal donors
(P 0.29). The total aspirate volume was significantly
higher in OA patients with PRG4-deficient SF (mean

RESTORATION OF CARTILAGE-LUBRICATING ABILITY WITH PRG4 SUPPLEMENTATION

SEM 17.2 6.2 ml versus 4.5 1.3 ml; P 0.01), as was

the total protein concentration (mean SEM 28.8 2.0
mg/ml versus 15.6 1.3 mg/ml; P 0.001).
Concentration of PRG4. The PRG4 concentration
varied across normal and OA samples (Figure 2); this
figure is not intended to portray that a certain proportion of OA SF is PRG4-deficient. The PRG4 concentration in normal SF averaged 287.1 31.8 g/ml (mean
SEM). The PRG4 concentration in OA SF samples
identified as PRG4-deficient and selected for lubrication
testing averaged 146.5 28.2 g/ml; these samples were
significantly deficient in PRG4 relative to normal SF
(P 0.05) (Figure 2).
Concentration of HA. The HA concentrations
were similar in normal and OA SF samples (Figure 3A).
In normal SF, the mean SEM HA concentration was
0.54 0.09 mg/ml (range 0.110.96). Concentrations in
PRG4-deficient OA SF samples were not significantly
different from those in normal SF samples (0.73 0.08
mg/ml; P 0.26).
MW distribution of HA. The MWD of HA was
shifted toward the lower MW range in PRG4-deficient
OA SF compared to normal SF (Figure 3B). The relative
HA concentration (as a percentage of the total concentration) in the 6.1-Md range tended to be lower in
PRG4-deficient OA SF (mean SEM 0.7 0.4%) than
in normal SF (2.8 1.0%; P 0.05). In the 3.16.1-Md
range, the relative HA concentration in PRG4-deficient
OA SF (33.6 2.9%) was significantly lower than that in
normal SF (49.1 3.6%; P 0.05). In the 1.13.1-Md,

Figure 2. PRG4 concentrations in normal and PRG4-deficient OA

SF samples. OA SF samples found to be deficient in PRG4 were
selected for friction testing (see Materials and Methods for details).
Horizontal black line shows the mean PRG4 concentration in normal
(NL) SF samples (n 13). Horizontal gray line shows the mean PRG4
concentration in PRG4-deficient OA (OA-LO) SF samples (n 5).
This figure is not intended to imply that a certain proportion of OA SF
is PRG4 deficient. Values are the mean SEM. P 0.05. See
Figure 1 for other definitions.

3967

Figure 3. Characterization of hyaluronan (HA) in normal and PRG4deficient OA SF samples. A, Concentrations of HA in normal and
PRG4-deficient OA SF samples. B, Molecular weight distribution of
HA in normal SF samples (n 8) and in PRG4-deficient OA
(OA-LO) SF samples (n 5). Values are the mean SEM. P
0.05. See Figure 1 for other definitions.

0.51.1-Md, and 0.5-Md ranges, relative HA concentrations in PRG4-deficient OA SF were significantly

higher than those in normal SF (31.1 1.7 versus 24.7
1.2%, 21.7 1.1 versus 13.4 1.3%, and 12.9 2.0
versus 7.1 0.8%, respectively; P 0.05 for each
comparison.)
Cartilage-lubricating ability. In all experiments,
friction was modulated by the test lubricant and Tps. In
all test lubricants, the static, Neq decreased with decreasing Tps and appeared to approach the kinetic, Neq
asymptotically as the Tps decreased from 120 seconds
toward 0 seconds. The static, Neq values were consistently
highest in PBS, ranging from a mean SEM of 0.143
0.011 at Tps 1.2 seconds to 0.242 0.013 at Tps 120
seconds; values were lower and similar for normal and
supplemented SF samples, ranging from 0.026 0.002
at Tps 1.2 seconds to 0.096 0.007 at Tps 120
seconds for normal SF. In all test lubricants, the
kinetic, Neq values increased only slightly with increasing Tps, with the mean SD values at Tps 1.2
seconds being on average within 13 1% of values at
Tps 120 seconds. Therefore, as presented previously
(8) and for brevity and clarity, kinetic, Neq data are
shown at Tps 1.2 seconds only. The mean SEM
equilibrium stress for all tests was 0.209 0.026 MPa.
OA SF deficient in PRG4 failed to lubricate as well
as normal SF. Both the static, Neq and the kinetic, Neq
values varied with the test lubricant and Tps, with an
interaction effect (P 0.001 for each comparison)
(Figure 4). The kinetic, Neq at Tps 1.2 seconds also
varied with the test lubricant (P 0.001) (Figure 4B).
The kinetic, Neq for PRG4-deficient OA SF was

3968

LUDWIG ET AL

SF supplemented with PRG4 plus HA (0.024 0.002;

P 0.05).
In general, no additional restoration of lubricating ability was provided by subsequent HA supplementation. The kinetic, Neq in PRG4-deficient OA SF
supplemented with PRG4 and in PRG4-deficient OA SF
supplemented with both PRG4 and HA did not differ
from each other or from normal SF (P 0.9961).
DISCUSSION

Figure 4. Effect of hyaluronan (HA) and proteoglycan 4 (PRG4)

supplementation on the cartilage boundarylubricating ability of
PRG4-deficient osteoarthritic (OA) synovial fluid (SF) samples, as
determined by cartilage-on-cartilage friction testing. Two friction
coefficients (), static (static, Neq) (A) and kinetic (kinetic, Neq; at a
presliding duration of 1.2 seconds) (B), in phosphate buffered saline
(PBS; negative control lubricant), PRG4-deficient OA (OA-LO) SF
alone, PRG4-deficient OA SF plus PRG4, PRG4-deficient OA SF plus
PRG4 and HA, and normal SF (NL; positive control lubricant) were
calculated. Values are the mean SEM. P 0.05. Neq represents
equilibrium axial load; angle brackets indicate that the value is an
average.

significantly higher than that for normal SF (0.043

0.008 versus 0.025 0.002; P 0.05).
Friction coefficients in PRG4-deficient OA SF
samples were restored to normal levels with PRG4
supplementation (Figure 4). The kinetic, Neq in
PRG4-deficient OA SF (0.043 0.008) was significantly reduced in PRG4-deficient OA SF supplemented
with PRG4 (0.023 0.003; P 0.05). In addition, the
kinetic, Neq in PRG4-deficient OA SF (0.043
0.008) was significantly reduced in PRG4-deficient OA

The findings of this study provide insight into

the molecular basis for altered cartilage boundary
lubricating ability of OA SF. These results are consistent
with the notion that PRG4 concentrations can vary
considerably among OA patients as well as among
normal donors. Furthermore, they indicate that normal
PRG4 levels may not be present in all SF from patients
with chronic OA and suggest that there is a subpopulation of OA patients whose SF is deficient in
PRG4, associated with diminished cartilage boundary
lubricating ability. These results further emphasize that
PRG4 is a critical boundary lubricant and is required for
normal joint lubrication.
The ELISA used to measure PRG4 levels extends previous PRG4 quantification methods. In this
assay, human SF was treated with Sialidase A-66 prior to
quantification. HRPPNA has previously been used as a
capture reagent in an SF sandwich ELISA (10,12),
without neuraminidase digestion. Due to 46% capping
of human PRG4 glycosylations with sialic acid (41), the
PRG4 concentration measured with and without neuraminidase digestion may differ. Digestion of SF and
control PRG4 with Sialidase prior to ELISA measurement increased the signal strength in both. The PRG4
concentration in samples not treated with Sialidase
could not be accurately determined from similarly
treated controls due to the very low signal obtained, as
the assay is optimized for controls and samples treated
with Sialidase. Potential HAPRG4 interactions that
may interfere with antibody recognition of PRG4 were
disrupted using hyaluronidase, as previously performed
in a quantitative Western blot method (11,13,14). Several antibodies have been used with previous PRG4
quantification methods (11,13,42). This ELISA recognizes high MW PRG4 species (345 kd, including
multimers, identified by LPN capture [36]) with glycosylations (identified by HRPPNA detection) (37), both
of which are important for functionality (41). Finally, SF
samples were stored with PIs before quantification;
sample storage without PIs may result in an underesti-

RESTORATION OF CARTILAGE-LUBRICATING ABILITY WITH PRG4 SUPPLEMENTATION

mate if PRG4 has degraded during storage. Addition of

PIs had no effect on the PRG4 signal as measured by
ELISA (data not shown).
The PRG4 concentrations obtained for normal
SF in this study are consistent with those measured in
previous studies. Furthermore, the range of PRG4 concentrations measured in normal SF (129450 g/ml)
reflects the previously reported wide range of PRG4
concentrations in normal SF (1015). Large variability
of these values in SF from patients with joint disease has
been reported (276762 g/ml) (1315) and was also
observed in the present study (range in all OA SF
samples examined 95426 g/ml). It should be noted
that none of the OA donors with PRG4-deficient SF
had a history of recent injury, which is known to affect
the PRG4 concentration (12). The PRG4 concentration
has previously been observed to increase with OA
(10,14,15), and several samples with normal to elevated
concentrations of PRG4 were also identified in this
study (data not shown).
While a decrease in PRG4 levels with OA has not
previously been reported in humans, a decrease in SF
PRG4 levels with secondary OA has been observed in
guinea pigs (16,17), as has a decreased presence of
PRG4-positive chondrocytes in the superficial zone after
meniscectomy in an ovine model (18). A decrease in
lubricating ability of SF from patients with rheumatoid
arthritis (RA) has been observed (19), as has a classification of RA patients based on high and low levels of
PRG4 expression in the synovium (43). Possible mechanisms for decreased PRG4 concentrations in the
PRG4-deficient OA SF samples identified in this study
include decreased expression/synthesis of PRG4, increased degradation of PRG4 (12), or increased loss of
PRG4 from the joint capsule through an inflamed
synovium (44,45). Further investigation into the characteristics of the study patients would contribute to the
understanding of the mechanism underlying PRG4 deficiency. Increased friction due to PRG4 deficiency is a
clinically relevant issue, as friction and wear are thought
to be coupled at the articular surface (21).
The normal HA concentration and shift to lower
MW HA observed in the PRG4-deficient OA SF samples is consistent with the findings of previous studies
(13,14,19,25). The HA concentrations measured are
lower than those observed in previous studies of human
SF. Concentrations ranged from 0.11 to 0.96 mg/ml
(normal) and from 0.23 to 2.69 mg/ml (OA, not friction
tested) in the present study, and from 1.8 to 3.33 mg/ml
(normal) (11,13,14,19,21,22) and from 0.1 to 1.3 mg/ml
(diseased) (22,24) in the literature. There was no statis-

3969

tically significant difference in the HA concentration

between OA SF and normal SF, as previously reported
(14,19,25). HA concentrations measured for bovine SF
(range 0.320.79 mg/ml) (data not shown) are consistent
with previously measured values (0.5 mg/ml) (46).
Both PRG4 deficiency and a shift toward a lower
MW of HA in some SF samples from patients with
chronic OA were observed in the current study. Previous
studies have demonstrated that the boundary-lubricating
ability of HA alone increases with increasing MW (30);
however, the synergistic boundary-lubricating ability of
HA with PRG4 is not dependent on MW (29). These
studies together suggest that treatment with PRG4 could
negate the deleterious effects of a shift toward HA of
low MW in OA SF and prevent alterations in boundarylubricating ability (29). Completing the biochemical and
biomechanical characterization on human SF samples
with normal and elevated PRG4 concentrations (identified but not described) will help to clarify this relationship.
In this study, a statistically significant effect of
additional supplementation with HA on the boundarylubricating ability of PRG4-deficient OA SF was not
observed. However, as PRG4 supplementation of
PRG4-deficient samples was of interest and was performed first, the effect of HA supplementation alone in
human SF remains to be fully elucidated. Other studies
have shown that HA supplementation of acute-injury
equine SF deficient in HA restored compromised
boundary-lubricating ability (30). Alterations in the
boundary-lubricating ability of human SF are of great
interest, as small increases in friction have been observed to be associated with increased wear at the
articular surfaces (21).
This study is unique in that both normal cartilage and normal SF were obtained for use as controls.
Normal cartilage was obtained from macroscopically
normal areas of femurs from donors who had not been
taking antiinflammatory drugs. The coefficients of friction for boundary lubrication obtained for normal SF
on normal cartilage (kinetic, Neq 0.025) are consistent with the coefficients of friction measured for
bovine SF on bovine cartilage in an identical test
(kinetic, Neq 0.025) (8); this supports the use of
normal cartilage. Furthermore, total protein concentrations measured in normal SF were consistent with
previously reported values (range 1828 mg/ml) (44,45)
and were lower than those measured in OA SF. The
volumes of normal SF obtained in the present study
were generally within the reported range of normal
(0.54 ml) (45). The OA SF volumes were significantly

3970

higher, as expected. It should be noted that in this study,

no correlation between the volume of SF aspirated and
the PRG4 concentration was observed.
Previous studies using this in vitro cartilage-oncartilage friction test confirmed that up to 5 sequential
tests could be conducted on a single osteochondral pair
over 5 days, with overnight storage at 4C between tests,
without degradation of the samples. To account for any
potential carryover effect of test lubricants and to isolate
the effect of PRG4 supplementation, the test sequence
we used was chosen according to the order of presumed
increasing lubricity. The HA and PRG4 used in this
study were representative of those in native human SF
and have been used in other studies (29). The concentration for PRG4 supplementation was selected based
on values previously observed to provide boundary
lubrication (8), values previously reported in human SF
(1015), and preliminary measurements in normal SF by
ELISA (as additional normal SF samples are obtained
and characterized on an ongoing basis). The HA concentration for supplementation was selected based on
preliminary measurements in normal SF by ELISA, and
a MW of 1.5 Md was selected as it is in the range of
commercially available formulations of HA for intraarticular injection (31,32). Furthermore, 1.5-Md HA has
previously been shown to provide boundary lubrication
(29).
These findings support and significantly extend
the observation that human SF deficient in PRG4 demonstrates decreased boundary-lubricating ability. The
PRG4-deficient OA SF samples identified had normal
HA concentration, altered HA MWD, and decreased
lubricating ability. This suggests that the MWD of HA
may be important and that low MW HA alone is not
sufficient to provide normal boundary lubrication.
Moreover, it provides further motivation to study
PRG4HA interactions in SF. PRG4 has been observed
to exist in both a disulfide-bonded multimeric form and
a monomeric form, which may affect its lubricating
function (36). Future studies determining the multimerto-monomer composition of PRG4 in normal SF and its
alterations with OA will provide further insight into this
fundamental joint lubrication mechanism.
Altered glycosylation patterns in OA, as observed
between RA and OA, could be another source of
variation in boundary-lubricating ability (37). The observations of this study are supported by in vivo studies
by other research groups demonstrating that intraarticular injection of PRG4 into a rat model of injuryinduced OA can prevent cartilage degeneration (33,34).
These results taken together with those of the present

LUDWIG ET AL

study suggest that in addition to postinjury patients,

some chronic OA patients who have PRG4-deficient SF
may benefit from PRG4 supplementation as a biotherapeutic agent to restore lubrication and maintain healthy
joints.
ACKNOWLEDGMENTS
The authors thank the University of Calgary Joint
Transplantation Program for access to the normal human
tissue, the Sports Medicine Centre at the University of Calgary
for collecting the OA SF, Mrs. Sue Miller and Dr. Roman
Krawetz at the McCaig Institute for Bone and Joint Health for
assistance with collecting normal SF, and Dr. Roman Krawetz
for assistance with the HA ELISA.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Schmidt had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Ludwig, Schmidt.
Acquisition of data. Ludwig, McAllister, Lun, Wiley.
Analysis and interpretation of data. Ludwig, Wiley, Schmidt.

REFERENCES
1. Ikegawa S, Sano M, Koshizuka Y, Nakamura Y. Isolation, characterization and mapping of the mouse and human PRG4 (proteoglycan 4) genes. Cytogenet Cell Genet 2000;90:2917.
2. Swann DA, Slayter HS, Silver FH. The molecular structure of
lubricating glycoprotein-I, the boundary lubricant for articular
cartilage. J Biol Chem 1981;256:59215.
3. Schumacher BL, Block JA, Schmid TM, Aydelotte MB, Kuettner
KE. A novel proteoglycan synthesized and secreted by chondrocytes of the superficial zone of articular cartilage. Arch Biochem
Biophys 1994;311:14452.
4. Jay GD, Britt DE, Cha DJ. Lubricin is a product of megakaryocyte
stimulating factor gene expression by human synovial fibroblasts.
J Rheumatol 2000;27:594600.
5. Swann DA, Silver FH, Slayter HS, Stafford W, Shore E. The
molecular structure and lubricating activity of lubricin isolated
from bovine and human synovial fluids. Biochem J 1985;225:
195201.
6. Schumacher BL, Hughes CE, Kuettner KE, Caterson B, Aydelotte
MB. Immunodetection and partial cDNA sequence of the proteoglycan, superficial zone protein, synthesized by cells lining
synovial joints. J Orthop Res 1999;17:11020.
7. Ateshian GA, Mow VC. Friction, lubrication, and wear of articular
cartilage and diarthrodial joints. In: Mow VC, Huiskes R, editors.
Basic orthopaedic biomechanics and mechano-biology. 3rd ed.
Philadelphia: Lippincott Williams & Wilkins; 2005. p. 44794.
8. Schmidt TA, Gastelum NS, Nguyen QT, Schumacher BL, Sah RL.
Boundary lubrication of articular cartilage: role of synovial fluid
constituents. Arthritis Rheum 2007;56:88291.
9. Fam H, Bryant JT, Kontopoulou M. Rheological properties of
synovial fluids. Biorheology 2007;44:5974.
10. Neu CP, Reddi AH, Komvopoulos K, Schmid TM, Di Cesare PE.
Increased friction coefficient and superficial zone protein expression in patients with advanced osteoarthritis. Arthritis Rheum
2010;62:26807.

RESTORATION OF CARTILAGE-LUBRICATING ABILITY WITH PRG4 SUPPLEMENTATION

11. Ballard BL, Antonacci JM, Temple-Wong MM, Hui AY,

Schumacher BL, Bugbee WD, et al. Effect of tibial plateau
fracture on lubrication function and composition of synovial fluid.
J Bone Joint Surg Am 2012;94:e64.
12. Elsaid KA, Fleming BC, Oksendahl HL, Machan JT, Fadale PD,
Hulstyn MJ, et al. Decreased lubricin concentrations and markers
of joint inflammation in the synovial fluid of patients with anterior
cruciate ligament injury. Arthritis Rheum 2008;58:170715.
13. Hansen BC, Temple-Wong MM, Antonacci JM, Cai MZ, Grissom
MJ, Love RE, et al. Internal derangement of the knee is associated
with impaired synovial fluid lubricant function and composition
[abstract]. Trans Orthop Res Soc 2010;56:1986.
14. Temple-Wong MM, Hansen BC, Grissom MJ, Cai MZ, Noori NB,
Roberts JM, et al. Effect of knee osteoarthritis on the boundary
lubricating molecules and function of human synovial fluid [abstract]. Trans Orthop Res Soc 2010;56:340.
15. Schmid T, Lindley K, Su J, Soloveychik V, Block J, Kuettner K,
et al. Superficial zone protein (SZP) is an abundant glycoprotein in
human synovial fluid and serum [abstract]. Trans Orthop Res Soc
2001;26:82.
16. Wei L, Fleming BC, Sun X, Teeple E, Wu W, Jay GD, et al.
Comparison of differential biomarkers of osteoarthritis with and
without posttraumatic injury in the Hartley guinea pig model.
J Orthop Res 2010;28:9006.
17. Teeple E, Elsaid KA, Fleming BC, Jay GD, Aslani K, Crisco JJ,
et al. Coefficients of friction, lubricin, and cartilage damage in the
anterior cruciate ligament-deficient guinea pig knee. J Orthop Res
2008;26:2317.
18. Young AA, McLennan S, Smith MM, Smith SM, Cake MA, Read
RA, et al. Proteoglycan 4 downregulation in a sheep meniscectomy
model of early osteoarthritis. Arthritis Res Ther 2006;8:R41.
19. Jay GD, Elsaid KA, Zack J, Robinson K, Trespalacios F, Cha CJ,
et al. Lubricating ability of aspirated synovial fluid from emergency department patients with knee joint synovitis. J Rheumatol
2004;31:55764.
20. Marcelino J, Carpten JD, Suwairi WM, Gutierrez OM, Schwartz S,
Robbins C, et al. CACP, encoding a secreted proteoglycan, is
mutated in camptodactyly-arthropathy-coxa vara-pericarditis syndrome. Nat Genet 1999;23:31922.
21. Jay GD, Torres JR, Rhee DK, Helminen HJ, Hytinnen MM,
Cha CJ, et al. Association between friction and wear in diarthrodial joints lacking lubricin. Arthritis Rheum 2007;56:36629.
22. Mazzucco D, Scott R, Spector M. Composition of joint fluid in
patients undergoing total knee replacement and revision arthroplasty: correlation with flow properties. Biomaterials 2004;25:
443345.
23. Asari A, Miyauchi S, Sekiguchi T, Machida A, Kuriyama S,
Miyazaki K, et al. Hyaluronan, cartilage destruction and hydrarthrosis in traumatic arthritis. Osteoarthritis Cartilage 1994;2:7989.
24. Dahl LB, Dahl IM, Engstrom-Laurent A, Granath K. Concentration and molecular weight of sodium hyaluronate in synovial fluid
from patients with rheumatoid arthritis and other arthropathies.
Ann Rheum Dis 1985;44:81722.
25. Dunn S, Kolomytkin OV, Marino AA. Pathophysiology of osteoarthritis: evidence against the viscoelastic theory. Pathobiology
2009;76:3228.
26. Lee HG, Cowman MK. An agarose gel electrophoretic method
for analysis of hyaluronan molecular weight distribution. Anal
Biochem 1994;219:27887.
27. Laurent TC, Laurent UB, Fraser JR. Structure and function of
hyaluronan: an overview. Immunol Cell Biol 1996;74:A17.
28. Ghosh P, Guidolin D. Potential mechanism of action of intraarticular hyaluronan therapy in osteoarthritis: are the effects
molecular weight dependent? Semin Arthritis Rheum 2002;32:
1037.

3971

29. Kwiecinski JJ, Dorosz SG, Ludwig TE, Abubacker S, Cowman

MK, Schmidt TA. The effect of molecular weight on hyaluronans
cartilage boundary lubricating abilityalone and in combination
with proteoglycan 4. Osteoarthritis Cartilage 2011;19:135662.
30. Antonacci JM, Schmidt TA, Serventi LA, Cai MZ, Shu YL,
Schumacher BL, et al. Effects of equine joint injury on boundary
lubrication of articular cartilage by synovial fluid: role of hyaluronan. Arthritis Rheum 2012;64:291726.
31. Waddell DD. Viscosupplementation with hyaluronans for osteoarthritis of the knee: clinical efficacy and economic implications.
Drugs Aging 2007;24:62942.
32. Watterson JR, Esdaile JM. Viscosupplementation: therapeutic
mechanisms and clinical potential in osteoarthritis of the knee.
J Am Acad Orthop Surg 2000;8:27784.
33. Flannery CR, Zollner R, Corcoran C, Jones AR, Root A, RiveraBermudez MA, et al. Prevention of cartilage degeneration in a rat
model of osteoarthritis by intraarticular treatment with recombinant lubricin. Arthritis Rheum 2009;60:8407.
34. Jay GD, Fleming BC, Watkins BA, McHugh KA, Anderson SC,
Zhang LX, et al. Prevention of cartilage degeneration and restoration of chondroprotection by lubricin tribosupplementation in
the rat following anterior cruciate ligament transection. Arthritis
Rheum 2010;62:238291.
35. Jay GD, Elsaid KA, Kelly KA, Anderson SC, Zhang L, Teeple E,
et al. Prevention of cartilage degeneration and gait asymmetry by
lubricin tribosupplementation in the rat following anterior cruciate
ligament transection. Arthritis Rheum 2012;64:116271.
36. Schmidt TA, Plaas AH, Sandy JD. Disulfide-bonded multimers of
proteoglycan 4 (PRG4) are present in normal synovial fluids.
Biochim Biophys Acta 2009;1790:37584.
37. Estrella RP, Whitelock JM, Packer NH, Karlsson NG. The
glycosylation of human synovial lubricin: implications for its role
in inflammation. Biochem J 2010;429:35967.
38. Lee HG, Cowman MK. An agarose gel electrophoretic method
for analysis of hyaluronan molecular weight distribution. Anal
Biochem 1994;219:27887.
39. Schmidt TA, Sah RL. Effect of synovial fluid on boundary
lubrication of articular cartilage. Osteoarthritis Cartilage 2007;15:
3547.
40. McDonald JH. Handbook of biological statistics. 2nd ed. Baltimore: Sparky House Publishing; 2009.
41. Jay GD, Harris DA, Cha CJ. Boundary lubrication by lubricin
is mediated by O-linked (13)Gal-GalNAc oligosaccharides.
Glycoconj J 2001;18:80715.
42. Su JL, Schumacher BL, Lindley KM, Soloveychik V, Burkhart W,
Triantafillou JA, et al. Detection of superficial zone protein in
human and animal body fluids by cross-species monoclonal antibodies specific to superficial zone protein. Hybridoma 2001;20:
14957.
43. Ungethuem U, Haeupl T, Witt H, Koczan D, Krenn V, Huber H,
et al. Molecular signatures and new candidates to target the
pathogenesis of rheumatoid arthritis. Physiol Genomics 2010;42A:
26782.
44. Hui AH, McCarty WJ, Masuda K, Firestein GS, Sah R. A systems
biology approach to synovial joint lubrication in health, injury, and
disease. Wiley Interdiscip Rev Syst Biol Med 2012;4:1537.
45. Koopman WJ, Moreland LW, editors. Arthritis and allied conditions: a textbook of rheumatology. 15th ed. Philadelphia: Lippincott Williams & Wilkins; 2005.
46. Ballard BL, Antonacci JM, Temple-Wong MM, Chen AC,
Schumacher BL, Schwartz AK, et al. Tibial plateau and tibial pilon
fractures disrupt the lubrication function and composition of
synovial fluid [abstract]. Trans Orthop Res Soc 2009;55:1539.