Diminished Cartilage-Lubricating Ability of Human

8/9/2019 Diminished Cartilage-Lubricating Ability of Human

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ARTHRITIS & RHEUMATISM

Vol. 64, No. 12, December 2012, pp 3963–3971

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

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) and2) to assess the cartilage boundary–lubricating ability

of PRG4-deficient OA SF as compared to that of nor-

mal 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, PRG4-deficient OA SF supplemented with PRG4 and/or HA,

and normal SF was assessed using a cartilage-on-

cartilage friction test. Two friction coefficients () were

calculated: static (static, N eq) and kinetic ()

(where N eq represents equilibrium axial load and angle

brackets indicate that the value is an average).

Results. The mean SEM PRG4 concentration

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 distri-

bution of HA was shifted toward the lower range. The

cartilage boundary–lubricating ability of PRG4-

deficient OA SF was significantly diminished as com-

pared to normal (mean SEM

0.043 0.008 versus 0.025 0.002; P < 0.05) and was

restored when supplemented with PRG4 (

0.023 0.003; P < 0.05).

Conclusion. These results indicate that some OA

SF may have decreased PRG4 levels and diminished

cartilage boundary–lubricating ability as compared to

normal SF and that PRG4 supplementation can restore

normal cartilage boundary lubrication function to these

OA SF.

The proteoglycan 4 (PRG4) gene (1) encodes formucin-like O-linked glycosylated proteins, including lu-bricin (2) and superficial zone protein (3). PRG4 pro-teins, collectively referred to as PRG4, are synthesizedand secreted by cells within articular joints, includingsuperficial zone articular chondrocytes (3) and synovio-cytes (4). PRG4 is present in synovial fluid (SF) (5) and

at the articular cartilage surface (6). PRG4 acts as aboundary lubricant; it mediates friction during cartilage-on-cartilage contact between the articular surfaces,

where lubrication is provided by molecular interactionsat the surface (7). While PRG4 alone is an effectiveboundary lubricant, it also acts synergistically with hya-luronan (HA) to further reduce friction to levels ap-proaching that of whole SF (8). HA, a linear polymer of

Supported by the National Science and Engineering ResearchCouncil of Canada, the Canadian Arthritis Network, Alberta Inno-

vates Technology Futures, Alberta Innovates Health Solutions (OA Team Grant), and the University of Calgary (funding from the Facultyof Kinesiology and from the Center for Bioengineering Research andEducation, Schulich School of Engineering).

Taryn E. Ludwig, BSc, Jenelle R. McAllister, MSc, VictorLun, MSc, MD, J. Preston Wiley, MPE, MD, Tannin A. Schmidt, PhD:University of Calgary, Calgary, Alberta, Canada.

Address correspondence to Tannin A. Schmidt, PhD, Facultyof Kinesiology, University of Calgary, 2500 University Drive NW,KNB 426, University of Calgary, Calgary, Alberta T2N 1N4, Canada.E-mail: [email protected].

Submitted for publication March 23, 2012; accepted in revisedform August 9, 2012.

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repeating disaccharides composed of D-glucuronic acidand D- N -acetylglucosamine (9), is another boundarylubricant that is present in SF (8). It appears that bothPRG4 and HA are critical to the boundary-lubricatingfunction of human SF.

Changes in the PRG4 composition of human SFafter acute injury and in osteoarthritis (OA) have beenobserved. Average concentrations of PRG4 in normalSF between 35 and 250 g/ml (10–15) have been re-ported. PRG4 concentrations have been observed todecrease significantly after anterior cruciate ligamentinjury, returning to normal within 1 year (12). Con-centrations have been observed to increase after intra-articular fracture (11), remain normal after internalderangement (13), and be elevated in late-stage OA (10,14). However, animal models have suggested thatthe PRG4 concentration in SF and its presence in thesuperficial zone can decrease in secondary OA (16–18).

Along with an altered lubricant composition, compro-mised boundary-lubricating ability was observed afterintraarticular fracture (11). However, no difference be-tween the steady-state boundary-lubricating ability of OA and normal SF has been observed (14,19). Muta-tions in the PRG4 gene in humans cause an autosomal-recessive disorder known as camptodactyly-arthropathy–coxa vara–pericarditis (CACP) syndrome (20). SF fromthese patients is void of PRG4 and fails to lubricate (21).Collectively, these findings in normal, injured, and dis-eased human SF suggest that SF deficient in PRG4 lacksnormal boundary-lubricating ability.

The HA composition of human SF has also beenobserved to change with injury and disease. Averagenormal concentrations of HA in human SF samplesrange between 1.8 and 3.33 mg/ml (11,13,14,19,21,22).The HA concentration in human SF has been observedto remain normal in internal derangement injuries (13),to significantly decrease with intraarticular fracture (11),effusive joint injury, and arthritic disease (22–24), and toremain normal during OA (14,19,25) and CACP syn-drome (21). The HA concentration has also been ob-served to be correlated with the age of the patient (25).The molecular weight distribution (MWD) of HA hasbeen shown to range continuously between 27 kd and 10

Md in normal SF, peaking between 6 and 7 Md (25–28).The MWD of HA has been observed to shift to the lowerrange during injury (13) and OA (14), but has also beenobserved to remain constant between normal SF andOA SF (25). The HA MWD in SF is of interest for thepotential difference in lubricating ability and interaction

with PRG4 of different MW species of HA (29). It hasbeen observed that HA supplementation of HA-

deficient equine SF after acute injury was able to restorecompromised boundary-lubricating ability (30).

Intraarticular injection of HA is currently used totreat OA. Commercially available formulations of intra-articular HA range from 0.5 to 6 Md and from 8 to

15 mg/ml (31,32). It has been demonstrated in injurymodels of OA in rats that intraarticular injection of PRG4 protects against cartilage degeneration (33–35).The potential application of PRG4 as a new and im-proved therapy for postinjury and OA knee joints, as

well as for maintenance of healthy joints, is promising.However, it is unclear if PRG4 concentrations remainnormal in OA SF, and the biomechanical effects of supplemental PRG4 on the boundary-lubricating abilityof SF, especially SF deficient in PRG4, in normal humancartilage are unknown.

The objectives of this study were therefore toquantify the PRG4 and HA content in SF samples fromnormal donors and patients with chronic OA and toassess the human cartilage boundary–lubricating abilityof PRG4-deficient OA SF as compared to that of normalSF, with and without supplementation with PRG4and/or HA.

MATERIALS AND METHODS

Materials. Materials for the PRG4 enzyme-linkedimmunosorbent assay (ELISA) (36) and PRG4 preparationand lubrication testing (8) were obtained as described previously. In addition, disodium EDTA, benzamidine HCl, N -ethylmaleimide, and a bicinchoninic acid (BCA) protein assay

kit were obtained from Thermo Fisher Scientific. Phenylmeth- ylsulfonyl fluoride was from Bio Basic. Costar EIA/RIA highbinding plates were from Corning. Horseradish peroxidase(HRP)–conjugated peanut agglutinin (PNA), 3,3,5,5-tetramethylbenzidine (TMB) tablets, DMSO, hydrogen perox-ide (30%), dibasic sodium phosphate, citric acid, H2SO4(95.0–98.0%), and Stains-All were obtained from Sigma- Aldrich. A hyaluronan DuoSet ELISA development kit wasobtained from R&D Systems, proteinase K was from Roche Applied Science, and MegaLadder and HiLadder HA molec-ular weight markers were from Hyalose. Sodium hyaluronate(1.5 Md) was from Lifecore Biomedical. Materials and equip-ment for sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS-PAGE), Western blotting, and protein staining were obtained from Invitrogen.

Samples. Collection of all human tissues and fluids wasapproved by the University of Calgary Conjoint Health Re-search Ethics Board. OA SF was aspirated from patients withsymptomatic chronic knee OA requiring aspiration (performedprior to therapeutic injection). Patients were diagnosed ashaving knee OA by 2 sports medicine physicians (VL andJPW) following a review of the patient’s symptoms, a physicalexamination, and plain-film radiography. OA SF was aspiratedusing standard sterile knee aspiration technique. As much fluidas possible was aspirated with each attempt.

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Normal SF samples and the distal portion of normalfemurs were obtained through the Joint Transplantation Pro-gram at the University of Calgary and had been harvested within 4 hours of the death of the donors. Femurs were storedat 80°C until used. Articular cartilage was macroscopicallynormal (International Cartilage Repair Society grade 1–2), as

assessed at time of use.Samples of normal and OA SF were clarified by

centrifugation (3,000 g for 30 minutes at 4°C [11,12,19]) priorto storage at 80°C with protease inhibitors (PIs) and whensufficient volume was available, without PIs for HA MWanalysis. Sixteen OA SF samples were screened for PRG4concentrations. Samples with low levels of PRG4 (defined asan average PRG4 concentration below the average in normalSF) were selected for lubrication testing and were assessed asa distinct group. Patients had no history of therapeutic injec-tion or injury within 4 months of aspiration.

Biochemical characterization of human SF. Bio-chemical characterization was performed on 16 OA and 13normal SF samples. Since this is an ongoing study, PRG4-deficient samples were selected for lubrication testing as they

were identified. PRG4-deficient samples were selected if pa-tients had no recent history of injury or prior therapeuticinjection, sufficient volume for lubrication testing, and no visible contamination with blood after clarification. The num-ber of PRG4-deficient samples selected is not intendedto be an indicator of the actual proportion of the OA popula-tion that has low levels of PRG4. The total protein concentra-tion in SF samples was measured in duplicate by BCA assay insamples diluted 30 and 60 in deionized H2O.

Measurement of PRG4 concentrations. PRG4 concen-trations in human SF samples were measured in triplicate by acustom sandwich ELISA. An antipeptide capture antibody(LPN) recognizing amino acids 1356–1373 at the C-terminal of the full-length PRG4 molecule (36) was used, followed bydetection with HRP–PNA (37). SF was digested with Strepto-

myces hyaluronidase (1 unit/ml for 3 hours at 37°C) and sub-sequently with Sialidase A-66 (overnight at 37°C) prior toquantification. Purified PRG4 controls (described below) werealso treated with Sialidase A-66.

Purified control PRG4 for the ELISA was preparedfrom culture medium conditioned with bovine cartilage ex-plants, as described previously (8). PRG4 standards used todetermine SF PRG4 concentrations were purified by DEAE-Sepharose anion exchange chromatography and Superose 6size-exclusion chromatography, verified for purity by Westernblot analysis, and quantified by BCA assay. An appropriatediluent was used so that the slopes of the control and sampleabsorbance curves were equivalent in the linear range of thesigmoidal curve.

High-binding ELISA plates were coated overnight at4°C with capture antibody (50 l of LPN at 2 g/ml). Plates were then washed and blocked for 1 hour at 37°C with 5% milkin phosphate buffered saline (PBS). After the block wasremoved, SF samples diluted to 4 and PRG4 controls at320 g/ml were loaded in triplicate, serially diluted (2), andincubated for 1 hour at 37°C with nutation. The plates werethen washed and incubated for 1 hour at 37°C with detectionby HRP–PNA (50 l at 5 g/ml). Plates were washed, devel-oped with TMB, and the development was stopped with 2 M H2SO4. Plates were read at 450 nm and 540 nm; readings at

540 nm were subtracted from those at 450 nm to correct foroptical properties of the plastic, according to the manufactur-er’s recommendation.

The assay was able to detect PRG4 to 10 g/ml in 90 lof SF diluted to 4. The coefficient of variation for triplicatesaveraged 12 9% (mean SD). Variation between plates

averaged 17 9% (mean SD). ELISA specificity for highMW PRG4 that was immunoreactive to both LPN and HRP–PNA was confirmed by Western blotting on purified PRG4and SF following 3–8% Tris–acetate SDS-PAGE and transferto PVDF membrane (Figure 1).

Measurement of the HA concentration. The HA concen-tration in human SF was measured in triplicate using acommercially available sandwich ELISA, which provided re-combinant human aggrecan as a capture reagent and biotinyl-ated 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 MWDof HA in SF samples stored without PIs and treated with

proteinase K was measured in duplicate by 1% agarose gelelectrophoresis, as described previously (38). The HA MWD was measured in 8 normal SF samples and the 5 PRG4-deficient OA SF samples. Briefly, HiLadder (0.5–1.5 Md) andMegaLadder (1.5–6.1 Md) MW markers were used as HA controls. One blank lane was left between samples for back-ground measurement. After electrophoresis for 3 hours at

Figure 1. Characterization of the proteoglycan 4 (PRG4) enzyme-

linked immunosorbent assay control by protein staining ( A ) andcharacterization of high molecular weight PRG4 immunoreactivity in

PRG4 control, normal (NL) human synovial fluid (hSF), and osteo-

arthritic (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 3–8% sodium dodecyl sulfate–polyacrylamide gel electro-

phoresis, followed by protein staining or Western blotting as describedin Materials and Methods. PRG4 controls treated with neuraminidase

and SF treated with hyaluronidase and neuraminidase were probed

with LPN and with HRP–conjugated PNA.

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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 cartilageboundary–lubricating ability of the SF samples was evaluatedin a cartilage-on-cartilage friction test in the boundary lubri-cation regimen using normal human osteochondral cores, asdescribed previously (39). Briefly, annulus and core-shapedosteochondral samples were harvested from macroscopicallynormal areas of the patellofemoral groove of the distal femursamples (3 donors, mean SD age 64 4 years). Samples were shaken vigorously overnight at 4°C in 40 ml of PBS torinse residual SF from the articular surface (previously con-firmed by lubrication testing [8,39]). Samples were bathedovernight at 4°C in the subsequent test lubricant prior tolubrication testing; the cartilage surface was completely im-

mersed in 0.1 ml (annulus) and 0.2 ml (core).Cartilage boundary lubrication tests were performedon an ELF 3200 instrument (Bose-EnduraTEC) as describedpreviously (39). Samples were first compressed at 0.002 mm/ second to 18% of the total cartilage thickness, followed by a40-minute stress relaxation period to allow for an interstitialfluid depressurization period. Using an exponential decaycurve fit for load during stress relaxation confirmed that63.2% of the equilibrium load was reached after an averagetime constant of 6.7 minutes and 98.1% was reached at 27minutes. Furthermore, predicted values of load at 40 minutesand 60 minutes were within 0.002N of one another. Thisindicates that fluid depressurization was achieved at 40 min-utes, nearly 6 times the time constant. Without removingcompression, samples were rotated 2 revolutions and 2revolutions at 0.3 mm/second with presliding durations (Tps;the duration of time the samples are stationary prior torotation) of 120, 12, and 1.2 seconds. The test sequence wasthen repeated in the opposite direction of rotation. This frictiontest has been shown to maintain boundary lubrication at adepressurized cartilage–cartilage interface (39).

In all experiments, each osteochondral pair (annulusand 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 (negativecontrol lubricant), 2) PRG4-deficient OA SF alone, 3) PRG4-

deficient OA SF plus PRG4, 4) PRG4-deficient OA SF plusPRG4 plus HA, and 5) normal SF (positive control lubricant).Normal SF from 1 donor (left and right knee, mean SEMPRG4 concentration 254.7 118.5 g/ml, mean SEM HA concentration 0.23 0.12 mg/ml, age 59 years) was used for allexperiments. PRG4-deficient OA SF was supplemented withPRG4 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 weredried and resuspended in PRG4-deficient OA SF. Two frictioncoefficients () were calculated (39): static (static, N eq), rep-resenting resistance to the onset of motion, and kinetic(kinetic, N eq; N eq represents equilibrium axial load andangle brackets indicate that the value is an average), repre-senting resistance to steady motion.

Statistical analysis. Data are presented as the mean SEM except where indicated otherwise. Repeated-measuresanalysis of variance (ANOVA) was used to assess the effects of lubricant solution and Tps (as repeated factors) on static, N eqand kinetic, N eq. The effect of test lubricant on kinetic, N eqat Tps 1.2 seconds was assessed by ANOVA with Tukeypost hoc testing. ANOVA was used to assess differences inPRG4 and HA composition. Arcsine square root transforma-tion was used to improve uniformity of the variance for theproportional (%) distribution of the MW of HA (40). Statis-tical analysis was performed with Systat 12 software.

RESULTS

Biochemical characteristics of SF. OA SF sam-ples identified as PRG4-deficient and selected for fric-tion testing were similar to normal samples in terms of the characteristics of the donors (Table 1). There was nosignificant difference between the ages of the OA pa-tients with PRG4-deficient SF and the normal donors( P 0.29). The total aspirate volume was significantlyhigher in OA patients with PRG4-deficient SF (mean

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

Age,

years Sex

Aspirate volume,

ml

Total protein,

mg/ml

OA patients

PRG4-deficient SFSample 1 56 Male 9 22.2Sample 2 79 Male 12 33.2Sample 3 54 Male 42 30.8Sample 4 62 Male 10 31.4Sample 5 66 Female 13 26.6

Mean SEM or total 63 4 4 male, 1 female 17.2 6.2† 28.8 2.0†Normal subjects

Mean SEM or total 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.

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SEM 17.2 6.2 ml versus 4.5 1.3 ml; P 0.01), as wasthe total protein concentration (mean SEM 28.8 2.0mg/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 propor-tion of OA SF is PRG4-deficient. The PRG4 concentra-tion in normal SF averaged 287.1 31.8 g/ml (mean SEM). The PRG4 concentration in OA SF samplesidentified as PRG4-deficient and selected for lubricationtesting averaged 146.5 28.2 g/ml; these samples weresignificantly 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 was0.54 0.09 mg/ml (range 0.11–0.96). Concentrations inPRG4-deficient OA SF samples were not significantlydifferent from those in normal SF samples (0.73 0.08mg/ml; P 0.26).

MW distribution of HA. The MWD of HA wasshifted toward the lower MW range in PRG4-deficientOA SF compared to normal SF (Figure 3B). The relativeHA concentration (as a percentage of the total concen-tration) in the 6.1-Md range tended to be lower inPRG4-deficient OA SF (mean SEM 0.7 0.4%) thanin normal SF (2.8 1.0%; P 0.05). In the 3.1–6.1-Mdrange, the relative HA concentration in PRG4-deficientOA SF (33.6 2.9%) was significantly lower than that innormal SF (49.1 3.6%; P 0.05). In the 1.1–3.1-Md,

0.5–1.1-Md, and 0.5-Md ranges, relative HA concen-trations in PRG4-deficient OA SF were significantlyhigher 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 eachcomparison.)

Cartilage-lubricating ability. In all experiments,friction was modulated by the test lubricant and Tps. Inall test lubricants, the static, N eq decreased with decreas-ing Tps and appeared to approach the kinetic, N eqasymptotically as the Tps decreased from 120 seconds

toward 0 seconds. The static, N eq values were consistentlyhighest in PBS, ranging from a mean SEM of 0.143 0.011 at Tps 1.2 seconds to 0.242 0.013 at Tps 120seconds; values were lower and similar for normal andsupplemented SF samples, ranging from 0.026 0.002at Tps 1.2 seconds to 0.096 0.007 at Tps 120seconds for normal SF. In all test lubricants, thekinetic, N eq values increased only slightly with in-creasing Tps, with the mean SD values at Tps 1.2seconds being on average within 13 1% of values atTps 120 seconds. Therefore, as presented previously(8) and for brevity and clarity, kinetic, N eq data areshown 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, N eq and the kinetic, N eq values varied with the test lubricant and Tps, with aninteraction effect ( P 0.001 for each comparison)(Figure 4). The kinetic, N eq at Tps 1.2 seconds also

varied with the test lubricant ( P 0.001) (Figure 4B).The kinetic, N eq for PRG4-deficient OA SF was

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.

Figure 3. Characterization of hyaluronan (HA) in normal and PRG4-

deficient OA SF samples. A, Concentrations of HA in normal andPRG4-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.



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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 SFsamples were restored to normal levels with PRG4supplementation (Figure 4). The kinetic, N eq inPRG4-deficient OA SF (0.043 0.008) was signifi-cantly reduced in PRG4-deficient OA SF supplemented

with PRG4 (0.023 0.003; P 0.05). In addition, thekinetic, N eq in PRG4-deficient OA SF (0.043 0.008) was significantly reduced in PRG4-deficient OA

SF supplemented with PRG4 plus HA (0.024 0.002; P 0.05).

In general, no additional restoration of lubricat-ing ability was provided by subsequent HA supplemen-tation. The kinetic, N eq in PRG4-deficient OA SF

supplemented with PRG4 and in PRG4-deficient OA SFsupplemented with both PRG4 and HA did not differfrom each other or from normal SF ( P 0.996–1).

DISCUSSION

The findings of this study provide insight intothe molecular basis for altered cartilage boundary–lubricating ability of OA SF. These results are consistent

with the notion that PRG4 concentrations can varyconsiderably among OA patients as well as amongnormal donors. Furthermore, they indicate that normalPRG4 levels may not be present in all SF from patients

with chronic OA and suggest that there is a subpop-ulation of OA patients whose SF is deficient inPRG4, associated with diminished cartilage boundary–lubricating ability. These results further emphasize thatPRG4 is a critical boundary lubricant and is required fornormal joint lubrication.

The ELISA used to measure PRG4 levels ex-tends previous PRG4 quantification methods. In thisassay, human SF was treated with Sialidase A-66 prior toquantification. HRP–PNA has previously been used as acapture reagent in an SF sandwich ELISA (10,12),

without neuraminidase digestion. Due to 46% capping

of human PRG4 glycosylations with sialic acid (41), thePRG4 concentration measured with and without neur-aminidase digestion may differ. Digestion of SF andcontrol PRG4 with Sialidase prior to ELISA measure-ment increased the signal strength in both. The PRG4concentration in samples not treated with Sialidasecould not be accurately determined from similarlytreated controls due to the very low signal obtained, asthe assay is optimized for controls and samples treated

with Sialidase. Potential HA–PRG4 interactions thatmay interfere with antibody recognition of PRG4 weredisrupted using hyaluronidase, as previously performedin a quantitative Western blot method (11,13,14). Sev-

eral antibodies have been used with previous PRG4quantification methods (11,13,42). This ELISA recog-nizes high MW PRG4 species (345 kd, includingmultimers, identified by LPN capture [36]) with glyco-sylations (identified by HRP–PNA detection) (37), bothof which are important for functionality (41). Finally, SFsamples were stored with PIs before quantification;sample storage without PIs may result in an underesti-

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

supplementation on the cartilage boundary–lubricating ability of PRG4-deficient osteoarthritic (OA) synovial fluid (SF) samples, as

determined by cartilage-on-cartilage friction testing. Two frictioncoefficients (), static (static, N eq) ( A ) and kinetic (kinetic, N eq; at apresliding 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 plusPRG4 and HA, and normal SF (NL; positive control lubricant) were

calculated. Values are the mean SEM. P 0.05. N eq represents

equilibrium axial load; angle brackets indicate that the value is an

average.

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mate if PRG4 has degraded during storage. Addition of PIs had no effect on the PRG4 signal as measured byELISA (data not shown).

The PRG4 concentrations obtained for normalSF in this study are consistent with those measured in

previous studies. Furthermore, the range of PRG4 con-centrations measured in normal SF (129–450 g/ml)reflects the previously reported wide range of PRG4concentrations in normal SF (10–15). Large variabilityof these values in SF from patients with joint disease hasbeen reported (276–762 g/ml) (13–15) and was alsoobserved in the present study (range in all OA SFsamples examined 95–426 g/ml). It should be notedthat none of the OA donors with PRG4-deficient SFhad a history of recent injury, which is known to affectthe PRG4 concentration (12). The PRG4 concentrationhas previously been observed to increase with OA (10,14,15), and several samples with normal to elevatedconcentrations of PRG4 were also identified in thisstudy (data not shown).

While a decrease in PRG4 levels with OA has notpreviously been reported in humans, a decrease in SFPRG4 levels with secondary OA has been observed inguinea pigs (16,17), as has a decreased presence of PRG4-positive chondrocytes in the superficial zone aftermeniscectomy in an ovine model (18). A decrease inlubricating ability of SF from patients with rheumatoidarthritis (RA) has been observed (19), as has a classifi-cation of RA patients based on high and low levels of PRG4 expression in the synovium (43). Possible mech-

anisms for decreased PRG4 concentrations in thePRG4-deficient OA SF samples identified in this studyinclude decreased expression/synthesis of PRG4, in-creased degradation of PRG4 (12), or increased loss of PRG4 from the joint capsule through an inflamedsynovium (44,45). Further investigation into the charac-teristics of the study patients would contribute to theunderstanding of the mechanism underlying PRG4 de-ficiency. Increased friction due to PRG4 deficiency is aclinically relevant issue, as friction and wear are thoughtto be coupled at the articular surface (21).

The normal HA concentration and shift to lowerMW HA observed in the PRG4-deficient OA SF sam-

ples is consistent with the findings of previous studies(13,14,19,25). The HA concentrations measured arelower than those observed in previous studies of humanSF. Concentrations ranged from 0.11 to 0.96 mg/ml(normal) and from 0.23 to 2.69 mg/ml (OA, not frictiontested) 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-

tically significant difference in the HA concentrationbetween OA SF and normal SF, as previously reported(14,19,25). HA concentrations measured for bovine SF(range 0.32–0.79 mg/ml) (data not shown) are consistent

with previously measured values (0.5 mg/ml) (46).

Both PRG4 deficiency and a shift toward a lowerMW of HA in some SF samples from patients withchronic OA were observed in the current study. Previousstudies have demonstrated that the boundary-lubricatingability of HA alone increases with increasing MW (30);however, the synergistic boundary-lubricating ability of HA with PRG4 is not dependent on MW (29). Thesestudies together suggest that treatment with PRG4 couldnegate the deleterious effects of a shift toward HA of low MW in OA SF and prevent alterations in boundary-lubricating ability (29). Completing the biochemical andbiomechanical characterization on human SF samples

with normal and elevated PRG4 concentrations (identi-fied but not described) will help to clarify this relation-ship.

In this study, a statistically significant effect of additional supplementation with HA on the boundary-lubricating ability of PRG4-deficient OA SF was notobserved. However, as PRG4 supplementation of PRG4-deficient samples was of interest and was per-formed first, the effect of HA supplementation alone inhuman SF remains to be fully elucidated. Other studieshave shown that HA supplementation of acute-injuryequine SF deficient in HA restored compromisedboundary-lubricating ability (30). Alterations in the

boundary-lubricating ability of human SF are of greatinterest, as small increases in friction have been ob-served to be associated with increased wear at thearticular surfaces (21).

This study is unique in that both normal carti-lage and normal SF were obtained for use as controls.Normal cartilage was obtained from macroscopicallynormal areas of femurs from donors who had not beentaking antiinflammatory drugs. The coefficients of fric-tion for boundary lubrication obtained for normal SFon normal cartilage (kinetic, N eq 0.025) are con-sistent with the coefficients of friction measured forbovine SF on bovine cartilage in an identical test

(kinetic, N eq 0.025) (8); this supports the use of normal cartilage. Furthermore, total protein concentra-tions measured in normal SF were consistent withpreviously reported values (range 18–28 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(0.5–4 ml) (45). The OA SF volumes were significantly



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higher, as expected. It should be noted that in this study,no correlation between the volume of SF aspirated andthe PRG4 concentration was observed.

Previous studies using this in vitro cartilage-on-cartilage friction test confirmed that up to 5 sequential

tests could be conducted on a single osteochondral pairover 5 days, with overnight storage at 4°C between tests,

without degradation of the samples. To account for anypotential carryover effect of test lubricants and to isolatethe effect of PRG4 supplementation, the test sequence

we used was chosen according to the order of presumedincreasing lubricity. The HA and PRG4 used in thisstudy were representative of those in native human SFand have been used in other studies (29). The concen-tration for PRG4 supplementation was selected basedon values previously observed to provide boundarylubrication (8), values previously reported in human SF(10–15), and preliminary measurements in normal SF byELISA (as additional normal SF samples are obtainedand characterized on an ongoing basis). The HA con-centration for supplementation was selected based onpreliminary measurements in normal SF by ELISA, anda MW of 1.5 Md was selected as it is in the range of commercially available formulations of HA for intraar-ticular injection (31,32). Furthermore, 1.5-Md HA haspreviously been shown to provide boundary lubrication(29).

These findings support and significantly extendthe observation that human SF deficient in PRG4 dem-onstrates decreased boundary-lubricating ability. The

PRG4-deficient OA SF samples identified had normalHA concentration, altered HA MWD, and decreasedlubricating ability. This suggests that the MWD of HA may be important and that low MW HA alone is notsufficient to provide normal boundary lubrication.Moreover, it provides further motivation to studyPRG4–HA interactions in SF. PRG4 has been observedto exist in both a disulfide-bonded multimeric form anda monomeric form, which may affect its lubricatingfunction (36). Future studies determining the multimer-to-monomer composition of PRG4 in normal SF and itsalterations with OA will provide further insight into thisfundamental joint lubrication mechanism.

Altered glycosylation patterns in OA, as observedbetween RA and OA, could be another source of

variation in boundary-lubricating ability (37). The ob-servations of this study are supported by in vivo studiesby other research groups demonstrating that intraartic-ular injection of PRG4 into a rat model of injury-induced OA can prevent cartilage degeneration (33,34).These results taken together with those of the present

study suggest that in addition to postinjury patients,some chronic OA patients who have PRG4-deficient SFmay benefit from PRG4 supplementation as a biothera-peutic agent to restore lubrication and maintain healthy

joints.

ACKNOWLEDGMENTS

The authors thank the University of Calgary JointTransplantation Program for access to the normal humantissue, the Sports Medicine Centre at the University of Calgaryfor collecting the OA SF, Mrs. Sue Miller and Dr. RomanKrawetz at the McCaig Institute for Bone and Joint Health forassistance with collecting normal SF, and Dr. Roman Krawetzfor assistance with the HA ELISA.

AUTHOR CONTRIBUTIONS

All au thors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe 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 thedata 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.

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Diminished Cartilage-Lubricating Ability of Human