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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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 boundary–lubricating 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, 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 (μ) were calculated: static (μstatic, Neq) and kinetic (<μkinetic, Neq>) (where Neq 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 μg/ml. OA SF samples deficient in PRG4 (146.5 ± 28.2 μg/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 boundary–lubricating ability of PRG4-deficient OA SF was significantly diminished as compared to normal (mean ± SEM <μkinetic, Neq> = 0.043 ± 0.008 versus 0.025 ± 0.002; P < 0.05) and was restored when supplemented with PRG4 (<μkinetic, Neq> = 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 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 cartilage-on-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 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 μg/ml (10–15) 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 (16–18). 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 autosomal-recessive disorder known as camptodactyly-arthropathy–coxa vara–pericarditis (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 (22–24), 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 (25–28). 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-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 (33–35). 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 boundary–lubricating 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

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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, N-ethylmaleimide, 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′,5,5′-tetramethylbenzidine (TMB) tablets, DMSO, hydrogen peroxide (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 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 sulfate–polyacrylamide 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 patient's 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.

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 −80°C until used. Articular cartilage was macroscopically normal (International Cartilage Repair Society grade 1–2), as assessed at time of use.

Samples of normal and OA SF were clarified by centrifugation (3,000g for 30 minutes at 4°C [11, 12, 19]) prior to storage at −80°C 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, PRG4-deficient 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 1356–1373 at the C-terminal of the full-length PRG4 molecule (36) was used, followed by detection with HRP–PNA (37). SF was digested with Streptomyces hyaluronidase (1 unit/ml for 3 hours at 37°C) and subsequently with Sialidase A-66 (overnight at 37°C) 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 DEAE-Sepharose 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 4°C with capture antibody (50 μl of LPN at 2 μg/ml). Plates were then washed and blocked for 1 hour at 37°C with 5% milk in phosphate buffered saline (PBS). After the block was removed, SF samples diluted to 4× and PRG4 controls at 320 μg/ml were loaded in triplicate, serially diluted (2×), and incubated for 1 hour at 37°C with nutation. The plates were then washed and incubated for 1 hour at 37°C with detection by HRP–PNA (50 μl at 5 μg/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 540 nm were subtracted from those at 450 nm to correct for optical properties of the plastic, according to the manufacturer's recommendation.

The assay was able to detect PRG4 to 10 μg/ml in 90 μl 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 3–8% Tris–acetate SDS-PAGE and transfer to PVDF membrane (Figure 1).

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Figure 1. Characterization of the proteoglycan 4 (PRG4) enzyme-linked 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 3–8% sodium dodecyl sulfate–polyacrylamide 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 HRP–conjugated PNA.

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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 PRG4-deficient OA SF samples. Briefly, HiLadder (0.5–1.5 Md) and MegaLadder (1.5–6.1 Md) MW markers were used as HA controls. One blank lane was left between samples for background measurement. After electrophoresis for 3 hours at 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 boundary–lubricating 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 4°C 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 4°C 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 cartilage–cartilage 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) PRG4-deficient 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 μg/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 μg/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 (μ) were calculated (39): static (μstatic, Neq), representing resistance to the onset of motion, and kinetic (<μkinetic, 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 μstatic, Neq and <μkinetic, Neq>. The effect of test lubricant on <μkinetic, 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

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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 ± 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).

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 groupAge, yearsSexAspirate volume, mlTotal protein, mg/ml
  • *

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

  • P < 0.05 versus normal subjects.

OA patients    
 PRG4-deficient SF    
  Sample 156Male922.2
  Sample 279Male1233.2
  Sample 354Male4230.8
  Sample 462Male1031.4
  Sample 566Female1326.6
 Mean ± SEM or total63 ± 44 male, 1 female17.2 ± 6.228.8 ± 2.0
Normal subjects    
 Mean ± SEM or total58 ± 310 male, 3 female4.5 ± 1.315.6 ± 1.3

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 μg/ml (mean ± SEM). The PRG4 concentration in OA SF samples identified as PRG4-deficient and selected for lubrication testing averaged 146.5 ± 28.2 μg/ml; these samples were significantly deficient in PRG4 relative to normal SF (P < 0.05) (Figure 2).

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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.

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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 0.11–0.96). 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).

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

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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 3.1–6.1-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 1.1–3.1-Md, 0.5–1.1-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 μstatic, Neq decreased with decreasing Tps and appeared to approach the <μkinetic, Neq> asymptotically as the Tps decreased from 120 seconds toward 0 seconds. The μstatic, 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 <μkinetic, 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, <μkinetic, 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 μstatic, Neq and the <μkinetic, Neq> values varied with the test lubricant and Tps, with an interaction effect (P < 0.001 for each comparison) (Figure 4). The <μkinetic, Neq> at Tps = 1.2 seconds also varied with the test lubricant (P < 0.001) (Figure 4B). The <μkinetic, Neq> for PRG4-deficient OA SF was significantly higher than that for normal SF (0.043 ± 0.008 versus 0.025 ± 0.002; P < 0.05).

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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 friction coefficients (μ), static (μstatic, Neq) (A) and kinetic (<μkinetic, 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.

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Friction coefficients in PRG4-deficient OA SF samples were restored to normal levels with PRG4 supplementation (Figure 4). The <μkinetic, 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 <μkinetic, Neq> 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 lubricating ability was provided by subsequent HA supplementation. The <μkinetic, 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 = 0.996–1).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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. HRP–PNA 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 HA–PRG4 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 HRP–PNA 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 underestimate 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 (129–450 μg/ml) reflects the previously reported wide range of PRG4 concentrations in normal SF (10–15). Large variability of these values in SF from patients with joint disease has been reported (276–762 μg/ml) (13–15) and was also observed in the present study (range in all OA SF samples examined 95–426 μg/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 statistically 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 0.32–0.79 mg/ml) (data not shown) are consistent with previously measured values (∼0.5 mg/ml) (46).

Both PRG4 deficiency and a shift toward a 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 boundary-lubricating 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 boundary-lubricating 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 (<μkinetic, Neq> = 0.025) are consistent with the coefficients of friction measured for bovine SF on bovine cartilage in an identical test (<μkinetic, 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 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 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-on-cartilage friction test confirmed that up to 5 sequential tests could be conducted on a single osteochondral pair over 5 days, with overnight storage at 4°C 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 (10–15), 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 PRG4–HA 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 multimer-to-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 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 SF may benefit from PRG4 supplementation as a biotherapeutic agent to restore lubrication and maintain healthy joints.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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