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Abstract

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

Objective

To determine whether the synovial fluid (SF) constituents hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPL) contribute to boundary lubrication, either independently or additively, at an articular cartilage–cartilage interface.

Methods

Cartilage boundary lubrication tests were performed with fresh bovine osteochondral samples. Tests were performed using graded concentrations of SF, HA, and PRG4 alone, a physiologic concentration of SAPL, and various combinations of HA, PRG4, and SAPL at physiologic concentrations. Static (μstatic, Neq) and kinetic (<μkinetic, Neq>) friction coefficients were calculated.

Results

Normal SF functioned as an effective boundary lubricant both at a concentration of 100% (<μkinetic, Neq> = 0.025) and at a 3-fold dilution (<μkinetic, Neq> = 0.029). Both HA and PRG4 contributed independently to a low μ in a dose-dependent manner. Values of <μkinetic, Neq> decreased from ∼0.24 in phosphate buffered saline to 0.12 in 3,300 μg/ml HA and 0.11 in 450 μg/ml PRG4. HA and PRG4 in combination lowered μ further at the high concentrations, attaining a <μkinetic, Neq> value of 0.066. SAPL at 200 μg/ml did not significantly lower μ, either independently or in combination with HA and PRG4.

Conclusion

The results described here indicate that SF constituents contribute, individually and in combination, both at physiologic and pathophysiologic concentrations, to the boundary lubrication of apposing articular cartilage surfaces. These results provide insight into the nature of the boundary lubrication of articular cartilage by SF and its constituents. They therefore provide insight regarding both the homeostatic maintenance of healthy joints and pathogenic processes in arthritic disease.

Articular cartilage is the lubricious, load-bearing tissue at the end of long bones in synovial joints that normally facilitates low-friction and low-wear articulation. When healthy, it provides low-friction properties to the synovial joint through a combination of lubrication mechanisms (1). Pressurized fluid, within the tissue and between the surfaces, such as in a fluid film, can bear significant portions of the load. Lubricant molecules within a surface layer or film at the articular surface also mediate load bearing, particularly the surface-to-surface contact in the boundary mode of lubrication. This mode of lubrication has been proposed to be important for the protection and maintenance of articular surfaces since the apposing cartilage layers within the joint make contact over ∼10% of the total area, where much of the friction may occur (2). Synovial fluid (SF) contains the molecules hyaluronan (HA) (3), proteoglycan 4 (PRG4) (the name assigned by the Human Genome Organization Gene Nomenclature Committee for proteins also known as lubricin, superficial zone protein, and megakaryocyte-stimulating factor) (4, 5), and surface-active phospholipids (SAPL) (6), each of which interacts with and adsorbs to the articular surface. Such molecules are all ideally positioned to contribute to boundary lubrication.

SF, as well as HA, PRG4, and SAPL, have each demonstrated boundary-lubricating ability at various test interfaces. SF was recently shown to function as an effective boundary lubricant at an interface between apposed articular cartilage surfaces using an annulus-on- disc configuration (7). These results were consistent with findings of several previous studies and extended them using native cartilage surfaces in similar (8, 9) and different (10, 11) test configurations, as well as using nonbiologic surfaces (8, 12, 13). The lubricating ability of HA has been assessed at cartilage–cartilage (14–18), cartilage–steel (19), and cartilage–glass interfaces (18, 20), as well as at a latex–glass interface under boundary lubrication conditions (12, 21), with variable conclusions, possibly due to the different test surfaces and configurations and the various resulting operative modes of lubrication. Conversely, PRG4 proteins (22), which are synthesized and secreted by cells lining the synovial cavity (4, 5), have consistently demonstrated boundary-lubricating ability at both cartilage–glass (23) and latex–glass interfaces (12, 21, 24, 25). Studies examining the lubricating ability of SAPL at a cartilage–cartilage interface (in combination with HA) (19) and at a cartilage–steel interface (19, 26), as well as at a latex–glass interface under boundary lubrication conditions (13), suggest that SAPL may also possess boundary-lubricating ability. Collectively, these studies suggest that HA, PRG4, and SAPL each may contribute to the boundary-lubricating ability of SF at a cartilage–cartilage interface.

Consequently, the boundary-lubricating ability of SF may be altered in joint injury and arthritis due to the alteration in concentrations of HA, PRG4, and SAPL. The concentration of HA in human SF ranges from 1–4 mg/ml in healthy individuals (27, 28) and decreases after effusive joint injury (29) and in arthritic disease to ∼0.1–1.3 mg/ml (28, 30). The concentration of PRG4 in human SF ranges from 52 μg/ml to 350 μg/ml postmortem and from 276 μg/ml to 762 μg/ml in SF obtained from patients undergoing arthrocentesis procedures (31). Conversely, using a rabbit knee injury model, the concentration of PRG4 in SF decreased from 280 μg/ml to 20–100 μg/ml 3 weeks after injury (32). The majority of the lipids in human SF are phospholipids, the concentration of which ranges from ∼0.1 mg/ml to ∼0.2 mg/ml in normal individuals, increases in osteoarthritis to ∼0.2–0.3 mg/ml (28), and can decrease following traumatic injury to ∼0.02–0.08 mg/ml (33). While most phospholipids are surface active, dipalmitoyl-phosphatidylcholine (DPPC) is particularly so and is the most abundant form present in SF at ∼45% (6, 34).

The governing hypothesis of the present study was that SF constituents contribute to the boundary lubrication of articular cartilage. The specific objective of this study was to determine whether the SF constituents HA, PRG4, and SAPL contribute to boundary lubrication, either independently or additively, at an articular cartilage–cartilage interface. To achieve this objective, the effect of graded dilutions of SF on cartilage boundary lubrication was first determined. Then, the independent effects of graded concentrations of HA and PRG4, and of a physiologic concentration of SAPL, on cartilage boundary lubrication were determined. Finally, the combined effect of physiologic concentrations of HA, PRG4, and SAPL on cartilage boundary lubrication was determined.

MATERIALS AND METHODS

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

Materials.

Materials for lubrication testing were obtained as described previously (7). In addition, high molecular weight (MW) sodium hyaluronate (HA; SUPARTZ, 10 mg/ml, 620–1,170 kd) was obtained from Seikagaku Corporation (Tokyo, Japan), and DPPC was obtained from Sigma-Aldrich (St. Louis, MO).

Lubricant preparation and characterization.

The concentrations of HA, PRG4, and phospholipids in the test lubricants were determined by the carbazole reaction for uronic acid (35), by enzyme-linked immunosorbent assay (ELISA) (36), and by phospholipid assay using Phospholipids B Standard Solution and Color Reagent (Wako, Richmond, VA) (28), respectively.

HA.

The concentration of HA in the SUPARTZ HA stock solution was confirmed prior to storage at −20°C.

PRG4.

PRG4 was prepared from ∼300 cartilage discs (6 mm diameter and ∼0.3 mm thick, including the articular surface) harvested from 6 immature bovine stifle joints. Cartilage discs were incubated for 6–15 days in Dulbecco's modified Eagle's medium with 0.01% bovine serum albumin, 25 μg/ml ascorbic acid, and 10 ng/ml recombinant human transforming growth factor β1 (PeproTech, Rocky Hill, NJ). Culture medium was changed every 3 days and collected for processing. To purify PRG4, the conditioned culture medium was saved, pooled, and fractionated by anion-exchange chromatography essentially as described previously (4). Briefly, the sample was applied onto DEAE-Sepharose, previously equilibrated with 0.15M NaCl, 0.005M EDTA, and 0.05M sodium acetate, pH 6.0. The 0.3–0.6M NaCl eluate was collected, concentrated with a Centricon Plus 100-kd MW cutoff filter, and then quantified by ELISA (36) using monoclonal antibody (mAb) 3A4 (a gift from Dr. Bruce Caterson) (37) prior to storage at −20°C. Control studies indicated that the DEAE buffer used for PRG4 purification did not alter boundary-lubricating ability, since SF samples (described below) retained lubricating ability after dialysis against the buffer.

The size distribution of immunoreactive PRG4 was characterized by Western blot using mAb 3A4 after electrophoresis on a 4–20% sodium dodecyl sulfate–polyacrylamide gel and transfer to a polyvinylidene difluoride membrane. A single immunoreactive band at ∼345 kd was visualized by ECL-Plus detection and digital scanning with a STORM 840 Imaging System (Molecular Dynamics, Fairfield, CT). The concentrations of HA (determined from uronic acid concentration) and phospholipids in the PRG4 preparation (at 450 μg/ml) were 30 μg/ml and <0.5 μg/ml, respectively.

SAPL.

A 10× solution of DPPC at 2,000 μg/ml (∼106 times the concentration of phospholipids at which micelles can be formed [38]) was sonicated (Sonics & Materials, Danbury, CT) (13) in phosphate buffered saline (PBS) for 15 minutes to solubilize the DPPC, a standard technique used to produce liposomes (39) resulting in an opaque solution for which the concentration of SAPL was confirmed. The SAPL solution was stored at −20°C prior to use.

SF.

Normal bovine SF pooled from 5 animals was obtained as described previously (7) and clarified by centrifugation (10,000g for 60 minutes at 4°C) prior to storage at −80°C. The concentrations of HA and phospholipid were ∼1,000 μg/ml and ∼100 μg/ml, respectively. The concentration of the major immunoreactive PRG4 band at ∼345 kd, visualized by Western blot (described above) after hyaluronidase treatment, was calculated to be ∼450 μg/ml by quantitative comparison to a similar molecular weight band of a known amount of purified bovine PRG4 (4), as determined by ELISA (36) (described above).

Sample preparation.

Fresh osteochondral samples (n = 40) were prepared for friction testing from the patellofemoral groove of 10 skeletally mature bovine stifle joints (∼1 year old, ∼0.86 moles pyridinoline/mole collagen [40]), as described previously (7). Briefly, each sample consisted of an osteochondral core (radius 6 mm) and an apposed osteochondral annulus (outer radius [Ro] 3.2 mm, inner radius [Ri] 1.5 mm), both with central holes (radius 0.5 mm) drilled down into and exiting the bone to facilitate fluid depressurization. In addition, samples were first rinsed vigorously overnight in ∼40 ml of PBS to deplete the articular surface of residual SF prior to lubrication testing in PBS. (Pilot studies confirmed that the glycosaminoglycan content within the articular cartilage [41] of rinsed samples was similar [within 1%] to that of nonrinsed samples [P = 0.83] [n = 4].) Samples were then bathed in ∼0.5 ml of the subsequent test lubricants, completely immersing the cartilage, at 4°C for 24 hours prior to lubrication testing.

Lubrication test.

Cartilage boundary lubrication tests (Figure 1) were performed on an ELF 3200 (Bose EnduraTEC, Minnetonka, MN) essentially as described previously (7). Briefly, samples of articulating cartilage were preconditioned by compressing at a constant rate of 0.002 mm/second to a compression level (1 − ΛZ, where ΛZ is the stretch ratio [42]) equal to 18% of the total cartilage thickness, rotated +2 revolutions and then −2 revolutions at an effective sliding velocity (veff; equal to ωReff, where ω is the angular frequency in radians/second and Reff is the effective radius, calculated to be 2/3 × [(Ro3 – Ri3)/(Ro2 – Ri2)] = 2.4 mm [9]) equal to 3 mm/second, and then unloaded to 0%. This sequence was then repeated twice more. Samples were then tested by first compressing to 1 − ΛZ = 18% and allowing a 60-minute stress relaxation duration (Tsr) for interstitial fluid depressurization. Then, samples were rotated +2 revolutions and then −2 revolutions at a veff of 0.3 mm/second (which is slow enough to maintain a boundary mode of lubrication at a depressurized articular cartilage–cartilage interface [7] and is on the order of that used in other test configurations [43]) with presliding durations (Tps; the duration the sample is stationary prior to rotation) of 1,200, 120, 12, and 1.2 seconds. The test sequence was then repeated in the opposite direction of rotation.

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Figure 1. Boundary lubrication test protocol. A, Osteochondral annulus and core samples were compressed axially to a compression level (1 − ΛZ, where ΛZ is the stretch ratio) equal to 18% of the total cartilage thickness. B, A rotational test protocol with preconditioning, a stress relaxation duration (Tsr) of 3,600 seconds, and an effective sliding velocity (veff) of 0.3 mm/second was then used to determine the effects of test lubricants and presliding durations (Tps) of 1,200, 120, 12, and 1.2 seconds on the boundary lubrication of articular cartilage.

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Experimental design.

To determine whether HA, PRG4, and SAPL contribute to cartilage boundary lubrication either independently or additively, test lubricants were prepared in PBS. In all experiments, samples of articulating cartilage substrate were tested 3–5 times, in PBS (serving as the negative control test lubricant) on the first day of lubrication testing, in various test lubricant(s), and then in SF (serving as the positive control test lubricant) on the last day of lubrication testing.

Graded dilutions of SF.

To determine the effect of graded dilutions of SF on cartilage boundary lubrication, tests were performed in PBS, 3.3% SF, 10% SF, 33% SF, and then 100% SF.

SF constituents alone.

To determine the independent effects of graded concentrations of HA and PRG4 and of a physiologic concentration of SAPL on cartilage boundary lubrication, 3 sequences of tests were performed. For HA, tests were performed in PBS, 110 μg/ml HA, 1,100 μg/ml HA, 3,300 μg/ml HA, and then SF. For PRG4, tests were performed in PBS, 4.5 μg/ml PRG4, 45 μg/ml PRG4, 450 μg/ml PRG4, and then SF. For SAPL, tests were performed in PBS, 200 μg/ml SAPL, and then SF.

SF constituents in combination.

To determine the additive effect of physiologic concentrations of HA (3,300 μg/ml), PRG4 (450 μg/ml), and SAPL (200 μg/ml) in various combinations on cartilage boundary lubrication, tests were performed in PBS, HA or PRG4, HA plus PRG4, HA plus PRG4 plus SAPL, and then SF.

Statistical analysis.

To evaluate the boundary lubrication properties of test lubricants, 2 friction coefficients (μ) were determined, as described previously (7). These parameters were calculated from the expression μ = τ/(Reff × Neq), where τ is torque, Neq is the equilibrium axial load after the 60-minute Tsr, and Reff is the effective radius of the annulus sample described above (9). Briefly, a static friction coefficient, μstatic, Neq, was calculated using the peak | τ |, measured just after (within 10° of) the start of rotation, and the equilibrium axial load at the end of the 60-minute stress relaxation period, Neq. A kinetic friction coefficient, <μkinetic, Neq> (the brackets indicate that the value is an average), was calculated using the | τ | averaged during the second complete revolution of the test sample and Neq. Then, μstatic, Neq and <μkinetic, Neq> were averaged for the + and − revolutions in each test.

Unless indicated otherwise, data are presented as the mean ± SEM. The effects of test lubricant and Tps (as a repeated factor) on each of the 2 friction coefficients, μstatic, Neq and <μkinetic, Neq>, were assessed by analysis of variance (ANOVA). In all test lubricants, <μkinetic, Neq> increased slightly with increasing Tps, with mean ± SD values at Tps = 1.2 seconds being on average within 9 ± 11%, or 0.009 ± 0.015, of values at Tps = 1,200 seconds (which ranged from ∼0.02 to ∼0.3) (P < 0.001). Therefore, for brevity and clarity, <μkinetic, Neq> data are presented at Tps = 1.2 seconds only. Accordingly, the effect of test lubricant on <μkinetic, Neq> at Tps = 1.2 seconds was assessed by ANOVA, with Tukey post hoc testing. Statistical analysis was implemented with Systat 10.2 (Systat, Richmond, CA).

RESULTS

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

Lubrication test characterization.

In all experiments, friction was modulated by test lubricant (e.g., differing at least between SF, the positive control, and PBS, the negative control) and Tps. In all test lubricants, μstatic, Neq increased with increasing Tps and appeared to approach <μkinetic, Neq> asymptotically as Tps decreased from 1,200 seconds toward 0 seconds (to 1.2 seconds). Mean ± SD values of μstatic, Neq were consistently highest in PBS, ranging from 0.34 ± 0.06 to 0.58 ± 0.03 with increasing Tps; mean ± SD values of μstatic, Neq were consistently lowest in SF, ranging from 0.037 ± 0.008 to 0.22 ± 0.02 with increasing Tps. Similarly, with <μkinetic, Neq>, mean ± SD values were highest in PBS (0.24 ± 0.04) and lowest in SF (0.028 ± 0.006). Equilibrium compressive load was not affected by test lubricants (P = 0.15) with mean ± SD Neq = 2.7 ± 0.6 N.

Effect of graded dilutions of SF.

Friction coefficients were reduced by SF in a dose-dependent manner (Figure 2). μstatic, Neq varied with test lubricant (P < 0.001) and Tps (P < 0.001), with an interaction effect (P < 0.001) (Figure 2A). Values of μstatic, Neq decreased with increasing concentrations of SF at all Tps. At Tps = 120 seconds, values of μstatic, Neq decreased from 0.40 ± 0.03 in PBS to 0.117 ± 0.006 in 33% SF and 0.120 ± 0.006 in 100% SF. <μkinetic, Neq> at Tps = 1.2 seconds also varied with test lubricant (P < 0.001) (Figure 2B). Values of <μkinetic, Neq> decreased significantly with increasing concentrations of SF, from 0.20 ± 0.02 in PBS to 0.11 ± 0.02 in 10% SF (P < 0.01), 0.029 ± 0.002 in 33% SF (P < 0.01), and 0.025 ± 0.005 in 100% SF (P < 0.01).

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Figure 2. Effect of graded concentrations of synovial fluid (SF) on the boundary lubrication of articular cartilage. Shown are static (μstatic, Neq) (A) and kinetic (<μkinetic, Neq> [the brackets indicate that the value is an average]) (B) friction coefficients in phosphate buffered saline (PBS) and various concentrations of SF. In B, the presliding duration was 1.2 seconds. Values are the mean and SEM (n = 4–8).

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Effect of SF constituents alone.

HA.

Friction coefficients were reduced by HA in a dose-dependent manner (Figure 3). μstatic, Neq varied with test lubricant (P < 0.001) and Tps (P < 0.001), with an interaction effect (P < 0.01) (Figure 3A). Values of μstatic, Neq decreased with increasing concentrations of HA at all Tps. At Tps = 120 seconds, values of μstatic, Neq decreased from 0.46 ± 0.02 in PBS to 0.22 ± 0.02 in 3,300 μg/ml HA, with the value in SF being the lowest at 0.11 ± 0.01. <μkinetic, Neq> at Tps = 1.2 seconds also varied with test lubricant (P < 0.001) (Figure 3B). Values of <μkinetic, Neq> decreased significantly with increasing concentrations of HA, from 0.26 ± 0.01 in PBS to 0.21 ± 0.02 in 110 μg/ml HA (P < 0.05) and to 0.118 ± 0.009 in 3,300 μg/ml HA (P < 0.01), which was greater than the value of 0.031 ± 0.004 in SF (P < 0.01).

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Figure 3. Effect of graded concentrations of hyaluronan (HA) on the boundary lubrication of articular cartilage. Shown are static (A) and kinetic (B) friction coefficients in PBS, various concentrations of HA, and SF. In B, the presliding duration was 1.2 seconds. Values are the mean and SEM (n = 4–8). See Figure 2 for other definitions.

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PRG4.

Friction coefficients were reduced by PRG4 in a dose-dependent manner (Figure 4). μstatic, Neq varied with test lubricant (P < 0.001) and Tps (P < 0.001), with no interaction effect (P = 0.72) (Figure 4A). Values of μstatic, Neq decreased with increasing concentrations of PRG4 at all Tps. At Tps = 120 seconds, values of μstatic, Neq decreased from 0.48 ± 0.02 in PBS to 0.27 ± 0.03 in 450 μg/ml PRG4, with the value in SF being the lowest at 0.12 ± 0.02. <μkinetic, Neq> at Tps = 1.2 seconds also varied with test lubricant (P < 0.001) (Figure 4B). Values of <μkinetic, Neq> decreased significantly with increasing concentrations of PRG4, from 0.23 ± 0.02 in PBS and 0.20 ± 0.01 in 4.5 μg/ml PRG4 to 0.10 ± 0.02 in 450 μg/ml PRG4 (both P < 0.001), which was greater than the value of 0.04 ± 0.01 in SF (P < 0.05).

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Figure 4. Effect of graded concentrations of proteoglycan 4 (PRG4) on the boundary lubrication of articular cartilage. Shown are static (A) and kinetic (B) friction coefficients in PBS, various concentrations of PRG4, and SF. In B, the presliding duration was 1.2 seconds. Values are the mean and SEM (n = 8). See Figure 2 for other definitions.

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SAPL.

Friction coefficients were not affected by SAPL (Figure 5). μstatic, Neq varied with test lubricant (P < 0.001) and Tps (P < 0.001), with an interaction effect (P < 0.001) (Figure 5A). Values of μstatic, Neq appeared to decrease slightly with the addition of SAPL compared with PBS at all Tps. At Tps = 120 seconds, values of μstatic, Neq ranged from 0.39 ± 0.02 in PBS to 0.34 ± 0.02 in 200 μg/ml SAPL, with the value in SF being the lowest at 0.110 ± 0.009. <μkinetic, Neq> at Tps = 1.2 seconds also varied with test lubricant (P < 0.001) (Figure 5B). Values of <μkinetic, Neq> did not decrease significantly from 0.21 ± 0.02 in PBS to 0.17 ± 0.01 in 200 μg/ml SAPL (P = 0.17), which was greater than the value of 0.031 ± 0.002 in SF (P < 0.001).

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Figure 5. Effect of surface-active phospholipids (SAPL) on the boundary lubrication of articular cartilage. Shown are static (A) and kinetic (B) friction coefficients in PBS, 200 μg/ml SAPL, and SF. In B, the presliding duration was 1.2 seconds. Values are the mean and SEM (n = 8). See Figure 2 for other definitions.

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Effect of SF constituents in combination.

Friction coefficients were reduced by certain combinations of HA, PRG4, and SAPL at physiologic concentrations of 3,300 μg/ml, 450 μg/ml, and 200 μg/ml, respectively (Figure 6). μstatic, Neq varied with test lubricant (P < 0.001) and Tps (P < 0.001), with an interaction effect (P < 0.001) (Figure 6A). Values of μstatic, Neq decreased with the addition of HA and/or PRG4 at all Tps. At Tps = 120 seconds, values of μstatic, Neq decreased from 0.51 ± 0.02 in PBS to 0.22 ± 0.02 in HA and 0.23 ± 0.02 in PRG4 and to 0.18 ± 0.01 in HA plus PRG4, with the value in SF being the lowest at 0.130 ± 0.008. <μkinetic, Neq> at Tps = 1.2 seconds also varied with test lubricant (P < 0.001) (Figure 6B). Values of <μkinetic, Neq> decreased significantly with the addition of HA and/or PRG4, from 0.29 ± 0.01 in PBS to 0.12 ± 0.01 in HA (P < 0.01) and 0.11 ± 0.01 in PRG4 (P < 0.01) and to 0.066 ± 0.003 in HA plus PRG4 (P < 0.05 and P < 0.01, respectively), which was greater than the value of 0.024 ± 0.003 in SF (P < 0.05). The addition of SAPL did not significantly lower the value of <μkinetic, Neq> (0.062 ± 0.002) from its value in HA plus PRG4 (P = 0.99).

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Figure 6. Effect of hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPL) in combination on the boundary lubrication of articular cartilage. Shown are static (A) and kinetic (B) friction coefficients in PBS, 3,300 μg/ml HA, 450 μg/ml PRG4, 3,300 μg/ml HA plus 450 μg/ml PRG4, 3,300 μg/ml HA plus 450 μg/ml PRG4 plus 200 μg/ml SAPL, and SF. In B, the presliding duration was 1.2 seconds. Values are the mean and SEM (n = 4–8). See Figure 2 for other definitions.

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DISCUSSION

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

The results described here indicate that SF constituents contribute, individually and in combination, both at physiologic and pathophysiologic concentrations, to the boundary lubrication of apposing articular cartilage surfaces. Normal SF functioned as an effective boundary lubricant at the articular cartilage–cartilage interface tested here (<μkinetic, Neq> = 0.025), even with a 3-fold decrease in constituent concentration (<μkinetic, Neq> = 0.029) (Figure 2B). Both HA and the PRG4 preparation used here contributed independently to a low μ value in a dose-dependent manner. Values of <μkinetic, Neq> decreased from ∼0.24 in PBS to 0.12 in 3,300 μg/ml HA (Figure 3B) and 0.11 in 450 μg/ml PRG4 (Figure 4B). HA and PRG4 in combination lowered the μ value further at these concentrations, attaining a <μkinetic, Neq> value of 0.066 (Figure 6B). SAPL at 200 μg/ml did not significantly lower the μ value, either independently or in combination with HA and PRG4. Collectively, these results suggest that the SF constituents PRG4 and HA contribute individually and in combination to the effective boundary lubrication of articular cartilage.

The SF constituents used in the present study were representative of those in native SF. SF is composed of HA ranging from 2,000 kd to 10,000 kd (30, 44), PRG4 proteins ranging from ∼14 kd to ∼345 kd (4, 45), and SAPL of different types with DPPC being a major form (33). HA has been shown to lubricate at a cartilage–cartilage interface on a joint scale equally well at 1,030 kd and 1,930 kd (14), and certain other properties of HA do not depend on molecular weight either (in the range of 500–6,000 kd) (46). Therefore, the use of SUPARTZ HA (average MW 800 kd) in the present study was reasonable for studying the boundary-lubricating ability of HA. The boundary-lubricating ability of PRG4 is associated with its large central mucin-like domain (43), which is present in various forms of PRG4 with MW >∼220 kd (25); therefore, an ∼345-kd form of PRG4 was prepared from conditioned media and used. Finally, DPPC was chosen since it is the major component of SAPL in SF (34).

Similar SF constituents have been used in several previous studies examining their lubricating function (12, 18, 21, 23–26), and future studies may examine the friction-lowering effects of other specific forms of SF constituents. Additional studies examining the structure–function relationship of SF constituents contributing to boundary lubrication, both alone and in combination, may provide the framework for the potential complete recapitulation of the boundary-lubricating ability of whole SF. In the present study, the observed friction-lowering effect of the constituents, used at physiologic concentrations, suggests that they are sufficient for much of the boundary lubrication of articular cartilage that is naturally mediated by SF. The friction coefficient values, and their variation, were determined here to focus on boundary lubrication mechanism, and thus may not represent those values for human articular cartilage in normal SF. Both normal human cartilage and normal SF are difficult to obtain in a sufficient quantity and controlled manner for the types of experiments performed here.

The dose-dependent boundary-lubricating abilities of SF, as well as those of PRG4 individually, are consistent with (and extend) the findings of several previous studies. Swann et al demonstrated a dose-dependent effect of SF at a cartilage–glass interface (23). Using the test configuration and protocol used in the present study (7), SF was previously demonstrated to be an effective boundary lubricant with a similar value of <μkinetic, Neq> (∼0.02). In the present study, the values of <μkinetic, Neq> in PBS (∼0.24) were considerably higher than those reported previously (∼0.07) (7). As has been noted (17), this can be attributed to the rinsing of samples in PBS after harvest to remove residual SF from the articular surface prior to testing in PBS. The low values of <μkinetic, Neq> in SF (∼0.025) indicate that the rinsing did not affect the ability of SF to effectively lubricate the cartilage samples.

The dose-dependent effect of PRG4 is consistent with findings in a previous study by Jay (24), using a similar test configuration with a latex–glass interface, in which lubrication function occurred at concentrations >200 μg/ml. However, the absolute value of <μkinetic, Neq> in 450 μg/ml PRG4 observed here (∼0.10) (Figure 4B) is slightly more than the range of μ values reported in several other studies by Jay et al (∼0.047–0.018 in 250–400 μg/ml PRG4) (12, 25, 43). This difference may be due to the way in which PRG4 interacts with native articular cartilage surfaces, used in the present study, compared with other test surfaces. Nevertheless, the results of the present study indicate that PRG4 contributes to the boundary lubrication of articular cartilage, as previously concluded from studies at a latex–glass interface. These contributions appeared specific to PRG4, since the HA and SAPL content in the preparation was low, and control proteins (albumins and globulins) at a physiologic concentration did not independently lower the μ value (data not shown).

The significant contribution of HA to the boundary lubrication of apposed articular cartilage surfaces reported here extends and clarifies the findings of previous studies examining the lubricating ability of HA with test protocols and/or configurations, particularly where a boundary mode of lubrication was dominant. Bell et al (16) demonstrated that Arthrease, a fermentation-derived sodium hyaluronate with an MW of 3,000 kd, functioned as an effective lubricant at a cartilage–cartilage interface, but only under static conditions in which the intrinsic biphasic lubrication was depleted. Despite the absolute values of μstatic for both HA and PBS being ∼3-fold less than those reported here, which may be attributable to differences in the test configuration and protocols, the study by Bell et al and the present study both show that HA contributes to boundary lubrication. Similarly, Jay et al (12) demonstrated that Healon, an uncrosslinked form of HA, lowered the μ value from ∼0.14 in PBS to ∼0.07 at 3,340 μg/ml, but not to the level in SF (∼0.02), at a latex–glass interface under boundary-lubricating conditions. This trend is similar to that observed in the present study, although again, the absolute value of μ is somewhat different from the <μkinetic, Neq> in 3,300 μg/ml HA reported here (∼0.12) (Figure 3B). Such differences may be due to interactions of HA with test surfaces, as postulated for PRG4 above, since the boundary lubrication function of HA is facilitated by binding to the test surfaces (47).

Investigators in several other studies have reported HA to be both effective (14, 18) and ineffective (15, 17) as a boundary lubricant, using different whole-joint test apparatuses in which several modes of lubrication were likely operative. The conflicting results of those studies support the disposition that characterization of a test configuration, surfaces, and mode of lubrication is important when analyzing the mechanism of boundary lubrication of articular cartilage. Accordingly, the test configuration and protocol used in the present study were characterized previously to achieve a boundary mode of lubrication (7). Thus, HA does appear to contribute, in a dose-dependent manner, to the boundary lubrication of articular cartilage. Additional pilot studies indicated that HA adsorbed to the articular surface of samples was able to contribute to boundary lubrication even without HA in the test bath, since samples soaked in HA, then rinsed and tested in PBS, still had a low μ value. These studies suggest that HA may function by being retained at or between the articular cartilage surfaces under relative motion during testing. Such adsorbed layers of HA at the articular surface may have facilitated sliding (16), due to their inherent slipperiness and ease of disentangling (48), and therefore reduced friction between asperities in contact.

The results indicating that SAPL, in the form of DPPC, do not significantly contribute to the boundary lubrication of articular cartilage, either alone or in combination with other SF constituents tested here, provide additional insight into the controversial role of SAPL as physiologic boundary lubricants of articular cartilage. DPPC at ∼0.35 mg/ml has been shown to slightly lower friction at a cartilage–steel interface (26), and phosphatidylcholine at 10 mg/ml dramatically reduced the μ value to ∼0.016 (compared with a μ value of ∼0.028 in bovine SF) at a latex–glass interface under boundary-lubricating conditions (13). In the present study, SAPL in the form of DPPC at a physiologic concentration of 200 μg/ml did not significantly lower <μkinetic, Neq> alone (Figure 5B), with <μkinetic, Neq> for DPPC remaining ∼5-fold greater than that for SF, or in combination with HA and PRG4 (Figure 6B). Additionally, because the PRG4 preparation tested in the present study was free of SAPL in appreciable quantities (<0.5 μg/ml), SAPL were not indirectly contributing to the boundary-lubricating ability of PRG4, as has been postulated as a complicating consideration (49).

However, the boundary-lubricating ability of additional forms of SAPL (phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins [6]) at a cartilage–cartilage interface, in various combinations, and the potential effect of the mode of delivery of the SAPL, still remain to be determined. The state in which endogenous lipids predominantly exist in SF (lamellae, micelles, or vesicles) also remains to be determined, although lamellar bodies have been detected by electron microscopy (39).

HA and PRG4 synergistically lowered friction at the cartilage–cartilage interface tested here, presumably due to molecular interactions facilitating a molecular distribution of shear at the articular surfaces. Jay et al (21) previously reported that HA and PRG4 acted synergistically, with HA enabling PRG4 to lubricate under higher contact pressures at a latex–glass interface under boundary-lubricating conditions. In that study, hydrophobic attraction of PRG4 molecules along the length of HA was suggested as a possible mechanism for this interaction, although PRG4 does contain other putative binding domains as well (25). In the present study, the interaction between HA and PRG4 appeared specific, since the subsequent addition of control proteins (albumins and globulins) at a physiologic concentration did not further lower the μ value (data not shown). Collectively, these results suggest that the repulsive force generated at the individual contact asperities, on the articular cartilage surfaces, coated in HA and PRG4 was greater than that at asperities coated in either HA or PRG4 alone. This repulsive force may have been provided by the structuring of water at the articular surface by the aggregation and/or interaction of PRG4 and HA (5). Regardless of how the repulsive force is generated, it may indirectly provide protection to chondrocytes from wear and mechanical disturbances in vivo by reducing surface tissue shear.

The role of boundary lubrication relative to other operative modes of lubrication mechanisms in vivo remains to be fully elucidated. The lubrication mechanisms associated with pressurized fluid, within cartilage and between its surfaces, likely contribute substantially to the low friction and low wear articulation within synovial joints. Extremely low μ values, ∼0.004–0.024, have been reported (for natural articular cartilage with SF as a lubricant) under test conditions in which bulk fluid pressurization is significant (11, 17) compared with those presented here. However, with increasing loading time and dissipation of hydrostatic pressure, the lubricant-coated surfaces of articular cartilage bear an increasingly higher portion of the load relative to pressurized fluid, and, consequently, μ values can become increasingly dominated by the boundary mode of lubrication (50, 51). Indeed, μstatic, Neq increasing with increasing Tps (as observed previously [7]) is suggestive of a time-dependent interdigitation of surface molecules (52) at the articular cartilage–cartilage interface. Early signs of articular cartilage wear have recently been associated with a loss of the boundary lubrication function of SF postinjury (32). Accordingly, boundary lubrication has been postulated to be critical to cartilage homeostasis by facilitating low friction and low wear (2). Future studies of the postulated role of boundary and other modes of lubrication in arthritic disease are needed.

The collective results of this study provide insight into the nature of the boundary lubrication of articular cartilage by SF and its constituents. The maintenance of the boundary-lubricating ability of SF, at the cartilage–cartilage interface tested here, even at a 3-fold dilution, suggests that the lubricant molecules in SF are normally present in excess. However, the rapid decline in boundary-lubricating ability with a further decrease in constituent concentration suggests that such an alteration, which can occur in the settings of both injury and disease (28–30, 32, 33, 53), can impair lubricating function. The combination of the SF constituents HA, PRG4, and SAPL at physiologic concentrations approaching, but not fully replicating, the boundary-lubricating ability of SF suggests that additional lubricant molecules and/or complexes remain to be identified. More than one specific molecule contributing to the boundary lubrication of articular cartilage is not particularly surprising given the variety of interactions that can occur between the many molecules present in SF and at the articular surface (for example, see refs.5, 21, 54, 55).

AUTHOR CONTRIBUTIONS

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

Dr. Sah 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 design. Schmidt, Nguyen, Sah.

Acquisition of data. Schmidt, Gastelum, Nguyen, Schumacher.

Analysis and interpretation of data. Schmidt, Gastelum, Nguyen, Sah.

Manuscript preparation. Schmidt, Sah.

Statistical analysis. Schmidt, Sah.

REFERENCES

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