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.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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 ) 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 ) 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).