Friction between articular cartilage surfaces in motion is mediated through a combination of lubrication mechanisms. During fluid film lubrication, cartilage surfaces are separated by a fluid layer, while during boundary lubrication friction is mediated by interactions between lubricant molecules adsorbed to the surface [
]. The boundary lubrication mode becomes increasingly dominant as loading time is increased and interstitial fluid is depressurized [
]. Furthermore, opposing cartilage surfaces make contact over only approximately 10 % of the total area, making these areas of contact vulnerable to high friction [
]. Synovial fluid (SF) constituents proteoglycan 4 (PRG4) and hyaluronan (HA) are the primary contributors to its cartilage boundary lubricating ability [
]. PRG4 [
] is a mucin-like O-linked glycosylated protein present in SF [
] and at the articular cartilage surface [
]. HA, a linear polymer of D-glucuronic acid and D-N-acetylglucosamine [
], is also present in SF. Alone, solutions of PRG4 or HA reduce friction in a dose-dependent manner at a cartilage-cartilage biointerface in a boundary mode of lubrication compared to phosphate buffered saline (PBS). When combined at physiological concentrations, PRG4 + HA further reduce friction synergistically towards that of whole SF. [
] Both PRG4 and HA are critical to the cartilage boundary lubricating function of SF, and decreased boundary lubricating ability of SF has been linked with increased wear at the articular surface [
While the molecular mechanism of the PRG4 + HA synergism at a cartilage-cartilage biointerface in a boundary mode of lubrication remains to be fully understood, some characterization of potential factors affecting the synergism
has previously been performed. In solutions of HA alone, friction coefficients decrease with increasing HA concentration [
], and slightly with increasing molecular weight (MW, from 20 kDa to 5 MDa, at a concentration of 3.3 mg/ml) [
]. However, upon addition of PRG4 at 450 μg/mL the dependence of friction coefficient on HA MW is no longer observed [
] and friction is reduced to a similar value by addition of PRG4 over the range of MW of the 3.3 mg/ml HA solutions. Some SF from patients with osteoarthritis (OA) is deficient in PRG4, has normal HA concentration, an HA MW distribution shifted towards the lower range over all sizes from 6 MDa to 0.5 MDa, and fails to lubricate as well as normal SF. Normal cartilage boundary lubricating ability could be restored with addition of PRG4 to the SF [
], as evidenced by a measured reduction in friction. A similar decrease in SF HA concentration and HA MW, although with an increase in PRG4 concentration, has been observed in an equine acute injury model; this SF also fails to lubricate, though cartilage boundary lubricating ability could be restored by supplementation with high MW HA (4 MDa), but not low MW HA (800 kDa) [
]. These studies collectively demonstrate that both PRG4 and HA, particularly high MW HA, are necessary contributors to the cartilage boundary lubricating function of SF. However, the potential concentration dependence of PRG4 and/or high MW HA, both of which can be diminished in diseased SF, of the functional friction-reducing PRG4 + HA synergism at a cartilage-cartilage biointerface remains to be fully clarified.
The effects of injury and disease on PRG4 structure in SF, including relative composition of multimers:monomers and fragments of PRG4 [
], remain to be fully elucidated. As PRG4 is known to be degraded by enzymes such as neutrophil elastase, which can be up-regulated in inflammatory conditions such as post-anterior cruciate ligament tear [
], the ability of its fragments to maintain their ability to interact with HA may be of functional significance. The lubricating ability of PRG4 is decreased after it is reduced and alkylated (R/A) to break both inter- and intra-molecular disulfide bonds [
], and preparations of PRG4 enriched in disulfide-bonded multimeric species provide enhanced lubricating ability compared to preparations enriched in monomeric PRG4 [
]; this demonstrates the functional importance of inter-molecular disulfide bonds specifically, as reduced preparations of monomers appear to lubricate as well as non-reduced monomers [
]. Furthermore, R/A decreases the ability of PRG4 to adsorb to cartilage surfaces [
]. However, the effect of loss of disulfide-bonded structure, which may occur in diseased SF, by R/A on PRG4’s ability to interact with HA and synergistically reduce friction in a boundary mode at a cartilage-cartilage biointerface is also unknown.
Lastly, the MW of HA has also been linked to its efficacy as an intra-articular viscosupplement. Intra-articular HA injections are currently used to treat pain in OA patients, and it is thought that increasing the MW of HA by cross-linking increases joint residence time [
]. Increased MW may also contribute to pain relief by increased protection of nerve endings via increased viscosity [
]. Hylan G-F 20 (“Synvisc”, Genzyme) is one such example of a cross-linked HA preparation currently available and used clinically for intra-articular injections [
]. Given the clinical utility of cross-linked HA preparations, and evidence for intra-articular administration of PRG4’s potential efficacy in preventing joint degradation in animal models of OA [
], the ability of cross-linked HA to functionally interact with PRG4 synergistically to reduce friction in a boundary mode at a cartilage biointerface, towards that of whole SF, is of significant interest and currently unknown.
Given the limited level of understanding pertaining to the concentration and structural dependency of PRG4 + HA synergistic cartilage boundary lubrication function, and the clinical correlations of SF cartilage boundary lubricant composition and function to joint health and disease, the objectives of this study were as follows: to evaluate cartilage boundary lubricating ability of 1) PRG4 + HA in solution at constant HA concentration in a range of PRG4 concentrations, 2) constant PRG4 concentration in a range of HA concentrations, 3) HA + R/A PRG4, and 4) hylan G-F 20 + PRG4.
The results described here demonstrate that concentration of both PRG4 and high MW HA can affect the ability of PRG4 + HA solutions to reduce friction in the boundary mode at a cartilage-cartilage biointerface. The lubricating ability provided by the PRG4 + HA solutions tested here approached that of whole SF except for very low PRG4 (4.5, 45 μg/mL) concentrations in physiologically normal HA concentrations. This diminished cartilage boundary lubricating ability was enhanced when low PRG4 concentrations (45, 150 μg/mL) were added to low HA concentrations (0.3, 1.0 mg/mL); in this case physiological levels of PRG4 reduced friction, but not to the same level as when combined with higher HA concentrations. These results demonstrate that both PRG4 and high MW HA concentration can be limiting in achieving reduction of friction in the boundary mode at a cartilage-cartilage biointerface, and that both are necessary contributors to the cartilage boundary lubricating ability of SF. Furthermore, the addition of R/A PRG4 to HA was unable to significantly reduce friction, indicating that PRG4’s tertiary and quaternary protein structure is important in its friction reducing synergism with HA at a cartilage-cartilage biointerface. Lastly, PRG4 + hylan G-F 20 demonstrated improved lubricating ability compared to hylan G-F 20 alone, indicating that the HA + PRG4 cartilage boundary lubrication synergism is also maintained with a clinically relevant preparation of cross-linked HA. Collectively, these results demonstrate that both PRG4 and HA are necessary for effective friction reduction towards the level of whole SF and suggest that deficiency of either or both may be detrimental to SF cartilage boundary lubricating function.
The in vitro friction test used here is able to quantify contributions of PRG4 and HA to friction reduction in the boundary mode at a physiologically relevant cartilage-cartilage biointerface. The test geometry, protocol, and physiological surfaces allow for friction in a boundary mode of lubrication to be measured, even in viscous HA solutions - as indicated by the observation that PRG4 is able to reduce friction in a dose dependent manner in high MW HA solutions (3.3 mg/mL). While traditional Stribeck curve analysis, originally developed for steel surfaces, is not possible here given the rotational test geometry that facilitates the depressurized, stationary area of contact, its application to biointerfaces composed of porous, hydrogels [
] (e.g. cartilage) has recently been demonstrated to be not appropriate for biological tissues; it is not able to account for the macromolecules present at the deformable cartilage surfaces and in the non-Newtonian lubricant solutions that contribute to friction forces [
]. Though model surfaces provide the advantage of well-defined sample surfaces and modes of lubrication, and have been used to study wear prevention (previous studies have shown that friction and wear are linked at the articular surface [
]) as well as the order in which PRG4 and HA are adsorbed to surfaces [
], they may not allow for all the operative physiological interactions at a cartilage-cartilage biointerface to occur. The precise molecular mechanism through which boundary lubrication is provided by PRG4 and HA in SF at the cartilage surfaces (viscous boundary layer [
], adaptive mechanical control [
]) remains to be fully clarified. However, the results presented here are in general consistent with PRG4 + HA functioning synergistically to reduce friction at a cartilage surface through thick film boundary lubrication as proposed by the adaptive multimodal mechanism [
This study used preparations of PRG4 and HA that are representative of their composition within SF. The PRG4 preparation contained both multimeric and monomeric PRG4 species typically found in SF [
], and the R/A preparation was deficient in the multimeric PRG4 which could potentially occur in OA SF. A single high MW HA preparation was used, with 1.5 MDa being within the range of previously reported HA MW distribution in normal and OA SF. [
] Future studies could examine the friction reducing ability of each PRG4 multimeric/monomeric species with HA at a cartilage-cartilage biointerface, as well as an HA solution composed of a mixtures of various MW HA at (patho)physiological concentrations to further examine the potential concentration/MW dependence of the PRG4 + HA synergism. Lastly, while a smaller number of replicates has previously been used to assess differences between lubricants [
], as the lubricating ability of the solutions of interest become more similar in composition and low-friction function, a higher number of replicates may help elucidate if the apparent subtle differences observed here are in fact functionally important.
The coefficients of friction obtained here are consistent with previously measured values for purified solutions of PRG4 and HA, alone and in combination, at a cartilage-cartilage biointerface. <μ
> for PRG4 at 4.5 and 45 μg/mL observed in previous studies was on the order of 0.2, while PRG4 at 450 μg/mL was 0.10 [
for PRG4 at 450 μg/mL observed in previous studies was on the order of 0.4 [
]. The < μ
> obtained here for HA at 0.3 mg/mL with PRG4 at 45 and 450 μg/mL (0.152, 0.073) are lower than previously obtained for PRG4 alone, demonstrating friction reduction compared to PRG4 or HA alone even when low concentrations of high MW HA are added to low concentrations of PRG4. <μ
> for 1.5 MDa HA alone at 3.3 mg/mL was 0.080 in this study, and has been observed to be approximately 0.09 [
]; the values observed here with PRG4 (even 45 μg/mL, 0.072) appear to be similar to 1.5 MDa HA alone, indicating that very low concentrations of PRG4 can limit the boundary lubricating ability of HA + PRG4 solutions. Previous measurements of < μ
> for 450 μg/mL PRG4 + 3.3 mg/mL 1.5 MDa HA (0.046 [
]) are consistent with the values observed in this study (0.054). Note that μ
is presented here as a representation of start-up friction, and is calculated from the peak torque measurement at start-up of motion. <μ
> is calculated from the average torque in the final 2 revolutions of the testing protocol, and is representative of steady-state lubricating ability. Differences is trends between < μ
> and μ
(ie. HA at 3.3 mg/mL + PRG4 at 150 μg/mL appears to be equivalent to 45 and 450 μg/mL for μ
but not for < μ
>) could be due to the fact that there are differences in how friction is reduced in start-up versus steady state motion.
This study provides insight into the effects that PRG4 tertiary and quaternary structure, which may be altered during injury and disease, have on its functional interaction with HA. However, potential changes in PRG4 structure, including relative composition of multimers:monomers and fragments of PRG4 [
], remain to be clarified. Previous preliminary results have demonstrated that < μ
> of native PRG4 alone at 450 μg/mL is increased 34 % upon R/A, providing evidence that the disulfide-bonded structure of PRG4 itself is important for boundary lubricating ability [
]. In this study, despite a slight reduction in friction, the cartilage boundary lubricating ability of HA alone and HA + R/A PRG4 were not significantly different, suggesting that degradation of PRG4 structure and/or assembly in SF could potentially impact SF boundary lubricating ability by altering the PRG4 + HA interaction. This suggests that PRG4’s tertiary and quaternary protein structure is important in the interaction with HA. Future studies examining the role of PRG4 multimer/monomer interaction with HA to reduce friction will help clarify this issue.
These results also demonstrate that PRG4 can further reduce friction at a cartilage-cartilage biointerface, under boundary mode lubrication, beyond that of a cross-linked HA clinical product alone. Indeed, the < μ
> obtained for hylan G-F 20 at 3.3 mg/mL and PRG4 at 450 μg/mL (0.048) is very close to those discussed above for PRG4 and 1.5 MDa HA. These results contrast with previous observations using a similar
cartilage boundary lubrication test, where it was observed that hylan G-F 20 failed to lubricate as well as SF, and failed to prevent chondrocyte apoptosis compared to SF. [
] Subsequent work demonstrated that addition of purified PRG4 to PRG4-void SF was able to decrease chondrocyte apoptosis, and lower < μ
> beyond that of PRG4 alone, suggesting again that the PRG4 + HA interaction is critical for normal SF function [
]. While the studies investigating chondrocyte apoptosis and boundary lubrication used a similar
boundary lubrication test setup as this study, overall values may differ due to test parameter differences (no annular geometry, less time for stress relaxation, live explants, 12 continuous cycles vs. start and stop). The observation that PRG4 + HA friction reduction is not disrupted by the cross-linking procedure is consistent with previous evidence suggesting that the PRG4 + HA interaction is not a specific site-dependent binding, but rather a physical interaction [
]. The hylan G-F 20 used in this study was diluted to 3.3 mg/mL from its clinical concentration of 8 mg/mL to provide consistency with previous studies characterizing the PRG4 + HA interaction and investigate the effect of cross-linking. The effect of this dilution on PRG4 + HA interaction is currently unclear, and future studies elucidating the mechanism of the PRG4 + HA interaction will provide insight into the effects of supra-physiological HA concentrations and their influence on interaction with PRG4
The authors have no competing interests to disclose.
TL: Study design, data acquisition, analysis and interpretation of data, and manuscript preparation. MH: data acquisition, analysis and interpretation of data, manuscript editing. TS: study design, analysis and interpretation of data, manuscript editing. All authors were involved in revising the article and approved the final submitted version.