Abstract
BackgroundProteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; however, the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown. The objectives of this study were 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 + reduced/alkylated (R/A) PRG4, and 4) hylan G-F 20 + PRG4.MethodsStatic and kinetic friction coefficients (μstatic,Neq, <μkinetic,Neq>) were measured using a previously characterized cartilage-cartilage boundary mode friction test for the following concentrations of purified PRG4 and HA: Test 1: HA (1.5 MDa, 3.3 mg/mL) + PRG4 from 4.5 – 1500 μg/mL; Test 2: PRG4 (450, 150, 45 μg/mL) + HA (1.5 MDa) from 0.3 – 3.3 mg/mL. Test 3: hylan G-F 20 (3. 3 mg/mL) + PRG4 (450 μg/mL). Test 4: HA (3.3 mg/mL) + R/A PRG4 (450 μg/mL). ANOVA was used to compare lubricants within (comparing 6 lubricants of interest) and between (comparing 3 lubricants of interest) test sequences, with Tukey and Fishers post-hoc testing respectively.ResultsThis study demonstrates that both PRG4 and HA concentration, as well as PRG4 disulfide-bonded structure, can alter the cartilage boundary lubricating ability of PRG4 + HA solutions. The boundary lubricating ability of high MW HA + PRG4 solutions was limited by very low concentrations of PRG4. Decreased concentrations of high MW HA also limited the cartilage boundary lubricating ability of HA + PRG4 solutions, with the effect exacerbated by low PRG4 concentrations. The reduction of friction by addition of PRG4 to a cross-linked HA viscosupplement product, but not with addition of R/A PRG4 to HA, is consistent with a non-covalent mechanism of interaction where tertiary and quaternary PRG4 structure are important.ConclusionsCollectively, these results demonstrate that deficiency of either or both PRG4 and HA, or alterations in PRG4 structure, may be detrimental to SF cartilage boundary lubricating function. This study provides further insight into the nature of cartilage boundary lubrication and advancement towards potential formulation of new intra-articular biotherapeutic treatments for osteoarthritis using PRG4 ± HA.
Highlights
Proteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown
Lubrication Testing proteoglycan 4 (PRG4) dose response in HA In constant [HA] = 3.3 mg/mL, coefficients of friction appeared to decrease towards that of Synovial fluid (SF) as [PRG4] increased, decreasing towards a plateau between 45 and 150 μg/mL. μstatic,equilibrium load (Neq) varied with test lubricant and pre-sliding duration (Tps) in both the low dose sequence (p < 0.001, p < 0.05, Fig. 2a) and Lubricant [PRG4], [HA]
reduced and alkylated (R/A) PRG4 Addition of R/A PRG4 at 450 μg/mL to 1.5 MDa HA at 3.3 mg/mL appeared to slightly, but not significantly, lower friction compared to HA alone. μstatic,Neq varied with test lubricant and Tps. at Tps = 1.2 s varied with test lubricant (p < 0.0001, Fig. 4b). for HA alone was significantly higher than SF (p = 0.001)
Summary
Proteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown. Friction between articular cartilage surfaces in motion is mediated through a combination of lubrication mechanisms. PRG4 [6] is a mucin-like O-linked glycosylated protein present in SF [7] and at the articular cartilage surface [8]. [5] 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 [10] When combined at physiological concentrations, PRG4 + HA further reduce friction synergistically towards that of whole SF. [5] 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 [10]
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