Direct Visualization of Lubricin-Mediated Enhancement of Hyaluronic Acid Viscosity Near the Cartilage Surface

Introduction

Viscosupplementation is one of the most common forms of osteoarthritis therapy. It involves injecting a lubricant, typically hyaluronic acid (HA), into the joint to restore viscosity of the synovial fluid. Friction is a better predictor of clinical performance than bulk viscosity [1]. Therefore, therapies that target friction reduction may be more effective than therapies focused on increasing bulk viscosity. 

An increase in viscosity leads to a decrease in friction via hydrodynamic effects described by the Stribeck framework [2]. Interestingly, some HA formulations exhibit friction coefficients lower than expected based on bulk viscosity alone, implying that a higher “local viscosity” may exist at the cartilage interface [3]. Notably, this interaction between HA and cartilage was shown to be mediated by lubricin, a glycoprotein that localizes to the articular surface. Specifically, the presence of lubricin facilitated the formation of a 50 μm-thick aggregated HA layer at the articular surface. Another study showed that functionalizing a rheometer with cartilage increased measured viscosity compared to measurements taken on metal or glass surfaces, again suggesting interaction between HA and cartilage [4]. 

Collectively, these studies all hint at increased viscosity of HA near the articular surface of cartilage. However, this phenomenon has not been directly visualized. The aims of this study were to: a) measure localized changes in viscosity of HA as a function of distance from the articular surface using microrheology; and b) to assess the role of lubricin in mediating this phenomenon.

Methods

Tissue harvest and processing: Cartilage explants (6 mm diameter, 2 mm high) were harvested from femoral condyles of neonatal bovids and stored at 4C in phosphate-buffered saline (PBS) for 3 days before imaging. The lubricin-removed group (n = 5) was incubated in 1.5 M NaCl for 1 hour, followed by a 1 hour PBS wash [2, 3].The lubricin-intact group (n = 7) was incubated in PBS for the same duration. 

Microrheology: Explants were cut into hemicylinders, incubated with HA (Hyvisc) mixed with polystyrene fluorescent beads (194 nm diameter) at a 1:1 volume ratio, yielding a final 4 mg/mL HA solution. Samples were incubated in HA solution for 15 minutes and imaged using a Zeiss i880 confocal microscope. To measure localized viscosity, videos of the beads’ Brownian motion were recorded at five distances from the articular surface - 0, 25, 50, 100, and 1000 μm (n = 3 videos per distance). Each video examined a 42.51 μm x 42.51 μm area for 94.86 seconds (2000 frames, 21.08 frames per second). Bead trajectories were analyzed using Trackpy. Diffusivity was calculated using the 2D anomalous diffusivity equation [5]. Viscosity was calculated using the Stokes-Einstein equation [6]. 

Statistics: A linear mixed-effects model was created with distance and condition as fixed effects and cartilage explant as a random effect. A 2-way ANOVA was run, followed by pairwise comparison using estimated marginal means. 

Results

Brownian motion tracking showed a distinctive reduction in root mean-square distance (DRMS) traveled by the fluorescent beads in the lubricin-intact samples compared to the lubricin-removed samples. The largest difference in DRMS was at the surface, and the magnitude of the differences decreased moving away from the articular surface. 

The presence of lubricin had a dramatic effect on the spatial profile of viscosity near the cartilage surface (figure 7D). The lubricin-removed group exhibited a viscosity of ~2250 mPa*s for all distances measured, while the lubricin-intact group exhibited an increased viscosity of 3293 mPa*s at 0 μm (p = 0.0005) that decayed until it converged to the bulk viscosity of 2318 mPa*s at 1000 μm (p = 0.7207). The viscosity of the lubricin-intact group was higher than the lubricin-removed group for distances up 50 μm (p = 0.0354). 

Discussion

This study demonstrated that lubricin increases the viscosity of HA near the articular surface. These results align with previous findings reporting a 50 μm-thick aggregated HA layer that matches the length of the region of elevated HA viscosity. The 38% increase in surface viscosity noted here could account for 27% decrease of friction seen in a previous study [2]. 

Lubricin-HA synergy is most likely mechanically mediated, arising from either entanglements or hydrophobic interactions [2]. It is important to note that while the length-scale of lubricin is 200 nm, the observed effects extend up to 50 um - over 250x greater. This suggests that lubricin is capable of mediating the self-aggregation of HA at much larger distances than its size would predict. Further studies are needed to determine the intrinsic properties of lubricin that enable the formation of macromolecular networks extending far beyond its own dimensions.

Conclusion

This is the first study to directly visualize localized changes in viscosity of HA near the articular surface of cartilage. HA experiences increased localized viscosity within the first 50 μm of the articular surface due to interactions with native lubricin.

References

[1] Bonnevie, E. D., Galesso, D., Secchieri, C., & Bonassar, L. J. (2019). Frictional characterization of injectable hyaluronic acids is more predictive of clinical outcomes than traditional rheological or viscoelastic characterization. PLOS ONE, 14(5), e0216702. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216702. 

[2] Bonnevie, E. D., Galesso, D., Secchieri, C., Cohen, I., & Bonassar, L. J. (2015). Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid. PLOS ONE, 10(11), e0143415–e0143415. https://journals.plos.org/plosone/articleid=10.1371/

journal.pone.0143415. 

[3] Trujillo, R. J., Tam, A. T., Bonassar, L. J., & Putnam, D. (2023). Effective viscous lubrication of cartilage with low viscosity microgels. Materialia, 33, 102000. https://www.sciencedirect.com/science/

article/pii/S2589152923003277?via%3Dihub.

[4] Cook, S. G., & Bonassar, L. J. (2020). Interaction with Cartilage Increases the Viscosity of Hyaluronic Acid Solutions. ACS Biomaterials Science & Engineering, 6(5), 2787–2795. https://doi.org/10.1021/acsbiomaterials.0c00100. 

[5] Metzler, R., & Klafter, J. (2000). The random walk’s guide to anomalous diffusion: a fractional dynamics approach. Physics Reports, 339(1), 1–77. https://doi.org/10.1016/S0370-1573(00)00070-3. 

[6] Walkthrough — Trackpy Documentation. Github. https://softmatter.github.io/trackpy/dev/tutorial/

walkthrough.html.