Synovial Fluid Constituents Are Not Unique In Their Ability To Drive Articular Cartilage Superlubricity

Introduction: Articular cartilage is an unmatched lubricating material, easily sustaining friction coefficients (μ) < 0.005 in vivo1, making it exceptional in its ability to support superlubricity (i.e., μ < 0.01).2 While such lubricity has historically been attributed to cartilage’s unique structure and/or its bathing (synovial) fluid (SF) composition, only recently have such μ been replicated on the benchtop.3-6 Indeed, we have shown that by coupling our unique convergent stationary contact area (cSCA) testing configuration with tribological tests performed at physiological sliding speeds (i.e., >60mm/s, thereby facilitating the generation of interfacial hydrodynamics) and with SF as a bathing lubricant, truly in vivo μ ( < 0.004) can, for the first time, be sustained on the benchtop.5,6
SF, the bathing lubricant of our joints, contains numerous macromolecules implicated in modulating cartilage lubrication.3,6,7 However, our recent work indicates that one constituent of SF, hyaluronic acid (HA), can, by itself, sustain biofidelic cartilage μ < 0.004 in our cSCA configuration under physiological sliding conditions.6,8 Previously, HA’s “unique” lubricating capacity has been interpreted within the context of either viscous or boundary lubrication; yet it remains unclear if HA’s ability to support biofidelic μ is specific to HA or to more generalizable lubricant properties.7,9-12 Therefore, we investigated potential interactions between hydrodynamics (titrated through sliding speed) and lubricant composition within the cSCA configuration on cartilage lubrication behaviors by testing contributions to superlubricity via [1] individual constituents of normal SF and [2] “synthetic” or non-natural lubricants having similar structural and/or rheological properties as HA.

Materials and

Methods: 19mm osteochondral explants (n=5-9/group) from skeletally mature bovine stifle femoral condyles were extracted5,6,8 and stored in 1X PBS + protease inhibitors + sucrose (to approximate SF’s 400mOsm osmolarity13); hereon referred to as PBS. The effect of various lubricants on superlubricity within the cSCA lubrication were investigated using a custom reciprocating tribometer.5,6,8 All explants underwent loading and sliding preconditioning before tribological characterization at sliding speeds ranging from 80-1mm/s at 0.2MPa contact stress.8,14
In a first study, SF constituents (HA, albumins, gamma-globulins (GG), phospholipids, and mucin [lubricin mimetic]) were investigated in isolation at physiological compositions (with PBS as the solvent) to isolate each constituent’s ability to support superlubricity in the cSCA contact.7 Explants were tested sequentially in PBS followed by the SF constituent in isolation. Sliding conditions (e.g., sliding speed or bath solution/lubricant) were only changed upon achieving both deformation and friction equilibria.8
In a second study, non-natural “synthetic” lubricants exhibiting comparable structural and/or rheological properties as HA (polyethylene oxides [PEO], GG [at high concentrations], mucins, dextrans, and glycerol) were used to further investigate superlubricity in cartilage.15-19 Explants were tested, sequentially, at five increasing MW/concentrations of each non-natural lubricant to investigate if (and how) the lubricant of interest modulates superlubricity within the cSCA. Equilibrium compression (Eq) and kinetic friction coefficients (Eq) under each condition were recorded in LabVIEW for analysis in MATLAB.5,6,8

Results, Conclusions, and Discussions: The cSCA configuration, with its ability to titrate bulk FLS and hydrodynamics, continues to highlight the role of hydrodynamics in further uncovering the mechanism/s of cartilage lubrication. In study 1, we found HA to be the only individual SF constituent capable of replicating SF-lubricated cSCA Eq at physiological composition. At a sliding speed of 80mm/s, which is comparable to speeds experienced within the knee during walking, only HA (at 3mg/mL; 1400kDa) supported superlubricity (Figure 1A).20 This work further highlights the role of HA (within the lubricant) in modulating cartilage lubrication, as no other SF lubricant (at physiological compositions) investigated reduced Eq (relative to PBS) nor achieved superlubricity at any sliding speed tested.2,7
Intriguingly, in study 2, PEO, mucin, dextrans, glycerol, & GGs drove concentration and MW-dependent reductions in Eq at most sliding speeds, despite being decidedly non-physiological. At 80mm/s, all lubricants significantly reduced Eq (p < 0.05), with each having at least one composition supporting superlubricity (Figure 1B).2
At our slower sliding speeds, lubricant effects on Eq varied. While PEO and dextran promoted significant reductions in Eq (relative to PBS) across all sliding speeds, glycerol did so only at 10mm/s. GGs had no effect on Eq at slower sliding speeds. It is also noteworthy that while most of our non-natural lubricants exhibited their lowest Eq (and superlubricity) at 80mm/s, for both 100 and 300mg/mL dextrans, superlubricity occurred at intermediate sliding speeds with Eq subsequently increasing at our fastest sliding speed. This work suggests that cartilage’s remarkable ‘superlubricity’ may not be unique to its natural bathing solution but can be replicated using “non-natural” lubricants of similar structural and/or rheological properties. These first-of-their kind observations provide new insights into possible mechanisms for cartilage’s phenomenal (indeed super) lubricity.

Acknowledgements (Optional): [1] Linn, J Biomech, 1968; [2] Muser, NanoSci Tech, 2015; [3] Bonnevie, J Biomech Eng, 2020; [4] Ateshian, J Biomech, 2009 ; [5] Moore, OA&C, 2017; [6] Farnham, Trib Let, 2021; [7] Fam, Biorheol, 2007; [8] Lambeth, BMES Ann Meet, 2023; [9] Krause, Biomacromol, 2001; [10] Miyazaki, J Appl Polym Sci, 1998; [11] Ogsten, J Physiol, 1953; [12] Bonnevie, PloS One, 2015; [13] Baumgarten, J Bo Join Surg, 1985; [14] Chan, Sci Rep, 2016; [15] Ebagninin, J Coll Interf Sci, 2009; [16] VROC, Application Note; [17] Meldrum, Sci Rep, 2018; [18] Tirtaatmadja, J Non-Newt Fluid Mech, 2001; [19] Segur, Ind Eng Chem, 1951; [20] Graham, Conn Tis Res, 2020.