Synovial fluid microrheology to identify potential candidates for viscosupplementation

Abstract

Purpose: Hyaluronic acid (HA) is a large, hydrophilic glycosaminoglycan that functions as the primary viscous lubricant in synovial fluid. Studies suggest that HA can be decreased, increased, or unchanged in concentration and/or molecular weight (MW) distribution in osteoarthritis (OA). Although HA viscosupplementation can be used to treat OA-related pain, it is difficult to predict which patients will respond best to HA supplementation. One potential explanation for the variable clinical response to HA is the presence of distinct OA phenotypes, some of which may benefit from HA supplementation whereas others may not. Here, we first asked whether HA concentration and MW distribution were altered in naturally occurring equine carpal osteoarthritis as a model for naturally occurring OA in humans. Multiple particle-tracking microrheology (MPTM) was performed to determine whether viscosity correlated with HA concentration and/or molecular weight. Methods: All protocols were approved by the university IACUC. Synovial fluid sampling: Synovial fluid samples were obtained from healthy research horses and clinical cases with carpal OA (n=87; 25 healthy and 62 OA joints). HA ELISA: Synovial fluid HA concentrations were quantified for all samples using both a sandwich ELISA (HA DuoSet ELISA, R&D Systems) and a competitive ELISA (HA ELISA, Echelon Biosciences). HA agarose gel electrophoresis: PBS-diluted and proteinase K-digested SF samples were run on 0.5% agarose gels, stained with 0.005% Stains-All and de-stained with 10% ethanol. Densitometric scans of stained gels were used to detect relative HA molecular weight distribution into four categories: >6.1MDa, 6.1-3.1MDa, 3.1-1.5MDa and <1.5MDa. Microrheology: Synovial fluid viscosity was measured using MPTM (n=24; 12 healthy and 12 OA joints). 15μl of synovial fluid was loaded with 0.5 nm yellow-green fluorescent beads and imaged on a custom epifluorescence microscope with a high-speed EMCCD detector. Statistical analysis: Data was log-transformation to achieve normality, and t-tests were used to compare healthy and OA groups. Pearson product-moment correlation coefficients were calculated. Results: Full cohort (n=87): Synovial fluid HA concentrations varied substantially between individual animals (range: 0.04 to 0.9 mg/mL, R&D Systems; 0.14 to 2.74 mg/mL, Echelon Biosciences), but did not differ between healthy and OA joints (median: 0.35 and 0.29 mg/mL, R&D Systems; 0.70 and 0.70 mg/mL, Echelon Biosciences, Fig. 1A). The proportion of high MW (>3.1MDa) to low MW (<3.1MDa) HA was significantly decreased in OA samples (median: 87% and 66%, p=0.009, Fig. 1B). MPTM cohort (n=24): Synovial fluid HA concentrations did not differ between healthy and OA joints. There was a trend for decreased synovial fluid viscosity in OA joints (median: 60 cP, IQR: 34-106) as compared to healthy joints (median: 120 cP, IQR: 68-263, p=0.07, Fig. 2A). HA concentration and viscosity were highly correlated (r=0.75, p<0.001, Fig. 2B). Conclusions: HA concentrations did not differ between healthy and OA joints due to significant inter-individual variability, suggesting that HA viscosupplementation may not be indicated in all OA cases. However, the trend for decreased synovial fluid viscosity in OA joints indicates that MPTM may be a more sensitive assay for detecting pathological changes in synovial fluid compared to measurements of HA total concentration or molecular weight distribution. HA concentration was a better predictor of synovial fluid viscosity than MW distribution. An inherent limitation of HA measurements is that neither HA ELISAs nor gel electrophoresis are able to measure the lowest MW HA isoforms. Multiple particle-tracking microrheology (MPTM) may hold potential promise as a method to identify joints that would be most likely to benefit from HA viscosupplementation. Additional work is needed to assess the rheologic and tribologic behavior of HA-deficient synovial fluid following HA supplementation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)