No association between local levels of molecular biomarkers and knee cartilage volumes early after anterior cruciate ligament injury

Abstract

Purpose: Trauma induced cytokine response in knee injuries may play a role in the development of osteoarthritis. We hypothesize that the cartilage volume affects the release of cartilage proteins and/or the presence of cytokines in the acute stage after knee injuries. Primary aim of our study was to compare knee cartilage volumes, derived from magnetic resonance images (MRI), with concentrations of biomarkers in biofluids at baseline (early after injury) in patients with anterior cruciate ligament (ACL) injuries. Secondary aim was to examine the correlation of confounders on biomarker levels or cartilage volumes. Methods: The KANON cohort consists of patients with an acute ACL tear to a previously uninjured knee (n = 121; age 26 years at injury, 74% males). In biofluids, collected at a median of 17 days (range 1-44 days) after the ACL injury, biomarkers were assessed using immunoassays: cartilage biomarkers aggrecan ARGS neoepitope (ARGS; in-house assay), cartilage oligomeric matrix protein (COMP; BioVendor) and cross-linked C-telopeptide of type II collagen (CTX-II; IDS); bone biomarker cross-linked N-telopeptide of type I collagen (NTX-I; Osteomark); cytokines (multiplex assay, MSD) interleukin (IL) 6, 8, 10, tumor necrosis factor α (TNF) and interferon gamma (INF-γ). Cartilage volumes were obtained from sagittal 3D SPGR MRI (1.5 T Philips Gyroscan; resolution: 0.29x0.29x3.0mm) based on quality-controlled, manual segmentations in three different cartilage compartments: tibiofemoral joint (TFJ), patellofemoral joint (PFJ) and total knee joint (TFJ + PFJ). MR images were acquired at the same day as the serum and urine sampling, but on average 11 days (range 1-24 days) after collection of synovial fluid. In linear regression models, we estimated cartilage volume as explanatory for molecular biomarker concentrations adjusted for age and time between injury and sampling. Models including both men and women were additionally adjusted for sex. Since both sex and BMI were associated with cartilage volume (see below) and BMI associated with sex (data not shown), we decided to adjust only for sex. 46 synovial fluid, 117 serum and 115 urine samples from individuals with corresponding MRI assessed cartilage volumes were used in linear regression analyses. Biomarkers with values below lower limit of quantification (LLOQ) were imputed with LLOQ divided by 2, (N% with levels above LLOQ): i.e. synovial fluid INF-γ (87%), serum IL-10 (46%) and urine CTX-II (99%). Unadjusted correlation (Pearson), Mann-Whitney U-test and student’s t-test were used for estimation of confounders. P values less than 0.05 were considered significant. Results: None of the synovial fluid biomarkers associated with cartilage volumes: Total knee joint (Table 1), TFJ or PFJ (data not shown); additional adjustment for differences in time between MRI assessment and synovial fluid sampling gave similar results (data not shown). In analyses stratified by sex, serum ARGS and urine CTX-II associated positively with total knee joint and TFJ cartilage volume in men, while in women, serum TNF associated negatively with total knee joint cartilage volume; none of the other biomarkers showed any association to any of the cartilage compartment volumes. Effects of confounders (Table 2): age associated negatively with serum ARGS, urine CTX-II and urine NTX-I concentrations, and positively with serum COMP levels; BMI associated positively with cartilage volumes, serum COMP levels and negatively with urine CTX-II; time between injury and sampling associated negatively with synovial fluid cytokine concentrations and positively with serum ARGS; compared to women, men had 1.3-fold higher serum COMP levels (Mann-Whitney: p = 0.002; men n = 89, women n = 32) and 1.3- and 1.4-fold more cartilage volume (t-test: p < 0.001, men n = 89, women n = 32). No other associations were found between confounders and molecular biomarker levels or cartilage volumes (Table 2). Conclusions: We found no evidence in support of local cytokines or cartilage biomarkers in the synovial fluid to be cross-sectionally associated with cartilage volume in the early phase after ACL injury. An association between cartilage volume and serum ARGS-aggrecan or urine CTX-II concentrations was only found in men. Several confounders affect biomarker levels and the cartilage volume creating a complex pattern of causalities that must be considered in analysis models.View Large Image Figure ViewerDownload Hi-res image Download (PPT)