Synovial fluid lubricin increases in canine cruciate ligament rupture

Abstract

Purpose: Loss of effective joint lubrication contributes to cartilage breakdown in post-traumatic osteoarthritis (PTOA). There are two major lubricants in synovial fluid: hyaluronic acid (HA), which confers viscous properties, and lubricin, which enables low friction boundary lubrication. The absence of functional lubricin results in severe, early onset of polyarthropathy in humans and lubricin knockout mice. Several studies have reported that lubricin decreases in experimental rodent models of PTOA; however, it is unclear whether similar losses are observed in naturally occurring, large animal models of joint injury and osteoarthritis (OA) progression. Naturally occurring cranial cruciate ligament rupture in the dog closely mimics anterior cruciate ligament injury in humans with respect to the development of joint instability and secondary osteoarthritic changes, making this a valuable translational model to study the role of lubricin in PTOA. Here, we investigated lubricin and pro-inflammatory cytokines in canine cranial cruciate ligament rupture (RCCL) and the relationship of these molecules to radiographic evidence of osteoarthritis. Methods: Stifle synovial fluid samples from 30 adult dogs (age 2.5-11 yrs; n=13 neutered males, 14 neutered females, 2 intact males and 1 intact female) sustaining rupture of the cranial cruciate ligament were obtained at the time of surgery and stored in liquid N2 for biobanking purposes. Medical histories, physical examination, and surgical data were obtained from patient medical records, including age, duration of injury prior to presentation, presence of concurrent meniscal injury and whether or not meniscectomy was performed. Stifle synovial fluid samples were also obtained from a cohort of 13 clinically healthy research Beagles (age 2.5 to 5 yrs; intact males). Synovial fluid lubricin levels were quantified using a lubricin sandwich ELISA, and pro-inflammatory cytokines (IL-2, IL-6, IL-8, TNF-α) were measured using a multiplex cytokine panel (Meso Scale Diagnostics, LLC). Pre-operative lateromedial and craniocaudal stifle radiographs were randomized, blinded and scored by two independent observers. A stifle OA scoring system was modified slightly from Innes et al, which evaluated: joint effusion, osteophytosis, intra-articular mineralization, and a global OA score. T-tests were used to analyze biochemical differences between healthy and RCCL joints (α=0.05), and Spearman’s rank-order correlations were performed to determine associations between lubricin and pro-inflammatory cytokines. Results: Lubricin concentrations were nearly six-fold greater in RCCL joints (mean ± SEM = 0.07 ± 0.02 mg/mL) as compared to healthy controls (mean ± SEM = 0.68 ± 0.06mg/mL) (Fig 1A), and this elevation persisted for more than one year after injury (Fig 1B). No significant differences in synovial fluid pro-inflammatory cytokines (IL-2, IL-6, IL8, and TNF-α) were detected between RCCL and healthy joints; however, pro-inflammatory cytokines were all correlated with one another. Duration of injury did not correlate with lubricin concentrations (Spearman’s test, ρ=-0.0205). Lubricin concentrations correlated with the presence of OA, but no associations between the severity of radiographic OA and lubricin levels were detected. Additionally, lubricin levels were not correlated with any of the pro-inflammatory cytokines measured (Table 1). Conclusions: Lubricin was significantly and persistently elevated in joints with RCCL. Interestingly, although pro-inflammatory cytokines in synovial fluid levels were correlated with one another, lubricin levels did not correlate with any pro-inflammatory cytokines. A limitation of this study is that dogs presented with varying degrees of OA and durations of injury prior to surgery, with many animals presenting with a history of lameness or injury of longer than one month. However, to our knowledge, this is the first study to demonstrate that synovial fluid lubricin is significantly increased in a translational, large animal model of knee joint instability. Lubricin may have value as a potential biomarker for knee instability (and perhaps osteoarthritis progression). Lubricin may also be elevated as a protective mechanism in joint instability; however, more mechanistic studies are required to determine the role of elevated synovial fluid lubricin in knee injuries.View Large Image Figure ViewerDownload Hi-res image Download (PPT)