Canine Cruciate Ligament Rupture Research Articles

Immunoexpression of Relaxin and Its Receptors in Stifle Joints of Dogs with Cranial Cruciate Ligament Disease.

Simple SummarySpontaneous cranial cruciate ligament rupture is one of the most frequently encountered joint diseases in dogs, often leading to disabling chronic progressive osteoarthritis. The cause of the progressive intra-articular collagen matrix degradation, leading to tear and mechanical failure, is unknown. A variety of contributing factors has been found, however, an initiating mediator triggering the collagen degrading cascade remains to be identified. Our finding of strong relaxin- and relaxin receptor expression on intra-articular target tissues, such as on ligament fibrocytes and synovial membranes, renders relaxin a candidate for pathogenetic involvement, for collagen lysis, and progressive ligament fiber disruption. If confirmed, this opens the way for medical treatment of the disease in its early stages. In addition, further proof of relaxin involvement in canine osteoarthritis and ligament rupture would constitute a useful spontaneous animal model for human disease.The etiology of spontaneous cranial cruciate ligament rupture in dogs is unknown despite being one of the most impacting orthopedic diseases in dogs. Numerous studies have contributed to the understanding of a multifactorial pathogenesis, this, however, without identifying a pivotal link to explain progressive collagen degeneration and osteoarthritic changes. In human medicine, recent reports have identified relaxin as a triggering factor in ligament ruptures in knee and metacarpal joints. We thus hypothesized that relaxin might also play a role in canine cruciate ligament rupture. Relaxin’s primarily known property is connective tissue remodeling through collagenolysis. We therefore investigated relaxin and its cognate receptors LGR7/LGR8 in 18 dogs with cranial cruciate ligament disease (CCLD) and compared them to a group of dogs with normal stifle joints. Applying immunohistochemistry (IHC), double immunofluorescence (dIF), and western blot analysis (WB), we found strong and significantly increased expression of both relaxin and its receptors in ruptured cruciate ligaments, and in synovial membranes. Pattern of immuno-staining on dIF strongly suggests relaxin binding to primed receptors and activation of signaling properties, which in turn may have affected collagen matrix metabolism. Thus, in canine cranial cruciate ligament disease, relaxin/receptor signaling may be a primary trigger for collagen fiber degradation and collagen lysis, eventually followed by ligament rupture.

Synovial fluid lubricin increases in spontaneous canine cruciate ligament rupture

Lubricin is an important boundary lubricant and chondroprotective glycoprotein in synovial fluid. Both increased and decreased synovial fluid lubricin concentrations have been reported in experimental post-traumatic osteoarthritis (PTOA) animal models and in naturally occurring joint injuries in humans and animals, with no consensus about how lubricin is altered in different species or injury types. Increased synovial fluid lubricin has been observed following intra-articular fracture in humans and horses and in human late-stage osteoarthritis; however, it is unknown how synovial lubricin is affected by knee-destabilizing injuries in large animals. Spontaneous rupture of cranial cruciate ligament (RCCL), the anterior cruciate ligament equivalent in quadrupeds, is a common injury in dogs often accompanied by OA. Here, clinical records, radiographs, and synovial fluid samples from 30 dogs that sustained RCCL and 9 clinically healthy dogs were analyzed. Synovial fluid lubricin concentrations were nearly 16-fold greater in RCCL joints as compared to control joints, while IL-2, IL-6, IL-8, and TNF-α concentrations did not differ between groups. Synovial fluid lubricin concentrations were correlated with the presence of radiographic OA and were elevated in three animals sustaining RCCL injury prior to the radiographic manifestation of OA, indicating that lubricin may be a potential biomarker for early joint injury.

Synovial fluid lubricin increases in canine cruciate ligament rupture

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)

Immunological changes in canine cruciate ligament rupture

Samples of sera (39) and synovial fluid (36) from 58 dogs suffering from inflammatory arthropathy of the stifle joints, 47 of them in combination with unilateral or bilateral rupture of the anterior cruciate ligaments (CLR), were investigated for C1q-binding immune complexes (IC) using a solid phase radioimmunoassay. In 31 out of 39 sera (79 per cent) and 25 out of 36 synovial fluids (69 per cent) significant IC concentrations could be detected. As controls 21 healthy dogs were used. The radioimmunoassay was standardised with aggregated dog gamma globulin. The sera of the dogs were also investigated for anticollagen antibodies to denaturated type 1 or type 2 dog collagens. No evidence of the presence of these antibodies could be obtained. In a small percentage of sera and, or, synovial fluids rheumatoid factor could be demonstrated. These results indicate that the CLR in dogs is accompanied by immunological mechanisms.