Radiographic synovial effusion and osteophytosis in dogs with unilateral complete cruciate ligament rupture and contralateral partial cruciate ligament rupture.

<p>Orthogonal stifle radiographic views of the unstable stifle with complete cruciate ligament rupture (CR) (<b>A,B</b>) and the contralateral stable stifle with partial CR (<b>C,D</b>) in a four old mixed breed dog at trial entry. Synovial effusion with compression of the infrapatellar fat pat and caudal bulging of the joint capsule is evident in both stifles. Peri-articular osteophytosis is present in both stifles and is particularly evident on the distal pole of the patella, the trochlea ridges of the distal femur, and the margins of the tibial plateau. Osteophytosis is more severe in the stifle with complete CR in this dog. Overall, at diagnosis and trial entry, stifle synovial effusion (<i>P</i><0.05) and osteophytosis (<i>P</i> = 0.01) were increased in the unstable stifle with complete cruciate ligament rupture (CR), when compared with the contralateral stable stifle with partial CR. Stifle radiographs were graded subjectively used a standardized scoring method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159095#pone.0159095.ref001" target="_blank">1</a>].</p

Phenotype variance was explained to a large degree by the associated genomic loci.

<p>Loci identified by linear mixed model (LMM) analysis were broadly defined as SNPs with r²>0.5 within 5Mb of the peak SNP. (<b>a</b>) For GCTA, 36 loci in 72.7Mb of the genome explained 48.09% of the phenotypic variance. (<b>b</b>) For GEMMA, 47 loci in 82.7Mb of the genome explained 55.88% of the phenotypic variance. (<b>c</b>) For PUMA, 65 loci in 86.58Mb of the genome explained 50.28% of the phenotypic variance in the ACL rupture trait. Whiskers represent the standard error of the mean.</p

Genetic risk scoring [42] using GWAS associated loci from linear mixed model analysis with GEMMA segregates ACL rupture disease risk in case and control Labrador Retriever dogs.

<p>(<b>a</b>) Distribution of the number of ACL rupture risk loci in case and control groups of Labrador Retriever dogs. The number of risk alleles in cases and controls is significantly different (<i>P</i><2.2E-16) (<b>b</b>) ACL rupture odds ratios of weighted genetic risk scores (wGRS) relative to the first quartile. Vertical bars represent the 95% confidence intervals. * Odds ratio is significantly different from the reference first quartile.</p

The Labrador Retriever is a dog breed with a relatively low amount of inbreeding.

<p>Whiskers represent the standard error of the mean for each analysis method (n = 237 dogs).</p

Autologous Bone Marrow-Derived Mesenchymal Stem Cells Modulate Molecular Markers of Inflammation in Dogs with Cruciate Ligament Rupture

<div><p>Mid-substance rupture of the canine cranial cruciate ligament rupture (CR) and associated stifle osteoarthritis (OA) is an important veterinary health problem. CR causes stifle joint instability and contralateral CR often develops. The dog is an important model for human anterior cruciate ligament (ACL) rupture, where rupture of graft repair or the contralateral ACL is also common. This suggests that both genetic and environmental factors may increase ligament rupture risk. We investigated use of bone marrow-derived mesenchymal stem cells (BM-MSCs) to reduce systemic and stifle joint inflammatory responses in dogs with CR. Twelve dogs with unilateral CR and contralateral stable partial CR were enrolled prospectively. BM-MSCs were collected during surgical treatment of the unstable CR stifle and culture-expanded. BM-MSCs were subsequently injected at a dose of 2x10⁶ BM-MSCs/kg intravenously and 5x10⁶ BM-MSCs by intra-articular injection of the partial CR stifle. Blood (entry, 4 and 8 weeks) and stifle synovial fluid (entry and 8 weeks) were obtained after BM-MSC injection. No adverse events after BM-MSC treatment were detected. Circulating CD8⁺ T lymphocytes were lower after BM-MSC injection. Serum C-reactive protein (CRP) was decreased at 4 weeks and serum CXCL8 was increased at 8 weeks. Synovial CRP in the complete CR stifle was decreased at 8 weeks. Synovial IFNγ was also lower in both stifles after BM-MSC injection. Synovial/serum CRP ratio at diagnosis in the partial CR stifle was significantly correlated with development of a second CR. Systemic and intra-articular injection of autologous BM-MSCs in dogs with partial CR suppresses systemic and stifle joint inflammation, including CRP concentrations. Intra-articular injection of autologous BM-MSCs had profound effects on the correlation and conditional dependencies of cytokines using causal networks. Such treatment effects could ameliorate risk of a second CR by modifying the stifle joint inflammatory response associated with cranial cruciate ligament matrix degeneration or damage.</p></div

ACL rupture associated loci identified by GWAS in the Labrador Retriever, a dog breed with a high disease prevalence.

<p>ACL rupture associated loci identified by GWAS in the Labrador Retriever, a dog breed with a high disease prevalence.</p

Synovial fluid/serum C-reactive protein (CRP) ratio in stifle joints of dogs with stable partial cruciate ligament rupture (CR) before and after treatment with bone marrow-derived mesenchymal stem cell treatment.

<p>At diagnosis, synovial fluid/serum CRP ratio was elevated in the partial CR stifles of dogs that developed a second CR (n = 3) within the study period, when compared with the dogs that did not develop a second CR (n = 8). Synovial CRP was not measured in one dog because of insufficient sample volume. Synovial fluid/serum CRP ratio values were higher at 8 weeks in the dogs that did not experience CR within the study period.</p

Functional annotation clustering of genes in regions associated with anterior cruciate ligament rupture identified by GWAS in the Labrador Retriever.

<p>Functional annotation clustering of genes in regions associated with anterior cruciate ligament rupture identified by GWAS in the Labrador Retriever.</p

Effect of autologous bone marrow-derived mesenchymal stem cell (BM-MSC) treatment on serum and stifle synovial fluid chemokine [C-X-C motif] ligand 8 (CXCL8) and stifle synovial fluid interferon gamma (IFN-γ) and interleukin 6 (IL-6).

<p>(<b>A</b>) Median serum CXCL8 concentration was significantly increased at 8 weeks after MSC treatment, compared with diagnosis. Median serum CXCL8 was also higher at 4 weeks, compared with diagnosis. (<b>B</b>) Synovial CXCL8 concentrations were similar at diagnosis and at 8 weeks in both stifles. (<b>C</b>) Suppression of synovial fluid IFNγ was evident in both stifles after intra-articular and intravenous injection of BM-MSCs, particularly the partial cruciate rupture stifle that was injected with BM-MSCs. Serum IFNγ was not significantly influenced by treatment. (<b>D</b>) High concentrations of synovial IL-6 were identified in the stable partial cruciate rupture stifles, when compared with the unstable stifles with cruciate ligament rupture. Median concentrations of synovial IL-6 were lower after BM-MSC treatment in both stifles. As the y axis is logarithmic in C and D, only values greater than zero are plotted.</p

Linear mixed model GWAS corrects for population structure and identifies 99 ACL associated loci explaining a large proportion of phenotypic variance.

<p>For each linear mixed model (LMM), the QQ plots show no evidence of population stratification relative to the expected distribution. Permutation testing with each model determined genome-wide significance at (<b>a</b>) <i>P</i><3.63E-7 for GCTA (Genome-wide Complex Trait Analysis) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173810#pone.0173810.ref033" target="_blank">33</a>], λ = 0.987 (<b>b</b>) <i>P</i><6.097E-7 for GEMMA (Genome-wide Efficient Mixed Model Association) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173810#pone.0173810.ref034" target="_blank">34</a>], λ = 0.994 and (<b>c</b>) <i>P</i><4.01E-7 for PUMA (Penalized Unified Multiple-locus Association) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173810#pone.0173810.ref035" target="_blank">35</a>], λ = 1.012. The plots represent analysis of 118,992 SNPs from 98 cases and 139 phenotype-negative controls. (<b>d</b>) With GCTA, 36 loci have <i>P</i><5E-4, with the most significant locus located in CFA 24, which did not meet genome-wide significance defined by minimum p-values from permutation testing. (<b>e</b>) With GEMMA, 47 loci have <i>P</i><5E-4, with the locus on CFA 24 meeting genome-wide significance defined by minimum p-values from permutation testing. (<b>f</b>) With PUMA, 65 loci were significant at <i>P</i><5E-4 and the locus on CFA 24 exceeded genome-wide significance defined by minimum p-values from permutation testing. The single SNP that met genome-wide significance lies within the gene <i>PPP1R16B</i>.</p