Analysis of copy number variation in dogs implicates genomic structural variation in the development of anterior cruciate ligament rupture.

Anterior cruciate ligament (ACL) rupture is an important condition of the human knee. Second ruptures are common and societal costs are substantial. Canine cranial cruciate ligament (CCL) rupture closely models the human disease. CCL rupture is common in the Labrador Retriever (5.79% prevalence), ~100-fold more prevalent than in humans. Labrador Retriever CCL rupture is a polygenic complex disease, based on genome-wide association study (GWAS) of single nucleotide polymorphism (SNP) markers. Dissection of genetic variation in complex traits can be enhanced by studying structural variation, including copy number variants (CNVs). Dogs are an ideal model for CNV research because of reduced genetic variability within breeds and extensive phenotypic diversity across breeds. We studied the genetic etiology of CCL rupture by association analysis of CNV regions (CNVRs) using 110 case and 164 control Labrador Retrievers. CNVs were called from SNPs using three different programs (PennCNV, CNVPartition, and QuantiSNP). After quality control, CNV calls were combined to create CNVRs using ParseCNV and an association analysis was performed. We found no strong effect CNVRs but found 46 small effect (max(T) permutation P<0.05) CCL rupture associated CNVRs in 22 autosomes; 25 were deletions and 21 were duplications. Of the 46 CCL rupture associated CNVRs, we identified 39 unique regions. Thirty four were identified by a single calling algorithm, 3 were identified by two calling algorithms, and 2 were identified by all three algorithms. For 42 of the associated CNVRs, frequency in the population was <10% while 4 occurred at a frequency in the population ranging from 10-25%. Average CNVR length was 198,872bp and CNVRs covered 0.11 to 0.15% of the genome. All CNVRs were associated with case status. CNVRs did not overlap previous canine CCL rupture risk loci identified by GWAS. Associated CNVRs contained 152 annotated genes; 12 CNVRs did not have genes mapped to CanFam3.1. Using pathway analysis, a cluster of 19 homeobox domain transcript regulator genes was associated with CCL rupture (P = 6.6E-13). This gene cluster influences cranial-caudal body pattern formation during embryonic limb development. Clustered genes were found in 3 CNVRs on chromosome 14 (HoxA), 28 (NKX6-2), and 36 (HoxD). When analysis was limited to deletion CNVRs, the association was strengthened (P = 8.7E-16). This study suggests a component of the polygenic risk of CCL rupture in Labrador Retrievers is associated with small effect CNVs and may include aspects of stifle morphology regulated by homeobox domain transcript regulator genes.

Proinflammatory polarization of stifle synovial macrophages in dogs with cruciate ligament rupture.

OBJECTIVE: To determine polarization of synovial macrophages during development of cruciate ligament rupture (CR) and determine whether differences in synovial macrophage polarization in CR, osteoarthritis (OA), and healthy joints exist. STUDY DESIGN: Prospective case-controlled study. ANIMALS: Client-owned dogs with unstable stifles with CR (n = 22), paired stable contralateral stifles with partial CR (pCR; n = 7), joints with OA not related to CR (n = 6), and clinically normal (Normal; n = 7) joints. METHODS: Synovial fluid samples were collected. Smears were made for differential cytology counts and estimated total nucleated cell counts. Cytospin preparations were made, and immunocytochemical staining was performed with the pan-macrophage marker CD68, M1 macrophage markers inducible nitric oxide synthase (iNOS) and chemokine (C-C motif) receptor 7 (CCR7), and M2 macrophage markers arginase 1 and CD163. Positively stained cells were counted. RESULTS: Numbers of lymphocytes were increased in the CR group compared with the OA and Normal groups (P < .05). Numbers of CD68+ , CCR7+ , and iNOS+ cells in the CR and OA groups were increased compared with the Normal group (P < .05). Globally, the ratio of positively stained M1 polarized CD68+ cells to M2 polarized CD68+ cells was highest for the OA group (2.49), followed by the pCR (2.1), CR (1.63), and Normal (0.7) groups. CONCLUSION: Polarization of synovial macrophages toward an M1 proinflammatory phenotype is an early event in the development of canine CR. CLINICAL SIGNIFICANCE: M1 polarization in pCR stifles provides evidence of a possible role for macrophages in progressive development of cruciate ligament fiber damage. Lymphocytes may play a role in the synovitis found in CR joints. Our findings provide evidence that these cells are therapeutic targets.

Use of a platelet-rich plasma-collagen scaffold as a bioenhanced repair treatment for management of partial cruciate ligament rupture in dogs.

Dogs are commonly affected with cruciate ligament rupture (CR) and associated osteoarthritis (OA), and frequently develop a second contralateral CR. Platelet rich plasma (PRP) is a component of whole blood that contains numerous growth factors, which in combination with a collagen scaffold may act to promote bioenhanced primary repair of ligament. This study tested the hypothesis that treatment of partial stable CR stifles with an intra-articular collagen scaffold and PRP would decrease the disease progression, synovitis and risk of complete CR over a 12-month study period. We conducted a prospective cohort study of 29 client-owned dogs with an unstable stifle due to complete CR and stable contralateral stifle with partial CR. All dogs were treated with tibial plateau leveling osteotomy (TPLO) on the unstable stifle and a single intra-articular application of PRP-collagen in the stable partial CR stifle. Dogs were evaluated at the time of diagnosis, and at 10-weeks and 12-months after treatment. We evaluated correlation between both development of complete CR and time to complete CR with diagnostic tests including bilateral stifle radiographs, 3.0 Tesla magnetic resonance (MR) imaging, and bilateral stifle arthroscopy. Additionally, histologic evaluation of synovial biopsies, C-reactive protein (CRP) concentrations in serum and synovial fluid, and synovial total nucleated cell count, were determined. Results indicated that a single application of PRP-collagen in partial CR stifles of client owned dogs is not an effective disease-modifying therapy for the prevention of progression to complete CR. Radiographic effusion, arthroscopic evaluation of cranial cruciate ligament (CrCL) damage, and MR assessment of ligament fiber tearing in partial CR stifles correlated with progression to complete CR over the 12-month follow-up period. We determined that the best predictive model for development of complete CR in PRP-collagen treated partial CR stifles included variables from multiple diagnostic modalities.

Multivariate genome-wide association analysis identifies novel and relevant variants associated with anterior cruciate ligament rupture risk in the dog model.

BACKGROUND: Anterior cruciate ligament rupture (ACLR) is a debilitating and potentially life-changing condition in humans, as there is a high prevalence of early-onset osteoarthritis after injury. Identification of high-risk individuals before they become patients is important, as post-treatment lifetime burden of ACLR in the USA ranges from $7.6 to $17.7 billion annually. ACLR is a complex disease with multiple risk factors including genetic predisposition. Naturally occurring ACLR in the dog is an excellent model for human ACLR, as risk factors and disease characteristics in humans and dogs are similar. In a univariate genome-wide association study (GWAS) of 237 Labrador Retrievers, we identified 99 ACLR candidate loci. It is likely that additional variants remain to be identified. Joint analysis of multiple correlated phenotypes is an underutilized technique that increases statistical power, even when only one phenotype is associated with the trait. Proximal tibial morphology has been shown to affect ACLR risk in both humans and dogs. In the present study, tibial plateau angle (TPA) and relative tibial tuberosity width (rTTW) were measured on bilateral radiographs from purebred Labrador Retrievers that were recruited to our initial GWAS. We performed a multivariate genome wide association analysis of ACLR status, TPA, and rTTW. RESULTS: Our analysis identified 3 loci with moderate evidence of association that were not previously associated with ACLR. A locus on Chr1 associated with both ACLR and rTTW is located within ROR2, a gene important for cartilage and bone development. A locus on Chr4 associated with both ACLR and TPA resides within DOCK2, a gene that has been shown to promote immune cell migration and invasion in synovitis, an important predictor of ACLR. A third locus on Chr23 associated with only ACLR is located near a long non-coding RNA (lncRNA). LncRNA's are important for regulation of gene transcription and translation. CONCLUSIONS: These results did not overlap with our previous GWAS, which is reflective of the different methods used, and supports the need for further work. The results of the present study are highly relevant to ACLR pathogenesis, and identify potential drug targets for medical treatment.

Temporal mechanically-induced signaling events in bone and dorsal root ganglion neurons after in vivo bone loading.

Mechanical signals play an integral role in the regulation of bone mass and functional adaptation to bone loading. The osteocyte has long been considered the principle mechanosensory cell type in bone, although recent evidence suggests the sensory nervous system may play a role in mechanosensing. The specific signaling pathways responsible for functional adaptation of the skeleton through modeling and remodeling are not clearly defined. In vitro studies suggest involvement of intracellular signaling through mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and mammalian target of rapamycin (mTOR). However, anabolic signaling responses to bone loading using a whole animal in vivo model have not been studied in detail. Therefore, we examined mechanically-induced signaling events at five time points from 0 to 24 hours after loading using the rat in vivo ulna end-loading model. Western blot analysis of bone for MAPK's, PI3K/Akt, and mTOR signaling, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to estimate gene expression of calcitonin gene-related protein alpha (CGRP-α), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), c-jun, and c-fos in dorsal root ganglion (DRG) of the brachial intumescence were performed. There was a significant increase in signaling through MAPK's including extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) in loaded limbs at 15 minutes after mechanical loading. Ulna loading did not significantly influence expression of the genes of interest in DRG neurons. Bone signaling and DRG gene expression from the loaded and contralateral limbs was correlated (SR>0.40, P<0.05). However, bone signaling did not correlate with expression of the genes of interest in DRG neurons. These results suggest that signaling through the MAPK pathway may be involved in load-induced bone formation in vivo. Further characterization of the molecular events involved in regulation of bone adaptation is needed to understand the timing and impact of loading events, and the contribution of the neuronal signaling to functional adaptation of bone.

Radiographic and magnetic resonance imaging predicts severity of cruciate ligament fiber damage and synovitis in dogs with cranial cruciate ligament rupture.

Cruciate ligament rupture (CR) and associated osteoarthritis (OA) is a common condition in dogs. Dogs frequently develop a second contralateral CR. This study tested the hypothesis that the degree of stifle synovitis and cranial cruciate ligament (CrCL) matrix damage in dogs with CR is correlated with non-invasive diagnostic tests, including magnetic resonance (MR) imaging. We conducted a prospective cohort study of 29 client-owned dogs with an unstable stifle due to complete CR and stable contralateral stifle with partial CR. We evaluated correlation of stifle synovitis and CrCL fiber damage with diagnostic tests including bilateral stifle radiographs, 3.0 Tesla MR imaging, and bilateral stifle arthroscopy. Histologic grading and immunohistochemical staining for CD3+ T lymphocytes, TRAP+ activated macrophages and Factor VIII+ blood vessels in bilateral stifle synovial biopsies were also performed. Serum and synovial fluid concentrations of C-reactive protein (CRP) and carboxy-terminal telopeptide of type I collagen (ICTP), and synovial total nucleated cell count were determined. Synovitis was increased in complete CR stifles relative to partial CR stifles (P<0.0001), although total nucleated cell count in synovial fluid was increased in partial CR stifles (P<0.01). In partial CR stifles, we found that 3D Fast Spin Echo Cube CrCL signal intensity was correlated with histologic synovitis (SR = 0.50, P<0.01) and that radiographic OA was correlated with CrCL fiber damage assessed arthroscopically (SR = 0.61, P<0.001). Taken together, results of this study show that clinical diagnostic tests predict severity of stifle synovitis and cruciate ligament matrix damage in stable partial CR stifles. These data support use of client-owned dogs with unilateral complete CR and contralateral partial CR as a clinical trial model for investigation of disease-modifying therapy for partial CR.

Genome-wide association analysis in dogs implicates 99 loci as risk variants for anterior cruciate ligament rupture.

Anterior cruciate ligament (ACL) rupture is a common condition that can be devastating and life changing, particularly in young adults. A non-contact mechanism is typical. Second ACL ruptures through rupture of the contralateral ACL or rupture of a graft repair is also common. Risk of rupture is increased in females. ACL rupture is also common in dogs. Disease prevalence exceeds 5% in several dog breeds, ~100 fold higher than human beings. We provide insight into the genetic etiology of ACL rupture by genome-wide association study (GWAS) in a high-risk breed using 98 case and 139 control Labrador Retrievers. We identified 129 single nucleotide polymorphisms (SNPs) within 99 risk loci. Associated loci (P<5E-04) explained approximately half of phenotypic variance in the ACL rupture trait. Two of these loci were located in uncharacterized or non-coding regions of the genome. A chromosome 24 locus containing nine genes with diverse functions met genome-wide significance (P = 3.63E-0.6). GWAS pathways were enriched for c-type lectins, a gene set that includes aggrecan, a gene set encoding antimicrobial proteins, and a gene set encoding membrane transport proteins with a variety of physiological functions. Genotypic risk estimated for each dog based on the risk contributed by each GWAS locus showed clear separation of ACL rupture cases and controls. Power analysis of the GWAS data set estimated that ~172 loci explain the genetic contribution to ACL rupture in the Labrador Retriever. Heritability was estimated at 0.48. We conclude ACL rupture is a moderately heritable highly polygenic complex trait. Our results implicate c-type lectin pathways in ACL homeostasis.

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

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.

Role of calcitonin gene-related peptide in functional adaptation of the skeleton.

Peptidergic sensory nerve fibers innervating bone and periosteum are rich in calcitonin gene-related peptide (CGRP), an osteoanabolic neurotransmitter. There are two CGRP isoforms, CGRPα and CGRPß. Sensory fibers are a potential means by which the nervous system may detect and respond to loading events within the skeleton. However, the functional role of the nervous system in the response of bone to mechanical loading is unclear. We used the ulna end-loading model to induce an adaptive modeling response in CGRPα and CGRPß knockout mouse lines and their respective wildtype controls. For each knockout mouse line, groups of mice were treated with cyclic loading or sham-loading of the right ulna. A third group of mice received brachial plexus anesthesia (BPA) of the loaded limb before mechanical loading. Fluorochrome labels were administered at the time of loading and 7 days later. Ten days after loading, bone responses were quantified morphometrically. We hypothesized that CGRP signaling is required for normal mechanosensing and associated load-induced bone formation. We found that mechanically-induced activation of periosteal mineralizing surface in mice and associated blocking with BPA were eliminated by knockout of CGRPα signaling. This effect was not evident in CGRPß knockout mice. We also found that mineral apposition responses to mechanical loading and associated BPA blocking were retained with CGRPα deletion. We conclude that activation of periosteal mineralizing surfaces in response to mechanical loading of bone is CGRPα-dependent in vivo. This suggests that release of CGRP from sensory peptidergic fibers in periosteum and bone has a functional role in load-induced bone formation.

Computed tomographic imaging of subchondral fatigue cracks in the distal end of the third metacarpal bone in the thoroughbred racehorse can predict crack micromotion in an ex-vivo model.

Articular stress fracture arising from the distal end of the third metacarpal bone (MC3) is a common serious injury in Thoroughbred racehorses. Currently, there is no method for predicting fracture risk clinically. We describe an ex-vivo biomechanical model in which we measured subchondral crack micromotion under compressive loading that modeled high speed running. Using this model, we determined the relationship between subchondral crack dimensions measured using computed tomography (CT) and crack micromotion. Thoracic limbs from 40 Thoroughbred racehorses that had sustained a catastrophic injury were studied. Limbs were radiographed and examined using CT. Parasagittal subchondral fatigue crack dimensions were measured on CT images using image analysis software. MC3 bones with fatigue cracks were tested using five cycles of compressive loading at -7,500N (38 condyles, 18 horses). Crack motion was recorded using an extensometer. Mechanical testing was validated using bones with 3 mm and 5 mm deep parasagittal subchondral slots that modeled naturally occurring fatigue cracks. After testing, subchondral crack density was determined histologically. Creation of parasagittal subchondral slots induced significant micromotion during loading (p<0.001). In our biomechanical model, we found a significant positive correlation between extensometer micromotion and parasagittal crack area derived from reconstructed CT images (SR = 0.32, p<0.05). Correlations with transverse and frontal plane crack lengths were not significant. Histologic fatigue damage was not significantly correlated with crack dimensions determined by CT or extensometer micromotion. Bones with parasagittal crack area measurements above 30 mm2 may have a high risk of crack propagation and condylar fracture in vivo because of crack micromotion. In conclusion, our results suggest that CT could be used to quantify subchondral fatigue crack dimensions in racing Thoroughbred horses in-vivo to assess risk of condylar fracture. Horses with parasagittal crack arrays that exceed 30 mm2 may have a high risk for development of condylar fracture.

Arthroscopic assessment of stifle synovitis in dogs with cranial cruciate ligament rupture.

Cranial cruciate ligament rupture (CR) is a degenerative condition in dogs that typically has a non-contact mechanism. Subsequent contralateral rupture often develops in dogs with unilateral CR. Synovitis severity is an important factor that promotes ligament degradation. Consequently, we wished to evaluate the utility of arthroscopy for assessment of stifle synovitis in dogs with CR. Herein, we report results of a prospective study of 27 dogs with unilateral CR and bilateral radiographic osteoarthritis. Arthroscopic images and synovial biopsies from the lateral and medial joint pouches were obtained bilaterally and graded for synovial hypertrophy, vascularity, and synovitis. Synovial tartrate-resistant acid phosphatase-positive (TRAP+) macrophages, CD3(+) T lymphocytes, Factor VIII+ blood vessels, and synovial intima thickness were quantified histologically and related to arthroscopic observations. Risk of subsequent contralateral CR was examined using survival analysis. We found that arthroscopic scores were increased in the index stifle, compared with the contralateral stifle (p<0.05). Numbers of CD3+ T lymphocytes (SR = 0.50, p<0.05) and TRAP+ cells in joint pouches (SR = 0.59, p<0.01) were correlated between joint pairs. Arthroscopic grading of vascularity and synovitis was correlated with number density of Factor VIII+ vessels (SR>0.34, p<0.05). Arthroscopic grading of villus hypertrophy correlated with numbers of CD3(+) T lymphocytes (SR = 0.34, p<0.05). Synovial intima thickness was correlated with arthroscopic hypertrophy, vascularity, and synovitis (SR>0.31, p<0.05). Strong intra-observer and moderate inter-observer agreement for arthroscopic scoring was found. Dog age and arthroscopic vascularity significantly influenced risk of contralateral CR over time. We conclude that arthroscopic grading of synovitis is a precise tool that correlates with histologic synovitis. Arthroscopy is useful for assessment of stifle synovitis in client-owned dogs, and could be used in longitudinal clinical trials to monitor synovial responses to disease-modifying therapy.

Functional adaptation in female rats: the role of estrogen signaling.

BACKGROUND: Sex steroids have direct effects on the skeleton. Estrogen acts on the skeleton via the classical genomic estrogen receptors alpha and beta (ERα and ERß), a membrane ER, and the non-genomic G-protein coupled estrogen receptor (GPER). GPER is distributed throughout the nervous system, but little is known about its effects on bone. In male rats, adaptation to loading is neuronally regulated, but this has not been studied in females. METHODOLOGY/PRINCIPAL FINDINGS: We used the rat ulna end-loading model to induce an adaptive modeling response in ovariectomized (OVX) female Sprague-Dawley rats. Rats were treated with a placebo, estrogen (17ß-estradiol), or G-1, a GPER-specific agonist. Fourteen days after OVX, rats underwent unilateral cyclic loading of the right ulna; half of the rats in each group had brachial plexus anesthesia (BPA) of the loaded limb before loading. Ten days after loading, serum estrogen concentrations, dorsal root ganglion (DRG) gene expression of ERα, ERß, GPER, CGRPα, TRPV1, TRPV4 and TRPA1, and load-induced skeletal responses were quantified. We hypothesized that estrogen and G-1 treatment would influence skeletal responses to cyclic loading through a neuronal mechanism. We found that estrogen suppresses periosteal bone formation in female rats. This physiological effect is not GPER-mediated. We also found that absolute mechanosensitivity in female rats was decreased, when compared with male rats. Blocking of adaptive bone formation by BPA in Placebo OVX females was reduced. CONCLUSIONS: Estrogen acts to decrease periosteal bone formation in female rats in vivo. This effect is not GPER-mediated. Gender differences in absolute bone mechanosensitivity exist in young Sprague-Dawley rats with reduced mechanosensitivity in females, although underlying bone formation rate associated with growth likely influences this observation. In contrast to female and male rats, central neuronal signals had a diminished effect on adaptive bone formation in estrogen-deficient female rats.

Contralateral cruciate survival in dogs with unilateral non-contact cranial cruciate ligament rupture.

BACKGROUND: Non-contact cranial cruciate ligament rupture (CrCLR) is an important cause of lameness in client-owned dogs and typically occurs without obvious injury. There is a high incidence of bilateral rupture at presentation or subsequent contralateral rupture in affected dogs. Although stifle synovitis increases risk of contralateral CrCLR, relatively little is known about risk factors for subsequent contralateral rupture, or whether therapeutic intervention may modify this risk. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a longitudinal study examining survival of the contralateral CrCL in client-owned dogs with unilateral CrCLR in a large baseline control population (n = 380), and a group of dogs that received disease-modifying therapy with arthroscopic lavage, intra-articular hyaluronic acid and oral doxycycline (n = 16), and were followed for one year. Follow-up in treated dogs included analysis of mobility, radiographic evaluation of stifle effusion and arthritis, and quantification of biomarkers of synovial inflammation. We found that median survival of the contralateral CrCL was 947 days. Increasing tibial plateau angle decreased contralateral ligament survival, whereas increasing age at diagnosis increased survival. Contralateral ligament survival was reduced in neutered dogs. Our disease-modifying therapy did not significantly influence contralateral ligament survival. Correlative analysis of clinical and biomarker variables with development of subsequent contralateral rupture revealed few significant results. However, increased expression of T lymphocyte-associated genes in the index unstable stifle at diagnosis was significantly related to development of subsequent non-contact contralateral CrCLR. CONCLUSION: Subsequent contralateral CrCLR is common in client-owned dogs, with a median ligament survival time of 947 days. In this naturally occurring model of non-contact cruciate ligament rupture, cranial tibial translation is preceded by development of synovial inflammation. However, treatment with arthroscopic lavage, intra-articular hyaluronic acid and oral doxycycline does not significantly influence contralateral CrCL survival.

Lymphocyte populations in joint tissues from dogs with inflammatory stifle arthritis and associated degenerative cranial cruciate ligament rupture.

OBJECTIVE: To evaluate lymphocyte populations in stifle synovium and synovial fluid of dogs with degenerative cranial cruciate ligament rupture (CCLR). STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs (n=25) with stifle arthritis and CCLR, 7 dogs with arthritis associated with cartilage degeneration (osteoarthritis [OA]), and 12 healthy Beagle dogs with intact CCL. METHODS: Arthritis was graded radiographically in CCLR dogs. After collection of joint tissues, mononuclear cells were isolated and subsequently analyzed using flow cytometry for expression of CD3, CD4, CD8, and CD21. RESULTS: The proportions of CD4(+) T helper lymphocytes, CD8(+) cytotoxic T lymphocytes, and CD3(+) CD4(-) CD8(-) T lymphocytes were increased in synovium from dogs with CCLR compared with synovium from healthy Beagle dogs (P<.05). The proportion of CD3(+) CD4(-) CD8(-) T lymphocytes in synovial fluid was increased in dogs with CCLR compared with dogs with OA (P<.05). In dogs with CCLR, the proportion of CD3(+) CD4(-) CD8(-) T lymphocytes in synovial fluid was inversely correlated with radiographic arthritis (S(R) =-0.68, P<.005). CONCLUSION: Lymphocytic inflammation of stifle synovium and synovial fluid is an important feature of the CCLR arthropathy. Lymphocyte populations include T lymphocytes expressing CD4 and CD8, and CD3(+) CD4(-) CD8(-) T lymphocytes. Presence of CD3(+) CD4(-) CD8(-) T lymphocytes was associated with development of stifle synovitis. Further work is needed to fully identify the phenotype of these cells.

Role of calcitonin gene-related peptide in bone repair after cyclic fatigue loading.

BACKGROUND: Calcitonin gene related peptide (CGRP) is a neuropeptide that is abundant in the sensory neurons which innervate bone. The effects of CGRP on isolated bone cells have been widely studied, and CGRP is currently considered to be an osteoanabolic peptide that has effects on both osteoclasts and osteoblasts. However, relatively little is known about the physiological role of CGRP in-vivo in the skeletal responses to bone loading, particularly fatigue loading. METHODOLOGY/PRINCIPAL FINDINGS: We used the rat ulna end-loading model to induce fatigue damage in the ulna unilaterally during cyclic loading. We postulated that CGRP would influence skeletal responses to cyclic fatigue loading. Rats were fatigue loaded and groups of rats were infused systemically with 0.9% saline, CGRP, or the receptor antagonist, CGRP(8-37), for a 10 day study period. Ten days after fatigue loading, bone and serum CGRP concentrations, serum tartrate-resistant acid phosphatase 5b (TRAP5b) concentrations, and fatigue-induced skeletal responses were quantified. We found that cyclic fatigue loading led to increased CGRP concentrations in both loaded and contralateral ulnae. Administration of CGRP(8-37) was associated with increased targeted remodeling in the fatigue-loaded ulna. Administration of CGRP or CGRP(8-37) both increased reparative bone formation over the study period. Plasma concentration of TRAP5b was not significantly influenced by either CGRP or CGRP(8-37) administration. CONCLUSIONS: CGRP signaling modulates targeted remodeling of microdamage and reparative new bone formation after bone fatigue, and may be part of a neuronal signaling pathway which has regulatory effects on load-induced repair responses within the skeleton.

Are bacterial load and synovitis related in dogs with inflammatory stifle arthritis?

It has been proposed that small quantities of microbial material within synovial joints may act as a trigger for development of synovitis. We have previously identified an association between intra-articular bacteria and development of inflammatory stifle arthritis and cranial cruciate ligament rupture (CCLR) in dogs, and now wished to quantify bacterial load and markers of synovitis in dogs with and without stifle arthritis and CCLR. Joint tissues were collected from dogs with CCLR (n=51) and healthy dogs with normal stifles (n=9). Arthritis was assessed radiographically in CCLR dogs. Bacterial load was assessed using qPCR and broad-ranging 16S rRNA primers. qRT-PCR was used to estimate expression of the T lymphocyte antigen receptor (TCR Vß), CD3ɛ, tartrate-resistant acid phosphatase (TRAP), IL-4, IL-17, and TNF-α genes. Severity of synovitis was assessed histologically. Bacterial load was increased in arthritic stifles, when compared with healthy stifles. Histologic synovitis in arthritic stifles was mononuclear and was significantly correlated with bacterial load (1 of 2 primer sets) (S(R)=0.49, p<0.001). In arthritic stifles, expression of TRAP in synovium was increased relative to healthy stifles. Expression of pro-inflammatory genes was not correlated with bacterial load, histologic inflammation, or radiographic arthritis. Translocation of bacterial material to the canine stifle is related to the presence of joint inflammation. The lack of a strong positive correlation suggests that bacterial load is unlikely to be a primary pro-inflammatory factor. However, dysregulation of immune responses within synovial tissues may be dependent upon an environmental microbial trigger.

Systemic effects of ulna loading in male rats during functional adaptation.

Functional skeletal adaptation is thought to be a local phenomenon controlled by osteoctyes. However, the nervous system also may have regulatory effects on adaptation. The aim of this study was to determine the effects of loading of a single bone on adaptation of other appendicular long bones and whether these responses were neuronally regulated. Young male Sprague-Dawley rats were used. The right ulna was loaded to induce a modeling response. In other rats, a second regimen was used to induce bone fatigue with a mixed modeling/remodeling response; a proportion of rats from each group received brachial plexus anesthesia to induce temporary neuronal blocking during bone loading. Sham groups were included. Left and right long bones (ulna, humerus, tibia, and femur) from each rat were examined histologically 10 days after loading. In fatigue- and sham-loaded animals, blood plasma concentrations of TNF-α, RANKL, OPG, and TRAP5b were determined. We found that loading the right ulna induced an increase in bone formation in distant long bones that were not loaded and that this effect was neuronally regulated. Distant effects were most evident in the rats that received loading without bone fatigue. In the fatigue-loaded animals, neuronal blocking induced a significant decrease in plasma TRAP5b at 10 days. Histologically, bone resorption was increased in both loaded and contralateral ulnas in fatigue-loaded rats and was not significantly blocked by brachial plexus anesthesia. In young, growing male rats we conclude that ulna loading induced increased bone formation in multiple bones. Systemic adaptation effects were, at least in part, neuronally regulated.

Seasonal variation in detection of bacterial DNA in arthritic stifle joints of dogs with cranial cruciate ligament rupture using PCR amplification of the 16S rRNA gene.

An underappreciated cause and effect relationship between environmental bacteria and arthritis may exist. Previously, we found that stifle arthritis in dogs was associated with the presence of environmental bacteria within synovium. Cranial cruciate ligament rupture (CCLR) is often associated with stifle arthritis in dogs. We now wished to determine whether seasonal variation in detection of bacterial material may exist in affected dogs, and to also conduct analyses of both synovium and synovial fluid. We also wished to analyze a larger clone library of the 16S rRNA gene to further understanding of the microbial population in the canine stifle. Synovial biopsies were obtained from 117 affected dogs from January to December 2006. Using PCR, synovium and synovial fluid were tested for Borrelia burgdorferi and Stenotrophomonas maltophilia DNA. Broad-ranging 16S rRNA primers were also used and PCR products were cloned and sequenced for bacterial identification. Overall, 41% of arthritic canine stifle joints contained bacterial DNA. Detection of bacterial DNA in synovial fluid samples was increased, when compared with synovium (p<0.01). Detection rates were highest in the winter and spring and lowest in the summer period, suggesting environmental factors influence the risk of translocation to the stifle. Organisms detected were predominately Gram's negative Proteobacteria, particularly the orders Rhizobiales (32.8% of clones) and Burkholderiales (20.0% of clones), usually as part of a polymicrobial population. PCR-positivity was inversely correlated with severity of arthritis assessed radiographically and with dog age. Bacterial translocation to the canine stifle may be associated with changes to the indoor environment.

The effect of early hyaluronic acid delivery on the development of an acute articular cartilage lesion in a sheep model.

BACKGROUND: Partial-thickness articular cartilage lesions occur with knee trauma and may progress to osteoarthritis. This study evaluates the effectiveness of hyaluronic acid on cartilage healing after acute knee injury in sheep. HYPOTHESIS: Early administration of hyaluronic acid to an acute cartilage injury will prevent chondrocyte death and improve cartilage metabolism. STUDY DESIGN: Controlled laboratory study. METHODS: A 10 x 10 mm partial-thickness articular cartilage lesion was created on the medial condyle of 16 adult sheep stifles (hindlimbs). Eight sheep received intra-articular hyaluronic acid injections at days 0, 8, and 15, and 8 controls received saline. Contralateral stifles were nonoperated controls. All sheep were sacrificed at 12 weeks after surgery. Synovial fluid was drawn before surgery and after euthanasia for collagen II, nitric oxide, and interleukin-1 beta analysis. The medial condyle was analyzed by gross appearance, confocal laser microscopy for cell viability, histologic analysis for cartilage morphology, and dimethylmethylene blue assay for proteoglycan. RESULTS: At 12 weeks, histologic analysis revealed that the hyaluronic acid group had significantly better scores than the saline group (P = .001). The hyaluronic acid group had significantly greater glycosaminoglycan content than the saline group (P = .011), and showed a trend of reduced chondrocyte death compared with the saline group (P = .07). Synovial fluid showed no significant differences between the groups in collagen II, nitric oxide, and interleukin-1 beta levels. CONCLUSION: The results demonstrated that early administration of hyaluronic acid shows a significant improvement in cartilage histologic analysis and increased glycosaminoglycan content after acute traumatic cartilage injury. CLINICAL RELEVANCE: Early hyaluronic acid treatment for acute partial-thickness articular cartilage lesions may decrease or delay articular degeneration.

Functional adaptation to loading of a single bone is neuronally regulated and involves multiple bones.

Regulation of load-induced bone formation is considered a local phenomenon controlled by osteocytes, although it has also been hypothesized that functional adaptation may be neuronally regulated. The aim of this study was to examine bone formation in multiple bones, in response to loading of a single bone, and to determine whether adaptation may be neuronally regulated. Load-induced responses in the left and right ulnas and humeri were determined after loading of the right ulna in male Sprague-Dawley rats (69 +/- 16 days of age). After a single period of loading at -760-, -2000-, or -3750-microepsilon initial peak strain, rats were given calcein to label new bone formation. Bone formation and bone neuropeptide concentrations were determined at 10 days. In one group, temporary neuronal blocking was achieved by perineural anesthesia of the brachial plexus with bupivicaine during loading. We found right ulna loading induces adaptive responses in other bones in both thoracic limbs compared with Sham controls and that neuronal blocking during loading abrogated bone formation in the loaded ulna and other thoracic limb bones. Skeletal adaptation was more evident in distal long bones compared with proximal long bones. We also found that the single period of loading modulated bone neuropeptide concentrations persistently for 10 days. We conclude that functional adaptation to loading of a single bone in young rapidly growing rats is neuronally regulated and involves multiple bones. Persistent changes in bone neuropeptide concentrations after a single loading period suggest that plasticity exists in the innervation of bone.