Determining predictive metabolomic biomarkers of meniscal injury in dogs with cranial cruciate ligament rupture.

OBJECTIVES: This study used hydrogen nuclear magnetic resonance spectroscopy for the first time to examine differences in the metabolomic profile of stifle joint synovial fluid from dogs with cranial cruciate ligament rupture with and without meniscal injuries, in order to identify biomarkers of meniscal injury. Identifying a biomarker of meniscal injury could then ultimately be used to design a minimally invasive diagnostic test for meniscal injuries in dogs. MATERIALS AND METHODS: Stifle joint synovial fluid was collected from dogs undergoing stifle joint surgery or arthrocentesis for lameness investigations. We used multi-variate statistical analysis using principal component analysis and univariate statistical analysis using one-way analysis of variance and analysis of co-variance to identify differences in the metabolomic profile between dogs with cranial cruciate ligament rupture and meniscal injury, cranial cruciate ligament rupture without meniscal injury, and neither cranial cruciate ligament rupture nor meniscal injury, taking into consideration clinical variables. RESULTS: A total of 154 samples of canine synovial fluid were included in the study. Sixty-four metabolites were annotated to the hydrogen nuclear magnetic resonance spectroscopy spectra. Six spectral regions were found to be significantly altered (false discovery rate adjusted P-value <0.05) between groups with cranial cruciate ligament rupture with and without meniscal injury, including three attributed to nuclear magnetic resonance mobile lipids [mobile lipid -CH3 (P=0.016), mobile lipid -n(CH3 )3 (P=0.017), mobile unsaturated lipid (P=0.031)]. CLINICAL SIGNIFICANCE: We identified an increase in nuclear magnetic resonance mobile lipids in the synovial fluid of dogs with meniscal injury which are of interest as potential biomarkers of meniscal injury.

Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Investigation of fibrillin microfibrils in the canine cruciate ligament in dogs with different predispositions to ligament rupture.

Cranial cruciate ligament disease (CCLD) is the most common cause of pelvic limb lameness in dogs but its precise aetiopathogenesis is uncertain. Fibrillin microfibrils (FM) are complex macro-molecular assemblies found in many tissues including ligaments, where they are thought to play an important mechanical role. We hypothesised that FM ultrastructural variation correlates with the differing predisposition of canine breeds to CCLD. Non-diseased cranial and caudal cruciate ligaments (CCLs and CaCLs) were obtained from Greyhound (GH) and Staffordshire Bull Terrier (SBT) cadavers. Fibrillin microfibrils were extracted from the ligaments by bacterial collagenase digestion, purified by size-exclusion chromatography and subsequently visualized by atomic force microscopy (AFM). With AFM, FMs have a characteristic beads-on-a-string appearance. For each FM, periodicity (bead-bead distance) and length (number of beads/FM) was measured. Fibrillin microfibril length was found to be similar for GH and SBT, with non-significant inter-breed and inter-ligament differences. Fibrillin microfibril periodicity varied when comparing GH and SBT for CCL (GH 60.2 ± 1.4 nm; SBT 56.2 ± 0.8 nm) and CaCL (GH 55.5 ± 1.6 nm; SBT 61.2 ± 1.2 nm). A significant difference was found in the periodicity distribution when comparing CCL for both breeds (P < 0.00001), further, intra-breed differences in CCL vs CaCL were statistically significant within both breeds (P < 0.00001). The breed at low risk of CCLD exhibited a periodicity profile which may be suggestive of a repair and remodelling within the CCL.

Canine total hip replacement using a cementless threaded cup and stem: a review of 55 cases.

OBJECTIVES: To determine the long-term results and complications associated with the Helica® cementless hip endoprosthesis system. MATERIALS AND METHODS: Retrospective study of 55 consecutive Helica total hip replacements performed between January 2010 and February 2015. RESULTS: A total of 55 total hip replacements were performed in 50 dogs; 45 dogs had unilateral hip replacements, and five had staged bilateral replacements. A total of 23 first-generation short femoral stems were implanted in 22 dogs, of which nine cases (39%) experienced complications and 19 (86%) recovered satisfactorily when revisions were included; 31 second-generation femoral stems were implanted in 28 dogs, of which 10 cases (32%) experienced complications, and 26 (93%) made satisfactory recoveries, including revisions. In 36 dogs for which data were available, postoperative Liverpool Osteoarthritis in Dogs scores were significantly reduced compared to preoperative scores. Complications occurred in a total of 19 cases (34·5%; 95% confidence interval: 22 to 47·1%); the most common was aseptic loosening of the femoral stem [11 cases (20%; 95% confidence interval: 9·4 to 30·6%)]. Following multivariable analysis, no risk factors were identified for overall complications, but there was a significant association of implant type (first-generation short stem) with loosening (odds ratio 4·9, 95% confidence interval: 1·1 to 22·1, P=0·034). CLINICAL SIGNIFICANCE: This study found the Helica hip endoprosthesis system to be effective in the management of hip dysplasia and osteoarthritis in dogs. Aseptic loosening of the femoral stem remains the most common complication but appears to have been significantly reduced with the introduction of the second-generation stem.

Variations during ageing in the three-dimensional anatomical arrangement of fascicles within the equine superficial digital flexor tendon.

BiTendons are constructed from collagenous fascicles separated by endotenon/interfascicular matrix (IFM). Tendons may be specialised for precision movement or to store energy during locomotion and for the latter the elasticity of the endotenon/IFM is particularly important. The equine superficial digital flexor tendon (SDFT) is a dedicated energy-storing tendon with a similar function to the human Achilles tendon. Classical anatomical descriptions portray fascicles as longitudinally arranged distinct anatomical structures. In the present study, using three-dimensional reconstruction from whole tissue slices and histological sections, the fascicles of the equine SDFT were found to adopt a complex interweaved arrangement. Fascicles were found to fully and partially converge and diverge within the tendon and fascicle bundles were observed. Fascicle morphology was not homogenous with narrowing, broadening and twisted fascicles observed in addition to relatively straight fascicles. The number of fascicle bundles observed in cross-section increased from the proximal to the distal end of the tendon, whilst the number of fascicles decreased with age in the proximal region. Fascicular patterns were not similar between the left and right limbs, across different regions or at different ages. A decrease in thickness of the endotenon/IFM between fascicles with age was found in the distal tendon region. The results provide a rationale for considering fascicular organisation when diagnosing and treating tendon injuries, for bioengineering tendon and when modelling tendon function.

Mechanical performance in axial compression of a titanium polyaxial locking plate system in a fracture gap model.

OBJECTIVE: To evaluate the bending strength of the VetLOX® polyaxial locking plate system. MATERIALS AND METHODS: Thirty-five 3.5 mm 12-hole titanium VetLOX® plates were used to stabilize seven different construct designs in a 1 cm fracture gap simulation model. Each construct was subjected to axial compression. Mean bending stiffness (BS) and yield load (YL) of each construct design were analysed using a one-way ANOVA and Tukey post-hoc analysis. Screw angulation was measured on reconstructed computed tomography (CT) images. RESULTS: Reducing plate working length for fixed-angle constructs significantly increased BS (p <0.01) and YL (p <0.01). For a constant plate working length, increasing screw number did not significantly affect BS (p = 1.0) or YL (p = 0.86). Screw angulation measurement technique was validated by intra-class correlation coefficients (ICC) (ICC >0.9 for inter- and intra-observer measurements). An average screw angle of 13.2° did not significantly affect mechanical performance although incomplete screw head-plate engagement was noted on some reconstructed CT images when angulation exceeded 10°. Prefabricated screw-head inserts did not significantly increase mechanical performance. A 4 mm bone-plate stand-off distance significantly reduced BS and YL by 63% and 69% respectively. CLINICAL RELEVANCE: The VetLOX® system allows the benefits of polyaxial screw insertion whilst maintaining comparable bending properties to fixed angle insertion. The authors recommend accurate plate contouring to reduce the risk of plate bending.

Elastin content is high in the canine cruciate ligament and is associated with degeneration.

Cruciate ligaments (CLs) are primary stabilisers of the knee joint and canine cranial cruciate ligament disease (CCLD) and rupture is a common injury. Elastin fibres, composed of an elastin core and fibrillin containing microfibrils, are traditionally considered minor components of the ligament extracellular matrix (ECM). However, their content and distribution in CLs is unknown. The purposes of this study were to determine the elastin content of canine CLs and to ascertain its relationship to other biochemical components and histological architecture. Macroscopically normal CLs were harvested from Greyhounds (n=11), a breed with a low risk of CCLD. Elastin, collagen and sulfated glycosaminoglycan content were measured and histological scoring systems were developed to quantify ECM changes using a modified Vasseur score (mVS) and oxytalan fibre (bundles of microfibrils) staining. Elastin contents were 9.86 ± 3.97% dry weight in the cranial CL and 10.79 ± 4.37% in the caudal CL, respectively, and did not alter with advancing histological degeneration. All CLs demonstrated mild degenerative changes, with an average mVS score of 11.9 ± 3.3 (maximum 24). Increasing degeneration of the ligament ECM showed a positive correlation (r=0.690, P<0.001) with increased oxytalan fibre staining within the ECM. Elastin is an abundant protein in CLs forming a greater proportion of the ligament ECM than previously reported. The appearance of oxytalan fibres in degenerative CL ECM may reflect an adaptive or reparative response to normal or increased loads. This finding is important for future therapeutic or ligament replacement strategies associated with cranial CL injury.

Intra-articular morphine, bupivacaine or no treatment for postoperative analgesia following unilateral elbow joint arthroscopy.

OBJECTIVES: To compare intra-articular morphine or bupivacaine against no treatment following unilateral elbow joint arthroscopy using force plate analysis and pain scoring. METHODS: Thirty-one dogs were randomly allocated to receive 0·1 mg/kg intra-articular morphine, 0·5 mg/kg bupivacaine or no treatment following elbow arthroscopy. Force plate analysis, pain scoring and kinematic evaluation were performed before anaesthesia, 4 and 24 hours after surgery. Peak vertical force index, symmetry index, rate of loading, rate of unloading, stance time and range of motion were obtained from gait analysis. Pain scoring was performed every 4 hours and interventional analgesia (0·3 mg/kg methadone) was administered if necessary. RESULTS: Of 29 dogs analysed, peak vertical force index (P<0·001), symmetry index (P=0·01) and rate of unloading (P=0·01) decreased significantly over time in each group; however, this was not affected by treatment. No significant differences were observed in stance time or rate of loading over time. Kinematic (range of motion) evaluation was not complete for all dogs. Pain scores increased significantly at both time points postoperatively in the no treatment group (P=0·007) and in morphine-treated dogs at 4 hours compared to baseline (P=0·03). For intra-articular bupivacaine significant increases in pain scores were not detected (P=0·28). CLINICAL SIGNIFICANCE: No benefit to intra-articular bupivacaine or morphine was detected using peak vertical force index from force plate analysis. Bupivacaine prevented increases in pain scores at both time points as did morphine at the 24-hour evaluation, compared to no treatment.

Variations in cell morphology in the canine cruciate ligament complex.

Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology.

Bilateral angular carpal deformity in a dog with craniomandibular osteopathy.

A four-month-old West Highland White Terrier was presented to the Small Animal Teaching Hospital at the University of Liverpool with the complaint of a bilateral angular carpal deformity. A 20° valgus deformity was present in both thoracic limbs, centred on the distal radial physes. Both distal ulnas were grossly thickened and there was concomitant thickening of the rostral mandible and calvarium. The dog exhibited signs of resentment on palpation of the mandible and signs of pain were elicited on flexion and extension of both elbow joints. No signs of pain were evident on palpation of the ulnas or calvarium. Radiographic images of both ulnas showed marked amorphous periosteal new bone formation. The distal ulnar physes were closed centrally and both elbow joints had humeroulnar subluxation. Radiographic changes to the calvarium and mandibular rami were consistent with a diagnosis of craniomandibular osteopathy. A bilateral ulna ostectomy was performed to correct the angular limb deformity and elbow subluxations. Histology of the ostectomised pieces showed changes consistent with craniomandibular osteopathy.

Computed tomographic findings and treatment of an unusual fracture of the proximal ulna in a mature dog.

This report describes the diagnosis and treatment of an unusual fracture of the proximal ulna in a four-year-old male neutered Labrador Retriever dog, the orientation of which has not been reported in the veterinary literature. Computed tomography allowed fracture visualisation and aided surgical planning. Fixation was achieved using a lag screw and an anti-rotational Kirschner wire. Six month follow-up indicated satisfactory outcome with complete return to normal function.

Update on the aetiopathogenesis of canine cranial cruciate ligament disease.

Cranial cruciate ligament disease (CCLD) is the most common cause of hindlimb lameness in the dog, being associated with and eventually leading to stifle osteoarthritis. Canine cranial cruciate ligament disease is a gradual degeneration of the ligament extracellular matrix (ECM) leading to ligament rupture. The aetiopathogenesis of this condition is still poorly understood but several risk factors have been identified such as breed, bodyweight, gender and conformation. Recent developments in this area include the role of genetics, stifle joint conformation, ligament ECM metabolism, and inflammation associated with immune-mediated disease within the stifle joint. A genetic mode of inheritance has been demonstrated in the Newfoundland which is predisposed to CCLD. Increased cellular metabolism within the cranial cruciate ligament has been directly associated with increased craniocaudal stifle joint laxity in dog breeds at high risk of CCLD. Conformation abnormalities, such as a narrowed distal femoral intercondylar notch, in high risk breeds have been shown to be associated with alterations in cranial cruciate ligament ultrastructure. Increased production of inflammatory cytokines, such as cathepsins and interleukins, by the stifle synovial cells may occur secondary to or may be an inciting cause of ligament degeneration. Future research endeavours will focus on the association between immune-mediated response and fibrocartilaginous metaplasia and matrix degradation within the cranial cruciate ligament, and whether this can be altered in all susceptible dogs or only certain breeds.

Effect of surgical technique on radiographic fusion of the anconeus in the treatment of ununited anconeal process.

OBJECTIVES: The aim of this study was to determine if internal fixation of the anconeus combined with a proximal ulnar osteotomy was more likely to result in fusion of the anconeus to the ulna compared with a proximal ulnar osteotomy alone. METHODS: A total of 12 orthopaedic referral clinics reviewed their clinical databases for cases of ununited anconeal process. Demographic and clinical parameters were collected along with radiographic follow-up at a minimum of four weeks. Cases treated with proximal ulnar osteotomy alone were compared with those treated with proximal ulnar osteotomy + internal fixation. Both groups were compared for background and disease variables. We tested for an association between treatment method and whether radiographic anconeal union had occurred. RESULTS: A total of 47 elbows (44 dogs) were identified. Of these, 28 cases (average age 7.6 months) were treated with proximal ulnar osteotomy (of which eight were stabilised with an intramedullary pin) alone. Nineteen cases (average age 7.1 months) were treated with proximal ulnar osteotomy + internal fixation. The two groups were not significantly different in age (P=0.638, Mann-Whitney U test). Fourteen of 28 cases with proximal ulnar osteotomy alone displayed anconeal union at follow-up compared with 16 of 19 cases of proximal ulnar osteotomy + internal fixation, and this difference was statistically significant (P=0.029, Fisher's exact test). CLINICAL SIGNIFICANCE: These data suggest that use of a lag screw to stabilise and compress the ununited anconeal process in addition to proximal ulnar osteotomy produces a better radiographic outcome. It is argued that radiographic union of the anconeus is likely to be associated with better long-term clinical outcome but further studies are required to confirm this.

Digital analysis of ulnar trochlear notch sclerosis in Labrador retrievers.

OBJECTIVES: To compare ulnar trochlear notch bone radiopacity in Labrador retrievers with and without fragmented medial coronoid process using quantitative analysis of film density on digitised radiographs. METHODS: Mediolateral view elbow radiographs from Labrador retrievers (n=34) aged between six and 18 months were obtained and digitised. Images from dogs with an arthroscopic diagnosis of fragmentation of the medial coronoid process (n=17) were compared with that of a control population (n=17), and this data subject to statistical analysis. RESULTS: A statistically significant relationship between the presence of increased trochlear notch radiopacity and a fragmented medial coronoid process was identified. Fractional analysis of this area shows the region of greatest difference in radiopacity between normal and fragmented medial coronoid process cohorts to be in the trochlear region of the medial coronoid process of the ulna. A decrease in radiopacity values in the dysplastic group versus the normal cohort was observed for the region of the proximo-caudal ulnar trochlear notch. CLINICAL SIGNIFICANCE: An increase in ulnar trochlear notch radiopacity is a finding associated with fragmentation of the medial coronoid process in Labrador retrievers.

Carpal derangement and associated carpal valgus in a dog.

This case report describes derangement of the numbered carpal bones resulting in a valgal growth deformity in the right carpus of a seven-month-old dog. Radiographic assessment of the right carpus revealed abnormalities in the size and shape of the numbered carpal bones and carpal valgus. Surgical correction of the growth deformity was planned by partial carpal arthrodesis; however medial collateral laxity associated with the carpal valgus necessitated a pancarpal arthrodesis to achieve correct limb alignment.

Inherited myopathy of great Danes.

A hereditary, non-inflammatory myopathy occurring in young great Danes with distinctive histological features in muscle biopsy specimens is reviewed. Onset of clinical signs is usually before one year of age and both sexes are affected. Clinical signs are characterised by exercise intolerance, muscle wasting, and an exercise-induced tremor. Although most affected dogs have a severe form of the disease, occasional dogs may have a less pronounced form and survive into adulthood with an acceptable quality of life. Litters containing affected puppies are born to clinically unaffected parents, and an autosomal recessive pattern of inheritance is likely. All recorded cases have had fawn or brindle coat coloration. Elevated serum creatinine kinase concentrations and spontaneous electrical activity in skeletal muscles are frequently found. While originally reported (Targett and others 1994) as a central core myopathy in this breed, the histochemical characteristics of the distinct cytoarchitectural structures differ from those of the well-characterised central core myopathy in human beings. In fact, these structures differ from any known myopathy in human beings and likely represents a unique non-inflammatory myopathy affecting dogs. Until this myopathy is characterised further, the name inherited myopathy in great Danes is suggested.

Ultrastructural differences in cranial cruciate ligaments from dogs of two breeds with a differing predisposition to ligament degeneration and rupture.

Cranial (anterior) cruciate ligament (CCL) samples were obtained from dogs of the Labrador retriever (LR) and greyhound (GH) breeds, of which the former but not the latter is predisposed to CCL rupture. Electron microscopy revealed that the collagen fibril diameters of GHs were larger than those of LRs (P=0.03). Histological examination revealed a "fibrocartilaginous" appearance of CCLs in seven of eight GHs, and, to a lesser extent, in three of eight LRs. The formation of fibrocartilage is clearly not a disadvantage to the healthy racing GH, and cannot be regarded as a pathological degeneration in this breed. It is suggested that fibrocartilage is formed as a beneficial physiological adaptation to the compression of CCLs caused by tensile stress as a result of the tightening of two twisted bands. Fibrocartilage would appear to protect CCLs in the GH, but it may be indicative of a mild degenerative change, which may eventually lead to rupture in the LR.

Distal femoral intercondylar notch dimensions and their relationship to composition and metabolism of the canine anterior cruciate ligament.

OBJECTIVE: To determine the relationship between the dimensions of the distal femoral intercondylar notch (ICN) and the composition and metabolism of the anterior cruciate ligament (ACL) in three dog breeds with different relevant risks to ligament rupture and subsequent osteoarthritis (OA). DESIGN: ICN measurements were obtained from the femurs of Golden Retrievers (high risk), Labrador Retrievers (high risk) and Greyhounds (low risk). Femoral condyle width and height, ICN height and width indices, and notch shape index were measured using Vernier callipers in all dogs. Intact ACLs were obtained from the same dog breeds for a study of the impinged areas and were analysed for collagen content, collagen cross-links, and sulphated glycosaminoglycan (GAG) content, matrix metalloproteinase (MMP)-2 and the tissue inhibitors of metalloproteinases (TIMPs)-1 and -2. RESULTS: Femoral condyle width and height and ICN width indices were significantly greater in the low risk compared to the high risk breeds (P<0.01 for all parameters). In contrast, the pro (P=0.003) and active (P=0.007) forms of MMP-2 and sulphated GAGs (P=0.0002) were significantly greater in the impinged areas of the ACLs of the rupture predisposed breeds. CONCLUSIONS: Impingement by the ICN on the ACLs of the high risk breeds may result in increased collagen remodelling and increased sulphated GAG deposition, causing reduced structural integrity of the ligament. Altered ACL composition may predispose the ligament to increased laxity leading to joint degeneration and OA. This may have a comparative implication for pathogenesis of ACL rupture in humans.