Measurement of humeroradial and humeroulnar transarticular joint forces in the canine elbow joint after humeral wedge and humeral slide osteotomies

Objective— To determine the effect of humeral wedge and humeral slide osteotomies on force distribution between the articular surfaces of the humerus and the radius and ulna in normal canine thoracic limbs.
Study Design— In vitro mechanical testing.
Sample population— Cadaveric canine right thoracic limbs (n=12).
Methods— Transarticular elbow force maps were measured using a tactile array pressure sensor in elbow joints of axially aligned limbs under 200 N axial load before and after humeral wedge and humeral slide osteotomies.
Results— Loading induced 2 distinct areas of high forces that corresponded with the proximal articular surfaces of the radius and ulna. Mean force on the proximal articular surface of the ulna was reduced by 25% and 28% after 4 and 8 mm sliding osteotomies, respectively. Statistically significant differences were not observed for the wedge osteotomies.
Conclusion— Humeral slide osteotomy significantly decreases force on the proximal articular surface of the ulna.
Clinical Relevance— The proximal articular surface of the ulna contributes significantly to load transfer through the canine elbow joint. Abnormalities that significantly increase this force might contribute to canine elbow dysplasia, specifically fragmentation of the medial coronoid process and osteochondritis dissecans of the medial aspect of the humeral condyle. Under the conditions studied, the overall reduction in mean joint surface force across the proximal articular surface of the ulna after humeral slide osteotomy indicates that this technique merits further investigation for potential use in medial compartmental osteoarthritis of the canine elbow joint.     

The jump behavior of the humeroradial and tarsocrural joints of the horse

Osteoligamentous preparations of the elbow and hock joints of the horse show “jump” behavior when flexed and extended. This behavior is not seen in undissected anatomical specimens or in the live horse. The hypothesis is that energy is stored in the collateral ligaments during the middle range of joint displacement and quickly released nearing full flexion or extension. This energy storage mechanism has the attribute of negative work. In the undissected specimen and the live horse energy is stored in muscles and tendons as well as in the collateral ligaments, and the obvious jump phenomenon is not observed although the principle of negative work still pertains.     

In vitro susceptibility of canine and feline Escherichia coli to fosfomycin

Therapeutic options for multi-drug resistant (MDR) Escherichia coli in dogs or cats are limited. The objective of this study was to establish in vitro susceptibility of canine and feline E. coli to fosfomycin. Two sources of isolates were categorized based on susceptibility as to no resistance (NDR), single drug resistance (SDR), multidrug resistance (MDR) or extreme drug resistance (XDR). Clinical isolates were collected from throughout the US from dogs (n=157) or cats (n=43) with naturally occurring infection between March 2008 and January 2010. Experimental isolates were collected from fecal samples of dogs treated with no drug (NDR), amoxicillin (expressing SDR) or enrofloxacin (expressing MDR or XDR). Fosfomycin minimum inhibitory concentrations (MIC) were determined using E-Test(?). For clinical isolates, most (165/200) originated from the urinary tract, with the number of isolates per resistant category being: NDR (N=44, 22%), SDR (N=65, 32.5%), MDR (N=74, 37%), and XDR (N=17, 8.5%). Of these isolates, 99% (197/200) were susceptible to fosfomycin with the MIC(90) and MIC(50) being 2 and 1 μg/ml, respectively (range: 0.25-196 μg/ml). The number of experimental isolates in each category was NDR (3), SDR (23), MDR (38), and XDR (11) (29.3, 44, and 14.7%, respectively). Of these, 100% were susceptible to fosfomycin with MIC(90) and MIC(50) being 1.5 and 1 μg/ml (range: 0.38-4 μg/ml), respectively. The susceptibility of canine and feline MDR and XDR E. coli to fosfomycin at concentrations well below the susceptible breakpoint supports further investigation for its use when treating E. coli resistant to alternative antimicrobials.     

In vitro function of canine neutrophils during experimental inflammatory disease

The effect of acute inflammation on neutrophil function in the dog was studied by measuring in vitro phagocytosis and killing of Staphylococcus aureus. Phagocytosis was not impaired after 30 or 60 minutes and bactericidal activity was not impaired after 60 minutes incubation. However, average bactericidal activity after 30 minutes incubation was diminished significantly (P less than 0.01). Wide variations in bactericidal activity after 30 minutes incubation during the course of the inflammation did not correlate with neutrophil count, number of toxic neutrophils, or clinical course of the inflammation. These results indicate that a defect in bactericidal activity can occur in dogs with severe inflammatory disease, and that repeated assays, rather than single determinations, may be needed to detect this dysfunction.     

In vitro properties of diffuse cytoplasmic esterase-positive canine mononuclear leukocytes

Depletion of cytoplasmic esterase-positive canine peripheral blood monocytes from mononuclear cell suspensions was attempted using plastic adherence, carbonyl iron ingestion and/or Sephadex G-10 filtration. An esterase-positive, nonadherent, nonphagocytic subpopulation was identified and further characterized by the presence or absence of cell membrane receptors for the Fc portions of immunoglobulin and the activated third component of complement. The majority of these nonadherent cells lacked these receptors. The data suggests that canine peripheral blood monocytes are a heterogenous cell population.     

In vitro retinoid-induced growth inhibition and morphologic differentiation of canine osteosarcoma cells

OBJECTIVE: To determine differentiation and growth inhibition effects of retinoids on canine osteosarcoma cells. SAMPLE POPULATION: 3 osteosarcoma cell lines established from osteosarcomas in dogs. PROCEDURE: Osteosarcoma cells were incubated with various concentrations of all-trans-retinoic acid and 9-cis-retinoic acid or control medium, counted daily for 10 days, and evaluated for morphologic changes. Synthesis of DNA was measured by use of a cell proliferation ELISA. To analyze effect of retinoids on colony formation on plastic dishes, cells were cultured for 14 days, fixed, and stained; number of colonies was counted. RESULTS: In a dose-dependent manner, both retinoids induced morphologic differentiation and growth inhibition in the 3 osteosarcoma cell lines and inhibited each cell's ability to form anchorage-dependent colonies. CONCLUSION AND CLINICAL RELEVANCE: Retinoids induced differentiation of osteosarcoma cells of dogs, resulting in altered expression of their malignant phenotype. Induction of differentiation by retinoids may have potential as an adjunctive treatment for osteosarcoma in dogs.     

In vitro and in vivo bioactivity of recombinant canine hepatocyte growth factor

Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.     

In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional culture

OBJECTIVE: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture. SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities. PROCEDURE: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined. RESULTS: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected. CONCLUSIONS: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.     

In vitro biomechanical study of rotational stabilizers of the canine elbow joint

OBJECTIVE: To develop a model for measuring rotary stability of the canine elbow joint and to evaluate the relative contribution of the anconeal process (AN), lateral collateral ligament (LCL), and medial collateral ligament (MCL). SAMPLE POPULATION: 18 forelimbs from 12 canine cadavers. PROCEDURE: Forelimbs were allocated to 3 experimental groups (6 forelimbs/group). Each intact forelimb was placed in extension at an angle of 135 degrees and cycled 50 times from -16 degrees (pronation) to +28 degrees (supination) in a continuous manner at 2.0 Hz. Cycling was repeated following sectioning of the structure of interest (group 1, AN; group 2, LCL; and group 3, MCL). Torque at -12 degrees (pronation) and +18 degrees (supination) was measured for each intact and experimentally sectioned limb. A Student t test was performed to compare torque values obtained from intact verses experimentally sectioned limbs and for comparison with established criteria for differentiation of primary (> or = 33%), secondary (10 to 33%), and tertiary rotational stabilizers (< 10%). RESULTS: In pronation, the AN was the only primary stabilizer (65%). For supination, the LCL was a primary stabilizer (48%), AN was a secondary stabilizer (24%), and MCL was a tertiary stabilizer (7%). CONCLUSIONS AND CLINICAL RELEVANCE: With the elbow joint in extension at an angle of 135 degrees, the AN is a primary rotational stabilizer in pronation, and the LCL is a primary stabilizer in supination. Disruption of the AN or LCL may affect rotary range of motion or compromise stability of the elbow joint in dogs.     

In vitro characteristics of normal and dystrophic skeletal muscle from dogs

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (post-fusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.     

In vitro antimicrobial resistance of staphylococci isolated from canine urinary tract infection

This study determined the diversity of species and antimicrobial resistance of staphylococci isolated from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 348 dogs with clinical signs of UTI, according to clinical examination and urinalysis, were processed for isolation of Staphylococcus. Colonies in pure culture were identified by biochemical reactions and tested for susceptibility to 15 antimicrobials. Seventy isolates of staphylococci were obtained (20.1%). Staphylococcus pseudintermedius was the most frequent species (32.8%), followed by S. epidermidis (18.6%), S. simulans (15.7%), S. schleiferi schleiferi (11.4%), S. aureus (11.4%), S. schleiferi coagulans (7.2%) and S. saprophyticus (2.9%). All the isolates were resistant to at least 1 drug and 77.1% were multiresistant. The study reports the alarming antimicrobial resistance of members of the Staphylococcus genus isolated from canine UTI and highlights the importance of coagulase-negative staphylococci in its etiology.     

In vitro generation of mature neutrophils from canine Lin- bone marrow cells

The major goal of this work was to describe the in vitro generation of mature functional neutrophils derived from a canine enriched haematopoietic progenitor cell population. We have utilised lineage depletion by immunomagnetic selection to isolate a canine haematopoietic progenitor cell population. The physical, immunological, metabolical and morphological methodologies employed in this study have permitted us to isolate and define a cell population enriched in Rh-123low and CD34+ cells. Irradiated pre-established long-term bone marrow cultures (LTBMC) were utilised to determine the self-renewal ability of lineage negative (Lin-) cells, as well as their capacity to differentiate into mature functional neutrophils. The authors demonstrate for the first time that canine neutrophils derived from Lin- cells are able to produce oxyradicals, express a specific neutrophil surface antigen, and contain gelatinase granules. These characteristics enable them to migrate through basement membranes to act as a first line defence mechanism. The fact that these cells are able to differentiate into functional mature cells, and give rise to long-term culture-initiating cells (LTC-IC) after 35 days of culture, allows the authors to assure that the isolated canine enriched haematopoietic cell population exhibit functional characteristics, associated with primitive haematopoietic cells.     

In vitro differentiation of canine celiac adipose tissue-derived stromal cells into neuronal cells

To investigate in vitro differentiation of canine adipose tissue-derived stromal cells (ATSCs) into neuronal cells, ATSCs from celiac adipose tissue in clinically healthy beagle dogs were treated with 100 muM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 muM isobuthylmethylxanthine (IBMX). ATSCs were morphologically changed into differentiated ATSCs from spindle-shaped cells to neuron-like cells with numerous processes after the treatment. Expression of neuron-specific enolase (NSE) as an early neuron specific marker protein was detected in both ATSCs and differentiated ATSCs, however diachronic increase of NSE expression was observed in differentiated ATSCs after the treatment with dbcAMP/IBMX. In addition, neurofilament-68 (NF-68) as an early to mature neuron specific marker protein was weakly expressed in differentiated ATSCs. Neuron specific glutamate and glucose transporter (EAAC1 and GLUT-3, respectively) mRNAs were strongly expressed in differentiated ATSCs compared with those in ATSCs, although glia specific glutamate transporter mRNA (GLT-1) was also detected in differentiated ATSCs. ATSCs can differentiate into early to mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, although additional research is needed to examine functional characteristics of differentiated ATSCs.     

In vitro effects of meloxicam with or without doxorubicin on canine osteosarcoma cells

Cyclooxygenase (COX) inhibitors, already widely used to reduce fever, inflammation and pain, are under increasing consideration as potential agents for the prevention and treatment of neoplasia. As COX-2 was detected in human and canine osteosarcomas, we have evaluated the effect of the preferential COX-2 inhibitor meloxicam on an established D-17 canine osteosarcoma cell line, which expressed, as well as COX-1 and COX-2 also COX-3 (as demonstrated by Western blot). An XTT proliferation kit was used to assess surviving cells after drug treatment. At low concentrations (1, 2, 4 and 10 microm) meloxicam caused an increase in cell numbers while a marked anti-proliferative effect was observed at higher concentrations (100, 200 microm) after 3 days and also 3 weeks of incubation. The chemotherapeutic drug doxorubicin showed a cytotoxic effect at all concentrations (60-1920 nm). Exposure of tumour cells to combinations of meloxicam and doxorubicin revealed synergistic effects (with 240 nm doxorubicin), as well as sub-additive and antagonistic results, especially if combined with concentrations of meloxicam typically found in serum. Care should be taken in concluding, on the basis of one in vitro study, that meloxicam does not have a role in the treatment of canine osteosarcomas given that the results from in vivo studies may differ.     

In vitro evaluation of screws and suture anchors in metaphyseal bone of the canine tibia

OBJECTIVE: To compare ease of insertion, load to failure, and mode of failure of cortical and cancellous screws, BoneBiter, IMEX, and TwinFix suture anchors in canine metaphyseal tibial bone. STUDY DESIGN: Experimental biomechanical study. ANIMALS: Canine cadaveric tibias. METHODS: One investigator inserted all anchors and subjectively evaluated ease of placement. Anchor systems were loaded to failure along axis of insertion with audio-video recording to determine failure mode. RESULTS: BoneBiter was the most difficult anchor to insert successfully. Mean+/-SD loads to failure were cancellous screw (711+/-193 N), IMEX 4.7 mm 18 g wire (661+/-163 N), IMEX 4.0 mm 18 g wire (661+/-165 N), cortical screw (635+/-184 N), BoneBiter #5 Kevlar suture (393+/- 109 N), and TwinFix 5.0 mm #2 polyester (267+/-73 N). No significant differences were noted among the cortical screw, cancellous screw, IMEX 4.7 and 4.0 mm, all of which were significantly (P<.001) greater than BoneBiter and TwinFix . Failure modes were pullout of bone, suture-wire breakage, eyelet breakage, or no failure to 1000 N: screws (18,0,0,2), IMEX (18,1,1,0), BoneBiter (2,8,0,0), and TwinFix (0,10,0,0). CONCLUSIONS: Fixation devices were user friendly, with the exception of BoneBiter. Mode of failure is dependent on suture material and anchor design. Cortical and cancellous screws, and IMEX anchors with 18 g wire have significantly greater load to failure compared with BoneBiter and TwinFix suture anchors. CLINICAL RELEVANCE: Based on load to failure, ease of use, design characteristics, and cost, IMEX anchors may have advantages over other comparable soft tissue fixation devices.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

In vitro evaluation of canine and feline calcium oxalate urolith fragility via shock wave lithotripsy

OBJECTIVE: To test the hypothesis that feline calcium oxalate uroliths are intrinsically more resistant to comminution via shock wave lithotripsy (SWL) than canine calcium oxalate uroliths through comparison of the fragility of canine and feline uroliths in a quantitative in vitro test system. SAMPLE POPULATION: Calcium oxalate uroliths (previously obtained from dogs and cats) were matched by size and mineral composition to create 7 pairs of uroliths (1 canine and 1 feline urolith/pair). PROCEDURE: Uroliths were treated in vitro with 100 shock waves (20 kV; 1 Hz) by use of an electrohydraulic lithotripter. Urolith fragmentation was quantitatively assessed via determination of the percentage increase in projected area (calculated from the digital image area of each urolith before and after SWL). RESULTS: After SWL, canine uroliths (n = 7) fragmented to produce a mean +/- SD increase in image area of 238 +/- 104%, whereas feline uroliths (7) underwent significantly less fragmentation (mean image area increase of 78 +/- 97%). The post-SWL increase in fragment image area in 4 of 7 feline uroliths was < 50%, whereas it was > 150% in 6 of 7 canine uroliths. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that feline calcium oxalate uroliths are less susceptible to fragmentation via SWL than canine calcium oxalate uroliths. In some cats, SWL may not be efficacious for fragmentation of calcium oxalate nephroliths or ureteroliths because the high numbers of shock waves required to adequately fragment the uroliths may cause renal injury.     

In vitro development of canine somatic cell nuclear transfer embryos in different culture media

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.