RESUMEN

BACKGROUND: There is a need to improve therapies for osteoarthritis in horses. OBJECTIVES: To assess the efficacy of equine allogeneic chondrogenic-induced mesenchymal stem cells combined with equine allogeneic plasma as a novel therapy for osteoarthritis in horses. STUDY DESIGN: Randomised, double-blinded, placebo-controlled experiment. METHODS: In 12 healthy horses, osteoarthritis was induced in the metacarpophalangeal joint using an osteochondral fragment-groove model. Five weeks after surgery, horses were randomly assigned to either an intra-articular injection with chondrogenic-induced mesenchymal stem cells + equine allogeneic plasma (= intervention) or with 0.9% saline solution (= control). From surgery until the study end, horses underwent a weekly joint and lameness assessment. Synovial fluid was collected for cytology and biomarker analysis before surgery and at Weeks 5, 5 + 1d, 7, 9 and 11. At Week 11, horses were subjected to euthanasia, and the metacarpophalangeal joints were evaluated macroscopically and histologically. RESULTS: No serious adverse events or suspected adverse drug reactions occurred during the study. A significant improvement in visual and objective lameness was seen with the intervention compared with the control. Synovial fluid displayed a significantly higher viscosity and a significantly lower glycosaminoglycan concentration in the intervention group. Other biomarkers or cytology parameters were not significantly different between the treatment groups. Significantly less wear lines and synovial hyperaemia were present in the intervention group. The amount of cartilage oligomeric matrix protein, collagen type II and glycosaminoglycans were significantly higher in the articular cartilage of the intervention group. MAIN LIMITATIONS: This study assessed the short-term effect of the intervention on a limited number of horses, using an osteoarthritis model. This study also included multiple statistical tests, increasing the risk of type 1 error. CONCLUSIONS: Equine allogeneic chondrogenic-induced mesenchymal stem cells combined with equine allogeneic plasma may be a promising treatment for osteoarthritis in horses. The Summary is available in Spanish - see Supporting Information.

Asunto(s)

Condrogénesis , Enfermedades de los Caballos/terapia , Trasplante de Células Madre Mesenquimatosas/veterinaria , Células Madre Mesenquimatosas/fisiología , Osteoartritis/veterinaria , Animales , Método Doble Ciego , Femenino , Caballos , Masculino , Osteoartritis/terapia , Prueba de Estudio Conceptual

RESUMEN

To the date, no reports exist of the pharmacokinetics (PK) of betamethasone (BTM) sodium phosphate and betamethasone acetate administered intra-articular (IA) into multiple joints in exercising horses. The purpose of the study was to determine the PK of BTM and HYD concentrations in plasma and urine after IA administration of a total of 30 mg BTM. Eight 4 years old Thoroughbred mares were exercised on a treadmill and BTM was administered IA. Plasma and urine BTM and HYD were determined via high performance liquid chromatography spectrometry for 6 weeks. Concentration-time profiles of BTM and HYD in plasma and urine were used to generate PK estimates for non-compartmental analyses and comparisons among times and HYD concentrations. BTM in plasma had greater Tmax (Tmax 0.8 h) vs. urine (Tmax 7.1 h). Urine BTM concentration (ng/mL) and amount (AUClast ; h × ng/mL) were greater than plasma. HYD was suppressed for at least 3 days (<1 ng/mL) for all horses. The time of last quantifiable concentration of BTM (Tlast ; hour) was not significantly different in plasma than urine. Use of highly sensitive HPLC-MS/MS assays enabled early detection and prolonged and consistent determination of BTM in plasma and urine.

Asunto(s)

Betametasona/análogos & derivados , Glucocorticoides/farmacocinética , Caballos/fisiología , Hidrocortisona/farmacocinética , Animales , Área Bajo la Curva , Betametasona/administración & dosificación , Betametasona/sangre , Betametasona/farmacocinética , Betametasona/orina , Femenino , Glucocorticoides/administración & dosificación , Semivida , Caballos/sangre , Hidrocortisona/sangre , Hidrocortisona/orina , Inyecciones Intraarticulares , Metatarso , Condicionamiento Físico Animal , Tarso Animal

RESUMEN

A computer-aided gait analysis system was used to contrast two guinea pig strains with differing propensity for osteoarthritis (OA), with/without administration of a nonsteroidal anti-inflammatory drug. Walking speed and static/dynamic gait parameters were determined at baseline. Flunixin meglumine was given and animals were evaluated 4, 24, and 72 hours after treatment. Body weight was compared using unpaired t-tests. Knee joints were histologically evaluated using species-specific criteria; indices were analyzed using one-way ANOVA, Kruskal-Wallis test, followed by Dunn's multiple comparisons. A generalized linear model followed by Tukey's posttests juxtaposed gait parameters; walking speed was a covariate for other outcome measures. Body weight was not different between strains; OA-prone animals demonstrated more progressive chondropathy. At baseline, OA-prone animals had slower walking speeds, narrower hind limb bases of support, shorter stride lengths, and slower limb swing speeds relative to OA-resistant animals. These differences were not detected 4 or 24 hours after treatment. By 72 hours, OA-prone animals had returned to baseline values. These findings indicate a distinct voluntary gait pattern in a rodent model of bilateral primary OA, modification of which may allow rapid screening of novel therapies. Flunixin meglumine temporarily permitted OA-prone animals to move in a manner that was analogous to OA-resistant animals.

RESUMEN

OBJECTIVES: To compare the clinical and inflammatory joint responses to intra-articular injection of bone marrow-derived mesenchymal stem cells (MSC) including autologous, genetically modified autologous, allogeneic, or xenogeneic cells in horses. METHODS: Six five-year-old Thoroughbred mares had one fetlock joint injected with Gey's balanced salt solution as the vehicle control. Each fetlock joint of each horse was subsequently injected with 15 million MSC from the described MSC groups, and were assessed for 28 days for clinical and inflammatory parameters representing synovitis, joint swelling, and pain. RESULTS: There were not any significant differences between autologous and genetically modified autologous MSC for synovial fluid total nucleated cell count, total protein, interleukin (IL)-6, IL-10, fetlock circumference, oedema score, pain-free range-of-motion, and soluble gene products that were detected for at least two days. Allogeneic and xenogeneic MSC produced a greater increase in peak of inflammation at 24 hours than either autologous MSC group. CLINICAL SIGNIFICANCE: Genetically engineered MSC can act as vehicles to deliver gene products to the joint; further investigation into the therapeutic potential of this cell therapy is warranted. Intra-articular MSC injection resulted in a moderate acute inflammatory joint response that was greater for allogeneic and xenogeneic MSC than autologous MSC. Clinical management of this response may minimize this effect.

Asunto(s)

Enfermedades de los Caballos/terapia , Inflamación/veterinaria , Trasplante de Células Madre Mesenquimatosas/veterinaria , Células Madre Mesenquimatosas/clasificación , Osteoartritis/veterinaria , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Femenino , Regulación de la Expresión Génica , Ingeniería Genética , Caballos , Inflamación/etiología , Inyecciones Intraarticulares , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Trasplante de Células Madre Mesenquimatosas/métodos , Osteoartritis/terapia , Líquido Sinovial/química , Líquido Sinovial/citología

RESUMEN

OBJECTIVE: Diminish interleukin-1ß (IL-1ß) signaling in a model of primary osteoarthritis by RNA interference-based transcript reduction or receptor blockade, and quantify changes incurred on transcript expression of additional mediators. METHODS: Knees of Hartley guinea pigs were collected at 120 and 180 days of age following injection with viral vectors (N = 4/treatment group/date) at 60 days. Two groups received either adeno-associated viral serotype 5 vector containing a knockdown sequence (TV), or adenoviral vector encoding for IL-1 receptor antagonist protein (Ad-IRAP); treatments were contrasted with opposite knees administered corresponding vector controls. A third group evaluated TV relative to saline-only injected knees. Chondropathy and immunohistochemistry findings were compared to untreated guinea pigs. Transcript expression levels in cartilage were calculated using the comparative CT (2(-ΔΔCT)) method and analyzed by one-way analysis of variance (ANOVA) with pairwise comparisons using Tukey 95% confidence intervals. RESULTS: Vector transduction was confirmed at both harvest dates. TV and Ad-IRAP, relative to vector controls, significantly decreased IL-1ß. Inflammatory mediators [tumor necrosis factor-α (TNF-α), IL-8, interferon-γ (IFN-γ)], and catabolic matrix metalloproteinase 13 (MMP13) were also decreased, while anabolic transforming growth factor-ß1 (TGF-ß1) was increased. IL-1ß was also decreased by TV vs saline, with a decrease in MMP13 and increase TGF-ß1; TNF-α, IL-8, and IFN-γ were transiently increased. CONCLUSIONS: This work confirmed that a reduction in IL-1ß signaling was accomplished by either method, resulting in decreased expression of three inflammatory mediators and one catabolic agent, and increased expression of an anabolic molecule. Thus, evidence is provided that IL-1ß serves a role in vivo in spontaneous osteoarthritis and that these translational tools may provide beneficial disease modification.

Asunto(s)

Artritis Experimental/metabolismo , Cartílago Articular/metabolismo , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Interleucina-1beta/antagonistas & inhibidores , Osteoartritis de la Rodilla/metabolismo , ARN Interferente Pequeño/genética , Animales , Artritis Experimental/patología , Cartílago Articular/patología , Condrocitos/metabolismo , Condrocitos/patología , Cobayas , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Masculino , Osteoartritis de la Rodilla/patología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

OBJECTIVE: To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1ß (IL-1ß) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. METHODS: Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1ß transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify in vitro transcript changes of IL-1ß and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2(-ΔΔCT)) method. RESULTS: Statistically significant decreases in IL-1ß expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-ß (TGF-ß) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P=0.0045) or >90% (P=0.0001) of the IL-1ß transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. CONCLUSIONS: Successful reduction of the IL-1ß transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1ß transcript in vivo are warranted.

Asunto(s)

Artritis Experimental/patología , Condrocitos/metabolismo , Interleucina-1beta/biosíntesis , Osteoartritis/patología , Interferencia de ARN , Animales , Artritis Experimental/metabolismo , Carbazoles/metabolismo , Células Cultivadas , Dependovirus/genética , Regulación de la Expresión Génica , Vectores Genéticos , Cobayas , Mediadores de Inflamación/metabolismo , Interleucina-1beta/genética , Masculino , Osteoartritis/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos

RESUMEN

OBJECTIVE: To evaluate healing of surgically created large osteochondral defects in a weight-bearing femoral condyle in response to delayed percutaneous direct injection of adenoviral (Ad) vectors containing coding regions for either human bone morphogenetic proteins 2 (BMP-2) or -6. METHODS: Four 13mm diameter and 7mm depth circular osteochondral defects were drilled, 1/femoral condyle (n=20 defects in five ponies). At 2 weeks, Ad-BMP-2, Ad-BMP-6, Ad-green fluorescent protein (GFP), or saline was percutaneously injected into the central drill hole of the defect. Quantitative magnetic resonance imaging (qMRI) and computed tomography (CT) were serially performed at 12, 24, and 52 weeks. At 12 (one pony) or 52 weeks, histomorphometry and microtomographic analyses were performed to assess subchondral bone and cartilage repair tissue quality. RESULTS: Direct delivery of Ad-BMP-6 demonstrated delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and histologic evidence of greater Glycosaminoglycan (GAG) content in repair tissue at 12 weeks, while Ad-BMP-2 had greater non-mineral cartilage at the surface at 52 weeks (p<0.04). Ad-BMP-2 demonstrated greater CT subchondral bone mineral density (BMD) by 12 weeks and both Ad-BMP-2 and -6 had greater subchondral BMD at 52 weeks (p<0.05). Despite earlier (Ad-BMP-6) and more persistent (Ad-BMP-2) chondral tissue and greater subchondral bone density (Ad-BMP-2 and -6), the tissue within the large weight-bearing defects at 52 weeks was suboptimal in all groups due to poor quality repair cartilage, central fibrocartilage retention, and central bone cavitation. Delivery of either BMP by this method had greater frequency of subchondral bone cystic formation (p<0.05). CONCLUSIONS: Delivery of Ad-BMP-2 or Ad-BMP-6 via direct injection supported cartilage and subchondral bone regeneration but was insufficient to provide long-term quality osteochondral repair.

Asunto(s)

Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 6/farmacología , Regeneración Ósea/fisiología , Cartílago Articular/efectos de los fármacos , Terapia Genética/métodos , Adenoviridae/genética , Animales , Densidad Ósea , Proteína Morfogenética Ósea 2/uso terapéutico , Proteína Morfogenética Ósea 6/uso terapéutico , Regeneración Ósea/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Modelos Animales de Enfermedad , Fémur/fisiología , Gadolinio DTPA , Vectores Genéticos/administración & dosificación , Glicosaminoglicanos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Miembro Posterior/fisiología , Caballos , Humanos , Imagen por Resonancia Magnética/métodos , Tomografía Computarizada por Rayos X , Soporte de Peso

RESUMEN

OBJECTIVE: To provide a comprehensive immunohistochemical (IHC) map of the temporal expression and tissue distribution of interleukin-1ß (IL-1ß) through progression of osteoarthritis (OA) in two strains of guinea pigs with varying propensity for spontaneous knee joint disease. METHODS: OA-prone Hartley and OA-resistant Strain 13 guinea pigs were collected at 60, 120, 180, 240, 360, and 480 days of age (N=4 animals per strain per date). IHC was performed on whole joint preparations; the distribution of IL-1ß expression on coronal sections was mapped, semi-quantitatively scored, and correlated to OA grade using Mankin criteria with guinea pig-specific modifications. OA and IHC indices were compared among times and between strains using the Kruskal-Wallis one-way analysis of variance by ranks followed by Dunn's post test. RESULTS: OA indices for both strains increased from 60 to 480 days of age; a statistically higher score (P ≤ 0.01) was found in Hartley animals at 180, 240, 360, and 480 days. At 60 days of age, IL-1ß expression was detected in cartilage, menisci, synovium, and subchondral bone in both strains. Persistent and statistically increased (P<0.05) IL-1ß expression was found in these same tissues in Hartley animals at 120 and 180 days, while Strain 13 animals demonstrated a significant reduction in positive immunostaining. Statistical differences in IHC indices between strains beyond 240 days of age were restricted to synovium (days 240 and 480) and subchondral bone (days 360 and 480). CONCLUSIONS: As expected, histologic OA proceeded in an accelerated manner in Hartley animals relative to Strain 13 animals. The OA-prone strain did not demonstrate reduced IL-1ß expression during adult maturity as occurred in the OA-resistant strain, and this persistent expression may have corresponded to early incidence of OA. Future interventional studies are warranted to explore whether dysregulation of IL-1ß expression may contribute to premature onset of spontaneous disease in the Hartley guinea pig.

Asunto(s)

Interleucina-1beta/metabolismo , Articulación de la Rodilla/metabolismo , Osteoartritis de la Rodilla/metabolismo , Animales , Cartílago Articular/metabolismo , Cobayas , Inmunohistoquímica , Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/patología , Membrana Sinovial/metabolismo

RESUMEN

Cell-mediated and direct adenoviral (Ad) vector gene therapies can induce bone regeneration, including dermal fibroblasts (DFbs). We compared two effective therapies, DFb-mediated and direct Ad vector delivery of bone morphogenetic protein-2 (BMP2), for relative efficacy in bone regeneration. Equine rib drill defects were treated by percutaneous injection of either DFb-BMP2 or an Ad-BMP2 vector. At week 6, both DFb-BMP2- and Ad-BMP2-treated rib defects had greater bone filling volume and mineral density, with DFb-BMP2 inducing greater bone volume and maturity in the cortical bone aspect of the defect than Ad-BMP2. The transplantation of DFb alone induced modest bone formation. Increased mineral density and bone turnover were evident in the cortical and cancellous bone directly adjacent to the healing drill defects treated with either DFb-BMP2 or Ad-BMP2. Using our cell/vector dosage and model, BMP2, whether delivered by the DFb vector or direct Ad vector, induced greater and robust bone regeneration. DFb-mediated BMP2 therapy promoted greater cortical bone regeneration than did direct gene delivery, possibly because of an increased cellularity of the bone healing site. BMP2 delivery, regardless of gene delivery method, increased the mineral density of the neighboring bone, which may be beneficial clinically in repairing or weak bone.

Asunto(s)

Proteína Morfogenética Ósea 2/genética , Regeneración Ósea/genética , Fibroblastos/trasplante , Técnicas de Transferencia de Gen , Osteogénesis/genética , Costillas/lesiones , Piel/citología , Adenoviridae , Animales , Terapia Genética/métodos , Vectores Genéticos , Caballos , Transducción Genética

RESUMEN

This study evaluated healing of rabbit bilateral ulnar osteotomies 6 and 8 weeks after surgery in response to percutaneous injection of transgenic adenoviral (Ad) bone morphogenetic protein-6 (BMP-6) vector or green fluorescent protein vector control (Ad-GFP) administered 7 days after surgery compared to untreated osteotomy controls. The amount, composition and biomechanical properties of the healing bone repair tissue were compared among groups and to historical data for intact rabbit ulnae obtained from similar studies at the same institution. Quantitative computed tomography was used to determine area, density and mineral content of the mineralized callus in the harvested ulnae. Maximum torque, torsional stiffness, and energy absorbed to failure were determined at 1.5 degrees /s. Calcified sections of excised ulnae (5 microm) were stained with Goldner's Trichrome and Von Kossa, and evaluated for callus composition, maturity, cortical continuity, and osteotomy bridging. Radiographic assessment of bone formation indicated greater mineralized callus in the ulnae injected with Ad-hBMP-6 as early as 1 week after treatment (2 weeks after surgery) compared to untreated osteotomy ulnae (p < 0.006) and Ad-GFP treated osteotomy ulnae (p < 0.002). Quantitative computed tomography confirmed greater bone area and bone mineral content at the osteotomy at 6 weeks in Ad-BMP-6 treated osteotomy as compared to untreated osteotomy ulnae (p < 0.001) and Ad-GFP treated osteotomy ulnae (p < 0.01). Ad-BMP-6 treated osteotomy ulnae were stronger (p < 0.001 and 0.003) and stiffer (p < 0.004 and 0.003) in torsion at 6 weeks than untreated osteotomy ulnae or Ad-GFP treated osteotomy ulnae, respectively. Maximum torque, torsional stiffness, and energy absorbed to failure were greater in Ad-BMP-6 treated osteotomy ulnae compared to their respective untreated contralateral osteotomy ulnae at 8 weeks [p < 0.03]. Maximum torque and torsional stiffness in the Ad-BMP-6 treated osteotomy ulnae were not different to intact ulnae values at 6 and 8 weeks. These experiments confirm that BMP-6 can be potently osteoinductive in vivo resulting in acceleration of bone repair.

Asunto(s)

Adenoviridae/genética , Proteínas Morfogenéticas Óseas/genética , Curación de Fractura/fisiología , Osteotomía/métodos , Cúbito/lesiones , Animales , Proteína Morfogenética Ósea 6 , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Terapia Genética , Humanos , Masculino , Conejos , Ratas , Ratas Sprague-Dawley , Tomografía Computarizada por Rayos X , Anomalía Torsional , Cúbito/diagnóstico por imagen , Cúbito/cirugía

RESUMEN

OBJECTIVE: To evaluate the value of various synovial fluid cytokines and eicosanoids to diagnose joint disease or categories of joint disease. STUDY DESIGN: Prospective acquisition of clinicopathologic data. ANIMALS OR SAMPLE POPULATION: Client-owned or donated horses: 50 joints with no evidence of disease; 28 joints with acute disease; 32 joints with chronic disease; 9 joints with cartilage damage and no other signs of joint disease. METHODS: Concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), prostaglandin F1-alpha (PGF(1)-alpha), and leukotriene B(4) (LTB(4)), were measured in equine synovial fluid by immunoassay and categorized according to duration and degree of joint disease. Any test value for a given category that was different from normal was further analyzed for sensitivity (S), specificity (Sp), and operating point (most valid test cutoff value). Likelihood ratios and predictive values were calculated at the operating point. Mediator concentrations were correlated to synovial fluid white blood cell count. Tests were reported as poor, fair, good, or excellent based on predictive values of <.25,.25-.5,.5-.75, or >.75, respectively. RESULTS: TNF synovial fluid concentration as a predictor of joint disease was good, and the value of TNF (maximum S and Sp) indicating joint disease was >36 pg/mL. IL-1beta as a predictor of joint disease was good, and the value of IL-1beta indicating joint disease was >4.5 pg/mL. IL-6 concentration was an excellent predictor of joint disease. Any IL-6 in synovial fluid indicated joint disease and correlated highly with synovial fluid white blood cell count (P <.0001). PGE(2) was a good-excellent predictor of disease (positive predictive value [PPV] = 0.75), and the concentration indicating joint disease was >22.5 pg/mL. The diagnostic PGF(1)-alpha concentration indicating severe chronic joint disease was identified to be >16.5 pg/mL with very high sensitivity (S = 1) and specificity (Sp =.89). PGF(1)-alpha concentrations > 9.5 pg/mL had a good PPV (.69) and NPV (.6) for any joint disease. TBX(2) concentrations below 31.5 pg/mL (S =.57; Sp =.61) were a very good predictor of joint disease (PPV =.72). LTB(4) concentration appeared to be greater in severe acute joint disease than normal joints; this was not significant (P =.15) and correlated highly with synovial fluid white blood cell count (P =.0001). CONCLUSIONS: The ability of a single value from a joint in an adult horse predicting the presence of joint disease was often good (.5-.75), and was excellent (> or =.75) for IL-6 and PGE(2). TNF-alpha and IL-1beta were no more effective than white blood cell count in screening for joint disease. IL-6 was the most sensitive and specific for joint disease and could be an excellent screening test for the presence of joint disease when lameness is difficult to identify or is intermittent. PGE(2) would be a functional screening test for the presence of any joint disease and offers a differentiating feature because values were not influenced by white blood cell count. PGF(1)-alpha values > 16.5 pg/mL identified chronic severe joint disease and may be clinically useful when there are minimal radiographic changes but substantial articular cartilage degradation.

Asunto(s)

Citocinas/metabolismo , Eicosanoides/metabolismo , Enfermedades de los Caballos/metabolismo , Artropatías/veterinaria , Líquido Sinovial/metabolismo , Enfermedad Aguda , Animales , Biomarcadores , Enfermedad Crónica , Dinoprostona/metabolismo , Caballos , Interleucina-6/metabolismo , Artropatías/metabolismo , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y Especificidad

RESUMEN

Paclitaxel is a chemotherapeutic agent that suppresses cellular proliferation and angiogenesis and has been effective in suppressing proliferative synovitis in animal models. Local joint delivery ofpaclitaxel is being pursued as a treatment for rheumatoid arthritis in humans, to avoid systematic toxicity of the drug. We used an extracorporeal, isolated metacarpophalangeal joint preparation that uniquely permitted the simultaneous evaluation of codependent hemodynamic, microvascular, and transsynovial flow responses of a joint. Specifically in this study, the isolated joint preparation provided quantitative assessment of vascular flow, transsynovial flow, and morphologic changes in response to intraarticular injection of paclitaxel (50 ng) in poly-(DL)-lactide co-glycolide 50:50 microspheres (50 microm diameter) to assess initial intra-articular biocompatibility. Control joints were isolated but not injected. Serial hemodynamic measurements, transsynovial fluid forces, synovial fluid analysis, synovial and capillary permeability, and oxygen metabolism were measured every 30 min during a subsequent 3-h isolation period. At termination, synovium and cartilage were harvested from bilateral metacarpophalangeal joints for histopathologic assessment. Intra-articular injection of this formulation of paclitaxel did not significantly affect hemodynamic parameters in the joint during this short-term study, and early joint inflammatory reaction was minimal. However, transsynovial fluid forces were significantly greater in treated joints as evidenced by greater synovial fluid flow, intra-articular pressure, transitional microvascular pressure, and permeability to fluid transport. Gross and histologic morphology of synovium and articular cartilage were normal in all isolated joints. In conclusion, this extracorporeal in vivo isolated joint model permitted investigation of the early changes in joint physiology induced by this microsphere formulation and dose ofpaclitaxel in joints and could provide a more physiologic and dynamic model for study of the pharmacokinetics of drug absorption following intra-articular administration. Due to the minimal inflammation and lack of evidence of gross or histologic change in the joint, this formulation of paclitaxel should be adequately biocompatible for use in an in vivo animal model for further study of its feasibility for human use.

Asunto(s)

Antineoplásicos Fitogénicos/farmacocinética , Artropatías/tratamiento farmacológico , Paclitaxel/farmacocinética , Animales , Cartílago/patología , Modelos Animales de Enfermedad , Caballos , Inyecciones Intraarticulares , Artropatías/patología , Articulaciones/irrigación sanguínea , Articulaciones/patología , Microcirculación , Microesferas , Presión Osmótica , Oxígeno/metabolismo , Poliglactina 910 , Flujo Sanguíneo Regional , Líquido Sinovial/metabolismo , Sinovitis/tratamiento farmacológico , Sinovitis/patología

RESUMEN

OBJECTIVE: To evaluate the effects of anti-inflammatory drugs on lipopolysaccharide (LPS)-challenged and -unchallenged equine synovial membrane in terms of production of prostaglandin E2 (PGE2) and hyaluronan, viability, and histomorphologic characteristics. SAMPLE POPULATION: Synovial membranes were collected from the carpal, tarsocrural, and femoropatellar joints of 6 adult horses. PROCEDURE: Synovial membranes from each horse were minced and pooled and explants were treated with one of the following: no drug (control), drug, LPS alone, or LPS and drug. Treatment drugs were phenylbutazone (PBZ), flunixin meglumine (FNX), ketoprofen (KET), carprofen (CRP), meloxicam (MEL), low-concentration methylprednisolone (METH), high-concentration METH, dimethyl sulfoxide (DMSO), or an experimental COX-2 inhibitor (dissolved in DMSO). Following 48 hours of culture, medium was assayed for PGE2 and hyaluronan concentration. Synovial explants were assessed for viability and histomorphologic characteristics. RESULTS: For the LPS-challenged explants, PBZ, FNX, KTP CRF MEL, and low-concentration METH suppressed PGE2 production, compared with LPS challenge alone. Only MEL suppressed PGE2 production from LPS-challenged explants, compared with unchallenged explants. Synovial explants maintained > 90% viability and there was no significant difference in viability or hyaluronan production among explants. Histomorphologic scores were significantly decreased for explants treated with low-concentration METH or DMSO. CONCLUSIONS AND CLINICAL RELEVANCE: PBZ, FNX, KTP, CRFP MEL, and low-concentration METH suppressed PGE2 production in LPS-challenged explants. Meloxicam appeared to have more selective suppression of COX-2 activity. Histomorphologic scores suggest detrimental effects of METH, DMSO, and the experimental COX-2 inhibitor. Commonly used nonsteroidal anti-inflammatory drugs suppress induced synovial membrane PGE2 production without detrimental effects on synovial membrane viability and function.

Asunto(s)

Antiinflamatorios/farmacología , Lipopolisacáridos/toxicidad , Membrana Sinovial/efectos de los fármacos , Animales , Carbazoles/farmacología , Clonixina/análogos & derivados , Clonixina/farmacología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Caballos , Ácido Hialurónico/metabolismo , Isoenzimas/metabolismo , Cetoprofeno/farmacología , Meloxicam , Metilprednisolona/farmacología , Técnicas de Cultivo de Órganos , Fenilbutazona/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Membrana Sinovial/citología , Membrana Sinovial/metabolismo , Tiazinas/farmacología , Tiazoles/farmacología

RESUMEN

OBJECTIVE: To evaluate the effects of orally administered phenylbutazone on proteoglycan synthesis and chondrocyte inhibition by IL-1beta in articular cartilage explants of horses. ANIMALS: 11 healthy 1- to 2-year-old horses. PROCEDURE: Horses were randomly assigned to the control (n = 5) or treated group (4.4 mg of phenylbutazone/kg of body weight, p.o., q 12 h; n = 6). Articular cartilage specimens were collected before treatment was initiated (day 0), after 14 days of treatment, and 2 weeks after cessation of treatment (day 30). Proteoglycan synthesis and stromelysin concentration in cartilage extracts were assessed after 72 hours of culture in medium alone or with recombinant human interleukin-1beta (IL-1beta; 0.1 ng/ml). RESULTS: On day 0, proteoglycan synthesis was significantly less in cartilage explants cultured in IL-1beta, compared with medium alone. Mean proteoglycan synthesis in explants collected on days 14 and 30 was significantly less in treated horses, compared with controls. However, incubation of explants from treated horses with IL-1beta did not result in a further decrease in proteoglycan synthesis. Significant differences in stromelysin concentration were not detected between or within groups. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of phenylbutazone for 14 days significantly decreased proteoglycan synthesis in articular culture explants from healthy horses to a degree similar to that induced by in vitro exposure to IL-1beta. Phenylbutazone should be used judiciously in athletic horses with osteoarthritis, because chronic administration may suppress proteoglycan synthesis and potentiate cartilage damage.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Cartílago Articular/efectos de los fármacos , Caballos/metabolismo , Fenilbutazona/farmacología , Proteoglicanos/biosíntesis , Administración Oral , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Cartílago Articular/metabolismo , Interleucina-1/farmacología , Metaloproteinasa 3 de la Matriz/análisis , Metaloproteinasa 3 de la Matriz/biosíntesis , Fenilbutazona/administración & dosificación , Proteoglicanos/análisis , Distribución Aleatoria , Líquido Sinovial/química

RESUMEN

OBJECTIVE: To determine the effects of phenylbutazone (PBZ) on bone activity and bone formation in horses. ANIMALS: 12 healthy 1- to 2-year-old horses. PROCEDURES: Biopsy was performed to obtain unicortical bone specimens from 1 tibia on day 0 and from the contralateral tibia on day 14. Fluorochromic markers were administered IV 2 days prior to and on days 0, 10, 15, and 25 after biopsy was performed. Six horses received PBZ (4.4 mg/kg of body weight, PO, q 12 h) and 6 horses were used as controls. All horses were euthanatized on day 30 and tissues from biopsy sites, with adjacent cortical bone, were collected. Osteonal density and activity, mineral apposition rate (MAR), and percentage of mineralized tissue filling the biopsy-induced defects in cortical bone were assessed. Serum samples from all horses were analyzed for bone-specific alkaline phosphatase activity and concentration of PBZ. RESULTS: MAR was significantly decreased in horses treated with PBZ. Regional acceleratory phenomenon was observed in cortical bone in both groups but was significantly decreased in horses treated with PBZ. Osteonal activity was similar at all time points in all horses. In control horses, percentage of mineralized tissue filling the cortical defects was significantly greater in defects present for 30 days, compared with defects present for 14 days. Differences in percentage of mineralized tissue were not detected in horses treated with PBZ. CONCLUSIONS AND CLINICAL RELEVANCE: PBZ decreased MAR in cortical bone and appeared to decrease healing rate of cortical defects in horses.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Curación de Fractura/efectos de los fármacos , Enfermedades de los Caballos/tratamiento farmacológico , Fenilbutazona/farmacología , Fosfatasa Alcalina/sangre , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Biopsia/veterinaria , Densidad Ósea/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Cromatografía Líquida de Alta Presión/veterinaria , Femenino , Fluoresceínas/química , Colorantes Fluorescentes/química , Fracturas Óseas/tratamiento farmacológico , Fracturas Óseas/cirugía , Fracturas Óseas/veterinaria , Osteón/patología , Caballos , Procesamiento de Imagen Asistido por Computador , Inmunoensayo/veterinaria , Masculino , Oxitetraciclina/química , Fenilbutazona/sangre , Fenilbutazona/uso terapéutico , Antagonistas de Prostaglandina/farmacología , Antagonistas de Prostaglandina/uso terapéutico , Distribución Aleatoria , Tibia/patología , Tibia/fisiología

RESUMEN

OBJECTIVE: To evaluate clinical safety of administration of injectable enrofloxacin. DESIGN: Randomized controlled clinical trial. ANIMALS: 24 adult horses. PROCEDURES: Healthy horses were randomly allocated into 4 equal groups that received placebo injections (control) or IV administration of enrofloxacin (5 mg/kg [2.3 mg/lb], 15 mg/kg [6.8 mg/lb], or 25 mg/kg [11.4 mg/lb] of body weight, q 24 h) for 21 days. Joint angles, cross-sectional area of superficial and deep digital flexor and calcaneal tendons, carpal or tarsal osteophytes or lucency, and midcarpal and tarsocrural articular cartilage lesions were measured. Physical and lameness examinations were performed daily. Measurements were repeated after day 21, and articular cartilage and bone biopsy specimens were examined. RESULTS: Enrofloxacin did not induce changes in most variables during administration or for 7 days after administration. One horse (dosage, 15 mg/kg) developed lameness and cellulitis around the tarsal plantar ligament during the last week of administration. One horse (dosage, 15 mg/kg) developed mild superficial digital flexor tendinitis, and 1 horse (dosage, 25 mg/kg) developed tarsal sheath effusion without lameness 3 days after the last administration. High doses of enrofloxacin (15 and 25 mg/kg) administered by bolus injection intermittently induced transient neurologic signs that completely resolved within 10 minutes without long-term effects. Slower injection and dilution of the dose ameliorated the neurologic signs. Adverse reactions were not detected with a 5 mg/kg dose administered IV as a bolus. CONCLUSIONS AND CLINICAL RELEVANCE: Enrofloxacin administered IV once daily at the rate of 5 mg/kg for 3 weeks is safe in adult horses.

Asunto(s)

Antiinfecciosos/farmacología , Fluoroquinolonas , Caballos/fisiología , Músculo Esquelético/efectos de los fármacos , Quinolonas/farmacología , Animales , Antiinfecciosos/administración & dosificación , Antiinfecciosos/efectos adversos , Biopsia/veterinaria , Recuento de Células Sanguíneas , Análisis Químico de la Sangre , Huesos/patología , Cartílago Articular/patología , Enrofloxacina , Estudios de Evaluación como Asunto , Femenino , Inyecciones/veterinaria , Articulaciones/diagnóstico por imagen , Articulaciones/fisiología , Cojera Animal , Masculino , Quinolonas/administración & dosificación , Quinolonas/efectos adversos , Radiografía/veterinaria , Ultrasonografía

RESUMEN

OBJECTIVE: This study investigated two biodegradable drug delivery systems (BDDS) for elution of gentamicin and elimination of synovial membrane infection. STUDY DESIGN: The effect of BDDS on control and infected synovial explants was determined. ANIMALS OR SAMPLE POPULATION: Synovial explants from four adult equine cadavers. METHODS: First, BDDS were placed in phosphate buffered saline for 14 days. Eluent was tested for gentamicin concentration (G) and bioactivity. Second, synovial explants were divided into four groups (n = 14/group): Group 1 (control); Group 2 (infected control) 405 cfu Staphylococcus aureus added at 6 hours; Group 3 (antibiotic BDDS [Ab-BDDS]) Ab-BDDS added at 24 hours; Group 4 (infected Ab-BDDS) 405 cfu S. aureus added at 6 hours, Ab-BDDS added at 24 hours. Both types of Ab-BDDS were used (n = 7/type/group). Explants were incubated in standard medium for 4 days. Medium was cultured and analyzed for (G) and hyaluronic acid concentration (HA). Explants were analyzed for viability and morphologic changes. RESULTS: The Ab-BDDS released >500 microg/mL of active gentamicin for 10 days. In Group 3, infection was eliminated within 24 hours, but histologic scores did not return to normal. Viability was significantly reduced by infection, but if eliminated, viability tended to return to normal. In Group 3, the Ab-BDDS had no significant effect on viability or (HA). Histopathologic scores were significantly higher for infected synovium. Infection, even if treated, significantly reduced (HA). CONCLUSIONS: Both Ab-BDDS eliminated infection within 24 hours. However, synovial morphology, viability and function did not return to normal. CLINICAL RELEVANCE: The Ab-BDDS may be useful for treatment of synovial membrane infection.

Asunto(s)

Implantes Absorbibles/veterinaria , Antibacterianos/administración & dosificación , Sistemas de Liberación de Medicamentos/veterinaria , Gentamicinas/administración & dosificación , Enfermedades de los Caballos/tratamiento farmacológico , Enfermedades de los Caballos/cirugía , Infecciones Estafilocócicas/veterinaria , Animales , Cadáver , Caballos , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/patología

RESUMEN

OBJECTIVE: To determine whether fracture fragment dimensions, suspensory ligament damage, and racing status at the time of injury were associated with outcome in Standardbred horses with apical fracture of the proximal sesamoid bone. DESIGN: Retrospective study. ANIMALS: 43 Standardbred racehorses. PROCEDURE: Medical records, racing records, and radiographs were reviewed, and ultrasonographic findings were scored. Measurements of the fractured portion of the proximal sesamoid bone were made. RESULTS: Seventy-four percent (32/43) of horses were pacers, and 26% (11/43) were trotters. Statistical differences between trotters and pacers regarding ability to start, number of starts, or amount of money earned after injury were not detected. Females earned significantly more money per start after injury than males. Eighty-six percent (37/43) of fractures involved hind limbs and 14% (6/43) involved forelimbs. Horses with forelimb injuries earned less money per start. Severity of suspensory ligament damage did not affect postinjury racing performance. A higher proportion of horses that had raced before injury returned to racing after surgery than horses that had not raced before injury, although a significant difference between these groups was not detected. Eighty-eight percent of horses that raced before injury raced after injury. Fifty-six percent of horses that did not race before injury raced after injury. Fracture fragment dimensions did not affect outcome. CLINICAL IMPLICATIONS: Dimensions of the apical fracture fragment of the proximal sesamoid bone in Standardbred horses and degree of suspensory ligament damage did not affect outcome. Prognosis for return to racing soundness is good in horses that had raced before injury and fair in horses that had not raced before injury.

Asunto(s)

Fracturas Óseas/veterinaria , Caballos/lesiones , Huesos Sesamoideos/lesiones , Animales , Artroscopía/veterinaria , Femenino , Miembro Anterior , Fracturas Óseas/economía , Marcha , Miembro Posterior , Ligamentos/diagnóstico por imagen , Masculino , Condicionamiento Físico Animal , Pronóstico , Estudios Retrospectivos , Deportes , Factores de Tiempo , Ultrasonografía

RESUMEN

OBJECTIVE: To quantitate nitric oxide synthase (NOS) activity in healthy and interleukin 1beta (IL-1beta)-exposed equine synovial membrane. ANIMALS: 6 healthy horses, 2 to 8 years old. PROCEDURE: Recombinant human IL-1beta (0.35 ng/kg of body weight) was injected intra-articularly into 1 metacarpophalangeal joint of each horse. The contralateral joint served as an unexposed control. All horses were euthanatized 6 hours after injection of IL-1beta, and synovial membrane specimens were assayed for NOS activity by measuring conversion of arginine to citrulline. Severity of inflammation was semiquantitated by analysis of synovial fluids and histologic examination of synovial membrane. RESULTS: Equine synovial membrane had minimal NOS activity. A significant difference was not detected in NOS activity between control and IL-1beta-exposed specimens. Histologic examination revealed a neutrophilic infiltrate in synovial membrane exposed to IL-1beta. Synovial fluid from IL-1beta-exposed joints had a moderate inflammatory response and significantly greater concentrations of IL-1beta and interleukin-6 than fluid from healthy joints. CONCLUSION: Healthy equine synovial membrane had low NOS activity that was not affected by exposure to IL-1beta.

Asunto(s)

Interleucina-1/farmacología , Óxido Nítrico Sintasa/metabolismo , Membrana Sinovial/enzimología , Animales , Caballos , Humanos , Inflamación/fisiopatología , Inyecciones Intraarticulares , Interleucina-1/administración & dosificación , Interleucina-6/metabolismo , Leucocitos/citología , Leucocitos/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Valores de Referencia , Líquido Sinovial/química , Líquido Sinovial/citología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/inmunología

RESUMEN

OBJECTIVE: To determine whether enrofloxacin has detrimental, dose-dependent effects on equine articular cartilage in vitro. ANIMALS: Cartilage explants were developed from 6 healthy horses between 0 and 96 months old. PROCEDURE: Patellar cartilage explants were incubated in 5 concentrations of enrofloxacin (2 microg/ml, 10 microg/ml, 1,000 microg/ml, 10,000 microg/ml, and 50,000 microg/ml) for 72 hours. Proteoglycan synthesis (Na35SO4 incorporation for 24 hours), proteoglycan degradation (Na35SO4 release for 72 hours), endogenous proteoglycan content (dimethylmethlene blue assay), and total protein content were determined. Cartilage explants were evaluated by use of histomorphologic and histomorphometric techniques (toluidine blue stain) for cytologic and matrix characteristics. Quantitative data were analyzed with a one-way ANOVA to compare results among various enrofloxacin concentration groups and the control group. A general linear model was used to determine whether age had an effect. RESULT: Proteoglycan synthesis was excellent in control specimens and in specimens incubated in low concentrations of enrofloxacin (2 microg/ml and 10 microg/ml). High concentrations of enrofloxacin (> 1,000 microg/ml) effectively eliminated proteoglycan synthesis regardless of horse age. Proteoglycan degradation at low concentrations (2 microg/ml and 10 microg/ml) was not different than control. High concentrations of enrofloxacin (> 1,000 microg/ml) caused significant degradation. Different concentrations of enrofloxacin did not affect endogenous proteoglycan. High concentrations of enrofloxacin were associated with a significant increase in number of pyknotic nuclei. CONCLUSION: Concentrations of enrofloxacin that might be achieved following systemic administration did not suppress chondrocyte metabolism in vitro. High concentrations of enrofloxacin (> 1,000 microg/ml) were toxic to chondrocytes.

Asunto(s)

Antiinfecciosos/farmacología , Cartílago Articular/efectos de los fármacos , Fluoroquinolonas , Proteoglicanos/metabolismo , Quinolonas/farmacología , Análisis de Varianza , Animales , Cartílago Articular/citología , Cartílago Articular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Relación Dosis-Respuesta a Droga , Enrofloxacina , Caballos , Técnicas de Cultivo de Órganos , Proteoglicanos/biosíntesis , Sulfatos/metabolismo , Radioisótopos de Azufre