Conditioned serum in vitro treatment of chondrocyte pellets and osteoarthritic explants.

BACKGROUND: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. OBJECTIVES: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1ß and cartilage explants with mild osteoarthritis. STUDY DESIGN: In vitro experimental study. METHODS: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h] and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1ß and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1ß prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6 and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. RESULTS: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS-treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated-compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. MAIN LIMITATIONS: Results from in vitro models cannot be directly translated to in vivo situations. CONCLUSIONS: In vitro treatment with conditioned serum did not alleviate IL-1ß-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.

HISTORIAL: Suero autólogo acondicionado (ACS) es usado para tartar osteoartritis en caballos, aunque sus efectos no han sido completamente investigados. OBJETIVOS: Investigar los efectos de suero equino y suero acondicionado en condrocitos estimulados con interleukina (IL)-1ß y explantes de cartílago con osteoartritis leve. DISEÑO DEL ESTUDIO: Estudio experimental in vitro. MÉTODOS: El efecto de tres preparaciones séricas diferentes (control no incubado (PS), suero incubado 24 h (PS24h), y suero incubado 24 h en frascos ACS (PCS)) combinados y obtenidos de caballos cojos fueron probados en dos modelos in vitro. Las concentraciones de IL-1ß y de receptor antagonista de IL-1 (IL-1Ra) fueron medidas en todos los sueros. En el modelo 1, los cultivos de pellets de condrocitos fueron estimulados con IL-1ß antes de ser tratados con las preparaciones séricas durante 2 y 48 h. Se realizaron análisis de micromatrices, reacciones de polimerasa en cadena y de matriz de metalopeptidasa-13. En el modelo 2, explantaciones de cartílago proveniente de caballos con osteoartritis estructural fueron tratados con PS o PCS en los días 0, 6 y 12, o dejados sin tartar, y evaluados al día 24 usando la escala de graduación OARSI para evaluación histológica de cartílago articular. RESULTADOS: La concentración de IL-1Ra en PS24h y PCS fue significativamente mayor que en PS. En el modelo 1, los genes relacionados a la inflamación y a la degradación de la matriz cartilaginosa estaban aumentados después de 48 h en todos los grupos tratados en comparación a los controles no tratados. Las moléculas de matriz cartilaginosa, agrecanos y colágenos estaban disminuidos en los pellets PS24h y PCS versus los controles no tratados. Los genes de señales de factores de crecimiento FGF7 estaban aumentados en todos los grupos tratados, BMP2 en PS24h y INHBA in PCS en comparación con los controles no tratados. En el modelo 2, la escala OARSI al día 24 no fue significativamente distinta entre los grupos de tratamientos. LIMITACIONES PRINCIPALES: Los resultados de modelos in vitro no pueden ser directamente aplicados a situaciones in vivo. CONCLUSIONES: El tratamiento in vitro con suero acondicionado no alivió las respuestas inducidas por IL-1ß en pellets de condrocitos o llevo a mejoramiento morfológico en explantes de cartílago con osteoartritis.

Conditioned serum in vitro treatment of chondrocyte pellets and osteoarthritic explants.

BACKGROUND: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. OBJECTIVES: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1ß and cartilage explants with mild osteoarthritis. STUDY DESIGN: In vitro experimental study. METHODS: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h], and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1ß and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1ß prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6, and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. RESULTS: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS- treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated- compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. MAIN LIMITATIONS: Results from in vitro models cannot be directly translated to in vivo situations. CONCLUSIONS: In vitro treatment with conditioned serum did not alleviate IL-1ß-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.

In vivo joint synovial fluid disposition of a novel sustained-release formulation of diclofenac and hyaluronic acid in horses.

Intra-articular administration of sustained-release anti-inflammatory drugs is indicated in horses suffering from joint inflammation, but no such drugs are labelled for veterinary use. To obtain initial data on synovial disposition and safety of a new sustained-release formulation of diclofenac (SYN321) in the joints of horses, an experimental interventional study of elimination and side effects of intra-articular administration of SYN321 was conducted. Nine clinically sound horses were included in the study, and SYN321 was administered by the intra-articular route. Dose ranges and sampling intervals were established in a pilot study with two horses, and then applied in a main study involving seven horses treated in the fetlock joint. Diclofenac was detected above lower limit of quantification (LOQ: 0.5 ng/ml) in synovial fluid throughout the study period (14 days), and below LOQ (0.1 ng/ml) in plasma after 4 days and in urine after 14 days. No obvious clinical side effects were detected. Clinical examination and objective lameness evaluation suggested that SYN321 has potential as a local joint NSAID treatment with sustained release in horses, but further studies on synovial fluid exposure, safety and clinical efficacy are warranted.

Effect of meloxicam treatment on movement asymmetry in riding horses in training.

Quantitative gait analysis has revealed that a large proportion of horses in training, perceived as free from lameness by their owners, show movement asymmetries of equal magnitude to horses with mild clinical lameness. Whether these movement asymmetries are related to orthopaedic pain and/or pathology has yet to be further investigated. Therefore, the objective of this study was to determine whether movement asymmetries in riding horses in training are affected by anti-inflammatory treatment with meloxicam. In a crossover design, horses were treated with meloxicam or placebo for four days respectively, with a 14-16 day washout period between treatments. Objective movement analysis utilising body mounted accelerometers was performed on a hard and a soft surface before and on day four of each treatment. A trial mean was calculated for the differences between the two vertical displacement minima and maxima of head (HDmin, HDmax) and pelvis (PDmin, PDmax) per stride. Horses (n = 66) with trial mean asymmetries greater than 6 mm for HDmin or HDmax, or more than 3 mm for PDmin or PDmax, at baseline were included. The difference before and after each treatment in the measured movement asymmetry was assessed with linear mixed models. Treatment with meloxicam did not significantly affect the movement asymmetry in any of the models applied (all p>0.30). These results raise new questions: are the movement asymmetries in riding horses in training simply expressions of biological variation or are they related to pain/dysfunction that is non-responsive to meloxicam treatment?