Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.     

In vitro stimulation of equine articular cartilage proteoglycan synthesis by hyaluronan and carprofen.

The effects of hyaluronan and carprofen (both racemic mixture and separate R and S enantiomers) on proteoglycan (PG) synthesis by equine cultured chondrocytes and cartilage explants were examined. Hyaluronan stimulated PG synthesis in both cell and explant cultures. The concentration-response curve of the latter was bell-shaped. Racemic carprofen and R and S enantiomers also stimulated PG synthesis, although concentration-response relationships varied for each preparation and high concentrations inhibited synthesis. It was concluded that (a) hyaluronan exerts a stimulatory effect on PG synthesis at low concentrations and (b) stimulatory effects of carprofen on PG synthesis are, to some degree, enantioselective with the carprofen S-enantiomer exerting the greatest effect. Hyaluronan and carprofen are used clinically despite incompletely understood mechanisms of action. These results suggest (a) hyaluronan and carprofen might exert an anti-arthritic action through stimulation of PG synthesis and (b) there is possible justification for therapeutic administration of enantiomeric rather than racemic carprofen.     

In vitro evaluation of the effect of dimethyl sulfoxide on equine articular cartilage matrix metabolism

OBJECTIVE: To evaluate the effects of dimethyl sulfoxide (DMSO) on equine articular cartilage matrix metabolism. STUDY DESIGN: Using a cartilage explant culture system, proteoglycan (PG) synthesis, PG release, lactate metabolism, chondrocyte viability, and metabolism recovery were determined after cartilage exposure to DMSO. SAMPLE POPULATION: Cartilage harvested from metacarpophalangeal and metatarsophalangeal joints of 12 horses (age range, 1 to 10 years). METHODS: Explants were exposed to concentrations of DMSO (1% to 20%) for variable times (3 to 72 hours). PG synthesis and release were determined by a radiolabel incorporation assay and dimethylmethylene blue (DMMB) dye assay, respectively. Lactate released into culture media was measured, and chondrocyte viability was assessed using the Formizan Conversion Assay and a paravital staining protocol. Metabolism recovery was assessed in explants that were allowed to recover in maintenance media after exposure to DMSO. RESULTS: PG synthesis and lactate metabolism were inhibited in a dose- and time-dependent manner after exposure to DMSO concentrations > or = 5%; there was no significant alteration in PG release. No change in chondrocyte viability was detected after incubation with DMSO. PG synthesis and lactate metabolism returned to baseline rates when allowed a recovery period after exposure to DMSO. CONCLUSIONS: DMSO concentrations > or = 5% suppress equine articular cartilage matrix metabolism. Suppression of PG synthesis and lactate metabolism is reversible and does not appear to be the result of chondrocyte death. CLINICAL RELEVANCE: Equine clinicians adding DMSO to intraarticular lavage solutions should be aware that DMSO may have deleterious effects on equine articular cartilage matrix metabolism.     

In vitro characterization of porcine juvenile articular cartilage

Dispersed cell cultures were established from the articular cartilage of the proximal portion of the humerus of young pigs. Articular and epiphyseal portions of the cartilage were separated, minced, and enzymatically dispersed, using bacterial collagenase. Morphologically, 2 cell types were observed, using phase-contrast microscopy. Smaller polygonal cells (32.5 +/- 3.5 microns diameter) containing cytoplasmic granules were found in both areas of the cartilage. In cultures from the articular region, cells grew as monolayer cultures and initially did not demonstrate contact inhibition. In cultures from the epiphyseal region, cells grew in a multilayered manner in a colonial arrangement with cells being released from the center of the colony into the culture medium. Small granular particles (0.03 to 0.08 micron diameter) were secreted by cells in both culture systems. Particle secretion was greater in epiphyseal cultures than in articular cultures with the rate decreasing as confluency was approached. These particles stained positively for lipid and alkaline phosphatase. Acridine orange was also incorporated into the granules. The 2nd cell type, a stellate-shaped cell (60 +/- 7.6 micron diameter), was found mainly surrounding the outside of colonial areas in epiphyseal cultures. These cells did not secrete small granular particles and stained positive for factor VIII. Evaluation of cultures by scanning and transmission electron microscopy further supported the presence of 2 cell types. With scanning electron microscopy, the smaller polygonal cell was characterized by varying sizes of blebs (0.03 to 0.1 micron diameter) associated with the cell membrane and small cytoplasmic processes projecting from the cell's surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of sodium heparin on equine articular cartilage.

This study compared the effect of sodium heparin and gentamicin sulphate on equine articular cartilage (AC) explants in order to investigate the possible use of sodium heparin in the treatment of infectious arthritis. Six concentrations of sodium heparin and gentamicin sulphate were tested. The supernatant and explant digest were assayed for glycosaminoglycan (GAG) content with the dimethyl-methylene blue assay and the per cent loss of GAG was calculated. A significant (P< 0.001) increase in percentage GAG loss was noted for the sodium heparin groups when compared to the control, whilst no significant increase was found among the treatment groups (P =0.782). For gentamicin, no significant difference in percentage GAG loss was found between the control and three of the five treatment groups (P =0.667). The percentage GAG loss in the sodium heparin treated AC explants was greater than for any of the gentamicin-treated AC explants. It can be concluded that sodium heparin sulphate stimulates an increase in GAG release from equine articular cartilage explants, though no firm conclusions can be drawn on its use in treating equine infectious arthritis. Copyright Harcourt Publishers Ltd.     

In vitro studies of a photo-oxidized bovine articular cartilage

Bovine articular cartilage was photo-oxidized and cultured with native articular bovine cartilage and synovial membrane to study the interaction between these tissues mimicking the physiological situation in the joint. The photo-oxidation was applied as a pretreatment of cartilage for future use in cartilage resurfacing procedures in joints. Properties of the transplant were assessed by testing the production of local mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and neutral metalloproteinase activities under normal conditions and after stimulation with various stimulants representative of inflammatory changes in pathophysiological conditions. Unlike normal cartilage photo-oxidized cartilage did not release significant amounts of NO and PGE2 and showed less gelatinolytic and caseinolytic activity compared to native bovine articular cartilage. Enzyme activity of the combined cultures was at a level intermediate between that of photo-oxidized cartilage and native cartilage cultures alone. In contrast to normal cartilage, living chondrocytes were not visible in photo-oxidized cartilage using live/dead staining. These results indicate, that the photo-oxidized cartilage may have a beneficial effect on adjacent native host cartilage and therefore be a suitable transplant for use in in vivo experiments.     

Influence of polysulfated glycosaminoglycan on equine articular cartilage in explant culture.

Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis. Cultures were exposed to concentrations of 0, 50, or 200 micrograms of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage. In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days. Subsequently, the amount of remaining labeled proteoglycan was determined. Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment. Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure. Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug. Proteoglycan monomer size was similar in all treatment groups. It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture.     

Effects of lesion size and location on equine articular cartilage repair.

The mechanisms and completeness of equine articular cartilage repair were studied in ten horses over a nine month period. Large (15 mm square) and small (5 mm square) full-thickness lesions were made in weight bearing and nonweight bearing areas of the radiocarpal, middle carpal and femoropatellar joints. The horses were euthanized in groups of two 1, 2.5, 4, 5 and 9 months later. Gross pathology, microradiography, and histopathology were used to evaluate qualitative aspects of articular repair. Computer assisted microdensitometry of safranin-O stained cartilage sections was used to quantitate cartilage matrix proteoglycan levels. Structural repair had occurred in most small defects at the end of nine months by a combination of matrix flow and extrinsic repair mechanisms. Elaboration of matrix proteoglycans was not complete at this time. Statistically better healing occurred in small weight bearing lesions, compared to large or nonweight bearing lesions. Synovial and perichondrial pannus interfered with healing of osteochondral defects that were adjacent to the cranial rim of the third carpal bone. Clinical and experimental experience suggests that these lesions are unlikely to heal, whereas similar lesions in the radiocarpal and femoropatellar joints had satisfactory outcomes. Observations made in this study support the use of early postoperative ambulation, passive flexion of operated joints, and recuperative periods of up to a year for large cartilage defects.     

In vitro effect of ventriculocordectomy before laryngoplasty on abduction of the equine arytenoid cartilage

Objective: To determine whether ventriculocordectomy (VCE) performed before prosthetic laryngoplasty (PL) results in increased rima glottidis size compared with PL alone. Study Design: Experimental study. Animals: Equine cadaver larynges (n=13). Methods: Right arytenoid cartilages were maximally abducted using a standard PL technique. Standard PLs were then performed on the left side and the force required to maximally abduct the left arytenoid cartilage recorded (F_max). Photographs were taken of the rima glottidis at zero force and at five equal levels of force up to F_max. The force applied was released, left VCE performed, and photographs repeated. Arytenoid left:right angle quotients (LRQ) and glottic cross‐sectional area ratios (CSAR) were calculated at each force level in each condition (PL and VCE‐PL). Results: Mean LRQ and CSAR for both PL and VCE‐PL increased with increasing force, initially rapidly before plateauing at ～50% of F_max. LRQ and CSAR were significantly greater for VCE‐PL than for PL (P<.001). When VCE was performed before PL, 12% less force was required to achieve an LRQ of 0.8, and 45% less for a CSAR of 0.8. Conclusions: In vitro, VCE performed before PL enables the arytenoid cartilage to be abducted to a greater degree for a given PL suture force.     

Effects of intra-articular administration of methylprednisolone acetate on normal equine articular cartilage

The effects of the corticosteroid 6-alpha-methylprednisolone acetate on normal equine articular cartilage were evaluated, using the middle carpal joint in 4 clinically normal young horses. One middle carpal joint of each horse was injected 3 times with 100 mg of 6-alpha-methylprednisolone acetate, at 14-day intervals. The opposite middle carpal joint (control) was injected with 2.5 ml of lactated Ringer solution at the same intervals. Effects were studied until 8 weeks after the first injection. Evaluation included clinical and radiographic examination, and gross, microscopic, and biochemical evaluation of joint tissues. Horses remained clinically normal during the study, and significant radiographic changes were not observed. Safranin-0 matrix staining intensity and uronic acid content were significantly (P less than 0.05) lower and hydroxyproline content was significantly (P less than 0.05) higher in articular cartilage of corticosteroid-injected joints vs control joints.     

Effects of intramuscular polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage.

The effect of intramuscular polysulfated glycosaminoglycan (PSG) on repair of cartilage injury was evaluated in eight horses. In each horse, one middle carpal joint had both a partial-thickness and a full-thickness articular cartilage defect created. In the contralateral middle carpal joint, chemical articular cartilage injury was created by intra-articular injection of 50 mg sodium monoiodoacetate (MIA). Horses were divided into two groups for treatment. Group 1 horses (control) received an intramuscular injection of normal saline every four days for a total of seven injections starting seven days after cartilage injury. Group 2 horses received 500 mg of PSG intramuscularly every four days for seven treatments starting seven days after cartilage injury. Horses were maintained for 12 weeks. Horses were evaluated clinically, and their middle carpal joints were evaluated radiographically and arthroscopically at the end of the study. Joint tissues were also collected and examined microscopically. The only significant difference between groups was slightly greater matrix staining intensity for glycosaminoglycans in the radiate articular cartilage layer in MIA injected and PSG treated joints. Partial-thickness defects had not healed and the predominant repair tissue in full-thickness defects was fibrous tissue. It was concluded that using this joint injury model, 500 mg PSG administered intramuscularly had no effect on the healing of articular cartilage lesions, and minimal chondroprotective effect from chemically induced articular cartilage degeneration.     

Cellular heterogeneity in cathepsin D distribution in equine articular cartilage

The distribution of cathepsin D in normal equine growth cartilage has been examined immunocytochemically using an antiserum raised against human cathepsin D. The cross-reactivity and specificity of the antiserum for equine cathepsin D was confirmed, and its lysosomal localisation was demonstrated in horse skin fibroblasts by confocal scanning microscopy. Cultured horse chondrocytes were heterogenous in their expression of cathepsin D. Heterogeneity of distribution of the enzyme was also seen in chondrocytes in cartilage from different anatomical sites. A high level of cathepsin D was observed in the deep layer of cartilage from the lateral trochlear ridge of the distal femur. Cathepsin D was absent in the hypertrophic zone of the distal radial growth plate.     

Effects of polysulfated glycosaminoglycan on chemical and physical defects in equine articular cartilage

The effect of intra-articular polysulfated glycosaminoglycan (PSG) on repair of chemical and physical articular cartilage injuries was evaluated in 8 horses. In each horse, a partial- and a full-thickness articular cartilage defect was made on the distal articular surface of the radial carpal bone. In the contralateral middle carpal joint, a chemical articular cartilage injury was induced by injecting 50 mg of Na monoiodoacetate (MIA). Four of the 8 horses were not treated (controls), and 4 horses were treated by intra-articular injection of 250 mg of PSG into both middle carpal joints once a week for 5 treatments starting 1 week after cartilage injury. Horses were maintained for 8 weeks. There was less joint circumference enlargement in PSG-treated horses in MIA-injected and physical defect carpi, compared with that in controls. In MIA-injected joints, there was less articular cartilage fibrillation and erosion, less chondrocyte death, and greater safranin-O staining for glycosaminoglycans in PSG-treated horses. Evaluation of joints in which physical defects were made revealed no differences between control and PSG-injected joints. None of the partial-thickness defects had healed. Full-thickness defects were repaired with fibrous tissue (which was more vascular and cellular in PSG-injected joints) and occasionally small amounts of fibrocartilage. Seemingly, PSG had chondroprotective properties in a model of chemically induced articular cartilage damage, whereas PSG had no obvious effect in a physical articular cartilage-defect model.     

The vasomotor effects of 5-hydroxytryptamine on equine basilar arteries In vitro

The vasomotor effects of 5-hydroxytryptamine (5-HT) on isolated equine basilar arteries were studied. 5-HT induced contractions of equine basilar arteries in a concentration-dependent manner, with a pEC₅₀ value (with 95% confidence limits) of 7.35 (7.08–7.62). Similar results were obtained with endothelium-denuded basilar arteries. Contractions were not competitively inhibited by the 5-HT₂ receptor antagonist ketanserin at low concentrations of 5-HT. Conversely, at high concentrations of 5-HT, contractions were inhibited by ketanserin in a concentration-dependent manner, with a pA ₂ value of 8.91 (8.62–9.20). The 5-HT₁ and 5-HT₂ receptor antagonist methiothepin shifted the concentration-response curve of 5-HT downwards and to the right in a concentration-dependent manner. In the presence of 10^-6 mol/L ketanserin, however, methiothepin antagonized 5-HT-induced contractions competitively with a pA ₂ value of 7.95 (7.59–8.31). The 5-HT₃ receptor antagonist MDL 72222 had no effect on 5-HT-induced contractions. The findings of this study indicate that 5-HT₁ and 5-HT₂ receptors are located in equine basilar arterial smooth muscle cells, and that stimulation of these receptors results in contraction.Abbreviations CR concentration ratio - EC₅₀ concentration producing 50% of the maximal response - 5-HT 5-hydroxytryptamine - MDL 72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - pA ₂ negative logarithm of the molar concentration of antagonist that produces a 2-fold rightward shift of the concentration-response curve - pEC₅₀ negative logarithm of EC₅₀ - PGF₂ prostaglandin F₂     

The effects of radial shock waves on the metabolism of equine cartilage explants in vitro

AIM: To investigate, in vitro, the effects of radial shock waves on the release of nitric oxide (NO) and synthesis of prostaglandin E2 (PGE2) and glycosaminoglycan (GAG), and liberation of GAG, from equine articular cartilage explants. METHODS: Equine cartilage from normal metacarpophalangeal and metatarsophalangeal joints was exposed to radial shock waves at various impulse doses and then maintained as explants in culture for 48 h. Shock waves were delivered at 1,876 Torr pressure and a frequency of 10 Hz. Treatment groups consisted of a negative control group, or application of 500, 2,000, or 4,000 impulses by use of either a convex handpiece (Group A) or concave handpiece (Group B). Synthesis of GAG was measured using incorporation of 35S-labelled sodium sulphate. Additionally, the synthesis of NO and PGE2, and content of GAG of the explants and media were determined. RESULTS: No significant effects (p>0.05) of radial shock-wave treatment were evident on the synthesis of NO or PGE2, or release of GAG by cartilage explants. However, radial shock waves decreased synthesis of GAG measured 48 h after exposure for all treatment groups other than the 500-impulse Group-A explants (p<0.05). CONCLUSIONS: Radial shock waves impact the metabolism of GAG in chondrocytes in equine articular cartilage. Further studies will be required to fully investigate the impact of this effect on the health of joints, and to elucidate the clinical impact.     

Comparison of inhibitory effects of glucosamine and mannosamine on bovine articular cartilage degradation in vitro

OBJECTIVE: To compare the inhibitory effects of glucosamine and mannosamine on articular cartilage degradation and the effects on chondrocyte viability in vitro. SAMPLE POPULATION: Bovine articular cartilage explants. PROCEDURES: Explants were cultured in commercial medium for 48 hours. Cartilage was exposed to medium containing 10% fetal bovine serum, 10 microg of lipopolysaccharide/mL, and 0.5, 1.0, 2.5, 5.0, and 10.0 mg of glucosamine or mannosamine/mL for 24 hours. Nitric oxide (NO) production (nitrite concentration) and proteoglycan (PG) release (PG concentration) in media were measured. Cartilage extracts were analyzed via zymography to detect gelatinolytic activity. At the end of the experiment, explants were assessed for chondrocyte viability. RESULTS: Addition of lipopolysaccharide resulted in increased NO production and PG release, but no increase in gelatinolytic activity, compared with controls. Glucosamine and mannosamine at concentrations as low as 0.5 mg/mL inhibited NO production. Glucosamine inhibited PG release at a minimum concentration of 1.0 mg/mL, whereas mannosamine inhibited PG release at a concentration of 0.5 mg/mL. Concentrations of glucosamine < or = 5.0 mg/mL did not adversely affect chondrocyte viability; however, at a concentration of 10.0 mg/mL, cell death was evident. Mannosamine had a toxic effect at a concentration of 5.0 mg/mL and was associated with pronounced chondrocyte death at a concentration of 10.0 mg/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Glucosamine and mannosamine inhibit selected indices of bovine articular cartilage degradation at concentrations that do not affect chondrocyte viability. The potential for cytotoxic effects at higher concentrations underscores the importance of establishing appropriate dosage regimens for these aminomonosaccharides.     

Effects of dosage titration of methylprednisolone acetate and triamcinolone acetonide on interleukin-1-conditioned equine articular cartilage explants in vitro

REASONS FOR PERFORMING STUDY: Osteoarthritis is a frequent sequela of joint disease, especially with severe injuries or if attempts at therapy are unsuccessful. Negative and positive effects of corticosteroid treatment of articular cartilage have been demonstrated by in vitro and in vivo studies. OBJECTIVES: To assess the metabolic effects of varying dosages of methylprednisolone acetate (MPA) and triamcinolone acetonide (TA) on interleukin-1alpha (IL-1) conditioned equine cartilage explants. Our hypothesis was that lower dosages of corticosteroids would be less detrimental to cartilage metabolism than higher dosages. TA would be less detrimental to cartilage metabolism than MPA. METHODS: Treatment groups included articular cartilage explants with no IL-1 (control), IL-1 alone, and IL-1 plus 10, 5, 1 and 0.5 mg/ml MPA or 1.2, 0.6, 0.12 and 0.06 mg/ml TA. Explants were labelled with 35SO4 prior to the beginning and end of the experiment to assess glycosaminoglycan (GAG) degradation and synthesis, respectively. Total GAG content in media and explants and total cartilage DNA were also analysed. RESULTS: MPA and TA reduced GAG synthesis compared to control and IL-1 alone. The highest dosage of MPA (10 mg/ml) reduced GAG synthesis less than lower dosages of MPA and all dosages of TA. Compared to IL-1 alone, all dosages of TA and lower dosages of MPA increased GAG degradation. MPA at 10 mg/ml reduced GAG degradation. Both MPA and TA increased media GAG content compared to control and IL-1 explants. Total cartilage GAGs were unchanged with MPA, but reduced with TA, compared with IL-1 alone. Total cartilage DNA was decreased with MPA and increased with TA compared to IL-1 and control explants. CONCLUSIONS: MPA and TA did not counteract the negative effects of IL-1 and did not maintain cartilage metabolism at control levels. Lower dosages of MPA and TA were not less detrimental to cartilage metabolism than higher dosages. TA did not appear to be less harmful than MPA on cartilage metabolism. The results of this study differ from the findings of comparable in vivo studies. POTENTIAL RELEVANCE: The low numbers of horses used in this study limits extrapolation of these findings to the equine population; however, this study also questions the clinical relevance of this in vitro model.