The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples.

Bilateral osteochondral defects (10 mm2 X 3 mm deep) were created on the distal articular surface of the radial carpal bone of ten, 2- to 3-year-old horses. One defect of each horse was repaired, using a sternal cartilage autograft (treated), and the other was left untreated (control). The horses were exercised on a high-speed treadmill at incrementally increased speed and duration over the course of 12 months. Horses were evaluated arthroscopically at 6 to 7 weeks, and clinical examinations were conducted weekly at exercise. Twelve months after surgery, carpuses of each horse were radiographed and clinically examined prior to euthanasia. A gross pathologic evaluation of each joint was conducted, and samples were collected for histologic, histochemical, histomorphometric, and biochemical evaluation. Radiographically, the grafted joints had more extensive evidence of arthropathy, and clinically, 8 of the 10 horses were more lame in the grafted limb. On the basis of histomorphometry, the repair tissue of the grafted defects contained a greater median percentage of hyaline cartilage (45%) than that of control defects 4.5%), and the control defects contained a greater percentage of fibrocartilage (82%) than did grafted defects (28.5%). A greater median percentage of repair tissue stained with safranin-O in the grafted defects (24.5%) than in the control defects (3.5%). On gross pathologic and histologic evaluation, repair tissue of the control defects had better continuity and was more firmly attached to the subchondral bone than was repair tissue of the grafted defects. Repair tissue of the grafted defects had extensive fissure and flap formation. Histologically, subchondral bone reactivity and fibroplasia was extensive in grafted joints. Repair tissue of grafted defects had a greater percentage of type II collagen (mean sem, 83.5 +/- 2.95%) than did controls (mean, 79.4 3.87%) that was not statistically significant. Hexosamine content was significantly higher (P < 0.05) in repair tissue of the grafted defect (mean, 28.9 +/- 3.00 mg/g of dry weight) vs control (mean, 20.6 +/- 1.85 mg/g of dry weight). On the basis of this experimental model, sternal cartilage autografts cannot be recommended at this time for repair of osteochondral defects in athletic horses.

Collagen type I was purified from equine skin and flexor tendon, and type II collagen was purified from equine articular cartilage. The proteoglycans in these tissues were extracted, using guanidine HCl; the collagens were solubilized, using pepsin digestion, then were selectively precipitated with Nacl. Gel electrophoresis indicated that the precipitates contained only type I or type II collagen. Amino acid analysis indicated that collagen constituted > 97% of the total protein in the precipitates. Hydroxylation of proline was 42.0 t 0.6% (mean SEM) in alpha 1(I) and alpha 2(I), and was 48.1 +/- 1.3% in alpha 1(II) chains. The hydroxylation of lysine was 23.2 +/- 0.7% in alpha 1(I) and 34.1 0.9% in alpha 2(I) chains from tendon, and 49.6 +/- 4.3% in alpha 1(II) chains from cartilage. The cyanogen bromide (CB)-peptide patterns of chromatographically purified equine alpha 2(I) and alpha 1(II) chains were similar to those published previously for rat, bovine, and human alpha 2 and alpha 1 chains. However, the CB-peptide pattern of the equine alpha 1(I) chain resembled the guinea pig alpha 1(I) chain, which has no methionine between CB7 and CB6. Purified equine alpha 1(I)CB7,6 contained no methionine, methionine sulfoxide, or homoserine lactone. Mass of 42.26 kd was determined by use of mass spectrometry, and N-terminal sequence analysis established that the first 12 amino acids of this CB7,6 were identical to the sequence of human alpha 1(I)CB7. Because of this species specific difference in structure of the alpha 1(I) chain, equine Cb-peptides should be used as standards in studies of variations in the proportions of type I and type II collagens in equine tissues expressing the phenotype of fibrous tissue and cartilage.

The effect of interleukin 1 (IL-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of IL-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to IL-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metalloproteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The IL-1-induced induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of IL-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses. Articular cartilage from affected joints of horses with naturally acquired osteoarthritis did not have metabolic alterations identical to those of IL-1-stimulated normal articular cartilage from the same individual, necessitating reevaluation of the validity of the IL-1-induced model of osteoarthritis. Osteoarthritis is a common, naturally acquired disease of horses, and tissue from animals of all ages and stages of osteoarthritis is available. The equine model of osteoarthritis may afford an important means of studying the alterations in articular cartilage metabolism as a function of age and disease severity.

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples. However, similar analysis of data on GAG release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of LPS derived from different bacterial sources made a significant difference in the response of explants to incubation with LPS.