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in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association

Abstract

Objective—To assess the effects of supraphysiologic concentrations of insulin-like growth factor-I (IGF-1) on morphologic and phenotypic responses of chondrocytes.

Sample Population—Articular cartilage obtained from 2 young horses.

Procedure—Chondrocytes were suspended in fibrin cultures and supplemented with 25, 12.5, or 0 mg of IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic expression were assessed histologically, using H&E and Alcian blue stains, immunoreaction to collagen type I and II, and in situ hybridization. Proteoglycan content, synthesis, and monomer size were analyzed. The DNA content was determined by bisbenzimide-fluorometric assay, and elution of IGF-1 into medium was determined by IGF-1 radioimmunoassay.

Results—Both 12.5 and 25 µg of IGF-1/ml enhanced phenotypic expression of chondrocytes without inducing detrimental cellular or metabolic effects. Highest concentration of IGF-1 (25 µg/ml) significantly increased total DNA content, glycosaminoglycan (GAG) content, GAG synthesis, and size of proteoglycan monomers produced, compared with cultures supplemented with 12.5 µg of IGF-1/ml or untreated cultures. Histologic examination confirmed these biochemical effects. Matrix metachromasia, type-II collagen in situ hybridization and immunoreaction were increased in cultures treated with 25 µg of IGF-1/ml, compared with cultures supplemented with 12.5 µg of IGF-1/ml or untreated cultures.

Conclusions and Clinical Relevance—Chondrocytes exposed to high concentrations of IGF-1 maintained differentiated chondrocyte morphology and had enhanced synthesis of matrix molecules without inducing apparent detrimental effects on chondrocyte metabolism. These results suggest that application of such composites for in vivo use during cartilage grafting procedures should provide an anabolic effect on the grafted cells. (Am J Vet Res 2002;63:301–305)

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in American Journal of Veterinary Research

Summary

Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 × 106 cells/g of cartilage (wet weight), compared with a yield of 17.83 × 106 cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham’s F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, α-ketoglutarate, and l-glutamine. The medium was buffered with hepes, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at −196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.

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in American Journal of Veterinary Research

Summary

Because articular chondrocytes are a target for drugs that can influence the integrity of cartilage, we investigated the effects of 3 antiarthritic drugs, glycosaminoglycan polysulfate, diclofenac-Na, and S-adenosylmethionine sulfate p-toluenesulfonate on total protein, fibronectin, and dna synthesis, as well as on extradomain-A fibronectin and keratan sulfate content. Glycosaminoglycan polysulfate stimulated dose-dependent incorporation of [35S]methionine into protein and fibronectin, whereas incorporation of [3H]thymidine into dna was unaffected. Total fibronectin, extradomain-A fibronectin, and keratan sulfate content were high in chondrocyte cultures treated with glycosaminoglycan polysulfate. In contrast, fibronectin and dna synthesis, as well as extradomain-A fibronectin and keratan sulfate content were unaffected by diclofenac-Na. S-Adenosylmethionine decreased dose-dependently the synthesis of fibronectin, as well as the content of fibronectin and keratan sulfate. At the highest concentration of S-adenosylmethionme tested, findings suggest that cell viability was impaired as assessed by the release of lactate dehydrogenase into the media.

Free access
in American Journal of Veterinary Research

Summary

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 ± 0.6% (mean ± sem) in α1(I) and α2(I), and was 48.1 ± 1.3% in α1(ID chains. The hydroxylation of lysine was 23.2 ± 0.7% in α1(I) and 34.1 ± 0.9% in α2(D) chains from tendon, and 49.6 ± 4.3% in a1( chains from cartilage. The cyanogen bromide (CB)-peptide patterns of chromatographically purified equine α2(I) and α1(II) chains were similar to those published previously for rat, bovine, and human α2 and α1 chains. However, the CB-peptide pattern of the equine α1(I) chain resembled the guinea pig α1(I) chain, which has no methionine between CB7 and CB6. Purified equine α1(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 α1(I)CB7. Because of this species specific difference in structure of the α1(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.

Free access
in American Journal of Veterinary Research

Summary

Equine neonatal chondrocytes were cultured in three-dimensional fibrin matrices under conditions of immediate implantation or implantation following monolayer culture for 6 days, and 3 cell concentrations (1 × 105, 1 × 106, and 5 × 106 chondrocytes/ cm3). Equine fibrinogen was collected by cryoprecipitation and polymerized by use of activated bovine thrombin. The fibrin implants were harvested and analyzed histologically and biochemically at 3, 7, and 14 days after the chondrocytes were implanted in fibrin. The differentiation ratio (ratio of rounded, chondrocyte-like cells to stellate, fibroblast-like cells) was statistically higher for implants that received 5 × 106 precultured cells at all time periods than for implants that received 1 × 105 or 1 × 106 precultured cells. The differentiation ratio was statistically higher for implants that received 5 × 106 immediately implanted cells than for other implants at 7 days after implantation. At 14 days, implants that received 5 × 106 precultured chondrocytes had a higher differentiation ratio than did implants that received 5 × 106 chondrocytes that had not been precultured. Among implants that received precultured chondrocytes, total glycosaminoglycan and chondroitin sulfate content was lowest for implants that received only 1 × 105 cells. Among implants that received chondrocytes that had not been precultured, glycosaminoglycan content was not significantly different among the 3 cell concentrations, and chondroitin sulfate content was different only between implants that received 5 × 106 vs 1 × 106 cells. Only after the longest incubation period and at the highest cell concentration studied did preculturing of chondrocytes improve maintenance of phenotype. Preculturing did not appear to influence proteoglycan synthesis.

Free access
in American Journal of Veterinary Research

SUMMARY

We tested the hypothesis that treatment of growing, susceptible (to hip dysplasia) pups by im administration of glycosaminoglycan polysulfates would mitigate the signs of incipient hip dysplasia. In 1 experiment, 7 pups, selected at random from 2 litters, were administered glycosaminoglycan polysulfates (2.5 mg/kg of body weight, im) twice weekly, and 7 control pups from the same litters were given sterile buffered 0.9% saline solution from the age of 6 weeks to 8 months. Hip joints were examined by radiography, with pups in the standard, limbs-extended position. At 8 months of age, all pups in this experiment did not manifest femoral head subluxation radiographically. The Norberg angle, a measure of coxofemoral congruity, improved from a mean ± sem value of 102° ± 1° in controls to 106° ± 1° in treated pups (P = 0.008). Pups were not subjected to necropsy.

In the second experiment, 8 pups were selected at random from 2 litters and were administered 5 mg of glycosaminoglycan polysulfates/kg, im, twice weekly from 6 weeks to 8 months of age. Similarly, 8 control pups were administered saline solution. At 8 months of age, hip joints were examined by radiography with pups in the standard position; at necropsy, intra-articular tissues were evaluated macroscopically and biochemically. Of 8 treated pups, none had subluxation radiographically, whereas 4 of 8 control dogs had femoral head subluxation. Mean Norberg angle on the radiographs was 109.7° ± 1.6° for the treated group and was 101.5° ± 1.6° for controls, representing a mean improvement in coxofemoral congruity of 8.2° in the treated pups. The radiographic diagnosis (normal vs dysplastic) and the Norberg angle measurements were significantly (P = 0.04 and 0.002, respectively) different for treated and control pups.

At necropsy, 1 of 8 treated pups had cartilage degeneration, whereas 4 of 8 control pups had cartilage degeneration. The mean pathologic score determined for the hip joints of treated pups was 1.6 ± 0.8, whereas for those of controls, the score was 3.3 ± 1.2 (P = 0.09). Normal (disease-free) pups had hip pathologic scores of zero. The mean fibronectin content of femoral head articular cartilage was reduced from 2.19 ± 0.61 μg/mg in nontreated pups to 0.59 ± 0.56 μg/mg for treated pups (P = 0.04).

Fibronectin content was used as a measure of the extent of cartilage degeneration, and the cartilage of disease-free hip joints contained 0.32 ± 0.03 μg/mg. The mean proteoglycan content of the cartilage was unaffected by drug treatment. A trend was evident for lower synovial fluid volume and lower ligament volume (more normal volumes) in treated pups, but the differences were not statistically significant.

Hip joint laxity was assessed by use of a distraction method during radiogaphy of pups in experiments 1 and 2. The differences in laxity determinations between the treated and control pups were not statistically significant.

Taken together, the data indicated that im administration of gycosaminoglycan polysulfates from 6 weeks to 8 months of age in growing pups that were susceptible to hip dysplasia resulted in less subluxation, as determined from the standard radiographic projection. Treated pups had closer coxofemoral congruity when they were 8 months old (P < 0.05); at necropsy, the joint pathologic scores of treated pups indicated a trend toward improvement (P < 0.09), but the differences were not statistically significant. The mechanism of action for this drug effect is unknown.

Free access
in American Journal of Veterinary Research

Objective

To determine whether keratan sulfate concentrations in plasma or synovial fluid from clinically normal horses were different from concentrations in horses with joint disease and whether concentrations varied with type of joint disease.

Design

Case-control study.

Animals

67 clinically normal horses, 10 clinically normal foals, and 160 horses with joint disease.

Procedure

ELISA was used to measure keratan sulfate concentrations.

Results

Mean plasma keratan sulfate concentration (mean ± SEM, 580 ± 124 ng/ml) in foals peaked at 10 weeks of age. Mean plasma keratan sulfate concentration in clinically normal horses was 200 ng/ml (95% confidence interval, 157 to 251 ng/ml). Horses with osteochondral (chip) fractures, other closed intraarticular fractures, inflammatory arthritis (synovitis), infectious arthritis, or osteochondrosis had significantly higher plasma keratan sulfate concentrations than did clinically normal horses, but horses with osteoarthritis did not.

Breed, gender, and type of joint disease affected keratan sulfate concentration in synovial fluid. Standardbreds with chip fractures of the metacarpophalangeal/ metatarsophalangeal joints had significantly higher keratan sulfate concentrations in synovial fluid than did Thoroughbreds. Keratan sulfate concentrations in synovial fluid from osteoarthritic carpal joints were lower than concentrations in normal carpal joints and tarsocrural joints with inflammatory joint disease.

Clinical Implications

Keratan sulfate concentration alone was not a specific marker of joint disease but was affected by various joint diseases. (J Am Vet Med Assoc 1997;210:369–374

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in Journal of the American Veterinary Medical Association