Phenotypic expression of equine articular chondrocytes grown in three-dimensional cultures supplemented with supraphysiologic concentrations of insulin-like growth factor-I

Lisa A. Fortier DVM, PhD1, Alan J. Nixon BVSc, MS2, and George Lust PhD3
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  • 1 Comparative Orthopaedics Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
  • | 2 Comparative Orthopaedics Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
  • | 3 James A. Baker Institute for Animal Health, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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)

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)