Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor β-1

Lisa A. Fortier From the Comparative Orthopaedics Laboratory (Fortier, Nixon) and the Section of Epidemiology (Mohammed), Department of Clinical Sciences, and the James A. Baker Institute for Animal Health (Lust), College of Veterinary Medicine, Cornell University, Ithaca, NY 14583.

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Alan J. Nixon From the Comparative Orthopaedics Laboratory (Fortier, Nixon) and the Section of Epidemiology (Mohammed), Department of Clinical Sciences, and the James A. Baker Institute for Animal Health (Lust), College of Veterinary Medicine, Cornell University, Ithaca, NY 14583.

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Hussni O. Mohammed From the Comparative Orthopaedics Laboratory (Fortier, Nixon) and the Section of Epidemiology (Mohammed), Department of Clinical Sciences, and the James A. Baker Institute for Animal Health (Lust), College of Veterinary Medicine, Cornell University, Ithaca, NY 14583.

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George Lust From the Comparative Orthopaedics Laboratory (Fortier, Nixon) and the Section of Epidemiology (Mohammed), Department of Clinical Sciences, and the James A. Baker Institute for Animal Health (Lust), College of Veterinary Medicine, Cornell University, Ithaca, NY 14583.

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 PhD

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Abstract

Objective

To determine the effects of transforming growth factor-β1 (TGF-β1) on the synthesis of DNA, collagen, and proteoglycans (PG) by equine chondrocytes.

Sample Population

Articular cartilage obtained from multiple joints of a 4-month-old foal.

Procedure

Chondrocytes were isolated by collagenase digestion, cultured in monolayer, trypsinized, and implanted at a cellular density of 10 × 106 chondrocytes/ml in a three-dimensional fibrin matrix. Chondrocytes in culture were supplemented with TGF-β1 at concentrations of 0, 1, 5, or 10 ng/ml in serum-free medium or medium containing fetal bovine serum (FBS). Total PG accumulation, [35S]-labeled PG synthesis, PG monomer hydrodynamic size, type II collagen production, total DNA content, and [3H]thymidine incorporation into DNA were determined at 7 and 14 days of culture.

Results

Chondrocytes maintained a rounded phenotype, dedifferentiating slightly to a more fibroblastic appearance only in medium containing FBS and 10 ng of TGF-β1/ml. Type II collagen immunoreaction on day 14 was decreased in the pericellular matrix in cultures containing FBS and 1, 5, and 10 ng of TGF-β1/ml, and in all serum-free culture conditions compared to FBS and 0 ng of TGF-β1/ml. Total proteoglycan accumulation and [35S]-labeled proteoglycan synthesis in cultures on days 7 and 14 were increased by the addition of exogenous TGF-β1 in serum-free conditions and decreased by TGF-β1 in FBS-supplemented conditions. Calculation of the partition coefficients for PG indicated that there was synthesis of low molecular weight PG in serum-free conditions and larger sized proteoglycans in FBS-supplemented conditions. Proteoglycan molecular size was unchanged by the addition of TGF-β1. Total DNA content of chondrocytes increased with the addition of TGF-β1 in FBS-supplemented conditions and decreased in serum-free conditions.

Conclusions

In a solid three-dimensional fibrin matrix, the effects of TGF-β1 on chondrocyte biological activity depend on the culture duration and on the presence of FBS in the medium. Stimulatory effects of TGF-β1 were most pronounced in serum-free culture conditions with high concentration of TGF-β1 (5 and 10 ng/ml) on day 7 and with low concentration of TGF-β1 (1 ng/ml) on day 14.

Clinical Relevance

TGF-β1 may not be a suitable growth factor for enhancement of equine articular grafting in sites exposed to serum. (Am J Vet Res 1997;58:66–70)

Abstract

Objective

To determine the effects of transforming growth factor-β1 (TGF-β1) on the synthesis of DNA, collagen, and proteoglycans (PG) by equine chondrocytes.

Sample Population

Articular cartilage obtained from multiple joints of a 4-month-old foal.

Procedure

Chondrocytes were isolated by collagenase digestion, cultured in monolayer, trypsinized, and implanted at a cellular density of 10 × 106 chondrocytes/ml in a three-dimensional fibrin matrix. Chondrocytes in culture were supplemented with TGF-β1 at concentrations of 0, 1, 5, or 10 ng/ml in serum-free medium or medium containing fetal bovine serum (FBS). Total PG accumulation, [35S]-labeled PG synthesis, PG monomer hydrodynamic size, type II collagen production, total DNA content, and [3H]thymidine incorporation into DNA were determined at 7 and 14 days of culture.

Results

Chondrocytes maintained a rounded phenotype, dedifferentiating slightly to a more fibroblastic appearance only in medium containing FBS and 10 ng of TGF-β1/ml. Type II collagen immunoreaction on day 14 was decreased in the pericellular matrix in cultures containing FBS and 1, 5, and 10 ng of TGF-β1/ml, and in all serum-free culture conditions compared to FBS and 0 ng of TGF-β1/ml. Total proteoglycan accumulation and [35S]-labeled proteoglycan synthesis in cultures on days 7 and 14 were increased by the addition of exogenous TGF-β1 in serum-free conditions and decreased by TGF-β1 in FBS-supplemented conditions. Calculation of the partition coefficients for PG indicated that there was synthesis of low molecular weight PG in serum-free conditions and larger sized proteoglycans in FBS-supplemented conditions. Proteoglycan molecular size was unchanged by the addition of TGF-β1. Total DNA content of chondrocytes increased with the addition of TGF-β1 in FBS-supplemented conditions and decreased in serum-free conditions.

Conclusions

In a solid three-dimensional fibrin matrix, the effects of TGF-β1 on chondrocyte biological activity depend on the culture duration and on the presence of FBS in the medium. Stimulatory effects of TGF-β1 were most pronounced in serum-free culture conditions with high concentration of TGF-β1 (5 and 10 ng/ml) on day 7 and with low concentration of TGF-β1 (1 ng/ml) on day 14.

Clinical Relevance

TGF-β1 may not be a suitable growth factor for enhancement of equine articular grafting in sites exposed to serum. (Am J Vet Res 1997;58:66–70)

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