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

Abstract

Objective: To determine the effects of transforming growth factor-beta 1 (TGF-beta 1) 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 x 10(6) chondrocytes/ml in a three-dimensional fibrin matrix. Chondrocytes in culture were supplemented with TGF-beta 1 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-beta 1/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-beta 1/ml, and in all serum-free culture conditions compared to FBS and 0 ng of TGF-beta 1/ml. Total proteoglycan accumulation and [35S]-labeled proteoglycan synthesis in cultures on days 7 and 14 were increased by the addition of exogenous TGF-beta 1 in serum-free conditions and decreased by TGF-beta 1 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-beta 1. Total DNA content of chondrocytes increased with the addition of TGF-beta 1 in FBS-supplemented conditions and decreased in serum-free conditions.

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

Clinical relevance: TGF-beta 1 may not be a suitable growth factor for enhancement of equine articular grafting in sites exposed to serum.