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


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


METHODS
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.


CONCLUSIONS
TGF-beta 1 may not be a suitable growth factor for enhancement of equine articular grafting in sites exposed to serum.
Fortier
L A
LA
Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14583, USA.
Nixon
A J
AJ
Mohammed
H O
HO
Lust
G
G
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D003470:Culture Media / Q000032:analysis / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D005337:Fibrin; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006736:Horses / Q000378:metabolism*; D007150:Immunohistochemistry; D011509:Proteoglycans / Q000096:biosynthesis*; D013462:Sulfur Radioisotopes; D013936:Thymidine / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000494:pharmacology*; D014316:Tritium
D003470:Culture Media; D006025:Glycosaminoglycans; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D016212:Transforming Growth Factor beta; D014316:Tritium; D005337:Fibrin; D003094:Collagen; D004247:DNA; D013936:Thymidine