Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor beta-1.
Abstract: 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.
Publication Date: 1997-01-01 PubMed ID: 8989499
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research investigated the impact of the protein Transforming Growth Factor-beta 1 (TGF-beta 1) on cartilage cells from horses. The results suggest that the protein’s effects on the cells depend on the conditions under which they are grown, and that it may not be suitable for improving healing in horse joint grafts.
Objective
The research aimed to examine how TGF-beta 1 influences the DNA, collagen, and proteoglycan (PG) synthesis of equine chondrocytes, which are cells in cartilage.
Methods
- Cartilage was procured from several joints of a 4-month-old foal. Using collagenase, an enzyme that breaks down collagen, chondrocytes were isolated.
- The isolated cells were cultivated in a single layer, treated with trypsin (an enzyme used in cell culture), and embedded in a three-dimensional fibrin matrix, a protein network that aids cell growth.
- The cell cultures were supplemented with TGF-beta 1 in varying concentrations (0, 1, 5, or 10 ng/ml), either in a serum-free medium or in a medium containing fetal bovine serum (FBS), a common supplement in cell culture media due to its growth-promoting substances.
- Over 7 and 14 days, researchers tracked several markers of cell activity, such as PG accumulation and synthesis, collagen production, and DNA content.
Results
- The chondrocytes retained their rounded phenotype, only showing signs of “dedifferentiation” (changing into a more fibroblast-like appearance) when grown in medium with FBS and the highest concentration of TGF-beta 1.
- On the 14th day, type II collagen production was reduced when cultured with FBS and various concentrations of TGF-beta 1, and in all serum-free conditions.
- Proteoglycan accumulation and synthesis were influenced by the presence of TGF-beta 1. In serum-free conditions, they increased with additional TGF-beta 1, while in conditions with FBS, they decreased.
- DNA content of chondrocytes increased with the addition of TGF-beta 1 in the FBS-supplement conditions but decreased in serum-free conditions.
Conclusions
The effects of TGF-beta 1 on chondrocytes grown in a three-dimensional fibrin matrix are reliant on the duration of the culture and the presence of FBS in the medium. The stimulatory effects of TGF-beta 1 were noticeable in serum-free conditions on day 7 with a high TGF-beta 1 concentration, and on day 14 with a low TGF-beta 1 concentration. The study concluded that TGF-beta 1 may not be a fitting growth factor for enhancing equine articular grafting in sites exposed to serum.
Cite This Article
APA
Fortier LA, Nixon AJ, Mohammed HO, Lust G.
(1997).
Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor beta-1.
Am J Vet Res, 58(1), 66-70.
Publication
Researcher Affiliations
- Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14583, USA.
MeSH Terms
- Animals
- Cartilage, Articular / cytology
- Cartilage, Articular / drug effects
- Cartilage, Articular / metabolism
- Cells, Cultured
- Collagen / biosynthesis
- Culture Media / analysis
- Culture Media / pharmacology
- DNA / biosynthesis
- Dose-Response Relationship, Drug
- Fibrin
- Glycosaminoglycans / biosynthesis
- Horses / metabolism
- Immunohistochemistry
- Proteoglycans / biosynthesis
- Sulfur Radioisotopes
- Thymidine / metabolism
- Transforming Growth Factor beta / analysis
- Transforming Growth Factor beta / pharmacology
- Tritium
Citations
This article has been cited 7 times.- Ongchai S, Somnoo O, Kongdang P, Peansukmanee S, Tangyuenyong S. TGF-β1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes. J Vet Sci 2018 Nov 30;19(6):735-743.
- Athanasiou KA, Responte DJ, Brown WE, Hu JC. Harnessing biomechanics to develop cartilage regeneration strategies. J Biomech Eng 2015 Feb 1;137(2):020901.
- Responte DJ, Arzi B, Natoli RM, Hu JC, Athanasiou KA. Mechanisms underlying the synergistic enhancement of self-assembled neocartilage treated with chondroitinase-ABC and TGF-β1. Biomaterials 2012 Apr;33(11):3187-94.
- Gunja NJ, Uthamanthil RK, Athanasiou KA. Effects of TGF-beta1 and hydrostatic pressure on meniscus cell-seeded scaffolds. Biomaterials 2009 Feb;30(4):565-73.
- Izal I, Ripalda P, Acosta CA, Forriol F. In vitro healing of avascular meniscal injuries with fresh and frozen plugs treated with TGF-beta1 and IGF-1 in sheep. Int J Clin Exp Pathol 2008 Jan 1;1(5):426-34.
- Elder BD, Athanasiou KA. Systematic assessment of growth factor treatment on biochemical and biomechanical properties of engineered articular cartilage constructs. Osteoarthritis Cartilage 2009 Jan;17(1):114-23.
- Akens MK, Hurtig MB. Influence of species and anatomical location on chondrocyte expansion. BMC Musculoskelet Disord 2005 May 17;6:23.
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