J Vet Sci.  2018 Nov;19(6):735-743. 10.4142/jvs.2018.19.6.735.

TGF-β1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes

• Ongchai S
• Somnoo O
• Kongdang P
• Peansukmanee S
• Tangyuenyong S

• KMID: 2427021
• DOI: http://doi.org/10.4142/jvs.2018.19.6.735

Abstract

We investigated the effect of transforming growth factor beta 1 (TGF-β1) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with TGF-β1 at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of TGF-β1. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by TGF-β1 stimulation was dose and time dependent. TGF-β1 was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in TGF-β1 treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the TGF-β1-treated scaffolds. Together, our results suggest that TGF-β1 has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

Keyword

chondrocytes; horses; hyaluronan; hyaluronan synthases; transforming growth factors

MeSH Terms

Bisbenzimidazole
Cartilage
Cell Proliferation
Chondrocytes*
Culture Media, Conditioned
DNA
Enzyme-Linked Immunosorbent Assay
Extracellular Matrix
Gelatin
Gene Expression
Horses
Hyaluronic Acid*
Immunohistochemistry
Microscopy, Electron, Scanning
Polymerase Chain Reaction
Reverse Transcription
RNA, Messenger
Transforming Growth Factor beta
Transforming Growth Factors
Up-Regulation
Bisbenzimidazole
Culture Media, Conditioned
DNA
Gelatin
Hyaluronic Acid
RNA, Messenger
Transforming Growth Factor beta
Transforming Growth Factors

Figure

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  Fig. 1 Transforming growth factor beta 1 (TGF-β1) upregulated hyaluronan synthase 2 (HAS2) mRNA levels and enhanced hyaluronan (HA) synthesis in monolayer cultures of equine articular chondrocytes. Chondrocytes treated with different concentrations of TGF-β1 for 3 days (A) and 3 h (B). HA levels in the conditioned media and HAS2 mRNA levels were determined by enzyme-linked immunosorbent assay and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. (C) HAS2 mRNA levels determined by semi-quantitative RT-PCR, measured from chondrocytes cultured in the absence or presence of 2.5 ng/mL of TGF-β1 for the times shown. GAPDH, glyceraldehydes-3-phosphate dehydrogenase. Asterisks denote significant differences at *p < 0.05 and **p < 0.01.

• 

  Fig. 2 Transforming growth factor beta 1 (TGF-β1) tended to increase cell proliferation in monolayer cultures of equine articular chondrocytes. Chondrocytes were treated with TGF-β1 in different concentrations. Cell proliferation was analyzed on days 0, 4, and 7 using the alamarBlue assay.

• 

  Fig. 3 Transforming growth factor beta 1 (TGF-β1) enhanced hyaluronan (HA) synthesis in three-dimensional cultures of equine articular chondrocytes. Chondrocytes embedded in scaffold were cultured for 21 days in the absence or presence of 10 ng/mL of TGF-β1. (A) Gross appearance of cultured scaffold at day 21. Scale bars = 5 mm. (B) Cumulative amount of HA in conditioned media was measured by enzyme-linked immunosorbent assay (ELISA) at indicated time intervals. The contents of HA (C) and uronic acid (D) in scaffold structures were determined at day 21 using ELISA and a colorimetric assay, respectively. Asterisks denote significant differences at *p < 0.05.

• 

  Fig. 4 Transforming growth factor beta 1 (TGF-β1) decreased cell proliferation and DNA content in three-dimensional cultures of equine articular chondrocytes. (A) Proliferation of equine chondrocytes was determined using the alamarBlue assay. (B) DNA content in scaffolds was analyzed based on two independent experiments using Hoechst dye 33258 assays. Cultures were maintained in the absence (control) or presence of 10 ng/mL TGF-β1. Asterisks denote significant differences at *p < 0.05.

• 

  Fig. 5 Transforming growth factor beta 1 (TGF-β1) enhanced extracellular matrix synthesis as visible in scanning electron micrographs of scaffolds from three-dimensional cultures of equine articular chondrocytes. The acellular scaffold has a highly porous appearance. Cellular scaffolds were cultured in the absence (control) or presence of 10 ng/mL of TGF-β1 for 21 days. 35× (upper), 1,000× (bottom).

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  Fig. 6 Transforming growth factor beta 1 (TGF-β1) enhanced extracellular matrix and hyaluronan (HA) synthesis as shown by histological and immunohistochemical staining of scaffolds from three-dimensional cultures of equine articular chondrocytes. H&E staining and immunohistochemical localization of HA in scaffolds embedded with equine chondrocytes in the absence (A, C, E, and G) or presence (B, D, F, and H) of 10 ng/mL of TGF-β1 for 21 days. 200× (A and B), 400× (C, D, G, and H), 40× (E and F).

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