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Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems.

Taylor SE, Vaughan-Thomas A, Clements DN, Pinchbeck G, Macrory LC, Smith RK, Clegg PD - BMC Musculoskelet Disord (2009)

Bottom Line: Significantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage.High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype.While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Clinical Science, University of Liverpool, Neston, South Wirral, UK. Sarah.Taylor@liv.ac.uk

ABSTRACT

Background: There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis.

Methods: Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative PCR (qPCR). Expression levels of genes associated with tendon phenotype were then evaluated in healthy, including developmental, and diseased equine tendon tissue and in tendon fibroblasts maintained in both monolayer culture and in three dimensional (3D) collagen gels.

Results: Significantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage. High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype. While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001). Relative scleraxis gene expression levels in tendon cell monolayer and 3D cultures were significantly lower than in normal adult tendon (P = 0.002, P = 0.02 respectively).

Conclusion: The findings of this study indicate that high expression of both COL1A2 and scleraxis, and low expression of tenascin-C is representative of a tensional tendon phenotype. The in vitro culture methods used in these experiments however, may not recapitulate the phenotype of normal tensional tendon fibroblasts in tissues as evidenced by gene expression.

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Gene expression of normal tendon, bone and cartilage samples. (a) Gene expression of COL1A2, scleraxis, tenascin-C, COMP and decorin in matched samples of tendon, cartilage and bone harvested from normal adult horses. (b) Gene expression of COL2A1, COL10A1 and SOX9 in matched samples of tendon, cartilage and bone harvested from normal adult horses. Relative gene expression data is represented graphically as log transformed values. P values were generated using a mixed effects linear regression model to allow for clustering within individual donors. (c) Gene expression of osteopontin, osteonectin and osteomodulin in matched samples of tendon, cartilage and bone harvested from normal adult horses.
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Figure 1: Gene expression of normal tendon, bone and cartilage samples. (a) Gene expression of COL1A2, scleraxis, tenascin-C, COMP and decorin in matched samples of tendon, cartilage and bone harvested from normal adult horses. (b) Gene expression of COL2A1, COL10A1 and SOX9 in matched samples of tendon, cartilage and bone harvested from normal adult horses. Relative gene expression data is represented graphically as log transformed values. P values were generated using a mixed effects linear regression model to allow for clustering within individual donors. (c) Gene expression of osteopontin, osteonectin and osteomodulin in matched samples of tendon, cartilage and bone harvested from normal adult horses.

Mentions: Figure 1 shows the data obtained for expression levels of genes presumed to be associated with tendon (a), bone (b) and cartilage (c). Scleraxis showed significantly higher expression in tendon than in bone (P = 0.002) (Figure. 1a), and whilst higher levels of expression were identified in tendon than cartilage this did not reach statistical significance. As expected, significantly higher COL1A2 was expressed in both tendon and bone than in cartilage (P = 0.01 and P = 0.008, respectively) (Figure 1a). Tenomodulin was identified in both tendon and bone but could not be detected in cartilage. No significant difference could be identified in the levels tenomodulin expression in tendon and bone. Tenascin-C expression was significantly lower in tendon than in bone (P = 0.02). COMP expression was significantly higher in tendon than in bone (P = 0.02) but no difference in expression was identified between tendon and cartilage (Figure. 1a). Decorin expression was highest in cartilage (P = 0.03) and lowest in bone (P = 0.02). Osteopontin, Runx2 and osteomodulin were able to discriminate between tendon and bone (P = 0.003, P = 0.03 and P = 0.01, respectively) (Figure. 1b) but Runx2 was the only hypothesised bone marker that distinguished between bone and cartilage (P = 0.007). Of the cartilaginous markers, only COL2A1 was significantly lower in tendon than in both bone and cartilage (P = 0.005 and P = 0.0003). Cartilage showed higher expression of COL10A1 and SOX9 than tendon (P = 0.01 and P = 0.005 respectively) (Figure. 1c). From the matched normal tissue samples a panel of COL1A2, scleraxis and tenascin-C were selected as the most discriminating genes of tendon phenotype.


Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems.

Taylor SE, Vaughan-Thomas A, Clements DN, Pinchbeck G, Macrory LC, Smith RK, Clegg PD - BMC Musculoskelet Disord (2009)

Gene expression of normal tendon, bone and cartilage samples. (a) Gene expression of COL1A2, scleraxis, tenascin-C, COMP and decorin in matched samples of tendon, cartilage and bone harvested from normal adult horses. (b) Gene expression of COL2A1, COL10A1 and SOX9 in matched samples of tendon, cartilage and bone harvested from normal adult horses. Relative gene expression data is represented graphically as log transformed values. P values were generated using a mixed effects linear regression model to allow for clustering within individual donors. (c) Gene expression of osteopontin, osteonectin and osteomodulin in matched samples of tendon, cartilage and bone harvested from normal adult horses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651848&req=5

Figure 1: Gene expression of normal tendon, bone and cartilage samples. (a) Gene expression of COL1A2, scleraxis, tenascin-C, COMP and decorin in matched samples of tendon, cartilage and bone harvested from normal adult horses. (b) Gene expression of COL2A1, COL10A1 and SOX9 in matched samples of tendon, cartilage and bone harvested from normal adult horses. Relative gene expression data is represented graphically as log transformed values. P values were generated using a mixed effects linear regression model to allow for clustering within individual donors. (c) Gene expression of osteopontin, osteonectin and osteomodulin in matched samples of tendon, cartilage and bone harvested from normal adult horses.
Mentions: Figure 1 shows the data obtained for expression levels of genes presumed to be associated with tendon (a), bone (b) and cartilage (c). Scleraxis showed significantly higher expression in tendon than in bone (P = 0.002) (Figure. 1a), and whilst higher levels of expression were identified in tendon than cartilage this did not reach statistical significance. As expected, significantly higher COL1A2 was expressed in both tendon and bone than in cartilage (P = 0.01 and P = 0.008, respectively) (Figure 1a). Tenomodulin was identified in both tendon and bone but could not be detected in cartilage. No significant difference could be identified in the levels tenomodulin expression in tendon and bone. Tenascin-C expression was significantly lower in tendon than in bone (P = 0.02). COMP expression was significantly higher in tendon than in bone (P = 0.02) but no difference in expression was identified between tendon and cartilage (Figure. 1a). Decorin expression was highest in cartilage (P = 0.03) and lowest in bone (P = 0.02). Osteopontin, Runx2 and osteomodulin were able to discriminate between tendon and bone (P = 0.003, P = 0.03 and P = 0.01, respectively) (Figure. 1b) but Runx2 was the only hypothesised bone marker that distinguished between bone and cartilage (P = 0.007). Of the cartilaginous markers, only COL2A1 was significantly lower in tendon than in both bone and cartilage (P = 0.005 and P = 0.0003). Cartilage showed higher expression of COL10A1 and SOX9 than tendon (P = 0.01 and P = 0.005 respectively) (Figure. 1c). From the matched normal tissue samples a panel of COL1A2, scleraxis and tenascin-C were selected as the most discriminating genes of tendon phenotype.

Bottom Line: Significantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage.High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype.While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Clinical Science, University of Liverpool, Neston, South Wirral, UK. Sarah.Taylor@liv.ac.uk

ABSTRACT

Background: There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis.

Methods: Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative PCR (qPCR). Expression levels of genes associated with tendon phenotype were then evaluated in healthy, including developmental, and diseased equine tendon tissue and in tendon fibroblasts maintained in both monolayer culture and in three dimensional (3D) collagen gels.

Results: Significantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage. High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype. While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001). Relative scleraxis gene expression levels in tendon cell monolayer and 3D cultures were significantly lower than in normal adult tendon (P = 0.002, P = 0.02 respectively).

Conclusion: The findings of this study indicate that high expression of both COL1A2 and scleraxis, and low expression of tenascin-C is representative of a tensional tendon phenotype. The in vitro culture methods used in these experiments however, may not recapitulate the phenotype of normal tensional tendon fibroblasts in tissues as evidenced by gene expression.

Show MeSH
Related in: MedlinePlus