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

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.

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.

Figure 2

Gene expression of developing and diseased tendon and in vitro gene expression. (a) Gene expression of COL1A2, scleraxis, tenascin-C in SDFT collected from foetuses, yearlings and adults. (b) Gene expression of COL1A2, scleraxis, tenascin-C in acute and chronic disease in comparison to normal adult tendon. (c) Gene expression of COL1A2, scleraxis, tenascin-C in 2D and 3D in vitro culture in comparison to normal adult tendon. (2D P1 = passage 1 monolayer culture, 2D P5 = passage 5 monolayer culture, 3D = tendon fibroblasts cultured in collagen gels). P values were generated using a two sample student's t-test for normally distributed log transformed data.