Tenascin-C Expression in Equine Tendon-derived Cells During Proliferation and Migration

Abstract

In vitro cell studies might be a useful tool for studying tendon pathology, but no suitable in vitro models exist for tendon disorders. The purpose of this study was to confirm whether cell scratch culture using tendon-derived fibroblasts can provide a suitable in vitro tendon disorder model. Extracellular matrix components were examined immunohistochemically in tendon tissue, and then their related gene expression levels were analyzed by conventional reverse transcription polymerase chain reaction (RT-PCR) and/or quantitative real-time RT-PCR in tissues and cells. Collagen type I (Col I), collagen type III (Col III), tenascin-C (TN-C) and cartilage oligomeric matrix protein (COMP) were detected in tendon tissue sections, and RT-PCR confirmed their expression in tendon tissue and cells. Cells that had been cultured from explanted tendon tissue maintained the characteristics of in vivo tendon cells. The combination of TN-C and COMP might be a useful marker of tendon cells because they display more tendon-specific expression than Col I and III. In particular, the significant increase of TN-C mRNA expression in the scratch wound assay, at 12 hr after scratching, concomitant with the regeneration of the cell sheet, indicates its crucial role in tendon cell proliferation and migration. Thus, TN-C appears to be a key factor in tendon wound healing. In vitro cell scratch assays using tendon cells appear to mimic the repair of tendon tissue after injury.

Keywords: cartilage oligomeric matrix protein; collagen; equine tendon; scratch wound assay; tenascin-C.

Fig. 1.

Immunohistochemical detection of matrix proteins. Immunoreactivity of Col I (A), Col III (B), TN-C (C) and COMP (D) in longitudinal sections of the equine tendon.

Fig. 2.

Vimentin and TN-C immunoreactivity in cultured equine tendon cells. A: Vimentin staining, B: TN-C staining. Different specimens were used independently for each staining method; the same magnification was used in all cases.

Fig. 3.

Analysis of TN-C expression in various tissues. RNA was extracted from the tendon, connective tissue, skeletal muscle, patella, ventricular myocardium, atrioventricular valve, and liver. The primer sets used for the RT-PCR are described in Table 2.

Fig. 4.

Quantitative expression of Col I, Col III, TN-C, and COMP mRNA in scratch wound healing assay. Col I, Col III and COMP did not display significant increases in their expression at any time point, but TN-C expression was significantly increased at 12 hr compared with that observed at 0 hr (control). The data are the averages of triplicate experiments. ** P<0.01, n=3, mean ± SD.