Abstract
Purpose: Tenomodulin (Tnmd) is the best known mature tendon factor for tendons and ligaments tissue with reported important regulatory roles. In addition, Tnmd C-terminal cysteine-rich domain has been described to exert anti-angiogenic functions in in vitro angiogenic assays as well as in vivo models of tendon injury and age- associated cardiac valve diseases. Interestingly, Tnmd expression in the intervertebral disc (IVD), which is normally avascular tissue, has been also suggested. Hence, the purpose of this study was first, to map the exact expression pattern of Tnmd during IVD development and aging and second, by implementing Tnmd-knockout mouse model to examine if Tnmd plays a role in IVD homeostasis. Methods: Lumbar IVDs of Tnmd knockout and wild type mice (n = 3–5) were first investigated by immunohistology for Tnmd expression in seven different postnatal stages. For detection of degenerative tissue changes, histological scoring and various stainings, namely hematoxylin/eosin (HE), Safranin O (SO), CD31 (endothelium), TUNEL (apoptosis) and type X collagen and Runx2 (hypertrophy markers) were carried out. Statistical testing by two-tailed non-parametric Mann-Whitney test was performed with the quantitative data on histlogical scoring, blood vessels and apoptotic cell count. Results: Immunofluorescence staining of Tnmd protein in developing (newborn, day 15, 1 and 2 months), adult (6 and 12 months) and aged (18 months) IVDs of WT mice showed that Tnmd is mainly expressed in the outer annulus fibrosus (AF) and nucleus pulposus (NP) throughout development as well as retained in the adult and aged stages. However, a marked downregulation in Tnmd expression was observed at 12 and 18 months of age. HE and SO stainings revealed clear degenerative changes such as lower IVD high, loose and disarranged fibers in the outer AF and presence of chondrocyte-like cells in the inner AF and NP of Tnmd -/- IVDs. Histological scoring at 6 and 12 months confirmed significant degeneration grade (p<0.01 and p<0.001, respectively). Immunofluorescence analysis for CD31-labelled vessels revealed significantly increased vessel count in the OAF of Tnmd-/- than WT mice (p<0.05). TUNEL staining demonstrated also increased number of apoptotic cells in the OAF of Tnmd-/- compared to WT mice (p<0.05). Last, immunofluorescence analysis of Col X and Runx2 protein expression suggested upregulation of both hypertrophic markers in the NP of Tnmd-/- mice to that of WT. Conclusions: Taken together, the above findings provide the first evidence that Tnmd is an important factor in prevention of IVD degeneration.
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