Abstract
Introduction
The ability of tendons to withstand stress generally decreases with age, often resulting in increased tissue degeneration and decreased regeneration capacity. However, the underlying molecular and cellular mechanisms of tendon senescence remain poorly characterized. Therefore, the aim of the current study was to identify genes showing an age-dependent altered expression profile in tendons.
Materials and Methods
A suppression-subtractive-hybridization (SSH) screen comparing cDNA libraries generated from Achilles tendons of mature-adult (3 months) and old (18 months) female C57BL/6 mice was conducted. Subsequently, the differential expression of the identified genes was validated by RT-qPCR and selected genes were then further analysed by immunohistochemistry and Western blot. To investigate age-related structural alterations in the collagenous extracellular matrix we applied SHG-microscopy and TEM. In vitro experiments with young and old tendon derived stem/progenitor cells (TDSCs) involved wounding assays, tendon-like constructs as well as collagen gel contraction assays.
Results
Among 168 identified genes, several ECM genes showed a differential expression, including Col1a1, Col3a1, fibronectin, fibromodulin, thrombospondin-1, decorin, biglycan, lysyl oxidase, and Sparc. As evidenced by RT-qPCR the mRNA levels of these genes were down-regulated in old tendons and in old TDSCs. Additionally, protein content of SPARC and Lysyl oxidase was diminished in vitro in cellular extracts from old TDSCs. The impact of Sparc on tendon ageing was further analysed in young and old Sparc−/− C57BL/6 as well as in age-matched wildtype mice. Tendons of Sparc−/− mice are generally thinner and TEM revealed thinner collagen fibrils and a larger interfibrillar area. Further, TDSCs of old and Sparc−/− tendons formed thinner in vitro tendon constructs, showed a higher collagen gel contraction capacity, and display altered cell-ECM adhesion and cell migration properties when compared to young wildtype cells.
Employing SHG-microscopy we further observed age-related changes in the collagenous structure of Achilles tendons.
Discussion
The decreased expression of ECM proteins and modulators thereof in old tendons in combination with structural changes is potentially associated with an increased risk of tendon injury in the elderly, since structure and composition of the tendon are directly related to its function.
