Changes in tendon extracellular matrix composition with age

Abstract

Introduction A major aspect of the normal ageing process is the loss of suppleness of tissues; skin becomes wrinkled and there is a decline in joint flexibility. These gradual changes predominantly result due to long‐term alterations in the extracellular matrices (ECMs) of structures including skin, tendons/ligaments, bones, cartilage and blood vessels. ECMs are generally remodelled during an individual's life, but for tendons/ligaments this occurs at a slow rate. Parameters such as slow turnover, long‐term post‐translational modifications and extensive cell–matrix interactions are three aspects of ECM biology, which influence the mechanisms of ageing. Ageing of tendon/ligament is a major problem affecting the mobility of an increasingly ageing population, and age‐related changes lead to numerous musculoskeletal pathologies in old age. Therefore, the initial objective of these studies was to compare the biochemical composition of young vs. old tendon, with an aim of elucidating the mechanisms controlling the ageing process in tendon ECM.Materials and methods Tail tendons were dissected from 2‐, 9‐, 12‐ or 22‐month‐old C57/Blacks, and both protein and RNA were analysed for differences in ECM tendon composition with age (n = 4). Proteoglycans were extracted in 4 m guanidine hydrochloride and sulphated glycosaminoglycans measured using the DMMB assay. Collagens were extracted by acid hydrolysis and total collagen measured using the hydroxyproline assay; additionally, the ratio of collagen types in the ageing tail tendons was analysed by Western blotting with antibodies raised against collagen types I, III and V. The degree of tissue hydration was determined by measuring the water content of the tendons. Matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 expression and activation were detected by gelatin substrate zymography, and the presence of their inhibitors was assessed by reverse zymography. To assess age changes at the transcriptional level, RNA was extracted from tendons, labelled with biotin and cDNA arrays (Affymetrix) performed.Results The results indicate that there is a change in tendon matrix composition with increasing age (Table 1). There is a significant loss in tissue hydration and a reduction in the amount of sulphated GAGs present in the 22‐month tendons compared with young tissue, whilst the amount of collagen present does not significantly alter. 2 months 9 months 12 months 22 months sGAG (µg/mg)* 49.6 ± 8.7 — 40.1 ± 4.6 28.9 ± 0.9 Collagen (µg/mg)† 48.4 ± 5.3 39.9 ± 3.4 40.2 ± 4.7 47.2 ± 4.3 Water content (%) 80.4 ± 3.9 74.6 ± 2.4 74.5 ± 3.8 69.3 ± 2.8 µg/mg wet weight tissue. µg/mg dry weight tissue. Analysis of MMP expression by gelatin zymography demonstrated that there was a significant loss of MMP‐9 expression in the tendons of 9‐ and 12‐month‐old animals. MMP‐9 was only evident in the 2‐ and 22‐month‐old tendons indicative of developmental turnover and a remodelling response, respectively. MMP‐2 expression and activation was evident in all tendon ages analysed, as were the MMP inhibitors – TIMP‐1 and TIMP‐2.Discussion The data demonstrate that during the ageing process, the composition of tendon ECM is modified. This may compromise the response of the tissue to application of mechanical loads hence the increased incidences of musculoskeletal injuries, e.g. tendon/ligament sprains in elderly people. The reduction in both water and sGAGs, and the increased expression of MMP‐9 may be responsible for changes observed in biomechanical properties where the tendons become more brittle and less able to withstand load. Understanding changes in the biochemical composition may allow us to manipulate cell activity and ultimately ECM structure and function to combat these age‐related effects and the musculoskeletal pathologies incurred.