Overuse patellar tendinopathy can contribute to 30% of injuries in individuals participating in exercise and sports (1). Patellar tendinopathy is characterized by proximal patellar tendon pain and tenderness that inhibits an individual's ability to exercise. The predominant feature of tendinopathy is a change in the appearance and organization of the extracellular matrix of the tissue, especially the disorganization of collagen fibers (2, 3).
Matrix disorganization may be the result of degenerative changes or a failed healing process associated with mechanical overloading (4). Degenerative changes may be at the end of a continuum of change that is proposed to include early cell-driven changes (reactive tendinopathy) through increasing extracellular matrix disorganization (tendon disrepair and degenerative tendinopathy) (5). Although the condition is likely to be driven by local levels of cytokines, tendinopathy does not appear to involve wider inflammatory processes (4, 6).
In normal tendons, the tenocytes are embedded in an extracellular matrix made up of type I collagen fibers arranged in parallel bundles with smaller amounts of other collagens, proteoglycans, hyaluronan, and noncollagenous proteins. Proteoglycans play a role in tissue hydration and regulate collagen integrity, while collagen provides the tissue with its tensile strength. Tenocytes are responsible for the synthesis and degradation of all of the macromolecular components of tendon. In normal tendons, the metabolism of tenocytes is reflective of the maintenance of the extracellular matrix where there is a balance between synthesis and catabolism of matrix macromolecules. Yet normal tendon has a limited potential to respond to changes in mechanical loading and to initiate repair of the extracellular matrix in response to overloading.
We have previously demonstrated increased levels of the large aggregating proteoglycans, aggrecan and versican, and of the small proteoglycans, biglycan and fibromodulin, in the extracellular matrix in human patellar tendinopathy compared with normal tissue (7). Furthermore, both the aggrecan and versican macromolecules in the matrix of abnormal tissue are present in significantly degraded states (7). In that study (7), no significant change was observed in the levels of type I collagen within the extracellular matrix of the abnormal tissue, nor were there significant changes in the expression of genes for the major collagens and proteoglycans associated with the tissue.
Previous studies of normal and abnormal human Achilles tendons have investigated gene expression for major matrix macromolecules and proteinases responsible for the catabolism of matrix molecules (8–10). This led to the suggestion that tendinopathy may result from a change in the metabolism of the tissue (11, 12).
No previous studies have directly investigated the dynamics of the metabolism of matrix macromolecules in normal and diseased human tendons. In this study, we determined the rates of catabolism and synthesis of newly synthesized proteoglycans in normal human patellar tendons and those with chronic overuse tendinopathy. The present study also established expression of matrix proteinases (matrix metalloproteinase [MMP] and ADAMTS) as well as tissue inhibitor of metalloproteinases (TIMP) in normal and abnormal tendons.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
The rate of synthesis of proteoglycans was ∼25-fold greater in abnormal patellar tendons than in normal tissue. This was due to the significant increase in the synthesis of the large proteoglycans aggrecan and versican in abnormal tissue, whereas negligible levels of these proteoglycans were synthesized in normal tissue (Figure 2). Normal tissue predominantly synthesized small leucine-rich proteoglycans (Figure 2). The increase in the synthesis of large proteoglycans by abnormal tissue was reflected in greater tissue levels of large proteoglycans in the matrix of tendons in human patellar tendinopathy (Figure 3). This is consistent with the results of previous studies showing increased levels of large proteoglycans associated with overuse tendinopathy (7, 21). In addition, the higher cellularity of the abnormal tissue is likely to contribute to the higher incorporation of the radiolabel observed in this tissue (discussed below).
The overall rate of loss of 35S-labeled proteoglycans from the extracellular matrix of abnormal human patellar tendons was greater than the rate measured in normal tendons (Figure 1). This was primarily due to the loss of the radiolabeled large aggregating proteoglycans, which were more abundant in the abnormal tissue, and the majority of these were lost in the first 4 days of the culture period. These results do not differ from those in normal tissue where large proteoglycans are catabolized rapidly in normal tendons and other dense fibrous connective tissues (13, 22). In contrast, small proteoglycans are lost at a much slower rate in these tissues, which was also observed in both abnormal and normal tendons in the present study. In abnormal samples, the rate of loss of small proteoglycans was detected after the first 4 days of culture (Figures 1 and 2). These results indicate that the main difference between the normal and abnormal tendons is in the rate of synthesis of large proteoglycans in particular, whereas the rates of loss of both the large and the small proteoglycans follows the pattern observed in the normal tissue.
This study also shows that large proteoglycans are rapidly degraded so that mainly fragments remain in cultured tissue (Figures 2 and 3). To elucidate whether in overuse patellar tendinopathy there is a change in the gene expression of matrix-degrading enzymes and their inhibitors, which have been shown to or are likely to play a part in the proteolytic processing of proteoglycans (10, 17, 23, 24), the expression of genes for a number of proteinases (MMPs and ADAMTS) and their tissue inhibitors (TIMPs) was determined in normal and abnormal tissue. Of these, only MMP-9 and TIMP-1 were found to have been significantly up-regulated in abnormal tendons (Figure 4). This suggests that the proteolysis of proteoglycans in the abnormal tissue is not likely to have been affected at the gene expression level of proteinases and their inhibitors.
These observations are consistent with those of Jones et al (9), who showed that there was no difference in the expression of ADAMTS-1, ADAMTS-4, and ADAMTS-5 genes between human Achilles tendons with chronic pain and normal tendons. Corps et al (10) also reported that there was no significant change in the expression of ADAMTS-4 between normal Achilles tendons and Achilles tendons with chronic pain. In the latter study, Corps et al analyzed the nature of ADAMTS-4 that was present in the matrix and showed an increase in the level of the processed active form of this proteinase in painful Achilles tendons. Taken together, the results of the present study and those of the studies by Jones et al (9) and Corps et al (10) support the notion that in painful tendons ADAMTS-4 activity is elevated by the activation of the proteinase and not by increased gene expression.
It should be noted that Jones et al (9) and Corps et al (10) showed that in ruptured Achilles tendons there was a significant up-regulation of the expression of ADAMTS-4 compared with that in painful or normal Achilles tendons, and that Western blot analysis showed that the mature, inactive form of ADAMTS-4 was present in the matrix (10). In the present study, the elevated expression of MMP-9 and TIMP-1 genes in abnormal tendons likely reflects changes in collagen metabolism in abnormal tendons. Jones et al (9) showed no changes in the expression of MMP-9 and TIMP-1 in painful Achilles tendons, but a significant increase in the expression of these genes, in addition to a significant increase in MMP-1 and MMP-19 expression, was observed in ruptured Achilles tendons.
A number of studies have shown that increased cellularity is associated with overuse tendinopathy (7, 10, 19, 20), and this would explain the increase in total levels of mRNA measured in abnormal tendons in the present study. It is likely that, as the result of the underlying pathologic process, more cells are present in the tissue, thereby producing a net increase in tissue proteoglycans. However, the results of the analysis of the radiolabeled proteoglycans that were synthesized by abnormal tissue strongly suggest that more large proteoglycans are synthesized and retained within the matrix of abnormal tissue than normal tissue. Since gene expression of large proteoglycans does not differ significantly between normal and abnormal tendons (7), this suggests that transcriptional regulation may not play a direct role in the pathologic mechanism, and that regulation may be occurring at the level of translation of the appropriate mRNA into protein. Regulation of catabolism appears to be the outcome of the activation of ADAMTS and possibly MMPs. The details of the mechanism of these regulatory processes warrant further investigation.
The absence of inflammatory cells has brought into question the role of inflammation in the pathologic mechanism, but it is likely that changes in the phenotype and metabolism of tendon cells in overuse tendinopathy may be driven by locally produced cytokines. Indeed, increased synthesis of proteoglycans in abnormal tendons may be driven by increases in growth factors and cytokines such as interleukin-1α (IL-1α), IL-1β, transforming growth factor β, and/or tumor necrosis factor α (25–27), possibly as a result of repetitive mechanical stress. These growth factors and cytokines may also stimulate the activation of matrix-degrading enzymes such as ADAMTS and MMP, eventually leading to the breakdown and disorganization of the extracellular matrix that is observed in overuse tendinopathy (28). Notably, in the present study, tissue levels of the small proteoglycans decorin and fibromodulin were maintained throughout the culture period in abnormal tendons (Figures 3C and D, lanes d–f and j–l), whereas tissue levels of the large proteoglycans decreased with time in culture (Figures 3A and B, lanes d–f, and j–l).
Overall, these data support the concept that tendons exhibiting overuse tendinopathy are characterized by an altered extracellular matrix that has a different composition, structure, and metabolism compared with normal tendons, and that these changes result in part from the increased synthesis of large proteoglycans that are rapidly lost from the extracellular matrix of abnormal tendons. These findings indicate that the extracellular matrix of the tendon exhibiting overuse tendinopathy is a dynamic structure characteristic of an attempted adaptive or healing process.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Samiric had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Parkinson, Samiric, Ilic, Cook, Feller, Handley.
Acquisition of data. Parkinson, Samiric, Ilic, Cook, Feller, Handley.
Analysis and interpretation of data. Parkinson, Samiric, Ilic, Cook, Feller, Handley.