Overuse injuries and trauma in tendon often involve acute or chronic pain and eventual matrix destruction. Anti-inflammatory drugs have been used as a treatment, however, the cellular and molecular mechanisms of the destructive processes in tendon are not clearly understood. It is thought that an inflammatory event may be involved as an initiating factor. Mediators of the inflammatory response include cytokines released from macrophages and monocytes. Interleukin-1 beta (IL-1 p) is a candidate proinflammatory cytokine that is active in connective tissues such as bone and cartilage. We hypothesized that tendon cells would express receptors and respond to IL-1 b in an initial "molecular inflammation" cascade, that is, connective tissue cell expression of cytokines that induce matrix destructive enzymes. This cascade results in expression of matrix metalloproteinases (MMPs) and aggrecanases that may lead to matrix destruction. Normal human tendon cells from six patients were isolated, grown to quiescence and treated with human recombinant IL-lP in serum-free medium for 16 h. Total RNA was isolated and mRNA expression assessed by semiquantitative RT-PCR. IL-lP (1 nM) induced mRNAs for cyclooxygenase 2 (COX2), MMP-I, -3, -13 and aggrecanase-1 as well as IL-1 j3 and IL-6, whereas mRNAs for COX1 and MMP-2 were expressed constitutively. The IL-lj3-treated tendon cells released prostaglandin E2 (PGE2) in the medium, suggesting that the inducible COX2 catalyzed this synthesis. Induction of PGE2 was detectable at 10 pM IL-1 j3. IL-10 also stimulated MMP-1 and -3 protein secretion. Induction of MMP-1 and -3 was detectable at 10 pM IL-1 fi. Post-injury or after some other inciting events, exogenous IL-1 P released upon bleeding or as leakage of local capillaries may drive a proinflammatory response at the connective tissue cell level. The resulting induction of COX2, MMP-1 and -3 may underscore a potential for nonlymphocyte-mediated cytokine production of MMPs that causes matrix destruction and a loss of tendon biomechanical properties. Endogenous IL-1 b might contribute to the process through a positive feedback loop by stimulating expression and accumulation of MMPs in the tendon matrix.

ABSTRACT:Mesenchymal stems cells have a demonstrated ability to differentiate into muscle, bone, and fat. Determining whether these same cells have the ability to differentiate into tendon-like fibroblasts has been hampered by the lack of specific tendon cell marker genes. In order to identify molecular markers of mature tendon, expression profiling was used to identify genes expressed in adult rat and human tendon tissue compared to other musculoskeletal tissues. Using this technique, approximately 1,600 transcripts appeared to be selectively expressed in rat tendon tissue and approximately 300 transcripts appeared to be selectively expressed in human tendon tissue, with 20 genes selectively expressed in both human and rat tendon tissue. Of these common tendon-selective genes, thrombospondin-4 (THBS4) and tenomodulin (TNMD) were found to have the highest tendon-selective expression compared to other tissues examined. Interestingly, expression of these tendon-selective genes, which are present in primary tendon fibroblasts, is lost when these cells are placed in two-dimensional culture systems. In conclusion, this study has defined a set of tendon-selective genes present in both adult rat and human tendons. Identification of tendon-selective genes provides potential molecular tools to facilitate a better understanding of tendon development and tendon repair. ß

Little is known about the factors that initiate and propagate tendon overuse injuries, but chronic inflammation and matrix destruction have been implicated. The purpose of this study was to evaluate the production of cyclooxygenase 11 (COX-2) and matrix metalloproteinases (MMPs) by tendon cells exposed to cyclic strain and inflammatory cytokines in vitro. Rabbit Achilles tendon cells were subjected to a stretching protocol with 5% elongation at 0.33 Hz for 6 h, or treated with 1000 pM interleukin1b (IL-lB), or exposed to IL-I 0 and stretching together. Gene expression was evaluated by RT-PCR and production of stromelysin was quantified with an ELISA. IL-I@ induced the expression of the collagenase-1 and stromelysin-I genes. Production of stromelysin proenzyme by cells stimulated with IL-ID was 17 times higher than production by control cells. Cells exposed to IL-lp and stretching produced 20 times more stromelysin than control cells. Cells subjected to stretching alone did not produce more stromelysin than control cells. The synergistic effect of IL-1 p and stretching was observed at doses of IL-lp ranging from 10 to 1000 pM. These data suggest that mechanical load and inflammatory cytokines can initiate a matrix destructive pathway in tendon that is more pronounced than with mechanical loading or inflammation alone.

The goals of this study were to investigate the response of the rat supraspinatus tendon to overuse at the molecular level using transcriptional profiling, and to identify potential markers of tendinopathy. Adult rats were subjected to an overuse protocol that consists of downhill running (10% grade) at 17 m/min for 1 h/day, 5 days/week, for a total of either 1, 2, or 4 weeks. Another group of rats served as nonrunning time 0 controls. Transcriptional profiling was performed on the supraspinatus and patellar tendons using an Affymetrix rat genome array. A gene was considered to be differentially expressed if the p value from an ANOVA test was less than 0.01 and the difference between runners and controls was at least twofold at any time point. The supraspinatus tendon had increased expression of well-known cartilage genes such as col2a1, aggrecan, and sox9. These genes were not regulated in the patellar tendon, an internal comparator. Few genes associated with inflammation, or angiogenesis, were differentially expressed, and no significant change in the regulation of matrix metalloproteinases was detected. The results of this study suggest that by expressing more cartilage genes, the tendon is converting toward a fibrocartilage phenotype as a result of the repetitive loading and repeated compression of the tendon as it passes through the acromial arch. ß

Delivery of rhBMP-12 in several sponge carriers has the potential to accelerate healing of rotator cuff repairs. Accelerated repair may allow shorter rehabilitation and an earlier return to occupational and recreational activities.