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Ciprofloxacin-induced tendinopathy and tendon rupture have been previously described, principally affecting the Achilles tendon. This study was designed to investigate the effect of ciprofloxacin on expressions of matrix metalloproteinases (MMP)-2 and -9, tissue inhibitors of metalloproteinase (TIMP)-1 and -2 as well as type I collagen in tendon cells. Tendon cells intrinsic to rat Achilles tendon were treated with ciprofloxacin and then underwent MTT (tetrazolium) assay. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis were used, respectively, to evaluate the gene and protein expressions of type I collagen, and MMP-2. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and -9. Reverse zymography was used to evaluate TIMP-1 and -2. Immunohistochemical staining for MMP-2 in ciprofloxacin-treated tendon explants was performed. Collagen degradation was evaluated by incubation of conditioned medium with collagen. The results revealed that ciprofloxacin up-regulated the expression of MMP-2 in tendon cells at the mRNA and protein levels. Immunohistochemistry also confirmed the increased expressions of MMP-2 in ciprofloxacin-treated tendon explants. The enzymatic activity of MMP-2 was up-regulated whereas that of MMP-9, TIMP-1 or TIMP-2 was unchanged. The amount of secreted type I collagen in the conditioned medium decreased and type I collagen was degraded after ciprofloxacin treatment. In conclusion, ciprofloxacin up-regulates the expressions of MMP-2 in tendon cells and thus degraded type I collagen. These findings suggest a possible mechanism of ciprofloxacin-associated tendinopathy. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:67–73, 2011
The fluoroquinolone class of antibiotics (e.g., ciprofloxacin, levofloxacin, moxifloxacin) has been used to treat a wide range of infections. It was reported in the literature that quinolone-induced tendinopathy or even tendon rupture principally affected the Achilles tendon.1, 2 However, the mechanisms by which ciprofloxacin predisposes tendinopathy or even tendon rupture remain to be investigated.
In animal studies with fluoroquinolone-treated rats, disorganization of the extracellular matrix (ECM), inflammation of the paratenon and degenerative changes in tendon cells have been demonstrated.3, 4 Besides, fluoroquinolone class of antibiotics has been documented to exert a number of effects on various cell types in vitro, including reduced expression of some ECM proteins,5, 6 decreased mitochondrial activity,6 enhanced matrix metalloproteinase (MMP) expression5, 7 noncytotoxic inhibition of tendon cell proliferation5, 8 and inhibition of tendon cell migration.9 Further matrix degradation or repeated micro-trauma to a tendinopathic tendon might inevitably lead to a tendon rupture.
The basic constituent of a tendon is collagen, which accounts for 70% of the dry weight of a tendon.10 Approximately 90% of collagen in normal tendons is type I and less than 10% is type III collagen.11 Type I collagen is organized into fibrils grouped in parallel to form organized bundles while type III collagen is almost completely confined to the endotendineum which surrounds the bundles.12 Tendon cells (fibroblasts), which are its basic cellular component of a tendon, are the source of collagen production, protein mediators of repair, and matrix proteoglycans.10 It appears that the physiologic response of tendon cells to trauma induces production of both types I and III collagen.13 The biomechanical properties of a tendon are primarily a feature of the ECM (mainly collagen), which is in a state of dynamic equilibrium between synthesis and degradation.14
Gelatinase such as MMP-2 and MMP-9 are MMPs with collagenolytic activity.15, 16 The activity of MMP is inhibited by tissue inhibitors of MMPs (TIMPs)17, 18 and the balance in MMPs and TIMPs regulated tendon remodeling. The expression of MMP-2 was up-regulated in Achilles tendinopathy and MMP-9 expression was also up-regulated in the ruptured area of Achilles tendon.19, 20 It is concerned that the potential combination of increased local matrix-degrading activity by enhanced MMP expression and/or decreased ECM production in tendon cells after ciprofloxacin treatment might induce the occurrence of tendinopathy or tendon rupture. The aim of this study is to investigate the effect of ciprofloxacin on expressions of type I collagen, MMP-2, MMP-9, TIMP-1, and TIMP-2 of tendon cells.
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There are evidences indicating that the increased expressions of certain MMPs are associated with tendinopathy or tendon rupture. Immunohistochemistry studies have shown MMP-1 expressed at the tear edge in ruptured supraspinatus tendons22 and in patellar tendinosis.23 Meanwhile, there is an increased amount of denatured collagen and MMP-1 in ruptured supraspinatus tendon which implied the increase in MMP-1 activity and degradation of the collagen fibril network might be a potential cause of a tendon rupture.24 The up-regulation of MMP-13 at both the mRNA and protein levels was demonstrated in patients with complete tears of rotator cuff tendons.25 In accordance with this study, an animal model revealed that expression of MMP-2 at the edges of an acute tear in the supraspinatus tendon.26 Besides, the expression of MMP-2 was up-regulated in Achilles tendinopathy.19, 20
Previous study using canine tenocytes with treatment of ciprofloxacin has demonstrated an increase of matrix-degrading activity.5 The present study further confirms that MMP-2 is up-regulated both in vitro and ex vivo after ciprofloxacin treatment. Collagenase which cleaves collagen at a single locus in the collagen triple helix, creating 3/4 and 1/4 fragments which can then be further degraded by a variety of proteinases including the gelatinases, such as MMP-2 and MMP-9.27 A study of synovial fluids from the glenohumeral joint of patients with rotator cuff pathology showed no change in the levels of TIMP-1.28 This study also revealed that ciprofloxacin exerted no effect on TIMP-1 or TIMP-2 expression.
The results of this study implied that ciprofloxacin might exert a negative effect on tendon structure or its healing process through the mechanism by up-regulating gelatinase (MMP-2) expression. This finding is further supported by the result that decreased amount of type I collagen in the conditioned medium and increased degradation of type I collagen. Blockage of MMPs has been demonstrated to enhance tendon-bone healing of anterior cruciate ligament grafts that further supports the hypothesis of this study.29
In studies of human tenocytes pretreated with interleukin-1 (IL-1), it revealed an up-regulation of MMP-1 and MMP-3 expression by ciprofloxacin.7 As compared with this previous study, the present study documented that ciprofloxacin could directly up-regulate expressions of MMP-2 without the presence of IL-1. Furthermore, to our knowledge, the result of immunohistochemical staining for MMP-2 in this study is the first one to document the ex vivo effect of ciprofloxacin on up-regulating MMP-2. Besides, this study specifically documents that type I collagen might be degraded by MMPs which are up-regulated by ciprofloxacin. Because fluoroquinolones may also stimulate inflammatory pathways in or around the tendon,30 the combined effect on tendon matrix turnover may account for the mechanisms of tendinopathy in some patients treated with ciprofloxacin.
An analysis of the results obtained from this study suggests that enhanced MMP expression might significantly compromise the integrity of the tendon ECM and thus induce the occurrence of tendinopathy or tendon rupture. The peak serum concentrations of ciprofloxacin given orally or intravenously were reported to range from 0.5 to 10 µg/mL31–34 and 5 µg/mL ciprofloxacin was the initial concentration to induce the expressions of MMP-2. Although, the concentration of ciprofloxacin in tendon tissue after standard dosing regimens remains unknown, the result of this study suggest a potential link between ciprofloxacin-associated tendinopathy and increased dosage of ciprofloxacin.
Because of the small size of the explants (about 0.5 cm in length, 0.2 cm in diameter), it is difficult to perform a biomechanical test to evaluate the changes in mechanical properties of a tendon after ciprofloxacin treatment. Further animal studies to investigate if there is a deterioration of the mechanical properties of a tendon after ciprofloxacin treatment are needed to validate the findings of this study.
In conclusion, ciprofloxacin up-regulates the expression of MMP-2 in tendon cells with concomitant degradation of type I collagen. These findings provide novel molecular mechanisms of ciprofloxacin-induced tendinopathy or tendon rupture.