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DOI: 10.1177/0363546514545858

2014 42: 2851 originally published online August 20, 2014Am J Sports MedSusannah L. Gilbert, Joseph T. Nguyen, Salma Chaudhury, Russell F. Warren and Scott A. Rodeo

Alice J.S. Fox, Michael O. Schär, Florian Wanivenhaus, Tony Chen, Erik Attia, Nikolaus B. Binder, Miguel Otero,Fluoroquinolones Impair Tendon Healing in a Rat Rotator Cuff Repair Model: A Preliminary Study

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Fluoroquinolones Impair Tendon Healingin a Rat Rotator Cuff Repair Model

A Preliminary Study

Alice J.S. Fox,*y MSc, Michael O. Schär,y MD, Florian Wanivenhaus,y MD,Tony Chen,z PhD, Erik Attia,y BS, Nikolaus B. Binder,y MD, PhD, Miguel Otero,y PhD,Susannah L. Gilbert,§ MS, Joseph T. Nguyen,|| MPH, Salma Chaudhury,y MD, PhD,Russell F. Warren,y MD, and Scott A. Rodeo,y MDInvestigation performed at the Hospital for Special Surgery, New York, New York, USA

Background: Recent studies suggest that fluoroquinolone antibiotics predispose tendons to tendinopathy and/or rupture. How-ever, no investigations on the reparative capacity of tendons exposed to fluoroquinolones have been conducted.

Hypothesis: Fluoroquinolone-treated animals will have inferior biochemical, histological, and biomechanical properties at thehealing tendon-bone enthesis compared with controls.

Study Design: Controlled laboratory study.

Methods: Ninety-two rats underwent rotator cuff repair and were randomly assigned to 1 of 4 groups: (1) preoperative (Preop),whereby animals received fleroxacin for 1 week preoperatively; (2) pre- and postoperative (Pre/Postop), whereby animalsreceived fleroxacin for 1 week preoperatively and for 2 weeks postoperatively; (3) postoperative (Postop), whereby animalsreceived fleroxacin for 2 weeks postoperatively; and (4) control, whereby animals received vehicle for 1 week preoperativelyand for 2 weeks postoperatively. Rats were euthanized at 2 weeks postoperatively for biochemical, histological, and biomechan-ical analysis. All data were expressed as mean 6 standard error of the mean (SEM). Statistical comparisons were performed usingeither 1-way or 2-way ANOVA, with P \ .05 considered significant.

Results: Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) analysis revealed a 30-fold increase in expres-sion of matrix metalloproteinase (MMP)-3, a 7-fold increase in MMP-13, and a 4-fold increase in tissue inhibitor of metalloprotei-nases (TIMP)-1 in the Pre/Postop group compared with the other groups. The appearance of the healing enthesis in all treatedanimals was qualitatively different than that in controls. The tendons were friable and atrophic. All 3 treated groups showed sig-nificantly less fibrocartilage and poorly organized collagen at the healing enthesis compared with control animals. There was a sig-nificant difference in the mode of failure, with treated animals demonstrating an intrasubstance failure of the supraspinatus tendonduring testing. In contrast, only 1 of 10 control samples failed within the tendon substance. The healing enthesis of the Pre/Postopgroup displayed significantly reduced ultimate load to failure compared with the Preop, Postop, and control groups. There was nosignificant difference in load to failure in the Preop group compared with the Postop group. Pre/Postop animals demonstratedsignificantly reduced cross-sectional area compared with the Postop and control groups. There was also a significant reductionin area between the Preop and control groups.

Conclusion: In this preliminary study, fluoroquinolone treatment negatively influenced tendon healing.

Clinical Relevance: These findings indicate that there was an active but inadequate repair response that has potential clinicalimplications for patients who are exposed to fluoroquinolones before tendon repair surgery.

Keywords: fluoroquinolone; tendon healing; rotator cuff repair; fleroxacin; tendinopathy

Fluoroquinolones (FQs) are an important class of antimi-crobial agents commonly used to treat infections fromgram-negative organisms, gram-positive organisms, andanaerobic bacteria. Because of their favorable

pharmacokinetic properties, excellent bactericidal activity,and broad antimicrobial spectrum,17,28 FQs are widelyadministered for urinary tract, upper respiratory, and intes-tinal infections and in the treatment of certain musculoskel-etal infections such as osteomyelitis and septic arthritis.

Side effects associated with the use of FQs have beenreported in the literature, however most are not severe.Gastrointestinal problems (nausea, vomiting, and diar-rhea) are the most frequent side effects, followed by mild

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neurological disorders, skin reactions, myalgia, arthralgia,and arthritis.16,25 Since 1983, cases of FQ-induced tendin-opathy have been reported in the literature.{ Although theexact incidence of FQ-induced tendinopathy is unknown,some authors haveestimated it to range from 0.14% to 0.4%.11,23,24,50 The inci-dence rate increases to 12.2% to 15.6% in transplant recip-ients.11,24 Although the Achilles tendon is most commonlyaffected after FQ treatment, inflammation and involve-ment of other tendons such as supraspinatus and bicepsbrachii also have been reported.27 The symptoms afterFQ treatment range from mild pain around the affectedtendon to complete rupture requiring surgical interven-tion. In 2008, the US Food and Drug Administration man-dated that all FQ products have a ‘‘black-box’’ warningindicating the increased risk of FQs in adverse eventssuch as tendinopathy and tendon rupture.41

Direct toxic effects and ischemic injury,23,39,46 or alter-ations in the synthesis or breakdown of extracellularmatrix (ECM) components,5,8,29,39,48 may contribute toFQ-induced tendon damage. Alterations and dysfunctionof cellular components, combining localized matrix-degrading activity and deficient ground substanceproduction, may lead to changes in the biomechanicalproperties of tendon matrix, resulting in tendinopathyand subsequent rupture.48 Inflammation of the paratenonand degenerative changes in tendon cells have also beenreported in FQ-treated animals,21,38 and FQs have beenshown to have a number of effects on various mammaliancell types in culture, including both increased anddecreased expression of inflammatory mediators,32,49

reduced expression of ECM proteins,4,48 reduced mito-chondrial activity,4 and noncytotoxic inhibition of caninetendon cell proliferation.48

These observations notwithstanding, the pathophysio-logical mechanisms underlying FQ-induced tendinopathyare still poorly understood, and very little is known aboutthe reparative capacity of tendons exposed to FQ. There-fore, the purpose of this study was to investigate the effectof the FQ fleroxacin on rotator cuff injury and repair in anestablished rat model. Fleroxacin, as well as the dosageused, was chosen because of its toxic potential to inducetendinopathy, as reported by a previous study20 and fur-ther confirmed by a pilot (unpublished) study at our insti-tution. We hypothesized that FQ-treated animals wouldshow inferior biochemical, histological, and biomechanicalproperties at the healing enthesis compared with controlanimals.

MATERIALS AND METHODS

This study was approved by our institutional animal careand use committee.

Study Design

Because previous studies have demonstrated anatomic simi-larities with the human shoulder, a rat model was selected tostudy rotator cuff tendon healing after surgical repair.6 Atotal of 92 male Sprague-Dawley rats (obtained at 275-300 g; Harlan Laboratories) underwent unilateral detach-ment of the supraspinatus tendon from the greater tuberos-ity followed by immediate anatomic repair withtransosseous fixation as described previously in detail.3

Rats were treated with either fleroxacin or 13 phosphate-buffered saline (PBS) vehicle and were randomly assignedto 1 of 4 groups: (1) preoperative (Preop), whereby animalsreceived fleroxacin for 1 week preoperatively; (2) pre- andpostoperative (Pre/Postop), whereby animals received flerox-acin for 1 week preoperatively and for 2 weeks postopera-tively; (3) postoperative (Postop), whereby animals receivedfleroxacin for 2 weeks postoperatively; and (4) control,whereby animals received 13 PBS for 1 week preoperativelyand for 2 weeks postoperatively. Fleroxacin (900 mg/kg) or13 PBS was administered by oral gavage every 24 hours.This particular FQ and dose was chosen based on its toxicpotential to induce tendinopathy.20 All animals were eutha-nized by overexposure to carbon dioxide at 2 weeks postoper-atively for biochemical (n = 5), histological (n = 8), orbiomechanical analysis (n = 10).

Drug Administration

Fleroxacin (TCI America) was prepared daily in a solutionof 13 PBS (Gibco; Invitrogen Life Technologies) and NaOH(Sigma-Aldrich) to achieve a dose of 900 mg/kg. Then, 1-NHCl was used to achieve a pH between 8 and 8.5. Ratsreceived the suspended fleroxacin solution or 13 PBS dailyby orogastric gavage. A standard dose volume of 2.5 mL/kgwas used throughout. All animals were lightly anesthe-tized with 2% isoflurane (Baxter Inc) for purposes ofadministering orogastric treatments.

Surgical Technique

The rats were anesthetized with an intraperitoneal injec-tion of ketamine (80 mg/kg; Fort Dodge Animal Health)and xylazine (5 mg/kg; Akorn Inc). Anesthesia was main-tained with 2% isoflurane (Baxter Inc). All operationswere performed by use of sterile technique with the ratin the lateral decubitus position. A deltoid-splitting

{References 1, 12, 19, 29, 31, 35, 43, 44, 47, 50.

*Address correspondence to Alice J.S. Fox, MSc, Laboratory for Soft Tissue Research, Hospital for Special Surgery, 535 East 70th Street, New York,NY 10021, USA (e-mail: [email protected]; [email protected]).

yLaboratory for Soft Tissue Research, Hospital for Special Surgery, New York, New York, USA.zLaboratory for Soft Tissue Research, Department of Biomechanics, Hospital for Special Surgery, New York, New York, USA.§Department of Biomechanics, Hospital for Special Surgery, New York, New York, USA.||Healthcare Research Institute, Hospital for Special Surgery, New York, New York, USA.

One or more of the authors has declared the following potential conflict of interest or source of funding: R.F.W. receives royalties from Biomet and ownsstock in Ivy Sports. S.A.R. is a consultant for Smith and Nephew. The study was funded by the Russell F. Warren Research Chair Fund.

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incision was made, and the acromioclavicular joint wasdivided, allowing visualization of the rotator cuff tendons.The supraspinatus tendon was isolated and a modifiedMason-Allen stitch was placed by use of 4-0 Ethibond(Johnson & Johnson Inc) nonabsorbable suture. The ten-don was then sharply detached from the greater tuberosityand the footprint gently decorticated with a scalpel bladeto ensure complete debridement of the native enthesis.Crossed bone tunnels were drilled at the anterior and pos-terior margins of the footprint and 2 mm lateral to thearticular surface by use of a 22-gauge needle (BectonDickinson). Suture ends were then passed through thebone tunnels and firmly tied over the humeral metaphysealcortex, anatomically repairing the supraspinatus tendon toits native footprint.6 The deltoid split and wound were subse-quently closed in a standard layered fashion with absorbablesutures. Buprenorphine (0.05 mg/kg; Reckitt Benckiser Phar-maceuticals Inc) was administered subcutaneously foranalgesia during the postoperative period. Ad libitumweightbearing and cage activity were allowed postopera-tively. The animals were observed daily for abnormal clinicalsigns (lethargy, loss of appetite) and changes in body weight.

Reverse-Transcriptase QuantitativePolymerase Chain Reaction Analysis

Previous studies have suggested that FQ-induced tendondamage involves cytotoxicity, inflammatory-like reactions,and alterations in matrix deposition and remodeling. Thus,to better understand the mechanisms underlying FQ actionsin tendon repair, we performed reverse-transcriptase quanti-tative polymerase chain reaction (RTqPCR) analysis on totalRNA isolated from supraspinatus tendon, assessing changesin messenger RNA (mRNA) expression of genes involved inthese processes. For RT-qPCR analysis, total RNA was iso-lated from the supraspinatus tendon (n = 5 per group) byuse of TRIzol reagent (Life Technologies) followed by DNaseI treatment and column clean-up (Qiagen). Total RNA wasreverse transcribed with the QuantiTect Reverse Transcrip-tion Kit (Qiagen) according to the manufacturer’s instruc-tions. Amplifications were carried out with SYBR Green I–based RT-PCR on the Opticon 2 Real-Time PCR DetectorSystem (BioRad). Amplification efficiencies were calculatedfor all primers by use of serial dilutions of pooled complemen-tary DNA samples. The data were calculated as the ratio ofeach gene to expression of b-actin (ACTB), and glyceralde-hyde 3-phosphate dehydrogenase (GAPDH) was used as anadditional housekeeping gene control. Melting curves weregenerated to ensure a single gene-specific peak, and no-template controls were included for each run and each setof primers to control for nonspecific amplifications. Dataare represented as relative expression of each gene comparedwith ACTB mRNA expression.

Determination of Serum Fleroxacin Levels

Analysis of fleroxacin in serum was conducted by use ofreverse high-performance liquid chromatography (n = 23per group; University of Tennessee, College of VeterinaryMedicine). Enrofloxacin, a structurally related compound,

was used as the internal standard. The compounds wereextracted from plasma using acetonitrile to precipitateplasma proteins. The system consisted of a 2695-separationmodule and a 2475-fluorescence detector (Waters Corp).Separation was attained on a Waters Atlantis dC18 3.9 3150–mm (5 mm) column preceded by a 5-mm Atlantis guardcolumn. Standard curves for plasma analysis were preparedby spiking untreated plasma with fleroxacin, which produceda linear concentration range of 50 to 7500 ng/mL. The methodwas accurate and precise across this concentration range.

Histological Analysis

Histological analysis of the healing enthesis was performedat 2 weeks postoperatively (n = 8 per group). The right supra-spinatus muscle, supraspinatus tendon, and humerus werecarefully dissected free of all remaining soft tissues immedi-ately after euthanasia. Tissue samples were fixed in 10%neutral-buffered formalin (Decal Chemical Corp) for 48hours. After fixation, tissues were decalcified in EDTA(Sigma-Aldrich) for 72 hours and washed in PBS solution.The tissues were then dehydrated and embedded in paraffin;5 mm–thick coronal sections of the repaired supraspinatustendon and the greater tuberosity were mounted on silane-coated slides and stained with safranin-O, hematoxylin andeosin, and picrosirius red. The greater tuberosity, repairedtendon-bone insertion, and midsubstance of the supraspina-tus tendon were examined under light and polarized lightmicroscopy at 340 and 3100, respectively, to assess fibrocar-tilage and collagen organization (Eclipse E800; Nikon).

All digital images were captured with a SPOT RT camera(Diagnostic Instruments) and imported into ImageJ(National Institutes of Health). The area of new fibrocartilageformation at the healing enthesis was determined by outlin-ing the area of metachromasia with safranin-O staining.Total area for each specimen was measured by use of ImageJsoftware.22 Collagen deposition and maturation at the heal-ing enthesis were semiquantitatively assessed by measuringbrightness on picrosirius red–stained slides viewed underpolarized light microscopy. Measurements were obtained byrotating the polarization plane until maximum brightnesswas obtained to control for variations in specimen orientationon the slide. To facilitate comparisons between groups, all tis-sues were embedded and cut in exactly the same orientation,and sections were cut to a uniform thickness. After digitalcapture, images were imported into ImageJ, whereby theimages underwent 8-bit digitization with a resolution of640 (horizontal) 3 480 (vertical) pixels. This produced imagesin which noncollagenous material was dark (zero) and collag-enous material was depicted in gray scales from 1 to 255.Five rectangular areas measuring 50 3 50 mm were ran-domly selected at the tendon region adjacent to the healingenthesis, and the gray scales were measured. The light inten-sities were measured under exactly the same conditions ofillumination for all specimens.

Biomechanical Testing

Biomechanical testing of the repaired tendon-bone inter-face was performed at 2 weeks postoperatively (n = 10

Vol. 42, No. 12, 2014 Fluoroquinolones Impair Tendon Healing 2853



per group). On the day of testing, each shoulder was thawedat room temperature, and the humerus with attachedsupraspinatus tendon was meticulously dissected undermagnification. All dissections were performed in a blindedfashion with respect to treatment group. At this time, thequalitative appearance (tendon swelling, thickening, discol-oration) of the supraspinatus tendon and its humeral inser-tion was evaluated, and both were digitally photographedby 2 blinded observers (A.J.S.F. and M.O.S.). The dimen-sions of each supraspinatus tendon were measured witha digital micrometer, and the cross-sectional area was deter-mined. The specimen was then placed into a custom-designed uniaxial testing system. The tendon was securedin a screw grip by use of 280-grit silicon carbide sandpaper,Insta-set accelerator, and Insta-cure1 cyanoacrylate(Bob Smith Industries). The humerus was secured intoa custom-designed vise grip that prevented fracture throughthe humeral physis. The supraspinatus tendon was securedto a 111-N load cell attached to a linear bearing that allowedalignment of the tendon in the direction of its pull. Thehumeral jig was secured to the linear stage, and grip-to-grip distance was standardized across all specimens. Thespecimen was preloaded to 0.1 N and then loaded to failureat a rate of 14 mm/s, corresponding to approximately 0.4%strain. The ultimate load to failure, stiffness, the Youngmodulus, maximum stress, energy to failure, and mode offailure were calculated from tensile tests. Displacementwas measured by use of a 1 mm–resolution micrometer sys-tem attached to the linear stage. The linear region of theload-displacement curve was used to calculate the stiffnessfor each specimen.

Statistical Analysis

Statistical analysis was performed with SPSS Statistics forWindows v 20.0 (IBM Corp), with P\ .05 considered signif-icant. One-way analysis of variance (ANOVA) was used tocompare serum fleroxacin levels. Two-way ANOVAs wereused to compare RTqPCR activity, histomorphometricmeasures, and biomechanical data between the 4 groups.All data are expressed as mean 6 standard error of themean (SEM).

RESULTS

Serum Fleroxacin Levels

High-performance liquid chromatography levels of bothexperimental and control groups provided a quantitativeindex of the fleroxacin concentration. Mean fleroxacin lev-els of the Pre/Postop and Postop groups were 64,132 64974 ng/mL and 67,124 6 9010 ng/mL, respectively.Mean fleroxacin level of the Preop and control groupswas 0 ng/mL.

Gross Examination

Five animals receiving fleroxacin died and were subse-quently replaced. Necropsy revealed that these deaths

were not due to the fleroxacin treatment itself but ratherto accidental endotracheal intubation, a result of techni-cian error. The Pre/Postop and Postop animals lost weightover the duration of the study, while the Preop and controlanimals gained weight. Analysis of body weight revealedthat all FQ-treated groups gained significantly less weightthan did control animals, with the most significantdecrease in body weight observed in the Postop and Pre/Postop groups (2.4 6 3.2 g [Preop] vs 265.5 6 3 g [Pre/Postop] vs 277.4 6 3.7 g [Postop] vs 20.4 6 4.2 g [control];P \ .01).

Our initial macroscopic evaluation showed that all rota-tor cuff repairs were grossly intact at the time of collection.No failed repairs, proximal humeral physeal fractures, orsuture pullouts from the transosseous tunnels wereencountered. However, the healing enthesis of the Pre/Postop animals was qualitatively different from the othergroups, and the supraspinatus tendon was friable and atro-phic. No observable differences were seen between theother treatment groups.

RTqPCR Analysis

Our results showed a significant upregulation of interleu-kin (IL)-1b mRNA in the Pre/Postop group comparedwith control and Preop groups (Figure 1A), while tumornecrosis factor (TNF)-a expression was significantly upre-gulated in the FQ-treated Postop and Pre/Postop groupswhen compared with vehicle-treated controls (Figure 1B).Further, the mRNA levels of the matrix-degradingenzymes matrix metalloproteinase (MMP)-3 and MMP-13were significantly upregulated in the Pre/Postop groupwhen compared with all other conditions (Figure 1, Cand D). Reduced expression of tissue inhibitor of metallo-proteinases (TIMP)-2 levels was accompanied by anincreased expression of MMP-3 and MMP-13 levels in allFQ-treated groups, with significant differences in Postopversus control groups (Figure 1F). However, the TIMP-1levels were significantly increased in the Pre/Postop condi-tion when compared with all other groups (Figure 1E). Norelevant alterations in the expression levels of the apopto-sis markers p53 and caspase 3 were detected in our analy-sis (data not shown), and we found no conclusive orsignificant results when evaluating the expression levelsof scleraxis and of various collagens (type I a2, type II a1,and type III a1) (data not shown).

Histological Analysis

Quantitative histomorphometry revealed that the fleroxacin-treated animals had significantly reduced fibrocartilage atthe healing enthesis compared with the control animals. Ani-mals in the Preop, Pre/Postop, and Postop fleroxacin-treatedgroups had a mean area of new fibrocartilage of 323,946 649,139 mm2, 316,984 6 42,819 mm2, and 350,771 668,899 mm2, respectively, whereas control animals hada mean fibrocartilage area of 628,691 6 97,163 mm2 (P \.05) (Figure 2A). Analysis of collagen birefringence withpolarized light microscopy revealed significantly less orga-nized collagen at the healing enthesis in all 3 fleroxacin-




treated groups (Preop, Pre/Postop, and Postop) comparedwith control animals (65.09 6 4.05 vs 78.91 6 10.91 vs68.90 6 9.16 vs 99.13 6 11.67, respectively; P \ .05) (Figure2, B-D).

Biomechanical Testing

There were significant differences in the mode of failurebetween the experimental and control animals. The FQ-treated animals in the Pre/Postop group displayed intra-substance failure of the supraspinatus tendon during bio-mechanical testing, whereas only 1 of 10 control samplesfailed within the tendon substance, with the remainingcontrol samples failing at the healing enthesis. In thePreop group, 6 of 10 samples failed within the tendon

substance, while the remaining 4 samples avulsed fromthe supraspinatus muscle. In the Postop group, 5 of 10samples failed within the tendon substance, while 4avulsed from the supraspinatus muscle and 1 failed atthe healing enthesis.

The healing enthesis of the Pre/Postop group displayedsignificantly reduced ultimate load to failure comparedwith the Preop, Postop, and control groups (3.60 6 0.72 Nvs 9.39 6 0.64 N vs 8.82 6 1.61 N vs 7.72 6 0.84 N, respec-tively; P \ .05) (Figure 3A). There was no significant differ-ence in load to failure in the Preop group compared with thePostop group. Pre/Postop animals demonstrated signifi-cantly reduced cross-sectional area compared with thePostop group (1.91 6 0.41 vs 3.40 6 0.45 mm2, respectively)(P \ .05) and control group (4.49 6 0.38 mm2) (P \ .001).

Figure 1. Results of reverse-transcriptase quantitative polymerase chain reaction analyses on total RNA isolated from supraspi-natus tendon: (A) Interleukin (IL)-1b expression. The Pre/Postop group showed a significant upregulation of IL-1b messenger RNA(mRNA) compared with control and Preop animals. (B) Tumor necrosis factor (TNF)-a expression. Postop and Pre/Postop groupsshowed a significant upregulation of TNF-a mRNA compared with control and Preop animals. (C) Matrix metalloproteinase(MMP)-3 expression. The Pre/Postop group showed a significant upregulation of MMP-3 mRNA compared with all other groups.(D) MMP-13 expression. The Pre/Postop group showed a significant upregulation of MMP-13 mRNA compared with all othergroups. (E) Tissue inhibitor of metalloproteinases (TIMP)-1 expression. The Pre/Postop group showed a significant increase inTIMP-1 mRNA expression compared with all other groups. (F) TIMP-2 expression. The Postop group showed a significantdecrease in TIMP-2 expression levels compared with the control group. Data are shown as mean 6 standard error of themean (SEM) (error bars). ACTB, b-actin. *P \ .05. **P \ .01. ***P \ .001.




There was also a significant reduction in cross-sectionalarea between the Preop group (2.31 6 0.31 mm2) and con-trol group (P \ .01) (Figure 3B). There were no significantdifferences between groups for stiffness, Young modulus,maximum stress, or energy to failure (data not shown).

DISCUSSION

Fluoroquinolones are commonly administered antimicrobialagents that target bacterial DNA gyrase (topoisomerase II)activity17 and are characterized by good tissue penetration,

broad antimicrobial spectrum, and a relatively low inci-dence of serious side effects. Deleterious effects of FQ ontendons have been documented since the 1980s,1 and whilestudies have explored the cellular and tissue responses ofFQ-induced tendinopathy, there have been no investiga-tions on the reparative capacity of tendons exposed toFQs. Therefore, the purpose of this study was to determinethe effect of FQ on tendon healing by using an establishedrodent rotator cuff repair model.6

Although the underlying mechanism of FQ-induced ten-dinopathy and tendon rupture remains unclear, recentstudies suggest that it is likely multifactorial. Inflammation

Figure 2. Histological results. (A) Area of fibrocartilage: All 3 fluoroquinolone (FQ)-treated groups showed significantly less fibro-cartilage compared with control rats. (B) Collagen organization: All 3 FQ-treated groups showed significantly less organized col-lagen at the healing enthesis compared with control rats. Safranin-O staining of the (C) control and (D) Pre/Postop groups. Therewas a significantly reduced area of fibrocartilage in the Pre/Postop group compared with control rats (340). All data expressed asmean 6 standard error of the mean (SEM) (error bars). *P \ .05.

A B

Figure 3. Biomechanical results. (A) Load to failure: The healing enthesis of the Pre/Postop group displayed significantly reducedultimate load-to-failure compared with the Preop (P \ .01), Postop (P \ .01), and control groups (P \ .05). (B) Cross-sectionalarea measurement: Pre/Postop animals demonstrated significantly reduced cross-sectional area compared with the Postopand control groups (P \ .05 and P \ .001, respectively). There was also a significant reduction in area between the Preopand control groups (P \ .01). Data expressed as mean 6 standard error of the mean (SEM) (error bars).




of the paratenon, degenerative changes in tenocytes, andalterations in collagen were reported in studies of FQ-treated animals.21,29,38 Fluoroquinolones have been shownto have a number of effects on various mammalian cell typesin culture, including both increased and decreased expres-sion of inflammatory mediators,32,49 reduced expression ofsome ECM proteins (type I collagen, elastin, fibronectin,and b1 integrin),

4,8,36,37,48 reduced mitochondrial activity,4

direct toxicity on collagen,23 elevated levels of activated cas-pase 3 (an apoptosis marker),36 and noncytotoxic inhibitionof canine tendon cell proliferation.48 Jorgensen et al19

attributed the histological changes seen in FQ-damagedAchilles tendons to an ischemic process. The investigatorshypothesized that tendon rupture may be due to a vascularphenomenon leading to ischemia. However, the plausibilityof this theory is called into question as the majority of ten-don ruptures occur at sites that are relatively avascular.15

Tendon pain and degeneration have been associated withan increase in the normal turnover of matrix proteins.2,18,33

It has been reported that FQs alter soft tissue structuresand MMP expression in both in vitro7-10,42,48 and in vivomodels.37,39 In agreement with these studies, our biochemi-cal findings suggest that alterations in inflammatory-drivenabnormal ECM remodeling may contribute to FQ-inducedtendon damage. Our findings also suggest that FQs haveno effect on ECM production but rather promote the produc-tion of a lower quality matrix (eg, type III collagen instead oftype I collagen). The increased IL-1b and TNF-a expressioncould contribute to the upregulation in MMP-3 and MMP-13levels in the Pre/Postop FQ-treated group, indicating a pro-nounced alteration in the tendon ECM homeostasis. Arecent study of 24 patients with full-thickness rotator cufftears reported an upregulation of TIMP-1 and MMP-3expression, suggesting an association with MMP expressionand rupture and retear of the rotator cuff tendons.14 Theseabnormal patterns in gene expression could account for thechanges in histological and biomechanical properties seen inthe FQ-treated specimens. Furthermore, this model of FQ-induced tendinopathy may provide scientists and clinicianswith a novel paradigm for evaluating effects of MMPs andTIMPs on tendon.

The rat supraspinatus tendon has a unidirectionalarrangement of perforating collagen (Sharpey) fibers atthe fibrocartilaginous enthesis. Successful tendon repairdepends, in part, on the integrity of the enthesis and is rec-ognized as the earliest sign of osseous integration.34

Composed mainly of proteoglycans and collagen, the enthe-sis facilitates the transmission and dissipation of tensile,compressive, and shear forces. Our histological resultsindicate that fibrocartilage formation and collagen organi-zation were deleteriously altered by the administration ofFQ, regardless of whether it was administered preopera-tively, postoperatively, or both. The FQ-treated animalshad a significant reduction in fibrocartilage and Sharpeycollagen fibers at the healing enthesis, indicating inferiorosseous integration of the tendon to bone. Calcified fibro-cartilage protects the bone from excessive shear, and colla-gen protects the tendon from compression. Without a well-established enthesis, the structural integrity of the repairwill be compromised, resulting in reduced biomechanical

properties. With all FQ-treated groups displaying inferiormaterial properties, this may explain the anecdotal evi-dence in the literature of tendinopathy and tendon rupturewith exposure to FQ.

In the present study, we found that the FQ-treated ten-dons had a significantly reduced load to failure and cross-sectional area compared with control specimens. We alsoobserved a predilection for midsubstance tendon rupturein the FQ-treated animals, which is in contrast to the con-trol specimens that failed at the healing enthesis. Thesefindings indicate that FQ alters the microstructural prop-erties of tendons and their enthesis and may predisposethem to degeneration, findings that are supported in theliterature.# Not surprisingly, the Pre/Postop group hadthe most significantly altered biomechanical properties.With a significantly reduced ultimate failure force andcross-sectional area, our data suggest that FQ may havea negative effect on tendon ECM degradation, which isexacerbated after repair. Sustained exposure (ie, pre- andpostoperatively) to FQ is likely to be detrimental, as itmay lead to increased susceptibility to injury or failureas a result of a degenerated tendon and decreased visco-elasticity. Interestingly, despite the significant differencesin load to failure, mode of failure, and cross-sectional areabetween groups, we did not detect any differences in stiff-ness, Young modulus, maximum stress, or energy to fail-ure. The influence of geometry may account for the lackof observable differences in the Young modulus betweengroups, indicating that structural properties are impairedbut material properties are not. Longer time periods arerequired to further examine these biomechanical findings.

Although the Pre/Postop group demonstrated the mostdramatic changes histologically, biomechanically, and bio-chemically, the findings of the Preop and Postop groups areimportant to note. Despite the absence of any detectableserum fleroxacin level at time of harvest, the Preop FQ-treated group demonstrated a statistically significantreduction in both tendon cross-sectional area and thearea of fibrocartilage at the healing enthesis as well asa predilection for intrasubstance failure during biome-chanical testing. Such findings suggest a structural andcompositional degeneration of the tendon ECM. Interest-ingly, the changes in the Postop group were more pro-nounced, with a statistically significant reduction incollagen organization, fibrocartilage, and TIMP-2; a signif-icant increase in TNF-a; and a range of failure locationsduring biomechanical testing. Down-regulated TIMP-2has been reported to be associated with tendinopathy.18,26

These observations suggest that the effects of FQ adminis-tration are much more pronounced in the postoperativeperiod, when the sequence of inflammation, repair, andremodeling occurs, compared with the preoperative period,when no injury has yet occurred.

Our results have important clinical implications.Awareness of the association between tendon disordersand FQ should lead to a careful assessment of patients.Our results indicate that the risk of structural failure after

#References 1, 12, 19, 29-31, 35, 43, 44, 47, 50.




soft tissue repair or reconstructive procedures in patientswho have taken FQ may be higher than in patients whohave not taken FQ. The risk-benefit ratio of FQ shouldbe considered, and patients should be carefully advised ofthe possibility of tendon disorders, during or after FQtreatment, and counseled to immediately suspend treat-ment at the earliest suspicion of tendinopathy.13,27 We rec-ommend delaying surgery (by at least 6 weeks) if a patienthas recently taken an FQ antibiotic. If a patient is takingFQ preoperatively and surgery is absolutely necessary,we suggest using a different antibiotic postoperatively.These recommendations may appear somewhat arbitrary;however, due to the paucity of data regarding the underly-ing cause of FQ-induced tendinopathy and tendon rupture,no absolute recommendations can be provided. Future clin-ical studies, in addition to focusing on postsurgery drugprotocols, should provide more definitive guidelines forclinicians on how to treat patients with FQ-induced tendin-opathy and/or tendon rupture.

The limitations of this study are important. First, fler-oxacin was selected because of its toxic potential to inducetendinopathy, as reported by a previous study and con-firmed by a pilot study at our institution.20 Nonetheless,fleroxacin is not representative of the most commonlyadministered FQ, nor is it the most commonly reportedFQ associated with inducing tendinopathy. In this regard,we recognize that this study examines a worst-case sce-nario of acute toxicity. Second, fleroxacin induced a numberof metabolic alterations throughout the body that led toa less robust condition in the treated animals comparedwith the controls. The loss of weight and increased leth-argy displayed by the FQ-treated rats may be caused bya generalized catabolic effect and/or an inflammation-related systemic effect, which could influence the enthesisin a nonspecific way such as reduced mechanical load oraltered drug metabolism. This finding of poor healthwhen FQs are used in animal models, however, is notunique to our study.40,45 Third, although the rat is anappropriate animal model for studying the rotator cuffbased on anatomic considerations,6 the model approxi-mates an acute rotator cuff injury and does not representthe chronic degenerative effects of long-standing FQ ther-apy. Longer time periods may provide valuable informa-tion regarding the effect of sustained FQ use on thetendon and healing enthesis. Fourth, our study may havebenefited from immunohistochemical analysis of variouscytokines, which could help to further define the underly-ing mechanism of disrupted tendon-bone healing.

SUMMARY

The results of this preliminary study suggest that there isa significant association between FQ use and impaired ten-don healing. All FQ regimens, whether administered pre-operatively, postoperatively, or both, have the potentialto cause a detrimental effect on tendon structure and itsability to heal. Therefore, we advise that an alternativeantibiotic be administered, particularly when soft tissuerepair is required. Additional studies are necessary to

elucidate the histochemical, histological, and biomechani-cal changes reported here and to provide potential targetsfor therapeutic intervention.

ACKNOWLEDGMENT

The authors thank the Center for Laboratory Animal Serv-ices staff and Orla O’Shea, MSc, for their assistance withanimal care and histology, respectively.

REFERENCES

1. Bailey RR, Kirk JA, Peddie BA. Norfloxacin-induced rheumatic dis-

ease. N Z Med J. 1983;96:590.

2. Bank RA, TeKoppele JM, Oostingh G, Hazleman BL, Riley GP. Lysyl-

hydroxylation and non-reducible crosslinking of human supraspina-

tus tendon collagen: changes with age and in chronic rotator cuff

tendinitis. Ann Rheum Dis. 1999;58:35-41.

3. Bedi A, Fox AJS, Kovacevic D, Deng X-H, Warren RF, Rodeo SA.

Doxycycline-mediated inhibition of matrix metalloproteinases

improves healing after rotator cuff repair. Am J Sports Med. 2010;

38:308-317.

4. Bernard-Beaubois K, Hecquet C, Hayem G, Rat P, Adolphe M. In

vitro study of cytotoxicity of quinolones on rabbit tenocytes. Cell

Biol Toxicol. 1998;14:283-292.

5. Burkhardt JE, Hill MA, Lamar CH, Smith GN, Carlton WW. Effects of

difloxacin on the metabolism of glycosaminoglycans and collagen in

organ cultures of articular cartilage. Fundam Appl Toxicol. 1993;20:

257-263.

6. Carpenter JE, Thomopoulos S, Flanagan CL, DeBano CM, Soslow-

sky LJ. Rotator cuff defect healing: a biomechanical and histologic

analysis in an animal model. J Shoulder Elbow Surg. 1998;7:599-605.

7. Chang H-N, Pang J-HS, Chen CPC, et al. The effect of aging on

migration, proliferation, and collagen expression of tenocytes in

response to ciprofloxacin. J Orthop Res. 2012;30:764-768.

8. Corps AN, Harrall RL, Curry VA, Fenwick SA, Hazleman BL, Riley GP.

Ciprofloxacin enhances the stimulation of matrix metalloproteinase 3

expression by interleukin-1beta in human tendon-derived cells:

a potential mechanism of fluoroquinolone-induced tendinopathy.

Arthritis Rheum. 2002;46:3034-3040.

9. Corps AN, Harrall RL, Curry VA, Hazleman BL, Riley GP. Contrasting

effects of fluoroquinolone antibiotics on the expression of the colla-

genases, matrix metalloproteinases (MMP)-1 and -13, in human ten-

don-derived cells. Rheumatology (Oxford). 2005;44:1514-1517.

10. Deng Y, Chen B, Qi Y, Magdalou J, Wang H, Chen L. The effects of

levofloxacin on rabbit anterior cruciate ligament cells in vitro. Toxicol

Appl Pharmacol. 2011;257:67-73.

11. Donck JB, Segaert MF, Vanrenterghem YF. Fluoroquinolones and

Achilles tendinopathy in renal transplant recipients. Transplantation.

1994;58:736-737.

12. Franck JL, Bouteiller G, Chagnaud P, Sapene M, Gautier D. Achilles ten-

don rupture in 2 adults treated with pefloxacin, one of the cases with

bilateral involvement [in French]. Rev Rhum Mal Osteoartic. 1991;58:904.

13. Gabutti L, Stoller R, Marti HP. Fluoroquinolones as etiology of tendin-

opathy [in German]. Ther Umsch. 1998;55:558-561.

14. Gotoh M, Mitsui Y, Shibata H, et al. Increased matrix metallopro-

tease-3 gene expression in ruptured rotator cuff tendons is associ-

ated with postoperative tendon retear. Knee Surg Sports Traumatol

Arthrosc. 2013;21:1807-1812.

15. Harrell RM. Fluoroquinolone-induced tendinopathy: what do we

know? South Med J. 1999;92:622-625.

16. Hayem G, Carbon C. A reappraisal of quinolone tolerability: the expe-

rience of their musculoskeletal adverse effects. Drug Saf. 1995;13:

338-342.

17. Hooper DC, Wolfson JS. Fluoroquinolone antimicrobial agents.

N Engl J Med. 1991;324:384-394.




18. Ireland D, Harrall R, Curry V, et al. Multiple changes in gene expression

in chronic human Achilles tendinopathy. Matrix Biol. 2001;20:159-169.

19. Jorgensen C, Anaya JM, Didry C, et al. Arthropathy with Achilles ten-

don involvement induced by pefloxacin. Apropos of a case [in

French]. Rev Rhum Mal Osteoartic. 1991;58:623-625.

20. Kashida Y, Kato M. Toxic effects of quinolone antibacterial agents on

the musculoskeletal system in juvenile rats. Toxicol Pathol.

1997;25:635-643.

21. Kato M, Takada S, Kashida Y, Nomura M. Histological examination

on Achilles tendon lesions induced by quinolone antibacterial agents

in juvenile rats. Toxicol Pathol. 1995;23:385-392.

22. Koike Y, Trudel G, Curran D, Uhthoff HK. Delay of supraspinatus

repair by up to 12 weeks does not impair enthesis formation: a quan-

titative histologic study in rabbits. J Orthop Res. 2006;24:202-210.

23. Le Huec JC, Schaeverbeke T, Chauveaux D, Rivel J, Dehais J, Le

Rebeller A. Epicondylitis after treatment with fluoroquinolone antibi-

otics. J Bone Joint Surg Br. 1995;77:293-295.

24. Leray H, Mourad G, Chong G, Marcelli C, Borderie P, Mion C. [Spon-

taneous ruptures of the Achilles tendon after kidney transplantation:

use of fluoroquinolones]. Presse Med. 1993;22:1834.

25. Lietman PS. Fluoroquinolone toxicities. An update. Drugs. 1995;

49(suppl 2):159-163.

26. Lo IK, Marchuk LL, Hollinshead R, Hart DA, Frank CB. Matrix metal-

loproteinase and tissue inhibitor of matrix metalloproteinase mRNA

levels are specifically altered in torn rotator cuff tendons. Am J Sports

Med. 2004;32:1223-1229.

27. McGarvey WC, Singh D, Trevino SG. Partial Achilles tendon ruptures

associated with fluoroquinolone antibiotics: a case report and litera-

ture review. Foot Ankle Int. 1996;17:496-498.

28. Moellering RC. The place of quinolones in everyday clinical practice.

Chemotherapy. 1996;42(suppl 1):54-61.

29. Movin T, Gad A, Guntner P, Foldhazy Z, Rolf C. Pathology of the

Achilles tendon in association with ciprofloxacin treatment. Foot

Ankle Int. 1997;18:297-299.

30. Ng WF, Naughton M. Fluoroquinolone-associated tendinopathy:

a case report. J Med Case Rep. 2007;1:55.

31. Pierfitte C, Royer RJ. Tendon disorders with fluoroquinolones. Ther-

apie, 1996;51:419-420.

32. Riesbeck K, Sigvardsson M, Leanderson T, Forsgren A. Superinduc-

tion of cytokine gene transcription by ciprofloxacin. J Immunol.

1994;153:343-352.

33. Riley GP, Curry V, DeGroot J, et al. Matrix metalloproteinase activi-

ties and their relationship with collagen remodelling in tendon pathol-

ogy. Matrix Biol. 2002;21:185-195.

34. Rodeo SA, Arnoczky SP, Torzilli PA, Hidaka C, Warren RF. Tendon-

healing in a bone tunnel: a biomechanical and histological study in

the dog. J Bone Joint Surg Am. 1993;75:1795-1803.

35. Royer RJ, Pierfitte C, Netter P. Features of tendon disorders with flu-

oroquinolones. Therapie. 1994;49:75-76.

36. Sendzik J, Shakibaei M, Schafer-Korting M, Stahlmann R. Fluoroqui-

nolones cause changes in extracellular matrix, signalling proteins,

metalloproteinases and caspase-3 in cultured human tendon cells.

Toxicology. 2005;212:24-36.

37. Shakibaei M, de Souza P, van Sickle D, Stahlmann R. Biochemical

changes in Achilles tendon from juvenile dogs after treatment with

ciprofloxacin or feeding a magnesium-deficient diet. Arch Toxicol.

2001;75:369-374.

38. Shakibaei M, Stahlmann R. Ultrastructure of Achilles tendon from

rats after treatment with fleroxacin. Arch Toxicol. 2001;75:97-102.

39. Simonin MA, Gegout-Pottie P, Minn A, Gillet P, Netter P, Terlain B.

Pefloxacin-induced Achilles tendon toxicity in rodents: biochemical

changes in proteoglycan synthesis and oxidative damage to colla-

gen. Antimicrob Agents Chemother. 2000;44:867-872.

40. Takizawa T, Hasimoto K, Itoh N, Yamashita S, Owen K. A compara-

tive study of the repeat dose toxicity of grepafloxacin and a number

of other fluoroquinolones in rats. Human Exp Toxicol. 1999;18:38-45.

41. Tanne JH. Virginity pledge ineffective against teen sex despite gov-

ernment funding, US study finds. BMJ. 2008;337:a3168.

42. Tsai W-C, Hsu C-C, Chen CPC, et al. Ciprofloxacin up-regulates ten-

don cells to express matrix metalloproteinase-2 with degradation of

type I collagen. J Orthop Res. 2011;29:67-73.

43. van der Linden PD, Sturkenboom MCJM, Herings RMC, Leufkens

HGM, Stricker BHC. Fluoroquinolones and risk of Achilles tendon

disorders: case-control study. BMJ. 2002;324:1306-1307.

44. van der Linden PD, van Puijenbroek EP, Feenstra J, et al. Tendon dis-

orders attributed to fluoroquinolones: a study on 42 spontaneous

reports in the period 1988 to 1998. Arthritis Rheum. 2001;45:235-

239.

45. von Keutz E, Schluter G. Preclinical safety evaluation of moxifloxacin,

a novel fluoroquinolone. J Antimicrob Chemother. 1999;43(suppl

B):91-100.

46. Waterston SW, Maffulli N, Ewen SW. Subcutaneous rupture of the

Achilles tendon: basic science and some aspects of clinical practice.

Br J Sports Med. 1997;31:285-298.

47. West MB, Gow P. Ciprofloxacin, bilateral Achilles tendonitis and uni-

lateral tendon rupture—a case report. N Z Med J. 1998;111:18-19.

48. Williams RJ, Attia E, Wickiewicz TL, Hannafin JA. The effect of cipro-

floxacin on tendon, paratenon, and capsular fibroblast metabolism.

Am J Sports Med. 2000;28:364-369.

49. Yoshimura T, Kurita C, Usami E, et al. Immunomodulatory action of

levofloxacin on cytokine production by human peripheral blood

mononuclear cells. Chemotherapy. 1996;42:459-464.

50. Zabraniecki L, Negrier I, Vergne P, et al. Fluoroquinolone induced

tendinopathy: report of 6 cases. J Rheumatol. 1996;23:516-520.

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