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J Shoulder Elbow Surg (2010) 19, 384-391

1058-2746/2010

doi:10.1016/j.jse

www.elsevier.com/locate/ymse

BASIC SCIENCE

The effect of matrix metalloproteinase inhibition ontendon-to-bone healing in a rotator cuff repair model

Asheesh Bedi, MDa, David Kovacevic, MDb, Carolyn Hettrich, MDb,Lawrence V. Gulotta, MDb, John R. Ehteshami, MDb, Russell F. Warren, MDc,Scott A. Rodeo, MDa,*

aSports Medicine and Shoulder Surgery, Laboratory for Soft Tissue Research, Hospital for Special Surgery, New York,NY, USAbLaboratory for Soft Tissue Research, Hospital for Special Surgery, New York, NY, USAcHospital for Special Surgery, New York, NY, USA

Hypothesis: Recent studies have demonstrated a potentially critical role of matrix metalloproteinases(MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the pathophysiology of rotatorcuff tears. We hypothesize that local delivery of a MMP inhibitor after surgical repair of the rotatorcuff will improve healing at the tendon-to-bone surface interface.Materials and methods: Sixty-two male Sprague-Dawley rats underwent acute supraspinatus detachmentand repair. In the control group (n¼ 31), the supraspinatus was repaired to its anatomic footprint. In theexperimental group (n¼ 31), recombinant a-2-macroglobulin (A2 M) protein, a universal MMP inhibitor,was applied at the tendon-bone interface with an identical surgical repair. Animals were sacrificed at 2 and4 weeks for histomorphometry, immunohistochemistry, and biomechanical testing. Statistical comparisonswere performed using unpaired t tests. Significance was set at P < .05.Results: Significantly greater fibrocartilage was seen at the healing enthesis in the A2 M-treated speci-mens compared with controls at 2 weeks (P < .05). Significantly greater collagen organization wasobserved in the A2 M-treated animals compared with controls at 4 weeks (P < .01). A significant reductionin collagen degradation was observed at both 2 and 4 weeks in the experimental group (P < .05). Biome-chanical testing revealed no significant differences in stiffness or ultimate load-to-failure.Conclusion: Local delivery of an MMP inhibitor is associated with distinct histologic differences at thetendon-to-bone interface after rotator cuff repair. Modulation of MMP activity after rotator cuff repairmay offer a novel biologic pathway to augment tendon-to-bone healing after rotator cuff repair.Level of evidence: Basic science study� 2010 Journal of Shoulder and Elbow Surgery Board of Trustees.

Keywords: Matrix metalloproteinase; inhibitor; rotator cuff repair; tendon-bone healing

uests: Scott A. Rodeo, MD, Attending Surgeon and Co-

edicine and Shoulder Surgery, Laboratory for Soft Tissue

ital for Special Surgery, 535 E 70th St, New York, NY

ss: rodeos@hss.edu (S.A. Rodeo).

/$36.00 - see front matter � 2010 Journal of Shoulder and Elbo

.2009.07.010

Rotator cuff tears are among the most common causes ofshoulder pain and disability in adults. Despite significantadvances in arthroscopic techniques for surgical repair,clinical series of large rotator cuff tears have demonstrateda significant incidence of structural failure after surgicalrepair when evaluated objectively with imaging studies.10-12

w Surgery Board of Trustees.

MMP inhibition after rotator cuff repair 385

In addition, the overall structure, composition, and organi-zation of the rotator cuff insertion is not reproduced aftersurgical repair and reflects an inability to recapitulate theevents that occur during embryonic development withcurrent surgical techniques.

Rather than regenerating the 4 organized zones of directinsertion, the tendon heals with an interposed zone offibrovascular scar tissue between the tendon and bone. Ifhealing is successful, this interface tissue gradually maturesuntil its matrix consists of oriented Sharpey-like collagenfibers that bridge the tendon-to-bone surface.25,26 Althoughpain relief may be achieved despite structural failure ofa rotator cuff repair, optimal long-term outcomes thatachieve both pain relief and restoration of strength arepredicated on successful tendon-bone healing and thereconstitution of a robust enthesis.10-12

The native rotator cuff insertion is a highly organized,complex interface between tendon and bone. Microscopicexamination of the enthesis shows interdigitation of thecollagen fibers with bone though 4 distinct histologic zones:tendon, unmineralized fibrocartilage, mineralized fibro-cartilage, and bone.31 This graduated change in stiffnessallows for transmission of complex mechanical loads fromsoft tissue to bone while minimizing peak stresses at anysingle point along the tendon.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases that collectively degrade componentsof tissue extracellular matrix. Recent studies have identifieda critical role of these enzymes in tumor growth, vascularaneurysmal disease, embryonic development, and normaltissue remodeling.3 The tissue inhibitors of matrix metal-loproteinases (TIMPs) are natural, endogenous inhibitors ofMMPs that provide a check-and-balance system to modu-late reparative and degradative processes and maintaina dynamic homeostasis of the extracellular matrix.3

Loss of homeostasis resulting in increased MMP activityhas been associated with degenerative tendinopathy androtator cuff disease.17,18 An imbalance of MMP activity hasbeen demonstrated in pathologic Achilles and patellar ten-dinosis.1,7,8,13-15,18,20,23,24 Lo et al17 recently demonstratedstatistically significant alterations in messenger RNA(mRNA) levels of specific MMPs and TIMPs in patients withfull-thickness rotator cuff tears compared with matchedcontrols, suggesting that biologic modulation of endogenousMMP activity to basal levels may reduce pathologic tissuedegradation and favorably influence healing after rotator cuffrepair.9,21,22

The purpose of this study was to determine the effect ofa broad-spectrum MMP inhibitor on tendon-to-bone surfacehealing after acute rotator cuff repair. We used anestablished rat rotator cuff repair model to evaluate tendon-to-bone surface healing with immediate postoperativeinhibition of MMP. We hypothesized that local inhibition ofMMPs would result in (1) improved collagen fiberorganization at the healing tendon-bone interface and(2) increased failure load at the tendon attachment site.

Materials and methods

This study was approved by our Institutional Animal Care andUses Committee (IACUC), with assigned protocol # 08-07-09R.

Study design

We selected a rat model to study rotator cuff tendon healing aftersurgical repair because previous studies have demonstratedanatomic similarities with the human shoulder. We obtained62 male Sprague-Dawley rats (weight, 250-300 grams). Eachanimal underwent detachment and immediate repair of the rightsupraspinatus tendon using bone tunnel and suture fixation asdescribed by Carpenter et al.4 Postoperatively, they were housed inpairs and allowed ad libitum activity. The animals were dividedinto 2 experimental groups: (1) local application of a-2-macro-globulin (A2 M), a universal MMP inhibitor, at the tendon-boneinterface after repair, or (2) surgical repair only. The animals weresacrificed at 2 and 4 weeks for biomechanical and histologicanalysis.

Surgical technique

The rats were anesthetized with an intraperitoneal injection ofketamine (80 mg/kg) and xylazine (5 mg/kg). Anesthesia wasmaintained using 2% isoflurane (Baxter Inc, Deerfield, IL). Alloperations were performed using a sterile technique, with the rat inthe lateral decubitus position. A deltoid-splitting incision was made,and the acromioclavicular joint was divided, allowing visualizationof the rotator cuff tendons. The supraspinatus tendon was isolatedand a modified Mason-Allen stitch was placed using 4-0 Ethibond(Johnson and Johnson, Piscataway, NJ) nonabsorbable suture(Figure 1).

The tendon was then sharply detached from the greatertuberosity and the footprint gently decorticated with a scalpelblade to ensure complete debridement of the native enthesis.Crossed bone tunnels were drilled at the anterior and posteriormargins of the footprint and 2 mm lateral to the articular surfaceusing a 22-gauge needle. Suture ends were passed through thebone tunnels and firmly tied over the humeral metaphyseal cortex,anatomically repairing the supraspinatus tendon to its nativefootprint (Figure 1).

In the experimental group, 25 mL of recombinant A2 M (1 IU/Kg; Roche Applied Science, Indianapolis, IN), an endogenousplasma glycoprotein and universal inhibitor of MMPs, was directlyapplied to the tendon-bone interface without a carrier vehicle. Thedose was selected based on work by Demirag et al9 that demon-strated efficacious MMP inhibition with local administration at thisconcentration in a rabbit model. The deltoid split and wound wasclosed in a standard, layered fashion with absorbable sutures.Buprenorphine (0.05 mg/kg) was administered subcutaneously foranalgesia during the postoperative period. Ad libitum weightbearing and cage activity was allowed postoperatively.

Biomechanical testing

The shoulders of 30 animals were used for biomechanical testingat 4 weeks postoperatively. On the day of testing, each shoulderwas thawed at room temperature, and the humerus with the

Figure 1 Acute rotator cuff repair model. (A) The acromioclavicular joint is divided and the deltoid is split in line with its fibers toallow atraumatic exposure and isolation of the supraspinatus tendon. (B) A modified Mason-Allen stitch is placed with a 4-0 Ethibondsuture (Johnson and Johnson, Piscataway, NJ) in the distal supraspinatus tendon. (C) The tendon is divided directly off the greater tuberosityand the footprint is defined and gently debrided of any residual debris. In the experimental group, a-2-macroglobulin protein is applied tothe tendon-bone interface before the transosseous sutures are tied. (D) The sutures are passed by way of the crossed transosseous bonetunnels and tied over the humeral metaphysis to achieve an anatomic rotator cuff repair.

Figure 2 Safranin-O staining of healing enthesis at 2 weeks postoperatively is shown in (A) control and (B) a-2-macroglobulin (A2 M)protein-treated specimens (original magnification �40). The area of new fibrocartilage formation at the tendon-bone surface interface wasdetermined by outlining the area of metachromasia. The black boxes represent areas of interest at the healing tendon-bone interface. Thetotal area of metachromasia (mean � standard deviation) for each specimen was measured and recorded using Image J computerizedimaging software (National institutes of Health, Bethesda, MD).

386 A. Bedi et al.

attached supraspinatus was meticulously dissected under magni-fication. Dissections were performed starting from normal tissueplanes medially, unequivocally isolating the supraspinatus muscleand its tendon above the scapular spine and dividing the rotatorinterval anteriorly. All dissections were performed in a blindedfashion with respect to treatment group.

The dimension of each supraspinatus tendon was measured usinga digital micrometer. The specimen was then placed into a custom-designed uniaxial testing system. The tendon was secured in a screwgrip using sandpaper and ethyl cyanoacrylate (Elmer’s Products Inc,Columbus, OH). The humerus was secured into a custom-designedvice grip that prevented fracture through the humeral physis. The

Figure 3 Significantly greater new fibrocartilage formationwas noted at the healing enthesis of a-2-macroglobulin (A2 M)protein-treated specimens compared with control specimens at 2weeks postoperatively (P < .05).

MMP inhibition after rotator cuff repair 387

supraspinatus tendon was secured to 45-N load cell attached toa linear bearing that allowed alignment of the tendon in the directionof its pull. The humeral jig was secured to the linear stage. Thespecimen was preloaded to 0.10 N and then loaded to failure at a rateof 1.4 mm/ corresponding to approximately 0.4% strain.4,28 Sampleswere tested in air at room temperature, and saline spray was used toensure specimens remained moist throughout testing. The maximumload-to-failure and failure location were recorded. Displacement wasmeasured using a 1-mm resolution micrometer system attached to thelinear stage. The linear region of the stress-strain curve was used tocalculate the stiffness for each specimen.

Histologic analysis

Thirty-two animals were used for histologic analysis. Eight rats inthe experimental and control groups were sacrificed at 2 weeksand 4 weeks after surgery. The tissue specimens were fixed in 10%neutral buffered formalin for 48 hours. After fixation, tissues weredecalcified in Immunocal (Decal, Congers, NY) for 48 hours andwashed in phosphate-buffered saline solution. The tissues weredehydrated and embedded in paraffin. Sections 5-mm thick,including the repaired supraspinatus tendon and the greatertuberosity, were cut in the coronal plane and stained with hema-toxylin and eosin, safranin-O, and picrosirius red. Six slides weremade from each specimen (48 slides/group). The qualitativeappearance of the repair site was evaluated in a blinded fashion.The greater tuberosity, repaired tendon-bone insertion site, andmidsubstance of the supraspinatus tendon were examined underlight and polarized light microscopy (Eclipse E800; Nikon, Mel-ville, NY). Digital images were captured using a SPOT RT camera(Diagnostic Instruments, Sterling Heights, MI).

Picrosirius red staining and examination with monochromaticpolarized microscopy was used for semiquantitative analysis ofcollagen organization at the healing enthesis. The sulfonic acidgroups on the picrosirius red molecules bind to the amino groups incollagen in an oriented fashion, resulting in a 7-fold increase incollagen birefringence. By quantifying the birefringence of collagenunder polarized light (based on brightness), time-dependent differ-ences in collagen deposition and maturation in the healing tendoncould be detected.6 Measurements were obtained by rotating thepolarization plane until maximum brightness was obtained tocontrol for variations in specimen orientation on the slide.

To facilitate comparisons between groups, all tissues wereembedded and cut in exactly the same orientation, and sectionswere cut to a uniform thickness. Digital images obtained under�100 magnification underwent 8-bit digitization by ImageJ software (National Institutes of Health, Bethesda, MD), witha resolution of 640 (horizontal)� 480 (vertical) pixels. Themicroscope fields were digitized using a computer-video system,yielding an image in which noncollagenous material was dark(gray level 0) and collagenous material was depicted by grayscales from 1 to 255. The gray scale measurements were per-formed at the tendon-bone interface using the Image J Softwareprogram.6 Five rectangular areas (2500 mm2 each) were randomlyselected, and gray scales were measured (mean � standard devi-ation). The light intensities were measured under exactly the sameconditions of illumination for all specimens.

The area of new fibrocartilage formation at the tendon-boneinterface was determined by outlining the area of metachromasiawith safranin-O staining at �40 magnification. The total area ofmetachromasia (mean � standard deviation) for each specimen

was measured using Image J computerized imaging software. Theconsensus of 3 independent observers was used for histo-morphometric measurements.

For immunohistochemical analysis, serial sections were treatedwith 3% H2O2 to quench endogenous peroxidase activity, nonspecificantibody-binding was blocked with 5% goat serum, and 1% bovineserum albumin in PBS was used as a negative secondary reagentcontrol. The sections were stained using a monoclonal antibody to a-smooth muscle actin (a-SMA) and factor VIII, a marker of myofi-broblasts and vascular endothelium (Sigma, St. Louis, MO). Eachmonoclonal antibody was applied to separate serial sections for60 minutes at 37�C, and bound antibodies were visualized usinga goat avidin-biotin peroxidase system with 3,30-diaminobenzidine(DAB, Dako Corp, Carpinteria, CA) as a substrate.

Collagen degradation was assessed with immunofluorescenceanalysis using COL-2/3 short, a monoclonal antibody specific tocleaved type I or II collagen fragments (generously donated byDr C.T. Chen).32 Collagen fragments were detected by greenstaining of the pericellular matrix at the tendon-bone interface.Ethidium bromide (red) was used as a nonspecific nuclear coun-terstain. Semiquantitative analysis was performed at �100 magni-fication by counting the ratio of chondrocytes with pericellulargreen staining to the overall number of chondrocytes (ie, red nuclei)in the area of metachromasia for each tendon-bone interface andprovided an indirect assay of MMP activity.

Statistical analysis

Statistical analysis was performed using unpaired t tests withSigmaStat software (Systat Software Inc, Chicago, IL). Statisticalsignificance was set at P < .05. Comparison of histomorphometricmeasures of fibrocartilage formation, collagen organization, andcollagen fragmentation between the control and experimentalgroups was performed using an unpaired t test at 2 and 4 weekspostoperatively. An unpaired t test was also used to compare theload-to-failure and stiffness of the healing enthesis of the controland experimental groups at 4 weeks.

Figure 4 Picrosirius red staining of healing enthesis at 4 weeks postoperatively in (A) control and (B) a-2-macroglobulin (A2 M)-treated specimens illuminated with monochromatic polarized light (original magnification �100). The white boxes represent areas ofinterest at the healing tendon-bone interface. Measurements were obtained by rotating the polarization plane until maximum brightness wasobtained to control for variations in specimen orientation on the slide.

Figure 5 Collagen fibers at the healing enthesis weresignificantly more organized in the specimens treated with a-2-macroglobulin (A2 M) compared with control specimens at 4weeks postoperatively (P < .01). The error bars designate thestandard deviation.

388 A. Bedi et al.

Results

Gross and histologic examination

All rotator cuff repairs were noted to be grossly intact at thetime of sacrifice. No humeral physeal fractures or blow-outs of the transosseous tunnels were encountered.

Histomorphometric analysis demonstrated a signifi-cantly greater area of new fibrocartilage at the healingenthesis in the A2 M-treated compared with the controlgroup at 2 weeks (P < .05; Figures 2 and 3). No significantdifference in metachromasia was detected by 4 weeks.Analysis of collagen birefringence revealed significantlygreater collagen organization in the A2 M-treated groupcompared with control animals at 4 weeks (P < .01;Figures 4 and 5). No significant difference in collagenorganization was noted at 2 weeks postoperatively.

Immunofluorescence analysis for cleaved collagen I andII fragments demonstrated a significant reduction incollagen degradation at the tendon-bone interface of theA2 M-treated group at both 2 and 4 weeks compared withcontrol specimens (P < .05; Figures 6 and 7).

Staining with monoclonal antibodies for a-SMA andfactor VIII revealed neoangiogenesis to be predominantlylocalized to the musculotendinous junction just proximal tothe healing enthesis in both groups. Organized bloodvessels were observed by 4 weeks in both groups, with noquantitative differences evident between control and A2 M-treated specimens.

Biomechanical testing

Biomechanical testing at 4 weeks revealed an ultimateload-to-failure of 23.3� 6.2 N for the control and23.2� 7.6 N for A2 M-treated groups. Mean stiffness ofthe constructs was 12.0� 4.0 N/mm and 9.0� 4.0 N/mm,

respectively. No significant difference in stiffness orultimate load-to-failure was detected between groups.

Discussion

Recent studies have demonstrated a potentially critical roleof MMPs and their inhibitors in the pathophysiology ofrotator cuff tears. We hypothesized that local delivery of anMMP inhibitor after surgical repair of the rotator cuff willimprove healing at the tendon-to-bone surface interface.Our results demonstrate that local administration of a-2-macroglobulin, an endogenous MMP inhibitor, at thegreater tuberosity footprint is associated with distinct

Figure 6 Immunofluorescence analysis using COL-2/3 short, a monoclonal antibody specific to cleaved type I or II collagen fragments,of the healing enthesis at 4 weeks postoperatively in (A) control and (B) a-2-macroglobulin (A2 M)-treated specimens (original magni-fication �40). Collagen fragments were detected by green staining of the pericellular matrix at the tendon-bone interface. Ethidium bromide(red) was used as a nonspecific nuclear counterstain in the assay.

Figure 7 A significant reduction in cleaved collagen fragments at the healing enthesis was detected in specimens treated with a-2-macro-globulin (A2 M) compared with control specimens at 2 and 4 weeks postoperatively (P < .05). The error bars designate the standard deviation.

MMP inhibition after rotator cuff repair 389

histologic differences at the healing enthesis after rotatorcuff repair. A statistically significant reduction in localcollagen degradation was achieved and maintained at both2 and 4 weeks postoperatively. This reduction in MMPactivity was associated with greater new fibrocartilageformation at 2 weeks and increased collagen organization at4 weeks in the healing enthesis.

The MMPs and their endogenous inhibitors play a criticalrole in maintaining the dynamic homeostasis and integrity ofthe extracellular matrix. Loss of this balance resulting inelevated MMP activity has been associated with a number ofpathologic conditions of connective tissue, including degen-erative tendinopathy and rotator cuff tears.1,7,8,13-15,18,23,24

Lo et al17 demonstrated statistically significant alterations inmRNA levels of MMPs and TIMPs in patients with full-thickness rotator cuff tears.17 Choi et al5 found MMP-2 to beexpressed and activated during the healing process of an acutesupraspinatus tendon tear. Yoshihara et al32 and Zhen et al33

have demonstrated elevated levels of MMP-1, MMP-3, andglycosaminoglycans in the synovial fluid of patients withmassive rotator cuff tears. These studies have provided

increasing evidence to suggest that biologic modulation ofendogenous MMP activity to basal levels may reduce patho-logic tissue degradation and favorably influence healing afterrotator cuff repair.9,17,21,22

The results from our study suggest that local adminis-tration of an MMP inhibitor in the perioperative period mayresult in a more mature, organized tendon-bone interfaceand is consistent with other recent studies that have eval-uated the role of MMPs in tendon-bone healing.9 Demiraget al9 investigated the effect of local MMP inhibition ontendon healing in a bone tunnel in a rabbit model ofanterior cruciate ligament reconstruction. Similar to ourstudy design, the A2 M was administered as a single, directinjection without a carrier vehicle in the perioperativeperiod. The A2 M-treated group demonstrated less scartissue and a greater number of Sharpey-like fibers at thetendon-bone interface and a significantly greater biome-chanical load-to-failure relative to control animals.9

Our study failed to demonstrate a greater load-to-failureor stiffness of the A2 M-treated repairs compared withcontrol specimens at 4 weeks postoperatively. Our data

Figure 8 Staining of the healing enthesis at 4 weeks for a-smooth muscle actin at (A) original magnification �40 and (B)� 100revealed neoangiogenesis to be predominantly localized to the musculotendinous junction just proximal to the healing enthesis (blackarrow) in both groups. No quantitative differences in vascularity were evident between control and a-2-macroglobulin (A2 M)-treatedspecimens. The black box in panel A marks the tendon-to-bone interface that is magnified in panel B.

Acknowledgment

We would like to acknowledge Lilly Ying for herexpertise and technical assistance with preparation ofslides for histologic analysis and Dr Chris T. Chen(Hospital for Special Surgery) for the generous donationof the COL 2/3-short monoclonal antibody.

Disclaimer

None of the authors, their immediate family, and anyresearch foundation with which they are affiliatedreceived any financial payments or other benefits fromany commercial entity related to the subject of thisarticle.

390 A. Bedi et al.

suggest that improvement in collagen organization at themacrostructural level is not by itself sufficient to improveattachment strength. Other important structural and envi-ronmental factors that are likely to affect biomechanicalfunction include development of mineralized fibrocartilage,collagen cross-linking, and proteoglycan content.2,16,19,29,30

The lack of a significant difference in the A2 M-treatedgroup could reflect that the rotator cuff heals expeditiouslyin a rodent model without augmentation, such that differ-ences could not be detected by 4 weeks. On the other hand,it is possible that the improved structural outcomes in theA2 M-treated group may not have had time to manifestthemselves by 4 weeks and that this group might in factshow mechanical differences from control specimens atlater times. Regardless, we suspect that evaluation at earlierand later times is necessary to identify the differences instructural integrity of the repair that are associated withlocal administration of a MMP inhibitor.

This study is not without limitations. Local drugdelivery is challenging, and our model provides no infor-mation on the bioavailability or duration of A2 M activityat the repair site. The binding and retention of A2 M at therepair site without a carrier protein in our model isunknown. Although various scaffolds and infusion pumpscan be used to provide sustained local delivery of biologicagents, each technique is associated with additionalcomplications and confounding variables from the morecomplex surgical procedure that limit the applicability ofthe results.27

In addition, we did not perform assays to directlyquantify enzymatic activity. However, immunofluorescencestaining for collagen cleavage products was performed toprovide an indirect assessment of local MMP activity in thetreatment and control groups. Ongoing studies using agentswith more predictable pharmacokinetics and with directmeasures of local MMP activity are in progress to furtherelucidate the role of MMPs in the postoperative healing

process at the enthesis. Our biomechanical testing was alsoperformed using an actuator displacement system, and weacknowledge that the reliability and reproducibility ofresults might have been improved with an optically basedsystem. In addition, the biology of healing in our acutedetachment and repair model may be significantly differentfrom what is observed in the setting of rotator cuff tendi-nosis and secondary degenerative tears.28

In conclusion, local inhibition of MMP activity afterrotator cuff repair is associated with significant histologicdifferences at the healing enthesis. Further investigationsare necessary to correlate these findings with changes in thestructural integrity of the repair. Modulation of MMPactivity in the perioperative period may offer a novel bio-logic pathway to augment tendon-to-bone healing afterrotator cuff repair.

MMP inhibition after rotator cuff repair 391

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