J Shoulder Elbow Surg (2010) 19, 384-391
www.elsevier.com/locate/ymse
BASIC SCIENCE
The effect of matrix metalloproteinase inhibition on tendon-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,* a
Sports Medicine and Shoulder Surgery, Laboratory for Soft Tissue Research, Hospital for Special Surgery, New York, NY, USA b Laboratory for Soft Tissue Research, Hospital for Special Surgery, New York, NY, USA c Hospital 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 rotator cuff tears. We hypothesize that local delivery of a MMP inhibitor after surgical repair of the rotator cuff will improve healing at the tendon-to-bone surface interface. Materials and methods: Sixty-two male Sprague-Dawley rats underwent acute supraspinatus detachment and repair. In the control group (n ¼ 31), the supraspinatus was repaired to its anatomic footprint. In the experimental group (n ¼ 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 and 4 weeks for histomorphometry, immunohistochemistry, and biomechanical testing. Statistical comparisons were 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 specimens compared with controls at 2 weeks (P < .05). Significantly greater collagen organization was observed in the A2 M-treated animals compared with controls at 4 weeks (P < .01). A significant reduction in collagen degradation was observed at both 2 and 4 weeks in the experimental group (P < .05). Biomechanical 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 the tendon-to-bone interface after rotator cuff repair. Modulation of MMP activity after rotator cuff repair may offer a novel biologic pathway to augment tendon-to-bone healing after rotator cuff repair. Level of evidence: Basic science study Ó 2010 Journal of Shoulder and Elbow Surgery Board of Trustees. Keywords: Matrix metalloproteinase; inhibitor; rotator cuff repair; tendon-bone healing
*Reprint requests: Scott A. Rodeo, MD, Attending Surgeon and CoChief, Sports Medicine and Shoulder Surgery, Laboratory for Soft Tissue Research, Hospital for Special Surgery, 535 E 70th St, New York, NY 10021. E-mail address:
[email protected] (S.A. Rodeo).
Rotator cuff tears are among the most common causes of shoulder pain and disability in adults. Despite significant advances in arthroscopic techniques for surgical repair, clinical series of large rotator cuff tears have demonstrated a significant incidence of structural failure after surgical repair when evaluated objectively with imaging studies.10-12
1058-2746/2010/$36.00 - see front matter Ó 2010 Journal of Shoulder and Elbow Surgery Board of Trustees. doi:10.1016/j.jse.2009.07.010
MMP inhibition after rotator cuff repair In addition, the overall structure, composition, and organization of the rotator cuff insertion is not reproduced after surgical repair and reflects an inability to recapitulate the events that occur during embryonic development with current surgical techniques. Rather than regenerating the 4 organized zones of direct insertion, the tendon heals with an interposed zone of fibrovascular scar tissue between the tendon and bone. If healing is successful, this interface tissue gradually matures until its matrix consists of oriented Sharpey-like collagen fibers that bridge the tendon-to-bone surface.25,26 Although pain relief may be achieved despite structural failure of a rotator cuff repair, optimal long-term outcomes that achieve both pain relief and restoration of strength are predicated on successful tendon-bone healing and the reconstitution of a robust enthesis.10-12 The native rotator cuff insertion is a highly organized, complex interface between tendon and bone. Microscopic examination of the enthesis shows interdigitation of the collagen fibers with bone though 4 distinct histologic zones: tendon, unmineralized fibrocartilage, mineralized fibrocartilage, and bone.31 This graduated change in stiffness allows for transmission of complex mechanical loads from soft tissue to bone while minimizing peak stresses at any single point along the tendon. Matrix metalloproteinases (MMPs) are a family of zincdependent proteases that collectively degrade components of tissue extracellular matrix. Recent studies have identified a critical role of these enzymes in tumor growth, vascular aneurysmal disease, embryonic development, and normal tissue remodeling.3 The tissue inhibitors of matrix metalloproteinases (TIMPs) are natural, endogenous inhibitors of MMPs that provide a check-and-balance system to modulate reparative and degradative processes and maintain a dynamic homeostasis of the extracellular matrix.3 Loss of homeostasis resulting in increased MMP activity has been associated with degenerative tendinopathy and rotator cuff disease.17,18 An imbalance of MMP activity has been demonstrated in pathologic Achilles and patellar tendinosis.1,7,8,13-15,18,20,23,24 Lo et al17 recently demonstrated statistically significant alterations in messenger RNA (mRNA) levels of specific MMPs and TIMPs in patients with full-thickness rotator cuff tears compared with matched controls, suggesting that biologic modulation of endogenous MMP activity to basal levels may reduce pathologic tissue degradation and favorably influence healing after rotator cuff repair.9,21,22 The purpose of this study was to determine the effect of a broad-spectrum MMP inhibitor on tendon-to-bone surface healing after acute rotator cuff repair. We used an established rat rotator cuff repair model to evaluate tendonto-bone surface healing with immediate postoperative inhibition of MMP. We hypothesized that local inhibition of MMPs would result in (1) improved collagen fiber organization at the healing tendon-bone interface and (2) increased failure load at the tendon attachment site.
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Materials and methods This study was approved by our Institutional Animal Care and Uses Committee (IACUC), with assigned protocol # 08-07-09R.
Study design We selected a rat model to study rotator cuff tendon healing after surgical repair because previous studies have demonstrated anatomic similarities with the human shoulder. We obtained 62 male Sprague-Dawley rats (weight, 250-300 grams). Each animal underwent detachment and immediate repair of the right supraspinatus tendon using bone tunnel and suture fixation as described by Carpenter et al.4 Postoperatively, they were housed in pairs and allowed ad libitum activity. The animals were divided into 2 experimental groups: (1) local application of a-2-macroglobulin (A2 M), a universal MMP inhibitor, at the tendon-bone interface after repair, or (2) surgical repair only. The animals were sacrificed at 2 and 4 weeks for biomechanical and histologic analysis.
Surgical technique The rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg). Anesthesia was maintained using 2% isoflurane (Baxter Inc, Deerfield, IL). All operations were performed using a sterile technique, with the rat in the lateral decubitus position. A deltoid-splitting incision was made, and the acromioclavicular joint was divided, allowing visualization of the rotator cuff tendons. The supraspinatus tendon was isolated and 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 greater tuberosity and the footprint gently decorticated with a scalpel blade to ensure complete de´bridement of the native enthesis. Crossed bone tunnels were drilled at the anterior and posterior margins of the footprint and 2 mm lateral to the articular surface using a 22-gauge needle. Suture ends were passed through the bone tunnels and firmly tied over the humeral metaphyseal cortex, anatomically repairing the supraspinatus tendon to its native footprint (Figure 1). In the experimental group, 25 mL of recombinant A2 M (1 IU/ Kg; Roche Applied Science, Indianapolis, IN), an endogenous plasma glycoprotein and universal inhibitor of MMPs, was directly applied to the tendon-bone interface without a carrier vehicle. The dose was selected based on work by Demirag et al9 that demonstrated efficacious MMP inhibition with local administration at this concentration in a rabbit model. The deltoid split and wound was closed in a standard, layered fashion with absorbable sutures. Buprenorphine (0.05 mg/kg) was administered subcutaneously for analgesia during the postoperative period. Ad libitum weight bearing and cage activity was allowed postoperatively.
Biomechanical testing The shoulders of 30 animals were used for biomechanical testing at 4 weeks postoperatively. On the day of testing, each shoulder was thawed at room temperature, and the humerus with the
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Figure 1 Acute rotator cuff repair model. (A) The acromioclavicular joint is divided and the deltoid is split in line with its fibers to allow atraumatic exposure and isolation of the supraspinatus tendon. (B) A modified Mason-Allen stitch is placed with a 4-0 Ethibond suture (Johnson and Johnson, Piscataway, NJ) in the distal supraspinatus tendon. (C) The tendon is divided directly off the greater tuberosity and the footprint is defined and gently de´brided of any residual debris. In the experimental group, a-2-macroglobulin protein is applied to the tendon-bone interface before the transosseous sutures are tied. (D) The sutures are passed by way of the crossed transosseous bone tunnels 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 was determined by outlining the area of metachromasia. The black boxes represent areas of interest at the healing tendon-bone interface. The total area of metachromasia (mean standard deviation) for each specimen was measured and recorded using Image J computerized imaging software (National institutes of Health, Bethesda, MD). attached supraspinatus was meticulously dissected under magnification. Dissections were performed starting from normal tissue planes medially, unequivocally isolating the supraspinatus muscle and its tendon above the scapular spine and dividing the rotator interval anteriorly. All dissections were performed in a blinded fashion with respect to treatment group.
The dimension of each supraspinatus tendon was measured using a digital micrometer. The specimen was then placed into a customdesigned uniaxial testing system. The tendon was secured in a screw grip using sandpaper and ethyl cyanoacrylate (Elmer’s Products Inc, Columbus, OH). The humerus was secured into a custom-designed vice grip that prevented fracture through the humeral physis. The
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supraspinatus tendon was secured to 45-N load cell attached to a linear bearing that allowed alignment of the tendon in the direction of its pull. The humeral jig was secured to the linear stage. The specimen was preloaded to 0.10 N and then loaded to failure at a rate of 1.4 mm/ corresponding to approximately 0.4% strain.4,28 Samples were tested in air at room temperature, and saline spray was used to ensure specimens remained moist throughout testing. The maximum load-to-failure and failure location were recorded. Displacement was measured using a 1-mm resolution micrometer system attached to the linear stage. The linear region of the stress-strain curve was used to calculate the stiffness for each specimen.
Histologic analysis Thirty-two animals were used for histologic analysis. Eight rats in the experimental and control groups were sacrificed at 2 weeks and 4 weeks after surgery. The tissue specimens were fixed in 10% neutral buffered formalin for 48 hours. After fixation, tissues were decalcified in Immunocal (Decal, Congers, NY) for 48 hours and washed in phosphate-buffered saline solution. The tissues were dehydrated and embedded in paraffin. Sections 5-mm thick, including the repaired supraspinatus tendon and the greater tuberosity, were cut in the coronal plane and stained with hematoxylin and eosin, safranin-O, and picrosirius red. Six slides were made from each specimen (48 slides/group). The qualitative appearance of the repair site was evaluated in a blinded fashion. The greater tuberosity, repaired tendon-bone insertion site, and midsubstance of the supraspinatus tendon were examined under light and polarized light microscopy (Eclipse E800; Nikon, Melville, NY). Digital images were captured using a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI). Picrosirius red staining and examination with monochromatic polarized microscopy was used for semiquantitative analysis of collagen organization at the healing enthesis. The sulfonic acid groups on the picrosirius red molecules bind to the amino groups in collagen in an oriented fashion, resulting in a 7-fold increase in collagen birefringence. By quantifying the birefringence of collagen under polarized light (based on brightness), time-dependent differences in collagen deposition and maturation in the healing tendon could be detected.6 Measurements were obtained by rotating the polarization plane until maximum brightness was obtained to control for variations in specimen orientation on the slide. To facilitate comparisons between groups, all tissues were embedded and cut in exactly the same orientation, and sections were cut to a uniform thickness. Digital images obtained under 100 magnification underwent 8-bit digitization by Image J software (National Institutes of Health, Bethesda, MD), with a resolution of 640 (horizontal) 480 (vertical) pixels. The microscope 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 gray scales from 1 to 255. The gray scale measurements were performed at the tendon-bone interface using the Image J Software program.6 Five rectangular areas (2500 mm2 each) were randomly selected, and gray scales were measured (mean standard deviation). The light intensities were measured under exactly the same conditions of illumination for all specimens. The area of new fibrocartilage formation at the tendon-bone interface was determined by outlining the area of metachromasia with safranin-O staining at 40 magnification. The total area of metachromasia (mean standard deviation) for each specimen
Figure 3 Significantly greater new fibrocartilage formation was noted at the healing enthesis of a-2-macroglobulin (A2 M) protein-treated specimens compared with control specimens at 2 weeks postoperatively (P < .05). was measured using Image J computerized imaging software. The consensus of 3 independent observers was used for histomorphometric measurements. For immunohistochemical analysis, serial sections were treated with 3% H2O2 to quench endogenous peroxidase activity, nonspecific antibody-binding was blocked with 5% goat serum, and 1% bovine serum albumin in PBS was used as a negative secondary reagent control. The sections were stained using a monoclonal antibody to asmooth muscle actin (a-SMA) and factor VIII, a marker of myofibroblasts and vascular endothelium (Sigma, St. Louis, MO). Each monoclonal antibody was applied to separate serial sections for 60 minutes at 37 C, and bound antibodies were visualized using a goat avidin-biotin peroxidase system with 3,30 -diaminobenzidine (DAB, Dako Corp, Carpinteria, CA) as a substrate. Collagen degradation was assessed with immunofluorescence analysis using COL-2/3 short, a monoclonal antibody specific to cleaved type I or II collagen fragments (generously donated by Dr C.T. Chen).32 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. Semiquantitative analysis was performed at 100 magnification by counting the ratio of chondrocytes with pericellular green staining to the overall number of chondrocytes (ie, red nuclei) in the area of metachromasia for each tendon-bone interface and provided an indirect assay of MMP activity.
Statistical analysis Statistical analysis was performed using unpaired t tests with SigmaStat software (Systat Software Inc, Chicago, IL). Statistical significance was set at P < .05. Comparison of histomorphometric measures of fibrocartilage formation, collagen organization, and collagen fragmentation between the control and experimental groups was performed using an unpaired t test at 2 and 4 weeks postoperatively. An unpaired t test was also used to compare the load-to-failure and stiffness of the healing enthesis of the control and experimental groups at 4 weeks.
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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 of interest at the healing tendon-bone interface. Measurements were obtained by rotating the polarization plane until maximum brightness was obtained to control for variations in specimen orientation on the slide.
Results Gross and histologic examination All rotator cuff repairs were noted to be grossly intact at the time of sacrifice. No humeral physeal fractures or blowouts of the transosseous tunnels were encountered. Histomorphometric analysis demonstrated a significantly greater area of new fibrocartilage at the healing enthesis in the A2 M-treated compared with the control group at 2 weeks (P < .05; Figures 2 and 3). No significant difference in metachromasia was detected by 4 weeks. Analysis of collagen birefringence revealed significantly greater collagen organization in the A2 M-treated group compared with control animals at 4 weeks (P < .01; Figures 4 and 5). No significant difference in collagen organization was noted at 2 weeks postoperatively. Immunofluorescence analysis for cleaved collagen I and II fragments demonstrated a significant reduction in collagen degradation at the tendon-bone interface of the A2 M-treated group at both 2 and 4 weeks compared with control specimens (P < .05; Figures 6 and 7). Staining with monoclonal antibodies for a-SMA and factor VIII revealed neoangiogenesis to be predominantly localized to the musculotendinous junction just proximal to the healing enthesis in both groups. Organized blood vessels were observed by 4 weeks in both groups, with no quantitative differences evident between control and A2 Mtreated specimens.
Biomechanical testing Biomechanical testing at 4 weeks revealed an ultimate load-to-failure of 23.3 6.2 N for the control and 23.2 7.6 N for A2 M-treated groups. Mean stiffness of the constructs was 12.0 4.0 N/mm and 9.0 4.0 N/mm,
Figure 5 Collagen fibers at the healing enthesis were significantly more organized in the specimens treated with a-2macroglobulin (A2 M) compared with control specimens at 4 weeks postoperatively (P < .01). The error bars designate the standard deviation.
respectively. No significant difference in stiffness or ultimate load-to-failure was detected between groups.
Discussion Recent studies have demonstrated a potentially critical role of MMPs and their inhibitors in the pathophysiology of rotator cuff tears. We hypothesized that local delivery of an MMP inhibitor after surgical repair of the rotator cuff will improve healing at the tendon-to-bone surface interface. Our results demonstrate that local administration of a-2macroglobulin, an endogenous MMP inhibitor, at the greater tuberosity footprint is associated with distinct
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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 magnification 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-macroglobulin (A2 M) compared with control specimens at 2 and 4 weeks postoperatively (P < .05). The error bars designate the standard deviation.
histologic differences at the healing enthesis after rotator cuff repair. A statistically significant reduction in local collagen degradation was achieved and maintained at both 2 and 4 weeks postoperatively. This reduction in MMP activity was associated with greater new fibrocartilage formation at 2 weeks and increased collagen organization at 4 weeks in the healing enthesis. The MMPs and their endogenous inhibitors play a critical role in maintaining the dynamic homeostasis and integrity of the extracellular matrix. Loss of this balance resulting in elevated MMP activity has been associated with a number of pathologic conditions of connective tissue, including degenerative tendinopathy and rotator cuff tears.1,7,8,13-15,18,23,24 Lo et al17 demonstrated statistically significant alterations in mRNA levels of MMPs and TIMPs in patients with fullthickness rotator cuff tears.17 Choi et al5 found MMP-2 to be expressed and activated during the healing process of an acute supraspinatus tendon tear. Yoshihara et al32 and Zhen et al33 have demonstrated elevated levels of MMP-1, MMP-3, and glycosaminoglycans in the synovial fluid of patients with massive rotator cuff tears. These studies have provided
increasing evidence to suggest that biologic modulation of endogenous MMP activity to basal levels may reduce pathologic tissue degradation and favorably influence healing after rotator cuff repair.9,17,21,22 The results from our study suggest that local administration of an MMP inhibitor in the perioperative period may result in a more mature, organized tendon-bone interface and is consistent with other recent studies that have evaluated the role of MMPs in tendon-bone healing.9 Demirag et al9 investigated the effect of local MMP inhibition on tendon healing in a bone tunnel in a rabbit model of anterior cruciate ligament reconstruction. Similar to our study design, the A2 M was administered as a single, direct injection without a carrier vehicle in the perioperative period. The A2 M-treated group demonstrated less scar tissue and a greater number of Sharpey-like fibers at the tendon-bone interface and a significantly greater biomechanical load-to-failure relative to control animals.9 Our study failed to demonstrate a greater load-to-failure or stiffness of the A2 M-treated repairs compared with control specimens at 4 weeks postoperatively. Our data
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Figure 8 Staining of the healing enthesis at 4 weeks for a-smooth muscle actin at (A) original magnification 40 and (B) 100 revealed neoangiogenesis to be predominantly localized to the musculotendinous junction just proximal to the healing enthesis (black arrow) in both groups. No quantitative differences in vascularity were evident between control and a-2-macroglobulin (A2 M)-treated specimens. The black box in panel A marks the tendon-to-bone interface that is magnified in panel B.
suggest that improvement in collagen organization at the macrostructural level is not by itself sufficient to improve attachment strength. Other important structural and environmental factors that are likely to affect biomechanical function 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-treated group could reflect that the rotator cuff heals expeditiously in a rodent model without augmentation, such that differences could not be detected by 4 weeks. On the other hand, it is possible that the improved structural outcomes in the A2 M-treated group may not have had time to manifest themselves by 4 weeks and that this group might in fact show mechanical differences from control specimens at later times. Regardless, we suspect that evaluation at earlier and later times is necessary to identify the differences in structural integrity of the repair that are associated with local administration of a MMP inhibitor. This study is not without limitations. Local drug delivery is challenging, and our model provides no information on the bioavailability or duration of A2 M activity at the repair site. The binding and retention of A2 M at the repair site without a carrier protein in our model is unknown. Although various scaffolds and infusion pumps can be used to provide sustained local delivery of biologic agents, each technique is associated with additional complications and confounding variables from the more complex surgical procedure that limit the applicability of the results.27 In addition, we did not perform assays to directly quantify enzymatic activity. However, immunofluorescence staining for collagen cleavage products was performed to provide an indirect assessment of local MMP activity in the treatment and control groups. Ongoing studies using agents with more predictable pharmacokinetics and with direct measures of local MMP activity are in progress to further elucidate the role of MMPs in the postoperative healing
process at the enthesis. Our biomechanical testing was also performed using an actuator displacement system, and we acknowledge that the reliability and reproducibility of results might have been improved with an optically based system. In addition, the biology of healing in our acute detachment and repair model may be significantly different from what is observed in the setting of rotator cuff tendinosis and secondary degenerative tears.28 In conclusion, local inhibition of MMP activity after rotator cuff repair is associated with significant histologic differences at the healing enthesis. Further investigations are necessary to correlate these findings with changes in the structural integrity of the repair. Modulation of MMP activity in the perioperative period may offer a novel biologic pathway to augment tendon-to-bone healing after rotator cuff repair.
Acknowledgment We would like to acknowledge Lilly Ying for her expertise and technical assistance with preparation of slides for histologic analysis and Dr Chris T. Chen (Hospital for Special Surgery) for the generous donation of the COL 2/3-short monoclonal antibody.
Disclaimer None of the authors, their immediate family, and any research foundation with which they are affiliated received any financial payments or other benefits from any commercial entity related to the subject of this article.
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