Botulinum Toxin is Detrimental to Repair of a Chronic Rotator Cuff Tear in a Rabbit Model Mohit Gilotra, Thao Nguyen, Matthew Christian, Derik Davis, R. Frank Henn III, Syed Ashfaq Hasan Department of Orthopaedics, University of Maryland, Maryland, Baltimore Received 3 May 2014; accepted 19 January 2015 Published online 28 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22836
ABSTRACT: Re-tear continues to be a problem after rotator cuff repair. Intramuscular botulinum toxin (Botox) injection can help optimize tension at the repair site to promote healing but could have an adverse effect on the degenerated muscle in a chronic tear. We hypothesized that Botox injection would improve repair characteristics without adverse effect on the muscle in a chronic rotator cuff tear model. The supraspinatus tendon of both shoulders in 14 rabbits underwent delayed repair 12 weeks after transection. One shoulder was treated with intramuscular Botox injection and the other with a saline control injection. Six weeks after repair, outcomes were based on biomechanics, histology, and magnetic resonance imaging. Botox-treated repairs were significantly weaker (2.64 N) than control repairs (5.51 N, p ¼ 0.03). Eighty percent of Botox-treated repairs and 40% of control repairs healed with some partial defect. Fatty infiltration of the supraspinatus was present in all shoulders (Goutallier Grade 3 or 4) but was increased in the setting of Botox. This study provides additional support for the rabbit supraspinatus model of chronic cuff tear, showing consistent fatty infiltration. Contrary to our hypothesis, Botox had a negative effect on repair strength and might increase fatty infiltration. ß 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1152–1157, 2015. Keywords: chronic rotator cuff tear; rotator cuff repair; botulinum toxin; fatty infiltration; rabbit model
Rotator cuff symptoms are the most commonly presenting complaints regarding the shoulder.1 Failure of rotator cuff repair continues to be a problem that has both clinical and economic implications.2 Chronic tears often lead to fatty degeneration of muscle, which is a poor prognostic factor for healing.3–5 Modulating the mechanical environment has been postulated as a way of overcoming rotator cuff biology and improving results after chronic repair. Botulinum toxin A (Botox) has been explored as a means of bioprotection or partial paralytic internal immobilization in the setting of tendon healing, with conflicting results.6–9 In the setting of a rodent acute rotator cuff model, however, healing occurs quickly and it is difficult to assess a re-tear.9 No published studies to date have explored the effect of Botox on chronic rotator cuff repair. Our main goal was to explore the effects of a single intramuscular Botox injection at the time of chronic rotator cuff repair in a well-established rabbit model.10,11 We hypothesized that Botox injection into the rabbit supraspinatus muscle reduces the active contractile force of the muscle, allowing for improved healing at the rotator cuff insertion in a chronic repair model. We also thought that the muscle with baseline fatty atrophy after chronic injury would not suffer further permanent injury after the chemically denervating effects of botulinum toxin.
MATERIALS AND METHODS Animal Model All animal procedures were approved by the Institutional Animal Care and Use Committee before the study was conducted. Fourteen skeletally mature New Zealand White Conflicts of interest: None. Correspondence to: Syed Ashfaq Hasan (T: þ1-781-388-8550; F: þ1-781-338-8550; E-mail: [email protected]) # 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
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JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2015
rabbits were obtained with a preoperative weight of 7–9 lbs. After induction and prone positioning, a 45-mm incision was made over the anterolateral aspect of the shoulder. The supraspinatus tendon was identified and sharply detached from its insertion with a scalpel10 and then sutured using a 5–0 Prolene suture (Ethicon, Somerville, NJ) to a Penrose drain (Grafco, Memphis, TN) to prevent healing. The second procedure was performed 12 weeks after the first to simulate chronic repair and to allow for fatty infiltration (Fig. 1).11–13 Using the previous incision and exposure, the supraspinatus stump was identified and Penrose drain removed. A bony trough approximately 2 2 5 mm was made at the medial aspect of the greater tuberosity. Three 1mm drill holes were created from the lateral aspect of the greater tuberosity through the trough. The supraspinatus tendon was repaired to its original insertion site with two horizontal mattress sutures with 2–0 nonabsorbable braided suture (Arthrex Fiberwire, Naples, FL). The suture was then passed through the drill holes, thus reapproximating the tendon to the trough. Botox (6 U/kg of body weight; Allergan, Irvine, CA) was injected into the supraspinatus muscle 2 cm from the myotendinous junction. The same volume of normal saline was injected into the contralateral shoulder, serving as a control. Botox dose was determined based on previous bioprotection literature, in which 6 U/kg was shown to be the optimal dose.6,9 Rabbit shoulders were not immobilized postoperatively. Animals were euthanized at 6 weeks, and bilateral shoulder girdles were harvested en bloc. A power analysis was conducted based on pilot data in a rat model because no comprehensive rabbit rotator cuff repair biomechanics study data are currently available. The primary outcome measure was ultimate load. Ten rabbits and 20 shoulders were needed to detect a 30% difference in ultimate load at 80% power with a p value of 0.05. Four animals were reserved for histological analysis. Gross Inspection Repair sites were examined macroscopically for evidence of healing scar and complete repair integrity (nominal result). Histology The supraspinatus muscle was subperiosteally dissected from its fossa and isolated with its repair site on the
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Figure 1. Fourteen rabbits underwent bilateral supraspinatus transections that were repaired after 12 weeks. Supraspinatus muscle was injected with either Botox or saline at time of repair.
proximal humerus. After fixation and decalcification, specimens were embedded in paraffin, sectioned with a microtome into 3- to 4-mm sections, and dried. Samples were stained with toluidine blue for fibrocartilage detection, Masson trichrome blue for collagen orientation, and hematoxylin and eosin staining to assess muscle morphology and fatty infiltration. Specimens were evaluated by three blinded investigators who graded level of muscle fatty infiltration on a scale analogous to Goutallier classification (Table 1)14 and collagen organization based on the Soslowsky score (Table 2).15,16 Biomechanics Twenty rabbit supraspinatus tendons were biomechanically tested for repair strength and tendon viscoelastic properties based on a well-established protocol.17–19 The muscle was bluntly removed from the tendon, and the humerus–tendon unit was stored in phosphate-buffered saline at 4 ˚C overnight for testing the next day. The biomechanics loading protocol that was used is based on a well-established model for testing small-animal rotator cuff repair.7,20 Biomechanical testing was conducted in a mini-materials testing machine (ElectroForce TestBench system; Bose Corporation, Eden Prairie, MN). Specimens were initially preloaded to 0.5 N, and then five cycles of preconditioning at 0.2%/s were applied to a strain of 5%. The specimen was stress-relaxed at 5% strain for 5 min and then loaded to failure. Stress was calculated as the tensile force/ initial area, stiffness as force/linear displacement, and tendon-scar modulus as linear portion of the stress–strain curve.
Ultimate load was determined based on point of failure and was the primary end point. Magnetic Resonance Imaging (MRI) Seven rabbits (14 shoulders) were randomly analyzed with a high-resolution 3-Tesla MRI scanner (Siemens, Washington, DC) on the same day they were euthanized. Both threedimensional proton density-weighted T2-weighted SPACE sequences and three-dimensional fat-saturated T1-weighted SPACE sequences were obtained for analysis. A blinded musculoskeletal radiologist graded the level of fatty infiltration of the supraspinatus based on Goutallier classification (Table 1)14 and determined repair integrity after reviewing all three-dimensional reconstructions. Statistical Analysis The Botox study group and saline control group were compared with the use of a paired t-test (p < 0.05) regarding biomechanics, histological grading, and radiological grading. The integrity of the suture repair was analyzed with Chisquare analysis and Fisher exact test.
RESULTS Gross Inspection Based on visual inspection, all repairs had healed. Sixty percent of the Botox-treated repairs (6 of 10 repairs) experienced partial failures, and 20% of the
Table 2. Soslowsky Scoring System Grade
Table 1. Goutallier Classification Grade 0 1 2 3 4
Findings Normal muscle Some fatty streaks 50% fat
0 1 2 3
Findings Normal collagen orientation tangentially Mild changes with collagen fibers
ABSTRACT: Re-tear continues to be a problem after rotator cuff repair. Intramuscular botulinum toxin (Botox) injection can help optimize tension at the repair site to promote healing but could have an adverse effect on the degenerated muscle in a chronic tear. We hypothesized that Botox injection would improve repair characteristics without adverse effect on the muscle in a chronic rotator cuff tear model. The supraspinatus tendon of both shoulders in 14 rabbits underwent delayed repair 12 weeks after transection. One shoulder was treated with intramuscular Botox injection and the other with a saline control injection. Six weeks after repair, outcomes were based on biomechanics, histology, and magnetic resonance imaging. Botox-treated repairs were significantly weaker (2.64 N) than control repairs (5.51 N, p ¼ 0.03). Eighty percent of Botox-treated repairs and 40% of control repairs healed with some partial defect. Fatty infiltration of the supraspinatus was present in all shoulders (Goutallier Grade 3 or 4) but was increased in the setting of Botox. This study provides additional support for the rabbit supraspinatus model of chronic cuff tear, showing consistent fatty infiltration. Contrary to our hypothesis, Botox had a negative effect on repair strength and might increase fatty infiltration. ß 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1152–1157, 2015. Keywords: chronic rotator cuff tear; rotator cuff repair; botulinum toxin; fatty infiltration; rabbit model
Rotator cuff symptoms are the most commonly presenting complaints regarding the shoulder.1 Failure of rotator cuff repair continues to be a problem that has both clinical and economic implications.2 Chronic tears often lead to fatty degeneration of muscle, which is a poor prognostic factor for healing.3–5 Modulating the mechanical environment has been postulated as a way of overcoming rotator cuff biology and improving results after chronic repair. Botulinum toxin A (Botox) has been explored as a means of bioprotection or partial paralytic internal immobilization in the setting of tendon healing, with conflicting results.6–9 In the setting of a rodent acute rotator cuff model, however, healing occurs quickly and it is difficult to assess a re-tear.9 No published studies to date have explored the effect of Botox on chronic rotator cuff repair. Our main goal was to explore the effects of a single intramuscular Botox injection at the time of chronic rotator cuff repair in a well-established rabbit model.10,11 We hypothesized that Botox injection into the rabbit supraspinatus muscle reduces the active contractile force of the muscle, allowing for improved healing at the rotator cuff insertion in a chronic repair model. We also thought that the muscle with baseline fatty atrophy after chronic injury would not suffer further permanent injury after the chemically denervating effects of botulinum toxin.
MATERIALS AND METHODS Animal Model All animal procedures were approved by the Institutional Animal Care and Use Committee before the study was conducted. Fourteen skeletally mature New Zealand White Conflicts of interest: None. Correspondence to: Syed Ashfaq Hasan (T: þ1-781-388-8550; F: þ1-781-338-8550; E-mail: [email protected]) # 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
1152
JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2015
rabbits were obtained with a preoperative weight of 7–9 lbs. After induction and prone positioning, a 45-mm incision was made over the anterolateral aspect of the shoulder. The supraspinatus tendon was identified and sharply detached from its insertion with a scalpel10 and then sutured using a 5–0 Prolene suture (Ethicon, Somerville, NJ) to a Penrose drain (Grafco, Memphis, TN) to prevent healing. The second procedure was performed 12 weeks after the first to simulate chronic repair and to allow for fatty infiltration (Fig. 1).11–13 Using the previous incision and exposure, the supraspinatus stump was identified and Penrose drain removed. A bony trough approximately 2 2 5 mm was made at the medial aspect of the greater tuberosity. Three 1mm drill holes were created from the lateral aspect of the greater tuberosity through the trough. The supraspinatus tendon was repaired to its original insertion site with two horizontal mattress sutures with 2–0 nonabsorbable braided suture (Arthrex Fiberwire, Naples, FL). The suture was then passed through the drill holes, thus reapproximating the tendon to the trough. Botox (6 U/kg of body weight; Allergan, Irvine, CA) was injected into the supraspinatus muscle 2 cm from the myotendinous junction. The same volume of normal saline was injected into the contralateral shoulder, serving as a control. Botox dose was determined based on previous bioprotection literature, in which 6 U/kg was shown to be the optimal dose.6,9 Rabbit shoulders were not immobilized postoperatively. Animals were euthanized at 6 weeks, and bilateral shoulder girdles were harvested en bloc. A power analysis was conducted based on pilot data in a rat model because no comprehensive rabbit rotator cuff repair biomechanics study data are currently available. The primary outcome measure was ultimate load. Ten rabbits and 20 shoulders were needed to detect a 30% difference in ultimate load at 80% power with a p value of 0.05. Four animals were reserved for histological analysis. Gross Inspection Repair sites were examined macroscopically for evidence of healing scar and complete repair integrity (nominal result). Histology The supraspinatus muscle was subperiosteally dissected from its fossa and isolated with its repair site on the
BOTULINUM TOXIN, ROTATOR CUFF TEAR
1153
Figure 1. Fourteen rabbits underwent bilateral supraspinatus transections that were repaired after 12 weeks. Supraspinatus muscle was injected with either Botox or saline at time of repair.
proximal humerus. After fixation and decalcification, specimens were embedded in paraffin, sectioned with a microtome into 3- to 4-mm sections, and dried. Samples were stained with toluidine blue for fibrocartilage detection, Masson trichrome blue for collagen orientation, and hematoxylin and eosin staining to assess muscle morphology and fatty infiltration. Specimens were evaluated by three blinded investigators who graded level of muscle fatty infiltration on a scale analogous to Goutallier classification (Table 1)14 and collagen organization based on the Soslowsky score (Table 2).15,16 Biomechanics Twenty rabbit supraspinatus tendons were biomechanically tested for repair strength and tendon viscoelastic properties based on a well-established protocol.17–19 The muscle was bluntly removed from the tendon, and the humerus–tendon unit was stored in phosphate-buffered saline at 4 ˚C overnight for testing the next day. The biomechanics loading protocol that was used is based on a well-established model for testing small-animal rotator cuff repair.7,20 Biomechanical testing was conducted in a mini-materials testing machine (ElectroForce TestBench system; Bose Corporation, Eden Prairie, MN). Specimens were initially preloaded to 0.5 N, and then five cycles of preconditioning at 0.2%/s were applied to a strain of 5%. The specimen was stress-relaxed at 5% strain for 5 min and then loaded to failure. Stress was calculated as the tensile force/ initial area, stiffness as force/linear displacement, and tendon-scar modulus as linear portion of the stress–strain curve.
Ultimate load was determined based on point of failure and was the primary end point. Magnetic Resonance Imaging (MRI) Seven rabbits (14 shoulders) were randomly analyzed with a high-resolution 3-Tesla MRI scanner (Siemens, Washington, DC) on the same day they were euthanized. Both threedimensional proton density-weighted T2-weighted SPACE sequences and three-dimensional fat-saturated T1-weighted SPACE sequences were obtained for analysis. A blinded musculoskeletal radiologist graded the level of fatty infiltration of the supraspinatus based on Goutallier classification (Table 1)14 and determined repair integrity after reviewing all three-dimensional reconstructions. Statistical Analysis The Botox study group and saline control group were compared with the use of a paired t-test (p < 0.05) regarding biomechanics, histological grading, and radiological grading. The integrity of the suture repair was analyzed with Chisquare analysis and Fisher exact test.
RESULTS Gross Inspection Based on visual inspection, all repairs had healed. Sixty percent of the Botox-treated repairs (6 of 10 repairs) experienced partial failures, and 20% of the
Table 2. Soslowsky Scoring System Grade
Table 1. Goutallier Classification Grade 0 1 2 3 4
Findings Normal muscle Some fatty streaks 50% fat
0 1 2 3
Findings Normal collagen orientation tangentially Mild changes with collagen fibers
